Deglycosylation patterns of isoflavones in soybean extracts inoculated with two enzymatically different strains of lactobacillus species

Journal Pre-proof Deglycosylation patterns of isoflavones in soybean extracts inoculated with two enzymatically different strains of Lactobacillus species You Jin Lim, Bora Lim, Hae Yeong Kim, Soon-Jae Kwon, Seok Hyun Eom

PII:

S0141-0229(19)30132-2

DOI:

https://doi.org/10.1016/j.enzmictec.2019.109394

Article Number:

109394

Reference:

EMT 109394

To appear in:

Enzyme and Microbial Technology

Received Date:

12 April 2019

Revised Date:

2 August 2019

Accepted Date:

4 August 2019

Please cite this article as: Lim YJ, Lim B, Kim HY, Kwon S-Jae, Eom SH, Deglycosylation patterns of isoflavones in soybean extracts inoculated with two enzymatically different strains of Lactobacillus species, Enzyme and Microbial Technology (2019), doi: https://doi.org/10.1016/j.enzmictec.2019.109394

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Deglycosylation patterns of isoflavones in soybean extracts inoculated with two enzymatically different strains of Lactobacillus species

You Jin Lima, Bora Limb, Hae Yeong Kimb, Soon-Jae Kwonc, Seok Hyun Eoma,*

Department of Horticultural Biotechnology, College of Life Sciences, Kyung Hee University,

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Yongin 17104, Republic of Korea b

Department of Food Science & Biotechnology, College of Life Sciences, Kyung Hee University,

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Yongin 17104, Republic of Korea

Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup

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56212, Republic of Korea

*Corresponding author at: Department of Horticultural Biotechnology, College of Life

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Sciences, Kyung Hee University, Yongin 17104, Republic of Korea

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E-mail address: [email protected] (SH Eom)

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Highlights 

Enzymetically different lactobacilli were evaluated to isoflavone bioconversion.

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Daidzein glucosides were most affective in bioconversion pattern between lactobacilli.

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Malonyl genistin was most degradable isoflavone glucosides by lactobacilli.

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Accumulation patterns of glycitein were reversely presented from those of daidzein and genistein.

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Abstract Microorganism selection is critical to deglycosylation in soybean fermentation for producing beneficial phytochemicals. This study investigated isoflavone bioconversion in

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soybean extract inoculated with Lactobacillus plantarum K2-12 and Lactobacillus curvatus JD0-31 exhibiting different enzyme activities. L. plantarum showed higher esterase (C4),

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esterase (C8), β-galactosidase, α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminase activities. We found that isoflavone bioconversion was distinguished into isoflavone backbone

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structure types. Malonyl- and acetyl- types of isoflavones except for malonyl daidzin were not significantly differed their contents between lactobacilli. Deglycosylating severity was

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observed in malonyl genistin in both lactobacilli, resulting mass production of genistein. On the other hand, daidzein glycosides were dependable to lactobacilli, in which L. plantarum

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efficiently degraded malonyl daidzin and daidzin in fast time. Glycitein was most degradable among the three aglycones by fermentation. These results suggest that efficient control of

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isoflavone deglycosylation by Lactobacillus species should be controlled to the inoculation period and select target isoflavones.

Keywords: bioconversion; β-glucosidase; enzymatic hydrolysis; fermentation

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1. Introduction Isoflavones, which are produced almost exclusively by plants of the family Fabaceae, most often occur as glycosyl groups in plants. Isoflavones in soybean (Glycine max) are distributed throughout the plant [1]. Twelve forms of isoflavone consisting of three aglycones (daidzein, glycitein, genistein) and their β-glycosides (daidzin, glycitin, genistin), acetylconjugated β-glycosides (6”-O-acetyl daidzin, acetyl glycitin, acetyl genistin), and malonyl-

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conjugated β-glycosides (6”-O-malonyl daidzin, malonyl glycitin, malonyl genistin) have been studied as major isoflavones in soybean (Fig. 1) [2]. These isoflavones exist predominantly in the form of glucosides, especially malonyl daidzin and malonyl genistin. Aglycones are found

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in low concentrations in raw soybean [3]. They are known to have higher physiological activity than their glycoside forms due to their high uptake in the human body [3,4]. Although

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aglycones have higher activity, the various types of isoflavones have also their own physiological functions [5,6]. Large amounts of soybeans are consumed as processed products.

from glycoside forms.

