CN103981115B - 一株高抗逆性屎肠球菌及其应用 - Google Patents
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Abstract
本发明涉及一株高抗逆性屎肠球菌及其应用。屎肠球菌(Enterococcus faecium)WEI‑9CGMCC No.7455分离自60日龄仔猪粪便，能够耐受低pH环境和高浓度猪胆盐，可以在胃肠消化液中长时间保持活性。本发明的屎肠球菌(Enterococcus faecium)WEI‑9CGMCCNo.7455对大肠杆菌、沙门氏菌、藤黄微球菌、金黄色葡萄球菌均有抑菌作用，抑菌圈直径达到30‑40mm。借此开发的微生态活菌制剂具有显著的益生特性，可以用于调节畜禽肠道微生态平衡，并在预防或辅助治疗畜禽细菌性腹泻有着广阔的应用前景。
Description
技术领域
本发明涉及一种屎肠球菌，特别是涉及一株高抗逆性屎肠球菌及其应用。
背景介绍
食品安全已是人们越来越关注的问题。饲料作为动物的食品和人类的间接食品，成为食品安全工程的重要一环。我国已启动“饲料安全工程”，并禁用了一批毒性和残留较大的饲料添加剂产品。加快安全性较高的新型饲料添加剂产品的开发与生产，对于实现健康养殖及养殖业可持续发展具有重大的现实意义。微生态制剂作为一种绿色、安全的饲料添加剂受到人们的广泛关注，对于动物健康和养殖业的促进作用正在逐渐的显现出来。
然而，目前市售微生态制剂产品质量参差不齐，很多产品所用菌种对胃肠消化液、胆盐耐受能力差，真正能够到达小肠发挥功效的菌种很少。为了提高产品效果，不少企业推出微囊化的微生态制剂产品，虽然提高了进入小肠的活菌数，但也大幅增加了生产成本。开发对胃肠消化液、胆盐有高耐受性的益生菌菌株能大大简化生产工艺，降低生产成本。
屎肠球菌是农业部《饲料添加剂品种目录》所列菌种，可以在微生态制剂中安全使用。屎肠球菌具有很强的产酸能力，是动物体内天然存在的有益乳酸菌之一，能够维持宿主肠道微生态平衡，抑制肠道病原菌的大量增殖，能够刺激肠道粘膜免疫，提高机体的抗病能力，产生有益的代谢产物，防止有害物质产生，改善机体健康水平。
因此，开发一种能够耐受胃肠消化液和胆盐，并对病原微生物有较强抑菌活性的屎肠球菌菌株，对于降低微生态制剂生产成本，强化微生态制剂功效具有重要的意义。
发明内容
本发明的目的是提供一株可用于调节畜禽肠道微生态平衡及预防畜禽细菌性腹泻的高抗逆性屎肠球菌(Enterococcus faecium)WEI-9CGMCC No.7745。
从健康仔猪粪便中分离、筛选得到一株乳酸菌，经16S rDNA测序鉴定为屎肠球菌(Enterococcus faecium)WEI-9，并于2013年6月19日送中国微生物菌种保藏管理委员会普通微生物中心保藏，编号为CGMCC No.7745，保藏地址：北京市朝阳区北辰西路1号院中科院微生物研究所邮编：100101电话：86-10-64807596。
屎肠球菌(Enterococcus faecium)WEI-9CGMCC No.7745属革兰氏阳性细菌，圆形或椭 圆形，呈链状排列，无芽孢，无鞭毛。生长温度范围在30-40℃，适宜pH值的范围6.0-7.5，是兼性厌氧乳酸菌。该菌株在常规和改良MRS培养基中生长良好，繁殖速度快，在改良MRS培养基中的生长曲线如图1所示，2h后进入对数期，14h菌数增殖达到最高，稳定期活菌数为0.98×109-1.2×109CFU／mL。所述改良MRS配方为：每1000mL水中加入蛋白胨10g，牛肉粉5g，酵母粉5g，葡萄糖20g，吐温801mL，K2HPO4·7H2O2g，NaAC·3H2O5g，柠檬酸铵2g，MgSO4·7H2O0.58g，MnSO4·H2O0.25g，pH6.8。
屎肠球菌(Enterococcus faecium)WEI-9CGMCC No.7745在pH为2-3的人工胃液中保持3h，存活率80％；在人工胃肠消化液中保持26h，活菌数可以维持在107CFU／mL，这个时间符合肠道生理周期，能够保证菌体发挥益生作用；在猪胆盐浓度为20g／L的人工猪胆盐溶液中，保持3h，存活率为51％，这个胆盐浓度远远高于动物体内的0.3g／L的平均胆盐浓度。
上述特性评价试验中，所述人工胃液包括如下成分：每1000mL水中含有胃蛋白酶3g，NaCl2g，调pH至2-3；所述人工胃肠消化液包括如下成分：人工胃液pH调至3，人工肠液包括如下成分：每1000mL水中含有胰蛋白酶10g，KH2PO47g，调pH至7.5，测试过程中，菌体在人工胃液中保持2h，然后接入人工肠液中保持24h；所述人工猪胆盐溶液成分包括：每1000mL水中含有胰蛋白酶1g，猪胆盐20g，NaCl5g，调pH至7.0。
屎肠球菌(Enterococcus faecium)WEI-9CGMCC No.7745对大肠杆菌、沙门氏菌、藤黄微球菌、金黄色葡萄球菌均有抑菌作用，抑菌圈直径达到30-40mm。
基于上述特性，本发明提供的屎肠球菌(Enterococcus faecium)WEI-9CGMCCNo.7745具有显著的益生特性，可以在畜禽肠道定殖，调节畜禽肠道微生态平衡，并在预防或辅助治疗畜禽细菌性腹泻有着广阔的应用前景。
