Dynamic changes of nutrients, isoflavone composition and antioxidant activities during liquid-state fermentation of soybean embryo homogenate by Ganoderma lucidum

Abstract

This study aims to explore compositional changes of oligosaccharides, amino-type nitrogen, free amino acids volatile compounds, isoflavones, total phenol, total flavonoid, and antioxidant capacities during liquid-state fermentation (LSF) of soybean embryo homogenate (SEH) by Ganoderma lucidum to improve usability as functional food material. Raffinose and stachyose were almost completely hydrolyzed into monosaccharides such as fructose, glucose and galactose after 2 days of fermentation. After 5 days of fermentation, the contents of amino-type nitrogen and total free amino acids increased 7.0-fold and 2.4-fold, respectively, compared to the control group. Most of isoflavone glycosides were almost hydrolyzed into aglycones after 2 days and thereafter some of isoflavone aglycones were transformed into ortho-hydroxyisoflavones (OHIs). Total phenol, total flavonoid and antioxidants activities were significantly increased during the fermentation period. The result indicates that LSF of soybean embryo by G. lucidum can potentially improve acceptability and usability of soybean embryo as functional food material.

Graphical abstract

Highlights

• •This study is explore the effect of Ganoderma lucidum-mediated fermentation on soybean embryo homogenate
• •Oligosaccharides and conjugated isoflavones were completely hydrolyzed within 2 days.
• •Free amino acids and antioxidant activities were significantly increased during the fermentation.

1. Introduction

Soybeans have traditionally been used to make soy-based fermented food products such as Doenjang (soy paste), Ganjang (soy sauce) and Cheonggukjang (fermented with Bacillus subtilis) as well as for making tofu (soy curd) and soymilk. In the industrial production of soymilk in Korea, only the cotyledon is used after removing the husk and embryo because the presence of embryo causes prolonged soaking time and increases in undesirable beany off-flavor as well as astringent and bitter taste in the product (Kim et al., 2013). Soybean embryo is not only rich in nutrients such as protein, unsaturated fatty acids, and vitamins, but also contains a large amount of bioactive ingredients, making it a good raw material for the development of functional foods (Kim et al., 2007; Yue, Abdallah, & Xu, 2010). However, soybean embryo, as an important byproduct in soymilk, soybean oil and soy protein production, has not been given enough attention and has been treated as waste due to various reasons, such as immature soybean embryo separation technology, immature market for soybean embryo products, and more importantly, the special bitter taste, astringent taste, and beany odor of soybean embryo has greatly limited its development and utilization. Soy isoflavones have a wide range of physiological activities, such as anti-aging and antioxidant effects (Kim et al., 2013; Tham, Gardner, & Haskell, 1998; Yeom, Kim, Kim, & Oh, 2012), preventing and treating menopausal syndrome, preventing osteoporosis (Clerici et al., 2007; Scheiber, Liu, Subbiah, Reba, & Setchell, 2001; Setchell, 1998) and cardiovascular and cerebrovascular diseases, preventing cancer and reducing the occurrence of tumors (Sarkar & Li, 2003; Setchell, 1998). The naturally occurring isoflavones in soybeans come in two forms: glucosidic forms (glucosides, acetylglucosides and malonylglucosides of isoflavones) and aglycones (mainly diadzein, glycitein and genistein). Glycoside forms account for 99 % of the total isoflavones, while aglycone forms have very little content. However, the biological absorption and utilization efficiency, as well as various physiological activities, of aglycones are stronger than their corresponding glycosides (Izumi et al., 2000; Sirilun, Sivamaruthi, Kesika, Peerajan, & Chaiyasut, 2017). Research has shown that the human gut microbiota produces β-glucosidase, which can hydrolyze glycosides into aglycones and greatly improve their absorption and utilization efficiency. Therefore, improving the bioavailability of soy isoflavones in the human body requires both increasing the intake of aglycones and improving gut microbiota through the intake of functional active probiotics. Lactic acid bacteria, as an important group of probiotics in the human body, .in the fermented food industry (Chien, Huang, & Chou, 2006; Hou, Yu, & Chou, 2000; Li et al., 2012). Furthermore, antinutrients such as phytate, oxalates, biogenic amines, tannins, trypsin inhibitors and allergens exist in soybean that may be transformed into SEH. These are plant compounds that reduce the body's ability to absorb essential nutrients (Mollakhalili-Meybodi, Arab, & Zare, 2022).

On the other hand, fermentation of soymilk with microorganisms have been considered as an alternative to improve the nutritional composition and reduce undesirable volatile compounds that contribute to flavor characteristics and various harmful compounds (Elhalis, Chin, & Chow, 2024; Hubert, Berger, Nepveu, Paul, & Daydé, 2008). Probiotics and Aspergillus spp. are extensively studied for enhancing soymilk quality (Chien et al., 2006; Hou et al., 2000; Hubert et al., 2008; Kaneko, Igarashi, & Aoyama, 2014; Marazza, Garro, & Giori, 2009; Zhao & Shah, 2014) and also attempts have been conducted to ferment soybean and soymilk using Basidiomycetes mushrooms such as Ganoderma spp. (Li, Xu, & Guo, 2023; Suruga, Tomita, & Kadokura, 2020; Yang & Zhang, 2009), Grifola frondosa (Yang, Zhang, Xiao, Feng, & Zhou, 2015), Cordyceps militaris (Dai, Zhou, Wang, Dong, & Xia, 2021), Pleurotus spp. (Sawada et al., 2023), Hericium erinaceus and Flammulina velutipes (Wang et al., 2023). The fruiting body and mycelia of mushrooms are highly nutritious natural resources that contain various bioactive compounds. Some mushroom species have long been used to promote health and prevent or treat diseases (Bakratsas, Polydera, Katapodis, & Stamatis, 2021). Additionally, mushrooms secrete diverse exoenzymes capable of hydroxylation, oxidation and transformation (Baldrian & Valášková, 2008; Singh & Singh, 2014) during their growth. Although some studies have been reported on the fermentation using whole soybean or soymilk prepared from whole soybean by mushrooms. Studies on LSF of soybean embryo using mushrooms are extremely rare. Furthermore, LSF of soybean embryo with G. lucidum can be considered more desirable than other fungi, considering rapid growth rate of the mycelia, the presence of various bioactive compounds, and its applicability for functional food and medicinal purposes. Therefore, this study aims to enhance the availability of soybean embryo as a raw material for food or health supplements by the LSF using G. lucidum. During the fermentation period, we investigated changes in nutritional components (oligosaccharides, amino nitrogen and free amino acid composition), volatile flavor compounds, isoflavones (glucosidic and free forms), and antioxidant activities.

