SSCP方法的条件优化与榨菜低盐腌制微生物多样性分析

Optimization of SSCP Condition and Diversity of Microbial Community in Pickled Mustard Tuber with Low Salinity

摘要:

应用单链构象多态性(SSCP)方法研究榨菜低盐腌制条件下的微生物群落结构与变化。研究了凝胶质量浓度、电泳前处理、银染操作条件对SSCP分析方法的影响。结果表明,在凝胶质量浓度为22 g/dL、丙烯酰胺与双丙烯酰胺质量比为29∶1,并加入体积分数6%的甘油,4℃条件下1×TBE缓冲液中250 V电泳18 h,通过强碱性显影液和甲醛作还原剂的银染方法显色,可以获得理想的SSCP图谱。对榨菜低盐腌制不同时期样品的SSCP图谱分析结果,发现在榨菜腌制初期优势菌群为肠膜明串珠菌(Leuconostoc mesenteroides),随后转变为植物乳杆菌(Lactobacillus plantarum)和短乳杆菌(Lactobacillus brevis)占优势;腌制后期主要存在的优势菌群为植物乳杆菌和Lactobacillus versmoldensis,结合感官分析证实这些优势乳酸菌对保持制品高品质具有功能作用。

Abstract:

Microbial community and its changes in pickled mustard tuber were investigated at the low salinity by using single-strand conformation polymorphism(SSCP) method.Effects of gel concentration,glycerol addition,foretreatment of electrophoresis and silver staining methods on SSCP pattern were also examined in this manuscript.The results demonstrated that an ideal SSCP method was obtained as the optimum conditions,gel concentration of 22 g/dL at the crosslinking degree of 29∶1(a ratio of acrylamide and bisacrylamide),glycerol concentration of 6%,voltage of 250 V,electrophoresis buffer of 1×TBE,electrophoresis at 4 ℃ for 18 hours,and using silver staining method of strong alkaline solution and formaldehyde as reducing agent.Based on the optimized SSCP method for pickled leaching liquid measurement,it was found that the Leuconostoc mesenteroides is the dominant microorganism in the initial stage of pickling,then Lactobacillus plantarum and Lactobacillus brevis grown up with the alteration of environmental conditions.At the later stage,the dominant microorganism are Lactobacillus plantarum and Lactobacillus versmoldensis,respectively.The contribution of lactic acid bacteria was confirmed by microbial community structure analysis in pickled mustard tuber with low-salinity,which is carried out by combining the sensory evaluation method.