Co-culture with Tetragenococcus halophilus improved the ethanol tolerance of Zygosaccharomyces rouxii by maintaining cell surface properties.

The accumulation of ethanol has a negative effect on the viability and fermentation performance of microorganisms during the production of fermented foods because of its toxicity. In this study, we investigated the effect of co-culture with Tetragenococcus halophilus on ethanol stress resistance of Zygosaccharomyces rouxii. The result showed that co-culture with T. halophilus promoted cell survival of Z. rouxii under ethanol stress, and the tolerance improved with increasing co-culture time when ethanol content was 8%. Physiological analysis showed that the co-cultured Z. rouxii cells maintained higher intracellular content of trehalose and amino acids including tyrosine, tryptophan, arginine and proline after 8% ethanol stress for 90 min. The membrane integrity analysis and biophysical analysis of the cell surface indicated that the presence of ethanol resulted in cell membrane damage and changes of Young's modulus value and roughness of cell surface. While the co-cultured Z. rouxii cells exhibited better membrane integrity, stiffer and smoother cell surface than single-cultured cells under ethanol stress. As for transcriptomic analyses, the genes involved in unsaturated fatty acid biosynthesis, trehalose biosynthesis, various types of N-glycan biosynthesis, inositol phosphate metabolism, MAPK signaling pathway and tight junction had higher expression in co-cultured Z. rouxii cells with down-regulation of majority of gene expression after stress. And these genes may function in the improvement of ethanol tolerance of Z. rouxii in co-culture.

The in vitro immunomodulatory effect of extracellular products (ECPs) of Vagococcus fluvialis L21 on European sea bass (Dicentrarchus labrax) leucocytes.

The immune associated genes, interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor-α (TNF-α), ciclo-oxigenase-2 (COX-2), and Mx gene were studied by real-time PCR in head-kidney leucocytes of sea bass after incubation with the extracellular products (ECPs) of the probiotic strain Vagococcus fluvialis L21 and polyinosinic:polycytidylic acid (POLY I:C), at different times (T1.5, T6, T12, T24, T48 and T72). In general, we can observe how pro-inflammatory cytokines IL-1ß, TNF-α, IL-6 and COX-2 studied displayed a strong peak after stimulation with 1.5 h of ECPs of V. fluvialis L21, significant differences (P < 0.05) exist with other periods and with the POLY I: C at the same time. Similarly to the case of IL-10 also produced a statistically significant (P < 0.05) peak of expression on leukocytes that were stimulated with the ECPs of V. fluvialis L21. In the case of Mx gene expression, we note that in almost all sampling times there is an up-regulation of the Mx gene in leucocytes incubated with ECPs and POLY I:C compared to the control and Mx expression was higher in leucocytes that were stimulated with the ECPs of V. fluvialis for all times, except in T24. With these results we can consider that the ECPs of V. fluvialis L21 have a great power of stimulating the in vitro expression of immune-related genes and may even be useful as adjuvants for vaccine in aquaculture.

Cytokine expression in head-kidney leucocytes of European sea bass (Dicentrarchus labrax L.) after incubation with the probiotic Vagococcus fluvialis L-21.

The European sea bass (Dicentrarchus labrax L.) is one of the most extensively farmed marine fish in the Mediterranean sea. Under the high-density condition, common in aquaculture, the infectious diseases can cause significant economic losses. Probiotics are presented as an alternative to antibiotics for the control of aquaculture diseases. This study used real-time PCR to investigate in vitro the dynamic of expression of immune-related genes in sea bass after incubation with live and inactivated (heat and Uv-light) probiotic Vagoccus fluvialis L-21 at different times (T1, T12, T24, T48). The immune associated genes, interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin 10 (IL-10), TumourTumour necrosis factor- (TNF-), ciclo-oxigenase-2 (COX-2), caspase-3 (Casp-3) and Mx were studied in head-kidney (HK) leucocytes of sea bass after incubation with the probiotic strain. Transcript of proinflammatory cytokines (IL-1, TNF-, COX-2) was highly up-regulated after 1 h of incubation with the probiotic strain V. fluvialis L-21. We found statistically significant difference in pick of expression of TNF-, after 1 h of incubation with Uv-light inactivated probiotic strain. The COX-2 expression was highly up-regulated at all times studied, with the exception of 12 and 24 h post incubation for the Uv-light inactivated bacteria. Transcript of IL-10 and Casp-3 showed the higher statistically significant differences of expression after 48 h post incubation with live bacteria. In the contrast, sea bass HK leucocytes expressed Mx at 12 and 48 h without statistically differences among treatments. Our results suggest that V. fluvialis L-21 is able to stimulate in vitro some immune-related genes associated with the early inflammatory response. Future studies in vivo are necessary to clarify this process in sea bass.

Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His(6)-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K(m) = 0.35 ± 0.03 mm for L-aspartate, K(m) = 0.098 ± 0 mm for D-aspartate, K(m) = 26 ± 2 mm for L-alanine, K(m) = 3.3 ± 0.2 mm for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT.