Article pubs.acs.org/JAFC

Effects of Phytase-Assisted Processing Method on Physicochemical and Functional Properties of Soy Protein Isolate Hongjian Wang, Yeming Chen, Yufei Hua,* Xiangzhen Kong, and Caimeng Zhang State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, People’s Republic of China S Supporting Information *

ABSTRACT: Phytate is an important antinutritional factor in food products. In this study, a phytase-assisted processing method was used to produce low-phytate soybean protein isolate (SPI) samples, and their physicochemical and functional properties were examined. Hydrolysis condition at low temperature (room temperature) and pH 5.0 was better than that recommended by manufacturer (pH 5.0, 55 °C) at keeping the properties of SPI, so the former condition was selected to prepare SPI samples with phytate contents of 19.86−0.11 mg/g by prolonging hydrolysis time (0 (traditional method), 5, 10, 20, 40, and 60 min). Ash content (R2 = 0.940), solubility (R2 = 0.983), ζ-potential value (R2 = 0.793), denaturation temperatures (β-conglycinin, R2 = 0.941; glycinin, R2 = 0.977), emulsifying activity index (R2 = 0.983), foaming capacity (R2 = 0.955), and trypsin inhibitor activity (R2 = 0.821) of SPI were positively correlated with phytate content, whereas protein content (R2 = 0.876), protein recovery (R2 = 0.781), emulsifying stability index (R2 = 0.953), foaming stability (R2 = 0.919), gel hardness (R2 = 0.893), and in vitro digestibility (R2 = 0.969) were negatively correlated with phytate content. Simulated gastrointestinal digestion and subsequent dialysis showed that percentages of dialyzable Zn and Ca were increased with decreasing phytate content, whereas the amounts of dialyzable Zn and Ca revealed different behaviors: the former was increased and the latter was decreased. Circular dichroism spectra showed that secondary structure of SPI was changed by phytase. Compared with traditional processing method, the phytase-assisted processing method could produce SPI with lower phytate and higher protein contents, which had better in vitro digestibility and could be used to prepare gels with higher hardness by partially losing some other functional properties. KEYWORDS: soybean protein isolate, phytase, phytate, hydrolysis condition, solubility, emulsifying, foaming, gelation, digestibility

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wheat rolls,11 cereal porridges,12 wheat bran flour, glandless cotton flour,13 bread,14 corn wet milling,15 and rice bran protein isolate.16 Han and Wilfred17 reported that 85% of phytate in soybean meal and 67% of phytate in cottonseed meal could be hydrolyzed by Aspergillus ficuum phytase at 50 °C and pH 4− 5.5. However, the effects of phytase on the physicochemical and functional properties of the obtained products were seldom examined. Soybean protein is the predominant vegetable protein in the diet of oriental and Western countries and may be the most inexpensive source of protein for nutritional or technological properties.18 As one important product of soybean protein, soybean protein isolate (SPI) is widely used as an ingredient in meat products, baby foods, beverages, and wheat flour products. To our best knowledge, there are very limited data available about the effects of phytase on SPI processing and the physicochemical and functional properties of the obtained SPI products. SPI processing mainly includes three steps: (1) extracting soybean flour by water; (2) removing the insolubles by centrifugation; and (3) precipitating at pH 4.5 to obtain SPI. According to our experiments, it was found that the addition of phytase in the first step could cause a low yield of SPI, and the

INTRODUCTION Phytates (myo-inositol-1,2,3,4,5,6-hexakisphosphates) are widely thought to play a significant role in decreasing the bioavailability of minerals by chelating with cations to form complexes, which may be insoluble or otherwise unavailable under physiological conditions.1 Hitherto, massive numbers of research studies, including animal experiments and human trials, have been carried out on the negative aspects of phytate, which clarify that dietary phytate has a negative impact on the bioavailability of mineral ions (i.e., Zn2+, Fe2+/3+, Ca2+, Mg2+, Mn2+, and Cu2+).2−6 Additionally, phytate has also been reported to form complexes with proteins at both low and high pH values, and the complex formations may alter the protein structure, which perhaps induces the changes of physicochemical and functional properties, enzymatic activity, and proteolytic digestibility of proteins (or enzymes).7 Hence, it is necessary to partially, or even completely, eliminate the antinutritional effect of phytate when phytate-rich products are produced or used. There are several processing techniques, such as soaking, germination, malting, fermentation,8 ultrafiltration,9 and ion exchange column,10 that can be used to decrease the inhibitory effect of phytate on mineral absorption. Besides, phytase (myo-inositol hexakisphosphate phosphohydrolase), which catalyzes the conversion of phytate to inositol and inorganic phosphate, also can be used to reduce phytate in food processing.7,11 There are some research studies describing the application of phytase in food processing to reduce phytate, such as white © 2014 American Chemical Society

Received: Revised: Accepted: Published: 10989

August 16, 2014 October 11, 2014 October 21, 2014 October 21, 2014 dx.doi.org/10.1021/jf503952s | J. Agric. Food Chem. 2014, 62, 10989−10997

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addition of phytase into the precipitated SPI would make the SPI processing have an additional step (the addition of water for phytase treatment). Therefore, the addition of phytase into the water extract of soybean flour should be the best strategy. In addition, the phytase used in this study is recommended to hydrolyze phytate at 55 °C and pH 5.0, which is well used in the phytase treatment. To examine whether this condition could produce SPI with high quality, the hydrolysis conditions (25−55 °C, pH 5.0−6.5) for phytase were examined, and the optimal condition was selected to prepare SPI samples with different phytate contents by prolonging the hydrolysis time. The effects of phytate content on the yield, components, and physicochemical (solubility, ζ-potential, denaturation temperature, and secondary structure) and functional properties (emulsifying activity, foaming activity, gelation, and in vitro digestibility) of SPI were systematically examined. This study will provide some valuable information for the application of phytase on SPI processing.

