Improved emulsifying capabilities of hydrolysates of soy protein isolate pretreated with high pressure microfluidization

Improved emulsifying capabilities of hydrolysates of soy protein isolate pretreated with high pressure microfluidization

LWT - Food Science and Technology 69 (2016) 1e8 Contents lists available at ScienceDirect LWT - Food Science and Technology journal homepage: www.el...

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LWT - Food Science and Technology 69 (2016) 1e8

Contents lists available at ScienceDirect

LWT - Food Science and Technology journal homepage:

Improved emulsifying capabilities of hydrolysates of soy protein isolate pretreated with high pressure microfluidization Lin Chen a, Jianshe Chen b, Lin Yu a, Kegang Wu a, * a b

College of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, China School of Food Science and Nutrition, University of Leeds, LS2 9JT Leeds, UK

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 October 2015 Received in revised form 27 December 2015 Accepted 11 January 2016 Available online 13 January 2016

Soy protein isolate (SPI) was treated by high-pressure microfluidization and pancreatin hydrolysis in this work. Results showed that microfluidization substantially enhanced pancreatin hydrolysis of SPI in terms of degree of hydrolysis (DH), with a preferable treatment condition at 120 MPa and 30 g/L SPI concentration. SDS-PAGE conducted under reducing conditions showed that microfluidization increased the accessibility of some subunits (a0 -7S, A-11S and B-11S) in SPI to pancreatin hydrolysis, resulting in changes in protein solubility (PS), surface hydrophobicity (H0), and molecular weight distributions for hydrolysates. Emulsion systems (20 vol.% oil, 20 g/L protein samples, pH 7.0) formed by control SPI and SPIH (SPI hydrolysates) were unstable due to fast coalescence and bridging flocculation during homogenization, while that formed by MSPIH (microfluidization pretreated SPIH) with 5.8% DH was more stable and showed smaller mean droplet size (d43). Compared with SPIH, MSPIH showed a stronger increase in PS and a more moderate change in H0 during pancreatin hydrolysis, suggesting the production of more surface-active soluble peptides, which may explain their markedly improved emulsifying capabilities. This work showed that modified SPI could be an effective food emulsifier with microfluidization pre-treatment and limited proteolysis leading to desirable functional modifications of globular proteins. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Soy protein isolate Pancreatin hydrolysis High-pressure microfluidization Emulsification functionality Surface-active peptides

1. Introduction Soy proteins have become increasingly popular because of their high nutritional quality and low price, which has been a driving force for increased soy research and commercial development of new soy protein products. Soy protein isolate (SPI) is the most refined soy protein products, containing >90% protein on dry weight basis, and has been used as emulsifier in food emulsions. It is preferred because of the surface-active properties of its constitutive proteins: 7S (b-conglycinin) and 11S (glycinin) globulins. Although SPI is characterized as less capable as an emulsifier when compared to milk proteins, it outperforms many other plantsourced proteins (Palazolo, Sorgentini, & Wagner, 2004). Moreover, considering the large molecular mass and a large amount of hidden hydrophobicity groups for soy proteins, SPI is prospectively of great potential to become an efficient food emulsifier if appropriately modified (Nishinari, Fang, Guo, & Phillips, 2014).

* Corresponding author. E-mail address: [email protected] (K. Wu). 0023-6438/© 2016 Elsevier Ltd. All rights reserved.

Protein modification based on proteolysis has a broad potential for designing protein functionality for specific applications, and the effects of proteolysis are determined by factors such as protease specificity, degree of hydrolysis (DH) and substrate characteristics (Tavano, 2013). Pancreatin, mainly composing of trypsin and chymotrypsin, has a broad specificity to peptide bonds, and preferentially cleaves C-terminal hydrophobic regions (Su et al., 2012). Previous studies showed that soy protein hydrolysates prepared with pancreatin hydrolysis exhibited improved emulsifying capabilities over the original proteins (de la Barca, Ruiz-Salazar, & JaraMarini, 2000; Qi, Hettiarachchy, & Kalapathy, 1997). Limited proteolysis exposes hydrophobic and hydrophilic residues, enhances the amphiphilic characteristics of proteins, and improves emulsification (Tavano, 2013). However, previous studies found that soy proteins were generally resistant to proteolysis (Govindaraju & Srinivas, 2007; Henn & Netto, 1998; Qi et al., 1997). The intrinsic difficulty is that globular soy proteins have compact quaternary and tertiary structures that protect many of the peptide bonds (Govindaraju & Srinivas, 2007). Moreover, protein aggregation during the processing of SPI may result in the burying of cleavage


