Metabolomic Differences between Viable but Nonculturable and Recovered Lacticaseibacillus paracasei Zhang

Abstract

The fermentation process can be affected when the starter culture enters the viable but nonculturable (VBNC) state. Therefore, it is of interest to investigate how VBNC cells change physiologically. Lacticaseibacillus (L.) paracasei Zhang is both a probiotic and a starter strain. This study aimed to investigate the metabolomic differences between VBNC and recovered L. paracasei Zhang cells. First, L. paracasei Zhang was induced to enter the VBNC state by keeping the cells in a liquid de Man-Rogosa-Sharpe (MRS) medium at 4 °C for 220 days. Flow cytometry was used to sort the induced VBNC cells, and three different types of culture media (MRS medium, skim milk with 1% yeast extract, and skim milk) were used for cell resuscitation. Cell growth responses in the three types of recovery media suggested that the liquid MRS medium was the most effective in reversing the VBNC state in L. paracasei Zhang. Metabolomics analysis revealed 25 differential metabolites from five main metabolite classes (amino acid, carbohydrate, lipid, vitamin, and purine and pyrimidine). The levels of L-cysteine, L-alanine, L-lysine, and L-arginine notably increased in the revived cells, while cellulose, alginose, and guanine significantly decreased. This study confirmed that VBNC cells had an altered physiology.

Keywords: cell recovery; cell sorting; lactobacilli; probiotic; viable but nonculturable state (VBNC).

Figure 1

Flow cytometry combined with fluorescence labeling to differentiate live, dead, and viable but nonculturable (VBNC) Lacticaseibacillus paracasei Zhang. Dot plots of cells of (A) side scatter (SSC) against forward scatter (FSC); (B) propidium iodide (PI) staining; (C) SYTO 9 staining; (D) SYTO 9/PI double staining. R1 in (A), R2 in (B), R3 in (C), and R4 in (D) indicate bacteria at all physiological states, dead, live, and VBNC cells, respectively.

Figure 2

The growth curves of normal and recovered viable but nonculturable Lacticaseibacillus paracasei Zhang. Error bars represent SD.

Figure 3

Fluorescent micrographs of cell activity and viability staining by the LIVE/DEAD BacLight^TM assay kit. Lacticaseibacillus paracasei Zhang in (A) normal state, recovered in (B) liquid de Man–Rogosa–Sharpe medium, (C) skim milk with 1% yeast extract, and (D) skim milk; magnification ×1000.

Figure 4

Orthogonal projections to latent structures discriminant analysis (OPLS-DA) of metabolomics data of viable but nonculturable (VBNC) and recovered Lacticaseibacillus paracasei Zhang (FS). OPLS-DA plots and results of permutation tests (200 random permutations) of metabolomics data generated in the (A,C) positive and (B,D) negative ion modes, respectively. In (A,B), the subfix letter of the sample code represents the specific replicate sample. The two indicators, R²Y and Q², represent the model interpretation rate and predictive ability, respectively. In (C,D), R² and Q² from 200 permutation tests in the OPLS-DA model are plotted. The y-axis shows R² and Q², whereas the x-axis shows the correlation coefficient of permuted and observed data. The two points on the right represent the observed R² and Q². The cluster of points on the left represents 200 permuted R² and Q². Dashed lines denote corresponding fitted regression lines for observed and permutated R² and Q².

Figure 5

Metabolic pathway enrichment analysis based on the differential metabolites identified between viable but nonculturable (VBNC) and recovered Lacticaseibacillus paracasei Zhang cells. The size and color of the circles represent the number of differential metabolites in the specifically enriched pathway and its extent of statistical significance, respectively. A darker circle represents a greater magnitude of significant difference.