Journal of Food Engineering 96 (2010) 97106

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Journal of Food Engineering

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Amaranth, quinoa and oat doughs: Mechanical and rheological behaviour, polymeric protein size distribution and extractability
C. Lamacchia a,b,*, S. Chillo a, S. Lamparelli b, N. Suriano b, E. La Notte a,b, M.A. Del Nobile a,b
a

Istituto per la Ricerca e le Applicazioni Biotecnologiche per la Sicurezza e la Valorizzazione dei Prodotti Tipici e di Qualit, Universit degli Studi di Foggia, Via Napoli, 25-71100 Foggia, Italy b Dipartimento di Scienze degli Alimenti, Universit degli Studi di Foggia, Via Napoli, 25- 71100 Foggia, Italy

a r t i c l e

i n f o

a b s t r a c t
The rheological characteristics, static and dynamic mechanical properties of amaranth, quinoa and oat doughs and the relative size distribution of their polymeric proteins were evaluated. For the sake of comparison, semolina dough rheological and mechanical properties and the relative size distribution of proteins were also determined. From rheological results it was inferred that the tenacity of amaranth, oat and quinoa dough samples was lower than that of semolina dough. The elastic modulus (Ec) of amaranth, oat and quinoa doughs was higher than that of semolina dough. Amaranth and quinoa G0 was found to be similar and signicantly higher (p < 0.05) with respect to that of oat dough at a moisture of 30%. The G00 of amaranth, quinoa and oat doughs showed different values. The highest G00 value was recorded for the amaranth dough while the lowest one was shown by oat. For semolina dough, the G0 and G00 values were signicantly lower than those of all the other dough samples. Moreover, at low and medium frequencies, tan d values of oat and quinoa doughs were statistically comparable and signicantly lower (p < 0.05) than that of amaranth and semolina doughs. At high frequencies, tan d values of investigated samples were different among them and the highest value was detected for amaranth, followed by semolina, quinoa and oat. Results of the size distribution of proteins in amaranth, quinoa, oat and semolina doughs were expressed as the proportion of unextractable polymeric protein (UPP). Unextractability of semolina dough proteins (61%) was greater with respect to the others, followed by amaranth (40.7%), oat (24%) and quinoa (10.1%). 2009 Elsevier Ltd. All rights reserved.

Article history: Received 10 September 2008 Received in revised form 1 July 2009 Accepted 3 July 2009 Available online 9 July 2009 Keywords: Mechanical properties Rheological properties Polymeric proteins Amaranth Quinoa Oat

1. Introduction Wheat our is the only cereal our that can form a threedimensional viscoelastic dough when mixed with water. This unique ability of wheat to suit the production of leavened and pasta products is due to the gluten, a cohesive, viscoelastic proteinaceous material prepared as a by-product of starch isolation from wheat our. The proteins that form gluten are storage proteins which consist of two major fractions: the monomeric gliadins and the polymeric glutenins (Schoeld, 1994). The latter are known to be the most important determinants of pasta and bread-making quality (DOvidio and Masci, 2004; Lindsay and Skerritt, 1999) and one group of these, of 36 proteins, is largely responsible for the elastic properties (Thatam et al., 2001). The high Mr subunits, in fact, possess the characteristics of a putative elastomer, with N- and

* Corresponding author. Address: Istituto per la Ricerca e le Applicazioni Biotecnologiche per la Sicurezza e la Valorizzazione dei Prodotti Tipici e di Qualit, Universit degli Studi di Foggia, Via Napoli, 25-71100 Foggia, Italy. Tel.: +39 0881 589 117; fax: +39 0881 740 211. E-mail address: c.lamacchia@unifg.it (C. Lamacchia). 0260-8774/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2009.07.001

