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Effects of limited enzymatic hydrolysis with pepsin and high-pressure homogenization on the functional properties of soybean protein isolate
LWT - Food Science and Technology 46 (2012) 453e459
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Effects of limited enzymatic hydrolysis with pepsin and high-pressure homogenization on the functional properties of soybean protein isolate Boen Yuan, Jiaoyan Ren, Mouming Zhao*, Donghui Luo, Longjian Gu College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, Guangdong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 May 2011 Received in revised form 30 October 2011 Accepted 4 December 2011
Effects of limited enzymatic hydrolysis with pepsin and/or high-pressure homogenization in acid condition on the functional properties and structure characteristic of soybean protein isolate (SPI) were investigated. Functional properties, including protein solubility, surface hydrophobicity, particle size distribution, the ability of resisting freezing/thawing, foaming properties and dynamic surface properties were evaluated. Result showed that, single acid treatment could improve functional properties of SPI, but was not as effective as single or combined pepsin hydrolysis and high-pressure homogenization in acid condition. Emulsibility, the ability of resisting freezing/thawing and foaming capacity of soybean proteins were remarkably improved by the combination treatment, but no improvement of foaming stability was detected. Changes of structures were detected by surface hydrophobicity, and hydrodynamic diameter. It was found that aggregates existed in all treated samples. Besides, more flexible and soluble aggregates were observed in samples treated by limited pepsin proteolysis, high-pressure homogenization and the combination treatments, which might contribute to their functional properties. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Soybean protein isolate Acidic treatment Pepsin High-pressure homogenization Functional properties Structures
1. Introduction Soybean proteins have been widely used in many protein-based food formulations, such as cream, whipped topping, breads, cakes, meat and dairy alternatives, noodles, soups and a variety of nutritional foods and supplements. Reports show that nearly six processed foods out of ten contain ingredients from soybeans (Liu, Wang, Ma, & Xiao, 2008). The content of globulin in total soybean proteins is 0.9 g/g, which mainly consists of glycinin and b-conglycinin. The glycinin, also known as 11S protein, consists of acidic (ca. 38 kDa) and basic polypeptides (ca. 20 kDa). b-conglycinin, also called 7S protein, is a trimeric glycoprotein including three types of subunits (a, a0 , b with molecular masses of ca. 65, 62 and 47 kDa, respectively) (Staswick, Hermodson, & Nielsen, 1981). At ambient temperatures, glycinin forms hexameric complexes (11S) at pH 7.6, while at pH 3.8 it is mainly present in trimeric complexes (7S) (Catsimpoolas, Campbell, & Meyer, 1969; Koshiyama, 1972; Wolf & Briggs, 1958). Compared with b-conglycinin, native glycinin has impact globular conformation and low molecular weight, which caused poor functional properties of soybean proteins. Physical, chemical and enzymatic modifications have been carried out to improve the functional properties of soy proteins. * Corresponding author. Tel./fax: þ86 20 87113914. E-mail address: [email protected] (M. Zhao). 0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.12.001
Generally, single modification treatment has relative low efficiency. Therefore, most researches focus on the combination of different modification methods to improve the functional properties of protein (An, Zhou, & Zhu, 2009; Penas, Prestamoa, & Gomezb, 2004). For example, it has been reported that heat treatment combined with chymotrypsin hydrolysis could enhance the gelling properties of whey proteins (Rickert, Johnson, & Murphy, 2004). It was also found that acid treatment before enzymatic modification could dissociate or even unfolded the protein structure and thus enhance the efficiency of the following treatment (Tang, Wang & Yang, 2009). The combination of acid treatment and pepsin hydrolysis modification is a good choice to be used for improvement of protein functional properties, since the pH of protein solution after acid modification (around pH 1.5e2.0) is suitable for the working condition of pepsin (Zakaria & McFeeters, 1978). High-pressure homogenization is now extensively used in food engineering. Cavitation, shear, turbulence and temperature rise are involved simultaneously during high-pressure homogenization processing. Reports showed that high-pressure homogenization could affect the tertiary and quaternary structures of most globular proteins but had little influence on the secondary structure (Subirade, Loupil, Allain, & Paquin, 1981). However, limited proteolysis could be used as complementary since it could change secondary structure to a certain extent (Panyam & Kilara, 1996). Functional properties, such as solubility and emulsification
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capability, can be enhanced by limited proteolysis with proteases (Govindaraju & Srinivas, 2007; Rickert, Johnson, &Murphy, 2004; Tsumura et al., 2005).We recently reported that there was a remarkable improvement on functional properties of glycinin by using limited Alcalase proteolysis combined with high-pressure homogenization (Luo et al., 2010). In this paper, we investigated the effects of limited proteolysis by pepsin and/or high-pressure homogenization in acid condition on the solubility, emulsification and foaming properties of soybean proteins. Moreover, the changes on the structure of soybean proteins were studied by surface hydrophobicity and mean hydrodynamic diameter. 2. Materials and methods 2.1. Chemicals and standards Defatted soy flour (protein content 0.45 g/g in soy flour) was purchased from Yuwang Soy Company (Shandong, China). Pepsin (catalog no. P7000, 3800 units/mg) was purchased from Suoyi Chemical Co. (Henan, China). ANS was obtained from Sigma Chemical Co. (Shanghai, China). All other reagents were of analytical grade. 2.2. Sample preparation Soybean proteins treated with acid, limited proteolysis, highpressure homogenization and their combination were prepared according to the method of Puppo et al. (2004), Deak, Murphy, and Johnson (2007) with a slight modification. As shown in Fig.1 (A and B), samples were prepared from defatted low-heat soybean meal. Soy flour was dispersed in distilled water (1:l5 g:mL). The dispersion was adjusted to pH 8.5 with 2 mol/L NaOH, stirred at room temperature for 1 h, and centrifuged at 10,000 g for 20 min at 20 C. The supernatant was equally divided into 2 parts. As Fig. 1 showed, one part of the supernatant was then adjusted to pH 4.5 with 2 mol/L HCl, centrifuged at 10,000 g for 20 min, resuspended with distilled water, then adjusted to pH 7.0 with 2 mol/L NaOH, dialyzed against deionized water and freeze-dried (the Fig. 1 B final phase). This was referred to SPI. The other part of supernatant was adjusted to pH 2.0, fractionated to four parts and were processed according to the following procedures (Fig. 1 A): AT treatment: the pH 2.0 solution stirred for 1 h at 25 C is referred to AT. AT-HPH treatment: the pH 2.0 solution treated with highpressure homogenization is referred to as AT-HPH. AT-LP treatment: the pH 2.0 solution was hydrolysed for 15 min with Pepsin at 37 C and it is referred to as AT-LP. AT-LPCHPH treatment: the pH 2.0 solution treated with the combination of limited proteolysis and high-pressure homogenization is referred to as AT-LPCHPH. This treatment was conducted under the conditions used for limited proteolysis and high-pressure homogenisation alone. All these four samples were treated following the final phase (Fig. 1 B). 2.3. Solubility Protein dispersions (10 mg:mL, dissoved in deionized water) were adjusted to a specific value within the range of pH 2e9 by 0.5 mol/L HCl or NaOH. The dispersions were agitated with a magnetic stirrer for 1 h at room temperature, and then centrifuged at 12,000 g for 20 min to obtain the supernatants. Protein contents of the supernatant were determined according to Lowry,
Fig. 1. Scheme of sample preparation. The major phase (A) and the final phase (B).
