纳豆芽孢杆菌胞壁肽聚糖提取工艺优化及其结构初步鉴定
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摘要
为探究纳豆芽孢杆菌细胞壁肽聚糖提取工艺及其结构,本实验以纳豆芽孢杆菌为研究材料,采用响应面优化超声波破碎法,提取纳豆芽孢杆菌细胞壁,然后经三氯乙酸、乙酸钠、氯仿、甲醇、胰蛋白酶处理等操作分离得到较为纯净的肽聚糖并对其组成成分和结构进行分析。结果表明:用超声波破碎法提取的纳豆芽孢杆菌细胞壁得率为45.8%,纯化后得到的肽聚糖得率为8.5%。所得纳豆芽孢杆菌胞壁肽聚糖中总糖占39.77%,蛋白质占54.47%,总脂含量占4.5%。溶菌酶溶解实验9 h后眼观变为澄清。经红外光谱(IR)和高效液相分析提取的肽聚糖样本与肽聚糖已知结构特征相一致。经过聚丙烯酰胺凝胶电泳(SDS-PAGE)分析经溶菌酶水解后的肽聚糖分子量约为60 ku。
The main purpose of this study was to investigate the optimization of the extraction process ofpeptidoglycan from Bacillus subtilis cell wall and its structural identification.In this experiment,Bacillus natto was used as the research material.The response surface method was used to optimize the ultrasonic disruptionmethod to extract the Bacillus natto cell wall.The purified peptides were then separated by trichloroacetic acid,sodium acetate,chloroform,methanol,trypsin and other operations.Glycans and their composition and structurewere analyzed.Conclusions were drawn:the extraction rate of Bacillus natto by ultrasonic wave was 45.8%,andthe extraction rate of purified peptidoglycan was 8.5%.The total sugar in Bacillus natto was 39.77%,the proteinwas 54.47%,and the total lipid content was 4.5%.Visually,the lysozyme dissolution became clear after 9 h.Thepeptidoglycan samples extracted by infrared spectroscopy(IR)and high performance liquid phase analysiswere consistent with known structural characteristics of peptidoglycan.The molecular weight of peptidoglycanafter hydrolysis by lysozyme was about 60 ku by SDS-PAGE analysis.
The main purpose of this study was to investigate the optimization of the extraction process ofpeptidoglycan from Bacillus subtilis cell wall and its structural identification.In this experiment,Bacillus natto was used as the research material.The response surface method was used to optimize the ultrasonic disruptionmethod to extract the Bacillus natto cell wall.The purified peptides were then separated by trichloroacetic acid,sodium acetate,chloroform,methanol,trypsin and other operations.Glycans and their composition and structurewere analyzed.Conclusions were drawn:the extraction rate of Bacillus natto by ultrasonic wave was 45.8%,andthe extraction rate of purified peptidoglycan was 8.5%.The total sugar in Bacillus natto was 39.77%,the proteinwas 54.47%,and the total lipid content was 4.5%.Visually,the lysozyme dissolution became clear after 9 h.Thepeptidoglycan samples extracted by infrared spectroscopy(IR)and high performance liquid phase analysiswere consistent with known structural characteristics of peptidoglycan.The molecular weight of peptidoglycanafter hydrolysis by lysozyme was about 60 ku by SDS-PAGE analysis.
引文
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[2]彭程远,解俊,王锡波,等.鰤鱼诺卡氏菌全肽聚糖的提取及鉴定[J].宁波大学学报(理工版),2015,28(1):21-24.Peng C Y,Xie J,Wang X B,et al.Studies on the extraction and iden tification of whole peptidoglycan from Nocardia seriolae[J].Journal of Ningbo University(NSEE),2015,28(1):21-24.
[3]向思琴.细菌细胞壁肽聚糖的结构与作用[J].技术与市场,2017,24(1):169.Xiang S Q.Extraction and identification of Nocardia total peptidoglycan[J].Technology and Market,2017,24(1):169.
