Application of Transcriptomics in the Analysis of Community Structure of Food Fermented with Lactic Acid Bacteria

Yanrui MA Guangpeng LIU Yuexun ZHANG Yan ZHAO Fengtao ZHU Miaomiao SUN Ling GAO

Abstract Revealing the structural characteristics, succession changes and functional genes of the microbial community in lactic acid bacteria-fermented food has been the focus of scientific research and industrial production for many years. In recent years, high-throughput sequencing technology has become an important tool for the research of lactic acid bacteria-fermented food microorganisms due to its advantages of high efficiency and being relatively cheap. In this paper, starting from the research on the genome, transcriptome and metabonomic levels of high-throughput sequencing technology, we reviewed the progress of transcriptomics in the study on microbial community structure, interaction, and functional gene mining of lactic acid bacteria-fermented food, and analyzed and discussed the main problems and development trends it faces, providing a certain reference for the scientific research and industrial production of fermented food in the future.

Key words Transcriptomics; Lactic acid bacteria; Colony structure

Received: June 17, 2021  Accepted: August 19, 2021

Supported by High-efficiency Ecological Agriculture Innovation Project of Taishan Industry Leading Talent Project (LJNY202001); Yantai Science and Technology Program (2019ZDCX013).

Yanrui MA (1996-), female, P. R. China, devoted to research about fruit and vegetable fermentation.

*Corresponding author.

Lactic acid bacteria-fermented food usually refers to the sour fragrant umami food formed by the process of microbial fermentation with lactic acid bacteria as the main body, which degrades proteins and carbohydrates and other macromolecular substances in raw materials into small molecular substances, and the main metabolite of which is lactic acid. As one of the main microbial groups of traditional lactic acid bacteria-fermented food, lactic acid bacteria can be divided into homolactic fermentation and heterolactic fermentation. The degradation product of carbohydrates by lactic acid bacteria in the process of homolactic fermentation is only lactic acid, while in the process of heterolactic fermentation, in addition to lactic acid, lactic acid bacteria also produce volatile compounds such as alcohols, aldehydes, ketones, etc., which give fermented food a unique taste and fragrance. Meanwhile, lactic acid bacteria form an acidic environment through ecological niche competition, and produce antagonistic metabolites, which can better inhibit the spoilage microorganisms in the product. Therefore, lactic acid bacteria play an important role in the flavor and safety of products, and traditional lactic acid bacteria-fermented foods are inseparable from the important role of lactic acid bacteria. In our daily life, there are many types of foods that are fermented by lactic acid bacteria, which can be divided into fermented vegetables, fermented condiments, fermented sourdough, fermented dairy products, fermented meat products, etc.［1］. The changes of microbial communities in fermented foods can be studied with transcriptomics technology, which can systematically analyze the metabolic changes and response responses of entire microbial communities. In this paper, we introduced the application of transcriptomics from the perspective of different fermented foods.

Fermentation Vegetables

Lactic acid bacteria-fermented vegetables are mainly Northeast sauerkraut, Sichuan-style pickled vegetables, and Korean kimchi. These fermented vegetables are not only sour and crunchy, appetizing and palate-cleansing, but also have special effects on digestion and other special effects. For example, Northeast sauerkraut contains a lot of cellulose, minerals and organic compounds indispensable for human metabolism, including lactic acid, choline, acetylcholine, Vitamin C, Vitamin B12, etc. Northeast sauerkraut is not only unique in flavor, but also has physiological effects such as regulating cholesterol, regulating blood balance, and preventing atherosclerosis, and it is thus deeply loved by people.