Rosebrough, Farr, and Randall (1951). Using bovine serum albumin as the standard. Percent protein solubility was calculated as nitrogen solubility index (NSI) ¼ (protein content of supernatant/amount of proteins added) 100%. 2.4. Surface hydrophobicity Surface hydrophobicity was determined according to the method of Kato and Nakai (1980), using the hydrophobicity fluorescence probe L-anilino-8-naphthalenesulfonate, without SDS. Protein dispersions (10 mg/ml) in 0.01 mol/L phosphate buffer (pH 7.0) were stirred for 2 h at 20 C and centrifuged at 8000 g for 20 min. The protein concentration in the solution was measured by the method the same in Section 2.3. Each supernatant was serially diluted with the same buffer to obtain protein concentrations ranging from 2 to 10 mg/mL. Then 20 mL of ANS (8.0 mmol/L in the same buffer) was added to 4 ml of sample. Fluorescence intensity was measured with a Hitachi F4500 fluorescence spectrometer (Tokyo, Japan), at wavelengths of 390 nm (excitation) and 470 nm (emission). The initial slope of fluorescence intensity vs. protein concentration plot (calculated by linear regression analysis) was used as the index of protein hydrophobicity.
B. Yuan et al. / LWT - Food Science and Technology 46 (2012) 453e459
2.5. Particle size distribution According to Luo et al. (2010), the oil/water (20:80 mL:mL) emulsion was prepared by adding the corn oil to the protein solution (20 mg:mL) with the aid of a mechanical stirrer (Shanghai Specimen Model Co., China). The pH of the aqueous phase was adjusted to 7.0. The resulting crude emulsion was then homogenized (30 MPa, two passes) using a laboratory homogenizer (APV Gaulin, Abvertslund, Denmark) to obtain aimed emulsions. 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). Measurements were carried out at room temperature and the volume fraction of emulsion in the diluted deionized water was approximately 1:1000. Calculation from 0.02 to 100 ml was expressed in different volume. The average particle size (d3, 2, volume-surface average diameter) was measured. We also stored the emulsion at 20 C for 24 h and determined the particle size distribution after thawing, in order to investigate the ability of resisting freezing/thawing. 2.6. Foaming properties According to the method of Fernandez and Macarulla (1997), we determined the foaming properties including foaming capacity (FC) and foam stability (FS). 6 mL of the sample solutions (10 mg/mL) at pH 7.0 in measuring cylinder (25 mL) were homogenized with an FJ-200 high-speed homogenizer with the condition of 10,000 rpm for 1 min. FC was calculated as the percent increase in volume of the protein dispersion upon mixing, while FS was estimated as the percentage of foam remaining after 30 min. 2.7. Measurement of mean hydrodynamic diameter The mean hydrodynamic diameter of the proteins in different samples was measured by a dynamic light scattering technique using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK) equipped with a 4 mW helium/neon laser at a wavelength output of 633 nm. Droplet sizing was performed at 25 C at 10-s intervals in a particle-sizing cell using backscattering technology. The intensity of light scattered from the proteins in the dispersions was used to calculate the mean hydrodynamic diameter (Z-average mean), based on the StokeseEinstein equation by assuming the proteins to be spherical. For each sample, the mean and standard deviation were calculated from at least five measurements. The samples were also filtered through 0.45 mm Millipore membrane prior to analysis.
455
replicated at least four times. It was found that peq could be reproduced to 0.2 mN/m. 2.9. Statistical analysis Data were expressed as mean standard deviation for triplicate determinations. An analysis of variance (ANOVA) was performed on the data, and a least significant difference (LSD) test with a confidence interval of 95% was used to compare the means. 3. Results and discussion 3.1. Protein solubility Solubility is an important functional property of proteins, since it can affect other functional properties, such as rheological, hydrodynamic and surface-active properties. Good solubility is crucial for many protein-based formulations. The solubility of the soybean protein samples at different pH level are shown in Fig. 2. Around the isoelectric point of pH 4e5, AT-LP and AT-LPCHPH showed the highest solubility among all the samples, because limited proteolysis treatment released low molecular weight peptides and improved the flexibility of protein. The additional effect of high-pressure homogenization made the solubility of ATLPCHPH higher than AT-LP. The pH-dependent solubility of proteins was important for other associated functional properties and applications in food systems (Kinsella, 1979), especially at pH<4 or >7. AT and AT-LP had the lowest solubility in all pH range except at pH >7, due to that with the effect of acid and limited proteolysis, proteins would unfold and disassociate, exposing more hydrophobic residues. Except SPI, samples treated by high-pressure homogenization (ATHPH & AT-LPCHPH) demonstrated higher solubility at pH < 7. ATLPCHPH showed the highest solubility at 2 < pH < 4. But no improvement was found at pH < 2. SPI had the best solubility reaching 62.2%. At pH 7.0, the solubility of SPI, AT, AT-HPH, AT-LP and AT-LPCHPH reached 62.8%, 59.3%, 67.6%, 54.0% and 63.2%, respectively. Due to the demolishment of the tight structure of proteins and insoluble aggregates, exposure of more charged and polar groups to the surrounding water, and the formation of soluble aggregates by the effect of high-pressure homogenization, the solubility of AT-HPH was the highest (Panyam & Kilara, 1996).
80 70 60
2.8. Dynamic surface properties Solubility(%)
50
The modified and native soybean proteins absorption at the oilewater interface was determined by monitoring the evolution of surface pressure (p) parameters with time. The protein concentration was maintained constant at 1 mg/ml in all samples and the temperature was maintained at 25 C during the process of analysis. Dynamic surface pressure (p) measurements of modified and native soybean proteins adsorbed films at the oilewater interface were determined using an optical contact angle meter, OCA-20 (Dataphysics Instruments GmbH, Germany). Details of this apparatus are given elsewhere (Caseli, Masui, Furriel, Leone, & Zaniquelli, 2005). Surface activity was expressed by the surface pressure, peq ¼ p0 peq, where, p0 and peq are the aqueous subphase surface pressure and the surface pressure of the protein solutions at 5000 s, respectively. Some experiments were
40 30 20 10 0 1
2
3
4
5
6
7
8
9
10
pH Value Fig. 2. Protein solubility of the five samples, (-) SPI, (C) at, (:) AT-HPH, (+) AT-LP and (B) AT-LPCHPH.
