Bioconversion of Daidzin to Daidzein by Lactic Acid Bacteria in Fermented Soymilk

Abstract

The purpose of this study is to select LAB that produce daidzein during soymilk fermentation from among 32 strains isolated from Japanese traditional fermented sushi and pickles. Fourteen strains showed more than 0.5% titratable acidity at 48 h fermentation in soymilk. These strains were applied to a daidzein production test. Seven strains including 5 Lactobacillus plantarum strains, 1 Lb. sakei subsp. sakei strain, and 1 Lb. coryniformis strain yielded more than 4.0 µg/g daidzein in soymilk after 48 h fermentation. Lb. plantarum JAB2001 showed the highest titratable acidity (0.5%) and the highest daidzein yield (10.10 µg/g) during soymilk fermentation at 40°C for 12 h. Strain JAB2001 could be used as a starter culture of fermented soymilk products.

Introduction

The intake of isoflavones in soy foods is thought to be associated with the low incidence of breast cancer in Japanese women, and these compounds have many functions for human health such as increasing bone mass density and reducing symptoms of postmenopausal syndrome (Chang 2002). Isoflavones are present as glucoside conjugates in soy-based foods. Glucosides such as daidzin, genistin, and glycitin are biologically inactive and are not bioavailable (King and Bignell 2000). Ingested glucoside isoflavones are hydrolyzed into aglycones by intestinal microflora; these aglycones are biologically active and bioavailable. Aglycones such as daidzein, genistein, and glycitein have an analogous chemical structure with estradiol, and exhibit weak estrogenic activity. Aglycones such as daidzein are linked to the prevention and potential treatment of hormone-dependent disorders including osteoporosis, cardiovascular disease, and cancer (Setchell and Cassidy 1999). Equol, which is derived from daidzein, is the most effective among the isoflavone derivatives in stimulating an estrogenic response (Setchell and Cassidy 1999). Equol is converted from daidzein by specific microflora; however, only 30–50% of individuals can produce equol from the ingested glucoside isoflavones in soy foods (Arai et al., 2000, Frankenfeld et al., 2005). Thus, more people would receive the health benefits of soy food products, via their estrogenic activities, that are enriched with daidzein or equol.

Fermented soymilk products are similar to fermented milk products such as yoghurt. Soymilk is fermented with lactic acid bacteria (LAB) and is then solidified. The isoelectric point of soymilk is approximately pH 4.5, and soymilk is solidified around pH 4.5, at approximately 0.6% titratable acidity.

The purpose of this study is to select LAB that produce daidzein during soymilk fermentation. The effects of fermentation temperature and time on the daidzein yield of the LAB strain were also investigated. LAB strains isolated from Japanese traditional fermented sushi and pickles were used in this study.

Materials and Methods

Bacterial strains    Thirty-two LAB strains, 10 cocci and 22 lactobacilli, were used in this study (Table 1). These strains were isolated from Japanese traditional fermented food, and were identified based on morphological and physiological characteristics and by 16S rRNA sequence analysis (Tsuda et al., 2012, 2015). LAB strains were subcultured in 5 mL MRS broth (de Man et al., 1960) at 30°C. Cultures were centrifuged (1,000 × g, 10 min), and the cells were washed twice with 5 mL sterile 0.85% NaCl solution. The cell suspensions were used as the inoculants, and 1% inoculants were used in all tests.

Table 1. LAB strains used in this study.

