LWT - Food Science and Technology 44 (2011) 511e519

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LWT - Food Science and Technology

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Optimization of the rheological properties of probiotic yoghurts supplemented with milk proteins
Ana Paula Marafon a, Adriana Sumi a, Maria Regina Alcntara b, Adnan Y. Tamime c, Maric Nogueira de Oliveira a, *
a b c

Faculty of Pharmaceutical Sciences, So Paulo University, Av Prof Lineu Prestes, 580, Bloco 16, 05508-900 So Paulo, Brazil Chemistry Institute, So Paulo University, Av Professor Lineu Prestes, 580, Bloco 3, 05508-900 So Paulo, Brazil 24 Queens Terrace, Ayr KA7 1DX, Scotland, United Kingdom

a r t i c l e i n f o
Article history: Received 27 November 2009 Received in revised form 1 September 2010 Accepted 3 September 2010 Keywords: Yoghurt Probiotic Acidication Rheology

a b s t r a c t
This study aimed to optimize the rheological properties of probiotic yoghurts supplemented with skimmed milk powder (SMP), whey protein concentrate (WPC) and sodium caseinate (Na-Cn) by using an experimental design type simplex-centroid for mixture modeling. It included seven batches/trials: three were supplemented with each type of the dairy protein used, three corresponding to the binary mixtures and one to the ternary one in order to increase protein concentration in 1 g 100 g1 of nal product. A control experiment was prepared without supplementing the milk base. Processed milk bases were fermented at 42  C until pH 4.5 by using a starter culture blend that consisted of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Bidobacterium animalis subsp. lactis. The kinetics of acidication was followed during the fermentation period as well the physico-chemical analyses, enumeration of viable bacteria and rheological characteristics of the yoghurts. Models were adjusted to the results (kinetic responses, counts of viable bacteria and rheological parameters) through three regression models (linear, quadratic and cubic special) applied to mixtures. The results showed that the addition of milk proteins affected slightly acidication prole and counts of S. thermophilus and B. animalis subsp. lactis, but it was signicant for L. delbrueckii subsp. bulgaricus. Partially-replacing SMP (45 g/100 g) with WPC or Na-Cn simultaneously enhanced the rheological properties of probiotic yoghurts taking into account the kinetics of acidication and enumeration of viable bacteria. 2010 Elsevier Ltd. All rights reserved.

1. Introduction The manufacture of probiotic yoghurt involves milk supplementation with dairy ingredients in order to increase protein concentration to 40e50 g of protein kg1 (Sodini, Montella, & Tong, 2005). Afterwards, the fortied milk is homogenized (17 MPa at 60  C), heated (90  C for 10 min), cooled to fermentation temperature (42  C) and inoculated with yoghurt starter culture (i.e. consisting of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus) and probiotic bacteria. Two types of yoghurt could be produced e stirred- or set-type, which differ according to the characteristic of the gel being broken or not, respectively. Gel formation is one of the main properties in yoghurt manufacture. The rheological properties of the gel are affected by milk composition, time and temperature of heat treatment, type and quantity of culture, fermentation temperature, gel breaking or not, and
* Corresponding author. E-mail address: monolive@usp.br (M. Nogueira de Oliveira). 0023-6438/$ e see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.09.005

storage conditions until the end of the products shelf-life (Kckcetin, 2008; Lucey & Singh, 1998; Xu, Emmanouelidou, Raphaelides, & Antoniou, 2008). Certain approved strains of Lactobacillus acidophilus and bidobacteria are the most widely used bacteria during the manufacture of probiotic yoghurts. It is well documented that probiotic bacteria grow slowly in milk because they are devoid of proteolytic enzymes, and contribute poor sensory and rheological characteristics to the product; hence, the practice is to blend these organisms with yoghurt starter culture (Damin, Minowa, Alcantara, & Oliveira, 2008; Lucas, Sodini, Monnet, Jolivet, & Corrieu, 2004; Oliveira, Sodini, Remeuf, & Corrieu, 2001). Moreover, the cultures must remain viable during the storage, distribution and retailing of the product to assure the demonstrated health benets (Bajaj et al., 2008; Donkor, Henriksson, Vasiljevic, & Shah, 2006; Fukuda et al., 2008; Lourens-Hattingh & Viljoen, 2001; Oliveira, Sodini, Remeuf, Tissier, & Corrieu, 2002; Sachdeva & Nagpal, 2009; Sanders, 2008; Sarkar, 2008; Tamime, Saarela, Sondergard, Mistry, & Shah, 2005; Uyeno, Sekiguchi, & Kamagata, 2008).

