A potential flavor culture: Lactobacillus harbinensis M1 improves the organoleptic quality of fermented soymilk by high production of 2,3-butanedione and acetoin.

Lactic acid bacteria (LAB) are commonly used in soymilk fermentation to improve health-related functionality, but their contribution to sensory qualities is less valued. We characterized Lactobacillus harbinensis M1, Lactobacillus mucosae M2, Lactobacillus fermentum M4, Lactobacillus casei M8 and Lactobacillus rhamnosus C1 from naturally-fermented tofu whey, along with Streptococcus thermophilus ST3 from kefir XPL-1 fermented soymilk, to investigate their potential as starter cultures of fermented soymilk. They were characterized for antibiotic susceptibility, probiotic potential and their performance as starter cultures. All the LABs showed sensitivity to the tested antibiotics. L. casei M8 had strongest tolerance to synthetic gastrointestinal juice (<1.0 log CFU/mL loss), as well as antagonistic effects towards five food-borne pathogens. GC/MS analysis showed that L. harbinensis M1 produced significantly higher abundance (P < 0.05) of 2,3-butanedione (2.45 ppm) and acetoin (44.30 ppm), thus improving the overall sensory acceptability of fermented soymilk. The coding genes for the synthesis of 2,3-butanedione/acetoin (alsS, alsD, butA) were predicted from the whole-genome. A co-culture of L. harbinensis M1 and L. casei M8 produced a fermented soymilk product with both markedly improved flavor and good probiotic potential. It appears that L. harbinensis M1 has much potential for improving the organoleptic properties of fermented soymilk.

High-throughput sequencing and culture-based approaches to analyze microbial diversity associated with chemical changes in naturally fermented tofu whey, a traditional Chinese tofu-coagulant.

Naturally fermented tofu whey (NFTW) has been used as traditional tofu coagulant in China for hundreds of years. In this study, the microbial diversity in NFTW was firstly analyzed with high-throughput sequencing and its effect on chemical contents of tofu whey (TW) was investigated. Lactobacillus with 95.31% was the predominant genus in the microbial community of NFTW while Picha, Enterococcus, Bacillus and Acetobacter occupied about only 0.90%, 0.04%, 0.02% and 0.09%, respectively. Besides, Lactobacillus amylolyticus were determined to be one of the dominated species with metagenomic analysis and culture method. Lactobacillus with α-galactosidase activities played leading role in metabolizing the soybean oligosaccharides of TW to produce lactic acid. And acetic acid produced by genus of Acetobacter was another main organic acid attributed to the acidification of TW except lactic acid. Meanwhile, the bioconversion of isoflavone glucosides into aglycones could also be promoted by Lactobacillus with the help of ß-glucosidase activity. Moreover, the production of equol in NFTW was confirmed, which might be jointly converted from daidzein by several strains. Therefore, our results indicated that Lactobacillus was the dominated microorganism and mainly affected the chemical changes of NFTW. This study help provide basic theory and technical references for the production of tofu and its derivative products (like sufu) with NFTW as coagulator.

Metagenomic analysis of core differential microbes between traditional starter and Round-Koji-mechanical starter of Chi-flavor Baijiu.

Xiaoqu starter serves as the saccharifying and fermenting agent in the production of Cantonese soybean-flavor (Chi-flavor) Baijiu, and the complex microbial communities determine the flavor and quality of the product. Round-Koji-mechanical starter (produced by using an automated starter-making disk machine) is advantageous as it decreases operator influence, labor costs, and fermentation time, but the product quality is lower compared to traditional starter. Thus, two types of starters (traditional and Round-Koji-mechanical starter) from a Cantonese Baijiu factory were compared in a metagenomic analysis to investigate the differences in microbial community composition and core microbes. The results showed that several core microbes related to carbohydrate metabolism, amino acid metabolism and lipid metabolism, were differentially enriched in the traditional starter. Mucor lusitanicus and Rhizopus delemar were significantly positively correlated with the three key metabolic pathways. Saccharomyces cerevisiae, Cyberlindnera fabianii, Kluyveromyces marxianus, Lactobacillus fermentum, Mucor ambiguous, Rhizopus microspores, Rhizopus azygosporus, Mucor circinelloides, and Ascoidea rubescens were significantly positively correlated with two of the three key metabolic pathways. The results of this study provide a basis for understanding the differential core microbes in traditional and Round-Koji-mechanical starters of Chi-flavor Baijiu, and they also provide guidance for improving Round-Koji-mechanical starter.