C-terminal domains containing residues for covalent cross-linking and a central domain that can potentially undergo deformation (Shewry and Thatam, 1990; Belton, 1999). In terms of its nutritional value, gluten (or wheat proteins) is considered to be poorer than proteins from animal sources and can cause allergic reaction and intolerances (Gallagher et al., 2004). Amaranth, quinoa and oat have attracted many interest because of their high nutritional value and for the absence of gluten. In spite of this, the absence of gluten, in these ours, results in major problems for many pasta and bakery products. Their utilization as food ingredients in the production of pasta and bakery products depends largely on their functional properties, which are related to protein structural characteristics. Attempts to use proteins from alternative ours as a partial substitute in wheat products have generally been unsuccessful, because of the contrasting differences between proteins such as the water-solubility, differences in primary structure and their size distributions, accounted for viscoelastic properties that are unique to wheat gluten proteins. Lorimer et al. (1991) reported that the addition of non-gluten forming proteins (e.g. bean-seed proteins) causes a dilution effect and consequent weakening of wheat dough. They suggested several issues that cause weakening,

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such as competition between legume proteins and gluten for water molecules, the disruption of starchprotein complexes by the foreign proteins and disruption of SS interchange by the non-gluten proteins. The major seed protein fraction of amaranth, oat and quinoa is represented by globulin, which does not possess the requisites to confer dough elasticity (Tatham et al., 2001; Belton, 1999) and of these only the amaranth one have been extensively studied. Amaranth globulins are composed of 11S-globulin, globulin-P and a small amount of 7S-globulins (Marcone, 1999; Martinez et al., 1997; Segura-Nieto et al., 1994). It was shown that the 11S-globulins have molecular characteristics similar to those of other legumes (Chen and Paredes-Lopez, 1997; Marcone et al., 1994, 1997; Segura-Nieto et al., 1994). Most of cysteine residues of the globulin-S are involved in disulde bridges required to maintain the quaternary structure, although their cleavage does not mainly affect the protein secondary structure (Marcone and Yada, 1997). In addition, globulin-P is composed of unitary molecules of molecular weight and polypeptide composition similar to those of 11Sglobulin, but it tends to polymerize, thus showing different solubility (Castellani et al., 1998; Martinez et al., 1997). Furthermore, globulin-P molecules have been reported as being composed of dimeric subunits linked by disulde bonds, since their polymers are stabilized by SS linkages (Martinez et al., 1997). Oat globulins are mainly composed of salt soluble globulin (11S-globulin), and in contrast to other cereals such as wheat, barley and rye, whose storage proteins are generally alcohol soluble prolamins, they represent the major protein fraction. Oat also contains prolamins, called avenins, that account for only 1015% of total protein, whereas those of wheat, rye and barley account for 4050%, 30 50% and 3545% of total protein, respectively (Moulton, 1959; Peterson and Brinegar, 1986). Quinoa globulins represent $77% of total proteins while the percentage of prolamins is low (0.50.7%) (Koziol, 1992). In this type of ours, the absence of gluten represents a formidable challenge to the cereal technologist in pasta products preparation. An effective instrument in predicting the processing behaviour and in controlling the quality of nal pasta is the characterization of rheological properties of non-conventional doughs. Farinograph, mixograph and extensograph are the most common empirical instruments used for characterizing dough rheology. Tests based on these instruments are useful for providing practical information for the pasta industries, while they are not sufcient for interpreting the fundamental behaviour of dough processing and pasta quality. Dynamic rheological testing, especially in the linear viscoelastic region, has been used to follow the structure and properties of doughs and to study the functions of dough ingredients (Janssen et al., 1996; Miller and Hoseney, 1999). This testing simultaneously measures the viscoelastic parameters of dough expressed in storage and loss moduli, G0 and G00 , and loss tangent tan d. It is generally found that doughs made from good quality our have tan d values lower than doughs made from poor quality our. High G0 and G00 values in pasta dough can be related to good structure (Song and Zheng, 2007). The technological properties of doughs and the quality of the nal products are affected by both the modication of polymeric protein size distribution and the protein polymerization through cross-linkage and it is well known that polymers aggregation leads to a signicant rise in elastic plateau modulus G0 N of the network (Cornec et al., 1994; Popineau et al., 1994). Two types of polymeric proteins can be separated by their solubility in SDSphosphate buffer: the soluble fraction and unextractable polymeric proteins (UPP). Only UPP percentage is well correlated with dough strength (Rmax and extensograph tests) and with mixograph peak time (MPT), indicating that the highest polymeric fraction is the major