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Milk solids supplementation is a good practice to improve probiotic growth during the fermentation period and favor bacterial viability in the product (Dave & Shah, 1997; Sodini, Lucas, Tissier, & Corrieu, 2005). The addition of proteins, peptides and amino acids is a better choice to fortify the milk base, but the published studies are not conclusive on the amounts of these substances to be used. As a consequence, different substances of milk supplementation have been studied by researchers aiming to reduce the fermentation time and improve the sensory characteristics of the product (Lucas et al., 2004; McComas & Gilliland, 2003; Oliveira et al., 2001). Traditionally, skimmed milk powder (SMP) is used to fortify the milk solids before fermentation. However, the availability and quality of other dairy ingredients may provide a cost-effective alternative to SMP. Among these ingredients are whey protein concentrates (WPC) (protein contents varied from 340 to 800 g kg1), and caseinates (casein salts of sodium and ammonium) that improve the texture and functional properties of the product (Sverin & Wenshui, 2005; Vasiljevic & Shah, 2008). The effect of replacement of SMP by WPC or caseinate on textural and physical properties of yoghurts has been studied by many researchers (Akalin, Gonc, Unal, & Fenderya, 2007; Bhullar, Uddin, & Shah, 2002; Damin et al., 2008; Oliveira et al., 2001). Some authors have reported not conclusive results (Saint-Eve et al., 2006; Sodini, Lucas, et al., 2005; Sodini, Montella, et al., 2005), whilst few studies reported their simultaneous effects in the manufacture of probiotic yoghurt (Kaur, Mishra, & Kumar, 2009; Kudelka, 2008; Torriani, Gardini, Guerzoni, & Dellaglio, 1996). In addition, studies using more than one ingredient simultaneously in the milk base will ultimately reduce the individual amount of each ingredient and, when appropriate statistical tools are employed, it is possible to optimize these quantities. Therefore, the objectives of the study were: (i) to analyze the optimum concentrations of SMP, WPC and sodium caseinate (Na-Cn) to be used to enhance the rheological properties of probiotic yoghurts, and (ii) to characterize the rheological properties of the products taking into account the kinetics of acidication and enumeration of viable bacteria in the product 24 h after storage of the product at 4  C. 2. Material and methods 2.1. Milk and milk proteins Three ingredients were used to increase in 1 g the protein contents of the yoghurt products: (i) skimmed milk powder (SMP,

Cooperativa Taquarense de Laticnios Ltda, Taquara, Brazil), (ii) whey protein concentrate (WPC, Lacprodan-35, Aria Food Ingredients, Viby, Germany) and (iii) sodium caseinate (Na-Cn, EM 7, DMV International, Veghel, Netherlands). WPC (WPC35), Na-Cn and SMP contained 36.6 g protein 100 g1, 86.5 g protein 100 g1 and 32.3 g protein 100 g1, respectively, as determined by micro Kjeldahl analysis (AOAC, 1995, p. 1141). 2.2. Experimental design and statistical analysis A simplex-centroid design was applied to model the kinetics of acidication of milk by yoghurt and probiotic bacteria, the physicochemical and rheological characteristics of the products supplemented with three different types of milk proteins [WPC (x1), Na-Cn (x2) and SMP (x3)]. Residual analysis, the coefcient of determination (adjusted R2), the signicance of the models and the lack of t were used to check the quality of the model. The results from all parameters were evaluated to check the homogeneity of the variances (Hartley Fmax) and submitted to one-way analysis of variance (ANOVA). The results were also expressed as mean SD. The T-test for dependent samples was used to compare the data of the milk proteins mixture. The P value of 0.05 was xed, and calculations were performed using the Statistica version 8.0 software (Statsoft Inc., Tulsa, USA). The results presented are the averages of two complete and independent experiments. 2.3. Lactic starter cultures Three strains of pure commercial starter cultures for direct inoculation of the processed milk were used for the manufacture of yoghurt products: (i) S. thermophilus (S.), (ii) L. delbrueckii subsp. bulgaricus (L.) (Delvo-Yog, CY-340 DSL, DSM, Heerlen, Netherlands), and (iii) Bidobacterium lactis subsp. animalis (B.) (B94 LAFTI, DSM, Heerlen, Netherlands). The former two organisms are the classical yoghurt starter culture, whilst the bidobacteria is a probiotic one. 2.4. Yoghurt manufacture 2.4.1. Milk base preparation Control milk base was prepared as follows: 12 g of SMP was added to 88 g of potable water at 25  C. Experimental trials were prepared mixing 12 g of SMP which was later supplemented with

Table 1 Chemical composition* of the control yoghurt and experimental products prepared with different proportions of whey protein concentrate (WPC), sodium caseinate (Na-Cn) and skimmed milk powder (SMP) either singly, in pairs or in a mixture. Milk proteins mixture Ingredients proportion in the mixture Amounts of added protein (g protein) WPC (x1) Control WPC Na-Cn SMP WPC Na-Cn WPC SMP Na-Cn SMP WPC Na-Cn SMP e 1.00 0.00 0.00 0.50 0.50 0.00 0.33 Na-Cn (x2) e 0.00 1.00 0.00 0.50 0.00 0.50 0.33 SMP (x3) e 0.00 0.00 1.00 0.00 0.50 0.50 0.33 Ingredient amounts (g ingredient.100 g1 milk base) WPC (x1) 0.00 2.75 0.00 0.00 1.37 1.37 0.00 0.91 Na-Cn (x2) 0.00 0.00 1.16 0.00 0.58 0.00 0.58 0.39 SMP (x3) 0.00 0.00 0.00 3.10 0.00 1.55 1.55 1.03 3.98 4.90 4.93 4.92 4.96 4.98 5.01 4.96 0.00a 0.01a 0.07a 0.07a 0.05a 0.04a 0.04a 0.07a 11.04 13.57 12.14 13.89 12.85 13.73 13.02 13.20 0.09a 0.05a 0.06a 0.04a 0.04a 0.05a 0.03a 0.05a 4.45 4.47 4.52 4.51 4.52 4.53 4.53 4.53 0.01a 0.01a 0.01b 0.00b 0.01b 0.01b 0.01b 0.01b 0.35 0.36 0.41 0.35 0.39 0.36 0.38 0.38 0.00a 0.00a 0.00a 0.00a 0.04a 0.00a 0.02a 0.02a 0.94 1.08 1.07 1.09 1.08 1.12 1.13 1.15 0.05a 0.01bc 0.01b 0.01bc 0.00bc 0.01bc 0.01bc 0.00c Chemical composition Proteina (g 100 g1) Total solidsa (g 100 g1) pHb Protein/Total solids Titrable acidityb (g lactic acid)