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During processing, the forms of isoflavones are changed, including aglycones deglycosylated

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Soy isoflavone content and composition are changed by heat and pressure treatments, pH changes during processing or by microbial enzymatic hydrolysis during fermentation [7,8].

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In general, soy isoflavones are converted from glycoside forms to aglycones by deglycosylation

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during fermentation [8,9]. The conversion depends on the type of microorganisms present, which may include Aspergillus, Bacillus, Lactobacillus, Streptococcus, Bifidobacteria, Enterococcus, and Weissella [5,10-12]. Lactobacillus, Streptococcus, Bifidobacteria, Enterococcus, and Weissella are considered as probiotics whereas Aspergillus and Bacillus are commonly occurred and used in the fermentation of soy paste in food industry [13]. There have been many recent studies reporting the change of isoflavones in soybean 3

fermentation using probiotics, especially the use of Lactobacillus [10-12]. However, the destination of deglycosylating isoflavones using Lactobacillus are still not clearly identified. In this experiment, we selected Lactobacillus because it remains in fermented food sources like kimchi and is quite clearly identified its enzymatic characteristics, while other probiotic microorganisms are not mainly found in food sources. β-glucosidase mainly contributes to deglycosylation of isoflavone glycosides [5,14]. Most studies on isoflavone bioconversion using microorganisms have focused on daidzein,

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genistein, and their glycoside forms relating to β-glucosidase because the compounds are considered as major isoflavones. Few studies exist on bioconversion or degradation of soybean’s 12 individual isoflavones, especially glycitein-conjugated forms, due to their

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relatively lesser content. In addition, microbial soybean fermentation has been conducted using

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soybeans treated by soaking, heat processing or autoclaving for sterilization, resulting in changed isoflavone forms and contents prior to fermentation. Thermal and pressure treatments

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induce cleavage of malonyl or acetyl residues and sugars from isoflavone glycosides prior to fermentation, making it difficult to evaluate distinct interactions between microorganism

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enzyme activities and innate soybean isoflavones. Therefore, the study of fermentation with non-processed raw soybean should be helpful to better understand individual isoflavone

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destination interacted to lactic acid bacteria. In this study, soybean seed extracts were inoculated by two enzymatically different

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Lactobacillus species to investigate the specific bioconversion of each of twelve isoflavones from raw soybean seeds. Individual isoflavones were also inoculated into two different strains of lactobacilli, L. plantarum K2-12 and L. curvatus JD0-31, and the biomass of isoflavone were measured to determine if the interfering phenomena of soybean extract in the bioconversion process exists. 4

2. Materials and Methods 2.1. Chemicals Analytical standards of isoflavone aglycones (daidzein, genistein, and glycitein) and β-glycosides (daidzin, genistin, and glycitin) were purchased from LC Laboratories (Woburn, MA, USA). Isoflavone standards of malonyl-conjugated glycosides (malonyl daidzin, malonyl

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genistin, and malonyl glycitin) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

2.2. Soybean extraction

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Glycine max ‘Pung won’ seeds were ground using a commercial blender. The ground powder of the samples (30 g) was combined with 300 mL of 58% aqueous acetonitrile (v/v)

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and shaken at 25 ºC for 24 h a total of three times. The solvent extracts were then combined,

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concentrated and lyophilized.

