추자도 멸치액젓의 Microbiom 분석 및 신종 미생물 특성 규명

Abstract

Fermentation is one of the most economical and classical methods for producing and preserving foods by humans. Besides preserving, fermentation also changes the organoleptic characteristics of foods through developing a wide diversity of flavors, aromas and textures. These fermented foods have been separately developed according to various fermentation conditions such as raw materials, combination of various microorganisms including seed bacteria, temperature, and pH. In particular, various microorganisms in fermented foods are the main factors that determine the taste and flavor of fermented foods, and the degree of fermentation can be determined depending on interactions among microbial communities. Therefore, microbiome analysis of fermented foods has recently emerged as a very important study field from a microbiological point of view in conjunction with the scientification of fermented foods. Therefore, with NGS technique, we want to find out the various microorganisms that exist in the fermented anchovy sauce of Chuja Island. Chuja Island became famous for anchovy, it is because when a spawning season of anchovy, many anchovies gather in Chuja island. In Chuja Island used anchovies to make salted anchovy sauce that can be used instead of soy sauce. The anchovy sauce for the Chuja Island is made by mixing salt and anchovies without adding any chemical seasoning and fermenting them for about three years. This MyeolchI-Aekjeot, traditional fermented anchovy sauce, has been consumed as an umami-tasting, seasoning ingredient. The umami of Myeolchi-Aekjeot is formed by exogenous enzymes of microorganisms, and despite the high concentration (20-30%) of salty environment, various microorganisms exist in fish sauces. Therefore, with NGS technique, we found out the various microorganisms that exist in the fermented anchovy sauce of Chuja Island. The fermented anchovy sauce, Myeolchi-Aekjeot, of Chuja island, which was used for the analysis of bacterial communities, was made by Chujado Federation of Fisheries Cooperatives in Jeju. It showed pH 6.1, 27% salinity, and 37.9 brix. To identify bacterial community, total DNA was extracted from fermented anchovy sauce and analyzed by an Illumina MiSeq platform. So total valid reads of 36,376 were obtained. Among total valid reads, reads of the species level was 19,661 reads (54%) and found 132 species. In results of analysis the bacterial community of fermented anchovy sauces of Chuja island, bacteria of phylum Firmicutes (82.5%) dominated at sample. And the Class Bacilli (76.4%), Order Bacillales (64.6%), Family Bacillaceae (64.3%) were the dominant in taxon, respectively. At the Genus level, the sample were dominated Lentibacillus (46.4%), Tetragenococcus (11.7%), and Pseudogracilibacillus (10.6%). Although there is no study on analyzed the bacterial community of fermented anchovy sauce in Jeju region, the results of the analysis of the bacterial community in 9 types of fermented anchovy sauce of Korea showed that unlike the results of this study, the bacteria of genus Tetragenococus and Halanaerobium in phylum Firmicutes were dominant in all samples. And study of analyzed the bacterial community of fermented anchovy salted fish (Myeolchi-Jeotgal) in jeju region, genus Tetragenococcus (90%) in phylum Firmicutes were dominant. According to the results of these studies, including this study, it can be confirmed that all phylum Firmicutes dominate. This is thought to be because a large number of bacteria belonging to this taxon form endospores and have the ability to exist in extreme environments. In addition, it appears to be the result because it contains a large number of the fermentation-related bacteria in this taxon. The difference in the results of these studies is thought to be due to the fermentation period. The fermentation period of fermented anchovy sauce of Chuja island analyzed in this study is 3 to 5 years, but the fermentation period of other fermented anchovy sauces and fermented anchovy salted fish is about 1 year, which is short. Through this, At the beginning of fermentation, bacteria that actively participate in fermentation, such as bacteria of genus Tetragenococus, appeared predominantly, and at the latter period of fermentation, genus Lentibacillus, which has a lategrowth characteristic, appears to be predominant. For this reason, it could be seen that the difference in dominant bacteria occurs over the fermentation period. In the future, if you conduct a bacterial community analysis by fermentation period of fermented anchovy sauce of Chuja island, we will see more accurate results. As a result of this analysis, it can be seen that fermented anchovy sauce of Chuja island is a well-fermented fish sauce. It is known that fish sauce such as fermented anchovy sauce, is rich in essential nutrients and tastes better with a longer fermentation period. It is thought that the presence of genus Lentibacillus in fish sauce can be used as an indicator of fermentation period. And in this study, strain JNUCC-1T was characterized and classified and this study proposed that strain JNUCC-1T isolated from fermented anchovy sauce in Chuja island is novel species via polyphasic taxonomy. Cells of strain JNUCC-1T are Gram-positive, aerobic, motile by means of polar flagella, and spore-forming rods (0.3-0.8 μm wide and 1.2-1.9 μm length). Spherical endospores form at the cell-terminal position. Colonies are circular with opaque of beige and below 1.0 mm in diameter. Growth occurs in 0-20% (w/v) NaCl (optimal in 5%) and pH 4.0-10.0 (optimal at pH 7.0) at 