2. Materials and methods

2.1. Materials

The soybean embryo used in this study was kindly donated by Yeonse Milk Company (Asan-si, Chungnam, Republic of Korea) in 2022. The samples were washed with distilled water, and then followed by dried for 12 h at 60 °C. The samples were powdered using a household grinder (Shinil SMX-M41KP, Shinil Electronic Co., Ltd., Cheonan-si, Chungnam, Republic of Korea), and the powdered samples were kept in a cold room (4 °C) until used. The G. lucidum strain (KACC 42231) was kindly donated from the Korean Agricultural Culture Collection (KACC) of Rural Development Administration, (Jeonju, Jeonbuk, Republic of Korea).

2.2. Reagents

Glucose, fructose, galactose, sucrose, raffinose, stachyose, daidzin, glycitin, genistin, daidzein, glycitein, genistein, 8-hydroxydaidzein (8-OHD), 8-hydroxygenistein (8-OHG), 8-hydroxyglycitein (8-OHGL), n-alkane standard mixture (C₆–C₃₀), gallic acid, rutin, 0.1 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-sulphonic acid (ABTS) and 2,4,6-triphyridyl-s-triazine (TPTZ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Silica gel (230–400 mesh) was purchased from Merck KGaA (Darmstadt, Germany). High performance chromatography (HPLC)-grade acetonitrile and methanol were purchased from J.T. Baker (Center Valley, PA, USA), and deionized water was prepared using a water purification system (model New Human Power I, Human Corp., Songpa-gu, Seoul, Republic of Korea). The other reagents (guaranteed grade) were purchased from Daihan Scientific Co. Ltd. (Wonju-si, Gangwon-do, Republic of Korea).

2.3. Cultivation of the seed culture

Seed culture of G. lucidum was cultivated as follows: the strain was pre-incubated onto potato dextrose agar media (Becton, Dickinson & Company, Sparks, MD, USA) at 25 °C for 6 days. The pre-incubated strain was inoculated into germinated-malt media (11° Brix) saccharified for 12 h at 65 °C with tap water (4-fold, v/v), and cultured at 25 °C for 2 weeks with gentle shaking (120 rpm). For large-scale culture, 1 L of saccharified malt medium (11° Brix) was added to a 4-L Erlenmeyer flask, inoculated with pre-cultured G. lucidum, and cultured at 25–26 °C for 2 weeks with shaking (120 rpm).

2.4. Preparation of SEH and fermentation

Soybean embryo powder was mixed with tap water (1:7, w/v) and homogenized for 1 min. Using a household blender. After heating at 80 °C for 30 min with gentle stirring, the homogenate was filtered using two layers of cheesecloth, and tap water was added to adjust the solid content to approximately 7° Brix. Each 300 mL of SEH and 15 mL (5 % v/v of SEH) of the seed culture of G. lucidum was dispensed in a 1 L- Erlenmeyer flask and the mixtures were fermented at 26 °C for 7 days with shaking (120 rpm). The sample was collected at regular intervals and followed by freeze drying. Before inoculation of the strain, all culture media and SEH were sterilized at 121 °C for 30 min.

2.5. Analysis of oligosaccharide and free sugar

Freeze-dried powder (1.0 g) of fermented SEH was extracted with 10 mL of 80 % ethanol using an ultrasonicator (Hwa-Shin Instrument Co., Seoul, republic of Korea) for 30 min at room temperature and centrifuged (4500 rpm) for 10 min. The residue was extracted one more with same solvent, followed by centrifugation as the described above. The supernatants were combined and evaporated under reduced pressure at 45 °C. Sugar composition was analyzed HPLC. The HPLC system consisted of a Waters 600E system controller, 626 pump, 717 plus autosampler, and 2414 refractive index detector (Waters Corp., Milford, MA, USA) with a Zorbax carbohydrate analysis column (150 cm × 4.6 mm, Agilent Technologies, Inc., Santa Clara, CA, USA) was used for separation. The analysis was performed at 40 °C with a flow rate of 1.2 mL/min using isocratic elution with acetonitrile (75 %):water (25 %) as a mobile phase. Each component was quantified using a calibration curve based on serial dilutions (0–1000 μg/mL) of standard compounds, and the content was expressed as mg/g on a dry weight basis (DW).

2.6. Free amino nitrogen content

Fifty mL of the fermented SEH was centrifuged at 4500 rpm for 10 min. Then, 25 mL of the supernatant was collected for analysis. Free amino nitrogen content was determined using the formol titration method (Taylor & H., 1957), and the amino nitrogen content (%) was calculated using the following formula:

$$\text{Free amino nitrogen}\left(\%\right)=\left(\text{V1}-\text{V0}\right)\times \mathrm{F}\times 0.0014\times \mathrm{D}\times 100/\mathrm{S}$$

V1: Volume of titrant consumed in the sample test (mL).

V0: Volume of titrant consumed in the blank test (mL).

F: Factor of 0.1 N-NaOH solution.

D: Dilution factor.

S: Amount of sample (mL).

0.0014: Amount of nitrogen (g) corresponding to 1 mL of 0.1 N-NaOH.

2.7. Free amino acid determination.

Freeze-dried powder (0.5 g) was extracted twice with 5 mL of 80 % methanol using an ultrasonicator at room temperature for 30 min. The combined extracts were concentrated under reduced pressure and dissolved in 2.0 mL of dilution buffer (pH 2.1) for amino acid analysis. Free amino acids were analyzed using a Sykam S433 automatic amino acid analyzer (Sykam GmbH, Eresing, Germany), equipped with an LCA K06/NA cation separation column (4.6 × 250 mm). The flow rate of the mobile phase was set to 0.45 mL/min, and ninhydrin was applied at a flow rate of 0.4 mL/min. The amino acids were quantified using the physiological amino acid standards mixture (Sykam GmbH, Eresing, Germany).

2.7. Volatile flavor compounds

One hundred mL of the fermented SEH, 1 mL of ethyl octanoate solution (500 μg/mL) as an internal standard, and 1.5 L of distilled water were placed in a 3 L round-bottom flask. The volatile compounds were extracted using the Likens-Nickerson type simultaneous steam distillation and extraction apparatus (Schultz, Flath, Mon, Eggling, & Teranishi, 1977) with 50 mL of n-pentane and diethyl ether (1:1, v/v). After steam distillation for 2 h, the solvent fraction was dried over with anhydrous sodium sulfate for 12 h, filtered, and concentrated under mild reduced pressure to about 5 mL. The sample was further concentrated to about 1 mL under a nitrogen gas stream at room temperature and analyzed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS).

