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Effect of Monascus aged vinegar on isoflavone conversion in soy germ by soaking treatment
Food Chemistry 186 (2015) 256–264
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Effect of Monascus aged vinegar on isoflavone conversion in soy germ by soaking treatment Ji-Cheng Chen a,c, Jie Wang a, Zhi-Jiang Wang a, Yan-Jie Li a, Jie Pang d, He-Tong Lin a,⇑, Shou-Wei Yin b,⇑ a
College of Food Science, Fujian Agriculture and Forest University, Fuzhou, Fujian 350002, China Research and Development Center of Food Proteins, Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, China c Department of Food Science, Cornell University, Ithaca, NY 14853, USA d Department of Physics, Jefferson Physical Lab, Harvard University, Cambridge, MA 02138, USA b
a r t i c l e
i n f o
Article history: Received 13 January 2015 Received in revised form 16 February 2015 Accepted 19 February 2015 Available online 26 February 2015 Keywords: Conversion Isoflavone Soaking Soy germ Monascus vinegar Aglycone
a b s t r a c t Soy germ rich in isoflavones has attracted much attention for health-promoting characteristics. An effective approach via Monascus aged vinegar soaking was adopted to enhance the aglycone amount. The profiles and interconversion of soy germ isoflavones via Monascus aged vinegar soaking were investigated, and the distribution in vinegars were also explored. The aglycones were dramatically increased by 40.76 times. Concomitantly, b-glycosides and malonylglycosides were significantly decreased. The proportion of aglycones presented a sharp increase with the endogenous b-glucosidase activity at the initial 4 h incubation. There appeared to be correlations between b-glucosidase activity and the hydrolysis of conjugated isoflavones. The results demonstrated that the reactions of decarboxylation, de-esterification and de-glycosylation were involved in the Monascus aged vinegar soaking, supporting synergistic effects of enzymolysis by endogenous b-glucosidase from soy germ and acid hydrolysis of vinegars. Soaking by vinegar is a promising pathway for preparing aglycone-rich soy germ. Ó 2015 Published by Elsevier Ltd.
1. Introduction Soybean (Glycine max Merrill) was originally cultivated in China 5000 years ago. It has been widely consumed in Asian countries (e.g., China, India, Japan and Korea) for thousands of years. Nowadays, soy products are not only consumed by Asian populations, but also encouraged for western diets. This phenomenon is mainly due to their nutritional properties and the presence of health-promoting functional ingredients in soy products such as isoflavone, a well-known phytoestrogen (Phommalth, Jeong, Kim, Dhakal, & Hwang, 2008). Isoflavone has been reported to be 1– 5 mg/g in dry-soybean (Murphy et al., 1999). Isoflavone contents vary in various parts of soybeans, and its contents in the soy germ are about 6–10 times higher than that in the cotyledon (Murphy, Barua, & Hauck, 2002). Therefore, soy germ, the richest source of
Abbreviations: A-daidzin, 6-O-acetyldaidzin; A-glycitin, 6-O-acetylglycitin; Agenistin, 6-O-acetylgenistin; M-daidzin, 6-O-malonyldaidzin; M-glycitin, 6-Omalonylglycitin; M-genistin, 6-O-malonylgenistin; RP-HPLC, reversed-phase high performance liquid chromatography; TFA, trifluoroacetic acid; p-NPG, p-nitrophenyl-b-D-glucopyranoside; UV, ultraviolet; p-NP, para-nitrophenol. ⇑ Corresponding authors. E-mail addresses: [email protected] (H.-T. Lin), [email protected] (S.-W. Yin). http://dx.doi.org/10.1016/j.foodchem.2015.02.099 0308-8146/Ó 2015 Published by Elsevier Ltd.
soy isoflavones, can be utilized as a health promoting ingredient in food supplement markets (Schryver, 2002). Isoflavones are known for their biological activities including estrogenic, antifungal, antioxidant activities (Matsuura, Sasaki, & Murao, 1995). Soy isoflavones consist of 15 chemical forms, and are classified into two groups, the aglycones (daidzein, genistein, and glycitein) and b-glucoside conjugates. The succinyl-b-glucosides derivatives are only found in some soybean fermented products, such as natto (Bacillus subtilis). In unprocessed soybean or soybean germ, malonylglycoside contents were the highest, followed by b-glycosides, aglycones, and acetylglycosides (Hsieh, Kao, & Chen, 2005). Concentrations of acetylglycosides, b-glycosides, and aglycones tend to increase during the commercial processing (Charron, Allen, Johnson, Pantalone, & Sams, 2005; Coward, Smith, Kirk, & Barnes, 1998; Franke et al., 1999; Yu, Liu, Qiu, & Wang, 2007). Chemical forms of isoflavones can affect their stability during various processing conditions, as well as their bioavailability. The aglycones are structurally similar to the mammalian estrogen and, therefore, mimic the function of estradiol in the human body, whereas the b-glucosides have less estrogenic activity (Brouns, 2002; Izumi et al., 2000; Setchell, 1998). Additionally, the b-glucosides absorbed more slower and in less amounts than their aglycones by humans due to their hydrophilic
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nature and higher molecular weight (Izumi et al., 2000). In particular, isoflavone aglycones have been associated with the prevention and treatment of hormone-dependent disorders based on epidemiological (Messina, Persky, Setchell, & Barnes, 1994) and small-scale human clinical studies (Kurzer, 2000). Therefore, it would greatly increase isoflavone bioactivities and bioavailability if b-glucosides forms were converted to aglycone forms. Therefore, the enrichment of isoflavone aglycones in soybean foods before consumption attracts growing attention. Several methods for the transformation of isoflavone glycosides to isoflavone aglycones, including water soaking, acid hydrolysis, fermentation, heat, and enzymatic transformation, have been attempted (Chen, Lo, Su, Chou, & Cheng, 2012; Chien, Hsieh, Kao, & Chen, 2005; Hubert, Berger, Nepveu, Paul, & Daydé, 2008; Liu, Zhang, Wu, Wang, & Wang, 2013; Toda, Sakamoto, Takayanagi, & Yokotsuka, 2000). The acidic hydrolysis method has advantages, low cost, simple technology and high hydrolytic percentage. But it also produces side reactions which causes high purification cost, and generates environmental pollution (Lee, Seo, & Oh, 2013). Fortunately, vinegar soaking approach would get out of this trouble. Vinegar soaked soybeans were authorized as functional food status by China Food and Drug Administration (CFDA) (License WEISHIJIANZI (1997) No. 823) mainly due to the presence of soy isoflavones, particularly aglycone forms. However, little information is available on the changes of isoflavone compositions and the interconversion of soy isoflavones of soy germ during soaking in vinegars. Yongchun Monascus aged vinegar, one of the four famous China-style fermented vinegars, was brewed from sticky rice in the traditional way and could be traced back to more than 2000 years ago (the early years of the North Song Dynasty). Various b-glycosidases may be produced during the fermentation process via microorganisms such as acetic acid bacteria (Acetobacter sp.), lactic acid bacteria (Lactobacillus plantarum), yeast (Dekkera sp.), Monascus purpureus. Additional, rare report took full advantage of that soy germ was rich in endogenous b-glucosidase. Therefore, the combination of endogenous b-glucosidase and acid hydrolysis may facilitate the transformation of isoflavone forms from glucosides to the aglycones. Thus, the objectives of this study was to understand the conversion of isoflavone forms of soy germ during the soaking by Monascus aged vinegar, investigate the possible mechanism of the transformation and provide a basis for developing functional foods of soy germ. 2. Materials and methods 2.1. Chemicals and reagents Acetonitrile, methanol, and acetic acid, HPLC grade, were purchased from Fisher Chemical Co. Ltd. (USA). The 12 standards (daidzin, glycitin, genistin, acetyldaidzin, acetylglycitin, acetylgenistin, malonyldaidzin, malonylglycitin, malonylgenistin, daidzein, glycitein and genistein) were purchased from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). HPLC-grade water was prepared with a Milli-Q Water Purification System (Millipore Corporation, Billerica, MA, USA). p-Nitrophenyl-b-D-glucopyranoside (p-NPG) and para-nitrophenol (p-NP) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). All chemicals were analytical grade.
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Heilongjiang province of China). The soybean germ was selected again to pick out the cotyledon, seed coat and broken germ, then dried at 40 °C under vacuum condition for 6 h. It was stored at 25 °C in sealed and dry condition for use. Yongchun Monascus aged vinegar without sterilization was kindly provided by Fujian Yongchun Shundetang Food Co., Ltd. (Fujian province, China). 2.3. Sample treatment 2.3.1. Different treatments on soy germ Soybean germs were soaked by Yongchun Monascus aged vinegar or Recovered Yongchun Monascus aged vinegar at 4 times their weight and incubated for 72 h at 25 °C. The soybean germs and the resultant soaking liquor (Yongchun Monascus aged vinegar) were then lyophilized, ground to uniformity by a coffee mill, and prepared as samples for isoflavone analysis by high performance liquid chromatography (HPLC). Soaking in water and cooking were as the controls. Soaking in water: soybean germ was soaked by adding distilled water (natural pH) at 4 times their weight and incubated at 25 °C for 72 h. Recovered vinegar soaking: soybean germ was soaked by the recovered Yongchun Monascus aged vinegar (the vinegar has used for soaking soy germ once, and then the resolution was recovered from the last time soking) at 4 times their weight and incubated at 25 °C for 72 h. Cooking: soybean germ was soaked by distilled water and cooked at 121 °C for 30 min. Finally, the soybean germs and soaking liquor were lyophilized to yield soybean germ powder for isoflavone analysis by HPLC. 2.3.2. The ratio of solid to liquid in vinegar soaking Soybean germ was soaked in different volume of Yongchun Monascus aged vinegar (vinegar:soy germ = 1:2, 1:4, 1:6, 1:8, 1:12, respectively, w/w) and incubated at 25 °C for 24 h. 2.3.3. Effects of soaking time on the soy germ Soybean germ was soaked by adding Yongchun Monascus aged vinegar at 4 times (w/w) their weight and incubated at 25 °C for different time (0, 4, 8, 12, 16, 20 and 24 h, respectively). After treatment, the soybean germ was dried, ground to uniformity by a coffee mill, and prepared as samples for isoflavones analysis by HPLC. 2.4. Isoflavone extraction method The extraction method of soy germ isoflavones was performed according to the procedure described by Rostagno, Palma, and Barroso (2003), with some modifications. In brief, 1 g soybean germ sample was added in 10 mL 80% aqueous methanol and incubated in ultrasonic bath (B.H.A. instruments Co., Ltd., Germany) for 35 min at 20 °C. The ultrasound power density and frequency were set to 150 W and 35 kHz, respectively. The extraction was centrifuged at 8000g for 15 min using SIGMA 1–15 K instrument (BHA instruments Co., Ltd., Germany) and then the supernatant was filtered by 0.45 lm filters for HPLC analysis. Each experiment was repeated at least three times. 2.5. High performance liquid chromatography (HPLC) The HPLC equipment consisted of an Agilent 1200 series Rapid Resolution LC System (Agilent Technologies, Palo Alto, CA) equipped with an automatic injector and PhotoDiode Array detector (DAD) detector. Data were collected on Agilent Chemstation software.
