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Isoflavone during protease hydrolysis of defatted soybean meal
Food Chemistry 118 (2010) 328–332
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Isoflavone during protease hydrolysis of defatted soybean meal Jianping Wu *, Alister D. Muir Agriculture and Agri-Food Canada, Saskatoon Research Station, 107 Science Place, Saskatoon, SK, Canada S7N 0X2
a r t i c l e
i n f o
Article history: Received 31 December 2008 Received in revised form 20 April 2009 Accepted 29 April 2009
Keywords: Protease Soybean flour pH Isoflavones Aglycone b-Glucosidase
a b s t r a c t The study examined the effects of proteases on the isoflavones during enzymatic hydrolysis of soybean flour. Protease itself did not affect the isoflavones during hydrolysis, whereas the applied conditions and contaminated b-glucosidase in the enzyme could greatly affect the content and composition of isoflavones. Soybean flour hydrolysis by ENZECO Alkaline Protease L-FG at high pH (10) resulted in complete loss of malonylglucosidic and acetylglucosidic conjugates in the hydrolysate. However, these conjugates such as 600 -O-malonylgenistin and 600 -O-malonyldaidzin remained as the principal compounds accounting for 66.2, 58.3 and 70.5% of the total isoflavones in the Protease M ‘‘Amano”, Alcalase 2.4L, and neutral enzyme ENZECO Neutral Protease-NBP-L hydrolysates, respectively, compared to that of 57.8% in the original soybean flour. The residue prepared by Protease M contained 10 times higher aglycones than that of soy flour, which was due to the contaminated b-glucosidase activity in the enzyme preparation. Our result showed that b-glucosidase contaminated in Protease M has a unique selectivity compared to that of the purified almond b-glucosidase. Results from the study indicated that hydrolysis of soybean flour may provide another alternative approach to enrich aglycone isoflavones in soybean-containing products. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Soybeans have been used for centuries as an important part of diet in many Asian countries. Consumption of soybean products has been seen an explosive growth worldwide, driven primarily by the US Food and Drug Administration (FDA) health claim concerning the role of soy protein in reducing the risk of heart disease (FDA, 1999). In addition to protein, isoflavones are also touted as one of the most important components responsible for preventing and attenuating cardiovascular diseases, certain cancers and osteoporosis (Brouns, 2002; Messina 1999; Setchell 1998; Setchell & Cassidy, 1999). The content and composition of aglycones in soy foods are largely decided by the genotype, environment, storage and processing conditions (Coward, Smith, Kirk, & Barnes, 1998; Eldridge & Kwolekm, 1983; Hoeck, Fehr, Murphy, & Welke, 2000; Hou & Chang, 2002; Nurmi, Mazur, Heinonen, Kokkonen, & Adlercreutz, 2002). It is believed that isoflavone aglycones, the predominant form in fermented soyfoods (Coward et al., 1998), display greater bioavailability than their corresponding glycosides (Izumi et al., 2000; Watanabe et al., 1998). Setchell et al. (2002) concluded that isoflavone b-glycosides do not cross the intestine of healthy humans, and their bioavailability requires initial hydrolysis of the sugar moiety by intestinal b-glucosidase for uptake to the periph-
eral circulation. Attempts have been made to increase purposely the aglycone forms mediated by b-glucosidase or microbial fermentation (Obata et al., 2002; Tsangalis, Ashton, Mcgill, & Shah, 2003; Xie, Hettiarachchy, Cai, Tsuruhami, & Koikeda, 2003). Enzymatic modifications of soybean proteins are extensively explored to improve protein functionalities, nutritional quality, or produce specific population diets/clinical supplements, or as a flavour enhancer (Aaslyng et al., 1998; Clemente, 2000; Lahl & Braun, 1994; Were, Hettiarachchy & Kalapathy, 1997; Wu & Cadwallader, 2002). Recent studies have shown that soybean-derived peptides may have antihypertension (Wu & Ding, 2001), anti-cancer (Galvez, Chen, Macasieb, & de Lumen, 2001; Hellerstein, 1999), lowering cholesterol (Chen, Chiou, Shieh, & Yang, 2002) and immunostimulation activities (Chen, Suetsuna, & Yamauchi, 1995). Compared with many reports on the effect of enzymes on the functional and biological properties of soybean proteins, there is limited information on the effect of proteases on isoflavones that are associated with many soybean products and ingredients. The objectives of this study were to study the effect of proteases on the content and composition of isoflavones. 2. Materials and methods 2.1. Materials
* Corresponding author. Present address: Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada T6H 2V8. Tel.: +1 780 492 6885; fax: +1 780 492 4346. E-mail address: [email protected] (J. Wu). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.04.129
Nutrisoy 7B flour (Lot# 991201C) was obtained from Archer Daniels Midland Co. (Decatur, IL, USA). Protease M (Aspergillus oryzae, Amano Enzyme USA Co., Ltd. Elgin, IL, USA), ENZECO
J. Wu, A.D. Muir / Food Chemistry 118 (2010) 328–332
Neutral Protease-NBP-L (Bacillus subtilis, Enzyme Development Co., New York, NY, USA), ENZECO Alkaline Protease L-FG (B. subtilis, Enzyme Development Co., New York, NY, USA) and Alcalase 2.4L (Bacillus licheniformis, Novozymes North America, Inc., Franklinton, NC, USA) were gifts upon request. Almond-derived b-glucosidase was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Isoflavone standards (daidzin, 600 -O-acetyldaidzin, daidzein, genistein, 600 -O-acetylgenistin, 600 -O-malonylgenistin, genistin, glycitin, 600 -Oacetylglycitin and glycitein,) were purchased from LC Labs (Woburn, MA, USA). HPLC-grade acetonitrile, acetic acid and spectranalyzed-grade dimethyl sulphoxide (DMSO) were obtained from Fisher Scientific (Pittsburgh, PA, USA). HPLC-grade water generated by Milli-Q system (Millipore, Bedford, MA, USA) was used for the preparation of the mobile phase. 2.2. Enzymatic hydrolysis of soybean flour Enzymatic hydrolysis of soybean flour was performed in a temperature- and pH-controlled reaction vessel equipped with a stirrer. Nutrisoy 7B flour was mixed thoroughly using a magnetic stirrer with distilled water into a 5% (w/w, protein/water) slurry. The pH and temperature of the slurry was adjusted to the conditions of individual enzyme, such as 4.5 and 50 °C for Protease M (PM), or 7 and 40 °C for ENZECO Neutral Protease-NBP-L (EN), or 10 and 50 °C for ENZECO Alkaline Protease L-FG (EA), or 8 and 50 °C for Alcalase 2.4L; enzyme was then added at the level of 4% (w/w, on the basis of protein content of the slurry) to initiate the digestion. Temperature was kept stable using a circulating water bath and pH was kept constant during protein digestion with continuous addition of 0.5 M NaOH or 0.5 M HCl if necessary. After 3 h incubation, residue was separated from the slurry after centrifugation at 6,000g for 25 min; the residue was re-suspended in 100 mL water and centrifuged under the same condition above. The hydrolysates combined as the resulting clear supernatants and the residues were freeze dried and kept 20 °C till further use.