contributing factor to variations in dough properties (MacRitchie and Laandra, 1997; Weegels, 1996). Dynamic rheological and static-mechanical tests are good ways to fundamentally study the changes in product characteristics due to both processing and formulations. Moreover, the dough components (starch, proteins and water) and their interactions play an important role on the conformational structure as well as the rheological properties (Shiau and Yeh, 2001). The dynamic viscoelastic behaviour of doughs can be understood by taking into account the dual role of water that behaves as an inert ller reducing the rheological properties proportionally and as a lubricant enhancing the relaxation (Masi et al., 1998). Starch is able to form a continuous network of particles together with the macromolecular network of hydrated gluten. This interaction gives rise to rheological properties of doughs. Though the interaction plays an important role, the relative contributions of the two sources are difcult to resolve. The component interactions depend on stress level. The starch starch interactions dominate over proteinprotein interactions at low stresses, while the proteinprotein interactions play a dominant role at large deformations (Khatkar and Schoeld, 2002). Gluten contributes to the viscoelastic properties of dough to varying degrees depending on its source differing with both gliadin/glutenin ratio and LMWGS (Edwards et al., 2001, 2003). Gliadin enhances viscous ow of dough. Glutenin addition results in a more elastic dough in comparison with gluten and gliadin additions (Edwards et al., 2001). Increasing the glutenin/gliadin ratio improves maximum shear viscosity and dough strength (Uthayakumaran et al., 2000). At our knowledge there are no works about the inuence on the rheology of proteins different from gluten. The aim of this work was to study the rheological characteristics of amaranth, quinoa and oat crumbly dough for pasta making. In addition, the molecular size distribution of the non-conventional dough polymeric proteins and their extractability were also evaluated.

2. Materials and methods 2.1. Materials and preparation of dough samples Amaranth, quinoa, oat wholemeal ours and semolina were purchased from Bongiovanni Mill (Molino Bongiovanni, Mondov, Cuneo, Italy). For each our, 300 g of dough crumbly samples were prepared using ordinary tap water and a fresh pasta home appliance (Pastamatic, Simac 1400N, Treviso, Italy). The kneading time was 15 min for non-conventional dough samples and 20 min for semolina ones. The water added to non-conventional ours and semolina to prepare dough samples was of 30% (Chillo et al., 2008). The quantity of water added and the mix times were those recommended by the pasta manufacturer involved in this work. Preliminary trials were also carried out to conrm the quantity of water used and the mix times in order to be sure that the dough samples were sufciently hydrated and suitable for the extrusion process. Three batches of dough for each non-conventional our were produced and compared with three batches of semolina crumbly dough made only of durum semolina. 2.2. Chemical analysis Flours were analyzed by standard procedures (ICC1995) in duplicate: moisture (Method 110/1), crude proteins (N 5.7) (Method 105/2), ash (Method 104/1) and total dietary ber content was quantied by a commercial assay kit (Megazyme, Astori s.n.c., Italy) based on an enzymatic gravimetric procedure of AACC (Method 32-07) (Fares et al., 2008).