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filtered through qualitative filter papers (Whatman No.1, Maidstone, UK), vacuum

2.3. Isolation of Lactobacillus and inocula preparation

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Lactobacillus plantarum K2-12 and Lactobacillus curvatus JD0-31 identified by 16S

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rRNA gene sequencing, MALDI-TOF mass spectrometry, and PCR-DGGE were isolated from kimchi and jeotgal, traditional Korean salted and fermented foods [15, 16]. Colonies were grown on de Man, Rogosa, and Sharpe (MRS) agar plates at 30 ºC for 48 h under anaerobic conditions using an Anaeropack system (Mitsubishi Gas Chemical, Japan). Single colonies were subcultured in MRS broth at 30 ºC for 24 h. For soybean extract inoculation, cells were diluted to 2.5 × 108 cfu/mL (OD600 = 2.0) with MRS broth. 5

2.4. Inoculation of soybean extract and isoflavone standards with lactobacilli Five milligrams of soybean extract were dissolved in 9 mL of distilled water. The extracts dissolved in 9 mL were inoculated with 1 mL of either L. plantarum K2-12 or L. curvatus JD0-31. To investigate the degradation of isoflavone standards by lactobacilli, 200 µg of 9 isoflavone standards were each dissolved in 9 mL of distilled water. The 1 mL of two

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bacterial cell solutions was added to the isoflavone standard solutions. The inocula were incubated in a 30 °C shaking incubator and were harvested for analysis on days 1, 2, 3, 4 and 5, respectively. The obtained inocula were centrifuged at 12,000 rpm for 10 min. The

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supernatants were filtered with a syringe filter (0.45 µm, Futecs Co., Ltd, Daejeon, Korea) and

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2.5. HPLC analysis of isoflavone

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used for HPLC analysis.

Extracts were analyzed using a reversed-phase HPLC (Waters 2695 Alliance HPLC;

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Waters Inc., Milford, MA, USA) with an octadecylsilane column (Prontosil 120-5-C18-SH 5.0 μm (250×4.6 mm), Bischoff, Leonberg, Germany). The flow rate of the mobile phase was 1.0

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mL/min. Sample injection volume was 5 μL. The mobile phases were determined to be a

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combination of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. Gradient elution was performed using 15% of solvent B at initial running time and increasing the concentration to 34% until 60 min of running time. Peaks were monitored at 254 nm with a Waters 996 photodiode array detector (PDA; Waters Inc.).

2.6. Determination of enzyme activity 6

Enzyme activities were determined by the semi-quantitative commercial API ZYM kit (bioMérieux, Marcy- l'Etoile, France). Briefly, a cell suspension (65 μL) was dispensed into each capsule of the API-ZYM strips. Enzyme activities were graded as positive (black block), negative (gray block), and weak positive (white block) with the API ZYM color reaction chart after 4 h of incubation in anaerobiosis at 37 °C.

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2.7. Statistical analysis

Regression analysis and t-test were carried out using MS Excel (Microsoft, Redmond, WA, USA). Relationships between different activities shown to 6 enzymes of Lactobacilli and

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isoflavone changes in soybean extracts at either 3 or 9 days of inoculation were expressed by

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regression analysis. For the regression analysis, the different enzyme activities between Lactobacilli were simply scored as “zero” value when an activity was shown weaker expression

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and “one” value when an activity was shown stronger expression between enzymes. The values of isoflavone changes used in the regression were calculated by subtracting the value of

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isoflavone content in soybean extracts at either 3 or 9 d after inoculation from the value of the

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content before inoculation.

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3. Results and discussion 3.1. Determination of enzymatic activities from two Lactobacillus species. The activities of 19 enzymes found in L. plantarum K2-12 and L. curvatus JD0-31 were determined by API Zym kit (Fig. 2). Activities of 13 enzymes matched between the two strains of lactobacilli. The remaining six, including esterase (C4), esterase (C8), βgalactosidase, α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminase, exhibited stronger

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activities in L. plantarum K2-12. In isoflavone bioconversion during soybean fermentation, βglucosidase is known as a main enzyme that hydrolyzes glycoside forms to aglycones. Several previous studies have reported that microorganisms with high β-glucosidase activity showed

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rapid conversion of β-glycoside forms into aglycones inducing deglycosylation [5,17].

The two lactobacilli selected in this study showed a distinct difference in β-glucosidase

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activity, with higher activity in L. plantarum K2-12 compared with L. curvatus JD0-31. β-

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galactosidase and α-glucosidase are cleavage enzymes for β-galactoside and α-glucoside, respectively. They were known to exhibit a slight hydrolytic effect of β-glucoside [17, 18]. β-

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galactosidase contributes to the hydrolyzation of β-glucosidic bonds in isoflavone glycosides though hydrolysis with less efficiency than β-glucosidase [17]. The β-galactosidase of L.