2.2. Sample collection Soybean germ (from a soybean variety of Zhongdou-27) was purchased from Jiusan Oil and Fat Chemical Factory in
2.5.1. HPLC determination conditions for isoflavones The HPLC conditions were adopted to the method developed by Rostagno, Villares, Guillamón, García-Lafuente, and Martinez
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(2009), with some modification in the chromatographic conditions to achieve good isoflavone separation. Separations were performed in an ODS C18 column (4.6 mm 150 mm, 3.9 lm) fitted with a guard column. The mobile phase was composed of trifluoroacetic acid (TFA) 0.1% in water (A) and TFA 0.1% in acetonitrile (B). The elution was performed with a linear gradient from 100% to 58% (v/v) of eluent A in 24 min, holding these conditions for 10 min and returning to 100% in 10 min. The chromatographic analysis was performed at 35 °C, the flow rate was 1.0 mL/min and detection wavelength was 254 nm. Injections of 5lL were effected with the automatic injector. Twelve standards of soy isoflavones were dissolved in absolute HPLC-grade methanol and the concentration of each standard was from 15 to 50 lg/mL. 2.6. Determination of b-glucosidase activity One hundred milligrams solid samples (soybean germ), or 500 lL liquid samples (vinegar) were taken, and 1.5 mL of 0.05 M citrate buffer (pH 4.5) containing 0.1 M NaCl was maintained for 1 h at room temperature. The samples were centrifuged (12,000g for 5 min), and the supernatant was used as enzyme source for activity analysis. The b-glucosidase activity was assayed by a modified procedure, based on the method of Matsuura and Obata (1993). Two milliliters of the substrate 1 mM p-nitrophenyl-b-D-glucopyranoside (p-NPG) in phosphate-citrate buffer (0.1 M pH 5.0) were transferred to a test tube and kept in a water bath at 30 °C for 10 min; then 0.5 mL of the supernatant was added and the tube left in the water bath at 30 °C for 30 min. After incubation at 37 °C for 30 min, the reaction was stopped with 2.5 mL of 0.05 M sodium carbonate. The activity of b-glucosidase was estimated by reading the absorbance of the liberated p-nitrophenol at 405 nm. The blank solution was composed of 2.5 mL of 0.05 M sodium carbonate solution, 2.0 ml of substrate solution and 0.5 mL of 0.05 M citrate buffer (pH 4.5) containing 0.1 M NaCl. One unit (U) of b-glucosidase activity was defined as the amount of enzyme required for the hydrolysis of 1 lmol of substrate (pNPG)/min, under the assay conditions. 2.7. Statistical analysis Statistical analysis was performed using one-way analysis of variance (ANOVA). All results were expressed as means ± SD, and significant differences were determined by Tukey HSD test. p < 0.05 is considered to be statistically significant. 3. Results 3.1. Validation of the HPLC method for analysis of the isoflavones We adopted a simplified gradient reversed phase HPLC method for the separation and determination of 12 isoflavones extracted from soy germ. Fig. 1 showed the typical HPLC chromatograms of the 12 isoflavones standard (A), and 12 isoflavones from untreated soy germ (B) and vinegar soaked soy germ (C). Isoflavones all absorb UV light, and UV detection may offer sufficient selectivity and sensitivity for the determination of isoflavones (Yuan, Liu, Peng, Wang, & Liu, 2009). The identifications of isoflavones were achieved by comparing their retention time and spectra against the known standards. As expected, the separation of 12 isoflavones standard was achieved in the HPLC chromatograms (Fig. 1A), and the calibration curves of the peak area (A) against the concentration (C) for these isoflavone standards at 254 nm gave good linear responses over a wide range (5–50 mg/L) of concentrations (Table S1), indicating that this HPLC method was sensitive for qualitative and quantitative determination of these isoflavones.
The isoflavones of extracts from untreated or soaked soy germ were also well separated via the HPLC procedure (Fig. 1B and C), indicating that the proposed method can be effectively applied for separation and determination of 12 forms of isoflavones in soy germ.
3.2. The changes of isoflavone forms in soy germ soaked by vinegars The incubation of soy germ with Yongchun Monascus aged vinegar led to considerable change in the isoflavone forms. Comparing chromatogram B with chromatogram C (Fig. 1B and C), it is obvious that the height of three peaks (peak 9, peak 10 and peak 12) in the soaked soy germ sample, namely, the aglycones (daidzein, glycitein and genistein, respectively), were significantly increased, whereas the corresponding malonyglycosides peaks (peak 4, peak 5 and peak 8) decreased accordingly.
3.3. The conversion of isoflavone forms by different treatments Table 1 presents the isoflavone profiles of soy germ after Monascus aged vinegar soaking, water soaking and cooking as the control. In the raw soy germs, the isoflavones consisted of 63.34% malonylglycosides, 31.79 % b-glycosides, 3.69% acetylglycosides and 1.18% aglycones. In general, isoflavones are not destroyed by soaking in water, acid hydrolytic, fermentation and heat, but rather are subjected to the interconversion between the different forms. The changes of isoflavone profiles in soy germ were investigated after Monascus aged vinegar soaking for 72 h at 25 °C, and the related results were listed in Table 1. The data indicated that malonylglycosides in soy germ could readily be converted to their respective acetylglycosides and nonconjugated b-glycosides through the decarboxylation and deesterification, respectively. Concomitantly, b-glycosides forms were transformed to aglycone forms by endogenous b-glucosidase (Table 1). The deesterification reaction of malonyl glycosides was faster than the decarboxylation reaction under the environment, that is, the conversion from malonyl glycosides to glycosides was faster than to acetylglycosides. The Monascus aged vinegar soaking resulted in aglycone-rich soy germ, and aglycones were further increased from 36.8% to 51.34% by the soaking with recovered Monascus aged vinegar (Table 1). Soaking in water and cooking are important procedure in the soybean food processing. The changes in isoflavone profiles during the processes were compared with the counterparts in the Monascus aged vinegar soaking. In the case of water soaking, the proportion of free isoflavones increased from 1.18% to 17.16%, whereas that of isoflavone glucosides decreased depending on the type of b-glucosides. The proportion of malonylated isoflavone glucosides decreased slightly from 63.34% to 52.38%, while acetylated isoflavone glucosides increased from 3.69% to 7.35%. Cooking approach decreased the proportion of malonylglycosides from 63.34% to 3% by the transformation to acetylglycosides and nonconjugated b-glycosides via deesterification and decarboxylation route, and the aglycone was increased to 48.6% after 30 min cooking at 121 °C (Yuan et al., 2009). The amount of aglycones after soaked with Monascus aged vinegar fell in between water soaking and cooking (Table 1). When the recovered Monascus aged vinegar was used for soaking, aglycone forms were further increased. The concentration of aglycones exceeded that of the cooked one (Table 1). The treatment by the recovered Monascus aged vinegar was the best advantageous to aglycone formation, and the highest contain of the aglycones reached 1134.74 ± 39.72 mg/100 g. The cooking control group is one of the best ways for the degradation of the malonylglucosides, de-carboxylation.
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Fig. 1. The HPLC chromatogram of the 12 isoflavone isomers. The HPLC chromatogram of the isoflavone standards (A), the chromatogram of untreated sample of soybean germs (B), the chromatogram of soaking sample of soybean germs soaked in the recovered vinegar (C). The peaks from 1 to 12 mean: 1-daidzin, 2-glycitin, 3-genistin, 4malonydaidzin, 5-malonylglycitin, 6-acetyldaidzin, 7-acetylglycitin, 8-malonylgenistin, 9-daidzein, 10-glycitein, 11-acetylgenistin, 12-genistein, respectively.
