Proceedings 10th Mountain Cheese Def

10th INTERNATIONAL Meeting on Mountain cheese 14-15 september 2011 dronero (cn), italy
farming systems, milk and cheese quality and implications for the future
Dairy Production in Mountain:
Proceedings of the 10th International Meeting on Mountain cheese
14-15 September 2011, Dronero, italy
Edited by University of Turin, Italy
Organizing Committee: Giorgio Borreani (University of Turin - Italy), Mauro Coppa (INRA, University of Turin - Italy), Daniele Giaccone (ARAP - Italy), Davide Veglia (Tecnogranda s.p.a.), Bruno Martin (INRA - France), Marie-Christine Montel (INRA - France) Scientific Committee: Donato Andueza (France), Marta Bertolino (Italy), Giorgio Borreani (Italy), Milena Brasca (Italy), Solange Buchin (France), Mauro Coppa (Italy), Paolo Cornale (Italy), Anne Ferlay (France), Florian Leiber (Switzerland), Bruno Martin (France), Marie-Christine Montel (France), Eric Mosimann (Switzerland), Andrea Revello Chion (Italy), Manuela Renna (Italy), Ernesto Tabacco (Italy) Scientific Secretary: Mauro Coppa (INRA, University of Turin - Italy) With the support of the following organisation: Dep. Agroselviter, University of Turin
Associazione Regionale Allevatori Piemonte (ARAP)
Comunit Montana Valli Grana e Maira
Comune di Canosio
Agrilab s.r.l.
Banca Cassa di Risparmio di Savigliano
Camera di Commercio di Cuneo
Tecnogranda s.p.a. Edited by: Mauro Coppa & Giorgio Borreani - Dep. Agroselviter, University of Turin ISBN 978-88-902754-5-6 Copyright 2011 Universit di Torino Printed by: Universit degli Studi di Torino - Via Verdi, 8 - 10124 Torino
Foreword
Welcome to the 10th International Meeting on Mountain Cheese. For its 10th edition, the International Meeting on Mountain Cheese is back in the Italian Alps. It takes place in Dronero, Piedmont region, September 14 and 15, 2011. The first seminars were initiated by Jacques Olivier Bosset from Switzerland who is now retired. They gathered an average of 50 scientists interested in the specific qualities of mountain cheeses. They took place in various European mountains; lEtivaz, Switzerland (1999), Luz Saint Sauveur, France (2000), Bella, Italy (2001), Aosta, Italy (2002), Arches, France (2003), Ragusa, Italy (2004), Valle de Joux, Switzerland (2005), Metsovo, Greece (2006), Saint Eulalie, France (2009). This 10th meeting is a great success: about 80 scientists from 7 European and non-European countries attend Mountain Cheese 2011! The countryside around Dronero at the bottom of the Maira Valley, one of the most beautiful alpine valley of Cuneo province is delightful in late summer and certainly contributes to create a stimulating and fruitful atmosphere for exchanging scientific information and deepening mutual friendships. The programme of this 10th Meeting on Mountain Cheeses includes 23 oral communications and 12 posters presented in the full session. These communications are spilt into two complementary sessions dealing with milk and cheese quality in relation to production conditions and mountain cheese microbiology. Each session will end with an invited lecture devoted to promote a general discussion on these key issues for the mountain cheeses. All the contributions are published into the following pages of these proceedings. The programme of this 10th Mountain Cheese covers a wide range of topics all related to the dairy production in mountains. They deal with the mountain farming systems, the milk and cheese qualities and their implications for the future. The ultimate goal of the meeting is to provide a comprehensive view of the current knowledge on the specific mountain cheese qualities and finally to draw perspectives for mountain farming. As in previous Mountain Cheese meetings, all the participants share the same idea that the challenge for mountain farming is to convert the natural handicaps linked to climate, topography or resources into strengths offered by the extraordinary diversity and originality of its productions. The contributions deal with the global quality of mountain cheeses, including environmental sustainability, organoleptic, nutritional and microbial qualities as well as their possible authentication. The scientific and organizing committees of Mountain Cheeses 2011 hope that the proceedings will be a tool and an inspiration for future progress in the field of the mountain cheese production. We would like to express our warm thanks to the numerous contributors in the organisation and in particular, to the scientific committee and chairpersons, whose expertise was essential for the publication and discussion of the scientific contributions. Finally, this meeting would not have taken place without the specific contribution of the University of Turin (Dep. Agroselviter), the Associazione Regionale Allevatori Piemonte (ARAP), the Communit Montana Valli Grana e Maira, the Comune di Canosio, the Agrilab s.r.l. and the Banca Cassa di Risparmio di Savigliano. We address a special thank to the farmers of the Maira Valley for their kindness and mobilisation for the organisation of the visit of their alpine farms. The organizing committee wish you a fruitful meeting on the fascinating topics related to mountain cheese.
On behalf of the organisation committee Bruno MARTIN (INRA-France)
Table of contents
Introductive talk The mountain dairy production in Piedmont Giaccone D., Valperga T. Session 1: Mountain milk and cheese quality in relation to production conditions Oral presentations Milk fatty acid composition of tank milk according to the origin and livestock farming practices in France Ferlay A., Martin B., Delavaud C., Sibra C., Constant I., Chilliard Y., Chassaing C. Effects of farming system and cheese making technology on quality traits and fatty acid profile of Caciocavallo Palermitano cheese at different ripening time Tornamb G., Di Grigoli A., Bellina V., Mazza F., Bonanno A. . Cheese chemical profile from Zebu cows grazing on two systems - pastoral and silvopastoral - in the highlands of Colima, Mexico Galina M.A., Claps S., Rubino R., Pizzillo M., Guerrero M., Pineda L.J. . The cheese quality as tool to safeguard autochthonous sheep breeds and mountain environment Claps S., Sepe L., Caputo A.R., Di Napoli M.A.,. Morone G., Annicchiarico G., Fedele V. . Traditional milking of Salers cows: influence of removing calf on cheese making ability of milk in comparison to Holstein cows Guiadeur M., Verdier-Metz I., Monsallier F., Agabriel J., Ciri C., Montel M.C., Martin B. A down-up approach to organise the variability of commercial, nutritional and aromatic properties of cow milk and its grading into quality classes Rubino R., Masoero G., Pizzillo M. .. Sensorial characterization of Raschera PDO cheese produced by different factories in the production area Galassi L., Bianchi P., Giaccone D., Revello Chion A., Tabacco E., Borreani G. . Influence of native pasture on compositional characteristics and aroma compounds of traditional Feta cheese by multiple dynamic headspace extraction and Gas Chromatography-Mass Spectrometry Bozoudi D., Caputo A.R., Claps S., Litopoulou-Tzanetaki E. ................ Variation in volatile profile of plants grazed by sheep and the produced milk at two different areas of mountainous Greece Bozoudi D., Parisi Z., Caputo A.R., Claps S., Belibasaki S., Litopoulou-Tzanetaki E. .. Phenolic content of forage, milk, whey and cheese from goats fed Avena sativa Sepe L., Cornu A., Graulet B.,Claps S., Rufrano D. . Comparative analysis of milk and cheese produced in the Plardon PDO production zone based on their phenolic profiles Gkouma M., Cornu A., Massouras T., Lemaire M., Graulet B. .. Vitamin B9 and B12 contents in cow milk according to production system Chassaing C., Graulet B., Agabriel C., Martin B., Girard C.L......................................................... Evolution of milk calcium content during the year Hurtaud C., Johan M., Leurent S., Gallard Y., Delaby L. .............................................................. Dairy production potential of the Jura meadows Mosimann E., Belot P.E., Parguel P. ............................................................................................... Raising Rivals Costs strategy and localized agro-food systems in Europe Barjolle D., Jeanneaux P., Meyer D., Donars M. .. Invited lecture The added nutritional value of mountain dairy products Leiber F. ........................................................................................................................................... Posters Study on the combined effects of i) milk production conditions and ii) cheese making process, on sensorial characteristics of Reblochon PDO cheese Convert T. ......................................................................................................................................... Carotenoids, Vitamin A and E concentrations in Saint Nectaire cheeses according to process and season Graulet B., Chatelard C., Lepetit M., Blay B., Hulin S., Duriot B., Martin B. ................................ 9
13 15 17 19 21 23 25 27 29 31 33 35 37 39 41
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Fatty acid profile of Raschera PDO cheeses produced by different cheese factories during winter and summer Revello Chion A., Battelli G, Giaccone D., Tabacco E., Borreani G. ... Using NIR spectroscopy on cheeses for the authentication of cows feeding and geographical origin Andueza D., Constant I., Agabriel C., Lucas A., Martin B. . Kinetics of cow milk fatty acids during rapid and progressive transitions from hay- to pasture-based diets Coppa M., Gorlier A., Lonati M., Lombardi G. . An European dataset to predict milk fatty acid profile from production conditions Coppa M., Martin B., Glasser F., Chilliard Y., Chassaing C., Agabriel C., Borreani G., Barcarolo R., Ferlay A. ... Fatty acid profile of milk from Fiurin goats grazing Alpine pasture Renna M., Cornale P., Lussiana C., Battaglini L.M., Mimosi A. .. Suitability of GAEC Standard 4.6 of Cross compliance on stocking rate in the Italian mountain grazing system Sepe L., Claps S., Fedele V. .............................................................................................................. Session 2: Mountain cheese microbiology Oral presentations Microbial diversity of raw milk and influence of farm practices Mallet A., Kauffman F., Chesneau C., Sesboue A., Desmasures N. .. Biodiversity and factors of variation of microbial flora on the teats of dairy cows Verdier-Metz I., Monsallier F., Gagne G., Bornes S., Delbs-Paus C., Veisseire P., Agabriel C., Martin B., Montel M.C. ... Application of Biolog methodology for the evaluation of terpenoids influence on lactic acid bacteria Belviso S., Ambrosoli R., Giordano M., Minati J.L., Zeppa G. ... Thermophilic starters, milk composition and technological parameters affect the composition of hardcooked cheeses Buchin S., Salmon J.C., Duboz G., Faurie F., Palme R., Duployer M.H. ...................................... Volatile organic compounds produced in milk by Enterococcus faecalis Battelli G., Silvetti T., Decimo M., Brasca M. Benefits and risks associated with Gram-negative bacteria within cheese microbial communities Delbs-Paus C., Irlinger F., Coton M., Miszczycha S., Pochet S., Helinck S., Veisseire P., Callon C., Thvenot D., Coton E., Montel M.C. ............................................................................... Invited lecture Microbial ecology in service of raw milk mountain cheeses Montel M.C. ...................................................................................................................................... Posters Microbiological characteristics of traditional Caciocavallo Palermitano cheese making Settanni L., Tornamb .G, Di Grigoli A., Noto F., Bellina V., Moschetti G., Bonanno A. .... Analysis of microbiological variation in PDO Vastedda della valle del Belce cheese during the storage period Scatassa M.L., Cardamone C., Todaro M. ....................................................................................... Implantation of lactic acid bacteria originated from raw milk in artisanal PDO Ossau-Iraty cheeses manufactured with different starters. Feutry .F, Torre P., Berthier F. ....................................................................................................... Evaluation of NSLAB isolates from Graviera Kritis cheese as adjuncts according to technological and probiotic criteria Tsafrakidou P., Zdragas A., Pavlidou S., Bozoudi D., Litopoulou-Tzanetaki E. ............................. Index of authors
53 55 57 59 61 63
67 69 71 73 75 77
79
85 87 89 91 93
Introductive Talk
D. Giaccone 1, T. Valperga1 1 Regional Breeders Association of Piedmont , Via Livorno, 60-10144, Torino (TO) *arap.giaccone@envipark.com Abstract The Piedmont is one of the most important Italian Region with a long term livestock activity. The dairy productions are characterized by the production of high quality milk and by several typical and Protected Designation of Origin cheeses. The cow, goat and sheep breeding has been developed throughout the regional territory, both in the plains and in the mountain environments. The Piedmont livestock has been continuously increased over the years, both for cows and for sheep and goats. The cow milk production in Piedmont in 2010 was about 800,000 tons, used both for alimentary milk and for the production of cheeses. The Piedmont livestock and the Regional Breeders Association The Piedmont Breeders system is structured with six Provincial Breeders Association (APA) which are coordinated by the Piedmont Regional Breeders Association (ARAP). The APAs engaged technical and genetic assistance for the farmers and working for the valorisation of livestock productions (milk, meat and cheese). The ARAP based its activity on territorial coordination, on the organisation of the animal shows and managed the accredited Laboratory analysing over one million milk samples a year for quality assurance, payment on quality, dairy herd management, and advisory purposes. The Piedmont Breeders System control only the cows, the sheep and goats that are members of the National Genealogical books. This means that Piedmont Breeders System control about 2/3 of the regional livestock. Piedmont has a livestock inventory of 825,000 cattle (Table 1): this number included both animals members of the Genealogic Books and not members for milk and meat production. The most important dairy breeds for milk production are the Italian Holstein, the Italian or Alpine Brown and the Italian Red Pied (Table 2). The traditional breeds for the production both of milk and meat are the Bar Pustertaler, the Oropa Red Pied and the Valdostana cow. The famous Piemontese breed is the most important cow for the meat production in Piedmont, but in some areas, such as Alpine environment, it is also milked. Its milk production is not important as quantity (about 5-6 kg/day per cow), but it is of high quality for the cheese making because of a high level of protein and casein. The sheep breeding is most important in mountain and hill areas with the production of milk and meat. The most important dairy sheep breads in Piedmont are the Langhe sheep and Biellese sheep, whereas the main breed for the meat production is the Sambucana sheep. The goat breeding is more concentrated in the hilly regions of Langhe and Monferrato, in the provinces of Cuneo, Asti and Alessandria, but is also present in the alpine valleys of the province of Turin. The two most important bread for milk production are the Sambucana and the Camosciata, whereas the Roccaverano goat is a typical Piedmont bread, important for the milk and meat production. Table 1. Number of cattle, goats and sheep breeded in Piedmont in 2010 (Source: The Piedmont Region Assessorato alla Sanit). Breed animal Head number Bovine Italian Holstein 207,000 Italian ad Alpine Brown 6,500 Italian Red Pied 5,800 Oropa Red Pied 8,100 Valdostana Red Pied 7,100 Valdostana Black Pied 400 Piemontese 350,000 Crossbreeds 240,000 Sheep 115,000 Goat 70,000 Table 3. Production of the six PDO cheeses produced in Piedmont in 2010 (Source: CLAL). Cheese Production 2010 (t) Toma Piemontese 1,422 Raschera 836 Bra 783 Castelmagno 227 Robiola di 109 Roccaverano Murazzano 16
The mountain dairy production in Piedmont
10th International Meeting on Mountain cheese 14-15 September 2011 dronero (cn), italy
Table 2. Farms and animal controlled by the Piedmont Breeders System in 2010 (Source: Italian Breeders Association AIA). Milk Milk Average milk Number Number Medium number Protein fat Breed production of farms of cows of cows per farm (%) (%) (kg/year per cow) Italian Holstein 841 85,239 101 9,399 3.68 3.36 Italian Brown 185 2,876 15.5 6,507 3.85 3.58 Italian Red Pied 200 4,013 20.1 6,266 3.81 3.45 Oropa Red Pied 130 2,524 19.4 2,013 3.62 3.41 Valdostana Red Pied 72 1,089 15.0 2,943 3.48 3.27 The dairy production in Piedmont The dairy productions of Piedmont are famous throughout the world. Several cheeses are known and appreciate in many foreign countries. Several typologies of cheeses can be found in Piedmont produced with cows milk, sheeps milk, goats milk or mixed milk. The cheeses produced could be fresh (with maturity of 0 to 10 days), medium ripened (30-45 days), long ripened (over 90 days), blue cheeses, ect. The cheese making is present all over the regional territory from the plains to the mountains. We can found cheese factory from the valleys of the province of Cuneo, bordering to Liguria Region, till the mountains bordering France. Climbing the Alps in the province of Turin you will find several pasture areas where typical cheeses are make. In the North of the Piedmont in the area of Verbano-Cusio-Ossola, bordering Switzerland, there are also many pastures till 2,000 m (a.s.l.). The South-east of Piedmont (in the famous hills of the Langhe and Monferrato) is characterized by traditional breeding , with an important production of sheep and goat cheeses. Each zone of the Piedmont is known for the production of typical or Protected Designation of Origin (PDO) cheeses, and for some cheeses the summer production is made from animal grazing pasture. The PDO cheeses of Piedmont are nine, three of them (Grana Padano, Taleggio, and Gorgonzola) are also produced in the neighbouring Regions, whereas the other six are only produced in Piedmont (Bra, Raschera, Castelmagno, Murazzano, Robiola di Roccaverano, and Toma Piemontese) (Table 3). Bra PDO is a fat cheese product shapes weighing from 6 to 8 kg, with raw or pasteurised cow's milk (possibly supplemented with small amounts of sheep's milk and / or goat). There are two types: Bra Duro (180 days of ripening) and Bra Tenero (45 days of ripening). The Bra is produced and ripening exclusively in the province of Cuneo. Raschera PDO is a cheese made from raw cow milk, also in the typical square forms. It is produced exclusively in the province of Cuneo, can boast of the Alpine Summer Certificate when it is produced above 900 m a.s.l, in some municipalities and respecting some specific conditions defined by the Production Specifications. Castelmagno PDO is the most famous Piedmont cheese known all over the world; It is produced in only three municipalities of the Grana Valley (Pradleves, Monterosso Grana and Castelmagno) in the province of Cuneo. It is a pressed semi-hard cheese rennet and typically brittle made from raw milk of cows. Its making is characterized by the traditional "double rupture" of the curd. Possibly supplemented with small amounts of sheep's milk and/or goat, is a flat cylindrical shape with a diameter of 15-25 cm. The minimum ripening period is 60 days. Castelmagno PDO is also expected to Alpine Summer Certificate where the milk comes only from cows, goats and sheep grazing pastures in the period from early May to the end of October. Murazzano PDO is produced from sheep's milk, pure or mixed (at least 60% sheeps milk). Its only produced by the municipalities belonging to the Production Specifications, situated in the Langhe region in the province of Cuneo. Murazzano is a fresh cheese (aged at least 6 days) produced in small forms of the weight of 250-300 g. Robiola di Roccaverano PDO is made from raw goat milk, pure or mixed (at least 50% goat milk). It is produced in the hills of Asti and Alessandria provinces. It is a fresh cheese, made with the use of lactic curd, subject to different maturation periods: fresh (4-9 days), middle matured (10-29 days) or mature (over 30 days). The Toma Piemontese PDO is produced in almost all the territory of Piedmont with only cow milk, whole or skimmed, raw or pasteurised. Among other typical Piedmont cheeses, should be remembered: the Bettelmat, cow's milk cheese produced in the mountains of Verbano-Cusio-Ossola whose origin is back to mid-1800; the Maccagno cheese typical production of Biella and valley of the Val Sesia, made from raw cows milk; the Murianengo cheese is one of the blue cheese and it is produced with cow or mixed milk, in the Moncenisio area (Torino); and others such as the Toma di Lanzo, the Testun, the Tomino of Melle, Robiola d'Alba. The ricotta cheese Seirass del Fen, are product with the whey of cow or mixed milk, and traditionally it is ripening in the hay, and is product in the Turins valley.
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Session 1: Mountain Milk and Cheese Quality in Relation to Production Conditions Oral presentations
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Fatty acid composition of tank milk according to the origin and livestock farming practices in France
A. Ferlay1*, B. Martin1, C. Delavaud1, C. Sibra2-3, I. Constant1, Y. Chilliard1, C. Chassaing2-3 1 INRA, UR1213 Herbivores, 63122, Saint Gens-Champanelle, France 2 Clermont Universit, VetAgro Sup, UR 2008.03.102 EPR, BP10448, F-63000, Clermont-Ferrand, France 3 INRA, USC 2005, 63370 Lempdes, France *anne.ferlay@clermont.inra.fr Abstract The aim of this study was to link the variability of the milk fatty acid (FA) composition of tank milk to the production conditions of different farms in France. The milk samples were collected from 10 tours located in lowland using either corn (CL, n=5) or grass (GL, n=5) based diets and 10 tours located in mountain using either corn (CM, n=5) or grass (GM, n=5) based diets. Tank milk samples were collected 5 periods across the year for analysis of milk FA composition. In order to characterize the production system on the day of sampling, surveys were carried out with each farmer involved in the experiment. The period had a significant effect on milk FA composition. The saturated FA (SFA) content decreased during spring whereas monounsaturated FA (MUFA) and 18:3n-3 content increased. Total trans 18:1 content increased during spring and more markedly in G systems. The SFA content was higher for C than for G systems. Keywords: fatty acids, tank milk, dairy cows, feeding practices, Introduction Milk FA composition has gained attention in recent years because of its potential nutritional implication in human health. Although milk fat is highly saturated, which could be related to coronary heart disease risk (Givens, 2010), n-3 FA, conjugated linoleic acid (CLA), odd- and branched-chain FA (OBCFA) could have potential antiatherogenic, antiobesity, or anticarcinogenic effects (Shingfield et al., 2008). Cow nutrition is the major factor affecting milk FA composition (Chilliard et al., 2007). Several experiments have been conducted under controlled conditions whereas information under real farming conditions is scarce. Moreover, it becomes important for the dairy industry to characterize tank milks in terms of FA composition according to the growing consumer demand to have dairy products with high nutritional quality. The aims of this study were on the tour scale to characterize across the year the variability of the milk FA composition in different production systems according to the nature of forage and the altitude, and to correlate farm feeding practices with milk FA composition. Material and methods The study was conducted on 20 tours (each of 5 farms) divided between 4 production systems characterized by their forage system (grassland (G) vs. corn silage (C)) and altitude (lowland (L) vs. mountain (M)). For each tour, the tank milk was collected 5 periods across the year: twice during the winter period with diets based on conserved forages (January and February) and 3 times during the grazing period (May, July and September). In order to characterize the milk production conditions (feeding and herd characteristics) corresponding to the day of sampling, surveys were carried out with each farmer involved in the experiment. Details on diet composition for each tour are described in a companion paper (Chassaing et al., 2011). The FA in lyophilized milk were methylated and analyzed by gas chromatography (Thermo Finnigan, Les Ulis, France). Data were analyzed using the mixed procedure for repeated measures (SAS Version, 9.2, SAS Institute Inc., Cary, NC) with production system, period and the interaction as fixed effects, and tour as random effect. Results and discussion Period had significant effect on milk FA concentrations. The milk SFA decreased strongly from February (70.04 g/100g of total FA) to early spring (66.74) and then increased slightly until September (P <0.001). The SFA concentrations tended to differ according the production systems, with higher values for C than for G systems (70.0 vs. 68.0, respectively). The total cis 18:1 increased between winter and spring (19.3 vs. 20.3). The total trans 18:1 increased between February (2.21) and May (4.09), with the highest values for the G systems (3.44 vs 2.65). The milk 18:3n-3 and c9t11-CLA were higher for G than for C systems across the year (0.76 vs 0.49 and 1.04 vs 0.57, respectively). These PUFA concentrations increased with grazing period, in agreement with 13
Chilliard et al. (2007) and Ferlay et al. (2008). The odd and branched-chain FA (OBCFA) concentrations were higher for G (5.29) than for C systems (4.39). One explanation could be that the microbial synthesis of these FA had been enhanced by diets rich in fibre (Vlaemincks et al., 2006). Figure 1: Evolution of milk selected fatty acid concentrations (g/100g of total FA) according to production system and period
Conclusions The tank milks from G systems contained less SFA and had higher levels of nutritionally desirable FA such as c9t11-CLA and 18:3n-3, but also trans 18:1, than those from C systems. Nevertheless, data reported in this study contribute to provide evidence on the advantages of the grazing systems on milk FA composition. Acknowledgements This work was funded by TRUEFOOD project (FOOD-CT-2006-016264). The authors are very grateful to the partners of the dairy sector and farmers having participated to this study. References Chassaing C., Graulet B., Agabriel C., Martin B., Girard C.L. 2011. 10th International Meeting on Mountain Cheese, September 14-15, 2011, in press. Chilliard Y., Glasser F., Ferlay A., Bernard L., Rouel J., Doreau M. 2007. Eur. J. Lipid Sci. Technol. 109:828855. Ferlay A., Agabriel C., Sibra C., Journal C., Martin B., Chilliard Y. 2008. Dairy Sci. Technol. 88:193-215. Givens D.I. 2010. Animal 4:1941-1952. Shingfield K.J, Chilliard Y., Toivonen V., Kairenius P., Givens D.I. 2008. Adv. Exp. Medicine and Biol. 606:365. Vlaeminck B., Fievez V., Cabrita A.R.J., Fonseca A.J.M., Dewhurst R.J. 2006. Anim. Feed Sci. Technol. 131:389417.
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Effects of farming system and cheese making technology on quality traits and fatty acid profile of Caciocavallo Palermitano cheese at different ripening time
G. Tornamb*, A. Di Grigoli, V. Bellina, F. Mazza, A. Bonanno Dipartimento DEMETRA, settore di Produzioni Animali, Universit degli Studi di Palermo, Viale delle Scienze, 90128 Palermo, Italy *gtornambe@unipa.it Abstract The Caciocavallo Palermitano is a typical stretched curd cheese made from cow milk, mainly in the Western Sicily. The aim of this investigation was to verify the influence of farming system and cheese making process (traditional with wood tools and natural endemic flora vs. innovative with modern stainless steel tools and selected lactic bacteria) on qualitative characteristics, including fatty acid (FA) profile, of Caciocavallo Palermitano at different ripening time. Cheeses were obtained from bulk milk from two farms: one extensive rearing a local breed fed at pasture and one intensive rearing a specialized dairy breed fed mainly hay and concentrate. Milk and cheese properties and FA profile were greatly affected by farming system: feeding cows exclusively at pasture seemed to promote a more health cheese FA profile. Also the cheese making process significantly influenced the quality of cheese; indeed, traditional technology led to a production of cheese higher in fat and with a more cohesive and yellow paste. The results of this study highlight an influence of both farming system and cheese making technology on the quality characteristics and FA profile of Caciocavallo Palermitano cheese. Keywords: typical cheese, local breed, pasture, traditional cheese making, cheese fatty acids Introduction The Caciocavallo Palermitano is a typical stretched curd (pasta filata) cheese, produced with cow whole milk coming from two daily milkings; it has a firm paste, a characteristic parallelepiped shape and a straw-coloured hue which tends to ochre during the maturing process (Bonanno et al., 2004). The Caciocavallo Palermitano is historically bound to the inland farming cows of indigenous breeds, especially Cinisara, which are able to exploit productively the natural pasture located in the hills or even in the semi-arid areas of the island (Di Grigoli et al., 2008; Tornamb et al., 2009). Therefore, this cheese is produced traditionally in small size farms, where local cows are fed at pasture, according to an artisanal manufacturing procedure based on the use of wood tools and the action of native microflora. In the last years, the agriculture and animal production in the area of Palermo has undergone an evolution, and this dairy product is obtained also in more intensive farming systems; here, high milk-producing cows of selected allochthonous breeds are reared intensively and, in compliance with the EU rules regarding the requirements of dairy factories, a more advanced cheese making process is adopted, using stainless steel equipment and, often, commercial starter culture. The objective of this experiment was to evaluate the effects of farming system (estensive vs. intensive) and the processing technology based on the use of traditional wooden tools or innovative steel equipment and addition of selected lactic bacteria, on the characteristics of Caciocavallo Palermitano cheese. Material and methods Bulk milk from two farms (A = estensive, where autochthonous cows mainly fed at pasture; B = intensive, where Brown cows fed hay and concentrate with a small proportion of pasture) was processed during 12 cheese making trials in May 2009. Cheese making was performed in parallel with traditional technology (TR), which involves the use of wooden tools, and innovative technology (INN) based on the use of stainless steel equipment and the addition of selected microflora. The cheeses were ripened up to 120 days. On milk and cheese samples collected at 1, 30, 60 and 120 days (covering the cut cheese surface by liquid paraffin) were determined the main chemical and rheological parameters. Fatty acids (FA) were methylated in lyophilized cheese and fatty acid methyl esters (FAME) analysed by HP 6890 GC system equipped with a flame ionization detector. GLM procedure of SAS 9.1.2 was used for the statistical analysis considering farm (f), cheese making technology (t), ripening time (r) and the interactions ft, fr and tr as fixed factors. Results and discussion Milk quality was affected by farming system: milk produced in A farm was richer in protein (3.65 vs. 3.39%, P<0.001) and casein (2.84 vs. 2.54%, P<0.001) and lower in urea (31.7 vs. 43.9 mg/dl, P<0.001), fat (3.35 vs. 3.80%, P<0.001) and somatic cells (5.28 vs. 5.88 log/ml, P<0.001), whereas there were no significant differences in coagulation parameters (r, k20, a30). The cheese yield as well as the cheese chemical and colorimetric parameters, shown in table 1, were strongly influenced by the farming system. Cheese making technology and ripening time also affected the cheese yield, chemical composition and color (L*, a*, b*) of the cheese. Indeed, the traditional cheese making technology reduced cheese yield, protein and soluble nitrogen, and increased fat in comparison with the innovative technology. The traditional cheese also resulted higher in lightness (L*) and 15
yellow colour index (b*). The innovative technology resulted in a more marked increase in soluble nitrogen during ripening (0.43, 0.63, 0.92, 1.05 % of DM for TR and 0.53, 1.13, 1.44, 1.68 for INN respectively at 1, 30, 60, 120 days of ripening; P<0.05) indicating a more pronounced proteolysis induced by selected microflora, while in the traditional process the native microflora leads to an increase in the intensity of yellow colour during ripening (28.30, 29.45, 30.60 b* for TRD and 27.40, 27.41, 26.72 for INN respectively at 30, 60 and 90 days of ripening; P<0.01). Table 1: Chemical (% DM) and colorimetric parameters of cheese.
Farm A B Cheese making technology TR INN 1 Ripening time (d) 30 60 120 Significance (1) Yield % 8.98 7.86 8.20 8.63 8.88 8.49 8.29 8.01 f***, t**, r*** DM % 58.4 60.6 61.6 57.4 52.7 D 60.0 C 61.6 B 63.8 A f***, t***, r***, ft*, fr*** Protein 49.6 48.5 48.4 49.7 51.0 A 48.1 B 48.6 B 48.5 B f**, t**, r***, ft* Fat 38.9 40.5 40.7 38.7 40.3 39.2 39.6 39.8 f**, t*** Ash 7.74 7.92 7.40 8.26 5.01 C 9.33 A 8.06 B 8.91 A t***, r*** SN/TN 11.6 13.9 10.0 15.5 6.01Cd 11.65Bc 15.51Ab 17.93Aa f*, t***, r***, tr* L* 81.4 80.1 83.0 78.4 -82.5 A 81.8 A 77.9 B f*, t***, r***, ft*** a* - 4.63 - 4.97 - 4.48 - 5.11 --4.55 A -4.46 A -5.38 B f**, t***, r***, ft** b* 26.6 30.0 29.4 27.2 -27.8 28.4 28.7 f***, t***, tr** (1) f = farm; t = cheese making technology; r = ripening time; ft, fr, tr = interactions; *** = P 0.001; ** = P 0.01; * = P 0.05
Also the cheese FA profile (table 2) was strongly affected by farming system, while the technology and ripening time did not show relevant effects. The cheese from A farm resulted richer in C18:1 trans11, C18:3 n-3 and Conjugated Linoleic Acid (CLA C18:2 cis9, trans11), and consequently showed higher values of polyunsaturated FA (PUFA), omega-3 FA and odd and branched chain FA (OBCFA) than farm B cheese. These results could be explained by the different cows feeding regimen which was mainly based on green forage from natural pasture in the farm A, and on hay and concentrate in the farm B. Indeed, pasture-based diets promote an enrichment of PUFA and CLA in dairy products (Chilliard et al., 2001). On the contrary, B cheese showed higher values for linoleic acid (C18:2 n-3), monounsaturated FA and omega-6 FA. The higher level of linoleic FA in the farm B could be a consequence of the consumption of a concentrate particularly rich in this acid. Table 2:Fatty acid profile of cheese (% FAME).
Farm Cheese making technology Ripening time (d) A B TR INN 1 30 60 120 C18:1 trans11 2.57 1.32 1.88 2.02 1.96 1.97 1.79 2.06 C18:2 n-6 1.62 2.45 2.12 1.96 1.97 2.20 2.18 1.81 C18:3 n-3 1.19 0.62 0.90 0.92 0.85 1.02 0.90 0.86 CLA C18:2 cis9, trans11 1.10 0.65 0.90 0.85 0.07 0.09 0.07 0.07 Monounsaturated FA 26.8 28.4 27.9 27.4 26.8 28.3 27.9 27.4 Polyunsaturated FA 4.97 4.40 4.85 4.52 4.93 4.95 4.48 4.39 Saturated FA/Unsaturated FA 2.12 2.02 2.02 2.11 2.12 1.98 2.05 2.12 1.47 0.84 1.18 1.14 2.13 2.39 2.64 2.27 omega-3 1.72 2.65 2.29 2.08 2.16 2.33 2.29 1.95 omega-6 6.12 4.68 5.11 5.69 6.48 4.97 5.02 5.13 OBCFA (2) (1) f = farm; *** = P 0.001; ** = P 0.01; * = P 0.05; + = P 0.10; (2) OBCFA = odd and branched chain FA. Significance (1) f*** f*** f*** f*** f** f** f+ f*** f*** f*
Conclusions The quality of Caciocavallo Palermitano cheese has been strongly influenced by both the farming system and the cheese making technology, which led to variation in cheese yield and chemical and colorimetric parameters. Moreover, the technology induced a different evolution of proteolysis during the ripening time, due to the different kind of microflora implicated in the processing. Conversely, cheeses from farming system, in which autochthonous breed cows were fed a pasture-based diet, showed a more beneficial FA profile for human health, being richer in PUFA, omega-3 and odd and branched chain FA, as well as in CLA C18:2 cis9, trans11. Acknowledgements This research was conducted within the Project PROLACTIS founded by the Regione Siciliana. References Bonanno A., Di Grigoli A., Tornamb G., Formoso B., Alicata M.L., Procida G., Manzi P., Marconi S., Pizzoferrato L. 2004. pp 43-50 in Proc. 6th Int. Meeting on Mountain Cheeses. 1-2 giugno, Ragusa, Italy. Chilliard Y., Ferlay A., Doreau M. 2001. Liv Prod. Sci. 70:31-48. Di Grigoli A., Bonanno A., Cifuni G.F., Tornamb G., Alicata M.L. 2008. Sci. Tecn. Latt-Cas 59 457-461. Tornamb G., Di Grigoli A., Alicata M.L., De Pasquale C., Bonanno A. 2009. Pag. 153-156 in proc. 15th Meeting of the FAO-CIHEAM Mountain Pastures Network, 7th-9th October 2009, Les Diablerets, Switzerland. http://fao09.adcf.ch/attachments/File/proceeding.pdf. 16
Cheese chemical profile from Zebu cows grazing on two systems - pastoral and silvopastoral - in the highlands of Colima, Mexico
M.A. Galina1*, S. Claps2, R. Rubino2, M. Pizzillo2, M. Guerrero1, L.J. Pineda1 1 FES-CUAUTITLN, UNIVERSIDAD NACIONAL AUTNOMA DE MXICO. Km 3.5 Carretera Cuautitln-Teoloyucan, Cuautitln Izcalli 54700, Mxico 2 CRA-ZOE, Unit di ricerca per la Zootecnia Estensiva, Via Appia; Bella Scalo - 85054 Muro Lucano (PZ), Italy *miguelgalina@hotmail.com Abstract A study was conducted on the Mexican tropical highlands to measure zebu cattle cheese quality under two grazing systems. Lactic cheeses were made from Zebu milk through fermentation with a liquid starter. Cattle were divided into two groups: 32 cows grazing 24 hr on regional tropical grasses (GR); and 27 females on silvopasture (SP). FAME, VOC and Terpenes were measured. Feeding system affected fatty acid profile and terpen content but differences were not significant. Terpenes were respectively 520 and 410 ng/kg (SP/GR). Total cheese fat and cholesterol contents in SP were 25 g/kg cheese and 187 mg/100 g fat, compared to GR 32 g/kg cheese and 245 mg/100 g fat. GR accounted for a higher tocopherol in cheese with 127 mg/100 g DM compared to 77mg/100 g DM for SP. The highest VOC was found in GR cheese (243,9 a.u) and the lowest in SP (182,3 a.u). Most VOC represented in GR were: ketones 56,7%, hydrocarbons 20,3%, esters 10,2% and terpenes 7,1% and in SP cheese were: ketones 32,4%, hydrocarbons 21,0%, esters 23.1%, and terpenes 14,2%. Nutritional characteristics in all results favoured the SP system, producing better cheese quality, though both systems lactic products (GR/SP) could improve human health. Keywords: milk, cheese, cows, quality, grazing Introduction Vegetation in dry tropics contains a number of interesting metabolites with benefits for human health including alkaloids, glycosides, fatty acids, terpenes, saponins, tannins, and flavonoids (Seigler, 2003). Nutritionally interesting and beneficial components are found to be at significantly superior levels in cheese made from milk under grazing with legumes compared to an indoor feeding system regardless of animal species (Galina et al., 2007). Very few studies have detailed the nutritional qualities of cheese made from milk of Zebu cattle managed in a tropical silvopastoral system, particularly, with the biodiversification of plant life offered on the High mountain regions (Claps et al., 2011). Material and methods The trial was carried out in the dry tropics of Colima, Mexico, on a ranch located at 1923N; 10341W, 400 m asl. The total grazing area was 45.8 ha divided into 17.8 ha of Cynodon plectostachyus and 18.0 ha of Brachiaria brizanhta bordering 10 ha of tropical forest. Two experimental herds were formed: first with 32 cows which grazed 24 hrs on regional tropical grasses (GR); and second of 27 females feeding on part of a silvopastoral environment (SP). Grazing area was subdivided for the first group into 8.8 ha of Cynodon and 8 ha of Brachiaria and for the second 9 ha of Cynodon and 10 ha of Brachiaria. The first group had access to 10 ha of tropical forest. Direct observation by following the animals grazing and/or browsing during 4 hours allowed cutting of vegetation similar to the ones consumed and permitted the identification of browsed vegetation. Trees consumed were Mimosa pudica, Plumera rubra, Bunchosia palmeri, Cordia alliodora, C. dentata, Platymiscium fasiocarpum, Erythroxylum mexicanum, E.rotundifolium, Caesalpina plumeria, Guttarda elliptica, Randia capitata, Caesalpina coriaria and Desmodium spp. Main tropical forest browsed by ruminants had an average nitrogen content of 20.72% while grasses were only 2.52%. Animals browsed ad libitum and were offered 2 kg of slow intake urea supplement (SIUS). Milk was collected from each group and cheese made collectively. Lactic cheese was made from Zebu milk through fermentation with a liquid starter. FAME, fatty acid methyl ester (FAME), Volatile Organic Compounds (VOC) and aromatic acid analysis (Terpenes) were measured (Galina et al., 2007; Claps et al., 2011). Volatile compounds were chromatographically measured by gas and terpenes were selectively detected by mass spectrometry. The CLA isomers composition in solidly frozen milk or cheese fats was analyzed using silver-ion high performance liquid chromatography (AG+-HPLC). Electron impact of ionisation at 70 eV was used. FA milk and cheese profiles were compared based on the different milk production conditions and species after determining by gas chromatography the FA methyl esters. Statistical analysis included both cheese FAME and VOC data, was processed by Analysis of Variance which included the management system as variable (SAS, 1989).
17
Results and discussion Table 1. Metabolic profile of cheese made from milk of grazing cattle GR and silvopasture. The metabolic profile is reported in Table 1, comparing the two feeding systems and illustrating important variations in cheese quality. In particular, they have influenced significantly the 2.52 20.72 nutritional and aromatic cheese characteristics. These results, 410 520 though not significantly different, confirms that these feeding systems allowed each cow to structure a diet according to their 32 25 own requirements, and therefore affect their milk and cheeses 245 187 nutritional characteristics, particularly, when grazing a mountain silvopastoral system which affected the fatty acid profile. The 127 77 data from the nutritional characteristics was always slightly in 243.9 182.3 favour of SP and could suggest that that diet was more 56.7 32.4 diversified. The higher average nitrogen content, of the main 20.3 21.0 tropical forest browsed by the ruminants stimulated brush 10.2 23.1 consumption (Claps et al., 2011). 7.1 14.2 The highest content of trans fatty acids was present in silvopastoral cheese and very low content in the grazing product. Until very recently, the negative effect of trans fatty acids on the human health, such as on coronaric and cytotoxic pathologies, came from observations of hydrogenated fatty acids produced during industrial processes and were considered similar to the ones documented for saturated fatty acids (Pedersen, 2001 and Secchiari, 2008). The trans derived from the biohydrogenetion processes, like those produced in the rumen, have shown instead positive effects regarding the human health. However, no significant differences were found when animals are kept grazing GR or SP with both showing the presence of beneficial compounds (Galina et al., 2007). They accounted for the majority of C18: 1 trans vaccenic. This last one, through the action of the delta 9 desaturisition, becomes metabolized in C18: 2 9cis 11trans that represents one of the more important precursors of the beneficial CLA (Claps et al., 2011). Even if a slight difference was demonstrated between the two systems (GR/SP), the values of CLA and the omega 6/omega 3 were superior when compared to values reported in the literature for indoor feeding systems, high in concentrates, (Pedersen, 2001).Therefore in the light of this new knowledge, the role of trans fatty acids in ruminants feeding system on free pasture has to be revaluated its ability to better milk (or cheese) for the consumer. Health in Mexico is a major concern due to high population density suffering from obesity or overweight translating into coronary malfunctions (Claps et al., 2011). This important value in both systems could elevate cheese product to a functional food.
Caracteristic Nitrogen content (%) Terpenes (ng/kg) Total cheese fat (g/kg cheese) Cholesterol (mg/100 g fat) Tocopherol (mg/100 g DM) VOC (a.u.) Ketones (%) Hydrocarbons (%) Esters (%) Terpenes (%) Pasturing Silvopasturing
Conclusions The results have shown that the two feeding systems demonstrated important variations in cheese quality. They have influenced significantly the products nutritional and aromatic characteristics. The result reduced the role of the terpens in favour of other metabolites which are less studied. In fact other compounds like ketones, aromatic hydrocarbons and esters, partially derivate from grass, play a more important role on the final characteristics of the product. The data of the nutritional characteristics always in favour of the SP system demonstrated better cheese quality, although both systems (GR/SP) could play a role improving human health through the milk. An important aspect is the complexity or the aromatic intensity of SP demonstrating that when forage diversification was fed there are additional possibilities for new and more compounds, enhancing cheeses quality related to human health. Acknowledgements Project funding by PAPPIT IN200809 UNAM References Claps S., Galina, M.A., Rubino R., Pizzillo M., Morone G., Di Napoli M.A., Caputo A.R., Pineda L.J. 2011. In press. Galina, M.A., Osnaya, F., Cuchillo, H.M., Haenlein G.F.W. 2007 Small Rum Res 71:264-272 Pedersen, J.I. 2001. Br. J.Nutr., 85 (3):249-250 Secchiari, P.L. 2008. Ital.J. Agronom., 1:73-101 Seigler, D.S. 2003. Biochemical systematics and ecology. 31: 845-873