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plantarum K2-12 showed positive activity, whereas this enzyme in L. curvatus JD0-31 was not active. Although studies on the isoflavone conversion of α-glucosidase are rare, Kang et al [18]

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reported that α-glucosidase may also hydrolyze glycoside forms of isoflavone [18]. Activity of α-glucosidase was positive in L. plantarum K2-12 and negative in L. curvatus JD0-31. Isoflavone forms of β-glycosides are easily hydrolyzed by β-glucosidases, however, malonyl and acetyl conjugated glycosides have ester bonds and are poorly hydrolyzed by β-glucosidases [9,19,20]. Esterase is an enzyme that splits an ester bond and has no desaccharification effect. The cleavage of the malonyl group might be promoted by microbial esterases [21,22]. L. 8

curvatus JD0-31 had no esterase activity, whereas L. plantarum K2-12 had weak positive esterase activity.

3.2. Changes to malonyl- and acetyl-conjugated isoflavone glycosides in soybean extract by inoculation with two Lactobacillus species. Changes to twelve isoflavones in soybean extract by L. plantarum K2-12 and L.

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curvatus JD0-31 were investigated for 9 d post-inoculation as shown in Figures 3 and 4. The bio-converting changes of malonyl- and acetyl-conjugated isoflavone glycosides showed different patterns among isoflavone types rather than between lactobacilli, except for malonyl

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daidzin (Fig. 3). In the case of inoculation with L. plantarum K2-12, malonyl daidzin degraded

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faster during the initial 3 d and maintained a consistent level until day 9; for L. curvatus JD031, the content decreased slightly after inoculation during the initial 3 d and decreased again

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between days six and nine. The final content of malonyl daidzin after 9 d was not significantly different between lactobacilli and there were no significant differences in other malonyl- and

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acetyl-conjugated glucosides at the end of inoculation. After the bacterial inoculation, malonyland acetyl-conjugated glycosides in soybean extract were degraded by both lactobacilli (Fig.

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3). Malonyl genistin and acetyl daidzin were the most degradable components, exhibiting approximately 50% degradation compared with the other components. Interestingly, malonyl-

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and acetyl-conjugated glycitins showed less degradable patterns over the 9 d following lactobacilli inoculation.

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3.3. Changes to β-glycosides and aglycones in soybean extract during inoculation with two Lactobacillus species The aglycones (daidzein, genistein, and glycitein) and their respective β-glycosides showed differences between the two lactobacilli fermentations (Fig. 4). Most of β-glycosides and aglycones in their contents were changed more by L. plantarum with higher level of enzyme activities (Table 1). Highly enzymatically active L. plantarum K2-12 rapidly degraded

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daidzin within 3 d and a large amount of daidzein accumulated as a byproduct. Daidzin content gradually increased over the remaining 6 d, resulting in byproducts due to malonyl- and acetylconjugated glucoside degradation.

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The amount of daidzein after 3 d was maintained at a relatively high concentration. Alternately, in the case of L. curvatus JD0-31, daidzin content gradually increased until 7 d and

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rapidly decreased between days 7 and 9 (Fig. 4A). Daidzein content also gradually increased

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until 7 d and dramatically increased between days 7 and 9 (Fig. 4B), and there appeared to be an inverse relationship between daidzin and daidzein content changes. Similar results

glycosides [5,23].

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describing this phenomenon have been shown in previous studies on deglycosylation of β-

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Malonyl genistin, acetyl genistin, and genistin content showed no differences between extracts inoculated with the two lactobacilli, but the accumulation rate of genistein was

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significantly different between the two (Fig. 3, Fig. 4). The accumulation pattern of genistein was similar to that of daidzein. It is something unique pattern that the genistein glucoside bioconversions were not significantly different between lactobacilli but differed in the accumulation of the aglycone when we used soybean extract. Therefore, further studies using standard isoflavones may helpful to understand the results. As our data were shown deglycosylation patterns (Fig. 3, Fig. 4), it is generally observed that different microorganisms 10

produce different deglycosylation patterns between daidzein- and genistein-glucosides during inoculation period [24]. Interestingly, the degradation of glycitin and accumulation of glycitein were promoted by L. curvatus JD0-31, which has a weak enzymatic activity compared with L. plantarum K212. The glycitin content rapidly decreased over 3 d and then leveled out in extract inoculated with L. curvatus JD0-31. The glycitin content in L. plantarum K2-12 inoculated extract

