Chun Cui, Mouming Zhao, Boen Yuan, Yuanhong Zhang, and Jiaoyan Ren
Effects of limited enzymatic hydrolysis with pepsin on the functional properties and structure characteristics of soybean proteins were investigated. Hydrolysates with different incubation time (10 to 900 min) were prepared. Results showed that SPI hydrolyzed for 60 min exhibited the best emulsibility and the ability of resisting freezing/thawing. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis proved that pepsin can degrade glycinin but had little effect on the α’ subunit of β-conglycinin. The structure unfolding reached the largest extent after incubation for 60 min and the soluble and flexible aggregates were formed. After 120 min, glycinin was degraded totally and β-conglycinin formed insoluble aggregates. Moreover, 2 methods were applied for the deactivation of pepsin to obtain final hydrolysates at pH 2.0 and 7.0, respectively. The structure analysis revealed that the unfolding extent and structure characteristic were different in these 2 conditions. When adjusting the pH value from 2.0 to 7.0, the unfolding protein molecular would reaggregate again at pH 7.0 due to the charge neutralization, and the hydrodynamic diameter and λmax absorbance decreased compared to pH 2.0. Moreover, some of the insoluble aggregates formed at pH 2.0 became soluble at pH 7.0, because of the salt-in phenomenon.
Abstract:
Keywords: functional properties, limited proteolysis, pepsin, soybean protein, structure change
Globulin, mainly including components of glycinin and β-conglycinin, makes up approximately 90% of soybean proteins. Due to the compacted globular conformation of glycinin, the functional properties of soybean proteins are limited. Hence, methods to modify glycinin structure and improve its functional properties are a very large technological challenge to expand the use of soybean proteins. In this study, we found that pepsin could effectively degrade glycinin and after pepsin treatment, the soybean protein showed better solubility at around isoelectrict point (pH 4 to 5), offering a potential use in the dairy industry for the manufacture of products such as yogurt.
Practical Application:
Introduction
ner and Gu´eguen 1995). So how to improve the glycinin structure is the priority in the development of modified soybean proteins. Limited enzymatic hydrolysis shows many advantages, including high effectiveness at low enzyme concentration, mild reaction condition and high safety, compared with other modification methods. Therefore limited enzymatic hydrolysis has been widely used for the improvement of protein functionality (Yin and others 2008a; Tang and others 2009; Luo and others 2010). The proteolytic enzymes most commonly used in protein hydrolysates production include Alcalase, pepsin, papain, trypsin, bacterial, and fungal proteases (Qi and others 1997). It has been reported that pepsin improves the functional properties of whey and casein protein (Chobert and others 1988). But little research has been done on the modification of soybean proteins structure and functional properties by the use of pepsin. We previously have found that soybean proteins dissociated in acid condition (pH 2.0 to 3.0) could induce the unfolding of glycinin structure (Zhao and others 2011). So, acid treatment before enzymatic modification could facilitate the exposure of the hidden sites inside the protein molecules, which might enhance the hydrolysis efficiency (Tang and others 2009). Since pepsin has MS 20131197 Submitted 8/22/2013, Accepted 10/5/2013. Authors are with Col- the optimum working pH of 2.0, it could be used as a good strategy lege of Light Industry and Food Sciences, South China Univ. of Technology, Guangzhou to improve soybean protein functionality (Zakaria and McFeeters 510640, China. Direct inquiries to author Ren (E-mail: [email protected]). 1978).
Soybean proteins have excellent functional properties and have been widely used in meat, beverage, baking, and other nutritional foods (Liu and others 2008). The consumption of soybean proteinbased food is increasing worldwidely each year, which expands the demand for manufacturing modified soybean proteins with desirable processing properties. Globulin, mainly including components of glycinin and β-conglycinin, makes up approximately 90% of total soybean proteins. The glycinin (referred to as 11S protein) consists of 2 polypeptide components, the acidic and the basic chains of 38 and 20 kDa, respectively (Staswick and others 1984). It has been reported that at acid condition (pH 1.5 to 2.0), the structure of glycinin could unfold due to the electrostatic repulsions. βconglycinin (also known as 7S protein) is a trimeric glycoprotein composed of 3 subunits: α (∼65 kDa), α’(∼62 kDa), and β (47 kD) (Thanh and Shibasaki 1977). Compared with β-conglycinin, native glycinin has more compacted globular conformation and thus caused limited functional properties of soybean proteins (Wag-
R C 2013 Institute of Food Technologists
doi: 10.1111/1750-3841.12309 Further reproduction without permission is prohibited
Vol. 78, Nr. 12, 2013 r Journal of Food Science C1871
C: Food Chemistry
Effect of pH and Pepsin Limited Hydrolysis on the Structure and Functional Properties of Soybean Protein Hydrolysates
Soybean protein hydrolyzed by pepsin . . .
C: Food Chemistry
In the present study, the effects of pepsin hydrolysis on the functional properties of soybean proteins were investigated. The structure change of soybean proteins were studied by electrophoresis, surface hydrophobicity, mean hydrodynamic diameter, and emission fluorescence spectroscopy. Moreover, we investigated the structure change of the hydrolysates at pH 2.0 (the optimal pH condition for pepsin) and at pH 7.0 (the final pH for the hydrolysates), respectively.
Materials and Methods Chemicals and standards Defatted soy flour was obtained from Yuwang Soy Co. (Shandong, China). Pepsin (catalog nr. P7000, 3800 units/mg), l-anilino-8-naphthalenesulfonate (ANS), and Pepstatin A were purchased from Sigma Chemical Co. (Shanghai, China). All other reagents were of analytical grade. Preparation of soybean proteins isolate (SPI) The SPI were prepared from defatted low-heat soybean meal according to the method of Puppo and others (2004) with a slight modification. A dispersion of soy flour was prepared by adding distilled water (1: l5, w/v) with final protein concentration of 3.1% (w/w). Then, 2 mol/L NaOH was used to adjust the dispersion to pH 8.5. The dispersion was stirred for 1 h at room temperature and then centrifuged (10000 × g, 20 min). The supernatant was adjusted to pH 4.5 with 2 mol/L HCl and centrifuged (10000 × g, 20 min). The obtained sediment was resuspended with distilled water (1: 5, v/v) and adjusted to pH 7.0 with 2 mol/L NaOH. Then it was dialyzed against deionized water and freeze-dried. The preparation of SPI hydrolysates The SPI dispersion (4% w/v) was adjusted to pH 2.0 with 1 mol/L HCl, and incubated at 37 ˚C for 30 min. The dispersion was equally divided into 7 fractions. Then, pepsin was added to each part at an enzyme to substrate ratio of 0.3% (w/w) to start the enzymatic hydrolysis reaction. Each fraction was incubated at 37 ◦ C for different time (10, 30, 60, 120, 300, 600, and 900 min). After the incubation, each sample was divided into 3 parts and then the enzyme was inactivated by 3 different methods. Part 1: The enzymatic hydrolysis was stopped by adjusting pH to 7.0 with 2 mol/L NaOH. The hydrolysates were freeze-dried for analysis of the functional properties and marked as final samples. SPI treated by pH 2.0 and neutralization were used as control. Part 2: The enzymatic hydrolysis was stopped by adding a 10fold molar amount of Pepstatin A to pH 2.0 and marked as the pH 2.0 series. SPI in pH 2.0 condition was used as control. The salt concentration of the pH 2.0 series was 0.13%. Part 3: The enzymatic hydrolysis was stopped by adjusting pH to 7.0 with 2 mol/L NaOH and marked as the pH 7.0 series. SPI in pH 2.0 condition and adjusted to pH 7.0 was used as control. The salt concentration of the pH 7.0 series was 0.51%. Part 2 and Part 3 were stored at 4 ◦ C for further detection of structural changes.