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MATERIALS AND METHODS

Materials. Defatted low-heat soybean meal (protein content, 52.9%, dry basis; moisture, 7.9%) was purchased from Shandong Wonderful Industrial Group Co., Ltd. (Dongying, China). Phytase from Aspergillus niger with an activity of 45000 U/g was purchased from Sukahan Bio-Technology Co., Ltd. (Weifang, China), and hydrolysis condition of pH 5.0 and 55 °C was recommended by the manufacturer. Its activity was about 200000 PU/g after simple purification. Purification of Phytase and Determination of Phytase Activity. The phytase purchased from Sukahan was dissolved in 50 mM sodium acetate buffer (pH 5.5), stirred for 30 min at 4 °C, and then centrifuged (9690g, 20 min, 4 °C) to obtain supernatant and precipitate. The supernatant was subjected to ammonium sulfate precipitation (80% saturation) with constant stirring. The precipitate was collected by centrifugation (9690g, 20 min, 4 °C) and dissolved in a minimum volume of 50 mM sodium acetate buffer (pH 5.5). The enzyme solution was dialyzed overnight at 4 °C against deionized (DI) water and freeze-dried to obtain purified phytase. Phytase activity was analyzed according to the method described by Quan et al.19 and Harland et al.20 with minor modifications, and one phytase unit (PU) was the amount of enzyme that released 1 μmol of inorganic phosphorus from 5 mM sodium phytate in 1 min at pH 5.5 and 37 °C. Phytase Treatment. Defatted soybean meal was ground three times at a 30 s interval and sieved through an 80 mesh sieve to obtain soybean flour. The procedure for extracting soybean protein isolate (SPI) is shown in Figure 1. SPI samples with different contents of phytate were obtained at room temperature (about 25 °C) and pH 5.0 by prolonging hydrolysis time (5, 10, 20, 40, and 60 min). There were two control samples in this study: SPI0 was prepared under the same condition without phytase according to the traditional method; SPI2 was prepared under the same condition with phytase at the optimal condition (pH 5.0, 55 °C) of phytase recommended by the manufacturer. SPI samples were sealed in polythene bags and stored at 4 °C until further analysis. Determination of Phytate Content. Phytate content was determined as described by Gao et al.21 with minor modifications. Approximately 0.5 g of SPI was extracted with 10 mL of 2.4% HCl, magnetically stirred at 220 rpm for 2 h at room temperature, and centrifuged at 1000g at 10 °C for 20 min. Crude extract was transferred into a 25 mL beaker containing 1 g of NaCl, which was magnetically stirred at 350 rpm for 20 min to dissolve the salt and then allowed to settle at 4 °C for 60 min. The mixture was centrifuged at 1000g at 10 °C for 20 min, and supernatant was transferred into a 200 mL volumetric flask followed by the addition of DI water to the final volume. The diluted solution (5 mL) was thoroughly mixed with 4 mL of Wade reagent (0.03% FeCl3·6H2O + 0.3% sulfosalicylic acid) and centrifuged (1000g, 10 min, 10 °C). Absorbance of color reaction products for both samples and standards was read at 500 nm. Sodium

Figure 1. Procedure for preparation of soybean protein isolate from defatted low-heat soybean flour. phytate solutions (0.1−0.5 mg/mL; Sigma-Aldrich, Shanghai, China) were used to make the standard curve (R2 > 0.99). ζ-Potential. The ζ-potentials of SPI suspensions (0.05%, w/v) were measured by using a Zetasizer Nano ZS (Mastersizer X, Malvern Instruments Ltd., Malvern, UK). Protein Solubility. Protein solubility of SPI was determined according to the method of Li et al.18 with minor modifications. Aliquots (200 mg) of SPI samples were dispersed and stirred in 20 mL of DI water (pH 6.8) at 200 rpm for 30 min at room temperature. Then, the suspensions were centrifuged at 4000g for 30 min. Nitrogen contents of the supernatants were measured by using the microKjeldahl method (N × 6.25), and the measurements were performed in duplicate. Percent nitrogen solubility (NS) was calculated as follows (eq 1): NS (%) = (N in supernatant/total N in 200 mg sample) × 100%