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maintained a consistent level over the 9 d post-inoculation. The glycitein that was detected over the first 3 d produced by L. curvatus JD0-31 was twice as much as for L. plantarum K2-12. The glycitin pattern, in which the content changed in the first 3 d and then maintained a constant

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level over the final 6 d, was a major change of the 12 isoflavones in soybean extract inoculated with the two lactobacilli.

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In extract inoculated with L. curvatus JD0-31, daidzein and genistein showed lower

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content over 7 d when compared to L. plantarum K2-12 inoculated extract, but the amount of those aglycones increased over the final 2 d such that the amounts on 9 d for both lactobacilli inoculations were not significantly different. The lower content of glycitein in L. plantarum

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K2-12 over the initial 7 d also increased to match the content in L. curvatus JD0-31 on 9 d, resulting in no significant differences on that day. Our results showed unique patterns compared

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to previous studies. Chien et al [5] showed the increasing trend of glycitein by four kinds of

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probiotics was like that of other aglycones; however, our study showed only glycitein and its glycoside forms were converted more rapidly in extracts inoculated with L. curvatus JD0-31. Glycitein and glycitin substituting by methoxy group (-OCH3) at the 6-position (Fig. 1) appears to be specific for both lactobacilli. Several studies have shown that glycitein conversion is distinct from other isoflavones. Da Silva et al [11] indicated that glycitin can be distinguished from other β-glycosides by microbial reaction, since all three aglycone amounts increased but 11

only glycitin increased among three β-glycoside forms during fermentation with Aspergillus. Further, in the fermentation of 10 kinds of steamed soybean by L. plantarum [25], all aglycones increased except for glycitein, which decreased in two kinds of soybeans. In 9 d of inoculation, only glycitin showed no significant difference with enzyme activity level (Table 1). Although the key enzyme of isoflavone bioconversion is known to be β-glucosidase, other factors may influence the glycosides of glycitein. Xu et al [26] noted that a methoxy group of glycitin

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contributed to structural degradation, and when three β-glycosides were heat-treated, glycitin remained in the smallest concentration. Structural instability of glycitin would result in differences with other aglycones.

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In the summarization of relevant isoflavone changes in significant vonversion, six isoflavones (malonyl daidzin, daidzin, daidzein, genistein, glycitin, and glycitein) in soybean

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showed significant differences in conversion patterns between the two lactobacilli. Malonyl daidzin and daidzin rapidly decreased within 3 d by L. plantarum K2-12 accompanying rapid

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increase in daidzein. Daidzin in L. curvatus JD0-31 inoculum was gradually accumulated for 7 d supposing the slow degradation of daidzin and continuous accumulation of daidzin by

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degradation of malonyl- and acetyl- daidzins. It was not predicted that the accumulation of genistein showed differences between lactobacilli where the decrease of malonyl genistin,

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acetyl genistin, and genistin did not significantly differ between lactobacilli. It may be due to

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the different degree of metabolic degradations among isoflavones by lactobacilli. Deglycosylation of glycitein glucosides was shown unique patterns as it was compared to deglycosylation of other isoflavone types. Glycitin was degraded faster by L. curvatus JD0-31 than by L. plantarum K2-12. The accumulation of glycitein was promoted by L. curvatus JD031 unlike other aglycones. Changes to β-glucosides and aglycones differed between the two lactobacilli-treated 12

extracts in terms of timing, but the ultimate content did not differ during the inoculation except for glycitin. It was determined that the distribution and quantitative determination of isoflavones could be controlled by both lactobacilli and their inoculation time depending on what the target isoflavone is.

