戊糖片球菌表面蛋白性质及抑菌作用研究
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摘要
以戊糖片球菌(Pediococcus pentosaceus) F28-8为研究对象,菌株的生长情况与产酸能力分析表明,F28-8具有良好的酸适应能力,透射电镜(TEM)观察表明其存在蛋白层结构。应用5 mol/L LiCl提取菌株表面蛋白并进行SDS-PAGE和MALDI-TOF-MS分析,发现该菌株表面含有主要组分为分子量83.5 kDa的N-乙酰基胞壁酿-L-丙氨酸酰胺酶和分子量51. 1 kDa的LysM肽聚糖结合域蛋白。进一步的菌株表面性质测试、胶原蛋白黏附实验以及抑菌实验结果显示,表面蛋白的剥离显著降低了菌株F28-8的表面性质(自动聚集能力、表面疏水性和表面电荷),降幅达到19.0%～56. 1%;LiCl提取的F28-8表面蛋白能显著降低沙门氏菌(Salmonella enterica subsp. CICC 21513)和金黄色葡萄球菌(Staphylococcus aureus CICC 21600)对胶原蛋白的黏附力,降幅分别为74. 59%和81. 16%;LiCl提取的F28-8表面蛋白对沙门氏菌和金黄色葡萄球菌的抑菌率分别达到31. 5%和15. 6%。研究结果表明,戊糖片球菌F28-8表面蛋白能改变菌体表面性质,增强其抑制致病菌黏附的能力并表现出一定的抑菌能力,在抗菌剂开发领域具有一定的应用潜力。
The growth condition and analysis of acid production capacity showed that Pediococcus pentosaceus F28-8 had a good acid adaptability, and the surface structure was observed with transmission electron microscopy. The S-layer was extracted with 5 mol/L LiCl and analysed by SDS-PAGE and MALDI-TOFMS. It was found that the S-layer of F28-8 contained a kind of surface protein with molecular weight of83. 5 kDa and 51. 1 kDa. The further surface property test, collagen adhesion test and antibacterial experiment showed that the stripping of surface protein significantly reduced the surface properties( automatic aggregation ability, surface hydrophobicity and surface charge), with a decrease of 19. 0%-56. 1%.The surface protein of F28-8 extracted by LiCl significantly reduced the adhesion of Salmonella and Staphylococcus aureus to collagen, with a decrease of 74. 59% and 81. 16%. The inhibitory rate of surface protein of F28-8 extracted by LiCl against Salmonella and Staphylococcus aureus reached 31. 5% and15. 6%, respectively. The results indicate that surface protein of P. pentosaceus F28-8 can change the surface properties of cells, enhance their ability to inhibit the adhesion of pathogenic bacteria and exhibit certain antibacterial ability, and has certain application potential in the field of antibacterial agents.
The growth condition and analysis of acid production capacity showed that Pediococcus pentosaceus F28-8 had a good acid adaptability, and the surface structure was observed with transmission electron microscopy. The S-layer was extracted with 5 mol/L LiCl and analysed by SDS-PAGE and MALDI-TOFMS. It was found that the S-layer of F28-8 contained a kind of surface protein with molecular weight of83. 5 kDa and 51. 1 kDa. The further surface property test, collagen adhesion test and antibacterial experiment showed that the stripping of surface protein significantly reduced the surface properties( automatic aggregation ability, surface hydrophobicity and surface charge), with a decrease of 19. 0%-56. 1%.The surface protein of F28-8 extracted by LiCl significantly reduced the adhesion of Salmonella and Staphylococcus aureus to collagen, with a decrease of 74. 59% and 81. 16%. The inhibitory rate of surface protein of F28-8 extracted by LiCl against Salmonella and Staphylococcus aureus reached 31. 5% and15. 6%, respectively. The results indicate that surface protein of P. pentosaceus F28-8 can change the surface properties of cells, enhance their ability to inhibit the adhesion of pathogenic bacteria and exhibit certain antibacterial ability, and has certain application potential in the field of antibacterial agents.