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Individual isoflavone standards were used for peak identification according to elution time, UV spectra and spiking tests. Isoflavone quantification was based on calibration curves for each of the 10 isoflavone standards; quantification of 600 -O-malonyldaidzin and 600 -O-malonylglycitin, however, was based on their corresponding glucosides adjusted for the molecular weight difference (Kudou et al., 1991). Each sample was analysed in duplicate and mean values were reported. 2.5. b-Glucosidase activity analysis b-Glucosidase activity in four proteases was determined according to the method of Grover (1977) by measuring the rate of hydrolysis of p-nitrophenyl-b-D-glucopyranoside (pNPG). One unit of the enzyme activity was defined as the amount of b-glucosidase that release 1 lmol of p-nitrophenol from the substrate pNPG per milliliter per minute. 2.6. Standard isoflavone incubation Isoflavone standards (a mixture of 0.2 mg of daidzin, acetyldaidzin, malonyldaidzin, genistin, acetylgenistin and malonylgenistin) were dissolved by 1.0 mL of DMSO and extracted solvent mixture (1:1, v/v), diluted 10 times with 0.2 M acetic acid buffer (pH 4.5). Equal volume isoflavone standard solution was incubated with Protease M ‘‘Amano” or with b-glucosidase (15 U) at the same condition used for protein hydrolysis as stated above. Samples (0.6 mL) were taken at various incubation intervals at 0, 0.25, 1, 3, 6 and 24 h and the enzyme was inactivated by adding 0.9 mL of acetonitrile whilst stirring under room temperature. After mixed with 1.5 mL HPLC-grade water, the samples were filtered through a 0.45 lm filter prior to HPLC analysis.
3. Result and analysis
2.3. Isoflavones extraction
3.1. Isoflavones during protease hydrolysis
Approximately 0.5 g of soy flour was accurately weighted into a screw cap test tube (125 mm 20 mm) and was extracted at room temperature for 2 h on a rotary mixer with 10 mL of methanol/acetonitrile/water (4:3:2, v/v/v). After a brief centrifugation (2000g for 10 min), a portion of the supernatant was removed with a syringe, filtered through a 0.45-lm filter into a sample vial, and analysed by HPLC.
Four proteases with different pH optimums were used to study their effects on the isoflavones during hydrolysis. Compared to the soybean flour, the content and composition of isoflavones in both the hydrolysate and resultant residues were significantly affected by the proteases and their associated conditions (Table 1). The resultant residues tended to contain higher amount of isoflavones than those of the hydrolysates; the amount of isoflavones in the Protease M residue was more than three times higher than that of the hydrolysate. With the exception of EA hydrolysis, whose recovery of total isoflavones was only 71.6%, the average recovery of isoflavones after hydrolysis was 91.3%; the lower recovery percentage in EA hydrolysis was probably due to the high pH value (10) applied. Hydrolysis under high pH conditions such as in ENZECO Alkaline Protease L-FG resulted in complete loss of malonylglucosidic and acetylglucosidic conjugates both in the hydrolysate and residue (Table 1). However, these conjugates, such as 600 -O-malonylgenistin and 600 -O-malonyldaidzin remained as the principal compounds accounting for 66.2% in Protease M hydrolysate, 58.3% in Alcalase 2.4L hydrolysate, 70.5% in ENZECO Neutral Protease-NBP-L (EN) hydrolysate, respectively, compared with 57.8% in soybean flour and 0% in EA hydrolysate (Table 1). It is well known that malonylglucosidic and acetylglucosidic conjugates are the most fragile compounds among these isoflavone chemicals and their stability were vulnerable to environmental factors and processing conditions (Coward et al., 1998; Griffith & Collison, 2001; Kudou et al., 1991). With the exception of EA hydrolysis, there were relative high total amount of malonylglucosidic and acetylglucosidic conjugates left in hydrolysates and residues compared
2.4. Isoflavones analysis Separation and quantification of isoflavones were performed on a symmetryÒ C18 reverse-phase column (4.6 250 mm, 5 lm particle size, Waters, Milford, MA, USA) according to Griffith and Collison (2001) with slight modifications as reported (Wu & Muir, 2008). All samples were run automatically via sample management system at a fixed injection volume of 10 lL on an HPLC WatersTM 2690 Separation Module System (Waters Inc.; Milford, MA, USA). UV absorbance was scanned from 200 to 350 nm using a Model Waters 996 Photodiode Array Detector, and peak area was integrated automatically with the supplied software at 254 nm. The sample compartment temperature and column temperature were kept at 15 and 34 °C, respectively. Column was eluted by a two-solvent system: (A) 0.1% acetic acid in water and (B) 0.1% acetic acid in acetonitrile. The column was held constantly at 15% B for the first 10 min, increased to 35% B over 45 min, then to 55% B over 10 min. The concentration of B was brought back to 15% over 5 min and held for another 5 min before the next run.
