Dairy Streptococcus thermophilus improves cell viability of Lactobacillus brevis NPS-QW-145 and its γ-aminobutyric acid biosynthesis ability in milk

Abstract

Most high γ-aminobutyric acid (GABA) producers are Lactobacillus brevis of plant origin, which may be not able to ferment milk well due to its poor proteolytic nature as evidenced by the absence of genes encoding extracellular proteinases in its genome. In the present study, two glutamic acid decarboxylase (GAD) genes, gadA and gadB, were found in high GABA-producing L. brevis NPS-QW-145. Co-culturing of this organism with conventional dairy starters was carried out to manufacture GABA-rich fermented milk. It was observed that all the selected strains of Streptococcus thermophilus, but not Lactobacillus delbrueckii subsp. bulgaricus, improved the viability of L. brevis NPS-QW-145 in milk. Only certain strains of S. thermophilus improved the gadA mRNA level in L. brevis NPS-QW-145, thus enhanced GABA biosynthesis by the latter. These results suggest that certain S. thermophilus strains are highly recommended to co-culture with high GABA producer for manufacturing GABA-rich fermented milk.

Figure 1. Amplification of GAD gene(s) and 16S rRNA gene from L. brevis 145 and eight dairy starters.

(a) detection of GAD gene using degenerate primers DP1 and DP2; (b) amplification of GAD gene(s) in L. brevis 145 using degenerate primers PGDG-2F and PGDG-4R; (c) specificity of primers s-Lbre-F and s-Lbre-R for amplifying 16S rRNA gene from L. brevis. Denotation: M, DNA ladders (Promega 1 kb DNA ladder in Fig. 1a; Invitrogen 1 Kb Plus DNA Ladder in Fig. 1b and Fig. 1c); B, amplification without DNA; Lane 1, L. brevis 145; Lane 2, S. thermophilus ASCC 1275; Lane 3, S. thermophilus ASCC 1303; Lane 4, S. thermophilus YI-B1; Lane 5, S. thermophilus YI-N1; Lane 6, S. thermophilus YI-M1; Lane 7, L. bulgaricus ASCC 756; Lane 8, L. bulgaricus ASCC 859; Lane 9, L. bulgaricus YI-B2.

Figure 2. Alignment of the amino acids of full-length glutamate decarboxylases from nine Lactobacillus brevis strains.

The conserved regions [NAIDKSEYPR(K)TA] and [GWQVPA(T)YPLPKN] were used to design degenerate primers. This figure was generated from BioEdit (version 7.2.5) after ClustalW multiple alignment.

Figure 3. The pH of fermented milks after co-culturing of L. brevis 145 with S. thermophilus or/and L. bulgaricus.

ST, S. thermophilus; Lbu, L. bulgaricus; Lbre 145, L. brevis 145; Blank milk, 10% (w/v) skimmed milk.

Figure 4. Cell viabilities of S. thermophilus and L. bulgaricus in fermented milks.

ST, S. thermophilus; Lbu, L. bulgaricus; Lbre 145, L. brevis 145.

Figure 5. Cell counts of L. brevis 145 in fermented milks.

ST, S. thermophilus; Lbu, L. bulgaricus; Lbre 145, L. brevis 145. Star (*P < 0.05) is for the comparison of data between fermentation with and without the supplementation of MSG; Capital letters (A, B, C and D) are designated to indicate the significance of the group data of L. brevis counts, the same letter among each group indicates no significance (P ≥ 0.05). The initial cell counts of L. brevis 145 after inoculation in milk was ~3 × 10⁷ CFU/mL (7.48 Log₁₀ CFU/mL).

Figure 6. Relative gene expression of gadA and gadB in L. brevis 145 after co-cultured with S. thermophilus in milk supplemented with 2 g/L of MSG.

The levels of gadA and gadB mRNA from L. brevis 145 after co-cultured with S. thermophilus YI-M1 was used as a reference for comparision. Comparative critical threshold method (R = 2^–ΔΔCт) was carried out for data analysis of three indendent exeriments, a positive value indicates up-regulation while a negative value indicates down-regulation. Lowercase letters (a & b) are designated to indicate the significance of gadB and gadA mRNA levels, the same letter above/below each bar indicates no significance (P ≥ 0.01). ST, S. thermophilus; Lbre 145, L. brevis 145.