B. Yuan et al. / LWT - Food Science and Technology 46 (2012) 453e459
Similarly, it has been reported that ultrasonic treatment could improve the protein solubility of commercial SPI (Tang, Wang, Yang, & Li, 2009). In this study, we found that the solubility of AT-HPH was higher than AT-LPCHPH and AT-LP. Implying that highpressure homogenization and limited proteolysis didn’t have coordination efficiency on improving solubility and the highpressure homogenization was more efficiency. However, Luo et al. (2010) found that the combination of high-pressure homogenization and limited proteolysis with Alcalase had coordination efficiency on improving solubility of glycinin. The reason for this phenomenon was that the suitable condition of pepsin is pH 2.0 and proteins may dissociate or even unfold in structure.
10
8
Volumn(%)
456
6
4
2
3.2. Surface hydrophobicity Glycinin known as 11S would unfold and disassociate into the 7S or 3S form at low pH. It has been reported that fluorescence spectroscopy shows a shift of w2 nm of lmax to longer wavelengths when the pH is lowered from 7.6 to 3.8 (Lakemond, Jongh, Hessing, Gruppen, & Voragen, 2000). In the present work, we had the same conclusion that single acid treatment could remarkably enhance the surface hydrophobicity compared with SPI (Table 1). AT-LP had the highest surface hydrophobicity, because limited proteolysis caused the exposure of hydrophobic groups in the inner part of protein (Panyam & Kilara, 1996). High-pressure homogenization involved treatments showed low surface hydrophobicity, due to the formation of aggregation caused by hydrophobic interaction (Kella, Barbeau, & Kinsella, 1986). Because the limited proteolysis treatment induced the releasing of much more hydrophobic residues from protein molecules, the following re-aggregation caused by high-pressure homogenization presented a larger decrease in surface hydrophobicity. The results of surface hydrophobicity were consistent with the solubility (Fig. 2). 3.3. Particle size distribution The comparative particle size distribution at pH 7.0 for all the samples was showed at Fig. 3. Emulsions with SPI, AT, AT-HPH, ATLP and AT-LPCHPH showed monomodal distribution of particle size and the maximum d3,2 values for each sample were found at 1.374 mm, 1.086 mm, 1.047 mm, 0.844 mm and 0.770 mm, respectively. Obviously, the emulsion of AT-LPCHPH showed the smallest particle size distribution. Proteins dispersed in low pH would disassociate and result in the exposure of hydrophobic groups, which might improve the hydrophilicelipophilic balance for better emulsification. Additional treatment, high-pressure homogenization or limited proteolysis, showed the coordination efficiency with acid treatment. High-pressure homogenization could improve the functional properties due to the modification of protein structure. Limited proteolysis of proteins with pepsin at pH 2.0 caused the unfolding of protein structure exposing more hydrophobic groups, so AT-LP showed better emulsibility than AT. High-pressure
Table 1 Surface hydrophobicity of the five samples in 0.01 mol/L phosphate buffer (pH 7.0). Samples
Surface hydrophobicity
SPI AT AT-HPH AT-LP AT-LPCHPH
4352.2 4981.4 4785.2 5208.5 3882.4
3.36a 1.99b 4.33c 1.06d 1.51e
Means standard deviations of triplicate analyses are given. Different letters (aee) on the tops of columns indicate significant (p < 0.05) differences among samples in the same storage time.
0 0 .1
1
10
1 00
Diameter(nm) Fig. 3. Particle size distribution of emulsions made with(-) SPI, (C) at, (:) AT-HPH, (+) AT-LP and (B) AT-LPCHPH.
homogenization at acid condition could form the soluble aggregate and attribute to the diversity of structure flexibility. The combined modification could induce significant improvements on soybean proteins emulsification characteristics. The reason was that more flexible aggregates were formed by treating with highpressure homogenization after limited proteolysis in acid solution. The aggregates could easily attach to the interface and form a thicker adsorbed layer. No visible aggregates were found in all samples except SPI (data not shown), implying that the modification samples could make the emulsion stabler than the native soybean proteins. When freezing the emulsion, icy crystal forms and interrupts the proteineprotein interaction, inducing the generation of larger particles after thawing. Therefore, the ability of resisting freezing/ thawing is very important for the application of soybean proteins. Table 2 shows the comparative particle size distribution after freezing at 20 C for 24 h and thawing. The result was consistent with the comparative particle size distribution determined at ambient, and the AT-LPCHPH showed the smallest particle size distribution. The reason might be that flexible soluble aggregates formed by combination treatment could attach to the interface of oilewater and form a gel-like elasticity film which was strong enough to resist the interruption of icy crystal. Single treatment with acid, high-pressure homogenization or limited proteolysis could also improve the ability of resisting freezing/thawing, although they were not as effective as the combination treatment. We concluded that combined modification could integrate the advantages of these three treatments, and improve nearly all related characteristics of emulsibility significantly.
Table 2 The ability of resisting freezing and thawing of the five samples. D [3,2] represents surface area weighted average; D [4,3] represents volume weighted average. Samples
D [3,2]
SPI AT AT-HPH AT-LP AT-LPCHPH
25.668 21.382 19.711 12.685 7.496
D [4,3]
1.13a 0.071b 0.092c 0.43d 0.49e
94.031 81.234 73.040 52.083 34.006
0.57a 0.85b 1.44c 0.64d 0.71e
Each value is the mean and standard deviation of duplicate measurements. Different letters (aee) on the tops of columns indicate significant (p < 0.05) differences among samples in the same storage time.