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The cheese quality as tool to safeguard autochthonous sheep breeds and mountain environment
S. Claps*, L. Sepe, A.R. Caputo, M.A. Di Napoli, G. Morone, G. Annicchiarico, V. Fedele CRA-ZOE, Consiglio per la Ricerca e la sperimentazione in Agricoltura, Unit di Ricerca per la Zootecnia Estensiva, Via Appia, Bella Scalo, 85054 Muro Lucano (PZ), Italy *salvatore.claps@entecra.it Abstract In the past, in mountain areas of South and Centre of Italy, indigenous sheep purebreds were widespread and typical and PDO Pecorino cheeses (i.e. Canestrato pugliese, Pecorino di Filiano, etc.) were made almost exclusively by autochthonous sheep breeds, grazing on mountain pasture. In the recent decades, the end of transhumance (from mountain to plain) and the partial substitution of the autochthonous sheep breeds with Sarda breed and others, has determined unfavourable consequences both in terms of cheese quality and for mountain environment (animal and shepherd with function of monitoring and habitat maintenance are disappearing from the mountain). Aim of this study was to evaluate the breed effect on nutritional quality of Canestrato pugliese cheese (four months ripened) made from two sheep breeds: Altamurana (A) and Sarda (S), as far as CLA content, Atherogenic (AI) and Thrombogenic Index (TI) and Health Promoting Index (HPI). The results showed lower value of CLA (0.52 vs. 0.57 g/100 g of FA), higher value of AI and lower value of HPI in the Canestrato pugliese cheese from Sarda breed (S) rather than from Altamurana breed (A). The nutritional quality of Canestrato pugliese cheese varied according to sheep breed. The autochthonous breed emerged for distinguishable characteristics. Keywords: Mountain, sheep breeds, cheese, quality Introduction Many natural mountain ecosystems have resulted from a dynamic equilibrium between wildlife and human activities (Olea and Mateo-Toms, 2009). The transhumance, with the movement of livestock between winter and summer pastures (from mountain to plain and vice versa) in South and Centre of Italy (Abruzzo region) with the autochthonous sheep breeds, was very common. This practice ensured the preservation of both environment and breeds. The rapid changes occurred in agriculture (i.e. intensification, mechanization, abandonment) in the last decades have been altered and amended the composition, structure and function of mountain ecosystem (MacDonald et al., 2000). Several indigenous breeds such as Gentile di Puglia and Altamurana were substituted by the Sarda breed. In particular, no more than 10,000 local multi-attitude Gentile di Puglia and Altamurana breeds are now reared; while up to 1963 they represented the most important sheep resource in South-Eastern Italy, numbering about 1 million head each breed (Signorelli et al., 2008). This work is based on the assumption that evidencing the breed effect in the quality of cheese could be a hint for the sustainable use, in mountain areas, of indigenous sheep breeds, and aims at identifying difference in cheese FA composition between Altamurana and Sarda breeds. Material and methods The experiment was carried out at CRA ZOE experimental farm of Foggia (Apulia region - South of Italy), 76 m asl, during the spring period. For this experiment, two mono-breed groups, Altamurana (A) and Sarda breed (S) sheep, homogeneous for milking day (906) and body condition score (2.750.05). Animals were fed on native pasture plus 400 g DM/head/day of concentrate supplementation, offered in two equal meals at milking. One cheese-making (Canestrato Pugliese cheese) for each breed was carried out for three consecutive days, using technology and tools almost equivalent to those traditional described in the rules of Protected Denomination of Origin (PDO). Nine cheeses for each breed, after four months ripening, were analysed, in duplicate, for FAME content (Fatty Acid Methyl Esters). Cheese lipid fraction was extracted according to Blingh and Dyer (1959) method and FAME were obtained according to Di Trana et al. (2004): FAME were separated and quantified using a gas chromatograph (Varian Mod. 3800) fitted with an automatic sampler (CP 8410) and equipped with a FID detector. FAME peaks were identified by comparison of retention times with those of known mixture of standard fatty acids (Sigma-Aldrich). Atherogenic Index (AI) and Trombogenic Index (TI) were calculated on the basis of the fatty acid profile as described by Ulbricht and Southgate (1991). The Health Promoting Index (HPI) was calculated as the sum of unsaturated fatty acid divided by the sum of lauric acid (C12), palmitic acid (C16) and 4*miristic acid (C14), according to Chen et al. (2004). Results and discussion As shown in Table 1, the fatty acid profile of Canestrato Pugliese cheeses from the milk of different breeds, at four months of ripening, was significantly influenced by breed, except to the content of total trans. The cheese from S breed had the highest value of SCFA (11.72 g/100 g of FA) significantly higher (P<0.05) than A breed. Cheese from the same breed (S) showed significantly higher content of MCFA (31.35 g/100 of FA vs. about 28 19
g/100 of FA) and the lowest value of LCFA (56.93 g/100 g of FA). The Canestrato Pugliese from Sarda (S) breed contained the highest value of SFA (73.44 g/100 g of FA) and the lowest MUFA (22.32 g/100 g of FA) and PUFA (4.24 g/100 g of FA) contents. Also -3 and -6 content were influenced by the breed. The lowest 3 value was detected in the Canestrato Pugliese cheese derived from Sardas milk (S). The highest content of CLA (0.57 vs. 0.52 g/100 g of FA) was found in the cheese from Altamurana breed (A). The differences observed for the CLA content are very important: in the recent years, CLA aroused much attention because several studies gave evidence for its anti-carcinogenic, anti-atherogenic, anti-obesity and immune-stimulating properties (Mele et al., 2010). Highly significant differences (P<0.01) were found for the nutritional index. In terms of nutritional index, the cheese from Sarda breed (S) showed the worst value. In S breed cheese, in fact, higher value of AI (2.45) and TI (3.03) were recorded and the lowest value of HPI (0.41), significantly different from Altamurana breed (A). Cheese from Sarda breed (S) contained high content of those fatty acids thought to be dangerous for human health. This is well marked by the lower Health promoting Index (HPI) and higher Atherogenic Index (AI) and Thrombogenic (TI). This result agrees with those of Di Trana et al. (2009). Table 1: Effect of ovine breed on fatty acid content (g/100 g of FA) and nutritional index of Canestrato Pugliese cheese - four months ripened -from Altamurana (A) and Sarda (S) breed A S SEM Effect Fatty acids SCFA 10.84 b 11.72 a 0.51 * MCFA 28.09 b 31.35 a 0.23 *** LCFA 61.09 a 56.93 b 0.57 * SFA 70.23 b 73.44 a 0.48 * MUFA 25.24 a 22.32 b 0.40 ** PUFA 4.56 a 4.24 b 0.058 * -3 0.57 a 0.52 b 0.05 *** -6 2.63 a 2.32 b 0.06 * CLA 0.57 a 0.52 b 0.05 ** Total trans 0.97 0.93 0.03 ns Nutritional Index AI 2.05 b 2.45 a 0.06 *** TI 2.69 b 3.03 a 0.08 *** HPI 0.49 a 0.41 b 0.02 *** Short Chain Fatty acid (SCFA); Medium Chain Fatty Acid (MCFA); Long Chain Fatty Acid (LCFA); Saturated Fatty Acids (SFA); Monounsaturated Fatty Acids (MUFA); Polyunsaturated Fatty Acids (PUFA); Conjugated linoleic acid (CLA); Standard Error of the Mean (SEM); ns = not significant; *P<0.05; **P<0.01;***P<0.001; means with different superscripts with letters (a ,b) in each row differ significantly for P<0.05. Conclusions The experiment confirmed that fatty acid profile and Nutritional Index of cheese are affected by sheep breeds. The Canestrato Pugliese cheese produced from Altamurana breed exhibited distinguishable and better characteristics, in terms of FA profile and HPI compared to the other breed. These characteristics could be used as a means for the valorisation and the reintroduction of autochthonous sheep breed in mountain areas, with benefit both to human health and safeguard of mountain areas and pure breeds. Acknowledgements This work was funded by Italian project Collezioni E A-OR of MiPAAF. References Bligh E. and Dyer W. J. 1959. Canad. Biochem. and Phys., 37 911-917. Chen S., Bobe G., Zimmerman S., Hammond E., Luhman C., Boylston T., Freeman A , Beitz D. (2004). J. Agri. Food Chem., 2004, 52 (11), 3422-3428. Olea P., Mateo-Toms P. 2009. Biol. Conserv., 142: 1848-1853. Di Trana A., Cifuni G.F., Fedele V., Braghieri A., Claps S., Rubino R. 2004. Progr. in Nutr., 6: (2), 109-115. Di Trana A., Claps S., Pizzillo M., Impemba G., Annicchiarico G., Cifuni G.F., Di Napoli M.A., Caputo A.R. 2009. Pag. 1-5 in proc. 17th International Congress of Mediterranean Federation for Health and Production of Ruminant. 27-30 May, Perugia, Italy. Mele M., Serra A., Conte G. 2010. Ital. J. Anim. Dci., 2:3-28. MacDonald D., Crabtree J.R., Weisinger G., Dax T., Stamou N., Fleury P., Gutierrez-Lazpita J., Gibon A. 2000. J. Env. Manag., 59: 47-49. Signorelli F., Contarini G., Annicchiarico G., Napoletano F., Orr L., Catillo G., Haenlein G.F.W., Moioli B. 2008. Small Rum. Res., 78: 24-31. Ulbricht T.L.V., Southgate D.A.T. 1991. The Lancet, 338:9 20
Traditional milking of Salers cows: influence of removing calf on cheese making ability of milk in comparison to Holstein cows
M. Guiadeur1*, I. Verdier-Metz2, F. Monsallier2, J. Agabriel1, C. Ciri3, M.C. Montel2, B. Martin1 1 INRA Clermont-Theix, UR1213 Herbivores, F-63122 St-Gens-Champanelle, France 2 INRA, UR545 Fromagres, 20 Cte de Reyne, F-15000 Aurillac, France 3 INRA Clermont-Theix, UE1296 UEMA, F-15190 Marcenat, France *marlene.guiadeur@wanadoo.fr Abstract The aim of this trial was to study the effect of milking Salers cows without their calf on milk composition, clotting ability and cheese quality. Holstein cows were considered as control. The study involved 24 primiparous Salers (S) and 12 Holstein (H) cows milked with their calf (C, 15 S and 6 H) or without (, 9 S and 6 H). Seven Cantal cheeses were made with raw milk from each lot (SC, S, HC, H) and analyzed after a 22-week ripening period. For H cows, no effect of the milking without their calf was observed on milk composition and clotting ability, and cheeses characteristics. S cheeses could not be analyzed because of an abnormal coagulation of the milk of 3 cows from this lot. In comparison to H cheeses (HC, H), SC cheeses had a lower fat content, a firmer and less melting texture and their flavour developed more slowly. Those specific characteristics are mainly linked to the low fat content of the milk of the Salers milked with the calf. This latter result raises the question of possible losses of Salers cheeses typicality when Salers cows are milked without their calf because in this case, fat content of the milk increases and reaches values similar to Holstein cows. Keywords: Salers breed, milking, calf, Cantal cheese, clotting ability Introduction The Salers cows are native from Massif Central (France) where they are mainly reared for meat. About 4000 cows, (2 % of the national herd) are still milked by 80 farmers in a traditional way, which requires the presence of the calf to stimulate milk ejection. The number of cows and farmers decreases due to the low production of this breed and the labour induced by the traditional milking. In this context, the milking of the cows without their calf seems important for safeguarding Salers milked. Joined studies (Guiadeur et al., 2011) evidenced that it seems possible to milk 43 % of the primiparous cows without their calf, and to obtain a milk richer in fat (+ 7.4 g/kg). As most of the Salers milk is transformed in local PDO cheeses (Cantal, Salers), it is important to check the influence of milking Salers cows without their calf on milk cheese making ability. The aim of the trial made in order to simplify the traditional way to milk Salers cows, was to study the effect of milking Salers cows without their calf on milk composition, clotting ability and cheese quality. Material and methods The experiment was carried out at the INRA experimental farm of Marcenat (mountain area of central France). It involved 24 primiparous Salers (S) and 12 Holstein (H) cows milked with their calf (C, 9 S and 6 H) or without (, 14 S and 6 H). Holstein cows were used as control. Individual milk of each cow was sampled twice (February and June, respectively about 126 and 210 days in milk) for chemical analysis and coagulation tests made with a formagraph. From the 15th December to the 20th of January, 7 petit Cantal cheeses were made with the bulk milk of each lot of cows (SC, S, HC and H). Three cheese makings were made from 100 L of full fat raw milk and 4 with a fat to protein ratio standardised at 1.0 by partial skimming. After a 22-week ripening period, chemical composition of cheeses was analyzed and sensory profiles were determined by an expert panel. Main microbial groups were counted on milk and cheeses (except S milks) as described by Millet et al. (2006). For milk, data were processed by analysis of variance (Proc GLM, SAS) including breed (H, S), treatment (C, ) and the interaction. The model for cheese included group of cheeses (HC, H and SC), standardization of the fat/protein ratio of the milk and the interaction. Results and discussion Individual cow milk characteristics The clotting time of 3 of the 9 S milks was higher than 30 min and considered as non-clotting. The reason is still not clear because fat, protein, casein, calcium and phosphorus content, somatic cell count, pH, and variants of - and -caseins of non-clotting S milks were not different from clotting S milks (results not shown). Table 1 reports characteristics of milk which had a clotting time lower than 30 min. As expected, the protein and casein (result not shown) content of individual milk from Salers cows was higher than Holstein cows (+ 4.0 and + 3.5 g/kg milk respectively, P<0.01). We did not observe any difference according to breed and treatment for the milk clotting ability. As curd firmness is closely related to casein content (Macheboeuf et al., 1993), the fact the Salers cows did not provide a firmer curd is surprising. It reveals a real specificity of the milks from Salers regarding their clotting ability, which is coherent with farmers know-how (Agabriel et al., 2009). Levels of microbial groups did not vary according to breed and treatment, except for Enterococcus slightly 21
higher (+0.55 log10CFU/ml) in SC and HC milks. Table 1: Effects of milking Holstein (H) and Salers (S) cows with (C) or without () their calf on individual milk properties.
H C C S Breed Effects Treatment ns ns ns Breed x Treatment ns ns ns
Protein (g/kg) 32.9ac 32.2a 35.7bc 37.0b ** Clotting time (min) 12.5 10.3 12.6 12.3 ns Curd firmness (mm) 92.88 109.20 92.17 101.00 ns Values with different superscripts differ at P<0.05; ns: P>0.05, *: P<0.05; **: P<0.01, ***: P<0.001.
Ripened cheese characteristics Because of a too long clotting time of the bulk milk from S cows, S cheeses characteristics were not analyzed. By comparison with Holstein cheeses (both HC and H), SC cheeses, made without any standardization of the fat/protein ratio of milk, had a very low fat in dry matter content (- 8.4 %, Table 2). This result is in agreement with the low fat content of SC milk. As a consequence, SC cheeses were rated by the panellists as firmer (result not shown, P<0.5) and less melting than the Holstein cheeses (Table 2). Salers cheeses had also a lower flavour intensity (P<0.05). When the fat/protein ratio of the milk was standardized prior cheese making, fat in dry matter content of the SC cheeses remained lower in average (- 2.9 %, P>0.05) because of higher fat losses in the whey and a slightly lower fat/protein ratio of the milk (0.95 for SC and 1.04 for HC and H). Differences in cheeses texture and flavour observed with unstandardized milk were lower than when the milk was standardized. SC cheeses had higher levels in total bacteria and Lactococcus in comparison to H (+0.56 and + 0.68 log10CFU/ml, P<0.05). No other significant differences for other microbial flora were observed. Milking the Holstein cows with or without the calf did not influence milk or cheese characteristics. Table 2: Effects of milking Holstein (H) and Salers (S) cows with (C) or without () their calf on bulk milk and cheese composition and sensory properties (scores 0-10).
HC Non standardized H SC 26.5 32.2b HC 28.9 29.1a Standardized H 30.0 30.1a 63.8 47.4bc 5.35 2.64b 5.38b 5.17 raw milk; SC 29.3 32.5b Lot *** *** Effects Stand. Lot x stand. *** ns *** ns ns ** ns *** ns ns differ at
Milk characteristics 33.3 35.6 Fat (g/kg) 30.0a 29.3a Protein (g/kg) Cheese Characteristics 63.5 64.2 Dry Matter (%) 50.9b 49.2b Fat in Dry Matter (%) 5.44 5.37 pH 2.97c 2.74c Melting texture (0-10) 5.53b 5.46b Flavour intensity (0-10) 5.23 5.24 Flavour persistency (0-10) Stand: Standardized; Standardized: fat / protein ratio = 1.0; P<0.05; ns: P>0.05, *: P<0.05; **: P<0.01; ***: P<0.001.
62.9 63.9 41.6a 47.2bc 5.53 5.48 1.65a 2.25cb 4.72a 5.46ab 4.76 5.17 non standardized: full fat
62.6 ns ns 44.4ac *** ns 5.51 * ns 2.39abc *** ns 5.14ab ** ns 5.06 ns ns values with different superscripts
Conclusion Our results underlined the very specific characteristics of Salers milk and cheese in comparison to Holstein ones, even when Holstein cows were milked with their calf. Salers cheeses were considerably firmer, less melting and had lower flavour intensity. The softer flavour of Salers cheeses, when analyzed at the same age, indicated that Salers cheeses could had been ripened longer. The Salers cheeses typicality seems partly related to the low fat content of the Salers milk: when fat to protein ratio of the milk was standardized, the effect of breed on cheese properties was considerably decreased. This latter result raises the question of possible losses of Salers cheeses typicality when Salers cows are milked without their calf because in this case, fat content of Salers milk increases and reaches values similar to Holstein cows. Further studies will complete those results. Acknowledgments We gratefully thank the staff from UEMA-Marcenat for animal care, R. Lavigne and R. Didienne for cheese making and I. Constant for valuable help during milk and cheese analyses. This work was funded within the program Pour et Sur le Dveloppement Rgional Salers (2007-2011). References Agabriel C., Brard L., Marchenay P., Casabianca F., Bouche R., Montel M.C. 2009. In Proc. of Mountain Cheese, 14-15 September, Saint Eulalie, France. Millet L., Saubusse M., Didienne R., Tessier L., Montel M.C. 2006. Int. J. Food Microbiol., 108 (1) : 105-114. Guiadeur M., Martin B., Juttier G., Ciri C., Agabriel J. 2011. In proc. 18th Renc. Rech. Ruminants, 7-8 December, Paris, France. Macheboeuf D., Coulon J.B., Dhour P. 1993. INRA Prod. Anim., 6: 333 344. 22
A down-up approach to organise the variability of commercial, nutritional and aromatic properties of cow milk and its grading into quality classes
R. Rubino1, G. Masoero2*, M. Pizzillo1 1 CRA-ZOE, Bella Scalo, 85054, Muro Lucano (PZ), Italia. 2 CRA-PCM, Via Pianezza 115 - 10151, Torino (TO) *giorgio_masoero@alice.it Abstract In a survey of 106 cow herds in Puglia concerning 4 farming Types, 18 key chemical variables were standardized, then algebraically added to a five-index quality system (Commercial, AntiOxidant, FattyAcids, Aromatic, Total). The Indexes, which resulted to be positively interrelated, were then graded into quartile classes. The 4 farming Types were differentiated according the Total Quality Index (r=0.56), which was less than the multivariate PLS of the 18 variables (0.73) and the direct fitting of FTMIR (0.91) or Electronic-nose (0.88). These rapid systems are able to efficiently classify a wide variability of milk and grade it into quality classes. Keywords: cow milk, quality classes, grading, FTMIR, electronic nose. Introduction Producers are concerned about the hygienic, health and raw Commercial properties of milk (fat, protein, somatic cells, microbes, urea); small cheese-makers, large dairies and factories are aware of the importance of the technical parameters (pH, acidity, lactose, casein, cheese-making, colouring, oxidative stability, rancidity, aroma, foaming); consumers have different opinions, but some are willing to pay more for top quality products, which should to labelled with the objective parameters. The aim of this work was to focus on the usefulness of grading milk in a very rapid way on the basis of multifaceted features that would be useful for the producers, transformers and consumers. Material and methods A total of 106 dairy farms in Puglia have been surveyed. The farms belong to four characteristics dairy Types: 1-SCH: (Silage Concentrate-High, n=25); 2-HCH (Hay Concentrate-High, n=33); 3-HCL (Hay ConcentrateLow, n=32); 4-PCZ (Pasture Concentrate Zero, with Podolic cows, n=16). The samples were analysed in the laboratory for their: Commercial properties (CO) ; Fatty Acid composition (FA); AntiOxidant (AO) properties measured by DAP (Degree of Antioxidant Protection, according Pizzoferrato et al., 2007); Aromatic profile (AR) of the volatile compounds, which were pooled as the main species. In parallel to the laboratory tests two rapid analysis systems were set up: MILKOSCAN FT120 (FTMIR) gave IR interferograms (1195 points) in triplicate while the rapid aromatic profile from ten MOS sensors for a 60 sec flush was obtained by means of an Electronic Nose (AirSense PEN3), which is in use at CRA-ZOE. The indexing key was simply to choose the most meaningful variables and to assign a positive or negative weight according to the quality dimension. Table 1 reports the positive and negative variables that contribute to the partial Indexes. The FAIndex (FAI) depended on the positive contributions of CLA and ISO C14-C15, while the negative ones depended on the Saturated FA, 6/3 and on the Trombogenic Index. All the chemical species determined were retained positive into the Aromatic Index. The Total Quality Index was then obtained from the algebraic sum of the four partial Indices. Table 1: Scheme of the calculations of the 5-Indexes and of the Grading in quartiles. Indexes + + + COI AOI FAI ARI TQI Commercial Index AntiOxydant Index FattyAcids Index Aromatic Index Fat Protein DAP CLA ISO-(C14+C15) Saturated 6/ 3 Tromboge nic_Index Lactose pH Somati c CC
Linear Score
Grades
COG AOG FAG ARG
Total-Quality TQG Index TQI = Total Quality-Index of (COI, AOI, FAI, ARI) Index = (X-X.avg)/Standard Dev.; Grade = Index Quart iles: 1=<25%,2:26-50%,3=51.75%,4>75%; DAP = i1AO / OT Where AC is the AntiOxidant compound (-tocopherol and carotene), OT is the oxidation target (cholesterol) and i is the number of components. Trombogenic Index = TI _ (C14:0 + C16:0 + C18:0) / [0.5*MUFA + 0.5*PUFAn-6 + 3* PUFA n-3 + PUFAn-3 /SPUFAn-6]
Aldeides, Chetones, Esthers, Hydrocharbon Alchools Therpene s s of (COI, AOI, FAI, ARI)
Results and discussion The Indexes all resulted to be positively correlated (Table 2), with the exception of the Aromatic Index, which was only weakly related to the AntiOxidant properties (r=0.22). The Total Quality Index depended mainly on the 23
Fatty Acids (r=0.79) and then on the Commercial Qualities (0.70) and AntiOxidant compounds (0.67), but the contribute to the Total Index from the Aromatic properties was minimum (0.54). The distributions of the Indexes were normal, while the Quartile transformation of the Index in TQG, instead resulted in a four class plate distribution. FTMIR was better at predicting TQI (RPD=2.5; r=0.92) than TQG (1.6), but the discrimination between the extreme quartiles (Q1-low vs. Q4-high ) remained very obvious (RPD 2.1, corresponding to r=0.88). FTMIR interferograms and ten-E-nose traces are in fact more easily adapted to a continuous variation than to a scale of discrete values. The E-nose rapid instrument was surprisingly better at grading the partial and the total qualities than FTMIR at predicting the class of Quality of the extremes in TQQ (2.3) and in FAQ (2.1). The success of the down-up approach depended to a great extent on the inclusion of Type-4-PCZ farms. The milk from this Type was graded as top class for all the indexes, even for the Commercial properties and this is a sign of strong opposition between the quality and the raw yield (Table 3). However a further differentiation was shown in the Total Index between Type 2 and the other intensive Types, 1 and 3. As reported in Table 4, the FTMIR and E-nose discrimination of the Types was higher (r=0.91 and 0.88) than all the other Indexes and Grades, whose maximum correlation was 0.56 with the TQI. This means that other information is available in the signals from rapid instruments, which can be linearly related to the class of farms. The down-up approach used to organise the variability and to grade the quality in the present work can be considered quite simplicistic since it is a raw algebraic sum of a restricted set of main, favourable or unfavourable variables which are not unrelated to each other. Thus, if a multivariate PLS approach had been elaborated, instead of the algebraic set of the 18 variables, the correlation of TQI with Types would have substantially increased from 0.56 to 0.73 (Table 4). Table 2: Correlations between the Indexes and performances of rapid analyses (FTMIR and E-nose) pertaining to the Indexes, Grades from quartiles and discrimination of the extreme Quartiles (Q1 vs. Q4).
Simple correlation coefficients between the Indexes r COI AOI FAI ARI TQI COI AOI FAI ARI 1 0.36 0.35 0.10 0.36 1 0.60 0.22 0.35 0.60 1 0.16 0.10 0.22 0.16 1 0.70 0.67 0.79 0.54 Significant r values >0.21 TQI 0.70 0.67 0.79 0.54 1 Rapid analyses: RPD = Relative Predicted deviation = SD / Standard Error in Cross-Validation Index Grade Quart-1th vs. Quart-4 th FTMIR E-nose FTMIR E-nose FTMIR E-nose 2.5 1.4 COG 1.7 1.5 COQ 2.0 2.4 1.9 1.3 AOG 1.8 1.3 AOQ 1.6 1.4 2.0 1.8 FAG 1.5 1.4 FAQ 1.9 2.1 1.0 1.5 ARG 1.1 1.1 ARQ 1.2 1.5 2.5 1.9 TQG 1.6 1.6 TQQ 2.1 2.3
Table 3: Partial and Total Indexes and Percentiles of the four Types of farms.
Index COI AOI FAI ARI TQI r2 0.28 0.45 0.60 0.04 0.58 1-SCH -0.03b -0.36b -1.70b -0.187 -2.3bc 2-HCH 3-HCL -1.84c 0.004b -0.36b -0.14b -1.12b -0.89b -0.532 0.0358 -3.85c -0.99bc 4-PCZ 3.72a 1.57a 6.75a 1.28 13.3a Grades COG AOG FAG ARG TQG r2 0.24 0.28 0.29 0.08 0.34 1-SCH 2.64b 2.20b 2.12b 2.40b 2.28b 2-HCH 1.84c 2.12b 2.28b 2.20b 1.91c 3-HCL 2.52b 2.42b 2.30b 2.50b 2.55b 4-PCZ 3.56a 3.88a 3.94a 3.19a 3.94a
Table 4: Correlations between the Type of farms (1-4), the five Indexes (I), the Grades (G) and prediction of the four Types through the use of rapid analyses (FTMIR and E-nose), r values.
R COI Type1-4 0.34 COG 0.26 AOI 0.52 EAOG FAI FAG ARI ARG TQI TQG FTMIR nose 0.41 0.58 0.41 0.15 0.21 0.56 0.44 0.91 0.88 MPLS 18 variables 0.73
Conclusions This road map of the externalisation of the intrinsic qualities of milk is based on a three dimensional path: Commercial, Nutritional (divided into Fatty Acids and fat soluble AntiOxidants) and Aromatic (an index which needs to be further investigated), the three-Grades finally being summarised in a four-Grade system of Total Quality. This work has proved that heuristic methods can be provided through rapid instruments, such as Enoses and especially FTMIR. This pioneer experience in Pugliesi cow herds has shown that the AntiOxidants, Fatty Acids and VOCs of milk were mainly able to distinguish one of the four studied farming Types; at the same time, it allowed the milk to be graded into four quartile classes of combined merits for each main criterion. The method worked well in a very special sample of herd types, which was different as far as management and genetic resources are concerned, and thus very rich in variation of the milk characteristics. The challenge will now be to verify this grading in a real scale with more uniform technologies and animals. Rapid systems can efficiently organise a wide variability of milk and grade it into quality classes. References Pizzoferrato L., Manzi P., Marconi S., Fedele V., Claps S., Rubino R. 2007. J. Dairy Sci. 90:45694574. 24
Sensorial characterization of Raschera PDO cheese produced by different factories in the production area
L. Galassi1, P. Bianchi1, D. Giaccone2, A. Revello-Chion3*, E. Tabacco3, G. Borreani3 1 ERSAF Via Pilla, 25/b, I-46100, Mantova 2 Associazione Regionale Allevatori del Piemonte Via Livorno 31, I-10144, Torino 3 Dep. of Agronomy, Forest and Land Management, University of Turin Via L. da Vinci 44, I-10095, Grugliasco (TO) *andrea.revellochion@unito.it Abstract The aim of this work was to evaluate the different sensory properties of Raschera PDO cheeses produced in winter and summer seasons from ten cheese factories. The cheeses were subjected to a Quantitative Descriptive Analysis (QDA) to determine their sensory properties. Ten expert panellists were selected to form the panel, involved in the definition of a list of attributes for texture, odour, taste and aroma, which were scored in an intensity scale. For winter and summer cheese productions were identified 30 and 36 attributes, respectively, showing a strong difference in sensory properties between cheese produced in summer and in winter. Results showed also significant differences between cheesemakers and highlighted that sensory properties of cheese were affected by a combination of several factors, which are involved in the whole dairy production chain. Keywords: sensory properties; Raschera PDO cheese; attributes. Introduction The Raschera cheese is a typical semi-fat, semi-cooked and pressed cheese, which is ripened for a minimum period of 60 days to be granted with the Protected Designation of Origin (PDO) label. It is produced within the Cuneo province (North-West Italy) from raw or pasteurised milk and its certified production standards do not include specifications on the cows dietary sources, which varied from extensive to intensive dairy systems. Consequently, the sensory properties of the resulting cheese can be expected to vary widely due to the broad scope of milk production and cheese-making practices used (Cornu et al., 2009). It is known that the cheesemaking technology, the chemical and microbial composition of milk associated to the conditions of milk production, such as geographical area, animal breed, animal diet and season have an important impact on sensory characteristics of cheeses perceived by consumers (Coulon et al., 2004). Unambiguously, precise and "scientific" standard cheese sensory descriptors could be a useful tool for cheesemakers to commercial exploitation of cheese, especially for typical PDO cheese such as Raschera. The aim of this work was to evaluate the different sensory properties of Raschera PDO cheese produced by different cheese factories in the production area. Material and methods Ten factories that produced more than 80% of total Raschera PDO cheese production were selected based on different characteristics to obtain the widest range of variability on sensory properties. To describe the sensory properties of cheese, a Quantitative Descriptive Analysis (QDA) was performed on two cheeses ripened for 60 days of each factory for both winter and summer seasons. Ten expert panellists were selected to form the panel, subjected to a training period for QDA and involved in the definition of a list of attributes for texture, odour, taste and aroma of cheese. Attributes generated from each assessor were characterised and agreed on a round table-meeting involving sampling taste and collective discussion to create the final list of attributes which described the cheese sensory profile. Subsequently, in the test, attributes were evaluated on an intensity scale from 1, when the attribute was defined as not perceptible, to 9, when the attribute had the maximal expression. Statistical analyses were performed with Senstools software (v 3.3.2, OP&P, Utrecht, The Netherlands). Results were analysed according to generalized procustes analysis and using a mixed model analysis of variance, where cheese factory, season and panellist were considered as fixed factors and replication as random factors. Results and discussion The assessors identified 30 attributes to describe the winter production of Raschera PDO cheese, which comprise 4 descriptors for texture, 11 for odour, 4 for taste, and 11 for aroma (Figures 1 and 2). Figure 2 shows that the summer cheeses were characterized by a sensory profile with more complex attributes and higher intensities of some descriptors compared to sensory profile of winter cheeses (Figure 1). The summer production of Raschera PDO cheese was described by 36 attributes, showing three odour (grass, hazelnut and smoked) and three aroma descriptors (of grass, hazelnut and smoked) more than the cheeses produced during winter. The analysis of variance showed a significant difference in 24 attributes for cheeses between the ten factories produced in winter, whereas summer cheeses presented a significant difference in 29 attributes between the ten factories. These differences were partly expected and could be due to several factors during the whole cheese production chain. For example, the use of raw or pasteurized milk has an effect on microbiological communities of cheese, and consequent effects on sensory profiles of cheese, which can also change with different composition of 25
animal diets (Cornu et al., 2009). Conclusions The results confirmed that sensory profiles of cheeses are affected by a combination of several factors, which are involved in the whole dairy production chain. Variations of sensory properties are also possible inside the PDO cheese, which have to follow a restricted and specified production rules. Figure 1: Sensory profiles of Raschera PDO cheese produced in winter by the ten cheese factories; RE = extensive system, raw milk; RI = intensive system, raw milk; PI = intensive system pasteurized milk
boiled vegetable aroma bread aroma soft toasted aroma garlic aroma barn aroma silage aroma strong toasted aroma rennet aroma whey aroma butter aroma cream aroma solubility adhesive elasticity cream odour butter odour 6 whey odour 5 rennet odour 4 3 2 1 0 soft toasted odour bread odour boiled vegetable odour sweet taste salty taste acid taste bitter taste strong toasted odour silage odour barn odour garlic odour
PI RI RE
hardness
Figure 2: Sensory profiles of Raschera PDO cheese produced in summer by the ten cheese factories; RE = extensive system, raw milk; RI = intensive system, raw milk; PI = intensive system pasteurized milk
cream odour grass aroma butter odour 6 hazelnut aroma whey odour rennet odour smooked aroma 5 boiled vegetable aroma strong toasted odour 4 bread aroma silage odour soft toasted aroma garlic aroma barn aroma silage aroma strong toasted aroma rennet aroma whey aroma 3 2 1 0 barn odour garlic odour soft toasted odour bread odour boiled vegetable odour smooked odour hazelnut odour
PI RI RE
grass odour butter aroma sweet taste cream aroma salty taste solubility acid taste adhesive elasticity bitter taste hardness
Acknowledgements The research was funded by the Regione Piemonte Project: Qualit degli alimenti, gestione degli animali e tecnologia di caseificazione: esempio di filiera produttiva di alcuni formaggi DOP tipici piemontesi in zona montana. All the authors contributed equally to this paper. References Coulon J.-B., Delacroix-Buchet A., Martin B., Pirisi P. 2004. Lait. 84:221-241. Cornu A., Rabiau N., Kondjoyan N., Verdier-Metz I., Pradel P., Tournayre P., Berdagu J.L., Martin B. 2009. Int. Dairy J. 19:588-594. 26
Influence of native pasture on compositional characteristics and aroma compounds of traditional Feta cheese by multiple dynamic headspace extraction and Gas Chromatography-Mass Spectrometry
D. Bozoudi1, A. R. Caputo2, S. Claps2, E. Litopoulou-Tzanetaki1* Laboratory of Food Microbiology and Hygiene, Faculty of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece 2 CRA-ZOE, Consilio per la Ricerca e Sperimentazione in Agricoltura, Unita di Ricerca per la Zootecnia Estensiva, Bella (PZ), Italy * ganet@agro.auth.gr Abstract Feta cheese samples from three different Greek mountainous areas, manufactured from sheep milk produced by animals consuming native pasture plants, were studied. Bulk samples of pastures were also studied, two from each area. The objective was to determine the influence of consumption of native plants on the compositional characteristics and aroma compounds of Feta cheese. The cheese pH and the WSN(%TN) of NW area (~800m altitude) was significantly lower (P<0.05) and the moisture content higher (P<0.05) than the respective values of SW areas (at ~800 and 1300-1500m altitude). A total of 73 VOC were determined in Feta cheese. Hexanal was found only in mature cheeses of NW area and 1-methyl-propyl ester-acetic acid, was detected only in cheeses of SW area (~800 altitude). Common compounds for cheeses from all areas were nonanal, decanal, 2-heptanone, 6methyl-5-hepten-2-one, butanoic acid ethyl ester, hexadienoic acid-bis-(1-methylethyl)-ester, 1-methyl-4-(1methylethyl)-benzene and the terpenoids -pinene, camphene, l-limonene and camphor. Terpenes found in all samples of pasture plants were -pinene, camphene, -pinene, eucalyptol, camphor and neryl acetone. Common compounds in pasture and cheese could be used as markers to characterize mountainous Greek Feta cheese. Keywords: pasture, milk, Feta cheese, VOC Introduction Animal feeding is a very important factor in cheese characterization, due to its action on milk bacteria and milk compounds, such as fats, proteins, flavors and so on (Grandison et al., 1986). Moreover, feeding is considered an important factor because it is expressed not only by the diet ingested but also by the notion of terroir (Coulon et al., 2004). The influence of different types of pastures on milk and cheese volatile composition has been studied (Bugaud et al., 2001), but usually with the aim of detecting useful markers to link the product to its origin. In the present research, the influence of three different pastures grazed by ewes at three different areas on the compositional characteristics and volatile compounds of Feta cheese was investigated. Materials and Methods The pasture plants were collected at the flowering stage of plant development. Six bulk pasture samples were obtained, two from each, NW area at ~800m altitude and SW ~850m as well as ~1300-1500m height. Mature cheeses from three cheesemaking trials for each area were used for analyses. The pH, NaCl content, total nitrogen and fat content and proteolysis of the cheese were determined and the VOC composition was estimated by multiple dynamic headspace extraction and Gas Chromatography-Mass Spectrometry (Ciccioli et al., 2004). Results and Discussion The cheese pH and the WSN (%TN) of NW area (~800m altitude) was significantly lower (P<0.05) and the moisture content higher (P<0.05) than the respective values of SW areas (at ~800 and 1300-1500m altitude; Table1). Table 1: Compositional characteristics of mature Feta cheese from three different areas.
pH Moisture % Salt-in-moisture Fat dry mater Protein N%TN WSN%TN o-PA NW Greece (~800m) 3.89a 0.06 52.7a 4.85 3.54a 0.62 54.99a 1.17 12.76a 1.09 0.22a 0.02 21.30a 0.22 SW Greece (~850m) 4.60b 0.18 45.30b 3.48 2.88a 1.13 57.29a 4.71 17.12b 2.66 0.38b 0.11 18.01a 2.87 SW Greece (~1500m) 4.09b 0.281 42.02b 1.51 2.88a 0.86 53.88a 7.26 18.85b 1.10 0.39b 0.08 5.43b 0,42
1
Mean of three cheesemaking trials (x SD) a, b: means of the same row with different superscripts differ (P<0.05). Mature cheeses coming from SW Greece contained similar amounts of aldehydes, alcohols, ketons and esters; however, the cheese coming from the lower altitude contained lower levels of hydrocarbons and terpenes (Table 2). 27
Table 2: VOC content1 of mature Feta cheese from three different areas.
Group of substances Aldehydes Alcohols Ketons Esters Hydrocarbons
1
NW Greece (~800m) 10.59 7.19 2.81 171.78 17.25 48.08
SW Greece (~850m) 3.52 221.81 8.33 217.95 20.72 17.89
SW Greece (~1300m) 3.07 376.14 5.23 192.26 31.98 93.38
Terpens
Amounts in arbitrary units (A.U.)
A total of 73 VOC were determined in Feta cheese. Hexanal was found only in mature cheeses of NW area and 1-methyl-propyl ester-acetic acid, was detected only in cheeses of SW area (~800 altitude). Common compounds for cheeses from all areas were nonanal, decanal, 2-heptanone, 6-methyl-5-hepten-2-one, butanoic acid ethyl ester, hexadienoic acid-bis-(1-methylethyl)-ester, 1-methyl-4-(1-methylethyl)-benzene and the terpenoids pinene, camphene, l-limonene and camphor (data not shown). VOC of bulk forage samples from the three areas differed in respect of the VOC content (Table 3). Even samples of the same area were found to contain considerably different amounts of the compounds, such as aldehydes (samples A, B), alcohols (samples E, F) and/or terpenes. Table 3: VOC content1 of bulk forage samples from three different areas.
Aldehydes Ketons Hydrocarbons Alcohols Terpenes
1
NW Greece (~800m) A B 17.01 31.42 1.75 11.20 20.26 21.07 3.11 11.19 20.69 15.46
SW Greece (~850m) C D 13.56 12.07 10.65 11.98 19.30 23.49 4.53 7.34 11.76 14.31
SW Greece (~1500m) E F 33.81 43.27 5.37 8.07 55.52 17.07 3.83 13.20 9.61 11.01
Amounts in arbitrary units (A.U.)
Terpenes found in all samples of pasture plants were -pinene, camphene, -pinene, eucalyptol, camphor and neryl acetone. Common terpenes most abundantly found in mature cheeses of all areas were -pinene, camphene and L-limonene. Conclusions This study suggests that common compounds in pasture and cheese could be used as markers to characterize mountainous Greek Feta cheese. References Bugaud C., Buchin S., Noel Y., Tessier L., Pochet S., Martin B., and Chamba J. F.. 2001. Lait, 81: 593-607. Coulon J.B., Delacroix-Buchet A., Martin B., Pirisi A. 2004. Lait, 84: 221-241. Grandison A. S., Brooker B. E., Young P., Ford G. D., Underwood H. M. 1986. J. SOC. Dairy Technol., 39: 119-123.
28
Variation in volatile profile of plants grazed by sheep and the produced milk at two different areas of mountainous Greece
D. Bozoudi1, Z. Parisi2, A. R. Caputo3, S. Claps3, S. Belibasaki4, E. Litopoulou-Tzanetaki1* Laboratory of Food Microbiology and Hygiene, Faculty of Agriculture Laboratory of Range Science (236), School of Forestry and Natural Environment Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece 3 CRA-ZOE, Consilio per la Ricerca e Sperimentazione in Agricoltura, Unita di Ricerca per la Zootecnia Estensiva, Bella (PZ), Italy 4 Veterinary Research Institute, NAGREF Campus, 57001, Thessaloniki, Greece * ganet@agro.auth.gr
2 1
Abstract The objective of this study was to characterize the volatile profile of plants grazed by sheep and of milk produced at two different mountainous areas, where traditional Feta cheese is made as a main dairy product. For this purpose, twenty three plants growing at two different mountainous areas at 800m (NW Greece; 15 plant species) and 1300m (SW Greece; 8 plant species) above sea level and six milk samples, three from each area, were collected and their volatile organic compounds (VOC) content was analyzed by gas chromatography and mass spectrometry (GC-MS). The plants collected in NW area were richer in aldehydes, hydrocarbons and terpenes. Milk samples of both areas were poor in aldehydes and less rich in alcohols than plants. Thirty two terpenoid compounds were isolated from the different plant species, with -pinene, camphene, camphor and fenchyl acetate being common constituents in plants of both areas. dl-Limonene was detected more frequently from the plant species of SW area and eucalyptol and -terpinene were almost exclusively traced in plants collected from the NW area. Common terpenoid compounds for plants and milk were -pinene, camphene, dllimonene and camphor. The above substances may be used as markers for linking dairy products to grazing pasture. Keywords: green pasture, milk, aroma compounds, GC-MS, VOC Introduction Traditional Feta cheese is produced in small dairies or on farms and the mountain farming system is based on local forage resources, with a combination of preserved feeds and fresh pastures. The sensory properties of the cheeses produced in this way often reflect the characteristics of the fresh pasture plants (Buchin et al., 1999; Carpino et al., 2004). The objective of the grazing study presented in this paper, was to identify the particular plant species and establish the composition of aroma compounds contained in sheep pastures and the produced milk at two different areas and altitudes of mountainous Greece. Materials and Methods The plants were collected at the flowering stage of plant development as follows: A wooden frame of one m2 was put on the ground and all the different plant types were hand collected from NW and SW area. Three samples of milk were also examined from each area. The aroma compounds were extracted from the plant samples by steam distillation (Carpino et al., 2004). Volatile organic compound (VOC) profile and content in the plant extracts (2ml in ethyl ether) and milk (50ml) were determined using the modified dynamic headspace technique (Fedele et al., 2000; Ciccioli et al., 2004). Results and Discussion The species composition of the grazing areas is presented in Table 1. Results of the MS-GC analysis showing the profile of each plant species in respect of the amounts in AU of aldehydes, ketons, esters, hydrocarbons, alcohols and terpenes are shown in Figure 1. In general, comparison of the composition of the plants obtained from the two different heights revealed, that the plants collected in NW area at ~800m altitude were richer in aldehydes, ketons, hydrocarbons and terpenes. In the majority of the plants collected from NW area terpenes predominated over the other VOC. Comparison of plant samples of the same botanical species collected at the two different locations (K1 and B5, Aegilops sp.; K4 and B6, Taehiatherum caput-medusae) and in the same development stage had a different VOC composition. Common aldehyde found in all plants was hexanal. The majority of the plants also contained 2-hexanal (E)-, benzaldehyde, nonanal and decanal. It is also worthwhile noting that the aldehyde composition of the same plants of Taeniatherum caput-medusae (K4, B6) and Aegilops sp. (K1 and B5), collected from the two different locations, was different.
29
Table 1: Plant species collected from two mountainous Greek areas.

