Journal of Food Engineering 96 (2010) 97–106

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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 Qualità, Università degli Studi di Foggia,Via Napoli, 25-71100 Foggia, Italyb Dipartimento di Scienze degli Alimenti, Università 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

Article history:Received 10 September 2008Received in revised form 1 July 2009Accepted 3 July 2009Available online 9 July 2009

Keywords:Mechanical propertiesRheological propertiesPolymeric proteinsAmaranthQuinoaOat

0260-8774/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.jfoodeng.2009.07.001

* Corresponding author. Address: Istituto per laBiotecnologiche per la Sicurezza e la Valorizzazione dUniversità degli Studi di Foggia, Via Napoli, 25-71100589 117; fax: +39 0881 740 211.

E-mail address: c.lamacchia@unifg.it (C. Lamacchia

The rheological characteristics, static and dynamic mechanical properties of amaranth, quinoa and oatdoughs and the relative size distribution of their polymeric proteins were evaluated. For the sake of com-parison, semolina dough rheological and mechanical properties and the relative size distribution of pro-teins were also determined.

From rheological results it was inferred that the tenacity of amaranth, oat and quinoa dough sampleswas lower than that of semolina dough. The elastic modulus (Ec) of amaranth, oat and quinoa doughs washigher than that of semolina dough. Amaranth and quinoa G0 was found to be similar and significantlyhigher (p < 0.05) with respect to that of oat dough at a moisture of 30%. The G00 of amaranth, quinoaand oat doughs showed different values. The highest G00 value was recorded for the amaranth doughwhile the lowest one was shown by oat. For semolina dough, the G0 and G00 values were significantly lowerthan those of all the other dough samples. Moreover, at low and medium frequencies, tan d values of oatand quinoa doughs were statistically comparable and significantly lower (p < 0.05) than that of amaranthand semolina doughs. At high frequencies, tan d values of investigated samples were different amongthem and the highest value was detected for amaranth, followed by semolina, quinoa and oat. Resultsof the size distribution of proteins in amaranth, quinoa, oat and semolina doughs were expressed asthe 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%).

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1. Introduction

Wheat flour is the only cereal flour that can form a three-dimensional viscoelastic dough when mixed with water. This un-ique ability of wheat to suit the production of leavened and pastaproducts is due to the gluten, a cohesive, viscoelastic proteinaceousmaterial prepared as a by-product of starch isolation from wheatflour. The proteins that form gluten are storage proteins whichconsist of two major fractions: the monomeric gliadins and thepolymeric glutenins (Schofield, 1994). The latter are known to bethe most important determinants of pasta and bread-making qual-ity (D’Ovidio and Masci, 2004; Lindsay and Skerritt, 1999) and onegroup of these, of 3–6 proteins, is largely responsible for the elasticproperties (Thatam et al., 2001). The high Mr subunits, in fact,possess the characteristics of a putative elastomer, with N- and

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C-terminal domains containing residues for covalent cross-linkingand a central domain that can potentially undergo deformation(Shewry and Thatam, 1990; Belton, 1999). In terms of its nutri-tional value, gluten (or wheat proteins) is considered to be poorerthan proteins from animal sources and can cause allergic reactionand intolerances (Gallagher et al., 2004). Amaranth, quinoa and oathave attracted many interest because of their high nutritional va-lue and for the absence of gluten. In spite of this, the absence ofgluten, in these flours, results in major problems for many pastaand bakery products. Their utilization as food ingredients in theproduction of pasta and bakery products depends largely on theirfunctional properties, which are related to protein structural char-acteristics. Attempts to use proteins from alternative flours as apartial substitute in wheat products have generally been unsuc-cessful, because of the contrasting differences between proteinssuch as the water-solubility, differences in primary structure andtheir size distributions, accounted for viscoelastic properties thatare unique to wheat gluten proteins. Lorimer et al. (1991) reportedthat the addition of non-gluten forming proteins (e.g. bean-seedproteins) causes a dilution effect and consequent weakening ofwheat dough. They suggested several issues that cause weakening,

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such as competition between legume proteins and gluten for watermolecules, the disruption of starch–protein complexes by the for-eign proteins and disruption of SS interchange by the non-glutenproteins.

The major seed protein fraction of amaranth, oat and quinoa isrepresented by globulin, which does not possess the requisites toconfer dough elasticity (Tatham et al., 2001; Belton, 1999) and ofthese only the amaranth one have been extensively studied. Ama-ranth globulins are composed of 11S-globulin, globulin-P and asmall amount of 7S-globulins (Marcone, 1999; Martinez et al.,1997; Segura-Nieto et al., 1994). It was shown that the 11S-globu-lins have molecular characteristics similar to those of other le-gumes (Chen and Paredes-Lopez, 1997; Marcone et al., 1994,1997; Segura-Nieto et al., 1994). Most of cysteine residues of theglobulin-S are involved in disulfide bridges required to maintainthe quaternary structure, although their cleavage does not mainlyaffect the protein secondary structure (Marcone and Yada, 1997).In addition, globulin-P is composed of unitary molecules of molec-ular weight and polypeptide composition similar to those of 11S-globulin, but it tends to polymerize, thus showing different solubil-ity (Castellani et al., 1998; Martinez et al., 1997). Furthermore,globulin-P molecules have been reported as being composed of di-meric subunits linked by disulfide bonds, since their polymers arestabilized by SS linkages (Martinez et al., 1997). Oat globulins aremainly composed of salt soluble globulin (11S-globulin), and incontrast to other cereals such as wheat, barley and rye, whose stor-age proteins are generally alcohol soluble prolamins, they repre-sent the major protein fraction. Oat also contains prolamins,called avenins, that account for only 10–15% of total protein,whereas those of wheat, rye and barley account for 40–50%, 30–50% and 35–45% of total protein, respectively (Moulton, 1959; Pet-erson and Brinegar, 1986).

