Soybean Paste Research Articles

Protective Effects of Fermented Soybeans (Cheonggukjang) on Dextran Sodium Sulfate (DSS)-Induced Colitis in a Mouse Model.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease, and the incidence of IBD is increasing every year owing to changes in dietary structure. Although the exact pathogenesis of IBD is still unclear, recent evidence suggests that gut dysbiosis is closely associated with IBD pathogenesis. Cheonggukjang is a traditional Korean fermented soybean paste produced using traditional and industrial methods, and contains probiotics, which affect the gut microbiota composition. However, the protective effect of Cheonggukjang against IBD is unknown. In this study, we investigated the bacterial community structure of traditional and commercial Cheonggukjang samples, as well as the protective effect of Cheonggukjang on a dextran sulfate sodium (DSS)-induced colitis mouse model. Traditional and commercial Cheonggukjang were found to contain various type of useful probiotics in their bacterial community structure. Cheonggukjang reduced the progression of DSS-induced symptoms, such as body weight loss, colonic shortening, disease activity index, and histological changes. Further, Cheonggukjang improved the intestinal epithelial barrier integrity on DSS-induced colitis mice. In addition, Cheonggukjang suppressed the expression of proinflammatory cytokines and inflammatory mediators through the inactivation of NF-κB and MAPK signaling pathways. These results indicate that Cheonggukjang exerts protective effects against DSS-induced colitis, suggesting its possible application as a functional food for improving inflammatory diseases.

Effects of Tartary buckwheat on physicochemical properties and microbial community of low salt natural fermented soybean paste

It is a valuable attempt to use Tartary buckwheat as the raw material of soybean paste due to its functional composition. In this research, the soybean pastes were prepared with Tartary buckwheat flour and wheat flour by low salt natural fermentation (10%, w/v NaCl), respectively; the physicochemical properties and microbial community structure of samples prepared from the two groups were investigated. The results showed that Tartary buckwheat promoted the accumulation of protease, total acid (TA), amino acid nitrogen (AAN) significantly compared with the control group during fermentation (p < 0.05). Both groups had similar bacterial communities, Klebsiella, unidentified_Chloroplast, Bacillus, Staphylococcus, Enterococcus, Acinetobacter, Weissella were present in all samples. But the fungal communities of the two groups were significantly different. The dominant fungi in all samples were Rhizopus and Aspergillus. In moromi fermentation, the Issatchenkia presented only in buckwheat group, Stenocarpella and Gibberella were characteristic fungi of wheat flour group. The changes of biodiversity and community structure showed that the microbial communities of the two groups experienced different succession patterns. The results can provide a reference for the further development of low salt Tartary buckwheat soybean paste and the study of microbial community succession in soybean paste fermentation.

Effect of Different Pretreatment of Raw Materials on Flavor Quality of Zhaotong Soybean Paste

Effect of Different Pretreatment of Raw Materials on Flavor Quality of Zhaotong Soybean Paste

Ingestion of miso regulates immunological robustness in mice.

In Japan, there is a long history of consumption of miso, a fermented soybean paste, which possesses beneficial effects on human health. However, the mechanism behind these effects is not fully understood. To clarify the effects of miso on immune cells, we evaluated its immunomodulatory activity in mice. Miso did not alter the percentage of B and T cells in the spleen; however, it increased CD69+ B cells, germinal center B cells and regulatory T cells. Anti-DNA immunoglobulin M antibodies, which prevent autoimmune disease, were increased following ingestion of miso. Transcriptome analysis of mouse spleen cells cultured with miso and its raw material revealed that the expression of genes, including interleukin (IL)-10, IL-22 and CD86, was upregulated. Furthermore, intravital imaging of the small intestinal epithelium using a calcium biosensor mouse line indicated that miso induced Ca2+ signaling in a manner similar to that of probiotics. Thus, ingestion of miso strengthened the immune response and tolerance in mice. These results appear to account, at least in part, to the salubrious effects of miso.