*Means (n 5) standard deviation with different letters in the same column are signicantly different (P The protein contents (g.100 g1) of WPC35, Na-Cn, and SMP were 36.6, 86.5 and 32.3, respectively. a Before fermentation. b After fermentation.

0.01).

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the amounts of milk protein ingredients either singly, in pairs or in a mixture as shown in Table 1, and potable water was added at 25  C in order to obtain 100 g of milk base. After reconstitution and fortication, the milk bases were preheated to 55  C in a plate heat exchanger (PHE) (Alfa Laval, type A3HRB-Lund, Sweden), homogenized at 15 MPa in double-stage equipment (i.e. 1st stage at 10 MPa and 2nd stage at 5 MPa) (Treu, Rio de Janeiro, Brazil). Subsequently, all the milk bases were heated to 95  C for 5 min in closed circuit, and then cooled to 10  C in the same PHE. 2.4.2. Starter and probiotic cultures preparation and fermentation Two inocula were separately prepared: (i) S. thermophilus and L. delbrueckii subsp. bulgaricus or classical yoghurt starter culture: 10 g of Delvo-Yog CY-340 DSL was weighed and rehydrated in 150 mL sterilized milk (121  C for 10 min); (ii) Bidobacterium animalis subsp. lactis or probiotic culture: 10 g of B94 LAFTI was weighed and rehydrated in 500 mL sterilized milk (121  C for 10 min). Both were tempered to 42  C for 15 min before inoculation. Yoghurt (0.2 mL) and probiotic (2.5 mL) cultures were added to 250 mL milk, and subsequently incubated at 42  C in a thermostatically controlled water bath until the pH reaches 4.5. The rate of acidication of each trial was monitored by using the Cinac (Cintique dacidication) system (Ysebaert Frpillon, France). After reaching pH 4.5, each fermentate was agitated manually using a stainless steel plunger (i.e. consisting of a rod and perforated disc) that was moved upwards and downwards for 60 s, dispensed into 50 mL polypropylene cups (heat sealed using Selopar equipment e BrasHolanda, Pinhais, Brazil), quickly cooled in an ice bath and stored at 4  C until required for analysis. The samples were prepared in duplicates, and the experiment was replicated twice on different days. Five kinetic parameters were considered: (a) Vmax (maximum acidication rate, measured in pH units per min (upH min1)), (b) tVmax (time in h to reach the maximum acidication rate), and (c) pH corresponding to Vmax, and (d) tpH 5.0 and tpH 4.5 (time in h to reach pH 5.0 and 4.5, respectively). 2.5. Physico-chemical determinations Protein and total solids contents of the milk bases were determined with an ultrasonic milk analyzer Ekomilk (Eon Trading, Bulgaria), and replicated ve times (Venturoso, Almeida, Rodrigues, Damin, & Oliveira, 2007). The pH of the yoghurts were determined with a pH meter model Q-400M1 (Quimis, Brazil), and the titratable acidity expressed as g lactic acid 100 g1 according to the method of Instituto Adolfo Lutz (Lutz, 1985). Analyses were performed in duplicate after storing the product for 24 h at 4  C. 2.6. Counts of yoghurt cultures and probiotic bacteria Enumeration of the yoghurt and probiotic microorganisms was made in duplicate after 24 h of production. Each sample (1.0 mL) was added to 9.0 mL sterile peptone water (0.1 g 100 mL1), and then appropriate dilutions were made. Subsequently, S. thermophilus and L. delbrueckii subsp. bulgaricus were plated into M17 agar and MRS agar (Oxoid Ltd., Basingstoke, UK), respectively; the latter agar was previously acidied to pH 5.4 with acetic acid, and afterwards the plates were incubated at 37  C for 48 h. B. animalis subsp. lactis was enumerated in RCA agar plus 1 mL mL1 dicloxacillin (pH 7.1) and 0.3 g 100 g1 of aniline, and incubation under anaerobic at 37  C for 72 h. Anaerobic condition was achieved by using AnaeroGen (Oxoid Ltd., Basingstoke, UK). The enumeration of the microbial counts was performed on plates containing 30e300

