为评估表层蛋白对嗜酸乳杆菌自聚集、生长及代谢的影响,通过测定菌体自聚集率、菌体生长、GC-MS分析表层蛋白去除前后嗜酸乳杆菌胞内小分子代谢物的变化。结果表明,表层蛋白去除前后,嗜酸乳杆菌的胞内氨基酸类如组氨酸、酪氨酸、鸟氨酸,有机酸类如乳酸、苹果酸等代谢物具有显著差异,这些代谢物直接影响了表层蛋白相关的主要生化反应和关键代谢通路。经过代谢通路富集分析,主要影响了精氨酸和脯氨酸代谢、赖氨酸生物合成、精氨酸生物合成、氨基糖和核苷酸糖代谢、丙氨酸,天冬氨酸和谷氨酸代谢这5条代谢通路。总之,表层蛋白的剥离影响了氨基酸合成的代谢通路,氨基酸合成会竞争生长所需的能量,进而影响嗜酸乳杆菌的生长,该研究为进一步认识表层蛋白对嗜酸乳杆菌胞内小分子化合物代谢的影响提供了依据。
In order to evaluate the effect of surface layer protein on the self-aggregation, growth and metabolism of Lactobacillus acidophilus NCFM, the self-aggregation rate and growth of L. acidophilus were measured, and GC-MS was used to analyze the changes of the intracellular small molecule metabolites of L. acidophilus before and after the removal of the surface layer protein. The intracellular amino acids and organic acids of L. acidophilus such as histidine, tyrosine, ornithine, lactic acid, malic acid and other metabolites changed significantly. These metabolites directly affected the major biochemical reactions and important metabolic pathways related with surface layer protein. Enrichment analysis of metabolic pathways showed that it mainly affected arginine and proline metabolism, lysine biosynthesis, arginine biosynthesis, amino sugar and nucleotide sugar metabolism, alanine, aspartate and glutamate metabolism. In conclusion, the removal of surface layer protein affected the metabolic pathways of amino acid synthesis. The synthesis of amino acids competed energy required for growth, and then affected the growth of L. acidophilus. This study provides a basis for further understanding the effects of surface layer protein on the metabolism of intracellular small molecule compounds in L. acidophilus.
[1] 王慧芳.乳杆菌S-层蛋白的肠道免疫调节功能及作用机制研究[D].无锡:江南大学, 2020.
WANG H F.Study on the intestinal immunomodulatory function and mechanism of Lactobacillus surface-layer proteins[D].Wuxi:Jiangnan University, 2020.
[2] WANG H F, ZHANG L, LI Q P, et al.Surface-layer protein produced by Lactobacillus crispatus JCM 2009 ameliorates lipopolysaccharide-induced inflammation through autophagy cross-talk with the NF-κ B signaling pathway[J].International Journal of Biological Macromolecules, 2021, 166:633-640.
[3] ZHANG Y C, XIANG X L, LU Q H, et al.Adhesions of extracellular surface-layer associated proteins in Lactobacillus M5-L and Q8-L[J].Journal of Dairy Science, 2016, 99(2):1 011-1 018.
[4] ABRAMOV V M, KOSAREV I V, PRIPUTENEVICH T V, et al.S-layer protein 2 of Lactobacillus crispatus 2029, its structural and immunomodulatory characteristics and roles in protective potential of the whole bacteria against foodborne pathogens[J].International Journal of Biological Macromolecules, 2020, 150:400-412.
[5] 牛钰涵.表层蛋白对乳杆菌益生性质的影响及其抑菌功能[D]:无锡:江南大学, 2019.
NIU Y H.Effect of surface layer proteins from Lactobacillus on the strains' probiotic properties and their antibacterial function[D].Wuxi:Jiangnan University, 2019.
[6] PALOMINO M M, WAECHNER P M, MARTIN J F, et al.Influence of osmotic stress on the profile and gene expression of surface layer proteins in Lactobacillus acidophilus ATCC 4356[J].Applied Microbiology and Biotechnology, 2016, 100(19):8 475-8 484.
[7] KLOTZ C, GOH Y J, O'FLAHERTY S, et al.Deletion of S-layer associated Ig-like domain protein disrupts the Lactobacillus acidophilus cell surface[J].Frontiers in Microbiology, 2020, 11(14):1-14.
[8] PATTI G J, YANES O, SIUZDAK G.Metabolomics:The apogee of the omics trilogy[J].Nature Reviews Molecular Cell Biology, 2012, 13(4):263-269.