50 mL and referred as the OPA solution. To assay proteolysis, 50 μL of Part 1 samples dispersed in deionized water (40 mg/mL w/v) was directly added to 1 mL of OPA solution. The solution was thoroughly mixed and then stood for 2 min at room temperature before subjected to spectrophotometric assay at 340 nm. DH was calculated as the following equation (Adler-Nissen 1986), DH = hht o t × 100%, where h is number of peptide bonds cleaved and htot is total number of peptide bonds. As reported, htot was calculated to be 7.8 meqv/g for SPI. The h was calcu2 −β meqv/g , where lated as the following equation: h = SerineNH α SerineNH2 is meqv serine NH2 /g protein, calculated as, ODsample −ODblank /g × 0.9516meqv/l × 0.1×100l , where SerineNH2 = ODstandard −ODblank X×P X is the mass of the sample and P is protein concentration of the sample. For SPI, α and β were reported to be 0.970 and 0.342, respectively, according to Adler-Nissen (1986).
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) The Part 1 samples were subjected to SDS-PAGE analysis for the change of SPI subunits after pepsin hydrolysis. Two buffer systems, 12% separating gel (pH 8.8) and 5% stacking gel (pH 6.8), were applied for SDS-PAGE analysis. Fifteen microliters of protein sample were loaded per well and the whole process was done according to the method of Laemmli (1970). Surface hydrophobicity Surface hydrophobicity of Part 1 was detected according to the method of Yuan and others (2012). Protein dispersions (10 mg/mL) were prepared and centrifuged at 8000 × g for 20 min. The protein concentration of the supernatant was measured according to Lowry and others (1951). After the addition of ANS, the fluorescence intensity of the solution was measured with a Hitachi F 7000 fluorescence spectrometer (Tokyo, Japan). The initial slope of fluorescence intensity compared with protein concentration plot was used as the index of protein hydrophobicity. Solubility Protein dispersions (10 mg/mL) of the Part 1 samples were prepared and centrifuged at 12000 × g for 20 min to obtain the supernatants (Yuan and others 2012). Protein contents of the supernatant were determined according to Lowry and others (1951). Percent protein solubility was calculated as nitrogen solubility index (NSI, %) = (protein content of supernatant/amount of proteins added) × 100%. Particle size distribution Particle size distribution of Part 1 was detected according to Luo and others (2010). The oil/water (20: 80 v/v) emulsion was prepared and then homogenized (30 MPa, 2 passes) using a laboratory homogenizer (APV Gaulin, Abvertslund, Denmark). The particle size distribution was determined immediately after emulsion preparation by an integrated laser light scattering instrument (Mastersizer Micro Particle Analyser, Malvern Instruments Ltd., Worcestershire, UK).
Determination of the degree of hydrolysis (DH) The DH was assayed by o-phthaldialdehyde (OPA) method according to the report of Church and others (1983). Twenty-five Solubility after ultra-centrifugation The effect of centrifugation speed on the solubility of protein milliliter of sodium tetraborate (100 mmol/L), 2.5 mL of sodium dodecyl sulfate (20%), 100 μL of β-mercaptoethanol, and 40 mg dispersions was studied. The Part 2 and Part 3 samples were disof OPA in 1 mL methanol were mixed to a total volume of persed in deionized water (10 mg/mL), agitated with a magnetic C1872 Journal of Food Science r Vol. 78, Nr. 12, 2013
Figure 1–SDS-PAGE analysis of soybean proteins incubated with pepsin at 37 ◦ C for different time. Fifteen microliters of protein sample were loaded per well. SPI represents the soybean protein isolate; control represents soybean protein treated at pH 2.0 and neutralization (marked as CK); Lanes 1 to 7 represent soybean proteins incubated for 10, 30, 60, 120, 300, 600, and 900 min, respectively. AS stands for acidic subunit both; and BS stands for basic subunit.
stirrer for 1 h at room temperature, and centrifuged at 12000 × g for 20 min or 650000 × g for 45 min, respectively, to obtain the supernatants. Then the solubility was detected according to Lowry’s methods described in 2.6 (Lowry and others 1951). Surface hydrophobicity
Measurement of mean hydrodynamic diameter Protein dispersions (10 mg/mL, dissolved in deionized water) of the Part 2 and Part 3 samples were determined for average hydrodynamic diameter by a dynamic light scattering technique using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK). The measurement was conducted using the same method mentioned in our previous work (Yuan and others 2012).
b
1000
e
800
c 600
a
Statistical analysis The preparation of SPI hydrolysates was replicated twice at different day using new SPI samples. Data were processed using the general linear model’s procedure of the SPSS 13.0 software (SPSS Inc, Chicago, Ill., U.S.A.) to identify the effect of different incubation time (10 to 900 min) on the functional properties and structure characteristics of SPI and its hydrolysates. A least significant difference test with a confidence interval of 95% was used to compare the means.
Result and Discussion Determination of DH and SDS-PAGE As Figure 1 shows, DH of samples incubated for 0 (control), 10, 30, 60, 120, 300, 600, and 900 min were 2.49%, 3.94%, 4.08%, 4.48%, 5.96%, 6.30%, 7.49%, and 7.52%, respectively. The DH increased very slowly and the degradation of SPI almost stopped after 600 min. SDS-PAGE was conducted to analyze the subunits of the Part 1 samples. As reported, the protein structure of soybean proteins would dissociate or even unfold at pH 1.5 to 2.0 (Tang and others 2009). Compared with native SPI, no obvious difference in
f
a
g
g
400
200
0 SPI
Intrinsic fluorescence emission spectra According to the method of Yin and others (2008b), intrinsic emission fluorescence spectra of the Part 2 and Part 3 samples were determined in a Hitachi F 7000 fluorescence spectrometer (Tokyo, Japan). Protein solutions were excited with 290 nm light, and the emission spectra were recorded from 300 to 400 nm.
d
CK
10min 30min 60min 120min 300min 600min 900min
Samples Figure 2–Surface hydrophobicity of SPI, control, and the hydrolysates incubated for various time in 0.01 mol/L phosphate buffer (pH 7.0). Different letters (a to g) on the tops of columns indicate significant (P < 0.05) differences among samples in the same storage time.
the subunits of single acid treatment (control) was found, implying that only acid treatment did not disrupt the subunits. Similarly, Jiang and others (2011) also found no obvious change in the electrophoretic pattern of 7S in low-pH (1.5) samples, or even by the 1-h pH-shifting treatment at extremely low pH (1.5) or high pH (12) (Jiang and others 2010). However, during enzymatic hydrolysis, both glycinin acidic subunit (AS) and basic subunit (BS) degraded after incubation for 10 min and disappeared totally at 120 min (see lanes 1 to 7). As for β-conglycinin, the α subunit degraded with the increase of incubation time, while little effect was observed on the α’ and β subunits of β-conglycinin, implying that pepsin has selective proteolysis effect on glycinin and the α subunit of β-conglycinin.
Surface hydrophobicity As shown in Figure 2, the surface hydrophobicity of SPI was 465.3 and as for the SPI with pH 2.0 acid treatment, its surface hydrophobicity increased up to 947.6 (control). This result signified that single acid treatment could notably enhance the surface hydrophobicity of SPI, which was consistent with the report of Lakemond and others (2000). As for SPI with pepsin treatment, it Vol. 78, Nr. 12, 2013 r Journal of Food Science C1873
C: Food Chemistry
Soybean protein hydrolyzed by pepsin . . .