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2.3. Mechanical properties A rectangular sample of dough (length 25 mm) was used to determine the elastic modulus in tension (Ec). The sample was submitted to stressstrain tests using a dynamic mechanical analyzer (DMA-Q 800, TA Instruments, New Castle, DE, USA) equipped with tension lm clamp. The tension lm clamp included one set of smooth clamp xtures for testing materials in a tensile mode. It had a length range from 5 to 30 mm, width to 6.5 mm and thickness to 2 mm. One end of the strand was attached to a superior mobile clamp and the other end attached to a lower xed clamp. Tests were carried out at 25 C, with a preload of 103 N and a force ramp of 0.1 N/min. From each type of our, three batches of dough were prepared and ten replicates for each batch of dough were carried out. The elastic modulus was evaluated from the initial slope of the obtained stressstrain curve using the following exponential equation (Del Nobile et al., 2007):

(SDS) phosphate buffer (pH 6.9) and mixed, initially in a vortexmixer and later kept at room temperature (24 C) for 30 min. The suspension was then centrifuged for 10 min at 17,000g to obtain supernatant (extractable or SDS-soluble proteins). The resulting residue was extracted with 0.9 ml 0.5% SDSphosphate buffer by sonication for 30 s using a Microson Ultrasonic cell distributor, ensuring that the sample was completely dispersed within the rst 5 s. Then the sample was treated at 30 C for 30 min and the supernatant, after centrifugation for 10 min at 17,000g, was termed unextractable protein. All extracts were ltered through a 0.45 lm PVDF lter prior to SE-HPLC analysis. 2.7. SE-HPLC analysis Polymeric proteins from doughs were fractionated through size exclusion high-performance liquid chromatography (SE-HPLC) (LC 10 AD Shimadzu; Shimadzu Corporation Instruments Division, Kyoto, Japan) using a Phenomenex Biosep TM SEC 4000 column (Phenomenex) (Kuktaite et al., 2000, 2003). The extracted proteins were separated on SE-HPLC according to Gupta et al. (1993). Three replicates of each samples were used for the investigation of protein composition. The percentage of unextractable polymeric protein (UPP) was calculated as described by Gupta et al. (1993). The percentages of total UPP were calculated as [peak 1 + 2 area (unextractable)/peak 1 + 2 area (total)] 100. Peak 1 + 2 area (total) refers to the total of peak 1 + 2 area (extractable) and peak 1 + 2 area (unextractable) (Johansson et al., 2001; Kuktaite et al., 2000, 2003). The percentage of large polymeric protein (LPP) was calculated [peak 1 area (unextractable)/peak 1 area (total)] 100. Peak 1 area (total) refers to the total of peak 1 area (extractable) and peak 1 area (unextractable). 2.8. Statistical analysis The results were compared by one-way variance analysis (ANOVA). Duncans multiple range test, with the option of homogeneous groups (p < 0.05), was used to determine signicance between the dough samples. STATISTICA 7.1 for Windows (StatSoft, Inc., Tulsa, OK, USA) was used.

rT Ec eT exp eT K

where eN and rN are the true strain and the true stress, respectively, calculated according to Mancini et al. (1999), Ec is the elastic modulus (i.e., the tangent to the stress strain curve at the origin), K is a constant value, regarded as a tting parameter. The measure of dough samples tenacity was obtained by numeric integration of the area under the stressstrain curve. The area under the stressstrain curve is the energy stored in the sample until fracture (Thorvaldsson et al., 1999). Moreover, it describes the ruggedness of a dough against crack growth or break. 2.4. Rheological properties Rheological properties for each crumbly dough were investigated using a controlled-strain rotational rheometer (ARES model, TA Instruments, New Castle, DE, USA) equipped with a force rebalance transducer (model 1 K-FRTN1, 11000 g cm, 200 rad/s, 2 2000 gmf) and parallel plates (superior plate diameter of 25 mm). The gap between the plates was of 3 mm. Steady temperature was ensured with an accuracy of 0.1 C by means of a controlled uid bath unit and an external thermostatic bath. Three measurement replicates were performed for each sample. The experiments were carried out at 25 C. In order to prevent water evaporation, a suitable cover tool (accessory provided by TA Instruments) sealing the top of the superior plate was used during testing. Storage modulus (G0 ), loss modulus (G00 ) and phase angle (tan d) were determined in a frequency range of 0.0130 Hz. G0 is a measure of the energy stored and recovered per cycle, whereas G00 is a measure of the energy dissipated or lost as heat per cycle of sinusoidal deformation (Ferry, 1980). Tan delta is directly related to the energy lost per cycle divided by the energy stored per cycle that can vary from zero to innity (Steffe, 1996). The strain value was obtained from preliminary strain sweep (0.1%) oscillatory trials to determine the linear viscoelastic region. 2.5. Dough preparation for protein extraction

