CN114854647B - 发酵乳杆菌及其培养和应用 - Google Patents
发酵乳杆菌及其培养和应用 Download PDFInfo
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- CN114854647B CN114854647B CN202210683693.3A CN202210683693A CN114854647B CN 114854647 B CN114854647 B CN 114854647B CN 202210683693 A CN202210683693 A CN 202210683693A CN 114854647 B CN114854647 B CN 114854647B
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- lactobacillus fermentum
- fermentation
- culture
- lactobacillus
- lactic acid
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Classifications
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明公开了发酵乳杆菌(Lactobacillus fermentum)SS‑31。研究结果显示,本发明的发酵乳杆菌SS‑31能够减轻细胞中NO的分泌,以及炎症因子IL‑1β、IL‑6、IL‑10。由此可见,发酵乳杆菌SS‑31具有较强抗炎活性。此外,发明人还研制了专门用于发酵乳杆菌SS‑31的高密度发酵培养基及其培养方法,该培养基成分简单无须加入牛肉粉和蛋白胨,且配制简便,操作快捷,具有很好的应用前景;该培养方法提高发酵设备的利用效率,可以实现高密度富集培养,从而使得发酵成本显著降低。综上,本发明实现了发酵乳杆菌SS‑31的高密度发酵培养,以便今后应用于进一步开发药物、保健品、食品或饲料等。
Description
技术领域
本发明属于微生物发酵技术领域,尤其涉及一株发酵乳杆菌及其培养和应用。
背景技术
炎症性肠病是一种慢性复发性疾病,因起病缓慢、病因不详、易反复发作且大多难以完全治愈。治疗炎症性肠病的手段主要有药物治疗、手术治疗。其中,药物治疗虽然能有效缓解症状,但长期的药物治疗会导致致病菌对抗生素有耐药性,而手术治疗价格昂贵。因此,探索新的安全可靠的治疗方法成为了亟待解决的问题。随着研究的深入发展,益生菌制剂被认为是一种新型辅助疗法。益生菌能够发挥调节肠道菌群,对炎症性肠病预防和治疗效果显著,且不会产生耐药性和不良反应等优势,益生菌有望成为预防或辅助治疗炎症性肠病的重要手段之一。
乳酸菌能够调节肠道菌群平衡、诱导宿主免疫***的非特异性激活、抗氧化、降血糖、抗肠炎、免疫调节等生理活性。近年来开发具有益生功能并对人类安全的乳酸菌株的研究报道很多,而且已有将这些菌株应用于药物或功能性食品。例如:
中国专利“植物乳杆菌CQPC02在制备预防肝脏氧化损伤的食品或药品中的应用”(专利号201811639687.8公开日2019年04月16日)
中国专利“乳酸菌、来源于该乳酸菌的天然免疫活化剂、感染症预防/治疗剂和饮食品”(专利号201980004577.0公开日2020年06月23日)
中国专利“一株可缓解高尿酸血症的植物乳杆菌及其用途”(专利号202011515393.1公开日2021年05月14日)
中国专利“新颖植物乳杆菌、乳酸菌组合物及其用以治疗或预防重金属相关疾病的用途”(专利号201911052816.8公开日2020年05月26日)