Quinoa globulins represent �77% of total proteins while thepercentage of prolamins is low (0.5–0.7%) (Koziol, 1992).

In this type of flours, the absence of gluten represents aformidable challenge to the cereal technologist in pasta productspreparation. An effective instrument in predicting the processingbehaviour and in controlling the quality of final pasta is thecharacterization of rheological properties of non-conventionaldoughs.

Farinograph, mixograph and extensograph are the most com-mon empirical instruments used for characterizing dough rheol-ogy. Tests based on these instruments are useful for providingpractical information for the pasta industries, while they are notsufficient for interpreting the fundamental behaviour of doughprocessing and pasta quality. Dynamic rheological testing, espe-cially in the linear viscoelastic region, has been used to followthe structure and properties of doughs and to study the functionsof dough ingredients (Janssen et al., 1996; Miller and Hoseney,1999). This testing simultaneously measures the viscoelasticparameters of dough expressed in storage and loss moduli, G0

and G00, and loss tangent tan d. It is generally found that doughsmade from good quality flour have tan d values lower than doughsmade from poor quality flour. High G0 and G00 values in pasta doughcan be related to good structure (Song and Zheng, 2007).

The technological properties of doughs and the quality of the fi-nal products are affected by both the modification of polymericprotein size distribution and the protein polymerization throughcross-linkage and it is well known that polymers aggregation leadsto a significant rise in elastic plateau modulus G0

N of the network(Cornec et al., 1994; Popineau et al., 1994). Two types of polymericproteins can be separated by their solubility in SDS–phosphatebuffer: 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 (MacRitchieand Lafiandra, 1997; Weegels, 1996).

Dynamic rheological and static-mechanical tests are good waysto fundamentally study the changes in product characteristics dueto both processing and formulations. Moreover, the dough compo-nents (starch, proteins and water) and their interactions play animportant role on the conformational structure as well as the rhe-ological properties (Shiau and Yeh, 2001). The dynamic viscoelasticbehaviour of doughs can be understood by taking into account thedual role of water that behaves as an inert filler reducing the rhe-ological properties proportionally and as a lubricant enhancing therelaxation (Masi et al., 1998). Starch is able to form a continuousnetwork of particles together with the macromolecular networkof hydrated gluten. This interaction gives rise to rheological prop-erties of doughs. Though the interaction plays an important role,the relative contributions of the two sources are difficult to resolve.The component interactions depend on stress level. The starch–starch interactions dominate over protein–protein interactions atlow stresses, while the protein–protein interactions play a domi-nant role at large deformations (Khatkar and Schofield, 2002). Glu-ten contributes to the viscoelastic properties of dough to varyingdegrees depending on its source differing with both gliadin/glute-nin ratio and LMW–GS (Edwards et al., 2001, 2003). Gliadin en-hances viscous flow of dough. Glutenin addition results in a moreelastic dough in comparison with gluten and gliadin additions (Ed-wards et al., 2001). Increasing the glutenin/gliadin ratio improvesmaximum shear viscosity and dough strength (Uthayakumaranet al., 2000).

At our knowledge there are no works about the influence on therheology of proteins different from gluten.

The aim of this work was to study the rheological characteris-tics of amaranth, quinoa and oat crumbly dough for pasta making.In addition, the molecular size distribution of the non-conventionaldough polymeric proteins and their extractability were alsoevaluated.

2. Materials and methods

2.1. Materials and preparation of dough samples

Amaranth, quinoa, oat wholemeal flours and semolina werepurchased from Bongiovanni Mill (Molino Bongiovanni, Mondovì,Cuneo, Italy). For each flour, 300 g of dough crumbly samples wereprepared using ordinary tap water and a fresh pasta home appli-ance (Pastamatic, Simac 1400N, Treviso, Italy). The kneading timewas 15 min for non-conventional dough samples and 20 min forsemolina ones. The water added to non-conventional flours andsemolina to prepare dough samples was of 30% (Chillo et al.,2008). The quantity of water added and the mix times were thoserecommended by the pasta manufacturer involved in this work.Preliminary trials were also carried out to confirm the quantityof water used and the mix times in order to be sure that the doughsamples were sufficiently hydrated and suitable for the extrusionprocess. Three batches of dough for each non-conventional flourwere produced and compared with three batches of semolinacrumbly dough made only of durum semolina.