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Table 1 Effect of proteases on the isoflavones (values are the mean of duplicate determinations, expressed in the form of lg aglycone/g of dry weight) distribution in the hydrolysates and their residues. Enzyme
Alcalase 2.4L
Isoflavone
Hydrolysate
Daidzein Daidzin 600 -O-Acetyldaidzin 600 -O-Malonyldaidzin Genistein Genistin 600 -O-Acetylgenistin 600 -O-Malonygenistin Glycitein Glycitin 600 -O-Acetylglycitin 600 -O-Malonylglycitin Total a
Residue
Protease M ‘‘Amano”
ENZECO Neutral Protease
ENZECO Alkaline Protease
Hydrolysate
Residue
Hydrolysate
Hydrolysate
Residue
Flour
Residue
67.7 180.4 412.8 –a 49.4 186.7 395.5 5.1 17.8 31.7 40.1 12.4
344.0 112.1 210.0 0.9 590.4 227.4 420.4 16.6 57.6 22.1 19.2 3.9
67.2 51.2 329.8 5.4 52.9 43.1 250.2 3.6 11.9 21.8 29.7 9.3
604.1 48.3 310.6 7.6 1012.5 90.9 515.5 27.6 95.5 14.1 34.4 –
79.2 66.5 537.6 35.6 60.9 63.9 408.8 0.0 15.9 17.9 53.3 2.7
317.7 44.5 305.0 – 547.4 82.5 520.9 17.2 57.8 8.4 29.0 5.9
42.9 334.1 – – 27.2 576.8 0.0 0.0 9.2 63.3 – –
240.4 156.6 – – 365.6 624.4 0.0 0.0 40.3 36.6 – –
37.5 252.4 482.5 12.5 47.5 288.4 557.1 11.4 – 36.7 48.6 23.3
1399.6
2024.5
876.3
2761.0
1342.3
1936.3
1053.6
1463.9
1797.9
Not detected.
with their original values in soybean flour (Table 1). In contrast with the total loss of malonylglucosidic and acetylglucosidic conjugates under extreme pH (10.0) hydrolysis conditions, hydrolysis under mild enzymatic conditions had relatively small influence on the stability of these isoflavones (Table 1). The reaction temperature is 50 °C for both alkaline enzymes, but the pH was 8 in Alcalase 2.4L hydrolysis whereas 10 in ENZECO Alkaline Protease L-FG hydrolysis; these results indicated that the difference in pH played an important role in deciding the stability of the conjugated isoflavones. We previously explained that the high water content and the buffering environment formed during enzymatic hydrolysis might be the reasons for the enhanced stability of conjugated chemicals (Wu & Muir, 2008). Interestingly, 600 -O-malonyldaidzin and 600 -O-malonygenistin were no longer the predominant chemicals in the PM residue. There was a large increase in aglycone content in the residue, e.g. the contents of daidzein, genistein and glycitein in PM residue were 604.1, 1012.5 and 95.5 lg aglycone/g compared to those of 37.5, 47.5 and 0 lg aglycone/g in the soybean flour; these three aglycones accounted for 62% of the total isoflavones in PM residue compared to that of 4.7% in soy flour. Similar trend existed in other enzymatic residues where the aglycone fraction accounted for 49.0% in Alcalase 2.4L residue, 44.1% in EA residue and 44.7% in EN residue of the total isoflavone content. The increase in aglycone content in our residues compared favourably with lactic acid fermented soymilk, showing a 63–67% increase in aglycone (Otieno & Shah, 2007). Using b-glycosidase from Penicillium multicolour, Xie et al. (2003) reported that the amount of genistein, daidzein, and glycitein in b-glycosidase treated soymeal were increased from 1.23, 1.25, and 1.51 mmol/g to 3.21, 2.02, and 2.12 mmol/g, respectively. Our results suggested that the major portion of aglycones formed during hydrolysis was remained in their residues after centrifugal separation; the residues accounted for 84.1, 90.9 and 73.3% of the total aglycones produced during the hydrolysis of soybean meal by EN, PM and Alcalase 2.4L, respectively. The increase in aglycone content in EA treated sample could be attributed to the high pH applied in enzymatic hydrolysis; however, the remarkable increase in their aglycones under mild conditions could be attributed to the contaminated b-glucosidase present in these protease preparations rather than the temperature and pH applied during incubation. It was reported that b-glucosidase has superior activity for conversion of isoflavones glycosides to aglycones (Matsuura & Obata, 1993; Xie et al., 2003). b-Glucosidase from microorganisms such as bifidobacteria and lactic acid bacteria have also been studied in hydrolysing isoflavone glycosides into aglycones (Matsuyama, Setoguchi, Arai, & Kiyosawa,
1990; Tsangalis et al., 2003). Microbially derived protease preparations tend to contaminate more or less amount of b-glucosidase; the activity of b-glucosidase in PM, EN, Alcalase 2.4L and EA were determined to be 18.5, 0.09, 0.03, 0 U/g, respectively. The contaminated b-glucosidase in these bacterial proteases contributed greatly to the isoflavones conversion observed during hydrolysis with the exception of EA. Since the optimal pH for b-glucosidase is acidic pH, it is in a good alignment with the result that PM shows the highest b-glucosidase activity among the enzymes tested. However, the increase in aglycones could not be attributed totally to the conversion of their manyolglucosidic and acetylglucosidic conjugates, since the increase magnitude in daidzein (from 546 lg total aglycones in soy flour to 4207.1 lg total aglycones in hydrolysate and residue) was far greater than the total manyolglucosidic (from 7030.0 in soybean flour to 4483.4 lg total aglycones in hydrolysate and residue) and acetylglucosidic conjugates (from 182.1 to 88.5 lg total aglycones) in PM treated samples; furthermore, a significant higher conversion percentage was observed from glucosidic isoflavones to aglycones than malonylglucosidic and acetylglucosidic conjugates to aglycones. It was calculated that 81.1% of daidzin was converted into daidzein, 78.8% of genistin into genistein, compared with 36.2% of 600 -O-malonygenistin into daidzein, 51.4% of 600 -O-acetyldaidzin into daidzein, and 37.1% of 600 -O-malonygenistin into genistein. These results suggested that the activity of b-glucosidase in PM might have a different enzyme selectivity from the reported b-glucosidase (P. multicolour) (Xie et al., 2003). 3.2. Standard isoflavone incubation In order to address the unique enzyme specificity of b-glucosidase in the Protease M, we tested its activity on authenticated isoflavone standards; purified b-glucosidase from almond was also used for comparison. Fig. 1 shows the chromatograms of these standard isoflavones under different incubation intervals using Protease M or purified almond b-glucosidase. Daidzin was completely converted into aglycone in the first 15 min in both enzymes; genistin was not detected in PM treated samples whereas there was trace amount of genistin left in the b-glucosidase treated samples. Matsuura and Obata (1993) reported that b-glucosidase from soybean had a conversion percentage of 16.9% from genistin to genistein and 14.3% for daidzin to daidzein in soymilk, which was much less than the results we reported. The content of malonyldaidzin decreased gradually with the incubation time; percentage conversion reached 74.5 and 61.1% for PM and b-glucosidase treated samples at 24 h, respectively (Fig. 2). It appeared that
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J. Wu, A.D. Muir / Food Chemistry 118 (2010) 328–332 A. Gin
B 0hr
M.Gn
A. Gin
A
Daidzin
0hr Daidzin
Genistin
M. Din
A. Din
M.Gin
Genistin
M. Din
A. Din
Genistein
0.25hr
Daidzein
Genistein
0.25hr Daidzein
1hr 1hr
3hr 3hr
6hr
6hr
24h
24h
0
10
20
30
Min
40
50
60
70
0
10
20
30
40
50
60
70
Min
Fig. 1. Chromatograms of standard isoflavones under different incubation intervals with Protease M ‘‘Amano” (A) and purified b-glucosidase (B).