附图说明
图1为屎肠球菌(Enterococcus faecium)WEI-9CGMCC No.7745在改良MRS培养基中的生长曲线。
图2为屎肠球菌(Enterococcus faecium)WEI-9CGMCC No.7745对人工胃肠消化液的耐受性。图中前2h是菌株在人工胃液中的存活曲线，随后的24h是菌株从人工胃液转接至人工肠液后的存活曲线。
图3为屎肠球菌(Enterococcus faecium)WEI-9CGMCC No.7745对金黄色葡萄球菌的抑菌效果，图中是两个相同剂量实验组。
具体实施方式
实施例一：屎肠球菌(Enterococcusfaecium)WEI-9CGMCC No.7745的分离和鉴定
称取60日龄健康仔猪的粪便1g，放入盛有99mL灭菌的0.85％生理盐水的三角瓶中(三角瓶中预先放入20粒无菌玻璃珠)，37℃，180rpm震荡30min，吸取1mL悬浊液做十倍递增梯度稀释，选取2-3个适宜稀释度，吸取1mL稀释液于无菌培养皿中，每个稀释度做两个平行；向加有稀释液的培养皿中加入融化后冷却至46℃的MRS琼脂培养基，小心转动培养皿使试样与培养基充分混合，37℃培养48-72h。从培养皿上挑选不同形态的菌落划线纯化，分别保藏并进行16S rDNA的测序鉴定。
实施例二：屎肠球菌(Enterococcus faecium)WEI-9CGMCC No.7745耐受低pH胃液、人工胃肠消化液和猪胆盐
从屎肠球菌(Enterococcus faecium)WEI-9CGMCC No.7745的保藏斜面(常规MRS培养基)挑一环菌苔接于装有100mL改良MRS培养基的250mL三角瓶中，于37℃，180rpm振荡培养14h，将菌液在6000rpm的条件下离心10min，弃去上清，用灭菌的0.85％的生理盐水洗涤一次，再用同体积生理盐水悬浮。吸取10mL悬浮菌液分别接于装有90mL pH分别为1、2、3、4、5的人工胃液和90mL人工猪胆盐溶液的250mL三角瓶中，猪胆盐浓度分别为3g/L、5g／L、10g／L、15g／L、20g/L，以不加胆盐的样品为对照。然后在37℃，静置培养180min，分别于初始接种、30min、60min、120min、180min对培养物进行活菌计数，绘制活菌数变化曲线，计算菌株在不同pH人工胃液和不同浓度人工胆盐中保温180min时的存活率。菌液在人工胃液中保温2h后吸取10mL菌悬液接于装有90mL人工肠液的250mL三角瓶中，同样在37℃，静置培养，分别于初始接种、3h、6h、21h、24h对培养物进行活菌计数，绘制活菌数变化曲线。测定结果显示，屎肠球菌(Enterococcus faecium)WEI-9CGMCC No.7745在pH高于1的人工胃液中保持180min(3h)，存活率达到80％以上；在浓度为20g/L的人工猪胆盐溶液中保持180min(3h)，存活率超过50％；在人工胃肠消化液中保持26h，活菌数可以维持在107CFU／mL，这个时间符合肠道生理周期，能够保证菌体发挥益生作用。上述结果表明，屎肠球菌(Enterococcus faecium)WEI-9CGMCC No.7745能够耐受酸性环境，耐受高浓度猪胆盐，并且能够在消化液中保持足够长的存活时间，说明屎肠球菌(Enterococcus Faecium)WEI-9CGMCC No.7745是一株高抗逆性益生乳酸菌，可以经过畜禽的胃、十二指肠后保持活性，并最终进入小肠定殖。
实施例三：屎肠球菌(Enterococcusfaecium)WEI-9CGMCC No.7745的抑菌活性
1.培养液上清的制备
从屎肠球菌(Enterococcus Faecium)WEI-9CGMCC No.7745的保藏斜面(常规MRS培养基)挑一环菌苔接种于液体改良MRS培养基中，37℃，180rpm培养14h，取1mL菌液12000rpm离心1min，取上清液。
2.抑菌活性检测平板的制备
将15mL完全熔化的LB琼脂培养基倒入培养皿中，待其凝固后垂直放上牛津杯；另取完全熔化的LB琼脂培养基，待其冷却到55℃时加入指示菌混匀，取10mL混有指示菌的培养基倒入放有牛津杯的培养皿中，待完全凝固后拔出牛津杯，制成检测平板。所述指示菌包括大肠杆菌、沙门氏菌、藤黄微球菌、金黄色葡萄球菌。
3.抑菌活性的测定
取200μL培养液上清加入检测平板的孔中，4℃放置24h后放入37℃培养箱中培养20h，测量抑菌圈直径大小为30-40mm。
Claims (3)
1.一株高抗逆性屎肠球菌 (Enterococcus faecium)WEI-9，保藏编号为CGMCC No.7745。
2.根据权利要求1所述的高抗逆性屎肠球菌，其特征在于，所述屎肠球菌WEI-9可在人工胃液或人工胃肠消化液或猪胆盐中存活，并对大肠杆菌、沙门氏菌、藤黄微球菌和金黄色葡萄球菌有抑菌作用。
3.权利要求1所述保藏编号为CGMCC No. 7745的屎肠球菌WEI-9的应用，其特征在于，屎肠球菌WEI-9可用于制备调节畜禽肠道微生态平衡的制剂。
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