The main fermentation strains of Northeast sauerkraut are lactic acid bacteria. Studies have shown that, in the long fermentation process, Leuconostoc is the dominant bacteria in the early fermentation of Northeast sauerkraut, which is then followed by the fermentation by Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus fermentum to produce acids, and the final fermentation process is mainly finished by L. plantarum. It can be seen that the dominant bacteria in the early stage of sauerkraut fermentation are Leuconostoc, and the dominant bacteria in the middle and late stages of fermentation is L. plantarum［2］. When applying the metagenomics method in the study of the distribution of microbial flora in Sichuan-style pickled vegetables, it was found through the analysis of the diversity of bacteria in the fermentation process of Sichuan-style pickled vegetables that the microbial flora in Sichuan-style pickled vegetables is also mainly based on lactic acid bacteria as the main body of fermentation. Weissella bacteria could be as high as 74.5% at the beginning of the fermentation, and remain at about 10% in the later stage when the dominant bacteria were Lactobacillus, the content of which could reach 80%-85%. It is proved that in the fermentation process of Sichuan-style pickled vegetables, Weissella is the promoter, and the key bacteria in the fermentation process is Lactobacillus［3］.

Korean kimchi is a model of Korean fermented vegetables, and there are about 23 kinds of lactic acid bacteria involved in the fermentation process of Korean kimchi［4-5］. Jung et al.［5］ used the method of RNA-Seq sequencing and macrotranscriptomics to study the subtle changes and metabolic changes in the microbial colony structure of Korean kimchi and the genetic characteristics of the microbial community during the 29 d fermentation process. The Korean kimchi samples obtained through 454 sequencing 701 556 reads, the average length of which was 438 bp. From the 16S rRNA sequencing data, it was found that the microbial community of Korean kimchi was dominated by three genera, namely Leuconostoc, Lactobacillus and Weissella.  Transcriptomics sequencing data coverage showed that Leuconostoc mesenteroides subspecies ATCC8293 and Lactobacillus sakei 23K were highly expressed, showing the importance of these two strains in Korean kimchi. Leuconostoc mesenteroides is most active in the early stage of fermentation, while L. sakei and Weissella koreensis are more active in the late stage of fermentation［6］. In addition, some phage DNA sequences were found, which proved that the strains in the fermentation process had been contaminated by phage. In short, the use of transcriptomics for exploring the evolution of the microbial community of fermented vegetables has certain practical significance for the development of industrial fermented vegetables.

Fermented Condiments

The most common traditional fermented condiments such as brewed soy sauce, mature vinegar, fermented bean curd, soybean paste, etc., are rich in lactic acid bacteria during the fermentation process, which plays an important role in their quality and taste. Lactic acid bacteria can ferment sugars to produce organic acids such as lactic acid and citric acid, which may improve the flavor of condiments and make them softer. Meanwhile, organic acids can undergo an esterification reaction with acetic acid produced by ethanol fermentation to produce esters and other flavors. Therefore, it is speculated that Lactobacillus may be related to the formation of organic acids and ester flavors, and may contribute to the formation of the fragrance in fermented condiments. Secondly, the acid production of Lactobacillus in the ethanol fermentation stage can form a synergistic effect with ethanol to prevent contamination by other bacteria in the ethanol fermentation stage. The lactic acid bacteria in soy sauce are mainly L. plantarum, L. mesenteroides, Lactobacillus brevis, Pediococcus acidilactici and Tetragenococcus halophilus［7］. The lactic acid bacteria in vinegar explored by metagenomics means are mainly Lactobacillus, Pediococcus and Lactococcus［8］. T. halophilus and salt-tolerant lactic acid bacteria such as Lactobacillus curvatus and Lactobacillus casei are the main fermentative strains of fermented bean curd［9］.

Korean Cheonggukjang is a local Korean delicacy. It has a strong smell and smells like dead corpses, hence the name. Such sauce is thick and delicious. Transcriptomics studies have found that the dominant bacteria in this cuisine are Bacillus and Lactobacillus［10］. Duan et al.［11］ used a combination of macrotranscriptomics and 16S RNA sequencing to explore the correlation between flavor formation and flora structure during shrimp paste fermentation. Tetragenococcus accounted for 95.1%. After searching the Nr database, it was found that among 588 transcripts, 520 T. halophilus transcripts were found. The citric acid cycle and oxidative phosphorylation of T. halophilus are activated, but the lactate dehydrogenase gene is not expressed. It proves that T. halophilus mainly undergoes aerobic metabolism during the fermentation of shrimp paste. The gene expression of amino acid metabolism, peptidase production, dipentene and pinene degradation pathways of T. halophilus is also very active.

