A comparative study on flavour components and therapeutic properties of unfermented and fermented defatted soybean meal extract

Microbial fermentation of plant material alters the composition of volatile and non-volatile plant natural products. We investigated the antioxidant, anticancer, and antiviral properties of extracts of defatted soybean meal fermented with Aspergillus fumigatus F-993 or A. awamori FB-133 using in vitro methods. Gas chromatography–mass spectrometry analysis of soybean meal fermented with A. awamori FB-133 and A. fumigatus F-993 identified 26 compounds with 11,14-octadecadienoic acid and methyl ester (63.63%) and 31 compounds with butylated hydroxytoluene (66.83%) and δ-myrcene (11.43%) as main constituents, respectively. The antioxidant activities of DSM extract were 3.362 ± 0.05 and 2.11 ± 0.02 mmol TE/mL, FDSM treated with A. awamori FB-133 were 4.763 ± 0.05 and 3.795 ± 0.03 mmol TE/mL and FDSM treated with A. fumigatus F-993 were 4.331 ± 0.04 and 3.971 ± 0.02 mmol TE/mL as determined by ABTS and FRAP assays, respectively. Both fermented extracts had better antioxidant activity than the unfermented extract as shown by multiple antioxidant activity assays. The concentration of fermented extracts required for 50% inhibition of cell viability was significantly lower than that of the unfermented extract when tested against the human liver cancer cell line HepG2 as shown by cell viability assays, indicating strong anticancer activity. The IC50 values for DSM, FDSM with A. fumigatusF-993 and FDSM with A. awamori FB-133 were27, 16.88 and 8.60 μg/mL, respectively. The extract of FDSM with A. awamori FB-133 showed the strongest anticancer activity, compared to DSM and FDSM with A. FumigatusF-993 extracts. Fermented extracts also reduced hepatitis A virus titres to a greater extent than unfermented extracts, thus showing strong antiviral property. Hepatitis A virus titres were reduced by 2.66 and 3 log10/0.1 mL by FDSM with A. fumigatusF-993 and FDSM by A.awamori FB-133, respectively, compared to DSM (5.50 log10/0.1 mL). Thus, the fermentation of soybean meal with A. fumigatusF-993 or A. awamori FB-133 improves the therapeutic effect of soybean extracts, which can be used in traditional medicine.

fermentation of defatted soybean. The fermentation was carried out under solid state fermentation in 250 mL Erlenmeyer flasks containing 5 g of defatted soybean moistened to 50% with distilled water and inoculated with 1 mL spore suspension (10 6 spores). The cultures were incubated at 30 °C for 3 days for solid state fermentation 24 . Defatted soybean extraction. Volatile compounds were extracted from fermented defatted soy bean DSM samples according to previously reported protocols with some modifications 25,26 . The DSM and fermented DSM (FDSM) samples were ground individually to a powder using a coffee bean blender. Five grams of the ground powder was transferred into a 25 mL test tube and extracted with 15 mL ethanol. The supernatant was separated from the residue by centrifuging at 2000 × g for 10 min in a centrifuge and transferred to a clean test tube. The residue was re-extracted with 15 mL ethanol; the separated ethanol layers were combined, and dried using a vacuum evaporator at less than 50 °C. The dried soybean extract was weighed and stored at −20 °C.
Gas chromatography-mass spectrometry (GC-MS) analysis. About 2 μl of each extract was used for GC-MS analysis using a HP 5890 GC coupled to HP5970 MS. The MS was set to an ionization voltage of 70 eV and the mass range of m/z 39-400 amu was scanned. The GC oven temperature was maintained initially at 50 °C for 5 min and then programmed to rise to 250 °C at a rate of 4 °C/min. Helium was used as the carrier gas at a flow rate of 1.1 mL/min. The injector and detector temperatures were set to 220 and 250 °C, respectively. The isolated (2020) 10:5998 | https://doi.org/10.1038/s41598-020-62907-x www.nature.com/scientificreports www.nature.com/scientificreports/ peaks were identified by matching mass spectra with data in the National Institute of Standard and Technology library. Quantitation was carried out by peak area integration.
Determination of antioxidant activity. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. The DPPH assay was performed according to a previously reported protocol 27 . The antioxidant activity was determined by a calibration curve prepared with ascorbic acid and expressed as mg of ascorbic acid equivalent (AAE)/mL of sample 28 .
2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. The ABTS assay was performed as described previously 29 and expressed in mMof Trolox equivalents (TE)/mL of extract. When the measured ABTS value was over the linear range of the standard curve, values of additional dilutions were recorded 30 .
Ferric reducing antioxidant power (FRAP) assay. The FRAP assay was performed according to the Benzie and Strain method, and expressed in mM TE/mL extract. Values of additional dilutions were measured when the FRAP value was over the linear range of the standard curve 9 .
Anticancer activity. Cell culture and treatments. Human liver cancer cell lineHepG2 was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were grown in RPMI-1640 medium supplemented with 10% foetal bovine serum, 1% MEM non-essential amino acid solution, and 1% penicillin streptomycin solution (10,000 units of penicillin and 10 mg of streptomycin in 0.9% (NaCl) in a humidified atmosphere having 5% CO 2 at 35 °C. The passage number range was maintained between 20 and 25. The cells were cultured in 75 cm 2 cell culture flasks. For experimental purposes, the cells were cultured in 96-well plates (0.2 mL of cell solution/well). The optimum cell concentration, as determined by the cell line growth profile, was 2 × 10 5 cells/mL. The cells were allowed to attach for 24 h prior to treatment with the extracts. The stock solution of each extract was filtered with 0.22 µm Minisart Filters (Sigma-Aldrich) before applying to cells. To test toxicity, 11 dilutions of each extract were tested by addition to cell monolayers washed with phosphate buffer saline (PBS) 10 .