2.2. Chemical analysis

Flours were analyzed by standard procedures (ICC1995) induplicate: moisture (Method 110/1), crude proteins (N � 5.7)(Method 105/2), ash (Method 104/1) and total dietary fiber contentwas quantified 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 todetermine the elastic modulus in tension (Ec). The sample was sub-mitted to stress–strain tests using a dynamic mechanical analyzer(DMA-Q 800, TA Instruments, New Castle, DE, USA) equipped withtension film clamp. The tension film clamp included one set ofsmooth clamp fixtures for testing materials in a tensile mode. Ithad a length range from 5 to 30 mm, width to 6.5 mm and thick-ness to 2 mm. One end of the strand was attached to a superiormobile clamp and the other end attached to a lower fixed clamp.Tests were carried out at 25 �C, with a preload of 10�3 N and a forceramp of 0.1 N/min. From each type of flour, three batches of doughwere prepared and ten replicates for each batch of dough were car-ried out. The elastic modulus was evaluated from the initial slopeof the obtained stress–strain curve using the following exponentialequation (Del Nobile et al., 2007):

rT ¼ EceT exp �eT � Kð Þ ð1Þ

where eN and rN are the true strain and the true stress, respectively,calculated according to Mancini et al. (1999), Ec is the elastic mod-ulus (i.e., the tangent to the stress strain curve at the origin), K is aconstant value, regarded as a fitting parameter.

The measure of dough samples tenacity was obtained by nu-meric integration of the area under the stress–strain curve. Thearea under the stress–strain curve is the energy stored in the sam-ple until fracture (Thorvaldsson et al., 1999). Moreover, it describesthe ruggedness of a dough against crack growth or break.

2.4. Rheological properties

Rheological properties for each crumbly dough were investi-gated using a controlled-strain rotational rheometer (ARES model,TA Instruments, New Castle, DE, USA) equipped with a force rebal-ance transducer (model 1 K-FRTN1, 1–1000 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 temperaturewas ensured with an accuracy of ±0.1 �C by means of a controlledfluid bath unit and an external thermostatic bath. Three measure-ment replicates were performed for each sample. The experimentswere carried out at 25 �C. In order to prevent water evaporation, asuitable cover tool (accessory provided by TA Instruments) sealingthe top of the superior plate was used during testing. Storage mod-ulus (G0), loss modulus (G00) and phase angle (tan d) were deter-mined in a frequency range of 0.01–30 Hz. G0 is a measure of theenergy stored and recovered per cycle, whereas G00 is a measureof the energy dissipated or lost as heat per cycle of sinusoidaldeformation (Ferry, 1980). Tan delta is directly related to the en-ergy lost per cycle divided by the energy stored per cycle thatcan vary from zero to infinity (Steffe, 1996). The strain value wasobtained from preliminary strain sweep (0.1%) oscillatory trialsto determine the linear viscoelastic region.

2.5. Dough preparation for protein extraction

Dough samples were placed in sterile plastic bottles after eachsampling; nitrogen was quickly flushed into the bottles beforefreezing to avoid further reaction. Samples were freeze-dried andlater ground using the hammer mill (0.8 mm sieve) (Munson Ham-mer 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 ex-tracted following the method of Gupta et al. (1993). Samples(10 mg) were suspended in 1 mL of 0.5% sodium dodecyl sulphate

(SDS) phosphate buffer (pH 6.9) and mixed, initially in a vortex-mixer and later kept at room temperature (24 �C) for 30 min. Thesuspension was then centrifuged for 10 min at 17,000g to obtainsupernatant (‘‘extractable” or ‘‘SDS-soluble” proteins).

The resulting residue was extracted with 0.9 ml 0.5% SDS–phos-phate buffer by sonication for 30 s using a Microson Ultrasonic celldistributor, ensuring that the sample was completely dispersedwithin the first 5 s. Then the sample was treated at 30 �C for30 min and the supernatant, after centrifugation for 10 min at17,000g, was termed ‘‘unextractable” protein. All extracts were fil-tered through a 0.45 lm PVDF filter prior to SE-HPLC analysis.

2.7. SE-HPLC analysis

Polymeric proteins from doughs were fractionated through sizeexclusion high-performance liquid chromatography (SE-HPLC) (LC10 AD Shimadzu; Shimadzu Corporation Instruments Division,Kyoto, Japan) using a Phenomenex Biosep TM SEC 4000 column(Phenomenex) (Kuktaite et al., 2000, 2003). The extracted proteinswere separated on SE-HPLC according to Gupta et al. (1993). Threereplicates of each samples were used for the investigation of pro-tein composition. The percentage of unextractable polymeric pro-tein (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 (to-tal) refers to the total of peak 1 + 2 area (extractable) and peak1 + 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 1area (unextractable).

2.8. Statistical analysis

The results were compared by one-way variance analysis (ANO-VA). Duncan’s multiple range test, with the option of homogeneousgroups (p < 0.05), was used to determine significance between thedough samples. STATISTICA 7.1 for Windows (StatSoft, Inc., Tulsa,OK, USA) was used.

3. Results and discussion

3.1. Chemical analysis

The flours examined had very similar protein content while sig-nificant differences could be detected for the ash and total fibercontent (Table 1). The lowest ash content value was recorded forsemolina sample (0.68%) while the highest were shown by ama-ranth (2.38%) and quinoa (2.17%) flours. On the contrary, oat flourshowed the highest total fiber content (11.33%) followed by quinoa(9.86%), amaranth (8.83%) and semolina (3.8%) samples.

3.2. Static and dynamic mechanical properties

Fig. 1 reports the stress–strain curves for amaranth, oat, quinoa,and semolina dough samples. As can be inferred from this figure,the semolina dough is much more extensible to break than ama-ranth, oat and quinoa dough samples.