120 PM A-Gin
conversion (%)
100 PM A-Din
80
PM M-Gin
beta A-Din
60 beta M-Gin
40
PM M-Din
beta M-Din
20 beta A-Gin
0 0
4
8
12
16
20
24
Time (hr) Fig. 2. The conversion percentage of standard isoflavone under various incubation intervals with PM and almond b-glucosidase at the same activity of b-glucosidase. Abbreviations: PM, Protease M ‘‘Amano”; b, almond purified b-glucosidase; A-Gin, acetylgenistin; A-Din, acetyldaidzin; M-Din, malonyldaidzin; M-Gin, malonylgenistin.
of acetyldaidzin left in b-glucosidase treated sample. It seemed that b-glucosidase in PM had a higher efficiency than that of almond-derived b-glucosidase and therefore, the conversion percentage from acetylgenistin and malonylgenistin into genistein with PM was higher than that of almond-derived b-glucosidase. The difference between their specificity and efficiency for the conversion of glucosides to aglycones had a great impact on the content of aglycones. Consequently, the contents of daidzein and genistein in PM treated samples were significantly higher than those of almond b-glucosidase treated samples (Fig. 1). The content of daidzein in PM treated sample was 237.1 lg daidzein/mL compared with 156.2 lg daidzein/mL in almond b-glucosidase treated sample at 0.25 h and this difference remained significant within 6 h but not at 24 h incubation. It appeared that the conversion from glucosides to aglycones with PM was higher and faster than that of the almond b-glucosidase (Fig. 2); the greatest conversion happened within 3 h, reached a plateau after 6 h incubation. The content of genistein in PM treated sample increased rapidly to 659.6 lg aglycone/mL at 15 min, compared with 161.4 lg aglycone/mL at 15 min and 356.8 lg glycone/mL at 24 h incubation in almond b-glucosidase. 4. Conclusions
b-glucosidase from both PM and almond could not convert all malonyldaidzin into daidzein under the condition tested. Generally speaking, acetyldaidzin showed a higher conversion percentage than that of malonyldaidzin (Fig. 2). At 24 h, there was no acetyldaidzin left in PM treated sample whereas there was 36%
This study showed that protease hydrolysed soybean products contained significant amount of isoflavones, and the content and composition of isoflavones were greatly affected by the applied reaction conditions and presence of b-glucosidase activity in the
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commercial protease enzyme. The microbial conversion of isoflavone forms has been reported in soybean products during fermentation efforts to convert conjugated isoflavones into their aglycone forms using bacterial b-glucosidase to develop aglycone-enriched products have been reported (Otieno & Shah, 2007, 2008; Xie et al. 2003). Our results indicated that the b-glucosidase present in commercial protease enzyme preparations has a unique specificity for the conversion of isoflavone chemicals, which was different from almond-derived b-glucosidase. Previous studies also showed that strain-dependent b-glucosidase activity in soymilk (Otieno & Shah 2007, 2008). In addition to b-glucosidase activity, enzyme specificity should be taken into consideration in bioconversion of isoflavones. PM hydrolysis of soybean flours not only resulted in potent bioactive peptides, but also produced a residue with an exceptional high content of aglycones. Our results indicated that protease hydrolysed residues contained more than 10 times higher concentration of aglycones than that of soy flour, thus provided another alternative approach to enrich aglycone isoflavones in soy-containing products. Our further study aims to develop an integrated technology to produce bioactive peptides and enriched aglycone isoflavones from soybean meal. Further research on the purification of the b-glucosidase from the PM preparation is needed to characterise its properties and compare with other sources of b-glucosidase.
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Hoeck, J. A., Fehr, W. R., Murphy, P. A., & Welke, G. A. (2000). Influence of genotype and environment on isoflavone contents of soybean. Crop Science, 40, 48–51. Hou, H. J., & Chang, K. C. (2002). Interconversions of isoflavones in soybeans as affected by storage. Journal of Food Science, 67, 2083–2089. Izumi, T., Piskula, M. K., Osawa, S., Obata, A., Tobe, K., Saito, M., et al. (2000). Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. Journal of Nutrition, 130, 1695–1699. Kudou, S., Fleury, Y., Welti, D., Magnolato, D., Uchida, T., Kitamura, K., et al. (1991). Malonyl isoflavone glucosides in soybean seeds (Glycine max Merrill). Agricultural and Biological Chemistry Journal, 55, 2227–2233. Lahl, W. J., & Braun, S. D. (1994). Enzymatic production of protein hydrolysates for food use in overview: Outstanding symposia in food science and technology. Food Technology, 48, 68–71. Matsuura, M., & Obata, A. (1993). The b-glucosidases from soybeans hydrolyze daidzin and genistin. Journal of Food Science, 58, 144–147. Matsuyama, J., Setoguchi, T., Arai, C., & Kiyosawa, I. (1990). Hydrolytic profiles of soybean isoflavone glycosides with b-glycosidases in the cultures of bifidobacteria and lactic acid bacteria. Bulletin of the Faculty of Agriculture Tamagawa University (Japan), 30, 33–42. Messina, M. J. (1999). Legumes and soybeans: Overview of their nutritional profiles and health effects. American Journal of Clinical Nutrition, 70, 439S–450S. Nurmi, T., Mazur, W., Heinonen, S., Kokkonen, J., & Adlercreutz, H. (2002). Isoflavone content of the soy base supplements. Journal of Pharmaceutical and Biomedical Analysis, 28, 1–11. Obata, A., Manaka, T., Tobe, K., Izumi, T., Saito, M., & Kikuchi, M. (2002). Process for producing isoflavone aglycone-containing composition. US Patent, 6,444, 239 Otieno, D. O., & Shah, N. P. (2007). Comparison of changes in the transformation of isoflavones in soymilk using