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sites (Henn & Netto, 1998). Therefore, pre-treatment is necessary to modify the structural characteristics of soy proteins, in order to increase their accessibility to protease and obtain enhanced functionalities. Microfluidization is a state-of-the-art dynamic high-pressure techniques, which uses the combined forces of ultrahigh pressure, high frequency vibration, instantaneous pressure drop, intense shear, and cavitation, and thus exhibits a much larger energy density compared with conventional valve homogenization (Ciron, Gee, Kelly, & Auty, 2010). Moreover, microfluidization has a variety of advantages including no exogenous chemicals, little nutritional loss and very short processing time. Recently, several studies have reported that microfluidization was highly efficient in modulating physico-chemical and structural properties of whey proteins (Dissanayake & Vasiljevic, 2009; Liu et al., 2011), peanut proteins (Hu, Zhao, Sun, Zhao, & Ren, 2011) and soy proteins (Shen & Tang, 2012). It was demonstrated that microfluidization could not only alter the structure of globular proteins, but also could disrupt insoluble heat-induced protein aggregates into smaller soluble aggregates, resulting in the exposure of inner groups buried inside the folded structure (Liu et al., 2011; Shen & Tang, 2012). Therefore, microfluidization treatment might have the potential to alter the accessibility of SPI to proteolysis and caused enhanced functionalities for hydrolysates. However, little work has been done so far to investigate this possibility. Hence, this work aims to investigate the effects of high-pressure microfluidization pre-treatment on the proteolysis pattern of SPI and on the emulsifying capabilities of its hydrolysates. Some key physico-chemical and structural properties of hydrolysed products were measured accordingly to better understand the underlying mechanisms of improved emulsifying capabilities for SPIH and MSPIH.

2. Materials and methods

2.3. Preparation of SPIH and MSPIH Dispersions of SPI and MSPI were proteolysed using pancreatin at pH 7.0 and 50  C. Based on preliminary experiments, appropriate E/S ratios (g enzyme/100 g substrate) varied from 0.025 to 0.5 were selected to reach different required DH for SPIH and MSPIH. The hydrolysis reaction was performed in a water bath with continuous stirring. The pH of sample dispersions was maintained during hydrolysis using a TIM840 Auto-titrator (Radiometer Analytical co., Villeurbanne, France) loaded with (0.1e1) mol/L NaOH solution. After 120 min of hydrolysis, the consumption of NaOH solution was recorded for the determination of DH using the pH-stat method described by Adler-Nissen (1986). Pancreatin hydrolysis was stopped by the immediate addition of PMSF to a concentration of 1 mmol/L. Serine protease inhibitor PMSF instead of thermal treatment was used to inactivate pancreatin, so that protein functionalities would not be influenced by thermal changes (Luo et al., 2010). The hydrolysates were lyophilized and ground to produce a powder, which was then stored in a desiccator. Control SPI and control MSPI were prepared using the same incubation conditions and enzyme inactivation treatment, but without pancreatin added. Samples were coded according to the profiles of microfluidization treatment, followed by the DH value. For example, MSPIH-5.8% means hydrolysate of SPI was pretreated by microfluidization and hydrolysed to a DH of 5.8%. 2.4. Determination of protein solubility (PS) PS was determined according to the method of Shen and Tang (2012). Sample dispersions (10 g/L, pH 7.0) were stirred at ambient temperature for 2 h and were centrifuged at 12,000  g for 20 min to obtain the supernatants. Protein content of the supernatants was determined by the micro-Kjeldahl method (N  6.25). The PS was calculated as the percentage ratio of soluble protein content in supernatant against total amount of protein presented in samples.

