Rheological properties of soy protein hydrolysates obtained from limited enzymatic hydrolysis

Rheological properties of soy protein hydrolysates obtained from limited enzymatic hydrolysis

ARTICLE IN PRESS LWT 40 (2007) 1215–1223 www.elsevier.com/locate/lwt Rheological properties of soy protein hydrolysates obtained from limited enzyma...

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ARTICLE IN PRESS

LWT 40 (2007) 1215–1223 www.elsevier.com/locate/lwt

Rheological properties of soy protein hydrolysates obtained from limited enzymatic hydrolysis B.P. Lamsal, S. Jung, L.A. Johnson Department of Food Science and Human Nutrition, and Center for Crops Utilization Research, Iowa State University, Ames, IA, USA Received 23 February 2006; received in revised form 24 August 2006; accepted 31 August 2006

Abstract Soy protein products hexane-defatted soy flour, extruded-expelled soy flour, soy protein concentrate and soy protein isolate, were modified by using the enzyme bromelain to 2% and 4% degrees of hydrolysis (DH). Peptide profiles, water solubility, and rheological properties including dynamic shear, large deformation, and apparent viscosities of resulting hydrolysates were determined. Protein subunits profiles for the hydrolysed isolates and concentrates were extensively altered by the treatment while only minor changes were observed for the hydrolysed flours. Water solubility profiles of all hydrolysates in the pH range of 3.0–7.0 were enhanced by hydrolysis. For the unhydrolysed controls, the isolate had the highest storage modulus (G0 ), followed by the concentrate, the extruded-expelled flour and the hexane-defatted flour. The hydrolysates retained some of their gelling ability even though the losses in storage modulus (G0 ) were substantial. After heating step to 95 1C, the G0 values of all substrates at 25 1C decreased with increase in DH. Texture profile analyses of the soy protein gels were also lower in hardness after hydrolysis. The Power Law model provided excellent fit to hydrolysate dispersions flow ðR2 40:99Þ. Hydrolysis decreased the consistency coefficients of dispersion and increased flow behavior index resulting in thinner dispersions. These results suggest that limited protease hydrolysis of various soy protein meals with bromelain produce soy protein ingredients with modified rheological properties. r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Soy proteins; Limited hydrolysis; Rheology; Gelation; Texture analysis

1. Introduction Food proteins, both animal and plant proteins, play critical roles in human nutrition. This traditional role aside, proteins in food formulations are increasingly expected to perform functional roles that are important to consumer food acceptance. Food proteins possess physico-chemical properties that govern their performance and behavior in food systems during processing, storage and consumption that are collectively termed functional properties. Such properties also provide the basis for utilizing an ingredient in food applications. Some examples of novel uses include use in snack and energy bars, hard pretzels, sport beverages, whole soy ingredients, egg replacements in bakery products, infant formulae, coating systems, tortillas

Corresponding author. Tel.: +1 515 294 2544; fax: +1 515 294 8181.

E-mail address: [email protected] (S. Jung).

and breads (Pszczola, 2005). Soy proteins undergo harsh processing conditions involving heat, shear, and exposure to acid, and may lose much of their functionalities. Limited or controlled enzymatic hydrolysis of soy proteins could provide ingredients with desired or restored functionalities (Panyam & Kilara, 1996; Surowka, Zmudzinski, & Surowka, 2004; Surowka, Zmudzinski, Fik, Macura, & Lasocha, 2004; Jung, Murphy, & Johnson, 2005). Ingredients that form strong gels and give high viscosity are preferred for use in comminuted meat products and gravies, while yogurts, soups, and infant food formulations require less viscous product mix and weaker gelling properties. The extent of proteolysis during enzymatic hydrolysis can be established with the degree of hydrolysis (DH). Hydrolysates intended for nutritional formulations, e.g., hypoallergenic hydrolysates, are extensively hydrolysed, with 90% of peptides being o500 Da in molecular weight (Mahmoud, 1994). Hydrolysates intended for use as nutritional supplements undergo slight

0023-6438/$30.00 r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2006.08.021