B. Yuan et al. / LWT - Food Science and Technology 46 (2012) 453e459
3.4. Foaming properties Fig. 4 shows the foaming capacity (FC) and foaming stability (FS) of SPI and the modification samples. The foaming capacities of all the modification samples were improved compared with that of SPI. The SPI exposed less hydrophobic groups and the tighter conformation of the globular than the modification samples, so the speed of SPI molecular transferring to the airewater interface was very slow and dropped the surface tension inefficiently (Hettiarachchy & Ziegler, 1994). Due to the high surface hydrophobicity, AT and AT-LP could enhance the foaming capacity up to 52.5% and 57.5%, respectively. The foaming capacity of AT-LP was better than AT, which might be due to that limited proteolysis could expose more hydrophobic groups from the inner of proteins than acid treatment. For the AT-HPH, its foaming capacity was equal with that of AT, implying that the aggregate formed by highpressure homogenization in acid condition had no improvement on the FC. The AT-LPCHPH showed the highest foaming capacity up to 65%. Because more flexible and low-molecular-weight aggregates were formed by the combination treatment, they could transfer to the airewater interface more rapidly and the proteineprotein interaction improved the strength of the viscoelastic cohesive film, dropping the surface tension efficiently. This result implied that the advantages of these three treatments could be integrated together. The results of FS indicated that no improvement on foaming stability of the modification samples was found compared with SPI. AT exhibited the lowest foaming stability, 70
d c
60
Foaming Capacity/%
b
b
50 40
a
30 20 10 0 SPI
AT
AT-HPH
AT-LP
AT-LPCHPH
Samples 100
457
which might be due to that the adsorbing film formed by the covalent and non-covalent bonds couldn’t stop the air bubble reaggregate. Compared with single acid treatment, the highpressure homogenization in acid condition could induce the formation of disulfide bonds, while the limited proteolysis with pepsin could expose much more hydrophobic groups to improve the hydrophobic interaction. So compared with AT, AT-HPH showed better foaming stability to 88.8%, which was more efficient than ATLP (85.5%). The combination treatment also improved the foaming stability in acid solution but was less efficient than high-pressure homogenization in acid condition. 3.5. Mean hydrodynamic diameter The hydrodynamic diameter of native or modified soybean proteins was evaluated by dynamic light scattering technique in order to investigate protein aggregation during modification(Tang & Ma, 2009). As shown in Table 3, the average particle size of untreated soybean protein (SPI) dispersion was about 56.94 nm. As expected, the average particle size of the protein in modified samples was considerably increased, indicating aggregation and unfolding of protein occurred during the process of treatments. Glycinin in acid solution would unfold and disassociated, so average particle size of AT (77.07 nm) was larger than native soybean proteins (56.94 nm). Moreover, due to the unfolding of protein induced by limited proteolysis, average particle size of AT-LP was larger than that of AT. We concluded that the increase of hydrodynamic diameter of AT and AT-LP could be attributed to the unfolding and disassociation of proteins. The increase of surface hydrophobicity was consistent with this conclusion. The decrease of surface hydrophobicity and the increase of hydrodynamic diameter for AT-HPH and AT-LPCHPH samples implied the formation of aggregation, but the extent of protein aggregation varied with different treatment. The average particle size of aggregates induced by single high-pressure homogenization (107.8 nm) was larger than that induced by combination treatment (99.5 nm), respectively. Since the average particle size of AT-HPH was larger than that of the AT-LPCHPH and the surface hydrophobicity was also higher, it implied that more but smaller aggregates were formed by the combination treatment. So we speculated that aggregates were formed by high-pressure homogenization. Moreover, single high-pressure homogenization in acid solution trended to form larger aggregates and the combination treatment trended to form smaller aggregates. This result was in agreement with the results of surface hydrophobicity and surface pressure (Table 4).
a c
d
d
3.6. Dynamic surface properties
Foaming Stability/%
b 80
Being surface-active, proteins can act as effective emulsifying agents, and the interfacial behavior of adsorbed proteins plays a crucial role in determining the stability of foams and emulsions
60
40
Table 3 Mean hydrodynamic diameter of the five samples.
20
0 SPI
AT
AT-HPH
AT-LP
AT-LPCHPH
Samples Fig. 4. Foaming capacity (FC) and foaming stability (FS) of SPI and the modified samples. Different letters (aed) on the tops of columns indicate significant (p < 0.05) differences among samples in the same storage time.
Samples
Average diameter/nm
SPI AT AT-HPH AT-LP AT-LPCHPH
56.94 77.07 107.8 86.64 99.95
0.21a 0.90b 1.12c 1.07d 1.03e
Each value is the mean and standard deviation of duplicate measurements. Different letters (aee) on the tops of columns indicate significant (p < 0.05) differences among samples in the same storage time.
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B. Yuan et al. / LWT - Food Science and Technology 46 (2012) 453e459 Table 4 The adsorption of time dependence of surface pressure (p) for SPI and the modified samples adsorbed films at the oilewater interface. samples
Surface pressure (p) mN/m
SPI AT AT-HPH AT-LP AT-LPCHPH
16.60 16.96 17.05 17.22 17.35
0.014a 0.007b 0.007c 0.012d 0.014e
Each value is the mean and standard deviation of duplicate measurements. Different letters (aee) on the tops of columns indicate significant (p < 0.05) differences among samples in the same storage time.
(Baeza, Carrera, Rodríguez Patino, & Pilosof, 2005). There are three stages for protein to be absorbed to the airewater and oilewater interface. The first stage is called the induction period. The second stage is protein diffusion to the interface and adsorption, and the last stage is penetration at the interface. In the induction period, proteins transferred to the oilewater interface but few were adsorbed on the film, so no increasing of surface pressure was detected during the induction period (Kim & Kinsella, 1987). Some authors attribute the existence of induction period to the molecular flexibility of the protein and its susceptibility to conformational changes (Graham & Phillips, 1979). As Table 4 has shown, the surface pressure of SPI, AT, AT-HPH, AT-LP and AT-LPCHPH reached 16.60 mN/m, 16.96 mN/m, 17.05 mN/m, 17.22 mN/m and 17.35 mN/ m, respectively. Because the disassociation in acid solution and limited proteolysis increased surface hydrophobicity, the protein could be more efficiently absorbed to the oilewater interface. Since the re-arrangement of the flexible and soluble aggregates on the oilewater interface were easier, single high-pressure homogenization in acid solution could increase the surface pressure but not as efficient as the limited proteolysis treatment. The combination treatment produced the highest surface pressure among all samples, implying that more flexible aggregates were formed by combination treatment. Moreover, we concluded that highpressure homogenization and limited proteolysis with pepsin had coordination efficiency on hastening the adsorption of proteins to the oilewater interface. This result was consistent with the emulsibility and the foaming properties. Because water is polar phase while oil and air is nonpolar phase, the oilewater and airewater system have the same behavior of proteins adsorption. 4. Conclusions Due to the dissociation and unfolding of the soybean protein structure in acid condition, the following treatments including pepsin proteolysis, high-pressure homogenization or even their combination, could enhance the interaction between protein molecules and the functional properties. With the analysis of surface hydrophobicity, hydrodynamic diameter, and surface pressure, we concluded that limited proteolysis with pepsin and/or high-pressure homogenization in acid condition could induce the formation of soluble and flexible aggregation. Moreover, smaller size aggregates were formed by the combination treatments in acid condition. In conclusion, the combination of acid treatment, pepsin proteolysis and high-pressure homogenization was a good choice to improve emulsibility, ability of resisting freezing/thawing and foaming activity of SPI. Acknowledgments We appreciate the support from National Natural Science Foundation of China (No. 31000759), National Natural Science Foundation
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Panyam, D., & Kilara, A. (1996). Enhancing the functionality of food proteins by enzymatic modification. Trends in Food Science and Technology, 7, 120e125. Peñasa, E., Préstamo, G., & Gomezb, R. (2004). High pressure and the enzymatic hydrolysis of soybean whey proteins. Food Chemistry, 85(4), 641e648. Puppo, C., Chapleau, N., Speroni, F., Lamballerie-Anton, M., de Michel, F., & Anon, C. (2004). Physicochemical modifications of highpressure-treated soybean protein isolates. Journal of Agricultural and Food Chemistry, 52(6), 1564e1571. Rickert, D. A., Johnson, L. A., & Murphy, P. A. (2004). Functional properties of improved glycinin and b-conglycinin fractions. Journal of Food Science, 69, 303e311. Staswick, P. E., Hermodson, M. A., & Nielsen, N. C. (1981). Identification of the acidic and basic subunit complexes of glycinin. Journal of Biological Chemistry, 256, 8752e8755. Subirade, M., Loupil, F., Allain, A. F., & Paquin, P. (1981). Effect of dynamic high pressure on the secondary structure of b-lactoglobulin and on its conformational properties as determined by Fourier transform infrared spectroscopy. International Dairy Journal, 8, 135e140. Tang, C. H., & Ma, C. Y. (2009). Effect of high pressure treatment on aggregation and structural properties of soy protein isolate. LWT - Food Science and Technology, 42, 606e611. Tang, C. H., Wang, X. S., & Yang, X. C. (2009). Enzymatic hydrolysis of hemp (Cannabis sativa L.) protein isolate by various proteases and antioxidant properties of the resulting hydrolysates. Food Chemistry, 114, 1484e1490.
B. Yuan et al. / LWT - Food Science and Technology 46 (2012) 453e459 Tang, C. H., Wang, X. Y., Yang, X. C., & Li, L. (2009). Formation of soluble aggregates from insoluble commercial soy protein isolated by means of ultrasonic treatment and their gelling properties. Journal of Food Engineering, 92, 432e437. 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 Science and Technology, 38, 255e261.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Effects of limited enzymatic hydrolysis with pepsin and high-pressure homogenization on the functional properties of soybean protein isolate Boen Yuan, Jiaoyan Ren, Mouming Zhao*, Donghui Luo, Longjian Gu College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, Guangdong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 May 2011 Received in revised form 30 October 2011 Accepted 4 December 2011
Effects of limited enzymatic hydrolysis with pepsin and/or high-pressure homogenization in acid condition on the functional properties and structure characteristic of soybean protein isolate (SPI) were investigated. Functional properties, including protein solubility, surface hydrophobicity, particle size distribution, the ability of resisting freezing/thawing, foaming properties and dynamic surface properties were evaluated. Result showed that, single acid treatment could improve functional properties of SPI, but was not as effective as single or combined pepsin hydrolysis and high-pressure homogenization in acid condition. Emulsibility, the ability of resisting freezing/thawing and foaming capacity of soybean proteins were remarkably improved by the combination treatment, but no improvement of foaming stability was detected. Changes of structures were detected by surface hydrophobicity, and hydrodynamic diameter. It was found that aggregates existed in all treated samples. Besides, more flexible and soluble aggregates were observed in samples treated by limited pepsin proteolysis, high-pressure homogenization and the combination treatments, which might contribute to their functional properties. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Soybean protein isolate Acidic treatment Pepsin High-pressure homogenization Functional properties Structures
1. Introduction Soybean proteins have been widely used in many protein-based food formulations, such as cream, whipped topping, breads, cakes, meat and dairy alternatives, noodles, soups and a variety of nutritional foods and supplements. Reports show that nearly six processed foods out of ten contain ingredients from soybeans (Liu, Wang, Ma, & Xiao, 2008). The content of globulin in total soybean proteins is 0.9 g/g, which mainly consists of glycinin and b-conglycinin. The glycinin, also known as 11S protein, consists of acidic (ca. 38 kDa) and basic polypeptides (ca. 20 kDa). b-conglycinin, also called 7S protein, is a trimeric glycoprotein including three types of subunits (a, a0 , b with molecular masses of ca. 65, 62 and 47 kDa, respectively) (Staswick, Hermodson, & Nielsen, 1981). At ambient temperatures, glycinin forms hexameric complexes (11S) at pH 7.6, while at pH 3.8 it is mainly present in trimeric complexes (7S) (Catsimpoolas, Campbell, & Meyer, 1969; Koshiyama, 1972; Wolf & Briggs, 1958). Compared with b-conglycinin, native glycinin has impact globular conformation and low molecular weight, which caused poor functional properties of soybean proteins. Physical, chemical and enzymatic modifications have been carried out to improve the functional properties of soy proteins. * Corresponding author. Tel./fax: þ86 20 87113914. E-mail address: [email protected] (M. Zhao). 0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.12.001
Generally, single modification treatment has relative low efficiency. Therefore, most researches focus on the combination of different modification methods to improve the functional properties of protein (An, Zhou, & Zhu, 2009; Penas, Prestamoa, & Gomezb, 2004). For example, it has been reported that heat treatment combined with chymotrypsin hydrolysis could enhance the gelling properties of whey proteins (Rickert, Johnson, & Murphy, 2004). It was also found that acid treatment before enzymatic modification could dissociate or even unfolded the protein structure and thus enhance the efficiency of the following treatment (Tang, Wang & Yang, 2009). The combination of acid treatment and pepsin hydrolysis modification is a good choice to be used for improvement of protein functional properties, since the pH of protein solution after acid modification (around pH 1.5e2.0) is suitable for the working condition of pepsin (Zakaria & McFeeters, 1978). High-pressure homogenization is now extensively used in food engineering. Cavitation, shear, turbulence and temperature rise are involved simultaneously during high-pressure homogenization processing. Reports showed that high-pressure homogenization could affect the tertiary and quaternary structures of most globular proteins but had little influence on the secondary structure (Subirade, Loupil, Allain, & Paquin, 1981). However, limited proteolysis could be used as complementary since it could change secondary structure to a certain extent (Panyam & Kilara, 1996). Functional properties, such as solubility and emulsification
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capability, can be enhanced by limited proteolysis with proteases (Govindaraju & Srinivas, 2007; Rickert, Johnson, &Murphy, 2004; Tsumura et al., 2005).We recently reported that there was a remarkable improvement on functional properties of glycinin by using limited Alcalase proteolysis combined with high-pressure homogenization (Luo et al., 2010). In this paper, we investigated the effects of limited proteolysis by pepsin and/or high-pressure homogenization in acid condition on the solubility, emulsification and foaming properties of soybean proteins. Moreover, the changes on the structure of soybean proteins were studied by surface hydrophobicity and mean hydrodynamic diameter. 2. Materials and methods 2.1. Chemicals and standards Defatted soy flour (protein content 0.45 g/g in soy flour) was purchased from Yuwang Soy Company (Shandong, China). Pepsin (catalog no. P7000, 3800 units/mg) was purchased from Suoyi Chemical Co. (Henan, China). ANS was obtained from Sigma Chemical Co. (Shanghai, China). All other reagents were of analytical grade. 2.2. Sample preparation Soybean proteins treated with acid, limited proteolysis, highpressure homogenization and their combination were prepared according to the method of Puppo et al. (2004), Deak, Murphy, and Johnson (2007) with a slight modification. As shown in Fig.1 (A and B), samples were prepared from defatted low-heat soybean meal. Soy flour was dispersed in distilled water (1:l5 g:mL). The dispersion was adjusted to pH 8.5 with 2 mol/L NaOH, stirred at room temperature for 1 h, and centrifuged at 10,000 g for 20 min at 20 C. The supernatant was equally divided into 2 parts. As Fig. 1 showed, one part of the supernatant was then adjusted to pH 4.5 with 2 mol/L HCl, centrifuged at 10,000 g for 20 min, resuspended with distilled water, then adjusted to pH 7.0 with 2 mol/L NaOH, dialyzed against deionized water and freeze-dried (the Fig. 1 B final phase). This was referred to SPI. The other part of supernatant was adjusted to pH 2.0, fractionated to four parts and were processed according to the following procedures (Fig. 1 A): AT treatment: the pH 2.0 solution stirred for 1 h at 25 C is referred to AT. AT-HPH treatment: the pH 2.0 solution treated with highpressure homogenization is referred to as AT-HPH. AT-LP treatment: the pH 2.0 solution was hydrolysed for 15 min with Pepsin at 37 C and it is referred to as AT-LP. AT-LPCHPH treatment: the pH 2.0 solution treated with the combination of limited proteolysis and high-pressure homogenization is referred to as AT-LPCHPH. This treatment was conducted under the conditions used for limited proteolysis and high-pressure homogenisation alone. All these four samples were treated following the final phase (Fig. 1 B). 2.3. Solubility Protein dispersions (10 mg:mL, dissoved in deionized water) were adjusted to a specific value within the range of pH 2e9 by 0.5 mol/L HCl or NaOH. The dispersions were agitated with a magnetic stirrer for 1 h at room temperature, and then centrifuged at 12,000 g for 20 min to obtain the supernatants. Protein contents of the supernatant were determined according to Lowry,
Fig. 1. Scheme of sample preparation. The major phase (A) and the final phase (B).