2.1. Materials Commercial SPI was obtained from Shandong Wonderful Industrial Co. (Yantai, China), containing (g/100 g of powder) 91.2 protein, 4.5 moisture, 2.8 ash, and 0.2 fat. Pancreatin (P7545; 8  standard USP unit), phenylmethanesulfonyl fluoride (PMSF), 1Anilino-8-naphthalenesulfonate (ANS) and Nile Red were purchased from SigmaeAldrich (St. Louis, MO, USA). All other chemicals used were of analytical grade and commercially available. Water used to prepare aqueous solutions was purified with a MilliQ filtration unit (Millipore, Bedford, UK).

2.2. Microfluidization pre-treatment of SPI SPI powder was dispersed into deionized water to reach different concentrations (10 g/L, 30 g/L, 50 g/L) and pH was adjusted to 7.0 using (0.1e1.0) mol/L HCl solutions, with magnetically stirring at ambient temperature for 2 h to fully disperse the SPI powder. The resultant dispersions were microfluidized directly without centrifugation using an M-110EH microfluidizer (Microfluidics Co., Newton, MA, USA) operating at different levels of pressure (40e160 MPa). Each sample passed through the system twice. The machine was equipped with two interaction chambers with different entry point diameter: 200 mm (IC200) and 75 mm (IC75). The IC75 was placed downstream from the IC200. Samples had an initial temperature of 15  C before microfluidization but increased to approximately 25  C after two passes. All samples were used for proteolysis directly after microfluidization treatment.

2.5. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed on a discontinuous Tris-glycine-SDS buffer system under reducing conditions using a TriseHCl Ready Gel with 15% resolving gel and 4% stacking gel (Bio-Rad Laboratories, Hercules, CA) according to the method of Jung, Murphy, and Johnson (2005), with slight modifications. Protein samples were dispersed into a Bio-Rad Laemmli sample buffer (62.5 mmol/L TriseHCl buffer (pH ¼ 6.8), containing 20 g/L SDS, 5 mL/100 mL 2mercaptoethanol, 25 mL/100 mL glycerol and 0.1 g/L bromophenol blue) (Bio-Rad Laboratories) to a concentration of 1 mg/mL. Then samples were shaken in a vortex for 10 s, heated at 95  C in a water bath for 5 min, and centrifuged at 12,000  g for 10 min. The gels were calibrated with marker with molecular weights (MW) ranging from 6.5 to 200 kDa (M8445, Sigma Chemical Co.). 15 mL of supernatant and 5 mL of marker were loaded into the lanes. All gels were run in a Mini-protean tetra system (Bio-Rad Laboratories). After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250. 2.6. Determination of surface hydrophobicity (H0) H0 was determined according to the method of Luo et al. (2010). Sample dispersions were diluted (0.05e0.2 g/L) in phosphate buffer (0.01 mol/L, pH 7.0) and were centrifuged (12,000  g, 20 min) to obtain the supernatants. 20 mL of ANS solution (8.0 mmol/L in the same buffer) was added to 4 mL of each dilution, and the

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2.7. High-performance size exclusion chromatography (HPSEC) € HPSEC was performed using an AKTA UPC10 protein purification chromatography fitting with a Superdex Peptide HR 10/300 GL column (Amersham Biosciences Co., Buckinghamshire, UK). Each sample was dispersed into the mobile phase (0.01 mol/L sodium phosphate buffer containing 0.1 mol/L NaCl, pH 7.2) to 5 mg/mL. After centrifugation (12,000  g, 20 min), the supernatants were filtered through a 0.45 mm PVDF membrane. 100 mL of supernatant was loaded on the column, and elution was performed isocratically at 0.5 mL/min. Because SPIH and MSPIH both had a high protein content, the UV absorbance at 220 nm (characteristic absorbance wavelength of peptide bond) was used to monitor protein elution in order to give a comprehensive demonstration of molecular weight distributions of proteins or peptides in samples. A molecular calibration curve was prepared from the average elution volume of standard proteins with MW ranging from 6.5 to 75 kDa (284038-41, Amersham Biosciences Co.), and a linear correlation was obtained: Kav ¼ 0.424 lgMW þ 2.163, R2 ¼ 0.988; where Kav is gelphase distribution coefficient, MW is sample molecular weight in Da.