Behavior study of Bifidobacterium longum using solid state fermentation from commercial soybean meal

The development of alternatives to dairy products has gained great importance using plant-based foods. In this study, efforts have been made to develop vegetable alternatives from soybean paste without any supplement using single potential probiotic culture. Commercial soybean meal was inoculated with Bifidobacterium (B.) longum under previously fitted fermentation conditions by experimental design. Carbohydrates, organic acids, isoflavones, and amino acids profiles were studied in nine conditions of moisture and temperature, and also at several times in optimized conditions. Kinetics and technological parameters, effect of simulated gastric conditions on bacterium survival in optimized soybean paste, and their relationship with soybean matrix using scanning electron microscopy (SEM) were also evaluated. B. longum was able to use the main carbohydrates in soybean producing organic acids as was expected (acetic/lactic ∼ 1.5) and to enhance bioactive isoflavones (maximum concentration: 32.6 mg/50 g of soybean paste). Furthermore, B. longum showed high tolerance at simulated saliva solution and gastric juice, but the pancreatic solution deeply affected its viability in MRSs and also in soybean paste. Soybean paste represents an excellent vegetable candidate to be used as potential probiotic carrier food for B. longum and an attractive alternative to develop new functional foods.

Impact of salt concentration on bacterial diversity and changes in biogenic amines during fermentation of farmhouse soybean paste in Northeast China

Farmhouse soybean paste in Northeast China is a traditional fermented product made from soybean, and more than 11% (w/w) salt is usually added during production to control the fermentation process. In this study, the variations in bacterial diversity, biogenic amines(BAs) and physicochemical properties during the natural fermentation of soybean paste with different salt concentrations (8%, 9%, 10%, 11%, and 12%) were studied. The results show that at 0 days (0 d) of fermentation in soybean paste, the dominant genera included Staphylococcus, unidentified Clostridiales, and Sporolactobacillus. During fermentation from 30 d to 90 d, the dominant genera were Tetragenococcus and Staphylococcus. However, the proportions of the dominant genera were different depending on the salt concentration. Putrescine(Put), tryptamine(Try), β-phenethylamine(Phe), cadaverine(Cad), histamine(His), and tyramine(Tyr) showed negative correlations with salt concentration. The amino type nitrogen(ANN), titratable acidity(TTA) and total number of colonies were also negatively correlated with salt concentration. Analysis of the correlation between genera and BAs showed that 12 genera were positively correlated with BAs, and 4 genera were negatively correlated with BAs. The results of this study indicated that salt has a significant impact on bacterial diversity during the fermentation of soybean paste, which in turn affects the changes in bacterial metabolites. From the perspective of food safety, the amount of salt added in the soybean paste can be reduced to 10% under the existing fermentation conditions.

Processing optimization and quality assessment for the innovative product of canned soybean paste oyster

Oysters have high nutritional value but are rare in processing products. In this study, we developed an innovative product of oyster combined with soybean paste. The processing conditions and flavoring formulations of canned oysters were optimized using orthogonal experiments. Headspace solid-phase microextraction (HP-SPME) combined with gas chromatography-mass spectrometry (GC-MS) was applied to evaluate the volatile compounds related to the aroma. The results showed that the optimal canned oyster was oyster dried at 90 °C for 20 min and oil-fried at 150 °C for 6 min with the adding flavoring formula of 20% soybean paste, 0.5% vinegar, and 0.01% I+G (disodium 5'-presenting nucleotide) based on the weight of oyster. Canned soybean paste oysters enrich volatile components related to aroma including 2-octanol, 2-furanmethanol, benzaldehyde, and 2-pentylfuran. The canned soybean paste oyster is a high-valued nutrient including protein, fat, zinc, and taurine. This study will provide processing conditions for favorite instant processed oysters with soybean paste supplementary and deep insight into the flavor formation.

Comparative Evaluation of Quality and Metabolite Profiles in Meju Using Starter Cultures of Bacillus velezensis and Aspergillus oryzae.

The production of good Meju soybean paste primarily depends on the selection of raw materials and fermenting microorganisms, which together influence its characteristic metabolites, taste, and aroma. In this study, we analyzed the relationship between properties and metabolites in Meju samples fermented by Aspergillus oryzae alone or with Bacillus velezensis. We developed fast-stable processing techniques to obtain Meju from A. oryzae and B. velezensis using the inoculation method, thereby ensuring safety in the production of soybean paste. The amino-type nitrogen content increased from an initial 180–228 mg% to a final 226–776 mg% during fermentation and was higher in Meju inoculated separately with the fungi and bacteria (C group) than in Meju co-inoculated with both the starters concurrently (D group). The levels of metabolites such as glucose, myo-inositol, glycerol, and fatty acids (palmitic, stearic, oleic, and linoleic acids) in Meju fermented by A. oryzae with B. velezensis were higher than those in Meju fermented by A. oryzae alone. Fungal growth was affected by the inoculated bacteria, which often occurs during the fermentation of co-inoculated Meju.

Characterization of selected Chinese soybean paste based on flavor profiles using HS-SPME-GC/MS, E-nose and E-tongue combined with chemometrics

Headspace solid-phase microextraction gas chromatography–mass spectrometry (HS-SPME-GC/MS) with electronic nose (E-nose) and electronic tongue (E-tongue) was applied for flavor characterization of traditional Chinese fermented soybean paste. Considering geographical distribution and market representation, twelve kinds of samples were selected to investigate the feasibility. A total of 57 volatile organic compounds (VOCs) were identified, of which 8 volatiles were found in all samples. Linear discrimination analysis (LDA) of fusion data exhibited a high discriminant accuracy of 97.22%. Compared with partial least squares regression (PLSR), support vector machine regression (SVR) analysis exhibited a more satisfying performance on predicting the content of esters, total acids, reducing sugar, salinity and amino acid nitrogen, of which correlation coefficients for prediction (Rp) were about 0.803, 0.949, 0.960, 0.896, 0.923 respectively. This study suggests that intelligent sensing technologies combined with chemometrics can be a promising tool for flavor characterization of fermented soybean paste or other food matrixes.

Comparative evaluation of the effects of natural and artificial inoculation on soybean paste fermentation

Soybean pastes fermented with natural (NIS) and artificial (AIS) inoculation were prepared to evaluate their fermentation characteristics, flavor compounds and microbial community compositions across an entire fermentation period. The results showed that the initial concentrations of free amino acids and aroma compounds were higher in NIS than in AIS, whereas it was opposite at the end of fermentation. A larger variation of flavor compounds was observed in AIS, which was associated with higher level of microbial metabolism during AIS fermentation. Penicillium and Tetragenococcus were the dominant microbial genera in NIS, while Aspergillus and Pediococcus were predominant in AIS. Correlation analysis of flavor compounds and microbial community dynamics during NIS and AIS fermentation revealed the specific role of microbial taxa in metabolite transformations. Pediococcus acidilactici could be the key microorganism strongly associated with 23 flavor compounds during AIS fermentation. Further, Aspergillus oryzae and Pediococcus acidilactici were primarily linked to amino acid metabolism in AIS, whereas Penicillium and Yaniella were found associated with aroma compounds, Tetragenococcus_sp_JNURIC_D6 and Candida solani with that of organic acids in NIS. These findings provide substantial data on the development of flavor-enhancing fermentation starter culture and the better control of flavor ripening in soybean paste production.

Comparison of laxative effects of fermented soybeans (Cheonggukjang) containing toxins and biogenic amines against loperamide-induced constipation mouse model.

BACKGROUND/OBJECTIVES Cheonggukjang is a traditional fermented soybean paste with significant health-promoting effects. On the other hand, there have been insufficient studies on the safety and efficacy of Cheonggukjang, which is produced using traditional methods containing toxins and biogenic amines (BAs). This study compared the laxative effect of Cheonggukjang, containing high or low levels of toxins and BAs (HTBC or LTBC) in a loperamide (Lop)-induced constipation mouse model.MATERIALS/METHODSTo induce constipation, Lop (5 mg/kg) was administered orally to ICR mice twice a day for 4 days, and the dose was increased to 8 mg/kg after a 3-day rest period. Cheonggukjang (500 mg/kg, HTBC, or LTBC respectively) was administered for four weeks before the Lop treatment.RESULTSThe number of stools, fecal weight, water contents, gastrointestinal transit, and histological alterations were recovered significantly in the HTBC or LTBC groups. HTBC and LTBC administration did not induce significant changes in body weight, dietary intake, and behavior. The opioid-receptor downstream signaling pathway in colon tissues was also evaluated. The c-Kit, stem cell kinase, and mitogen-activated protein kinases subfamilies, including extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinases, and p38, were all downregulated in the HTBC or LTBC-administered mice colon compared to the Lop group.CONCLUSIONThese results show that Cheonggukjang, containing high levels of toxins and BAs, have a similar laxative effect in a mouse model of Lop-induced constipation.