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(about 90% peptides 45000 Da) or moderate (about 46% peptides 45000 Da) hydrolysis. Enzymatic hydrolysis of protein employs various proteases, bromelain being one such protease. Bromelain is an endoprotease chiefly used for muscle tenderization (Melendo, Beltran, & Roncales, 1997; Teran, 2003; Kolle, McKenna, & Savell, 2004), and producing acid-coagulation resistant milk (Christensen, Florin, & Harris, 2002). Studies using bromelain to improve physicochemical and functional properties of proteins have focused on emulsion formation and water-holding capacity (Karakaya & Ockerman, 2002), umami chicken flavor development (Maehashi, Matsuzaki, Yamamoto, & Udaka, 1999), soy protein solubility and foaming properties (Molina-Ortiz & Wanger, 2002), and controlling of gluten network formation in low fat pastry products (Hart, 1999). Native, globular proteins are generally resistant to enzyme hydrolysis due to their compact tertiary structures that protect many of the peptide bonds (Adler-Nissen, 1976). Denatured proteins, like most processed soy proteins, have exposed peptide bonds available for enzymatic cleavage. Enzymatic hydrolysis can be responsible for (1) a decrease in hydrolysate molecular weight, (2) an increase in ionizable group number, and (3) exposure of previously concealed hydrophobic groups (Panyam & Kilara, 1996). Changes in functional properties, including rheological characteristics, are a direct result of the abovementioned changes. Structural modifications to the two major soy protein components, namely glycinin and b-conglycinin, by proteases (endo or exo-peptidases) dictate the resultant hydrolysate properties (Jung et al., 2005). Gel hardness and fracturability of soy proteins are attributed to glycinin, whereas b-conglycinin contributes to gel elasticity (Utsumi, Matsumura, & Mori, 1997). Extensive literature is available on the enzymatic modification of soy proteins from moderate to high DH by proteolytic enzymes and effects on hydrolysate functional properties (Kim, Park, & Rhee, 1990; Achouri, Zhang, & Shiying, 1998; Calderon-de-la Barca, RuizSalazar, & Jara-Maarini, 2000; Hrckova, Rusnakova, & Zemanovic, 2002; Tsumura et al., 2005). The effects of limited hydrolysis on rheological properties of soy proteins, however, are not understood. The aim of the present study was to determine the rheological properties (gelation and viscosity) of soy protein products, which have undergone limited protease hydrolysis (up to 4% DH). 2. Materials and methods 2.1. Substrates Four commercial soy protein products were used as starting materials; a hexane-defatted soy flour (SF, Nutrisoy 7B), a soy protein concentrate (SPC, Acron S) and a soy protein isolate (SPI, Profam 955), which were acquired from Archer Daniels Midland Co. (Decatur, IL) and an extruded-expelled soy flour (EEF), which was

acquired from Iowa Soy Specialities (Vinton, IA). The crude protein contents for the substrate as determined in our laboratory were: SF, 54 g/100 g, EE, 49 g/100 g, SPC, 77 g/100 g, and SPI, 93 g/100 g on as is basis. Protein dispersibility index (PDI) was determined externally by Eurofins Scientific Inc. (Des Moines, IA) and were 88, 68, 71, and 11 for SF, EE, SPC, and SPI, respectively. 2.2. Enzymatic hydrolysis The food-grade enzyme bromelain, an endopeptidase derived from pineapple stems (Bio-Cat Inc., Troy, VA) was used to carry out the hydrolysis. The enzyme activity was specified as 2000 gelatin digestion units (GDU)/g protein. Hydrolysis was performed at 50 1C and pH 7.0. DH was controlled and determined by using the pH-Stat method (Adler-Nissen, 1986), which determines the %DH on the basis of the number of free titratable amino groups produced by hydrolysis of peptide bonds. Compared to other methods, the pH stat method allows direct monitoring of DH in real time. DH was calculated using the following equation: DH ¼ ½ðVNaOH  NNaOH Þ=ða  MP  htot Þ  100%, where a is the degree of dissociation of a-amino groups, MP is the mass of protein (g), htot is the number of peptide bonds in the substrate (meqv/g protein), concentration of the base (NaOH) was 2 mol/l, a value was 0.44, and htot was 7.8 (Adler-Nissen, 1986). Hydrolysis of the 10 g/100 g substrate dispersion was carried out in a 250-mL temperature-controlled glass reactor with constant stirring. pH during hydrolysis was controlled by using a titrator (718 STAT Trition, Brinkmann, Westbury, NY). Appropriate enzyme-to-substrate ratios were selected to reach 2% or 4% DH values, wherein a plateau in DH over time was achieved. Freezing the hydrolysates, which were later freeze-dried, quickly stopped enzymatic reaction. The unhydrolysed control dispersion was prepared at the same pH, temperature and reaction time as was necessary to achieve 4% DH and similarly freeze-dried. Crude protein contents of the freezedried hydroylsates were the same as those of the corresponding substrates. 2.3. Sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed on unhydrolysed controls and enzyme hydrolysates to determine the effects of the enzyme treatments on the protein polypeptide profiles. SDS-PAGE was carried out using a SDS-Tris-glycine buffer system with 4% stacking gels and 13% resolving gels (Mini Protean II Gel, Biorad Inc., Hercules, CA) following methods of Jung et al. (2005). A low-range molecular weight (MW) marker ranging from 66 to 6.5 kDa (M3913, Sigma Chemical Co., St. Louis, MO) and laboratorypurified b-conglycinin and glycinin were used as standards.

