Enhancement of antioxidant activity and improvement of the bright color of fermented soybean using ergothioneine biosynthesized by Aspergillus oryzae

Even though fermented soybean (FS) contains various antioxidant compounds, this long‐time processing still faces various oxidations caused by enzymes or heat. To elucidate the possibility of the oxidations as well as the level of enhancement of antioxidant properties to limit such oxidation, ergothioneine (ESH) concentrated extract (CE) from Aspergillus oryzae was used to interfere during FS processing. So, the supplementation of ESH CE (0%, 2%, 4%, and 6%) to one of three main stages such as soaking stage (SK), steaming (SM), or fermentation (FM) of the processing before measuring the quality changes of the final fermented product was done. The result showed that when added 4%–6% of ESH CE to the SK, ESH was slightly increased while 2,2‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activities and total phenolic compounds (TPC) increased considerably after fermentation by Bacillus subtilis. Total lipid was also slightly increased, whereas free fatty acid (FFA), peroxide value (PV), and thiobarbituric acid reactive substances (TBARs) decreased sharply compared with the control sample. Although supplemented at SK, L*‐value increased sharply from 0% to 4% and then decreased slightly at 6% of ESH CE, higher than at SM, and subsequent FM, ∆E‐value and a* and b* value changed in the opposite direction when these parameters decreased the sharpest at SK, lower than at SM and then at FM. The results indicate that supplementing with 4%–6% ESH CE at the early SK of FS processing shows a compatible difference with other stages, not only helping to prevent lipid oxidation but also improving the bright color of the final FS.

multiple functions including lowering blood pressure and preventing cardiovascular disease, stroke, Alzheimer's disease, atherosclerosis, and calcium loss as well as inhibiting markers related to obesity and Type 2 diabetes (Dajanta et al., 2013;Flores-Medellín et al., 2021;Ju et al., 2019;Li et al., 2020;Liu et al., 2022;Nagata et al., 2017). There are some changes between total glycoside and total aglycone during fermentation by B. subtilis. The increasing total aglycone and malonylglycoside isoflavone make DPPH activity higher but decrease lipid oxidation (LO) (Cho et al., 2011;Kwak et al., 2007). The genistein and daidzein are increased strongly after fermentation together with the increase in DPPH radical scavenging activities (Hu et al., 2010;Wu & Chou, 2009). This makes FS a promising functional food for humans (Gopikrishna et al., 2021). These pieces of evidence indicate that FS is a super healthy food because various useful biological changes take place during fermentation by B. subtilis.
However, FS processing contains various stages that affect the quality of FS products. A long time for steaming can reduce the time for fermentation and ammonia content (Wei et al., 2001). During FS processing, high temperature of steaming and a long time of fermentation at room temperature affect color changes caused by Maillard reaction and PPO enzyme reaction (Halloin, 1982;Taghinezhad et al., 2015). Different conditions of fermentation such as pH, temperature, and FT changes in crude protein, crude fat, and carbohydrate content of FS (Tham et al., 2022). Fatty acids (FAs) from FS are produced during fermentation. Various FAs are determined in FS products such as palmitic, stearic, oleic, linoleic, and eicosanoic acids (Kanghae et al., 2017). N atto branched-chain fatty acids (BCFA) are also detected in FS such as C14-17 iso-and anteiso-BCFA. The saturates, monounsaturates, and major polyunsaturates are the more stable foundation (Wang et al., 2019). Soybeans are nutrient-dense food ingredients and also serve as a good source of lipoxygenase (Shi et al., 2020). Hence, LO may happen to produce polyunsaturated FA into hydroperoxides. Unfortunately, there are no studies about the oxidation of lipids during FS processing. Because FS contains a high level of unsaturated fat and total polyphenols, the oxidation of lipid as well as color change might happen.
Ergothioneine is 2-mercaptohistidine trimethyl betaine (ESH) is a naturally occurring amino acid analog that is soluble in water, heat resistant, and safe as a nutraceutical and antioxidant (Borodina et al., 2020;Ha et al., 2021;Pramvadee et al., 2012). The thione form of ESH makes the antioxidant property of such chemical stronger (Bao et al., 2010). It is biosynthesized in some bacteria and fungi including Aspergillus oryzae but not in animals (Kerley et al., 2018;Nguyen et al., 2013;Takusagawa et al., 2018). ESH is also synthesized in actinomycetes, cyanobacteria, methylobacteria, some fungi, and mushrooms (Nguyen et al., 2013). ESH can be biosynthesized when the medium contains three kinds of amino acids such as cysteine, methionine, and histidine (Ey et al., 2007). So, applying ESH to FS processing could help to co-limit LO and color changes and then improve the quality of the final FS product.
During the FS processing, under a long time of soaking, steaming, and fermentation, LO and color changes caused by enzymatic and chemical reactions might happen that reduce the quality of the product.
In this study, the hypothesis is that ESH extracted from A. oryzae could enhance the antioxidant activity of FS, so the supplementation of ESH CE to exactly the stage that shows the most effective for preventing LO and color changes of FS should be clarified. Then, it could help to improve FS quality. Therefore, the supplementation of various ratio of ESH CE from A. oryzae to one of the three different stages of FS processing to measure the limitation of lipid and color changes to improve the quality of soybean fermentation product was done.