尽管如此,目前还没有针对乳酸菌预防或治疗炎症性肠病的相关报道,并且乳酸菌要在体内的数量达到一定水平时,才能产生一定的益生效果。而当前乳酸菌的较低发酵水平限制了其扩大应用,因此,利用菌株的高密度培养技术实现乳酸菌作为主要成分的功能性食品、饲料中达到添加益生菌至相应数量。实现菌株的高密度培养的核心是发酵培养基及发酵条件的优化。乳酸菌的生长繁殖需要碳源、氮源、无机盐、营养因子等营养物质,并且在生长过程需要不断与外界进行物质能量交换,受到外界环境(温度、pH等)影响。高密度培养技术是通过一定培养技术和设备,改变培养条件,或者外加其他试剂使得液体培养的菌体密度超过普通培养的方法。与普通培养相比,高密度培养既可以更快速提高菌体密度,缩短生产周期,又能降低设备生产成本。因此,乳酸菌的高密度发酵富集培养是当前开发具有抗炎活性益生菌产品研发过程的关键技术。
发明内容
本发明要解决的技术问题是提供一株发酵乳杆菌及其培养和应用,具体是一株具有抗炎活性的乳酸菌和发酵培养,以及将其用于开发功能保健食品、饲料、饲料发酵剂。
为解决上述技术问题,本发明采用以下技术方案:
一株发酵乳杆菌,为发酵乳杆菌(Lactobacillus fermentum)SS-31,保藏编号为CGMCC NO:24925。
上述发酵乳杆菌16S rDNA基因具有序列表SEQ.ID.NO.1的碱基序列。
上述发酵乳杆菌的高密度发酵培养基,培养基中碳源为葡萄糖、麦芽糖、蔗糖、乳糖中一种或多种,氮源为蛋白胨、酵母浸粉、牛肉胨、胰蛋白胨中一种或多种,无机盐分别为磷酸氢二钾、磷酸二氢钾、柠檬酸铵、醋酸钠中一种或多种。
培养基中碳源为麦芽糖,氮源为酵母浸粉,无机盐为磷酸氢二钾。
上述发酵乳杆菌的高密度发酵培养基,为MRS液体培养基,每1L MRS液体培养基由以下含量成分组成:麦芽糖15.00g、酵母浸粉20.00g、磷酸氢二钾9.00g、硫酸锰0.50g,硫酸镁1.00g,吐温80 1.00g,水1000mL,调节pH至6.5,115℃高压灭菌20min。
上述发酵乳杆菌的高密度发酵培养方法,将发酵乳杆菌种子液接种到权利要求5的高密度发酵培养基中,接种量1%-5%(v/v),调整初始发酵pH值为5.8-7.8,发酵后采用15%-25%的氨水溶液控制发酵液pH值6.8±0.02,32-42℃培养12-24h。
上述发酵乳杆菌在药物、保健品、食品或饲料中的应用。
药物为抗炎药物。
乳酸菌饮料,含有上述发酵乳杆菌,其活菌数达到108CFU/mL以上。
生物饲料发酵剂,含有上述发酵乳杆菌的发酵液,其发酵培养液中碳源为麦芽糖和蔗糖复合物并含有纤维素酶。
发明人在前期研究中,从广西柳州酸笋中分离得到了本发明的发酵乳杆菌(Lactobacillus fermentum)SS-31。通过采用脂多糖诱导RAW264.7巨噬细胞建立细胞炎症模型,采用CCK-8法测定细胞活力,ELISA试剂盒测定NO、IL-1β、IL-6、IL-10释放量评价抗炎活性。结果显示,本发明的发酵乳杆菌SS-31能够减轻细胞中NO的分泌,以及炎症因子IL-1β、IL-6、IL-10。由此可见,发酵乳杆菌SS-31具有较强抗炎活性。此外,发明人还研制了专门用于发酵乳杆菌SS-31的高密度发酵培养基及其培养方法,该培养基成分简单无须加入牛肉粉和蛋白胨,且配制简便,操作快捷,具有很好的应用前景;该培养方法提高发酵设备的利用效率,可以实现高密度富集培养,从而使得发酵成本显著降低。
综上,本发明丰富了柳州酸笋源乳酸菌的功能研究,在发挥益生菌功效的同时,有利于推广广西特色发酵食品的菌株资源;同时,本发明实现了发酵乳杆菌SS-31的高密度发酵培养,以便今后应用于进一步开发药物、保健品、食品或饲料等。
附图说明
图1为发酵乳杆菌SS-31的16S rDNA PCR扩增产物的琼脂糖凝胶电泳图,图中:1Marker,2发酵乳杆菌SS-31。
图2为不同浓度发酵乳杆菌SS-31和LPS处理对RAW.264.7细胞存活率的影响结果图。
图3为发酵乳杆菌SS-31对LPS刺激RAW264.7细胞产生的NO含量结果图。
