CN106967630A - 一种发酵乳杆菌及其应用 - Google Patents
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- GOLXNESZZPUPJE-UHFFFAOYSA-N spiromesifen Chemical compound CC1=CC(C)=CC(C)=C1C(C(O1)=O)=C(OC(=O)CC(C)(C)C)C11CCCC1 GOLXNESZZPUPJE-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明公开了一种发酵乳杆菌及其应用。其保藏编号：GDMCC No.60112。本发明的发酵乳杆菌(Lactobacillus fermentum)DJ8A具有较强的耐酸和耐胆盐能力，对大肠杆菌及金黄色葡萄球菌均有明显抑菌效果，因此能用于制备抗菌药物，具有广阔的市场前景。
Description
技术领域
本发明属于微生物领域，具体涉及一种发酵乳杆菌及其应用。
发明背景
饲料中添加抗生素能够抵抗疾病，促进动物的生长，加快集约化畜牧业的发展。但是随着人们对绿色、安全和环保理念的不断加强和对健康程度的不断重视，抗生素添加剂在给人们带来巨大经济利益的同时其负面效应也逐渐凸显，最典型的特征就是抗生素引起的耐药性、内源性感染、二重感染以及药物残留，这些将对养殖业、饲料工业和动物带来严重威胁，进而通过食物链危及人类健康。因此，限制乃至禁止饲料行业和养殖业使用抗生素的呼声越来越高，研究和开发新的抗生素替代品刻不容缓。
近年来，益生菌因其安全、无毒、无残留、不诱发耐药性且对动物生长具有显著作用而被作为抗生素替代品的首要选择。乳酸杆菌是益生菌中的主要菌种，它是动物消化道栖生微生物，在防治胃肠道感染、调节免疫功能和调节肠道的微生物菌群平衡中具有较好作用，是一种很有前途的益生素生产菌种。
目前，乳酸菌类产品的开发利用已成为我国饲料行业发展的一个新趋势，但也存在一些亟待解决的问题。如益生效果不佳、产品质量不稳定、活菌数量不够、抗逆性差等。因此，筛选益生性能好、耐受能力强、繁殖速度快、质量稳定的乳酸杆菌类产品，对提高产品的市场竞争力以及促进畜牧业的发展都非常有意义。
发明内容
本发明的第一个目的是提供一种具有较强的耐酸和耐胆盐能力，对大肠杆菌及金黄色葡萄球菌均有明显抑菌效果的发酵乳杆菌(Lactobacillus fermentum)DJ8A。
本发明的发酵乳杆菌(Lactobacillus fermentum)DJ8A，其于2016年11月15日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：中国广州市先烈中路一百号省微生物所实验楼五楼，邮编：510070，保藏编号：GDMCC No.60112。
本发明的第二个目的是提供发酵乳杆菌(Lactobacillus fermentum)DJ8A在制备抗菌药物中的应用。
所述的抗菌药物是抗金黄色葡萄球菌或大肠杆菌的药物。
本发明的发酵乳杆菌(Lactobacillus fermentum)DJ8A具有较强的耐酸和耐胆盐能力，对大肠杆菌及金黄色葡萄球菌均有明显抑菌效果，因此能用于制备抗菌药物，具有广阔的市场前景。
本发明的发酵乳杆菌(Lactobacillus fermentum)DJ8A，其于2016年11月15日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：中国广州市先烈中路一百号省微生物所实验楼五楼，邮编：510070，保藏编号：GDMCC No.60112。
附图说明：
图1为本发明的发酵乳杆菌(Lactobacillus fermentum)DJ8A在MRS培养基上培养的菌落形态特征。
图2为本发明的发酵乳杆菌(Lactobacillus fermentum)DJ8A显微形态特征。
图3为本发明的发酵乳杆菌(Lactobacillus fermentum)DJ8A的发酵上清液对大肠杆菌的抑菌能力，1：发酵乳杆菌(Lactobacillus fermentum)DJ8A的发酵上清液；2：菌株DJ9B的发酵上清液；②：pH 4.45MRS空白培养基；③：pH 6.60MRS空白培养基。
图4为本发明的发酵乳杆菌(Lactobacillus fermentum)DJ8A的发酵上清液对金黄色葡萄球菌的抑菌能力，1：发酵乳杆菌(Lactobacillus fermentum)DJ8A的发酵上清液；2：菌株DJ9B 的发酵上清液；②：pH 4.45MRS空白培养基；③：pH 6.60MRS空白培养基。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制，根据本发明的实质对本发明进行的简单改进都属于本发明要求保护的范围。