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Ten ml of protein in sample buffer and 5 ml of standards, both at 1 mg/ml concentration, were loaded per lane.

offered by the gel during compression was taken as gel hardness.

2.4. Solubility profile

2.7. Apparent viscosity

Solubility profiles of the protein hydrolysates in water over the pH range of 3.0–7.0 were determined. Freeze-dried hydrolysates were suspended in deionized water at 1 g/100 g protein concentration. The pH of the dispersion was adjusted to 3, 4, 5, 6, and 7 with 2 mol/l NaOH or 2 mol/l HCl and stirred at room temperature for 1 h, readjusting the pH if necessary. The dispersions were then centrifuged at 10,000  g for 15 min at 20 1C (Avanti J-20, fixed-angle rotor JLA 10.5, Beckman Coulter, Fullerton, CA). Crude protein content in the supernatant was determined by using the Biuret method with bovine serum albumin (A-7906, Sigma Chemical Co., St. Louis, MO) as standard. Solubility was expressed as the percentage of original protein present in the supernatant.

Shear stresses developed with applied shear rates ranging from 10 to 500 s1 for 10 g/100 g protein dispersions were measured with the same cone-and-plate probe previously described. These flow curves were modeled using the Power Law model:

2.5. Steady shear rheology A 20.0 ml 10 g/100 g protein dispersion in deionized water at pH 7 was prepared for each soy protein hydrolysate. The dispersion was allowed to equilibrate overnight at 4 1C and stirred for 30 min at room temperature before analysis. Degassing the dispersions was done in 100-ml vacuum flasks. Small-amplitude oscillatory shear tests were performed with a dynamic rheometer (RS 150 Haake, Karlsruhe, Germany) using a cone-and-plate probe (60 mm  21) and 0.105 mm gap. Two to 3 ml of hydrolysate dispersion was loaded and excess sample was wiped off the bottom plate. A thin layer of silicone oil and lubricated O-ring was put around the bottom plate edge to prevent drying. Oscillatory strain of 1% was applied at 0.1 Hz frequency while the dispersion was heated in situ from 25 to 95 1C (2 1C min1), held for 3 min, and cooled to 25 1C at 2 1C min1. Evolution of storage modulus (G0 ) with temperature was monitored. At least three measurements were performed for duplicate dispersions prepared for each hydrolysate and mean values reported. The stock dispersion was kept stirring during measurement period. 2.6. Texture profile analysis (TPA) Fifteen ml of a 10 g/100 g protein dispersion at pH 7.0 was poured into three glass bottles (2.5 cm ID  4.8 cm height) and tightly closed. They were shaken for 20 min, immersed in a 96 1C water bath for 30 min, chilled in a 20 1C water bath for 30 min, and stored overnight at 4 1C. Gels were equilibrated to room temperature for 2 h before compression with an acrylic probe (1.2 mm ID  35 mm height) in a texture analyzer (TA-XT2, Texture Technologies Corp., Scarsdale, NY). The uniaxial compression at the rate of 0.5 mm/s was applied twice to the depth of 12 mm (37% deformation). The maximum resistance