colonies, and the counts were expressed as log10 cfu mL1 of yoghurt. Microscopic examination of the cells in each bidobacteria colony was conrmed using a light microscope. 2.7. Rheological properties The yoghurts packed in cups and stored at 4  C were gently stirred 5 times in clockwise direction prior to rheological analysis. The viscoelastic properties were determined by small amplitude oscillatory measurement (SAOM) using a controlled stress rate rheometer (Physica MCR 300, Anton Paar, GmbH, Germany) equipped with cone-plate geometry (CP25-1, 12.5 mm diameter, 1 angle and 0.05 mm gap, Anton Paar, GmbH, Germany). The temperature of the rheometer was controlled at 26 1  C by a viscotherm VT2 circulating bath and a Peltier system (Anton Paar, GmbH, Germany). Yoghurt sample was loaded on the inset plate and 5 min were allowed for the sample temperature to equilibrate to 26  C prior SAOM was performed. Subsequently, each sample was subjected to a shear stress sweep test (shear stress ramp from 1.0 to 50 Pa) at a constant frequency of 1 Hz to ascertain the storage modulus (G0 ), loss modulus (G00 ) and damping factors (tan d). The complex viscosity (h) expressed in Pa s was calculated at 1 Pa, which was in the range of linear viscoelastic behavior. The limit of linearity as the end point of the linear region was considered the point at which G0 deviated more than 5% from its maximum value (LVR). Gamma or fracture strain e the value of the strain at which point the network starts to breakdown (Lucey, 2001) was also determined. For all test samples, six replicates were performed, and a fresh sample was used for each replicate. 3. Results and discussion 3.1. Chemical composition The chemical composition of the control (not supplemented with milk proteins) and the experimental milk bases (i.e. fortied with WPC, Na-Cn or SMP in different proportions e either singly, in pairs or in a mixture) is shown in Table 1. On average, the total solids and protein contents were 12.93 0.26 g 100 g1 and 4.83 0.04 g 100 g1, respectively, with no signicant differences between the trials (P 0.01). The pH level and titratable acidity content of the experimental milk bases before fermentation averaged 6.57 0.28 and 0.26 0.03 g lactic acid, respectively (Table 1). No signicant differences were observed in pH of all the milk bases including the control before fermentation (P 0.05). The relationship between total solids and protein contents averaged 0.37 0.01 with no signicant differences between the trials (P 0.01). According to Fox (2001), Tamime, Robinson, and Latrille (2001) the fortication of milk base to 5 g protein 100 g1 improved the rheological properties of the yoghurt. It is very difcult to study separately the effect of protein or total solids contents, as these two variables are not modied independently of the formulations composition. By increasing the total solids content, the texture of the yoghurt improves the sensory proling and instrumental measurements of the product, even if the total solids content is increased by the addition of sucrose or other stabilizing agents. The levels of total solids content in yoghurt were studied by (Tamime & Robinson, 2007) and they reported that the consistence of yoghurt was improved by increasing the total solids content of the milk base from 12 to 20 g 100 g1. The main effect was observed from 12 to 14 g 100 g1; however, levels higher than 16 g 100 g1 resulted in lower changes. Prentice (1992) reported that the increase in protein is the principal factor that affects texture and, fortifying the milk

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Table 2 Kinetic acidication parameters* of the control milk, and milk supplemented with different proportions of whey protein concentrate (WPC), sodium caseinate (Na-Cn) and skimmed milk powder (SMP) either singly, in pairs or in a mixture fermented at 42  C until pH4.5. Type of yoghurt Amounts of added protein (g protein) WPC (x1) Control WPC Na-Cn SMP WPC Na-Cn WPC SMP Na-Cn SMP WPC Na-Cn SMP 0.00 1.00 0.00 0.00 0.50 0.50 0.00 0.33 Na-Cn (x2) 0.00 0.00 1.00 0.00 0.50 0.00 0.50 0.33 SMP (x3) 0.00 0.00 0.00 1.00 0.00 0.50 0.50 0.33 Kinetic acidication parameters Vmax. (0.103upH.min1) 24.3 25.6 23.8 22.2 22.5 22.0 23.1 24.7 0.0d 0.0e 0.0cd 0.0ab 0.0ab 0.0a 0.0bc 0.0de Tvmax. (h) 2.40 2.60 2.58 2.69 2.60 2.75 2.67 2.33 0.00b 0.00c 0.04c 0.03de 0.00c 0.04e 0.00d 0.00a pHVmax 5.60 5.61 5.54 5.48 5.53 5.48 5.50 5.69 0.04ab 0.05ab 0.03ab 0.06a 0.05a 0.07a 0.06a 0.05b tpH
5.0

(h) 0.00a 0.03bc 0.00b 0.00bc 0.04b 0.00c 0.06bc 0.04a

tpH4.5 (h) 5.87 6.38 6.42 6.87 6.78 6.09 6.20 6.00 0.13a 0.43a 0.41a 0.46a 0.39a 0.38a 0.41a 0.35a

2.47 2.69 2.60 2.67 2.62 2.73 2.67 2.45

*Means (n 6) standard deviation with different letters in the same column are signicantly different (P 0.05). Vmax: maximum acidication rate; Tvmax: time to reach the maximum acidication rate; pHVmax: pH corresponding to Vmax; tpH5.0and tpH4.5: time in h to reach pH 5.0 and 4.5, respectively.

