Evaluation of the fermentation properties of different soybean (Glycine max L.) cultivars 

Sample preparation
 
Six Korean soybean cultivars (Glycine max L. ‘Taekwangkong’ [TKK], ‘Daechankong’ [DCK], ‘Misokong’ [MSK], ‘Seonpungkong’ [SPK], ‘Uramkong’ [URK] and ‘Chamolkong’ [COK]) were grown in the National Institute of Crop Science, Rural Development Administration (Miryang, South Korea) during the 2017 growing season and were stored at 4°C. Bacterial strains isolated from traditional Korean soybean paste were used as the starter culture.
 
Soybean fermentation
 
Soybeans were soaked for 15 h at room temperature; after draining the water, the soybeans were sterilized by autoclaving at 12°C for 30 min. Sterile soybeans were inoculated with a suspension of Bacillus amyloliquefaciens at a concentration of 106 CFU/g and approximately 200 g (wet weight) was placed in a sterile styrofoam cup that was incubated at 40°C for 24 h. The fermented soybeans were crushed or freeze dried and maintained at -70°C until use.
 
Measurement of EA and ATN and VS content
 
The protease activity assay was carried out as previously described (Anson, 1938), with some modification. The α-amylase activity was measured using the dextrinogenic unit of nagase method (Lee et al., 2017). The ATN content was measured using the Formol method (Lee et al., 2017). Viscous substances were weighed by evaporating a certain amount of the sample at 105°C and were marked as dry substances (%).
 
Measurement of OA content
 
One gram of freeze-dried sample was added to 80% MeOH and extracted using a sonicator (BKUP-600N; Biokonvision, Gyeonggi, South Korea) for 1 h. The extract was passed through a 0.2-μm membrane filter and the filtrate was analyzed by high-performance liquid chromatography (HPLC) (Ultimate 3000 with pump and autosampler; Dionex, Sunnyvale, CA, USA) using an HLB Sep-pak C18 cartridge (Waters, Milford, MA, USA) and Aminex 87H column (300 mm × 6.5 mm; Waters), with 0.01 N sulfuric acid (99%; Fluka, Ronkonkoma, NY, USA) as the mobile phase.
 
Measurement of FAA content
 
The FAA content was determined using a post reaction-type AA analyzer (L-8900; Hitachi, Tokyo, Japan). Freeze-dried powdered sample (1 g) was extracted in 50 mL of distilled water at 80°C for 15 min. After centrifugation (Ultra-4.0; Hanil, Gimpo, South Korea) of 1 mL of the extract in 5% (w/v) trichloroacetic acid at 10 000 rpm for 10 min, the supernatant was passed through a 0.2-μm membrane filter and analyzed as previously described (Yoo and Chang, 1999).

Measurement of VC content
 
VCs were extracted from fermented soybeans using the solid-phase microextraction (SPME) method with 75 mL of carboxen/polydimethylsiloxane fiber (Supelco, Bellefonte, PA, USA). The extract was concentrated by rotary evaporation (N-1000; Eyela, Tokyo, Japan), transferred to a vial, and concentrated to 100 μL under nitrogen gas. VCs were analyzed by gas chromatography (GC) (TRACE1310; Thermo Fisher Scientific, Waltham, MA, USA)/mass spectrometry (MS) (TSQ8000; Thermo Fisher Scientific using a DB-WAX column (60 m × 0.25 mm, 0.5-μm film thickness; Agilent Technologies, Santa Clara, CA, USA) (Park et al., 2017).
 
Statistical analysis
 
Experiments were carried out with triplicate samples and all data are expressed as mean ± standard deviation (SD). Significant differences among treatment means was determined by one-way analysis of variance. Data were analyzed using SPSS v.12.0 software (SPSS Inc., Chicago, IL, USA). Differences between groups were evaluated by Duncan’s multiple range test, and the significance level was set at 0.05.

EA and ATN and VS content in fermented soybeans
 
The EA of the extracts of six soybean cultivars was measured after 24 h of fermentation (Table 1). The protease activity was the highest in DCK (664.07 U/g), followed by SPK (650.14 U/g). α-Amylase is an important enzyme in fermented soybeans as it hydrolyzes carbohydrates to confer a sweet taste. The α-amylase activity was the highest in TKK and DCK at 21.56 and 16.57 U/g, respectively. These results demonstrate that there was a significant difference in the EA of fermented soybeans of different cultivars. The activity of protease from microorganisms involved in fermentation affects the taste of a product (Gil et al., 2016). Although the biochemical mechanisms of fermentation were similar among soybean cultivars, differences in nutritional value and quality exist due to variable activities of hydrolytic enzymes, indigenous microflora and fermentation conditions (Chukeatirote, 2015).
 

       
The protease activity during fermentation leads to the deamination of proteins and their breakdown into AAs; it serves as a quality indicator of soybean fermentation (Ghosh et al., 2018). The ATN content was the highest in DCK (Table 1), reflecting a high enzymatic activity (Eom et al., 2009). It has been reported that the amounts of sugar and AAs produced by microorganisms during soybean fermentation are highly correlated (Gil et al., 2016). VSs produced by fermentation are a polyglutamate mixture of levan and protein decomposed from fructan in soybeans (Bang et al., 2011). The VS content was higher in DCK than in other cultivars. These results suggest the difference in the composition of species due to genetic diversity depending on the growth environment conditions of soybean (Jain et al., 2018).
 
OA content in fermented soybeans
 
The OA content in fermented soybean varied among cultivars, as determined by HPLC (Table 2). The major OAs were lactic acid > acetic acid > fumaric acid. Citric acid was not detected in any of the samples. DCK had the highest OA content at 7034.58 mg/kg. Fig 1 shows the percentage of OAs according to cultivar; MSK had the highest lactic acid content (86.58%), whereas the acetic acid content was the highest in URK (34.30%).
 

 

       
A previous study reported differences in the OA content in eight soybean cultivars (Yoo and Chang, 1999). This is in agreement with our findings, which also showed that lactic acid and acetic acid were present when B. subtilis was used for fermentation. On the contrary, another study showed that the main OAs in soybeans fermented using Lactobacillus were lactic acid and oxalic acid (Lee et al., 2017). These differences may be due to the soybean cultivars and starter strains that were used. It is expected that the OA composition affects the antimicrobial activity and flavor of the products.
 
FAA content in fermented soybeans
 
FAAs in soybeans have a high nutritional value and have several health benefits (Gibbs et al., 2004). The FAA content varied with the soybean cultivar. The EAA contents tended to increase after fermentation in all of the tested cultivars, but were the highest in DCK and lowest in MSK (Fig 2). Notably, in DCK, the EAA content after fermentation was about seven times higher than that before fermentation. The most abundant EAA was Val, followed by Leu, Lys, Thr, Try, Phe, Ile and Met. Glu was the most abundant FAA in all the cultivars, with levels 2–10 times higher in fermented soybean than in raw soybean (Fig 3). DCK had the highest FAA content following fermentation, and also showed the greatest increase. In an earlier study, the Glu concentration showed the greatest increase among FAAs following fermentation (Jung et al., 2009). Although it depends on the cultivar, changes in the FAA content caused by fermentation generally increase the palatability, bitterness, and sweetness of soybean (Gil et al., 2016). This is because the AAs resulting from the breakdown of proteins during the fermentation process combine to produce a unique flavor (Katekan, et al., 2011). The increase in FAAs after fermentation of soybeans results in functional benefits to the health of humans (Luo et al., 2014; Shekar and Pushpendra, 2017).  
 

 

       
γ-Amino-n-butyric acid (GABA) and ornithine (Orn), which are AA metabolites produced by soybean fermentation, were the most abundant in TKK and DCK tetramethylpyrazine. An earlier study has showed that pyrazine compounds were present in soybean samples fermented using Bacillus but were absent in those fermented using Lactobacillus (Lee et al., 2017), which is in disagreement with the results of the present study.
       
The main compound responsible for the unpleasant odor that is sometimes associated with fermented soybean is tetramethylpyrazine generated by fermentation starter strains although a small amount of this VC can positively affect the taste. The amount of tetramethylpyrazine produced varies depending on fermentation conditions (e.g., microbial strain, water, salinity and temperature) and the type and amount of other compounds. Therefore, maintaining an appropriate VC content is important for increasing consumer preference.

DCK soybean cultivar showed excellent fermentation characteristics including the highest OA, FAA and Orn content. Furthermore, the VC content, which can confer an unpleasant odor when present in excess amount, was also higher in DCK than in MSK. Therefore, although DCK has suitable characteristics to produce fermented foods, the fermentation process may require optimization in order to achieve a VC content that is acceptable by consumers.

This study was carried out with the support of the Cooperative Research Program for Agriculture Science and Technology Development (Project no. PJ01350803) of the Rural Development Administration, Republic of Korea.