[9] JOHNSON B, SELLE K, O'FLAHERTY S, et al.Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM[J].Microbiology-Sgm, 2013, 159:2 269-2 282.
[10] CHEN X Y, XU J J, SHUAI J B, et al.The S-layer proteins of Lactobacillus crispatus strain ZJ001 is responsible for competitive exclusion against Escherichia coli O157:H7 and Salmonella typhimurium[J].International Journal of Food Microbiology, 2007, 115(3):307-312.
[11] LU H Q, CHEN H Q, TANG X, et al.Evaluation of metabolome sample preparation and extraction methodologies for oleaginous filamentous fungi Mortierella alpina[J].Metabolomics, 2019, 15(4):50.
[12] DUNN W B, BROADHURST D, BEGLEY P, et al.Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry[J].Nature Protocols, 2011, 6(7):1 060-1 083.
[13] KIND T, WOHLGEMUTH G, LEE D Y, et al.FiehnLib:Mass spectral and retention index libraries for metabolomics based on quadrupole and time-of-flight gas chromatography/mass spectrometry[J].Analytical Chemistry, 2009, 81(24):10 038-10 048.
[14] CHONG J, SOUFAN O, LI C, et al.MetaboAnalyst 4.0:Towards more transparent and integrative metabolomics analysis[J].Nucleic Acids Research, 2018, 46(1):486-494.
[15] LORTAL S, VANHEIJENOORT J, GRUBER K, et al.S-Layer of Lactobacillus-Helveticus ATCC-12046 - isolation, chemical characterization and re-formation after extraction with lithium-chloride[J].Journal of General Microbiology, 1992, 138:611-618.
[16] HYMES J P, JOHNSON B R, BARRANGOU R, et al.Functional analysis of an S-layer-associated fibronectin-binding protein in Lactobacillus acidophilus NCFM[J].Applied and Environmental Microbiology, 2016, 82(9):2 676-2 685.
[17] 程新, 赵延胜, 董英, 等.Mn2+对植物乳杆菌影响的代谢组学分析[J].中国食品学报, 2019, 19(2):258-265.
CHENG X, ZHAO Y S, DONG Y, et al.Metabolomics analysis of Mn2+ effect on the metabolic pathways in Lactobacillus plantarum[J].Journal of Chinese Institute of Food Science and Technology, 2019, 19(2):258-265.
[18] BOULESTEIX A L, STRIMMER K.Partial least squares:A versatile tool for the analysis of high-dimensional genomic data[J].Briefings in Bioinformatics, 2007, 8(1):32-44.
[19] 石轩峰.植物乳杆菌乳酸脱氢酶的发酵优化、制备及酶促反应研究[D]:无锡:江南大学, 2005.
SHI X F.Studies on optimization, preparation and enzyme catalytic reaction of lactate dehydrogenase by Lactobacillus plantarum[D].Wuxi:Jiangnan University, 2005.
[20] 孟珺, 王艳阳, 康媛媛, 等.乳杆菌表层蛋白理化性质与生物功能的研究进展[J].河南工业大学学报(自然科学版), 2020, 41(4):129-138.
MENG J, WANG Y Y, KANG Y Y, et al.Advances in research on physicochemical properties and biological functions of Lactobacillus surface layer proteins[J].Journal of Henan University of Technology(Natural Science Edition), 2020, 41(4):129-138.
[21] MENG J, ZHU X, GAO S M, et al.Characterization of surface layer proteins and its role in probiotic properties of three Lactobacillus strains[J].International Journal of Biological Macromolecules, 2014, 65:110-114.
[22] 胡斌, 田丰伟, 赵建新, 等.嗜酸乳杆菌NCFM表层蛋白的提取及其与菌体酸和胆盐耐受及粘附性能的关系[J].食品工业科技, 2014, 35(5):118-121.
HU B, TIAN F W, ZHAO J X, et al.Extraction of surface layer protein from Lactobacillus acidophilus NCFM and the relationship among SLP and strain acid,bile resistance and cell adhesion[J].Science and Technology of Food Industry, 2014, 35(5):118-121.
[23] 孟珺.乳杆菌表层蛋白的理化性质和生物学功能研究[D]:无锡:江南大学, 2016.
MENG J.Study of the physicochemical properties and biological functions of Lactobacillus surface layer proteins[D].Wuxi:Jiangnan University, 2016.