3.4. Changes in nine single isoflavone standards by inoculation with two Lactobacillus

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species

The conversion profiles of nine isoflavone standards (three malonyl glycosides, three β-glycosides, and three aglycones) inoculated with two lactobacilli are shown in Figures 5 and

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6. The three acetyl glycosides were not selected because they occur in trace amounts in

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soybeans and are undetectable. Further, their concentrations in extracts inoculated with the two lactobacilli were not significantly different. L. plantarum K2-12 degraded more isoflavone

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standards, except for malonylglycitin and glycitein, than L. curvatus JD0-31 (Table 2). All three malonyl glycosides were reduced during inoculation, but concentrations of malonyl daidzin

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and malonyl genistin were more rapidly reduced by L. plantarum K2-12 with weak positive activities of esterases inducing the cleavage of malonyl group, especially malonyl genistin

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(Table 2, Fig. 5). Malonyl glycitin showed no significant differences between extracts inoculated to lactobacilli although L. curvatus JD0-31 has no esterases activities (Table 2, Fig.

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5). The compound had the highest reduction rate for both lactobacilli and coincided with the highest β-glycoside production rate (Fig. 5C). Production of by-products by degradation of malonyl glycosides was significantly different between lactobacilli. The accumulation of βglycosides was lower and the accumulation of aglycones was higher in extracts inoculated with L. plantarum K2-12 compared with L. curvatus JD0-31, indicating that L. plantarum K2-12 rapidly converted β-glycosides produced from malonyl glycosides to aglycones. Acetyl 13

glycosides were not detected in the inoculation of malonyl glycoside standards by lactobacilli. Thermal treatment or pH control can convert malonyl glycosides to acetyl glycoside forms [27], but lactobacilli had no effect on this conversion. Decarboxylation of malonyl glycosides did not occur in either lactobacilli inocula. The inoculation by-product profiles of single malonyl glycosides in soybean extract varied. Because certain plant extracts can inhibit enzyme activities, bioconversion of endogenous isoflavones in raw soybean extract could affect profiles

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of by-products such as organic acids and phenolic acids during lactobacilli inoculation [28]. A large amount of hydrolysis was observed in β-glycoside standards rather than malonyl-conjugated glycosides. Three β-glycoside standards in the inoculation of L. plantarum

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K2-12, showing positive β-galactosidase, α-glucosidase, and β-glucosidase, were dramatically degraded with 88 to 100% of degradation rate (Fig. 2, Table 2). On the other hand, the standards

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in the inoculation of L. curvatus JD0-31, having only weak positive β-glucosidase activity,

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were weakly degraded with 12 to 36% of degradation rate (Fig. 2, Table 2). L. plantarum K2-12 completely degraded daidzin and genistin within 1 d with the production of aglycone (Fig. 6A). In contrast, the hydrolysis rate of daidzin and genistin for L.

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curvatus JD0-31 was 11% and 42% in 1 d, respectively (Table 2). No further hydrolysis of βglycosides occurred after 2 d for either lactobacillus inoculation. Raimondi et al. [23] reported

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that conversion of daidzin into daidzein depends on the presence of β-glucosidase. L. plantarum

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K2-12, with high β-glucosidase activity, more rapidly hydrolyzed the three β-glycosides. Mahungu et al. [29] reported that genistein was less stable to heat than daidzin, and in our study, genistin was found to be more susceptible to bacterial hydrolysis than daidzin. This rapid conversion of genistin to genistein would result in the flat line pattern of genistin content in lactobacilli inoculated soybean (Fig. 4A). Daidzein and genistein produced from their respective β-glycosides by L. plantarum K2-12 were degraded after 2 d inoculation. 14

Degradation of glycitin also proceeded rapidly in β-glycoside standards inoculated with L. plantarum K2-12, but the amount of glycitein produced from glycitin was unexpectedly higher for L. curvatus JD0-31. Inoculating with L. plantarum K2-12 resulted in a large amount of glycitin degradation, while accumulation of glycitein was observed at a very low level. These phenomena may be explained by the following reasons: First, the degradation rate of glycitein standard was 82%, which was significantly higher than that of other aglycones (Table

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2, Fig. 6B). According to Xu et al. [26], glycitein is the most heat-sensitive among the three aglycones. The aglycones produced from their glycoside degradation undergo subsequent degradation in dependent of microorganisms [30]. In this study, we found that glycitein was the most degraded among the aglycones, indicating that glycitein is the most vulnerable not