Effect of hydrolyzed soybean on the volatile flavors and microbial community in the traditional brewing process of chi-flavor Baijiu.

Addition of soybean in raw materials could improve the flavor of chi-flavor Baijiu (CFB) in production. For investigating the mechanism of flavor improvement during fermentation, the changes of volatile flavors and their relationship with microbial community were analyzed. The results showed that the average contents of lactic and acetic acid in EG (added with hydrolyzed soybean) samples were higher those of CK (without hydrolyzed soybean) samples. The contents of main volatile esters, including ethyl acetate, ethyl palmitate, and ethyl benzoate, in EG samples were higher than those in CK samples at the end of fermentation. The content of alcohols in EG sample was 140.55 mg/L, higher than that in CK sample at the end of fermentation. Especially, the average content of characteristic flavor ß-phenylethanol in EG samples increased 17.6% in comparison with that in CK samples during fermentation. Lactobacillus and Pediococcus were the dominant bacterial genera, whereas Saccharomyces, Mortierella, and Trichosporon were dominant fungal genera in both CK and EG samples. Lactobacillus and Weissella confusa showed strong positive correlation with ß-phenylethanol, ethyl acetate, and ethyl benzoate. This study provides an in-depth analysis of the effects of hydrolyzed soybeans on the volatile flavor compounds and microbial communities of CFB and theoretical guidance for improving the quality of CFB.

Screening ester-producing yeasts to fortify the brewing of rice-flavor Baijiu for enhanced aromas.

To enhance the aromas in Guangdong rice-flavor Baijiu, ester-producing yeast was selected to fortify Baijiu brewing. Among eight kinds of ester-producing yeasts selected, Saccharomyces cerevisiae CM15 (CM15) that showed both the stronger ability to utilize substrates to produce esters and the excellent tolerance to industrially relevant stress factors was chosen. When CM15 was synergistically fermented with six kinds of Kojis from distilleries of rice-flavor liquor in Guangdong, the enhanced total esters had happened to the liquors brewing with the fortified four kinds of Kojis, especially with Koji F. When Koji F was fortified with CM15, the resultant Baijiu showed a higher esters proportion and a lower higher alcohol ratio than that of Baijiu brewed only with Koji F, with the content of ethyl acetate and ethyl lactate increasing by 25% and 214%, respectively. This study suggested that CM15 can be used as a functional microorganism to fortify Baijiu brewing, which might also be suitable for other traditional fermented foods.

Characterization and Correlation of Microbiota and Higher Alcohols Based on Metagenomic and Metabolite Profiling during Rice-Flavor Baijiu Fermentation.

Higher alcohol, as an inevitable product of fermentation, plays an important role in the flavor and quality of Baijiu. However, the relationship between the complex microbial metabolism and the formation of higher alcohols in rice-flavor Baijiu was not clear. To investigate the relationship between microorganisms and higher alcohol production, two fermentation mashes inoculated with starters from Heyuan Jinhuangtian Liquor Co., Ltd. (Heyuan, China) as JM and Guangdong Changleshao Co., Ltd. (Meizhou, China) as CM, respectively, with significant differences in higher alcohol profiles during rice-flavor Baijiu fermentation were selected. In general, higher alcohols presented a rapid accumulation during the early fermentation stages, especially in JM, with higher and faster increases than those in CM. As for their precursors including amino acids, pyruvic acid and ketoacids, complex variations were observed during the fermentation. Metagenomic results indicated that Saccharomyces cerevisiae and Rhizopus microsporus were the microorganisms present throughout the brewing process in JM and CM, and the relative abundance of R. microsporus in JM was significantly higher than that in CM. The results of higher alcohol metabolism in JM may contribute to the regulation of higher alcohols in rice-flavor Baijiu.

Adaptive mechanism of Lactobacillus amylolyticus L6 in soymilk environment based on metabolism of nutrients and related gene-expression profiles.

