Diversity Of Lactic Acid Bacteria In Brine Of Traditional Sichuan Paocai

Sichuan paocai, one of the most famous Chinese fermented vegetables is produced and consumed primarily in southwestern China where nearly every household still maintains the custom of making paocai. As Sichuan paocai is characterized by lactic acid fermentation, Sichuan paocai is rich in lactic acid bacteria (LAB). Although LAB in Sichuan paocai has been studied a lot, many researches applied culture-dependent techniques. The information of a comprehensive bacterial community structure and LAB diversity is very insufficient. In this study, a total of 126 traditional Sichuan paocai brine samples produced by local household in Chongqing were collected. The full-length 16S rRNA gene single-molecule real-time (SMRT) sequencing with the combination of droplet digital PCR (ddPCR) were employed to explore the bacterial and LAB compositions and diversity of paocai brine samples, as well as any relevance between physiochemical indices and bacterial profiles of the paocai brine. The functional proteins and pathways of bacterial compositions in paocai brine samples were predicted and compared within different sample groups. The main findings of this work are introduced as following:(1) The basic physiochemical indices such as pH, total acid (TA), lactic acid, acetic acid, succinic acid, salt concentration and nitrite were measured as well as the taste indices of paocai brine samples. The results showed that there was a large variation in different physiochemical and taste indices among paocai brine samples, which reflected the differential taste preference of local households provided the samples.No distinguish cluster of paocai brine samples from various sampling places based on the taste indices,indicating the consistency of the overall taste property of paocai in Chongqing. The nitrite content of the tested samples was generally at a relatively low level, indicating low risk of nitrite in these samples. pH,salt concentration and nitrite content were negatively correlated with TA. Lactic acid was positively correlated with TA while negatively correlated with acetic acid. There was a significant negative correlation between nitrite and pH. Sourness positively related to bitterness, astringency or aftertaste-astringency, but negatively correlated with saltiness and umami.(2) A total of 240 LAB strains were isolated and identified from traditional Sichuan paocai brine samples, belonging to genera Lactobacillus (Lb., 13 species, 205 strains), Pediococcus (P., 2 species, 33 strains), Enterococcus (E., 1 strain) and Lactococcus (Lc., 1 strain). Lb. plantarum was the most dominant LAB strains with the number of 102, followed by Lb. buchneri (48 strains) and P. ethanolidurans (32 strains). Other low-abundance species were Lb. parafarraginis, Lb. brevis, Lb, alimentarius, Lb.paracasei,Lb. namurensis,Lb. versmoldensis, Lb. plajomi, Lb. acidifarinae, Lb. fermentum, Lb.harbinensis, Lb. parabrevis, P. acidilactici, E.faecium and Lc. lactis. It is the first time that Lb. plajomi was found in paocai brine.(3) Nearly half of the samples collected were analyzed by SMRT sequencing of full-length 16S rRNA gene. The results showed that there was a high richness of bacterial community in traditional Sichuan paocai brine samples, where 35 phyla, 71 classes, 118 orders, 204 families, 508 genera, and 961 species were detected. Firmicutes and Proteobacteria were the dominant bacteria phyla; Lactobacillus was the most predominant genus; Lb. acetotolerans and Lb. brevis were the most abundant species. Lb.acetotolerans, Lb. buchneri, Lb. brevis and P. ethanolidurans were the core species in the samples analyzed as they were found in over 90% samples. In addition, many low-abundance strains detected in this study have not been reported in previous researches on Sichuan paocai. The results from absolute quantification by ddPCR manifested Lb. acetotolerans instead of Lb. plantarum which was the most isolated species was the most dominant species in the samples studied.(4) Samples were grouped based on physiochemical indices. Results showed that TA affected the bacterial a diversity of samples most directly and positively correlated with a diversity estimators. The bacterial ? diversity were affected by acidity, salt concentration and raw materials. The higher the acidity the higher the relative abundance of LAB. The correlations between TA and Lb. acetotolerans or Lb.brevis were opposite. The high salt concentration did not reduce the relative abundance of undesirable bacteria in the samples, but indeed inhibited the presence of LAB. Not the longer time the use of paocai brine, the better bacteria compositions. No significant difference existed in dominant bacteria within major material groups, but the ? diversity were significantly different probably due to the differences in low abundance bacteria. The nitrite content was only positively correlated with Lb. versmoldensis in the samples, and had no significant correlation with Enterobacteriaceae bacteria and other dominant flora.(5) The metagenomes of bacterial community in paocai brine samples were predicted using PICRUSt and annotated with 4365 categories of functional proteins (or enzymes) in COG database, belonging to the cell processes and signals, information storage and processing, metabolism, complex function and poorly characterized categories. Most functional proteins were associated with the metabolism. And the predicted metagenomes were annotated with 5749 categories of KO and further assigned to hundreds of pathways, which were mainly in the categories of metabolism, genetic information, environmental information and cell processes. Pathways of carbohydrate metabolism, metabolism of terpenoids and polyketone compounds, metabolism of cofactors and vitamins, cellular motility, amino acid metabolism,energy metabolism, xenobiotics biodegradation and metabolism, lipid metabolism, energy metabolism,glycan biosynthesis and metabolism, other amino acid biosynthesis and metabolism, metabolism of other amino acids, membrane transport and so on were enriched in different sample groups. Differential metabolic pathways may cause the variations in brine tastes. In addition, low-abundance bacteria likely contributed to the significant differential pathways in different samples.