t ¼ Kgn , where t is shear stress, K is consistency coefficient, g is shear rate, and n is flow behavior index. Apparent viscosities were calculated at shear rates of 10 and 500 s1 from the fitted parameters. 2.8. Crude protein and moisture determination The nitrogen contents of the samples were determined with a combustion-type nitrogen analyzer (Rapid N-Analyzer, Elementar Americas, NJ). Nitrogen contents were converted to crude protein contents using a factor of 6.25. Moisture contents were determined by drying the samples in a forced-draft oven at 130 1C for 3 h. 2.9. Statistical analysis General linear model, PROC GLM, in SAS system (Version 8.2, SAS Institute, Inc., Cary, NC) was used to compare the means at po0:05. 3. Results and discussion 3.1. Soy protein hydrolysis Fig. 1 shows typical progression of hydrolysis (DH) with time for soy protein substrates when hydrolysed with bromelain. The appropriate enzyme-to-substrate (E/S) ratios were identified to produce a plateau at 2% or 4% DH. The reaction progressed rapidly for the first 15 min and then relatively slowly over time reaching a plateau. This exponential reaction was typical of protease hydrolysis (Kim et al., 1990). The no-enzyme control substrates displayed DH of 0.5%, 0.35%, 0.07%, and 0.12% DH for SF, EEF, SPC, and SPI, respectively. This DH was due to the solubilization of the proteins with time, which decreased the pH, therefore the DH value of the controls did not reflect any proteolytic activity (Jung et al., 2005). The reaction times to reach 2% and 4% DH with appropriate E/S ratio (g enzyme per g protein) for all substrates are presented in Table 1. For SF and EEF, 2% DH was achieved in less than one-half the time with about one-half the amount of enzyme than used to achieve 4% DH. For SPC and SPI, the times needed to reach 2% DH were more than those needed to reach 4% DH when E/S ratio was reduced to less than one-half.

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4.5 4

Degree ofhydrolysis, %

3.5 3 2.5 2 1.5 1 0.5 0 0

5

10

15

20 25 Time, min

30

35

40

45

Fig. 1. Typical progression of degree of hydrolysis with time for soy protein substrates (shown: extruded-expelled soy flour) when acted upon by bromelain. Symbols: (&) 4% DH, (B) 2% DH, and (n) unhydrolysed control.

Table 1 Mean ðn ¼ 2Þ hydrolysis reaction times and enzyme-to-substrate (E/S) ratios for each soy protein substrate Soy protein substrate

Degree of hydrolysis (%)

E/S ratio (%)

Time to plateau (min)

SF

4 2 4 2 4 2 4 2

0.51 0.24 0.27 0.12 0.37 0.17 0.61 0.14

65a 23d 39b 21.5d 39.5b 64a 22d 34.5c

EEF SPC SPI

Reaction times sharing same superscript are not significantly different ðpo0:05Þ. SF: soy flour; EEF: extruded-expelled soy flour; SPC: soy protein concentrate; and SPI soy protein isolate. The unhydrolysed control for a given substrate was treated for the longest hydrolysis reaction time without enzyme added. Enzyme-to-substrate ratio: g of enzyme per g of protein  100 (%).

The extent of protein denaturation (i.e., PDI value) probably affected the reaction time to reach a given DH. SPI had the lowest PDI value (i.e. higher extent of denatured protein), which seems to have promoted cleavage sites access of bromelain leading to 4% DH in about 22 min. For SF, which had the highest PDI value (i.e., less protein denaturation), 4% DH was attained after 65 min reaction time. Longer reaction times for higher PDI soy substrates have also been reported by Henn and Netto (1998) and Jung et al. (2005). 3.2. Hydrolysate peptide profiles Modifications to the peptide profiles were substratespecific, with major changes for SPI and SPC and minor