base with SMP, results in development of casein network and micelles. In addition, the type and level of protein content affects the texture and rheological characteristics of the product (Penna, Converti, & de Oliveira, 2006; Puvanenthiran, Williams, & Augustin, 2002; Sodini, Lucas, et al., 2005). Finally, the total solids affected the viability of Lactobacillus bulgaricus and L. acidophilus in milk and milkewhey mixture as reported by (Almeida, Tamime, & Oliveira, 2009). After fermentation, the pH of yoghurts ranged between 4.45 and 4.53 with slightly signicant statistical differences (P 0.05). However, the level of titratable acidity (g 100 g1) in all the yoghurts after 24 h storage at 4  C may be grouped as follows: (i) 0.94 e least in the control product, (ii) 1.08 e intermediate in WPC, Na-Cn, SMP and WPC Na-Cn yoghurts, and (iii) range between 1.12 and 1.15 e highest in WPC SMP, Na-Cn SMP and WPC NaCn SMP yoghurts. It is evident that the titratable acidity content in the yoghurts was inuenced by the type of protein used to fortify the total solids content of the milk base (Table 1). 3.2. Fermentation kinetics Kinetic parameters (Vmax., T Vmax, pHVmax, tpH 5.5 and tpH 4.5) of milk bases supplemented with WPC, Na-Cn and SMP and fermented at 42  C until pH 4.5 are show in Table 2. Maximum acidication rate varied from 22.0 103 upH.min (WPC SMP) to 25.6 103 upH.min (WPC), time to reach the maximum acidication rate was 2.33 h (Na-Cn SMP) or 2.75 h (WPC SMP), and pH corresponding to Vmax was on average 5.55. Signicant statistical differences were observed between the different trials for all kinetic parameters (P 0.05) except for fermentation time to reach pH 4.5. Time to reach pH 4.5 ranged between 6.00 and 6.87 h, which was for all the milk bases supplemented with dairy proteins with

no signicant differences (P 0.05). On average, the control milk (no addition of dairy proteins) reached pH 4.5 in 5.87 h 0.13. These results conrm that milk supplementation with dairy proteins affects slightly fermentation time (average increase in 31.5 min) as described by many researchers (Martinz et al., tpH4.5 ofe 2003; Oliveira et al., 2001; Sodini, Remeuf, Haddad, & Corrieu, 2004). In contrast, milk fortied only with SMP showed the highest time to reach pH4.5 (6.87 h). 3.3. Counts of viable bacteria Counts of the yoghurt starter culture (S. thermophilus and L. delbrueckii subsp. bulgaricus) and B. animalis subsp. lactis before fermentation, i.e. after inoculation, were 9.30 0.37, 5.52 0.19 and 10.10 0.46 log10 cfu mL1, respectively. The lactobacilli counts were lower as they tend to post-acidify the yoghurt during the storage period, which reduces the viability of the probiotic bacteria; thus, it is recommended to use yoghurt starter culture devoid or with lower counts of this species (Oliveira et al., 2001). Bacterial counts in yoghurts prepared with milks supplemented with WPC, Na-Cn and SMP (i.e. either singly, in pairs or in a mixture) and fermented at 42  C until pH 4.5 are shown in Table 3. Counts of S. thermophilus ranged between 8.98 log10 cfu mL1 (WPC yoghurt) and 9.60 log10 cfu mL1 (WPC NA-CN SMP yoghurt), and slightly signicant differences between the trials (P 0.05) were found. In contrast, the counts of streptococci in the control yoghurt averaged 9.29 0.10 log10 cfu mL1, which were similar to the counts in the experimental products. However, the counts of L. delbrueckii subsp. bulgaricus ranged between 3.86 log10 cfu mL1 (WPC yoghurt) and 9.11 log10 cfu mL1 (SMP yoghurt), and there were signicant statistical differences (P 0.05) (Table 3), whilst the count averaged 6.06 0.33 log10 cfu mL1 in the control yoghurt. In addition, good growth of L. delbrueckii subsp. bulgaricus was

Table 3 Counts* of viable bacteria in the control yoghurt and experimental products prepared with whey protein concentrate (WPC), sodium caseinate (Na-Cn) or skimmed milk powder (SMP) either singly, in pairs or in a mixture after 24 h of fermentation. Type of yoghurt Amounts of added protein (g protein) WPC (x1) Control WPC Na-Cn SMP WPC Na-Cn WPC SMP Na-Cn SMP WPC Na-Cn SMP 0.00 1.00 0.00 0.00 0.50 0.50 0.00 0.33 Na-Cn (x2) 0.00 0.00 1.00 0.00 0.50 0.00 0.50 0.33 SMP (x3) 0.00 0.00 0.00 1.00 0.00 0.50 0.50 0.33 Counts (log10 cfu mL1) S. thermophilus 9.29 8.98 9.31 9.11 9.35 9.23 9.00 9.60 0.10abc 0.30a 0.04abc 0.10ab 0.10bc 0.02ab 0.00ab 0.00c L. delbruckii subsp. bulgaricus 6.06 3.86 5.05 9.11 5.00 5.00 5.00 5.66 0.33c 0.21a 0.13b 0.10d 0.00b 0.00b 0.00b 0.12c B. animalis subsp. lactis 6.89 7.00 7.15 6.91 6.98 7.15 7.40 7.80 0.03a 0.07a 0.05ab 0.17a 0.33a 0.10ab 0.60ab 0.05b

*Means (n 4) standard deviation with different letters in the same column are signicantly different (P

0.05).