The tenacity of amaranth, oat, quinoa dough samples comparedwith semolina dough is presented in Fig. 2. The dough samples ofamaranth and quinoa showed significant differences (p < 0.05)with respect to that of oat. It is worth noting that the tenacity ofsemolina dough was higher than that of the other dough samplesand the difference was statistically significant (p < 0.001).

Table 1Chemical parameters of semolina and amaranth, quinoa and oat wholemeal flours.

Flour samples Water % Protein content % (d.b.) Ash % (d.b.) Total fiber % (d.b.)

Semolina 12.71 (±0.3)a 12.70 (±0.05)a 0.68 (±0.08)A 3.8 (±0.3)A

Amaranth 13.20 (±0.05)b 12.70 (±0.9)a,c 2.38 (±0.1)B,a 8.83 (±0.5)B,a

Quinoa 13.50 (±0.1)c 11.60 (±0.4)b,c 2.17 (±0.02)C,b 9.86 (±0.06)C,b

Oat 12.50 (±0.03)a 12.67 (±0.01)a 1.97 (0.03)D,c 11.33 (±0.1)D,c

a,b,c p < 0.05.A,B,C,D p < 0.001.

Fig. 1. Curve of stress–strain for the amaranth (j), oat ( ), quinoa (d), andsemolina (N) samples.

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

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

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

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Fig. 3 shows the values of Ec for amaranth, oat and quinoa doughsamples compared with the semolina dough. Ec (MPa) was calcu-lated as the slope of the initial portion of the stress–strain curve.It was noted that, among non-conventional doughs, oat showedan Ec value significantly lower (p < 0.001) with respect to that ofamaranth and quinoa samples. On the other side, the Ec values ofthe latter two samples were statistically comparable. The semolinadough presented a value of Ec significantly lower (p < 0.001) thanthat of the other investigated samples.

In Fig. 4 the G0 and G0 0 (Pa) values of the investigated dough sam-ples are reported. For semolina dough, G0 and G00 values were signif-icantly lower (p < 0.001) than those of all the other dough samples.Among non-conventional doughs, amaranth and quinoa G0 values

were found to be similar and significantly higher (p < 0.05) thanthat of oat. G00 of amaranth, quinoa and oat doughs showed differ-ent values. The highest G00 value was recorded for the amaranthdough while the lowest one was shown by oat. Moreover, it canbe inferred from Fig. 4 that the G0 values for all dough samplesare 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 domi-nant contribution of the elastic component to the viscoelasticity(Subramanian et al., 2006). Dough samples, shown in Fig. 4, hada solid-like gel behaviour with rheological spectra resembling thatof weak gel (Ross-Murphy, 1988; Richardson et al., 1989). Typicalweak gel characteristics were observed. G0 was greater than G00

throughout the frequency range and the modules showed a slightdependence on frequency.

Fig. 5 shows the tan d values vs the oscillatory frequency foramaranth, oat and quinoa dough samples compared with that ofthe semolina dough. As can be inferred from the figure, at lowand medium frequencies, the oat and quinoa tan d values were sta-tistically comparable and significantly lower (p < 0.05) than that ofwheat dough. At high frequencies, tan d values of the investigatedsamples were different among them and the highest value was de-tected for amaranth followed by semolina, quinoa and oat. It wasdemonstrated that dough with small tan d value reflects a rigidand stiff material and doughs characterized as moist and slack pos-sessed higher tan d values than those described as having a shorttexture and dry surface appearance (Weipert, 1990). Rao et al.(2000) found that the high G0 and low tan d values of doughsshowed that they were firmer and more elastic at small strainsand moderately rapid frequencies of measurements. Moreover, Ed-wards et al. (1999) found no significant correlation between tan dvalues and dough strength of durum semolina as measured byempirical methods, while the G0 values strongly correlated withthe dough strength. Although, these studies suggest that oscilla-tory measurements in the linear viscoelastic region can segregatesemolina doughs differing in strength (Rao et al., 2000; Edwardset al., 1999; Weipert, 1990).

3.3. Molecular weight distribution of semolina, amaranth, quinoa andoat dough polymeric proteins

The molecular weight distribution of polymeric proteins ofsemolina, amaranth, quinoa and oat doughs are depicted inFig. 6. The curves were drawn by measuring the areas of the peaksobtained by SE-HPLC using a Biosep-SEC-S-4000 Phenomenex col-umn and evaluating the molecular weights from a calibrationgraph using standard proteins. Semolina dough shows peaks repre-sented (Fig. 6a), in decreasing molecular weight order, by: largepolymeric proteins (with MW ranging from about 3980 to2500 kDa), small polymeric proteins (with MW ranging from 300to 200 kDa), large monomeric proteins (with MW of about50 kDa) and small monomeric proteins (with MW ranging from

