乳酸菌在自然发酵过程中受到多种环境胁迫的影响。群体感应系统能够介导细菌生物膜的形成从而提高其环境耐受性,这可能是提高乳酸菌发酵性能的潜在调控靶点。该研究比较了1株植物乳杆菌逆境驯化前后的AI-2/LuxS系统差异,进而解析外源群体感应干扰物质对菌株生理代谢的影响。结果显示,柠檬酸驯化后的植物乳杆菌D-840生物膜形成能力提高了485%(P<0.05)。2株菌均能产生群体感应信号分子AI-2,驯化后植物乳杆菌D-840在柠檬酸胁迫下单位菌体所产AI-2显著低于出发菌株(P<0.05)。在15 g/L柠檬酸质量浓度下,植物乳杆菌D和植物乳杆菌D-840中的luxS基因表达量均下调,pfs基因表达量则分别提高了71.4%和49.3%(P<0.05)。AI-2前体(S)-4,5-二羟基-2,3-戊二酮的添加对菌株生理代谢的影响并未呈现浓度依赖性。S-腺苷甲硫氨酸在添加量为1 g/L时植物乳杆菌D和植物乳杆菌D-840苯乳酸产量分别提高了12.2%和25.5%(P<0.05)。研究表明,AI-2/LuxS系统与植物乳杆菌抗逆性及代谢性能具有显著的相关性,并且通过添加AI-2前体上调AI-2/LuxS系统,可以实现其相关的代谢产物积累量的提高,研究结果为提高乳酸菌发酵性能提供了新思路。
As a kind of probiotics widely used in the food industry, lactic acid bacteria are affected by various environmental stresses in the fermentation process and digestive tract. The production of yogurt, kimchi, high-acid fruit and vegetable juices and other foods all put forward requirements for the stress resistance of the bacteria. Some microorganisms can form biofilms to improve their environmental tolerance ability. According to the previous reports, the quorum sensing systems is associated with the formation of bacterial biofilms, and the regulation of quorum sensing systems may be potential regulatory targets for improving the fermentation performance of lactic acid bacteria. Previously, we have obtained a citric acid tolerant strain - Lactobacillus plantarum LP-D840, which was acclimated from its parent strain- L. plantarum D by gradually increasing citric acid concentration in the medium. The original strain and the acclimated strain are suitable samples for understanding the correlation between quorum sensing and strain tolerance, due to their highly similar gene background. In this study, the difference regarding the physiological and metabolic properties and the AI-2/LuxS system between the parent and acclimated strains were compared. The effects of exogenous quorum sensing interferents, including (S)-4,5-dihydroxy-2,3-pentanedione (DPD) and S-adenosylmethionine(SAM), on the growth and metabolism of the strain were analyzed. The production of signal molecule AI-2 was detected by the bioluminescence method, the changes of gene transcription level were determined by reverse transcription real-time quantitative PCR (RT-qPCR), and metabolites were detected by HPLC. The results showed that the biofilm formation ability of acclimated strain - L. plantarum D-840 was 485% higher than that of parent strain - L. plantarum D, and the cell density of L. plantarum D-840 was 5.4 times higher than that of L. plantarum D under the stress of citric acid. The acclimated strain L. plantarum D-840 showed distinct capability of biofilm formation, whereas the original L. plantarum D did not show biofilm synthesis. By controlling the agitating condition, the planktonic and biofilm cells of L. plantarum D-840 were obtained separately. Compared with planktonic cells, the biofilm cells of L. plantarum D-840 had higher tolerance to citric acid, malic acid, and bile salts. The result showed that the higher stress tolerance to citric acid of L. plantarum D-840 was closely associated with its biofilm formation. The two strains entered the logarithmic growth phase at about 4 h, and the signal molecule AI-2 accumulated rapidly during 4-8 h. The AI-2 production of L. plantarum D-840 and L. plantarum D decreased slightly after reaching the highest value at 8 h and 16 h, respectively. During the fermentation process, the AI-2 synthesis ability of the two strains (AI-2/OD) showed a decreasing trend initially and then stabilized. Under citric acid stress, the AI-2 produced by L. plantarum D-840 and L. plantarum D showed a positive correlation with the concentration of citric acid, while the AI-2 activity of per unit absorbance produced by L. plantarum