Rosebrough, Farr, and Randall (1951). Using bovine serum albumin as the standard. Percent protein solubility was calculated as nitrogen solubility index (NSI) ¼ (protein content of supernatant/amount of proteins added) 100%. 2.4. Surface hydrophobicity Surface hydrophobicity was determined according to the method of Kato and Nakai (1980), using the hydrophobicity fluorescence probe L-anilino-8-naphthalenesulfonate, without SDS. Protein dispersions (10 mg/ml) in 0.01 mol/L phosphate buffer (pH 7.0) were stirred for 2 h at 20 C and centrifuged at 8000 g for 20 min. The protein concentration in the solution was measured by the method the same in Section 2.3. Each supernatant was serially diluted with the same buffer to obtain protein concentrations ranging from 2 to 10 mg/mL. Then 20 mL of ANS (8.0 mmol/L in the same buffer) was added to 4 ml of sample. Fluorescence intensity was measured with a Hitachi F4500 fluorescence spectrometer (Tokyo, Japan), at wavelengths of 390 nm (excitation) and 470 nm (emission). The initial slope of fluorescence intensity vs. protein concentration plot (calculated by linear regression analysis) was used as the index of protein hydrophobicity.
B. Yuan et al. / LWT - Food Science and Technology 46 (2012) 453e459
2.5. Particle size distribution According to Luo et al. (2010), the oil/water (20:80 mL:mL) emulsion was prepared by adding the corn oil to the protein solution (20 mg:mL) with the aid of a mechanical stirrer (Shanghai Specimen Model Co., China). The pH of the aqueous phase was adjusted to 7.0. The resulting crude emulsion was then homogenized (30 MPa, two passes) using a laboratory homogenizer (APV Gaulin, Abvertslund, Denmark) to obtain aimed emulsions. 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). Measurements were carried out at room temperature and the volume fraction of emulsion in the diluted deionized water was approximately 1:1000. Calculation from 0.02 to 100 ml was expressed in different volume. The average particle size (d3, 2, volume-surface average diameter) was measured. We also stored the emulsion at 20 C for 24 h and determined the particle size distribution after thawing, in order to investigate the ability of resisting freezing/thawing. 2.6. Foaming properties According to the method of Fernandez and Macarulla (1997), we determined the foaming properties including foaming capacity (FC) and foam stability (FS). 6 mL of the sample solutions (10 mg/mL) at pH 7.0 in measuring cylinder (25 mL) were homogenized with an FJ-200 high-speed homogenizer with the condition of 10,000 rpm for 1 min. FC was calculated as the percent increase in volume of the protein dispersion upon mixing, while FS was estimated as the percentage of foam remaining after 30 min. 2.7. Measurement of mean hydrodynamic diameter The mean hydrodynamic diameter of the proteins in different samples was measured by a dynamic light scattering technique using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK) equipped with a 4 mW helium/neon laser at a wavelength output of 633 nm. Droplet sizing was performed at 25 C at 10-s intervals in a particle-sizing cell using backscattering technology. The intensity of light scattered from the proteins in the dispersions was used to calculate the mean hydrodynamic diameter (Z-average mean), based on the StokeseEinstein equation by assuming the proteins to be spherical. For each sample, the mean and standard deviation were calculated from at least five measurements. The samples were also filtered through 0.45 mm Millipore membrane prior to analysis.
455
replicated at least four times. It was found that peq could be reproduced to 0.2 mN/m. 2.9. Statistical analysis Data were expressed as mean standard deviation for triplicate determinations. An analysis of variance (ANOVA) was performed on the data, and a least significant difference (LSD) test with a confidence interval of 95% was used to compare the means. 3. Results and discussion 3.1. Protein solubility Solubility is an important functional property of proteins, since it can affect other functional properties, such as rheological, hydrodynamic and surface-active properties. Good solubility is crucial for many protein-based formulations. The solubility of the soybean protein samples at different pH level are shown in Fig. 2. Around the isoelectric point of pH 4e5, AT-LP and AT-LPCHPH showed the highest solubility among all the samples, because limited proteolysis treatment released low molecular weight peptides and improved the flexibility of protein. The additional effect of high-pressure homogenization made the solubility of ATLPCHPH higher than AT-LP. The pH-dependent solubility of proteins was important for other associated functional properties and applications in food systems (Kinsella, 1979), especially at pH<4 or >7. AT and AT-LP had the lowest solubility in all pH range except at pH >7, due to that with the effect of acid and limited proteolysis, proteins would unfold and disassociate, exposing more hydrophobic residues. Except SPI, samples treated by high-pressure homogenization (ATHPH & AT-LPCHPH) demonstrated higher solubility at pH < 7. ATLPCHPH showed the highest solubility at 2 < pH < 4. But no improvement was found at pH < 2. SPI had the best solubility reaching 62.2%. At pH 7.0, the solubility of SPI, AT, AT-HPH, AT-LP and AT-LPCHPH reached 62.8%, 59.3%, 67.6%, 54.0% and 63.2%, respectively. Due to the demolishment of the tight structure of proteins and insoluble aggregates, exposure of more charged and polar groups to the surrounding water, and the formation of soluble aggregates by the effect of high-pressure homogenization, the solubility of AT-HPH was the highest (Panyam & Kilara, 1996).