changes for SF and EEF (Fig. 2). The protein subunit bands present in all unhydrolysed control samples, namely subunits of b-conglycinins, and acidic and basic subunits of glycinin, almost entirely disappeared in SPI and SPC hydrolysates at 2% and 4% DH. On the other hand, most of the a0 , a, and b subunits of b-conglycinin were hydrolysed in EEF and SF. Glycinin subunits were largely intact in EEF and SF at 2% DH, but acidic subunits were hydrolysed to some extent at 4% DH. The basic subunits of glycinin, however, were not hydrolysed in EEF, and SF, which was expected because these subunits reside in the interior of the undenatured glycinin complex and therefore are less exposed to enzymatic attack. This observation agreed with the findings of Lakemond, de Jongh, Hessing, Gruppen and Voragen (2000), who observed that the enzyme clostripain did not affect the basic polypeptides of the soy glycinin complex, whereas the acidic polypeptides, which predominantly face outside the complex, were degraded by the enzyme at ionic strengths below 0.5. These results confirm that the extent of substrate denaturation plays a major role in enzymatic hydrolysis (AdlerNissen, 1976; Jung, et al., 2005). Hydrolysis of ethanolwashed SPC (71 PDI), however, showed almost complete degradation of protein subunits. This hydrolysis behavior can be explained by the fact that this particular SPC had denatured glycinin and b-conglycinin as shown by Jung et al. (2005). EEF and SF hydrolysates showed new bands at approximately 25 and 30 kDa, whereas SPI showed very faint bands appearing below 20 kDa. Small peptides appeared at the bottom of the gel for all hydrolysates suggesting the presence of peptides o6:5 kDa.

3.3. Water solubility profiles Fig. 3 shows water solubility profiles for soy protein hydrolysates at 2% and 4% DH and corresponding unhydrolysed controls. The solubility profiles for all hydrolysates were the typical U-shaped curves with solubility being higher on either side of isoelectric point (around pH 4.5 for soy proteins). The unhydrolysed controls at pH 7 had protein solubilities close to their PDI values, except for SPC. Hydrolysis up to 4% DH increased their solubilities of all substrates compared to control samples (2% and 4% DH hydrolysates being similar in solubility). The percentages of increased solubility for the 4% DH hydrolysates at pH 7 over the corresponding unhydrolysed control were 360, 225, and 36 for SPI, SPC, and EE, with SF showing no change. The increased solubility of the hydrolysates over the unhydrolysed control were attributed to the production of soluble peptides (Tsumura et al., 2005) and increased number of exposed ionizable amino and carboxyl groups (Panyam & Kilara, 1996) during hydrolysis, which in turn depended on the degree of denaturation (Adler-Nissen, 1976). Therefore, it was not surprising that SPI with the lowest PDI had the highest increase in solubility. The increased solubility of the

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EEF MW (kDa) β-con Gly M

C 2%

SF 4%

C

1219

SPI

2%

4%

M

C 2%

SPC 4% C

2% 4%

66 44 36

A

29 24 20

B

14.2 6.5 Fig. 2. Peptide profiles for the unhydrolysed control and bromelain-modified soy protein substrates at 2% and 4% DH. MW, molecular weight; b-con, bconglycinin; Gly, Glycinin; M, MW Marker; C, control, A, Gly acidic, and B, Gly basic subunits; EEF, extruded-expelled soy flour; SF, hexane-defatted soy flour, SPI, soy protein isolate; and SPC, soy protein concentrate.

SF

SPI 100

100

80 Solubility (%)

Solubility (%)

80 60 40

60 40 20

20

0

0 2

3

4

5 pH

6

7

8

2

3

4

5 pH

7

8

SPC

100

100

80

80 Solubility (%)

Solubility (%)

EEF

6

60 40

60 40 20

20

0

0 2

3

4

5 pH

6

7

8

2

3

4

5 pH

6

7

8

Fig. 3. Water solubility profiles of control (n), and 2 (’), and 4% DH (~) bromelain hydrolysates from soy flour, SF (top left); soy protein isolate, SPI (top right); extruded-expelled soy meal, EEF (bottom left); and soy protein concentrate, SPC (bottom right).