(1) Differential Scanning Calorimetry (DSC). The thermal characteristics of SPI were examined by using a TA Q100-DSC thermal analyzer (TA Instruments, New Castle, DE, USA), according to the procedure of Wang et al.22 with some modifications. SPI (10.0 mg) was dispersed into 40 μL of 0.01 M phosphate buffer (pH 7.0), and 10 μL was hermetically sealed into a coated aluminum pan and heated from 20 to 110 °C at a rate of 5 °C/min. A sealed empty pan was used as control. Peak or denaturation temperature (Td) and total enthalpy of denaturation (ΔH, J/g dry matter) were computed from the thermograms by Universal Analysis 2000 software, version 4.7A (TA Instruments-Waters LLC). All experiments were conducted in duplicate. In all cases, the sealed pans were equilibrated at 25 °C for >6 h. Measurement of Circular Dichroism (CD). CD spectra were obtained using an MOS-450 spectropolarimeter (BioLogic Science Instrument, France). The CD spectroscopic measurement was done according to the method of Tang et al.,23 which was performed in a quartz cuvette of 2 mm with a protein concentration of 0.1 mg/mL in DI water at pH 6.8. The sample was scanned from 190 to 250 nm, and the spectra were an average of eight scans. The following parameters were used: step resolution, 1 nm; acquisition duration, 1 s; bandwidth, 10990

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0.5 nm; sensitivity, 100 mdeg. The cell was thermostated with a Peltier element at 25 °C, unless specified otherwise. Recorded spectra were corrected by subtraction of the spectrum of DI water. The secondary structure composition was estimated from the CD spectra using the CONTIN/LL program in CDPro software. Emulsifying Activity. The emulsifying activity index (EAI) and emulsion stability index (ESI) of SPI were determined according to the method of Wang et al.22 An SPI suspension (9 mL; 0.2% (w/v)) and 3 mL of soybean oil were homogenized using one high-speed homogenizer (FA25, Fluko Co., Germany) for 1 min at 10000 rpm to form an emulsion. An aliquot (50 μL) of the emulsion was collected from the bottom of the emulsion, immediately (0 min) or 10 min after homogenization, and diluted (1:100, v/v) in 0.1% (w/v) sodium dodecyl sulfate (SDS) solution. After thorough mixing, the absorbance of the diluted emulsion was read at 500 nm. EAI and ESI values were calculated by using eqs 2 and 3 EAI (m 2 /g) = 2 × 2.303 × A 0 × DF/[c × φ × (1 − θ )]

(2)

ESI (min) = A 0 /(A 0 − A10) × 10

(3)

(Sigma-Aldrich) was added and incubated for 2 h under low magnetic stirring. Then, the obtained emulsion was divided into two aliquots. One aliquot (10 g) was mixed with 10 mL of 20% (w/v) trichloroacetic acid (TCA). The mixture was kept for 10 min at room temperature and then centrifuged (4000g, 30 min) to obtain the precipitate. After a washing with 10 mL of TCA (20%, w/v), the final precipitate was obtained by centrifugation at the same parameters above. The nitrogen (N) content was determined by using the Kjeldahl method. The percent N release after the digestion was calculated as % N release = (N0 − Nt) × 100/Ntotal

Nt (mg) is the TCA-insoluble N after digestion, N0 (mg) is the TCAinsoluble N in SPI sample, and Ntotal (mg) is the total N of SPI sample. Measurements were performed in duplicate. The other aliquot (10 g) was dialyzed using a cellulose dialysis tube (cutoff 12 kDa) containing 25 mL of NaCl solution (9 mg/mL). The solution was dialyzed under stirring at 37 °C for 2 h. The dialyzed solution in the tube was then removed and stored at 4 °C in an amber bottle under a nitrogen blanket. The amounts of dialyzable calcium (Ca) and zinc (Zn) corresponded to those obtained outside the dialysis membrane (analyzed by atomic absorption spectrophotometer), expressed as percentages of total calcium and zinc present in the test sample, were used as an indicator of mineral bioavailability. Measurements were performed in duplicate. Determination of Ca and Zn. Ca and Zn contents in SPI were measured by atomic absorption spectrophotometer (SperctrAA220/ 22Z, Varian, USA) according to the method of Kamchan et al.27 SPI (0.5 g) was weighed into a preweighed porcelain crucible (predried in an oven at 105 °C overnight) and put into a muffle furnace (550 °C, 8 h). The resulting white ash was weighed, dissolved into 3 mL of concentrated nitric acid, and diluted with distilled water in a 25 mL volumetric flask. The solution was used to determine Ca and Zn contents. Trypsin Inhibitor Activity (TIA) Assay. TIA of SPI was measured according to the method of Xu et al.,28 with slight modifications. For the TIA standard curve, 0−1.0 mL of trypsin inhibitor standard solution (0.01%, w/v) was pipetted into a set of test tubes, and the volume was made up to 2 mL with DI water. The test tubes were placed and warmed in a 37 °C water bath. Then 5 mL of 0.04% (w/v) Nα-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPA) in pH 8.2 Tris-HCl buffer (1% dimethyl sulfoxide, v/v; 0.02 M CaCl2), which should be prepared freshly and prewarmed in 37 °C water bath, was pipetted into test tubes followed by 2 mL of 0.01% (w/v) trypsin in 0.04 mM HCl solution. Exactly 10 min later, the reaction was terminated by adding 1 mL of 30% acetic acid (v/v) into each tube. After mixing, the content of each tube was centrifuged at 3000 rpm for 10 min, and the absorbance of the filtrate was measured at 410 nm by using a UV-2100 spectrophotometer (UNICO, WFZ UV-2100) against a reagent blank. The reagent blank was prepared by adding 1 mL of 30% acetic acid (v/v) to a test tube containing trypsin and water before 5 mL of BAPA solution was added. The linearity of the calibration curve was r = 1. TIA of SPI should be diluted to the point where 1 mL produces trypsin inhibition of 40−60%. Then 1 mL of diluted sample was added to tubes, and it was done just as above. One TIA unit is arbitrarily defined as an increase of 0.01 absorbance unit at 410 nm per 10 mL of the reaction mixture under the conditions used. Statistical Analysis. Data reported are mean values ± standard deviations. Data were analyzed using SPSS for Windows (version 13.0, SPSS Inc. Chicago, IL, USA) following an analysis of variance (ANOVA) one-way linear model. Mean comparisons were performed using the Duncan test, and the significance level was established for P < 0.05.