3.4. Distribution of isoflavones in soybean germs The isoflavone profiles of soy germ were analyzed by the developed HPLC method. In the raw soy germ, isoflavones accumulated in the order of malonylglycosides, b-glycosides, acetylglycosides and aglycones, among which malonylglycosides were the most abundant form and had the highest concentrations (1498.32 mg/ 100 g), being followed by b-glycosides (751.89 mg/100 g). Acetylglycosides and aglycones forms were found in very small concentrations, 87.38 and 27.84 mg/100 g, respectively. Malonylglycosides constituted 63.34% of the total isoflavones in untreated soy germs. The result is in agreement with the previous reports that malonylglycosides accounted for 57–79% of total isoflavones (Kim et al., 2007; Yamabe, Kobayashi-Hattori, Kaneko, Endo, & Takita, 2007). The b-glycosides accounted for 31.79% of
total isoflavones in soy germs, which are in agreement with the previous reports that b-glycosides accounted for 26.7–40.6% (Kim et al., 2007). Xu et al. reported that the highest proportion at more than 76% of the total isoflavones was malonylglycosides, followed by b-glycosides at 19%, whereas acetylglycosides and aglycones occurred in only very small proportions (Xu & Chang, 2008). 3.5. Effects of solid/liquid ratio on modification of isoflavones Limited information is available on the changes of the isoflavone profiles and the interconversion of soy isoflavones in soy germ via soaking in vinegar. In this work, Yongchun Monascus aged vinegar could increase free isoflavones (aglycone forms) so as to enhance the nutritional values of soy germs. Effects of the solidto-liquid ratios on the interconversions of soy isoflavone forms
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Table 1 The changes in isoflavone compositions of soy germ during different treatments (mg/100 g). Three independent experiments (n = 3) and each performed in triplicate, mean ± SD. Isoflavones
Control
Water soaking
Vinegar soaking
Recovered vinegar soaking
Cooking
Daidzin Glycitin Genistin b-Glycosides %
391.68 ± 14.71a 247.04 ± 8.65a 113.17 ± 3.96a 751.89 ± 27.32a 31.79
263.68 ± 10.23b 166.40 ± 5.92c 79.36 ± 2.98 cd 509.44 ± 18.83c 23.11
262.74 ± 9.90b 159.49 ± 5.58c 92.12 ± 3.22b 514.35 ± 19.00c 25.63
184.75 ± 6.67d 149.94 ± 5.25c 77.40 ± 2.91d 412.09 ± 15.42d 18.64
249.86 ± 9.74bc 212.35 ± 8.43b 107.39 ± 4.76a 569.60 ± 19.94b 28.62
A-daidzin A-glycitin A-genistin Acetylglycosides %
45.39 ± 1.69b 31.94 ± 1.22c 10.05 ± 0.65d 87.38 ± 3.36d 3.69
27.90 ± 0.99d 24.70 ± 0.89d 109.44 ± 3.83c 162.05 ± 5.97c 7.35
54.14 ± 1.90a 44.84 ± 1.97b 177.28 ± 6.20b 276.27 ± 9.67b 13.77
26.14 ± 0.98d 44.20 ± 1.65b 296.52 ± 11.38a 366.86 ± 14.84a 16.60
56.32 ± 1.97a 59.90 ± 2.20a 277.38 ± 9.81a 393.60 ± 14.78a 19.78
M-daidzin M-glycitin M-genistin Malonylglycosides %
859.52 ± 35.38a 423.12 ± 17.92a 215.68 ± 9.63a 1498.32 ± 59.93a 63.34
647.68 ± 26.91b 337.92 ± 14.52b 168.96 ± 6.96b 1154.56 ± 47.18b 52.38
238.85 ± 9.55c 157.53 ± 6.80c 81.07 ± 3.64c 477.45 ± 19.10c 23.79
119.18 ± 4.97d 120.09 ± 4.89d 57.48 ± 2.70d 296.75 ± 12.87d 13.43
29.06 ± 1.76e 16.26 ± 0.95e 14.46 ± 0.79e 59.78 ± 2.89e 3.00
Daidzein Glycitein Genistein Aglycones %
16.64 ± 0.68f 7.10 ± 0.35d 4.10 ± 0.24f 27.84 ± 1.27e 1.18
243.20 ± 9.51e 78.00 ± 3.47b 56.96 ± 2.40e 378.16 ± 14.24d 17.16
545.02 ± 20.08d 83.87 ± 3.39b 109.57 ± 3.93d 738.46 ± 26.85c 36.80
848.71 ± 29.70a 96.12 ± 3.56a 189.91 ± 6.75a 1134.74 ± 39.72a 51.34
785.92 ± 27.51b 17.02 ± 0.60c 164.40 ± 5.75b 967.34 ± 33.86b 48.60
Total isoflavones %
2365.42 ± 83.79a 100.00
2204.21 ± 78.15ab 100.00
2006.52 ± 71.23bc 100.00
2210.44 ± 78.37ab 100.00
1990.32 ± 70.66c 100.00
Data with no letters in common in the same line are significantly different (p < 0.05). The percentages were the mean value, which based the proportion of aglycones, b-glycosides, acetylglycosides and malonylglycosides in different treatments, respectively.
were evaluated to optimize the vinegar soaking process. The soybean germ and soaking liquor were lyophilized to yield soybean germ powder for isoflavone analysis by developed HPLC. The isoflavone profiles of soy germs after the vinegar soaking at the selected solid/liquid ratios are shown in Fig. 2. Aglycone forms and b-glucoside conjugates showed a nearly opposite trend upon increasing soy germ-to-vinegar ratios from control (raw soy germ) to 1:12 (w/w). The amount of malonylglucosides decreased gradually from control, to germ-to-vinegar ratios of 1:2 and 1:4 due to the deesterification and decarboxylation reactions, then arrived at a plateaus upon further increase in soy germ-to-vinegar ratios. The b-glycosides profiles presented a similar trend upon varying the solid/liquid ratios (Fig. 2). Concomitantly, aglycones and acetylglycosides presented nearly opposite trends. In brief, the amount of aglycones increased sharply from 1.18% (control) to 30.0% through incubating soy germs in Monascus aged vinegar at the ratio of 1:4, and aglycones proportions remained unaffected for further increase of the solid/liquid ratios from 1:4 to 1:12. The present data showed that the high conversion efficiency of b-glucoside conjugates toward aglycones forms were achieved at the germ-to-vinegar ratios of 1:4 and higher, and there were no significant increase (p > 0.05) in aglycones proportion upon further heightening the germ-to-vinegar ratios. Therefore, the ratio of germ/vinegar of 1:4 was selected in the following experiments. 3.6. Effects of incubating time of soaking vinegar The conversion kinetics of isoflavone forms were investigated for 24 h incubation in Monascus aged vinegar. Soy germs and Monascus aged vinegar were taken out at 4 h intervals, and soybean germs were lyophilized to yield soybean germ powder. The isoflavone profiles in soybean germ powder and Monascus aged vinegar were analyzed by developed HPLC. Fig. 3 shows on the isoflavone forms in soy germ (left) and Monascus aged vinegar (right) as a function of incubation time in Monascus aged vinegar. In soy germs, total isoflavones consisted of 55.52% daidzein and its derivatives, 29.98% glycitein and its derivatives, and 14.50% genistein and it derivatives (Fig. 3).
Fig. 2. Effects of Ratio of solid to liquid in vinegar soaking on the soy germ isoflavone forms. Using the untreated soybean as raw materials control. Three independent experiments (n = 3) and each performed in triplicate, mean ± SD.
The conversion kinetics of various isoflavone forms varied depending on the type of isoflavone forms. On the whole, the proportion of free isoflavones increased gradually upon the increase of incubation times, whereas that of isoflavone glucosides, e.g., malonylglucosides and b-glucoside decreased accordingly (Fig. 3A–C). As expected, some isoflavone forms were transferred into Monascus aged vinegar in the isoflavone types and incubation time dependent manner (Fig. 3a–c). Effects of incubation times in Monascus aged vinegar on the isoflavone forms in soy germs were shown in the Fig. 3A–C. In the case of daidzein and its derivatives, the free isoflavone (daidzein) increase gradually from 16.64 mg/100 g to 507.10 mg/100 g, whereas malonyl derivatives and b-glucosides decreased gradually from 859.52 (M-daidzin) to 247.60 mg/100 g, and 391.68 (daidzin) to 208.00 mg/100 g, respectively. In addition, the amount of acetyl conjugates decreased slightly. Obviously, the changes of genistein and its derivatives presented a similar trend, with the exception for acetyl conjugates (A-genistin) which increased significantly upon the Monascus aged vinegar soaking. In the case of glycitin and it derivatives, malonyl derivative (M-glycitin) and b-glucoside
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Fig. 3. (I) Effects of incubation times in Monascus aged vinegar on the isoflavone forms in soy germ (left) and Monascus aged vinegar (right). Panel A and a: daidzin and its derivatives. Panel B and b: genistin and its deravitives. Panel C and c: glycitin and its deravitives. (II) Effects of incubation times in Monascus aged vinegar on the isoflavone forms of soy germ plus Monascus aged vinegar. The amount of isoflavone forms in soy germs and Monascus aged vinegar analyzed by the developed HPLC, and the sum of various isoflavone forms in soy germs and Monascus aged vinegar were reported. Three independent experiments (n = 3) and each performed in triplicate, mean ± SD.