SPC hydrolysate was attributed to the denaturation state of the glycinin and b-conglycinin, as previously reported. 3.4. Storage modulus (G0 ) and gelation temperature Storage modulus (G0 ) and loss modulus (G00 ) are two viscoelastic parameters indicating gel strength. After heating beyond a certain temperature, G0 values rise dramatically due to increased aggregation of proteins, indicating stronger and more elastic gels. This information

is useful in controlling texture and mouthfeel in various food applications such as comminuted meats and sausages, yoghurts and puddings, products in which soy proteins are used. Fig. 4 shows representative evolution of storage modulus (G0 ) with heating and cooling of 10 g/100 g soy protein hydrolysate dispersion. During heating, storage modulus G0 was small and almost constant until a certain temperature was reached, at which it rapidly increased indicating transition from a liquid-like state (sol) to a solid-like state. This temperature

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1220

7000

Table 2 Gel storage moduli for 10 g/100 g soy protein dispersions during heating and cooling

Storage modulus, G', Pa

6000

Soy protein substrate

5000 4000

SPI

Cooling 3000

SPC

2000 SF

Tgel

1000

EEF

0 20

30

40

50 60 70 Temperature, °C

80

90

100

Fig. 4. Representative storage modulus (G0 ) and temperature curve for 10% w/w soy protein dispersions during heating and cooling.

is usually taken as gelation temperature (Tgel) and corresponds to the temperature at which G0 increases and becomes greater than the background noise, which is one of the common methods of detecting the gelling point in the absence of a crossover between G0 and G00 (Matsumura & Mori, 1996; Ould Eleya & Gunasekaran, 2002). Gelation temperature is usually above the denaturation temperature and denaturation is a prerequisite for heat-induced gelation of globular proteins (Nagano, Hirotsuka, Mori, Kohyama, & Nishinari, 1992; Utsumi et al., 1997; Ould Eleya & Gunasekaran, 2002). Heat denaturation opens up buried hydrophobic patches inside protein molecules and facilitates subsequent peptide associations and formation of the gel network structure. Storage modulus further increases during subsequent cooling, which is seen in Fig. 4. Such an increase during cooling, called gel reinforcement (Ould Eleya & Gunasekaran, 2002), is typical of protein gels and generally attributed to consolidation of attractive forces such as van der Waals and hydrogen bonding between proteins within gel primary network. Table 2 shows Tgel, and G0 values during heating and cooling of hydrolysate dispersions. The Tgel values for untreated substrates ranged between 80 and 86 1C, which is within the reported range for soy proteins (Nagano et al., 1992; Nagano, Akasaka, & Nishinari, 1994; Utsumi et al., 1997). b-conglycinin starts denaturing at around 65 1C and peaks at 70 1C, whereas glycinin denaturation starts at around 80 1C and a gel network begins to form (Yamauchi, Yamagishi, & Iwabuchi, 1991). While onset of gelation coincides with onset temperature of denaturation of b-conglycinin and glycinin solutions, onset of gelation is greater than the denaturation temperature in soy protein preparations (Kang & Lee, 2005). The Tgel values for the 2% and 4% DH hydrolysates were slightly lower than for the control samples of SF and

Degree of hydrolysis (%)

Control 2 4 Control 2 4 Control 2 4 Control 2 4

Tgel, 1C mean  SE

82.570.3cd – – 80.670.4cde 79.170.7e 85.270.4ab 83.073bc 79.570.8de 77.971.2ef 86.470.3a 75.770.9f 79.171.4e

Storage modulus (G0 ), kPa 95 1C  SE, Heating

25 1C  SE, Cooling

5.671.5b 0.0870.01d 0.270.02d 36.677.3a 0.770.2cd 0.770.1cd 4.270.6bc 0.970.1cd 0.770.0cd 4.470.5bc 1.370.b1cd 1.270.1cd

3747120a 5.470.9d 4.670.3d 176.3730.4b 12.073.4d 6.170.8d 38.071.8c 6.070.3d 5.070.4d 94.0722.2c 6.270.3d 5.470.3d

SE: standard error of mean. Means sharing the same superscript in a given column are not significantly different at po0:05.