where DF is the dilution factor (100), c is the initial concentration of SPI (0.2 g/100 mL), φ is the optical path (0.01 m), θ is the fraction of oil used to form the emulsion (0.25), and A0 and A10 are the absorbance of the diluted emulsions at 0 and 10 min, respectively. Measurements were performed in triplicate. Foaming Activity. The foam capacity and stability were determined according to the method of Venktesh et al.24 with slight modifications. Two grams of SPI was dispersed in 100 mL of DI water (pH 6.8). Then, it was whipped in the high-speed homogenizer (FA25, Fluko Co.) at 10000 rpm for 1 min and poured into a 250 mL graduated cylinder. The volume of the foam after 30 s was recorded, and the volume increase was expressed as percent foam capacity. The foam stability was determined by measuring the decrease in foam volume as a function of time up to 30 min. foam capacity (%) = [vol after whipping (mL) − vol before whipping (mL)] × 100/vol before whipping (mL)

(5)

(4)

Measurements were performed in triplicate. Textural Measurements of SPI Gels. The methods of preparing SPI gels and determining the texture of SPI gels were according to the method of Li et al.18 Briefly, gels were prepared by heating 15% (w/v) SPI suspension in cylindrical glass molds with an inner diameter of 20 mm and a height of 40 mm. The molds were filled three-fourths full, and the air bubbles in suspension were removed using a vacuum pump. The sample was heated in a 95 °C water bath for 30 min and immediately cooled in an ice water bath. Then the sample was kept at 4 °C for 24 h for further analysis. Then, the gels were taken out of the molds and transferred to a Texture Analyzer (TMS-PRO, Food Technology Corp., USA). A cylinder probe with a diameter of 10 mm was chosen. The probe test speed, starting trigger force, depth, backoff distance, and holding time between two cycles were set as follows: 120 mm/min, 0.05 N, 10 mm, 40 mm, and 0 s, respectively. The tests were performed in triplicate at 25 °C. The hardness was analyzed by Rheology Advantage Data Analysis software. In Vitro Digestibility of Protein and Bioavailability of Minerals. The in vitro digestibility of protein and bioavailability of minerals were respectively evaluated according to the methods of Dinnella et al.25 and Lestinenne et al.26 with minor modifications. About 1 g of SPI was precisely weighed in an enzymolysis reactor and dispersed into 20 mL of DI water. After 10 min of conditioning in a shaking water bath at 37 °C, the pH was adjusted to 2.0 with 1 M HCl solution under magnetic stirring. Next, 2% (w/w) of pepsin (SigmaAldrich, Shanghai, China) was added, and the mixture was incubated for 2 h. The pH was then increased to about 5.3 with 1 M NaHCO3, 6% (w/w) of bile salts (Sigma-Aldrich) was added, and the pH was adjusted to 7.5 with 1 M NaOH. Then 4% (w/w) of pancreatin

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RESULTS AND DISCUSSION Hydrolysis Condition. The condition (pH 5.0 and 55 °C) was recommended by the manufacturer for the hydrolysis of phytate by phytase. In the beginning of this study, this condition was used to produce SPI samples, but it was found 10991

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acid-precipitated to obtain SPI1 samples. SPI0 was used as control. Table 2 shows that phytate contents of SPI samples are 19.86 (SPI0), 14.58 (5 min), 8.64 (10 min), 4.42 (20 min), 1.89 (40 min), and 0.11 (60 min) mg/g, respectively; interestingly, protein recovery (R2 = 0.781; 52.26−54.83%) and content (R2 = 0.876; 90.57−92.85%) in SPI are linearly and slightly increased, whereas ash content is linearly (R2 = 0.940) and significantly decreased from 5.15 to 3.62% with decreasing phytate content. Two important minerals (Zn and Ca) in SPI are also significantly decreased with prolonged hydrolysis time, which is in good agreement with the behavior of ash content. Ca content in SPI0 (818.6 μg/g) was in agreement with the results of Honig and Wolf30 (1.05−1.66 mg/g) and Kroll et al.31 (0.076%, w/w), and the Zn content in SPI0 (21 μg/g) was also consistent with the results of Honig and Wolf30 (29−36 μg/g). Generally, the interaction between phytate and protein was electrostatic, and both phytate and soybean proteins were negatively charged at neutral pH.32 As a result, phytate did not bind to soybean proteins at neutral pH,31 but the binding of phytate did happen in the presence of Ca2+ (or Zn2+ and some other divalent ions), which was considered that phytate− mineral (Ca2+, Zn2+, and some other divalent ions)−protein complexes were formed.33,34 By phytase treatment, phytate was hydrolyzed to phosphorus and inositol, and mineral−protein complexes remained. By deceasing the pH from neutral pH to acidic pH (acid-precipitation for SPI), the number of bound Ca2+ on soybean proteins was decreased,31 which might be the reason that ash content in SPI1 was decreased with prolonged hydrolysis time. For SPI0, some phytate−mineral complexes (insoluble) might be released from proteins by decreasing the pH, but they would be mixed with soybean proteins in the precipitate, which should be the reason that ash content in SPI0 was still larger than that in SPI0.35 Phytate is one kind of salt rich in negative charge, so it was considered that phytate−mineral−protein complexes would be more acidic than mineral−protein complexes, revealing that the former should be more hydrophilic than the latter, which might be one reason for the higher protein recovery induced by phytase. ζ-Potential and Protein Solubility. Figure 2a shows that the ζ-potential value of the SPI suspension is linearly (R2 = 0.793) and significantly increased from −26.1 to −35.4 mV with increasing phytate content, which is consistent with the hypothesis stated above. Figure 2b shows that the protein solubility of SPI is linearly (R2 = 0.983) and slightly increased from 94.81 to 98.89% with increasing phytate content, which is

that the obtained samples had bad solubility. By increasing the hydrolysis pH (pH 5.0, 5.5, 6.0, and 6.5; 25 °C), the degree of phytate degradation was decreased (Supporting Information Table S1); by decreasing the hydrolysis temperature (55, 45, 35, and 25 °C (room temperature); pH 5.0), it was found that the solubility of SPI sample was increased (Supporting Information Table S2). As a result, two hydrolysis conditions (pH 5.0, 25 °C, 60 min, 20 PU/g soybean flour; pH 5.0, 55 °C, 60 min, 10 PU/g soybean flour) were selected for the following research, and the obtained SPI samples were named SPI1 and SPI2, respectively. In addition, SPI was also produced by the traditional method without phytase (SPI0). Table 1 shows that SPI0 has a phytate content of 19.86 mg/ g, which is in agreement with the results of Saito et al. (20.5 Table 1. Physicochemical and Functional Properties of SPI with or without Phytase According to the Procedure Described in Figure 1a phytate content (mg/g) protein solubility (%) EAI (m2/g) ESI (min) FC (%) FS (%) hardness of gel (N)

SPI0b

SPI1c

SPI2d

19.86 ± 0.56a

0.11 ± 0.06b

0.11 ± 0.04b

98.89 ± 1.16a 24.99 ± 0.33a 40.01 ± 0.71b 75.1 ± 1.5a 53.0 ± 2.3b 0.51 ± 0.05b

95.57 ± 0.87b 15.26 ± 0.46b 55.93 ± 1.15a 60.2 ± 1.1b 70.0 ± 1.3a 0.68 ± 0.06a

72.36 ± 1.07c 4.12 ± 0.45c 15.05 ± 1.02c 39.2 ± 1.6c 35.0 ± 2.0c 0.25 ± 0.05c

a The data are shown as means ± SD. Means with different letters in the same row are significantly different at the 5% level. bSPI0, traditional method. cSPI1, pH 5.0, 20 PU/g soy flour, 25 °C, 60 min. d SPI2, pH 5.0, 10 PU/g soy flour, 55 °C, 60 min.

mg/g)29 and Kumar et al. (1−2%).1 Phytate contents of SPI1 (0.11 mg/g) and SPI2 (0.11 mg/g) are very low, revealing that phytate also can be efficiently hydrolyzed at 25 °C by increasing the addition of phytase. Table 1 also shows that SPI0 has the highest solubility, EAI, and FC, whereas SPI1 has the highest ESI, FS, and gel hardness; SPI2 shows the worst properties. The results revealed that phytase treatment at 55 °C had bad effects on the properties of low-phytate SPI. Therefore, room temperature (25 °C) was selected for the low-phytate SPI production. Effects of Phytase-Assisted Processing Method on the Components of SPI. To systematically examine the effects of phytase-induced phytate degradation on SPI, the water extract of soy flour was respectively hydrolyzed by phytase (pH 5.0, 25 °C, 20 PU/g soybean flour) for 5, 10, 20, 40, and 60 min and

Table 2. Components of SPI Treated by Phytase for Different Times (5, 10, 20, 40, 60 min; pH 5.0, 25 °C, 20 PU/g Soybean Flour)a SPI1

a

time (min)

SPI0

5

10

20

40

60

phytate content (mg/g) degree of phytate degradation (%) protein content (%) moisture (%) ash content (%) Ca (μg/g SPI) Zn (μg/g SPI) protein recovery (%)

19.86 ± 0.61a 0f 90.57 ± 0.62c 1.49 ± 0.21a 5.15 ± 0.11a 818.6 ± 18.2a 20.94 ± 0.65a 52.26 ± 1.11c

14.58 ± 0.20b 26.59 ± 0.94e 91.26 ± 0.51b 1.48 ± 0.14a 4.38 ± 0.12b 739.9 ± 20.4b 19.73 ± 0.48a 53.84 ± 1.14b