| Preparation of ESH CE from A. oryzae
The preparation of ESH from A. oryzae was based on the method of Ha et al. (2022) with some modifications. The biosynthesis of ESH by A. oryzae occurred in the by-product of purified starch industry (BPSI) medium supplemented by 120 mg histidine, 54.38 mg cysteine, and 83.4 mg methionine per 20 g medium. Twenty to twenty-five percent of rice husk was added into the medium to create porosity and create gaps for air to circulate inside the environment easily. The moisture content and pH value of the culture medium were adjusted to 40.0% and 5.5, respectively. A 20 g medium sample was put into a 250-ml flask, sterilized at 121 C for 15 min, and cooled to about 35 C before A. oryzae was inoculated at 10 6 CFU/g medium and incubated at 37 C for 3 days. After that, the culture was harvested and diluted 10 times with water. The extract from the mold culture product was heated at 90 ± 1 C for 10 min, centrifuged at 8000 g for 10 min, collected the crude extract, and evaporated under a vacuum at 60 C for 3 h to obtain a concentrated extract (ESH CE).

| Preparation of FS and supplementation of ESH CE to different stage of FS processing
The preparation of FS was based on the method of Tham et al. (2022) with some modifications.
Five hundred grams of soybeans was soaked in distilled water (w/v = 1:3) for 12 h at room temperature. After that, the samples were peeled and steamed for 30 min. The steamed beans were cooled down to room temperature, and B. subtilis was inoculated with a density of 10 6 (CFU/mL) and mixed well. The inoculated soybean was then spread evenly with a thickness of about 2 cm in a 100-ml glass and then closed with the lid before being placed in an incubator for 48 h fermentation at 37 C. FS products were vacuum packed and stored at À18 C. For supplementation experiments, ESH CE from 0% to 6% was added to one of three main stages of FS processing at the SK, before SM, and before FM. For the SK treatment, the ESH CE (0%, 2%, 4%, and 6%) was added to water containing soybean for 12 h before transfer to SK and FM. For SM treatment, the SK was done with water only; then the ESH CE was added to soaked soybean, mixed for 15 min before steamed for 30 min. Afterward, the steamed soybean was transferred to FM in an incubator for 48 h at 37 C. For FM treatment, all steps of soaking and steaming were done under normal conditions, then added 0%-6% ESH CE to this steamed soybean, and transferred to FM in the incubator for 48 h at 37 C. All parameters that indicated the quality of the final FS product related to antioxidant capacity, LO, and the color changes were analyzed to evaluate the effect of ESH CE on FS quality.
2.4 | Determination of 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity DPPH radical scavenging activity of ESH CE was determined according to Fu et al. (2002) with a slight modification. Briefly, 1-10 μl of extracted samples were mixed with a 0.5 ml portion of 0.1 mM DPPH ethanol solution and made to a final volume of 2 ml with distilled water. The mixtures were mixed and kept at 25 C for 30 min in the dark before being measured at 517 nm against a control without DPPH using a spectrophotometer (UV-1800 Shimadzu, Japan).