For GC analysis, an Agilent 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) was used, with a Supelcowax 10 fused silica capillary column (30 m × 0.32 mm, 0.25 μm film thickness, Supelco Inc., Bellefonte, USA). The column temperature was programmed from 50 °C to 230 °C at a rate of 2 °C/min and held at 230 °C for 20 min. The injector and flame ionization detector temperatures were set to 250 °C, respectively. Nitrogen gas was used as the carrier gas at a flow rate of 1.0 mL/min with a split ratio of 20:1. Linear retention indices (RI) were calculated using an n-alkane mixture (C₆–C₃₀) under the same GC conditions (Van Den Dool & Kratz, 1957). GC–MS analysis was conducted using, a Shimadzu QP2010 plus GC/MS (Shimadzu Corp., Osaka, Japan) under the same column and temperature programming condition as in GC analysis. Other conditions are as follows: injector and interface temperatures, 250 °C, respectively; carrier gas, helium (flow rate: 1.0 mL/min); split ratio, 60:1 and electron ionization mode, 70 eV. Volatile compounds were tentatively identified by comparing mass spectra with the Shimadzu mass spectral database and literature-reported GC retention indices (Babushok, Linstrom, & Zenkevich, 2011) and gas chromatographic retention data webbook (Chemistry Webbook, SRD 69) of the National Institute of Standard and Technology (NIST). The concentrations of volatile compounds were calculated as ethyl octanoate equivalent (assuming relative response factor of all analytes were 1.0) (Cicchetti, Merle, & Chaintreau, 2008; Tedone et al., 2011) and was expressed as μg/100 mL of the SEH liquid.

2.8. Determination of isoflavone content

Freeze-dried powder (0.5 g) of fermented SEH was extracted twice with 5 mL of 80 % methanol with ultrasonication for 30 min. The combined extracts were concentrated under reduced pressure, dissolved in 2 mL of methanol and filtered using a microsyringe filter (0.45 μm) for HPLC analysis. HPLC system consisted of a Waters 600E system controller, 626 pump, 717 plus autosampler, and 996 photodiode array detector (PDA) at 260 nm. An Eclipse XDB C₁₈ RS column (4.6 mm × 250 mm, 5 μm, Agilent Technologies, Inc., Santa Clara, CA, USA) was used for separation. A gradient mode was applied for elution, with 0.1 % phosphoric acid in deionized water (solvent A) and 100 % acetonitrile (solvent B) and 0.8 mL of flow rate. The elution program was set as follows: 83 % A/17 % B (0 min), 83 % A/17 % B (5 min), 60 % A/40 % B (15 min), 53 % A/47 % B (15 min) and 83 % A/17 % B (5 min). Each compound was quantified using a calibration curve prepared by authentic standard (0–1000 μg/mL in methanol), and the content was expressed as mg/g, DW.

2.9. Analysis of ortho-hydroxyisoflavone (OHI)

After fermenting with G. lucidum for 7 days, 30 g of freeze-dried SEH powder was extracted twice with 300 mL of 70 % methanol (1:10, w/v), respectively. The extracts were concentrated under reduced pressure at 45 °C and dissolved in 100 mL of distilled water. The solution was further extracted with water-saturated butanol (100 mL × 3), and the resulting butanol extract was concentrated under reduced pressure. The concentrate was dissolved in a small amount of methanol and loaded onto a silica gel column (20 × 5 cm). The column was eluted sequentially with mixtures of chloroform, methanol and distilled water in the following ratios; 90:10:1, 80:20:2, 70:30:3, 65:35:10 (lower phase), and 60:40:10 (v/v/v) using each 500 mL. Each eluate (30 mL/tube) was analyzed by HPLC under the same conditions as the isoflavone analysis. OHI-rich fraction was analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) (Waters Corp., Milford, MA, USA). The chromatographic separation by UPLC was carried out using an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm, Waters Corp.) with a column temperature of 40 °C and a flow rate of 0.5 mL/min, where the mobile phase contained solvent A (water +0.1 % formic acid) and solvent B (acetonitrile +0.1 % formic acid). The mobile phase was eluted using the following gradient elution conditions: 0–5 min (3 % B); 5–16 min (3–100 % B); 16–17 min (100 % B); 17–19 (100–3 % B); 19–25 min (3 % B). The constituents eluted from the column were detected by a high-resolution tandem mass spectrometer SYNAPT G2 Si HDMS QTOF (Waters Corp.) in negative ion mode with 1 kV and 40 V, respectively. Centroid MS^Emode was used to collect the mass spectrometry data. The primary scan ranged from 50 to 1200 Da and the scanning time was 0.2 s. The serial parameters for QTOF-MS analysis were set as follows; the nebulizer and auxiliary gas, nitrogen; flow rate of drying gas (nitrogen), 15 L/min; drying gas temperature, 225 °C; nebulizer pressure, 45 psi; flow rate and temperature of sheath gas, 11 L and 350 °C; capillary voltage, 3500 V; fragmentation voltage, 400 V; collision energy, 0 V; mass scan range, 50–1200 m/z. All the parent ions were fragmented using 20–40 eV. Data was processed on the Agilent MassHunter Workstation data acquisition software (ver. B. 02.01) and qualitative analysis software (ver. B. 03.01) and a UNIFI 1.8 software platform (Waters Corp.) was used for automatic matching analysis.

2.10. Total phenolic and total flavonoid contents (TPC and TFC)

Freeze-dried sample (0.5 g) was twice extracted with 5 mL of 80 % methanol under ultrasonication for 30 min at room temperature. The extraction mixtures were centrifuged at 5000 rpm for 10 min. The extracts were combined and evaporated under reduced pressure at 45 °C. The concentrate was re-dissolved in 5 mL of 80 % methanol and used for subsequent assays. TPC was determined according to the Folin-Ciocalteu method (Singleton & Russi, 1965) with some modification. Briefly, a 100 μL of sample extract was mixed with 2150 μL of 2 % sodium carbonate solution and incubation for 2 min at room temperature in the dark, followed by the addition of 250 μL of 50 % Folin-Ciocalteu reagent. After keeping it for 30 min at room temperature, the absorbance was measured at 725 nm using a UV–Vis spectrophotometer (Shimadzu UV-1601, Kyoto, Japan) against a blank (80 % methanol). TPC was calculated based on a calibration curve of gallic acid, The result was expressed as mg gallic acid equivalents per gram SEH powder (mg GAE/g, DW).