8.64 ± 0.42c 56.50 ± 1.32d 92.52 ± 0.40a 1.24 ± 0.16a 4.26 ± 0.09b 694.8 ± 28.7c 18.67 ± 0.51b 54.67 ± 1.02b

4.42 ± 0.51d 77.74 ± 1.07c 92.15 ± 0.61a 1.29 ± 0.17a 3.73 ± 0.14c 657.2 ± 19.5d 15.17 ± 0.76 c 54.37 ± 0.81a

1.89 ± 0.24e 90.50 ± 0.98b 92.45 ± 0.54a 1.32 ± 0.20a 3.63 ± 0.13c 619.9 ± 14.4e 14.08 ± 0.32c 54.54 ± 0.65a

0.11 ± 0.10f 99.56 ± 1.12a 92.85 ± 0.42a 1.31 ± 0.12a 3.62 ± 0.20c 562.2 ± 22.3f 12.71 ± 0.28d 54.83 ± 0.90a

The data are shown as means ± SD. Means with different letters in the same row are significantly different at the 5% level. 10992

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Figure 2. Effects of phytate content on ζ-potential (a) and protein solubility (b) of SPI. Means with different letters on the same line are significantly different at the 5% level.

molecules, and others, collectively known as ligands) would bind to proteins, and it was widely accepted that the liganded proteins were more thermally stable than the nonliganded ones. As the hypothesis stated above, the phytate in phytate− mineral−protein complexes was gradually hydrolyzed with prolonged hydrolysis time, and mineral−protein complexes remained. The ash content, including Ca and Zn, in SPI was also decreased with prolonged hydrolysis time (Table 2). These showed that SPI obtained from long hydrolysis time contained lower bound phytate and minerals than that from short hydrolysis time. Scilingo and Anon38 reported that the Ca content increase could cause an increase in thermal stability of SPI, and Martins et al.39 reported that the binding of phytate derivatives could also enhance the thermal stability of proteins. Therefore, it was reasonable that SPI with low phytate and ash contents was less thermally stable that that with high phytate and ash contents. Secondary Structure of Proteins in SPI. SPI0 and SPI1 (phytate content, 0.11 mg/g) were selected to be examined by CD spectrometry between 190 and 250 nm. The ratios of αhelix, β-sheet, β-turn, and random coil were calculated from their CD spectra and are shown in Table 4. It was found that

in agreement with the results in Figure 2a. This is because the ζ-potential value reflects the degree of repulsion between the adjoining charged colloids: colloids with higher ζ-potential values are more electrically stable and vice versa. Ishiguro et al.36 reported that the solubility of soy milk protein was positively correlated with soy milk phytate content and that a greater amount of coagulant was needed to coagulate highphytate soy milk to form tofu compared to low-phytate soy milk. In addition, Liu and Chang37 reported similar results as above. Thermal Behaviors. All SPI samples showed typical DSC thermograms of soybean isolates in which two endothermic transitions corresponded to the denaturation of β-conglycinin and glycinin, respectively. Td1 (denaturation temperature of βconglycinin), Td2 (denaturation temperature of glycinin), and ΔH (total enthalpy change of SPI) were obtained from DSC thermograms and are shown in Table 3. β-Conglycinin and Table 3. DSC Characteristics of SPI with Different Phytate Contentsa phytate content in SPI (mg/g) 19.86 14.58 4.42 0.11

± ± ± ±

0.61a 0.20b 0.51c 0.10d

Td1b (°C) 79.4 78.5 74.8 71.2

± ± ± ±

0.5a 0.3b 0.4c 0.1d

Td2c (°C) 96.2 94.5 92.8 92.1

± ± ± ±

0.7a 0.5b 0.4c 0.4d

ΔHd (J/g) 10.2 9.8 9.5 9.3

± ± ± ±

0.5a 0.6b 0.4c 0.1d

Table 4. Ratio of Secondary Structure of Proteins in SPI0 and SPI1 with Phytate Contents of 19.86 and 0.11 mg/g, Respectively

The data are shown as means ± SD. Means with different letters in the same column are significantly different at the 5% level. bTd1, denaturation temperature of β-conglycicin. cTd2, denaturation temperature of glycicin. dΔH, total enthalpy change of β-conglycicin and glycicin. a

SPI0 SPI1

α-helix

β-sheet

β-turn

random coil

total

0.324 0.321

0.167 0.185

0.199 0.211

0.308 0.280

0.998 0.997

the ratios of β-sheet and β-turn were increased, whereas that of random coil was decreased for SPI1 compared to SPI0; the ratio of α-helix was slightly decreased. Phytate could inhibit the activities of many kinds of digestive enzymes, revealing that it might change the conformations of those enzymes.1 Therefore, it was reasonable that phytate could affect the secondary structure of proteins in SPI. Similar to the digestive enzymes, it was considered that soybean proteins should be slightly denatured by the binding of phytate, and Wang et al.22 reported that the slight denaturation of soybean proteins by high hydrostatic pressure could increase

glycinin of SPI0 exhibited two endothermic peaks at about 79.4 (Td1) and 96.2 °C (Td2), and ΔH was 10.2 J/g. With decreasing phytate content, Td1 (R2 = 0.941), Td2 (R2 = 0.977), and ΔH (R2 = 0.963) were all linearly decreased. When SPI had a phytate content of 0.11 mg/g, Td1, Td2, and ΔH were 71.2 °C, 92.1 °C, and 9.3 J/g, respectively. These results showed that SPI with higher phytate content had higher denaturation temperatures than that with lower phytate content. In food processing and biological processes, many kinds of molecules (i.e., proteins, lipids, anions, metal ions, ions, odorant 10993