(glycitin) presented a sharp decrease for the initial 4 h, then reached a plateau, and the free isoflavone (glycitein) and acetyl derivative remain nearly unaffected in the soaking duration. Effects of incubation times in Monascus aged vinegar on the isoflavone forms in Monascus aged vinegar were shown in the Fig. 3a– c. The isoflavone profiles in the Monascus aged vinegar were different from that in soaked soy germs. Some selected isoflavone forms diffused toward the Monascus aged vinegar from soy germs. In the initial soaking stage (4 h), the sharp increases in b-glucoside conjugates were observed, while the aglycones were nearly unaffected upon incubating soy germ in the Monascus aged vinegar. This
phenomenon may be associated with the hydrophobic character of the isoflavone forms, and the aglycones were more hydrophobic when compared with b-glucoside conjugates, its solubility was restricted in the aqueous Monascus aged vinegar. The distribution of isoflavones in Monascus aged vinegar after soaking for 24 h, including 327.6 mg/100 g malonyl derivatives, 35.96 mg/100 g acetyl conjugates, 120.8 mg/100 g b-glucoside conjugates and 15.69 mg/100 g free isoflavones (aglycone forms). The proportions of various isoflavone forms were 65.51% malonyl derivatives, 7.19% acetyl conjugates, 24.16 b-glucoside, and 3.14% aglycone forms. The proportion of isoflavones forms was similar
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Fig. 3 (continued)
with that in the raw soy germs, and the total isoflavones in Monascus aged vinegar accounted for 21.41% of the total isoflavones in raw soy germs. The aforementioned total isoflavones constituted 55.52% daidzein and its derivatives, and the change in total isoflavone forms of soy germs plus Monascus aged vinegar during the incubation in the vinegar were similar with their main constituents (daidzein and its derivatives) (Fig. 3). 3.7. The role of endogenous b-glucosidase on isoflavone conversion Since the increase in free isoflavones generated during soaking in water or Monascus aged vinegar and the concomitant decrease in isoflavone glucosides during soaking can be restricted by the addition of the inhibitor of b-glucosidase, glucono-d-lacton, the changes are believed attributable to the action of endogenic b-glucosidase contained in soybeans (Matsuura, Obata, & Fukushima, 1989). Therefore, the activities of b-glucosidase during the soaking process were assayed herein using p-NPG as the substrate (Fig. 4). Endogenic b-glucosidase activity in both soaking liquors (water and Monascus vinegar) increased for an initial 4 h incubation, further increase in incubation time decreased the endogenous bglucosidase activity, especially for water (Fig. 4). This results was in agreement with the interconversion profiles of isoflavone forms during the soaking where the proportion of free isoflavone forms (aglycones) presented a sharp increase at an initial 4 h incubation, and the amount of b-glucoside conjugates (e.g., malonyl derivatives, b-glucoside showed concomitantly a sharp decrease (Fig. 3A–C)). In addition, the endogenous b-glucosidase activity in Monascus aged vinegar over 12 h soaking was higher than the counterparts in water soaking (Fig. 4). The phenomenon accounted for the differences in isoflavone forms of soy germs in both soaking processes. The Monascus aged vinegar was utilized to soak soy germ for 72 h at 25 °C, resulting in the increase of aglycones from 1.18% to 36.80%. In contrast, the water soaking at the same condition increased the proportion of the aglycones in soy germs to 17.16% (Table 1). 4. Discussion The bioavailability of isoflavones depended on their chemical forms (aglycones or glucosides) which influenced the solubility in the intestinal lumen and the absorption as well as on the metabolism by intestinal and microbial enzymes (Vitale, Piazza, Melilli, Drago, & Salomone, 2013). The enrichment of aglycone forms is a potential and promising strategy to enhance the bioavailability
Fig. 4. The effects of the incubation time on endogeneic b-glucosidase activities in the soaking liquids. Three independent experiments (n = 3) and each performed in triplicate, mean ± SD.
and physiological functions of soybean germs products. Several processing techniques were adopted to convert isoflavone glycosides to aglycones (Chen et al., 2012; Chien et al., 2005; Hubert et al., 2008; Liu et al., 2013; Toda et al., 2000). The common processing factors are thermal degradation, enzyme and acid (alkaline) hydrolysis, which involve in modification of soy-derived foods in terms of the content and distribution of the three isoflavone derivatives, daidzein, genistein and glycitein, and their respective conjugates. The higher levels of aglycones detected when soaking at approximately 45 °C were probably due to the greater activity of the endogenic b-glucosidase, which produced the hydrolysis of the glucoside conjugates to form the corresponding aglycones (Aguiar, Baptista, Alencar, Haddad, & Eberlin, 2007). Aglycones content in fermented soy products reached up to 90% of the total isoflavones due to the activation of the enzyme b-glucosidase during fermentation (Wang et al., 2007). In general, malonylglycosides in soybeans were labile to heat process and could readily be converted to their respective more heat-stable acetylglycosides and nonconjugated b-glycosides through decarboxylation and deesterification. The cooking process resulted in the variations of isoflavone distributions: the degradations of the malonyl conjugates and b-glycosides, meanwhile increasements of the three aglycones (Wang & Murphy, 1996). The acidic hydrolysis could effectively hydrolyze b-glycosides into aglycones (Liu et al., 2013). In this work, we adopted a facile approach via Monascus aged vinegar soaking and achieved high conversion efficiency of b-glycosides conjugates to aglycones forms, and the proportion of aglycones forms were further increased from 1.18% (raw soy germs), 36.8% (vinegar soaking) and 51.34% (recovered vinegar soaking) (Table 1). Herein, a schematic illustration of the interconversion pathway of isoflavones forms in soy germs via vinegar soaking approach was proposed (Fig. 5). The conversion kinetics of isoflavone forms were investigated for 24 h incubation in Monascus aged vinegar. The conversion kinetics of various isoflavone forms varied depended on the type of isoflavone forms. On the whole, the proportion of free isoflavones increased gradually upon the increase in incubation time, whereas that of isoflavone glucosides, e.g., malonylglucosides and b-glucoside decreased accordingly (Fig. 3A–C). The isoflavone forms in soy germ (left) and Monascus aged vinegar (right) as a function of incubation time in Monascus aged vinegar, reflecting that some selected isoflavone forms, e.g., malonylglucosides migarated toward the Monascus aged vinegar from soy germs. The total isoflavones in Monascus aged vinegar accounted for
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Decarboxylation
De-esterification
Deglycosylation
Ο HO
Ο
Ο
O O
HO
Ο
H
H H
R2
OH
R3
-glucosides
Aglycones
Acetyl- -glucosides
Soaking Germination Fermentation
Malonyl- -glucosides
Ο
ΟΗ
-glucosidase or fermentation Hydrolysis (acid and alkaline) Heat Treatment
Soaking Germination Fermentation Heat Treatment
Fig. 5. The interconversion of the 12 isoflavones isomers in soybeans and soy products occurred during the different processing.
21.41% of the total isoflavones in raw soy germs, and its proportion was similar with raw soy germs. This phenomenon may be associated with the hydrophobic character of the isoflavone forms. The aglycones in soy germ soaked with recovered vinegar (Table 1) were dramatically increased by 40.76 times, which was from 27.84 ± 1.27 mg/100 g to 1134.74 ± 39.72 mg/100 g (p < 0.05), at the same time, malonylglycosides were significantly reduced from 1498.32 ± 59.93 mg/100 g to 296.75 ± 12.87 mg/ 100 g (p < 0.05) and b-glycosides were significantly decreased from 751.89 ± 27.32 mg/100 g to 412.09 ± 15.42 mg/100 g (p < 0.05). In fact, if operated independently, b-glycosidase was not effective in hydrolyzing the conjugated glycosides to their respective aglycones, even with increased levels of the enzyme and prolonged incubation (Ismail & Hayes, 2005). In this work, the proportion of malonylated isoflavone glucosides decreased slight from 63.34% to 52.38% upon water soaking. Interestingly, malonylglycosides in soy germ could readily be converted to their respective acetylglycosides and nonconjugated b-glycosides, and its proportion decreased from 63.34% to 23.79% via Monascus aged vinegar soaking (Table 1). Yongchun Monascus aged vinegar, is one of the four famous China-style fermented vinegars, and various enzymes including b-glycosidases may be produced during the fermentation process via microorganisms such as acetic acid bacteria, lactic acid bacteria, yeast, M. purpureus. The soaking approach via Monascus aged vinegar can also be considered as after-fermentation stages in which various enzyme may induce the conversion of conjugated b-glycosides to free isoflavones forms. In brief, it was a combination of enzyme (b-glucosidase) and acid hydrolysis during the soaking with Monascus aged vinegar. These results are in agreement with previous works reporting that the glycosides isoflavones converted into aglycones in a reaction catalyzed by endogenous b-glucosidase of soybean in soaking water, and fermentation of soybean caused the hydrolysis of isoflavone malonyl- and b-glucosides to form aglycones (Góes-Favoni, CarrãoPanizzi, & Beleia, 2010; Tsangalis, Ashton, McGill, & Shah, 2002). It was highly anticipated that further research should be focus on the more detailed mechanism regulated by the potential microbes in Monascus aged vinegar during the soaking processing.
degradation of 12 isoflavone forms were determined. The possible degradation mechanisms varied with the different treatments. It was obvious that the aglycones were dramatically increased meanwhile the glycosides significantly reduced via Monascus aged vinegar approach. The proportion of aglycones presented a sharp increase with the endogenous b-glucosidase activity at the initial 4 h incubation. There appeared to be correlations between b-glucosidase activity and the hydrolysis of conjugated isoflavones. The present data indicated that the decarboxylation, de-esterification and de-glycosylation reactions were involved the interconversion of the isoflavones forms of soy germs during the soaking by Monascus aged vinegar, reflecting the synergistic effects of endogenous b-glucosidase activity of soy germ, acid hydrolysis and formation of the microorganisms. In addition, a schematic illustration of the interconversion pathway of isoflavones forms of soy germs via vinegar soaking approach was proposed. This study opens a promising safe pathway for preparing aglycone-rich soy germ as functional food ingredients via vinegar soaking. Conflict of interest The authors declare no competing financial interest. Acknowledgments This work was supported by China Postdoctoral Science Foundation (Grant No. 2012M521265), Science and Technology Planning Project of Fuzhou City (Grant No. 2013-N-47), the National Natural Science Foundation of China (Grant No. 31201350), and the Program for New Century Excellent Talents in Fujian Province University of China (Grant No. NCETFJ200720). I would appreciate the advices on the revision of Prof. Ruihai Liu in Cornell University and Prof. Qihe Chen in Zhejiang University. I would like to express my gratitude to all those who gave me the possibility to complete this manuscript. Appendix A. Supplementary data
5. Conclusion The isoflavone profiles were significantly changed in soy germ soaked by Yongchun Monascus aged vinegar. The conversion and
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. 02.099.