Fermented Sourdough

Traditional sourdough is a kind of noodle product made by microbial fermentation of wet noodles. It is a necessary fermentation strain in the process of making steamed bread. The main fermentation flora in the fermentation process of steamed bread comes from microorganisms in sourdough. These microorganisms play a very important role in fermented wheaten food. In other words, the fermentation process of sourdough is a process in which the microorganisms decompose proteins and carbohydrates in dough and interact to produce a variety of flavor substances including alcohols, phenols, aldehydes, and esters. The fermentation process can not only improve the texture of dough, form a unique flavor and mouthfeel, etc., but also form an acidic environment due to microbial fermentation, and can also prevent food spoilage caused by fungal or bacterial infection.

Sourdough is an extremely complex micro-ecological system. Studies have shown that there are no less than 50 kinds of lactic acid bacteria in sourdough, and about 20 kinds of yeasts, mainly Lactobacillus, Saccharomyces and Candida. Zotta et al.［12］ isolated 41 strains of lactic acid bacteria from the traditional sourdough "cornetto" in southern Italy, including L. plantarum, Leuconostoc, Lactobacillus paraplantarum, L. curvatus, Lactobacillus pentosus and Weissella cibaria. Metagenomic analysis of Mexican fermented maize dough "pozol" showed that it contained 14 species of bacteria, including Lactococcus, Streptococcus suis, L. plantarum, L. casei, Lactobacillus alimentarium, Lactobacillus delbrueckii and Clostridium［13］.

Macrotranscriptomics analysis of traditional "preferments" found that L. plantarum and L. fermentum were the most important lactic acid bacteria, followed by P. pentosaceus. In the entire flora ecosystem, Lactococcus lactis played a role in the early stage of fermentation［14］. Wu Sirguleng［15］ isolated and identified yeast and lactic acid bacteria in sourdough in western Inner Mongolia. A total of 85 Saccharomycetes and 108 lactic acid bacteria were isolated from 28 sourdough samples, including the following bacteria: 37 strains of L. plantarum, 14 strains of Leuconostoc citreum, 10 strains of W. cibafia, 8 strains of Lactobacillus alimentarius, 7 strains of Weissella confusa, 7 strains of Lactobacillus helveticus.

Fermented Dairy Products

Lactic acid bacteria-fermented dairy products are mainly divided into acid fermented milk (yogurt, sour camel milk), alcoholic fermented milk (kumiss, posset), cheese and sour cream. The use of lactic acid bacteria for fermenting dairy products has the functions of improving the quality of dairy products, enhancing the health effects of dairy products and extending the shelf life of the products, and the products are deeply loved by people all over the world.

Sun et al.［16］ isolated and identified the lactic acid bacteria in traditional yogurt made by herders in Xinjiang and Mongolia. They isolated lactic acid bacteria from 4 parts of yogurt camel milk, including 4 strains of L. helveticus, 2 strains of each of L. casei subsp. pseudoplantarum and Lactobacillus bulgaricus, 1 strain of each of Pediococcus acidilactici, Campylobacter, Pediococcus urinaeequi and Enterococcus faecalis. The separation and identification of kumiss fermentation bacteria was carried out using kumiss in the Xilinguole pastoral area as the source of separation, and a total of 47 strains of bacteria were identified, belonging to 18 genera including Lactobacillus, Enterococcus, Leuconostoc, and Lactococcus［17］. The leading strains of cheese fermentation are lactic acid bacteria, which decompose lactose into lactic acid, thereby lowering the pH of the fermentation environment, thus inhibiting the growth and reproduction of spoilage bacteria and prolonging the storage time of cheese. Ma et al.［18］ isolated and identified lactic acid bacteria in cheese products from different pastoral areas in Xinjiang and obtained 104 lactic acid bacteria species, of which 82 belonged to the genus Lactobacillus, 12 belong to the genus Enterococcus, and 10 belong to the genus Weissella. These Lactobacillus are respectively L. casei, L. plantarum, L. helveticus, Lactobacillus kefiranofaciens, W. cibaria and Enterococcus durans.