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Figure 3. Effects of phytate content on EAI and ESI (a) and FC and FS (b) of SPI. Means with different letters on the same line are significantly different at the 5% level.

the denaturation temperatures and solubility, which was consistent with the results in Figure 2b and Table 3. Unfortunately, they did not give the data concerning the secondary structure. Emulsifying and Foaming Properties. Figure 3a shows that the EAI of SPI is linearly (R2 = 0.983) and significantly increased from 15.26 to 25.00 m2/g, whereas the ESI of SPI is linearly (R2 = 0.953) and significantly decreased from 55.93 to 40.01 min, with increasing phytate content. Li et al.18 reported that high hydrostatic pressure could significantly increase solubility and EAI but significantly decrease ESI of SPI in the ranges of 0−300 MPa and 5−15 min, which was in agreement with the results above. Wang et al.22 used high hydrostatic pressure to treat SPI and obtained results similar to those of Li et al.18 It is known that protein solubility is a key factor for its emulsifying activity. Generally, high solubility corresponds to high emulsifying activity and vice versa. Table 3 shows that the denaturation temperature of SPI is increased with increasing phytate content, revealing that the molecular flexibility of SPI is decreased.40 It is known that molecular flexibility is positively correlated with its emulsion stability. In addition, phytate− mineral−protein complexes (high-phytate SPI) were more hydrophilic than mineral−protein complexes (low-phytate SPI), meaning that the former had larger surface hydrophilicity/hydrophobicity ratios (similar to the hydrophilic− lipophilic balance of a surfactant) than the latter, which could be well evidenced by the results in Figure 2b. Therefore, the balance between protein solubility and flexibility could be used to explain the different behaviors of EAI and ESI of SPI. Figure 3b shows that the FC of SPI is linearly (R2 = 0.955) and significantly increased from 60 to 75%, whereas the FS of SPI is linearly (R2 = 0.919) and significantly decreased from 70 to 53%, with increasing phytate content. Li et al.18 reported that high hydrostatic pressure also could significantly increase FC but significantly decrease FS of SPI in the ranges of 200−300 MPa and 5−15 min. Foaming activity, similar to emulsifying activity, belongs to the functional properties related to the protein surface. Therefore, the different behaviors of FC and FS of SPI could be explained by the same theory as above. Hardness of SPI Gels. SPI samples with different phytate contents were used to prepare gels, and the hardness, a typical parameter of gel, was determined and is shown in Figure 4. It was found that hardness was linearly (R2 = 0.893) and significantly decreased from 0.69 to 0.50 N with increasing

Figure 4. Effects of phytate content on hardness of SPI gel. Means with different letters are significantly different at the 5% level.

phytate content. Toda et al.41 prepared soy milks with different phytate contents by using different soybean varieties, and breaking stress (hardness) of filled tofu (coagulant: low concentration (0.25%) of MgCl2) was highly and negatively (P < 0.001, r = −0.73) correlated with soy milk phytate content. Ishiguro et al.37 prepared soy milks with different phytate contents by the addition of different levels of phytate, and filled tofu with higher phytate content had lower breaking stress. Hou and Chang42 prepared soy milks with different phytate contents by using phytase, and it was found that the yield of momen tofu was increased; breaking stress was decreased with decreasing phytate content, showing a different trend from the two research studies above. However, it was considered that the different trend should be induced by the different preparation methods of tofu. For momen tofu, the step of pressing was used to remove the whey fraction, and a lower yield of menon tofu was obtained from high-phytate soy milk compared to low-phytate soy milk, indicating that lowphytate momen tofu had a higher moisture content than highphytate momen tofu. It was considered that the high-moisture content glossed over the real effects of phytate on the hardness of tofu. Additionally, it is widely accepted that the gel network formation is the result of a combination of hydrophobic and 10994

