Effect of Pre-Stressing on the Acid-Stress Response in Bifidobacterium Revealed Using Proteomic and Physiological Approaches.

Weak acid resistance limits the application of Bifidobacteria as a probiotic in food. The acid tolerance response (ATR), caused by pre-stressing cells at a sublethal pH, could improve the acid resistance of Bifidobacteria to subsequent acid stress. In this study, we used Bifidobacterium longum sub. longum BBMN68 to investigate the effect of the ATR on the acid stress response (ASR), and compared the difference between the ATR and the ASR by analyzing the two-dimensional-PAGE protein profiles and performing physiological tests. The results revealed that a greater abundance of proteins involved in carbohydrate metabolism and protein protection was present after the ASR than after the ATR in Bifidobacterium. Pre-stressing cells increased the abundance of proteins involved in energy production, amino acid metabolism, and peptidoglycan synthesis during the ASR of Bifidobacterium. Moreover, after the ASR, the content of ATP, NH3, thiols, and peptidoglycan, the activity of H+-ATPase, and the maintenance of the intracellular pH in the pre-stressed Bifidobacterium cells was significantly higher than in the uninduced cells. These results provide the first explanation as to why the resistance of Bifidobacterium to acid stress improved after pre-stressing.

Integrated transcriptomic and proteomic analysis of the bile stress response in a centenarian-originated probiotic Bifidobacterium longum BBMN68.

Bifidobacteria are natural inhabitants of the human gastrointestinal tract and well known for their health-promoting effects. Tolerance to bile stress is crucial for bifidobacteria to survive in the colon and to exert their beneficial actions. In this work, RNA-Seq transcriptomic analysis complemented with proteomic analysis was used to investigate the cellular response to bile in Bifidobacterium longum BBMN68. The transcript levels of 236 genes were significantly changed (≥ threefold, p < 0.001) and 44 proteins were differentially abundant (≥1.6-fold, p < 0.01) in B. longum BBMN68 when exposed to 0.75 g l(-1) ox-bile. The hemolysin-like protein and bile efflux systems were significantly over produced, which might prevent bile adsorption and exclude bile, respectively. The cell membrane composition was modified probably by an increase of cyclopropane fatty acid and a decrease of transmembrane proteins, resulting in a cell membrane more impermeable to bile salts. Our hypothesis was later confirmed by surface hydrophobicity assay. The transcription of genes related to xylose utilization and bifid shunt were up-regulated, which increased the production of ATP and reducing equivalents to cope with bile-induced damages in a xylan-rich colon environment. Bile salts signal the B. longum BBMN68 to gut entrance and enhance the expression of esterase and sortase associated with adhesion and colonization in intestinal tract, which was supported by a fivefold increased adhesion ability to HT-29 cells by BBMN68 upon bile exposure. Notably, bacterial one-hybrid and EMSA assay revealed that the two-component system senX3-regX3 controlled the expression of pstS in bifidobacteria and the role of this target gene in bile resistance was further verified by heterologous expression in Lactococcus lactis. Taken altogether, this study established a model for global response mechanisms in B. longum to bile.

Mechanism analysis of acid tolerance response of bifidobacterium longum subsp. longum BBMN 68 by gene expression profile using RNA-sequencing.

To analyze the mechanism of the acid tolerance response (ATR) in Bifidobacterium longum subsp. longum BBMN68, we optimized the acid-adaptation condition to stimulate ATR effectively and analyzed the change of gene expression profile after acid-adaptation using high-throughput RNA-Seq. After acid-adaptation at pH 4.5 for 2 hours, the survival rate of BBMN68 at lethal pH 3.5 for 120 min was increased by 70 fold and the expression of 293 genes were upregulated by more than 2 fold, and 245 genes were downregulated by more than 2 fold. Gene expression profiling of ATR in BBMN68 suggested that, when the bacteria faced acid stress, the cells strengthened the integrity of cell wall and changed the permeability of membrane to keep the H(+) from entering. Once the H(+) entered the cytoplasm, the cells showed four main responses: First, the F(0)F(1)-ATPase system was initiated to discharge H(+). Second, the ability to produce NH(3) by cysteine-cystathionine-cycle was strengthened to neutralize excess H(+). Third, the cells started NER-UVR and NER-VSR systems to minimize the damage to DNA and upregulated HtpX, IbpA, and γ-glutamylcysteine production to protect proteins against damage. Fourth, the cells initiated global response signals ((p)ppGpp, polyP, and Sec-SRP) to bring the whole cell into a state of response to the stress. The cells also secreted the quorum sensing signal (AI-2) to communicate between intraspecies cells by the cellular signal system, such as two-component systems, to improve the overall survival rate. Besides, the cells varied the pathways of producing energy by shifting to BCAA metabolism and enhanced the ability to utilize sugar to supply sufficient energy for the operation of the mechanism mentioned above. Based on these reults, it was inferred that, during industrial applications, the acid resistance of bifidobacteria could be improved by adding BCAA, γ-glutamylcysteine, cysteine, and cystathionine into the acid-stress environment.

Changes in biogenic amines of silver carp (Hypophthalmichthys molitrix) fillets stored at different temperatures and their relation to total volatile base nitrogen, microbiological and sensory score.

BACKGROUND: Biogenic amines have received considerable attention owing to their undesirable effects in humans. There are few studies of changes in biogenic amine contents related to freshwater fish. Silver carp is an important freshwater fish species in China. This study aimed to investigate the changes in biogenic amines and their relation to total volatile base nitrogen (TVB-N), microbiological and sensory score of silver carp fillets stored at 0, 3 and 15 °C. RESULTS: The total biogenic amine contents of all silver carp fillets (regardless of storage time and temperature) ranged from 13.05 to 318.10 mg kg(-1). Putrescine and histamine were the main biogenic amines in silver carp fillets during storage. Cadaverine was only detected after 12 days at 3 °C and after 2 days at 15 °C. Spermidine and spermine contents increased during the early storage period and then slightly decreased. CONCLUSION: Low temperature could control the quality of silver carp fillets by inhibiting the contents of biogenic amines. Putrescine showed significant correlation with TVB-N, total aerobic content, sensory score, tryptamine and phenylethylamine. Putrescine was a good quality marker of silver carp fillets in the cold chain.