Lactococcus lactis is widely used in cheese production. At each stage of cheese maturation, L. lactis faces different stress environments. Cretenet et al.［19］ applied transcriptomics methods to study the expression of L. lactis in different processes of cheese making. In the fermentation process of cheese, pH was continuously lowered due to the production of lactic acid by L. lactis. In the process of acid stress, F0F1-ATPase acted on the elimination of cytoplasmic protons. Both the arginine deiminase and citrate decarboxylase pathways were involved in pH homeostasis and energy production. When cheese was fermented for 8 h, and its pH was about 5.9. Expression profile analysis showed that only 3 genes in the F0F1-ATPase system (7 in total) were overexpressed during the cheese fermentation process. Genes atpG and atpD were overexpressed in fermentation for 8 h, and atpD and atpF were overexpressed at 7 d. Genes citC, citD, citE and citF involved in the metabolism of citric acid were temporarily overexpressed after 8 h of fermentation. Transcriptomics methods proved that after 8 h of fermentation by L. lactis, the transcription of genes related to the F0F1-ATPase pathway did not change significantly, proving that the F0F1-ATPase pathway is not an acid stress pathway in L. lactis. The expression of arginine deiminase pathway activators ahrC, arcB, and arcC1 increased, while the expression of genes arcD1 and arcD2 of the arginine antitransport system decreased. The arginine deiminase pathway is involved in the pH stress of L. lactis.

Yanrui MA et al. Application of Transcriptomics in the Analysis of Community Structure of Food Fermented with Lactic Acid Bacteria

Bisanz et al.［20］ used the RNA-Seq technology for the first time to analyze the bacterial flora in two different flavors of yogurt, i.e., strawberry and vanilla flavored yogurt, and that in yogurt of the same flavor but fermented at different time points. They used the ABISOLiD4 sequencing platform to sequence the mRNA, and obtained 48 458 804 read lengths, with each sample reaching 37.2 times the average coverage. However, the sequence coverage of L. lactis was less than 39%; and meanwhile, they also compared the abundance of mRNA in yogurt with high sequencing coverage under different flavors and storage times. In each microorganism, cluster of orthologous groups of proteins (COG) of genes with RPKM value ≥ 200 and RPKM value ≥ 1 000 can find the function of this type of genes. Carbohydrate transport and metabolism, protein translation, amino acid transport and metabolism are the concentrated manifestations of highly expressed genes. According to statistics, the peak genes of most microorganisms except Streptococcus thermophilus can be classified into the functional area of "transport". S. thermophilus exhibits high abundance in both carbohydrate transport and metabolism functional areas, including the β-galactosidase gene annotated as "LacZ", which mainly performs the function of lactose degradation and is also highly expressed in Lactobacillus delbrueckii subsp. bulgaricus, but the opposite is true in animal Bifidobacterium animalis subsp. lactis. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the main genes involved in the carbon transport metabolic process and glycolysis process in S. thermophilus had high expression characteristics, while animal B. animalis subsp. lactis had high expression characteristics in energy production and transfer. Among the mRNA transcripts expressed by different products, protein metabolism of all microorganisms and more specific ribosomal protein components were the most representative, and after further analysis, it was found that prokaryotic bacteria can adapt to this fermentation environment.

The research team of Inner Mongolia Agricultural University also compared the growth of L. casei Zhang in cow milk and soymilk by RNA-Seq transcriptomics. Lactobacillus casei Zhang was grown and fermented in milk, and a total of 84 genes were significantly expressed in the stable growth phase of pH 4.5 and the logarithmic growth phase of pH 5.2, of which 59 genes were significantly up-regulated. 40.5% of these genes are associated with carbohydrate and energy metabolism. The main up-regulated genes were related to the PTC system and the phosphopentose pathway. In soymilk, the expression of 63 genes was significantly different in the stable growth phase of pH 4.5 and the logarithmic growth phase of pH 5.2. Comparing the logarithmic growth phase of pH 5.2 with the delayed growth phase of pH 6.4, there were significant differences in the expression of 162 genes. Among them, 48.6% of the genes were up-regulated in the logarithmic growth phase, and 48.8% of the genes were up-regulated in the stable growth phase, and all the genes were related to the transport and metabolism of amino acids. Among them, the main up-regulated genes were related to metabolism of proteolytic enzyme systems (extracellular proteases, oligopeptide transport systems and intracellular peptidases), amino acids (glutamic acid, lysine and methionine) and nucleotides (purine and pyrimidine), and especially the active expression of the proteolytic enzyme system enabled L. casei Zhang to decompose soy protein in soymilk and provide sufficient amino acids and nucleotides for its own growth. It was speculated that it might be the main reason why L. casei Zhang grew better in soymilk than in cow milk［21］.