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significantly increased from 12.71 to 20.94 μg/g and from 562.2 to 818.6 μg/g, respectively; percentages of dialyzable Zn and Ca are significantly decreased from 15.4 to 2.5% and from 27.64 to 20.13%, respectively; interestingly, the amount of dialyzable Zn is significantly decreased from 1.96 to 0.53 μg/g, whereas that of dialyzable Ca is significantly increased from 155.39 to 164.78 μg/g. These results showed that phytase should be able to increase the amount of bioavailable Zn, although it decreased the Zn content in SPI, and that phytase increased the percentage of bioavailable Ca, but decreased the amount of bioavailable Ca, which should result from the phytase-induced Ca content decrease in SPI. The results were in agreement with many research studies which revealed that the removal of phytate could enhance the bioavailability of minerals.1 Wise et al.48 reported that Ca-bound phytate increased chelation with trace minerals, especially Zn, to form coprecipitates that make the Zn unavailable, which might be used to explain the amount decrease of bioavailable Zn with increasing phytate content. This was because Ca content was decreased with decreasing phytate content. In addition, the low percentages of dialyzable Zn (2.5−15.4%) and Ca (20.13− 27.64%) may result from the binding of Zn and Ca to protein hydrolysates.49,50 In this study, it was found that hydrolysis condition at low temperature (25 °C) and pH 5.0 was better than that (55 °C, pH 5.0) recommended by the manufacturer for retaining the properties of SPI, so the former condition was used to prepare SPI sample with decreasing phytate content by prolonged hydrolysis time. The results showed that protein content, protein recovery, solubility, ζ-potential value, denaturation temperatures, EAI, FC, and TIA were highly and positively correlated, whereas ash content (including Ca and Zn), ESI, FS, gel hardness, and in vitro protein digestibility were highly and negatively correlated with phytate content of SPI. Simulated gastrointestinal digestion and subsequent dialysis showed that the percentages of dialyzable Zn and Ca were increased with decreasing phytate content in SPI but that the amounts of dialyzable Zn and Ca revealed different behaviors: the former was increased and the latter was decreased. CD spectra revealed that the phytase treatment could change the secondary structure of SPI. In all, these results showed that the phytaseassisted processing method could produce low-phytate SPI with better gelling and nutritional properties than the traditional method by slightly losing solubility and partially losing emulsifying activity and foaming activity. It was considered that this kind of SPI would be a good ingredient in the production of sausage and similar products.

electrostatic interactions.43 As stated above, SPI with a high phytate content was more acidic than that with a low phytate content (Figure 2a), revealing that proteins in the former SPI had stronger electrostatic repulsion, and phytate−mineral− protein complexes (high-phytate SPI) had lower surface hydrophobicity/hydrophilicity ratios than mineral−protein complexes. These could be used to well explain the results in Figure 4. In Vitro Digestibility of Protein and Bioavailability of Minerals. Figure 5 shows the in vitro protein digestibility and

Figure 5. Effects of phytate content on in vitro digestibility and TIA of SPI. Means with different letters on the same line are significantly different at the 5% level.

TIA of SPI with different phytate contents. It was found that the in vitro digestibility was linearly (R2 = 0.96) and significantly decreased from 72 to 59%, whereas the TIA was linearly (R2 = 0.821) and significantly increased from 71934 to 78500 U/g with increasing phytate content. It was calculated that the TIA in SPI with a phytate content of 0.11 mg/g was about 92% of that with a phytate content of 19.86 mg/g, revealing that phytate had some effects on inhibiting the activity of trypsin. Ritter et al.44 reported that low-phytate SPI obtained from sequential ion exchange process was more digestible than high-phytate SPI, and Knuckles et al.45 found that the inhibitory effect of the phytate on in vitro digestibility of casein and bovine serum albumin was positively correlated with phytate content. Borowsk et al.46 found that protein isolates from fava bean and soybean with low phytate contents also had low TIA, and Singh et al.47 reported that the activity of trypsin could be substantially inhibited by low levels of phytate. Table 5 shows the dialyzable Ca and Zn in SPI with different phytate contents by simulated gastrointestinal digestion. With increasing phytate content, Zn and Ca contents in SPI are

Table 5. Dialyzable Ca and Zn in SPI with Different Phytate Contents by Simulated Gastrointestinal Digestiona phytate content (mg/g SPI) 19.86 14.58 8.64 4.42 1.89 0.11 a

± ± ± ± ± ±

0.61a 0.20b 0.42c 0.51d 0.24e 0.10f

Zn content (μg/g SPI) 20.94 19.73 18.67 15.17 14.08 12.71

± ± ± ± ± ±

0.31a 0.16b 0.24b 0.33c 0.42c 0.35d

dialyzable Zn (μg/g SPI) 0.53 0.81 1.08 1.18 1.35 1.96

± ± ± ± ± ±

0.04e 0.12d 0.16c 0.11c 0.07b 0.15a

dialyzable Zn (%) 2.5 4.1 5.8 7.8 9.6 15.4

± ± ± ± ± ±

0.4f 0.2e 0.1d 0.4c 0.3b 0.5a

Ca content (μg/g SPI) 818.6 739.9 694.8 657.2 619.9 562.2

± ± ± ± ± ±

5.6a 4.2b 5.0c 3.7d 4.3e 2.8f

dialyzable Ca (μg/g SPI) 164.8 158.7 158.1 159.5 157.3 155.4

± ± ± ± ± ±

1.2a 2.2b 2.4b 1.5b 1.8b 1.0b

dialyzable Ca (%) 20.13 21.46 22.75 24.27 25.38 27.64

± ± ± ± ± ±

1.21e 0.62d 1.02d 0.48c 0.46b 0.63a

A cellulose dialysis tube (cutoff 12 kDa) was used. Means with different letters in the same column are significantly different at the 5% level. 10995

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ASSOCIATED CONTENT

S Supporting Information *

Tables S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(Y.H.) Phone: 0510-85917812. Fax: 0510-85329091. E-mail: [email protected]. Funding

The work received financial support from the National Natural Science Foundation of China (No. 21276107), the National Great Project of Scientific and Technical Supporting Programs funded by the Ministry of Science and Technology of China during the 12th five-year plan (No. 2012BAD34B04-1), and the 863 Program (Hi-tech research and development program of China, No. 2013AA102204-3). Notes

The authors declare no competing financial interest.

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