TFC was determined according to a method described by Zhishen, Mengcheng, and Jianming (1999). A 250 μL of sample extract was mixed with 1.6 mL of distilled water and 75 μL of 5 % NaNO₂ solution. After 5 min at room temperature, 75 μL of 10 % AlCl₃·6H₂O solution was added, followed by 500 μL of 1 M NaOH solution after 1 min. The absorbance was measured at 510 nm using a UV spectrophotometer. The TFC was calculated based on a calibration curve of rutin and the value was expressed as mg rutin equivalents (RE) per gram SEH powder (mg RE/g, DW).

2.11. Antioxidant activity assay

The prepared solutions for TPC and TFC assays were used to evaluate DPPH radical scavenging activity, ABTS radical scavenging activity, and ferric reducing antioxidant power (FRAP). DPPH activity was determined according to Blois's method (1958) with some modification. A sample extract (100 μL) was mixed with 500 μL of 0.1 M Tris-HCl buffer and 80 μL of 500 μM DPPH (in methanol). The mixture was kept for 20 min at room temperature in the dark and absorbance was measured at 517 nm using a UV spectrophotometer. The calibration curve was prepared by Trolox solution (15–1500 μM). The value was expressed as mM Trolox equivalent antioxidant capacity per gram of fermented SEH powder as a dry weight base (mM TEAC/g, DW). ABTS radical scavenging activity was determined according to the method of Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, and Byrne (2006) with slight modification. A 7.4 mM ABTS solution and a 2.6 mM potassium persulfate solution were mixed in a 1:1 (v/v) ratio and incubated for 12 h at room temperature in the dark. The solution was diluted with methanol to an absorbance of 1.1 ± 0.02 at 734 nm. Then, the ABTS solution (2800 μL) and the sample extract (200 μL) were mixed, incubated at 30 °C for 2 h in the dark, and the absorbance was measured at 734 nm. The calibration curve was linear between 25 and 400 μM Trolox concentration. The value was expressed as mM Trolox equivalent antioxidant capacity per gram of fermented SEH powder (mM TEAC/g, DW). Ferric reducing antioxidant power (FRAP) was determined to the method by Benzie and Strain (1996) with some modification. The working solution for FRAP assay was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ dissolved in 40 mM HCl, and 20 mM FeCl₃·6H₂O (10:1:1, v/v/v) and the pre-incubated at 37 °C for 15 min. A 150 μL of sample extract was mixed with 2850 μL of the pre-incubated FRAP reagent, and thereafter maintained for 15 min. The absorbance was measured at 593 nm using a UV spectrophotometer. The calibration curve was linear between 25 and 600 μM Trolox The result was expressed as mM Trolox equivalents per gram of SEH powder (mM TEAC/g, DW).

2.12. Statistical analysis

All experiments except of volatile flavor compounds were performed in triplicate. The results were expressed as mean ± standard deviation (SD). Statistical analysis was conducted with SPSS (ver. 10.1) for Windows SPSS Inc. Chicago, USA). One-way analysis of variance (ANOVA) and Duncan's multiple range test was carried out to test any significant differences among samples with different fermentation periods. Statistical significances were determined at the level of p < 0.05. A priori fixed value of p ˂ 0.05 was considered to be significantly different.

3. Results and discussion

3.1. Changes of oligosaccharides and free sugars

The main sugars present in soybeans are monosaccharides such as glucose, fructose, and galactose, sucrose and oligosaccharides such as raffinose and stachyose. Raffinose and stachyose, in particular, are non-digestible oligosaccharides (flatulence factors) found in legumes (Zhou, Kong, Yang, & Yin, 2012). These oligosaccharides are not broken down in the body because the α-galactosidase, required to hydrolyze their glycosidic bonds, is not secreted. As a result, they reach the colon undigested, where anaerobic bacteria break them down, producing gas and causing bloating or gastrointestinal discomfort, especially in sensitive individuals (Yang et al., 2015). The changes in sugar composition during the fermentation of SEH with G. lucidum are shown in Table 1. In the unfermented SEH (control), sucrose had the highest (p < 0.05) concentration (1289.9 ± 44.5 mg/g, DW), followed by stachyose (199.4 ± 10.3 mg/g), galactose (105.9 ± 2.6 mg/g), and fructose + glucose (96.4 ± 3.1 mg/g). Particularly, the non-digestible oligosaccharide stachyose was present at 199.4 ± 10.3 mg/g in the control, but after 2 days of fermentation, its concentration was significantly decreased (p < 0.05) by 82.3 % to 35.3 ± 3.7 mg/g. After 4 days-fermentation, stachyose was nearly not detected. Raffinose content in control was 42.0 ± 0.4 mg/g, but it was significantly increased (p < 0.05) to 114.2 ± 6.0 mg/g after 2 days-fermentation and was not detected in the samples after 4 days. These results indicate to be due to the hydrolysis of stachyose and raffinose into monosaccharides by enzymes secreted from G. lucidum mycelia in the early stage of fermentation. Hou et al. (2000) reported that, when soymilk was fermented for 48 h using Bifidobacterium sp. such as Bifidobacterium infantis and B. longum, the contents of raffinose and stachyose decreased while their hydrolysis products, fructose, glucose, and galactose increased. Dai et al. (2021) also reported that when soy whey was cultured in a liquid medium with Cordyceps militaris for 8 days, stachyose and raffinose started to decrease significantly from the early stage of fermentation and were not detected after 2 days.

Table 1.

Changes of sugar composition during LSF of SEH by G. lucidum.

Sugar	Fermentation time (day)
	0	2	4	5	7
Fructose  + glucose	96.4 ± 3.1a	210.2 ± 3.2c	273.7 ± 11.1d	117.5 ± 4.9b	121.9 ± 8.1b
Galactose	105.9 ± 2.6a	609.6 ± 7.4e	372.6 ± 12.4c	479.8 ± 34.2d	293.1 ± 4.3b
Sucrose	1289.9 ± 44.5d	1420.6 ± 26.8e	1076.4 ± 24.2c	855.2 ± 44.4b	667.9 ± 5.4a
Raffinose	42.01 ± 0.4a	114.2 ± 6.0b	ND	ND	ND
Stachyose	199.4 ± 10.3b	35.3 ± 3.7a	ND	ND	ND

Values (mg/g, DW) are mean ± SD (n = 3). Different superscript letters in the same rows indicate significant differences according to Duncan's multiple range test (p < 0.05); ND, not detected.