实施例1：菌株的分离纯化
购买广州先烈中路菜市场所售发酵风味食品(酸菜、酸豆角、梅菜)，加适当的生理盐水进行无菌研磨，用生理盐水将研磨液稀释至合适浓度，取3个适宜稀释度的菌液涂布CaCO3-MRS(蛋白胨10g，牛肉膏10g，酵母粉4g，磷酸氢二钾2g，柠檬酸三铵2g，乙酸钠5g，葡萄糖20g，碳酸钙20g，吐温-80 1.08g，硫酸镁0.2g，硫酸锰0.05g，三级水定容至1L，调pH至7.2，灭菌备用)平板，37℃厌氧培养48h。挑取溶钙圈较大的菌落在 MRS(配方如上，无碳酸钙)平板上划线分离，反复纯化。将纯化后的菌落进行革兰氏染色、镜检及H2O2接触酶试验，挑出无芽孢、接触酶阴性的革兰氏阳性菌株，置于10％的甘油管中， -80℃保存备用。本试验共分离到81株溶钙圈较大、接触酶阴性、无芽孢的革兰氏阳性菌株。
用浓度为6mol/L的盐酸调节MRS液体培养基(蛋白胨10g，牛肉膏10g，酵母粉4g，磷酸氢二钾2g，柠檬酸三铵2g，乙酸钠5g，葡萄糖20g，吐温-80 1.08g，硫酸镁0.2g，硫酸锰0.05g，三级水定容至1L，调pH至7.2，灭菌备用)pH至4.0、3.0、2.5。将初筛获得的乳酸菌进行活化并培养至对数生长期后，按体积比2％的接种量分别接种至pH为4.0、 3.0、2.5的MRS液体培养基中，37℃厌氧培养24h，以未接种的培养基为对照，测各组菌液的OD600nm值，选取耐酸性能较好的乳酸菌进行后续实验。
本试验发现48株乳酸菌可在pH 4.0的MRS液体培养基中生长，10株乳酸菌能够在pH 3.0 环境下生长，其中菌株DJ8A和DJ10C在pH 3.0环境下生长良好。
上述试验纯化所得的菌株均保存于广东省微生物菌种保藏中心。
实施例2：菌株耐酸、耐胆盐试验
选取可在pH 3.0生长的菌株，重新培养至对数生长期后，按体积比2％接种量接种至含有0.1％、0.2％、0.3％胆盐的MRS液体培养基中，37℃厌氧培养24h，以相应胆盐含量的未接种培养基为对照，测各组菌液的OD600nm值，选取耐胆盐性能较好的乳酸菌进行后续实验。
本试验发现，实施例1中所提到的10株可在pH 3.0环境下生长的菌株均能在0.1％的牛胆盐中生长，但均不能在0.2％及0.3％牛胆盐中生长，其中菌株DJ8A在0.1％的牛胆盐中生长良好。
实施例3菌种鉴定
(1)形态学鉴定：将筛选获得的菌株涂布分离于MRS平板上，37℃厌氧培养24h后观察其菌落特征；取单菌落涂片，革兰氏染色，显微镜下观察其个体形态。
菌株DJ8A在MRS平板上37℃培养48h后，菌落呈圆形、扁平、白色，表面光滑、湿润，边缘整齐，直径3-4mm(图1)；菌株DJ8A的显微形态特征为短杆状，单个、成对或成长链，不产芽孢、革兰氏阳性(图2)。
(2)16S rRNA分子鉴定：
采用CTAB法提取待鉴定菌株DJ8A的基因组DNA，采用细菌16S rRNA通用引物进行PCR扩增。将PCR产物送到上海美吉生物医药科技有限公司测序。将测序结果提交GenBank进行Blast比对分析。
菌株DJ8A的16S rRNA基因序列经测序(其序列如SEQ ID NO.1所示)、分析，比对，结果与已报道的发酵乳杆菌Lactobacillus fermentum CECT562(T)的16S rRNA基因序列同源性达到99.65％，鉴定其为发酵乳杆菌(Lactobacillus fermentum)DJ8A，其于2016年11月 15日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：中国广州市先烈中路一百号省微生物所实验楼五楼，邮编：510070，保藏编号：GDMCC No.60112。
实施例4发酵乳杆菌(Lactobacillus fermentum)DJ8A产酸能力及抑菌能力考察
(1)产酸能力：将发酵乳杆菌(Lactobacillus fermentum)DJ8A进行活化培养48h，挑取单菌落分别接种至pH 6.60和pH 3.85的MRS液体培养基中，37℃厌氧培养15h，4℃，10000 rpm离心10min，取上清液并测定pH值，以未接种的MRS液体培养基做同等处理，作为空白对照。
培养15h后，发酵乳杆菌(Lactobacillus fermentum)DJ8A可使pH 6.60和pH 3.85的 MRS液体培养基pH分别降至4.49和3.34。
(2)抑菌能力：取发酵乳杆菌(Lactobacillus fermentum)DJ8A的发酵上清液，以大肠杆菌及金黄色葡萄球菌作为指示菌，以调至相应pH的MRS培养基为对照，以NA作为培养基，使用牛津杯打孔法进行抑菌实验。按1％接种量重新转接大肠杆菌和金黄色葡萄球菌，28℃， 180rpm振荡培养17h，其菌体浓度约为107cfu/ml，取培养好的大肠杆菌2ml或金黄色葡萄球菌2ml与200ml经融化后保持在45℃左右的NA培养基混匀，在无菌平板上放置4个灭过菌的牛津杯，用无菌移液管吸取20ml覆盖平板。凝固后取出牛津杯，分别加入100uL的乳酸菌上清液样品或调至相应pH的MRS对照培养基，37℃培养24h，观察结果。
