Enhanced β-Glucosidase Activity of Lactobacillus plantarum by a Strategic Ultrasound Treatment for Biotransformation of Isoflavones in Okara

Abstract

The enhanced β-glucosidase of Lactobacillus plantarum BCRC 10357 was investigated by a strategic ultrasound treatment on bacteria at different growth stages to induce biological stress response for biotransformation of isoflavones from glucosides to aglycones in okara. Without ultrasound, the highest β-glucosidase activity was occurred in 12-h incubation as 24.45 U/mL; while after ultrasound treatment at death phase, the highest β-glucosidase activity was greatly enhanced to 48.80 U/mL in 24-h re-incubation, showing the bacteria at early death phase behaving the highest resistance to stress than at other stages. With strategically ultrasound-treated L. plantarum BCRC 10357 to ferment okara in the broth at 37 °C, the fraction of bioactive aglycones (daidzein and genistein) in isoflavones was increased from 57% in 12 h to 89% in 36 h. The effective method to improve β-glucosidase released from L. plantarum was developed for the valorization of okara as potential functional ingredients in food.

Introduction

Lactic acid bacteria (LAB) have been widely used as probiotics or fermenting agents in fermented vegetables, dairy products, and meat products to offer advantages in characteristics of food, such as special flavor of fermented food, improvement of nutritional value, and extension of the shelf life of food. LAB can be served as important members of the normal intestinal fungus group in human gastrointestinal tract by producing lactic acid as the main metabolic product (Nuraida, 2015). Among the LAB group, Lactobacillus, Streptococcus and Bifidobacterium are the most common probiotics for human health by decomposing macromolecule components into smaller ones that are easily absorbed by human body and producing vitamins B₁, B₂, B₆, B₁₂, etc. to increase nutritional values of food (Fooks et al., 1999; Chiang and Pan, 2012). In addition, the growth of LAB in fermented foods also affects their functions in fermented foods, and effective methods to improve the growth of LAB are useful (Horie et al., 2018).

One of the enzymes released by LAB is β-glucosidase, which plays an important role in the metabolism of carbohydrates by LAB in the fermentation of plant foods (Michlmayr and Kneifel, 2014). LAB used in the fermentation of soybean products or side-products can bio-transform isoflavones from glucosides into the more bioactive aglycones (Tsai et al., 2009; Zhao and Shah, 2014). S. thermophilus, L. acidophilus, L. delbrueckii ssp. bulgaricus, L. casei, and L. plantarum have been reported to be used in the fermentation of soymilk (Donkor and Shah, 2008; Michlmayr and Kneifel, 2014). Such biotransformation efficiency is subjected to the release of β-glucosidase available and often affected by the limitation of enzyme transported against cellular membrane. Therefore, the promotion in β-glucosidase released and transported is beneficial to improve the nutrient value of fermented foods using LAB.

The levels of enzyme released by bacteria are related to their specific behaviors as well as growth characteristics. With sufficient nutrients at optimal growth temperature, pH, and oxygen levels, microorganisms generally grow at a characteristic of maximum growth rate, and the ability of microorganisms to sense and respond to environmental stresses is crucial to their survival (Moat et al., 2002). Different stressors with various intensities of stresses induce different cellular responses, including cell repair with available resources, temporary adaptation to some stressors, autophagy, and cell death (Milisav, 2011). Hence, the degree of β-glucosidase released from LAB might be changed from the stress responses induced by an appropriate stressor.

Ultrasound as a high-frequency wave with frequency over 20 kHz is able to generate cavity and hot spots in liquid phase to promote chemical reactions. Ultrasound has been applied in food processing, such as cutting, sterilization, extraction, and so on (Wang et al., 2017; Zheng et al., 2014). When ultrasonic waves act on microbial cells, the cell membrane breaks from the shearing effect of liquid medium causing the release of cytoplasm contents more easily (Yeo and Liong, 2011; Lentacker et al., 2014). The high-intensity ultrasound was reported to promote the biochemical reactions, such as transglycosylation of bifidobacteria in milk (Nguyen et al., 2009), and extracting enzymes from cells to enhance enzyme activity by ultrasound (Kwiatkowska et al., 2011). It is supposed that ultrasound under appropriate conditions can be employed as an effective stressor to induce proper stress response of LAB.

