Metabolic Pathway Profiling in Intracellular and Extracellular Environments of Streptococcus thermophilus During pH-Controlled Batch Fermentations

Abstract

Elucidating the metabolite profiles during the growth of Streptococcus thermophilus is beneficial for understanding its growth characteristics. The changes in the intracellular and extracellular concentrations of carbohydrates, nucleotides, amino sugars, nucleoside sugars, fatty acids, and amino acids, as well as their metabolites over time, were investigated by metabolomics technology. Most metabolites of nucleotides were highly accumulated in the intracellular environment after the mid-exponential phase. Increases in the intracellular unsaturated fatty acids and N-acetyl-glucosamine and N-acetyl-muramoate recycling provided potential evidence that cell envelope remodeling occurred after the mid-exponential phase. At the later fermentation stages, potentially functional metabolite produced by glycine was highly accumulated in the intracellular environment. Additionally, potential toxic metabolites produced by phenylalanine and tyrosine could not be excreted into the extracellular environment in a timely basis. The accumulation of large amounts of these metabolites might be the primary cause of the overconsumption of amino acids and influence the growth of S. thermophilus.

Keywords: Streptococcus thermophilus; extracellular metabolite; intracellular metabolite; metabolic pathway; pH-controlled batch fermentations.

FIGURE 1

Growth curve of ST-MZ-2 during culture (A). Changes in the concentrations of metabolites associated with the carbohydrate (B) and nucleotide (C) metabolic pathways in ST-MZ-2 during culture. The x-axis displays the sampling time, and the y-axis displays the log10 peak area for metabolites. Values are the mean ± SEM, n = 3. The bars with different superscripts are significantly different (p < 0.05).

FIGURE 2

Principal component analysis (PCA) score plot based on LC-MS analysis in (A) positive ion mode and (B) negative ion mode for ST-MZ-2 intracellular samples (ST); (C) positive ion mode and (D) negative ion mode for ST-MZ-2 extracellular samples (FL).

FIGURE 3

Changes in the concentrations of metabolites associated with the amino sugar and nucleotide sugar (A) and fatty acid (B) metabolic pathways in ST-MZ-2 during culture. The x-axis displays the sampling time, and the y-axis displays the log10 peak area for metabolites. Values are the mean ± SEM, n = 3. The bars with different superscripts are significantly different (p < 0.05).

FIGURE 4

Transmission electronic microscopy (TEM) photograph of ST-MZ-2 at the (A) late-lag phase (×5000). (B) mid-exponential phase (×5000). 1, the invagination cells. 2, the cell division (C) late-exponential phase (×5000). 1, lysis in cell wall and cell membrane. (D) stationary phase (×5000). 1, the cytoplasmic membrane was separated from the cell wall. 2, complete lysis of the cell, leakage of cytoplasmic content.

FIGURE 5

Changes in the concentrations of metabolites associated with the proline, glycine, leucine, valine, histidine, phenylalanine, tyrosine, and tryptophan metabolic pathways in ST-MZ-2 during culture. The x-axis displays the sampling time, and the y-axis displays the log10 peak area for metabolites. Values are the mean ± SEM, n = 3. The bars with different superscripts are significantly different (p < 0.05).

FIGURE 6

The proposed metabolic profile model of ST-MZ-2 after the mid-exponential phase. Increased and decreased levels are indicated by plus symbol “+” and minus symbol “−,” respectively.