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only to heat but also to microorganisms. Thus, any glycitein that accumulated from degradation

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of glycitin would have been degraded in large amounts. Second, some of the degraded glycitin would have been converted to isoflavone derivatives other than glycitein. The production

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pattern of glycitein in the glycitin standard inoculated by the two lactobacilli was like those obtained with soybean extract inoculation. In contrast, the glycitin pattern in the inoculated

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soybean extract differed from that of the glycitin standard inocula (Fig. 4A), and may be caused

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by other endogenous factors found in the soybean extract.

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4. Conclusion

The present study reports several novel patterns on isoflavone bioconversion of

soybean extracts by two enzymatically different lactobacilli. In conclusion, the three structurally different types of aglycones and their glycosides had distinct conversion patterns each other by two lactobacilli. Also, fermentation time is important for the level of each isoflavone. Malonyl and acetyl glycosides showed no significant differences between two 15

lactobacilli except for malonyl daidzin. The rapid degradation of malonyl daidzin and daidzin by L. plantarum with high enzyme activity was observed, which resulted in the large production of daidzein. Malonyl genistin was the easiest compound that can be hydrolyzed by lactobacilli, exhibiting about 50% reduction of original yield. Genistein production pattern was similar to that of daidzein, but genistin was distinguished from daidzin by maintaining low levels during inoculation regardless lactobacilli. Numerous previous studies have rarely reported on glycitin

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and glycitein bioconversion during fermentation. In this study, it was observed that the production of glycitein resulting the degradation of glycitin was more promoted by L. curvatus than L. plantarum, while the production of other aglycones resulting degradation of their glucosides was shown oppositely. However, it was shown that the low production of glycitein

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during fermentation was detected despite the fair amount of glycitin degradation. The reason

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could be interpreted by high degradation of glycitein itself by lactobacilli. Our results with the fermentation of standard isoflavone individuals demonstrated that glycitein was the most

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degradable aglycone among three aglycones.

Acknowledgements

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This work was supported by a grant from the Radiation Technology R&D program (NRF-2017M2A2A6A5018538) through the National Research Foundation of Korea funded

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by the Ministry of Science and ICT.

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[10] J. Chun, G.M. Kim, K.W. Lee, I.D. Choi, G.-H. Kwon, J.-Y. Park, S.-J. Jeong, J.-S. Kim,

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phenolic compounds, J. Microbiol. Biotechnol. 27 (2017) 869-77.

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Spectrometry, and PCR-DGGE, J. Microbiol. Biotechnol. 28(7) (2018) 1112-1121.

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Bean (Rhynchosia Molubilis: Yak-Kong), Korean. J. Nutr. 36(1) (2003) 32-39.

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[25] J.H. Lee, B. Kim, C.E. Hwang, M.A. Haque, S.C. Kim, C.S. Lee, S.S. Kang, K.M. Cho, D.H. Lee. Changes in conjugated linoleic acid and isoflavone contents from fermented

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soymilks using Lactobacillus plantarum P1201 and screening for their digestive enzyme inhibition and antioxidant properties, J. Funct. Foods. 43 (2018) 17-28.

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[28] S.H. Eom, B.I. Reisch. Inhibitors of neomycin phosphotransferase II enzyme-linked

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immunosorbent assay in grapevine (Vitis vinifera L.) leaves, Vitis. 47(1) (2008) 7-9.

[29] S. Mahungu, S. Diaz-Mercado, J. Li, M. Schwenk, K. Singletary, J. Faller. Stability of isoflavones during extrusion processing of corn/soy mixture, J. Agric. Food Chem. 47(1)

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(1999) 279-284.

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[30] P. Gaya, Á. Peirotén, J.M. Landete. Transformation of plant isoflavones into bioactive

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isoflavones by lactic acid bacteria and bifidobacteria, J. Funct. Foods. 39 (2017) 198-205.

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Figure 1. Chemical structure of isoflavone in soybean, consisting of 12 forms.

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Figure 2. Determination of enzymatic activity from two Lactobacillus species by API Zym kit.