Bu renqiqige［22］ studied the community structure and functional gene expression in koumiss samples at different fermentation periods. Bu renqiqige［22］ studied the community structure and functional gene expression in kumiss samples at different fermentation periods. They used transcriptomics technology and metagenomics to optimize and adjust the community structure and community function, so as to provide a theoretical basis for the research on food safety, clinical application, and improvement of the controllability of traditional fermentation. The study selected six different stages to extract samples from kumiss during the fermentation process to obtain metagenomics, then amplified and performed pyrosequencing to analyze the succession of bacterial community structure during the fermentation process, and performed RNA-Seq high-throughput sequencing on the samples at the three fermentation stages during the fermentation process to construct a kumiss transcriptome library to obtain the expression of functional genes during the fermentation process. The main research conclusions are as follows: After sequencing the samples of kumiss at the three fermentation stages through high-throughput macrotranscriptome, a high-quality short sequence of 12.2 Gb was obtained; and meanwhile, with the extension of the fermentation time, the number of Lactobacillus first increased and then decreased, while the number of Lactococcus had been showing an upward trend as the fermentation progressed. In terms of phylum level, Proteobacteria and Firmicutes were the dominant phyla in kumiss, and Lactobacillus and Lactococcus were the dominant genera. In this study, 64210 merge-Unigenes were assembled, and the protein coding regions (CDSs) and sequence directions of 59339 Unigemes could be directly determined; and the remaining sequences were used to predict the coding regions, and the new protein coding sequences had 2230 Unigenes. The results of merge-Unigened functional classification analysis showed that the fermentation process was closely related to the processing, catalytic ability and metabolic process of microbial cells. The results of COG functional classification showed that genes involved in amino acid transcription and metabolism and genes related to replication, recombination and repair played a key regulatory role during the fermentation process of kumiss. From the analysis of the KEGG metabolic pathway, it could be seen that the metabolic pathway, the secondary metabolism of biosynthesis, and the microbial metabolic pathway in complex environment were the main signal pathways in the fermentation process. The results of GO (differentially expressed genes) functional enrichment analysis showed that the metabolic process, cell processing process, and organic matter metabolic process were the main biological processes from the initial stage to the middle stage of fermentation; and during this period, the expression of genes related to molecular functions such as organic cyclic compound binding, heterocyclic compound binding, and small molecule compound binding were active. Genes related to biological processes, such as cell processing, cell macromolecular metabolism, and single organism cell processing, played a decisive role in the middle to end of fermentation; and the expression of genes related to molecular functions, such as molecular structure activity, ribosomal structure composition, protein binding, and so on, were very active. KEGG differentially expressed gene pathway enrichment analysis showed that the ribosomal pathway was the dominant metabolic pathway in the early to mid-fermentation stage, while lipid metabolism, aminobenzoate degradation, steroid biosynthesis, mRNA monitoring pathways and other metabolic pathways played a major role in the mid- to late-fermentation stage.

The fermentation of yogurt currently on the market is dominated by S. thermophilus and L. delbrueckii subsp. bularicus. The two strains use their metabolites such as folic acid and carbon dioxide to stimulate each others growth. Sieuwerts et al.［23］ used a combination of transcriptomics and related metabolite determination to reveal the correlation between the growth and metabolism of these two strains, mainly involving the metabolism of purines, amino acids, and long-chain fatty acids. Formic acid, folic acid and fatty acids were mainly produced by S. thermophilus. L. delbrueckii subsp. bularicus had the effect of protein hydrolysis and provides amino acids needed for the growth of the two strains. However, it could be seen from the expression level of amino acid-related genes in the mixed system that the generated amino acids such as sulfur amino acids and branched chain amino acids were not enough to meet the growth requirements. Genes related to iron absorption and exopolysaccharide production in S. thermophilus were also affected by the mixed system, and their expression level was higher than that of the strains cultured in a single system. Through this research, we have a more in-depth understanding of the ecology of the mixed culture of strains in fermented food and their interaction mechanism.