3.2. Changes in amino nitrogen content

Amino nitrogen refers to the nitrogen present in the amino group of amino acids, indicating the degree to which proteins are broken down into amino acids due to the action of protease. Amino nitrogen is originally present in foods but can also be changed during food processing and aging. Since it significantly affects the taste of food, measuring the amino nitrogen content in food is often used to evaluate the sensory value, degree of aging, and digestibility of the food (Qin & Ding, 2007; Yanfang & Wenyi, 2009). In fermented foods, an increase in amino nitrogen due to protein hydrolysis is also used as an indicator to evaluate the degree of fermentation (Eom, Jung, & Oh, 2009). As shown in Fig. 1, the amino nitrogen content of unfermented soybean embryo (control) was approximately 0.06 %. However, it increased rapidly in the early stage of fermentation by G. lucidum, reaching about 0.49 % (p < 0.05) after 7 days-fermentation, which was approximately 8-fold higher than that of control.

Fig. 1.

Change of amino nitrogen content during LSF of SEH by G. lucidum.

Control (5), fermentation for 5 days without inoculation of G. lucium. Values (mg/g, DW) are mean ± SD (n = 3). Different small letters among the bars indicate significant differences according to Duncan's multiple range test (p < 0.05).

Jeong, Kim, Moon, and Park (2010) investigated the changes in amino nitrogen content during LSF of soymilk for 72 h with Bacillus subtilis KC-3. They reported that, while the free amino nitrogen content was 542.5 mg% (DW) in the control (0 day), it began to increase as fermentation progressed, reaching 882 mg% in the middle stage and 1190 mg% in the late stage of fermentation. Li et al. (2012) also reported that when soymilk was fermented for 22 h with six probiotic strains, the amino nitrogen content increased rapidly from 6 h, the early stage of fermentation, but the rate of increase varied significantly depending on the strain. In this study, the significant increase in amino nitrogen during the fermentation of SEH with G. lucidum may be due to the hydrolysis of proteins into free amino acids by protein-hydrolyzing enzymes secreted from G. lucidum mycelia.

3.3. Free amino acid composition

The changes in the free amino acid composition during fermentation of SEH with G. lucidum are shown in Table 2. In control (0 days), arginine (7.51 ± 0.34 mg/g), γ-aminobutyric acid (5.79 ± 0.30 mg/g), and lysine (1.59 ± 0.12 mg/g) were present in dominant amounts. However, as the fermentation progressed, the contents of most amino acids were significantly increased (p < 0.05) except only γ-aminobutyric acid. Notably, glutamic acid increased (p < 0.05) from 1.33 ± 0.11 mg/g in control to 14.28 ± 0.30 mg/g after 7 days, approximately a 10-fold increase. Lysine also increased (p < 0.05) from 1.59 ± 0.12 mg/g in control to 7.91 ± 3.76 mg/g after 7 days of fermentation. In addition, aspartic acid threonine, serine and glycine increased by approximately 8.5-, 11.8-, 11.8- and 9.7-fold, compare with that of control, respectively, and also the content of total free amino acid were increased significantly (p < 0.05) from 23.28 ± 0.67 mg/g in the control to 83.76 ± 4.26 mg/g after 7 days of fermentation. As reported by Sabotič, Trček, Popovič, and Brzin (2007), Basidiomycetes mushrooms have highly active proteolytic enzymes to hydrolyze proteins and Ganoderma sp. also secretes a wide range of extra-cellular proteases (Vidhya & V., 2019). The significant increase in free amino acids in fermented SEH can be attributed to the hydrolysis of proteins by the action of proteases secreted from G. lucidum mycelia. Overall, the alteration in the composition or content of amino acids affects the nutritional value and sensory attribute of fermented soybean embryo.

Table 2.

Change of free amino acid composition during LSF of SEH by G. lucidum.

Amino acid	Fermentation time (day)
	0	2	4	5	7
Aspartic acid	0.69 ± 0.08a	1.20 ± 0.07a	4.61 ± 0.86b	4.60 ± 0.21b	5.85 ± 0.21c
Threonine	0.49 ± 0.01a	1.84 ± 0.13b	4.72 ± 0.31c	5.23 ± 0.33d	5.79 ± 0.41e
Serine	0.44 ± 0.08a	0.98 ± 0.02b	3.05 ± 0.43c	3.74 ± 0.34d	4.73 ± 0.32e
Asparagine	0.33 ± 0.11a	0.67 ± 0.24a	1.68 ± 0.61b	1.99 ± 0.70b	2.44 ± 0.97b
Glutamic acid	1.33 ± 0.11a	4.59 ± 0.23b	10.82 ± 0.61c	11.87 ± 0.53d	14.28 ± 0.30e
Proline	ND	ND	0.56 ± 0.09a	0.55 ± 0.07a	0.50 ± 0.12a
Glycine	0.31 ± 0.08a	0.57 ± 0.11b	2.09 ± 0.13c	2.59 ± 0.13d	3.02 ± 0.03e
Alanine	0.57 ± 0.10a	1.23 ± 0.02b	2.89 ± 0.20d	2.47 ± 0.31c	2.38 ± 0.18c
Valine	0.26 ± 0.01a	0.68 ± 0.10b	1.86 ± 0.15d	1.76 ± 0.01d	0.99 ± 0.02c
Cystine	ND	0.81 ± 0.08a	1.03 ± 0.04b	1.13 ± 0.18b	1.30 ± 0.06c
Methionine	ND	0.50 ± 0.00a	0.75 ± 0.00b	1.00 ± 0.04c	1.50 ± 0.11d
Isoleucine	0.50 ± 0.00a	1.21 ± 0.05b	3.51 ± 0.01d	3.01 ± 0.02c	1.22 ± 0.04b
Leucine	0.70 ± 0.06a	2.02 ± 0.02c	4.41 ± 0.13e	3.52 ± 0.02d	1.49 ± 0.01b
Tyrosine	0.99 ± 0.02a	2.69 ± 0.09b	4.62 ± 0.18e	4.15 ± 0.21d	3.31 ± 0.08c
Phenylalanine	1.08 ± 0.24a	2.44 ± 0.09b	4.11 ± 0.20d	3.41 ± 0.23c	2.21 ± 0.06b
γ-Aminobutyric acid	5.79 ± 0.30d	4.02 ± 0.33c	2.78 ± 0.31b	2.50 ± 0.00b	2.09 ± 0.44a
Histidine	0.29 ± 0.06a	1.00 ± 0.01b	2.16 ± 0.12c	3.15 ± 0.14d	3.96 ± 0.41e
Carnosine	ND	1.23 ± 0.03a	1.94 ± 0.27b	1.82 ± 0.10b	ND
Ornithine	ND	0.76 ± 0.02a	1.40 ± 0.14b	1.71 ± 0.06c	1.69 ± 0.09c
Lysine	1.59 ± 0.12a	7.77 ± 0.73b	10.93 ± 3.08b	10.67 ± 2.36b	7.91 ± 3.76b
Arginine	7.51 ± 0.34a	10.65 ± 0.85b	12.80 ± 1.35c	12.64 ± 0.86c	9.75 ± 0.36b
Total	23.28 ± 0.67a	47.36 ± 0.91b	85.05 ± 2.55c	88.15 ± 2.68c	83.76 ± 4.26c

Values (mg/g, DW) are mean ± SD (n = 3). Different superscript letters in the same row indicate significant differences according to Duncan's multiple range test (p < 0.05). ND, not detected.