Origin	Species	Strain No.
Funazushi	Streptococcus salivarius	AB3002
	Lactobacillus plantarum	FM2001
	Lb. plantarum	FM2003
	Lb. plantarum	JAB2001
	Lb. plantarum	JAM2001
	Lb. plantarum	JAM3701
	Lb. casei	HM3701
	Lb. farciminis	HM2001
	Lb. buchneri	GM3701
Pickles	Pediococcus parvulus	SAB03
	Pc. parvulus	SAB07
	Pc. parvulus	SBB09
	Pc. parvulus	SBB11
	Pc. parvulus	TAB01
	Pc. parvulus	TAB02
	Pc. ethanolidurans	SBB10
	Pc. ethanolidurans	WAB06
	Pc. ethanolidurans	WAB13
	Lb. plantarum	WAB01
	Lb. plantarum	WAB04
	Lb. plantarum	WCB01B
	Lb. coryniformis	SAB01
	Lb. coryniformis	WBB05
	Lb. coryniformis	WBB07
	Lb. alimentarius	SBB06
	Lb. curvatus	WAB05
	Lb. sakei subsp. sakei	SBB08
	Lb. sakei subsp. sakei	SBB12
	Lb. sakei subsp. sakei	WCB01S
	Lb. sakei subsp. sakei	WCB04
	Lb. sakei subsp. sakei	WCB05
	Lb. sakei subsp. sakei	WCB11

Lactic acid production in soymilk    Pure soymilk (solid soybean conc.: 9%, protein conc.: 4.7 g/100 mL; Marusan-ai, Aichi, Japan) was purchased from local markets. The soymilk was inoculated with individual LAB strains and incubated at 30°C. The titratable acidity was determined at 0, 6, 12, 24, 36, and 48 h. All tests were performed in triplicate.

Cellobiose fermentation    LAB strains were incubated in MRS-C broth (MRS broth in which cellobiose was substituted for glucose and supplemented with 0.006% (w/v) bromocresol purple) at 30°C to select for LAB with β-glucosidase activity (Chun et al., 2007). The color change of bromocresol purple in the broth by acid production was observed until 5 d incubation.

Sample preparation for isoflavone analysis    The effect of fermentation temperature on isoflavone conversion was investigated. Pure soymilk was inoculated with individual LAB strains and incubated at 20, 30, and 40°C. Isoflavones were extracted from the fermented soymilk using a modified method of OMA 2001.10 (Ogita et al., 2015). A 5-g sample of the fermented soymilk was applied to the extraction method. The obtained extraction was mixed with an equal volume of an internal standard solution including daidzin (10 µg/mL), daidzein (5 µg/mL), and equol (20 µg/mL). All isoflavone standards were purchased from Nacalai Tesque, Kyoto, Japan. The mixture was applied to HPLC analysis. All tests were performed in triplicate.

HPLC analysis    Daidzin, daidzein, and equol in the sample were analyzed using a reverse-phase HPLC system (880 pump system, 875 UV detector, and 860 column oven, Jasco, Tokyo, Japan; column: TSKgel ODS-100V 5 µm, Tosoh, Tokyo, Japan). The mobile phase A was 85% water, 15% acetonitrile, and 0.1% glacial acetic acid, and the mobile phase B was 50% water, 50% acetonitrile, and 0.1% glacial acetic acid. Absorbance was measured at 254 nm for daidzin and daidzein, and at 254 and 280 nm for equol. The flow rate was 1.5 mL/min, and the column temperature was 40°C. A 20-µL sample was used for HPLC analysis. Daidzin and daidzein were analyzed by gradient elution as follows: 0 to 60% of B, 0 to 20 min; 60 to 100% of B, 20 to 50 min; 100 to 0% of B, 50 to 55 min. Equol was analyzed using an isocratic flow of 25% of mobile phase B for 50 min. Equol was detected at approximately 36 min, and it was confirmed that the peak was higher at 280 nm than that at 254 nm.

Statistical analysis    To identify differences in the daidzein concentration in the fermented soymilk, one-way analysis of variance (ANOVA) was applied to the means, and the Student-Newman-Keuls test (P<0.05) was applied using Statview 5.0 software (SAS Institute, Cary, NC, USA).

Results and Discussion

Acid production in soymilk    Fourteen strains produced more than 0.5% lactic acid at 48 h (Table 2). Lactobacillus plantarum JAB2001 produced the highest lactic acid in soymilk. The titratable acidity increased from 0.18% at the beginning of fermentation at 30°C to 0.78% at 48 h. Meanwhile, 12 strains produced lactic acid levels of less than 0.3% at 48 h.