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Effect of Monascus aged vinegar on isoflavone conversion in soy germ by soaking treatment Ji-Cheng Chen a,c, Jie Wang a, Zhi-Jiang Wang a, Yan-Jie Li a, Jie Pang d, He-Tong Lin a,⇑, Shou-Wei Yin b,⇑ a
College of Food Science, Fujian Agriculture and Forest University, Fuzhou, Fujian 350002, China Research and Development Center of Food Proteins, Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, China c Department of Food Science, Cornell University, Ithaca, NY 14853, USA d Department of Physics, Jefferson Physical Lab, Harvard University, Cambridge, MA 02138, USA b
a r t i c l e
i n f o
Article history: Received 13 January 2015 Received in revised form 16 February 2015 Accepted 19 February 2015 Available online 26 February 2015 Keywords: Conversion Isoflavone Soaking Soy germ Monascus vinegar Aglycone
a b s t r a c t Soy germ rich in isoflavones has attracted much attention for health-promoting characteristics. An effective approach via Monascus aged vinegar soaking was adopted to enhance the aglycone amount. The profiles and interconversion of soy germ isoflavones via Monascus aged vinegar soaking were investigated, and the distribution in vinegars were also explored. The aglycones were dramatically increased by 40.76 times. Concomitantly, b-glycosides and malonylglycosides were significantly decreased. The proportion of aglycones presented a sharp increase with the endogenous b-glucosidase activity at the initial 4 h incubation. There appeared to be correlations between b-glucosidase activity and the hydrolysis of conjugated isoflavones. The results demonstrated that the reactions of decarboxylation, de-esterification and de-glycosylation were involved in the Monascus aged vinegar soaking, supporting synergistic effects of enzymolysis by endogenous b-glucosidase from soy germ and acid hydrolysis of vinegars. Soaking by vinegar is a promising pathway for preparing aglycone-rich soy germ. Ó 2015 Published by Elsevier Ltd.
1. Introduction Soybean (Glycine max Merrill) was originally cultivated in China 5000 years ago. It has been widely consumed in Asian countries (e.g., China, India, Japan and Korea) for thousands of years. Nowadays, soy products are not only consumed by Asian populations, but also encouraged for western diets. This phenomenon is mainly due to their nutritional properties and the presence of health-promoting functional ingredients in soy products such as isoflavone, a well-known phytoestrogen (Phommalth, Jeong, Kim, Dhakal, & Hwang, 2008). Isoflavone has been reported to be 1– 5 mg/g in dry-soybean (Murphy et al., 1999). Isoflavone contents vary in various parts of soybeans, and its contents in the soy germ are about 6–10 times higher than that in the cotyledon (Murphy, Barua, & Hauck, 2002). Therefore, soy germ, the richest source of
Abbreviations: A-daidzin, 6-O-acetyldaidzin; A-glycitin, 6-O-acetylglycitin; Agenistin, 6-O-acetylgenistin; M-daidzin, 6-O-malonyldaidzin; M-glycitin, 6-Omalonylglycitin; M-genistin, 6-O-malonylgenistin; RP-HPLC, reversed-phase high performance liquid chromatography; TFA, trifluoroacetic acid; p-NPG, p-nitrophenyl-b-D-glucopyranoside; UV, ultraviolet; p-NP, para-nitrophenol. ⇑ Corresponding authors. E-mail addresses: [email protected] (H.-T. Lin), [email protected] (S.-W. Yin). http://dx.doi.org/10.1016/j.foodchem.2015.02.099 0308-8146/Ó 2015 Published by Elsevier Ltd.
soy isoflavones, can be utilized as a health promoting ingredient in food supplement markets (Schryver, 2002). Isoflavones are known for their biological activities including estrogenic, antifungal, antioxidant activities (Matsuura, Sasaki, & Murao, 1995). Soy isoflavones consist of 15 chemical forms, and are classified into two groups, the aglycones (daidzein, genistein, and glycitein) and b-glucoside conjugates. The succinyl-b-glucosides derivatives are only found in some soybean fermented products, such as natto (Bacillus subtilis). In unprocessed soybean or soybean germ, malonylglycoside contents were the highest, followed by b-glycosides, aglycones, and acetylglycosides (Hsieh, Kao, & Chen, 2005). Concentrations of acetylglycosides, b-glycosides, and aglycones tend to increase during the commercial processing (Charron, Allen, Johnson, Pantalone, & Sams, 2005; Coward, Smith, Kirk, & Barnes, 1998; Franke et al., 1999; Yu, Liu, Qiu, & Wang, 2007). Chemical forms of isoflavones can affect their stability during various processing conditions, as well as their bioavailability. The aglycones are structurally similar to the mammalian estrogen and, therefore, mimic the function of estradiol in the human body, whereas the b-glucosides have less estrogenic activity (Brouns, 2002; Izumi et al., 2000; Setchell, 1998). Additionally, the b-glucosides absorbed more slower and in less amounts than their aglycones by humans due to their hydrophilic
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nature and higher molecular weight (Izumi et al., 2000). In particular, isoflavone aglycones have been associated with the prevention and treatment of hormone-dependent disorders based on epidemiological (Messina, Persky, Setchell, & Barnes, 1994) and small-scale human clinical studies (Kurzer, 2000). Therefore, it would greatly increase isoflavone bioactivities and bioavailability if b-glucosides forms were converted to aglycone forms. Therefore, the enrichment of isoflavone aglycones in soybean foods before consumption attracts growing attention. Several methods for the transformation of isoflavone glycosides to isoflavone aglycones, including water soaking, acid hydrolysis, fermentation, heat, and enzymatic transformation, have been attempted (Chen, Lo, Su, Chou, & Cheng, 2012; Chien, Hsieh, Kao, & Chen, 2005; Hubert, Berger, Nepveu, Paul, & Daydé, 2008; Liu, Zhang, Wu, Wang, & Wang, 2013; Toda, Sakamoto, Takayanagi, & Yokotsuka, 2000). The acidic hydrolysis method has advantages, low cost, simple technology and high hydrolytic percentage. But it also produces side reactions which causes high purification cost, and generates environmental pollution (Lee, Seo, & Oh, 2013). Fortunately, vinegar soaking approach would get out of this trouble. Vinegar soaked soybeans were authorized as functional food status by China Food and Drug Administration (CFDA) (License WEISHIJIANZI (1997) No. 823) mainly due to the presence of soy isoflavones, particularly aglycone forms. However, little information is available on the changes of isoflavone compositions and the interconversion of soy isoflavones of soy germ during soaking in vinegars. Yongchun Monascus aged vinegar, one of the four famous China-style fermented vinegars, was brewed from sticky rice in the traditional way and could be traced back to more than 2000 years ago (the early years of the North Song Dynasty). Various b-glycosidases may be produced during the fermentation process via microorganisms such as acetic acid bacteria (Acetobacter sp.), lactic acid bacteria (Lactobacillus plantarum), yeast (Dekkera sp.), Monascus purpureus. Additional, rare report took full advantage of that soy germ was rich in endogenous b-glucosidase. Therefore, the combination of endogenous b-glucosidase and acid hydrolysis may facilitate the transformation of isoflavone forms from glucosides to the aglycones. Thus, the objectives of this study was to understand the conversion of isoflavone forms of soy germ during the soaking by Monascus aged vinegar, investigate the possible mechanism of the transformation and provide a basis for developing functional foods of soy germ. 2. Materials and methods 2.1. Chemicals and reagents Acetonitrile, methanol, and acetic acid, HPLC grade, were purchased from Fisher Chemical Co. Ltd. (USA). The 12 standards (daidzin, glycitin, genistin, acetyldaidzin, acetylglycitin, acetylgenistin, malonyldaidzin, malonylglycitin, malonylgenistin, daidzein, glycitein and genistein) were purchased from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). HPLC-grade water was prepared with a Milli-Q Water Purification System (Millipore Corporation, Billerica, MA, USA). p-Nitrophenyl-b-D-glucopyranoside (p-NPG) and para-nitrophenol (p-NP) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). All chemicals were analytical grade.
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Heilongjiang province of China). The soybean germ was selected again to pick out the cotyledon, seed coat and broken germ, then dried at 40 °C under vacuum condition for 6 h. It was stored at 25 °C in sealed and dry condition for use. Yongchun Monascus aged vinegar without sterilization was kindly provided by Fujian Yongchun Shundetang Food Co., Ltd. (Fujian province, China). 2.3. Sample treatment 2.3.1. Different treatments on soy germ Soybean germs were soaked by Yongchun Monascus aged vinegar or Recovered Yongchun Monascus aged vinegar at 4 times their weight and incubated for 72 h at 25 °C. The soybean germs and the resultant soaking liquor (Yongchun Monascus aged vinegar) were then lyophilized, ground to uniformity by a coffee mill, and prepared as samples for isoflavone analysis by high performance liquid chromatography (HPLC). Soaking in water and cooking were as the controls. Soaking in water: soybean germ was soaked by adding distilled water (natural pH) at 4 times their weight and incubated at 25 °C for 72 h. Recovered vinegar soaking: soybean germ was soaked by the recovered Yongchun Monascus aged vinegar (the vinegar has used for soaking soy germ once, and then the resolution was recovered from the last time soking) at 4 times their weight and incubated at 25 °C for 72 h. Cooking: soybean germ was soaked by distilled water and cooked at 121 °C for 30 min. Finally, the soybean germs and soaking liquor were lyophilized to yield soybean germ powder for isoflavone analysis by HPLC. 2.3.2. The ratio of solid to liquid in vinegar soaking Soybean germ was soaked in different volume of Yongchun Monascus aged vinegar (vinegar:soy germ = 1:2, 1:4, 1:6, 1:8, 1:12, respectively, w/w) and incubated at 25 °C for 24 h. 2.3.3. Effects of soaking time on the soy germ Soybean germ was soaked by adding Yongchun Monascus aged vinegar at 4 times (w/w) their weight and incubated at 25 °C for different time (0, 4, 8, 12, 16, 20 and 24 h, respectively). After treatment, the soybean germ was dried, ground to uniformity by a coffee mill, and prepared as samples for isoflavones analysis by HPLC. 2.4. Isoflavone extraction method The extraction method of soy germ isoflavones was performed according to the procedure described by Rostagno, Palma, and Barroso (2003), with some modifications. In brief, 1 g soybean germ sample was added in 10 mL 80% aqueous methanol and incubated in ultrasonic bath (B.H.A. instruments Co., Ltd., Germany) for 35 min at 20 °C. The ultrasound power density and frequency were set to 150 W and 35 kHz, respectively. The extraction was centrifuged at 8000g for 15 min using SIGMA 1–15 K instrument (BHA instruments Co., Ltd., Germany) and then the supernatant was filtered by 0.45 lm filters for HPLC analysis. Each experiment was repeated at least three times. 2.5. High performance liquid chromatography (HPLC) The HPLC equipment consisted of an Agilent 1200 series Rapid Resolution LC System (Agilent Technologies, Palo Alto, CA) equipped with an automatic injector and PhotoDiode Array detector (DAD) detector. Data were collected on Agilent Chemstation software.