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3.4. Rheology The mechanical spectra obtained within the linear viscoelastic zone for the various yoghurts are shown in Fig. 1. In general, the range of shear stress studied shows that the form of the mechanical spectra was typical of weak gels, and they could be grouped as follows: (i) SMP yoghurts had strong gels, (ii) WPC SMP and NaCn SMP yoghurts had weak gels, and (iii) the rest of yoghurts had intermediate gels. The fortication of the milk base with dairy proteins (SMP, Na-Cn, WPC Na-Cn, WPC Na-Cn SMP and WPC yoghurts) resulted in an increase in the value of both modulus (G0 and G00 ) when compared with the control yoghurt. In contrast, both modulus decreased when SMP was substituted in equal parts with WPC and Na-Cn (i.e. WPC SMP and Na-Cn SMP yoghurts) in the milk bases (Fig. 1). Similar results were reported by (Sanz, Salvador, & Jimnez, 2008), and where the milk base was fortied with functional asparagus ber. The storage and loss modulus (Pa), damping factors (at shear stress of 5.24 Pa), complex viscosities, LVR and gamma of the control yoghurt and experimental products prepared with the addition of milk proteins as obtained from their shear stress sweeps are shown in Table 4. In general, the loss modulus (G00 ) was lower than the storage modulus (G0 ) for all the yoghurts irrespective of the milk protein added to the milk base. The lower G00 values indicate that the yoghurt gels containing added protein had solidlike character, as was also observed by (Purwandari, Shah, & Vasiljevic, 2007). However, the damping factor (tan d) was similar (P ! 0.05) for all the experimental yoghurts except for the WPC SMP and Na-Cn products. Damping factor means for the yoghurts fortied with any type of dairy proteins were lower than the WPC SMP and Na-Cn products, which may indicate that the rearrangement of yoghurt structures occurred to a more solid-like gel (Doleyres, Schaub, & Lacroix, 2005). Lucey, Tamehana, Singh, and Munro (1998), suggested that extensive particle rearrangement during structure formation results in dense clusters of aggregates and lower G0 values. Similar observations regarding G00 , G0 and tan d have been reported by Hess, Roberts, and Ziegler (1997) and Purwandari et al. (2007). Complex viscosity varied between 2.29 and 28.17 Pa s for the yoghurts prepared with added milk proteins, whilst for the viscosity of the control yoghurt was 17.74 Pa s. In contrast, LVR measurements ranged between 2.37 and 3.14 Pa for the experimental yoghurts, but the control product was lower (1.85 Pa). Gamma measurement of the control yoghurt was 2.36%, whilst the protein fortied products ranged between 0.92% (e.g. WPC Na-Cn or WPC SMP) and 2.15% (milk supplemented with SMP) (Table 4). According to (Lucey, 2001), low values of gamma indicate a brittle or short texture.

Fig. 1. Mechanical spectra of control yoghurts and experimental products prepared with whey protein concentrate (WPC), sodium caseinate (Na-Cn) and skimmed milk protein (SMP) either singly, in pairs or in a mixture at 26  C. Storage modulus, G0 ; Control, WPC, (b) Loss modulus, G00 . Points are average of six replicates. Na-Cn, SMP, WPCNa-Cn, WPCSMP, Na-CnSMP, WPC Na-CnSMP.

observed in milk supplemented with SMP (i.e. an increase ofe 3.58 log cycles), but after higher fermentation time (see Table 2). The count of B. animalis subsp. lactis averaged 7.16 log10 cfu mL1 (i.e. ranged between 6.89 log10 cfu mL1 (control yoghurts)) and 7.80 log10 cfu mL1 (WPC SC SMP yoghurts) where there was a slight signicant difference (Table 3). These counts agree with the legislation requirements for probiotic products, but could be considered low for the rst day of the shelf-life of the yoghurts; however, it is safe to consider higher inoculation rate of the bidobacteria to achieve higher counts in probiotic yoghurts.

Table 4 Viscoelastic properties* (at 5.24 Pa shear stress) of the control yoghurts and experimental products prepared with whey protein concentrate (WPC), sodium caseinate (Na-Cn) and skimmed milk powder (SMP) either singly, in pairs or in a mixture fermented at 42  C until pH4.5. Milk proteins mixture Ingredients proportion in the mixture WPC (x1) Control WPC Na-Cn SMP WPC Na-Cn WPC SMP Na-Cn SMP WPC Na-Cn SMP 0.00 1.00 0.00 0.00 0.50 0.50 0.00 0.33 Na-Cn (x2) 0.00 0.00 1.00 0.00 0.50 0.00 0.50 0.33 SMP (x3) 0.00 0.00 0.00 1.00 0.00 0.50 0.50 0.33 Viscoelastic properties G0 (Pa) 90.97 0.00 109.38 0.00ab 121.95 10.98d 168.67 18.33cd 142.70 12.43ab 24.20 5.21a 10.47 2.90c 114.85 20.69b
ab

G00 (Pa) 35.07 0.00ab 37.33 0.00ab 40.82 2.65d 53.68 4.62cd 44.27 3.32ab 14.96 2.09a 9.80 1.46c 39.95 3.89b

Damping factor 0.43 0.34 0.34 0.32 0.32 0.62 1.00 0.35 0.01b 0.01ab 0.02ab 0.01ab 0.01a 0.05c 0.09d 0.00ab

Complex viscosity (Pa s) 17.74 0.00ab 23.40 3.33bc 22.40 2.21d 28.17 3.05cd 23.80 2.06a 4.87 0.61a 2.29 0.50bc 19.37 3.28ab

LVR (Pa) 1.85 2.78 2.78 2.82 3.14 2.59 2.37 2.51 0.03a 0.05bc 0.03bc 0.03bc 0.12c 0.08b 0.13ab 0.09b

Gamma (%) 2.36 1.50 1.60 2.15 0.92 0.93 1.00 1.59 0.18c 0.07b 0.11b 0.06c 0.06a 0.06a 0.05a 0.11b

*Means (n 6) standard deviation with different letters in the same column are signicantly different (P value of the strain at which point the network starts to breakdown.