varying concentrations of exogenous and probiotic-derived endogenous b-glucosidases. Journal of Applied Microbiology, 103, 601–612. Otieno, D. O., & Shah, N. P. (2008). Production of b-glucosidase and hydrolysis of isoflavone phytoestrogens by Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus casei in soymilk. Journal of Food Science, 73, M15–M20. Setchell, K. D. R. (1998). Phytoestrogens: The biochemistry, physiology, and implications for human health of soy isoflavones. American Journal of Clinical Nutrition, 68, 1333S–1346S. Setchell, K. D. R., Brown, N. M., Zimmer-Nechemias, L., Brashear, W. T., Wolfe, B. E., Kirschner, A. S., et al. (2002). Evidence for lack of absorption of soy isoflavone glycosides in humans, supporting the crucial role of intestinal metabolism for bioavailability. American Journal of Clinical Nutrition, 76, 447–453. Setchell, K. D. R., & Cassidy, A. (1999). Dietary isoflavones: Biological effects and relevance to human health. Journal of Nutrition, 129, 758S. Tsangalis, D., Ashton, J. F., Mcgill, A. E. J., & Shah, N. P. (2003). Biotransformation of isoflavones by bifidobacteria in fermented soymilk supplemented with Dglucose and L-cysteine. Journal of Food Science, 68, 623–631. Watanabe, S., Yamaguchi, M., Sobue, T., Takahashi, T., Miura, T., Arai, Y., et al. (1998). Pharmacokinetics of soybean isoflavones in plasma, urine and feces of men after ingestion of 60 g baked soybean powder (Kinako). Journal of Nutrition, 128, 1710–1715. Were, L., Hettiarachchy, N. S., & Kalapathy, U. (1997). Modified soy proteins with improved foaming and water hydration properties. Journal of Food Science, 62, 821–823. 850. Wu, J., & Ding, X. (2001). Hypotensive effect of angiotensin converting enzyme inhibitory peptides derived from defatted soybean meal on spontaneously hypertensive rats (SHR). Journal of Agricultural and Food Chemistry, 49, 501–506. Wu, Y. F. G., & Cadwallader, K. R. (2002). Characterization of the aroma of a meatlike process flavoring from soybean-based enzyme-hydrolyzed vegetable protein. Journal of Agricultural and Food Chemistry, 50, 2900–2907. Wu, J., & Muir, A. D. (2008). Isoflavone content and its potential contribution to the antihypertensive activity in soybean angiotensin I converting enzyme inhibitory peptides. Journal of Agricultural and Food Chemistry, 56, 9899– 9904. Xie, L., Hettiarachchy, N. S., Cai, R., Tsuruhami, K., & Koikeda, S. (2003). Conversion of isoflavone glycosides to aglycones in soyLife and soymeal using b-glycosidase. Journal of Food Science, 68, 427–430.
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Isoflavone during protease hydrolysis of defatted soybean meal Jianping Wu *, Alister D. Muir Agriculture and Agri-Food Canada, Saskatoon Research Station, 107 Science Place, Saskatoon, SK, Canada S7N 0X2
a r t i c l e
i n f o
Article history: Received 31 December 2008 Received in revised form 20 April 2009 Accepted 29 April 2009
Keywords: Protease Soybean flour pH Isoflavones Aglycone b-Glucosidase
a b s t r a c t The study examined the effects of proteases on the isoflavones during enzymatic hydrolysis of soybean flour. Protease itself did not affect the isoflavones during hydrolysis, whereas the applied conditions and contaminated b-glucosidase in the enzyme could greatly affect the content and composition of isoflavones. Soybean flour hydrolysis by ENZECO Alkaline Protease L-FG at high pH (10) resulted in complete loss of malonylglucosidic and acetylglucosidic conjugates in the hydrolysate. However, these conjugates such as 600 -O-malonylgenistin and 600 -O-malonyldaidzin remained as the principal compounds accounting for 66.2, 58.3 and 70.5% of the total isoflavones in the Protease M ‘‘Amano”, Alcalase 2.4L, and neutral enzyme ENZECO Neutral Protease-NBP-L hydrolysates, respectively, compared to that of 57.8% in the original soybean flour. The residue prepared by Protease M contained 10 times higher aglycones than that of soy flour, which was due to the contaminated b-glucosidase activity in the enzyme preparation. Our result showed that b-glucosidase contaminated in Protease M has a unique selectivity compared to that of the purified almond b-glucosidase. Results from the study indicated that hydrolysis of soybean flour may provide another alternative approach to enrich aglycone isoflavones in soybean-containing products. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Soybeans have been used for centuries as an important part of diet in many Asian countries. Consumption of soybean products has been seen an explosive growth worldwide, driven primarily by the US Food and Drug Administration (FDA) health claim concerning the role of soy protein in reducing the risk of heart disease (FDA, 1999). In addition to protein, isoflavones are also touted as one of the most important components responsible for preventing and attenuating cardiovascular diseases, certain cancers and osteoporosis (Brouns, 2002; Messina 1999; Setchell 1998; Setchell & Cassidy, 1999). The content and composition of aglycones in soy foods are largely decided by the genotype, environment, storage and processing conditions (Coward, Smith, Kirk, & Barnes, 1998; Eldridge & Kwolekm, 1983; Hoeck, Fehr, Murphy, & Welke, 2000; Hou & Chang, 2002; Nurmi, Mazur, Heinonen, Kokkonen, & Adlercreutz, 2002). It is believed that isoflavone aglycones, the predominant form in fermented soyfoods (Coward et al., 1998), display greater bioavailability than their corresponding glycosides (Izumi et al., 2000; Watanabe et al., 1998). Setchell et al. (2002) concluded that isoflavone b-glycosides do not cross the intestine of healthy humans, and their bioavailability requires initial hydrolysis of the sugar moiety by intestinal b-glucosidase for uptake to the periph-