Soybean protein hydrolyzed by pepsin . . .
SPI Control 10 min 30 min 60 min 120 min 300 min 600 min 900 min
100
C: Food Chemistry
Solubility/%
80
60
40
20
0
1
2
3
4
5
6
7
8
9
10
pH value
Figure 3–Solubility of the SPI, control, and the hydrolysates in pH 7.0 and freeze-drying from pH 2.0 to 9.0, ( ) SPI, ( ) control, ( ) 10 min, ( ) 30 min, ( ) 60 min, ( ) 120 min, ( ) 300 min, ( ) 600 min, ( ) 900 min.
was found that with the incubation time from 10 min to 60 min, the surface hydrophobicity of the hydrolysates was lower than that of the control, but higher than the other samples. The reason might be that soybean protein unfolded and dissociated under acid treatment followed by enzymatic hydrolysis, therefore, some of the exposed hydrophobic regions were degraded by pepsin and soluble fragments were formed (Molina and others 2001). At 120 min, all the subunits of glycinin degraded, and the pepsin began to degrade the α subunit of β-conglycinin, so the surface hydrophobicity obviously decreased, which was in agreement with the result of SDS-PAGE assay.
Solubility As shown in Figure 3, the solubility of the soybean protein exhibited typically U-shape trend at different pH levels, the lowest solubility was reached at around the isoelectric point and the highest solubility was found at pH 9.0 or pH 2.0. Compared to native SPI, the solubility of the proteins treated at pH 2.0 was lower, which might be due to the unfolding and exposure of hydrophobic groups from the inner part of proteins (Tsumura and others 2005). The hydrolysates showed better solubility around the isoelectrict point (pH 4 to 5). The solubility at pH 4.5 increased from 0.87% to 53.04% during the incubation time from 0 to 900 min. This was because enzymatic hydrolysis treatment could release peptides with relative low-molecular-weight and enhanced flexibility of the protein hydrolysate (Kong and others 2007). The high solubility around the isoelectric point is necessary for the use of soybean protein in dairy products such as yogurt. From pH 6 to 9, the solubility of the hydrolysates increased during the incubation time from 10 min to 120 min. But further incubation time from 120 min to 900 min, the solubility decreased. According to SDS-PAGE analysis, glycinin was degraded by pepsin and released low-molecular-weight peptides, improving protein flexibility and forming soluble aggregates, so the solubility was improved (Sheen and Sheen 1988). But after 120 min, the pepsin degraded the α subunit of β-conglycinin and the solubility decreased, implying that some insoluble aggregate formed (Yin and others 2008a). Particle size distribution Good emulsifying proteins can rapidly be absorbed to the oil– water interface and drop the surface tension efficiently, undergo rapid conformational change and rearrangement to form a visC1874 Journal of Food Science r Vol. 78, Nr. 12, 2013
coelastic cohesive film through intermolecular interactions at the interface. The comparative particle size distribution at pH 7.0 for each sample has shown in Table 1. Compared to SPI and control, the emulsion of hydrolysates incubated for 10, 30, and 60 min showed relative smaller particle size distribution and better emulsibility. The native SPI exposed less hydrophobic groups than the hydrolyzed samples and demonstrated much tighter globular conformation, which slowed down the speed for SPI transferring to the air–water interface and dropped the surface tension ineffectively. This finding was consistent with our previous study (Yuan and others 2012). So, the particle size distribution of SPI was larger than that of the hydrolysate. As the incubation time further increased from 120 min to 900 min, the particle size distribution of the hydrolysates increased. The reason might be that glycinin degraded totally and the soluble aggregate began to disassemble, so no efficient adsorbed layer formed on the oil–water interface. Moreover, insoluble aggregates formed from the β-conglycinin enlarged the distribution of the particle size. Table 1 (B) shows the comparative particle size distribution after freezing at –20 ˚C for 24 h and thawing, which was in accordance with that determined at ambient and thus confirmed that SPI hydrolyzed for 60 min had the strongest stability.
Solubility after ultra-centrifugation At common centrifugation condition such as 12000 × g for 20 min, only the insoluble fraction could be isolated. But at ultracentrifugation condition, not only the insoluble fraction, but also the soluble large-molecular-weight fraction would be isolated. In our previous study (Zhao and others 2011), centrifuging at 650000 × g for 45 min was effective to isolate some aggregate, though the molecular weight was still unknown. The solubility of the hydrolysates in different centrifugation condition is shown in Figure 4. As Figure 4A has shown, the solubility increased during the incubation time from 0 to 60 min and the solubility of the pH 2.0 series were higher than those of the pH 7.0 series. This was because the strong electrostatic repulsive force weakened the protein–protein interaction and strengthened the protein–water interaction. After incubation for 120 min, the solubility of pH 7.0 series was found to be higher than those of the pH 2.0 series. Because the salt concentration increased from 0.13% to 0.51%, when adjusting the pH from 2.0 to 7.0, we presumed that due to the salt-in phenomenon, the insoluble aggregates formed after 120 min of incubation with pepsin was resoluble under the higher ionic strength (Arogundade and others 2006). As Figure 4B showed, under the ultra-centrifugation condition, the solubility was much lower than the solubility in common centrifugation condition (Figure 4A), because not only the insoluble fraction but also some of the soluble fractions were isolated. The solubility of the hydrolysates increased as the incubation time increased, because pepsin hydrolysis released the low-molecularweight peptides which would not be isolated during the ultracentrifugation. During the incubation time from 0 to 60 min, the solubility of pH 2.0 series was nearly the same as that of pH 7.0 series, implying that the ionic strength had little influence on the low-molecular-weight peptides. After incubation for 120 min, the solubility of pH 2.0 series increased very slowly. This is because the glycinin had already been degraded totally and the pepsin only degraded some part of the β-conglycinin, inducing the formation of the insoluble aggregates and just a few low-molecular-weight peptides released. However, the solubility of the pH 7.0 series increased more remarkably after 120 min incubation. This result
C: Food Chemistry
15.28 ± 0.170i Means ± standard deviations of triplicate analyses are given. Different letters (a to i) in the same row and section indicate significant (P < 0.05) differences among samples in the same storage time.
16.43 ± 0.278h 6.725 ± 0.082g 1.392 ± 0.031e 2.535 ± 0.052d 8.408 ± 0.094c
4.505 ± 0.111f
2.790 ± 0.071i 2.822 ± 0.041h 1.589 ± 0.016g 0.360 ± 0.001e 0.647 ± 0.022d
Minus 20 ˚C freezing and thawing (B) 1.779 ± 0.028b D[3, 2] 1.442 ± 0.004a (μm) 6.735 ± 0.032b D[4, 3] 12.54 ± 0.101a (μm)
1.995 ± 0.017c
1.389 ± 0.030f
9.217 ± 0.229f 10.66 ± 0.340e 4.640 ± 0.066d 1.206 ± 0.064a 1.140 ± 0.010a 0.927 ± 0.020a
1.544 ± 0.012b
3.973 ± 0.073c
2.394 ± 0.037f 2.621 ± 0.107e 1.456 ± 0.025d 0.398 ± 0.001b 0.410 ± 0.002b 0.505 ± 0.002a
0.458 ± 0.001b
1.114 ± 0.010c
900 min 600 min 300 min 120 min 60 min 30 min 10 min Control SPI
At room temperature (A) D[3, 2] 0.550 ± 0.001a (μm) D[4, 3] 1.163 ± 0.003a (μm)
Samples
Table 1–Particle size distribution of emulsions of SPI, control, and the hydrolysates with different hydrolysis time at room temperature (A) and –20 ˚C freezing and thawing (B).