0.05). Damping factor: G0 /G00 ; LVR: limit of linearity; Gamma:

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The texture of yoghurt is inuenced by many factors, such as the level of fortication of the solids of the milk base, temperature of heat treatment applied, homogenization pressure used, strains of starter culture used and by the shearing (stirring) of the gel after fermentation (Sodini et al., 2004). Moreover, according to Renan et al. (2009), the pH on stirring of the gel appeared to be a key factor for the recovery of the structure, probably through electrostatic interactions either as repulsive or building attractive. Martinz et al. (2003) reported that the importance of added dairy protein to the milk base, especially those from whey, had different inuences on the gel structure. They observed that yoghurts enriched with WPC, milk protein concentrate (MPC) and SMP had different behaviors during fermentation, and the gel formed was different in terms of viscosity after breaking the gel and variation of syneresis level, which was due to different characteristics of protein source. Yoghurts enriched with MPC or SMP had higher viscosity and syneresis compared to those supplemented with WPC. Furthermore Sodini et al. (2004) reported that when the milk base was supplemented with proteins obtained from ultraltration (UF), WPC or caseinate, the rmness of the yoghurts increased compared to a product fortied with SMP; this was due to the increase of the protein content in the relation to total solids level. Finally, Damin, Alcantara, Nunes, and Oliveira (2009) showed that yoghurt made with milk supplemented with sodium caseinate resulted in signicant properties changes, which were decrease in fermentation time, and increase in yield stress, storage modulus, and rmness, with increases in supplement level. 3.5. Estimate of the polynomial predictive models The effect of each component of the milk base formulation can be observed by the magnitude, signicance (standard error) and signal (or ) associated with the respective coefcient in the tted model for each response. The value and signal of the linear coefcients obtained for each response showed that all components contributed to increasing the kinetic parameters (positive b1, b2 and b3 for tVmax and tpH5.5), as well as the counts of viable bacteria [positive b1, b2 and b3 for the counts of streptococci (S.), lactobacilli (L.) and bidobacteria (B.)] and viscoelastic properties (positive b1, b2 and b3 for G0 , G00 , complex viscosity and LVR) are shown in Table 5. The linear coefcients of polynomial model for Vmax showed that all interactions were counteracted. This means that the addition of WPC, Na-Cn or SMP had positive effect, i.e. by decreasing the maximum acidication rates, but had negative effects on other kinetic parameters, such as time to reach maximum acidication rate and time to reach pH 5.5.
Table 5 Coefcients of the polynomial models for the response variables. Factors Kinetic parameters Vmax. WPC (b1) Na-Cn (b2) SMP (b3) WPC Na-Cn (b12) WPC SMP (b13) Na-Cn SMP (b23) WPC Na-Cn SMP (b123) P (model) Adjusted R2 0.026 0.024 0.022 0.009 0.008 ns 0.071 <0.01 0.946 tvmax 2.60 2.58 2.69 ns 0.43 ns 0.51 <0.01 0.974 tph5.0 2.69 2.60 2.67 ns ns ns 6.30 <0.01 0.898

Although the protein contents of the experimental yoghurts were similar (Table 1), the acidication rates/proles were different (Table 2). Analyzing the binary coefcients of the models (b12, b13 and b23), the yoghurts fortied with SMP or Na-Cn contributed to increasing Vmax, counts of S. thermophilus and L. delbrueckii subsp. bulgaricus and viscoelastic properties. Furthermore, no signicant effects were observed when SMP Na-Cn were added to the milk base (P 0.05) for tVmax and tpH5.5 and counts of B. animalis subsp. lactis. However, when using WPC as a protein substitute of SMP, binary coefcients of polynomial model (b13) showed that these components contributed to increasing Vmax. In contrast, the interactions regarding the counts of L. delbrueckii subsp. bulgaricus and viscoelastic properties were antagonists i.e. they contributed in decreasing the counts of lactobacilli and viscoelastic properties. No signicant interactions were found (b13) in kinetic parameters and in tpH5.5, and counts of S. thermophilus and B. animalis subsp. lactis. Different effects were observed when SMP was replaced in equal proportion with Na-Cn, no signicant interactions were found (b23) regarding the kinetic parameters and counts of B. animalis subsp. lactis. However, the binary coefcients of polynomial model of S. thermophilus and L. delbrueckii subsp. bulgaricus counts and viscoelastic properties showed that all interactions were antagonists. Signicant interactions of the three milk proteins used on kinetic parameters were observed (b123), for example, affecting negatively the tVmax (augmenting it) and positively Vmax and tpH5.5 (decreasing it). Similar positive effects were observed for the counts of viable bacteria when the milk base was fortied with WPC Na Cn SMP, which increased the counts especially for L. delbrueckii subsp. bulgaricus (due to higher b123). Ternary coefcients of polynomial models for viscoelastic properties showed that all interactions were signicant for G0 , G00 and complex viscosity, but antagonist for LVR. Contour curves obtained for kinetics responses (Fig. 2) reinforce the signicant inuence observed of dairy proteins addition to the milk bases on maximum acidication rate, time to reach maximum acidication rate and time to reach pH 5.0. It can be observed that in the center of the triangular diagrams there is the optimum mixture compositions of the three dairy proteins used that increased the kinetic parameters at the beginning of the fermentation process. Any deviation of this point will allow a mixture composition that will result in decreasing the fermentation kinetic of the fortied milk bases by S. thermophilus, L. delbrueckii subsp. bulgaricus and B. animalis subsp. lactis.