Detection of the microbial diversity and flavour components of northeastern Chinese soybean paste during storage

A combination of 16S rDNA and GC-IMS was used to study the changes in the composition of microorganisms and volatile organic compounds (VOCs) during the storage of northeastern Chinese soybean paste. Firmicutes and Actinobacteriota dominated the microbial communities of the soybean paste at the phylum level, bacterial profiles of different samples were different at genus level. Fifty-one VOCs were identified from soybean paste, most of which existed in the early storage stage. Most esters and alcohols decreased with the extension of the storage time, while acids and pyrazines accumulated in the later period of storage. Esters, alcohols, acids and aldehyde compounds are the key substances in the volatile components of soybean paste, which give the soybean paste the sour, sweet, rose, mushroom and smoky flavor characteristics. The biomarker Bacillus-velezensis in soybean paste is directly related to ester features; Kroppenstedtia, Sporolactobacillus-nakayamae, and Corynebacterium-stationis are positively associated with the biosynthesis of aldehydes.

The microbial community, biogenic amines content of soybean paste, and the degradation of biogenic amines by Lactobacillus plantarum HM24.

Soybean paste was a traditional fermented product in Northeast China, mainly fermented by molds, yeast, Bacillus, and lactic acid bacteria. This study investigated the dynamic changes of the microbial community and biogenic amine content during the fermentation of the traditional soybean paste. The microbial diversity of soybean paste in different regions was analyzed by MiSeq sequencing technology. The results showed that Penicillium and Tetragenococcus were the dominant microorganisms responsible for the fermentation of soybean paste. Biogenic amine was found in the traditional soybean paste at different fermentation stages, putrescine, and tyramine were the mainly biogenic amines and their content increased with the extension of fermentation time. Serratia in the soybean paste was positively correlated with the formation of spermine, cadaverine (p < .01), and β‐phenethylamine (p < .05), Leuconostoc was negatively correlated with tyramine formation (p < .05), and Enterococcus was positively correlated with the formation of histamine, tryptamine and cadaverine (p < .01). Lactobacillus fermentum HM22, Lactobacillus plantarum HM24, and Enterococcus faecalis YF10042 with strong biogenic amine degrading capacity were inoculated into the koji. After 20 days of fermentation, the degradation rates of tryptamine, β‐phenylethylamine, putrescine, cadaverine, histamine, and tyramine in soybean paste inoculated with L. plantarum HM24 were 35.31%, 43.14%, 30.18%, 33.44%, 32.74%, and 39.91%, respectively, indicating that the use of L. plantarum HM24 as a starter culture in soybean paste fermentation might be a good strategy for biogenic amines reduction.

Contamination characteristics and risk assessment of aflatoxins in homemade soybean paste, a traditional fermented soybean food, in South Korea

Soybean paste is manufactured through microbial fermentation and may become contaminated with aflatoxins. Herein, we conducted nationwide large-scale monitoring (n = 1436) over three years (2018–2020) to investigate aflatoxin levels according to geographic, demographic, manufacturing, quality factors, and risk characteristics of homemade soybean paste produced through fermentation. The mean level of total aflatoxins was 5.88 μg/kg (range, 0.01–281.92), with the most common contaminating type being the B type. Aflatoxin levels significantly differed according to the region, age of the manufacturer, type of starter used, and the amino-type nitrogen content and pH of the homemade soybean paste (p < 0.05). Aflatoxin levels was significantly higher when starters were manufactured using the traditional method (inoculation with a naturally occurring strain in the surrounding environment). The aflatoxin exposure level estimated through the average intake of homemade soybean paste in all age groups was 0.1012 ng/kg body weight/day. The risk assessment for the genotoxic and carcinogenic potential of aflatoxins using the margin of exposure approach revealed values of 3705–3954 for average intake of homemade soybean paste, indicating public health concern. These results suggest that follow-up studies and safety management strategies are needed to reduce aflatoxin levels in homemade soybean paste.

Cheonggukjang-Specific Component 1,3-Diphenyl-2-Propanone as a Novel PPARα/γ Dual Agonist: An In Vitro and In Silico Study.

