Development of safe food using lactic acid bacteria

采用鸡和人粪便中分离的乳酸菌和双歧杆菌进行黏附实验，筛选出对鸡作物上皮、人肠道Caco-2细胞和HT-29细胞以及固定化细胞外基质蛋白具有较强黏附的菌株。为了快速鉴定crispatus乳杆菌，我们新开发了针对s层蛋白基因的PCR引物（Horie et al.2002）。黏附性乳酸菌和双歧杆菌抑制沙门氏菌等病原菌对Caco-2细胞的黏附。在厌氧条件下，平板培养可诱导L.crispatus和L.gasseri的黏附。鉴定出一种46.5kDa的s层蛋白LbsA为高黏附性crispatus菌株MH 315的黏附蛋白。对LbsA编码区进行克隆和测序。将预测的氨基酸序列与其他乳酸菌菌株的s层蛋白比对表明，LbsA中存在层粘连蛋白结合位点（Mukai et al.2003）。Western bolting法鉴定了一种58.7kDa的表面蛋白，该蛋白被认为是l.g assseri MH 411的粘附素。我们还选择了产生过氧化氢（H_2O_2）的乳酸乳球菌亚球菌。乳酸菌和植物芽孢杆菌来自食物。在测试条件下，他们生产出了百万分之三百的H_2O_2。其中一种菌株，乳酸乳酸杆菌亚种。乳酸菌AI62能有效抑制伊万诺氏李斯特菌、小肠结肠炎耶尔森菌和亲水气单胞菌等病原体（Ito et al.2003）。

Adhesion assay was performed using Lactobacillus and Bifidobacterium strains isolated from chicken and human faeces to select the strains which strongly adhered to chicken crop epithelium, human intestinal Caco-2 cells and HT-29 cells, and immobilized extracellular matrix proteins. For rapid identification of Lactobacillus crispatus we newly developed PCR primers targeting S-layer protein gene(Horie et al.2002). Adhesive lactobaciller and bifidobacterial strains inhibited the adhesion of pathogens such as Salmonella to Caco-2 cells Adhesion of L.crispatus and L.gasseri strains tested were induced on plate cultures under anaerobic conditions. A 46.5kDa S-layer protein(named as LbsA) was identified as an adhesin of highly adhesive L.crispatus MH 3 1 5 strain. The DNA region coding for LbsA was cloned and sequenced. Alignment comparisons of the predicted amino acid sequence with S-layer proteins of other Lactobacillus strains suggested the presence of laminin-binding site in LbsA (Mukai et al.2003). An 58.7kDa surface protein was identified an putative adhesin of L.gasseri MH 411 by Western bolting assay.We also selected hydrogen peroxide(H_2O_2)-producing Lactococcus lactis subsp.lactis and L.plantarum strains from foods. They produced >300ppm H_2O_2 under the condition tested. One of these strains, L.lactis subsp.lactis AI62 inhibited pathogens such as Listeria ivanovii, Yersinia enterocolitica and Aeromonas hydrophila efficiently(Ito et al.2003).