| Beer is one of the oldest and most consumed alcoholic drink in the world.Due to the long process and complex equipment,some bacteria species were able to survive or grow in beer,thus posing threats on beer production and quality.Therefore,efficient monitoring the spoilage microbes were one of the most important strategy to guarantee the production safety and product quality,which attracted extensive attention.Iso-?-acid is an important anti-bacterial substance which could inhibit the growth of vast majority of bacteria in beer.However,several bacteria strains can survive in the harsh environment.The spoilage bacteria obtained several traits to resist unfavorable environment through adaptive evolution during long-time exposure in beer environment.These reflect similar psychological features and belong to a sub-branch of the microbes evolved in microenvironment.Revealing the psychological and genetic traits of these spoilage bacteria could uncover the underlying mechanism these bacteria survived in beer,providing guidance for the monitor of beer spoilage bacteria in beer.The main conclusions were as follows:205 spoilage bacteria strains were isolated from samples collected from the equipment,environment,and brewing processes from 7 breweries in the Yangze Delta region and identified through M13-PCR fingerprint and 16S rDNA sequencing,thus constructing the beer spoilage bacteria library.Most isolated bacteria strains belong to Lactic acid bacteria,including Lactobacillus,Lactococcus and Leuconostoc species.Among them,the most bacteria strains were L.plantarum?39 strains?,L.lactis?34 strains?,L.brevis?33 strains?and L.casei?30strains?.The main spoilage bacteria were detected in environment and brewing processes which were closely related to beer.A new culture-dependent rapid test method based on iso-?-acid-gradient medium plate constructed and the minimum inhibitory concentrations of beer spoilage bacteria strains were tested using this method.Besides,through phytobacteria experiment and PCR analysis,the beer spoilage ability and iso-?-acid related genes were analyzed and correlated.The results showed that the ability to resist iso-?-acid is the prerequisite for the beer spoilage of Lactobacillus species bacteria.The adoption of horA and horC genes as markers to predict the iso-?-acid resistance ability and beer spoilage ability of Lactobacillus species bacteria has a high false positive rate and false negative rate.L.casei W2-9-5 with relatively lower iso-?-acid resistance ability was obtained through successive cultivation of L.casei 2-9-5 in MRS media without iso-?-acid and comparative transcriptomics analysis of L.casei 2-9-5 and W2-9-5 was conducted.The results showed that1613 genes were significant changed after adding hop bitter compounds,which were related to transmembrane transport,membrane composition,organic acids metabolism,amino acid metabolism and oxidative phosphorylation.Among all genes,mntA,mnt B and mntC genes belonging to ABC transporter family and ATPa,ATPb,ATPc and ATP?genes encoding F0/F1-ATP synthase subunit were significantly up-regulated and confirmed by RT-qPCR.The expression of mntA,mnt B and mntC genes in model bacteria L.lactis NZ3900 and the addition of exogenous Mn2+indicated that maintaining the intracellular Mn2+concentration is curcial for the resistance of L.casei strains to resist iso-?-acid while the proteins encoded by mntA,mntB and mntC genes were able to relieve the effect of H+/Mn2+exchange on L.casei strains.The ATP yielding ability of recombinant L.lactis NZ3900 was significantly higher than that of wild-type,indicating that L.casei strains with better iso-?-acid resistance ability had relatively stronger ATP yielding ability,indicating the important roles of ATP in the iso-?-acid resistance ability of L.casei strains.Proteomics analysis was conducted to further reveal the response of L.casei 2-9-5 strain towards the iso-?-acid pressure.After the optimization of 2D electrophoresis condition,68 up-regulated protein spots were identified and the significantly up-regulated proteins in L.casei 2-9-5 under 0.04%iso-?-acid mainly involved in carbohydrate metabolism,energy generation,organic acids metabolism and cell wall composition.Based on the proteomic analysis results,organic acids were found to be important for L.casei 2-9-5 to resist iso-?-acid.The extracellular organic acids concentrations in L.casei 2-9-5 under iso-?-acid pressure was significantly higher than control.Furthermore,the addition of oxalic acid,tartaric acid,pyruvate and ketoglutaric acid in media were able to enhance the growth of L.casei 2-9-5 under iso-?-acid pressure.These phenomena were also observed in different genus of Lactobacillus species bacteria with different iso-?-acid resistance ability since the addition of organic acids also promoted the growth of these bacteria strains.Together,our data supported that organic acids were closely related to the resistance ability of Lactobacillus species bacteria towards iso-?-acid. |
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