Lactobacillus amylolyticus L6 isolated from naturally fermented tofu-whey was characterized as potential probiotics. To give insight into the adaptive mechanism of L. amylolyticus L6 in soymilk, the gene-expression profiles of this strain and changes of chemical components in fermented soymilk were investigated. The viable counts of L. amylolyticus L6 in soymilk reached 1012 CFU/mL in the stationary phase (10 hr). The main sugars reduced gradually while the acidity value significantly increased from 45.33° to 95.88° during fermentation. About 50 genes involved in sugar metabolization and lactic acid production were highly induced during soymilk fermentation. The concentration of total amino acid increased to 668.38 mg/L in the logarithmic phase, and 45 differentially expressed genes (DEGs) in terms of nitrogen metabolism and biosynthesis of amino acid were detected. Other genes related to lipid metabolism, inorganic ion transport, and stress response were also highly induced. Besides, the concentration of isoflavone aglycones with high bioactivity increased from 14.51 mg/L to 36.09 mg/L during the fermentation, and the expression of 6-phospho-ß-glucosidase gene was also synchronously induced. This study revealed the adaptive mechanism of L. amylolyticus L6 in the soymilk-based ecosystem, which gives the theoretical guidance for the application of this strain in other soybean-derived products.

Cloning and expression of a novel α-galactosidase from Lactobacillus amylolyticus L6 with hydrolytic and transgalactosyl properties.

Lactobacillus amylolyticus L6, a gram-positive amylolytic bacterium isolated from naturally fermented tofu whey (NFTW), was able to hydrolyze raffinose and stachyose for the production of α-galactosidase. The cell-free extract of L. amylolyticus L6 was found to exhibit glycosyltransferase activity to synthesize α-galacto-oligosaccharides (GOS) with melibiose as substrate. The coding genes of α-galactosidase were identified in the genome of L. amylolyticus L6. The α-galactosidase (AglB) was placed into GH36 family by amino acid sequence alignments with other α-galactosidases from lactobacilli. The optimal reaction conditions of pH and temperature for AglB were pH 6.0 and 37°C, respectively. Besides, potassium ion was found to improve the activity of AglB while divalent mercury ion, copper ion and zinc ion displayed different degrees of inhibition effect. Under the optimum reaction condition, AglB could catalyze the synthesis of GOS with degree of polymerization (DP) ≥5 by using 300 mM melibiose concentration as substrate. The maximum yield of GOS with (DP) ≥3 could reach 31.56% (w/w). Transgalactosyl properties made AglB a potential candidate for application in the production of GOS.

Molecular characterization of Lactobacillus plantarum DMDL 9010, a strain with efficient nitrite degradation capacity.

Nitrites commonly found in food, especially in fermented vegetables, are potential carcinogens. Therefore, limiting nitrites in food is critically important for food safety. A Lactobacillus strain (Lactobacillus sp. DMDL 9010) was previously isolated from fermented vegetables by our group, and is not yet fully characterized. A number of phenotypical and genotypical approaches were employed to characterize Lactobacillus sp. DMDL 9010. Its nitrite degradation capacity was compared with four other Lactobacillus strains, including Lactobacillus casei subsp. rhamnosus 719, Lactobacillus delbrueckii subsp. bulgaricu 1.83, Streptococcus thermophilus 1.204, and lactobacillus plantarum 8140, on MRS medium. Compared to these four Lactobacillus strains, Lactobacillus sp. DMDL 9010 had a significantly higher nitrite degradation capacity (P<0.001). Based on 16S rDNA sequencing and sequence comparison, Lactobacillus sp. DMDL 9010 was identified as either Lactobacillus plantarum or Lactobacillus pentosus. To further identify this strain, the flanking regions (922 bp and 806 bp upstream and downstream, respectively) of the L-lactate dehydrogenase 1 (L-ldh1) gene were amplified and sequenced. Lactobacillus sp. DMDL 9010 had 98.92 and 76.98% sequence identity in the upstream region with L. plantarum WCFS1 and L. pentosus IG1, respectively, suggesting that Lactobacillu sp. DMDL 9010 is an L. plantarum strain. It was therefore named L. plantarum DMDL 9010. Our study provides a platform for genetic engineering of L. plantarum DMDL 9010, in order to further improve its nitrite degradation capacity.