3. Results and discussion 3.1. Chemical analysis The ours examined had very similar protein content while signicant differences could be detected for the ash and total ber content (Table 1). The lowest ash content value was recorded for semolina sample (0.68%) while the highest were shown by amaranth (2.38%) and quinoa (2.17%) ours. On the contrary, oat our showed the highest total ber content (11.33%) followed by quinoa (9.86%), amaranth (8.83%) and semolina (3.8%) samples. 3.2. Static and dynamic mechanical properties

Dough samples were placed in sterile plastic bottles after each sampling; nitrogen was quickly ushed into the bottles before freezing to avoid further reaction. Samples were freeze-dried and later ground using the hammer mill (0.8 mm sieve) (Munson Hammer Mill, Model 121, Munson Machinery, Co., Inc., Utica, NY, USA). 2.6. Extraction and fractionation of proteins Proteins from milled freeze-dried dough samples were extracted following the method of Gupta et al. (1993). Samples (10 mg) were suspended in 1 mL of 0.5% sodium dodecyl sulphate

Fig. 1 reports the stressstrain curves for amaranth, oat, quinoa, and semolina dough samples. As can be inferred from this gure, the semolina dough is much more extensible to break than amaranth, oat and quinoa dough samples. The tenacity of amaranth, oat, quinoa dough samples compared with semolina dough is presented in Fig. 2. The dough samples of amaranth and quinoa showed signicant differences (p < 0.05) with respect to that of oat. It is worth noting that the tenacity of semolina dough was higher than that of the other dough samples and the difference was statistically signicant (p < 0.001).

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Table 1 Chemical parameters of semolina and amaranth, quinoa and oat wholemeal ours. Flour samples Semolina Amaranth Quinoa Oat
a,b,c A,B,C,D

Water % 12.71 13.20 13.50 12.50 (0.3) (0.05)b (0.1)c (0.03)a

a

Protein content % (d.b.) 12.70 12.70 11.60 12.67 (0.05) (0.9)a,c (0.4)b,c (0.01)a
a

Ash % (d.b.) 0.68 2.38 2.17 1.97 (0.08) (0.1)B,a (0.02)C,b (0.03)D,c
A

Total ber % (d.b.) 3.8 (0.3)A 8.83 (0.5)B,a 9.86 (0.06)C,b 11.33 (0.1)D,c

p < 0.05. p < 0.001.

Fig. 1. Curve of stressstrain for the amaranth (j), oat ( ), quinoa (d), and semolina (N) samples.

Fig. 3. Ec values of amaranth, oat and quinoa dough samples compared with the semolina dough.

Fig. 2. Tenacity values of amaranth, oat and quinoa dough samples compared with the semolina dough.