2.2. Sample collection Soybean germ (from a soybean variety of Zhongdou-27) was purchased from Jiusan Oil and Fat Chemical Factory in
2.5.1. HPLC determination conditions for isoflavones The HPLC conditions were adopted to the method developed by Rostagno, Villares, Guillamón, García-Lafuente, and Martinez
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(2009), with some modification in the chromatographic conditions to achieve good isoflavone separation. Separations were performed in an ODS C18 column (4.6 mm 150 mm, 3.9 lm) fitted with a guard column. The mobile phase was composed of trifluoroacetic acid (TFA) 0.1% in water (A) and TFA 0.1% in acetonitrile (B). The elution was performed with a linear gradient from 100% to 58% (v/v) of eluent A in 24 min, holding these conditions for 10 min and returning to 100% in 10 min. The chromatographic analysis was performed at 35 °C, the flow rate was 1.0 mL/min and detection wavelength was 254 nm. Injections of 5lL were effected with the automatic injector. Twelve standards of soy isoflavones were dissolved in absolute HPLC-grade methanol and the concentration of each standard was from 15 to 50 lg/mL. 2.6. Determination of b-glucosidase activity One hundred milligrams solid samples (soybean germ), or 500 lL liquid samples (vinegar) were taken, and 1.5 mL of 0.05 M citrate buffer (pH 4.5) containing 0.1 M NaCl was maintained for 1 h at room temperature. The samples were centrifuged (12,000g for 5 min), and the supernatant was used as enzyme source for activity analysis. The b-glucosidase activity was assayed by a modified procedure, based on the method of Matsuura and Obata (1993). Two milliliters of the substrate 1 mM p-nitrophenyl-b-D-glucopyranoside (p-NPG) in phosphate-citrate buffer (0.1 M pH 5.0) were transferred to a test tube and kept in a water bath at 30 °C for 10 min; then 0.5 mL of the supernatant was added and the tube left in the water bath at 30 °C for 30 min. After incubation at 37 °C for 30 min, the reaction was stopped with 2.5 mL of 0.05 M sodium carbonate. The activity of b-glucosidase was estimated by reading the absorbance of the liberated p-nitrophenol at 405 nm. The blank solution was composed of 2.5 mL of 0.05 M sodium carbonate solution, 2.0 ml of substrate solution and 0.5 mL of 0.05 M citrate buffer (pH 4.5) containing 0.1 M NaCl. One unit (U) of b-glucosidase activity was defined as the amount of enzyme required for the hydrolysis of 1 lmol of substrate (pNPG)/min, under the assay conditions. 2.7. Statistical analysis Statistical analysis was performed using one-way analysis of variance (ANOVA). All results were expressed as means ± SD, and significant differences were determined by Tukey HSD test. p < 0.05 is considered to be statistically significant. 3. Results 3.1. Validation of the HPLC method for analysis of the isoflavones We adopted a simplified gradient reversed phase HPLC method for the separation and determination of 12 isoflavones extracted from soy germ. Fig. 1 showed the typical HPLC chromatograms of the 12 isoflavones standard (A), and 12 isoflavones from untreated soy germ (B) and vinegar soaked soy germ (C). Isoflavones all absorb UV light, and UV detection may offer sufficient selectivity and sensitivity for the determination of isoflavones (Yuan, Liu, Peng, Wang, & Liu, 2009). The identifications of isoflavones were achieved by comparing their retention time and spectra against the known standards. As expected, the separation of 12 isoflavones standard was achieved in the HPLC chromatograms (Fig. 1A), and the calibration curves of the peak area (A) against the concentration (C) for these isoflavone standards at 254 nm gave good linear responses over a wide range (5–50 mg/L) of concentrations (Table S1), indicating that this HPLC method was sensitive for qualitative and quantitative determination of these isoflavones.
The isoflavones of extracts from untreated or soaked soy germ were also well separated via the HPLC procedure (Fig. 1B and C), indicating that the proposed method can be effectively applied for separation and determination of 12 forms of isoflavones in soy germ.
3.2. The changes of isoflavone forms in soy germ soaked by vinegars The incubation of soy germ with Yongchun Monascus aged vinegar led to considerable change in the isoflavone forms. Comparing chromatogram B with chromatogram C (Fig. 1B and C), it is obvious that the height of three peaks (peak 9, peak 10 and peak 12) in the soaked soy germ sample, namely, the aglycones (daidzein, glycitein and genistein, respectively), were significantly increased, whereas the corresponding malonyglycosides peaks (peak 4, peak 5 and peak 8) decreased accordingly.
3.3. The conversion of isoflavone forms by different treatments Table 1 presents the isoflavone profiles of soy germ after Monascus aged vinegar soaking, water soaking and cooking as the control. In the raw soy germs, the isoflavones consisted of 63.34% malonylglycosides, 31.79 % b-glycosides, 3.69% acetylglycosides and 1.18% aglycones. In general, isoflavones are not destroyed by soaking in water, acid hydrolytic, fermentation and heat, but rather are subjected to the interconversion between the different forms. The changes of isoflavone profiles in soy germ were investigated after Monascus aged vinegar soaking for 72 h at 25 °C, and the related results were listed in Table 1. The data indicated that malonylglycosides in soy germ could readily be converted to their respective acetylglycosides and nonconjugated b-glycosides through the decarboxylation and deesterification, respectively. Concomitantly, b-glycosides forms were transformed to aglycone forms by endogenous b-glucosidase (Table 1). The deesterification reaction of malonyl glycosides was faster than the decarboxylation reaction under the environment, that is, the conversion from malonyl glycosides to glycosides was faster than to acetylglycosides. The Monascus aged vinegar soaking resulted in aglycone-rich soy germ, and aglycones were further increased from 36.8% to 51.34% by the soaking with recovered Monascus aged vinegar (Table 1). Soaking in water and cooking are important procedure in the soybean food processing. The changes in isoflavone profiles during the processes were compared with the counterparts in the Monascus aged vinegar soaking. In the case of water soaking, the proportion of free isoflavones increased from 1.18% to 17.16%, whereas that of isoflavone glucosides decreased depending on the type of b-glucosides. The proportion of malonylated isoflavone glucosides decreased slightly from 63.34% to 52.38%, while acetylated isoflavone glucosides increased from 3.69% to 7.35%. Cooking approach decreased the proportion of malonylglycosides from 63.34% to 3% by the transformation to acetylglycosides and nonconjugated b-glycosides via deesterification and decarboxylation route, and the aglycone was increased to 48.6% after 30 min cooking at 121 °C (Yuan et al., 2009). The amount of aglycones after soaked with Monascus aged vinegar fell in between water soaking and cooking (Table 1). When the recovered Monascus aged vinegar was used for soaking, aglycone forms were further increased. The concentration of aglycones exceeded that of the cooked one (Table 1). The treatment by the recovered Monascus aged vinegar was the best advantageous to aglycone formation, and the highest contain of the aglycones reached 1134.74 ± 39.72 mg/100 g. The cooking control group is one of the best ways for the degradation of the malonylglucosides, de-carboxylation.
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Fig. 1. The HPLC chromatogram of the 12 isoflavone isomers. The HPLC chromatogram of the isoflavone standards (A), the chromatogram of untreated sample of soybean germs (B), the chromatogram of soaking sample of soybean germs soaked in the recovered vinegar (C). The peaks from 1 to 12 mean: 1-daidzin, 2-glycitin, 3-genistin, 4malonydaidzin, 5-malonylglycitin, 6-acetyldaidzin, 7-acetylglycitin, 8-malonylgenistin, 9-daidzein, 10-glycitein, 11-acetylgenistin, 12-genistein, respectively.