Fermented Meat Products

Lactic acid bacteria-fermented meat products are more common in China as follows: cured bacon, sour meat, sausages, cured fish, dried duck and salted fish, etc. Proper curing will give meat products a richer taste, good color, and is conducive to the storage and preservation of meat.   Lactic acid bacteria play a very important role in the fermentation of meat products. For example, acidic environment prevents the growth of spoilage bacteria, promotes color formation, inhibits the formation of toxins, and improves nutritional value［24］.

Lactic acid bacteria are the dominant flora in fermented sausages, and they play a vital role in the fermentation, preservation and flavor formation of sausages. People have isolated three kinds of Lactobacillus from Dongs fermented sour meat, namely Pediococcus pentosaceus, Lactobacillus delbrueckii and Lactobacillus sake［25］. Drosinos et al.［26］ isolated L. plantarum, L. curvatus and L. sakei from Greek fermented sausages. L.sakei 23K is a psychrophilic bacterium widely used in fermented meat products［27］. In order to understand the gene expression of specific genes of L. sakei in the living environment of meat products, Xu et al.［28］ applied gene microarray transcriptomics technology to study their transcriptional expression under different culture conditions, and a total of 551 were detected. Compared with the expression of the strain on blank medium, the genes related to peptide chain hydrolysis were up-regulated to varying degrees in the media containing muscle fibers and sarcoplasm. The expression of oppB and oppC genes in the amino acid and peptide transport system was up-regulated. Except for glnA and metK genes, most genes related to the metabolism of peptides, amino acids and related molecules were overexpressed in the media containing muscle fibers and sarcoplasm. The stress-related genes were not induced to express in the muscle fiber culture medium.

Lactic acid bacteria participate in the fermentation of meat products, which is critical to improving the flavor and taste of meat products. However, a small number of lactic acid bacteria can lead to the spoilage of meat products. Lactococcus is a kind of psychrophilic bacteria, which is a kind of spoilage bacteria that causes the spoilage of meat products during the cryopreservation process and can produce an oily state and sour odor. Studies have shown that differences in the growth environment of Lactococcus can lead to great differences in the degree of spoilage. Through transcriptomics analysis of different periods of lactococcal glucose metabolism, it is found that as time progresses, glucose is continuously consumed, and the gene expression of lactococcal carbohydrate and glycerol metabolic pathways is up-regulated. Meanwhile, the expression of genes related to the pyruvate metabolism pathway related to the production of spoilage is abnormal. Through transcriptomics, it has been observed at the gene level that fish Lactococcus can maintain its survival through the up-regulation of related gene expression and the enhancement of metabolic pathway activity during the whole process of meat product spoilage［29］. Leuconostoc gelidum subsp. gasicomitatum is also a harmful microorganism that can cause spoilage of meat products, and usually brings a spoilage taste of butter to meat products. The source of the buttery taste of this strain under different carbon source growth conditions can be studied through the combination of gas chromatography-mass spectrometry (GC-MS) and transcriptomics methods can study.

Conclusions

As mentioned above, in recent years, transcriptomics based on high-throughput sequencing technology has made some progress in the analysis of food fermentation flora, but there are still many limitations. ① The read length of second-generation sequencing is relatively short, and the assembly of macrotranscriptome data can only reach the level of Scaffold, especially for complex genomes. The high content of repetitive sequences makes the genome size "shrink"; ② The assembly of sequencing data largely relies on the known microbial sequences in the existing database, which is not conducive to the timely discovery of new microbial species and will waste part of the sequencing data. ③ The sequencing data of metagenomics and macrotranscriptomes are large, and it is difficult to analyze them. And biological information analysis requires higher data processing systems, and different algorithms may have very different results. ④ The detection of low-abundance genes is more difficult. ⑤ Higher costs result in fewer duplicate samples currently used for sequencing, which reduces the reliability and reproducibility of the results.