Background: Cheonggukjang is a traditional fermented soybean paste that is mostly consumed in Korea. However, the biological activities of Cheonggukjang specific compounds have not been studied. Thus, we aimed to discover a novel dual agonist for PPARα/γ from dietary sources such as Cheonggukjang specific volatile compounds and explore the potential role of PPARα/γ dual agonists using in vitro and in silico tools. Methods: A total of 35 compounds were selected from non-fermented and fermented soybean products cultured with Bacillus subtilis, namely Cheonggukjang, for analysis by in vitro and in silico studies. Results: Molecular docking results showed that 1,3-diphenyl-2-propanone (DPP) had the lowest docking score for activating PPARα (1K7L) and PPARγ (3DZY) with non-toxic effects. Moreover, DPP significantly increased the transcriptional activities of both PPARα and PPARγ and highly activated its expression in Ac2F liver cells, in vitro. Here, we demonstrated for the first time that DPP can act as a dual agonist of PPARα/γ using in vitro and in silico tools. Conclusions: The Cheonggukjang-specific compound DPP could be a novel PPARα/γ dual agonist and it is warranted to determine the therapeutic potential of PPARα/γ activation by dietary intervention and/or supplementation in the treatment of metabolic disorders without causing any adverse effects.

Bacterial and fungal communities of traditional fermented Chinese soybean paste (Doujiang) and their properties.

Soybean paste (Doujiang) is one of the traditional fermented foods from China, fermented by various microorganisms. However, the microflora of Doujiang keeps little known. In this study, the microbial communities of seven kinds of representative Doujiang samples were investigated by both culture‐independent and culture‐dependent methods. We found that core OTUs among seven Doujiang samples were mainly from Bacillus, Pseudomonas, Candida, and Aspergillus according to Illumina sequencing. Every type of Doujiang sample harbored a different composition of microbial community. Doujiang LSJ and LBJ had the highest bacterial and fungal richness and diversity, respectively. The structure of microbial community was remarkably correlated with Doujiang properties—pH, and the content of total protein, soluble protein, amino acid, and total sugar (p < .05). Bacillus spp. were most frequently isolated bacterial species. Fungi of Monascus, Candida, and Aspergillus were also isolated. Eleven microbial strains showed high protease activities to degrade corn proteins, which can form obvious transparent hydrolytic circles in corn gluten meal medium plates. Therefore, microbial communities were supposed to tightly connect to Doujiang type and properties. It is possible to apply potential protein‐degrading microbial strains to corn byproducts for protein production in the future study.

Anti-Obesity and Anti-Inflammatory Effects of the Desalted &lt;i&gt;Salicornia europaea&lt;/i&gt; L. Fermented by &lt;i&gt;Bacillus velezensis&lt;/i&gt; K26

Salicornia europaea L. (SE) as a halophyte has been widely used as a seasoned vegetable with various biological activities, including anti-inflammatory and anti-diabetic activities. In particular, the desalted SE (DSE) has been considered an attractive alternative compared to SE for its anti-obesity effect. 1-Deoxynojirimycin (1-DNJ) is one of the known α-glucosidase inhibitors and has been reported to have similar biological activities with SE. This study reports the anti-obesity and anti-inflammatory activities of DSE fermented by Bacillus velezensis K26 (BVDSE) previously isolated from Korean fermented soybean paste and elucidated to produce 1-DNJ in 3T3-L1 adipocytes and RAW264.7 cells. BVDSE (5%) did not show cytotoxicity in 3T3-L1 cells and inhibited lipid accumulation, which was decreased to 23% in the differentiated cells. BVDSE did not inhibit the release of the pro-inflammatory cytokine, TNF-α, while it remarkedly inhibited IL-6 on lipopolysaccharide-induced RAW264.7 cells. Taken together, these results indicate that the fermentation of DSE with B. velezensis K26 as a 1-DNJ producer has more significant anti-obesity and anti-inflammatory effects than even non-fermented DSE, assuming the synergistic effects by 1-DNJ production from B. velezensis K26, by which it could be highlighted that BVDSE may be considered for use as a functional food supplement.