~800m, North West Greece Family Graminaceae Species Aegilops sp. Taehiatherum caput-medusae Bromus sp. 1 Brachypodium sp. Trifolium campestre Trifolium hirtum Vicia sp. Trifolium sp.1 Lathyrum sp. Vicia villosa Anthemis cf tinctoria ssp. parnassica Crepis sp. Euphrasia sp. Thymus sp. Rumex acetosella Code K1 K4 K6 K9 K2 K5 K7 K12 K14 K15 K3 K13 K8 K10 K11 1300-1700m, South West Greece Family Graminaceae Species Phleum sp. Aegilops sp. Taeniatherum medusae Petrorhagia sp.1 Petrorhagia sp.2 Ptentilla recta Rosa sp. Convolvulus sp.1 Code B3 B5 B6 B7 B8 B2 BB1 B4
caput-
Caryophyllaceae Rosaceae Convolvulaceae
Leguminosae
Compositae Scrophulariaceae Labiatae Polygonaceae
In this study, 32 different terpenoid compounds were isolated from the different plant species. The most widespread monoterpenes, -pinene and camphene were determined in the volatile profile of practically all plant species of both grazing sites and seem to be important plant constituents. Moreover, it is worthwhile noting, that plants from NW area were considerably richer in number of terpenoids (5-15 terpenes), with the highest and lowest numbers detected in Lathyrum sp. (K14) and Bromus sp.1 (K6), respectively. Differences in the volatile composition of milk samples were observed. The milk samples of both areas were poor in aldehydes, ketons, esters and alcohols (data not shown). However, 2-pentanal, benzaldehyde, decanal and 6-methyl-5-hepten-2-one were common constituents in milk samples. Their terpenoid profile was more or less similar to that of the plants, in respect of the common and predominant terpenes, -pinene, camphene, dl-limonene and camphor. Figure 1: Volatile Organic Compounds (VOC) profile of different plants from grazing areas at ~800m and 1300m altitude. K1-K15; BB1-B8: plant species explained in Table 1.
1000
Aldehydes
Ketons
Esters
Hydrocarbons
Alcohols
Terpens
100
Arbitrary Units (AU)
10
1 K1 K4 K6 K9 K2 K5 K7 K12 K14 K15 K3 K13 K8 K10 K11 B3 B5 B6 B7 B8 B2 BB1 B4
NW area ~800m altitude Plants
SW area 1300m altitude
Conclusions Common terpenoid substances in both, plants and milk, such as -pinene, camphene, dl-limonene and camphor, confirm that the type of diet is responsible for the presence of these marker compounds and could be used as biochemical indicators to characterize cheeses. References Buchin S., Martin B., Dupont D., Bornard A., Achilleos C. 1999. J. Dairy Res., 66: 579-588. Carpino S., Mallia S., Licitra G., Van Soest P.J., Acree T.E. 2004. Fl. and Fragr. J., 19: 293-297. Ciccioli P., Brancaleoni E., Frattoni M., Fedele V., Claps S., & Signorelli F. 2004. Ann. di Chim. 94: 669-678. Fedele V., Signorelli F., Brancaleoni E., Ciccioli P., Claps S. 2000. Pag. 152-153 in proc. 7th International Conference on Goats, IGA, France, Paris, France: INRA
30
Phenolic content of forage, milk, whey and cheese from goats fed Avena sativa
L. Sepe1*, A. Cornu2, B. Graulet2, S. Claps1, D. Rufrano1 1 CRA-ZOE, Consiglio per la Ricerca e sperimentazione in Agricoltura, Unit Zootecnia Estensiva, via Appia, 85054 Muro Lucano (PZ), Italy 2 INRA, UR 1213 Herbivores, F-63122 Saint-Gens-Champanelle, France *lucia.sepe@entecra.it Abstract Plant secondary metabolite (PSM) content in food attracts considerable attention for health consideration. As PSM in dairy products primarily depends on animal feeding, this study aimed to evaluate the phenolic content of milk and cheese from goats fed with plants commonly used as forage in mountain farming systems. Siriana goats were fed indoor with freshly cut Avena sativa in pureness for 13 days. Milk and whey tank samples were daily collected contextually with cheese-making (Caciotta cheese) and stored at 20C. Offered herbage was daily collected and stored at 20C, freeze-dried and ball milled. Phenolic compounds were analysed as UV-absorbing compounds (UAC), on plant, milk, whey and cheese samples using HPLC-DAD. Twenty-five peaks were found in plants, 26 in milk, 26 in whey and 26 in cheese. Milk and whey UAC profiles were similar with benzoic acid derivatives as major compounds, and different to that of cheese. Although several flavones were found in Avena sativa, none was found in the corresponding milk, whey and cheese. Keywords: phenolic compound, goat, milk, cheese, whey, Avena sativa Introduction There is an increasing interest from consumers about healthy food (CLA or 6- enriched milk, organic products, etc.) and for phenolic compounds, natural or as additive, for their antioxidant, anticarcinogens, antimutagens properties, or because they could affect the sensory properties of cheese and beverage. Phenolic compounds occur in milk at concentrations too low to present an interest in human health, except for those that have hormone activities, like isoflavones, coumestans and lignans (Andersen et al., 2009). Another interest of phenolic compounds in milk concerns their plant origin and their potential as tracers of animal feeding. Unlike terpenes, that are readily transferred from plant to milk (Viallon et al, 2000), phenolic compounds undergo modifications in the animal digestive tract (O'Connell and Fox, 2001). Therefore the link between their occurrence in milk and the plant ingested needs to be investigated. In this study, mono-specific feed was given to goats in order to get the phenolic profile corresponding to a given plant species. This communication will focus on the phenolic compounds found in fresh Avena sativa and in the corresponding milk, cheese and whey. Material and methods Ten Siriana goats were fed indoor with Avena sativa in pureness, given fresh for 10 days of adaptation and 3 days of trial. In these 3 days, herbage (the part of plant effectively ingested) was daily collected, freeze-dried and ball milled. Milk and whey samples were collected on days 2nd and 3rd, contextually with cheese-making (Caciotta cheese) and stored at 20C. Phenolic compounds were extracted from herbage with ethanol:water (80:20) at 90C on 200 mg samples (adapted from Fraisse et al., 2007), while from milk and whey by a procedure derived from King et al. (1998), consisting in a precipitation of 10 ml milk/whey in 22 ml acetonitrile and an overnight deconjugation using a glucuronidase-sulfatase enzyme mixture. Thawed cheese was homogenised in water using ultraturrax and centrifuged. The supernatant was used for phenolic compounds extraction by the same method as for milk. The extracts were analysed by HPLC on a reverse phase column (LiChroCART 125-4, Merck) eluted by 0.3 ml/min of a 0-100% gradient of acetonitrile in water, both containing 0.1% formic acid. Phenolic compounds were detected at 275 nm using a diode array detector and their spectra between 200 and 400 nm were stored. Peaks were integrated using the Agilent Chemstation software. For the preliminary study, the UV spectra were compared to those of standard compounds and classified into simple phenol, benzoic acid derivatives, cinnamic acid derivative and flavones spectral families. Results and discussion Cinnamic acid derivatives and flavones had recognisable, characteristic spectra with several intense absorption bands. Simple phenols had UV spectra resembling those of phloroglucinol, catechol or phenol, with only one absorption band at low intensity, and one must be aware that complex polyphenols like equol might be misclassified in this group. Likewise, benzoic acid derivatives had UV spectra resembling those of syringic or 4hydroxybenzoic acid, although some flavanols like catechin or flavanones like naringenin might be classified in this group. In Avena sativa, 25 peaks were obtained (Table 1). Among them, flavones largely dominated, in diversity as well as in quantity. Indeed, 7 peaks, comprising the largest one, had UV spectra similar to those of apigenin or luteolin. Avena can be considered close to other Poaceae like barley, in the leaves of which a wide variety of flavones have been described by Ferreres et al. (2008). Those which have been identified derived from apigenin, 31
luteolin or chrysoeriol with 1 or 2 sugar moieties linked by either C- or O-glycosidic linkages. The most important peak was isoorientin-7-O-glucoside (i.e. luteolin-6-C-glucoside-7-O-glucoside). Cinnamic acid derivatives were also abundant. In milk, only benzoic acid derivatives were found in significant amounts. Non-phenolic, UV-absorbing compounds such as hippuric acid, riboflavine and indole derivatives were also detected. Surprisingly, no flavonoid was detected in milk in our trial, although flavones had been ingested in high amount and despite flavonoids have been found in goat's milk (De Feo et al., 2006) even using the same method (Gkouma et al., 2011). Avena flavones were either not absorbed or were absorbed and degraded in the goat's body. Jordan et al. (2010) also observed that flavones were not transferred into goat milk while flavanones were. In cheese, although the more intense peaks still had benzoic acid derivatives' type spectra, there was a greater number of simple phenol peaks and they contributed to the third of the total area. Neither hippuric acid nor riboflavine were found. One of the indole derivatives found in milk was recovered in cheese. Phenolic profiles of whey were similar to those of milk, largely dominated by benzoic acid derivatives. Hippuric acid and riboflavine were also present, but no indole derivative. Table 1: Phenolic compounds in Avena sativa herbage and in milk, cheese and whey from goats fed the fresh herbage
Chemical family Flavones Cinnamic acid derivatives Simple phenols Benzoic acid derivatives Unclassified Total Plant Number of Total area compounds % 7 80 5 11 5 4 4 25 6 1 1 100 Milk Number of Total area compounds % 7 12 3 22 3 96 1 100 Cheese Number of Total area compounds % 15 5 5 25 33 41 24 100 Whey Number of Total area compounds % 6 15 6 27 3 97 1 100
Conclusions The Avena diet contained almost exclusively flavones as soluble phenolic compounds and could be used as a typical flavones diet in studies aiming at understanding the fate of the different families of phenolic compounds after ingestion. In this study, no flavone was recovered in milk. Either flavones were not absorbed in the digestive tract, or they were degraded. These preliminary results allowed proposing a method to analyse phenolic compounds in cheese and to observe that a variety of phenolic compounds are carried from milk to cheese. Nevertheless, phenolic compounds' profile of milk was much closer to whey profile than to cheese ones. The classification of compounds according to UV spectra gave little information, owing to the great number of peaks having the same spectra. Further studies including mass spectrometry need to be undertaken in order to identify compounds more precisely. Quantitative studies will also be necessary to measure phenolic compounds partition between whey and cheese. Acknowledgements This work was partially funded by MiPAAF grant for abroad stage for CRAs researchers and technologists. References Andersen C., Nielsen TS, Purup S, Kristensen T, Eriksen J., Soegaard K., Sorensen J, Frett X.C., 2009, Animal, 3, 1189-1195 De Feo V., Quaranta E., Fedele V., Claps S., Rubino R., Pizza C. 2006, Ital. J. Food Sci., 1 (18): 85-92. Ferreres F., Andrade P.B., Valentao P., Gil-Izquierdo A., 2008, J. Chromatography A, 1182, 56-64 Fraisse D., Carnat A., Viala D., Pradel P., Besle J.M., Coulon J.B., Felgines C., Lamaison J.L. 2007. J. Sci. Food Agric., 87: 2427-2435. Gkouma M., Cornu A., Massouras T., Lemaire M., Graulet B., 10th Int. Meeting on Mountain Cheese, Dronero, 14-15 september 2011 (in press). Jordan MJ, Monino M.I., Martinez C., Lafuente A., Sotomayor J.A., J. Agric. Food Chem., 2010, 58, 8265-8270 King R.A., Mano M.M., Head R.J. 1998. J. Dairy Res., 65, 479-489. O'Connell, J. E. and P. F. Fox (2001). Int. Dairy J. 11: 103-120. Viallon C., Martin B., Verdier-Metz I., Pradel P., Garel J.P., Coulon J.B., Berdagu J.L., 2000, Lait, 80, 635641.
32
Comparative analysis of milk and cheese produced in the Plardon PDO production zone based on their phenolic profiles
M. Gkouma1,2, A. Cornu1*, T. Massouras2, M. Lemaire3, B. Graulet1 1 Unit de Recherche sur les Herbivores (UR1213), INRA, 63122 Saint-Gens-Champanelle, France 2 Laboratory of Dairy Technology, Department of Food Science & Technology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece 3 Montpellier Supagro, Site de la Gaillarde, 2 place Pierre Viala, 34060 Montpellier cedex 02, France *Agnes.Cornu@clermont.inra.fr Abstract Goat milk and cheese samples were collected from two different areas in the Pelardon PDO production zone, in order to explore the diversity of their phenolic profiles and try to relate it to variations in feeding practices. The results showed a diversity in the phenolic profiles of both milk and cheese samples, which was attributed to the heterogeneity of the vegetation and soil as well as the diverse farming and grazing methods applied. Nevertheless, the statistical analysis failed to reveal a link between milk and cheese phenolic composition and the production area or the feeding system. Data will require a more detailed analysis using informations from the farmer inquiries, in order to try to elucidate the main factors of feeding practices acting on milk and cheese phenolic composition. Keywords: phenolic compounds, milk, cheese, Plardon Introduction Phenolic compounds (PCs) are a diverse group of chemicals which play a major role in the sensory attributes of many food products. The majority of PCs present in milk and dairy products are derived from the animal feed. Nevertheless, their occurrence in dairy products is also a consequence of the catabolism of proteins in cow or by bacteria in cheese. More rarely, phenolic compounds might arise from a contamination with sanitising agents, process-induced incorporation or their deliberate addition as specific flavouring or functional ingredients (OConnell et al., 2001). Due to their specific profiles in different plant families, phenolic compounds are considered as potential tracers of the geographical origin of milk (Besle et al., 2010; Prache et al., 2005). Plardon PDO is a traditional French goat cheese, produced in a large geographic zone extended across Languedoc-Roussillon. Rangeland grazing is among its main specifications of production. The Plardon PDO zone is spread over different geographical areas with different soils, vegetations and grazing methods that could strongly influence the PCs profile of cheeses, i.e. the lowland areas in the garrigues of Corbires and Pays Gardois, mainly with limestone soils, opposed to the northern mid-mountain, rather schistose area of Cvennes. The object of this study was to evaluate the diversity in PC composition in milk and cheese in relation to the grazing method and the geographical production area. These geographic areas Cvennes and Garrigues, hold about 70% of the members of the Plardon Farmers Union. Materials and methods Nine dairy farms from each of the 2 different areas were selected. Among them, 3 groups of farms were identified, (coded as type 1, 2 and 3), corresponding to different feeding systems: type 1 refers to a basal diet consisting of rangeland grazing in type 2 the basal diet mainly consisting of hay, but at least three hours of rangeland grazing per day and type 3 refers to hay with outdoor grazing on temporary grasslands. Milk and cheese samples from each farm were collected in July and stored at -20C until analysis. Phenolic compounds were extracted with acetonitrile followed by purification with methanol after enzymatic deconjugation. The final extracts were analyzed using an HPLC system, coupled with a photodiode array detector (DAD), for the separation and identification of phenolic compounds. Results and Discussion The HPLC-DAD system allowed a qualitative analysis of the phenolic compounds present in milk and cheese. Both the absorbance spectra and the retention times (RT) were used for the tentative identification of the compounds. The principal identified compounds detected in milk and cheese are presented in Table 1. In milk, thirteen UV absorbing compounds were found in sufficient concentration to obtain representative absorbance spectra. Of these, 5 could be flavonoids. Hippuric acid was the predominant compound in milk. The second most abundant compound (RT 11.01) was not identified. Its spectrum looked like a flavanone. This compound was only found in milk of types 1 and 2 (the more extensive farming systems, exploiting rangelands) of the Cvennes area, the highest value being in type 1. Benzoic acid is a breakdown product of aromatics that can derive both from endogenous amino acids or from the ingested plants. Two unidentified compounds (A and B) having the same absorption spectrum were detected with retention times 13.46 and 16.56. These compounds were significantly influenced by the production area: they were more abundant in milk from the Cvennes area. These compounds were also abundant in the milk of type 1, but the type effect was not significant. 33
In cheese, a larger number of compounds was found. This may reflect a concentration of these compounds during cheese making, but also a more efficient extraction from the cheese. Seven of them could be flavonoids and 6 could be phenolic acids. Seven spectra could not be classified, including those of compounds B and C, corresponding to compounds A and B found in milk. The hippuric acid was not recovered, probably removed with the whey (Sieber et al, 1994). The major compounds were catechol and benzoic acid and the compound B found in milk. Riboflavin was still present and new compounds like 4-hydroxyphenylacetic acid, 4hydroxybenzoic acid, m-crsol and 3,4-dimethylphenol have been identified, but these identifications need to be confirmed. The Analysis of Variance (ANOVA) proved that neither the production area nor the type of farming had a significant influence on phenolic compound concentrations, except for benzoic acid which was significantly lower in cheeses of type 2 compared to those of types 1 and 3, and compound B which was more abundant in the Cvennes area. Table 1 : principal identified compounds detected in milk and cheese in arbitrary area units (aau) at 275 nm. Effect of the production zone (C = Cvennes, G = Garrigues), type of farming (1,2 and 3), together with the combination of the two factors, estimated by ANOVA (ns: non significant ; * : p<0,05 ; ** : p<0,01 ; ***: p<0,001). RT : Retention time
RT Compounds / Chemical family C1 170 1191 306 1895 660 2716 480 676 1503 179 0 482 684 1795 0 2484 419 C2 265 1272 305 691 1800 813 112 27 1071 158 0 487 0 695 0 1021 312 Average Areas (aau) C3 G1 G2 662 1689 302 0 1887 1004 548 0 1383 108 0 741 0 1720 0 1099 698 451 2464 163 0 1936 175 0 89 1677 210 8 513 0 1501 109 456 165 292 1425 134 0 1195 704 166 61 1784 96 62 249 0 585 197 0 27 G3 489 1760 354 0 1817 2 13 79 1098 249 0 673 0 1873 0 330 71 Area ns ns ns * ns ** *** ns ns ns ns ns ns ns ns * ns Effect Type Area+Type ns ns ns ns ns ns ns ns ns ns ns ns ns * ns ns ns ns ns ns * ns *** *** ns ns ns ns ns ns ns ns ns ns Milk 3.45 Catechol 4.42 Hippuric acid 6.47 Riboflavin 10.38 Benzoic acid 11.01 flavanone 13.46 A 16.56 B Cheese 2.62 4-hydroxyphenylacetic id 3.26 Catechol 4.65 4- hydroxybenzoic acid 5.72 Indole-like 7.96 Riboflavin 11.96 m-cresol 13.15 benzoic acid 13.36 3,4-dimethylphenol 16.92 B 20.08 C
Conclusions This study confirmed the greater variety of phenolic compounds in cheese compared to milk. The phenolic profiles of milk and cheese finally seem to be just marginally influenced by farming practices and rangeland grazing, according to the different types defined. Their low concentrations made those UV-absorbing compounds difficult to identify. Extractions targeting the peaks of interest could be implemented. Moreover, the proposed identifications should be confirmed with standard additions and assayed by mass spectrometry.
Aknowledgements The authors thank the following organisms for supporting this study: Ple Fromager AOC Massif Central, Syndicat des Producteurs de Plardon, DATAR Massif Central, Conseil Rgional Languedoc Roussillon and the European Erasmus Program.
References Besle J.M., Viala D., Martin B., Pradel P., Meunier B., Berdagu J.L., Fraisse D., Lamaison J.L., Coulon J.B., 2010, J. Dairy Sci., 93: 2846-2856. OConnell J.E., Fox P.F. 2001.International Dairy Journal 11 (2001) 103120 Prache S., Cornu A., Berdagu J.L., Priolo A., 2005, Small Ruminant Research 59 (2005) 157168. Sieber R., Butikofer, U., Bosset, J.O. 1994. International Dairy Journal 5 (1995) 221-246.
34
Vitamin B9 and B12 contents in cow milk according to production system

C. Chassaing1,2*, B. Graulet3, C. Agabriel1,2, B. Martin3, C. L. Girard4. 1 Clermont Universit, VetAgro Sup, UR 2008.03.102 EPR, BP 10448, F-63000 Clermont-Ferrand, France 2 INRA, USC 2005, F-63370 Lempdes, France 3 INRA, UR1213 Herbivores, Theix, F 63122 Saint-Gens-Champanelle, France 4 Agriculture et Agroalimentaire Canada - Centre de Recherche et de Dveloppement sur le Bovin Laitier et le Porc, J1M 0C8, Sherbrooke, Canada *c.chassaing@vetagro-sup.fr Abstract The factors of variation for vitamin B9 and B12 content in cow's milk have not been studied intensively. Nevertheless, these vitamins are of great nutritional interest and the consumption of a large glass of milk could provides up to 15% of the recommended daily allowances for vitamin B9 and 90% for vitamin B12. The objective of this study was therefore to describe the variability of the levels of vitamins B9 and B12 in 100 bulk milk samples produced under four French major production systems (plain or mountain and forage system based on grass or maize silage). For each production system, milk was sampled and analyzed at five periods over the year 2008. The characteristics of these milks differed according to the production system. The highest levels of vitamin B9 were generally associated with diets based on hay in the winter period and with diets based on pasture during the grazing season. Conversely, the highest levels of vitamin B12 were principally measured in milks produced with diets based on maize silage. Keywords: B-complex vitamins; bulk tank milk; dairy cow; feeding practices Introduction Historically, a limited importance has been given to B vitamins in dairy cows because supply from the diet and/or from synthesis by rumen microorganisms was globally considered as sufficient (except vitamins B5 and B9) to cover the estimated requirements of dairy cows (National Research Council, 2001). However, this statement was mainly resulting from observations performed in the 50s and production levels considerably increased since then. It is obvious that requirements for B vitamins, which are involved in the main metabolic pathways (cell respiration, energy production, nucleic acid synthesis, amino-acid metabolism, neoglucogenesis...), increase proportionally to the rise in milk production. It is supported by the fact that increasing B vitamin supply for lactating dairy cows has beneficial effects including on milk performance (Girard et al., 2010). From a nutritional point of view, milk and dairy products are good sources not only of vitamin A (the most currently studied), but also of vitamins B12 (cobalamins), B2 (riboflavin)... However, if the factors regulating vitamin A concentration in milk and cheeses have been described (Nozire et al., 2006; Lucas et al., 2006), such is not the case for B-complex vitamins. Thus, the aim of this work was to determine if concentrations of vitamin B12 and B9 in bulk tank milk vary according to feeding practices of the dairy herd and/or to the period of the year. Material and methods The study was carried out in 20 groups of five farms divided among four production systems mainly characterized by their forage system and altitude : feeding systems based on grass in Mountain (GM) or in Lowland (GL) and maize silage feeding systems located in Mountain (MM) or in Lowland (ML). In each group of farms, bulk tank milk was sampled five times in the course of the year 2008 at key times in animal feeding patterns: twice in the over-wintering period with diets based on preserved forage (January and February), and three times during the grazing period: in May, July and September. During the week of each milk sampling, a survey was carried out to record herd characteristics and feeding. Average diets for each group were described on the basis of proportions of forages and concentrate in the diet calculated from the declared quantities the farmers dispensed. When the amount of one feed ingested was unknown (such as pasture), the estimation of the amount ingested was based on the energy requirements. The energy balance was supposed equal to zero. Milk folates (vitamin B9) and vitamin B12 were extracted according to DeVries et al. (2005) and Preynat et al. (2009), respectively. Vitamin concentrations were then measured in duplicate by radioassay in two different assays for each vitamin (SimulTRAC-S Radioasssay kit, Vitamin B12 [57Co] / Folate [125 I], MP Biomedicals, Diagnostics Division, Orangeburg, NY, USA). Data were processed using the repeated measures procedure (SAS Version 9.2, SAS Institute Inc., Cary, NC), introducing the production system, period, and the interaction into the model as fixed effects and the group of farms as random effect. Because the characteristics of the milk were correlated at different periods, period was considered a repeated factor. Results and discussion The use of grass decreased gradually from feeding systems based on grass (GM or GL) to maize silage (MM or ML) (figure 1). In GM and GL, forages were mainly hay during the wintering period and fresh grass during the grazing period. The proportion of maize in cow diets was lower in MM than in ML whatever the period of the 35
year. It was particularly marked from May to September when the proportion of maize silage was extremely reduced in MM at the benefit of pasture and grass silage (estimated to more than 50 % of the DM intake) or grass silage and hay. Figure 1: Diet evolution according to the production system and the period (% in the diet on a DM basis)
concentrate
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Jan Feb May Jul Sep Jan Feb May Jul Sep Jan Feb May Jul Sep Jan Feb May Jul Sep
maize silage
grass silage
hay
pasture
GrassMountain (GM)
GrassLowland(GL)
MaizeMountain(MM)
MaizeLowland(ML)
Vitamin B12 content in milk did not vary according to the period of the year, but the highest concentrations were observed in ML (figure 2). Vitamin B9 concentrations were higher in grass-based systems than in maize systems, but lower in May when the proportion of grazed grass in the ingested forages increased. Performing a principal component analysis shows that milk vitamin B12 or B9 concentrations were respectively related to the proportions of maize or hay in the diet (greater in grass-systems and during the indoor period or at the end of the outdoor period). This latter observation on vitamin B9 could be the result of a concentration effect linked to lower mean milk production levels in these conditions; indeed, the quantity of folates secreted per day in milk of MM or ML cows was higher than in milk of cows fed grass-based diet (data not shown). These original observations have to be explored in future studies to try to relate more precisely the production systems (diet composition, cobalt and folic acid dietary intakes, milk production and composition) and these B vitamin content in milk. Figure 2: Vitamin B9 and B12 contents in cow milk according to the production system and the sampling period
120 110 100 90 80 Jan Feb May Jul Sep
B9 (ng/g)
bc c ab a b a
GM
GL
MM
bc
ML
c bc
3800
B12 (pg/g)
c b b ab a ab ab a bc a a a a ab a ab b a ab ab
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ab
b a
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Sep
Production System : GM: Grass Mountain; GL: Grass Lowland; MM: Maize Mountain; ML: Maize Lowland Means within a period with different superscripts differ (P<0.05)
Conclusions The characteristics of these milks differed according to the production systems. From a nutritional point of view, vitamin B12 supply to the consumer would then be favoured by the consumption of milk produced by cows fed a maize-rich ration. Conversely, the highest levels of vitamin B9 in the milk were generally associated with diets based on hay in the wintering period and with diets based on pasture during the grazing season. Acknowledgements This work, included in TRUEFOOD project, was co-funded by the European Commission, within the 6th Framework Programme. We acknowledge C. Sibra (VetAgro Sup) and I. Constant (INRA), and all our partners of the dairy sector and the farmers involved in this study. References DeVries J. W., Rader J. I., Keagy P. M., Hudson C. A. 2005. Journal of AOAC International. 88:5-15. Girard C., Preynat A., Graulet B., Lapierre H. 2010. Pag. 235-243 in proc. 3rd EAAP International Symposium on Energy and Protein Metabolism and Nutrition, 6-10 September, Parma, Italy. Lucas A., Agabriel C., Martin B., Ferlay A., Verdier-Metz I., Coulon J.B., Rock E. 2006. Lait, 86, 177-202. National Research Council, 2001. 7th rev edn. National Academy Press, Washington, DC. Nozire P., Graulet B., Lucas A., Martin B., Grolier P., Doreau M. 2006. Anim. Feed Sci. Technol., 131: 418-450. Preynat, A., Lapierre H., Thivierge M. C., Palin M. F., Matte J. J., Desrochers A. Girard C. L. 2009. J. Dairy Sci., 92:677-689. 36
Evolution of milk calcium content during the year

C. Hurtaud1*, M. Johan1, S. Leurent2, Y. Gallard2, L. Delaby1 1 INRA Agrocampus Ouest, UMR1080 Production du Lait, F-35590 SAINT-GILLES, France 2 INRA, Domaine du Pin-au-Haras, F-61310 EXMES, France *Catherine.Hurtaud@rennes.inra.fr Abstract The calcium content of milk from 2 breeds of dairy cows (Holstein and Normande) was observed during a complete lactation. The cows were reared in 2 feeding systems providing 2 levels of feeding: an intensive system based on corn silage and pasture supplemented with concentrate and a grass system based on preserved grass silage and pasture and no concentrate. Normande cows had higher milk calcium content than Holstein cows. There was no significant variation in total milk calcium content according to system of feeding over the whole lactation. However, in winter, calcium content was reduced in the Grass system. Milk calcium content evolved according to stage of lactation and season, with minimum milk calcium content usually occurring in May and June. The effect of season may be related to the maximum daily temperature and the duration of the day. In conclusion, many factors (nature of forage, breed, season) seem to impact on milk calcium content. Keywords: dairy cow breed, feeding, season, milk calcium Introduction
Milk calcium content is a strongly heritable trait, which is related to milk protein and fat content (Alais, 1984). Calcium content is regarded as being relatively stable in cows milk during lactation. However, results from experimental and some commercial farms, have suggested that changes in milk calcium content can occur and relate in particular to changes in diets. The objective of this experiment was to compare the calcium content of milk from Holstein (Ho) and Normande (No) cows fed low input grass based systems or maize silage based systems.
Materials and methods The experiment took place on the INRA experimental farm of Le Pin-au-Haras (Orne, France). A total of 60 dairy cows (34 multiparous and 26 primiparous), comprising 25 Ho and 35 No cows were observed from calving to drying off. Table 1: Feeding systems during the year
Strategy System Winter feeding (100 days) At grazing (from April to autumn) Unsupplemented grazing period (days) Additional forage Concentrate (kg DM/cow/day) Expression of dairy potential Intensive Maize silage Dehydrated alfalfa 30% concentrate 0.35 ha /dairy cow 90 Maize silage (from July) 4 Autonomy Low input Grass Grass silage Haylage No concentrate 0.60 ha /dairy cow 210 Grass silage (during dry weather) 0
Two feeding systems were compared from January 2010 to January 2011. The Intensive system was designed to maximise individual performance, with a high energy diet fed during lactation. In the winter period, maize silage supplemented with 30% concentrate (12% wheat, 12% maize, 12% barley, 45% soybean meal, 11% beet pulp, 1% soya oil, 2.0% sugar cane molasses, 4% minerals, 1% salt) was fed. During the spring, summer and autumn periods, the Intensive system cows grazed pasture (0.35 ha/cow) supplemented with 4 kg/d of concentrate (21% wheat, 21% maize, 21% barley, 12% soybean meal, 21% beet pulp, 2% vegetable fat, 1% sugar cane molasses, 1% salt) and 250 g/d of minerals (Ca-P-Mg, 11-42-40) and were supplemented with maize silage from July (Table 1). The Grass system was designed to decrease inputs by feeding conserved grass in the form of silage (harvested by fine chop after wilting) or big bale silage (long, semi-wilted) and 300 g/d of minerals (Ca-P-Mg, 23-8-8) with no concentrate for the winter period. During spring, summer and autumn periods, cows in the Grass system grazed pasture (0.6 ha/cow) and had 500 g/d of minerals (Ca-P-Mg, 11-42-40) with no concentrate and were only supplemented with grass silage during dry weather (Table 1). In winter, cows were fed ad libitum with 10% of daily refusals. Eleven Ho and 18 No cows were allocated to the Intensive treatment and 14 Ho and 17 No cows were allocated to the Grass treatment. Individual milk yield was measured at each milking. Fat, protein and lactose contents were determined 3 times per week (i.e. on six milkings) by infrared analysis (Milkoscan, Foss Electric, Hillerd, Denmark). Once a month for the whole lactation, a detailed calcium analysis of milk samples was performed from a composite of evening and morning milkings. Total calcium was analysed by atomic spectrophotometry absorption on milk. Data were analysed using Proc Mixed procedure. 37
Results and discussion No significant interaction was detected between feeding system and breed for milk yield and composition. During the whole lactation, the Grass treatment reduced milk yield (- 5.0 kg/d, P<0.001), with no significant effect on protein and total calcium contents. The No cows produced less milk (- 4.6 kg/d, P<0.001), but with a higher protein content (2.55 g/kg, P<0.001) and total calcium content (86.5 mg/kg, P<0.001) compared to Ho cows (Table 2). Table 2: Effect of breed and feeding system on milk yield and composition during the whole lactation.
Ho Milk yield, kg/d Protein content, g/kg Total calcium, mg/kg Intensive 26.6 32.9 1297 Grass 20.9 31.6 1295 Intensive 21.3 35.0 1384 No Grass 17.0 34.6 1381 SEM 3.51 2.58 70.1 Breed ** ** ** Effect Feeding ** ns ns
During winter (January to March, 2010), the Grass treatment reduced milk yield, protein and total calcium contents (Table 3). During grazing period for all the cows, the Grass system only significantly reduced milk yield. According to Meschy (2010), calcium contained in conserved or fresh green forages is less available because it is in oxalate form. Moreover, calcium from hay (organic form) is less digestible than calcium from inorganic sources. Table 3: Effect of breed and feeding system on milk yield and composition during winter and grazing period.
Ho Intensive Winter Milk yield, kg/d Protein content, g/kg Total calcium, mg/kg 33.2 32.3 1303 Grass 23.9 29.6 1242 22.3 32.0 1235 Intensive 26.5 34.2 1403 23.2 35.2 1325 No Grass 18.5 32.4 1362 18.9 35.0 1331 SEM 3.55 2.22 58.1 3.05 1.98 58.0 Breed ** ** ** ** ** ** Effect Feeding ** ** ** ** ns ns
Grazing period (April to September) Milk yield, kg/d 28.4 Protein content, g/kg 33.2 Total calcium, mg/kg 1224
Figure 1: Seasonal variation of milk calcium (corrected for the effect of lactation)( Holstein cows, Normande cows)
Milk calcium content changed during the year. It decreased from January to June and largely increased after July irrespective of breed. During May and June, milk from Ho cows, had a calcium concentration below the French legal limit of 1.2 g/L for consumption milk. There was a significant effect of stage of lactation on milk calcium content (not shown). At the beginning of lactation, colostrum had an elevated calcium content. After 2 months, the calcium content stabilised and increased at the end of lactation (Gaucheron, 2005).
When the data were corrected for stage of lactation, there was a significant effect of month (Figure 1). Month included numerous significant factors such as maximum daily temperature, day-length and radiance duration. When daily temperatures increase, cows increase respiratory rate causing alkalosis. When blood pH increases, the chemical groups on blood proteins release hydrogen ions, which combine with HCO3 to form water and CO2. As hydrogen ions leave the protein, their absence creates a negative charge that attracts free ionized Ca. As a result blood calcium can be reduced (Sanchez et al, 1994), and as a consequence, milk calcium. Conclusions The results of this study indicate that numerous factors (nature of forage, breed, season) impact on milk calcium content. The biological mechanisms underlying these relationships require further investigation. Acknowledgements This work was funded by the INRA PHASE department. References Alais, 1984. Science du lait. Principes des techniques laitires. Ed. SEPAIC Gaucheron F., 2005, Reprod. Nutr. Develop., 45 :473-483 Meschy F., 2010, Nutrition minrale des ruminants, Versailles, ed Qu Sanchez W.K., Mc Guire M.A., Beede DK, 1994, J. Dairy Sci., 77: 2051-2079 38
Dairy production potential of the Jura meadows