eral circulation. Attempts have been made to increase purposely the aglycone forms mediated by b-glucosidase or microbial fermentation (Obata et al., 2002; Tsangalis, Ashton, Mcgill, & Shah, 2003; Xie, Hettiarachchy, Cai, Tsuruhami, & Koikeda, 2003). Enzymatic modifications of soybean proteins are extensively explored to improve protein functionalities, nutritional quality, or produce specific population diets/clinical supplements, or as a flavour enhancer (Aaslyng et al., 1998; Clemente, 2000; Lahl & Braun, 1994; Were, Hettiarachchy & Kalapathy, 1997; Wu & Cadwallader, 2002). Recent studies have shown that soybean-derived peptides may have antihypertension (Wu & Ding, 2001), anti-cancer (Galvez, Chen, Macasieb, & de Lumen, 2001; Hellerstein, 1999), lowering cholesterol (Chen, Chiou, Shieh, & Yang, 2002) and immunostimulation activities (Chen, Suetsuna, & Yamauchi, 1995). Compared with many reports on the effect of enzymes on the functional and biological properties of soybean proteins, there is limited information on the effect of proteases on isoflavones that are associated with many soybean products and ingredients. The objectives of this study were to study the effect of proteases on the content and composition of isoflavones. 2. Materials and methods 2.1. Materials
* Corresponding author. Present address: Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada T6H 2V8. Tel.: +1 780 492 6885; fax: +1 780 492 4346. E-mail address: [email protected] (J. Wu). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.04.129
Nutrisoy 7B flour (Lot# 991201C) was obtained from Archer Daniels Midland Co. (Decatur, IL, USA). Protease M (Aspergillus oryzae, Amano Enzyme USA Co., Ltd. Elgin, IL, USA), ENZECO
J. Wu, A.D. Muir / Food Chemistry 118 (2010) 328–332
Neutral Protease-NBP-L (Bacillus subtilis, Enzyme Development Co., New York, NY, USA), ENZECO Alkaline Protease L-FG (B. subtilis, Enzyme Development Co., New York, NY, USA) and Alcalase 2.4L (Bacillus licheniformis, Novozymes North America, Inc., Franklinton, NC, USA) were gifts upon request. Almond-derived b-glucosidase was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Isoflavone standards (daidzin, 600 -O-acetyldaidzin, daidzein, genistein, 600 -O-acetylgenistin, 600 -O-malonylgenistin, genistin, glycitin, 600 -Oacetylglycitin and glycitein,) were purchased from LC Labs (Woburn, MA, USA). HPLC-grade acetonitrile, acetic acid and spectranalyzed-grade dimethyl sulphoxide (DMSO) were obtained from Fisher Scientific (Pittsburgh, PA, USA). HPLC-grade water generated by Milli-Q system (Millipore, Bedford, MA, USA) was used for the preparation of the mobile phase. 2.2. Enzymatic hydrolysis of soybean flour Enzymatic hydrolysis of soybean flour was performed in a temperature- and pH-controlled reaction vessel equipped with a stirrer. Nutrisoy 7B flour was mixed thoroughly using a magnetic stirrer with distilled water into a 5% (w/w, protein/water) slurry. The pH and temperature of the slurry was adjusted to the conditions of individual enzyme, such as 4.5 and 50 °C for Protease M (PM), or 7 and 40 °C for ENZECO Neutral Protease-NBP-L (EN), or 10 and 50 °C for ENZECO Alkaline Protease L-FG (EA), or 8 and 50 °C for Alcalase 2.4L; enzyme was then added at the level of 4% (w/w, on the basis of protein content of the slurry) to initiate the digestion. Temperature was kept stable using a circulating water bath and pH was kept constant during protein digestion with continuous addition of 0.5 M NaOH or 0.5 M HCl if necessary. After 3 h incubation, residue was separated from the slurry after centrifugation at 6,000g for 25 min; the residue was re-suspended in 100 mL water and centrifuged under the same condition above. The hydrolysates combined as the resulting clear supernatants and the residues were freeze dried and kept 20 °C till further use.
329
Individual isoflavone standards were used for peak identification according to elution time, UV spectra and spiking tests. Isoflavone quantification was based on calibration curves for each of the 10 isoflavone standards; quantification of 600 -O-malonyldaidzin and 600 -O-malonylglycitin, however, was based on their corresponding glucosides adjusted for the molecular weight difference (Kudou et al., 1991). Each sample was analysed in duplicate and mean values were reported. 2.5. b-Glucosidase activity analysis b-Glucosidase activity in four proteases was determined according to the method of Grover (1977) by measuring the rate of hydrolysis of p-nitrophenyl-b-D-glucopyranoside (pNPG). One unit of the enzyme activity was defined as the amount of b-glucosidase that release 1 lmol of p-nitrophenol from the substrate pNPG per milliliter per minute. 2.6. Standard isoflavone incubation Isoflavone standards (a mixture of 0.2 mg of daidzin, acetyldaidzin, malonyldaidzin, genistin, acetylgenistin and malonylgenistin) were dissolved by 1.0 mL of DMSO and extracted solvent mixture (1:1, v/v), diluted 10 times with 0.2 M acetic acid buffer (pH 4.5). Equal volume isoflavone standard solution was incubated with Protease M ‘‘Amano” or with b-glucosidase (15 U) at the same condition used for protein hydrolysis as stated above. Samples (0.6 mL) were taken at various incubation intervals at 0, 0.25, 1, 3, 6 and 24 h and the enzyme was inactivated by adding 0.9 mL of acetonitrile whilst stirring under room temperature. After mixed with 1.5 mL HPLC-grade water, the samples were filtered through a 0.45 lm filter prior to HPLC analysis.
3. Result and analysis
2.3. Isoflavones extraction
3.1. Isoflavones during protease hydrolysis
Approximately 0.5 g of soy flour was accurately weighted into a screw cap test tube (125 mm 20 mm) and was extracted at room temperature for 2 h on a rotary mixer with 10 mL of methanol/acetonitrile/water (4:3:2, v/v/v). After a brief centrifugation (2000g for 10 min), a portion of the supernatant was removed with a syringe, filtered through a 0.45-lm filter into a sample vial, and analysed by HPLC.