80 70 60
2.8. Dynamic surface properties Solubility(%)
50
The modified and native soybean proteins absorption at the oilewater interface was determined by monitoring the evolution of surface pressure (p) parameters with time. The protein concentration was maintained constant at 1 mg/ml in all samples and the temperature was maintained at 25 C during the process of analysis. Dynamic surface pressure (p) measurements of modified and native soybean proteins adsorbed films at the oilewater interface were determined using an optical contact angle meter, OCA-20 (Dataphysics Instruments GmbH, Germany). Details of this apparatus are given elsewhere (Caseli, Masui, Furriel, Leone, & Zaniquelli, 2005). Surface activity was expressed by the surface pressure, peq ¼ p0 peq, where, p0 and peq are the aqueous subphase surface pressure and the surface pressure of the protein solutions at 5000 s, respectively. Some experiments were
40 30 20 10 0 1
2
3
4
5
6
7
8
9
10
pH Value Fig. 2. Protein solubility of the five samples, (-) SPI, (C) at, (:) AT-HPH, (+) AT-LP and (B) AT-LPCHPH.
B. Yuan et al. / LWT - Food Science and Technology 46 (2012) 453e459
Similarly, it has been reported that ultrasonic treatment could improve the protein solubility of commercial SPI (Tang, Wang, Yang, & Li, 2009). In this study, we found that the solubility of AT-HPH was higher than AT-LPCHPH and AT-LP. Implying that highpressure homogenization and limited proteolysis didn’t have coordination efficiency on improving solubility and the highpressure homogenization was more efficiency. However, Luo et al. (2010) found that the combination of high-pressure homogenization and limited proteolysis with Alcalase had coordination efficiency on improving solubility of glycinin. The reason for this phenomenon was that the suitable condition of pepsin is pH 2.0 and proteins may dissociate or even unfold in structure.
10
8
Volumn(%)
456
6
4
2
3.2. Surface hydrophobicity Glycinin known as 11S would unfold and disassociate into the 7S or 3S form at low pH. It has been reported that fluorescence spectroscopy shows a shift of w2 nm of lmax to longer wavelengths when the pH is lowered from 7.6 to 3.8 (Lakemond, Jongh, Hessing, Gruppen, & Voragen, 2000). In the present work, we had the same conclusion that single acid treatment could remarkably enhance the surface hydrophobicity compared with SPI (Table 1). AT-LP had the highest surface hydrophobicity, because limited proteolysis caused the exposure of hydrophobic groups in the inner part of protein (Panyam & Kilara, 1996). High-pressure homogenization involved treatments showed low surface hydrophobicity, due to the formation of aggregation caused by hydrophobic interaction (Kella, Barbeau, & Kinsella, 1986). Because the limited proteolysis treatment induced the releasing of much more hydrophobic residues from protein molecules, the following re-aggregation caused by high-pressure homogenization presented a larger decrease in surface hydrophobicity. The results of surface hydrophobicity were consistent with the solubility (Fig. 2). 3.3. Particle size distribution The comparative particle size distribution at pH 7.0 for all the samples was showed at Fig. 3. Emulsions with SPI, AT, AT-HPH, ATLP and AT-LPCHPH showed monomodal distribution of particle size and the maximum d3,2 values for each sample were found at 1.374 mm, 1.086 mm, 1.047 mm, 0.844 mm and 0.770 mm, respectively. Obviously, the emulsion of AT-LPCHPH showed the smallest particle size distribution. Proteins dispersed in low pH would disassociate and result in the exposure of hydrophobic groups, which might improve the hydrophilicelipophilic balance for better emulsification. Additional treatment, high-pressure homogenization or limited proteolysis, showed the coordination efficiency with acid treatment. High-pressure homogenization could improve the functional properties due to the modification of protein structure. Limited proteolysis of proteins with pepsin at pH 2.0 caused the unfolding of protein structure exposing more hydrophobic groups, so AT-LP showed better emulsibility than AT. High-pressure
Table 1 Surface hydrophobicity of the five samples in 0.01 mol/L phosphate buffer (pH 7.0). Samples
Surface hydrophobicity
SPI AT AT-HPH AT-LP AT-LPCHPH
4352.2 4981.4 4785.2 5208.5 3882.4
3.36a 1.99b 4.33c 1.06d 1.51e
Means standard deviations of triplicate analyses are given. Different letters (aee) on the tops of columns indicate significant (p < 0.05) differences among samples in the same storage time.
0 0 .1
1
10
1 00
Diameter(nm) Fig. 3. Particle size distribution of emulsions made with(-) SPI, (C) at, (:) AT-HPH, (+) AT-LP and (B) AT-LPCHPH.
homogenization at acid condition could form the soluble aggregate and attribute to the diversity of structure flexibility. The combined modification could induce significant improvements on soybean proteins emulsification characteristics. The reason was that more flexible aggregates were formed by treating with highpressure homogenization after limited proteolysis in acid solution. The aggregates could easily attach to the interface and form a thicker adsorbed layer. No visible aggregates were found in all samples except SPI (data not shown), implying that the modification samples could make the emulsion stabler than the native soybean proteins. When freezing the emulsion, icy crystal forms and interrupts the proteineprotein interaction, inducing the generation of larger particles after thawing. Therefore, the ability of resisting freezing/ thawing is very important for the application of soybean proteins. Table 2 shows the comparative particle size distribution after freezing at 20 C for 24 h and thawing. The result was consistent with the comparative particle size distribution determined at ambient, and the AT-LPCHPH showed the smallest particle size distribution. The reason might be that flexible soluble aggregates formed by combination treatment could attach to the interface of oilewater and form a gel-like elasticity film which was strong enough to resist the interruption of icy crystal. Single treatment with acid, high-pressure homogenization or limited proteolysis could also improve the ability of resisting freezing/thawing, although they were not as effective as the combination treatment. We concluded that combined modification could integrate the advantages of these three treatments, and improve nearly all related characteristics of emulsibility significantly.
Table 2 The ability of resisting freezing and thawing of the five samples. D [3,2] represents surface area weighted average; D [4,3] represents volume weighted average. Samples
D [3,2]
SPI AT AT-HPH AT-LP AT-LPCHPH
25.668 21.382 19.711 12.685 7.496
D [4,3]
1.13a 0.071b 0.092c 0.43d 0.49e
94.031 81.234 73.040 52.083 34.006
0.57a 0.85b 1.44c 0.64d 0.71e
Each value is the mean and standard deviation of duplicate measurements. Different letters (aee) on the tops of columns indicate significant (p < 0.05) differences among samples in the same storage time.