3.4. Composition of volatile flavor compounds

Although soymilk has various health benefits, its off-odor and off-taste, described as beany, green grassy, astringent, and bitter are factors that hinder its widespread consumption (Kaneko et al., 2014; Lozano, Drake, Benitez, & Cadwallader, 2007). Moreover, when soybean embryo is used as a raw material for soymilk production, these unpleasant off-flavors increase further in the product. Various volatile compounds have been identified in soymilk so far, and these compounds contribute directly or indirectly to the aroma characteristics of soymilk. Among them, the main compounds of the distinctive off-flavors in soymilk are compounds such as n-hexanal, cis-3-hexen-1-ol, n-hexanol, and trans-2-nonenal (Kaneko et al., 2014; Lozano et al., 2007; Yuan & Chang, 2007). In this study, the volatile compounds identified in control and fermented SEH with G. lucidum are shown in Table 3. Free fatty acids such as linoleic acid, palmitic acid, and oleic acid as well as β-phenylethyl alcohol, γ-dodecalactone, and trans, trans-2,4-decadienal were detected in significant amounts. Compared to the control, linoleic, palmitic, and oleic acids increased rapidly until 2 days after the start of fermentation but then decreased after 3 days. These results are thought to be due to the hydrolysis of triglycerides into free fatty acids by triacylglycerol hydrolase secreted by G. lucidum in the early stages of fermentation (Colak, Camedan, Faiz, Sesli, & Kolcuoglu, 2009). Additionally, 3-octanone, cis-2-heptenal, benzaldehyde, phenylacetaldehyde, and 2,4-decadienals increased steadily until the end of fermentation. Among these, 3-octanone, 1-octen-3-ol, 3-octanol, benzaldehyde, phenylacetaldehyde, benzyl alcohol, and phenylethyl alcohol are known to be present as volatile compounds of mushrooms, suggesting that they were originated from mushroom mycelia during the fermentation (Aisala, Sola, Hopia, Linderborg, & Sandell, 2019; Rapior, Marion, Pélissier, & Bessière, 1997). Particularly, C₈ alcohols such as 3-octanone, 3-octanol, 1-octen-3-ol, and 2-octen-1-ol are representative compounds found in fruiting bodies of mushrooms (Rapior et al., 1997; Aisala et al., 2014). In addition, compounds such as 3-octanone, cis-2-heptenal, benzaldehyde, phenylacetaldehyde, and 2,4-decadienals increased steadily until the end of fermentation. These compounds reached their maximum levels in the early stage of fermentation and thereafter tended to decline until 7-days fermentation.

Table 3.

Changes in volatile compounds during LSF of SEH by G. lucidum.

Peak no	RT (min)	RI		Compound	Fermentation time (day)
		Calc1)	Ref2)		0	2	4	7
1	4.44	1093	1082	n-Hexanal	33.84	40.26	22.95	25.73
2	8.13	1223	1206	3-Methyl-1-butanol	ND	86.98	79.60	84.13
3	9.80	1260	1255	3-Octanone	6.85	10.81	19.22	28.78
4	11.60	1300	1282	3-Hydroxy-2-butanone	26.63	21.92	19.22	24.56
5	12.51	1318	1322	cis-2-Heptanal	10.54	24.21	21.70	24.71
6	17.56	1421	1392	3-Octanol	8.82	10.54	10.87	26.93
7	19.41	1458	1444	1-Octen-3-ol	19.01	25.36	38.06	37.67
8	21.02	1490	1448	1-Heptanol	5.29	ND	10.20	14.55
9	22.34	1516	1518	Benzaldehyde	6.20	42.40	20.30	33.58
10	22.95	1529	1535	trans-2-Nonenal	12.06	19.40	18.83	46.31
11	23.82	1546	1547	cis-2-Octen-1-ol3)	15.50	17.89	28.14	42.85
12	27.30	1615	1641	Phenylacetaldehyde	ND	29.31	23.42	26.79
13	34.86	1759	1756	trans, cis-2,4-Decadienal	15.65	47.49	46.69	55.57
14	36.87	1800	1808	trans, trans-2,4-Decadienal	89.93	211.69	167.06	186.34
15	40.64	1879	1865	Benzyl alcohol	ND	75.52	136.00	99.01
16	42.09	1910	1904	β-Phenylethyl alcohol	113.75	81.38	64.72	155.16
17	46.36	2000	1992	Phenol	ND	21.81	37.01	35.74
18	48.45	2057	2057	γ-Nonalactone3)	ND	30.98	46.55	44.09
19	63.12	2400	2379	γ-Dodecalactone3)	ND	98.64	172.33	115.30
20	81.69	2900	2913	Palmitic acid	464.22	717.90	254.24	860.61
21	89.30	˃2900	3200	Oleic acid	25.22	340.17	162.91	403.59
22	90.97	˃2900	3290	Linoleic acid	72.67	494.47	180.29	1153.5

Values were expressed as μg/100 mL of fermented SEH; RT, retention time; RI, retention indices; ND, not detected. ¹⁾Calculated (calc)retention indices by a polar Supelcowax 10 capillary (30 m × 0.32 mm) column. ²⁾Referred from previously reported data by Babushok et al. (2011). ³⁾NIST standard reference database. Number 69 gas chromatography-retention indices (2023). Available online: https://webbook.nist.gov/cgi/cbook.