3.4. Distribution of isoflavones in soybean germs The isoflavone profiles of soy germ were analyzed by the developed HPLC method. In the raw soy germ, isoflavones accumulated in the order of malonylglycosides, b-glycosides, acetylglycosides and aglycones, among which malonylglycosides were the most abundant form and had the highest concentrations (1498.32 mg/ 100 g), being followed by b-glycosides (751.89 mg/100 g). Acetylglycosides and aglycones forms were found in very small concentrations, 87.38 and 27.84 mg/100 g, respectively. Malonylglycosides constituted 63.34% of the total isoflavones in untreated soy germs. The result is in agreement with the previous reports that malonylglycosides accounted for 57–79% of total isoflavones (Kim et al., 2007; Yamabe, Kobayashi-Hattori, Kaneko, Endo, & Takita, 2007). The b-glycosides accounted for 31.79% of
total isoflavones in soy germs, which are in agreement with the previous reports that b-glycosides accounted for 26.7–40.6% (Kim et al., 2007). Xu et al. reported that the highest proportion at more than 76% of the total isoflavones was malonylglycosides, followed by b-glycosides at 19%, whereas acetylglycosides and aglycones occurred in only very small proportions (Xu & Chang, 2008). 3.5. Effects of solid/liquid ratio on modification of isoflavones Limited information is available on the changes of the isoflavone profiles and the interconversion of soy isoflavones in soy germ via soaking in vinegar. In this work, Yongchun Monascus aged vinegar could increase free isoflavones (aglycone forms) so as to enhance the nutritional values of soy germs. Effects of the solidto-liquid ratios on the interconversions of soy isoflavone forms
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Table 1 The changes in isoflavone compositions of soy germ during different treatments (mg/100 g). Three independent experiments (n = 3) and each performed in triplicate, mean ± SD. Isoflavones
Control
Water soaking
Vinegar soaking
Recovered vinegar soaking
Cooking
Daidzin Glycitin Genistin b-Glycosides %
391.68 ± 14.71a 247.04 ± 8.65a 113.17 ± 3.96a 751.89 ± 27.32a 31.79
263.68 ± 10.23b 166.40 ± 5.92c 79.36 ± 2.98 cd 509.44 ± 18.83c 23.11
262.74 ± 9.90b 159.49 ± 5.58c 92.12 ± 3.22b 514.35 ± 19.00c 25.63
184.75 ± 6.67d 149.94 ± 5.25c 77.40 ± 2.91d 412.09 ± 15.42d 18.64
249.86 ± 9.74bc 212.35 ± 8.43b 107.39 ± 4.76a 569.60 ± 19.94b 28.62
A-daidzin A-glycitin A-genistin Acetylglycosides %
45.39 ± 1.69b 31.94 ± 1.22c 10.05 ± 0.65d 87.38 ± 3.36d 3.69
27.90 ± 0.99d 24.70 ± 0.89d 109.44 ± 3.83c 162.05 ± 5.97c 7.35
54.14 ± 1.90a 44.84 ± 1.97b 177.28 ± 6.20b 276.27 ± 9.67b 13.77
26.14 ± 0.98d 44.20 ± 1.65b 296.52 ± 11.38a 366.86 ± 14.84a 16.60
56.32 ± 1.97a 59.90 ± 2.20a 277.38 ± 9.81a 393.60 ± 14.78a 19.78
M-daidzin M-glycitin M-genistin Malonylglycosides %
859.52 ± 35.38a 423.12 ± 17.92a 215.68 ± 9.63a 1498.32 ± 59.93a 63.34
647.68 ± 26.91b 337.92 ± 14.52b 168.96 ± 6.96b 1154.56 ± 47.18b 52.38
238.85 ± 9.55c 157.53 ± 6.80c 81.07 ± 3.64c 477.45 ± 19.10c 23.79
119.18 ± 4.97d 120.09 ± 4.89d 57.48 ± 2.70d 296.75 ± 12.87d 13.43
29.06 ± 1.76e 16.26 ± 0.95e 14.46 ± 0.79e 59.78 ± 2.89e 3.00
Daidzein Glycitein Genistein Aglycones %
16.64 ± 0.68f 7.10 ± 0.35d 4.10 ± 0.24f 27.84 ± 1.27e 1.18
243.20 ± 9.51e 78.00 ± 3.47b 56.96 ± 2.40e 378.16 ± 14.24d 17.16
545.02 ± 20.08d 83.87 ± 3.39b 109.57 ± 3.93d 738.46 ± 26.85c 36.80
848.71 ± 29.70a 96.12 ± 3.56a 189.91 ± 6.75a 1134.74 ± 39.72a 51.34
785.92 ± 27.51b 17.02 ± 0.60c 164.40 ± 5.75b 967.34 ± 33.86b 48.60
Total isoflavones %
2365.42 ± 83.79a 100.00
2204.21 ± 78.15ab 100.00
2006.52 ± 71.23bc 100.00
2210.44 ± 78.37ab 100.00
1990.32 ± 70.66c 100.00
Data with no letters in common in the same line are significantly different (p < 0.05). The percentages were the mean value, which based the proportion of aglycones, b-glycosides, acetylglycosides and malonylglycosides in different treatments, respectively.
were evaluated to optimize the vinegar soaking process. The soybean germ and soaking liquor were lyophilized to yield soybean germ powder for isoflavone analysis by developed HPLC. The isoflavone profiles of soy germs after the vinegar soaking at the selected solid/liquid ratios are shown in Fig. 2. Aglycone forms and b-glucoside conjugates showed a nearly opposite trend upon increasing soy germ-to-vinegar ratios from control (raw soy germ) to 1:12 (w/w). The amount of malonylglucosides decreased gradually from control, to germ-to-vinegar ratios of 1:2 and 1:4 due to the deesterification and decarboxylation reactions, then arrived at a plateaus upon further increase in soy germ-to-vinegar ratios. The b-glycosides profiles presented a similar trend upon varying the solid/liquid ratios (Fig. 2). Concomitantly, aglycones and acetylglycosides presented nearly opposite trends. In brief, the amount of aglycones increased sharply from 1.18% (control) to 30.0% through incubating soy germs in Monascus aged vinegar at the ratio of 1:4, and aglycones proportions remained unaffected for further increase of the solid/liquid ratios from 1:4 to 1:12. The present data showed that the high conversion efficiency of b-glucoside conjugates toward aglycones forms were achieved at the germ-to-vinegar ratios of 1:4 and higher, and there were no significant increase (p > 0.05) in aglycones proportion upon further heightening the germ-to-vinegar ratios. Therefore, the ratio of germ/vinegar of 1:4 was selected in the following experiments. 3.6. Effects of incubating time of soaking vinegar The conversion kinetics of isoflavone forms were investigated for 24 h incubation in Monascus aged vinegar. Soy germs and Monascus aged vinegar were taken out at 4 h intervals, and soybean germs were lyophilized to yield soybean germ powder. The isoflavone profiles in soybean germ powder and Monascus aged vinegar were analyzed by developed HPLC. Fig. 3 shows on the isoflavone forms in soy germ (left) and Monascus aged vinegar (right) as a function of incubation time in Monascus aged vinegar. In soy germs, total isoflavones consisted of 55.52% daidzein and its derivatives, 29.98% glycitein and its derivatives, and 14.50% genistein and it derivatives (Fig. 3).
Fig. 2. Effects of Ratio of solid to liquid in vinegar soaking on the soy germ isoflavone forms. Using the untreated soybean as raw materials control. Three independent experiments (n = 3) and each performed in triplicate, mean ± SD.
The conversion kinetics of various isoflavone forms varied depending on the type of isoflavone forms. On the whole, the proportion of free isoflavones increased gradually upon the increase of incubation times, whereas that of isoflavone glucosides, e.g., malonylglucosides and b-glucoside decreased accordingly (Fig. 3A–C). As expected, some isoflavone forms were transferred into Monascus aged vinegar in the isoflavone types and incubation time dependent manner (Fig. 3a–c). Effects of incubation times in Monascus aged vinegar on the isoflavone forms in soy germs were shown in the Fig. 3A–C. In the case of daidzein and its derivatives, the free isoflavone (daidzein) increase gradually from 16.64 mg/100 g to 507.10 mg/100 g, whereas malonyl derivatives and b-glucosides decreased gradually from 859.52 (M-daidzin) to 247.60 mg/100 g, and 391.68 (daidzin) to 208.00 mg/100 g, respectively. In addition, the amount of acetyl conjugates decreased slightly. Obviously, the changes of genistein and its derivatives presented a similar trend, with the exception for acetyl conjugates (A-genistin) which increased significantly upon the Monascus aged vinegar soaking. In the case of glycitin and it derivatives, malonyl derivative (M-glycitin) and b-glucoside
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Fig. 3. (I) Effects of incubation times in Monascus aged vinegar on the isoflavone forms in soy germ (left) and Monascus aged vinegar (right). Panel A and a: daidzin and its derivatives. Panel B and b: genistin and its deravitives. Panel C and c: glycitin and its deravitives. (II) Effects of incubation times in Monascus aged vinegar on the isoflavone forms of soy germ plus Monascus aged vinegar. The amount of isoflavone forms in soy germs and Monascus aged vinegar analyzed by the developed HPLC, and the sum of various isoflavone forms in soy germs and Monascus aged vinegar were reported. Three independent experiments (n = 3) and each performed in triplicate, mean ± SD.