E. Mosimann1*, P.E. Belot2, P. Parguel3 Agroscope Changins-Wdenswil, 1260 Nyon, Switzerland 2 Jura Conseil Elevage, 39000 Lons-le-Saunier, France 3 Institut de lElevage, 25048 Besanon, France *eric.mosimann@acw.admin.ch
1
Abstract Six PDO cow cheeses of the Franco-Swiss Jura area are concerned by the Interreg project Creating values in PDO territories. The project aims at reinforcing economical and cultural values at three levels: territory, food chain and stock farm. Reflections and results about grasslands values are presented in this paper. An important issue is the implementation of a network (observatory) of technical references to support the dairy production based on grasslands forage. Grass dairy production potential (DPP) is estimated through botanical composition, phenology and utilization form. It ranges between 7 and 28 kg milk d-1 cow-1. During the season, grass growth, yield and nutritive values of hay are assessed. These data are used for estimating the dairy potential of the main grassland types (kg milk y-1 ha-1). Results are compared with those collected by official milk control board at the farm scale. In 2010, in Franche-Comt, dairy potential of grassland fodder ranged from 9 (winter hay) to 13 kg milk d-1 cow-1 (grazed pasture). These results reveal a shift between the theoretical grass dairy production potential and the effective valorisation of this resource. Some conclusions on use of concentrates and fodder autonomy are drawn. Keywords: grassland, meadow, dairy production potential, PDO cheese, Interreg Introduction The Franco-Swiss Interreg program Creating values in PDO territories results from questions asked by the official representatives in charge of PDO cheese management in the Jura area (see http://www.interregaocfromages.org). The identified needs relate to (i) the grasslands used by dairy cows, (ii) the development of milking technologies, (iii) the work in cheese dairies and (iv) the maintenance of SME on the territory. The project aims at supporting professional, technical and scientific partnerships between both regions. Issued from a wide part of the Jura range, PDO cheeses share territories and landscapes that belong to same bioclimatic zones. Reinforced exchanges will lead to a better comprehension of meadows and pastures potentialities. Grass is a meaningful element of the production system that raises the question of a right balance between the animal needs, the environmental safety (biodiversity, maintenance of the resource) and the product quality (specific characteristics, image). Grasslands are considered as a major key to the creation of values in the territories. Six PDO cow cheeses have been involved in the project: Bleu de Gex (F), Mont dOr (F), Comt (F), Vacherin Mont-dOr (CH), Tte de Moine (CH) and Gruyre (CH). In these systems, the basic diet primarily consists of grass in summer and hay in winter. Their specifications regarding fodder production mention that hay must be produced locally (PDO area, farm area). Comt has additional requirements, in particular a maximal grass production of 4600 kg milk ha-1 y-1. The working group aims at evaluating different methods allowing to establish references on the milk production potential of grass fodder. Increased valorisation of meadows and pastures from the lowland up to the mountain will drive to autonomous and traceable cheese production. Material and methods The dairy production potential (DPP) of grasslands has been tackled according to two approaches (fig. 1): 1. At the plot scale, the production of milk from grass is influenced by botanical composition, phenological stage and fodder conservation. In Switzerland, seven distinct botanical types are characterised according to their nutritive value (Daccord et al., 2006). Each year, their phenological stages are monitored in 60 sites located between 400 m and 1200 m leading to an actualised table of the forage quality according to harvest date (Meisser et al., 2008). DM yield corresponds to the harvested biomass on meadows and is estimated through grass height (Delaby et al., 2010) and DM yield measurements in fenced plots within pastures (Mosimann, 2005). Figure 1: two ways used to calculate the dairy production potential (DPP) of grassland
Botanical composition Phenological stage Grass DM yield (kg DM ha-1)
DPP grass (kg milk kg DM-1) PLOT SCALE Total milk production - DPP concentrate = DPP grass (kg milk)
DPP grassland (kg milk ha-1)
Fodder surface (ha) HERD SCALE
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2. At the herd scale, grass DPP is obtained by subtracting the quantity of milk produced with concentrates (2.2 kg milk per kg concentrate) from the total milk production. This amount is then related to the fodder surface. In France, data collected at the farm scale were analysed by means of regressions models and classified according to natural regions and altitude (Figuet, 2010). Results and discussion 1. Plot scale. The dairy production potential is very variable according to the grasslands types, ranging between 7 (mature hay) and 28 (spring grazed grass) kg milk day-1 cow-1. The grass intakes required to support the full productive potential of grass also depend on feeding/grazing conditions (16 kg DM d-1 cow-1 is considered as an average intake). On the other hand, grass grows irregularly through the season and according to the region (Table 1). For example, an intensive managed grazed pasture yields 9.2 t DM ha-1 y-1 with an average grass DPP of 23 kg milk day-1 cow-1. The corresponding grassland DPP reaches 13,225 kg milk y-1 ha-1. Table 1: Average daily growth rates and annual yield of Jura pastures
Country* - region F F F CH CH CH CH CH Lowland Intermediate Mountain Lowland (normal) Lowland (dry) Intermediate Mountain (South) Mountain (North) April (1/2) 18.1 39.2 27.7 5.1 April (2/2) 58.2 55.4 61.0 64.4 55.1 24.8 7.5 2.3 Daily grass growth (kg DM ha-1 d-1) May May June June (1/2) (2/2) (1/2) (2/2) 54.8 55.1 41.5 33.9 55.4 65.7 33.1 34.0 61.7 91.4 46.8 60.5 84.5 73.9 46.9 35.8 83.0 65.0 28.2 15.4 62.9 76.5 50.6 37.2 26.2 53.0 50.4 37.2 5.2 19.1 44.1 45.2 July 39.0 34.3 54.7 36.7 8.9 35.4 31.4 29.3 August 13.9 0.0 36.5 40.3 8.4 33.3 18.7 17.2 Sept 29.2 20.9 25.6 31.7 27.9 31.7 10.1 6.3 Annual yield (t DM ha-1) 6.9 5.7 9.0 9.2 6.3 7.6 4.6 3.3
* F: grass height measurement (Delaby et al., 2010), CH: harvested plots (Mosimann, 2005) 2. Herd scale. Two databases issued from the dairy control were built in Franche Comt (Figuet, 2010): (i) milk production (number of farms and cows, milk quantities and characteristics); (ii) dry fodder (results from analyses and enquiries on the feeding value of hays and renewals). Backward regressions were carried out on these data. A computerised simulation tool was set up in order to manage the coming winter cheese production and to calculate the dairy production based on grasslands forage. Table 2 presents some results for the Jura Department (F). In 2010-2011, the average grassland DPP ranged between 3200 and 4100 kg milk y-1 ha-1. Table 2: Estimated grass DPP calculated from data collected by Jura Conseil Elevage (2010-2011)
Parameter Concentrate annual consumption (kg DM y-1 cow-1) Concentrate daily consumption (kg DM d-1 cow-1) Grass annual yield (t DM y-1 ha-1) Grass dairy production potential per cow (kg milk d-1 cow-1) Grass intake (kg DM d-1 cow-1) Grass dairy production potential per hectare (kg milk y-1 ha-1) Jura department 1567 4.6 5.6 11.0 16.4 3751 Lowland 1574 4.6 6.4 10.5 16.4 4095 Highland 1406 4.1 5.1 10.7 16.9 3231
Conclusions Due to differentiated fertilisation and utilisation intensity, grasslands from the Jura Mountains have various dairy production potentials. The current debate relates to the use of concentrates in the diet. High productive cows fed with extensive low yielding hay need supplementary feedstuff. On one hand, cheese transformed from their milk beneficiates from the positive image of bio diverse meadows. On the other hand, the question of energy and proteins produced on external surfaces (hectares elsewhere) is eluded. The relevance of using the dairy production potential of grasslands to control cheese volumes is questioned. Strategy is very different from one country to another. A limitation of milk quantity per hectare is in contradiction with the target of producing milk with a maximum of grass. The Interreg project proposes to implement a network (observatory) of technical references on meadows and pastures. The data resulting from the various areas will contribute to an objective reflection on the grass management intensity. It should lead to a consolidation of the dairy production based on grasslands and higher fodder autonomy at farms level. Acknowledgements This work was funded by the Franco-Suisse Interreg program IVa. References Daccord R., Wyss U., Jeangros B., Meisser M. 2006. Fiche ADCF Agridea 2.7.1, 4 pages. Delaby L., Dubief F., Cassez M., Belot P.E., Frrejean L., Kerdoncuff M. 2010. Renc. Rech. Ruminants, 17 : 59. Figuet S. 2010. AgroSup Dijon, Jura conseil levage, 36 pages. Meisser M., Amaudruz M., Jeangros B. 2008. Grassland Science in Europe, Vol.13: 931-933. Mosimann E. 2005. Revue Suisse dAgriculture, 37(3), 99-106. 40
Raising Rivals Costs strategy and localized agro-food systems in Europe

D. Barjolle1*, P. Jeanneaux2*, D. Meyer2, M. Donars2 1 ETHZ Institute for Environmental Decisions, AFEE Group, CH-8032 Zurich 2 VETAGRO SUP Campus agronomique de Clermont/UMR Mtafort F-63370 Lempdes *barjolle@ethz.ch; p.jeanneaux@vetagro-sup.fr Abstract For some Localized AgroFood Systems (LAFS), e.g. Protected Designation of Origin (PDO) Comt in France, PDO Gruyre in Switzerland, the cheese and milk prices are above average whilst for some other LAFS, e.g. PDO Cantal in France, the prices are similar or even below average. This paper explores different strategies implemented by the LAFS to improve the specificity of their product, and increase the return of added value to the actors in the area of production. In particular, the objective of this paper is to assess to what extend the Raising Rivals' Costs Theory is helpful to better understand how works the chosen strategy. Two cases studies have been performed. In the first case (PDO Gruyre), the Raising Rivals Costs theory explains the observed situation. In that case, the production collective rules required to produce the PDO cheese correspond to a differentiation strategy and give power to protect the LAFS against competitors. In the second case (PDO Cantal), rules governing the cheese production are low, and few firms have taken control on the supply chain and have imposed with the time a Costs Leadership strategy. Keywords PDO, localized agro-food system, Raising Rivals Costs, governance Introduction Facing important changes of the CAP (ending of the milk quotas by the year 2015 in Europe, subsidies reallocation...), the future of the dairy production in the European mountain areas raises numerous questions for the policy-makers and the dairy farmers organisations. As a response to the decrease of the milk prices, some collective initiatives of farmers and processors have chosen a strategy of differentiation through the quality of the product, rather than a Costs Leadership strategy. To support the efforts related to the quality, especially for traditional products linked to a delimited geographic area, a European legal tool protects the traditional names and allows preserving the value based on their reputation (Barjolle and Sylvander, 2000). Dairy farmers and cheese-makers produce for PDO cheeses with variable results particularly concerning the price of the milk. Some LAFS are traditionally qualified as success stories (Gruyre PDO in Switzerland), whilst other PDOs (Cantal PDO in France) pay the same price for the milk as standard milk. The price difference may be 10 to 25% over a long period (Jeanneaux and al., 2009). These two cases focus on a product whose name is protected, and rely on agents sharing collective interests and structured in an interprofessional organization. These are interesting examples of collective action deploying a strategy of competitive advantage based on origin but with different economic results. Cantal focuses on a cost leadership strategy while Swiss Gruyre focuses on a differentiation strategy based on a specific quality linked to origin. To explore these different collective strategies, we use the Raising Rivals Costs Theory (Salop and Scheffman, 1983; Scheffman and Higgins, 2003). In its classical field, this theory explains how works the strategy of vertical integration or a situation in which pressure is applied to suppliers to create difficulties for competitors. The aim of this paper is to assess to what extend the Raising Rivals' Costs Theory is helpful to better understand how works the chosen strategy. Material and Methods To understand the collective strategies (relationship between breeders, cheese-makers, ripeners) concerning Gruyre and Cantal PDO, this research aims at gathering and treating data from several sources: Statistical and economic data (milk prices, cheese prices, costs, quantities) provided by the French and Swiss statistical services and each LAFS associations statistical services. Textual legal data contained in the specifications on both PDOs, in order to understand the organization of the production and the mechanisms of protection. In conducting the monographs of the PDO situations, the protagonists legal equipment is the focus of attention. Results and discussion This research introduces a double originality. Firstly, by focusing on collective and individual behaviour, we can scrutinize the processes used by the firms to increase their bargaining power in order to maintain their competitive advantage. The collective rules concern every step of the production process from dairy production to ripening (dairy cow breed, farm and cheese dairy location, workshop processing size, raw milk, ripening duration...). In the case of PDO gruyere, the collective strategy of the traditional producers (breeders, cheese-makers, ripeners) allows, through the application of 41
collective rules, to impose costs on new competitors (or even old one, who could wish to develop other way of production) and to set a minimal price for milk (which fits with the minimal requirements put in the collective rules). The governance sets the frame of the allocation of the value within the LAFS. The farmers who respect the collective rules are allowed to sell their milk to a cheese maker, who respect them as well, and both are increasing the specificity of the product, but at the same time, under certain conditions, they can reduce the pressure exerted by competitors (=rivals) in raising their costs at the same level as their own (=obliging them to respect the collective rules, which correspond to certain production costs). On the opposite in the case of PDO Cantal, few firms have taken control on the supply chain and have imposed with the time a model based on costs leadership. Quality refers not to strong link to the geographical origin and no collective management of quality takes place. The code of practices successively evolved towards fewer requirements to promote intensive practices in order to reduce production costs. Secondly, this research shows that the Raising Rivals Costs theory can be mobilized out of their classical fields, which are the vertical integration, the suppliers pressure and the collusion to impose production costs to the rivals. The traditional actors (farmers, cheese-makers, ripeners) of the LAFS define collective rules and exert a collusion power against their potential rivals. These rules are validated and enforced by the public authorities through the PDO legal framework. Under these particular conditions, it is possible to protect the LAFS from the pressures of competitors willing to develop a production based on Costs Leadership strategy. References Barjolle D., Sylvander B., 2000, "Some factors of success for origin labelled products in agri-food supply chains in Europe: market, internal resources and institutions ", in Sylvander B., Barjolle D., Arfini F. (dir.), The socioeconomics of origin labelled products in agri-food supply chains: spatial, institutional and co-ordination aspects, Paris, INRA-Editions, pp. 45-71 Jeanneaux P., Callois J.-M., Wouts C., 2009, "Durabilit dun compromis territorial dans un contexte de pression comptitive accrue : le cas de la filire AOC Comt", Revue d'conomie rgionale et urbaine, vol. 1, pp. 179-201 Salop S.C., Scheffman D.T., 1983, Raising Rivals Costs, American Economic Review, 73, p.267-271 Scheffman D., Higgins R.S., 2003, 20 Years of Raising Rivals Costs: History, Assessment, and Future , George Mason Law Review, 12(2), 371-387
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The added nutritional value of mountain dairy products