Counts of viable bacteria S. 8.98 9.31 9.10 0.79 ns 0.84 10.59 <0.01 0.790 L. 3.86 5.05 9.11 2.17 5.95 8.31 26.95 0.01 0.997 B. 7.00 7.15 6.91 Ns Ns Ns 15.48 0.01 0.631 and tpH
4.5:

Viscoelastic properties G0 109.38 121.95 168.67 108.14 459.29 539.34 2172.46 <0.01 0.967 G00 37.33 40.82 53.68 20.77 122.19 149.80 645.82 <0.01 0.974 Complex viscosity 23.40 22.40 28.17 ns 83.66 91.67 373.27 <0.01 0.957 LVR 2.78 2.78 2.82 1.43 0.85 1.71 4.29 <0.01 0.921

WPC: whey protein concentrate; Na-Cn: sodium caseinate; SMP: skimmed milk powder. Vmax: maximum acidication rate; tVmax:time to reach the maximum acidication rate; tpH S: S. thermophilus; L: L. delbrueckii subsp. bulgaricus; B: B. animalis subsp. lactis. G0 : storage modulus; G00 : loss modulus; LVR: limit viscoelastic region. Ns: no signicant.

5.0

time in hours to reach pH 5.0 and 4.5, respectively.

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517

Fig. 2. Contour plots showing the effects of whey protein concentrate (WPC), sodium caseinate (SC) and skimmed milk powder (SMP) on: (a) maximum acidication rate (vmax, upH min1), (b) time to reach the maximum acidication rate (tvmax, h) and (c) time to reach pH5.0 (tpH5.0, h). Each line corresponds to a given response value.

Fig. 3. Contour plots showing the effects of whey protein concentrate (WPC), sodium caseinate (Na-Cn) and skimmed milk powder (SMP) on counts (log10 cfu mL1) of: (a) S. thermophilus, (b) L. delbreuckii subsp. bulgaricus and (c) B. animalis subsp. lactis. Each line corresponds to a given response value.

Contour curves obtained for counts of viable bacteria responses (Fig. 3) indicate that the counts were affected by the addition of WPC, Na-Cn or SMP to the milk bases. In Fig. 3a, the counts of S. thermophilus were higher than 9.3 log10 cfu mL1, irrespective of the composition of the milk protein mixture. While counts of L. delbrueckii subsp. bulgaricus were higher when substituting SMP by WPC during the fortication of the milk base (Fig. 3b). In contrast, the counts of B. animalis subsp. lactis remained the same ( e 7.3 log10 cfu mL1) with a little displacement in the direction when Na-Cn was used to fortify the milk base (Fig. 3c).

Fig. 4 shows the inuence of milk proteins on viscoelastic properties of probiotic yoghurt. G0 , G00 and h showed an optimum when a mixture of all the proteins was used (WPC NaCn SMP) x1 0.19, x2 0.36 and x3 0.45 (Fig. 4a, b and c). In contrast, better LVR (i.e. lower limit viscoelastic region) was obtained in the region of x1 0.31, x2 0.41, and x3 0.28 (Fig. 4d). These results indicated that the optimal area obtained for viscoelastic properties, which corresponds to the percentage of ingredients x1 0.52 g 100 g1 (WPC), x2 0.42 g 100 g1 (NaCn) and x3 1.39 g 100 g1 (SMP), were satisfactory regarding the kinetic parameters, counts of viable bacteria and viscoelastic properties.

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Fig. 4. Contour plots showing the effects of whey protein concentrate (WPC), sodium caseinate (Na-Cn) and skimmed milk powder (SMP) on rheological properties of probiotic yoghurts. (a) storage modulus (G0 ); (b) loss modulus (G00 ); (c) complex viscosity; (d) limit of viscoelastic region (LVR). Each line corresponds to a given response value.

4. Conclusions The results of this study showed that fortication of the milk base with WPC, Na-Cn, or SMP to the same level of protein content affected the acidication prole, counts of viable bacteria and rheological properties of yoghurt. It was possible to estimate the acidication prole and counts of bacteria in milk supplemented with SMP, WPC or Na-Cn by response surface methodology as well as calculate approximately the viscoelastic properties of probiotic yoghurts. It is safe to conclude from these limited trials that optimum rheological properties of probiotic yoghurts were achieved when the milk bases were fortied with dairy proteins, but the type used is important. Finally, partially-replacing SMP (i.e. 45%) with WPC or Na-Cn simultaneously enhanced the rheological properties of probiotic yoghurts taking into account the kinetics of acidication and enumeration of viable bacteria; however, sensory proling studies in the future are required to conrm these results including industrial trials. Acknowledgements The authors wish to thank DSM Food Specialities for the donation of cultures, FAPESP (Process. 2007/03588-8) and CNPq for nancial support, and Ana Carolina R. Florence for gures improvement. References
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