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Grades of the activities are as follows: white, negative; gray, weak positive; black, positive.

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Figure 3. Malonyl- (A) and acetyl- (B) conjugated isoflavone glycosides in soybean extract during inoculation with L. plantarum K2-12 and L. curvatus JD0-31. Values marked with

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asterisks are significantly different between L. plantarum K2-12 and L. curvatus JD0-31

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inocula (Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicates no significance).

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Figure 4. β-glycosides (A) and aglycones (B) of isoflavones in soybean extract during

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inoculation with L. plantarum K2-12 and L. curvatus JD0-31. Values marked with asterisks are

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significantly different between L. plantarum K2-12 and L. curvatus JD0-31 inocula (Student’s

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t-test, *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicates no significance).

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Figure 5. Inoculation of malonyl isoflavone standards by L. plantarum K2-12 and L. curvatus

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JD0-31. A, malonyl daidzin and its by-products; B, malonyl genistin and its by-products; C, malonyl glycitin and its by-products. Values marked with asterisks are significantly different

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between L. plantarum K2-12 and L. curvatus JD0-31 inocula (Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicates no significance).

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Figure 6. Inoculation of β-glycoside (A) and aglycone (B) standards by L. plantarum K2-12

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and L. curvatus JD0-31. Values marked with asterisks are significantly different between L. plantarum K2-12 and L. curvatus JD0-31 inocula (Student’s t-test, *p < 0.05, **p < 0.01, ***p

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< 0.001, and ns indicates no significance).

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Table 1. Relationships between different activities shown to 6 enzymes of Lactobacilli and isoflavone changes in soybean extracts at either 3 or 9 days of inoculation.

3 days of inoculation

9 days of inoculation

Isoflavones p-value

r2

p-value

Malonyl daidzin

0.8339

0.0110*

0.0361

0.7182ns

Malonyl genistin

0.1153

0.5103ns

0.2329

0.3323ns

Malonyl glycitin

0.6947

0.0393*

0.0040

0.9050ns

Acetyl daidzin

0.3323

0.2311ns

0.4418

0.1498ns

Acetyl genistin

-

-

-

-

Acetyl glycitin

0.7893

0.0180*

0.2276

0.3387ns

Daidzin

0.9979

0.0000***

0.3241

0.2383ns

Genistin

0.5311

0.1004ns

0.3577

0.2098ns

Glycitin

0.8719

0.0064**

Daidzein

0.9388

0.0014**

Genistein

0.7702

Glycitein

0.8416

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r2

0.0092**

0.0718

0.6077ns

0.0216*

0.1633

0.4269ns

0.0100**

0.0671

0.6202ns

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0.8478

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respectively.

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Following signs *, **, ***, and ns indicate p < 0.05, p < 0.01, p < 0.001, and no significance,

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Table 2. The degradation rate (%) of isoflavone standards at either 1 or 5 d after inoculation of lactobacilli. L. curvatus JD0-31

1d

5d

1d

5d

Malonyl daidzin

8.89±1.30

32.48±2.15

6.18±1.75

19.11±0.01

Malonyl genistin

9.27±1.52

27.14±0.01

0.08±2.31

8.65±3.22

Malonyl glycitin

3.49±1.70

33.04±0.99

6.98±5.17

36.71±3.83

Daidzin

100.00±0.00

100.00±0.00

11.41±2.72

12.04±0.01

Genistin

97.10±0.83

100.00±0.00

41.86±1.27

36.00±6.92

Glycitin

64.51±1.19

88.32±0.97

22.95±2.80

19.68±0.74

Daidzein

66.48±8.19

52.11±2.24

53.12±1.25

43.43±4.04

Genistein

32.70±3.61

28.21±0.70

Glycitein

81.34±0.78

83.17±1.14

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L. plantarum K2-12

6.68±6.37

81.30±1.43

80.32±0.38

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35.15±2.13

The degradation rate (%) = (IsoC1-IsoC2)/IsoC1×100%

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IsoC1 : The initial content of isoflavone standard (µg·mL-1)

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IsoC2 : The content of isoflavone standard after inoculation (µg·mL-1)

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