引文
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[15] PRADOACOSTA M, RUZAL S M, ALLIEVI M C,et al. Synergistic effects of the Lactobacillus acidophilus surface layer and nisin on bacterial growth[J].Applied&Environmental Microbiology, 2010, 76(3):974-977.
[2]余萍,吴少勤.益生菌及其应用研究进展[C]//全国益生菌与健康及应用技术交流讨论会.沈阳:汉臣氏生物医药开发有限公司,2009:173-176.YU P, WU S Q. Advances in research on probiotics and their applications[C]//National Symposium on Probiotics and Health and Applied Technology Exchange. Shenyang:Hanson Biomedical Development Co Ltd, 2009:173-176.
[3]曹振辉,潘洪彬,张鲜焰,等.戊糖片球菌的益生功能及其在食品科学领域中的应用[J].安徽农业科学,2016(9):106-108.CAO Z H, PAN H B, ZHANG X Y, et al. Application of the beneficial function of Pediococcus pentosaceus in the field of food science[J]. Anhui Agricultural Sciences,2016(9):106-108.
[4]杨振泉,靳彩娟,张咪,等.高黏附性戊糖片球菌的筛选、标记及其表面疏水与自凝聚性特征[J].食品与生物技术学报,2015, 34(9):926-934.YANG Z Q, JIN C J, ZHANG M, et al. Screening and molecular marking of highly adhesive Pediococcus pentosaceus and the characteristics of their surface hydrophobicities and auto aggregation abilities[J]. Journal of Food Science and Biotechnology, 2015, 34(9):926-934.
[5]何名芳,陈卫平.乳酸菌抑制致病菌黏附作用机制研究进展[J].食品工业科技,2013, 34(20):386-389.HE M F, CHEN W P. The development of the Lactobacillus adhesive effects mechanism in inhibiting the pathogen microorganism[J]. Food Industry and Technology,2013, 34(20):386-389.
[6]佘银,罗芳,高婉茹,等.乳酸菌黏附特性的研究新进展[J].食品研究与开发,2018(4):218-224.SHE Y, LUO F, GAO W R, et al. The new advance of research on the adhesion of Lactobacillu.s[J]. Food Research and Development, 2018(4):218-224.
[7] ROOS S, JONSSON H. A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components.[J]. Microbiology, 2002, 148(2):433-442.
[8] SILLANPAA J, MARTINEZ B, ANTIKAINEN J, et al.Characterization of the collagen-binding S-layer protein,cbsA of Lactobacillus crispatus[J]. Journal of Bacteriology, 2000, 182(22):6440-6450.
[9] SUN Z, LI P, LIU F, et al. Synergistic antibacterial mechanism of the Lactobacillus crispatus surface layer protein and nisin on Staphylococcus saprophyticus[J]. Scientific Reports, 2017, 7(1):265.
[10] ZHANG B, ZUO F, YU R, et al. Comparative genomebased identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells[J]. Scientific Reports, 2015, 5(1):14109.
[11] JOHNSON-HENRY K C, HAGEN K E, GORDONPOUR M, et al. Surface-layer protein extracts from Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia coli 0157:H7 adhesion to epithelial cells[J].Cellular Microbiology, 2010, 9(2):356-367.
[12] RAHMAN M M, KIM W S, KUMURA H, et al. Autoaggregation and surface hydrophobicity of bifidobacteria[J]. World Journal of Microbiology&Biotechnology,2008, 24(8):1593-1598.
[13] KOS B, SUSKOVIC J, VUKOVIC S, et al. Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92[J]. Journal of Applied Microbiology,2003, 94(6):981-987.
[14] HORIE M, ISHIYAMA A, FUJIHIRA-UEKI Y, et al.Inhibition of the adherence of Escherichia coli strains to basement membrane by Lactobacillus crispatus expressing an S-layer[J]. Journal of Applied Microbiology, 2002,92(3):396-403
[15] PRADOACOSTA M, RUZAL S M, ALLIEVI M C,et al. Synergistic effects of the Lactobacillus acidophilus surface layer and nisin on bacterial growth[J].Applied&Environmental Microbiology, 2010, 76(3):974-977.