Optimizing Divalent Cation Supplementation to Enhance the Production of the Kimchi Starter Strain Latilactobacillus curvatus WiKim0094

Abstract

To support the industrial-scale production of kimchi starter cultures, this study aimed to enhance the cell density of Latilactobacillus curvatus WiKim0094 by optimizing the composition of divalent cations in a nutrient-limited medium. Screening experiments identified Mg²⁺, Mn²⁺, and Ca²⁺ as key factors that significantly promoted cell growth. Using a Box-Behnken design, their optimal concentrations were determined to be 3.0, 0.1, and 4.4 mM, respectively, resulting in a 5.7-fold increase in the maximum viable cell count compared with the control. Comparative transcriptomic analysis revealed that supplementation with these metal ions activated pathways associated with nucleic acid synthesis, energy metabolism, nutrient transport, cell structure formation, and stress resistance. These findings provide mechanistic insights into divalent cation-mediated enhancement of microbial growth and propose a cost-effective strategy for the large-scale cultivation of kimchi starter strains. Future studies will focus on validating this approach in large-scale bioreactors to facilitate industrial application.

Keywords: Latilactobacillus curvatus; divalent cation; kimchi starter; response surface methodology; transcriptomics.

Fig. 1. Effect of different divalent cations (MgSO₄, MnSO₄, CaCl₂, CuSO₄, ZnSO₄, and FeSO₄) at low, medium, and high concentrations on the relative viability of L. curvatus WiKim0094 compared with the control.

Colony-forming units after 24 h of incubation are expressed as a percentage of the control (1/5 MRS broth without added cations). Treatment concentrations were as follows: MgSO₄, 1, 10, and 20 mM; MnSO₄, 0.1, 1, and 5 mM; CaCl₂, 0.1, 1, and 10 mM; CuSO₄, 0.01, 0.1, and 1 mM; ZnSO₄, 0.01, 0.1, and 1 mM; FeSO₄, 0.005, 0.05, and 0.5 mM. Bars represent the mean ± standard deviation. Different letters indicate statistically significant differences among treatments (p < 0.05).

Fig. 2. Three-dimensional response surface plots from the Box-Behnken design showing the interactive effects of divalent cations on the growth of L. curvatus WiKim0094.

The plots show the interactions between colony-forming units (CFU) and concentrations of (A) MgSO₄ and CaCl₂, (B) MgSO₄ and MnSO₄, and (C) MnSO₄ and CaCl₂.

Fig. 3. Growth curve of L. curvatus WiKim0094 in optimized MMC medium (●, supplemented with 3.0 mM MgSO₄, 0.1 mM MnSO₄, and 4.4 mM CaCl₂) and control medium (■, 1/5 MRS broth).

Viable cell counts were measured every 3 h over a 24 h incubation period. Error bars represent standard deviations from triplicate experiments.

Fig. 4. Differential gene expression in L. curvatus WiKim0094 under divalent cation supplementation.

(A) Bar plot showing the number of differentially expressed genes (DEGs) in the T12/NC12 and T24/NC24 comparisons, categorized as upregulated, downregulated, or shared. (B) Venn diagram illustrating overlap between upregulated and downregulated DEGs across the two comparisons. T12 and T24, cultures supplemented with 3.0 mM Mg²⁺, 0.1 mM Mn²⁺, and 4.4 mM Ca²⁺ for 12 h and 24 h; NC12 and NC24, unsupplemented control cultures at the corresponding time points.

Fig. 5. Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in L. curvatus WiKim0094 under divalent cation supplementation.

(A, C) Enriched biological process terms for upregulated and downregulated DEGs in the T12/NC12 and T24/NC24 groups, respectively. (B, D) Enriched molecular function terms for upregulated and downregulated DEGs in the T12/NC12 and T24/NC24 groups, respectively. T12 and T24, cultures supplemented with 3.0 mM Mg²⁺, 0.1 mM Mn²⁺, and 4.4 mM Ca²⁺ for 12 h and 24 h; NC12 and NC24, unsupplemented control cultures at the corresponding time points.

Fig. 6. Functional annotation of differentially expressed genes (DEGs) in L. curvatus WiKim0094 based on EggNOG classifications under divalent cation supplementation.

DEGs from T12/NC12 (gray bars) and T24/NC24 (blue bars) comparisons were grouped into three major categories: information storage and processing, cellular processes and signaling, and metabolism. T12 and T24, cultures supplemented with 3.0 mM Mg²⁺, 0.1 mM Mn²⁺, and 4.4 mM Ca²⁺ for 12 h and 24 h; NC12 and NC24, unsupplemented control cultures at the corresponding time points.

Fig. 7. Validation of RNA-sequencing (RNA-seq) data by quantitative real-time PCR (qRT-PCR) analysis of eight selected genes in L. curvatus WiKim0094.

The recA, gyrB, and rpoB genes were used as internal reference genes. Error bars represent standard deviations from three biological replicates.