Fig. 3 shows the values of Ec for amaranth, oat and quinoa dough samples compared with the semolina dough. Ec (MPa) was calculated as the slope of the initial portion of the stressstrain curve. It was noted that, among non-conventional doughs, oat showed an Ec value signicantly lower (p < 0.001) with respect to that of amaranth and quinoa samples. On the other side, the Ec values of the latter two samples were statistically comparable. The semolina dough presented a value of Ec signicantly lower (p < 0.001) than that of the other investigated samples. In Fig. 4 the G0 and G00 (Pa) values of the investigated dough samples are reported. For semolina dough, G0 and G00 values were significantly lower (p < 0.001) than those of all the other dough samples. Among non-conventional doughs, amaranth and quinoa G0 values

Fig. 4. G0 (closed symbol) and G00 (open symbol) values as function of oscillatory frequency for the dough samples: semolina (N and 4), amaranth (j and h), oat ( and ), and quinoa (d and s).

were found to be similar and signicantly higher (p < 0.05) than that of oat. G00 of amaranth, quinoa and oat doughs showed different values. The highest G00 value was recorded for the amaranth dough while the lowest one was shown by oat. Moreover, it can be inferred from Fig. 4 that the G0 values for all dough samples are larger than G00 values (p < 0.001). This behaviour is typical of

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a viscoelastic solid (Rao and Steffe, 1992) which presents a dominant contribution of the elastic component to the viscoelasticity (Subramanian et al., 2006). Dough samples, shown in Fig. 4, had a solid-like gel behaviour with rheological spectra resembling that of weak gel (Ross-Murphy, 1988; Richardson et al., 1989). Typical weak gel characteristics were observed. G0 was greater than G00 throughout the frequency range and the modules showed a slight dependence on frequency. Fig. 5 shows the tan d values vs the oscillatory frequency for amaranth, oat and quinoa dough samples compared with that of the semolina dough. As can be inferred from the gure, at low and medium frequencies, the oat and quinoa tan d values were statistically comparable and signicantly lower (p < 0.05) than that of wheat dough. At high frequencies, tan d values of the investigated samples were different among them and the highest value was detected for amaranth followed by semolina, quinoa and oat. It was demonstrated that dough with small tan d value reects a rigid and stiff material and doughs characterized as moist and slack possessed higher tan d values than those described as having a short texture and dry surface appearance (Weipert, 1990). Rao et al. (2000) found that the high G0 and low tan d values of doughs showed that they were rmer and more elastic at small strains and moderately rapid frequencies of measurements. Moreover, Edwards et al. (1999) found no signicant correlation between tan d values and dough strength of durum semolina as measured by empirical methods, while the G0 values strongly correlated with the dough strength. Although, these studies suggest that oscillatory measurements in the linear viscoelastic region can segregate semolina doughs differing in strength (Rao et al., 2000; Edwards et al., 1999; Weipert, 1990). 3.3. Molecular weight distribution of semolina, amaranth, quinoa and oat dough polymeric proteins The molecular weight distribution of polymeric proteins of semolina, amaranth, quinoa and oat doughs are depicted in Fig. 6. The curves were drawn by measuring the areas of the peaks obtained by SE-HPLC using a Biosep-SEC-S-4000 Phenomenex column and evaluating the molecular weights from a calibration graph using standard proteins. Semolina dough shows peaks represented (Fig. 6a), in decreasing molecular weight order, by: large polymeric proteins (with MW ranging from about 3980 to 2500 kDa), small polymeric proteins (with MW ranging from 300 to 200 kDa), large monomeric proteins (with MW of about 50 kDa) and small monomeric proteins (with MW ranging from

