为获取新型食品级酸性菊粉酶,对前期构建的广西特色生榨米粉来源乳酸菌库进行了筛选、鉴定和酶学表征。采用终质量浓度为20 g/L菊粉作为唯一碳源的筛选培养基并结合菊粉酶酶活力测定,筛选出1株产酸性菊粉酶的乳酸菌,经过菌落形态观察及16S rDNA基因序列分析,鉴定为柠檬明串珠菌,命名为Leuconostoc citreum 1-1。以菊粉为底物开展酶学表征,结果表明,Leuconostoc citreum 1-1菊粉酶的最适温度为35 ℃,最适pH为4.0,在20~40 ℃,pH 3.5~9.0可保持80%的酶活力,薄层色谱法结果表明菊糖几乎100%被水解成果糖。该新型食品级酸性菊粉酶及其生产菌株L.citreum 1-1在高果糖浆生产等菊粉类果糖基资源利用领域具有良好的研究价值和应用潜力。
关键词:
菊粉酶; 酸性; 乳酸菌; 筛选; 菊粉
To obtain a novel acidic inulinase, an acidic inulinase producing lactic acid bacteria was screened and characterization of its enzymatic properties was conducted. By using selective medium containing inulin (20 g/L) as sole carbon source and inulin-hydrolyzing ability, a strain producing an acidic inulinase was screened out from the lactic acid bacteria collection of Guangxi's specialty raw rice flour constructed. It was identified as Leuconostoc citreum based on its morphology and 16S rDNA sequence analysis and was designated as L. citreum strain 1-1. Using inulin as substrate, inulinase produced by L. citreum 1-1 showed the maximum activity at 35℃ and pH 4.0, and maintained more than 80% of maximum activity at pH 3.5-9.0. The thin layer chromatography results showed that fructose was the sole product (100% conversion) of inulin hydrolysis by the inulinase. The novel acidic inulinase and its producing strain L.citreum 1-1 may have great scientific research value, and as well as application potentials in production of high fructose syrup and other utilizations of fructose-based resources like inulin.
[1] RONKART S N, BLECKER C S, FOURMANOIR H, et al.Isolation and identification of inulooligosaccharides resulting from inulin hydrolysis[J]. Analytic Chimica Acta, 2007, 606(1): 81-87.
[2] DE OLIVEIRA A J B, GONCALVES R A C, CHIERRITO T P C, et al. Structure and degree of polymerisation of fructooligosaccharides present in roots and leaves of Stevia rebaudiana (Bert.) Bertoni[J]. Food Chemistry, 2011, 129(2):305-311.
[3] 张泽生, 刘亚萍, 李雨蒙, 等. 菊粉的研究与开发[J]. 中国食品添加剂, 2017(10): 183-188.[4] 刘燕, 任欢欢, 马秀梅, 等 1株产菊粉酶菌株的筛选、鉴定及发酵条件优化[J]. 微生物学杂志, 2018, 38(1):70-75.
[5] 胥九兵, 王乃强, 刘宗利, 等. 菊芋资源开发利用研究进展[J]. 精细与专用化学品, 2012, 20(11):18-20.
[6] PANDEY A, SOCCOL C R, SELVAKUMAR P, et al.Recent developments in microbial inulinases[J]. Applied Biochemistry and Biotechnology, 1999, 81(1):35-52.
[7] 王翠. 产菊粉酶菌株的筛选及菊粉酶性质的研究[D]. 天津: 天津科技大学, 2010.
[8] LI Y, LIU G L, CHI Z M.Ethanol production from inulin and unsterilized meal of Jerusalem artichoke tubers by Saccharomyces sp. W0 expressing the endo-inulinase gene from Arthrobacter sp.[J]. Bioresource Technology, 2013, 147: 254-259.
[9] ZHANG T, GONG F, PENG Y, et al.Optimization for high-level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase[J]. Process Biochemistry, 2009,44(12):1 335-1 339.
[10] GONG F, SHENG J, CHI Z, et al.Inulinase production by a marine yeast Pichia guilliermondii and inulin hydrolysis by the crude inulinase[J]. Journal of Industrial Microbiology & Biotechnology, 2007, 34(3):179-185.
[11] PALUDAN-MÜLLER C, GRAM L, RATTRAY F P. Purification and characterisation of an extracellular fructan β-fructosidase from a Lactobacillus pentosus strain isolated from fermented fish[J]. Systematic & Applied Microbiology, 2002, 25(1):13-20.
[12] PESSONI R A B, FIGUEIREDO-RIBEIRO R C L, BRAGA M R. Extracellular inulinases from Penicillium janczewskii, a fungus isolated from the rhizosphere of vernonia herbacea (Asteraceae)[J]. Journal of Applied Microbiology, 1999, 87(1):141-147.
[13] 熊伍平, 王成华, 陆雁, 等. 一个外切菊粉酶基因的克隆表达及酶学性质[J]. 生物加工过程, 2013, 11(3): 40-45.
[14] WU D T, CHEONG K L, WANG L Y, et al.Characterization and discrimination of polysaccharides from different species of Cordyceps using saccharide mapping based on PACE and HPTLC[J]. Carbohydrate Polymers, 2014, 103:100-109.
[15] 王建华. 微生物菊粉酶基因结构、酶学性质与应用研究进展[J]. 天然产物研究与开发, 2001(1):83-89.
[16] 于春, 于基成, 张春红, 等. 灰平链霉菌Streptomyces griseoplanus S501产外切菊粉酶的分离纯化及酶学性质研究[J]. 食品与发酵工业, 2015, 41(8):42-47.
[17] 高威. 产菊粉酶耐热细菌的筛选及酶学性质研究[D]. 大连: 大连理工大学, 2008.