Soybean protein hydrolyzed by pepsin . . .
Figure 4–Solubility of SPI, control, and the hydrolysates incubated for different time after 12000 × g centrifuging for 20 min (A) and 650000 ) represents for pH 2.0 series; ( ) × g centrifuging for 45 min (B). ( represents pH 7.0 series. Different letters on the tops of columns in A (a to d and e to k) and B (a to g and h to o) indicate significant (P < 0.05) differences among samples in the same storage time.
implied that some insoluble aggregate became soluble and had strong protein–water interaction under the high ionic strength.
Mean hydrodynamic diameter Dynamic light scattering technique was applied to analyze the hydrodynamic diameter of the samples As Figure 5 has shown, the hydrodynamic diameter for soybean proteins (control) at pH 2.0 and 7.0 was 92.19 nm and 72.62 nm, respectively, due to that soybean proteins unfolded in acid condition and reaggregated when neutralization. The highest hydrodynamic diameter value for pH 2.0 series reached 129.42 nm at 120 min, while the highest value for pH 7.0 series was 98.43 nm at 60 min. The reason might be that under the acid and pepsin hydrolysis condition, the soybean proteins unfold and aggregate to a larger extent. However, when adjusting the pH to 7.0, the unfolding protein molecular would reaggregate again due to the salt concentration increasing from 0.13% to 0.51%. During the incubation time from 0 to 120 min, the hydrodynamic diameter increased, implying that the pepsin hydrolysis induced the glycinin unfolding and formed large Vol. 78, Nr. 12, 2013 r Journal of Food Science C1875
Soybean protein hydrolyzed by pepsin . . .
C: Food Chemistry Figure 5–The hydrodynamic diameter of SPI, control, and the hydrolysates incubated for different time, ( ) represents for pH 2.0 series; ( ) represents pH 7.0 series. Different letters (a to g and h to m) on the tops of Figure 6–The λmax of emission fluorescence spectroscopic of SPI, control, ) represents for pH columns indicate significant (P < 0.05) differences among samples in the and the hydrolysates incubated for different time, ( 2.0 series; ( ) represents pH 7.0 series. Different letters (a to f and g to same storage time. l) on the tops of columns indicate significant (P < 0.05) differences among samples in the same storage time.
and soluble aggregate. After the glycinin unfolded and aggregated to the largest extent, the aggregates began to degrade to lowmolecular-weight peptides and thus the hydrodynamic diameter degradation effect on the glycinin and a little effect on the α subunit of β-conglycinin. Moreover, soybean proteins unfolded decreased. and formed the soluble and flexible aggregation at first and to the largest extent when hydrolyzed for 60 min. But after 120 Intrinsic fluorescence emission spectra The polarity of the environment of the tryptophan and tyrosine min, the glycinin was degraded totally and the insoluble aggregaresidues determines the fluorescence spectrum and implies their tion of β-conglycinin was formed. As a result, for the functional specific interactions and the change of the proteins configuration. properties, SPI hydrolyzed by pepsin for 60 min showed the best When the chromophores become more exposed to solvent, the solubility, emulsibility, and the ability to resist freezing/thawing. fluorescence emission maximum (λmax ) suffers a red shift, transfer- Furthermore, because the reaction was performed at pH 2.0 and ring to the longer wavelength. When the chromophores become the final samples was pH 7.0, we found that the configuration of more embedded into the inner part of the protein molecule, λmax the sample was quite different in these 2 pH conditions. Since the transfers to the shorter wavelength showing a blue shift (Pallar`es salt concentration increased from 0.13% to 0.51% when adjusting the pH value from 2.0 to 7.0, the unfolding protein molecular and others 2004). As Figure 6 showed, the emission fluorescence spectroscopic would reaggregate again at pH 7.0 due to the charge neutralization, technique was used to analyze the conformational changes of the and the hydrodynamic diameter and λmax decreased compared to hydrolysates at pH 2.0 and pH 7.0. For soybean proteins in pH 2.0 those of the pH 2.0. Besides, some of the insoluble aggregates and 7.0, the λmax reached 338.6 nm and 337.6 nm, respectively. formed in pH 2.0 became soluble in pH 7.0, because of the salt-in The λ values of pH 2.0 series were larger than those of the pH phenomenon. max
7.0 series. The reason was that when adjusting pH to 7.0, soybean proteins would reaggregate and some of the chromophores embedded into the inner part of the proteins again. The λmax became larger with the increase of the incubation time, implying that pepsin hydrolysis caused more chromophores exposed to the solvent. The increase of λmax from 0 to 120 min was more remarkable than that from 120 min to 900 min, indicating that much more chromophores exposed during the former period and very few chromophores exposed during the latter period. The reason might be that pepsin only degraded the glycinin at first and when all the glycinin was degraded (after 120 min), pepsin had little effect on the β-conglycinin due to the formation of aggregate, hence the enzyme is not allowed to interact within the aggregates as is obtained in the single molecule state.
Conclusion With the analysis of SDS-PAGE, surface hydrophobicity, particle size distribution, hydrodynamic diameter, and emission fluorescence spectroscopic, we concluded that pepsin had strong C1876 Journal of Food Science r Vol. 78, Nr. 12, 2013
Acknowledgments We appreciate the support from Natl. Natural Science Foundation of China (Nr. 31171783 and 31000759) and Financial support from Guangdong Province (Nr. 2012BAD37B08-1 and Nr. 20100172120024 ).
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Kong XZ, Zhou HM, Qian HF. 2007. Enzymatic hydrolysis of wheat gluten by proteases and properties of the resulting hydrolysates. Food Chem 102:759–63. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–85. Lakemond CMM, Jongh HHJ, Hessing M, Gruppen H, Voragen AGJ. 2000. Soy glycinin: influence of pH and ionic strength on solubility and molecular structure at ambient temperatures. J Agric Food Chem 48:1985–90. Liu C, Wang XS, Ma H, Zhang ZQ, Gao WR, Xiao L. 2008. Functional properties of protein isolates from soybeans stored under various conditions. Food Chem 111:29–37. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–75. Luo DH, Zhao QZ, Zhao MM, Yang B, Long XT, Ren JY, Zhao HF. 2010. Effects of limited proteolysis and high-pressure homogenisation on structural and functional characteristics of glycinin. Food Chem 122:25–30. Molina E, Papadopoulou A, Ledward DA. 2001. Emulsifying properties of high-pressure treated soy protein isolates and 7S and 11S globulins. Food Hydrocolloids 15: 263–9. Pallar`es I, Vendrell J, Avil`es FX, Ventura S. 2004. Amyloid fibril formation by a partially structured intermediate state of a-chymotrypsin. J Mol Biol 342:321–31. Puppo MC, Chapleau N, Speroni F, Lamballerie-Anton MD, de Michel F, Anon MC. 2004. Physicochemical modifications of high pressure-treated soybean protein isolates. J Agric Food Chem 52:1564–71. Qi M, Hettiarachchy NS, Kalapathy U. 1997. Solubility and emulsifying properties of soy protein isolates modified by pancreatin. J Food Sci 62:1110–5. Sheen SJ, Sheen VT. 1988. Effect of chemical and enzymatic degradation on the functional properties of fraction-l-protein. J Agric Food Chem 36:445–50. Staswick PE, Hermodson MA, Nielsen NC. 1984.Identification of the cystines which link the acidic and basic components of glycinin subunits. J Biol Chem 259:13431–5.