D-840 was significantly lower than L. plantarum D. RT-qPCR results showed that the expression of luxS gene was down-regulated in two strains under citric acid stress, while the expression of pfs gene was up-regulated. At citric acid, the expression of luxS gene in L. plantarum D and L. plantarum D-840 decreased by 34.5% and 28.8% (P<0.05), respectively, while the pfs gene increased by 71.4% and 49.3% (P<0.05), respectively. Therefore, compared with luxS gene, pfs gene has a more significant regulatory effect on the synthesis of AI-2, and has a closer correlation with the citric acid tolerance of the strains in this study. The addition of AI-2 precursor DPD had no significant effect on the growth curves of the two strains, while it could affect the production of lactate and phenyllactate of L. plantarum D-840. When the concentration of DPD was greater than 0.5 μmol/L, the synthesis of lactic acid of L. plantarum D-840 was significantly inhibited. While the production of phenyllactic acid in L. plantarum D-840 was significantly increased by 27.1% at the addition of 0.1 μmol/L DPD. SAM inhibited the growth of the two strains, but promoted the synthesis of phenyllactic acid. When the concentration of SAM was 1 g/L, the production of phenyllactic acid in L. plantarum D and L. plantarum D-840 increased by 12.2% and 25.5%, respectively. The result indicated that the regulation effect of SAM on the physiological metabolism of the strain was better than that of DPD. This study indicated that the AI-2/LuxS system has a significant correlation with the stress resistance and metabolic properties of Lactobacillus plantarum. Via the addition of AI-2 precursors, the AI-2/LuxS system can be up-regulated, and the accumulation of relevant metabolites can be enhanced. This information provided a novel approach for improving the fermentation performance of Lactobacillus plantarum, and the results might be expanded to other lactic acid bacteria.
[1] MARTEAU P, LE NEVÉ B, QUINQUIS L, et al.Consumption of a fermented milk product containing Bifidobacterium lactis CNCM I-2494 in women complaining of minor digestive symptoms:Rapid response which is independent of dietary fibre intake or physical activity[J].Nutrients, 2019, 11(1):92.
[2] WANG J C, BAI X Y, PENG C T, et al.Fermented milk containing Lactobacillus casei Zhang and Bifidobacterium animalis ssp. lactis V9 alleviated constipation symptoms through regulation of intestinal microbiota, inflammation, and metabolic pathways[J].Journal of Dairy Science, 2020, 103(12):11 025-11 038.
[3] 陆文伟, 林炳谕, 尹一婷, 等.双歧杆菌生物膜成膜规律及特性研究[J].中国食品学报, 2017, 17(12):42-49.
LU W W, LIN B Y, YIN Y T, et al.The biofilm formation of Bifidobacterium and its characteristics[J].Journal of Chinese Institute of Food Science and Technology, 2017, 17(12):42-49.
[4] 吴旭光, 王红, 徐一凡, 等.猪源肠外致病性大肠埃希氏菌的生物膜形成能力及耐药性[J].微生物学通报, 2020, 47(1):162-171.
WU X G, WANG H, XU Y F, et al.Biofilm-forming capability and drug resistance of extraintestinal pathogenic Escherichia coli isolated from diseased swine[J].Microbiology China, 2020, 47(1):162-171.
[5] KUBOTA H, SENDA S, NOMURA N, et al.Biofilm formation by lactic acid bacteria and resistance to environmental stress[J].Journal of Bioscience and Bioengineering, 2008, 106(4):381-386.
[6] LIU L, WU R Y, ZHANG J L, et al.Overexpression of luxS promotes stress resistance and biofilm formation of Lactobacillus paraplantarum L-ZS9 by regulating the expression of multiple genes[J].Frontiers in Microbiology, 2018, 9:2628.
[7] LEBEER S, CLAES I J J, VERHOEVEN T L A, et al.Impact of luxS and suppressor mutations on the gastrointestinal transit of Lactobacillus rhamnosus GG[J].Applied and Environmental Microbiology, 2008, 74(15):4 711-4 718.