(glycitin) presented a sharp decrease for the initial 4 h, then reached a plateau, and the free isoflavone (glycitein) and acetyl derivative remain nearly unaffected in the soaking duration. Effects of incubation times in Monascus aged vinegar on the isoflavone forms in Monascus aged vinegar were shown in the Fig. 3a– c. The isoflavone profiles in the Monascus aged vinegar were different from that in soaked soy germs. Some selected isoflavone forms diffused toward the Monascus aged vinegar from soy germs. In the initial soaking stage (4 h), the sharp increases in b-glucoside conjugates were observed, while the aglycones were nearly unaffected upon incubating soy germ in the Monascus aged vinegar. This
phenomenon may be associated with the hydrophobic character of the isoflavone forms, and the aglycones were more hydrophobic when compared with b-glucoside conjugates, its solubility was restricted in the aqueous Monascus aged vinegar. The distribution of isoflavones in Monascus aged vinegar after soaking for 24 h, including 327.6 mg/100 g malonyl derivatives, 35.96 mg/100 g acetyl conjugates, 120.8 mg/100 g b-glucoside conjugates and 15.69 mg/100 g free isoflavones (aglycone forms). The proportions of various isoflavone forms were 65.51% malonyl derivatives, 7.19% acetyl conjugates, 24.16 b-glucoside, and 3.14% aglycone forms. The proportion of isoflavones forms was similar
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Fig. 3 (continued)
with that in the raw soy germs, and the total isoflavones in Monascus aged vinegar accounted for 21.41% of the total isoflavones in raw soy germs. The aforementioned total isoflavones constituted 55.52% daidzein and its derivatives, and the change in total isoflavone forms of soy germs plus Monascus aged vinegar during the incubation in the vinegar were similar with their main constituents (daidzein and its derivatives) (Fig. 3). 3.7. The role of endogenous b-glucosidase on isoflavone conversion Since the increase in free isoflavones generated during soaking in water or Monascus aged vinegar and the concomitant decrease in isoflavone glucosides during soaking can be restricted by the addition of the inhibitor of b-glucosidase, glucono-d-lacton, the changes are believed attributable to the action of endogenic b-glucosidase contained in soybeans (Matsuura, Obata, & Fukushima, 1989). Therefore, the activities of b-glucosidase during the soaking process were assayed herein using p-NPG as the substrate (Fig. 4). Endogenic b-glucosidase activity in both soaking liquors (water and Monascus vinegar) increased for an initial 4 h incubation, further increase in incubation time decreased the endogenous bglucosidase activity, especially for water (Fig. 4). This results was in agreement with the interconversion profiles of isoflavone forms during the soaking where the proportion of free isoflavone forms (aglycones) presented a sharp increase at an initial 4 h incubation, and the amount of b-glucoside conjugates (e.g., malonyl derivatives, b-glucoside showed concomitantly a sharp decrease (Fig. 3A–C)). In addition, the endogenous b-glucosidase activity in Monascus aged vinegar over 12 h soaking was higher than the counterparts in water soaking (Fig. 4). The phenomenon accounted for the differences in isoflavone forms of soy germs in both soaking processes. The Monascus aged vinegar was utilized to soak soy germ for 72 h at 25 °C, resulting in the increase of aglycones from 1.18% to 36.80%. In contrast, the water soaking at the same condition increased the proportion of the aglycones in soy germs to 17.16% (Table 1). 4. Discussion The bioavailability of isoflavones depended on their chemical forms (aglycones or glucosides) which influenced the solubility in the intestinal lumen and the absorption as well as on the metabolism by intestinal and microbial enzymes (Vitale, Piazza, Melilli, Drago, & Salomone, 2013). The enrichment of aglycone forms is a potential and promising strategy to enhance the bioavailability
Fig. 4. The effects of the incubation time on endogeneic b-glucosidase activities in the soaking liquids. Three independent experiments (n = 3) and each performed in triplicate, mean ± SD.
and physiological functions of soybean germs products. Several processing techniques were adopted to convert isoflavone glycosides to aglycones (Chen et al., 2012; Chien et al., 2005; Hubert et al., 2008; Liu et al., 2013; Toda et al., 2000). The common processing factors are thermal degradation, enzyme and acid (alkaline) hydrolysis, which involve in modification of soy-derived foods in terms of the content and distribution of the three isoflavone derivatives, daidzein, genistein and glycitein, and their respective conjugates. The higher levels of aglycones detected when soaking at approximately 45 °C were probably due to the greater activity of the endogenic b-glucosidase, which produced the hydrolysis of the glucoside conjugates to form the corresponding aglycones (Aguiar, Baptista, Alencar, Haddad, & Eberlin, 2007). Aglycones content in fermented soy products reached up to 90% of the total isoflavones due to the activation of the enzyme b-glucosidase during fermentation (Wang et al., 2007). In general, malonylglycosides in soybeans were labile to heat process and could readily be converted to their respective more heat-stable acetylglycosides and nonconjugated b-glycosides through decarboxylation and deesterification. The cooking process resulted in the variations of isoflavone distributions: the degradations of the malonyl conjugates and b-glycosides, meanwhile increasements of the three aglycones (Wang & Murphy, 1996). The acidic hydrolysis could effectively hydrolyze b-glycosides into aglycones (Liu et al., 2013). In this work, we adopted a facile approach via Monascus aged vinegar soaking and achieved high conversion efficiency of b-glycosides conjugates to aglycones forms, and the proportion of aglycones forms were further increased from 1.18% (raw soy germs), 36.8% (vinegar soaking) and 51.34% (recovered vinegar soaking) (Table 1). Herein, a schematic illustration of the interconversion pathway of isoflavones forms in soy germs via vinegar soaking approach was proposed (Fig. 5). The conversion kinetics of isoflavone forms were investigated for 24 h incubation in Monascus aged vinegar. The conversion kinetics of various isoflavone forms varied depended on the type of isoflavone forms. On the whole, the proportion of free isoflavones increased gradually upon the increase in incubation time, whereas that of isoflavone glucosides, e.g., malonylglucosides and b-glucoside decreased accordingly (Fig. 3A–C). The isoflavone forms in soy germ (left) and Monascus aged vinegar (right) as a function of incubation time in Monascus aged vinegar, reflecting that some selected isoflavone forms, e.g., malonylglucosides migarated toward the Monascus aged vinegar from soy germs. The total isoflavones in Monascus aged vinegar accounted for
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Decarboxylation
De-esterification
Deglycosylation
Ο HO
Ο
Ο
O O
HO
Ο
H
H H
R2
OH
R3
-glucosides
Aglycones
Acetyl- -glucosides
Soaking Germination Fermentation
Malonyl- -glucosides
Ο
ΟΗ
-glucosidase or fermentation Hydrolysis (acid and alkaline) Heat Treatment
Soaking Germination Fermentation Heat Treatment
Fig. 5. The interconversion of the 12 isoflavones isomers in soybeans and soy products occurred during the different processing.
21.41% of the total isoflavones in raw soy germs, and its proportion was similar with raw soy germs. This phenomenon may be associated with the hydrophobic character of the isoflavone forms. The aglycones in soy germ soaked with recovered vinegar (Table 1) were dramatically increased by 40.76 times, which was from 27.84 ± 1.27 mg/100 g to 1134.74 ± 39.72 mg/100 g (p < 0.05), at the same time, malonylglycosides were significantly reduced from 1498.32 ± 59.93 mg/100 g to 296.75 ± 12.87 mg/ 100 g (p < 0.05) and b-glycosides were significantly decreased from 751.89 ± 27.32 mg/100 g to 412.09 ± 15.42 mg/100 g (p < 0.05). In fact, if operated independently, b-glycosidase was not effective in hydrolyzing the conjugated glycosides to their respective aglycones, even with increased levels of the enzyme and prolonged incubation (Ismail & Hayes, 2005). In this work, the proportion of malonylated isoflavone glucosides decreased slight from 63.34% to 52.38% upon water soaking. Interestingly, malonylglycosides in soy germ could readily be converted to their respective acetylglycosides and nonconjugated b-glycosides, and its proportion decreased from 63.34% to 23.79% via Monascus aged vinegar soaking (Table 1). Yongchun Monascus aged vinegar, is one of the four famous China-style fermented vinegars, and various enzymes including b-glycosidases may be produced during the fermentation process via microorganisms such as acetic acid bacteria, lactic acid bacteria, yeast, M. purpureus. The soaking approach via Monascus aged vinegar can also be considered as after-fermentation stages in which various enzyme may induce the conversion of conjugated b-glycosides to free isoflavones forms. In brief, it was a combination of enzyme (b-glucosidase) and acid hydrolysis during the soaking with Monascus aged vinegar. These results are in agreement with previous works reporting that the glycosides isoflavones converted into aglycones in a reaction catalyzed by endogenous b-glucosidase of soybean in soaking water, and fermentation of soybean caused the hydrolysis of isoflavone malonyl- and b-glucosides to form aglycones (Góes-Favoni, CarrãoPanizzi, & Beleia, 2010; Tsangalis, Ashton, McGill, & Shah, 2002). It was highly anticipated that further research should be focus on the more detailed mechanism regulated by the potential microbes in Monascus aged vinegar during the soaking processing.
degradation of 12 isoflavone forms were determined. The possible degradation mechanisms varied with the different treatments. It was obvious that the aglycones were dramatically increased meanwhile the glycosides significantly reduced via Monascus aged vinegar approach. The proportion of aglycones presented a sharp increase with the endogenous b-glucosidase activity at the initial 4 h incubation. There appeared to be correlations between b-glucosidase activity and the hydrolysis of conjugated isoflavones. The present data indicated that the decarboxylation, de-esterification and de-glycosylation reactions were involved the interconversion of the isoflavones forms of soy germs during the soaking by Monascus aged vinegar, reflecting the synergistic effects of endogenous b-glucosidase activity of soy germ, acid hydrolysis and formation of the microorganisms. In addition, a schematic illustration of the interconversion pathway of isoflavones forms of soy germs via vinegar soaking approach was proposed. This study opens a promising safe pathway for preparing aglycone-rich soy germ as functional food ingredients via vinegar soaking. Conflict of interest The authors declare no competing financial interest. Acknowledgments This work was supported by China Postdoctoral Science Foundation (Grant No. 2012M521265), Science and Technology Planning Project of Fuzhou City (Grant No. 2013-N-47), the National Natural Science Foundation of China (Grant No. 31201350), and the Program for New Century Excellent Talents in Fujian Province University of China (Grant No. NCETFJ200720). I would appreciate the advices on the revision of Prof. Ruihai Liu in Cornell University and Prof. Qihe Chen in Zhejiang University. I would like to express my gratitude to all those who gave me the possibility to complete this manuscript. Appendix A. Supplementary data
5. Conclusion The isoflavone profiles were significantly changed in soy germ soaked by Yongchun Monascus aged vinegar. The conversion and
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. 02.099.
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