Four proteases with different pH optimums were used to study their effects on the isoflavones during hydrolysis. Compared to the soybean flour, the content and composition of isoflavones in both the hydrolysate and resultant residues were significantly affected by the proteases and their associated conditions (Table 1). The resultant residues tended to contain higher amount of isoflavones than those of the hydrolysates; the amount of isoflavones in the Protease M residue was more than three times higher than that of the hydrolysate. With the exception of EA hydrolysis, whose recovery of total isoflavones was only 71.6%, the average recovery of isoflavones after hydrolysis was 91.3%; the lower recovery percentage in EA hydrolysis was probably due to the high pH value (10) applied. Hydrolysis under high pH conditions such as in ENZECO Alkaline Protease L-FG resulted in complete loss of malonylglucosidic and acetylglucosidic conjugates both in the hydrolysate and residue (Table 1). However, these conjugates, such as 600 -O-malonylgenistin and 600 -O-malonyldaidzin remained as the principal compounds accounting for 66.2% in Protease M hydrolysate, 58.3% in Alcalase 2.4L hydrolysate, 70.5% in ENZECO Neutral Protease-NBP-L (EN) hydrolysate, respectively, compared with 57.8% in soybean flour and 0% in EA hydrolysate (Table 1). It is well known that malonylglucosidic and acetylglucosidic conjugates are the most fragile compounds among these isoflavone chemicals and their stability were vulnerable to environmental factors and processing conditions (Coward et al., 1998; Griffith & Collison, 2001; Kudou et al., 1991). With the exception of EA hydrolysis, there were relative high total amount of malonylglucosidic and acetylglucosidic conjugates left in hydrolysates and residues compared
2.4. Isoflavones analysis Separation and quantification of isoflavones were performed on a symmetryÒ C18 reverse-phase column (4.6 250 mm, 5 lm particle size, Waters, Milford, MA, USA) according to Griffith and Collison (2001) with slight modifications as reported (Wu & Muir, 2008). All samples were run automatically via sample management system at a fixed injection volume of 10 lL on an HPLC WatersTM 2690 Separation Module System (Waters Inc.; Milford, MA, USA). UV absorbance was scanned from 200 to 350 nm using a Model Waters 996 Photodiode Array Detector, and peak area was integrated automatically with the supplied software at 254 nm. The sample compartment temperature and column temperature were kept at 15 and 34 °C, respectively. Column was eluted by a two-solvent system: (A) 0.1% acetic acid in water and (B) 0.1% acetic acid in acetonitrile. The column was held constantly at 15% B for the first 10 min, increased to 35% B over 45 min, then to 55% B over 10 min. The concentration of B was brought back to 15% over 5 min and held for another 5 min before the next run.
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Table 1 Effect of proteases on the isoflavones (values are the mean of duplicate determinations, expressed in the form of lg aglycone/g of dry weight) distribution in the hydrolysates and their residues. Enzyme
Alcalase 2.4L
Isoflavone
Hydrolysate
Daidzein Daidzin 600 -O-Acetyldaidzin 600 -O-Malonyldaidzin Genistein Genistin 600 -O-Acetylgenistin 600 -O-Malonygenistin Glycitein Glycitin 600 -O-Acetylglycitin 600 -O-Malonylglycitin Total a
Residue
Protease M ‘‘Amano”
ENZECO Neutral Protease
ENZECO Alkaline Protease
Hydrolysate
Residue
Hydrolysate
Hydrolysate
Residue
Flour
Residue
67.7 180.4 412.8 –a 49.4 186.7 395.5 5.1 17.8 31.7 40.1 12.4
344.0 112.1 210.0 0.9 590.4 227.4 420.4 16.6 57.6 22.1 19.2 3.9
67.2 51.2 329.8 5.4 52.9 43.1 250.2 3.6 11.9 21.8 29.7 9.3
604.1 48.3 310.6 7.6 1012.5 90.9 515.5 27.6 95.5 14.1 34.4 –
79.2 66.5 537.6 35.6 60.9 63.9 408.8 0.0 15.9 17.9 53.3 2.7
317.7 44.5 305.0 – 547.4 82.5 520.9 17.2 57.8 8.4 29.0 5.9
42.9 334.1 – – 27.2 576.8 0.0 0.0 9.2 63.3 – –
240.4 156.6 – – 365.6 624.4 0.0 0.0 40.3 36.6 – –
37.5 252.4 482.5 12.5 47.5 288.4 557.1 11.4 – 36.7 48.6 23.3
1399.6
2024.5
876.3
2761.0
1342.3
1936.3
1053.6
1463.9
1797.9
Not detected.
with their original values in soybean flour (Table 1). In contrast with the total loss of malonylglucosidic and acetylglucosidic conjugates under extreme pH (10.0) hydrolysis conditions, hydrolysis under mild enzymatic conditions had relatively small influence on the stability of these isoflavones (Table 1). The reaction temperature is 50 °C for both alkaline enzymes, but the pH was 8 in Alcalase 2.4L hydrolysis whereas 10 in ENZECO Alkaline Protease L-FG hydrolysis; these results indicated that the difference in pH played an important role in deciding the stability of the conjugated isoflavones. We previously explained that the high water content and the buffering environment formed during enzymatic hydrolysis might be the reasons for the enhanced stability of conjugated chemicals (Wu & Muir, 2008). Interestingly, 600 -O-malonyldaidzin and 600 -O-malonygenistin were no longer the predominant chemicals in the PM residue. There was a large increase in aglycone content in the residue, e.g. the contents of daidzein, genistein and glycitein in PM residue were 604.1, 1012.5 and 95.5 lg aglycone/g compared to those of 37.5, 47.5 and 0 lg aglycone/g in the soybean flour; these three aglycones accounted for 62% of the total isoflavones in PM residue compared to that of 4.7% in soy flour. Similar trend existed in other enzymatic residues where the aglycone fraction accounted for 49.0% in Alcalase 2.4L residue, 44.1% in EA residue and 44.7% in EN residue of the total isoflavone content. The increase in aglycone content in our residues compared favourably with lactic acid fermented soymilk, showing a 63–67% increase in aglycone (Otieno & Shah, 2007). Using b-glycosidase from Penicillium multicolour, Xie et al. (2003) reported that the amount of genistein, daidzein, and glycitein in b-glycosidase treated soymeal were increased from 1.23, 1.25, and 1.51 mmol/g to 3.21, 2.02, and 2.12 mmol/g, respectively. Our results suggested that the major portion of aglycones formed during hydrolysis was remained in their residues after centrifugal separation; the residues accounted for 84.1, 90.9 and 73.3% of the total aglycones produced during the hydrolysis of soybean meal by EN, PM and Alcalase 2.4L, respectively. The increase in aglycone content in EA treated sample could be attributed to the high pH applied in enzymatic hydrolysis; however, the remarkable increase in their aglycones under mild conditions could be attributed to the contaminated b-glucosidase present in these protease preparations rather than the temperature and pH applied during incubation. It was reported that b-glucosidase has superior activity for conversion of isoflavones glycosides to aglycones (Matsuura & Obata, 1993; Xie et al., 2003). b-Glucosidase from microorganisms such as bifidobacteria and lactic acid bacteria have also been studied in hydrolysing isoflavone glycosides into aglycones (Matsuyama, Setoguchi, Arai, & Kiyosawa,