F. Leiber ETH Zurich, Institute of Agricultural Sciences, Animal Nutrition, 8092 Zurich, Switzerland fleiber@ethz.ch Abstract An overview over health relevant properties of mountain dairy products is given. The highest evidence appears to be linked to the fatty acid profile of these products, but also phenolic and terpene compounds appear to contribute to the particular nutritional value of these products. A clear relation to the pasture biodiversity is evident. A controversial field of discussion is the non-pasteurization of the milk, which, however seems to be very significant for the human health. Keywords: (human health, milk compounds, milk processing, biodiversity) Introduction In 1931, the US American dentist and nutritionist Weston Price examined hundreds of children in different regions of Switzerland regarding their health, particularly of the teeth (Price 1936/2006). He compared populations in the canton of Valaise, particularly the Ltschental, which was completely isolated from any foods coming from outside the valley and was based on a very traditional food production and consumption with the lowland region of St. Gallen, which had a totally different kind of nutrition. The most abundant problematic diseases in Switzerland at that time were, according to the Swiss authorities, caries and tuberculosis. In the Ltschental, however, Weston Price found tuberculosis not existent at all, and the incidence for caries was at 2.3% , while it was 25.5% in the region of St. Gallen. Further, he describes the Ltschental people as having some of the finest physiques in all Europe (Price 1936/2006) and being generally in a very high physical and mental health. One of the main reasons, which Price identified for the distinct healthiness of the alpine population was the high consumption of dairy products (cheese was together with rye the main constituent of the diet), which were found to be high in vitamins and much higher than the average samples of commercial dairy products in America and Europe, and in the lower areas of Switzerland (Price 1936/2006). Although without giving direct evidence for the underlying mechanism, Price points out that the St. Gallen cows were kept in barns the whole year and brought to pasture only in autumn for a short time, while the cows in the alpine valleys were grazing the whole summer. According to Price, the most valued food in the Ltschental was the butterfat from cows grazing on the highest pastures nearby the glaciers. This butter even had religious attributes in the population and was considered as a healing substance. In 2004, a Swiss scientific publication (Hauswirth et al., 2004) claimed the alpine cheese to be the reason for an alpine paradox, meaning that alpine people live very long despite a very high intake of bovine lipids, which had become nutritional bad guys in the scientific opinion, during the 20th century. The Hauswirth study claimed exactly these fats to be the reason for health, because the alpine cheese, which the authors had investigated, contained higher proportions of -linolenic acid (an omega-3 fatty acid) than industrial cheeses from the lowlands. The particular health value of mountain dairy products is nowadays increasingly pointed out by different scientific (Innocente et al., 2002; Leiber et al., 2005; Revello Chion et al., 2010; and many others) and commercial (Glarona, 2009; and many others) publications and many contemporary research work is carried out to proof and improve these properties. But already the work of Weston Price leads directly to main key-words related to these particular values of mountain dairy products: pasture, milk fat and vitamins. When screening the actual literature, these are clearly the most often investigated factors and variables, and the high correlation between them has been clearly established. Additionally, plant secondary compounds like polyphenols (Jayanegara et al., 2011), terpenes (Revello Chion et al., 2010) and etheric oils (Tava et al., 2011) are becoming more and more important, partly because they are assumed to mediate the pasture or herb effects on milk fatty acids (Leiber et al., 2005; Klber et al., 2011), partly because they directly appear in the milk and may exhibit beneficial nutritional or taste effects (Mariaca et al., 1997; Falchero et al., 2009). One further aspect, which also implicitly emerges from the findings of Weston Price is the processing of milk: many of the typical mountain cheeses in Europe are raw milk cheeses and the Ltschental cheese for sure has been. The consumption of raw milk and raw milk products is a problematic topic because of hygienic considerations, but on the other hand, the evidence for its role in the development of antiallergenic stability in children is high (Braun-Fahrlnder & von Mutius, 2010). Thus, if the added health value of mountain dairy products should be discussed, the main questions are: which nutrients are really enriched in mountain milk, and why? do all the recently discussed nutrients (particularly the fatty acids) have a role for human nutrition? what is the role of raw milk products?
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Functional fatty acids in dairy products from mountain production It has clearly and repeatedly been shown that pasture compared to concentrate rich or maize-silage based feeding increases -linolenic acid in milk fat (Innocente et al., 2002; Ferlay et al., 2006; Collomb et al., 2008; and many others). On the one hand this happens independently of the elevation of the pasture, on the other hand, an increasing effect with increasing altitude was also repeatedly found (Bugaud et al., 2001; Leiber et al., 2005a; Collomb et al., 2008). Aside from omega-3 fatty acids, also the conjugated linoleic acids in milk attracted high scientific interest in the recent years (Kraft et al., 2003; Collomb et al., 2006; and many others). Here mainly rumenic acid (c9,t11C18:2) is important, because of its considerable concentrations in ruminant products. For this fatty acid a general positive pasture effect is known but it is less clear, whether an additional effect of the mountain pastures exists (pro: Collomb et al., 2002a; contra: Leiber et al., 2005a). It appears, that as well vegetation type (Collomb et al., 2002b; Falchero et al., 2010) as also the grazing management (Coppa et al., 2011) influence the concentration of -linolenic acid in milk fat from mountain production systems. The effects are sometimes contrasting to those on rumenic acid (Coppa et al., 2011), which is well explainable by the different metabolic origin of these two fatty acids (Chilliard et al., 2007). How the nature of the pasture affects the milk fatty acid profile is, however, still unsolved. Different studies show that it is not generally a linear effect of -linolenic acid (ALA) in the forage which influences the same fatty acid in milk (Leiber et al., 2004, 2005a). Influences of specific forage species and pasture types were hypothesized to be due to different concentrations and compositions of plant secondary compounds, which might influence the digestive lipid metabolism by inhibition of different steps of biohydrogenation (Leiber et al., 2005a). The evidence for this hypothesis is growing (Vasta et al., 2009; Cabiddu et al., 2010; Klber et al., 2011), but also the lipid mobilisation due to nutritional energy deficiency might influence the milk fat composition (Leiber et al., 2005a; Leiber et al., 2011). The pathway from feed to milk is very long in ruminants and many steps of this pathway, like ruminal biohydrogenation, endogenous desaturation and chain-elongation or endogenous fatty acid synthesis may be influenced by nutritional and environmental factors. Thus, the above mentioned mechanisms may be important issues for understanding interactions between the animals metabolism and the feeding environment, but it does not appear realistic that a complete mechanism can ever be modelled, particularly under the very diverse conditions of mountain grazing. Functional fatty acids in dairy products from mountain production. Are they important? An often expressed critique towards the health claims on mountain dairy products is, that the concentrations of the relevant fatty acids are anyway so low that they are not relevant. This can be answered by the fact that dairy products are a very regular part of European diets and that e.g. a doubling of the ALA concentrations (which is possible in mountain pasture milk compared to conventional milk [Leiber et al., 2005]) would constantly increase the respective intake. Based on the average intake of ALA (Astorg et al., 2004) and the dairy product consumption by French men (ZMP, 2007) a complete change from conventional to alpine dairy products (based on the data of Leiber et al., 2005a) would increase the total daily intake of this fatty acid by 43%. Converted to the often recommended intake of fish, this would equal to nearly 800 g salmon per day (based on Hamilton et al., 2005). This is a very rough calculation but it demonstrates that the dairy lipids can be a very considerable source for omega-3 fatty acids. The second critique is, that the conversion of ALA to long-chain omega-3 PUFA, such as DHA is extremely limited (Pawlosky et al., 2001). However, high ALA diets were repeatedly demonstrated to increase DHA concentrations in heart and brain phospholipids (Abedin et al., 1999) and to decrease the incidence of cardiovascular diseases (Holy et al., 2011). Although this evidence is from rodents, also for humans the relevance of ALA for the production of long-chain omega-3 PUFA is stated (Barcelo-Coblijn & Murphy, 2009). A simple consideration also may guide this discussion: the human organism is equipped with all enzymes for chain-elongation and desaturation to convert ALA into long-chin omega-3 PUFA. If there is enough substrate (ALA) available, the expression of the relevant proteins for these processes should be possible to be optimized by the endogenous metabolism as for many other essential processes, too. One important criteria discussed in this context is the proper equilibrium between omega-3 and omega-6 fatty acids, because they may compete about the same enzymes (Contreas & Rapoport, 2002), which again demonstrates the relevance of increased ALA concentrations in food. The general importance of omega-3 fatty acids for human health is broad, including the above mentioned coronary heart complex, the development of the nervous system and anti-inflammatory processes (Sinclair et al., 2002). Also for rumenic acid (CLA), the potential increase in intake when the dairy production is converted to a pasture-based system is highly considerable, particularly when the vaccenic acid (t11C18:1), which can be converted to CLA, is also taken into account (van Wijlen & Colombani, 2010). A broad range of positive health effects is reported for CLA, however, the evidence for humans and the general positive value is not as clear as for omega-3 fatty acids (Benjamin & Spener, 2009). Fat soluble vitamins in dairy products from mountain pastures It seems logical that the grazing at high altitude should enhance the vitamin D-complex in the organism of the animals, due to the higher UV-B radiation, leading also to increased concentrations in the milk. However, an increase of the 25-hydroxy vitamin D in the plasma of cows could not be systematically explained by pasture altitude and the detection of Vitamin D was not possible in alpine milk at all (Leiber et al., 2005b). 44
Vitamin E and beta-carotenes are clearly increased by pasture (Lucas et al., 2006; La Terra et al., 2010), but a particular mountain effect could not be demonstrated (Leiber et al., 2005a). Plant secondary compounds in dairy products from mountain pastures As mentioned above, one role of the plant secondary compounds may be the modification of ruminal biohydrogenation and thus influence on the milk fatty acid. But these compounds also may appear in the milk, where they may enhance the antioxidative activity and microbial stability (OConnell & Fox, 2001). Terpenes in milk may be clearly enhanced by grazing (Revello Chion et al., 2010) and the concentrations differ depending on pasture types (Mariaca et al., 1997; De Noni et al., 2008). It is considered that natural mountain pastures contain particularly high proportions of forbs at a generally high biodiversity. These forbs are considered to contribute particular diversity and amount of plant secondary compounds, which is much higher than in cultivated, grassdominated lowland pastures. This was demonstrated for etheric oils (Tava et al., 2011), terpenes (Mariaca et al., 1997) and polyphenols (Jayanegara et al., 2011) in alpine pasture plants. For terpenes, a proportional transfer from forage to milk was shown (Viallon et al., 2000), which means that here a clear mountain effect on the dairy product should be possible. For polyphenols this remains to be established. Besides the aromatic effects of these compounds, particularly the antioxidative capacities of many of them has to be considered as a clear health advantage. The significance of raw milk products Mountain cheeses are very often processed without pasteurization of the milk. There is increasing evidence that the consumption of raw milk products by children is a key factor preventing them from allergic diseases, as reviewed by Braun-Fahrlnder & von Mutius (2010) but it the discussion is controverse and complex, the more in times of EHEC epidemies. It was shown that heat treatment of milk may severely impair the antioxidant capacity of milk (Calligaris et al., 2004) and that pasteurization clearly influences the composition of volatile compounds and reduces the indigenous microflora in cheeses (Buchin et al., 1998), which might be clear factors influencing the anti-allergic constitution of the consumers. The question of pasteurization may probably be one of the most important factors for the health-value of mountain dairy products, and the observations of Weston Price may well be linked to this topic, too. Conclusion The highest evidence for the health value of mountain dairy products is based on certain functional fatty acids, which are doubtless enriched by grazing of mountain pastures. For fat-soluble vitamins, the exceptional advantage over other pasture systems is not as clear. By contrast, it can be deduced that terpenes and essential oils are enriched in mountain milk, too. These compounds may bring also a health advantage mainly by antioxidant activity. All the mentioned compounds can be more or less clearly linked to the high biodiversity of the extensive pasture systems in the mountains. Another systematic particularity of the mountain dairy products is the raw milk based processing. Although controversially discussed, it may be a very important point for the overall health value of these products. References Abedin L, Lien EL, Vingrys AJ, Sinclair AJ 1999. Lipids 34: 475-482. Astorg P, Arnauld N, Czernichow S, Noisette N, Galan P, Hercberg S 2004. Lipids 39: 527-535. Barcelo-Coblijn G, Murphy EJ 2009. Progr. Lipid Res. 48 : 355-374. Benjamin S, Spener F 2009. Nutr. Metabol. 6 : 36. Braun-Fahrlnder C, von Mutius E 2010. Clin. Exper. Allergy 41: 29-35. Buchin S, Delague V, Duboz G, Berdague JL Beuvier E, Pochet S, Grappin R 1998. J. Dairy Sci. 3097-3108. Bugaud C, Buchin S, Coulon JB, Hauwuy A, Dupont D 2001. Lait 81: 401-414. Cabiddu A, Salis L, Tweed JKS, Molle G, Decandia M, Lee MRF 2010. J. Sci. Food Agric. 90: 829-835. Calligaris S, Manzocco L, Anese M, Nicoli MC 2004. Int. Dairy J. 14 : 421-427. Chilliard Y, Glasser F, Ferlay A, Bernard L, Rouel J, Doreau M 2007. Eur. J. Lipid Sci. Technol. 109: 828-855. Collomb M, Btikofer U, Sieber R, Jeangros B, Bosset JO 2002a. Int. Dairy J. 12: 649-659. Collomb M, Btikofer U, Sieber R, Jeangros B, Bosset JO 2002b. Int. Dairy J. 12: 661-666. Collomb M, Schmid A, Sieber R, Wechsler D, Ryhnen EL 2006. Int. Dairy J. 16: 1347-1361. Collomb M, Bisig W, Btikofer U, Sieber R, Bregy M, Etter L 2008. Dairy Sci. Technol. 88: 631-647. Contreas MA, Rapoport SI 2002. Curr. Opin. Lipidol. 13: 267-272. Coppa M, Ferlay A, Monsalier F, Verdier-Metz I, Pradel ,P, Didienne R, Faruggia A, Montel MC, Martin B 2011. J. Dairy Sci. 94: 1132-1145. De Noni I, Batelli G 2008. Food Chem. 109: 299-309. Falchero L, Sala G, Gorlier A, Lombardi G, Lonati M, Masoero G 2009. J. Dairy Res. 76: 365-371. Falchero L, Lombardi G, Gorlier A, Lonati M, Odoardi M, Cavallero A 2010. Dairy Sci.Technol. 90: 657-672. Ferlay A, Martin B, Pradel P, Coulon JB, Chilliard Y 2006. J. Dairy Sci. 89: 4026-4041. Glarona 2009. Ksegenossenschaft Glarus. http://www.glarona.ch/glarneralpkaese.html 45
Hamilton MC, Hites RA, Schwager SJ, Foran JA, Knuth BA, Carpenter DO 2005. Environ. Sci. Technol. 39: 8622-8629. Hauswirth CB, Scheeder MRL, Beer JH 2004. Circulation 109: 103-107. Holy EW, Forestier M, Richter EK, Akhmedov A, Leiber F, Camici GG, Mocharla P, Lscher TF, Beer JH, Tanner FC 2011. Artherioscl. Thromb. Vasc. Biol. 31: 1772-1780. Innocente N, Praturlon D, Corradini C 2002. Ital. J. Food Sci. 14:217-224. Jayanegara A, Marquardt S, Kreuzer M, Leiber F 2011. J. Sci. Food Agric. 91: 1863-1870. Kraft J, Collomb M, Mckel P, Sieber R, Jahreis G 2003. Lipids 38: 657-664. Klber T, Meier JS, Kreuzer M, Leiber F 2011. J. Dairy Sci. 94: 1477-1489. La Terra S, Marino VM, Maneti M, Licitra G, Carpino S 2010. Dairy Sci. Technol. 90: 687-698. Leiber F, Scheeder MRL, Wettstein HR, Kreuzer M 2004. J Anim. Feed Sci. 13 S1: 693-696. Leiber F, Kreuzer M, Nigg D, Wettstein HR, Scheeder MRL 2005a. Lipids 40: 191-202. Leiber F, Liesegang A, Kreuzer M, Wettstein HR 2005b. Pag. 309-311 in: Vitamine und Zusatzstoffe in der Ernhrung von Mensch und Tier. 10. Symposium. Germany, Friedrich Schiller-Universitt Jena und Bundesforschungsanstalt fr Landwirtschaft Braunschweig; R Schubert, G. Flachowsky, G. Jahreis, R, Bitsch eds, Braunschweig, Germany. Leiber F, Hochstrasser R, Wettstein HR, Kreuzer M 2011. Livest. Sci. 138 : 1-12. Lucas A, Agabriel C, Martin B, Ferlay A, Verdier-Metz I, Coulon JB, Rock E 2006. Lait 86 : 177-202. Mariaca RG, Berger TFH, Gauch R, Imhof MI, Jeangros B, Bosset JO 1997. J. Agric. Food Chem. 45 : 44234434. OConnell JE, Fox PF 2001. Int. Dairy J. 11 : 103-120. Pawlosky RJ, Hibbeln, JR, Novotny JA, Salem N 2001. J. Lipid Res. 42: 1257-1265. Price WA 2006/1939. Nutrition and physical degeneration. 7th Edition, page 23-43. Benediction Classics. Revello Chion A, Tabacco E, Giaccone D, Peiretti PG, Batelli G, Borreani G 2010. Food Chem. 121: 393-399. Sinclair AJ, Attar-Bashi NM, Li D 2002. Lipids 37:11131123. Tava A, Cecotti R, Grecchi M, Falchero L, Coppa M, Lombardi G 2011. Nat. Prod. Comm. 6: 101-105. Van Wijlen RPJ, Colombani PC 2010. Int. Dairy J. 20 : 433-448. Vasta V, Mele M, Serra A, Scerra M, Luciano G, Lanza M, Priolo A 2009. J. Anim. Sci. 87: 26742684. Viallon C, Martin B, Verdier-Metz I, Pradel P, Garel JP, Coulon JB, Berdague JL 2000. Lait 80 : 635-641. ZMP 2007. Milch 2007 ISBN 978-3-86720-027-1.
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Session 1: Mountain Milk and Cheese Quality in Relation to Production Conditions Posters
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Study on the combined effects of i) milk production conditions and ii) cheese making process, on sensorial characteristics of Reblochon PDO cheese
Convert T. GIS Alpes Jura, Suaci Alpes du Nord, 40 rue du Terraillet, 73190 Saint Baldoph, France, tconvert@suacigis.com Abstract The study dealt with the combined effects of i) milk production conditions and ii) cheese making processes, on sensorial characteristics of Reblochon, a French soft mountain cheese made from raw cow's milk granted with the Protected Designation of Origin (PDO) label. The experiment consisted in making Reblochon PDO cheeses, at a pilot scale, from 12 producers milks simultaneously following two typical and contrasted processes. Producers were surveyed for their practices, since these may actually influence the microbial or chemical milk composition. During 3 weeks prior to the cheese making and at different stages during process and maturing, fourteen microbial groups were quantified in milk and cheese. After 4 weeks maturing, an expert panel evaluated a cheese sample from each batch regarding its sensory profile. The preliminary results indicated a low level of total microorganisms in processed milks (11 on 12 milks contained less than 7000 cfu/mL). They also showed that composition and stability of microbial ecosystem depends on both producers and dates of sampling. Moreover, despite a heavy impact of technology, some links appeared between microbial composition of milk and sensory characteristics of cheese. Deeper data analyses are currently in progress. Keywords: cheese, raw milk, microbial ecosystem, sensory profile, farmers practices Introduction It is known that some producers practices can influence milk microbial composition (Coulon et al., 2004; Demarigny et al. 1997). That is why, in this study, we took into account the producers practices as a determinant of the milk microbial composition. The work presented here focused on 2 questions: i) what is the variability of milk microbial composition, depending on the date of sampling and the producer, and ii) despite the heavy impact of technology, is it possible to highlight links between milk microbial composition and sensory characteristics of Reblochon cheese? Reblochon is a soft washed-rind and smear-ripened cheese made from raw cow's milk, in the Haute-Savoie region of the French Alps, which is granted with PDO label. The global objective was to highlight the possible associations existing between milk production conditions and cheese making processes on sensory characteristics of cheeses. Material and methods Twelve producers from the Reblochon PDO area were selected for the variety of their practices regarding milking, housing, feeding practices. Cheeses productions were done by the cheesemongers of an experimental cheese making unit (Actilait, La Roche sur Foron) in order to minimize the variability between batches. The experiment took place between 24th January and 2nd February 2011. Each producers milk, from one milking, followed two typical and contrasted processes: a farmhouse (F) and a dairy plant (L) processing methods. They differed mainly by the type and quantity of starters, the duration of cheese making stages and maturing conditions. To reduce the impact of milk chemical composition on cheese sensory characteristics, milk were standardised in fat in dry matter by adding pasteurised cream made from each producers milk. Fourteen microbial analyses were done on raw milk and during production, to mainly count useful microflora but also some spoilage or pathogenic bacteria. One time a week, during 3 weeks before production, each producers milk was analysed for microbial composition. For each of the 24 production batches (12 producers x 2 processes) the same microbial analyses were done at 5 technologically significant stages: on standardised milk, after salting (day1), after modification of maturing conditions (day 4 to 7), half maturing (day 14) and at the end of maturing (day 28). After 28 days of ripening, the cheeses from each of the 24 batches were analysed by 12 trained panellists (Les Maisons du Gout, Rennes, Fr) for their sensory profiles. Twenty two criteria were notated, describing aspect, odour, texture and taste of cheeses. In order to work on the proportion of each microflora and not only on their level, a relative index (IR) was calculated for each microflora. IR associated with a microflora was defined as the microbial count on its selective medium divided by the sum of all microbial selective counts. IRs were used as indicator of microbial balance of milk. In order to identify links between microbial milk composition (IRs and counts) and sensory characteristics of cheeses, Pearsons correlations were calculated using Statgraphics statistical software. Two variables are considered correlated if Pearsons coefficient (r) is comprised between 0,5 and 1. Results and discussion The total microbial counts in processed milks were low, with 11 on 12 milks containing less than 7000 cfu/mL and 5 on 12 milks containing less than 1000 cfu/mL. This is not uncommon in milks produced in the Reblochon PDO area, since sanitary pressure is high due to the vulnerability of the Reblochon technology to pathogen 49
development. Despite the low level of total microbial counts, variability in milk microbial composition was observed (data not shown). For some producers, the milk microbial composition varied with sampling date, since other producers displayed a relative stability in terms of microbial balance during 4 weeks of sampling. Producers milks which composition remained stable over time could display various microbial compositions: as an example, milks from producer n7 always contained 3-10% of group I Lactobacillus (LbI), while milks from producer n5 contained 10 times less of LbI (0.2 to 0.6%). Twelve pairs of microbial and sensory variables were found moderately correlated. The correlated microbial variables were more often level variables than IR variables (10 pairs vs. 2 pairs of variables). The correlated sensory variables were mainly texture descriptors (flowing aspect, firmness) but also taste criteria (lactic aroma, salted flavour). As an example of such a correlation the Figure 1 shows that milks with a high ratio of Enterococci, known to be involved in curd ripening (Ogier and Serror, 2008), gave cheeses with a more flowing aspect.
Figure 1: correlation between flowing aspect of cheese and IR Enterococcus (Relative Index) in raw milk. Diamonds and squares symbols correspond respectively to farmhouse (F) and dairy plant (L) processing methods. Black bold line shows the regression line of all fabrications, small and large pointed lines correspond to F and L regression line, respectively. r indicates the value of Pearsons correlation coefficient. KF: selective medium for Enterococci.
8 Sensory note of experimental cheeses, for flowing aspect 7 r = 0,8603 6 r=0,6467 5 4 3 2 1 0 0 2 4 6 8 10 12 14 IR Enterocoques (% of KF agar count on sum of all 13 m edia counts) F Regression line F+L regression line L L regression line F r=0,3409
Conclusions Despite low levels of total count microflora in the processed milks, the experimental design highlighted links between milk microbial composition and some Reblochon PDO cheese sensory characteristics. Further data analyses will search for associations between producers practices, milk microbial composition and sensory profiles of Reblochon cheese. The evolutions of microbial dynamics through both contrasted processes will also be studied. Considering that producers practices and milk microbial compositions are dependant of a seasonal effect, the same experiment will be duplicate in summer 2011. Considering the low number of producers involved and the variability of their practices and milk microbial composition, this study is considered as an exploratory study. It will allow to identify interesting ways to deepen, at a larger scale. Acknowledgements This work was funded by CNIEL, FranceAgriMer, the Rgion Rhne Alpes (PEP Bovin Lait program) and the partners of the GIS Alpes Jura Research and Development convention. We also greatly thank the organisms involved in the experiment itself and/or in the management of the project (CA74, CL74, SIR, INRA Theix and Poligny, Vetagrosup, Actilait, CTFC). References Coulon J.B., Delacroix-Buchet A., Martin B., Pirisi A. 2004. Lait, 84: 221-241. Demarigny Y., Beuvier E., Buchin S., Pochet S., Grappin R. 1997. Lait, 77: 151-167. Ogier J.C., Serror P. 2008. Int. J. Food Microbiol. 126: 291-301.
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Carotenoids, Vitamin A and E concentrations in Saint Nectaire cheeses according to process and season
B. Graulet1*, C. Chatelard2, M. Lepetit3, B. Blay3, S. Hulin2, B. Duriot1, B. Martin1. 1 INRA Unit de Recherche sur les Herbivores 63122 Saint-Gens-Champanelle, France 2 Ple Fromager AOP du Massif Central, 15000 Aurillac, France 3 Interprofession Saint-Nectaire, 63610 Besse-en-Chandesse, France *benoit.graulet@clermont.inra.fr Abstract A study was settled with the Saint-Nectaire cheese in order to compare the concentrations in carotenoids and vitamins A and E (that take part of the nutritional density) between cheeses produced in farms (n=8) or dairies (n=5) and evaluate the annual changes (4 to 5 samplings during the year). The same carotenoids were quantified in Saint-Nectaire cheeses than in the literature for bovine milk but their relative proportions were different due to unexpected high concentrations in lutein (equivalent to those of -carotene). -carotene, vitamin A and E concentrations were lower than expected, but cheeses were largely richer in xanthophylls (lutein and zeaxanthin). Except vitamin A concentration, 11.5% higher in farmhouse cheeses, all the other lipophilic micronutrients were found at similar concentrations in cheeses made in dairy plants or farms, thus suggesting that cheesemaking practices found only in dairy plants (milk standardisation, pasteurisation or curd washing) only marginally influence liposoluble micronutrients in cheeses. Lutein and -carotene concentrations were higher in cheeses produced during summer reflecting pasture feeding, whereas, tocopherol concentration was higher in winter, probably due to the vitamin supplement distribution. Keywords: (feeding practices; carotenoids; vitamins; dairy cows ; cheese making process) Introduction Cheese composition in vitamins A and E and carotenoids varies according to feeding practices of dairy herds (Nozire et al., 2006; Lucas et al., 2006). Their concentrations are improved by grass-based diets, especially when cows are grazing on permanent and/or first-use pastures. This result suggests that specifications for milk production found in some traditional cheeses could give an added nutritional value to cheeses. However, the data available in literature refers mainly to farmhouse cheeses obtained from raw milk and it is obvious that some aspects of the process like milk standardisation, pasteurisation, massive starter addition or curd washing may also modulate the nutrient content in cheeses. Thus, the main objective of this work was to explore the potential effects of the cheese-making process (farmhouse vs dairy plant) on the composition in liposoluble micronutrients in cheese made in different seasons. The St-Nectaire cheese (one of the main PDO cheeses in France) was a convenient model since specifications of its production include farmhouses and dairy plant cheeses and require a diet based on grass (fresh or preserved) from mountain grassland. Material and methods The experimental design included the 5 dairy plants and 8 of the 240 farmers making Saint Nectaire. The latter were selected such as to represent the overall variability for altitude (850 to 1130 m), herd size (30 to 85 cows), breed characteristics (Montbliarde and/or primHolstein) and feeding practices. In this area, the indoor rations are based on hay and/or grass silage and outdoor rations are based on mountain pasture with or without conserved forage complementation. The cheeses were sampled after 30 days of ripening at 5 periods along the year. The 2 first samplings were performed, when animals were fed the winter indoor ration (cheeses made on 2nd February and 16th March) and the followings when cows were grazing during the spring-summer period (cheeses made on 2nd June, 6th August and 14th September). In the dairy plants, only the 4 first samplings were made since the fifth one corresponded to a transitional period between grazing and indoor feeding. Liposoluble vitamins and carotenoids were extracted from 750 mg of dried cheese powder combining ethyl alcohol deproteinisation, n-hexan-ethyl acetate (9/1) purification, and 2-hours at 60C saponification to release carotenes from the cheese fat matrix (Duriot and Graulet, in press). Extracts were analyzed using UPLC as reported for milk samples (Chauveau-Duriot et al., 2010). The ANOVA statistical analysis considered the technological process (farmhouse vs dairy plant), the season (indoor vs outdoor) and the interaction between these factors. Results and discussion Analysis of cheeses reveals 6 different carotenoids in variable proportions (table 1): 3 xanthophylls (37% lutein, 10% zeaxanthin, 4% -cryptoxanthin) and 3 isomers of -carotene (36% all-trans, 10% 13-cis, 3% 9-cis). These compounds are those classically found in milk using this analytical method that let a greater resolution and sensitivity owing good separation and quantification of lutein/zeaxanthin and of the 3 -carotene isomers that are traditionally difficult to separate (Chauveau-Duriot et al., 2010). However, it should be noted that all-transcarotene concentrations are usually largely higher in milk than those reported in cheeses (Graulet, personal 51
communication). Our results differ from those obtained by Lucas et al. (2006) in Tomme de Savoie, a pressed cheese with similar characteristics, who found xanthophylls concentrations 10-fold lower than those obtained in St-Nectaire cheeses, whereas carotene content was in the same range (Lucas et al., 2006). This difference could be related to a specific nutrient composition of St-Nectaire compared to Tomme de Savoie, but also to the optimization of the analytic methodology between the 2 studies. -tocopherol was almost marginal among the sum of vitamins E. Table 1: Micronutrient content in cheeses according to cheese-making process and season (g/g fat).
St-Nectaire Cheese Groups Indoor Farmhouse Carotenoids Zeaxanthin Lutein -cryptoxanthin 13-cis-carotene 9-cis-carotene all-trans-carotene Carotenoids sum Vitamins Retinol -tocopherol -tocopherol Tocopherols sum ns : not significant 4.36 0.12 3.13 3.25 4.35 0.02 2.74 2.76 4.55 0.16 2.64 2.80 3.63 0.24 3.05 3.29 ns 0.01 0.10 0.05 0.01 ns ns ns ns ns ns ns 0.32 1.06 0.14 0.26 0.08 1.09 2.95 0.36 1.41 0.14 0.34 0.12 1.12 3.50 0.36 1.35 0.16 0.30 0.08 1.06 3.32 0.32 1.19 0.13 0.36 0.12 1.25 3.37 ns 0.05 ns 0.10 ns 0.10 ns ns ns ns 0.10 ns ns ns ns 0.05 0.10 ns ns ns ns Dairy plant Outdoor Farmhouse Dairy plant Statistical significance (p<) Season Cheesemaking Interaction
The concentration in liposoluble micronutrients only marginally differed between dairy plant and farmhouse Saint-Nectaire cheese. Indeed, we only noted that 13-cis -carotene concentrations tended to be higher in dairy St-Nectaire, whereas retinol concentrations were lower in these cheeses, especially during outdoor period. Moreover, lutein concentrations were higher in dairy plant cheeses during indoor period but lower during outdoor period. This result suggests that cheesemaking practices, like milk standardisation, pasteurisation or curd washing, only marginally influence liposoluble micronutrients in cheeses. The lower retinol concentrations found in dairy cheese may be due to curd washing that could eliminate free retinol. The total carotenoids content was not significantly different between indoor and outdoor periods because only slight increases were observed during summer for 13-cis and all-trans-carotenes, and lutein in the farmhouse system only. This result is surprising because carotenoid contents in milk or cheese are known to be higher in grazing cows (Nozire et al., 2006 ; Lucas et al., 2006). However, due to the latency of transfer of carotenoids from forage to milk (Calderon et al., 2007), the 3rd period of sampling could have been too close to the end of the indoor period thus limiting carotenoids increase in milk fat as a result of fresh grass consumption. Moreover, due to the specifications of St-Nectaire production, the diet composition remains based on grass, provided fresh or preserved that supply minimal amounts of carotenoids, especially lutein (Nozire et al., 2006). Total vitamin E content decreased from indoor to outdoor period (due to its main form -tocopherol), whereas -tocopherol content increased probably as a result of a change in the form and amount of vitamin E dietary supply between season: as supplement of -tocopheryl-ester in winter or as both and -tocopherol in grass in summer Conclusions This study has indicated that Saint-Nectaire cheese is especially rich in lutein by comparison to data available on other cheeses. Moreover, the liposoluble micronutrient content is dairy plant and farmhouse cheese was very close, suggesting that cheesemaking practices like milk standardisation, pasteurisation or curd washing only marginally influence liposoluble micronutrients in cheeses. Acknowledgements We thank the farmers and the dairies who participated to this study and Conseil Rgional Auvergne, Conseil Gnral du Puy-de-Dme and Conseil Gnral du Cantal for funding. References Calderon F., Chauveau-Duriot B., Pradel P., Martin B., Graulet B., Doreau M., Nozire P., 2007. J. Dairy Sci. 90. 5651-5664. Chauveau-Duriot B., Doreau M., Nozire P., Graulet B. 2010. Anal. Bioanal. Chem. 397, 777-790. Duriot B., Graulet B., in press. In Vitamin A: chemistry, analysis, function and effects. RSC Publishing, London. Lucas A., Agabriel C, Martin B., Ferlay A., Verdier-Metz I., Coulon J.-B., Rock E. 2006. Lait, 86, 177-202. Nozire P., Graulet B., Lucas A., Martin B., et al. 2006. Anim. Feed Sci. Technol., 131, 418-450. 52
Fatty acid profile of Raschera PDO cheeses produced by different cheese factories during winter and summer
A. Revello-Chion1*, G. Battelli2, D. Giaccone3, E. Tabacco1, G. Borreani1 1 Dep. of Agronomy, Forest and Land Management, University of Turin Via L. da Vinci 44, I-10095, Grugliasco (TO) 2 Institute of Sciences of Food Production, National Research Council Via G. Celoria 2, I-20133, Milano Italy 3 Associazione Regionale Allevatori Piemonte Via Livorno 31, I-10144, Torino *andrea.revellochion@unito.it Abstract The fatty acids (FA) composition of Raschera cheeses granted with the protected designation of origin (PDO) label produced by ten cheese factories during winter and summer was studied. Cheese factories differed for type (raw or pasteurised), and origin of milk used to produce cheese (intensive or extensive production systems). Cheeses produced from raw or pasteurized milk did not show any difference on FA composition, whereas effects of season, production system and their interaction on FA profiles have been detected. Summer cheeses produced from milk of extensive production systems presented higher contents of human health FA, i.e. conjugated linoleic acid (CLA) and C18:3 c9 c12 c15 (-linolenic acid), as well as, higher contents of odd- and branchedchain FA (OBCFA), which could be a useful tool to discriminate the different origin of cheese. Keywords: Raschera cheese, fatty acid composition, dairy system, intensive, extensive Introduction Most of the milk produced in Northern Italy is transformed into traditional and typical cheeses, which are often granted with PDO label. Among them, Raschera cheese is a typical semi-fat, semi-cooked and pressed cheese, which is ripened for a minimum period of 60 days. It is produced within the Cuneo province (North-West Italy) from raw or pasteurized milk, and its certified production standards do not include restrictions on composition of animal diets. These production conditions can have an effect on sensory and nutritional properties of cheese, including the FA composition, which has increased in interest due to its impact on human health. Among several factors, the major impacts on cheese FA composition are associated with the type of rearing systems (i.e. animal breed, stage of lactation, animal diet), the geographical area of production, the season and interaction of these factors (Engel et al., 2007; Lucas et al., 2006). The aim of this research was to evaluate the variation in FA profile of Raschera PDO cheeses produced in different cheese factories in Piedmont. Material and methods Two cheeses from winter and summer productions, ripened for 60 days, were collected in ten cheese factories that produced more than 80% of total Raschera PDO cheese. Three cheese factories mainly transformed milk produced by extensive dairy farms and seven from milk produced by intensive dairy farms. Each cheese was sub-sampled and analysed for the FA methyl esters by gas chromatography. The cheese FA profiles were subjected to ANOVA using the GLM procedure of the SPSS software (v 16.0), considering production system and season, and their interaction as the fixed effects. Results and discussion The FA composition of the cheeses was significantly affected by the production systems adopted on the farms, season and their interaction (Table 1). Cheese produced in summer season from milk of extensive production systems presented the highest C18:2 c9 t11 (CLA), C18:1 t11 (vaccenic acid), C18:3 c9 c12 c15, monounsaturated FA (MUFA), polyunsaturated FA (PUFA), and OBCFA (C14:0i; C15:0i; C15:0a; C15:0; C16:0i; C17:0i; C17:0a; C17:0) contents, and the lowest values of saturated FA (SFA) and C18:2 c9 c12 (linoleic acid). C18:1 c9 (oleic acid) content was higher in summer cheeses than winter cheeses. OBCFA has been proposed to discriminate the origin of dairy products basing on animal diets and production zones (Engel et al., 2007). Figure 1 shows the discrimination obtained by plotting the cheeses according to the contents of C18:3 c9 c12 c15 and the sum of C14:0i and C16:0i (a) and the contents of CLA and sum of C14:0i and C16:0i (b). Cheese obtained from milk of extensive farms in summer season, characterized by use of high percentage of fresh forage in the cow diets, presented elevated amounts of CLA, C18:3 c9 c12 c15 and C14:0i+C16:0i, and clearly differed from cheese produced in winter season and from milk of intensive production systems regardless of season. Conclusions Extensive production system offers the opportunity of producing typical cheese with increasing content of FA that have beneficial effects for human health, such as CLA, C18:3 c9 c12 c15, PUFA and OBCFA. Furthermore, the FA profile could be a useful tool to trace the different origins of dairy products. 53
Table 1: Partial fatty acid profile of Raschera PDO cheeses (g 100 g-1 total fatty acids) produced from milk of intensive and extensive farms during winter and summer seasons; P = production system of farms; S = season; ns = not significant; * = P < 0.05; ** = P < 0.01; *** = P < 0.001.
Production system Intensive Extensive Winter Summer Winter Summer 28.89 28.41 29.03 25.60 1.79 1.82 1.78 1.74 0.43 0.43 0.44 0.58 0.47 0.45 0.45 0.60 0.58 0.59 0.56 0.91 0.29 0.29 0.27 0.45 9.62 10.41 9.53 9.80 21.48 23.14 21.47 22.51 1.67 1.81 1.71 3.48 2.64 2.54 2.86 1.92 0.50 0.61 0.50 1.56 0.36 0.36 0.46 0.95 0.05 0.04 0.05 0.09 0.06 0.06 0.06 0.11 0.13 0.12 0.14 0.20 0.15 0.16 0.16 0.27 68.14 66.24 67.72 64.16 28.25 30.08 28.35 30.87 3.61 3.68 3.94 4.98 0.34 0.34 0.39 0.59 SEM P ns ns ** * * ns ns * * ns ** *** * * ** * ns ns * ** Effect S ns ns ** * ** * ns ns * ** ** ** ns * * ** * * ns ** PxS ns ns ** ** * * ns ns * * ** ** * * * * ns ns ns *
C16:0 C16:1 C17:0i C17:0a C17:0 C17:1 C18:0 C18:1 c9 C18:1 t11 C18:2 c9 c12 C18:2 c9 t11 (CLA) C18:3 c9 c12 c15 C19:0 C19:1 C20:0 C20:1 SFA MUFA PUFA C14:0i+C16:0i
0.478 0.031 0.016 0.016 0.039 0.020 0.230 0.297 0.204 0.094 0.110 0.061 0.006 0.006 0.008 0.013 0.547 0.425 0.169 0.026
Figure 1: Discrimination of cheese from milk produced by intensive and extensive farms during winter and summer seasons based on their FA composition: (a) sum of C14:0i and C16:0i, C18:3 c9 c12 c15; (b) sum of C14:0i and C16:0i, C18:2 c9 t11 CLA. Fatty acids are expressed as g 100 g-1 total fatty acids; = extensive winter; = extensive summer; = intensive winter; = intensive summer.
2.50 2.50
a)
C18:2 c9 t11 CLA C18:3 c9 c12 c15 2.00 1.50 1.00 0.50 0.00 0.00 2.00 1.50 1.00 0.50 0.00 0.00
b)
0.20
0.40
0.60
0.80
0.20
0.40 C14:0i+C16:0i
0.60
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Acknowledgements The research was funded by the Regione Piemonte Project: Qualit degli alimenti, gestione degli animali e tecnologia di caseificazione: esempio di filiera produttiva di alcuni formaggi DOP tipici piemontesi in zona montana. All the authors contributed equally to this paper. References Engel E. Ferlay A., Cornu A., Chilliard Y., Agabriel C., Bielicki G., Martin B. 2007. J. Agric. Food Chem. 55:9099-9108. Lucas A., Rock E. Chamba J.-F., Verdier-Metz I., Brachet P., Coulon J.-B. 2006. Lait 86:21-41.
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Using NIR spectroscopy on cheeses for the authentication of cows feeding and geographical origin D. Andueza1, I. Constant1, C. Agabriel2,3, A. Lucas1, B. Martin1 1 INRA - UR1213 Herbivores, Site de Theix, F-63122 St Gens-Champanelle 2 VetAgro Sup, UR EPR 2008.03.102, F-63012 Clermont-Fd Cedex 1 3 INRA, USC 2005, F-63370 *donato.andueza@clermont.inra.fr Abstract An experiment was undertaken to evaluate the ability of NIR spectroscopy to distinguish pasture-fed from preserved forages cheeses, to predict the fresh herbage proportion in the regime (>75, 50-75, 0-50 and 0 %) and the origin of the cheeses (middle mountain vs. alpine pasture). The reflectance spectra of 308 cows cheeses samples were obtained from 3 cheeses varieties (Abondance, Cantal and Tomme de Savoie). The partial least square-discriminant analysis (PLS-DA) allowed to correctly classify 95% of the pasture vs. preserved forages samples but only 60 % of the samples according to the fresh herbage proportion in the regime. For cheeses obtained during the over-grazing period NIRS allowed to correctly classify 93% of the samples according to their origin (middle mountain vs. alpine pasture). Nevertheless, most of the misclassified cheeses belonged to alpine pasture group (80 %). NIRS was able to classify cheeses samples from different regimes (pasture-fed from preserved forages), but was not powerful enough for the prediction of the proportion of fresh herbage in the regime, at least when it was obtained by rapid surveys in the farms. Discrimination model should be improved for the authentication of cheeses according to the origin (mountain vs. alpine pasture). Keywords: traceability, cows feeding, geographical origin, preserved forages, fresh herbage proportion Introduction Recently growing consumer demand for knowledge about the quality and production conditions of dairy products has prompted the producers to improve information on milk and cheese origin and animal diet. It has been shown that detailed chemical analysis on dairy products, such as assay of terpenoids or fatty acids (Martin et al., 2005; Agabriel et al., 2004; Engel et al., 2007), can be used to authenticate its geographical origin and/or cow diet. However, these techniques are cumbersome and time-consuming, and their large-scale application for routine analysis is not feasible. Near-infrared spectroscopy (NIRS) has shown potential for use in quality assurance for a number of foodstuffs, being non-destructive, fast, and suitable for online measurements. The potential of NIRS for the prediction of the cheese composition in beta-carotene and fatty acid has been proved (Lucas et al., 2008a, b). Martin et al., (2006) successfully applied NIRS technology to the authentication of milk geographical origin (lowland vs. upland) when animal diet clearly differs in lowland (maize silage) and upland (fresh herbage (FH) based diets). The aim of this work was to test the ability of NIRS to discriminate cheeses derived from cows fed preserved forages, from different amount of pastured FH or from the geographical origin. Material and methods A total of 308 cow milk cheese of 3 cheese varieties were supplied by 52 farmhouse producers: Abondance cheese (pressed semi-cooked curd, n=92), Tomme de Savoie cheese (pressed uncooked curd, n=106) and Cantaltype cheese (pressed uncooked curd, n=110). The main characteristics of these cheeses have been previously described (Lucas et al., 2006). Cheese samples were selected in order to promote a wide compositional variability. Cheese aliquots were protected from light with aluminium foil and stored at -20C until analysis. During sampling periods, data on animal feeding were recorded by surveys that permitted to classify the cheeses in different categories according to the estimated amount of the different forages in the diet, as described in Lucas et al. (2006). Cheeses were classified, first in two different diets (preserved forages only and from FH based). Secondly, cheese samples were assigned to 4 categories according to the proportion of FH in the diet: no FH, less than 50% of forage dry matter (DM) derived from FH, FH DM proportion between 50 and 75 % and FH higher than 75% of total diet forages. Lastly, according to their geographical origin, FH-derived cheeses were classified as mountain (800-1500 m) or alpine (>1800 m) cheeses. Approximately 20 g of grated fresh cheese were placed in a 50 mm diameter ring cup and scanned in reflectance mode at 2 nm intervals from 400 to 2498 nm using a Foss NIRSystems model 6500 scanning VIS/NIR spectrometer (Foss NIRSystems, Silver Spring, MD, USA) equipped with an autocup module and controlled by ISIscan software version 2.21 (Infrasoft International, Port Matilda, PA, USA). Each spectrum was time averaged from 32 scans. The reflectance (R) values were converted into absorbance (A) values using the formula A=log (1/R). Spectra were transformed using the standard normal variate and detrend (SNVD) (Barnes et al., 1989) method as scatter correction and the first derivative. A principal components analysis was performed to rank the spectra according to their Mahalanobis distance (H statistic) from the average spectra. Two outlier detection passes were used to discard samples with H > 3. No samples were discarded. Discriminant analysis was performed using the PLS-DA technique, and the models were tested using a cross-validation procedure. 55
Results and discussion The performance of the classification models is summarised in Table 1. For the preserved forages vs. FH, analysis, the 95.5% of the samples were correctly classified. Similar results were obtained by Coppa et al., (2010) who concluded that NIRS was able to distinguish bulk milk according to cows feeding regime (pasture feeding vs. corn silage) using dehydrated milk samples. Table 1. Number of samples (N) and proportion of correctly classified cheeses (cc) according to cows feeding (fresh herbage vs. preserved forages), fresh herbage proportion in the regime (>75, 50-75, 0-50 and 0 %) and geographical origin (mountain vs. alpine pasture) from NIRS spectra.
Preserved forages vs. fresh herbage Fresh herbage in cow diet: 0% vs. 0-50% vs. 50-75 % vs. >75% Mountain vs. alpine pasture N 308 308 208 cc (%) 95.5 59.7 92.8
The performances of the discrimination model for cheeses classification according different proportion of fresh herbage shows that 40.3 % of samples were misclassified when samples from preserved forages were compared to samples from low, or medium or high proportion of FH in diet. This result indicated that NIRS does not permit to classify the cheeses according to the proportion of pastured FH in the diet when data was obtained by rapid surveys in the farms. The low precision of this method could explain these results. The application of PLS-DA model for discriminating the origin of cheeses (mountain vs. alpine pastures) provided 7.2% of error. This result showed that VIS/NIRS may represent a promising technology for classification of cheeses from different origins for screening purposes. However, the discrimination power of the model was not completely satisfactory because most of the misclassified samples belong to the alpine samples group (80%) (data not shown). These results do not completely agree with those obtained by Coppa et al., (2010), who obtained a high error rate when they tried to predict using milk samples, the pasture type (lowland fresh herbage lands vs. mountain pasture vs. alpine pastures) grazed by cows. Conclusions The low classification error of preserved forages- vs. FH-derived cheeses model showed the ability of NIR spectroscopy to distinguish cheeses according to the presence of FH in cow diet. However NIRS appear not powerful enough to classify the samples according to the proportion of FH by cows, at least when it is estimated roughly by rapid surveys. More research should be done in order to improve results for the authentication of cheeses according to the origin and to proportion of fresh herbage in the cows diet. Acknowledgements The authors thank Comit Interprofessionnel des Fromages du Cantal (Aurillac, France), Syndicat Interprofessionnel du Fromage dAbondance (Thonon-les-Bains, France) and Syndicat Interprofessionnel de la Tomme de Savoie (Annecy, France) for having supplied cheeses samples. We are grateful to M. Jestin from the INRA UR 1213 Herbivores for the technical assistance in near-infrared spectroscopy analysis. References Agabriel C., Ferlay A., Journal C., Sibra C., Teissier D., Grolier P., Bonnefoy J.C., Rock E., Chilliard Y., Martin B. 2004. Renc. Rech. Rum. 11: 51-54. Barnes, R. J., Dhanoa, M. S., Lister, S. J. 1989. Applied Spectroscopy. 43: 772777. Coppa, M., Martin, B., Agabriel, C., Constant, I., Lombardi, G., Andueza, D. 2010. Pag. 39-40. in Proceedings of the IV conference NIR on the GO, 27-28 May. Padova Italy. Engel E., Ferlay A., Cornu A., Chilliard Y., Agabriel C., Bielicki G. Martin B. 2007. J. Agric. Food Chem. 55: 90999108. Lucas, A., Rock, E., Chamba, J. F., Verdier-Metz, I., Brachet, P., Coulon, J. B. 2006. Lait. 86: 21-41. Lucas, A., Andueza, D., Rock, E., Martin, B. 2008a. J. Agric. Food Chem. 56: 68016808. Lucas, A., Andueza, D., Ferlay, A., Martin, B. 2008b. Int. Dairy J. 18: 595604. Martin B., Cornu A., Kondjoyan N., Ferlay A., Verdier-Metz I., Pradel P., Rock E., Chilliard Y., Coulon J.B. Berdagu J.L. 2005. Pag. 127-136. in EAAP Publication n112, Wageningen Academic Publishers, Wageningen, The Netherlands. Martin B., Jestin M., Constant I., Agabriel C. Andueza D. 2006. Renc. Rech. Rum. 13: 194.
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Changes in cow milk fatty acids during transition from hay- to pasture-based diets
M. Coppa1* , A. Gorlier1, M. Lonati1, G. Lombardi1 Dep. AGROSELVITER, University of Turin, Via L. da Vinci 44, I-10095, Grugliasco (TO) *mauro.coppa@unito.it
1
Abstract To understand the kinetics of milk fatty acids (FA) during rapid or progressive transitions from hay- to pasturebased diets, two farms in south-western Alps were selected. The milk of five multiparous Valdostana Red Pie cows from each farm was analysed on days 0, 1, 2, 3, 5, 7 (at day 1 herbage was introduced or remarkably increased in the diet), then twice a week. Changes in milk FA profile became significant only after days 3 or 5. The FA from C4:0 to C18:0, C18:2n-6, C18:3n-3, and C20:0 became stable after day 5 in both transitions. The cis9-C18:1, trans11-C18:1, and cis9trans11-CLA became stable respectively 3, 16, and 16 days after reaching the maximum fresh herbage intake. The C4:0 showed a peak 3 days after herbage introduction or increase in the diet, probably because of a high butyrate production in rumen due to a high carbohydrate concentration in fresh herbage. Keywords: milk fatty acids, alpine pasture, diet change, dairy cow. Introduction Fresh herbage in cow diet increases poly-unsaturated FAs (PUFA) in milk, in particular cis9trans11-conjugated linoleic acid (CLA) and C18:3n-3, the main -3 FAs, and reduces saturated FAs (SFA) (Chilliard et al., 2007). Several studies compared milk FA profile from different diets, but few of them focused on FA profile kinetics during transitions between diets. Moreover, these studies were carried out in experimental conditions and considered abrupt diet changes, e.g. Elgersma et al. (2004) (from a temporary grassland-based diet to a silagebased one) and Khanal et al. (2006) (from a total mixed ration diet to a temporary ryegrass grassland-based one). At our knowledge changes occurring in milk during transitions from a hay from alpine meadows- to an alpine highly biodiversified pasture-based diet have not been studied yet. Similarly, little is known concerning ordinary farming conditions in which diet changes are usually gradual. The aim of this work was to describe, in alpine ordinary farming condition, milk FAs kinetics during the transition from winter hay-based feeding to pasture when the diet change is rapid or progressive. Material and methods The experiment was carried out in the Orco valley, in south-western Alps (Italy). Two dairy farms representative of alpine valley floor pasture-based systems were selected. The transition from winter feeding (zero grazing) to pasture (full grazing - about 80 % od daily dry matter intake) was rapid in one farm (three days) and progressive in the other one (sixteen days). In both farms during spring, cows were turned out to pasture and managed under strip grazing, with new paddocks offered twice a day. Individual milk samples from morning milking of five multiparous Valdostana Red Pie cows were collected for FAs analysis in both farms: i) three times during a twoweek pre-experiment period, and ii) at days 1, 2, 3, 5, 7 and then twice a week, restarting the sequence at each herbage increase in cow diet in the progressive transition farm. In rapid transition herbage in cow diet increased from 0% to 65 % at day 1 and went to full grazing after day 3, while in progressive one it increased from 0 to 40 % at day 1, from 40% to 65 % at day 6, and from 65% to full grazing at day 20. A 28-day and a 34-day period were monitored for the rapid and the progressive farms respectively. Milk samples were immediately thawed and stored at -20C until FA analysis, performed by gas-chromatographic method. Data were processed using the Repeated Measure model of analysis of variance, using sampling day as the repeated factor. Results and discussion Figure 1 reports the kinetics of the main FAs of milk during time. No differences were observed between day 0 and day 2 for all FAs both in the rapid and in the progressive transition. Changes in milk FA profile became perceptible after day 2, as observed also by Elgersma et al. (2004), but they became significant only after day 3 or day 5 according to the FAs. The FA from C4:0 to C18:0 and C18:2n-6, C18:3n-3, and C20:0 became stable after day 5 in both transition types, showing concentrations that did not differ from those of all the following sampling days. On the contrary, by studying abrupt diet changes, Elgersma et al. (2004) and Khanal et al. (2008) showed stable FA concentrations after only two days. Our results could be explained by considering that both the rapid and the progressive transition were more gradual compared to the abrupt diet changes of the cited studies. Similar results were also found by Kelly et al. (1998). The cis9-C18:1 was stable three days after reaching the maximum fresh herbage intake (day 3 in the rapid transition and day 10 in the progressive one), whereas trans11-C18:1 and cis9trans11-CLA after sixteen days (day 16 in the rapid transition and day 24 in the progressive one). The increase in vaccenic and rumenic acids and their later stabilisation we observed in ordinary farming conditions are consistent to those reported by Khanal et al. (2008) at turning out cows at pasture, and by Roy et al. (2006) on cows fed a hay-based diet supplemented with linseed oil in controlled trials. These last 57
authors explained the prolonged increase in vaccenic and rumenic acid as a results of a high stability of rumen environment in forage-rich diet, which may account for the lack of increase in milk fat of trans10C18:1,replacing trans11-C18:1 as the predominant trans-C18:1 isomers leaving the rumen in starch-rich diets. A decreasing trend was observed for FAs between C4:0 and C17:0, while FA from C18:0 to C20:0 increased with time in both transition types. The sum of SFA, the n-6/n-3 ratio, the atherogenity and trombogenity indices decreased, whereas the sums of MUFA, PUFA, trans-FA, n-3 and n-6 FA, as well as the cis9-C18:1/C16:0 ratio decreased during time in both rapid and progressive transitions. The C4:0 showed a different trend compared to all other FAs. Its concentration increased rapidly after the introduction of fresh herbage in the diet, reaching the maximum value at day 3. the C4:0 concentration decreased at values lower than day 0 at day 16, after which it remained stable in rapid transition milk, while it increased again in day 10 and in day 24, three days after each herbage increase in cow diet. Between day 10 and day 24 it decreased, and after day 24 it remained stable. The peaks of C4:0 observed in our trial can be explained by considering that fresh herbage has a higher amount of readily degradable carbohydrates compared to hay, especially at young phenological stages. In fact, due to a high substrate availability, rumen bacteria responsible of readily degradable carbohydrates degradation may have a rapid increase, resulting in an increase of hydroxybuthyrate production and transfer to mammary glands. Similar peaks in ruminal -hydroxybuthyrate production have already been observed in sheep by Lettat et al. (2011) at an abrupt diet change from a hay-based diet to a readily fermentable concentrate-based one. Figure 1: Kinetics of the main milk FA at diet change form a winter hay-based diet to an alpine pasture-based one following rapid or progressive transition
Conclusions We presented original results obtained in alpine ordinary farming condition showing the kinetics of milk FA profile during a rapid transition and a progressive transition from winter hay-based to pasture. In the perspective of a change in milk payment criteria (at least in Europe), including FA profile among quality parameters, the knowledge of how and how rapidly milk FA profile changes by introducing fresh herbage in the diet could be a useful tool to manage diet change, so as to improve milk nutritional quality and farmers income. Acknowledgements The Gestmont project (P.I. prof. Andrea Cavallero) was funded by Regione Piemonte, Agricultural Department. References Chilliard Y, Glasser F, Ferlay A, Bernard L, Rouel J, Doreau M (2007) Eur. J. Lipid Sci. Tech. 109: 828-855. Kelly ML, Kolver ES, Bauman DE, Van Amburg ME, Muller LD (1998) J. Dairy. Sci. 81: 1630-1636. Khanal RC, Dhiman TR, Boman RL (2008) Livest. Sci. 114: 164-175. Elgersma A, Ellen G, Van der Horst H, Boer H, Dekker PR, Tamminga S. (2004) Anim. Feed Sci. Tech. 117: 13-27. Lettat A, Nozire P, Silberberg M, Morgavi DP, Berger C, Martin C (2010) J. Anim. Sci. 88: 3041-3046. Martin B., Verdier-Metz I., Buchin S., Hurtaud C., Coulon, J.B. (2005). Anim. Sci. 81: 205-212. Roy A, Ferlay A, Shingfield KJ, Chilliard Y (2006) Anim. Sci. 82:479-492. 58
An European dataset to predict milk fatty acid profile from production conditions
M. Coppa1-2*, B. Martin2, F. Glasser2, Y. Chilliard2, C. Chassaing3, C. Agabriel3, G. Borreani1, R. Barcarolo4, A. Ferlay2 1 Dep. AGROSELVITER, University of Turin, Via L. da Vinci 44, I-10095, Grugliasco (TO), Italy 2 INRA, UR 1213 Herbivores, F-63122 Saint-Gens-Champanelle, France 3 Clermont Universit, VetAgro Sup, UR 2008.03.102 EPR, BP 10448, F-63000 Clermont-Ferrand, France 4 INRA, USC 2005, F-63370 Lempdes, France 5 Veneto Agricoltura Ist. Qualit e Tecnologie Agroalimentari, Via S. Germano 74, I-36016, Thiene (VI) *mauro.coppa@unito.it Abstract The objective of this work was to build a dataset of bulk milk fatty acids (FA) profile and related farming practices, with a wide territorial representativeness, in order to develop models to predict of milk FA at a farm scale. Data from three research centres were collected. A total of 1118 bulk milk samples coming from France, Northern Italy, Norway, Slovakia, and Slovenia were included in the dataset. For each milk sample, cows diet and herd characteristics were collected by on farm surveys. The wide variability in production conditions and animal feeding was also reflected by a great variation in milk FA profile. Total saturated FA (SFA) ranged from 52.1 to 79.2 g/100 g of FA, total monounsaturated FA (MUFA) from 18.0 to 42.3 g/100 g of FA, polyunsaturated FA (PUFA) from 1.8 to 7.5 g/100 g of FA. Also cis9trans11-CLA and C18:3n-3 had a wide variability, ranging from 0.1 to 2.8 and from 0.02 to 1.7 g/100 g of FA, respectively. The present work will allow to better understand which parameters could play in central role on milk FA composition at a farm scale. Keywords: Dairy cows, Animal Feeding, Milk, Fatty Acids, Production Conditions. Introduction The effect of fatty acids (FA) intake on human health is well known (Shingfield et al, 2008; Givens, 2010). As in Europe dairy products consumption is about 92.9 kg/capita year (FAO, 2011), their FA profile could have a strong impact on human health. In fact, in some niche-areas of several European countries (i.e. France, Belgium, The Netherlands, etc.) an extra-premium for milk, having high concentration of healthy FA (i.e. omega-3 and PUFA), is already given. Thus farmers need to recover information about the attended FA profile of their milk, considering the current production conditions. Moreover, the identification of farm-scale strategies to increase the concentration of healthy FA in milk could be an useful tool to increase farmers income. Most of researches on milk FA have been conducted in controlled conditions (Leiber et al., 2005; Chilliard et al., 2007) or on a restricted territorial context when carried out at a farm scale (Ferlay et al., 2008). These studies focused on finding relations between FA profile and animal feeding. The aim of this work was to build a dataset of milk FA profile and their relative production conditions, with a wide territorial representativeness, to develop models for milk FA profile prediction according to cow feeding and production conditions. Material and methods Data from eighteen experiments have been collected from FA analyses performed in three different laboratories. A total of 1118 bulk milk samples, collected between 2001 and 2010 coming from about 1000 farms located in France, Northern Italy, Norway, Slovakia and Slovenia, were included in the dataset. For each milk sample, cow diet and herd characteristics were reordered by surveys in farm. Table 1: Farm and herd characteristics.
Latitude (N) Altitude (m a.s.l.) N of cows/farm Milk yield (kg/cow*day) Primiparous (%) Lactation rank (n) Day in milk Average 668 137 19.4 28.2 2.8 169 Min ~ 44 15 4 6.1 0.0 1.5 30 Max ~ 60 2000 711 40.0 58.2 5.0 381
Table 2: Dairy cow diets (n = 1118).