With the continuous advancement of sequencing technology, the third generation of sequencing technologies with more advantages have emerged. For example, PacBio RS has an ultra-long read length (10 kb) and extremely low guanine nucleic acid and cytidylic acid (guanylic acid and cytidylic acid, GC) preference, which is conducive to genome assembly. The combined use of the second-generation sequencing and optical mapping technologies has been applied in transcriptome research, providing a more accurate method for the study of microbial community structure in fermented foods. The increasingly complete genome database of microorganisms, especially fermented food microorganism species, provides more and more solid support for the splicing and annotation of metagenomic and macrotranscriptome data. The cross application of metagenomics, metatranscriptomics, metabolomics, proteomics and other technologies will help us understand more clearly the community structure succession and interaction of microorganisms in fermented foods and the contribution of each strain to flavor and unearth new functional genes. In the future, the combination of multidisciplinary theoretical knowledge such as microbiology, food chemistry, bioinformatics, and molecular biology, as well as new sequencing technologies, more complete gene databases, advanced data analysis tools, and a variety of omics methods will surely bring a new dawn to the research of fermented food microorganisms.

References

［1］ MIAO LH, BAI FL, LI JR. Advanced on ecological succession of lactic acid bacteria in traditional fermented food［J］. Food and Fermentation Industries, 2015, 41(1): 175-180. (in Chinese)

［2］ WU RN, YU ML, MENG LS, et al. PCR-DGGE analysis of the microbial diversity in naturally fermented suan-cai from northeast China［J］. Modern Food Science & Technology, 2014, 30(10): 8-12, 35. (in Chinese)

［3］ TONG TT, TIAN FW, WANG G, et al. Metagenomic analysis of bacterial diversity changes during vegetables fermentation using Sichuan pickle water as starter［J］. Science and Technology of Food Industry, 2015, 36(21): 173-177. (in Chinese)

［4］ NAM YD, CHANG HW, KIM KH, et al. Metatranscriptome analysis of lactic acid bacteria during kimchi fermentation with genome-probing microarrays［J］. International journal of food microbiology, 2009, 130(2): 140-146.

［5］ JUNG JY, LEE SH, KIM JM, et al. Metagenomic analysis of kimchi, a traditional korean fermented food［J］. Applied and Environmental Microbiology, 2011, 77(7): 2264-2274.

［6］ BOKULICH NA, LEWIS ZT, BOUNDY-MILLSET K, et al. A new perspective on microbial landscapes within food production［J］. Current Opinion in Biotechnology, 2016(37): 182-189.

［7］ ZHANG Q, LIN K, YUAN CH, et al. Separation and identification of lactic acid bacteria isolated from soy sauce mash during the low-salt-level solid state fermentation ［J］. Sichuan Food and Fermentation, 2014, 50(2): 1-4. (in Chinese)

［8］ WANG MY, ZHAO GZ, ZHAO JX, et al. Analysis of microbial community microecology in Shanxi aged vinegar fermentation process and isolation of lactic acid bacteria［J］. China Condiment, 2016, 41(6): 29-33, 38. (in Chinese)

［9］ HAN BZ, ROMBOUTS FM, NOUT MJR. A Chinese fermented soybean food［J］. International Journal of Food Microbiology, 2001, 65(1-2): 1-10.

［10］ NAM YD, YI SH, LIM SI. Bacterial diversity of cheonggukjang, a traditional Korean fermented food, analyzed by barcoded pyrosequencing［J］. Food Control, 2012, 28(1): 135–142.

［11］ DUAN S, HU X, LI M, et al. Composition and metabolic activities of bacterial community in shrimp sauce at the flavor forming stage of fermentation as revealed by metatranscriptome and 16S rRNA gene sequencing［J］. Journal of Agricultural & Food Chemistry, 2016, 64(12): 2591.

［12］ ZOTTA T, PIRAINO P, PARENTE E, et al. Characterization of lactic acid bacteria isolated from sourdoughs for Cornetto, a traditional bread produced in Basilicata (Southern Italy)［J］. World Journal of Microbiology & Biotechnology, 2008, 24(9): 1785-1795.