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay.
The MTT assay was performed using the protocol described previously 13 . Briefly, the cells were incubated for 4 h with 0.8 mg/mL of MTT dissolved in serum-free medium. After washing with 1 mL PBS, 1 mL DMSO was added and gently shaken for 10 min for complete dissolution. Aliquots (200 μL) of the resulting solution were transferred into 96-well plates and absorbance was recorded at 560 nm using the SpectraMax ® 190 Microplate Reader (Molecular Devices). The concentration required for 50% inhibition of cell viability (IC 50 ) was calculated as described 3 .

Antiviral assay. Cells and viruses. Vero and BHK-21 cells were grown in Eagle's minimal essential medium
(MEM, GIBCO) containing 5% inactivated calf serum and 50 mg/mL gentamicin. Maintenance medium at pH 7.5 consisted MEM supplemented with 1.5% inactivated calf serum and gentamicin. The vesicular stomatitis virus (VSV) was propagated in Vero cells. Virus stocks were plaque-assayed on Vero cells as described 31 . cytotoxicity assay. Vero cells grown in 96-well tissue culture plates were incubated with 12 concentrations (μg/mL) of each extract for 24 h at 37 °C. Cell viability was measured with the MTT assay. The cytotoxicity of each extract was expressed as the 50% cytotoxic concentration (CC 50 ), which is the concentration required to reduce cell viability to 50% of the control 32 .
Statistical analysis. The results are reported as mean ± standard deviation (SD) for at least three experiments. Statistical differences were analysed by the one-way ANOVA test.