Okara is a by-product of soymilk from soybeans in food industries, containing useful ingredients that are beneficial to human health, such as isoflavones, lignans, phytosterols, etc., and is worth reusing. One of the LAB, L. plantarum BCRC 10357, was reported to be used in the fermentation of Graptopetalum paraguayense E. Walther (Wu et al., 2011), and could be used in the fermentation of okara. In addition, an effective processing method for how to apply ultrasound to improve microbial fermentation would be useful in food industries. The aim of this study was to develop an effective method by modifying ultrasound treatment on L. plantarum BCRC 10357 at different growth phases, such that β-glucosidase released from L. plantarum BCRC 10357 could be maximized for biotransformation of glucoside isoflavones in the fermentation of okara. The effect of ultrasound stimulation on the viable cells count and β-glucosidase activity of bacteria as well as the fermentation of okara was performed.

Materials and Methods

Lactic acid bacteria culture and cell growth    L. plantarum BCRC 10357 was obtained from Food Industry Research and Development Institute (Hsinchu, Taiwan), and the culture was preserved at −80 °C by mixing with sterile 30% (v/v) glycerol in the ratio of 1:1. Prior to use, the bacterial culture was activated by inoculating in de Man, Rogosa and Sharpe (MRS) broth at 37 °C for 24 h for two times, and then inoculated in MRS agar at 37 °C for 48 h.

The pour plate method was used to determine the growth of bacteria. The dilution liquid was prepared from fermented liquid diluted with sterilized 0.1% (w/v) peptone water by using 10-fold serial dilution method. One millimeter of liquid was taken by pour plate method and introduced into sterilized MRS agar at 45 °C. The plate was incubated at 37 °C for 48–72 h for viable cell counts.

Ultrasound stimulation on L. plantarum BCRC 10357    The ultrasound stimulation on L. plantarum BCRC 10357 was performed for the selected time at log phase, stationary phase, and death phase. The liquid culture of L. plantarum BCRC 10357 was stimulated with ultrasound using sonicator (20 kHz, max. 700 W, 1–100% amplitude (intensity) controlled, model Q700, QSONICA, USA) with probe (1.27 cm × 12.7 cm) at different levels of ultrasonic amplitude for the selected periods (1, 2, and 3 min). During the sonication, the tip of probe was maintained at the position of 1 cm below the liquid culture level and the temperature was kept at 25 °C. After the ultrasound treatment, the liquid culture was inoculated to the new medium in 1% (v/v) to be incubated at 37 °C for 48 h. The experiment without ultrasound treatment was noted as the control group.

Preparation and fermentation of okara    The non-genetically modified soybeans Kaohsiung Sel. 10 used to prepare okara was purchased from Taichung Farmers' Association (Taichung, Taiwan). The soybeans were rinsed to clean thoroughly and soaked with 6 times in weight of distilled water for overnight. After water drained, the soybeans with distilled water in a ratio 1:10 (w/v) of soybean (dry weight) to water were homogenized for 3 min, and then filtered to leave the okara. The okara was freeze-dried to be fermented. The fermentation liquid was prepared by mixing lyophilized okara with MRS broth in the required proportions and sterilized at 121 °C for 15 min. After that, the liquid culture was inoculated in 1% (v/v) into the fermentation liquid to be fermented at 37 °C for 36 h.

Determination of β-glucosidase activity    The determination of β-glucosidase activity was based on the hydrolysis rate of p-nitrophenyl β-D-glucopyranoside (p-NPG) by measuring the amount of p-nitrophenol released. The measuring procedure followed the method of Otieno et al. (2006) with a slight modification. The quantity 0.1 mL of p-NPG (5 mM, prepared with 0.1 M phosphate buffer solution at pH 7.0) was added into 1 mL of okara fermentation liquid to react at 37 °C for 30 min. The reaction was stopped by adding 0.05 mL of Na₂CO₃ (1 M) at 4 °C. The mixture was then centrifuged at 12,000 ×g for 30 min. The supernatant was filtered by 0.45 µm membrane and analyzed by using spectrophotometer at 405 nm. One unit (U) of the enzyme activity was defined as the amount of β-glucosidase that released one nanomole of p-nitrophenol from p-NPG per milliliter per minute at 37 °C under assay condition (Otieno et al., 2006).