［13］ ESCALANTE A, WACHER C, AMELIA FARRIS. Lactic acid bacterial diversity in the traditional mexican fermented dough pozol as determined by 16S rDNA sequence analysis.［J］. International Journal of Food Microbiology, 2001, 64(1-2): 21-31.

［14］ WECKX S, VAN D MR, ALLEMEERSCH J, et al. Community dynamics of bacteria in sourdough fermentations as revealed by their metatranscriptome［J］. Applied & Environmental Microbiology, 2010, 76(16): 5402-5408.

［15］ WU SIRGULENG. Identification and biodiversity of yeast and LAB isolated from sourdoughs collected from western region of Inner Mongolia［D］. Inner Mongolia: Inner Mongolia Agricultural University, 2011. (in Chinese)

［16］ SUN TS, LIU HX, NI HJ, et al. Isolation and identification of lactic acid bacteria in Hogormag: A traditional fermented camel milk product ［J］. China Dairy Industry, 2006(9): 4-7. (in Chinese)

［17］ MA YL. Studies on biological activity and isolation and identification of lactic acid bacteria in the Kumiss［D］. Inner Mongolia: Inner Mongolia Agricultural University, 2005. (in Chinese)

［18］ MA Y, NI YQ, LU SL, et al. Isolation and identification of lactic acid bacteria from a traditional cheese in Xinjiang ［J］. China Brewing, 2011(8): 38-40. (in Chinese)

［19］ CRETENET M, VALIRIE LAROUTE, VINCENT ULVI, et al. Dynamic analysis of the Lactococcus lactis transcriptome in cheeses made from milk concentrated by ultrafiltration reveals multiple strategies of adaptation to stresses［J］. Applied and Environmental Microbiology, 2010, 77(1):247-257.

［20］ BISANZ JE, MACKLAIM JM, GLOOR GB, et al. Bacterial metatranscriptome analysis of a probiotic yogurt using an RNA-Seq approach［J］. International Dairy Journal, 2014, 39(2): 284-292.

［21］ WANG JC. Study on mechanisms of probioitc Lactobacillus casei Zhang growing bovine milk and soymilk based on transcriptomics and proteomics［D］. Inner Mongolia: Inner Mongolia Agricultural University, 2012. (in Chinese)

［22］ BU RENQIQIGE. Metatranscriptomics and bacteria community structure analysis of Koumisss fermentation process［D］. Inner Mongolia: Inner Mongolia Agricultural University, 2014. (in Chinese)

［23］ SIEUWERTS S, MOLENAAR D, HIJUM SAFT, et al. Mixed-culture transcriptome analysis reveals the molecular basis of mixed-culture growth in Streptococcus thermophilus and Lactobacillus bulgaricus［J］. Applied & Environmental Microbiology, 2010, 76(23): 7775-7784.

［24］ PAN RS. Application of lactic acid bacteria in the processing of meat products［J］. Journal of Henan Vocation 2 Technical Teachers College, 1997(3): 52-55. (in Chinese)

［25］ ZHANG DF, XU WM, XU XL. Isolation and identification of lactic acid bacteria from traditional sour meat［J］. Jiangsu Journal of Agricultural Sciences, 2008, 24(006): 986-988. (in Chinese)

［26］ DROSINOS, MATARAGAS EH, XIRAPHI M, et al. Characterization of the microbial flora from a traditional Greek fermented sausage, 2005, 69(2): 307-317.

［27］ CHAILLOU, STéPHANE, CHAMPOMIER-VERGIS, et al. The complete genome sequence of the meat-borne lactic acid bacterium Lactobacillus sakei 23K［J］. Nature Biotechnology, 2005, 23(12): 1527-1533.

［28］ XU HQ, GAO L, JIANG YS, et al. Transcriptome response of Lactobacillus sakei to meat protein environment［J］. Journal of Basic Microbiology, 2015, 55(4): 490-499. .

［29］ ANDREEVSKAYA M, JOHANSSON P, LAINE P, et al. Genome sequence and transcriptome analysis of meat-spoilage-associated lactic acid bacterium Lactococcus piscium MKFS47［J］. Applied & Environmental Microbiology, 2015, 81(11): 3800-3811.