Feeding (% daily DM intake) Pasture Grass silage Hay Maize silage Other feeding Concentrates Pasture diets Average Min 63 10 3 0 9 0 6 0 0 0 19 0 Max 100 58 61 65 5 67 Conserved forage diets Average Min Max 21 0 94 31 0 100 17 0 89 1 0 15 30 0 69
Results and discussion The FA profiles of different trials did not always reported all the same isomers, which identification varied depending on the laboratory and the year of experiment. FA analysis techniques have been improved in the last years, allowing the identification of much more isomers it was possible in the past. Also production conditions have been registered with different units or reported as different parameters. This lack of homogeneity was eliminated standardizing both data on FA profile and production condition to comparable parameters and units before integrating them in the dataset. As a result, the bulk milk analysed was representative of a wide variability of herd characteristics (Table 1). Farm were located from sea level to alpine regions. All farm sizes were represented: from small extensive farms to large intensive ones. More than ten dairy cow breeds with very 59
different milk yields were represented. Also cow diet was widely diversified (Table 2): from full grazing (with pasture grazed at all altitudes), to intensive diet with concentrates rising up to more than 50 % of DM intake. The wide variety of cow diets and production condition reflected also a wide range in milk FA profiles (Table 3). Total SFA ranged from 52.1 to 79.2, MUFA from 18.0 to 42.3, and PUFA from 2.0 to 7.5 g/100 g of FA. Also cis9trans11-CLA and C18:3n-3 had a wide variability, ranging from 0.1 to 2.8 and from 0.02 to 1.7 g/100 g of FA, respectively. Table 3: Milk Fatty acids profile
Fatty acid (g/100 g FA) C4:0 C6:0 C8:0 C9:0 C10:0 cis9-C10:1 C12:0 iso-C13:0 anteiso-C13:0 C13:0+cis9- C12:1 iso-C14:0 C14:0 iso-C15:0 anteiso-C15:0 cis9-C14:1 C15:0 iso-C16:0 C16:0 isoC17:0+trans9-C16:1 anteiso-C17:0 cis9-C16:1 C17:0 iso-C18:0 cis9-C17:1 C18:0 trans4-C18:1 trans5-C18:1 trans6/7/8-C18:1 trans9-C18:1 trans10/11-C18:1 trans12-C18:1 cis9/trans13-C18:1 cis10-C18:1 cis11-C18:1 cis12-C18:1 cis13-C18:1 N of data 1118 1118 1118 967 1118 997 1118 473 410 997 1020 1118 997 997 1046 1046 955 1118 1051 914 1106 1046 899 1069 1118 831 831 935 955 1118 935 1118 403 989 935 935 Average 3.24 2.18 1.32 0.03 3.01 0.31 3.56 0.08 0.01 0.19 0.16 11.83 0.32 0.60 0.97 1.25 0.33 28.99 0.51 0.39 1.44 0.69 0.05 0.24 9.46 0.01 0.01 0.22 0.19 2.11 0.26 19.33 0.50 0.62 0.16 0.06 Min 1.30 1.24 0.61 0.00 0.00 0.11 1.75 0.04 0.00 0.04 0.00 7.52 0.15 0.32 0.44 0.03 0.00 18.82 0.03 0.00 0.00 0.39 0.00 0.12 4.67 0.00 0.00 0.00 0.04 0.17 0.00 12.41 0.00 0.00 0.05 0.02 Max 5.30 3.68 3.22 0.09 5.52 0.64 6.33 0.13 0.09 0.50 0.30 14.94 0.62 1.33 1.99 2.20 1.40 39.13 2.21 1.23 2.82 1.31 0.17 0.79 16.86 0.06 0.04 0.72 0.60 6.22 0.78 29.27 1.09 3.09 0.72 0.16 Fatty acid (g/100 g FA) cis14/trans16-C18:1 cis15/trans17C18:1+C19:0 cis9trans13C18:2 trans11cis15-C18:2 cis9cis12-C18:2 C20:0 C18:3n-6 C18:3n-3 cis9trans11-CLA cis9cis11-CLA trans9trans13-CLA C20:2n-6 C22:0 C20:3n-6 C20:3n-3+cis13-C22:1 C20:4n-6 C20:5n-3 C24:0 cis9-C24:1 C22:4n-6 C22:5n-3 C22:6n-3 Even chain SFA Even & branched chain SFA MUFA PUFA UNSAT cis-C18:1isomers trans-C18:1isomers Odd chain FA Branched chain FA CLA isomers n-3 FA n-6 FA n-6/ n-3 all cis9-C18:1/C16:0 N of data 995 979 935 935 1118 1051 840 1118 1117 935 935 962 991 1033 944 1015 1012 973 919 905 935 855 1118 1118 1118 1118 1118 1118 1118 1046 1092 1118 1118 1118 1118 1118 Average 0.27 0.15 0.16 0.19 1.59 0.13 0.03 0.65 0.87 0.05 0.02 0.03 0.05 0.06 0.02 0.09 0.06 0.04 0.01 0.01 0.09 0.01 63.80 67.85 26.66 3.84 30.51 20.41 2.92 2.06 2.17 0.94 0.80 1.78 2.65 0.68 Min 0.00 0.00 0.00 0.00 0.77 0.00 0.00 0.02 0.10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 48.56 52.13 18.03 1.97 20.16 13.22 0.17 0.96 0.15 0.10 0.17 0.87 0.69 0.34 Max 0.52 0.29 0.43 0.91 4.48 0.29 1.77 1.69 2.80 0.60 0.11 0.13 0.14 0.19 0.19 0.49 0.24 0.21 0.41 0.45 0.40 0.35 74.82 79.15 42.34 7.53 48.36 32.36 7.53 3.27 4.13 3.03 1.96 4.89 10.91 1.36
Conclusions The present work will allow to better understand which parameters could play a central role on milk FA composition at a farm scale. A wide dataset of milk FA profile and related production conditions could be a useful resources to start the development of tool for dairy farming system improvements. Acknowledgements The data included in the dataset were obtained by different public funding obtained in the frame of different regional, national and European programs. We thank INRA, Phase Division for funding this work. References FAO. 2011. Statistical dataset: http://faostat.fao.org/site/610/DesktopDefault.aspx?PageID=610#ancor Chilliard Y., Glasser F., Ferlay A., Bernard L., Rouel J., Doreau, M. 2007. Eur. J. Lipid Sci. Tech. 109: 828-855. Ferlay A., Agabriel C., Sibra C., Journal C., Martin B., Chilliard, Y. 2008. Dairy Sci. Tech. 88: 193-215. Givens D.I. 2010. Animal. 4: 1941-1952. Leiber F., Kreuzer M., Nigg D., Wettstein H.R., Scheeder M.R.L. 2005. Lipids. 40: 191-202. Shingfield K.J., Chilliard Y., Toivonen V., Kairenius P., Givens D.D. 2008. Adv. Exp. Med. Biology. 606: 3-65. 60
Fatty acid profile of milk from Fiurin goats grazing Alpine pasture
M. Renna*, P. Cornale, C. Lussiana, L.M. Battaglini, A. Mimosi Dep. Animal Science, University of Turin, Via L. da Vinci 44, I-10095, Grugliasco (TO) *manuela.renna@unito.it Abstract A preliminary study on performance of a small goat population native of Piedmont, officially known as Capra Grigia delle Valli di Lanzo and locally named Fiurin, showed good milk yields and comparable gross composition if compared to other local goats reared in the Italian Alps. Increasing knowledge on milk characteristics is an essential task since valorisation of animal derived food products represents a fundamental support in safeguarding and recovering of livestock breeds facing extinction. For this reason, a subsequent investigation was carried out to assess the fatty acid profile of milk fat from Fiurin goat. Ninety individual milk samples were collected during the grazing season of year 2010 from a farm located in Tesso valley (1,580 m a.s.l.). Results showed that five fatty acids (C10:0, C14:0, C16:0, C18:0, and C18:1 c9) accounted for 73% of total fatty acids. The sum of caproic, caprylic, and capric acids averaged 12.74 g 100g-1 fat. Total trans fatty acids and conjugated linoleic acids were equal to 6.27 and 0.56 g 100g-1 fat, respectively. Information on chemical characteristics of milk from this breed will be essential to promote it through the production of a typical dairy product able to provide new sources of income for mountain areas. Keywords: Fiurin goat, milk fatty acids, conservation, livestock genetic diversity Introduction Local dairy goat breeds, especially those at risk of extinction, are characterised by typical traits and have an extraordinary ability to exploit high altitude pastures. The strong link existing between high-quality dairy products and autochthonous breeds could be seen as an effective tool to valorise them and to consequently help their genetic heritage conservation. In this scenario, increasing knowledge on raw milk chemical and sensory properties is an essential task. One of the most interesting aspects of milk from ruminants concerns the nature of its fat. Milk fatty acid profile could be used as an indicator of breed specificity since breed is considered one of the factors able to significantly affect fatty acids concentrations in milk (Signorelli et al., 2008). The goal of this work was to determine the fatty acid composition of milk from Fiurin, an endangered Italian goat breed recently identified in Piedmont (NW Italy). Material and methods Milk samples from ten Fiurin goats were collected every two weeks from May 9th to September 13th, 2010 in a farm located in Tesso valley, Piedmont (1580 m a.s.l.). Lactation number of the goats averaged 3.61.4. On the first sampling day, the goats were in mid lactation (10637 days in milk). Their milk production was equal to 2.91.1 L head-1 day-1, with an average fat content of 3.130.25%. The goats were fed fresh grass from pasture ad libitum during the whole sampling period. Milk samples were analysed for their fatty acid composition according to Collomb and Bhler (2000) by using a gas chromatograph (GC Shimadzu 17A) equipped with a CP-Sil 88 capillary column. Results are presented as mean and standard deviations (SD) of all collected samples. Results and discussion Results showed that five fatty acids (C10:0, C14:0, C16:0, C18:0, and C18:1 c9) accounted for 73% of total fatty acids (Table 1). Caproic, caprylic, and capric acids, generally associated with the characteristic flavour of goat cheeses, accounted on average for 2.33, 2.56, and 7.85 g 100g-1 fat, respectively. Lauric, myristic, palmitic, and trans fatty acids have been reported to increase risk factors for cardiovascular diseases (CVD); in Fiurin milk they accounted for 3.10, 7.89, 19.78, and 6.27 g 100g-1 fat, respectively. On the contrary, omega-3 fatty acids, which have been shown to exert protective effects on many widespread diseases including CVD, were equal to 1.12 g 100g-1 fat. A wide range of beneficial biological effects on human diseases have been attributed to conjugated linoleic acids (CLA) in animal models; results showed that total CLA was equal to 0.56 g 100g-1 milk fat. Total C18:1 and C18:2 fatty acids were equal to 16.12 and 2.38 g 100g-1 fat, respectively. The obtained results are in accordance with values previously reported for goat milk in the literature (Park et al., 2007). A remarkable difference was observed in -linolenic acid which averaged 0.96% of total fatty acid methyl esters; such value was approximately doubled compared to the mean value (0.42%) reviewed by Park et al. (2007). Conclusions It is well known that cheese characteristics are strongly affected by the fatty acid composition of the unprocessed raw milk. Differences in milk fatty acids among breeds could be consequently used as specific markers of derived dairy products. Such specificity could be also expected to contribute to the safeguard and recovery of livestock breeds at risk of extinction. The production of a typical dairy product manufactured with milk from Fiurin goat may combine both breed preservation and economic profitability of mountain areas. 61
Table 1: Individual (A) and groups (B) of fatty acids (g 100g-1 fat and % of total FAMEs) in Fiurin goat milk.
(A) C4 C5 C6 C7 C8 C10 C10:1 C12 C13 iso C13 aiso C12:1 c+C13 C14 iso C14 C15 iso C14:1 t C15 aiso C14:1 c C15 C16 iso C16 C17 iso C16:1 t C17 aiso C16:1 c C17 C18 iso C17:1 t C18 aiso C18 C18:1 t5 C18:1 t6-11 C18:1 t12-14+c6-8 C18:1 c9 C18:1 c11 C18:1 c12 C18:1 c14+t16 C19 C18:2 tt-NMID+t9t12 C18:2 c9t13+t8c12 C18:2 c9t12 C18:2 cc-MID+t8c13 C18:2 t11c15+t9c12 C18:2 c9c12 C18:2 c9c15 C20 C20:1 t C18:3 c6c9c12 C20:1 c5 C20:1 c9 C20:1 c11 C18:3 c9c12c15 CLA c9t11+t8c10+t7c9 CLA t11c13+c9c11 CLA t9t11 C20:2 cc (n6) C22 C20:3 (n6) C20:3 (n3) C20:4 (n6) C20:5 (n3) C22:5 (n3) C22:6 (n3) g 100g-1 fat mean SD 2.34 0.34 <0.01 <0.01 2.33 0.28 0.02 0.01 2.56 0.38 7.85 1.13 0.23 0.07 3.10 0.67 0.02 0.01 0.02 0.01 0.10 0.03 0.05 0.03 7.89 1.19 0.12 0.02 <0.01 <0.01 0.25 0.04 0.07 0.02 0.73 0.09 0.13 0.03 19.78 3.60 0.26 0.07 0.10 0.03 0.58 0.12 0.32 0.06 0.66 0.14 <0.01 <0.01 0.03 0.02 0.27 0.13 8.53 2.41 0.01 0.01 1.72 0.50 0.76 0.25 12.82 2.18 0.30 0.05 0.13 0.05 0.39 0.09 0.12 0.04 0.10 0.03 0.04 0.02 0.24 0.07 0.21 0.06 0.21 0.08 1.03 0.23 0.01 0.01 0.29 0.14 <0.01 <0.01 0.01 0.01 0.01 0.01 0.03 0.02 0.02 0.01 0.77 0.24 0.54 0.17 0.02 0.02 0.01 0.01 <0.01 <0.01 0.03 0.02 0.01 0.01 <0.01 <0.01 0.06 0.04 0.05 0.03 0.06 0.05 0.02 0.02 % of total FAMEs mean SD 3.00 0.46 <0.01 <0.01 3.01 0.53 0.03 0.02 3.31 0.65 10.10 1.66 0.30 0.10 3.99 0.89 0.02 0.01 0.03 0.02 0.14 0.05 0.06 0.03 10.09 1.29 0.15 0.02 <0.01 <0.01 0.31 0.05 0.09 0.03 0.93 0.08 0.17 0.04 25.16 3.27 0.33 0.10 0.13 0.04 0.74 0.12 0.41 0.08 0.84 0.13 <0.01 <0.01 0.03 0.02 0.35 0.17 10.81 2.60 0.01 0.01 2.19 0.60 0.97 0.33 16.40 2.51 0.38 0.06 0.17 0.06 0.50 0.10 0.15 0.04 0.13 0.03 0.05 0.02 0.31 0.11 0.26 0.07 0.26 0.08 1.30 0.20 0.01 0.01 0.36 0.16 0.01 0.01 0.01 0.01 0.02 0.01 0.04 0.02 0.03 0.02 0.96 0.24 0.68 0.21 0.02 0.02 0.01 0.01 <0.01 <0.01 0.03 0.02 0.01 0.01 <0.01 <0.01 0.08 0.05 0.06 0.04 0.08 0.06 0.02 0.02 (B) short chaina medium chainb long chainc saturatedd branched chaine monounsaturatedf C18:1g C18:1 transh polyunsaturatedi C18:2j C18:2 transk trans without CLAl n3m n6n n6/n3 CLAo unsaturatedp
a b
g 100g-1 fat mean SD 15.32 1.62 33.51 4.85 29.44 5.03 57.89 5.76 1.69 0.29 17.04 2.48 16.12 2.43 2.48 0.64 3.37 0.81 2.38 0.50 1.35 0.31 6.27 1.46 1.12 0.39 2.33 0.43 2.29 0.80 0.56 0.19 20.41 3.07
% of total FAMEs mean SD 19.74 2.87 42.74 4.28 37.49 4.66 73.96 3.02 2.16 0.35 21.78 2.72 20.61 2.67 3.17 0.79 4.26 0.73 3.03 0.47 1.72 0.34 8.01 1.74 1.41 0.39 2.97 0.47 3.00 1.23 0.71 0.23 26.04 3.02
g h i
o p
C4, C5, C6, C7, C8, C10, C10:1 C12, C13 iso, C13 aiso, C12:1 c+C13, C14 iso, C14, C15 iso, C14:1 t, C15 aiso, C14:1 c, C15, C16 iso, C16, C17 iso, C16:1 t, C17 aiso, C16:1 c C17, C18 iso, C17:1 t, C18 aiso, C18, C18:1, C19, C18:2, C20, C20:1 t, C18:3 c6c9c12, C20:1 c5, C20:1 c9, C20:1 c11, C18:3 c9c12c15, C18:2 c9t11+t8c10+t7c9, C18:2 t11c13+c9c11, C18:2 t9t11, C20:2 cc (n6), C22, C20:3 (n6), C20:3 (n3), C20:4 (n6), C20:5 (n3), C22:5 (n3), C22:6 (n3) C4, C5, C6, C7, C8, C10, C12, branched chain, C14, C15, C16, C17, C18, C19, C20, C22 C13 iso+aiso, C14 iso, C15 iso+aiso, C16 iso, C17 iso+aiso, C18 iso+aiso C10:1, C12:1 c+C13, C14:1 ct, C16:1 ct, C17:1 t, C18:1, C20:1 t, C20:1 c5, C20:1 c9, C20:1 c11 C18:1 t5, t6-11, t12-14+c6-8, c9, c11, c12, c14+t16 C18:1 t5, t6-11, t12-14+c6-8 C18:2, C18:3 c6c9c12, C18:3 c9c12c15, C20:2 cc (n6), C20:3 (n3), C20:3 (n6), C20:4 (n6), C20:5 (n3), C22:5 (n3), C22:6 (n3) C18:2 tt-NMID+t9t12, c9t13+t8c12, c9t12, cc-MID+t8c13, t11c15+t9c12, c9c12, c9c15, c9t11+t8c10+t7c9, t11c13+c9c11, t9t11 C18:2 tt-NMID+t9t12, c9t13+t8c12, c9t12, cc-MID+t8c13, t11c15+t9c12, c9t11+t8c10+t7c9, t11c13+c9c11, t9t11 C14:1 t, C16:1 t, C17:1 t, C18:1 t , C18:2 t (without CLA trans), C20:1 t C18:2 t11c15+t9c12, C18:2 c9c15, C18:3 c9c12c15, C20:3 (n3), C20:5 (n3), C22:5 (n3), C22:6 (n3) C18:1 t12, C18:1 c12, C18:2 tt-NMID+t9t12, C18:2 c9t12, C18:2 t9c12, C18:2 c9c12, C18:3 c6c9c12, C20:2 cc (n6), C20:3 (n6), C20:4 (n6) C18:2 c9t11+t8c10+t7c9, t11c13+c9c11, t9t11 C10:1, C12:1 c+C13, C14:1 ct, C16:1 ct, C17:1 t, C18:1, C18:2, C20:1 t, C18:3 c6c9c12, C20:1 c5, C20:1 c9, C20:1 c11, C18:3 c9c12c15, C18:2 c9t11+t8c10+t7c9, C18:2 t11c13+c9c11, C18:2 t9t11, C20:2 cc (n6), C20:3 (n6), C20:3 (n3), C20:4 (n6), C20:5 (n3), C22:5 (n3), C22:6 (n3)
Abbreviations: FAMEs, fatty acid methyl esters; c, cis; t, trans; NMID, non methylene interrupted diene; MID, methylene interrupted diene ; CLA, conjugated linoleic acid.
Acknowledgements This work was funded by Assessorato Agricoltura della Regione Piemonte. References Collomb M., Bhler T. 2000. Travaux de chimie alimentaire et dhygine 91: 306-332. Park Y.W., Jurez M., Ramos M., Haenlein G.F.W. 2007. Small Rumin. Res. 68: 88-113. Signorelli F., Contarini G., Annichiarico G., Napolitano F., Orr L., Catillo G., Haenlein G.F.W., Moioli B. 2008. Small Rumin. Res. 78: 24-31. 62
Suitability of GAEC Standard 4.6 of Cross compliance on stocking rate in the Italian mountain grazing system
L. Sepe*, S. Claps, V. Fedele CRA ZOE, Council of Agricultural Research, Research Unit of Extensive Animal Production, Via Appia, Bella Scalo, I-85054 Muro Lucano (PZ) *lucia.sepe@entecra.it Abstract The European Regulation adopted in 2003 within the Cross compliance mechanism with the aim to avoid habitat deterioration identified two limits of stocking rate (Standard 4.1): minimum 0.2 and maximum 4.0 LU/ha. Considering the wide variability of the Italian territory and livestock, the above limits may appear static and excessively generic. In fact, the Regulation does not consider the evolution of the grazing system (decrease of the number of farms, grazing animals and shepherds on one side and increase of intensive livestock on the other side) and the many other variables that influence the amount and quality of herbage from pastures: climate, soil, plant biodiversity, animal species. While the mentioned limits prevent habitat deterioration, they risk damaging the mountain grazing system in some areas. A zoning study which is able to find more suitable limits for wide habitats at the Regional level seems a viable way to preserve and safeguard not only the environment but also and firstly the breeders and their animals. Keywords: cross compliance, stocking rate, mountain, grazing system Introduction In 2003 the European Community, following up to a widespread overstocking problem, decided to identify a minimum and a maximum stocking rate (0.2 and 4 LU/ha), within the Cross compliance mechanism, in order to ensure a minimum level of lands maintenance and avoid habitat deterioration. Recently, the Cross Compliance Regulation has been applied in Italy with the MD 30125 (22/12/2009) concerning pasture and stocking rate, despite the deep changes that have occurred in the Italian zootechny in the last several decades. In fact, considering the total livestock in relation to grazing areas, the threshold of 4 LU/ha is rarely achieved in present times. In some areas we have, now, the opposite problem: a risk of under-grazing, with damages to plant biodiversity and habitat. In some Italian Regions special interventions have been adopted that take into account the local situation of zootechny and environment, but they are still few cases. The herdsmen are directly involved in the mountain habitat preservation but their rule is not highlighted enough. The aim of this paper is to offer elements for an evaluation of the impact of the application of the GAEC standard 4.6 in the Italian grazing system, with a special focus on the mountain system. The Italian pastoral system under the Cross Compliance mechanism After World War II the Italian zootechny has undergone deep changes in terms of total consistency and heads: from about 13 million heads of sheep and goats to the current 9 millions, while over 70% of bovines have been replaced with lower number of selected breed animals in intensive farming (Cavallero et al., 1997). In past times, under the dominant pastoral model, the pasture was the main source of feeding, with permanent, spontaneous, native sward and/or grazable forestland. Nowadays, the diffusion of intensive system and the decreased number of herds have led to a reduced use of grazing areas, from Alps to Southern Appennines. According to the two last Censuses in agriculture (ISTAT, 1991 and 2001), about 500 thousand hectares of mountain areas, mainly meadows and permanent pasture, are no longer used, which means that about 45% of the surface partially disappeared under shrublands, forests and crops. In this situation, the overgrazing problem (Fig. 1) (adapted from Costantini et al., 2007) that led to the Reg. of GAEC, is generally considered to have turned into its opposite: due to the low stocking rate in many Italian areas, the abandoned pasture have evolved towards shrub and less appetible vegetation, with a decrease of biomass quality. We are witnessing a paradox: the surface per animal increases, but the quality of vegetation on pasture, measured as the percentage of crude protein (7-8% in shrub and little trees) (unpublished data), decreases. The grazing animals, managed with seasonal stocking rates reduced up to 60% of balanced stocking rate (0.29-0.48 LU/ha), under a controlled grazing model, are able to limit or stop the growth of this vegetation (Cavallero et al., 2000). The animal species is an important parameter to consider, in fact each species has different ways to pick up herbage and all together contribute to maintain the balance of the mountain-hill environment. We can say that the herdsmen and their animals can be seen under a dual role: i) providers of services and products and ii) bio-processors of plant resource into foodstuffs of high functionality for human consumption, resources that would otherwise be lost. In fact, in addition to products (milk, cheese, meat, skin, etc.), they provide to communities those indirect services that too often offer modest income, compared to the large effort in terms of energy and passion, income that in a mere calculation does not justify the continuation of activities: conservation of animal and plant biodiversity (Argenti et al., 2000); preservation of plant and animal biodiversity of naturalistic interest; preservation of the landscape and tourist vocation. The reduction of farms with pastures (50% in N-W Italy and 21.9% in South) means disappearance of 63
man, which is the territory guard. Figure 1. On the left: little surface in Middle-Southern Italy risks of desertification because of overgrazing. On the right, forest and grazing lands characterize the mountain landscape in Abruzzo (Courtesy of D.A. Mancusi).
Discussion A generalisation at the national level, considering the great diversity of territory, soil, climate, animal and vegetable biodiversity, might lead to over or under estimation of the best values of stocking rate limits. Few Regions have adopted different stocking rate limits. Valle dAosta region, e.g., has approved higher minimum levels (0.5 LU when grazing over 50 days, 0.3 if over 3 months, 0.2 if over 5 months of grazing). Its regulation takes into account the short grazing period due to snow and cold temperature, the differentiation of the use of pasture and the real consistency of herds. Some other Regions have identified regimes alternative to grazing (one mowing/year on meadows) in case of deteriorated pastures. There are special Measures, such as 2111 in Liguria, that give compensatory grants to shepherds respecting other limits (0.5 and 3 LU/ha respectively, with 90 days minimum use of pasture): an example of recognition of the role of herdsman for habitat preservation. In order to avoid damages to herdsmen and their pastoral system, the intervention in all Regions is urgent, on different lines: a) study of the environment for the individuation of macro-areas (zoning) in order to define more suitable stocking rate limits, considering climate, soil, biomass availability and animal species using the pasture; b) promotion and adoption of special measures that support herdsmen in disadvantaged areas, such as creation of water points, policies against wolves; c) adoption of specific measures that recognize the role that population and animals living in the mountains play for habitat preservation. Conclusions The maintenance of the mountain habitat means, basically, the maintenance of the economic activities affecting its nature: agriculture, animal production, tourism and handicraft. A too generic regulation of cross compliance risks damaging the delicate environments where the herdsmen live and work, despite difficulties and low income. In this delicate period of general crisis, an intervention of zoning study at the Regional level with the aim of finding more suitable limits for wide habitats seems a viable way to preserve and safeguard not only the environment but also and firstly the breeders and their animals. Acknowledgements This work was carried out within the EFFICOND Italian Project2, Coordinator dr Paolo Bazzoffi. References Argenti G., Sabatini S., Staglian N., Talamucci P. 2000. Vegetazione prato pascoliva infraforestale e biodiversit di unarea alpina orientale. Proceedings of the 2nd Congress S.I.S.E.F; Applicazioni e prospettive per la ricerca forestale italiana; 20-22 October 1999; Bologna, Italy. p. 267-72 Cavallero A., Bassignana M., Iuliano G., Reyneri A. 1997. Sistemi semi intensivi e pastorali per lItalia Settentrionale: analisi di risultanze sperimentali e dello stato attuale dellalpicoltura. Riv. Agron; 31:482-504. Cavallero A., Reyneri A., Lombardi G. 2000. Impiego di diverse specie e carichi animali per la conservazione di pascoli subalpini invasi da arbusti. Riv. Agron.;34:174-7. Costantini E.A.C., Urbano F., Bonati G., Nino P., Fais A. 2007. Atlante Nazionale delle aree a rischio di desertificazione. INEA, Roma, p. 108. ISTAT. 1991. 4th General Census of Italian Agriculture, 1990. ISTAT. 2001. 5th General Census of Italian Agricolture, 2000.
The Measure has the primary objective of the maintenance of human presence through the continuation of farming in disadvantaged and marginal areas with function of territorial defence and safeguarding. 2 (EFF= environmental efficacy of COND = cross-compliance [Condizionalit in Italian lang.]), project of the CRA (Agricultural Research Council) started in 2009 to monitor and assess the environmental protection measures imposed by the CAP on the national agricultural policy.
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Session 2: Mountain Cheese Microbiology Oral presentations
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66
Microbial diversity of raw milk and influence of farm practices

A. Mallet1*, F. Kauffman2, C. Chesneau2, A. Sesboue2, N. Desmasures1 1 Unit MILA EA 3213. Universit de Caen Basse Normandie- IBFA. Esplanade de la Paix 14032 Caen. France. 2 Laboratoire de Mathmatique Nicolas Oresme. CNRS UMR 6139. UFR des Sciences. Universit de Caen Basse Normandie, 14032 Caen. France *adrien.mallet@unicaen.fr Abstract The objectives of this study were to investigate the microbial diversity of raw milk and practices that influence this diversity. The microbial diversity of raw milk of Basse-Normandie was evaluated in winter and in spring. Ten groups of microorganisms were quantified in raw milk from 130 farms. Qualitative analysis by 16S/26S rDNA sequencing was performed on raw milk samples from 12 farms. A survey was made to study the relationship between farm practices and milk microbial quality, using a statistical model. Profiles of milks were variable between producers and the proportion of microbial groups could be completely inverted and changed between the two periods of analysis for a given farm. A consequent number of isolates were sequenced (1500 bacteria and 90 yeasts). A substantial qualitative diversity was observed, with 53 genera and 112 species of bacteria and 9 genera and 17 species of yeasts identified. Moreover, some species that are poorly documented in raw milk (i.e. Ochrobactrum anthropi, Renibacterium salmoninarum and Enhydrobacter aerosaccus) have been isolated. Results suggest that raw milk is a source of microbial diversity and its quantitative diversity is practice-dependent (i.e. preparation of teat; the configuration of the milkline). Keywords: raw milk; microbial diversity, farms practices Introduction For many years, microbial communities levels in raw milk have decreased. As microbial diversity is important for the quality of raw milk cheeses, preserving or increasing this diversity has to be considered. The aim of the study was to investigate the quantitative and qualitative microbial diversity of raw milk produced in the BasseNormandie region in France and to explore relationships existing between quantitative results and farm practices. Material and methods Samples of collected raw milks (mixtures of two or four milkings) from 130 dairy farms in Basse-Normandie were collected in winter and in spring 2008. Quantification of 10 groups of micro-organisms was carried out on 8 selective media. To study the relationship between microbial flora of raw milk and farms practices, a survey, that compared the farms, the herd structure, the feeding practices and the milking and hygiene practices, was carried out. Moreover each sample of raw milk was inoculated on TSAYE (20g/L glucose + pimaricin) in five conditions: three temperatures (20C, 30C and 40C at pH 7) and two pHs (5,5 and 9 at 30C). After incubation (24h/72h), the Petri dishes were frozen at -80C for later qualitative analysis. Twelve farms were selected due to the higher level of interest flora and farm practices, in order to investigate their qualitative diversity, by identification of bacteria and yeasts based on 16S/26S rDNA sequencing. Yeasts collected from OGA medium, were identified directly by Genoscreen (Lille, France) with targeting the D1/D2 domain of 26S rDNA. DNAs from bacteria (collected from TSAYE) were extracted using a Wizard Genomic DNA purification Kit (Promega) and were amplified and sequenced using the universal primers W02 (5-GNTACCTTGTTACGACTT-3) and W18 (5GAGTTTGATCMTGGCTCAG-3) as previously described by Callon et al. (2004). Results and Discussion Mean of Aerobic Mesophilic Flora (4,8.103 cfu/ml) showed good hygienic quality of raw milk in BasseNormandie. Seasonal differences for lactobacilli, with the lowest counts in winter have been observed, this is in agreement with Salmeron et al (2002). Other microbial groups were constant between the two periods (Table1). Proportion of the different microbial groups is an indicator of diversity. Paucimicrobial milk (<1000 cfu/ml) had the most important proportion of microbial technological interest flora (notably ripening bacteria) compared to all milks. For the total of 130 milks, microbial proportions were similar between the two periods. Lactobacilli were somewhat higher in summer. The proportion of microbial groups could be completely inverted, and could sometimes change between the two periods of analysis for a given farm. Quantitative diversity was practice dependent. As expected, the mixture of four milkings had a higher level of Total Bacterial Count (TBC), lactic bacteria and yeasts than mixture of two milkings. Farms with a large herd (>60 cows) and/or a big tank (>4000 liters) had a lower count of Pseudomonas, Gram negative bacteria and yeast level. Preparation of teats with pre and post-dipping reduced the level of several groups of microorganisms and notably the positive floras. The configuration of the milkline could also influence the microbial diversity with a higher level of TBC and lactic bacteria with a dead-ended milkline. 67
Total, for the 12 selected farms (24 samples), 53 genera and 112 bacterial species, 8 genera and 17 species of yeasts have been listed. Contrary to lactic acid bacteria, Staphylococcus were frequently identified in the samples (Principal species found are presented in figure 1). Moreover, some species that are poorly documented in raw milk (i.e. Ochrobactrum anthropi, Renibacterium salmoninarum and Enhydrobacter aerosaccus) have been identified. So these results proved that raw milk analyzed had an important microbial diversity. The distribution by season was proximate with 85 species found in winter and 75 species in spring. Half of the species was detected only in one season. Gram positive bacteria were mostly found with 66% (72% in winter and 60% in spring). The number of species found was variable according to culture conditions, with the maximum of species observed at 30C (pH7). But the use of five different conditions increased the number of species found because some of them were only isolated in one or two conditions. Table 1: Microflora of raw milk:
Micro-organisms Total Bacterial Count Lactococci Lactobacilli Leuconostoc Ripening bacteria Gram-negative Pseudomonas Yeasts Mould Geometric mean (cfu/ml) Winter 2008 5,6 x 103 8,2 x 101 1,0 x 10 6,8 x 10 4,7 x 10 2,8 x 10
2
Spring 2008 4,1 x 103 7,4 x 101 2,0 x 102 1,1 x 102 5,3 x 102 7,2 x 102 5,9 x 102 9,3 x 101 2,4 x 100
9,8 x 101
2
7,9 x 102
2
7,3 x 101
0
Figure 1: Principal species found in the 24 milk samples collected in winter and in spring. (Gram positive: black; Gram negative: grey)
Conclusions and Perspectives A substantial quantitative and qualitative microbial diversity have been observed in raw milk of BasseNormandie. Perspectives of the study are to investigate some possible reservoirs of the raw milk bacterial diversity in the environment of milking. Acknowledgements This work was supported by CIFRE Grant from the Association Nationale de la Recherche Technique, by the Regional Council of Basse-Normandie, by European Funds for Regional Development and by private funds. References Salmeron, J., De Vega, C., Perez-Elortondo, F., Albisu, M., & Barron, L. 2002. Effect of pasteurization and seasonal variations in the microflora of ewe's milk for cheesemaking. Food Microbiology. 19, 167-174. Callon, C., Millet, L., & Montel, M.C. 2004. Diversity of lactic acid bacteria isolated from AOC Salers cheese, J. Dairy Res. 71, 231244.
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Biodiversity and factors of variation of microbial flora on the teats of dairy cows
I. Verdier-Metz1*, F. Monsallier1, G. Gagne2, S. Bornes2, C. Delbs-Paus1, P. Veisseire2, C. Agabriel3, B. Martin4, M.-C. Montel1 1 INRA UR545 Fromagres, 20 cte de Reyne, F 15000 Aurillac, France Clermont Universit, Universit d'Auvergne, Laboratoire de Biologie, IUT Aurillac, BP 10448, F-63000 Clermont-Ferrand, France 3 VetAgroSup, Unit Elevage et Productions des Ruminants, F-63370 Lempdes, France 4 INRA UR1213 Herbivores, F-63122 Saint-Gens-Champanelle, France *isabelle.verdier-metz@clermont.inra.fr Abstract To determine how farming practices and the intrinsic characteristics of individual dairy cows can influence the composition of the microbial community on teat skin, sixteen dairy farms in the Cantal region of France were studied. Microbial groups of the teat skin of 6 dairy cows per farm were counted using 10 dairy-specific media. Farms were classed into three groups according to the milking hygiene practices, the litter and the forage. Cows were also classed into three groups according to individual characteristics. Teat hygiene seems to be the factor that causes the greatest changes in the level of microbial flora on the teat. Whatever the type of practice, rather young cows are associated with lower levels of microbial flora on the teat skin. The microbial community on cow teat skin belonging to 3 groups of farms was evaluated by combining a culture-dependent method and a direct molecular approach. The diversity of this microbial community was large as 79.8% of clones corresponded to unidentified and 66 identified species belonging to most of those commonly found in raw milk. Then teat skin is an interesting source or vector of biodiversity for milk. Keywords: teat, milk, biodiversity, microbial flora, milking practices Introduction Teat skin has already been described as a potential reservoir of microbial diversity for milk (Piton and Richard, 1982; Michel et al., 2006) but the microbial community naturally present on dairy cow teat skin is not well documented (Vacheyroux et al., 2011). To take advantage of the natural microbial flora on teats, i.e. to maintain the flora of interest while eliminating pathogenic bacteria, it is necessary to improve knowledge of the flora and its variation factors. The aim of this study was to establish how farming practices and dairy cows' individual characteristics can influence the composition of the microbial community on teat skin. The composition of this microbial community was evaluated by combining a direct molecular approach using 16S rDNA cloning and sequencing methods with a culture-dependent method, based on culture on different media followed by molecular identification of isolates by 16S rDNA sequencing. Material and methods Sixteen dairy farms in the Cantal region in France were visited twice during the indoor confinement period. Farms were classed into three groups (FA, FB and FC) according to their practices and especially their milking hygiene practices. Cows were also classed into three groups (C1, C2, C3) according to individual characteristics. The teat skin of 6 dairy cows per farm was sampled with wet sterile swabs during milking, before the farmer washed the udder. Microbial groups were counted using 10 dairy-specific media (Millet et al., 2006). Relationships between farming practices, cow characteristics and teat skin microbial levels were investigated. Then the six teat skin samples of each farm were pooled. On one hand the six-cow pooled teat samples were plated on five culture media specific to the main bacterial groups and the colonies obtained were picked and preserved in sterile water. On the other hand total bacterial DNA of six-cow pooled teat samples was extracted. PCR amplification of 16S rRNA gene was carried out either directly from water suspension of each colony, either from the total DNA of teat samples by using the universal primers W02 and W18 as described by Callon et al. (2004). PCR products were directly sequenced (colonies) or cloned and sequenced using the W18 primer. The sequences obtained were compared to those of GeneBank database. Results and discussion The 16 dairy farms were classified in 3 groups (FA, FB, FC) by using a Multiple Correspondence Analysis (MCA). In group FA (6 farms), the winter feeding system was based on hay with a high proportion of milk produced by Montbliarde cows. Two third of the dairy cows were tied and the bedding material used was mats without straw. Milking was in most cases performed by pipeline (4/6). All FA farms applied intensive milking hygiene practices. Farms of group FB (5 farms) used a silage feeding system (corn and grass). Herds were composed entirely of Holstein cows, which were housed in free stalls with straw bedding. These farms used moderate milking hygiene practices: all always cleaned teats before milking and performed post-milking disinfection but they did not systematically wipe teats. On farms of group FC (5 farms) the breed, type of forage 69
and housing system were similar to group FA. This group was characterised by the use of straw bedding (3/5), a pipeline milking system and non-intensive milking hygiene practices. On average, the teat skin of group FA presented the lowest counts of total flora (4.9 log10 cfu/mL), Gram negative bacteria including Pseudomonas (1.6 log10 cfu/mL) and coliforms (88% of samples below the detection limit), yeasts, coagulase negative Staphylococci (3.6 log10 cfu/mL) and ripening bacteria (Staphylococci, Corynebacteria, Microcacceae, ; 3.9 log10 cfu/mL). The teat skin of group FC were characterised by the highest total flora (more than 6 log10 cfu/mL), Enterococcus (3.8 log10 cfu/mL), Pseudomonas (2.5 log10 cfu/mL), coagulase negative Staphylococci (5.98 log10 cfu/mL) and ripening bacteria counts (5.5 log10 cfu/mL). Group FB presented the highest Lactobacillus count (2.3 log10 cfu/mL). Overall, teats with high levels of microbial flora, especially Lactobacilli, Enterococci, ripening bacteria and yeasts, were associated with farming practices FB and FC, based on the use of straw bedding, rather low-intensity milking hygiene practices and multiparous cows with long, broad teats. The 96 dairy cows were classified in 3 groups (C1, C2, C3) by MCA according to their characteristics. The cows of group C1 (27 cows) were mainly primiparous (93%), with udders above hock level, short (41%) or normal (52%) teat length and normal teat shape. The cows of group C2 (34 cows) were multiparous (3/4 of cows were 4 lactations). Their udders were above hock level (59%) or hock-high (41%), with normal teat length and shape. The characteristics of the group C3 cows (35 cows) were close to those of group C2, with multiparous dairy cows (46% > 4 lactations), hock-high udders but with teats that were long (80%) and wide (77%). On average, there was no significant difference between the 3 groups of dairy cows for mould and coliform counts. But group C1 cows presented lower levels of Gram negative bacteria than group C2 and C3 cows but the differences were very slight (< 0.6 log10 cfu/mL). For Pseudomonas, the difference between C1 and C3 was also slight (<0.5 log10 cfu/mL). Low microbial levels on teat skin were mainly associated with primiparous cows and teats above hock level, particularly where milking hygiene practices were intensive. Whatever the farming practices and cow characteristics, healthy teats were reservoirs of microbial flora of interest for cheese making, especially ripening bacteria including coagulase-negative Staphylococci but also Micrococcaceae and Corynebacteriaceae The combination of the culture dependent and independent methods highlighted the large diversity of the bacterial community that may be found on teat skin with 79.8% of clones corresponded to unidentified and 66 identified species belonging to most of those commonly found in raw milk (Enterococcus, Pediococcus, Enterobacter, Pantoea ) with a dominance of Aerococcus and Staphylococcus. Then teat skin is an interesting source or a vector of biodiversity for milk. Staphylococcus auricularis, Staphylococcus devriesi, Staphylococcus arlettae, Streptococcus bovis, Streptococcus equinus, Clavibacter michiganensis, Coprococcus catus or Arthrobacter gandavensis commensal bacteria of teat skin, teat canal as well as human skin were also found in other environments. They have never been identified in milk suggesting that there is a breakdown of microbial flow from animal to milk. Our results confirm that teat skin can be considered as a possible microbial source for milk (Michel et al., 2001; Vacheyrou et al., 2011). Conclusions The original data obtained in this study shown that dairy cow characteristics can interact with farming practices to affect microbial flora counts on teat skin. Teat skin could be a particularly interesting source of biodiversity for milk. It may be noted that the composition of microbial community varied qualitatively and quantitatively from one farm to another as shown in other study (Vacheyrou et al., 2011). The results suggest that it may be possible to preserve a good balance between microbial groups by applying less intensive milking hygienic practices and using straw bedding. Further research on a larger number of cows is needed to verify the influence of different milking hygiene practices on microbial flora diversity on teat skin and in milk and to better understand the microbial flows from animal to milk. References Callon C., Millet L., Montel M.C. 2004. J. Dairy Res., 71: 231-244. Michel V., Hauwuy A., Chamba J.F. 2001. Lait, 81: 575-592. Michel V., Hauwuy A., Chamba J.F. 2006. Renc. Rech. Ruminants, 13: 309-312. Millet L., Saubusse M., Didienne R., Tessier L., Montel M.C. 2006. Int. J. Food Microbiol., 108 : 105-114. Piton C., Richard J. 1982. Lait, 62 : 67-74. Vacheyroux M., Normand A.C., Guyot P., Cassagne C., Piarroux R., Bouton Y. 2011. Int. J. Food Microbiol., 146 : 253-262.
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Application of Biolog methodology for the evaluation of terpenoids influence on lactic acid bacteria
S. Belviso, R. Ambrosoli, M. Giordano, J.L. Minati, G. Zeppa* Dep. DIVAPRA, University of Turin, Via L. da Vinci 44, I-10095, Grugliasco (TO) *giuseppe.zeppa@unito.it Abstract Terpenoids are plant metabolites with many biological properties, including a possible antimicrobial activity. Biolog methodology for metabolic characterization of microorganisms has been found suitable for the evaluation of antimicrobial activity of different plant components against bacterial consortia. With such methodology it is possible to ascertain the initial duration of the antimicrobial activity (if present) and its resulting effect on microorganisms viability, so allowing a precise screening of different substances vs. different microorganisms. An application of this methodology has been employed for the evaluation of the effect of 6 oxygenate terpenoids at different concentrations that could be normally found in raw milk, against a wide number of lactic acid bacterial strains, isolated from raw milk in previous researches and belonging to the usual physiological and metabolic groups of such microorganisms (thermophilic and mesophilic rods and cocci, homo- and heterofermenting). Initial results confirm the suitability of the adopted methodology for screening purposes, indicating that among the different groups tested various types of lactic acid bacteria responses to the presence of the same terpenoid can be detected. Keywords: terpenoids, antimicrobial activity, lactic acid bacteria, Biolog methodology. Introduction Terpenes are secondary metabolites produced by higher plants, algae, and fungi. These compounds can play many biological roles and have demonstrated various interesting properties, including antimicrobial activity (Ajikumar et al., 2008). As secondary plant metabolites, terpenes could also be ingested by animals and then transferred into associated milk and cheeses (Coulon et al., 2004). This property was employed in order to trace mountain dairy products (Coulon et al., 2004; Cornu et al., 2005; Favaro et al., 2005; Zeppa et al., 2005; Fernndez Garca et al., 2008, Revello Chion et al., 2010). Recently, the capability of lactic acid bacteria (LAB) to degrade terpenes was also showed (Belviso et al., 2011), underlining the bidirectional nature of the interaction terpenes-LAB. This could be taken into account during the manufacture of traditional raw milk cheeses, where interactions between LAB and terpenes, being both present, can occur. The aim of this research was then highlight the effect of different terpenoids on metabolic activity of some LAB strains. Materials and methods Six oxygenated terpenoids (geraniol, linalool, -terpineol, terpinen-4-ol, carvone, menthone) in two concentrations (0.1 and 1 mg/L), have been tested for their effect on LAB metabolism. Concentrations were in the range the most frequently found in milk of grazing animals. Tests were carried out on 36 LAB strains, mainly belonging to hetero-fermenting and homo-fermenting thermophilic and mesophilic cocci, isolated from cow and goat raw milk in previous researches. LABs metabolic activity in presence of the compounds was ascertained with an application of Biolog methodology of microbial characterization, previously developed for evaluating microbial response to various cultural conditions (DeNittis et al., 2010; Bertolone et al., 2011). Biolog MT2 microplates were used, in whose micro-wells, containing Tetrazolium salts as Redox indicator, each compound to be tested was added in suitable amounts to reach the planned concentrations. Blanks with no terpenoids in the wells were also prepared. The wells were then inoculated with a cell suspension in M17 broth (Oxoid, Italy) of each LAB strain. Suspensions were adjusted to give cell concentrations similar to the ones commonly found in good quality raw milk. In particular, two LAB inocula, for each concentration of each compound, were used (103-104 cells/ml). Each strain was tested in triplicate. Inoculated plates were incubated in the dark at 30C and periodically submitted to absorbance readings for the evaluation of Redox indicator colour changes, which are proportional to microbial activity and were taken as an intensity measure of microbial metabolism in the wells. Each temporal evolution of absorbance values was plotted in an experimental curve, which was considered as representative of the effect exerted on a certain LAB strain by a certain terpenoid at a certain concentration. Experimental curves were modelled (TableCurve 2D software) with Gompertzs modified equation (Zwietering et al., 1990) and the constant parameters of the equation taken as characteristic of each curve. Such parameters are (duration of lag-phase), (velocity in the exponential growth phase), and A (maximum growth value). Increase or decrease of was interpreted as a measure of immediate (antimicrobial or stimulating) terpenoids activity on microorganisms, while variations induced on or A were seen as terpenoids residual effects. All the parameters were submitted to four-way analysis of variance with strain, cell concentration, terpenoid and terpenoid concentration as factors and Duncan test with STATISTICA for Windows software (StatSoft Inc., Tulsa, OK, USA). 71
Results and discussion Preliminary results take into account the effect of all terpenoids under study on 10 LAB strains (6 mesophilic homo-fermenting cocci, two Enterococcus sp., one thermophilic homo-fermenting coccus, 1 obligate heterofermenting rod). The results obtained for all the parameters of Gompertzs modified equation are summarized in Table 1. In general, the results obtained show that some of the terpenoids under study interfere with the metabolic activity of some of the LABs tested, while terpenoids concentrations do not exert any influence on their activity. In particular, the interaction strain inoculum concentration terpenoid (see Table 1, 1 2 3) shows a statistically significant influence on lag-phase duration (). Duncan test (data not shown) indicated this result to be due to terpenoids geraniol and linalool, that resulted active on two homo-fermenting mesophilic cocci and two Enterococcus sp. strains, inducing a high increase of in their metabolic curves (as compared with the curves obtained without these terpenoids). This can be interpreted as the consequence of an antimicrobial activity by terpenoids. On the other hand, interaction strain terpenoid (see Table 1, 1 3) did not show any ability to affect the parameter , but resulted active on parameter . In this case, Duncan test (data not shown) indicated that terpenoids geraniol, linalool, -terpineol and terpinen-4-ol were able to induce an increase of that parameter on two Enterococcus sp. strains, suggesting a stimulation of microbial metabolism in this case. Table 1: Results of four-way analysis of variance within 10 LAB strains, 2 LAB inoculum concentrations, 6 terpenoids, 2 terpenoid concentrations, for duration of lag-phase (), velocity in exponential-phase () and curve asymptote ().
Effect and interactions (1) Strain (2) Inoculum concentration (3) Terpenoid (4) Terpenoid concentration 12 13 23 14 24 34 123 124 134 234 1234 **** 0.001; *** 0.005; ** 0.01; * 0.05 *** ns *** ns ns ns ns ns ns ns * ns ns ns ns Parameter **** ns * ns * ** * ns ns ns ns ns ns ns ns **** **** ns ns **** * ns ns ns ns ns ns ns ns ns
Conclusions The adopted Biolog methodology proved to be suitable for the purpose of the research. The number of strains on which the 6 terpenoids have been tested does not allow to draw general indications about their activity on LABs, but our preliminary results suggest that the already noted bidirectional nature of terpenes-LAB interaction is confirmed. While some compounds show a capacity to stimulate microbial metabolism, others have a strong antimicrobial effect, significantly increasing the lag-phase duration of LABs metabolic curves. Although strong, the latter seems to be a transitory effect, as no linked effect has been seen on microbial activity after the lag phase. Moreover, not all the LABs tested were affected in the same way. Further research is needed to elucidate this aspect and others related. References Ajikumar P.K., Tyo K., Carlsen S., Mucha O., Phon T.H., Stephanopoulos G., 2008. Mol. Pharm., 5:167190. Belviso S., Giordano M., Dolci P., Zeppa G., 2011. Dairy Sci. Technol., 91:227-236. Bertolone E., Minati J.L., Zanoni B., Ambrosoli R. 2011. I. J. Food Sci. in press on n.3. Cornu A., Kondjoyan N., Martin B., Verdier-Metz I., Pradel P., Berdargu J.L., Coulon J.B., 2005. J. Agric. Food Chem., 85:20402046. Coulon J.B., Delacroix-Buchet A., Martin B., Pirisi A., 2004. Lait, 84:221241. DeNittis M., Querol A., Zanoni B., Minati J.L, Ambrosoli R., 2010. J. Appl. Microbiol., 108, 4, 1199:1206. Favaro G., Magno A., Boaretto A., Bailoni L., Mantovani R., 2005. J. Dairy Sci., 88:34263434. Fernndez Garca E., Imhof M., Schlichtherle-Cerny H., Bosset J.O., Nuez M., 2008. Int. Dairy J., 18:147157. Revello Chion A., Tabacco E., Giaccone D., Peiretti P.G., Battelli G., Borreani G., 2010. Food Chem., 121:393399. Zeppa G., Giordano M., Bertolino M., Gerbi V., 2005. J. Chrom. A., 1071: 247-253. Zwietering M.H., Jongenburger I., Rombouts F.M., VanT Riet K., 1990. Appl. Environ. Microbiol., 56: 18751893.
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Thermophilic starters, milk composition and technological parameters affect the composition of hard-cooked cheeses
S. Buchin*,J.C. Salmon, G. Duboz, F. Faurie, R. Palme, M.H. Duployer UR342 INRA, Technologie et Analyses Laitires, rue de Versailles, F-39800 Poligny, France * solange.buchin@poligny.inra.fr Abstract The concomitant effects of thermophilic starters, milk composition and technological parameters on composition of hard-cooked cheeses were investigated. Twenty-four experimental raw milk cheeses were manufactured from the combinations of the 3 following factors: 2 starter combinations, made of the same strain of Streptococcus thermophilus, differing by the strain of Lactobacillus helveticus and the strain of Lactobacillus delbrueckii; 3 types of milk differing by their protein level (29, 32, 35 g/l); 3 levels of cooking temperature (53, 55, 57C). pH was monitored during cheese-making, microbial populations and volatile fatty acids (VFAs) in cheese until 5 months of ripening, the gross composition at unmoulding and 5 months of ripening. Starter combination had an effect mainly on starter growth, acidification kinetics during pressing and production of branched VFAs. Milk had an effect on pH before moulding, on growth of non-starter populations and on the gross composition of cheeses. Temperature in vat affected the growth of most of microbial populations, acidification kinetics during pressing and the amounts of many VFAs. Interactions were observed between these 3 factors. Keywords: milk composition, temperature in vat, thermophilic starters, hard-cooked cheeses Introduction Thermophilic starters used in the manufacture of hard-cooked cheeses can influence the characteristics of ripened cheeses (Buchin et al., 2006; Brodier et al., 2008; Charlet et al., 2009), maybe through an early effect on composition and bacterial dynamics in cheese. If so, this effect may be modulated by factors that influence the dynamics and/or activity of these bacteria. The aim of the present study was to evaluate how the physicochemical composition of milk or the technological parameters could interact with thermophilic starters in the determination of cheese characteristics. The present results will focus on acidification kinetics, microbial kinetics, gross composition and production of volatile fatty acids (VFAs). Material and methods Twenty-four experimental hard-cooked cheeses of 10 kg were manufactured from raw milk with the combinations of the 3 following factors: 2 levels of starter combination (S1=STH1D1, S2=STH2D2), including the same strain of Streptococcus thermophilus (ST) at 106 cfu/mL, 2 strains of Lactobacillus helveticus (H1 and H2) and 2 strains of Lactobacillus delbrueckii (D1 and D2) at 5.105 cfu/mL; 3 types of milk differing by their protein level (29, 32, 35 g/L); 3 levels of cooking temperature in vat (53, 55, 57C). One strain of Lactobacillus paracasei (FHL) and one strain of Propionibacterium freudenreichii (PAB) were added in all vats at 103 cfu/mL. They were ripened until 5 months (successively at 12, 17 and 7C during 1, 1.5 and 2.5 months respectively). pH was monitored during cheese-making, thermophilic starters in milk and in cheeses until 1 month, FHL, PAB and VFAs until 5 months, the gross composition at unmoulding and 5 months ripening, then 3 factors and interactions ANOVAs were done on data (Charlet et al., 2009; Bugaud et al., 2001). Results and discussion Microbial counts were in accordance with those previously found for cheeses (Depouilly et al., 2004). Thermophilic starters showed the highest counts at 20h (7.51.0 log cfu/g for ST, 9.00.3 log cfu/g for D, 7.70.7 log cfu/g for H), FHL at 2.5 months, the end of hot room (7.80.4 log cfu/g), and PAB between 2.5 and 5 months (7.80.5 log cfu/g).Temperature in vat (T) was an effective factor on all microbial populations. As expected, it affected positively thermophilic strains (starters) and negatively mesophilic ones (FHL and PAB). Milk (M) affected mainly mesophilic populations, probably via undetermined chemical or microbial compositional factor(s). Starter combination (S) affected mainly thermophilic lactobacilli. ST were mainly affected by T, showing higher counts with T55 and T57 at 20h, and with T57 at 1 month (P<0.001). D and H were affected both by M and S in milk (D: S2>S1 and M35>M32>M29 P<0.001 with M35T57 the highest P<0.05; H: S1>S2 and M35>M32 P<0.05). Afterwards, they were no more affected by M. D was higher with S1 at 20h (P<0.01) and 1 month (P<0.001), with interactions with T: at 20h, S1 counts were higher as temperature increased, whereas S2 had the lowest counts with T57 (P<0.001). At 1 month, counts were the highest with S1T53 then S1T55 (P<0.05). H was higher at 1 month with S2 (P<0.001) and T57 (P<0.001) in interaction, due to higher levels of S2T57 (P<0.001). FHL were affected by M in milk (M35>M32 P<0.01), then by T at 20h (T53> P<0.001), and by both in interaction until 5 months (M29T57<). PAB were also affected by T from 20h to the end of ripening (T53>T55>T57 P<0.001), and by M at 1 and 2.5 months (M29< P<0.001). It seems that there were some interactions between S and mesophilic populations, since S2 enhanced counts of LHF at 1 month and of PAB at 5 months (P<0.05). 73
Acidification kinetics depended on the 3 factors. At the beginning and until moulding, pH was higher in M29 (P<0.001). Between 6 and 9 hours after moulding, pH was higher in M29 than in M32 (P<0.05), showing a delay of acidification, maybe via the amount of nutrients for starters. S1 provided more acidic pH, at the beginning and from 2h after moulding, showing a higher acidifying activity. pH remained higher during moulding with T55 until 4h and with T57 until 20h (due to higher values of cheeses S2T57 at 20h). Gross composition was only affected by M, with same effects at 20h and 5 months (Table 1). Dry matter (DM) content was higher with M32 (P<0.05), maybe because of faster acidification during moulding. Contents of fat/DM and Ca/non fat DM were lower with M29 (P<0.001), i.e. with the lowest protein content, in accordance with Mietton et al. (1994). NaCl/water content was lower with M35 (P<0.001). The effect of the 3 factors on VFAs levels differed from one compound to another (Table 1). T affected acetic acid (C2) from 20h to 2.5 months (P<0.05), propionic (C3) (P<0.001), butyric (C4) (P<0.01), isobutyric (iC4) (P<0.01), isovaleric (iC5), butyric (C4) (P<0.01) and caproic (C6) (P<0.001) acids at 2.5 months, and C6 at 5 months (P<0.05). There were globally lower amounts with T57 (C2, C4, iC5) and/or higher amounts with T53 (all acids). As T53 favoured the growth of mesophilic strains, contrarily to T57, it may be supposed that the production of the above-cited VFAs is linked to the growth of such bacteria. There was an effect of S on VFAs, as already observed by Buchin et al. (2006), but only on branched VFAs, with S2 providing higher levels of iC4 at 2.5 months (P<0.01), and iC5 at 2.5 (P<0.01) and 5 (P<0.05) months. It is difficult to establish if there was a direct effect of one or more strains of S2, or an indirect effect of this combination. M had only few effects. Table 1: Gross composition and volatile fatty acids of cheeses
20h mean Gross composition (%) pH Dry matter (DM) Fat/DM Ca/non fat DM NaCl/water Volatile fatty acids VFAs (mg / 100 g) Acetic acid C2 Propionic acid C3 Butyric acid C4 Caproic acid C6 Isobutyric acid iC4 Isovaleric acid iC5 50 0.8 6.1 2.9 0.1 0.5 19 0.5 1.6 0.8 0.1 0.2 * * 53 0.8 7.3 3.2 0.5 1.2 16 0.4 1.3 0.7 0.2 0.3 * * 153 226 9.9 4.0 0.8 4.4 23 53 2. 4 0. 8 0. 3 2. 4 * *** *** * *** * * * *** *** 207 374 12 4.6 1.3 6.7 26 52 2 1.0 0.4 * 3.2 * 5.2 63 53 0.1 0.8 0.7 * *** 5.7 66 53 2.8 2.1 0.1 0.9 1 0.1 0.3 * *** *** *** std M S T mean 1 month std M S T mean 2.5 months std M S T mean 5 months std M S T
Results of ANOVA for factors Milk (M), Starters (S), Temperature (T): *: P<0.05, **: P<0.01,***: P<0.001
Conclusion Different effects of M seemed to occur, i.e. an effect of physico-chemical composition on cheese gross composition directly, an effect of nutrient contents or microbial composition on growth of mesophilic strains, and of nutrient contents on acidification by thermophilic starters. Low T was detrimental for thermophilic starters, but enhanced mesophilic strains, with consequences on production of VFAs. S affected growth of H and D, with consequences on acidification kinetics and the amounts of some VFAs. Acknowledgements This work was funded by the European 7th PCRD Program (Truefood). References Brodier F., Buchin S., Duboz G., Berthier F. 2008. Pag. 168 in proc. 5th IDF Symposium on Cheese Ripening, 9-13 March, Bern, Switzerland. Buchin S., Berthier F., Salmon J.C., Duboz G. 2006. Pag. 94 in proc. 14th Colloque des Bactries Lactiques, 1719 May Paris, France. Bugaud C., Buchin S., Coulon J.B., Hauwuy A. 2001. Lait, 81 : 757-773. Charlet M., Duboz G., Faurie F., Le Qur J. L., Berthier F. 2009. Int. J. Food Microbiol., 131: 10-19. Depouilly A., Dufrene F., Beuvier E., Berthier F. 2004. Lait, 84 : 155-167. Mietton B., Weber F., Desmazeaud M., De Roissart H.1994. Pag. 55-133 in Bactries lactiques. Vol 2 Lorica, Uriage, France.
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Volatile organic compounds produced in milk by Enterococcus faecalis