Tang CH, Wang XS, Yang XC. 2009. Enzymatic hydrolysis of hemp (Cannabis sativa L.) protein isolate by various proteases and antioxidant properties of the resulting hydrolysates. Food Chem 114:1484–90. Thanh VH, Shibasaki K. 1977. Beta-conglycinin from soybean proteins. Isolation and immunological and physicochemical properties of the monomeric forms. BBA – Protein Struct 490:370–84. Tsumura K, Saito T, Tsuge K, Ashidaa H, Kugimiya W, Inouye K. 2005. Functional properties of soy protein hydrolysates obtained by selective proteolysis. LWT – Food Sci Technol 38:255– 61. Wagner JR, Gu´eguen J. 1995. Effects of dissociation, deamidation, and reducing treatment on structural and surface active properties of soy glycinin. J Agric Food Chem 43:1993– 2000. Yin SW, Tang CH, Cao JS, Hub EK, Wen QB, Yang XQ. 2008a. Effects of limited enzymatic hydrolysis with trypsin on the functional properties of hemp (Cannabis sativa L.) protein isolate. Food Chem 106:1004–13. Yin SW, Tang CH, Wen QB, Yang XQ, Li L. 2008b. Functional properties and in vitro trypsin digestibility of red kidney bean (Phaseolus vulgaris L.) protein isolate: effect of high-pressure treatment. Food Chem 110:938–45. Yuan BE, Ren JY, Zhao MM, Luo DH, Gu LJ. 2012. Effects of limited enzymatic hydrolysis with pepsin and high-pressure homogenization on the functional properties of soybean protein isolate. LWT – Food Sci Technol 46:453–9. Zakaria F, McFeeters RF. 1978. Improvement of the emulsification properties of soy protein by limited pepsin hydrolysis. LWT – Food Sci Technol 11:42–4. Zhao MM, Yuan BE, Luo DH, Zhao QZ. 2011. Subunit dissociation of soybean protein isolates in acid conditions. J South China Univ Technol 39: 22–8.
Vol. 78, Nr. 12, 2013 r Journal of Food Science C1877
C: Food Chemistry
Soybean protein hydrolyzed by pepsin . . .
Effects of limited enzymatic hydrolysis with pepsin on the functional properties and structure characteristics of soybean proteins were investigated. Hydrolysates with different incubation time (10 to 900 min) were prepared. Results showed that SPI hydrolyzed for 60 min exhibited the best emulsibility and the ability of resisting freezing/thawing. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis proved that pepsin can degrade glycinin but had little effect on the α’ subunit of β-conglycinin. The structure unfolding reached the largest extent after incubation for 60 min and the soluble and flexible aggregates were formed. After 120 min, glycinin was degraded totally and β-conglycinin formed insoluble aggregates. Moreover, 2 methods were applied for the deactivation of pepsin to obtain final hydrolysates at pH 2.0 and 7.0, respectively. The structure analysis revealed that the unfolding extent and structure characteristic were different in these 2 conditions. When adjusting the pH value from 2.0 to 7.0, the unfolding protein molecular would reaggregate again at pH 7.0 due to the charge neutralization, and the hydrodynamic diameter and λmax absorbance decreased compared to pH 2.0. Moreover, some of the insoluble aggregates formed at pH 2.0 became soluble at pH 7.0, because of the salt-in phenomenon.
Abstract:
Keywords: functional properties, limited proteolysis, pepsin, soybean protein, structure change
Globulin, mainly including components of glycinin and β-conglycinin, makes up approximately 90% of soybean proteins. Due to the compacted globular conformation of glycinin, the functional properties of soybean proteins are limited. Hence, methods to modify glycinin structure and improve its functional properties are a very large technological challenge to expand the use of soybean proteins. In this study, we found that pepsin could effectively degrade glycinin and after pepsin treatment, the soybean protein showed better solubility at around isoelectrict point (pH 4 to 5), offering a potential use in the dairy industry for the manufacture of products such as yogurt.
Practical Application:
Introduction
ner and Gu´eguen 1995). So how to improve the glycinin structure is the priority in the development of modified soybean proteins. Limited enzymatic hydrolysis shows many advantages, including high effectiveness at low enzyme concentration, mild reaction condition and high safety, compared with other modification methods. Therefore limited enzymatic hydrolysis has been widely used for the improvement of protein functionality (Yin and others 2008a; Tang and others 2009; Luo and others 2010). The proteolytic enzymes most commonly used in protein hydrolysates production include Alcalase, pepsin, papain, trypsin, bacterial, and fungal proteases (Qi and others 1997). It has been reported that pepsin improves the functional properties of whey and casein protein (Chobert and others 1988). But little research has been done on the modification of soybean proteins structure and functional properties by the use of pepsin. We previously have found that soybean proteins dissociated in acid condition (pH 2.0 to 3.0) could induce the unfolding of glycinin structure (Zhao and others 2011). So, acid treatment before enzymatic modification could facilitate the exposure of the hidden sites inside the protein molecules, which might enhance the hydrolysis efficiency (Tang and others 2009). Since pepsin has MS 20131197 Submitted 8/22/2013, Accepted 10/5/2013. Authors are with Col- the optimum working pH of 2.0, it could be used as a good strategy lege of Light Industry and Food Sciences, South China Univ. of Technology, Guangzhou to improve soybean protein functionality (Zakaria and McFeeters 510640, China. Direct inquiries to author Ren (E-mail: [email protected]). 1978).
Soybean proteins have excellent functional properties and have been widely used in meat, beverage, baking, and other nutritional foods (Liu and others 2008). The consumption of soybean proteinbased food is increasing worldwidely each year, which expands the demand for manufacturing modified soybean proteins with desirable processing properties. Globulin, mainly including components of glycinin and β-conglycinin, makes up approximately 90% of total soybean proteins. The glycinin (referred to as 11S protein) consists of 2 polypeptide components, the acidic and the basic chains of 38 and 20 kDa, respectively (Staswick and others 1984). It has been reported that at acid condition (pH 1.5 to 2.0), the structure of glycinin could unfold due to the electrostatic repulsions. βconglycinin (also known as 7S protein) is a trimeric glycoprotein composed of 3 subunits: α (∼65 kDa), α’(∼62 kDa), and β (47 kD) (Thanh and Shibasaki 1977). Compared with β-conglycinin, native glycinin has more compacted globular conformation and thus caused limited functional properties of soybean proteins (Wag-
R C 2013 Institute of Food Technologists
doi: 10.1111/1750-3841.12309 Further reproduction without permission is prohibited
Vol. 78, Nr. 12, 2013 r Journal of Food Science C1871
C: Food Chemistry
Effect of pH and Pepsin Limited Hydrolysis on the Structure and Functional Properties of Soybean Protein Hydrolysates
Soybean protein hydrolyzed by pepsin . . .
C: Food Chemistry
In the present study, the effects of pepsin hydrolysis on the functional properties of soybean proteins were investigated. The structure change of soybean proteins were studied by electrophoresis, surface hydrophobicity, mean hydrodynamic diameter, and emission fluorescence spectroscopy. Moreover, we investigated the structure change of the hydrolysates at pH 2.0 (the optimal pH condition for pepsin) and at pH 7.0 (the final pH for the hydrolysates), respectively.