3.5. Changes in isoflavone composition

Isoflavones, which are important bioactive compounds found in soybeans, primarily exist in the forms of glucoside and, to some extent, in the free forms. These glucosides are hydrolyzed by β-glucosidase produced by intestinal microorganisms. Therefore, the ability to break down these glucosides depends on secretion capacity of β-glucosidase from the intestinal microorganisms (Chang, Kim, & Han, 2010; Setchell et al., 2002). Additionally, it is known that the free forms of isoflavone is more easily absorbed in the intestine and exhibits stronger bioactivity compared to the glucoside forms. Accordingly, pre-hydrolyzing and intaking of them could be more advantageous for the enhancement of bioavailability (Izumi et al., 2000; Setchell et al., 2002). Particularly, in fermented soybean foods, isoflavone glucosides are converted to their free forms by enzymes secreted by the microorganisms during fermentation. However, the hydrolysis of isoflavone glucosides varies depending on the type of microorganism used in fermentation (Li et al., 2012; Nurmilah, Frediansyah, Cahyana, & Utama, 2024). The changes in the composition of isoflavone compounds during the fermentation of SEH by G. lucidum are shown in Table 4.

Table 4.

Change of isoflavone contents during fermentation of SEH by G. lucidum.

FT (day)	Daidzin	Glycitin	Genistin	Daidzein	Glycitein	Genistein
0	0.98 ± 0.09a	1.00 ± 0.09a	0.62 ± 0.02a	0.65 ± 0.01a	0.42 ± 0.03a	0.31 ± 0.03a
2	ND	ND	ND	1.67 ± 0.02c	1.23 ± 0.13c	1.03 ± 0.02d
4	ND	ND	ND	1.68 ± 0.01c	1.27 ± 0.14c	1.01 ± 0.01d
5	ND	ND	ND	1.33 ± 0.00b	1.36 ± 0.02c	0.81 ± 0.01c
7	ND	ND	ND	0.66 ± 0.05a	1.08 ± 0.07b	0.41 ± 0.03b

Values (mg/g, DW) are mean ± SD (n = 3); FT, fermentation time; ND, not detected. Different superscript letters in the same column indicate significant differences according to Duncan's multiple range test (p < 0.05).

In the control, isoflavone glucosides (daidzin, glycitin, genistin) and their aglycones (daidzein, glycitein, genistein) were detected, with glucoside forms being quantitatively more abundant than the free forms. Isoflavones in soybean exist not only as glucosides but also as succinyl-, malonyl-, and acetylglucosides. After 2 days from the start of fermentation, most of the glucoside forms were converted into free forms. Additionally, after 4 days of fermentation, the free forms of isoflavone reached its maximum, thereafter, it decreased significantly (p < 0.05) until the 7 days of fermentation. These results indicate that some of isoflavones were utilized as a nutrient source during the growth of G. lucidum mycelia or it converted into other compounds during the fermentation progressing.

3.6. Analysis of OHI

New trace compounds that were not detected in the control began to form in the middle stage of fermentation, and after 7 days of fermentation, five new peaks were detected as shown in Fig. 2 (B). These compounds were presumed to be biotransformation products of the free forms of isoflavone, as these compounds increased with the fermentation extended. Therefore, structural identification was performed by HPLC and UPLC-QTOF-MS analyses after the isolation of OHI-rich fraction by SCC and the results are shown in Fig. 2 (C) and Fig. 2 (D). The newly formed compounds were mainly found in the fraction eluted with a mixture of CHCl₃:MeOH:DW (70:30:3) by SCC. Therefore, this fraction was analyzed by HPLC-PDA and UPLC-PDA-QTOF-MS and OHI compounds were identified on the mass spectral data by QTOF-MS (Table 5). Among these compounds, 8-OHD and 8-OHG were positively identified by comparing the QTOF-MS mass spectral data and retention times with those of the authentic standards in HPLC. Additionally, the compounds of peak no. 2, 3, and 4 in Table 5 were also considered to be OHIs with different hydroxylation sites on daidzein and glycitein based on their molecular masses. In fermented soybean foods such as miso and natto, multiple hydroxylated OHI compounds at the 6- and 3′-positions as well as at the 8-position of daidzein, glycitein, and genistein, have been previously found (Hirota et al., 2004). Suruga et al. (2020) identified two OHI compounds, including 8-OHD, in solid-state fermented soybean with G. lucidum for 4 weeks. One of these compounds was presumed to be 6- or 3’-OHD. However, it suggests that when soybeans are fermented with G. lucidum, the formation of OHI derivatives will be faster in LSF condition than in SSF.

Fig. 2.

Identification of OHI in 7 days-fermented SEH by G. lucidum.

Table 5.

Identification of OHI derivatives in fermented SEH by G. lucidum.

Peak no.	RT (min)	Proposed compounds	[M – H]−/ [M + HCOO]− (m/z)	Molecular formula	Molecular mass (Da)	Identi- fication
1	4.61	8-Hydroxydaidzein	269.0453	C15H10O5	270.24	MS, STD
2	4.83	Hydroxyglycitein1)	299.0559	C16H12O6	300.26	MS
3	4.89	Hydroxydaidzein1)	269.0418	C15H10O5	270.24	MS
4	5.06	Hydroxyglycitein1)	299.0519	C16H12O6	300.26	MS
5	5.51	8-Hydroxygenistein	285.0405	C15H10O6	286.24	MS, STD
6	5.61	Erysimoside	741.3282	C35H52O14	696.68	MS

¹Hydroxylation sites were unidentified. RT, retention time. MS, based on QTOF-MS spectral data. STD, comparison of retention times with those of authentic compounds in HPLC analysis.

A, unfermented SEH (control); B, after 7 days of fermentation; C, OHI-rich fraction isolated from 7 days fermented SEH by SCC; D, total ion chromatogram of UPLC-PDA-QTOF-MS of OHI-rich fraction at 260 nm. Da-G, daidzin; Gl-G, glycitin; Ge-G, genistin; Da, daidzein; Gl, glycitein; Ge, genistein.

It is also known that the number and position of hydroxyl groups attached to the isoflavone molecules have a significant impact on bioactivity and OHI derivatives possess various bioactivities, which have been reported to be stronger than those of glucosides or free forms of isoflavone (Chang, 2014; Hsiao, Ho, & Pan, 2020; Lee, Lee, Song, Choi, & Kim, 2018). Although the production of hydroxyisoflavones increased as the fermentation period extended, further fermentation beyond 7 days was deemed unsuitable due to the generation of strong mushroom-like odor and excessive mycelial growth leading to mycelial clumping.