G. Battelli*, T. Silvetti, M. Decimo, M. Brasca Institute of Sciences of Food Production, National Research Council (ISPA-CNR), via Celoria 2 *giovanna.battelli@ispa.cnr.it Abstract Enterococci can contribute positively to the development of flavour during cheese ripening as their presence is consistently reported in many raw milk cheeses. They can influence flavour taste and texture of cheeses as they produce several enzymes that interact with milk components, thus promoting important biochemical transformations. For this survey, 40 Enterococcus faecalis strains, collected in different valleys in northwest Italy, were inoculated in milk and submitted to head-space solid-phase-microextraction gas chromatographymass spectrometry analysis. The major volatile compounds detected were: ethanol, diacetyl, acetoin, acetic and benzoic acids. The variability was huge, demonstrating a very different enzymatic activity among strains. Apart from other important phenological parameters for characterizing strains that can be used as starter cultures, it seems useful to also give some information about their capability of enhancing flavour characteristics in the cheese. Keywords: Enterococcus faecalis, Flavour, SPME, milk, enzymatic activity Introduction Enterococci are ubiquitous LAB which constitute a component of indigenous microflora in both the raw and pasteurized milk. As NSLAB, they are present in many types of cheeses (Giraffa, 2003). This work aims to provide more information on aroma compounds produced by E. faecalis for improving cheese flavour. Material and methods Bacterial strains and growth conditions. Forty Enterococcus faecalis strains, from different northwest Italy regions belonging to the bacterial collection of ISPA-CNR, were used in this study. The strains, previously cultured overnight at 37C in M17 broth, were inoculated in 5 ml of UHT whole milk in 20 mL sterile headspace glass vial sealed with PTFE/silicone, obtaining a final concentration of approximately 108 CFU/mL of milk. After incubation at 37C for 48 h, the vials were immediately stored at -20C. Prior to the analysis 3.5 g NaCl were added to the vials. Volatile compound analysis was performed by means of a Head-Space Solid Phase Micro Extraction module (Combi-Pal automated sampler CTC Analytics, Zwingen, Switzerland) coupled to a gas chromatograph- mass spectrometer (Agilent Technologies, Inc., Wilmington, DE, USA). Conditioning: 10 min at 50C; stirring, 250 rpm; fiber, DVB/CAR/PDMS 50/30 m (Supelco, Bellefonte, USA); exposition, 40 min at 50C maintaining stirring; desorption at 260C for 10 min in the injection port of the GC. Chromatographic conditions were previously described (Revello et al., 2010). VOCs analysis was also performed on control milk containing M17 broth. Results and discussion Fig. 1. Total ion chromatogram of the volatiles in the headspace of a The principal VOCs (in terms of level) found in all milk inoculated with an E. faecalis strain (AA8) extracted by means milk samples were acetaldehyde, benzaldehyde, 2of SPME. In bold, compounds present mainly in inoculated milk, in heptanone, 2-nonanone, 2-undecanone, butyric, italic compounds present also in control milk. hexanoic, octanoic and decanoic acids plus other VOCs to a lesser extent (Fig. 1). In milk inoculated with the E. faecalis strains, ethanol, diacetyl, acetoin, acetic acid, and benzoic acid were also found. Consequently, only these volatiles were considered, assuming that they derive from the microbial metabolic activities in milk. Ethanol possesses limited influence on cheese aroma, but it contributes to the formation of esters (Vtov et al., 2006). Enterococci production of ethanol is related to citrate metabolism (Sarantinopoulos et al., 2001; Serio et al., 2010). In E. faecalis, ethanol is also produced through the partial reduction of acetyl phosphate needed to maintain the redox balance during the heterolactic fermentation of gluconate (Axelsson, 2004). Acetoin and especially diacetyl are important flavour compounds, responsible for the creamy, buttery aroma in many dairy products, being diacetyl perceivable at a very low concentration. Both compounds derive from the catabolism of citrate representing an important technological characteristic of many LAB. Even though citrate metabolism has been exhaustively documented in strains of Lactococcus, Lactobacillus, and Leuconostoc, more limited and 75
sporadic information concerning citrate metabolism in Enterococcus strains are available (Foulqui Moreno et al., 2006). For the Enterococcus genus, production of acetic acid was already observed, ascribing its origin to various sources (Freitas et al., 1999). It is well-documented that milk products can contain natural benzoic acid as some LAB are able to convert hippuric acid or tyrosine into benzoic acid (Garmiene et al., 2010). However, this work represents the first demonstration of benzoic acid production in milk by E. faecalis. The data regarding the 5 principal compound detected in the inoculated milk, were standardised and inserted in a radar plot in order to compare the resulting bouquet of the strains from the different valleys (Fig. 2). Strains from Val dAosta and Cuneo Valleys seemed to produce less benzoic acid than the strains from Lombardy, moreover, apparently they were capable to produce more diacetyl and acetoin.
Figure 2. Radar plot of the mean values for the main VOCs extracted by means of SPME-GC-MS of milk inoculated with the E. faecalis strains collected in different mountains valleys. Numbers in brackets refer to the number of strains isolated from milk curd or cheese of the different valleys.
Conclusion While aroma components formed by bacteria during cheese production have been extensively investigated, the general capability of microorganisms for the production of volatiles has not been thoroughly explored. The results of this study represent the first step in understanding the relevant role of Enterococci in flavour development in raw milk cheeses. Apart from other important phenological parameters for characterizing strains that can be used as starter cultures, it seems useful to also give some information about their capability of enhancing flavour characteristics in the cheese. To complete these data, a sensory evaluation would be necessary to investigate the real impact of VOCs produced by strains on the resulting aroma of dairy products. It will be also interesting to analyse the effect in interaction with other flora in cheese. References Axelsson, L.. 2004. Lactic acid bacteria: classification and physiology.In :S. Salminen, A. von Wright, A. Ouwehand (Eds.), The Lactic acid bacteria: Microbiology and Functional Aspects, third ed., Marcel Dekker, New York, 2004, pp. 1-65. Foulqui-Moreno, M. R., P. Sarantinopoulos, E. Tsakalidou, and L. De Vuyst. 2006. The role and application of enterococci in food and health. Int. J. Food Microbiol. 106: 1-24. Freitas, A. C., A. E. Pintado, M. E. Pintado, and F. X. Malcata. 1999. Organic acids produced by lactobacilli, enterococci and yeasts isolated from Picante cheese. Eur. Food Res. Technol. 209:434438. Garmiene, G., J. Salomskiene, I. Jasutiene, I. Macioniene, and I. Miliauskiene. 2010. Production of benzoic acid by lactic acid bacteria from Lactobacillus, Lactococcus and Streptococcus genera in milk. Giraffa, G. 2003. Functionality of enterococci in dairy products. Int. J. Food Microbiol. 88: 15222. Revello Chion A., Tabacco E., Giaccone D., Peiretti P.G., Battelli G., Borreani G. 2010. Variation of fatty acid and terpene profiles in mountain milk and Toma piemontese cheese as affected by diet composition in different seasons. Food Chemistry 121:393-399 Sarantinopoulos, P., C. Andrighetto, M. D. Georgalaki, M. C. Rea, A. Lombardi, T. M. Cogan, G. Kalanztopoulos, and E. Tsakalidou. 2001. Biochemical properties of enterococci relevant to their technological performance. Int. Dairy J. 11: 621-647. Serio, A., C. Chaves-Lpes, A. Paparella, and G. Suzzi. 2010. Evaluation of metabolic activities of enterococci isolated from Pecorino Abruzzese cheese. Int. Dairy J. 20: 459-464. Vtov, E., B. Loupancov, J. Zemanov, H. toudkov, P. Beziva, and L. Babk. 2006. Czech. J. Food Sci. 24: 268-274.Solid-phase microextraction for analysis of mould cheese aroma.
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Benefits and risks associated with Gram-negative bacteria within cheese microbial communities
C. Delbs-Paus1*, F. Irlinger2, M. Coton3, S. Miszczycha4, S. Pochet5, S. Helinck2, P. Veisseire6, C. Callon1, D. Thvenot4, E. Coton3, M.-C. Montel1 1 INRA, UR 545 Fromagres, F-15000 Aurillac, France 2 INRA AgroParisTech, UMR782 Gnie et microbiologie des procds alimentaires, 78850 Thiverval-Grignon, France, 3 ADRIA Normandie, 14310 Villers-Bocage, France 4 VetAgrosup, UR CALYTISS, Campus vtrinaire de Lyon, 69280 Marcy lEtoile 5 INRA, UR342 Technologie et Analyses Laitires, 39800 Poligny, France 6 Laboratoire de Biologie, Universit d'Auvergne, IUT Aurillac, F-63000 Clermont-Ferrand, France *celine.delbes@clermont.inra.fr Abstract Microbial diversity including yeasts, moulds, Gram-positive as well as Gram-negative bacteria is a core element in the cheese making processes, necessary to conserve sensory quality while ensuring microbiological safety. The safety status as well as the impact of most Gram-negative bacteria on the sensory characteritics of cheeses is poorly documented. Potential benefits and risks associated with these bacteria in cheese were investigated. Among 173 Gram negative isolates from various French cheeses, 26 genera comprising at least 69 species were identified. Around 50% of all tested isolates were resistant to several antibiotics. As regards biogenic amines, only 4 strains produced relatively low amounts of putrescine and some cadaverine in cheese. In the cheese core, Gram-negative bacteria did not impact the sensory quality of the cheeses. However on the surface, cheeses containing Hafnia alvei or Psychrobacter celer presented stronger aromatic intensities and a wider variety of colors and appearances were observed. Finally, in the presence of the technological consortium, H. alvei limited the growth of the pathogen E. coli O26: H11 in the cheese core. Keywords: cheese, biodiversity, Gram-negative bacteria, sensory quality, barrier effect Introduction Microbial communities in milks and cheeses correspond to a balance between lactic acid bacteria, catalasepositive bacteria, Gram-negative bacteria, yeasts and molds. Gram-negative populations are an important component as they can reach high levels (106-107 UFC / g) in cheeses and present high species diversity. They may constitute a health risk if pathogenic species are present, such as Shiga toxin-producing Escherichia coli (STEC: E. coli O157: H7 or O26: H11), but also through biogenic amine-production. On the other hand, they can positively or negatively contribute to the sensory quality of cheeses. Gram-negative bacteria may also participate to the inhibition of pathogens. Thus, the aims of this work were i) to assess the benefits and risks of a collection of Gram-negative bacteria of dairy origin in laboratory medium and cheese, ii) to evaluate the barrier effect of Gram-negative bacteria against STEC. Material and methods The EFSA QPS approach proposed to evaluate the safety of food strains in vitro was adopted to study the biodiversity of a collection of Gram-negative bacteria from milk and cheese from different French cheese producing regions and to assess several potential risks, in particular antibiotic resistance (AB) and biogenic amines-production (BA). To assess the benefits and risks of Gram-negative bacteria, strains representative of the observed biodiversity were selected and potential risks were evaluated. The impact of their interactions within a microbial technological consortium under different environmental conditions was studied in model cheeses of uncooked pressed or smear soft cheese types to determine their i) implantation, ii) BA-production, iii) synthesis of aromatic compounds related to sensory qualities and iv) inhibition of STEC. Results and discussion In total, 173 Gram-negative isolates were identified by ribosomal gene sequencing. A large biodiversity was observed with nearly half of all Gram-negative isolates belonging to the Enterobacteriaceae family. Overall, 26 different genera represented by 69 species including potential new species were identified among the studied Gram-negative isolates for both surface and milk or cheese core samples. The most frequently isolated genera corresponded to Pseudomonas, Proteus, Psychrobacter, Halomonas and Serratia and represented almost 54% of the dairy collection. Over 50% of all tested isolates showed resistances to several antibiotics belonging to the monobactam, cepheme, fosfomycin, colistin, phenicol, sulfamide. Thirty-six % of isolates were negative for in vitro BA production. Among BA-producers, cadaverine was the most frequently produced followed by isoamylamine, histamine and putrescine. The impact of Gram-negative bacteria on sensory characteristics and production of volatile compounds as well as biogenic amines in the core of an uncooked pressed type model cheese as well as on the surface of a smear 77
soft model cheese was investigated in the presence of a defined complex microbial consortium. The consortium consisted of 8 bacteria (Lactococcus lactis Tan4, Enterococcus faecalis SB1, Lactobacillus plantarum FH3, Leuconostoc mesenteroides MSE7, S. salivarius spp. thermophilus MY800 1a, Arthrobacter arilaitensis Mu107, Corynebacterium casei CRBM9, Staphylococcus equorum RPF6) and 2 yeasts (Debaryomyces hansenii OGA10, Yarrowia lipolytica Sn4Co3). It was chosen to mimic a representative community comprising the major microbial groups commonly found in raw milk cheese. Twenty strains of Gram-negative bacteria, selected on the basis of their biodiversity and in vitro BA production ability, were individually tested in cheese. Four out of 6 strains of Enterobacteriaceae initially inoculated at 3 log CFU g-1, reached counts close to 6 log CFU g-1 in the core of uncooked pressed cheese. Only slight differences in comparison to control cheeses were observed for microbial counts, acetate concentration and texture. Cheese core colour, odour and volatile compound composition were not modified. Conversely, on the surface of smear soft cheese, when initially inoculated at 6 log CFU g-1, Psychrobacter celer took over bacterial biodiversity from the 25th day to the end of ripening. This implantation led to a higher production of volatile aroma compounds such as aldehydes, ketones and sulphur compounds. Whatever its inoculation level, H. alvei did not affect too negatively the growth of the bacterial ecosystem and was subdominant at the end of ripening. It inuenced the production of total volatile aroma compounds with volatile sulphur compounds being most abundant. As regards BA production in cheese, putrescine was only detected in cheeses inoculated with H. alvei and never exceeded 200 mg/kg cheese dry matter in uncooked pressed model cheese and 2000-4000 mg/kg dry matter in smear soft model cheese. Cadaverine was only detected in cheeses inoculated with H. alvei, Klebsiella oxytoca, Halomonas venusta or Morganella morganii but at low concentrations (< 11 mg/100g cheese dry matter). Only insignificant amounts of the most detrimental amines (histamine, tyramine) or other BA were produced in the cheese models. The potential of three strains of H. alvei, in interaction with the technological consortium, to inhibit the growth of E. coli O26:H11 in cheese was evaluated. When initially inoculated at a concentration of 102 CFU ml-1 in milk, E. coli O26:H11 reached count around 105 CFU ml-1 on day 1 in uncooked pressed model cheese inoculated with the starter and the technological consortium. It was reduced by ~1 log CFU ml-1 when an H. alvei strain was inoculated at 106 CFU ml-1 into milk. Then the technological consortium and H. alvei operated synergistically to reduce it further by 0.5-1 log CFU ml-1 by the end of ripening. The inhibition of E. coli O26:H11 growth by H. alvei during the first day was not correlated with pH values nor production of organic acids. Conclusions This study highlighted the high biodiversity of Gram-negative bacteria among the complex microbial flora of raw milk and cheese. Potential risks associated with antibiotic resistances or in vitro production of biogenic amines by certain Gram-negative strains were unveiled. However, as regards cheese BA content, the risk for consumer health of the tested Gram-negative bacteria in cheese appeared quite low. This study also highlighted the impact of several Gram-negative strains, as H. alvei, on production of volatile aroma compounds during cheese ripening. Finally, the potential of H. alvei as a biopreservative strain in cheese was revealed. Future research should explore the flavouring role of Gram-negative strains and their interactions with pathogens within the complex microbial communities of raw milk cheeses. Acknowledgements This work was funded by the French Agence Nationale de la Recherche under ANR Gramme project nANR-07PNRA-010. References Coton, M., Delbs-Paus, C., Irlinger, F., Desmasures, N., Le Flche, A., Stahl, V., Montel, M.-C. and Coton, E. Food Microbiol. under review. Deetae, P., J. Mounier, P. Bonnarme, H. E. Spinnler, F. Irlinger, and S. Helinck. 2009. J. Appl. Microbiol. 107 (4): 1404-1413. Delbs-Paus C., Pochet S., Helinck S., Veisseire P., Bord C., Lebecque A., Coton M., Desmasures N., Coton E., Irlinger F., Montel M.-C. Food Microbiol. under review. Montet M. P., Jamet E., Ganet S., Dizin M., Miszczycha S., Dunire L., Thevenot D., and Vernozy-Rozand C. Int. J. Microbiol. Volume 2009, Article ID 653481, 10 pages. Tornadijo, M.E., Garcia, M.C., Fresno, J.M.and Carballo, J. 2001. Food Microbiology, 18, 499-509.
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Microbial ecology in service of raw milk moutain cheeses

M.C. Montel INRA UR545 Fromagres, 20 cte de Reyne, F 15000 Aurillac, France cmontel@clermont.inra.fr Introduction Raw milk products, part of the famous European patrimony in gastronomy and tourism, contribute to Europe reputation all over the world for the high quality and diversity of its food products. Raw milk products are most often produced in small dairy units, often located in difficult mountain areas. They participate to structure and maintain socio-economic activities in rural areas, where milk producers play also a role of guardianof nature, contributing to promote tourism. One of the specificity of raw milk cheeses, and at a lower level of traditional cheeses, relies on the diversities of the microbial ecosystems3 involved in their production. The future of raw milk productions is strongly linked to the ability of the producers to preserve diversity of microbial ecosystems of raw milk in combination with the preservation of the safety and quality of the raw milk products. Microbial diversity Core and rind of cheese are two ecosystems and microbial diversity is at the heart of elaboration of their qualities as it is recognised that microbial diversity contribute to sensorial qualities (Beuvier et Buchin, 2004), safety (barrier effect against pathogens) (Maoz et al, 2003 ; Millet et al, 2006; Roth et al, 2010); and health (Bertrand et al, 2007 ; Waser et al, 2007). Referring to the definition of microbial diversity (Journal officiel du 12 avril 2009), this can be assessed at different levels of organization; species diversity, genetic diversity, functional diversity or ecological diversity. a-Ecological diversity Multiple ecosystems -biofilms of milking machine, teat skins, environments around the animal (feed, pasture,litter, air, water, environnements during cheese manufacturing and ripening - overlap and contribute to the structure and activities of rind and core of cheeses. These ecosytems are more or less driven by the farmer or cheese producer. Unfortunately, the flow of micro-organisms between these different ecosystems is still poorly studied as their microbial composition is more or less described. Evaluation of biodiversity Even if the extent of biodiversity in dairy microbial ecosystems is complex, its evaluation is essential to understand how microbial systems inter and intraconnected together. It is yet unrealistic to deeply measure microbial biodiversity and its dynamic during process, we can only get different images according to the methods chosen for well defined objectives. The methods to explore this microbial diversity are constantly evolving. Traditional culture-based methods with identification typing and characterisation of the microoorgansims is the oldest and still largely used by food microbiologists. During the two last decade, direct molecular methods without culturing have merged (Juste et al, 2008). The microbial community is analysed through direct DNA extraction from the samples and the nucleic acid based omic analysis for monitoring microbial community diversity and population dynamics by means of rRNA gene abundances or rRNA molecules. However, these approaches are not always well suited for establishing microbial metabolic activity and their roles in ecosystem function. Metagenomic (pyrosequencing) metatranscriptomic and metabolomic techniques, have been developed as other ways to study microbial community assemblages, giving rise to exponentially increasing collections of informations from numerous environments requiring bioinformatic analysis (Masoud et al, 2011). Then, the cheese microbiologists may loose their way, so they should use the tool to validate very well defined hypothesis on the buiding of microbial structure and activities of cheeses. b-Species diversity Changes in species composition is the most usual and relevant indicator for identifying natural disturbances related to environnemental changes and human activity. The number of species is the most common indicator to assess the diversity of bacteria, yeasts, molds when they are cultivated. But our knowledge is currently mainly limited by the enumeration, closely dependent on the media used (cf Verdier Metz et al in this meeting). Although there is no official definition of the species for these organisms, there is a consensus. It is accepted that two bacteria whose gene sequences of 16S ribosomal RNA have a similarity less than 97% do not belong to the same species but different species may have the same sequence 16s RNAr. It is therefore essential to combine genomic traits with phenotypic characteristics. The concept of species is replaced by an Operational Taxonomic Units OTU for non-culturable microorganisms identifyed by the sequencing of 16S ribosomal RNA (Amman et al, 1995). While nearly 400 000 OTU were identified in the international databases (Ribosomal Database
3
Ecosytem is afunctional self supporting system that includes the organisms in a natural community and their environment. Ecology is the study of the inter relationships between organisms and their environement in microbial ecology Atlas , Bartha 1998 Ed Addison WesleyLlongmam 79
Project, the number of bacterial species identified would be only 8000 (bacterio.cict.fr website). Indeed, in most microbial ecosystems, only a small proportion of microorganisms are cultivable, for example, only 1% in soil. On the basis on published studies it would seem that over 90% milk and cheeses microbial population can be grown on laboratory media and more than two hundred of bacterial species and around fifty yeast species has been described. Lactic acid bacteria, the most studied and the most aboundant with an important function (acidification) in the core of cheese are not the most numerous in term of number of species. Gram negative bacteria represent a large group in term of species (cf. Delbes et al in this meeting). It is often argue that this diversity is underestimated as often only dominant populations are detected. Each farm milk harbors its own microbial microbiota and variability is associated with the species composition but also with their proportion. Its dynamic during the process will vary according to the technology and the ripening conditions. On teat community the porportion of non cultivable flora seems higher than milk and cheese but the media may be not suitable (Verdier-Metz et al, 2011). The source of milk inoculation is poorly known. Vacheyrou et al , 2011 described cultivable microbial communities in raw milk and potentical transfer from environnement stable (teat surface, air , hay, teats, dust) to milk. During this meeting the presentations of Mallet et al and Verdier-Metz et al, will provide new knoweldge about the effect of farm practices on milk and teat microbial diversity. Further studies at system level will be necessary to better identify the other sources (feed , pasture) of milk inoculation and to elucidate the formation of microbail biofilm in the milking machine. They must be conducted by multidisciplinary approaches associating agronomists, microbiologists, zooetchnicians, veterinaries. c-Strains diversity The number of species does not reflect the richeness of a community. Intraspecific diversity (diversity of strains within a species) must be taken into account even if it is more difficult to assess, referring at once to the genetic diversity and phenotypic diversity. It will be expressed in terms of genomic characteristics of individuals (strains) in link with the physical and microbial environments in which they live. Strain diversity has been extensively studied among species of lactic acid bacteria to select starter culture or probiotic (cf. Tsafrakidou et al ; Batteli et al in this meeting), to evaluate for example the implantation of starter among natural flora (cf. Feutry et al, in this meeting) or transfer of microorgansim from hay to milk (Bouton et al, 2005). Inversly little is know about strain diversity for microflora present at the cheese surface. Moreover typing at high speed is important goal to determine microbial flux without focusing only on a few species.
d-Diversity of functional groups

A major goal in microbial ecology is to link specific microbial populations to cheese processes. The cultivation and characterization of isolates using genetic, biochemical and physiological tests provide information about their potential activities, their abilities to adapt and survive but did not provide an understanding of the process networks in situ. The approach of biodiversity would not be complete without approaching the diversity of functional groups that determine the quality of cheeses. Cheeses are ecosytems and their properties can not be deduced simply by taking into account species or strains composition and their individual activities. The physiological characteristics of an individual can be quite different when grown in a culture medium or in its natural environment. Moreover, the micro-organsims can intract in positive (commensalism, synergism) or negative (amensalism, competition) ways. Negative relationship is interesting to limit the development of pathogens. Within a community, strains of the same species or different species can perform the same function in the metabolic pathways identified in the process-acidification, production of various aromatic compounds from amino acids or fatty acids, production of metabolites regulating microbial interactions. It can be defined functional groups. In an ecosytem ,it is necessary to have some functional redundance. But if the species involved in acidification are fairly well identified, those responsible for other functions (inhibition of pathogenic bacteria, production of aromatic compounds ), alone but mainly in interaction are not yet all known. It is questionnable how acidification at early stage of the process modulate some microbial actvities through all the process (cf. Buchin et al, 2011 in this meeting). The role of dead cells by relarguing enzymes or subdomiant populations is not yet fully understood. The biochemical composition of cheese matrix can also influence the development of microbial populations and their activities. The role of terpens on microbial growth represent a good example ( cf Belviso et al in this meeting). Should we preserve biodiversity ? This issue is often at the heart of the debate on the microbial diversity. It can be argued that we must maintain maximum diversity in natural environments of dairy systems because we do not know all the consequences of the reduction of biodiversity. In order undesirable species and first, the pathogenic ones are tracked by trying to eliminate them and minimize risk. Hygiene measures should definitively be taken but without forgetting their original meaning "all the principles and practices designed to preserve and improve health." Measures are often too drastic and reduce without discernment biodiversity. Their impact on the functioning of other ecosystems nested around the milk and cheese are rarely anticipated. For example, what effect of antibiotic use on antibiotics 80
resistance of micro-organisms? What risk of pathogens emergence after biodiverity reduction? In any action to reduce the biodiversity for which we do not perceive the value at time t, so we should ask ourselves if we do not eliminate genetic resources that could be exploited in the future for other purposes (synthesis of new molecules for example). In strategy for producing industrial and standardised production, it could be also decided to maintain only biodiversity absolutely necessary for standard sensorial and safety characteristics of cheese. But do not forget that functional redundancy is a an advantage for an ecosystem in the face of external disturbances. What strategies for the preservation of microbial diversity? The preservations in situ or ex situ are two complementary and not opposed strategies for biodiversity conservation. Collections of biological ressources are one on the way to preserve and exploit microbial diversity for varied purposes such as industrial cheese production. But to continually enrich them we need reservoirs of biodiversity in situ. The specificity of raw milk cheese production rely on the preservation of microbial diversity in situ. This protects the heritage value of genetic and functional basis for the preservation of know-how, even in a "black box". In the cheese sector, it must rely on the preservation of traditional practices that have proven themselves: culture on whey, use of wooden vats (cf. Settani et al in this meeting). It makes sense in industries as signs of recognition that the maintenance of biodiversity is expected by the International Union for Conservation of Nature as "the spearhead of an evolution of this sector. It must be reasoned, based on good science and exerting less selective pressure on microbial communities. Neverthless these microbial communities are not static and are continualy evolving, so some biodiversity observatories should be created to observe structural and functional changes. This is more and more realistic as the technics of investigation become more and more efficient. Acknowledgements I would like to thank all the scientists who contribute to the knowledge of microbial communities of moutain cheese and especially those of my laboratory, References Amman, R.L., Ludwing,W., Schleifer K.H. 1995. Microbiol.Rev., 59: 143169. Bertrand X, Dufour V, Millon L, et al. 2007. Journal of Applied Microbiology, 102 (4): 1052-1059. Beuvier, E. and Buchin, S. Raw milk cheeses. Fox, P. F.; McSweeney, P. L. H.; Cogan, T. M., and Guinee, T. P. Cheese: chemistry, physics and microbiology. 3rd ed. 2004; pp. 319-345. Bouton Y., Tessier L, Guyot P.., Beuvier E. 2005. Rencontre Recherche Ruminants 12 403-405. Just, A., B. P. H. J. Thomma, and B. Lievens. 2008 Recent advances in molecular techniques to study microbial communities in food-associated matrices and processes. Food Microbiology, 25 (6): 745-761. Masoud W., Takamiya M., Vogensena F. K, Lillevang S., Abu Al-Soud W., Srensen S.J. and Jakobsen M. 2011. International Dairy Journal, 21(3): 142-148. Millet, L., M. Saubusse, R. Didienne, L. Tessier, and M. C. Montel. 2006. International Journal of Food Microbiology, 108(1): 105-114. Maoz, A., R. Mayr, and S. Scherer. 2003. Applied and Environmental Microbiology, 69 (7): 4012-4018. Roth, E., Miescher Schwenninger, S., Hasler, M., Eugster-Meier, E., Lacroix, C. 2010. BMC Microbiology 10, 1 Vacheyrou M., Normand A-C., Guyot P., Cassagne C., Piarroux R. , Bouton Y. 2011. Int. J. Food Microbiol, 146: 253-262. Waser M., Michels, KB, et al. 2007. Europe Clin Exp Allergy, 37 (5): 661-670.
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Session 2: Mountain Cheese Microbiology Posters
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Microbiological characteristics of traditional Caciocavallo Palermitano cheese making