| Determination of total phenolic compounds
The total phenolic compound (TPC) was determined following the method of Carciochi et al. (2016). This measurement used gallic acid (GAE) as a standard. To construct the calibration curves, different concentrations from the standard stock solution of gallic acid (1 mg/ml) in the range of 1, 2, 4, 6, 8, and 10 μg/ml were prepared. TPC in FS was extracted twice with 80% ethanol, pooled, filtered, and stored at À18 C. 0.2 ml of extract was reacted with 0.8 ml of Folin-Ciocâlteu (1:10) reagent and 2 ml of 7.5% sodium carbonate and kept in the dark at ambient temperature for 30 min before being measured at the wavelength of 765 nm using UV-Vis spectrophotometer (UV-1800 Shimadzu, Japan). Distilled water was used as the blank sample. The total polyphenol content in the sample was expressed as gallic acid equivalent (mgGAE/mL).
The mobile phases include acetic acid (2.5%) and deionized water (50: 50) at a flow rate of 0.7 ml/min. 2.7 | Determination of L*, a*, b*, and ΔE value Approximately 100 g of FS samples was weighed, placed in a petri dish, set in the imaging chamber, and photographed with a camera (Sony N50, Japan). Then, the samples were immediately transferred to measure L*, a*, b*, and ΔE values using the FRU WR-10 colorimeter (Guangdong, China).

| Determination of TBARs
TBARs were determined following the method of Tarladgis et al. (1960). Briefly, 3 g of FS sample was mixed with 10 ml of 5% TCA. It was filtered and made up to 25 ml using TCA. Next, 3 ml of sample solution was mixed with 1 ml reagent TBARs and heated at 94 C for 5 min. The mixture was cooled down with cold water, followed by a measurement of absorbance at 530 nm using a UV-Vis spectrophotometer (UV-1800 Shimadzu, Japan). The results were equivalent mg of malonaldehyde/kg sample.
2.9 | Determination of total lipid, free fatty acid, and peroxide value Total lipid, peroxide value (PV), and free fatty acid FFA of the soybean and FS samples were measured following the methods in AOAC (2000).

| Statistical analysis
For ANOVA and LSD test at a 5% significance level (P ≤ 0.05) of data used the Statgraphics Centurion XV software (Version 15.2.11, Statgraphic Technologies, Inc. Virginia, USA). Mean and standard deviation were calculated in triplicate by Excel 2010 software.

| Characterization of ESH CE from A. oryzae using a BPSI-based medium
The results in Table 1 showed that three amino acids such as histidine, cysteine, and methionine affected the biosynthesis of ESH from A.
oryzae. The optimal ratio of histidine-cysteine-methionine concentration was found as 120:54.38:83.04 mg, respectively. With the percentage of mold, moisture content, and pH value of the culture medium of 0.5%:50%:5.5, it was possible to biosynthesize ESH content of 546.46 mg/100 g corresponding to the IC50 value of 118.49 μl/ml at the final density of fungal cell as 17.7 Â 10 8 CFU/g. A. oryzae was also much higher (Ha et al., 2021). Weiya et al. (2016) reported that using hot water at 87.5 C for 10 min can extract ESH with more than 97.1%. Under this experiment condition, this study showed that ESH was extracted by hot water at around 87.1%. Still, with such a high level of ESH achieved, ESH CE has a potent antioxidant property for enhancement of lipid antioxidation and limiting color change during FS processing.
3.2 | Change in ESH, DPPH, and TPC of FS when supplemented the concentrated extract to one of three different stages of processing The results in Figure 1 show Notes: All reported values are shown as mean ± standard error. Crude protein, carbohydrate, and fat of BPSI were 25.18 ± 0.53, 73.11 ± 0.70, and 0.61 ± 0.09, respectively. The ratio of histidine-cysteine-methionine concentration was 120:54.38:83.04 mg, respectively. The percentage of mold, moisture content, and pH value of the culture medium were 0.5%:50%:5.5, respectively. The medium after cultivation was diluted 10 times with water before heated at 90 ± 1 C for 10 min, then centrifuged at 8000 g for 10 min, and collected; the crude extract was evaporated under vacuum at 60 C for 3 h to obtain a concentrated extract (ESH CE). Abbreviation: BPSI, by-product of purified starch industry. temperature for 30 min has promoted a part of ESH penetrating the soybean, but another part can be lost due to the constant contact of steam on the soybean surface while soybeans steamed. It is also possible that some ESH is lost due to thermal transformation. However, ESH is known to be heat resistant (Pramvadee et al., 2012), and it can also reduce because part of it has become a nutrient for B. subtilis ferment for 48 h (Wolff, 1962 (Dajanta et al., 2013;Liu et al., 2022). The increasing total aglycones during fermentation increase antioxidant activity DPPH and decrease lipid peroxidation (Kwak et al., 2007). Therefore, the increase in TPC composition is due to the changes in aglycone composition produced during soybean fermentation by B. subtilis. In addition, the presence of ESH in the fermented product is due to the addition of ESH CE during processing, which can lead to elevated TPC and DPPH. As a result, this increased the DPPH free radical scavenging capacity of the product.

