食品科学 ›› 2019, Vol. 40 ›› Issue (22): 88-95.doi: 10.7506/spkx1002-6630-20181120-232

• 生物工程 • 上一篇    下一篇

传统奶酪中产转糖基活性β-半乳糖苷酶乳酸菌的筛选鉴定及其合成低聚半乳糖条件

王丽军,牟元珍,关波,万丽云,张艳,胡有贞,倪永清   

  1. (石河子大学食品学院,新疆 石河子 832003)
  • 出版日期:2019-11-25 发布日期:2019-12-02
  • 基金资助:
    国家自然科学基金地区科学基金项目(31560432);兵团国际科技合作项目(2017BC012); 石河子大学青年创新人才培育计划项目(CXRC201702)

Screening and Identification of Lactic Acid Bacteria Producing β-Galactosidases with Transglycosylation Activity from Traditional Cheese, and Optimization of Conditions for Galacto-Oligosaccharides Synthesis

WANG Lijun, MOU Yuanzhen, GUAN Bo, WAN Liyun, ZHANG Yan, HU Youzhen, NI Yongqing   

  1. (Food College, Shihezi University, Shihezi 832003, China)
  • Online:2019-11-25 Published:2019-12-02

摘要: 分离筛选产转糖基活性β-半乳糖苷酶的乳酸菌新菌株,为酶法高效合成低聚半乳糖(galacto-oligosaccharides,GOS)提供新的酶源。以乳糖为唯一碳源,碳酸钙溶钙圈和添加5-溴-4-氯-3-吲哚-β-D-半乳糖苷(X-Gal)的MRS培养基筛选平板进行初筛,以产酶菌株粗酶液催化乳糖反应产物的薄层色谱(thin layer chromatography,TLC)分析复筛,从新疆伊犁地区牧民手工制作的奶酪样品中筛选产转糖基活性β-半乳糖苷酶的乳酸菌。结合其形态学、生理生化特征及16S rRNA序列同源性分析对产转糖基活性β-半乳糖苷酶菌株进行鉴定。单因素试验确定产酶条件和转糖基反应条件,TLC结合高效液相色谱分析转糖基反应产物各组分含量。筛选获得产转糖基活性β-半乳糖苷酶的菌株6 株,其中Lactobacillus plantarum YLBGNL-S7所产β-半乳糖苷酶的转糖基活性最强。单因素试验结果表明,在温度50 ℃、pH 6.0、乳糖质量浓度300 mg/mL的条件下反应4 h,GOS得率可达质量分数43.40%,其中转移二糖和转移三糖质量分数分别为18.29%和12.95%。以上结果表明,L. plantarum YLBGNL-S7是一株产转糖基活性β-半乳糖苷酶的新菌株,在益生性GOS的合成领域具有应用前景。

关键词: β-半乳糖苷酶, 转糖基活性, 低聚半乳糖, 植物乳杆菌, 奶酪

Abstract: This study aimed to obtain a lactic acid bacterial strain producing β-galactosidase with high transglycosylation activity in order to provide a novel source of β-galactosidase for efficient synthesis of galacto-oligosaccharides (GOS). Lactic acid bacterial strains were isolated from traditional cheese samples collected from pastoral households in Yili, Xinjiang autonomous region. Primary screening on MRS plates with added 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) using lactose as the sole carbon source was performed based on calcium dissolution zone size, and secondary screening was carried out by thin layer chromatography (TLC) analysis of the lactose hydrolysates obtained with β-galactosidases produced by the selected strains. The isolate with the highest transglycosylation activity was identified by physiological and biochemical characteristics, together with sequence analysis of the 16S rRNA gene. The optimal temperature, pH and lactose concentration for the synthesis of GOS using lactose as substrate were investigated, and GOS produced under optimal reaction conditions were quantified by TLC and high performance liquid chromatography (HPLC). Six lactic acid bacterial strains producing β-galactosidases with transglycosylation activity were isolated, among which β-galactosidase produced by Lactobacillus plantarum YLBGNL-S7 showed the highest transglycosylation activity. An initial lactose mass concentration of 300 mg/mL, a reaction temperature of 50 ℃, an initial pH of 6.0 and a reaction time of 4 h were determined as the optimum conditions for GOS synthesis with β-galactosidase produced by YLBGNL-S7, which resulted in a GOS yield of 43.40%. The GOS mixture contained 18.29% diaccharides and 12.95% trisaccharides. These results demonstrated that L. plantarum YLBGNL-S7 is a novel promising source of β-galactosidase with good potential for prebiotic GOS synthesis.

Key words: β-galactosidase, transglycosylation activity, galacto-oligosaccharides, Lactobacillus plantarum, cheese

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