B. Yuan et al. / LWT - Food Science and Technology 46 (2012) 453e459
3.4. Foaming properties Fig. 4 shows the foaming capacity (FC) and foaming stability (FS) of SPI and the modification samples. The foaming capacities of all the modification samples were improved compared with that of SPI. The SPI exposed less hydrophobic groups and the tighter conformation of the globular than the modification samples, so the speed of SPI molecular transferring to the airewater interface was very slow and dropped the surface tension inefficiently (Hettiarachchy & Ziegler, 1994). Due to the high surface hydrophobicity, AT and AT-LP could enhance the foaming capacity up to 52.5% and 57.5%, respectively. The foaming capacity of AT-LP was better than AT, which might be due to that limited proteolysis could expose more hydrophobic groups from the inner of proteins than acid treatment. For the AT-HPH, its foaming capacity was equal with that of AT, implying that the aggregate formed by highpressure homogenization in acid condition had no improvement on the FC. The AT-LPCHPH showed the highest foaming capacity up to 65%. Because more flexible and low-molecular-weight aggregates were formed by the combination treatment, they could transfer to the airewater interface more rapidly and the proteineprotein interaction improved the strength of the viscoelastic cohesive film, dropping the surface tension efficiently. This result implied that the advantages of these three treatments could be integrated together. The results of FS indicated that no improvement on foaming stability of the modification samples was found compared with SPI. AT exhibited the lowest foaming stability, 70
d c
60
Foaming Capacity/%
b
b
50 40
a
30 20 10 0 SPI
AT
AT-HPH
AT-LP
AT-LPCHPH
Samples 100
457
which might be due to that the adsorbing film formed by the covalent and non-covalent bonds couldn’t stop the air bubble reaggregate. Compared with single acid treatment, the highpressure homogenization in acid condition could induce the formation of disulfide bonds, while the limited proteolysis with pepsin could expose much more hydrophobic groups to improve the hydrophobic interaction. So compared with AT, AT-HPH showed better foaming stability to 88.8%, which was more efficient than ATLP (85.5%). The combination treatment also improved the foaming stability in acid solution but was less efficient than high-pressure homogenization in acid condition. 3.5. Mean hydrodynamic diameter The hydrodynamic diameter of native or modified soybean proteins was evaluated by dynamic light scattering technique in order to investigate protein aggregation during modification(Tang & Ma, 2009). As shown in Table 3, the average particle size of untreated soybean protein (SPI) dispersion was about 56.94 nm. As expected, the average particle size of the protein in modified samples was considerably increased, indicating aggregation and unfolding of protein occurred during the process of treatments. Glycinin in acid solution would unfold and disassociated, so average particle size of AT (77.07 nm) was larger than native soybean proteins (56.94 nm). Moreover, due to the unfolding of protein induced by limited proteolysis, average particle size of AT-LP was larger than that of AT. We concluded that the increase of hydrodynamic diameter of AT and AT-LP could be attributed to the unfolding and disassociation of proteins. The increase of surface hydrophobicity was consistent with this conclusion. The decrease of surface hydrophobicity and the increase of hydrodynamic diameter for AT-HPH and AT-LPCHPH samples implied the formation of aggregation, but the extent of protein aggregation varied with different treatment. The average particle size of aggregates induced by single high-pressure homogenization (107.8 nm) was larger than that induced by combination treatment (99.5 nm), respectively. Since the average particle size of AT-HPH was larger than that of the AT-LPCHPH and the surface hydrophobicity was also higher, it implied that more but smaller aggregates were formed by the combination treatment. So we speculated that aggregates were formed by high-pressure homogenization. Moreover, single high-pressure homogenization in acid solution trended to form larger aggregates and the combination treatment trended to form smaller aggregates. This result was in agreement with the results of surface hydrophobicity and surface pressure (Table 4).
a c
d
d
3.6. Dynamic surface properties
Foaming Stability/%
b 80
Being surface-active, proteins can act as effective emulsifying agents, and the interfacial behavior of adsorbed proteins plays a crucial role in determining the stability of foams and emulsions
60
40
Table 3 Mean hydrodynamic diameter of the five samples.
20
0 SPI
AT
AT-HPH
AT-LP
AT-LPCHPH
Samples Fig. 4. Foaming capacity (FC) and foaming stability (FS) of SPI and the modified samples. Different letters (aed) on the tops of columns indicate significant (p < 0.05) differences among samples in the same storage time.
Samples
Average diameter/nm
SPI AT AT-HPH AT-LP AT-LPCHPH
56.94 77.07 107.8 86.64 99.95
0.21a 0.90b 1.12c 1.07d 1.03e
Each value is the mean and standard deviation of duplicate measurements. Different letters (aee) on the tops of columns indicate significant (p < 0.05) differences among samples in the same storage time.
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B. Yuan et al. / LWT - Food Science and Technology 46 (2012) 453e459 Table 4 The adsorption of time dependence of surface pressure (p) for SPI and the modified samples adsorbed films at the oilewater interface. samples
Surface pressure (p) mN/m
SPI AT AT-HPH AT-LP AT-LPCHPH
16.60 16.96 17.05 17.22 17.35
0.014a 0.007b 0.007c 0.012d 0.014e
Each value is the mean and standard deviation of duplicate measurements. Different letters (aee) on the tops of columns indicate significant (p < 0.05) differences among samples in the same storage time.
(Baeza, Carrera, Rodríguez Patino, & Pilosof, 2005). There are three stages for protein to be absorbed to the airewater and oilewater interface. The first stage is called the induction period. The second stage is protein diffusion to the interface and adsorption, and the last stage is penetration at the interface. In the induction period, proteins transferred to the oilewater interface but few were adsorbed on the film, so no increasing of surface pressure was detected during the induction period (Kim & Kinsella, 1987). Some authors attribute the existence of induction period to the molecular flexibility of the protein and its susceptibility to conformational changes (Graham & Phillips, 1979). As Table 4 has shown, the surface pressure of SPI, AT, AT-HPH, AT-LP and AT-LPCHPH reached 16.60 mN/m, 16.96 mN/m, 17.05 mN/m, 17.22 mN/m and 17.35 mN/ m, respectively. Because the disassociation in acid solution and limited proteolysis increased surface hydrophobicity, the protein could be more efficiently absorbed to the oilewater interface. Since the re-arrangement of the flexible and soluble aggregates on the oilewater interface were easier, single high-pressure homogenization in acid solution could increase the surface pressure but not as efficient as the limited proteolysis treatment. The combination treatment produced the highest surface pressure among all samples, implying that more flexible aggregates were formed by combination treatment. Moreover, we concluded that highpressure homogenization and limited proteolysis with pepsin had coordination efficiency on hastening the adsorption of proteins to the oilewater interface. This result was consistent with the emulsibility and the foaming properties. Because water is polar phase while oil and air is nonpolar phase, the oilewater and airewater system have the same behavior of proteins adsorption. 4. Conclusions Due to the dissociation and unfolding of the soybean protein structure in acid condition, the following treatments including pepsin proteolysis, high-pressure homogenization or even their combination, could enhance the interaction between protein molecules and the functional properties. With the analysis of surface hydrophobicity, hydrodynamic diameter, and surface pressure, we concluded that limited proteolysis with pepsin and/or high-pressure homogenization in acid condition could induce the formation of soluble and flexible aggregation. Moreover, smaller size aggregates were formed by the combination treatments in acid condition. In conclusion, the combination of acid treatment, pepsin proteolysis and high-pressure homogenization was a good choice to improve emulsibility, ability of resisting freezing/thawing and foaming activity of SPI. Acknowledgments We appreciate the support from National Natural Science Foundation of China (No. 31000759), National Natural Science Foundation
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