[4]付艳茹,龚虹,刘彦民,等.长双歧杆菌完整肽聚糖对小鼠免疫功能的影响[J].中国微生态学杂志,2010,22(3):217-219.Fu Y R,Gong H,Liu Y M,et al.Effect of the whole peptidoglycan from Bifidobacterium longum on the immune function ofmice[J].Chinese Journal of Microecology,2010,22(3):217-219.
[5]Sekine K,Toida T,Saito M,et al.A new morphologically characterized cell wall preparation(whole peptidoglycan)fromBifidobacterium infantis with a higher efficacy on the regression of an established tumor in mice[J].Cancer Research,1985,45(3):1300.
[6]金盼盼.乳酸杆菌DM8909胞壁肽聚糖对小鼠阴道感染的免疫调节作用[D].大连:大连医科大学,大连2014.Jin P P.Immunomodulatory effect of lactobacillus peptidoglycan on vaginal infection in mice[D].Dalian:Dalian MedicalUniversity,2014.
[7]王静华,赵洪涛,汪以真.细菌细胞壁肽聚糖的研究进展[J].中国兽药杂志,2004,38(1):38-40.Wang J H,Zhao H T,Wang Y Z.Advance in the research of peptidoglycan of the bacterial cell wall[J].Chinese Journal ofVeterinary Drug,2004,38(1):38-40.
[8]刘芳,杨跃寰.细菌细胞壁肽聚糖的研究[J].四川理工学院学报(自然科学版),2011,24(6):628-631.Liu F,Yang Y H.Research on peptidoglycan of bacterial cell wall[J].Journal of Sichuan University of Science&Engineer?ing(Natural Science Edition),2011,24(6):628-631.
[9]容艳筠,邓毛程.纳豆芽孢杆菌在食品中的研究现状和应用进展[J].技术与市场,2017,24(7):146-147.Rong Y J,Deng M C.Research status and application progress of Bacillus natto in food industry[J].Technology and Market,2017,24(7):146-147.
[10]付艳茹,靳烨.完整肽聚糖提取工艺的研究[J].中国乳品工业,2009,37(4):12-15.Fu Y R,Jin Y.Study on the optimum extraction process of whole peptidoglycan[J].China Dairy Industry,2009,37(4):12-15.
[11]Bradford M M.A rapid and sensitive method for the quantization of microgram of protein utilizing the principle of protein-dye binding[J].Anal Biochem,1976,72(1):248-254.
[12]施济人,贾守约.临床检验手册[M].济南:山东科学技术出版社,1988:894-896.Shi J R,Jia S Y.Manual of clinical examination[M].Ji’nan:Shandong Science and Technology Press,1988:894-896.
[13]Dubois M,Gilles D A,Hamilton J K,et al.Colorimetric method for the determination of sugars and related substances[J].Anal Chem,1956,28(3):350-356.
[14]张威,何红波,解宏图,等.不同水解方法对土壤中性糖和氨基糖含量测定的影响[J].生态环境,2006,15(5):1067-1071.Zhang W,He H B,Xie H T,et al.Hydrolysis and assay by gas chromatography for neutral and amino suars in soil[J].Ecolog?ical and Environment,2006,15(5):1067-1071.
[15]朱志春,郑毅,朱中南,等.超声波破碎法提取辅酶Q10研究[J].海峡科学,2012(2):17-19.Zhu Z C,Zheng Y,Zhu Z N,et al. Study on the extraction of coenzyme Q10 by ultrasonic breakage[J].Channel Science,2012(2):17-19.
[16]衣海龙.利用超声波法破碎啤酒酵母细胞壁的工艺研究[J].酿酒,2015,42(4):83-85.Yi H L.Study on the technology of breaking the cell wall of beer yeast by ultrasonic wave[J].Liquor Making,2015,42(4):83-85.
[17]余晓.功率超声破碎酵母细胞的影响因素研究[D].南京:南京农业大学,2015.Yu X.Ultrasonic disruption of yeast cells:effects of processing parameters[D].Nanjing:Nanjing Agricultural University,2015.