Determination of isoflavones    The extraction procedure of isoflavones from fermented okara-liquid for analysis followed the method of Coward et al. (1998) with a slight modification. The amount 0.5 g of the freeze-dried powder of fermented okara-liquid was added into 5 mL of 80% methanol solution with shaking at 100 rpm and 25 °C for 24 h. The mixture was centrifuged at 10,000 ×g for 20 min to get the supernatant, which was then filtered with 0.45 µm membrane and analyzed for the concentrations of isoflavones using high performance liquid chromatography (HPLC). The HPLC system was equipped with the diode Array-detector set at 260 nm and Mightysil RP-18 column (5 µm, 250 mm × 4.6 mm) with column temperature maintained at 30 °C. The mobile phase consisted of solvent A (acetonitrile) and solvent B (0.1% trifluoracetic acid) with the flow rate at 0.8 mL/min. The gradient was set as follows: solvent A 10% (0 min) → 10% (10 min) → 55% (35 min) → 10% (45 min) → 10% (50 min) → 10% (60 min). The standards of daidzin, genistin, daidzein, and genistein (Sigma, USA) were used to determine the isoflavones concentrations (µg/mL) in the fermented okara.

Statistical Analysis    The experimental data were expressed as mean ± S.D. (n=3). Statistical analysis was evaluated by one-way ANOVA with Duncan's multiple range tests using SPSS, and statistical significance was determined at P < 0.05.

Results and Discussion

Growth curve of L. plantarum BCRC 10357 before ultrasonic stimulation    The viable cell counts and pH values of L. plantarum BCRC 10357 incubated in MRS broth for 120 h are displayed in Fig. 1(A), and the corresponding values of β-glucosidase activity of L. plantarum BCRC 10357 are shown in Fig. 1(B). From viable cell counts after inoculation, the log phase, stationary phase, and death phase were identified to start at 2 h, 7 h, and 22 h, respectively. As shown in Fig. 1(A), the pH values declined quickly as the growth of L. plantarum BCRC 10357 entered into the log phase, at which the acids were largely produced. When the death phase reached, the pH value was reduced to 3.75 and maintained at that value, due to lacking additional acids generated. As illustrated in Fig. 1(B), the β-glucosidase activity of L. plantarum BCRC 10357 in the early stage of incubation was very low, and began to significantly increase in 5 h of incubation; the β-glucosidase activities were higher in the stationary phase from 8 h to 12 h of incubation (16.71–21.41 U/mL) than in other periods, and the β-glucosidase activity quickly reduced after 24 h of incubation. The characteristics of growth curve were then used to explore the effect of ultrasonic stimulation on stress responses of L. plantarum BCRC 10357 at various growth phases.

Fig. 1.

Variations of (A) viable cell counts and pH values, and (B) β-glucosidase activity of L. plantarum BCRC 10357 incubated in MRS broth at 37 °C for 120 h. Data were expressed as mean ± standard deviations from triplicate experiments.

Ultrasonic stimulation on L. plantarum BCRC 10357    L. plantarum BCRC 10357 has been generally used as the probiotic in fermented products. The main function of using appropriately ultrasonic stimulation on L. plantarum BCRC 10357 was to induce and promote the release of more β-glucosidase for subsequent bioconversion without separating β-glucosidase from the bacteria. Thus, L. plantarum BCRC 10357 incubated at log phase (5 h) was first selected to explore the effect of ultrasonic stimulation at different amplitudes (20%, 40%, and 60%) and durations (1, 2, and 3 min). The bacterial liquid culture was then inoculated at 1% (v/v) in new medium for re-incubation. The control group was performed without ultrasound treatment for comparison. The viable cell counts for various incubation times were determined, as shown in Table 1. From the results, initially the viable cell counts of groups after ultrasound treatment were lower than that of the control group, due to physical damage on the cells by ultrasound. However, the bacteria after stimulation with ultrasound grew more rapidly, giving the viable cell counts more than 10⁹ CFU/mL after 12 h of re-incubation. This indicated that L. plantarum BCRC 10357 had so high resistance to stress as to repair the damaged cells after ultrasonic stimulation. Table 2 shows the variation of pH values for different incubation periods after ultrasound treatment. The variation of pH values with ultrasound treatment was almost the same as that of the control group, at a pH value of 3.75 after 24 h of incubation, showing that the injury of cells had been repaired before entering the stage of death phase.

Table 1. Effect of ultrasound treatment with different intensities and durations on viable cell counts of L. plantarum BCRC 10357 in MRS broth after incubation at 37 °C for 48 h.

