戊糖乳杆菌发酵产乳酸及高产菌株诱变选育

生物技术通报 ›› 2014, Vol. 0 ›› Issue (11): 179-186.

戊糖乳杆菌发酵产乳酸及高产菌株诱变选育

王义强^1,2,3,王启业¹,马国辉¹,林丽云^1,

1.中南林业科技大学生物技术实验室,长沙 410004；2.经济林培育与保护教育部重点实验室,长沙 410004；3.国家林业局生物乙醇研究中心,长沙 410004

收稿日期:2014-03-27 出版日期:2014-11-07 发布日期:2014-11-07
作者简介:王义强,男,教授,博士生导师,研究方向：细胞工程与生物能源
基金资助:
国家林业局“948“项目（2011-4-13）

Lactobacillus pentosus Fermentation Lactic Acid Production and High Yield Strain Mutation Breeding

Wang Yiqiang^1,2,3,Wang Qiye¹,Ma Guohui¹,Lin Liyun¹

1. Biotechnology Laboratory of Central South University of Forestry and Technology, Changsha 410004；2. Key Lab of Non-wood Forest Nurturing and Protection of National Ministry of Education, Changsha 410004；3. Bio-ethanol Research Center of State Forestry Administration, Changsha 410004

Received:2014-03-27 Published:2014-11-07 Online:2014-11-07

摘要/Abstract

摘要： 戊糖乳杆菌（Lactobacillus pentosus）是能利用木质纤维素水解液发酵产乳酸的潜力菌株,发酵条件优化与高产菌株的选育是提高乳酸产量的重要手段。通过单因素试验、Plackett-Burman设计与响应面试验,对戊糖乳杆菌ATCC 8041产乳酸的发酵培养基及发酵条件进行了优化。结果表明,该菌株发酵培养基的最佳组合为葡萄糖93.11 g/L、酵母浸粉5.19 g/L、碳酸钙29.43 g/L、蛋白胨10.00 g/L、Na₂HPO₄?12H₂O 5.00 g/L、MgSO₄0.20 g/L、MnSO₄50 mg/L；最佳发酵条件为37℃、pH6.5、接种量6%、装液量80%。在此优化条件下,该菌株发酵产乳酸为54.12 g/L。进一步以戊糖乳杆菌ATCC 8041为出发菌株,通过原生质体进行紫外诱变,经多重筛选,最终获得一株遗传稳定性好的高产乳酸突变株,命名为戊糖乳杆菌Lactic UVC-02,由中国典型培养物保藏中心保存,注册号为CCTCC M 2013209。该突变株Lactic UVC-02经葡萄糖发酵,乳酸产量达64.17 g/L,比出发菌株ATCC 8041（54.12 g/L）提高18.6%。

关键词: 戊糖乳杆菌, 乳酸发酵, 紫外诱变, 响应面试验

Abstract: Lactobacillus pentosus is a potential strain which can make use of the lignocellulose hydrolysate fermentation to produce lactic acid, optimization of fermentation conditions and the breeding of high yield strain are the important means to improve lactic acid production. Fermentation medium and fermentation conditions were optimized for strain Lactobacillus pentosus ATCC 8041, through single factor experiment, Plackett-Burman design and response surface experiment. The results showed that the best combination of fermentation medium is 93.11 g/L glucose, 5.19 g/L yeast powder, 29.43 g/L calcium carbonate, 10.00 g/L peptone, 5.00 g/L Na₂HPO₄?12H₂O, 0.20 g/L MgSO₄, 50 mg/L MnSO₄. The best fermentation conditions is temperature 37℃, pH6.5, quantity of 6%, and fluid amount 80%. The lactic acid production is 54.12 g/L under the optimized fermentation medium and fermentation conditions. And then, Lactobacillus pentosus ATCC 8041 was taken as the original strain, treated with UV mutation ATCC 8041 protoplast, after through multiple screening, finally we have got a mutant which has high lactic acid production, named Lactobacillus pentosus lactic UVC-02 which conserved in China Center for Type Culture Collection, registration number for CCTCC M 2013209. The results for glucose fermentation showed that lactic UVC-02 produced 64.17 g/L lactic acid, which was 18.6% higher than the original strain.

Key words: Lactobacillus pentosus , Lactic acid fermentation, UV mutation, Response surface experiment

王义强,王启业,马国辉,林丽云. 戊糖乳杆菌发酵产乳酸及高产菌株诱变选育[J]. 生物技术通报, 2014, 0(11): 179-186.

Wang Yiqiang,Wang Qiye,Ma Guohui,Lin Liyun. Lactobacillus pentosus Fermentation Lactic Acid Production and High Yield Strain Mutation Breeding[J]. Biotechnology Bulletin, 2014, 0(11): 179-186.

