副干酪乳杆菌素的分离纯化及其在肉制品中的应用初探
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摘要
本课题组从L-乳酸酸菜发酵液中分离出一株乳酸杆菌,通过对该菌株的优化与鉴定,确定其为副干酪乳杆菌(Lactobacillus paracasei),命名为副干酪乳杆菌HD1.7。经过前期研究得知,副干酪乳杆菌HD1.7所产抑菌物质(副干酪乳杆菌素)具有抗菌活性强,抑菌范围广等优点,具有良好的应用前景。本试验对副干酪乳杆菌素进行了分离纯化、初步确定其相对分子量并对其在肉制品中的应用做了初步研究,为日后该种细菌素的大量生产及其在食品中的广泛应用奠定一定的理论基础。
本试验对副干酪乳杆菌素的分离纯化方法及条件进行了研究,确定了以30%～60%硫酸铵逐级盐析、截留相对分子量5,000和10,000的两种超滤膜进行超滤、SP-Sepharose Fast Flow离子交换层析、Sephadex G-50凝胶层析的操作流程对副干酪乳杆菌素进行分离纯化。同时,在分离纯化过程中进行效价和蛋白含量的测定。经分离纯化后的副干酪乳杆菌素,比活力为1725.3AU/mg,是原发酵液37.09倍,得率为10.46%。通过尿素-SDS-PAGE对副干酪乳杆菌素的相对分子量进行了测定,初步确定其相对分子量为7,800。
对副干酪乳杆菌素在蒸煮火腿和烟熏肉中的防腐应用做了初步的研究。结果表明副干酪乳杆菌素与某些化学防腐剂单独使用时均有一定的防腐作用。但如果将副干酪乳杆菌素与化学防腐剂联合使用其防腐的效果更佳、且能减少化学防腐剂的添加量,从而减小化学防腐剂对人体的副作用。试验结果表明,副干酪乳杆菌素粗制品添加量为100mg/kg与1%乳酸钠联合使用的防腐效果要优于其它防腐联合剂的组合。
One strain which can produce antimicrobial substance was isolated from the L-LAB sauerkraut broth fermentation by our lab. After optimizing and identifying this strain, it was identified as Lactobacillus paracasei, and it was named as Lactobacillus paracasei HD1.7. Through the research on the strain, we knowed that the antimicrobial substance produced by Lactobacillus paracasei HD1.7 had strong antimicrobial activity and very broad antimicrobial range, so it has great foreground in application. Isolation and purification of the bacteriocin produced by Lactobacillus paracasei HD1.7 were mainly studied in this paper; the comparative molecular weight of the bacteriocin was ascertained; some elementary research about its application in cooked meat food was done. Theory elements about mass-production of this kind of bacteriocin and the academic elements of extensive application of this bacteriocin in food antisepsis were established in this paper.
An effective experiment protocol was researched to isolate and purify the bacteriocin produced by Lactobacillus paracasei HD1.7, and the conditiaon of isolation and purification was ascertained. Ultrafiltration was conducted by using ultrafiltration membranes which comparative molecular weight was 5,000 and 10,000. It was confirmed that through ammonium sulphate precipitation (30-60%), SP-Sepharose Fast Flow ion exchange chromatography and Sephadex G-50 gel chromatography, the bacteriocin could be isolated and purified effectively. Determination of protein content and titer was operated during the process of isolation and purification. After isolation and purification, the specific activity of bacteriocin was 1725.3AU/mg, 37.09 fold than the initial culture and a recovery of 10.46% of the initial activity was obtained. The comparative molecular weight of the antimicrobial bacteriocin was determined to be 7,800 by Urea-SDS-PAGE.
Elementary research was done to make the bacteriocin which was produced by Lactobacillus paracasei HD1.7 be applied in braised ham and bacon. The results showed that the bacteriocin and some chemical preservatives all had certain antisepsis role when used singly. When the bacteriocin and chemical preservatives were mixed together, new preservatives would enhance the antisepsis effect, and the adding dose of chemical preservatives would be reduced. So side effect of chemical preservatives to human beings would be decreased. The experiment results demonstrated that the mixture of bacteriocin and 1% NaL had better antisepsis effect than other preservative combinations.
本试验对副干酪乳杆菌素的分离纯化方法及条件进行了研究,确定了以30%～60%硫酸铵逐级盐析、截留相对分子量5,000和10,000的两种超滤膜进行超滤、SP-Sepharose Fast Flow离子交换层析、Sephadex G-50凝胶层析的操作流程对副干酪乳杆菌素进行分离纯化。同时,在分离纯化过程中进行效价和蛋白含量的测定。经分离纯化后的副干酪乳杆菌素,比活力为1725.3AU/mg,是原发酵液37.09倍,得率为10.46%。通过尿素-SDS-PAGE对副干酪乳杆菌素的相对分子量进行了测定,初步确定其相对分子量为7,800。
对副干酪乳杆菌素在蒸煮火腿和烟熏肉中的防腐应用做了初步的研究。结果表明副干酪乳杆菌素与某些化学防腐剂单独使用时均有一定的防腐作用。但如果将副干酪乳杆菌素与化学防腐剂联合使用其防腐的效果更佳、且能减少化学防腐剂的添加量,从而减小化学防腐剂对人体的副作用。试验结果表明,副干酪乳杆菌素粗制品添加量为100mg/kg与1%乳酸钠联合使用的防腐效果要优于其它防腐联合剂的组合。
One strain which can produce antimicrobial substance was isolated from the L-LAB sauerkraut broth fermentation by our lab. After optimizing and identifying this strain, it was identified as Lactobacillus paracasei, and it was named as Lactobacillus paracasei HD1.7. Through the research on the strain, we knowed that the antimicrobial substance produced by Lactobacillus paracasei HD1.7 had strong antimicrobial activity and very broad antimicrobial range, so it has great foreground in application. Isolation and purification of the bacteriocin produced by Lactobacillus paracasei HD1.7 were mainly studied in this paper; the comparative molecular weight of the bacteriocin was ascertained; some elementary research about its application in cooked meat food was done. Theory elements about mass-production of this kind of bacteriocin and the academic elements of extensive application of this bacteriocin in food antisepsis were established in this paper.
