• Food Science and Biotechnology
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• v.15 no.1
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• pp.96-101
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• 2006

Isoflavone distribution and ${\beta}$-glucosidase activity in cheonggukjang, a traditional Korean whole soybean-fermented food prepared with or without addition of Bacillus subtilis, were analyzed every 6 hr for 36 hr. Thermal cooking of raw-soaked soybeans significantly increased ${\beta}$-glucoside isoflavone level by 57.1 % and decreased malonyl-${\beta}$-glucosides by 57.6% (p<0.05). Consistent changes of isoflavone profiles in cheonggukjang without B. subtilis addition (COB) and samples with addition of B. subtilis (CWB) were not observed during 36 hr fermentation. ${\beta}$-Glucosides of isoflavones are major forms in both COB and CWB. ${\beta}$-Glucosidase activity in cheonggukjang decreased significantly compared to that of soaked soybeans due to thermal denaturation, while recovery of enzyme activity in COB was observed. Two new unidentified peaks were detected, and their relative peak areas in CWB were significantly larger than those in COB with increasing fermentation period (p<0.05), which indicates both peaks could be associated with fermentation metabolites.

• Korean Journal of Food Science and Technology
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• v.42 no.2
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• pp.125-129
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• 2010

Isoflavone distribution and $\beta$-glucosidase activity in 16 commercially available doenjang samples were determined. Twelve of the samples were home-made doenjang (HMD) with a relatively long fermentation period and 4 of the samples were factory-produced doenjang (FPD) from four different manufactures. Total isoflavones (TI) in the HMD ranged from $370-723\;{\mu}g/g$, while those in FPD ranged from $179-537\;{\mu}g/g$. The isoflavone distribution in HMD was different from those in FPD. Generally, the TI in HMD was higher than those in FPD. The major isoflavone was aglycones, which ranged from 42.98 to 89.96% in HMD and from 35.51 to 93.48% in FPD. Isoflavones in the $\beta$-glucoside forms were not detected in tested FPD samples. Principal components analysis (PCA) of the isoflavone profiles showed that HMD were differentiated from FPD. First principal component (PC1) and second principal component (PC2) expressed 43.6 and 22.9% of the data variability, respectively. $\beta$-Glucosidase activity in doenjang was lower than that in raw soybeans. The results of this study can be used to understand the differences in the isoflavone distribution in traditionally manufactured and factory produced doenjang.

• Journal of Microbiology and Biotechnology
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• v.12 no.1
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• pp.8-13
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• 2002

The isoflavone glycosides are hydrolyzed by ${\beta}$-glucosidase from gut microbes to the bioactive aglycones. However, the specific bacteria from the human intestinal tract that are involved in the metabolism of these compounds are not known. This study was undertaken to develop a fermented soymilk which converts isoflavones to the more bioactive aglycones form using a Bifidobacterium strain. The ${\beta}$-glucosidase activity of 15 Bifidobacterium strains were measured during cell growth. Among them, Bifidobacterium sp. Int-57 was selected for this study, because it has the highest ${\beta}$-glucosidase activity. Growth, acid development, ${\beta}$-glucosidase activity, and the hydrolysis of daidzin and genistin were investigated in four soymilks inoculated with Bifidobacterium sp. Int-57. After 12 h of fermentation, the counts of viable Bifidobacterium sp. Int-57 in all the soymilks reached a level of more than $10^8$ cfu/ml, which was then maintained. The pH of soymilks started to decrease rapidly after 6 h of fermentation and leveled off after 18 h. The titratable acidity of BL# 1 soymilk, BL#2 soymilk, and JP#l soymilk increased from 0.18 to 1.21, 1.15, and $1.08\%$ over the fermentation period, respectively. After 24 h of fermentation, the $\beta$-glucosidase activity in BL#1 soymilk, BL#2 soymilk, JP#l soymilk, and JP#2 soymilk increased to 59.528, 40.643, 70.844, and 56.962 mU/ml, respectively. The isoflavone glycosides, daidzin and genistin, in soymilks were hydrolyzed completely in the relatively short fermentation time of 18 h. These results show that Bifidobacterium sp. Int-57 can be used as a potential starter culture for developing fermented soymilk which has completely hydrolyzed isoflavone glycosides.

• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.317-323
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• 2016

An isoflavone glucosidase that catalyzes the hydrolysis of isoflavone glucosides into glucose and corresponding aglycones was purified from Candida fermentati SI. The N-terminal sequence was determined to be GLNCDYCN. We designed degenerate primers on the basis of these amino acid sequences and successfully cloned the full structural gene sequence of the isoflavone glucosidase using inverse PCR. The exo-β-(1,3)-glucanase gene consists of 1227 base-pair nucleotides, encoding a 408-amino-acid sequence that shares 41–96% amino acid homology with other yeast exo-β-(1,3)-glucanases belonging to glycoside hydrolase family 5. The recombinant exo-β-(1,3)-glucanase was expressed in Pichia pastoris X-33, using a pPICZA vector system, and further characterized. The molecular mass of the purified exo-β-(1,3)-glucanase was estimated by SDS-PAGE to be 47 kDa. The optimal pH and temperature were pH 4.5 and 40℃, respectively. The K_m values of the purified exo-β-(1,3)-glucanase for daidzin and genistin were 0.12 mM and 0.14 mM, respectively. The V_max values of the purified isoflavone glucosidase were 945.03 U/mg for daidzin and 835.92 U/mg and for genistin.

• Food Science and Preservation
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• v.22 no.1
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• pp.119-128
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• 2015

In this study, the ${\beta}$-glucosidase activity and total phenolic and isoflavone contents and antioxidant activities during Cheonggukjang fermentation with bitter melon powder (BMP) were investigated and evaluation of the same was performed. The level of ${\beta}$-glucosidase activity was increased at 48 hr and decreased after 72 hr, and the total phenolic and isoflavone-malonylglycoside and aglycone contents increased, while the antioxidant activities increased, but the total isoflavone and isoflavone-glycoside contents decreased during the Cheonggukjang fermentation. In particular, the soybean with 5% BMP fermented at $37^{\circ}C$ for 72 hr displayed the highest antioxidant activities, among all the samples. The highest levels of total phenolic and daidzein contents and DPPH radical scavenging activity, ABTS radical scavenging activity and FRAP assay results after 72 hr fermentation in Cheonggukjang with 5% BMP were found to be 13.5 mg/g, $390.57{\mu}g/g$, 90.74%, 99.79%, and 1.705 (OD593 nm) respectively. In addition, the more BMP was added to the Cheoggukjang, the more the off-odor of the Cheonggukjang decreased. These results suggest that BMP can be used to come up with a new type of Cheonggukjang with improved palatability and antioxidant activity.

• Journal of Nutrition and Health
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• v.36 no.1
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• pp.32-39
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• 2003

Phytoestrogens, especially soy-derived isoflavones, are receiving great scrutiny as a food supplement for preventing hormone dependent disease such as postmenopausal osteoporosis. Their beneficial effects are derived from aglycone form of isoflavones, such as daidzein, genistein or glycitein. In contrast to the common usage of soybean, black bean (Rhynchosia Molubilis : Yak-kong) has been used as a supplement for preventing postmenopausal osteoporosis in oriental medicine. To investigate the effects of the saliva, artificial stomach fluid, and digestive enzymes on the conversion of glycosidic isoflavone to aglycone form, soybean and black bean were extracted with 70% methanol and freeze-dried. The recovery yield of methanol extracts of black bean was 14.1% which was higher than that of soybean, 13.5%. In terms of total isoflavones, we routinely obtained larger amount of isoflavones from black bean than those from soybean. By incubating methanol extracts of soybean and black bean with IN HCI for 180 min, the proportions of aglycones relative to the total isoflavone were significantly increased (32.4% and 52.4%, respectively). In vitro conversion, digestive enzymes ($\beta$-glucosidase and $\alpha$-glucosidase) may hydrolyze glycosidic bond of isoflavone more effectively than saliva or artificial stomach fluid did. It seems to say that the activity of $\beta$-glucosidase was higher than those of $\alpha$-glucosidase. The rate of conversion of glucoside form to aglycone form in black bean and soybean was low in physiological condition (pH) tested, although the enzymatic hydrolysis of glucoside was active. These results demonstrated that the composition of aglycone in food may be the important factors in terms of the bioavailability of isoflavones. (Korean J Nutrition 36(1): 32-39, 2003)

• Journal of Life Science
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• v.21 no.11
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• pp.1579-1585
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• 2011