Materials and Methods Chemicals and standards Defatted soy flour was obtained from Yuwang Soy Co. (Shandong, China). Pepsin (catalog nr. P7000, 3800 units/mg), l-anilino-8-naphthalenesulfonate (ANS), and Pepstatin A were purchased from Sigma Chemical Co. (Shanghai, China). All other reagents were of analytical grade. Preparation of soybean proteins isolate (SPI) The SPI were prepared from defatted low-heat soybean meal according to the method of Puppo and others (2004) with a slight modification. A dispersion of soy flour was prepared by adding distilled water (1: l5, w/v) with final protein concentration of 3.1% (w/w). Then, 2 mol/L NaOH was used to adjust the dispersion to pH 8.5. The dispersion was stirred for 1 h at room temperature and then centrifuged (10000 × g, 20 min). The supernatant was adjusted to pH 4.5 with 2 mol/L HCl and centrifuged (10000 × g, 20 min). The obtained sediment was resuspended with distilled water (1: 5, v/v) and adjusted to pH 7.0 with 2 mol/L NaOH. Then it was dialyzed against deionized water and freeze-dried. The preparation of SPI hydrolysates The SPI dispersion (4% w/v) was adjusted to pH 2.0 with 1 mol/L HCl, and incubated at 37 ˚C for 30 min. The dispersion was equally divided into 7 fractions. Then, pepsin was added to each part at an enzyme to substrate ratio of 0.3% (w/w) to start the enzymatic hydrolysis reaction. Each fraction was incubated at 37 ◦ C for different time (10, 30, 60, 120, 300, 600, and 900 min). After the incubation, each sample was divided into 3 parts and then the enzyme was inactivated by 3 different methods. Part 1: The enzymatic hydrolysis was stopped by adjusting pH to 7.0 with 2 mol/L NaOH. The hydrolysates were freeze-dried for analysis of the functional properties and marked as final samples. SPI treated by pH 2.0 and neutralization were used as control. Part 2: The enzymatic hydrolysis was stopped by adding a 10fold molar amount of Pepstatin A to pH 2.0 and marked as the pH 2.0 series. SPI in pH 2.0 condition was used as control. The salt concentration of the pH 2.0 series was 0.13%. Part 3: The enzymatic hydrolysis was stopped by adjusting pH to 7.0 with 2 mol/L NaOH and marked as the pH 7.0 series. SPI in pH 2.0 condition and adjusted to pH 7.0 was used as control. The salt concentration of the pH 7.0 series was 0.51%. Part 2 and Part 3 were stored at 4 ◦ C for further detection of structural changes.
50 mL and referred as the OPA solution. To assay proteolysis, 50 μL of Part 1 samples dispersed in deionized water (40 mg/mL w/v) was directly added to 1 mL of OPA solution. The solution was thoroughly mixed and then stood for 2 min at room temperature before subjected to spectrophotometric assay at 340 nm. DH was calculated as the following equation (Adler-Nissen 1986), DH = hht o t × 100%, where h is number of peptide bonds cleaved and htot is total number of peptide bonds. As reported, htot was calculated to be 7.8 meqv/g for SPI. The h was calcu2 −β meqv/g , where lated as the following equation: h = SerineNH α SerineNH2 is meqv serine NH2 /g protein, calculated as, ODsample −ODblank /g × 0.9516meqv/l × 0.1×100l , where SerineNH2 = ODstandard −ODblank X×P X is the mass of the sample and P is protein concentration of the sample. For SPI, α and β were reported to be 0.970 and 0.342, respectively, according to Adler-Nissen (1986).
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) The Part 1 samples were subjected to SDS-PAGE analysis for the change of SPI subunits after pepsin hydrolysis. Two buffer systems, 12% separating gel (pH 8.8) and 5% stacking gel (pH 6.8), were applied for SDS-PAGE analysis. Fifteen microliters of protein sample were loaded per well and the whole process was done according to the method of Laemmli (1970). Surface hydrophobicity Surface hydrophobicity of Part 1 was detected according to the method of Yuan and others (2012). Protein dispersions (10 mg/mL) were prepared and centrifuged at 8000 × g for 20 min. The protein concentration of the supernatant was measured according to Lowry and others (1951). After the addition of ANS, the fluorescence intensity of the solution was measured with a Hitachi F 7000 fluorescence spectrometer (Tokyo, Japan). The initial slope of fluorescence intensity compared with protein concentration plot was used as the index of protein hydrophobicity. Solubility Protein dispersions (10 mg/mL) of the Part 1 samples were prepared and centrifuged at 12000 × g for 20 min to obtain the supernatants (Yuan and others 2012). Protein contents of the supernatant were determined according to Lowry and others (1951). Percent protein solubility was calculated as nitrogen solubility index (NSI, %) = (protein content of supernatant/amount of proteins added) × 100%. Particle size distribution Particle size distribution of Part 1 was detected according to Luo and others (2010). The oil/water (20: 80 v/v) emulsion was prepared and then homogenized (30 MPa, 2 passes) using a laboratory homogenizer (APV Gaulin, Abvertslund, Denmark). The particle size distribution was determined immediately after emulsion preparation by an integrated laser light scattering instrument (Mastersizer Micro Particle Analyser, Malvern Instruments Ltd., Worcestershire, UK).
Determination of the degree of hydrolysis (DH) The DH was assayed by o-phthaldialdehyde (OPA) method according to the report of Church and others (1983). Twenty-five Solubility after ultra-centrifugation The effect of centrifugation speed on the solubility of protein milliliter of sodium tetraborate (100 mmol/L), 2.5 mL of sodium dodecyl sulfate (20%), 100 μL of β-mercaptoethanol, and 40 mg dispersions was studied. The Part 2 and Part 3 samples were disof OPA in 1 mL methanol were mixed to a total volume of persed in deionized water (10 mg/mL), agitated with a magnetic C1872 Journal of Food Science r Vol. 78, Nr. 12, 2013
Figure 1–SDS-PAGE analysis of soybean proteins incubated with pepsin at 37 ◦ C for different time. Fifteen microliters of protein sample were loaded per well. SPI represents the soybean protein isolate; control represents soybean protein treated at pH 2.0 and neutralization (marked as CK); Lanes 1 to 7 represent soybean proteins incubated for 10, 30, 60, 120, 300, 600, and 900 min, respectively. AS stands for acidic subunit both; and BS stands for basic subunit.
stirrer for 1 h at room temperature, and centrifuged at 12000 × g for 20 min or 650000 × g for 45 min, respectively, to obtain the supernatants. Then the solubility was detected according to Lowry’s methods described in 2.6 (Lowry and others 1951). Surface hydrophobicity
Measurement of mean hydrodynamic diameter Protein dispersions (10 mg/mL, dissolved in deionized water) of the Part 2 and Part 3 samples were determined for average hydrodynamic diameter by a dynamic light scattering technique using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK). The measurement was conducted using the same method mentioned in our previous work (Yuan and others 2012).
b
1000
e
800
c 600
a
Statistical analysis The preparation of SPI hydrolysates was replicated twice at different day using new SPI samples. Data were processed using the general linear model’s procedure of the SPSS 13.0 software (SPSS Inc, Chicago, Ill., U.S.A.) to identify the effect of different incubation time (10 to 900 min) on the functional properties and structure characteristics of SPI and its hydrolysates. A least significant difference test with a confidence interval of 95% was used to compare the means.