2.8. Preparation and characterization of emulsions Sample dispersions (20 g/L, pH 7.0) were stirred at ambient temperature for 2 h, with sodium azide (0.2 g/L) added as antimicrobial agent. Oil-in-water emulsions (20 vol.% sunflower oil) were prepared using a laboratory-scale jet homogenizer operating at 300 bar. Emulsion droplet-size distributions were measured using a Mastersizer 2000 static light-scattering analyser (Malvern Instruments, Worcestershire, UK) with absorption parameter of 0.001. The refractive index of 1.330 and 1.462 were used for water and sunflower oil, respectively. The mean droplet size was charP P acterized by d43 (mm), defined as d43 ¼ nid4i / nid3i , where ni is the number of droplets of diameter di. Emulsion microstructures were assessed qualitatively using a Leica TCS SP2 confocal laser scanning microscope (CLSM) (Leica, Heidelberg, Germany) in fluorescence mode. Samples were scanned using a 63 water-immersion objective lens with numerical aperture 1.20. The oil phase was stained with Nile Red dye (20 mL of 0.1 g/L dye in polyethylene glycol added to 5 mL emulsion) and excited at 488 nm. As Nile Red stains the oil phase, individual oil droplets and regions rich in emulsion droplets appear as bright patches, whereas the aqueous (water/protein) phase appears dark in the images.

3. Results and discussion 3.1. Effects of microfluidization pre-treatment on the enzymatic accessibility of SPI In order to assess the influence of microfluidization pretreatment on the enzymatic accessibility of SPI, the degree of hydrolysis (DH), SDS-PAGE profiles, and protein solubility (PS) of hydrolysates before and after microfluidization pre-treatment were investigated. Fig. 1 shows the effects of microfluidization pressure (40e160 MPa) and SPI concentration (10 g/L, 30 g/L, 50 g/L) on DH of hydrolysates. At relatively low microfluidization pressure of 40 MPa, there is no significant (p > 0.05) changes in DH for hydrolysates. However, with a further increase in microfluidization pressures, DH of MSPIH with different SPI concentrations all increased markedly up to 120 MPa, and then decreased slightly at higher pressures. The maximum DH obtained by MSPIH-(10 g/L), MSPIH-(30 g/L) and MSPH-(50 g/L) were 8.0%, 8.1% and 7.6%, respectively. In contrast, without microfluidization pre-treatment, the maximum DH of SPIH systems was only 5.7%. These results clearly demonstrated that the proteolysis of SPI could be enhanced by microfluidizaiton pre-treatment. On the other hand, the initial SPI concentration also posed an important effect on microfluidization pre-treatment, because the maximum DH obtained at high SPI concentration (50 g/L) was significantly (p < 0.05) lower than those obtained at low SPI concentration (10 g/L and 30 g/L). This may be because at high protein concentration, the unfolded proteins could associate more easily to form aggregates (Wang et al., 2008). Based on above results, it can be seen that microfluidizaiton treatment at pressure level of 120 MPa and SPI concentration of 30 g/L was a preferable condition to enhance the pancreatin hydrolysis of SPI than other conditions, and was therefore chosen to prepare MSPI for the following experiments. SDS-PAGE was performed to investigate the protein breakdown in samples caused by different treatments. Fig. 2A and B shows the progressive changes in peptide profiles of SPIH and MSPIH prepared with pancreatin hydrolysis at different E/S ratios (0e0.5 g/ 100 g). Control SPI and control MSPI showed similar SDS-PAGE patterns, which were mainly composed of a0 , a and b subunits of b-conglycinin (7S), and acidic (A) and basic (B) subunits of glycinin (11S). This finding was consistent with previous study and

8.4 7.8 7.2

DH (%)

fluorescence intensity was measured with a F4500 fluorescence spectrometer (Hitachi Co., Japan) at wavelengths of 390 nm (excitation) and 470 nm (emission). The initial slope of fluorescence intensity versus protein concentration plot was used as an index of H0.


6.6 6.0 5.4

2.9. Statistical analysis Data were expressed as mean ± standard deviation for triplicate determinations. The results were subjected to analysis of variance (ANOVA). Significant differences between means (p < 0.05) were identified by the least significant difference (LSD) test.






Pressure (MPa) Fig. 1. Effects of microfluidizaiton pre-treatment conducted at different pressures (40e160 MPa) and SPI concentrations (C: 10 g/L; *: 30 g/L; :: 50 g/L) on DH of hydrolysates prepared with pancreatin hydrolysis at E/S ¼ 0.5 g/100 g.


