Unravelling the key factors for the dominance of Leuconostoc starters during kimchi fermentation

Abstract

Recent studies aim to prevent kimchi spoilage and enhance the sensory and nutritional qualities using lactic acid bacteria, particularly Leuconostoc species, as kimchi starters. However, the factors enabling the successful adaptation and predominance of Leuconostoc species remain unclear. This study investigates the factors that contribute to the successful adaptation of Leuconostoc starter strains WiKim32, WiKim33, WiKim0121 and CBA3628 during kimchi fermentation using a comprehensive multi-omics approach. Our findings reveal that ATP-dependent molecular chaperones, which respond to cold and acidic kimchi environments, play crucial roles in successfully adapting Leuconostoc starter strains. Moreover, genes involved in carbohydrate metabolic pathways enhance ATP production, thereby supporting chaperone activity and bacterial growth. This study highlights the practical use of Leuconostoc starter strains WiKim32, WiKim33 and WiKim0121 and identifies essential factors for their successful adaptation and predominance during kimchi fermentation.

Fig. 1. Fermentation profiles of kimchi samples inoculated with different Leuconostoc starter strains.

Changes in a pH levels, b colony-forming units of lactic acid bacteria, and the concentrations of c fructose, d glucose, e acetate, f lactate, g mannitol, and h ethanol in the CTR, W32, W33, W0121, and C3628 samples during kimchi fermentation. The kimchi samples inoculated with strains WiKim32, WiKim33, WiKim0121, and CBA3628, and saline solution were labelled as 'W32', 'W33', 'W0121', 'C3628', and 'CTR', respectively. Data were presented as mean ± standard deviation. Experiments were performed in triplicate.

Fig. 2. Microbial composition and phylogeny of kimchi samples.

a Bacterial community profiles at amplicon sequence variant (ASV) level in the CTR, W32, W33, W0121, and C3628 samples during kimchi fermentation. ‘F’ indicates family level. b A phylogenetic tree of Leuconostoc species, including Leuconostoc ASV001, Leuconostoc ASV002, Leuconostoc ASV003, and four starter strains (WiKim32, WiKim33, WiKim0121, and CBA3628). The tree was inferred using the neighbour-joining method and bootstrap values were calculated from 1000 replicates. Weissella cibaria CBA3612 and Weissella koreensis KACC 15510 were used as the outgroup. Bar, 0.01 substitution per nucleotide position. The kimchi samples inoculated with strains WiKim32, WiKim33, WiKim0121, and CBA3628, and saline solution were labelled as 'W32', 'W33', 'W0121', 'C3628', and 'CTR', respectively.

Fig. 3. Functional classification of genes and CAZyme profiles in Leuconostoc starter strains.

a Clusters of orthologous groups (COG) functional categories display the number of genes in each starter strain, classified as follows: C, energy production and conversion; D, cell cycle control, cell division, chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, post-translational modification, protein turnover, and chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport, and catabolism; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; and V, defence mechanisms. b The number of CAZyme genes in each starter strain is classified as follows: GH glycoside hydrolases, GT glycosyl transferases, AA auxiliary activities, CE carbohydrate esterases, PL polysaccharide lyases, and CBM carbohydrate-binding molecules.

Fig. 4. Comparative metabolic pathways reconstructed from Leuconostoc starter strains.

Metabolic pathways of Leuconostoc mesenteroides WiKim32, Leuconostoc mesenteroides WiKim33, Leuconostoc mesenteroides WiKim0121, and Leuconostoc sp. CBA3628 were generated using the iPath v2 module based on Kyoto Encyclopaedia of Genes and Genomes (KEGG) Orthology (KO) numbers. The pathways are displayed in different colours depending on the presence and/or absence of genes in each starter strain, as depicted in the table inside the figure.

Fig. 5. Carbohydrate metabolic pathways and gene expression profiles of Leuconostoc starter strains during kimchi fermentation.

a Reconstructed carbohydrate metabolic pathways of Leuconostoc mesenteroides, Leuconostoc citreum, Weissella cibaria, and Weissella koreensis. The pathways are represented by different colours depending on the presence (P) and/or absence (A) of genes in each strain, as depicted in the table inside the figure. b Expression levels of genes in Leuconostoc mesenteroides, Leuconostoc citreum, Weissella cibaria, and Weissella koreensis in response to carbohydrate availability. Arabic numbers and KO numbers are positioned close to the lines in a reconstructed pathways and to b the heatmap, showing the expression levels of each gene across the strains. The kimchi samples inoculated with strains WiKim32, WiKim33, WiKim0121, and CBA3628, and saline solution were labelled as 'W32', 'W33', 'W0121', 'C3628', and 'CTR', respectively.

Fig. 6. Correlation network analysis and gene expression profiles of stress-related functions in Leuconostoc starter strains during kimchi fermentation.

The average transcripts per million (TPM) values of 1238 genes in 33 categories based on KEGG BRITE hierarchical classifications were calculated, and 83 genes that exhibited transcript levels above 2000 TPM were selected. Spearman correlation coefficients were calculated between the relative abundance of Leuconostoc ASV001 and the TPM values mapped to Leuconostoc mesenteroides ATCC 8293^T, Leuconostoc mesenteroides WiKim32, Leuconostoc mesenteroides WiKim33, and Leuconostoc mesenteroides WiKim0121 in the kimchi samples CTR, W32, W33, and W0121 samples, respectively, and between the relative abundance of Leuconostoc ASV002 and the TPM values mapped to Leuconostoc sp. CBA3628 in the C3628 sample. Then, a a network analysis was performed. The colours of nodes represent the following: red, bacterial relative abundances; yellow-green, KO number within oxidative phosphorylation category; grey, KO number within ribosome category; white-yellow, KO number within translation factors category; pink, KO number within chaperones and folding catalysts category; green, KO number within membrane trafficking category; blue, KO number within mitochondrial biogenesis category; apricot, KO number within exosome category; and sky-blue, KO number within the categories more than two. The edges represent Spearman correlations, with thicker edges indicating a stronger positive correlation and thinner edges displaying a weaker correlation or a negative correlation, as per the correlation scale ranging from −1.0 to 1.0. The highly ‘strong’ positive correlation of over 0.6 is visualised with red edges. b The TPM values of genes encoding F-type ATPase and chaperone proteins from five strains in each kimchi sample during fermentation were visualised using the heatmap. Gene expression levels are indicated by colour intensity, with red representing higher TPM values (0 to 50,000 or greater). The pH values at each time point are provided above the heatmap, with the intensity of colour (yellow–black) indicating the pH, as shown on the scale bar (4.06‒5.14). The boxes of gene names are filled with colours corresponding to each category described in the network analysis. The kimchi samples inoculated with strains WiKim32, WiKim33, WiKim0121, and CBA3628, and saline solution were labelled as 'W32', 'W33', 'W0121', 'C3628', and 'CTR', respectively.

Fig. 7. Arginine deiminase pathway activity in Weissella strains and associated metabolite changes during kimchi fermentation.

a Putative schematic diagram of arginine deiminase pathway of Weissella cibaria and Weissella koreensis with their TPM values observed in each pathway and visualised by the heatmap during kimchi fermentation. Changes in the concentrations of b arginine and c ornithine in the CTR, W32, W33, W0121, and C3628 kimchi samples. The kimchi samples inoculated with strains WiKim32, WiKim33, WiKim0121, and CBA3628, and saline were labelled as 'W32', 'W33', 'W0121', 'C3628', and 'CTR', respectively. Data were presented as mean ± standard deviation. Experiments were performed in triplicate.