| Change in lipid, peroxide, and TBARs of FS under normal condition of FS processing
The results from Figure 2 show that under normal conditions of FA showed a statistically significant difference (P < 0.05). Besides, lipid seems to be biosynthesized during FS fermentation because their content is almost higher than soybean material. These results indicated that fat oxidation occurred strongly during FS processing under normal conditions, especially during soaking and fermentation.
Soybeans (SB) are very nutritious and provide a healthy source of protein, low in fat and cholesterol (Middelbos & Fahey, 2008;Shibata et al., 2008;Thrane et al., 2017). However, the quality changes of SB can occur due to oxidation and browning during the distribution process as well as due to lower storage stability (Joung et al., 1997).
Among them, LO can decrease the nutritional value and shelf life of soybean (Kim et al., 2008). The oxidation of unsaturated FAs to form volatile compounds and hydroperoxides, which are catalyzed by the lipoxygenase (EC 1.13.11.12), results in rancidity (Priestley & Leopold, 1979). TBARs and PV were decreased profoundly at low-temperature storage (Park et al., 2018). So, LO is one of the problems in maintaining the quality of soybean. Besides, autoxidation that accelerated at a high temperature via a free radical chain reaction involving singlet oxygen can occur during processing (Lee et al., 2005;Lee & Choe, 2003). During the fermentation, the lipid content of FS could F I G U R E 2 Changes in total lipid, free fatty acid (a), peroxide, and TBARs (b) during fermented soybean processing under normal condition.
(a)-(d) indicated a statistically significant difference (P < 0.05) of total lipid, free fatty acid, peroxide, or TBARs between different stages of after soaking, after steaming and after fermentation in the FS processing under normal condition without supplementation of any ESH CE.
increase from 26% to 28%, depending on fermentation (Tham et al., 2022). So, two trends that cause lipid changes are an increase in lipid content due to fermentation and a decrease in lipid content due to oxidation. As a result, the similar lipid content of raw material and FS product was recorded. This study showed that there is apparent oxidation of lipid to FFA, PV, and FFA during the processing of FS caused decreasing the product quality, which needs an interfering antioxidant solution to improve the situation.

| Effect of ESH CE on lipid oxidation when added at different stages of FS processing
The results in Figure 3 show that lipid content increased with the addition of ESH CE from 0% to 6%. Supplementation at the SK increased lipid content to the highest, and there was no statistically significant difference at the two concentrations of 4%-6%. Although the sample was supplemented with ESH CE at the FM, the lipid content was the lowest. The lipid content increased slightly, and there was no statistically significant difference at the two concentrations of 4%-6% (P < 0.05). Besides, the FFA, TBARS, and peroxide values all decreased significantly and had a statistically significant difference (P < 0.05) according to the increase from 0% to 6% of the supplemented ESH CE content. The addition of ESH CE to the SK decreased these indicators, followed by the addition in the SM and finally the FM.
The results in Figure 3 show that, if the addition of ESH CE profoundly reduces oxidation indices such as PV, TBARs, and FFA, lipids will be reduced insignificantly due to oxidation. The tendency to increase lipids due to the addition of ESH CE while the decrease in lipids due to oxidation is not much because the oxidation process is overcome, which is very good for the product. There exist two mecha-