[8] CHEN X, SCHAUDER S, POTIER N, et al.Structural identification of a bacterial quorum-sensing signal containing boron[J].Nature, 2002, 415(6871):545-549.
[9] DE MAN J C, ROGOSA M, SHARPE M E.A medium for the cultivation of lactobacilli[J].Journal of Applied Bacteriology, 1960, 23(1):130-135.
[10] DEKEERSMAECKER S C J, VANDERLEYDEN J.Constraints on detection of autoinducer-2 (AI-2) signalling molecules using Vibrio harveyi as a reporter[J].Microbiology (Reading, England), 2003, 149(Pt 8):1 953-1 956.
[11] MOSLEHI-JENABIAN S, GORI K, JESPERSEN L.AI-2 signalling is induced by acidic shock in probiotic strains of Lactobacillus spp.[J].International Journal of Food Microbiology, 2009, 135(3):295-302.
[12] SUN L L, ZHANG Y, GUO X J, et al.Characterization and transcriptomic basis of biofilm formation by Lactobacillus plantarum J26 isolated from traditional fermented dairy products[J].LWT, 2020, 125:109333.
[13] WU L H, LU Z M, ZHANG X J, et al.Metagenomics reveals flavour metabolic network of cereal vinegar microbiota[J].Food Microbiology, 2017, 62:23-31.
[14] LI X F, JIANG B, PAN B L, et al.Purification and partial characterization of Lactobacillus species SK007 lactate dehydrogenase (LDH) catalyzing phenylpyruvic acid (PPA) conversion into phenyllactic acid (PLA)[J].Journal of Agricultural and Food Chemistry, 2008, 56(7):2 392-2 399.
[15] LIU L, GUO S Y, CHEN X, et al.Metabolic profiles of Lactobacillus paraplantarum in biofilm and planktonic states and investigation of its intestinal modulation and immunoregulation in dogs[J].Food & Function, 2021, 12(12):5 317-5 332.
[16] 刘蕾, 武瑞赟, 李军, 等.基于AI-2介导的类植物乳杆菌生物被膜态与浮游态抗胁迫能力比较与分析[J].食品科学, 2017, 38(22):41-47.
LIU L, WU R Y, LI J, et al.Stress resistance of biofilm and planktonic cells of Lactobacillus paraplantarum L-ZS9 regulated by autoinducer 2[J].Food Science, 2017, 38(22):41-47.
[17] LIVAK K J, SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods, 2001, 25(4):402-408.
[18] XIONG Q, LIU D, ZHANG H H, et al.Quorum sensing signal autoinducer-2 promotes root colonization of Bacillus velezensis SQR9 by affecting biofilm formation and motility[J].Applied Microbiology and Biotechnology, 2020, 104(16):7 177-7 185.
[19] MAN L L, MENG X C, ZHAO R H, et al.The role of plNC8HK-plnD genes in bacteriocin production in Lactobacillus plantarum KLDS1.0391[J].International Dairy Journal, 2014, 34(2):267-274.
[20] WINZER K, HARDIE K R, BURGESS N, et al.LuxS:its role in central metabolism and the in vitro synthesis of 4-hydroxy-5-methyl-3(2H)-furanone[J].Microbiology, 2002, 148(4):909-922.
[21] WANG Y, YI L, ZHANG Z C, et al.Overexpression of luxS cannot increase autoinducer-2 production, only affect the growth and biofilm formation in Streptococcus suis[J].The Scientific World Journal, 2013, 2013:924276.
[22] YANG X Y, LI J P, SHI G C, et al.Improving 3-phenyllactic acid production of Lactobacillus plantarum AB-1 by enhancing its quorum-sensing capacity[J].Journal of Food Science and Technology, 2019, 56(5):2 605-2 610.
[23] JIA F F, ZHENG H Q, SUN S R, et al.Role of luxS in stress tolerance and adhesion ability in Lactobacillus plantarum KLDS1.0391[J].BioMed Research International, 2018, 2018:4506829.