Result and Discussion Determination of DH and SDS-PAGE As Figure 1 shows, DH of samples incubated for 0 (control), 10, 30, 60, 120, 300, 600, and 900 min were 2.49%, 3.94%, 4.08%, 4.48%, 5.96%, 6.30%, 7.49%, and 7.52%, respectively. The DH increased very slowly and the degradation of SPI almost stopped after 600 min. SDS-PAGE was conducted to analyze the subunits of the Part 1 samples. As reported, the protein structure of soybean proteins would dissociate or even unfold at pH 1.5 to 2.0 (Tang and others 2009). Compared with native SPI, no obvious difference in
f
a
g
g
400
200
0 SPI
Intrinsic fluorescence emission spectra According to the method of Yin and others (2008b), intrinsic emission fluorescence spectra of the Part 2 and Part 3 samples were determined in a Hitachi F 7000 fluorescence spectrometer (Tokyo, Japan). Protein solutions were excited with 290 nm light, and the emission spectra were recorded from 300 to 400 nm.
d
CK
10min 30min 60min 120min 300min 600min 900min
Samples Figure 2–Surface hydrophobicity of SPI, control, and the hydrolysates incubated for various time in 0.01 mol/L phosphate buffer (pH 7.0). Different letters (a to g) on the tops of columns indicate significant (P < 0.05) differences among samples in the same storage time.
the subunits of single acid treatment (control) was found, implying that only acid treatment did not disrupt the subunits. Similarly, Jiang and others (2011) also found no obvious change in the electrophoretic pattern of 7S in low-pH (1.5) samples, or even by the 1-h pH-shifting treatment at extremely low pH (1.5) or high pH (12) (Jiang and others 2010). However, during enzymatic hydrolysis, both glycinin acidic subunit (AS) and basic subunit (BS) degraded after incubation for 10 min and disappeared totally at 120 min (see lanes 1 to 7). As for β-conglycinin, the α subunit degraded with the increase of incubation time, while little effect was observed on the α’ and β subunits of β-conglycinin, implying that pepsin has selective proteolysis effect on glycinin and the α subunit of β-conglycinin.
Surface hydrophobicity As shown in Figure 2, the surface hydrophobicity of SPI was 465.3 and as for the SPI with pH 2.0 acid treatment, its surface hydrophobicity increased up to 947.6 (control). This result signified that single acid treatment could notably enhance the surface hydrophobicity of SPI, which was consistent with the report of Lakemond and others (2000). As for SPI with pepsin treatment, it Vol. 78, Nr. 12, 2013 r Journal of Food Science C1873
C: Food Chemistry
Soybean protein hydrolyzed by pepsin . . .
Soybean protein hydrolyzed by pepsin . . .
SPI Control 10 min 30 min 60 min 120 min 300 min 600 min 900 min
100
C: Food Chemistry
Solubility/%
80
60
40
20
0
1
2
3
4
5
6
7
8
9
10
pH value
Figure 3–Solubility of the SPI, control, and the hydrolysates in pH 7.0 and freeze-drying from pH 2.0 to 9.0, ( ) SPI, ( ) control, ( ) 10 min, ( ) 30 min, ( ) 60 min, ( ) 120 min, ( ) 300 min, ( ) 600 min, ( ) 900 min.
was found that with the incubation time from 10 min to 60 min, the surface hydrophobicity of the hydrolysates was lower than that of the control, but higher than the other samples. The reason might be that soybean protein unfolded and dissociated under acid treatment followed by enzymatic hydrolysis, therefore, some of the exposed hydrophobic regions were degraded by pepsin and soluble fragments were formed (Molina and others 2001). At 120 min, all the subunits of glycinin degraded, and the pepsin began to degrade the α subunit of β-conglycinin, so the surface hydrophobicity obviously decreased, which was in agreement with the result of SDS-PAGE assay.
Solubility As shown in Figure 3, the solubility of the soybean protein exhibited typically U-shape trend at different pH levels, the lowest solubility was reached at around the isoelectric point and the highest solubility was found at pH 9.0 or pH 2.0. Compared to native SPI, the solubility of the proteins treated at pH 2.0 was lower, which might be due to the unfolding and exposure of hydrophobic groups from the inner part of proteins (Tsumura and others 2005). The hydrolysates showed better solubility around the isoelectrict point (pH 4 to 5). The solubility at pH 4.5 increased from 0.87% to 53.04% during the incubation time from 0 to 900 min. This was because enzymatic hydrolysis treatment could release peptides with relative low-molecular-weight and enhanced flexibility of the protein hydrolysate (Kong and others 2007). The high solubility around the isoelectric point is necessary for the use of soybean protein in dairy products such as yogurt. From pH 6 to 9, the solubility of the hydrolysates increased during the incubation time from 10 min to 120 min. But further incubation time from 120 min to 900 min, the solubility decreased. According to SDS-PAGE analysis, glycinin was degraded by pepsin and released low-molecular-weight peptides, improving protein flexibility and forming soluble aggregates, so the solubility was improved (Sheen and Sheen 1988). But after 120 min, the pepsin degraded the α subunit of β-conglycinin and the solubility decreased, implying that some insoluble aggregate formed (Yin and others 2008a). Particle size distribution Good emulsifying proteins can rapidly be absorbed to the oil– water interface and drop the surface tension efficiently, undergo rapid conformational change and rearrangement to form a visC1874 Journal of Food Science r Vol. 78, Nr. 12, 2013
coelastic cohesive film through intermolecular interactions at the interface. The comparative particle size distribution at pH 7.0 for each sample has shown in Table 1. Compared to SPI and control, the emulsion of hydrolysates incubated for 10, 30, and 60 min showed relative smaller particle size distribution and better emulsibility. The native SPI exposed less hydrophobic groups than the hydrolyzed samples and demonstrated much tighter globular conformation, which slowed down the speed for SPI transferring to the air–water interface and dropped the surface tension ineffectively. This finding was consistent with our previous study (Yuan and others 2012). So, the particle size distribution of SPI was larger than that of the hydrolysate. As the incubation time further increased from 120 min to 900 min, the particle size distribution of the hydrolysates increased. The reason might be that glycinin degraded totally and the soluble aggregate began to disassemble, so no efficient adsorbed layer formed on the oil–water interface. Moreover, insoluble aggregates formed from the β-conglycinin enlarged the distribution of the particle size. Table 1 (B) shows the comparative particle size distribution after freezing at –20 ˚C for 24 h and thawing, which was in accordance with that determined at ambient and thus confirmed that SPI hydrolyzed for 60 min had the strongest stability.
Solubility after ultra-centrifugation At common centrifugation condition such as 12000 × g for 20 min, only the insoluble fraction could be isolated. But at ultracentrifugation condition, not only the insoluble fraction, but also the soluble large-molecular-weight fraction would be isolated. In our previous study (Zhao and others 2011), centrifuging at 650000 × g for 45 min was effective to isolate some aggregate, though the molecular weight was still unknown. The solubility of the hydrolysates in different centrifugation condition is shown in Figure 4. As Figure 4A has shown, the solubility increased during the incubation time from 0 to 60 min and the solubility of the pH 2.0 series were higher than those of the pH 7.0 series. This was because the strong electrostatic repulsive force weakened the protein–protein interaction and strengthened the protein–water interaction. After incubation for 120 min, the solubility of pH 7.0 series was found to be higher than those of the pH 2.0 series. Because the salt concentration increased from 0.13% to 0.51%, when adjusting the pH from 2.0 to 7.0, we presumed that due to the salt-in phenomenon, the insoluble aggregates formed after 120 min of incubation with pepsin was resoluble under the higher ionic strength (Arogundade and others 2006). As Figure 4B showed, under the ultra-centrifugation condition, the solubility was much lower than the solubility in common centrifugation condition (Figure 4A), because not only the insoluble fraction but also some of the soluble fractions were isolated. The solubility of the hydrolysates increased as the incubation time increased, because pepsin hydrolysis released the low-molecularweight peptides which would not be isolated during the ultracentrifugation. During the incubation time from 0 to 60 min, the solubility of pH 2.0 series was nearly the same as that of pH 7.0 series, implying that the ionic strength had little influence on the low-molecular-weight peptides. After incubation for 120 min, the solubility of pH 2.0 series increased very slowly. This is because the glycinin had already been degraded totally and the pepsin only degraded some part of the β-conglycinin, inducing the formation of the insoluble aggregates and just a few low-molecular-weight peptides released. However, the solubility of the pH 7.0 series increased more remarkably after 120 min incubation. This result
C: Food Chemistry
15.28 ± 0.170i Means ± standard deviations of triplicate analyses are given. Different letters (a to i) in the same row and section indicate significant (P < 0.05) differences among samples in the same storage time.