Table 2. Titratable acidity (%) of fermented soymilk.

Species	Strain No.	Time (h)						Cellobiose fermentation
		0	6	12	24	36	48
Lb. plantarum	JAB2001	0.17 (0.01)*	0.20 (0.00)	0.34 (0.01)	0.58 (0.05)	0.65 (0.03)	0.79 (0.02)	+
Lb. plantarum	JAM2001	0.19 (0.00)	0.18 (0.02)	0.31 (0.06)	0.55 (0.08)	0.69 (0.02)	0.76 (0.04)	+
Lb. plantarum	FM2003	0.16 (0.01)	0.17 (0.00)	0.28 (0.01)	0.51 (0.05)	0.62 (0.06)	0.75 (0.01)	+
Lb. plantarum	FM2001	0.17 (0.01)	0.20 (0.03)	0.27 (0.05)	0.52 (0.02)	0.65 (0.02)	0.75 (0.01)	+
Lb. farciminis	HM2001	0.16 (0.01)	0.17 (0.01)	0.24 (0.01)	0.43 (0.02)	0.63 (0.01)	0.72 (0.05)	+
Lb. plantarum	JAM3701	0.19 (0.01)	0.18 (0.02)	0.33 (0.08)	0.56 (0.03)	0.67 (0.06)	0.72 (0.07)	+
Pc. ethanolidurans	WAB06	0.17 (0.01)	0.20 (0.01)	0.26 (0.04)	0.44 (0.08)	0.61 (0.02)	0.67 (0.01)	+
St. salivarius	AB3002	0.15 (0.01)	0.25 (0.01)	0.47 (0.00)	0.59 (0.02)	0.60 (0.01)	0.66 (0.00)	−
Lb. sakei subsp. sakei	SAB04	0.16 (0.01)	0.18 (0.03)	0.21 (0.14)	0.43 (0.12)	0.56 (0.09)	0.66 (0.04)	−
Lb. plantarum	WAB01	0.18 (0.01)	0.19 (0.00)	0.24 (0.01)	0.51 (0.05)	0.55 (0.08)	0.66 (0.01)	+
Pc. ethanolidurans	WAB13	0.17 (0.01)	0.21 (0.00)	0.27 (0.01)	0.42 (0.01)	0.50 (0.04)	0.61 (0.02)	+
Lb. plantarum	WAB04	0.18 (0.02)	0.18 (0.02)	0.26 (0.02)	0.45 (0.04)	0.60 (0.02)	0.61 (0.02)	+
Lb. plantarum	WCB01B	0.16 (0.00)	0.21 (0.01)	0.24 (0.03)	0.45 (0.06)	0.51 (0.08)	0.54 (0.07)	+
Lb. coryniformis	WBB07	0.17 (0.01)	0.18 (0.01)	0.19 (0.02)	0.42 (0.00)	0.45 (0.01)	0.52 (0.00)	-
Lb. sakei subsp. sakei	WCB04	0.16 (0.00)	0.18 (0.02)	0.20 (0.00)	0.22 (0.01)	0.45 (0.01)	0.48 (0.06)	−
Lb. curvatus	WAB05	0.16 (0.01)	0.16 (0.00)	0.25 (0.01)	0.35 (0.00)	0.41 (0.01)	0.46 (0.03)	−
Lb. sakei subsp. sakei	WCB01S	0.18 (0.02)	0.21 (0.03)	0.28 (0.17)	0.30 (0.07)	0.29 (0.05)	0.43 (0.02)	−
Pc. ethanolidurans	SBB10	0.15 (0.00)	0.14 (0.00)	0.15 (0.02)	0.23 (0.00)	0.28 (0.05)	0.35 (0.20)	+
Pc. parvulus	SBB09	0.16 (0.01)	0.17 (0.02)	0.21 (0.02)	0.21 (0.04)	0.29 (0.09)	0.34 (0.09)	+
Lb. alimentarius	SBB06	0.16 (0.00)	0.17 (0.00)	0.17 (0.01)	0.22 (0.01)	0.23 (0.01)	0.33 (0.01)	+
Lb. coryniformis	SAB01	0.16 (0.00)	0.17 (0.00)	0.18 (0.01)	0.20 (0.02)	0.26 (0.03)	0.31 (0.07)	−
Lb. casei	HM3701	0.17 (0.01)	0.19 (0.01)	0.20 (0.00)	0.23 (0.01)	0.24 (0.04)	0.30 (0.09)	+
Lb. sakei subsp. sakei	WCB05	0.16 (0.00)	0.19 (0.01)	0.19 (0.01)	0.20 (0.02)	0.26 (0.09)	0.30 (0.15)	−
Pc. parvulus	SAB07	0.16 (0.01)	0.17 (0.02)	0.18 (0.01)	0.18 (0.01)	0.21 (0.01)	0.22 (0.03)	+
Pc. parvulus	SAB03	0.16 (0.01)	0.17 (0.01)	0.17 (0.01)	0.20 (0.01)	0.20 (0.01)	0.21 (0.01)	+
Lb. buchneri	GM3701	0.16 (0.00)	0.16 (0.01)	0.17 (0.00)	0.18 (0.00)	0.19 (0.00)	0.19 (0.00)	−
Pc. parvulus	TAB01	0.16 (0.01)	0.14 (0.00)	0.16 (0.01)	0.16 (0.00)	0.17 (0.00)	0.19 (0.01)	+
Lb. sakei subsp. sakei	SBB12	0.16 (0.00)	0.17 (0.00)	0.17 (0.00)	0.17 (0.00)	0.18 (0.01)	0.18 (0.00)	−
Lb. coryniformis	WBB05	0.16 (0.00)	0.17 (0.02)	0.18 (0.01)	0.17 (0.01)	0.17 (0.01)	0.18 (0.01)	−
Pc. parvulus	SBB11	0.15 (0.00)	0.16 (0.01)	0.17 (0.00)	0.17 (0.00)	0.18 (0.00)	0.18 (0.00)	+
Pc. parvulus	TAB02	0.16 (0.00)	0.15 (0.01)	0.15 (0.01)	0.16 (0.00)	0.16 (0.00)	0.18 (0.00)	+
Lb. sakei subsp. sakei	SBB08	0.16 (0.00)	0.19 (0.03)	0.17 (0.01)	0.18 (0.00)	0.17 (0.00)	0.17 (0.00)	−
Lb. sakei subsp. sakei	WCB11	0.16 (0.00)	0.16 (0.01)	0.17 (0.01)	0.17 (0.01)	0.17 (0.02)	0.17 (0.00)	−