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enhanced hydrolysis of MSPI as compared with SPI in terms of DH. It was reported that microfluidization treatment could alter the structure of globular proteins and dissociate protein aggregates (Dissanayake & Vasiljevic, 2009; Shen & Tang, 2012). As a result, more hydrolysis sites could be exposed and become accessible to proteases. Table 1 shows the DH and PS of SPIH and MSPIH prepared with pancreatin hydrolysis at different E/S ratios (0e0.5 g/100 g). Control SPI showed a low PS of 39.4%. Because of the harsh processing conditions including heat and exposure to acid, protein aggregation usually occurs in commercial SPI, resulting in a rather poor PS ~o  n, 1991). After microfluidization (Arrese, Sorgentini, Wagner, & An treatment, PS of MSPI increased to 45.5%. This result was consistent with previous studies, which reported that microfluidization could improve the protein solubility of thermally denatured globular proteins, and the improvement was considered to be due to the disruption or disintegration of insoluble aggregates into smaller soluble aggregates by high pressure shearing during microfluidization (Dissanayake & Vasiljevic, 2009; Shen & Tang, 2012). Upon hydrolysis, DH and PS of both SPIH and MSPIH increased progressively with the increase of E/S ratios. The improvement in PS by proteolysis was attributed to the reduction in MW and increase in ionizable amino and carboxyl groups (Tavano, 2013). However, it is noteworthy that when the same E/S ratios were introduced, MSPIH obtained higher DH and PS than SPIH did. For example, at E/S ¼ 0.2 g/100 g, the DH and PS of MSPIH were 5.8% and 82.7% respectively, much higher than those of SPIH (DH ¼ 3.5% and PS ¼ 56.2%). These results suggest that microfluidization followed by pancreatin hydrolysis posed a striking effect on improving the PS of SPI. This finding may be explained by the fact that microfluidization pre-treatment increased the enzymatic accessibility of some subunits (a0 -7S, A-11S and B-11S) in SPI, which made the pancreatin hydrolysis of MSPI not only intensive (higher DH), but also extensive (higher PS): more soy proteins in MSPI could be readily hydrolysed and become soluble. 3.2. Surface hydrophobicity (H0) of SPIH and MSPIH

Fig. 2. Changes of SDS-PAGE profiles of SPIH (A) and MSPIH (B) prepared with pancreatin hydrolysis at different E/S ratios (0e0.5 g/100 g). a0 , a and b: subunits of bconglycinin; A and B: acidic and basic subunits of glycinin.

suggested that microfluidization did not cause the degradation of main subunits in soy proteins (Shen & Tang, 2012). After pancreatin hydrolysis, different subunits in SPIH showed different enzymatic accessibilities: a-7S and b-7S disappeared at E/S ¼ 0.05 g/100 g; A11S and a0 -7S disappeared at E/S ¼ 0.3 g/100 g and E/S ¼ 0.5 g/ 100 g, respectively; B-11S appeared highly resistant to pancreatin hydrolysis, and was still identifiable at E/S ¼ 0.5 g/100 g. However, in MSPIH, the progressive degradation pattern of the major subunits except a-7S and b-7S with the increase of E/S ratios was different from those in SPIH. The a0 -7S, A-11S and B-11S in MSPI turned out to be more readily hydrolysed because they underwent total degradation at E/S ¼ 0.1 g/100 g, E/S ¼ 0.2 g/100 g and E/ S ¼ 0.3 g/100 g, respectively. This finding suggests that microfluidization pre-treatment substantially increased the accessibility of these subunits to pancreatin hydrolysis, which may explain the