Incubation time (h)	Ultrasound amplitude (%)	Viable cell counts (log CFU/ml)
		1 min	2 min	3 min
0		Control : 7.26±0.00a
	20	7.08±0.02b	6.85±0.05c	6.76±0.02d
	40	7.00±0.01b	6.58±0.06e	6.41±0.04f
	60	6.72±0.10d	6.11±0.05g	5.83±0.07h
12		Control : 9.41±0.06a
	20	9.38±0.05a	9.43±0.04a	9.43±0.04a
	40	9.46±0.06a	9.41±0.03a	9.36±0.01a
	60	9.43±0.06a	8.51±0.08b	8.32±0.12c
24		Control : 9.18±0.06b
	20	9.18±0.04b	9.48±0.07a	9.46±0.04a
	40	9.43±0.02a	9.45±0.04a	9.43±0.03a
	60	9.49±0.02a	9.49±0.04a	9.49±0.01a
36		Control : 8.28±0.04b
	20	8.00±0.08de	8.26±0.01bc	8.30±0.13b
	40	7.92±0.08e	8.23±0.10bc	8.36±0.02b
	60	8.15±0.02cd	8.28±0.11b	8.60±0.06a
48		Control : 7.71±0.08c
	20	7.08±0.12d	7.96±0.03ab	7.92±0.02ab
	40	6.92±0.10e	7.83±0.04bc	7.94±0.05ab
	60	7.72±0.10c	7.91±0.15b	8.08±0.13a

Data were expressed as mean ± standard deviations from triplicate experiments.

Different superscript letters at the same time were significantly different (p < 0.05) by Duncan's test.

Table 2. Effect of ultrasound treatment with different intensities and durations on pH value of L. plantarum BCRC 10357 in MRS broth after incubation at 37 °C for 48 h.

Incubation time (h)	Ultrasound amplitude (%)	pH value
		1 min	2 min	3 min
0		Control : 6.05±0.01ab
	20	5.99±0.02cd	5.98±0.01d	6.05±0.01b
	40	5.99±0.01cd	6.01±0.02c	6.07±0.01ab
	60	6.07±0.02ab	6.08±0.01a	6.07±0.01ab
12		Control : 3.88±0.01g
	20	3.96±0.01f	4.02±0.02e	4.09±0.01d
	40	3.96±0.01f	4.08±0.00d	4.20±0.02c
	60	4.02±0.01e	5.00±0.03b	5.17±0.01a
24		Control : 3.75±0.02bc
	20	3.77±0.02bc	3.76±0.01bc	3.75±0.01c
	40	3.77±0.01b	3.76±0.01bc	3.76±0.01bc
	60	3.75±0.01bc	3.82±0.02a	3.84±0.01a
36		Control : 3.75±0.01b
	20	3.75±0.01b	3.76±0.01ab	3.75±0.02b
	40	3.73±0.01c	3.77±0.00a	3.75±0.00b
	60	3.76±0.01ab	3.75±0.02ab	3.75±0.01b
48		Control : 3.75±0.01ab
	20	3.74±0.01ab	3.76±0.01a	3.75±0.02ab
	40	3.73±0.01b	3.76±0.02a	3.75±0.01ab
	60	3.75±0.01ab	3.75±0.01ab	3.74±0.01ab

Data were expressed as mean ± standard deviations from triplicate experiments.

Different superscript letters at the same time were significantly different (p < 0.05) by Duncan's test.

The effects of ultrasonic stimulation at 60% amplitude for various treating times on β-glucosidase activity are displayed in Fig. 2(A). Without ultrasonic stimulation, the behavior of the control group in 12 h of re-incubation for β-glucosidase activity (24.45 ± 0.10 U/mL) was consistent with that shown in Fig. 1(B). It was found that, in 12 h of re-incubation, the β-glucosidase activity of control group was higher than those of other sonicated groups, 17.97 U/mL for 1 min of sonication, 1.74 U/mL for 2 min of sonication, and 1.78 U/mL for 3 min of sonication. This displayed that after sonication the level of the cells injured was dependent on the time and intensity of ultrasonic treatment. The heavier injury in cells like for 2 min and 3 min of sonication might require much longer time to repair and recover the cells, such that the β-glucosidase activities for sonication groups were less than that for the control group in just 12 h of re-incubation, which was the preferred incubation time for uninjured cells (control group). As increasing the re-incubation time, the highest β-glucosidase activity of L. plantarum BCRC 10357 at 40.22 U/mL occurred in 24 h of re-incubation after 2 min of sonication. The best re-incubation time was identified as 24 h to get the highest β-glucosidase activity. Besides, this enhanced β-glucosidase activity in 24 h of re-incubation after 2 min of sonication was about 65% higher than that of control group (24.45 U/mL) without ultrasound in 12 h of re-incubation. Such enhancement in β-glucosidase activity can be attributed to the increased permeability of cell membrane as well as the resistance-to-stress behavior of L. plantarum BCRC 10357 by ultrasound.