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

about 60 to 20 kDa). These proteins, described by Carceller andAussenac (2001) and Larroque et al. (1996), are represented byhigh molecular weight (HMW) and low molecular weight (LMW)glutenin proteins (large and small polymeric proteins), gliadins(large monomeric proteins) and albumins and globulins (smallmonomeric proteins). The amaranth dough profile, in Fig. 6b,showed the presence of polymeric proteins at higher molecularweight (ranging from about 3900 to 2500 kDa) represented byglobulin-P polymers (Glb-P) (Castellani et al., 1998; Martinezet al., 1997), followed by globulin-S (Glb-S) molecules (MW ofabout 200 kDa) characterized as a 11S type globulin (Chen andParedes-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 proteinsprofile (Fig. 6c) showed the presence of four peaks: a very smallpeak at higher molecular weight (ranging from about 3800 to2500 kDa), a peak with molecular weight included between 630and 398 kDa, a very large peak with a molecular weight includedbetween 50 and 39 kDa and a small peak with molecular weightincluded between 15 and 19 kDa. Albumins and globulins areknown to be the major protein fraction (44–77% of total proteins)in quinoa flour while the percentage of prolamins is low (0.5–0.7%)(Koziol, 1992). So, it is probable that the second and third peak cor-respond to globulin and albumin respectively, while the first peakcould contain polymerized globulin (Gb P) as for amaranth dough.This would be consistent with the fact that quinoa proteins showhigh levels of cysteine (Koziol, 1992; Van Etten et al., 1963). Theoat dough profile (Fig. 6d) showed five peaks and four of them lookvery similar in the protein proportion but different in the molecu-lar size distribution. In decreasing order, the first peak containsproteins with molecular weight included between 3980 and2500 kDa, followed by a peak containing proteins included be-tween 630 and 390 kDa, a peak containing proteins with molecularweight included between 300 and 200 kDa and two peaks contain-ing proteins with a molecular weight included between 150 and100 kDa, and 100 and 60 kDa, respectively.

These data show that all three non-conventional doughs havepolymeric proteins with a high molecular weight ranging between3000 and 2500 kDa such as semolina dough and as can be inferredby the Fig. 6, oat and amaranth doughs contain high amount ofthese proteins with respect to the quinoa dough. The semolina highmolecular weight polymeric proteins contain very large insolublepolymer molecules which have been positively and significantlycorrelated to flour processing (Lafiandra et al., 1999). These latterobservations are consistent with the known strong dependenceof rheological properties on molecular weight and molecularweight distribution for polymers in general (Mead, 1994). Kasarda(1999) has observed that the molecular weight distribution of glu-ten polymers is ‘‘fairly certain” to be a key factor in the variationsof dough strength and elasticity, but also that determination of theHMW distribution of gluten is confounded by the insolubility ofthe largest glutenin components. In addition, Huang and Khan(1997) have found a relationship between the total amount ofHMW glutenin subunits in the flour and dough mixing strengthand bread loaf volume, that strongly suggests that is the quantityof HMW glutenin subunits that determines wheat protein qualitydifferences of high resistent starch (HRS) wheats. At our knowledgethere are no reports that relate the molecular size distribution andrheological behaviour of flours different from wheat and furtherstudies using pure components instead whole flour dough arenecessary.

3.4. SE-HPLC profiles of wheat, amaranth, quinoa and oat doughs

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

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

102 C. Lamacchia et al. / Journal of Food Engineering 96 (2010) 97–106

and oat doughs. The elution profile for extractable proteins fromsemolina dough (Fig. 7a) showed a polymeric protein peak of glute-nin at the extreme left of the profile (peak 1, >100,000 Da), followedby a large peak of monomeric gliadin proteins (100,000 Da) and fi-nally small peaks of albumins and globulins (Carceller and Aussenac,2001). In contrast, the profile of unextractable semolina dough pro-teins showed a much greater proportion of protein in the first peak(Fig. 7b), in accordance with Gupta et al. (1993), Carceller and Ausse-nac (2001). The SE-HPLC elution profile for extractable proteins inamaranth dough showed few peaks, the greatest proportion beingproteins of intermediate size, in both the extractable and unextract-able preparations (Fig. 7c and d). The elution profile for extractableproteins from quinoa dough (Fig. 7e) showed three main peaks, thelarger containing proteins of intermediate and small size. The SE-HPLC profile for unextractable proteins in quinoa dough (Fig. 7f)

showed more peaks with respect the extractable profile, but alsoin this case, the major peaks were represented by those containingproteins of intermediate and small size. The oat dough showed sev-eral peaks in its SE-HPLC extractable profile (Fig. 7g), the greatestproportion being proteins of large size. In contrast, the profile ofthe unextractable proteins (Fig. 7h) showed much greater propor-tion of proteins of intermediate and small size.

3.5. Percentage of total UPP of wheat, amaranth, quinoa and oatdoughs

The percentage of unextractable polymeric proteins in total andlarge polymeric proteins in semolina, amaranth, quinoa and oatdoughs was calculated from chromatograms obtained by proteinsfractionation 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 great-er with respect to the others followed by amaranth (40.7% UPP and40.1% LPP), oat (24% UPP and 25.1% LPP) and quinoa (10.10% UPPand 19% LPP). Significant differences in extractability of the poly-meric proteins in the dough tested, could be explained by the dif-ferent degree of polymerization that would result in differences inthe availability of cysteine residues. The more the cysteine residuesare available, the more proteins polymerize, increase in size andbecome insoluble (Don et al., 2005). The amount of UPP, in fact,is influenced by different quaternary structures which result frompolymers (involving disulphide bridges) (Gobin et al., 1997; Rhaziet al., 2003) and aggregates (involving hydrogen bonding) (Ausse-nac et al., 2001) of different size.