EEF. Heat-set gels produced by non-hydrolysed SPI were the strongest (highest G0 value), followed by SPC, EEF and SF. Although the hydrolysates retained some gelling ability, hydrolysis of up to 4% DH caused important losses in gel strengths for all the substrates, ranging between 6- and 75-fold compared to the controls. The reduced hydrophobicity of protein hydrolysates caused by enzymatic hydrolysis (Fan et al., 2005; Jung et al., 2005) and the drop in sulfhydryl exchange reactions during gelation (Fan et al., 2005) may explain the inferior gel forming abilities of the hydrolysates. In addition, increased charge repulsion between peptides due to net charge increase upon hydrolysis may have occurred and contributed to the decreased gelling ability (Panyam & Kilara, 1996). The G0 values at 25 1C were similar for both 2% and 4% DH hydrolysates for a given substrate. This result was expected, as there were no dramatic differences in the peptide profiles of the 2% and 4% DH hydrolysates. Although some newer polypeptides formed in the 25 and 30 kDa molecular weight range for EEF and SF, they did not affect the final gel strength because the hydrolysates from all four substrates had similar G0 values at 25 1C. 3.5. Texture profile analysis (TPA) A typical force vs. time curve during uniaxial compression of gels is shown in Fig. 5 (top) and was used to evaluate the impact of enzyme treatment on gel hardness (Fig. 5, bottom). The SF control gels were the hardest followed by EEF, SPC, and SPI controls. This result suggested that when SF control was heated for 30 min, it formed a gel having the highest hardness. This result might be due to interactions between proteins and carbohydrates (oligosaccharides and fiber) that are in a higher amount in

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the SF sample, and/or the native state of the proteins in the SF and EEF samples. Denaturation of the proteins would indeed affect the gel characteristics of the substrate. Glycinin is related to hardness and fracturability of soy gels, whereas b-conglycinin contributes to their elasticity (Utsumi et al., 1997; Kang & Lee, 2005). The basic polypeptides of glycinin preferentially associate with the b-subunits of b-conglycinin via electrostatic interactions, 2.5

Hardness

2 Force, N

1.5 1 0.5 0 -0.5 0

0.25

0.5

-1

0.75 Time, min

1

1.25

1.5

1221

and glycinin and b-conglycinin interact non-covalently with each other to form composite aggregates during gel formation (Utsumi et al., 1997). Different hydrolysis of both glycinin and b-conglycinin, as observed in the SDS PAGE, certainly explained the changes occurring in the gel hardness of the hydrolysates. There was, however, no clear trend in the gel hardness depending on the DH level or nature of the substrate. Gels prepared from 4% DH hydrolysates were significantly ðpo0:05Þ less hard than the unhydrolysed controls and 2% DH hydrolysates for SF and EEF, the highest decrease being observed for SF. The hardness of the 2% DH SPC increased significantly compared to the control whereas no gel was obtained with the 2% DH SPI. The reduced gel-forming ability, i.e. reduced gel-hardness, may be related to reduced protein– protein interactions and low surface hydrophobicity of the hydrolysate (Babiker, 2000; Fan et al., 2005) and/or increased charge repulsion between hydrolysed peptides (Panyam & Kilara, 1996).

2.5 a

3.6. Apparent viscosity

Gel Hardness,N

2.0 1.5 1.0

b d

0.5

bc

c

d

d ef

e

f

g

SF

EEF

SPC

4% DH

Control

4% DH

2% DH

Control

4% DH

2% DH

Control

4% DH

2% DH

Control

0.0

SPI

Fig. 5. Typical texture profile analyses plot for soy protein gels (top) and gel hardness comparison (bottom). Error bars indicate standard error of mean. Values sharing the same letters are not statistically different ðpo0:05Þ. The 2% DH SPI hydrolysate did not produce a standing gel.

The Power Law parameters (consistency coefficient K, and flow behavior index, n), and calculated apparent viscosities at 10 and 500 s1 shear rates for bromelain soy hydrolysates are shown in Table 3. The Power Law provided excellent fit for the dispersion flow curves (shear stress vs. shear rate data, R2 40:99). The K value for the unhydrolysed control dispersion was highest for SPI, followed by SPC, EEF, and SF. The viscosities of the unhydrolysed controls at 10 s1 were 1.68, 1.55, 1.07, and 0.08 for SPI, SPC, EEF, and SF, respectively, and followed the same trend as consistency coefficient. This was expected because the consistency coefficient is related to apparent viscosity. Upon hydrolysis, the K values and apparent viscosities followed similar trends (i.e., decreased values with increased DH) resulting in thinner dispersions. This