3.7. TPC, TFC and antioxidant activity

The change of the TPC during the fermentation of SEH with G. lucidum was shown in Table 6. The TPC in the unfermented SEH (0 day) was 0.37 ± 0.05 mg/g, but after 2 days of fermentation, it increased (p < 0.05) to 0.58 ± 0.01 mg/g, approximately 1.57-fold higher than the control. After 5 days of fermentation, it further increased to approximately 0.82 ± 0.02 mg/g, representing an approximate 2.2-fold increase. Additionally, the TFC in unfermented SEH was 1.55 ± 0.14 mg/g, but after 5 days of fermentation, it reached a maximum of 3.18 ± 0.17 mg/g.

Table 6.

Changes of antioxidant capacities during LSF of SEH by G. lucidum.

FT (day)	TPC (mg/g as GAE)	TFC (mg/g as rutin)	Antioxidant capacities (mM TEAC/g, DW)
			DPPH	ABTS	FRAP
0	0.37 ± 0.05a	1.55 ± 0.14a	0.23 ± 0.04a	2.30 ± 0.10a	0.56 ± 0.03a
2	0.58 ± 0.01b	1.87 ± 0.11b	0.55 ± 0.11b	2.80 ± 0.09b	0.75 ± 0.05b
4	0.71 ± 0.02c	2.50 ± 0.08c	0.99 ± 0.05c	3.04 ± 0.04c	1.08 ± 0.01c
5	0.82 ± 0.02d	3.18 ± 0.17d	1.73 ± 0.10d	3.11 ± 0.03c	1.23 ± 0.06d
7	0.69 ± 0.01c	3.15 ± 0.14d	1.84 ± 0.04d	3.50 ± 0.08d	1.35 ± 0.01e

Values of TPC and TFC are mean ± SD (n = 3) as DW. FT, fermentation time; TPC, total phenol content; TFC, total flavonoid content; TEAC, Trolox equivalent antioxidant capacity; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-sulphonic acid; FRAP, ferric reducing antioxidant power. Different superscript letters in the same columns indicate significant differences according to Duncan's multiple range test (p < 0.05).

Many studies have been conducted on the antioxidant properties of fermented soybean products and the isoflavones contained in these fermented products (Do Prado, Pagnoncelli, de Melo, Karp, & Soccol, 2022; Hsiao et al., 2020). Various methods have been applied to evaluate the antioxidant activity of soybean-based and plant-based products. However, due to the different chemical reaction mechanisms, it is not sufficient to fully explain the antioxidant properties of a sample using only a single method (Xiao et al., 2014). For this reason, it is desirable to apply 2–3 different methods to explore the antioxidant properties of a sample. Among the methods, DPPH radical scavenging activity, ABTS radical scavenging activity, and the FRAP assay are commonly used for measuring the antioxidant activity of foods and plant-based products (Thaipong et al., 2006). The DPPH and ABTS methods measure radical scavenging activity, while the FRAP method measures reducing power. Antioxidant activity was measured for SEH fermented with G. lucidum by three different methods, namely, DPPH and ABTS radical scavenging activities, and reducing power by the FRAP method. The results were expressed as Trolox equivalent antioxidant capacity (TEAC) (Table 6). The TEAC value for DPPH radical scavenging activity in the control was 0.23 ± 0.04 mM TEAC/g. As fermentation progressed, it began to increase, reaching (p < 0.05) 1.84 ± 0.04 mM TEAC/g after 7 days of fermentation, which was approximately 8-fold higher than that in the control. ABTS radical scavenging activity in the control showed 2.30 ± 0.10 mM TEAC/g, while after 7 days of fermentation, it increased significantly (p < 0.05) to 3.50 ± 0.08 mM TEAC/g, approximately 1.5-fold higher than that in the control. The reducing power by FRAP was 0.56 ± 0.03 mM TEAC/g in the control, whereas it was 1.35 ± 0.01 mM TEAC/g in the SEH after 7 days-fermentation. The increase in antioxidant activity observed when SEH was fermented with G. lucidum is due to the hydrolysis of glucoside forms of isoflavone into their free forms, and the biotransformation of some of free forms into OHIs, which have enhanced antioxidant activities (Sheih, Fang, Wu, & Chen, 2014). Additionally, it is presumed that the G. lucidum mycelia secrete compounds that have strong antioxidant properties into the broth during the fermentation period (Ćilerdžić, Kosanic, Stajić, Vukojević, & Ranković, 2016).

4. Conclusion

Soy embryo, which is produced as a byproduct during the production on a factory scale of soymilk, has higher contents of phytochemicals such as isoflavones, soyasaponins and tocopherols compared to cotyledon. However, their use as raw materials for soymilk production has been limited due to the generation of undesirable factors such as green, beany off-flavors, astringent and bitter taste. Furthermore, harmful compounds including allergens, anti-nutritional factors and biogenic amines exist in soybean embryo may be transformed into soymilk. These compounds cause difficulties for expanding the consumption of soybean embryo as food raw materials. Microbial fermentation of soybean is considered as an alternative to enrich of nutritional values, health benefits and reduction of harmful ingredients. In this study, SEH, which are prepared in a similar process to the soymilk production using whole soybean, was subjected to LSF with G. lucidum for 7 days. The changes in compositions of sugars, amino nitrogen, free amino acids and isoflavones, and antioxidant activities of periodically taken samples during fermentation process were investigated. Indigestible oligosaccharides (stachyose and raffinose) were efficiently hydrolyzed into monosaccharides in the early stage of fermentation. The contents of free amino nitrogen and most of individual amino acids were significantly increased as the fermentation progressed. Additionally, isoflavone glucosides were completely hydrolyzed into their free forms, and thereafter, some of them were biotransformed into OHIs while overall antioxidant activities were also significantly increased as fermentation progressed. In conclusion, LSF of soybean embryo with G. lucidum can be improved its nutritional properties, bioavailability and antioxidant activities, thereby increasing its potential use as a food ingredient and functional health materials.

Funding

This research was funded by Korea Forest Service (Grant No. 2021381C10–2123-BD02).

Availability of data and materials

The data that support the findings of this study are available from the Y. H. Kim, one of co-authors, upon reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors have consent to the publication of the manuscript.

CRediT authorship contribution statement

Gi-Yoon Kim: Formal analysis. Ye Seul Kwon: Writing – original draft. Yoseph Asmelash Gebru: Writing – review & editing. Young-Hoi Kim: Writing – original draft. Dong Hyeon Kang: Writing – review & editing. Myung-Kon Kim: Resources, Methodology, Investigation, Conceptualization. Han-Seok Choi: Resources, Methodology, Investigation, Conceptualization.

Declaration of competing interest

The authors declare that there are no conflicts of interest or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.