仿刺参(Apostichopus japonicus)肠道源乳酸乳球菌(Lactococcus lactis)的分离鉴定及其益生特性分析

ISOLATION AND IDENTIFICATION OF LACTOCOCCUS LACTIS FROM THE INTESTINAL TRACT OF SEA CUCUMBER(APOSTICHOPUS JAPONICUS)AND ITS PROBIOTIC PROPERTIES IN VITRO

利用MRS固体培养基从南移仿刺参肠道中分离得到1株优势乳酸菌,命名为AJC-XP-15,其最适生长温度为30℃,最适生长pH为6.5;其表面疏水性和自聚率分别为34.56%和35.61%,经16S RNA基因序列分析,并结合形态学和生理生化特性将其鉴定为乳酸乳球菌(Lactococcus lactis)。拮抗实验结果显示,菌株AJC-XP-15及其发酵上清液对塔氏弧菌(Vibriotubiashii)、哈维氏弧菌(V.harveyi)、副溶血弧菌(V.parahaemolyticus,VP)和嗜水气单胞菌(Aeromonas hydrophila)等病原菌具有很好的抑制作用;体外益生实验表明,菌株AJC-XP-15对人工胃肠液的耐受性很好,在pH3.0的模拟人工胃液处理3 h后的存活率为71.43%,在pH 6.8的模拟人工肠液处理3 h后的存活率为92.1%;抗氧化能力结果显示,菌株AJC-XP-15的无细胞提取物对DPPH自由基和羟自由基的清除能力最强,分别为(82.67±6.92)%和(15.36±2.95)%,其发酵上清液对超氧阴离子自由基的清除能力最强,为(26.36±2.58)%;药物敏感性分析结果显示,菌株AJC-XP-15对四环素、恩诺沙星、盐酸多西环素等抗生素敏感,对氟苯尼考、复方新诺明、庆大霉素等抗生素不敏感。研究结果可为仿刺参肠道源乳酸菌的分离鉴定和候选益生菌种筛选提供参考依据。

To obtain candidate probiotic lactic acid bacteria that can be used for sea cucumber culture,a dominant lactic acid bacterium(Lactococcus lactis AJC-XP-15)was isolated from the intestinal tract of sea cucumber(Apostichopus japonicus)by de Man,Rogosa and Sharpe agar medium.The strain was identified based on its 16S rRNA gene,and morphological,physiological,and biochemical characteristics.The antiviral ability,antioxidant activity,and drug sensitivity of the bacterium were assessed in detail.Results show that the cells and fermented supernatant of the bacterium could inhibit the growth of Vibrio tubiashii,V.harveyi,V.parahaemolyticus,and Aeromonas hydrophila;the optimal growth condition was 30℃and pH 6.5.The surface hydrophobicity and self-polymerization rate were 34.56%and 35.61%,respectively.The survival rates were 71.43%(pH 3.0)and 92.1%(pH 6.8)after 3-h artificial exposure to gastric juice.Moreover,the strongest scavenging activity of cell-free extract of the bacterium to DPPH and hydroxyl radicals was(82.67±6.92)%and(15.36±2.95)%,respectively;while those of the fermented supernatant to superoxide anion radicals was(26.36±2.58)%.The strain was sensitive to tetracycline,enrofloxacin,and doxycycline hydrochloride,but insensitive to florfenicol,trimethoprim/sulfamethoxazole,and gentamicin.This study provided a reference for the isolation and identification of lactic acid bacteria from the intestinal tract of sea cucumber(Apostichopus japonicus)and the screening for candidate probiotics.