Antioxidant activity.
The mean values of the antioxidant activity measured for DSM, FDSM1, and FDSM2 are presented in Table 3. The antioxidant activity of DSM extract was 3.362 ± 0.05 and 2.11 ± 0.02 mmol TE/mL as determined by ABTS and FRAPS assays, respectively, and 0.511 ± 0.01 mg AAE/mL with the DPPH assay. The antioxidant activity of FDSM2 extract was 4.763 ± 0.05 and 3.795 ± 0.03 mmolTE/mL with ABTS and FRAPS assays, respectively, and 0.625 ± 0.02 mg AAE/mL by DPPH assay. The antioxidant activity of FDSM1 extract was 4.331 ± 0.04 and 3.971 ± 0.02 mmolTE/mL by ABTS and FRAP assays, respectively, and 0.692 ± 0.01 mg AAE/ mL with DPPH assay. Therefore, both FDSM extracts showed stronger antioxidant activity than the DSM extract.
Anticancer activity. Colorimetric MTT assay was used to determine the extracts' anticancer activity, utilizing human hepatic cancerous cell line "HepG2". The cytotoxic effects of DSM, FDSM2, and FDSM1 extracts, expressed asIC 50 , are summarized in f 4. The IC 50 values for FDSM2, FDSM1, and DSM extracts were 8.6, 16.88, and 27 μg/mL, respectively, indicating that the FDSM extracts were more cytotoxic than the DSM extract. Table 4 also represents the titres of hepatitis A virus expressed as log 10 /0.1 mL. Extracts of FDSM1 showed stronger antiviral activity (2.66 log 10 /0.1 ml) compared to that of the DSM and FDSM2 extracts (5.5 and 3 log 10 /0.1 ml, respectively). The results of the present study revealed no significant differences in hepatitis A virus titres for FDSM1 and DSM extracts. (2020) 10:5998 | https://doi.org/10.1038/s41598-020-62907-x www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Plant extracts contain many important phytoestrogens compounds, the most relevant to human health include: phytoestrogens genistein and daidzein (from soybean), formononetin (from clover), biochanin-A (from chickpeas) and coumestans and lignans (from flaxseed). However, the majority of such compounds are naturally found, in plants, in the glycosylated form. Moreover, the bioavailability of such glycoconjugates varies from their analogous unsubstituted aglycones. In mammals, it has been documented that phytoestrogens induce oestrogenic and anti-oestrogenic effects via their weak binding to the nuclear α and β oestrogen receptors (ER). In general, the relative binding affinity of phytoestrogens to β oestrogen receptors (ERβ) is higher than that to α oestrogen receptors (ERα). It has been reported that such phytoestrogens are associated with a lower rate of steroid-hormone-dependent cancers, such as colon, prostate and breast cancer.
It has been revealed that the role played by of polyphenols against flue virus, as antiviral mechanism, can be attributed to the inhibition of its adsorption to Madin-Darby canine kidney cells 33 , as well as the interference in viral membrane fusion 28 . It is a well-known fact that mechanism of oxidative stress, commonly associated viral infections including HSV-1, plays a major role in viral replication inside the viral-infected cells. Moreover, viral replication can be inhibited through restoring the intracellular redox conditions by the help of special antioxidants (e.g., glutathione "GSH" or n-butanoyl derivative "GSH-C4") which inhibit HSV-1 replication 34,35 . In addition, Ramadan et al. 2014 have concluded that the main ingredients of the volatile compounds in DSM extracts are 23.8% (2,3-dicyano-7,7-dimethyl-5,6-benzodiene) and 19.1% (9-ditertbutyl-1-oxaspiro-4,5-deca-6,9-diene-2,8-dione) 1 .
It has been concluded that the major volatile antioxidant compounds, in FDSM, were butylated hydroxytoluene and 11,14-octadecadienoic acid, methyl ester. Octadecadienoic acid has been reported to be present in Michelia champaca Linn extracts and impart antioxidant and anticancer activities to the plant extract 36 . Specific food additives prevent lipid oxidation and rancidity, for example Butylated hydroxyl toluene "BHT", which is the most commonly used synthetic antioxidant 37 . Studies have revealed that "BHT" has anticancer activities and tumour promotion effects. Moreover, its effect on other carcinogens depends on a number of factors including, the carcinogen, target organ, exposure parameters, as well as the animal being tested. The toxicity of butylated hydroxytoluene is suggested to be the result of it being an electrophilic metabolite [38][39][40][41][42][43] . Numerous  www.nature.com/scientificreports www.nature.com/scientificreports/ studies have revealed that Butylated hydroxyl toluene "BHT" acts as a potent antioxidant that significantly inhibits cytokine-induced inflammatory responses in both human and mouse cells 44 .
Although, it has been concluded that the potency of modified ester of epigallocatechin gallate against HSV-1 infections, in vitro, is greater than that of epigallocatechin gallate, yet further studies are still recommended to fully explain the exact mechanism of action taking place in humans. Though, the use of the appropriate natural    Table 3. Antioxidant activity of extracts of defatted soybean meal(DSM) and defatted soybean meal fermented with Aspergillus fumigatus F-993 (FDSM1) and A. awamori FB-113 (FDSM2) measured by ABTS, FRAP and DPPH assays. All data are presented as mean ± SD. Values in the same column with the different superscripts are significant at P < 0.05. *Results are expressed as inhibitory activity in mmol Trolox equivalent/mL (mM TE/ mL) of extract. **Results are expressed as inhibitory activity in mg ascorbic acid equivalent per mL (mg AAE/ mL) of extract.  Table 4. Cytotoxicity and antiviral activity of extracts of defatted soybean meal (DSM) and defatted soybean meal fermented with Aspergillus fumigatus F-993 (FDSM1) and A. awamori FB-113 (FDSM2) against HepG2 liver cancer cell line and hepatitis A virus. All data are presented as mean ± SD. Values in the same column with the different superscripts are significant at P < 0.05.