20-45 ℃ (optimal at 30 ℃). Positive results in tests for oxidase, catalase, nitrate reduction and hydrolysis of Tween 20 and 60. The major fatty acid (>20%) is anteiso-C15:0. The major polar lipids are diphosphatidylglycerol (DPG), phosphatidyl-glycerol (PG), and one unidentified phospholipid (PL). The predominant respiratory isoprenoid quinone is menaquinone-7 (MK-7). Comparative analysis of 16S rRNA gene sequences showed that strain JNUCC-1T is most closely related to Lentibacillus alimentarius M2024T and Virgibacillus kekensis YIM-kkny16T. However, phylogenetic analysis showed that strain JNUCC-1T forms a phyletic lineage with members of the genus Lentibacillus. The topologies of phylogenetic trees built using the maximum-likelihood, maximum-parsimony, and neighbor-joining methods also supported these results, indicating that strain JNUCC-1T formed a stable clade. The genome size is 3,687,469 bp, and GC content of the genomic DNA is 43.3 %. Protein-coding sequences were predicted by Prodigal version 2.6.2, which showed that the genome contained 3,833 genes. In addition, 66 tRNA genes were identified by tRNAscan-SE version 1.3.1, and 17 rRNA genes were identified by Rfam version 12.0. As a result of analyzing the genes classified according to the COG functional categories, the genome of strain JNUCC-1T represented higher proportions in the categories of Replication, recombination and repair (7.7%), Amino acid transport and metabolism (7.7%), Inorganic ion transport and metabolism (5.6%), Carbohydrate transport and metabolism (5.3%), Energy production and conversion (5.2%), Transcription (5.1%), Cell wall/membrane/envelope biogenesis (4.81), Translation, ribosomal structure and biogenesis (4.6%). Analysis of phenotypic and genetic traits through COG functional categories gave the same results. The strain JNUCC-1T found the presence of motility and flagella, and through genomic analysis, it was confirmed that it contained 43 predicted genes related to motility or flagella. And the genome of strain JNUCC-1T contains 169 predicted genes associated with sporulation, such as spore germination protein GerD, GerM, and YaaH, and 33 predicted genes associated with antibiotic resistance such as pbp2A, ampC and multidrug resistance genes. The results of other biochemical tests, such as API system and hydrolysis of macromolecule test also related genes in COG functional categories of Amino acid transport and metabolism, Carbohydrate transport and metabolism, and Lipid transport and metabolism could confirm. This may be the basis for supporting the results of previous biochemical tests. Genes that could support the results of the chemotaxonomic test were identified. In genome of strain could confirm 9 predicted genes associated with phosphatidylglycerol and diphosphatidylglycerol such as umpA, lgt, and ltaS, and 14 predicted genes associated with menaquinone biosynthesis such as bioC, fadD, and ubiE, and 19 predicted genes associated with fatty acids biosynthesis such as fabG, fabH, and fabI. The genome of strain JNUCC-1T contains 19 predicted genes associated with osmotic stress regulation, such as choline glycine betaine transporter BetT2, osmoprotectant import permease protein OsmW. These genes could be key factors allowing strain JNUCC-1T to adapt to high-salt environments via osmotic regulation and adjustment of cytoplasmic pH, respectively. Metabolites produced by extreme halophile bacteria such as the strain JNUCC-1T have been utilized in various industries. Compatible solutes produced to overcome osmotic stress are utilized in the bio industry, enzymes that have activity at high concentrations of salinity are utilized in eco-friendly industries such as decomposition of pollutants and toxic substances, and enzymes such as esterase added in fermentation food used to improve the flavor of food. As a result of analyzing the genome of the strain, the strain is expected to have the potential ability to be used in various industries, and in the future, it will be able to derive the ability of the strain through additional studies such as metabolite regulation, efficacy evaluation, and genetic manipulation. The genome sequence of strain JNUCC-1T has been deposited in GenBank under accession no. WHOH00000000 (BioProject accession no. PRJNA577786 and BioSample accession no. SAMN13037569). The public version of the strain JNUCC-1T genome sequence deposited in GenBank was annotated using the Prokaryotic Genome Annotation Pipeline (PGAP). In this study, Phylogenetic tree showed that strain JNUCC-1T forms a phyletic lineage with members of the genus Lentibacillus. The chemotaxonomic and phenotypic characterizations of strain JNUCC-1T showed typical features of members of the genus Lentibacillus, with MK-7 as the predominant respiratory isoprenoid quinone and major fatty acid including anteiso-C15:0. The strain JNUCC-1T is considered as a novel species of the genus Lentibacillus, for which the name Lentibacillus jejunsis sp. nov., (Type strain JNUCC-1T = KCTC 43912T) is proposed. This study analyzed the community of microorganisms present in fermented anchovy sauce of Chuja island using the Illumina Miseq platform to confirm that about species of 130 bacteria coexist. Among them, it was found that the bacteria in the genus Lentibacillus dominate, which means that fermentation is good for a long time, so that the essential nutrients are rich and the flavor is improved. In addition, the biodiversity was secured to respond to the implementation of the Nagoya Protocol by separating the new strains from the anchovy sauce and identifying their characteristics.