Table 3 Mean ðn ¼ 6Þ Power Law parameters and apparent viscosity for 10% w/w soy protein hydrolysate dispersions Soy substrate

SPI

SPC

SF

EEF

Degree of hydrolysis (%)

Control 2 4 Control 2 4 Control 2 4 Control 2 4

Power law parameters

Apparent viscosity (Pa s)

K (Pa sn)

n

10 s1

500 s1

7.5872.8a 0.0370.0d 0.0370.0d 4.9270.56b 0.4670.0d 0.4070.01d 0.1270.0d 0.0270.0d 0.0270.0d 3.0670.08c 0.2670.1d 0.170.01d

0.3570.06g 0.8870.01b 0.8670.0b 0.5070.02f 0.6670.0e 0.6670.01e 0.8470.0bc 0.9270.03a 0.9370.03a 0.5470.0f 0.7170.05d 0.8070.02c

1.55a 0.03d 0.02d 1.54a 0.21c 0.18cd 0.08cd 0.02d 0.02d 1.07b 0.13cd 0.06cd

0.11c 0.02gh 0.01h 0.22a 0.06d 0.05de 0.04def 0.01h 0.01h 0.18b 0.05ef 0.03fg

Means sharing the same superscript are not significantly different at po0:05. SE: standard error of mean; K: consistency coefficient; and n: flow behavior index.

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loss was very pronounced between the unhydrolysed control and 2% DH, but was not significantly different between 2% and 4% DH. This result agreed with observations of Tsumura et al. (2005) who reported that the apparent viscosity of b-conglycinin and glycinin papain hydrolysate from selective proteolysis was lower than that of unhydrolysed SPI. In general, lower apparent viscosity is observed in protein, as their molecular mass is reduced by proteolysis. Peptide profiles for the soy substrates were dramatically altered after hydrolysis as discussed earlier, and can explain the changes in viscosities. As previously reported (Puski, 1976; Jung et al., 2005), the observed decrease in viscosity after hydrolysis can be attributed in part to increased protein solubility, which was seen to hold for our hydrolysates also (Fig. 3). All hydrolysate dispersions exhibited shear-thinning non-Newtonian flow behavior ðno1Þ up to 500 s1 shear rate and tended towards Newtonian behavior with n values increasing with DH and approaching the value of 1. Small strain and large deformation rheology of enzymemodified soy protein products along with their apparent viscosities were investigated. Limited protease hydrolysis (up to 4% DH) of different soy protein substrates with endopeptidase bromelain resulted in thinner hydrolysate dispersions with weaker gelation properties. Soy protein hydrolysates retained some of their gelling ability even though the loss in G0 values were dramatic for different substrates. G0 values at 25 1C after heating to 95 1C were in the order of unhydrolysed control 42% DH 44% DH for all substrates. TPA of soy gels also showed loss in hardness after hydrolysis. Hydrolysis decreased the consistency coefficients and apparent viscosities, and increased the flow behavior indices of dispersions. Soy hydrolysates with such modified properties could find application in baby foods, yogurts, and soups. Acknowledgments This work was supported by USDA Special Grants 2003-34432-13326 and the Iowa Agricultural and Home Economics Experiment Station project number 6643. References Achouri, A., Zhang, W., & Shiying, X. (1998). Enzymatic hydrolysis of soy protein and effect of succinylation on the functional properties of resulting protein hydrolysates. Food Research International, 31(9), 617–623. Adler-Nissen, J. (1976). Enzymatic hydrolysis of proteins for increased solubility. Journal of Agricultural and Food Chemistry, 24(6), 1090–1093. Adler-Nissen, J. (1986). Enzymic hydrolysis of food proteins (pp. 116–125). New York: Elsevier. Babiker, E. E. (2000). Effect of transglutaminase treatment on the functional properties of native and chymotrypsin-digested soy protein. Food Chemistry, 70(2), 139–145. Calderon-de-la Barca, A. M., Ruiz-Salazar, R. A., & Jara-Maarini, M. E. (2000). Enzymatic hydrolysis and synthesis of soy protein to improve

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