*:  values in parentheses represent standard deviation.

+: positive; −: negative.

All 8 Lb. plantarum strains tested showed more than 0.5% acidity, while 5/6 Pediococcus parvulus strains tested showed less than 0.3% acidity. Acid production of the 7 Lb. sakei subsp. sakei strains tested varied according to the strain, from 0.17 to 0.66%.

Cagno et al. (2010) reported that a mixed starter culture of Lb. plantarum, Lb. fermentum, and Lb. rhamnosus showed a decrease in pH from 6.7 to 5.5 after 96 h incubation at 30°C with stirring. In this study, Lb. plantarum JAB2001 produced 0.78% lactic acid (approximately pH 3.9) after 48 h incubation at 30°C without stirring. Thus, this strain can be used as the single starter culture for soymilk fermentation.

Cellobiose fermentation    Cellobiose fermentation abilities of the LAB strains are shown in Table 2. Eleven strains fermented cellobiose among the 14 strains producing more than 0.5% acidity.

Isoflavone glucosides are hydrolyzed to bioavailable aglycones by intestinal β-glucosidases (Chun et al., 2007). Cellobiose is a disaccharide consisted of β1–4 linked glucoses. The cellobiose fermentation test was used as an indicator of β-glucosidase activity in this study. Eleven strains, which produced more than 0.5% acidity and fermented cellobiose, were applied to the daidzein production analysis.