Fig. 2.

Comparison of β-glucosidase activity of L. plantarum BCRC 10357 for 24 h of incubation at 37 °C with ultrasound at various amplitudes and durations: (A) sonication at 60% amplitude for various incubation times; (B) sonication at various amplitudes and durations. Control group: without ultrasound treatment. Data were expressed as mean ± standard deviations from triplicate experiments.

The ultrasonic stimulation at various amplitudes and durations on L. plantarum BCRC 10357 for 24 h of re-incubation was performed. As shown in Fig. 2 (B), with ultrasound at 40% amplitude for 3 min, the β-glucosidase activity was obtained at 36.86 U/mL, which was higher than that of control group (8.16 ± 0.35 U/mL) in 24 h of incubation and that with sonication at 60% amplitude for 3 min (26.32 ± 0.11 U/mL), but still lower than that with sonication at 60% amplitude for 2 min (40.22 ± 0.60 U/mL). This indicated that using a larger amplitude or intensity of ultrasonic stimulation for a longer duration would damage the cells more severe, leading to the reduction of β-glucosidase activity.

From the above results, the L. plantarum BCRC 10357 was able to significantly release more β-glucosidase via appropriate ultrasound stimulation and subsequently sufficient re-incubation. The previous studies indicated that L. plantarum was a species of Gram-positive bacteria encountered in many different environmental niches with flexible and adaptive behavior reflected by the large number of regulatory and transport functions (Kleerebezem et al., 2003), and the β-glucosidase of L. plantarum was localized as extracellular enzyme with the domain possibly involved in binding to cell wall (Kleerebezem et al., 2003; Sestelo et al., 2004; Brinster et al., 2007; Michlmayr and Kneifel, 2014). Thus, the more β-glucosidase produced after ultrasonic stimulation with re-incubation was speculated to result from the stress response of L. plantarum BCRC 10357 for survival to adapt to the changes in conditions encountered by regulating the physiological behavior to produce more β-glucosidase that was translocated across the cytoplasmic membrane to extracellular sites and the medium (Brinster et al., 2007). This described that the L. plantarum BCRC 10357 behaved a high adaptation to limited ultrasonic stress, and the appropriate level of ultrasonic stimulation with sufficient re-incubation was important to induce proper stress response of the bacteria for the release of more β-glucosidase.

Ultrasound stimulation on L. plantarum BCRC 10357 at various growth stages    As mentioned above, ultrasound treatment significantly affected the growth and survival of L. plantarum BCRC 10357 as well as β-glucosidase released, when ultrasound acting on L. plantarum BCRC 10357 incubated at log phase (5 h). However, the effects of ultrasound stimulating on the bacteria at other growth stages are still unknown. In this study, the cultures of L. plantarum BCRC 10357 at log phase (5 h), stationary phase (10 h), and death phase (24 h) were sonicated at 60% amplitude for 2 min, then inoculated in 1% (v/v) into the new culture medium and re-incubated. The results are shown in Fig. 3. The β-glucosidase activities of L. plantarum BCRC 10357 in 24 h of re-incubation after ultrasound treatment were 48.80 U/mL for death phase, 40.22 U/mL for log phase, and 36.62 U/mL for stationary phase, indicating that the bacteria at early time of death phase had the higher resistance to stress than at other stages. As a result, L. plantarum BCRC 10357 at log phase grew rapidly with strong metabolic activity, but having the lower resistance to stress, and was susceptible to changes in external or environmental pressure.

Fig. 3.

Comparison of β-glucosidase activity of L. plantarum BCRC 10357 for 48 h of incubation at 37 °C after ultrasonic stimulation (60% amplitude, 2 min) on various growth phases. Data were expressed as mean ± standard deviations from triplicate experiments.

To further compare the efficiency of operation modes of ultrasonic stimulation, L. plantarum BCRC 10357 incubated at death phase (24 h) was sonicated at 60% amplitude operating by either continuously (run for 2 min) or periodically (run for 1 min, stopped for 1 min, and run for 1 min). The group without ultrasound treatment was used as the control. As shown in Fig. 4, the highest β-glucosidase activity of the control group was only 9.34 U/mL in 12 h of re-incubation, while the group treated by continuous operation of ultrasound for 2 min gave the best β-glucosidase activity in 24 h of re-incubation at 48.80 U/mL, about 5.2 times of that of the control group and higher than that by periodical operation mode. This is due to the increasing permeability of cellular membrane by ultrasonic action after repairing of cells and the response of L. plantarum BCRC 10357 to ultrasonic stress for survival by regulating the physiological behavior making the bacteria able to release more β-glucosidase.