16.43 ± 0.278h 6.725 ± 0.082g 1.392 ± 0.031e 2.535 ± 0.052d 8.408 ± 0.094c
4.505 ± 0.111f
2.790 ± 0.071i 2.822 ± 0.041h 1.589 ± 0.016g 0.360 ± 0.001e 0.647 ± 0.022d
Minus 20 ˚C freezing and thawing (B) 1.779 ± 0.028b D[3, 2] 1.442 ± 0.004a (μm) 6.735 ± 0.032b D[4, 3] 12.54 ± 0.101a (μm)
1.995 ± 0.017c
1.389 ± 0.030f
9.217 ± 0.229f 10.66 ± 0.340e 4.640 ± 0.066d 1.206 ± 0.064a 1.140 ± 0.010a 0.927 ± 0.020a
1.544 ± 0.012b
3.973 ± 0.073c
2.394 ± 0.037f 2.621 ± 0.107e 1.456 ± 0.025d 0.398 ± 0.001b 0.410 ± 0.002b 0.505 ± 0.002a
0.458 ± 0.001b
1.114 ± 0.010c
900 min 600 min 300 min 120 min 60 min 30 min 10 min Control SPI
At room temperature (A) D[3, 2] 0.550 ± 0.001a (μm) D[4, 3] 1.163 ± 0.003a (μm)
Samples
Table 1–Particle size distribution of emulsions of SPI, control, and the hydrolysates with different hydrolysis time at room temperature (A) and –20 ˚C freezing and thawing (B).
Soybean protein hydrolyzed by pepsin . . .
Figure 4–Solubility of SPI, control, and the hydrolysates incubated for different time after 12000 × g centrifuging for 20 min (A) and 650000 ) represents for pH 2.0 series; ( ) × g centrifuging for 45 min (B). ( represents pH 7.0 series. Different letters on the tops of columns in A (a to d and e to k) and B (a to g and h to o) indicate significant (P < 0.05) differences among samples in the same storage time.
implied that some insoluble aggregate became soluble and had strong protein–water interaction under the high ionic strength.
Mean hydrodynamic diameter Dynamic light scattering technique was applied to analyze the hydrodynamic diameter of the samples As Figure 5 has shown, the hydrodynamic diameter for soybean proteins (control) at pH 2.0 and 7.0 was 92.19 nm and 72.62 nm, respectively, due to that soybean proteins unfolded in acid condition and reaggregated when neutralization. The highest hydrodynamic diameter value for pH 2.0 series reached 129.42 nm at 120 min, while the highest value for pH 7.0 series was 98.43 nm at 60 min. The reason might be that under the acid and pepsin hydrolysis condition, the soybean proteins unfold and aggregate to a larger extent. However, when adjusting the pH to 7.0, the unfolding protein molecular would reaggregate again due to the salt concentration increasing from 0.13% to 0.51%. During the incubation time from 0 to 120 min, the hydrodynamic diameter increased, implying that the pepsin hydrolysis induced the glycinin unfolding and formed large Vol. 78, Nr. 12, 2013 r Journal of Food Science C1875
Soybean protein hydrolyzed by pepsin . . .
C: Food Chemistry Figure 5–The hydrodynamic diameter of SPI, control, and the hydrolysates incubated for different time, ( ) represents for pH 2.0 series; ( ) represents pH 7.0 series. Different letters (a to g and h to m) on the tops of Figure 6–The λmax of emission fluorescence spectroscopic of SPI, control, ) represents for pH columns indicate significant (P < 0.05) differences among samples in the and the hydrolysates incubated for different time, ( 2.0 series; ( ) represents pH 7.0 series. Different letters (a to f and g to same storage time. l) on the tops of columns indicate significant (P < 0.05) differences among samples in the same storage time.
and soluble aggregate. After the glycinin unfolded and aggregated to the largest extent, the aggregates began to degrade to lowmolecular-weight peptides and thus the hydrodynamic diameter degradation effect on the glycinin and a little effect on the α subunit of β-conglycinin. Moreover, soybean proteins unfolded decreased. and formed the soluble and flexible aggregation at first and to the largest extent when hydrolyzed for 60 min. But after 120 Intrinsic fluorescence emission spectra The polarity of the environment of the tryptophan and tyrosine min, the glycinin was degraded totally and the insoluble aggregaresidues determines the fluorescence spectrum and implies their tion of β-conglycinin was formed. As a result, for the functional specific interactions and the change of the proteins configuration. properties, SPI hydrolyzed by pepsin for 60 min showed the best When the chromophores become more exposed to solvent, the solubility, emulsibility, and the ability to resist freezing/thawing. fluorescence emission maximum (λmax ) suffers a red shift, transfer- Furthermore, because the reaction was performed at pH 2.0 and ring to the longer wavelength. When the chromophores become the final samples was pH 7.0, we found that the configuration of more embedded into the inner part of the protein molecule, λmax the sample was quite different in these 2 pH conditions. Since the transfers to the shorter wavelength showing a blue shift (Pallar`es salt concentration increased from 0.13% to 0.51% when adjusting the pH value from 2.0 to 7.0, the unfolding protein molecular and others 2004). As Figure 6 showed, the emission fluorescence spectroscopic would reaggregate again at pH 7.0 due to the charge neutralization, technique was used to analyze the conformational changes of the and the hydrodynamic diameter and λmax decreased compared to hydrolysates at pH 2.0 and pH 7.0. For soybean proteins in pH 2.0 those of the pH 2.0. Besides, some of the insoluble aggregates and 7.0, the λmax reached 338.6 nm and 337.6 nm, respectively. formed in pH 2.0 became soluble in pH 7.0, because of the salt-in The λ values of pH 2.0 series were larger than those of the pH phenomenon. max
7.0 series. The reason was that when adjusting pH to 7.0, soybean proteins would reaggregate and some of the chromophores embedded into the inner part of the proteins again. The λmax became larger with the increase of the incubation time, implying that pepsin hydrolysis caused more chromophores exposed to the solvent. The increase of λmax from 0 to 120 min was more remarkable than that from 120 min to 900 min, indicating that much more chromophores exposed during the former period and very few chromophores exposed during the latter period. The reason might be that pepsin only degraded the glycinin at first and when all the glycinin was degraded (after 120 min), pepsin had little effect on the β-conglycinin due to the formation of aggregate, hence the enzyme is not allowed to interact within the aggregates as is obtained in the single molecule state.
Conclusion With the analysis of SDS-PAGE, surface hydrophobicity, particle size distribution, hydrodynamic diameter, and emission fluorescence spectroscopic, we concluded that pepsin had strong C1876 Journal of Food Science r Vol. 78, Nr. 12, 2013
Acknowledgments We appreciate the support from Natl. Natural Science Foundation of China (Nr. 31171783 and 31000759) and Financial support from Guangdong Province (Nr. 2012BAD37B08-1 and Nr. 20100172120024 ).
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Vol. 78, Nr. 12, 2013 r Journal of Food Science C1877
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Soybean protein hydrolyzed by pepsin . . .