Semolina polymers are represented by glutenins which arelinked by inter-chain disulphide bonds (Bietz and Wall, 1972; Fis-ichella et al., 2003; Grosch and Wieser, 1999; Schofield, 1986) thatare resistant to cleavage (Lindsay and Skerritt, 1998). The inherentability of glutenin subunits to form disulphide bonds is thought tobe determined by the primary and secondary structure of these

Fig. 7. SE-HPLC elution profiles for extractable proteins (a, c, e, g) and for unextract

proteins, which determines whether cysteine residues are presentand available to form disulphide bonds (Shewry et al., 1995). Ama-ranth, quinoa and oat proteins are mainly represented by globulinand of these only the globulin-P, which are known to be present inamaranth, have been reported to polymerize being composed of di-meric subunits linked by disulphide bonds (Martinez et al., 1997).

4. Conclusion

The tenacity of amaranth, oat and quinoa doughs was lowerthan that of semolina dough sample. The elastic modulus of ama-ranth, oat and quinoa dough samples was higher than that of sem-olina dough. The G0 values of amaranth and quinoa were similarbut significantly 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 lowestwas shown by oat. The semolina dough showed G0 and G00 valuessignificantly lower than those of all the other dough samples.The oat and quinoa doughs had similar tan d values, at low and

able proteins (b, d, f, h) of semolina, amaranth, quinoa and oat dough samples.

Fig. 7 (continued)

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

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

showed polymeric proteins with high molecular weight distribu-tion (about 3.000 kDa) such as in semolina dough. However, thepercentage of these proteins that was unextractable were signifi-cantly different among amaranth, quinoa and oat doughs and re-sulted 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-scaledeformation, will be carried out.

Acknowledgement

This research work was financially support by Italian Puglia Re-gion, Strategic Project ‘‘Process innovation for production of func-tional pasta”, PS_003.

References

Aussenac, T., Carceller, J.L., Kleiber, D., 2001. Change in SDS-solubility of gluteninpolymers during dough mixing and resting. Cereal Chemistry 78, 39–45.

Belton, P.S., 1999. On the elasticity of wheat gluten. Journal of Cereal Science 29,103–107.

Bietz, J.A., Wall, J.S., 1972. Wheat glutenin subunits: molecular weights determinedby sodium dodecylsulphate–polyacrylamide gel electrophoresis. CerealChemistry 49, 416–430.

Carceller, J.L., Aussenac, T., 2001. SDS-insoluble glutenin polymer formation indeveloping grains of hexaploid wheat: the role of the ratio of high to lowmolecular weight glutenin subunits and drying rate during ripening. AustralianJournal of Plant Physiology 26, 301–310.

Castellani, O., Martinez, E.N., Anon, M.C., 1998. Structural modifications of amaranthglobulin induced by pH and NaCl. Journal of Agricultural and Food Chemistry46, 4846–4853.

Chen, S., Paredes-Lopez, O., 1997. Isolation and characterization of the 11S globulinfrom amaranth seeds. Journal of Food Biochemistry 21, 53–65.

Chillo, S., Laverse, J., Falcone, P.M., Protopapa, A., Del Nobile, M.A., 2008. Influence ofthe addition of buckwheat flour and durum wheat bran on spaghetti quality.Journal of Cereal Science 47, 144–152.

Cornec, M., Popineau, Y., Lefebvre, J., 1994. Characterisation of gluten subfractionsby SE-HPLC and dynamic rheological analysis in shear. Journal of Cereal Science19, 131–139.

D’Ovidio, R., Masci, R., 2004. The low-molecular-weight glutenin subunits of wheatgluten. Journal of Cereal Science 39, 321–339.

Del Nobile, M.A., Chillo, S., Mentana, A., Baiano, A., 2007. Use of the generalizedmaxwell model for describing the stress relaxation behavior of solid-like foods.Journal of Food Engineering 78, 978–983.

Don, C., Lookhart, G., Naeem, H., MacRitchie, F., Hamer, R.J., 2005. Heat stress andgenotype affect the glutenin particles of the glutenin macropolymer-gelformation. Journal of Cereal Science 42, 69–80.

Edwards, N.M., Dexter, J.E., Scanlon, M.G., 2001. The use of rheological techniques toelucidate durum wheat dough stretch properties. In: The Fifth ItalianConference on Chemical Process Engineering, vol. 2. Florence, Italy, pp. 825–830.

Edwards, N.M., Dexter, J.E., Scanlon, M.G., Cenkowski, S., 1999. Relationship ofcreep-recovery and dynamic oscillatory measurements to durum wheatphysical dough properties. Cereal Chemistry 76, 638–645.

Edwards, N.M., Mulvaney, S.J., Scanlon, M.G., Dexter, J.E., 2003. Role of gluten and itscomponents in determining durum semolina dough viscoelastic properties.Cereal Chemistry 80, 755–763.

Fares, C., Codianni, P., Nigro, F., Platani, C., Scazzina, F., Pellegrini, N., 2008.Processing and cooking effects on chemical, nutritional and functionalproperties of pasta obtained from selected emmer genotypes. Journal of theScience of Food and Agriculture 88, 2435–2444.

Ferry, J.D., 1980. Viscoelastic Properties of Polymers. John Wiley and Sons, NY, USA.Fisichella, S., Alberghino, G., Amato, M.E., Lafiandra, D., Mantarro, D., Palermo, A.,

Savarino, A., Scarlata, G., 2003. Purification of wheat flour high-Mr gluteninsubunits by Reactive Red 120-Agarose and reactive yellow 86-Agarose resin.Journal of Cereal Science 38, 77–85.