L. Settanni1*, G. Tornamb2, A. Di Grigoli2, F. Noto1, V. Bellina2, G. Moschetti1, A. Bonanno1 1 Dip. DEMETRA, Settore di Microbiologia Agraria, Universit degli Studi di Palermo, Viale delle Scienze 4, 90128 Palermo, Italy 2 Dip. DEMETRA Settore di Produzioni Animali, Universit degli Studi di Palermo, Viale delle Scienze 4, 90128 Palermo, Italy *luca.settanni@unipa.it Abstract Traditional pasta filata Caciocavallo Palermitano cheese is manufactured from raw cows milk employing the wooden equipment typical of the Sicilian dairy tradition. This work was undertaken to evaluate the influence of the traditional dairy equipment on the microbiological characteristics of curds to be transformed into cheese. Traditional raw milk productions were performed concomitantly with standard manufactures carried out in a stainless steel vat inoculated with a commercial starter preparation. Milk from two different farms was separately processed and several samples were collected during traditional and standard productions to be microbiologically analysed. The wooden vat was found to be a reservoir of lactic acid bacteria, while unwanted (spoilage and/or pathogenic) microorganisms were not hosted or present at very low levels. Streptococcus thermophilus was the species found at the highest concentration in all samples, included the internal surface of the wooden vat. The comparison among traditional and standard productions did not produce any difference, since all curds were characterised by similar levels of pH and acidity, demonstrating that the indigenous S. thermophilus strains acted as a mixed starter culture. Keywords: lactic acid bacteria biodiversity, pasta filata cheese, Streptococcus thermophilus, wooden dairy plant equipment. Introduction Traditional Caciocavallo Palermitano cheese is manufactured within Palermo province (Sicily, Italy) from raw cows milk. This cheese represents one of the niche Sicilian food products that better links its history to the production area. In fact, this cheese is manufactured traditionally in small size farms of inland Sicily where cows of indigenous breeds, especially Cinisara, are fed mainly on poor natural pasture. The autochthonous microflora (starter and non starter) is important in the process of determination of cheese specificity (Piraino et al., 2005) and the wooden equipment may play an active role during production. The present work examined the effects of the wooden vat on the microbiological characteristics of curd during production, before the ripening process took place. In particular, the objectives pursued in this study were: (1) to estimate the number of several microbial groups at each step of production, from milk to stretched curd; (2) to isolate, identify and differentiate LAB at strain level; (3) to verify the presence of indigenous LAB strains on the internal surfaces of the wooden vat used for milk coagulation, adapted to the cheese factory and technological conditions, able to act as starter cultures; (4) to evaluate the persistence of the dominant strains during the production line; (5) to compare the traditional production with a standard production manufactured in a stainless steel vat inoculated with a commercial freezedried starter preparation. Material and methods Raw cows milk to be processed into cheese was collected from two farms (A and B) located within Palermo province (Sicily, Italy) and delivered to a local dairy factory. The bulk milk from the two farms were processed separately. Each bulk milk was delivered once a day; it comprised the milk from the evening milking, kept refrigerated under slow stirring, and the milk from the morning milking. Two traditional productions (TA and TB), each performed in triplicate in three consecutive weeks, were entirely followed to collect milk, curd and whey samples for analysis. Two standard Caciocavallo Palermitano cheese productions (SA and SB) were carried out in stainless steel vat inoculated with a commercial freeze-dried starter preparation. Microbiological analysis were carried out to evaluate total mesophilic microorganisms (TMM), total psychrotrophic microorganisms (TPM), coliforms, enterococci, pseudomonads, positive coagulase staphylococci, LAB, yeasts, clostridia, Salmonella spp. and Listeria monocytogenes. LAB were isolated, differentiated by random amplification of polymorphic DNA-PCR (RAPD-PCR) and identified by 16S rRNA gene sequencing. Samples of cooked and acidified curds were analysed for pH and total titratable acidity (TTA). Results and discussion The bulk milk from the two farms (A and B), employed in both traditional and standard transformations, was characterised by different levels of bacteria. Butyric clostridia were never detected (RCM). Except for coagulase positive staphylococci, found at the same level for both milk matrices, bulk milk B showed higher counts, of almost 1.5 2 log CFU/mL, than bulk milk A for all other microbial groups. This difference was even larger for coliforms counted at 1.7 log CFU/mL for bulk milk A and 5.0 for bulk milk B. TMM was 4.0 and 6.1 log 85
CFU/mL for bulk milk A and B, respectively. A similar trend was observed for pseudomonads, enterococci, yeasts and TPM which showed higher level for bulk milk B. In both matrices, the bacterial groups found at the highest numbers were LAB, which were as high as TMM. Furthermore, both bulk milk showed a ratio between mesophilic coccus and rod LAB closed to 1, whereas, the counts for thermophilic rod LAB were lower than those for thermophilic coccus LAB. Bulk milk A showed a number of microbial cells that can be retained common for raw cows milk to be processed into cheese (Franciosi et al., 2011), while higher values may indicate a massive microbial contamination of milk or low hygienic conditions of the farm or the milking system. In traditional cheese productions (TA and TB), after ca. 10 min of resting in the wooden vat, cell counts of bulk milk A changed for some bacterial groups, in particular, mesophilic rod and coccus LAB and thermophilic coccus LAB, which reached levels of 5.0, 5.4 and 5.3 log CFU/mL, respectively, increasing their concentration till 1.8 log CFU/mL. This trend was not observed for bulk milk B, whose LAB did not increase their levels in the same period. The results of the other microbial groups were almost unchanged after milk resting. These results could be explained by the counts obtained with sterile swabs streaked on given areas of the empty wooden vat before cheese production. The results showed that the microorganisms mainly found on those surfaces were LAB, while the other groups were less represented or absent. After curd cooking, the levels of LAB, including enterococci, as well as coliform bacteria, did not greatly vary for all four productions. TPM, pseudomonads and yeasts decreased their cell counts, while coagulase positive staphylococci disappeared. The acidification of curd determined higher concentration of almost all groups except pseudomonads, which remained at the same level or showed a negligible decrement in number. In particular, the acidified curds of the four productions were dominated by thermophilic coccus LAB which were identified as a strain consortium within the species S. thermphilus, a species found to dominate during manufacturing of other similar Italian pasta-filata cheeses (Baruzzi et al., 2002; Aponte et al., 2008). Both traditional and standard productions were characterised by similar pH and TTA values after cooking, as well as after acidification (Table 1). Table 1: Values of pH and total titratable acidity of curds produced during traditional and standard Caciocavallo Palermitano cheese manufacturing
Samples pH TTA (mg/100g) TA-cooked curd 5.65 0.06 117 34 TA-acidified curd 5.13 0.08 359 25 TB-cooked curd 5.62 0.05 126 28 TB-acidified curd 5.19 0.09 386 13 SA-cooked curd 5.70 0.04 136 41 SA-acidified curd 5.07 0.08 368 13 SB-cooked curd 5.62 0.08 136 24 SB-acidified curd 5.10 0.07 378 25 Abbreviations are as follows: TTA, total titratable acidity; TA, traditional production A; TB, traditional production B; SA, standard production A; SB, standard production B. Results indicate mean value S.D. of three independent measurements.
Conclusions With regards to the objectives of this study, five main conclusions can be draft: 1) the traditional Caciocavallo Palermitano cheese production is safe, since the wooden equipment does not contaminate milk with pathogenic species, such as Salmonella spp., L. monocytogenes and coagulase positive staphylococci, and the potentially spoilage species are kept at low levels; 2) several LAB are found during the whole transformation process of milk into cheese, with S. thermophilus being the dominant species; 3) the wooden vat analysed in this work acts as a reservoir of LAB for the inoculation of milk; 4) some S. thermophilus strains persist during the production line and may be considered autochthonous for this Caciocavallo Palermitano cheese production; 5) standard productions manufactured in the stainless steel vat inoculated with a commercial freeze-dried starter culture are characterised by a lower LAB biodiversity in comparison with the traditional Caciocavallo Palermitano cheese productions. References Aponte, M., Fusco, V., Andolfi, R., Coppola, S. 2008. Int. Dairy J. 18: 403-413. Baruzzi, F., Maturante, A., Morea, M., Cocconcelli, P.S. 2002. J. Dairy Sci., 85: 1390-1397. Franciosi, E., Settanni, L., Cologna, N., Cavazza, A., Poznanski, E. 2011. World J. Microbiol. Biotechnol., 27: 171-180. Piraino, P., Zotta, T., Ricciardi, A., Parente, E. 2005. Int. Dairy J., 15: 11381149.
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Analysis of microbiological variation in PDO Vastedda della valle del Belce cheese during the storage period
M.L. Scatassa1, C. Cardamone1, M. Todaro* 1 Istituto Zooprofilattico Sperimentale della Sicilia A. Mirri, via G. Marinuzzi, 4, - 90129 Palermo, Italy 2 Dep. DEMETRA, University of Palermo, Viale delle Scienze, 13 90128 Palermo, Italy *zootmax@unipa.it Abstract The PDO Vastedda della valle del Belce is a Sicilian pasta filata sheep cheese, made from raw milk without starter addition. It is a small round cheese without rind, weighing about 500-700 g. It is cheese is marketed also out of Sicily to allow its marketing and to prolong its shelf-life. The aim of this work was to evaluate the variation of microbiological parameters during the shelf-life period. Then 162 Vastedda cheeses from 18 cheese-making processes in 7 farms have been analysed at different times of storage at 4C (0, 15, 30, 45, 60, 75, 90, 105, 120 days). Coliforms and E. coli were detected into 7/18 cheese-making processes and their count decreased during the storage period. Enterococci were more resistant to high temperature achieved during the stretching and their were rather stable during storage period (105 cfu/g). The concentration of total Bacterial Count and Mesophilic Lactococci were around 107 cfu/g, while the concentration of Thermophilic Lactococci was higher (108 cfu/g) probably due to the stretching at 90C. In conclusion, this study shows the good sanitary conditions of cheeses. Overall, the lactic flora was kept alive and vital at high concentration (> 107 cfu/g) until 120 days of storage. Keywords: PDO Vastedda della valle del Belice cheese, microbiological variation, shelf-life. Introduction The PDO Vastedda della valle del Belice cheese is an unripened cheese obtained from raw ovine milk of the Valle del Belice breed ewes reared in the Belice area. This historical cheese is the only pasta filata cheese produced in Italy with raw sheep milk. The metabolic activity of the lactic microflora in the raw milk determines the dropping of pH in the curd, that happens after 6-48 h of curd maturation according to different environmental temperatures. Near to pH 5.5-5.2, the curd is stretched using water or whey at 90-95C and the shape is due to ceramics plates. It is a small round cheese without rind, weighing about 500-700 g. The PDO Vastedda della valle del Belce is marketed also out of Sicily, so to allow its marketing and to prolong its shelf-life, the cheese after 24-36 h of brine salting is sealed under vacuum with water vapour impermeable plastic film and stored at 4C up to 90 days. The microbiological content of the PDO Vastedda della valle del Belce was investigated by Reale et al. (2007) and Scatassa et al. (2007). Authors reported the dominant populations of microflora present in the Vastedda cheese and their variations during the cheese-making process. No data are available on microbiological variations during the storage until the sale; the aim of this work was to evaluate this variations during the shelf-life period. Material and methods A total of 162 Vastedda cheeses from 18 cheese-making batches in 7 farms were analysed at different times of storage at 4C (0, 15, 30, 45, 60, 75, 90, 105, 120 days). The samples were subjected to microbiological analyses using the following procedures: Coagulase-positive staphylococci (ISO 6888-1:1999/A1:2003 and ISO 6888-2:1999/A1:2003 and confirmation with API Staph); detection of Staphylococcus enterotoxin according to ELFA: VIDAS (Staph enterotoxin II e SET-RPLA (Oxoid) / ELISA test - Diffchamb); Enumeration of microorganisms at 30C (ISO 4833:2003); Coliforms (ISO 4832:1991); Escherichia coli (ISO 16649-2:2001); Enterococci on Rapid Enterococcus Agar (REA) incubated at 44C for 48 h, whose suspect colonies were tested for confirmation and biochemical identification (API 20Strep); Sulphite reducing bacteria growing under anaerobic conditions (ISO 15213:2003); Salmonella spp. (ISO 6579:2002); Listeria monocytogenes (ISO 11290-1:1996); mesophilic and thermophilic Lactococci on M17 medium, incubated at 22C for 48 h and at 44C for 72 h respectively; Lactobacilli on acidified MRS medium and incubated under micro-aerobic conditions (5% CO2) at 37C for 72 h. For statistical analysis, a two factors ANOVA model was utilised to estimate the means relative to each class of storage period, moreover least square means were plotted to observe the variations of microflora. Data were statistically analysed by GLM procedure of the SAS 9.1.2 software (2004). Results and discussion Coliforms and E. coli were observed in 7/18 cheese-making batches and they decreased during the storage period (Fig. 1). Only in two cheese-making batches, E. coli presented concentrations between 105 and 106 cfu/g. Enterococci were more resistant to the high temperature achieved during the stretching and their counts were 87
rather stable during the storage period (105 cfu/g). Coagulase-positive staphylococci showed a decrease until 6075 days to lightly go up later (Fig. 1). The concentrations of Total Bacterial Count and Mesophilic Lactococci were around 107 cfu/g, while the concentration of Thermophilic Lactococci was higher (108 cfu/g). This is probably due to bacterial selection during the cheese-making process of Vastedda and in particular during the stretching that uses hot water at 90C. Counts of CBT remained constant such as mesophilic Lactobacilli, while Lactococci (LATTOCM and LATTOCT) showed a rapid decline of their concentration during storage (Fig. 2). Otherwise, no pathogenic organisms (including sulfite reducing Clostridia, Listeria monocytogenes and Salmonella) were observed in the samples analysed. S. aureus was found in 5/18 cheese-making batches with maximum concentration around to 105 cfu/g but no enterotoxaemia activity was found. Figure 1: Variations of Coliforms (COLIFOR), E. coli (ECOLI), Coagulase-positive staphylococci (STAFF) and Enterococci bacterial (ENTERO) during the shelf life period Figure 2: Variations of Mesophilic Lactobacilli (LATTOBM), Mesophilic Lactococci (LATTOCM), Thermophilic Lactococci (LATTOCT) and Total Bacterial Count (CBT) during the shelf life period
Conclusions In conclusion this study has evidenced the good sanitary conditions of the PDO Vastedda della Valle del Belce cheese, without the presence of pathogenic bacteria. Overall, the non lactic microflora decreased during the storage (< 102 cfu/g) with the exception of Enterococci, that remain around 105 cfu/g; the lactic flora was kept alive and vital at high concentration (> 107 cfu/g) until 120 days of storage. Acknowledgements This work was funded by grant APQ Ricerca Dipartimento Industria, Assessorato Industria Regione Siciliana. References Reale S., Vitale F., Scatassa M.L., Caracappa S., Curr V., Todaro M. 2007. Ital. J. Anim. Sci. 6 (suppl. 1): 595597. SAS 2004. SAS 9.1.2 Qualification Tools Users Guide. Statistical Analysis System (SAS) Institute Inc., Cary, NC, USA. Scatassa M.L., Di Noto A.M., Todaro M., Caracappa S. 2007. Pag. 379-381 in proc. International Symposium FIL-IDF, Alghero, 2007.
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Implantation of lactic acid bacteria originated from raw milk in artisanal PDO OssauIraty cheeses manufactured with different starters.
F. Feutry1*, P. Torre2, F. Berthier3 Syndicat de dfense de lAOC Ossau-Iraty, 64120 Ostabat-Asme, France. 2 Laboratorio de lactologia, Area de Nutricin y Bromatologa, Departamento Ciencias del Medio Natural, Universidad Pblica de Navarra, 31006 Pamplona, Spain. 3 UR342 INRA, Technologie et Analyses Laitires, rue de Versailles, F-39801 Poligny, France. *fabienne.feutry@educagri.fr
1
Abstract The implantation of LAB originated from raw milks was monitored in six artisanal Ossau-Iraty cheeses throughout manufacture and ripening. They were manufactured in five farmhouse plants covering the cheesemaking practices used to manufacture Ossau-Iraty from raw milk. Plants S1(1), S1(2), S1(3) produced each one cheese with a common commercial starter S1; plant S1/S2 produced one cheese with a combination of the S1 starter plus another starter named S2; plant NS produced two cheeses, NS(1) and NS(2), without any starter nor back-slopping. LAB were monitored at 5 stages from vat milk to 6 months of ripening. Strains were typed by Repetitive bacterial DNA elements- PCR fingerprints. Between 35% and 100% of the LAB isolated from cheese, depending on sampling stage and cheese, came from the associated raw milks. Milks were the source of nearly all Lactobacillus paracasei. Except in cheese S1(3), the proportion of LAB coming from the raw milk increased during ripening. From the curd stage on, milk LAB always cohabited, except in cheese S1/S2, with adventitious LAB whose sources could not be ascertained. This study demonstrates the primary and irreplaceable role of raw milk in the building of microbial ecosystems of farmhouse Ossau-Iraty. A ripening time longer than 120 days could favor the dominance of the milk LAB. Keywords: Lactic acid bacteria, strain typing, raw milk, Ossau-Iraty, ewes cheese. Introduction Ossau-Iraty cheese is one of two French PDO ewes milk cheeses. It is a ripened, uncooked, pressed ewes milk cheese. It is produced in southwestern France, in a limited geographic area comprising the mountainous regions of Basque Country and the Bearn. It is manufactured from raw or pasteurized milk and coagulated by animal rennet. Curd is heated to 36-44C, lightly pressed, salted and ripened for at least 3 months. Its rind is yelloworange or grey. Ossau-Iraty cheesemakers are allowed to use commercial lactic starters to ensure regular and efficient acidification, but the use of such starters may modify the characteristics of the cheese microbiota during manufacture and ripening (Ortigosa et al., 1999). To monitor LAB strains dynamics in cheese requires a culturedependent approach combined with various molecular techniques. PCR amplification of repetitive bacterial DNA elements (Rep-PCR) has proven useful for differentiating a wide range of LAB isolated from cheeses at species, subspecies and strain level (Berthier et al., 2001 ; Callon et al., 2004). The aim of this study was to monitor the implantation of raw milk LAB in Ossau-Iraty cheeses manufacture with different starter cultures. Material and methods Five farmhouse cheese plants were selected: S1(1), S1(2), S1(3) produced each one, one cheese with a common commercial starter S1 (Lactococcus lactis, Streptococcus thermophilus); S1/S2 produced one cheese with a combination of the S1 starter and another named S2 ( Lb. rhamnosus); NS produced two cheeses, NS(1) and NS(2), at two different times, without any starter nor back-slopping. Each cheese was then manufactured according to the cheesemaking practices from each plant. The cheeses were either brined or dry salted and ripened at 10-12 C for 180 days. For each cheese, milk before addition of starter, curd before salting, ripened cheese without rind at days 8 (8D), 120 (120D) and 180 (180D) were analysed. LAB were isolated from the following media: MRS agar, pH 6.5 at 30 C for 48 h; FH agar with 50 mg/L vancomycin incubated at 30 C under anaerobic conditions for 48 h; MSE agar at 30C C for 48 h; SB agar at 44C for 48 h. Four to 20 colonies were picked out from each plate. After subculturing on MRS agar, all isolates were checked for Gram reaction and catalase activity and examined microscopically. Total DNA was extracted from Gram positive and catalase negative isolates using a rapid cold shock method (Gaya et al., 1999). Rep-PCR was used first to classify and then to assess the intraspecies diversity of the LAB isolates. Forty-nine LAB strains were used as reference strains. The primer pair REP-1R-Dt/Rep 2D (Versalovic et al., 1991) was used. PCR amplifications were performed as previously described (Berthier et al., 2001). After electrophoresis, the resulting fingerprintings were analysed by the Gel Compar II software, version 4.0 (Applied Maths, Kortrijk, Belgium). Calculation of similarity between the band profiles was based on the Pearson correlation coefficient. Results and discussion Rep-PCR was applied to 571 LAB (16 identified species), isolated from milks, cheeses samples and commercial starters. Accordingly to Rep-PCR fingerprinting, many strains isolated from the cheeses came from the associated raw milk between 35% and 100% depending on sampling stage and cheese (Fig. 1). Except in 89
cheese S1(3), the proportion of strains coming from the raw milk increased during ripening. Milks were the source of nearly all Lactobacillus paracasei (Fig. 2). From the curd stage on milk, strains always cohabited, except in cheese S1/S2, with adventitious strains whose sources could not be ascertained. They may also have been present in the milk but either not culturable or present at levels too low to be isolated. They may also come from the manufacturing environment as reported by Snchez et al. (2006) and Oneca et al. (2003). Figure 1: Relative proportions of strains from different origins through manufacture and ripening of each cheese. (D=day). Milk Commercial starters Other
S1(1)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CURD 8D 120D 180D
S1(2)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CURD 8D 120D 180D
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CURD
S1(3)
8D
120D
180D
S1/S2
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CURD 8D 120D 180D
NS(1)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CURD 8D 120D 180D
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CURD
NS(2)
8D
120D
180D
Figure 2: Genotypes (a-j) of the Lb. paracasei strains isolated from S1(1), S1(2), S1(3), S1/S2, NS(1) and NS(1) cheeses. A: distribution throughout manufacture and ripening; B: Rep-PCR fingerprintings (a-j) of Lb.paracasei isolated from milks.
A Milk Curd 8D 120D 180D S1(1) a,b a a a a, b S1(2) c c c c c S1(3) d d e*, f* e*, f* d, e*, f* S1/S2 g g NS (1) h, i h, j* i h, j* h NS(2) h, j h h h, j h, j B
In bold, gentotypes isolated from vat milks * Genotypes not detected from milks
Thus adventitious and raw milk strains represented (i) the totality of strains isolated from the NS and S1 cheeses, and (ii) the majority of strains isolated from the S1/S2 cheese from day 120. Strains from the S1 starter could be isolated until day 8 from all the cheeses inoculated with that starter and predominated, together with the Lb. rhamnosus strain of starter S2, in the S1/S2 cheese. The St. thermophilus strain from starter S1 could be detected neither in curds nor in the S1 and S1/S2 cheeses. Conclusions This study demonstrates the primary and irreplaceable role of raw milk in the building of microbial ecosystems of cheeses. Moreover, a ripening time longer than 120 days, could favor the dominance of the indigenous microbiota Acknowledgements This work was funded by the european IIIB Interreg program, the General Council of the Pyrnes Atlantiques, the Regional Council of Aquitaine, the Government of Navarra and by the trade union of the controlled label of origin Ossau Iraty. References Berthier, F., Beuvier, E., Dasen, A., Grappin R. 2001. Int. Dairy J., 11: 293-305. Callon, C., Millet, L., Montel, M.C., 2004.. J. Dairy Res., 71: 231-244. Gaya, P., Babn, M., Medina, M., Nuez, M., 1999. J. Appl. Microbiol., 87: 849-855. Oneca, M., Irigoyen, A., Ortigosa, M., Torre, P. 2003. FEMS Microbiol. Lett., 227: 271-277. Ortigosa, M., Brcenas, P., Arizcun, C., Prez-Elortondo, F., Albisu, M., & Torre, P. 1999. Food Microbiol, 16: 237-247. Snchez, I., Sesea, S., Poveda, J. M., Cabezas, L., & Plop, L. (2006). Int. J. Food Microbiol., 107: 265-273. Versalovic, J., Koeuth, T., & Lupski, J. R., 1991. Nucleic Acids Res., 19: 6823-6831. 90
Evaluation of NSLAB isolates from Graviera Kritis cheese as adjuncts according to technological and probiotic criteria
1
P. Tsafrakidou1 , A. Zdragas 2, S. Pavlidou 1, D. Bozoudi 1, E. Litopoulou-Tzanetaki1* Laboratory of Food Microbiology and Hygiene, Faculty of Agriculture 2 Veterinary Research Institute, NAGREF Campus, 57001, Thessaloniki, Greece * ganet@agro.auth.gr
Abstract A total of 54 isolates obtained from mature Graviera Kritis cheese manufactured at two different traditional dairies (three manufactures in each) were studied for their technological and probiotic properties. The isolates were characterized at species level by the SDS - PAGE of whole-cell proteins mainly as Lactobacillus paracasei (similarity 76.3%) and Leuconostoc pseudomesenteroroides (similarity 74.6%).Their genetic heterogeneity was also studied by PFGE. The 82% of the isolates from dairy I exhibited smaller acidification ability than those from dairy II. In addition, the 58.2% of the isolated strains degraded preferentially s-casein. The amino acid amounts accumulated in the milk were either very low (0-0.5mM L-glycine) for the isolates from one cheese of dairy I or high (3.5mM L-glycine) for strains from the two cheeses of the same dairy. 93% of the strains from dairy II were grouped in a large cluster of 35 isolates from both dairies, exhibiting proteolytic activity in the range of 0.69-3.46 mM of L-glycine. All the isolates were able to grow in the presence of bile at 0.3-2% and the 95.5% of them at low pH values (3 and 1.5 respectively). None of the strains exhibited antibacterial activities and/or haemolytic activity. Selected isolates could be used as adjuncts to make cheese and deserve further studies. Keywords: NSLAB, cheese, technological, probiotic properties Introduction To date, the search for starter cultures has relied on studying the properties of isolates, preferably obtained from natural and spontaneously fermented food products. NSLAB isolates, which possess health promoting effects are of particular interest (Bao et al., 2010). The aim of this work was to study the technological and probiotic properties of NSLAB from traditional Graviera Kritis cheese. Material and methods A total of 54 NSLAB isolates from mature cheeses made at two dairies (dairy I, 27 isolates; dairy II, 27 isolates) were characterized by the SDS-PAGE of whole-cell proteins; they were also studied in respect of their technological properties (acidification ability and/or proteolytic activity in milk by UREA-PAGE and o-PA methods) and genotypic heterogeneity (by PFGE). Their antibacterial activity against food-born pathogens (on MRS agar) and haemolytic activity (on blood agar) were also estimated (Pavlidou et al., 2011, Psoni et al., 2006). Their probiotic potential was also estimated (acid and bile tolerance in MRS broth). Three cheese samples from each of the two dairies, were obtained. Results and discussion The isolates were characterized at species level by the SDS-PAGE of whole-cell proteins, mainly as Lactobacillus paracasei (similarity 76.3%) and Leuconostoc pseudomesenteroroides (similarity 74.6%)(data not shown). The strains exhibited a small genotypic heterogeneity. However, groups of plant related isolates were discriminated (data not shown). The isolates produced very low amounts of acid, with the majority (82%) of the isolates from dairy I (group A) exhibiting smaller acidification ability (pH 0.08 0.15 pH units) than those from dairy II (pH 0.20 - 0.53 pH units) (group B), after growth in milk for 24 h (Table 1). Table 1: PH evolution in milk inoculated by isolates of NSLAB from dairy I (A, B, C) and II (indicated by numbers). Mean SD of three independent experiments.
Group A Strains B10 C2 B3 C4 A4 B1 A10 A8 B4 A9 B6 B2 A7 A2 C7 Difference in pH (pH) after 6h 16h 24h 0.040.015 0.040.006 0.080.015 0.050.030 0.050.010 0.090.010 0 0.220.307 0.090.040 0.080.035 0.080.035 0.110.012 0.020.015 0.120.060 0.110.031 0.010.012 0.140.081 0.110.035 0.070.096 0.140.050 0.120.030 0.040.038 0.130.046 0.120.015 0.040.045 0.130.067 0.130.025 0.010.012 0.120.070 0.130.020 0.060.040 0.070.020 0.130.010 0.180.278 0.140.066 0.130.026 0.010.012 0.120.064 0.130.015 0.040.040 0.130.030 0.140.047 0.010.006 0.100.000 0.140.010 Group B Strains 785 A6 715 802 812 729 B9 C1 813 760 716 781 C3 803 731 Difference in pH (pH) after 6h 16h 24h 0.010.010 0.100.000 0.200.046 0.150.032 0.190.093 0.220.076 0.040.035 0.120.031 0.220.055 0.060.015 0.110.031 0.220.030 0.050.036 0.170.046 0.230.040 0.010.006 0.100.046 0.230.087 0.060.039 0.150.015 0.280.012 0.060.036 0.070.006 0.280.231 0 0.170.035 0.290.058 0.050.045 0.140.021 0.290.079 0.040.015 0.140.046 0.290.066 0.010.006 0.170.029 0.290.035 0.070.021 0.120.015 0.300.025 0.070.012 0.210.015 0.320.031 0.040.036 0.160.040 0.320.075
91
A5 C5 C6 A3 B5 B7 A1 787
0 0.010.006 0.080.025 0.060.032 0.080.056 0.100.056 0.130.020 0.040.040
0.140.062 0.100.000 0.080.025 0.130.020 0.1500.07 0.080.006 0.160.067 0.090.035
0.140.012 0.140.040 0.140.060 0.150.040 0.150.060 0.150.010 0.170.067 0.170.052
766 718 786 B8 784 783 764 810 767 713 711 759 768 801 763 811
0.040.032 0.050.010 0 0.080.025 0.030.010 0.020.015 0.080.047 0.060.015 0.040.031 0.100.015 0.050.026 0.080.055 0.090.042 0.080.023 0.10.006 0.060.020
0.160.042 0.190.006 0.180.029 0.120.010 0.160.069 0.190.040 0.200.030 0.190.015 0.210.026 0.250.020 0.220.036 0.250.010 0.210.026 0.240.044 0.260.012 0.250.015
0.330.085 0.330.051 0.330.040 0.330.010 0.330.087 0.340.075 0.350.042 0.360.040 0.370.025 0.390.015 0.400.080 0.420.010 0.420.044 0.430.025 0.450.068 0.530.031
The 58.2% of the test strains degraded preferentially s-CN (data not shown). There was a great diversity even among isolates from the same dairy, concerning the amino acid amounts accumulated in the milk and three groups of isolates were discriminated. Group A included isolates from one cheese of dairy I with low activity (0-0.5 mM L-glycine), while in group B stains from two cheeses, mainly from dairy I, with high activity were allocated. The 93% of the strains from dairy II were grouped in a large cluster of 35 isolates from both dairies, exhibiting proteolytic activity in the range of 0.69-3.46 mM of L-glycine (Table 2). All the isolates were able to grow in the presence of bile at 0.3-2% and the 95.5% of them at low pH values (3 and 1.5 respectively)(data not shown). None of the strains exhibited antibacterial activities and/or haemolytic activity (data not shown). Selected isolates (for example B8, B9, C3) could be used as adjuncts to make cheese and deserve further studies. Table 2: Proteolytic activity (o-PA method; mM L-Glycine) of NSLAB from dairy I (A, B, C) and II (indicated by numbers). Mean SD of three independent experiments.
Strains Group A A4 A6 A7 A8 A9 A10 A5 A2 A3 A1 B1 B3 C2 C7 811 C6 C1 810 B7 mM L-Gly after 24h 7d 0 0 0 0 0 0 0 0 0 0 0 0 0 0.030.04 0 0.130.22 0 0.230.02 0 0.540.50 0 3.542.02 0 3.830.98 0.360.47 4.050.99 0.390.04 4.200.52 0.310.35 4.900.99 0.630.55 5.111.18 0.690.06 5.331.85 1.380.49 6.681.53 0.520.18 17.180.74 Strains Group C 785 787 C5 763 729 812 731 760 B5 C4 813 781 784 802 766 803 715 B10 B10 759 716 711 768 B2 B8 764 718 713 786 C3 783 B4 B9 767 B6 mM L-Gly after 24h 7d 0.270.27 0.690.27 0.850.42 0.710.07 0.280.27 0.860.43 0.320.32 0.960.13 1.380.18 1.000.29 1.921.09 1.060.63 1.620.13 1.260.67 0.560.19 1.260.76 1.320.45 1.290.23 0.290.15 1.320.50 1.080.54 1.430.09 1.170.52 1.440.47 0.530.72 1.480.24 1.020.18 1.530.23 0.210.07 1.680.16 1.320.19 1.710.29 1.640.26 1.710.40 0 1.840.32 0 1.840.32 0.480.15 1.840.15 1.760.46 1.860.64 0.910.16 1.940.48 0.790.09 1.960.09 0 2.010.79 0.880.55 2.051.19 0.240.42 2.130.12 1.680.45 2.150.61 1.190.15 2.310.70 1.031.02 2.440.70 0.290.08 2.490.54 0.480.50 2.741.39 0 2.811.96 0.490.48 2.990.14 0.900.05 3.210.41 0.150.14 3.230.39
Group B
References Bao Y., Zhang Y., Liu Y., Wang S., Dong X., Wang Y., Zhang H. 2010. Food Control, 21: 695-701. Psoni L., Kotzamanides C., Andrighetto C., Lombardi A., Tzanetakis N., Litopoulou-Tzanetaki E. 2006. Int. J. Food Microbiol., 109: 109-120. Pavlidou S., Bozoudi D., Hatzikamari M., Tzanetakis N., Litopoulou-Tzanetaki E. 2011. J.Food Sci.,76: M175M183. 92
Index of authors
93
A Agabriel C. Agabriel J. Ambrosoli R. Andueza D. Annicchiarico G. B Barcarolo R. Barjolle D. Battaglini L.M. Battelli G. Belibasaki S. Bellina V. Belot P.E. Belviso S. Berthier F Bianchi P. Blay B. Bonanno A Bornes S. Borreani G Bozoudi D. Brasca M. Buchin S. C Callon C. Caputo A.R. Cardamone C. Chassaing C. Chatelard C. Chesneau C. Chilliard Y. Ciri C. Claps S. Constant I. Convert T. Coppa M. Cornale P. Cornu A. Coton E. Coton M. D Decimo M. Delaby L. Delavaud C. Delbs-Paus C. Desmasures N.
35; 55; 59; 69 21 71 55 19
Di Grigoli A. Di Napoli M.A. Donars M. Duboz G. Duployer M.H. Duriot B. F Faurie F. Fedele V Ferlay A. Feutry .F G Gagne G. Galassi L. Galina M.A. Gallard Y. Giaccone D. Giordano M. Girard C.L Gkouma M. Glasser F. Gorlier A. Graulet B. Guerrero M. Guiadeur M. H Helinck S. Hulin S. Hurtaud C. I Irlinger F. J Jeanneaux P. Johan M. K Kauffman F. L Leiber F. Lemaire M. Lepetit M. Leurent S. Litopoulou-Tzanetaki E. Lonati M.
15; 85 19 41 73 73 51
59 41 61 53; 75 29 15; 85 39 71 89 25 51 15; 85 69 25; 53; 59 27; 29; 91 75 73
73 19; 63 13; 59 89
69 25 17 37 9; 25; 53 71 35 33 59 57 31; 33; 35; 51 17 21
77 19; 27; 29 87 13; 35; 59 51 67 13; 59 21 17; 19; 27; 29; 31; 63 13; 55 49 57; 59 61 31; 33 77 77
77 51 17
77
41 37
67
75 37 13; 69; 77 67
43 33 51 37 27; 29; 91 57
94
Lucas A. Lussiana C. M Mallet A. Martin B. Masoero G. Massouras T. Mazza F. Meyer D. Mimosi A. Minati J.L. Miszczycha S. Monsallier F. Montel M.C. Morone G. MoschettiaG. Mosimann E. N Noto F. P Palme R. Parguel P Parisi Z. Pavlidou S. Pineda L.J. Pizzillo M. Pochet S. R Renna M. Revello Chion A. Rubino R. Rufrano D. S Salmon J.C. Scatassa M.L. Sepe L. Sesboue A. Settanni L. Sibra C. Silvetti T.
55 61
67 13; 21; 35; 51;55; 59; 69 23 33 15 41 61 71 77 21; 69 21; 69; 77; 79 19 85 39
T Tabacco E. Thvenot D. Todaro M. Tornamb G. Torre P. Tsafrakidou P. V Valperga T. Veisseire P. Verdier-Metz I. Z Zdragas A. Zeppa G.
25; 53 77 87 15; 85 89 91
9 69; 77 21; 69
91 71
85
73 39 29 91 17 17; 23 77
61 25; 53 17; 23 31
73 87 19; 31; 63 67 85 13 73
95

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10th INTERNATIONAL Meeting on Mountain cheese 14-15 september 2011 dronero (cn), italy

Dairy Production in Mountain:

Proceedings of the 10th International Meeting on Mountain cheese

14-15 September 2011, Dronero, italy

Edited by University of Turin, Italy

Associazione Regionale Allevatori Piemonte (ARAP)

Comunit Montana Valli Grana e Maira

Banca Cassa di Risparmio di Savigliano

Camera di Commercio di Cuneo

On behalf of the organisation committee Bruno MARTIN (INRA-France)

The mountain dairy production in Piedmont

NW Greece (~800m) 10.59 7.19 2.81 171.78 17.25 48.08

SW Greece (~850m) 3.52 221.81 8.33 217.95 20.72 17.89

SW Greece (~1300m) 3.07 376.14 5.23 192.26 31.98 93.38

Amounts in arbitrary units (A.U.)

Amounts in arbitrary units (A.U.)

Table 1: Plant species collected from two mountainous Greek areas.

Caryophyllaceae Rosaceae Convolvulaceae

Compositae Scrophulariaceae Labiatae Polygonaceae

1 K1 K4 K6 K9 K2 K5 K7 K12 K14 K15 K3 K13 K8 K10 K11 B3 B5 B6 B7 B8 B2 BB1 B4

NW area ~800m altitude Plants

SW area 1300m altitude

Vitamin B9 and B12 contents in cow milk according to production system

Evolution of milk calcium content during the year

Dairy production potential of the Jura meadows

DPP grassland (kg milk ha-1)

Fodder surface (ha) HERD SCALE

Raising Rivals Costs strategy and localized agro-food systems in Europe

The added nutritional value of mountain dairy products

Table 2: Dairy cow diets (n = 1118).

Session 2: Mountain Cheese Microbiology Oral presentations

Microbial diversity of raw milk and influence of farm practices

Volatile organic compounds produced in milk by Enterococcus faecalis

Microbial ecology in service of raw milk moutain cheeses

d-Diversity of functional groups

Session 2: Mountain Cheese Microbiology Posters

Microbiological characteristics of traditional Caciocavallo Palermitano cheese making

0 0.010.006 0.080.025 0.060.032 0.080.056 0.100.056 0.130.020 0.040.040

0.140.062 0.100.000 0.080.025 0.130.020 0.1500.07 0.080.006 0.160.067 0.090.035

0.140.012 0.140.040 0.140.060 0.150.040 0.150.060 0.150.010 0.170.067 0.170.052

35; 55; 59; 69 21 71 55 19

59 41 61 53; 75 29 15; 85 39 71 89 25 51 15; 85 69 25; 53; 59 27; 29; 91 75 73

69 25 17 37 9; 25; 53 71 35 33 59 57 31; 33; 35; 51 17 21

67 13; 21; 35; 51;55; 59; 69 23 33 15 41 61 71 77 21; 69 21; 69; 77; 79 19 85 39

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