An effective experiment protocol was researched to isolate and purify the bacteriocin produced by Lactobacillus paracasei HD1.7, and the conditiaon of isolation and purification was ascertained. Ultrafiltration was conducted by using ultrafiltration membranes which comparative molecular weight was 5,000 and 10,000. It was confirmed that through ammonium sulphate precipitation (30-60%), SP-Sepharose Fast Flow ion exchange chromatography and Sephadex G-50 gel chromatography, the bacteriocin could be isolated and purified effectively. Determination of protein content and titer was operated during the process of isolation and purification. After isolation and purification, the specific activity of bacteriocin was 1725.3AU/mg, 37.09 fold than the initial culture and a recovery of 10.46% of the initial activity was obtained. The comparative molecular weight of the antimicrobial bacteriocin was determined to be 7,800 by Urea-SDS-PAGE.
Elementary research was done to make the bacteriocin which was produced by Lactobacillus paracasei HD1.7 be applied in braised ham and bacon. The results showed that the bacteriocin and some chemical preservatives all had certain antisepsis role when used singly. When the bacteriocin and chemical preservatives were mixed together, new preservatives would enhance the antisepsis effect, and the adding dose of chemical preservatives would be reduced. So side effect of chemical preservatives to human beings would be decreased. The experiment results demonstrated that the mixture of bacteriocin and 1% NaL had better antisepsis effect than other preservative combinations.
引文
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[2]蒋永福,张琨,黎碧娜.天然防腐剂-乳酸链球菌素的研究进展[J].精细化工, 2002, 19(8): 453~456.
[3]乔长晟,王玲,朱晓红等.新型天然食品防腐剂的发展现状与趋势[J].宁夏农学院学报, 2001, 22(4): 60~63.
[4]谢俊杰,佘世望,许杨等.食品防腐新领域-微生物天然防腐剂[J].食品科学, 1999, 1: 13~14.
[5] Broghton J.D. et al. Food Technol, 1990, 44: 100~106.
[6] Mattick A.T. et al. Lancet, 1997, 2: 5~7.
[7] Hrish A. NATURE (London), 1951, 167:1031~1032.
[8]李明春.乳球菌肽(Nisin)的研究进展[J].食品科学, 1999, 12: 10~12.
[9]赵玲艳,邓放明,杨细平等.生物防腐剂-乳酸菌素[J].中国食物与营养, 2005, 2: 27~29.
[10]张芳,徐学万.几种天然防腐剂及其在食品中的应用[J].食品工业, 2001, 3: 30~31.
[11] Tagg J R, Dajani A S and L W Wannamaker. Bacteriocins of gram-positive bacterial [J]. Bacteriology Review, 1976, 40: 722~756.
[12] Tramer J.G.G., Fowler.J. Estimation of Nisin in Food [J]. Sci.Food Agric, 1964, 15(8): 522~528.
[13] Wolf C.E., Gibbons W.R. Improved Method for Quantification of the Bacteriocin Nisin [J]. J of Appl Bacterio, 1996, 80: 453~457.
[14] Klaenhammer, T,R. Genetics of bacteriocins produced by lactic acid bacteria [J]. FEMS. Microbiol.Rev. 1993, 12: 39~86.
[15] Kanatani K, Oshimura M, Sano K. Appl Environ Microbiol. 1995, 61(3): 1061~1067.
[16]顾觉奋.分离纯化工艺原理[M].北京:中国医药科技出版社, 2000: 59, 115, 124, 135, 159.
[17]叶振华.吸着分离过程基础[M]第l版.北京:化学工业出版社, 1998.
[18]赵永芳.生物化学技术原理及应用[M].第三版.北京:科学出版社, 2002: 24, 161.
[19]汪家政,范明.蛋白质技术手册[M].北京:科学出版社, 2000.
[20] Jack R W, Tagg J R, Ray B. Microbiol Rev. 1995, 59: 171~200.
[21] Cheeseman G. C. and Berridge, N. J. An improved meehod of preparing nisin biochem [J], 1957, (65): 603~608.
[22] Gonzalez, B., Arca, P,Mayo, B. and Suarez, J.E. Detection, purification and partial characterization of plantaricin C, a bacteriocin produced by a Lactobacillus plantarum strain of dairy origin. Appl. Environ. Microbiol, 1994, 60: 2158~2163.
[23]陈秀珠,何松,龙力红,还连栋,薛禹谷.乳酸乳球菌AL2产生的乳链菌肽的提纯和性质[J].微生物学报, 1996, 36(4): 269~275.
[24] Suárez AM, Azcona JI, Rodríguez JM, Sanz B, Hernández P. One-step purification of nisin A by immunoaffinity chromatography. Appl Environ Microbiol, 1997, 63: 4990~4992.
[25]刘稳,朱文淼,马桂荣.乳链菌肽的分离纯化和部分生物学性质[J].生物化学杂志, 1996, 12(5): 588~592.
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