Daidzein production in soymilk    First, daidzin, daidzein, and equol concentrations in the fermented soymilk after 48 h incubation were investigated to select LAB with high daidzein yields (Table 3). Daidzin and daidzein concentrations in soymilk were almost the same as previous studies (Ishimi et al., 2009, Tsuangalis et al., 2005). Equol was not detected in any of the fermented soymilks.

Table 3. Daidzein concentration in fermented soymilk after 48 h fermentation.

		Isoflavon conc.(µg/g)
	Strain No.	Daidzin	Daidzein	Equol
Soymilk		45.40 (1.66)*	1.67 (0.06)	ND
Lb. plantarum	JAB2001	14.18 (3.39)	7.72a (0.28)	ND
Lb. plantarum	JAM2001	9.46 (1.50)	6.13c (0.25)	ND
Lb. plantarum	FM2003	12.33 (1.68)	7.21ab (0.36)	ND
Lb. plantarum	FM2001	15.59 (0.96)	3.64e (0.18)	ND
Lb. farciminis	HM2001	15.08 (1.06)	ND	ND
Lb. plantarum	JAM3701	ND	6.52bc (0.09)	ND
Pc. ethanolidurans	WAB06	41.90 (1.13)	ND	ND
St.salivarius	AB3002	37.99 (2.96)	2.85f (0.43)	ND
Lb. sakei subsp. sakei	SAB04	7.42 (0.53)	4.30d (0.68)	ND
Lb. plantarum	WAB01	37.62 (1.07)	ND	ND
Pc. ethanolidurans	WAB13	42.31 (3.76)	ND	ND
Lb. plantarum	WAB04	12.95 (0.14)	1.49g (0.09)	ND
Lb. plantarum	WCB01B	11.89 (0.48)	7.47a (0.51)	ND
Lb. coryniformis	WBB07	5.90 (0.94)	7.26ab (0.49)	ND

*:  values in parentheses represent standard deviation.

ND: not detected.

Means in daidzein row followed by different superscript are significantly different (P < 0.05).

Seven LAB strains including 5 Lb. plantarum strains, 1 Lb. sakei subsp. sakei strain, and 1 Lb. coryniformis strain yielded more than 4.0 µg/g daidzein after 48 h soymilk fermentation. The highest daidzein yield (approximately 7.5 µg/g) was obtained using Lb. plantarum JAB2001, FM2003, WCB01B and Lb. coryniformis WBB07, respectively. Five Lb. plantarum strains yielded daidzein in soymilk, while daidzein was not increased with the other 2 Lb. plantarum strains. All Lb. plantarum strains showed decreased daidzin concentration except for strain WAB01. Daidzin was not degraded with strain WAB01 after 48 h fermentation. Other than Lb. plantarum, Lb. sakei subsp. sakei SAB04 and Lb. coryniformis WBB07 showed daidzein accumulation. Although daidzin was decreased with Lb. plantarum WAB04 and Lb. farciminis HM2001, respectively, daidzein was not detected in the fermented soymilk.

Among 11 cellobiose-fermenting strains, Lb. plantarum WAB01, Pc. ethanolidurans WAB06 and WAB13 hardly hydrolyzed daidzin. These 3 strains did not degrade daidzin after 48 h fermentation, respectively. It appears that these strains have low daidzin hydrolase activity despite the fact that they can ferment cellobiose. On the other hand, 2 strains among the cellobiose non-fermenting 3 strains, Lb. sakei subsp. sakei SAB04 and Lb. coryniformis WBB07, hydrolyzed daidzin in soymilk. This suggests that the cellobiose fermentation test is not a suitable indicator of isoflavone glucosides hydrolysis in soymilk.

Both daidzin and daidzein were not accumulated using Lb. plantarum WAB04 and Lb. farciminis HM2001. These strains likely degraded daidzin or daidzein to other forms.