about 60 to 20 kDa). These proteins, described by Carceller and Aussenac (2001) and Larroque et al. (1996), are represented by high molecular weight (HMW) and low molecular weight (LMW) glutenin proteins (large and small polymeric proteins), gliadins (large monomeric proteins) and albumins and globulins (small monomeric proteins). The amaranth dough prole, in Fig. 6b, showed the presence of polymeric proteins at higher molecular weight (ranging from about 3900 to 2500 kDa) represented by globulin-P polymers (Glb-P) (Castellani et al., 1998; Martinez et al., 1997), followed by globulin-S (Glb-S) molecules (MW of about 200 kDa) characterized as a 11S type globulin (Chen and Paredes-Lopez, 1997; Romero-Zepeda and Paredes-Lopez, 1996; Segura-Nieto et al., 1994) and protein species of MW < 100 kDa (mainly composed by albumins). Quinoa dough polymeric proteins prole (Fig. 6c) showed the presence of four peaks: a very small peak at higher molecular weight (ranging from about 3800 to 2500 kDa), a peak with molecular weight included between 630 and 398 kDa, a very large peak with a molecular weight included between 50 and 39 kDa and a small peak with molecular weight included between 15 and 19 kDa. Albumins and globulins are known to be the major protein fraction (4477% of total proteins) in quinoa our while the percentage of prolamins is low (0.50.7%) (Koziol, 1992). So, it is probable that the second and third peak correspond to globulin and albumin respectively, while the rst peak could contain polymerized globulin (Gb P) as for amaranth dough. This would be consistent with the fact that quinoa proteins show high levels of cysteine (Koziol, 1992; Van Etten et al., 1963). The oat dough prole (Fig. 6d) showed ve peaks and four of them look very similar in the protein proportion but different in the molecular size distribution. In decreasing order, the rst peak contains proteins with molecular weight included between 3980 and 2500 kDa, followed by a peak containing proteins included between 630 and 390 kDa, a peak containing proteins with molecular weight included between 300 and 200 kDa and two peaks containing proteins with a molecular weight included between 150 and 100 kDa, and 100 and 60 kDa, respectively. These data show that all three non-conventional doughs have polymeric proteins with a high molecular weight ranging between 3000 and 2500 kDa such as semolina dough and as can be inferred by the Fig. 6, oat and amaranth doughs contain high amount of these proteins with respect to the quinoa dough. The semolina high molecular weight polymeric proteins contain very large insoluble polymer molecules which have been positively and signicantly correlated to our processing (Laandra et al., 1999). These latter observations are consistent with the known strong dependence of rheological properties on molecular weight and molecular weight distribution for polymers in general (Mead, 1994). Kasarda (1999) has observed that the molecular weight distribution of gluten polymers is fairly certain to be a key factor in the variations of dough strength and elasticity, but also that determination of the HMW distribution of gluten is confounded by the insolubility of the largest glutenin components. In addition, Huang and Khan (1997) have found a relationship between the total amount of HMW glutenin subunits in the our and dough mixing strength and bread loaf volume, that strongly suggests that is the quantity of HMW glutenin subunits that determines wheat protein quality differences of high resistent starch (HRS) wheats. At our knowledge there are no reports that relate the molecular size distribution and rheological behaviour of ours different from wheat and further studies using pure components instead whole our dough are necessary. 3.4. SE-HPLC proles of wheat, amaranth, quinoa and oat doughs

Fig. 5. Tan d values vs the oscillatory frequency for the dough samples: semolina (4), amaranth (h), oat ( ), and quinoa (s).

Fig. 7 shows the SE-HPLC proles for extractable (a, c, e, g) and unextractable (b, d, f, h) proteins in semolina, amaranth, quinoa

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Fig. 6. Molecular size distribution (MWD) of semolina (a), amaranth (b), quinoa (c) and oat (d).

and oat doughs. The elution prole for extractable proteins from semolina dough (Fig. 7a) showed a polymeric protein peak of glutenin at the extreme left of the prole (peak 1, >100,000 Da), followed by a large peak of monomeric gliadin proteins (100,000 Da) and nally small peaks of albumins and globulins (Carceller and Aussenac, 2001). In contrast, the prole of unextractable semolina dough proteins showed a much greater proportion of protein in the rst peak (Fig. 7b), in accordance with Gupta et al. (1993), Carceller and Aussenac (2001). The SE-HPLC elution prole for extractable proteins in amaranth dough showed few peaks, the greatest proportion being proteins of intermediate size, in both the extractable and unextractable preparations (Fig. 7c and d). The elution prole for extractable proteins from quinoa dough (Fig. 7e) showed three main peaks, the larger containing proteins of intermediate and small size. The SEHPLC prole for unextractable proteins in quinoa dough (Fig. 7f)