本实验结果表明，菌株DJ8A的发酵上清液(pH 4.49)对大肠杆菌及金黄色葡萄球菌均有明显抑菌效果(表1，图3，图4)。
表1发酵乳杆菌(Lactobacillus fermentum)DJ8A发酵上清液的抑菌能力
实施例5发酵乳杆菌(Lactobacillus fermentum)DJ8A耐酸和耐胆盐考察
配制pH 2.0无胆盐的MRS液体培养基和pH 6.6且含0.3％胆盐的MRS液体培养基，将在37℃已厌氧培养15h的发酵乳杆菌(Lactobacillus fermentum)DJ8A菌液，按体积比1％接种量分别转接至pH 2.0的MRS液体培养基和含0.3％胆盐的MRS液体培养基中，37℃厌氧培养3h和6h，取合适的稀释倍数进行涂布平板，每组重复三个平行实验，37℃培养48h后，选择菌落数30-300范围的平板进行菌落计数。
结果表明发酵乳杆菌(Lactobacillus fermentum)DJ8A可在pH 3.0或含0.1％胆盐环境下生长，在pH 2.0或含0.3％胆盐条件下处理3h，活菌数仍在107cfu/mL以上，处理6h，活菌数仍在106cfu/mL以上，说明菌株DJ8A具有较强的耐酸和耐胆盐能力(表2)。
表2发酵乳杆菌(Lactobacillus fermentum)DJ8A耐酸和耐胆盐特性
序列表
<110>广东省微生物研究所（广东省微生物分析检测中心）
<120>一种发酵乳杆菌及其应用
<160>1
<210>1
<211>861
<212>DNA
<213>发酵乳杆菌（Lactobacillus fermentum）DJ8A
<400> 1
TGCAGTCGAA CGCGTTGGCC CAATTGATTG ATGGTGCTTG CACCTGATTG ATTTTGGTCG 60
CCAACGAGTG GCGGACGGGT GAGTAACACG TAGGTAACCT GCCCAGAAGC GGGGGACAAC 120
ATTTGGAAAC AGATGCTAAT ACCGCATAAC AACGTTGTTC GCATGAACAA CGCTTAAAAG 180
ATGGCTTCTC GCTATCACTT CTGGATGGAC CTGCGGTGCA TTAGCTTGTT GGTGGGGTAA 240
TGGCCTACCA AGGCGATGAT GCATAGCCGA GTTGAGAGAC TGATCGGCCA CAATGGGACT 300
GAGACACGGC CCATACTCCT ACGGGAGGCA GCAGTAGGGA ATCTTCCACA ATGGGCGCAA 360
GCCTGATGGA GCAACACCGC GTGAGTGAAG AAGGGTTTCG GCTCGTAAAG CTCTGTTGTT 420
AAAGAAGAAC ACGTATGAGA GTAACTGTTC ATACGTTGAC GGTATTTAAC CAGAAAGTCA 480
CGGCTAACTA CGTGCCAGCA GCCGCGGTAA TACGTAGGTG GCAAGCGTTA TCCGGATTTA 540
TTGGGCGTAA AGAGAGTGCA GGCGGTTTTC TAAGTCTGAT GTGAAAGCCT TCGGCTTAAC 600
CGGAGAAGTG CATCGGAAAC TGGATAACTT GAGTGCAGAA GAGGGTAGTG GAACTCCATG 660
TGTAGCGGTG GAATGCGTAG ATATATGGAA GAACACCAGT GGCGAAGGCG GCTACCTGGT 720
CTGCAACTGA CGCTGAGACT CGAAAGCATG GGTAGCGAAC AGGATTAGAT ACCCTGGTAG 780
TCCATGCCGT AAACGATGAG TGCTAGGTGT TGGAGGGTTT CCGCCCTTCA GTGCCGGAGC 840
TAACGCATTA AGCACTCCGC C 861
Claims (3)
1.发酵乳杆菌(Lactobacillus fermentum)DJ8A，其保藏编号为GDMCC No.60112。
2.权利要求1所述的发酵乳杆菌(Lactobacillus fermentum)DJ8A在制备抗菌药物中的应用。
3.根据权利要求2所述的应用，其特征在于，所述的抗菌药物是抗金黄色葡萄球菌或大肠杆菌的药物。
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