Natural anti-diabetic semipurified ethyl acetate fraction was isolated from the submerged-liquid culture of Agaricus blaze Murill (AB) in a medium containing soybean flakes. Hot-water extract of AB (HEAB) was prepared by extraction at $121^{\circ}C$ for 60 min, followed by filtering through a filter presser filled with diatomate. The ${\beta}$-glucan-free HEAB, which was a supernatant fraction from HEAB by precipitation in an 80% ethanol solution, was fractionated into hexane, chloroform, ethyl acetate, and butanol fractions. The inhibition of the ${\alpha}$-glucosidase activity by fractions was 59.0, 17.0, 61.6, and 37.9%, respectively, suggesting that ethyl acetate fraction was the most active. A subfraction having a strong ${\alpha}$-glucosidase inhibitory activity (80.4%) was isolated from the ethyl acetate fraction. This subfraction contained isoflavones (genistin and daidzin) and their conjugates with sugars as potent inhibiters of ${\alpha}$-glucosidase activity. These results suggest that the ethyl acetate fraction or HEAB containing isoflavones and their sugars conjugates could be useful sources for controlling blood sugar levels in humans.

• Korean Journal of Food Science and Technology
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• v.31 no.6
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• pp.1405-1409
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• 1999

This study was conducted to investigate the isoflavone contents of soybeans, Meju, and Doenjang. $The\;{\beta}-glucosidase$ activities were also determined and characterized in soybeans, Meju, and Doenjang. The total concentration of daidzein was 406 mg/kg of soybeans, 433 mg/kg of Meju, and 538 mg/kg of Doenjang. Aglycones compose 26.03% of total isoflavones in soybeans, 61.96% in Meju, and 107.68% in Doenjang. The total concentration of genistein was 484, 200 and 538 mg/kg of soybeans, Meju and Doenjang, respectively. Aglycones compose 19.49% of total isoflavones in soybeans, 68.52% in Meju, and 85.26% in Doenjang. The ${\beta}-glucosidase$ activity was detected in soybeans (5.65 units/mg protein), Meju (2.04 units/mg protein), and Doenjang (0.69 units/mg protein). The optimal pH of ${\beta}-glucosidase$ activities were $6.5{\sim}7.0$ and the optimal temperature of ${\beta}-glucosidase$ activities was $50^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.25 no.4
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• pp.469-478
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• 2015

In spite of the reported probiotic effects, Bifidobacterium bifidum BGN4 (BGN4) showed no βglucosidase activity and failed to biotransform isoflavone glucosides into the more bioactive aglycones during soy milk fermentation. To develop an isoflavone-biotransforming BGN4, we constructed the recombinant B. bifidum BGN4 strain (B919G) by cloning the structural β-glucosidase gene from B. lactis AD011 (AD011) using the expression vector with the constitutively active promoter 919 from BGN4. As a result, B919G highly expressed β-glucosidase and showed higher β-glucosidase activity and heat stability than the source strain of the β-glucosidase gene, AD011. The biotransformation of daidzin and genistin compounds using the crude enzyme extract from B919G was completed within 4 h, and the bioconversion of daidzin and genistin in soy milk during fermentation with B919G also occurred within 6 h, which was much faster and higher than with AD011. The incorporation of this β-glucosidase-producing Bifidobacterium strain in soy milk could lead to the production of fermented soy milk with an elevated amount of bioavailable forms of isoflavones as well as to the indigenous probiotic effects of the Bifidobacterium strain.

• KSBB Journal
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• v.16 no.2
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• pp.174-178
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• 2001

Soybeans contain the phytoestrogens genistein and daidzein, their glucosides genistin and daidzin and coumesterol. These isoflavonoid compounds are capable of producing an estrogenic response in a number of diverse species. This study determined optimum conditions for the extraction of the main isoflavones(daidzin, genistin, daidzein, genistein) in defatted soybean meal using high-performance liquid chromatography. The best optimum extraction was achieved at 75% ethanol, $80^{circ}C$, pH4 and a three hour contact time. In addition, isoflavones with high purity were separated by adding up to 4%(w/v) of calcium chloride dihydrate. Most soybean extracts were composed of $beta$-glucosidic conjugate(daidzin, genistin) which is difficult to adsorb in body. Therefore, $beta$-glucosidase was used to convert as conjugate to aglycone form (daidzein, genistein) which is easy to adsorb. The optimal conditions of enzyme reaction involved to be 8.4 units of enzyme concentration, pH5.0, $40^{circ}C$ and 40 minutes.