In this paper, 3 Lb. plantarum and 1 Lb. coryniformis strains yielded approximately 7.5 µg/g daidzein after 48 h soymilk fermentation, respectively. Tsangalis et al. (2003) reported 6.2 µg/ mL daidzein yield after 48 h soymilk fermentation at 37°C with Bifidobacterium animalis. Cagno et al. (2010) reported a daidzein yield of 57.0 µmol/L (approximately 14.49 µg/mL) after 96 h soymilk fermentation at 30°C using a mixed starter culture, including Lb. plantarum, Lb. fermentum and Lb. rhamnosus, compared to 6.1 µmol/L (1.6 µg/mL) at 0 h. Although there are differences in the preparation and ingredients of the soymilks used in the studies, the daidzin concentration in our study approached 1.6 µg/mL. From these results, 3 Lb. plantarum and 1 Lb. coryniformis strains were capable of yielding daidzein when the strain was used as a single starter culture for soymilk fermentation. Notably, the effect that mixed cultures have on daidzein production remains to be investigated.

In this study, almost all Lb. plantarum strains could degrade daidzin, and half of the strains could accumulate daidzein in soymilk. Lb. plantarum species are potential candidates as daidzein-producing LAB, and could be used as starters for fermented soymilk products. Lb. plantarum JAB2001 successfully produced acid and accumulated daidzein during soymilk fermentation. Next, the effects of fermentation temperature and time on daidzein production were investigated in soymilk fermented with strain JAB2001.

The influence of fermentation temperature on daidzein production    The influence of fermentation temperature on the daidzein production of strain JAB2001 was investigated in soymilk (Fig. 1). The highest titratable acidity was at 40°C at all periods. The titratable acidity did not exceed 0.5% at 20°C. The highest daidzein yield (10.10 µg/g) was observed at 40°C, followed by 30°C and 20°C. At 40°C, the maximum daidzein concentration was reached at 12 h, decreasing thereafter. At 30°C, the maximum daidzein yield of 8.57 µg/g was reached at 24 h, decreasing thereafter. At 20°C, the daidzein concentration was slowly increased up to 48 h. The daidzein yields were almost equivalent (approximately 7.0 µg/g) at all tested temperatures after 48 h fermentation. At 30 and 40°C, the daidzin concentration was decreased over the first 12 h. The daidzin concentration then slowly decreased until 24 h, and then greatly decreased up to 48 h. Although there was an increase in daidzein concentration over the first 12 h concomitant with daidzin degradation, the daidzein concentration was almost level from 24 to 48 h.

Fig. 1.

Daidzin and daidzein concentraion in the fermented soymilk with Lb. plantarum JAB2001.

The highest titratable acidity and daidzein concentration were observed with 12 h fermentation at 40°C. Hence, an incubation temperature of 40°C should be applied to produce daidzein-reinforced fermented soymilk products.

The daidzin concentration rapidly decreased during the initial 12 h and the last 24 h of fermentation. However, the daidzein concentration increased only during the initial 12 h of fermentation and was almost level from 24 to 48 h. This indicates that while daidzin was initially degraded to daidzein, it was degraded to another form during the latter part of the fermentation with strain JAB2001. The factors responsible for this difference remain to be clarified. However, similar results have been previously reported (Cagno et al., 2010, Chun et al., 2007, Raimondi et al., 2009, Tsangalis et al., 2002). These reports showed that daidzein was increased at the early period of fermentation, and leveled off thereafter. It is possible that daidzein was produced from daidzin when the titratable acidity was below 0.5%. Daidzein was increased throughout the fermentation at 20°C, and the titratable acidity was always below 0.5%. Similar results have been shown previously (Chun et al., 2007). These authors reported that isoflavone aglycones rapidly increased during the initial 6 h of fermentation, while there was no significant increase in aglycones concentration in soymilk fermented with Lb. paraplantarum KM for more than 6 h. In that study, the titratable acidity was greater than 0.5% after 6 h fermentation. Further work is needed to determine the effect of titratable acidity on daidzein accumulation by LAB in soymilk.

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