Good emulsifying properties are positively correlated with surface hydrophobicity of proteins, because surface hydrophobicity plays an important role in the initial anchoring of a protein to the oil/water interface (Lam & Nickerson, 2013). As shown in Fig. 3, the H0 of control MSPI (1323.5) was significantly (p < 0.05) higher than that of control SPI (1099.8), suggesting the exposure of initially buried hydrophobic groups after microfluidization, as also reported by Shen & Tang, 2012. Another important implication of this result is that appropriate microfluidization treatment was proved to be an effective way to unfold globular soy proteins or dissociate protein aggregates, which might cause the exposure of not only hydrophobic groups but also hydrolysis sites initially buried inside the folded structure. Upon hydrolysis, H0 of SPIH increased to a maximum of 1838.6 at DH 1.9%, but then decreased sharply at higher DH. Limited proteolysis could cause the exposure of hydrophobic groups buried in the interior of protein molecules (Tavano, 2013). However, excessive proteolysis led to a decrease in H0, which might be due to (1) enzymatic cleavage of hydrophobic clusters resided at the protein surface where they contributed to surface hydrophobicity or (2) increased proteineprotein hydrophobic interactions that led to protein rearrangement, confining most of the hydrophobic clusters within the interior of aggregates (Jung et al., 2005). As a result, at high DH values, SPIH showed very low H0, although high PS. On contrast, it is somewhat surprising that H0 of MSPIH showed a continuous decrease with increasing DH, despite that the decrease was relatively moderate compared with that of SPIH. The

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Table 1 Degree of hydrolysis (DH) and protein solubility (PS) of SPIH and MSPIH prepared at different enzyme/substreate (E/S) ratio*. E/S ratio (g/100 g)

DH (%)

PS (%)

SPIH 0 0.025 0.05 0.1 0.2 0.3 0.4 0.5

0 1.1 1.9 2.6 3.5 4.3 5.1 5.7


(±0.03) (±0.04) (±0.03) (±0.03) (±0.05) (±0.06) (±0.05)

0 2.1 3.9 4.8 5.8 6.5 7.4 8.1

n m k j h f e


(±0.02) (±0.05) (±0.02) (±0.04) (±0.03) (±0.04) (±0.06)

l i g d c b a

39.4 44.2 47.4 52.3 56.2 58.3 61.8 63.2

MSPIH (±0.3) (±0.5) (±0.6) (±0.7) (±0.5) (±0.6) (±0.9) (±0.7)

n m k j i h g f

45.5 57.3 65.6 76.3 82.7 84.5 86.3 85.1

(±0.4) (±0.6) (±1.1) (±0.6) (±0.5) (±0.7) (±0.4) (±0.5)

l h e d c b a b

* In the comparison of the same type of index, data with the same letters are not significantly different (P > 0.05).

Surface hydrophobicity H0

2000 1750 1500 1250 1000 750 500 250 0










DH (%)

Fig. 3. Effects of DH on surface hydrophobicity (H0) of SPIH ( ) and MSPIH (C).

H0 of MSPIH with DH between 2.1% and 5.8% only showed marginal changes, decreasing from initial 1293.6 to 1021.7 at DH 5.8%. The reason for this is not clear, but it might be related to the increased enzymatic accessibility of MSPI, which made the exposure and breakdown of hydrophobic groups more evenly during hydrolysis. We can see that compared with control SPI, some MSPIH (DH ¼ 4.8e5.8%) had much increased PS, but still maintained comparably high H0. 3.3. Molecular weight distributions (MWD) of SPIH and MSPIH with selected DH values HPSEC was performed to investigate the MWD of protein samples in their original conformation (Fig. 4). According to the distributions of chromatogram peaks, the SEC profiles of all tested samples can be divided into three zones: zone I: >75.0 kDa, zone II: 75.0e2.0 kDa, and zone III: <2.0 kDa. Most of the soluble proteins in control SPI were distributed in zone II, and there were three major peaks referred to as P1 (68.7 kDa), P2 (43.5 kDa), and P3 (26.9 kDa). Compared with control SPI, control MSPI showed an obvious increase in zone I and P1eP3, suggesting that microfluidization treatment caused the release of soluble proteins or protein aggregates from thermally denatured soy proteins, as also reported by Shen & Tang, 2012. After pancreatin hydrolysis, the chromatogram peaks in zone II for both SPIH-3.5% and MSPIH-5.8% showed a tendency of shifting towards higher elution volumes (smaller MW) as compared with the control. And at relatively high elution volumes in zone II and zone III, some new peaks were detected, which might arise from the production of small peptides. These results

Fig. 4. HPSEC profiles of SPIH and MSPIH with selected DH values (d: control SPI; d: control MSPI; ┄: SPIH-3.5%; ┄: MSPIH-5.8%). Symbol ‘þ’ from left to right indicates protein markers of 75.0, 43.0, 29.0, 13.7, and 6.5 kDa, respectively. The Roman numerals indicate different zones: zone I, >75.0 kDa; zone II, 75.0e2.0 kDa; zone III, <2.0 kDa.