Probiotic potential and safety assessment of bacteriocinogenic Enterococcus faecium strains with antibacterial activity against Listeria and vancomycin-resistant enterococci

Enterococcus spp., known for their wide ecological distribution, have been associated with various fermented food products of plant and animal origin. The strains used in this study, bacteriocinogenic Enterococcus faecium previously isolated from artisanal soybean paste, have shown strong activity against Listeria spp. and vancomycin-resistant enterococci. Although their antimicrobial activity is considered beneficial, the potential application of enterococci is still under debate due to concerns about their safety for human and other animal consumption. Therefore, this study not only focuses on the screening of potential virulence factors, but also the auxiliary beneficial properties of the strains Ent. faecium ST651ea, ST7119ea, and ST7319ea. Phenotypic screening for gelatinase, hemolysin, and biogenic amine production showed that the strains were all safe. Furthermore, the antibiogram profiling showed that all the strains were susceptible to the panel of antibiotics used in the assessment except for erythromycin. Yet, Ent. faecium ST7319ea was found to carry some of the virulence genes used in the molecular screening for safety including hyl, esp, and IS16. The probiotic potential and other beneficial properties of the strains were also studied, demonstrating high aggregation and co-aggregation levels compared to previously characterized strains, in addition to high survivability under simulated gastrointestinal conditions, and production of numerous desirable enzymes as evaluated by APIZym, indicating diverse possible biotechnological applications of these strains. Additionally, the strains were found to carry genes coding for γ-aminobutyric acid (GABA) production, an auxiliary characteristic for their probiotic potential. Although these tests showed relatively favorable characteristics, it should be considered that these assays were carried out in vitro and should therefore also be assessed under in vivo conditions.

Characteristics of Bacterial Strains with Desirable Flavor Compounds from Korean Traditional Fermented Soybean Paste (Doenjang).

To identify and analyze the characteristics of the microorganisms involved in the formation of the desirable flavor of Doenjang, a total of 179 strains were isolated from ninety-four Doenjang collected from six regions in South Korea, and fourteen strains were selected through a sensory evaluation of the aroma of each culture. The enzyme activities of amylase, protease and lipase was shown in the various strains. Bacillus sp.-K3, Bacillus sp.-K4 and Bacillus amyloliquefaciens-J2 showed relatively high protease activity, at 317.1 U, 317.3 U and 319.5 U, respectively. The Bacillus sp.-K1 showed the highest lipase activity at 2453.6 U. In the case of amylase, Bacillus subtilis-H6 showed the highest activity at 4105.5 U. The results of the PCA showed that Bacillus subtilis-H2, Bacillus subtilis-H3, and Bacillus sp.-K2 were closely related to the production of 3-hydroxy-2-butanone (23.51%~43.37%), and that Bacillus subtilis-H5 and Bacillus amyloliquefaciens-J2 were significantly associated with the production of phenethyl alcohol (0.39% and 0.37%). The production of peptides was observed to vary among the Bacillus cultures such as Val-Val-Pro-Pro-Phe-Leu and Pro-Ala-Glu-Val-Leu-Asp-Ile. These peptides are precursors of related volatile flavor compounds created in Doenjang via the enzymatic or non-enzymatic route; it is expected that these strains could be used to enhance the flavor of Doenjang.

Characterization and correlation of microbial communities and metabolite and volatile compounds in doenjang fermentation

To investigate the general fermentation features of doenjang, a traditional Korean fermented soybean paste, eleven batches of doenjang were prepared. The bacterial and fungal communities and the metabolites such as free sugars, organic acids, amino acids, and volatile compounds were analyzed during fermentation. Tetragenococcus, Aspergillus, and Debaryomyces were the most common microbes; galactose, fructose, and glucose were the major sugars; and lactate and acetate were the major organic acids. Spearman correlation analyses showed that the quantity of meju was correlated with only Pediococcus and Halomonas abundance, while solar salt concentration was correlated with the relative abundance of many microbial taxa and the amount of glycerol and total volatile compounds. The abundance of heterolactic acid bacteria, such as Tetragenococcus, Pediococcus, Weissella, and Enterococcus, was positively correlated with the levels of lactate, acetate, and ethanol, suggesting that heterolactic acid fermentation may be a major metabolism pathway during the fermentation of doenjang. The abundance of Weissella, Hyphopichia, and Wickerhamomyces was positively correlated with ethyl acetate levels, whereas the abundance of Staphylococcus and Bacillus was positively correlated with the concentration of major volatile compounds, 3-methylbutanoic acid and tetramethylpyrazine, respectively, suggesting that they may play important roles in the production of flavor compounds during fermentation.