Mechanism analysis of methionine inhibiting growth of Bifidobacterium bifidum

Bifidobacterium bifidum rarely utilises yeast extract, which causes significant challenges in its industrial production. This study characterised the utilisation of amino acids by B. bifidum to improve the viability of the strain. In this study, the proliferation effect of B. bifidum in different nitrogen sources was determined, and the amino acid contents of different nitrogen sources were analysed, which indicated that hydrophobic amino acids may inhibit B. bifidum growth. Among different hydrophobic amino acids, methionine was identified as the strongest growth inhibitor. Furthermore, the intracellular pH and NH4+ contents of B. bifidum CCFM16 were higher in methionine-free medium. Among the NH3-producing enzymes related to amino acid metabolism, the metC gene was significantly transcribed in methionine-free medium. In addition, tcyP, metB, metC and metE genes encoding cysteine transporter proteins and the conversion pathway of cysteine to methionine were transcribed 2.03 -fold, 7.03 -fold, 3.05 -fold and 2.92 -fold, respectively, in methionine-free medium compared to methioninecontaining medium. Compared with methionine-containing medium, the activity of tricarboxylic acid cycle ratelimited enzyme isocitrate dehydrogenase in methionine-free medium also increased from 198.89 to 410.56 nmol/min/mg protein, which probably enhanced the carbon flux and promoted growth. Meanwhile, the activity of pyrroline-5-carboxylate reductase, an enzyme that synthesises proline, increased from 14.76 to 35.28 nmol/ min/mg protein, which promoted proline synthesis in response to osmotic stress. Cyclopropane fatty acids were also significantly higher in methionine-free medium, which may promote growth by decreasing cell membrane permeability to protons and enhancing cell membrane fluidity. This study provides a theoretical basis for highdensity culture of B. bifidum.