suggest that proteolysis caused a decrease in MWD for soy proteins. However, it should be noted that there was a considerable proportion of fraction with high MW for SPIH-3.5%, which might suggest the existence of unhydrolysed soy proteins, as also observed in SDS-PAGE profiles. In contrast, for MSPIH-5.8%, there was single one broad peak appeared in zone II, corresponding to the MW of ca. 13.6 kDa. On the other hand, the total integrated area of MSPIH-5.8% was distinctly larger than that of SPIH-3.5%, suggesting its higher content of soluble proteins. 3.4. Emulsifying capabilities of SPIH and MSPIH Under identical emulsification condition, the final droplet size of an emulsion is mainly dependent on the properties of emulsifiers used. A more effective emulsifier is one that stabilizes a smaller


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droplet size (McClements, 2007). The mean droplet size d43 of emulsions (20 vol.% oil, 20 g/L protein samples, pH 7.0) formed by SPIH and MSPIH is plotted as a function of DH in Fig. 5. Based on preliminary experiments, the sample concentration used to make emulsions was chosen to be 20 g/L. Because the emulsion formed by 20 g/L control SPI showed a relatively large droplet size (d43 z 27.4 mm), there was scope for substantial improvement on emulsifying capability. The emulsion formed by control MSPI showed a smaller d43 of 21.3 mm, suggesting that the microfluidization treatment improved the emulsifying capability of SPI. Upon hydrolysis, with increasing DH, d43 of emulsions formed by SPIH and MSPIH both decreased initially, and then increased sharply at high DH values, suggesting that limited pancreatin hydrolysis improved the emulsifying capability of soy proteins, whereas excessive hydrolysis was detrimental, as also reported in literature (Jung et al., 2005; Qi et al., 1997). Emulsions formed by SPIH showed a minimum d43 of 15.6 mm at DH 2.6%. In contrast, hydrolysed MSPI exhibited greater improvement, with a minimum d43 of ca. 6.0 mm at DH ¼ 4.8e5.8%. These results clearly showed that combined microfluidization pre-treatment and pancreatin hydrolysis was more efficient in improving the emulsifying capability of SPI than individual treatment. In order to further characterize the emulsifying capabilities of different samples, the microstructures of emulsions were visualized using CLSM. Droplet-size distributions (DSD) measured by light-scattering were superimposed on microimages to give a better description of emulsion microstructures. Fig. 6AeC shows CLSM images and DSD of emulsions formed by SPIH with different DH. As shown in Fig. 6A, the emulsion formed by control SPI appeared to have some big droplets and flocculate extensively. The reason for these instabilities may be that a limited SPI concentration of 20 g/L was used for making emulsions, which was not enough to fully cover the surfaces of oil droplets created during homogenization, and might result in fast coalescence and bridging flocculation (adsorbed protein molecules or protein aggregates are shared amongst adjacent droplets) of emulsion droplets (Dickinson, 2010a). The DSD of control SPI emulsion was mainly in the range of 10e100 mm. For SPIH-2.6% emulsion (Fig. 6B), although less strongly flocculated than in control SPI emulsion, flocculation was still apparent. On the other hand, the appearance of some small droplets (ranging from 0.1 to 1 mm) was clearly evident. It seems that pancreatin hydrolysis of SPI produced some soluble peptides that were very surface-active, enabling the generation and