Fig. 4.

Effect of operating mode of ultrasound (60% amplitude, 2 min) on β-glucosidase activity of L. plantarum BCRC 10357 at 37 °C for 48 h of incubation. Data were expressed as mean ± standard deviations from triplicate experiments.

As the growth behavior of L. plantarum BCRC 10357 described, without ultrasonic stimulation, the β-glucosidase activities showed higher in 8 h to 12 h of incubation than in other periods (Fig. 1(B)). However, using ultrasound to stimulate the L. plantarum BCRC 10357 for various conditions, the highest β-glucosidase activity occurred at sonication (60% amplitude) for 2 min and 24 h of re-incubation; the results indicated that with ultrasonic stimulation the stress response of the bacteria was effectively induced to generate more enzymes in the later period of re-incubation (24 h) due to that the injured cells required the longer time to repair and promote the β-glucosidase released. The previous study also reported that the physiology of lactic acid bacteria with ultrasonic treatment showed change of membrane permeability, recovery from injury of cellular membrane, and rehabilitating the proliferation ability on fermentation at 37 °C for 24 h (Yeo and Liong, 2011). Recently, Papadimitriou et al. (2016) indicated that the injured cells were metabolically active but had suffered damage to affect their proliferation capacity, so that those injured cells sometimes needed extended recovery times or special conditions to repair injury (Papadimitriou et al., 2016).

Fermentation of okra using L. plantarum BCRC 10357    L. plantarum BCRC 10357 with the enhanced β-glucosidase activity after ultrasound treatment was used to ferment the soymilk residue (okara) for the biotransformation of isoflavones from glucosides to bioactive aglycones. As literatures reported, the biotransformation rate is highly related to the level of β-glucosidase activity (Otieno et al., 2006; Donkor and Shah, 2008). After ultrasonic stimulation at 60% amplitude for 2 min in 24-h incubation, L. plantarum BCRC 10357 was inoculated at 1% (v/v) into the fermentation liquid with 3% okara and fermented for 36 h. The results of β-glucosidase activity and changes of daidzin, genistin, daidzein, and genistein in fermented okara-liquid during fermentation are displayed in Fig. 5.

Fig. 5.

The contents of isoflavones and β-glucosidase activity during fermentation by L. plantarum BCRC 10357 after ultrasound stimulation at 60% amplitude for 2 min with 3% okara in MRS broth at 37 °C. Data were expressed as mean ± standard deviations from triplicate experiments.

The highest β-glucosidase activity of 44.92 U/mL was observed to occur at 24 h of fermentation time, after which L. plantarum BCRC 10357 entered the stage of death phase and the β-glucosidase activity declined (Fig. 5). With the highest β-glucosidase activity at 24 h, the contents of daidzin and genistin were decreased from 8.04 and 8.81 µg/mL in 12 h to 1.71 and 2.65 µg/mL in 36 h, respectively; while the contents of daidzein and genistein were increased from 12.29 and 10.05 µg/mL in 12 h to 19.18 and 16.13 µg/mL in 36 h, respectively. The highest β-glucosidase activity appeared in 24 h of fermentation, making the fraction of aglycones (daidzein and genistein) in isoflavones increased from 57% in 12 h to as high as 89% in 36 h, so that the nutritional value of okara was inherently improved.

Conclusions

In this study, the effective method for enhancing β-glucosidase activity from L. plantarum BCRC 10357 by ultrasonic stimulation was developed. L. plantarum BCRC 10357 showed good stress response in releasing β-glucosidase when encountering external pressure. The highest β-glucosidase activity can be obtained by using ultrasound to act on L. plantarum BCRC 10357 at its early time of death phase in 24 h of incubation time. With strategic ultrasound-treated L. plantarum BCRC 10357, the enhanced β-glucosidase activity can be used in the fermentation of okara for the biotransformation of glucoside isoflavones to produce high fractions of bioactive aglycones (daidzein and genistein) in okara, which has the potential to be added in foods as functional ingredients, so as to valorize the utilization of okara.

Acknowledgement    The authors thank the Ministry of Science and Technology, R.O.C. for providing financial support for this research (project No. MOST 105-2320-B-030-001).

Declarations of interest: none.

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