真空冷冻干燥保护剂对植物乳杆菌67黏附能力的影响

Effect of vacuum freeze-drying protective agent on adhesion of Lactobacillus plantarum 67

黏附是乳酸菌在机体内发挥益生作用的关键,研究冻干保护剂对乳酸菌黏附能力的影响有助于益生菌产品的开发及其功能的进一步挖掘。研究单一保护剂对真空冷冻干燥后植物乳杆菌67的黏附率和存活率等的影响,筛选出保护效果较优的单一保护剂;并以黏附率和存活率等为指标优化保护剂配方,探究优化的复合保护剂对冻干后菌株67表面微观形态、黏附Caco-2细胞能力及其抑制肠道致病菌黏附Caco-2细胞能力的改善作用。结果表明,菌株67在分别去除了脱脂乳(skim milk,S)、菊粉(inulin,L)、蔗糖(sucrose,C)、谷氨酸钠(sodium glutamate,G)及海藻糖(trehalose,T)的5种复合保护剂中冻干后,对Caco-2细胞的黏附率均显著低于其他复合保护剂(P<0.05);在分别去除L、G和山梨醇后,存活率均显著低于其他复合保护剂(P<0.05)。菌株67分别在140 g/L S、80 g/L L、60 g/L C、90 g/L G及100 g/L T中冻干后对Caco-2细胞的黏附率均大于5.85%,显著高于各自保护剂的其他浓度(P<0.05);分别在120 g/L S、100 g/L L、100 g/L C、70 g/L G及80 g/L T中冻干后的存活率均大于18.34%,显著高于各自保护剂的其他浓度(P<0.05)。菌株67在含有160 g/L S、100 g/L L、40 g/L C、70 g/L G及120 g/L T的优化复合保护剂中冻干后,其黏附率和存活率分别为28.73%和93.52%,均显著高于未优化的20.47%和76.31%(P<0.05);较未优化的复合保护剂而言,菌体形态变化较小、未破裂、未塌缩,能更紧密地黏附于Caco-2细胞上,且冻干前后的黏附素均为表面蛋白;同时,菌株抑制金黄色葡萄球菌和大肠杆菌黏附Caco-2细胞的能力也得到了显著提高(P<0.05)。S、L、C、G及T对冻干后植物乳杆菌67的黏附率和存活率产生较大的影响,优化后的复合保护剂对冻干后植物乳杆菌67的黏附能力、存活能力及其抑制肠道致病菌黏附肠上皮细胞的能力有良好的保护作用。

Adhesion is the key for lactic acid bacteria(LAB)to play a probiotic role in the body,the research of the effect of freeze-dried protective agents on the adhesion of LAB will help to develop probiotic products and further explore their functions.The effects of the single protectant on the adhesion rate and survival rate of Lactobacillus plantarum 67 after vacuum freeze-drying were studied,and a single protective agent with better protection effect were selected,and the composite protective agent was optimized according to the adhesion rate and survival rate.The improvement effect of the optimized composite protective agent on the surface microscopic morphology,the ability to adhere to Caco-2 cells,and the ability to inhibit intestinal pathogens from adhering to Caco-2 cells of strain 67 after freeze-drying were explored.The results showed that the adhesion rate to Caco-2 cells of strain 67 was significantly decreased after freeze-drying in 5 kinds of composite protectors that removed skim milk(S),inulin(L),sucrose(C),sodium glutamate(G)and trehalose(T),respectively(P<0.05),and the survival rate was significantly decreased after freeze-drying in 3 kinds of composite protectors that removed L,G and sorbitol respectively(P<0.05).The adhesion rate of strain 67 to Caco-2 cells were greater than 5.85%after freeze-drying in 140 g/L S,80 g/L L,60 g/L C,90 g/L G and 100 g/L T respectively,which significantly higher than other concentrations of their respective protective agents(P<0.05),and the survival rate were greater than 18.34%after freeze-drying in 120 g/L S,100 g/L L,100 g/L C,70 g/L G and 80 g/L T respectively(P<0.05),which significantly higher than other concentrations of their respective protective agents.The adhesion rate and the survival rate of strain 67 were 28.73%and 93.52%respectively when the optimized composite protectant that contain 160 g/L S,100 g/L L,40 g/L C,70 g/L G and 120 g/L T,and which significantly higher than not optimization that were 20.47%and 76.31%(P<0.05).Compared with the not optimization composite protectant,the change of cell morphology was smaller,and the strain 67 adhere to Caco-2 cells was more closely and without breaking,without collapsing,and the adhesin was surface proteins before and after freeze-drying.At the same time,the ability of strain 67 to inhibit the adhesion of Staphylococcus aureus and Escherichia coli to Caco-2 cells was significantly improved(P<0.05).S,L,C,G and T have a great impact on the adhesion rate and the survival rate of L.plantarum 67,and the optimized composite protective agent has a good protective effect on the adhesion ability,the survival ability and the ability to inhibit intestinal pathogenic bacteria from adhering to intestinal epithelial cells of L.plantarum 67 after freeze-drying.