stabilization of small droplets. But considering that the PS of SPIH2.6% was relatively low (52.3%), still the content of these surfaceactive peptides was not sufficient for full coverage of droplet surfaces. Insoluble proteins may adsorb onto droplet surfaces, but comparing with soluble proteins, they are less capable of covering efficiently the droplets due to their much slower diffusivity and much higher saturation surface load, and tend to cause fast coalescence and bridging flocculation of emulsion droplets at relatively low concentration during homogenization (Dickinson, 2010b; Tcholakova, Denkov, & Lips, 2008). For SPIH-5.7% emulsion (Fig. 6C), droplet flocs appeared to mainly consist of relatively big droplets and DSD showed a shift to larger sizes in contrast with that of SPIH-2.6% emulsion. Fig. 6DeF shows CLSM images and DSD of emulsions formed by MSPIH with different DH. For control MSPI emulsion (Fig. 6D), flocculation appeared to be less strong than that in control SPI emulsion, and some small droplets (ranging from 0.1 to 1 mm) were stabilized. For MSPIH-5.8% emulsion (Fig. 6E), the most striking observation was the disappearance of emulsion flocculation. And in contrast with SPIH-2.6% emulsion, many more small droplets were stabilized in MSPIH-5.8% emulsion. As has been demonstrated, after microfluidization pre-treatment, some subunits (a0 -7S, A-11S and B-11S) in SPI that were resistant to pancreatin hydrolysis, could be more readily hydrolysed, causing a strong increase in PS and a moderate change in H0 for MSPIH. As a result, MSPIH-5.8% not only had markedly increased PS (82.7%), but also maintained high H0 (1021.7). These results may suggest that compared with SPIH, more soluble peptides with high surface activity were produced in MSPIH during pancreatin hydrolysis, resulting in a greater improvement on emulsifying capability. Moreover, the decrease in molecular weight for MSPIH-5.8% probably caused an increase in flexibility of protein structure, which had added advantage of faster adsorption kinetics, making it in principle easier to produce fine stable droplets (Adjonu, Doran, Torley, & Agboola, 2014). For MSPIH-8.1% emulsion (Fig. 6F), both CLSM microimage and DSD measurement showed the appearance of some extraordinary big droplets. This suggests that excessive proteolysis caused a decrease in emulsifying capability for MSPIH, as it did for SPIH. The reason for these results may be mainly due to the similar change in SPIH and MSPIH at high DH values: the strong decrease in H0 made the hydrolysates lose surface activity. In addition, it was reported that emulsion droplets formed by highly hydrolysed proteins had a thin and weak interfacial film, and were susceptible to coalescence (Ye, Hemar, & Singh, 2004). 4. Conclusions

Fig. 5. Effects of DH on mean droplet size (d43) of emulsions (20 vol.% oil, 20 g/L protein samples, pH 7.0) formed by SPIH ( ) and MSPIH (C).

To summarize, this work investigated the effects of highpressure microfluidization pre-treatment on pancreatin hydrolysis pattern of SPI and on emulsifying capabilities of its hydrolysates. Results demonstrated that microfluidization pre-treatment substantially enhanced pancreatin hydrolysis of SPI in terms of DH, with a preferable treatment condition at pressure level of 120 MPa and SPI concentration of 30 g/L. After microfluidization pretreatment, subunits of a0 -7S, A-11S and B-11S in SPI, that were resistant to pancreatin hydrolysis, became more readily hydrolysed. As a result, compared with SPIH, MSPIH showed a stronger increase in PS and a more moderate change in H0 during pancreatin hydrolysis, suggesting the production of more surface-active soluble peptides. Compared with control SPI, MSPIH-5.8% had much increased PS (82.7%), comparably high H0 (1021.7) and decreased MWD, which could be the main causes for its markedly improved emulsifying capability, and may explain its effectively use in protecting emulsions against fast coalescence and bridging flocculation during emulsification. In conclusion, modified SPI may provide

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Fig. 6. Microstructures of emulsions (20 vol.% oil, 20 g/L protein samples, pH 7.0) formed by SPIH and MSPIH with different DH values: (A) control SPI; (B) SPIH-2.6%; (C) SPIH-5.7%; (D) control MSPI; (E) MSPIH-5.8%; (F) MSPIH-8.1%. Droplet size distributions are superimposed on the microimages, with horizontal scale indicating particle size (mm).

a valuable new source of emulsifier for utilization in food industry. High-pressure microfluidization pre-treatment could be an effective way to increase the enzymatic accessibility of SPI, producing prominent benefits in improving functionalities of its hydrolysates. However, what changes actually happened to SPI after microfluidizaiton which make it become readily hydrolysed remains unknown, and a further study is currently undertaken in author's lab.

Acknowledgement This work was supported by National Research Fund for Doctoral Program of China (No. 20134420120008) and Guangdong Provincial Science and Technology Project (No. 2013B020311015 and 2013B020311014). L.C. acknowledges helpful discussions with Prof. E. Dickinson and Prof. B. Murray (University of Leeds).


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