[18]李春喜,宋红艳,王子镐.超声波在化工中的应用与研究进展[J].石油学报,2001,17(3):86-94.Li C X,Song H Y,Wang Z G.Recent advances of research and applications of ultrasolind in chemical engineering[J].ActaPetrolei Sinica(Petroleum Processing Section),2001,17(3):86-94.
[19]苑华宁,黄冬云,张晖,等.反复冻融和超声协同作用破碎酵母细胞[J].食品与发酵工业,2013,39(12):62-67.Yuan H N,Huang D Y,Zhang H,et al.Breaking of brewer yeast with repeated freeding and thawing combined with ultra?sound treatment[J].Food and Fermentation Industries,2013,39(12):62-67.
[20]姜玲,董庆霖,邢向英,等.雨生红球藻细胞破碎的工艺优化[J].食品研究与开发,2010,31(7):72-75.Jiang L,Dong Q L,Xing X Y,et al.Optimization of cell disruption technology of Haematococcus plurialis[J].Food Researchand Development,2010,31(7):72-75.
[21]李金忠,马海乐,吴沿友.超声波提取山药多糖的研究[J].粮油食品科技,2005,13(5):14-15.Li J Z,Ma H L,Wu Y Y.Study on the ultrasonic extraction of polysaccharide from Chinese yam[J].Science and Technologyof Cereals,Oil and Food,2005,13(5):14-15.
[22]易军鹏,朱文学,马海乐,等.牡丹籽油超声波辅助提取工艺的响应面法优化[J].农业机械学报,2009,40(6):103-110.Yi J P,Zhu W X,Ma H L,et al.Optimization on ultrasonic-assisted extraction technology of oil from Paeonia suffruticosa An?dr.seeds with response surface analysis[J].Transactions of The Chinese Society of Agricultural Machinery,2009,40(6):103-110.
[23]张玉霞,雍国新,黎渊珠,等.超声波协助提取绿豆分离蛋白的研究[J].食品研究与开发,2014,35(20):13-17.Zhang Y X,Yong G X,Li Y Z,et al.Study on ultrasonic extraction of mung bean protein isolates[J].Food Research and De?velopment,2014,35(20):13-17.
[24]沈柱英.纳豆糖肽的分离纯化、结构表征及其免疫活性研究[D].南昌:江西农业大学,2014.Shen Z Y.Studies on isolation,purification,identification and immune functions of polysaccharopeptide from the Natto[D].Nanchang:Jiangxi Agricultural University,2014.
[25]Marcotte L,Kegelaer G,Sandt C,et al.An alternative infrared spectroscopy assay for the quantification of polysaccharidesin bacterial samples[J].Anal Biochem,2007,361(1):7-14
[26]张悦,宋晓玲,黄倢.双歧杆菌肽聚糖结构及分子量的分析[J].微生物学报,2007,34(4):676-681.Zhang Y,Song X L,Huang J.Structure and molecular weight analysis Bifidobacterium peptidoglycan[J].Microbiology Chi?na,2007,34(4):676-681.
[27]杨媛.嗜酸乳杆菌肽聚糖的修饰及其对小鼠肠黏膜保护作用研究[D].宁波:宁波大学,2014.Yang Y.Modification of Lactobacillus peptidoglycan and its effect on the study of mucose intestinal mucosa protection[D].Ningbo:Ningbo University,2014.
[28]Binda E,Carrano L,Marcone G L,et al.Extraction and analysis of peptidoglycan cell wall precursors[J].Methods inMolecular Biology,2016,1440:153.
[29]Lebeer S,Claes I J,Vanderleyden J.Anti-inflammatory potential of probiotics:lipoteichoic acid makes a difference[J].Trends in Microbiology,2012,20(1):5-10.
[30]Rae C S,Geissler A,Adamson P C,et al.Mutations of the Listeria monocytogenes peptidoglycan N-deacetylase and O-acetylase result in enhanced lysozyme sensitivity,bacteriolysis,and hyper induction of innate immune pathways[J].Infection and Immunity,2011,79(9):3596-3606.
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