showed more peaks with respect the extractable prole, but also in this case, the major peaks were represented by those containing proteins of intermediate and small size. The oat dough showed several peaks in its SE-HPLC extractable prole (Fig. 7g), the greatest proportion being proteins of large size. In contrast, the prole of the unextractable proteins (Fig. 7h) showed much greater proportion of proteins of intermediate and small size. 3.5. Percentage of total UPP of wheat, amaranth, quinoa and oat doughs The percentage of unextractable polymeric proteins in total and large polymeric proteins in semolina, amaranth, quinoa and oat doughs was calculated from chromatograms obtained by proteins fractionation through SE-HPLC. Fig. 8 shows that the unextractabil-

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ity of semolina dough proteins (61% UPP and 60.4% LPP) was greater with respect to the others followed by amaranth (40.7% UPP and 40.1% LPP), oat (24% UPP and 25.1% LPP) and quinoa (10.10% UPP and 19% LPP). Signicant differences in extractability of the polymeric proteins in the dough tested, could be explained by the different degree of polymerization that would result in differences in the availability of cysteine residues. The more the cysteine residues are available, the more proteins polymerize, increase in size and become insoluble (Don et al., 2005). The amount of UPP, in fact, is inuenced by different quaternary structures which result from polymers (involving disulphide bridges) (Gobin et al., 1997; Rhazi et al., 2003) and aggregates (involving hydrogen bonding) (Aussenac et al., 2001) of different size. Semolina polymers are represented by glutenins which are linked by inter-chain disulphide bonds (Bietz and Wall, 1972; Fisichella et al., 2003; Grosch and Wieser, 1999; Schoeld, 1986) that are resistant to cleavage (Lindsay and Skerritt, 1998). The inherent ability of glutenin subunits to form disulphide bonds is thought to be determined by the primary and secondary structure of these

proteins, which determines whether cysteine residues are present and available to form disulphide bonds (Shewry et al., 1995). Amaranth, quinoa and oat proteins are mainly represented by globulin and of these only the globulin-P, which are known to be present in amaranth, have been reported to polymerize being composed of dimeric subunits linked by disulphide bonds (Martinez et al., 1997). 4. Conclusion The tenacity of amaranth, oat and quinoa doughs was lower than that of semolina dough sample. The elastic modulus of amaranth, oat and quinoa dough samples was higher than that of semolina dough. The G0 values of amaranth and quinoa were similar but signicantly higher (p < 0.05) respect to that of oat dough. G00 for the three non-conventional doughs showed different values: the highest was recorded for the amaranth dough while the lowest was shown by oat. The semolina dough showed G0 and G00 values signicantly lower than those of all the other dough samples. The oat and quinoa doughs had similar tan d values, at low and

Fig. 7. SE-HPLC elution proles for extractable proteins (a, c, e, g) and for unextractable proteins (b, d, f, h) of semolina, amaranth, quinoa and oat dough samples.

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Fig. 7 (continued)

Fig. 8. Percentage of total and large UPP in semolina, amaranth, quinoa and oat dough samples.

medium frequencies, and they resulted to be signicantly lower (p < 0.05) than that of amaranth and semolina doughs. At high frequencies, the investigated samples showed tan d values different among them and the highest value was detected for amaranth followed by semolina, quinoa and oat. The non-conventional doughs

showed polymeric proteins with high molecular weight distribution (about 3.000 kDa) such as in semolina dough. However, the percentage of these proteins that was unextractable were signicantly different among amaranth, quinoa and oat doughs and resulted to be higher for the amaranth one. In future work,

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rheological studies on these doughs in the range of the large-scale deformation, will be carried out. Acknowledgement This research work was nancially support by Italian Puglia Region, Strategic Project Process innovation for production of functional pasta, PS_003. References
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