参考文献

[1] 曹本昌, 徐建林, 匡群.根霉发酵L-乳酸[J].食品与发酵工业, 1991（1）：37-41.
[2] 金其荣, 金丰收. 乳酸衍生物发展应用新动向[J]. 山西食品工业, 2002, 3：2-4.
[3]钱志良, 胡军, 雷肇祖. 乳酸的工业化生产应用和市场[J]. 工业微生物, 2001（6）：49-54.
[4]曾炜, 陈丰秋, 詹晓力. 乳酸的生产技术及其研究进展[J]. 化工进展, 2006, 25（7）：746-747.
[5]张刚, 乳酸细菌——基础、技术和应用[M]. 北京：化学工业出版社, 2007.
[6]乐晓洁, 王昌禄, 顾晓波, 等. 细菌发酵生产L-乳酸高产菌株的选育[J]. 中国食品添加剂, 2004, 1：67-69.
[7]郑艳, 薛景珍, 刘长江. L-乳酸发酵菌株的选育[J]. 微生物学杂志, 2004, 24（3）：26-28.
[8]张秀凤, 李正英, 江均平. 细菌L-乳酸发酵的研究[J]. 食品与机械, 2005, 21（5）：74-76.
[9]白冬梅, 赵学明, 胡宗定. 反相HPLC双检测器法同时测定米根霉乳酸发酵液中的有机酸与葡萄糖[J]. 食品与发酵工艺, 2001, 27（1）：13-17.
[10]付晓芬, 江均平, 张洁, 等. 微生物利用木糖发酵L—乳酸代谢途径的研究[J]. 食品工业科技, 2009（8）：359-362.
[11]Masayuki T, Masahiro H, Tanabe S, et al. Production of L-lactic acid by simultaneous saccharification and fermentation using unsterilized defatted rice bran as a carbon source and nutrient components[J]. Food Sci Technol Res, 2005, 11：400-406.
[12]Zhu Y, Lee YY, Elander RT. Conversion of aqueous ammonia-treated corn stover to lactic acid by simultaneous saccharification and co-fermentation[J]. Appl Biochem Biotechnol, 2007（1-12）：137-140.
[13]Bustos G, Moldes AB, Cruz JM, et al. Production of fermentable media from trimming wastes and bioconversion into lactic acid by Lactobacillus pentosus[J]. Journal of the Science of Food and Agriculture, 2004, 84：2105-2112.
[14]张丽丽, 沈兆兵, 史吉平, 等. 紫外诱变和丁醇驯化复合选育高产丁醇菌株[J]. 中国酿造, 2013, 32（5）：129-133.
[15]Bustos G, Moldes AB, Cruz JM, et al. Influence of the metabolism pathway on lactic acid production from hemicellulosic trimming vine shoots hydrolyzates using Lactobacillus pentosus[J]. Biotechnol Prog, 2005, 21：793-798.
[16] 杨承剑, 黄兴国, 等. Plackett-Burman设计在益生菌生长主要影响因子筛选中的应用[J]. 饲料工业, 2007, 28（16）：31-33.
[17]Pan CM, Fan YT, Xing Y, et al. Statistical optimization of process parameters on biohydrogen production from glucose by Clostridium sp. Fanp2[J]. Bioresour Technol, 2008, 99（8）：3146-3154.
[18] Yuan LL, Li YQ, Wang Y, et al. Optimization of critical medium components using response surface methodology for phenazine-1-carboxylic acid production by Pseudomonas sp. M-18Q[ J]. J Biosci Bioeng, 2008, 105（3）：232-237.
[19]代志凯, 张翠, 阮征. 试验设计和优化及其在发酵培养基优化中的应用[J]. 微生物学报, 2010, 37（6）：894-903.
[20]白冬梅, 赵学明, 胡宗定. 高效液相色谱手性流动相添加剂分离乳酸对应体[J]. 分析化学, 2001, 29（4）：413-415.
[21]田泱源, 李瑞芳. 响应面法在生物过程优化中的应用[J]. 食品工程, 2010（2）：8-11.
[22]潘丽军, 庞锐, 吴学凤, 等. 葡萄糖和木糖共发酵生产L-乳酸的培养基和培养条件响应面优化[J]. 食品科学, 2011, 32（9）：140-145.
[23]丁涓, 魏敏, 张莉. 玉米浆发酵生产 L-乳酸的工艺优化[J]. 食品科学, 2011, 32（1）：127-130.
[24] 秦浩. 高产L-乳酸菌株的选育及其发酵条件的研究[D]. 无锡：江南大学, 2012.
[25]Naveena BJ, Altaf M, Bhadriah K, et al. Selection of medium components by Plackett-Burman design for production of l（+）lactic acid by Lactobacillus amylophilus GV6 in SSF using wheat bran[J]. Bioresource Technology, 2005, 96（4）：485-490.
[26]Coelho LF, De Lima CJB, Rodovalho CM, et al. Lactic acid production by new Lactobacillus plantarum LMISM6 grown in molasses：optimization of medium composition[J]. Brazilian Journal of Chemical Engineering, 2011, 28（1）：27-36.
[27]Gowdhaman D, Sugumaran KR, Ponnusami V. Optimization of lactic acid production from tea waste by Lactobacillus plantarum MTCC 6161 in solid state fermentation by central composite design[J]. Inter J Chemtech Res, 2012, 4（1）：143-148.
[28]石彩蕊, 王义强, 陈介南, 等. 产β-葡萄糖苷酶微生物育种研究进展[J]. 生物技术通报, 2011（3）：59-65.
[29]仲松, 张庆庆, 孙平平. 紫外诱变原生质体选育高产L-乳酸菌株的研究[J]. 安徽工程大学学报, 2011, 26（1）：28-30.
[30]吴慧昊, 牛锋. 乳酸菌低温菌株的复合诱变选育[J]. 微生物学通报, 2013, 40（4）：631-645.
[31]Yin L, Ruan Q, Fu Y. Strain improvement of Rhizopus oryzae for over-production of lactic acid by random mutations[J]. African Journal of Microbiology Research, 2013, 7（23）：2970-2975.