Gallagher, E., Gormeley, T.R., Arendt, E.K., 2004. Recent advances in the formulationof gluten-free cereal-based products. Trends in Food Science Technology 15,143–152.

Gobin, P., Ng, P.K.W., Buchanan, B.B., Kobrehel, K., 1997. Sulphydryl-disulfidechanges in proteins of developing wheat grain. Plant Physiology andBiochemistry 35, 777–783.

Grosch, W., Wieser, H., 1999. Redox reactions in wheat dough as affected byascorbic acid. Journal of Cereal Science 29, 1–16.

Gupta, R.B., Khan, K., MacRitchie, F., 1993. Biochemical basis of flour properties inbread wheats. I. Effects of variation in the quantity and size distribution ofpolymeric protein. Journal of Cereal Science 18, 23–41.

Huang, D.Y., Khan, K., 1997. Quantitative determination of high molecular weightglutenin subunits of hard red spring wheat by SDS–PAGE. I. Quantitative effectsof total amounts on breadmaking quality characteristics. Cereal Chemistry 74,781–785.

Janssen, A.M., van Vliet, T., Vereijken, J.M., 1996. Fundamental and empiricalrheological behavior of wheat flour doughs and comparison with bread makingperformance. Journal of Cereal Science 23, 43–54.

Johansson, E., Prieto-Linde, M.L., Jonsson, J.O., 2001. Effects of wheat cultivar andnitrogen application on storage protein composition and breadmaking quality.Cereal Chemistry 78, 19–25.

Kasarda, D.D., 1999. Glutenin polymers: the in vitro to in vivo transition. CerealFoods World 44, 566–571.

Khatkar, B.S., Schofield, J.D., 2002. Dynamic rheology of wheat flour dough. I. Non-linear viscoelastic behaviour. Journal of the Science Food and Agriculture 82,827–829.

Koziol, M.J., 1992. Chemical composition and nutritional evaluation of quinoa(Chenopodium quinoa Willd). Journal of Food and Compositional Analysis 5, 35–68.

Kuktaite, R., Johansson, E., Juodeikiene, G., 2000. Composition and concentration ofproteins in Lithuanian wheat cultivars: relationships with bread-makingquality. Cereal Research Communications 28, 195–202.

Kuktaite, R., Larsson, H., Johansson, E., 2003. Protein composition in different phasesobtained by the ultracentrifugation of dough. Acta Agronomica Hungarica 51,163–172.

Lafiandra, D., Masci, S., Blumenthal, C., Wrigley, C.W., 1999. The formation ofglutenin polymers in practice. Cereal Foods World 44, 572–578.

Larroque, O.R., Gianibelli, M.C., Batey, I.L., MacRitchie, F., 1996. Identification ofelution subfractions from the first peak in SE-HPLC chromatograms of wheatstorage proteins. In: Wrigley, C.W. (Ed.), Proceedings of Sixth InternationalGluten Workshop. Cereal Chemistry Division, Royal Australian ChemicalInstitute, North Melbourne, Australia, pp. 228–293.

Lindsay, M.P., Skerritt, J.H., 1998. Examination of the structure of the gluteninmacropolymer in wheat flour and dough by stepwise reduction. Journal ofAgricultural and Food Chemistry 64, 3447–3457.

Lindsay, M.P., Skerritt, J.H., 1999. The glutenin macropolymer of wheat flourdoughs: structure–function perspective. Trends in Food Science and Technology10, 247–253.

Lorimer, N., Zabik, M.E., Harete, J.B., Stchiw, N.C., Uebersax, M.A., 1991. Effect ofnavy bean protein flour and bean globulins on composite flour rheology,chemical bonding and microstructure. Cereal Chemistry 68, 213–220.

MacRitchie, F., Lafiandra, D., 1997. Structure–function relationship of wheatproteins. In: Food Proteins and Their Application. S. Marcel Dekker, New York,pp. 293–323.

Mancini, M., Moresi, M., Rancini, R., 1999. Mechanical properties of alginate gels:empirical characterization. Journal of Food Engineering 39, 369–378.

Marcone, M.F., 1999. Evidence confirming the exsistence of a 7S globulin-likestorage protein in Amaranthus hypocondriacus seed. Food Chemistry 65, 533–542.

Marcone, M.F., Yada, R.Y., 1997. Sulfhydryl and disulfide groups of the oligomericseed globulin from Amaranthus hypocondriacus K343. Journal of FoodBiochemistry 21, 255–277.

Marcone, M.F., Beniac, D., Harauz, G., Yada, R., 1994. Quaternary structure andmodel for the oligomeric seed globulin of Amaranthus hypocondriacus K343.Journal of Agricultural and Food Chemistry 42, 2675–2678.

Marcone, M.F., Kakuda, Y., Yada, R.Y., 1997. Salt-soluble seed globulins ofdicotyledonous and monocotyledonous plants II. Structural characterization.Food Chemistry 63, 265–274.

Martinez, E., Castellani, O., Anon, M.C., 1997. Common molecular features amongamaranth storage proteins. Journal of Agricultural and Food Chemistry 45,3832–3839.

Masi, P., Cavella, S., Sepe, M., 1998. Characterization of dynamic viscoelasticbehaviour of wheat flour doughs at different moisture contents. CerealChemistry 75, 428–432.