| Change in color of FS when supplementation of ESH CE to one of three different stages of FS processing
The results in Figure 4 show that the addition at the immersion stage from 0 to 4 shows that the colors of the FS are increased in brightness. Similarly, a* value is also reduced when increasing from 0%% to 4% and then increases again at 6%. The changing trend of b* value is similar to that of a* value. Unfortunately, as the increase continues to 6%, the color darkens again. ΔE value decreases. It can be seen that increasing the concentration to 6% ESH CE has made the product darker, which may be due to the amount of darkening of the CE solution that makes the color of the FS darker rather than the restriction of color processing being ineffective. Compared with the results from the effectiveness of the addition of concentrations of 0%-6% in limiting LO, 6% at the SK showed the most effectiveness. This is a testament to the point of the 6% ESH CE supplement.
There are two stages that affect the color change in natto during processing such as steaming and fermentation. The first stage is steaming. High temperature of this stage may lead to browning due to a non-enzyme browning reaction named the Maillard reaction (Taghinezhad et al., 2015;Tamanna & Mahmood, 2015). An environment that contains reducing sugars and high protein contents can lead to the Maillard reaction, which caused a decrease in L* value (Mwangwela et al., 2007). The second stage that affects the color change is fermentation by a browning reaction due to the Maillard reaction and PPO enzyme reaction caused by a long time at room temperature. This enzymatic browning reaction happened when an environment contains oxygen, polyphenols, and PPO (Garcia-Molina et al., 2005;Halloin, 1982). The intensity of melanosis in crustaceans varies by species due to differences in substrates and enzyme concentrations (Benjakul et al., 2003). PPO has different optimal molecular weights and operating conditions depending on its origin ( (Tepwong et al., 2012). Therefore, the results of this study indicate that with 4%-6% of ESSH CE added to SK, the brightness of color seems the best. L* value shows the brightness for natto and discoloration corresponding to darker (Iwahashi et al., 2015). Kubo et al. (2021) reported that when natto was stored from 10 to 20 C for 10 days, the brightness and yellowness decreased corresponding to L* value and b* decreased. This is caused by Maillard reaction due to high-temperature storage (Makino, 2007). Therefore, during fermentation of FS, browning reactions may happen due to both enzyme PPO and Maillard reaction as well. As a result, the color of FS changed during steaming and fermentation. And the 4%-6% ESH CE shows the practical impact of reducing the color changes caused by both the Maillard and PPO enzyme reactions.
F I G U R E 3 Changes in total lipid (a), free fatty acid (b), peroxide (c), and TBARs (d) of fermented soybean product when supplemented with 0%-6% ESH CE to the different stages of the processing. (a)-(d) indicated a statistically significant difference of total lipid, free fatty acid, peroxide, or TBARS between amount of additional extract at the same stage of ESH CE supplementation in the processing (P < 0.05). (a)-(d) indicated a statistically significant difference of total lipid, free fatty acid, peroxide, or TBARS between different stages of ESH CE supplementation in the processing at the same concentration of additional extract (P < 0.05).

| CONCLUSION
The addition of 4%-6% ESH CE from A. oryzae to FS processing, especially during the SK, showed a compatible difference with other stages. It contributed to higher antioxidant capacity that helped to limit enzyme-induced fat oxidation and auto-oxidation. Besides, it can also restrict the dark brown discoloration of the product. The results indicate that this is a great solution to improve the quality of FS products effectively.

ACKNOWLEDGMENTS
This is funded in part by the Can Tho University Improvement Project VN14-P6. And this is also supported in part by Tra Vinh University. Vietnam.
F I G U R E 4 Changes in L (a), a * (b), b * (c), and ΔE value (d) of fermented soybean product when supplemented with 0%-6% ESH CE to the different stages of the processing. (a)-(d) indicated a statistically significant difference of L, a * , b * , or ΔE between amount of additional extract at the same stage of ESH CE supplementation in the processing (P < 0.05). (a)-(d) indicated a statistically significant difference of L, a, * b * , or ΔE between different stages of ESH CE supplementation in the processing at the same concentration of additional extract (P < 0.05).