1990; Tsangalis et al., 2003). Microbially derived protease preparations tend to contaminate more or less amount of b-glucosidase; the activity of b-glucosidase in PM, EN, Alcalase 2.4L and EA were determined to be 18.5, 0.09, 0.03, 0 U/g, respectively. The contaminated b-glucosidase in these bacterial proteases contributed greatly to the isoflavones conversion observed during hydrolysis with the exception of EA. Since the optimal pH for b-glucosidase is acidic pH, it is in a good alignment with the result that PM shows the highest b-glucosidase activity among the enzymes tested. However, the increase in aglycones could not be attributed totally to the conversion of their manyolglucosidic and acetylglucosidic conjugates, since the increase magnitude in daidzein (from 546 lg total aglycones in soy flour to 4207.1 lg total aglycones in hydrolysate and residue) was far greater than the total manyolglucosidic (from 7030.0 in soybean flour to 4483.4 lg total aglycones in hydrolysate and residue) and acetylglucosidic conjugates (from 182.1 to 88.5 lg total aglycones) in PM treated samples; furthermore, a significant higher conversion percentage was observed from glucosidic isoflavones to aglycones than malonylglucosidic and acetylglucosidic conjugates to aglycones. It was calculated that 81.1% of daidzin was converted into daidzein, 78.8% of genistin into genistein, compared with 36.2% of 600 -O-malonygenistin into daidzein, 51.4% of 600 -O-acetyldaidzin into daidzein, and 37.1% of 600 -O-malonygenistin into genistein. These results suggested that the activity of b-glucosidase in PM might have a different enzyme selectivity from the reported b-glucosidase (P. multicolour) (Xie et al., 2003). 3.2. Standard isoflavone incubation In order to address the unique enzyme specificity of b-glucosidase in the Protease M, we tested its activity on authenticated isoflavone standards; purified b-glucosidase from almond was also used for comparison. Fig. 1 shows the chromatograms of these standard isoflavones under different incubation intervals using Protease M or purified almond b-glucosidase. Daidzin was completely converted into aglycone in the first 15 min in both enzymes; genistin was not detected in PM treated samples whereas there was trace amount of genistin left in the b-glucosidase treated samples. Matsuura and Obata (1993) reported that b-glucosidase from soybean had a conversion percentage of 16.9% from genistin to genistein and 14.3% for daidzin to daidzein in soymilk, which was much less than the results we reported. The content of malonyldaidzin decreased gradually with the incubation time; percentage conversion reached 74.5 and 61.1% for PM and b-glucosidase treated samples at 24 h, respectively (Fig. 2). It appeared that
331
J. Wu, A.D. Muir / Food Chemistry 118 (2010) 328–332 A. Gin
B 0hr
M.Gn
A. Gin
A
Daidzin
0hr Daidzin
Genistin
M. Din
A. Din
M.Gin
Genistin
M. Din
A. Din
Genistein
0.25hr
Daidzein
Genistein
0.25hr Daidzein
1hr 1hr
3hr 3hr
6hr
6hr
24h
24h
0
10
20
30
Min
40
50
60
70
0
10
20
30
40
50
60
70
Min
Fig. 1. Chromatograms of standard isoflavones under different incubation intervals with Protease M ‘‘Amano” (A) and purified b-glucosidase (B).
120 PM A-Gin
conversion (%)
100 PM A-Din
80
PM M-Gin
beta A-Din
60 beta M-Gin
40
PM M-Din
beta M-Din
20 beta A-Gin
0 0
4
8
12
16
20
24
Time (hr) Fig. 2. The conversion percentage of standard isoflavone under various incubation intervals with PM and almond b-glucosidase at the same activity of b-glucosidase. Abbreviations: PM, Protease M ‘‘Amano”; b, almond purified b-glucosidase; A-Gin, acetylgenistin; A-Din, acetyldaidzin; M-Din, malonyldaidzin; M-Gin, malonylgenistin.
of acetyldaidzin left in b-glucosidase treated sample. It seemed that b-glucosidase in PM had a higher efficiency than that of almond-derived b-glucosidase and therefore, the conversion percentage from acetylgenistin and malonylgenistin into genistein with PM was higher than that of almond-derived b-glucosidase. The difference between their specificity and efficiency for the conversion of glucosides to aglycones had a great impact on the content of aglycones. Consequently, the contents of daidzein and genistein in PM treated samples were significantly higher than those of almond b-glucosidase treated samples (Fig. 1). The content of daidzein in PM treated sample was 237.1 lg daidzein/mL compared with 156.2 lg daidzein/mL in almond b-glucosidase treated sample at 0.25 h and this difference remained significant within 6 h but not at 24 h incubation. It appeared that the conversion from glucosides to aglycones with PM was higher and faster than that of the almond b-glucosidase (Fig. 2); the greatest conversion happened within 3 h, reached a plateau after 6 h incubation. The content of genistein in PM treated sample increased rapidly to 659.6 lg aglycone/mL at 15 min, compared with 161.4 lg aglycone/mL at 15 min and 356.8 lg glycone/mL at 24 h incubation in almond b-glucosidase. 4. Conclusions
b-glucosidase from both PM and almond could not convert all malonyldaidzin into daidzein under the condition tested. Generally speaking, acetyldaidzin showed a higher conversion percentage than that of malonyldaidzin (Fig. 2). At 24 h, there was no acetyldaidzin left in PM treated sample whereas there was 36%
This study showed that protease hydrolysed soybean products contained significant amount of isoflavones, and the content and composition of isoflavones were greatly affected by the applied reaction conditions and presence of b-glucosidase activity in the
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commercial protease enzyme. The microbial conversion of isoflavone forms has been reported in soybean products during fermentation efforts to convert conjugated isoflavones into their aglycone forms using bacterial b-glucosidase to develop aglycone-enriched products have been reported (Otieno & Shah, 2007, 2008; Xie et al. 2003). Our results indicated that the b-glucosidase present in commercial protease enzyme preparations has a unique specificity for the conversion of isoflavone chemicals, which was different from almond-derived b-glucosidase. Previous studies also showed that strain-dependent b-glucosidase activity in soymilk (Otieno & Shah 2007, 2008). In addition to b-glucosidase activity, enzyme specificity should be taken into consideration in bioconversion of isoflavones. PM hydrolysis of soybean flours not only resulted in potent bioactive peptides, but also produced a residue with an exceptional high content of aglycones. Our results indicated that protease hydrolysed residues contained more than 10 times higher concentration of aglycones than that of soy flour, thus provided another alternative approach to enrich aglycone isoflavones in soy-containing products. Our further study aims to develop an integrated technology to produce bioactive peptides and enriched aglycone isoflavones from soybean meal. Further research on the purification of the b-glucosidase from the PM preparation is needed to characterise its properties and compare with other sources of b-glucosidase.
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