Mead, D.W., 1994. Determination of molecular weight distributions of linearflexible polymers from linear viscoelastic material functions. Journal ofRheology 38, 1769–1795.

Miller, K.A., Hoseney, R.C., 1999. Dynamic rheological properties of wheat starch–gluten doughs. Cereal Chemistry 76, 105–109.

Moulton, A.L.C., 1959. The place of oat in celiac diet. Archives Disease Childhood238, 687–691.

Peterson, D.M., Brinegar, C., 1986. Storage proteins in oats. In: Webster, F. (Ed.),Chemistry and Technology. AACC, St. Paul, MN, pp. 153–203.

Popineau, Y., Cornec, M., Lefebvre, J., Marchylo, B., 1994. Influence of high Mrglutenin subunits on glutenin polymers and rheological properties of glutensand gluten subfractions of near-isogenic lines of wheat sicco. Journal of CerealScience 19, 231–241.

Rao, M.A., Steffe, J.F., 1992. Viscoelastic Properties of Foods. Elsevier AppliedScience, New York, NY, USA.

Rao, V.K., Mulvaney, S.J., Dexter, J.E., 2000. Rheological characterization of long- andshort-mixing flours based on stress–relaxation. Journal of Cereal Science 31,159–171.

Rhazi, L., Cazalis, R., Lemelin, E., Aussenac, T., 2003. Changes in the glutathione thiol-disulphide status during wheat grain development. Plant Physiology andChemistry 41, 895–902.

Richardson, R.K., Morris, E.R., Ross-Murphy, S.B., Taylor, L.J., Dea, I.C.M., 1989.Characterisation of the perceived texture of the thickened systems by dynamicviscosity measurements. Food Hydrocolloids 3, 175–191.

Romero-Zepeda, H., Paredes-Lopez, O., 1996. Isolation and characterization ofamarantin, the 11S amaranth seed globulin. Journal of Food Biochemistry 19,329–339.

Ross-Murphy, S.B., 1988. Small deformation measurements. In: Blanshard, J.M.V.,Mitchell, J.R. (Eds.), Food Structure: Its Creation and Evaluation. Butterworths,London, UK, pp. 387–400.

Schofield, J.D., 1986. Flour proteins: structure and functionality in baked products.In: Blanshard, J.M.V. (Ed.), Chemistry and Physics of Baking. Royal Society ofChemistry Inc., London, pp. 14–28.

Schofield, J.D., 1994. In: Bushuk, W., Rasper, V.F. (Eds.), Wheat Production,Properties and Quality, first ed. Blackie, Glasgow, pp. 73–99.

106 C. Lamacchia et al. / Journal of Food Engineering 96 (2010) 97–106

Segura-Nieto, M., Barba de la Rosa, A.P., Paredes-Lopez, O., 1994. Biochemistry ofamaranth proteins. In: Pared-Lopez, O. (Ed.), Amaranth: Biology, Chemistry andTechnology. CRC Press, Boca Raton, FL, pp. 75–106.

Shewry, P.R., Thatam, A.S., 1990. The prolamin storage proteins of cereal seeds:structure and evolution. Biochemical Journal 267, 1–12.

Shewry, P.R., Sayanova, O., Tatham, A.S., Tamas, L., Turner, M., Richard, G., Hickman,D., Fido, R., Halford, N.G., Greenfield, J., Grimwade, B., Thomson, N., Miles, M.,Freedman, R., Napier, J., 1995. Structure, assembly and targeting of wheatstorage proteins. Journal of Plant Physiology 145, 620–625.

Shiau, S.Y., Yeh, A.I., 2001. Effect of alkali and acid on dough rheologicsl propertiesand characteristics of extruded noodles. Journal of Cereal Science 33, 27–37.

Song, Y., Zheng, Q., 2007. Dynamic rheological properties of wheat flour dough andproteins. Trends in Food Science and Technology 18, 132–138.

Steffe, J.F., 1996. Rheological Methods in Food Process Engineering, second ed.Freeman Press, East Lansing, MI, USA.

Subramanian, R., Muthukumarappan, K., Gunasekaran, S., 2006. Linear viscoelasticproperties of regular- and reduced-fat pasteurized process cheese duringheating and cooling. International Journal of Food Properties 9, 377–393.

Thatam, A.S., Hayes, L., Shewry, P.R., Urry, D.W., 2001. Wheat seed proteins exhibit acomplex mechanism of protein elasticity. Biochimica et Biophysica Acta 1548,187–193.

Thorvaldsson, K., Stading, M., Nilsson, K., Kidman, S., Langton, M., 1999. Rheologyand structure of heat-treated pasta dough: influence of water content andheating rate. Lebensmittel-Wissenschaft und -TechnologieLebnsm 32, 154–161.

Uthayakumaran, S., Newberry, M., Keentok, M., Stoddard, F.L., Bekes, F., 2000. Basicrheology of bread dough with modified protein content and glutenin-to-gliadinratios. Cereal Chemistry 77, 744–749.

Van Etten, C.H., Miller, R.W., Wolff, I.A., Jones, Q., 1963. Aminoacid composition ofseeds from 200 angiosperm plants. Journal of Agricultural and Food Chemistry11, 399–410.

Weegels, P., 1996. Functional properties of wheat glutenin. Journal of Cereal Science23, 1–17.

Weipert, D., 1990. The benefits of basic rheometry in studying dough rheology.Cereal Chemistry 67, 311–317.