图4为发酵乳杆菌SS-31对LPS刺激RAW264.7细胞产生的IL-1β含量结果图。
图5为发酵乳杆菌SS-31对LPS刺激RAW264.7细胞产生的IL-6含量结果图。
图6为发酵乳杆菌SS-31对LPS刺激RAW264.7细胞产生的IL-10含量结果图。
图2至图6中*P<0.05表示与正常组相比的差异
保藏信息说明
发酵乳杆菌(Lactobacillus fermentum)SS-31,保藏编号为CGMCC NO:24925,保藏日期:2022年_05_月_19_日,保藏地址为:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101,保藏单位:中国普通微生物菌种保藏管理中心。
保藏条件:将富集培养后的菌液与已灭好的甘油充分混合,使终浓度控制在20-30%之间,分装至甘油管中,最后置于-80℃冰箱或液氮罐保藏。
具体实施方式
实施例1发酵乳杆菌SS-31抗炎活性评价及鉴定
1.发酵乳杆菌SS-31的测序鉴定
(1)甘油管活化:将甘油管中保藏的发酵乳杆菌SS-31划线于MRS固体培养基平板上培养,37℃培养48h。每1L MRS固体培养基由以下含量成分组成:MRS液体培养基1000mL,琼脂15g。每1L MRS液体培养基由以下含量成分组成:碳源20.00g、胰蛋白胨20.00g、磷酸氢二钾9.00g、硫酸锰0.50g,硫酸镁1.00g,吐温80 1.00g,水1000mL,调节pH至6.5,115℃高压灭菌20min。
(2)液体活化:挑取单菌落接种于含有10mL MRS液体培养基的试管中静止培养,37℃培养24h进行一次活化,将菌悬液体以2%(v/v)的接种量接种于含有100mL MRS液体培养基中,37℃静止培养24h进行二次活化,得到种子液。
(3)鉴定:将上述种子液以2%(v/v)的接种量接种到增殖培养基种,调节初始pH为6.8,置于37℃恒温箱内培养24h,划线到MRS固体培养基培养48h,挑取单菌落进行PCR。16SrRNA基因扩增采用反向引物1492R(5’-AGAGTTTGATTTGATCCTGGCTAG-3’,SEQ.ID.NO.2)、正向引物27F(5’-GGTTACCTTGTTACGACTT-3’,SEQ.ID.NO.3)进行,以25μL反应体系进行PCR扩增,采取94℃预变性5min、94℃变性1min、64℃退火1min、72℃延伸2min,35个循环,4℃保存。扩增结束后取4μL PCR产物进行1%琼脂糖凝胶电泳观察,结果见图1。测序,将测序得到的序列与NCBI的GenBank数据库进行同源比对分析,结果表明本发明菌株SS-31为发酵乳杆菌,16s rDNA序列如SEQ.ID.NO.1所示2.乳酸菌抗炎活性的测定
(1)细胞的培养:RAW 264.7巨噬细胞在含有10%胎牛血清的DMEM中培养。将处于对数期的细胞以2.5×105个/mL接种于24孔板中,每孔500μL,并在37℃、5%CO2的加湿培养箱中过夜培养,用于后续实验。
(2)细胞活力的测定:将胰酶消化后的RAW264.7细胞悬液,通过血细胞计数法将浓度调整为1.0×104个/孔,均匀接种于96孔板,37℃、5%CO2培养箱中培养至融合态。加入1μg/mL LPS分别刺激24h后,加入10μL已活化的不同浓度(1×105、1×106、1×107、1×108、1×109CFU/mL)的菌液培养3h。每孔加入10μL CCK-8溶液,37℃孵育1h后于450nm处测定吸光度值,评估不同浓度乳酸菌对RAW264.7细胞的影响。细胞存活率计算公式如下:
式中:As实验组含有细胞的培养基、CCK-8、乳酸菌;Ac模型组含有细胞的培养基、CCK-8、无乳酸菌;Ab对照组不含细胞和乳酸菌的培养基、CCK-8。
(4)NO的测定:将胰酶消化后的RAW264.7细胞悬液,通过血细胞计数法将浓度调整为2.5×105cell/mL,均匀接种于24孔板,37℃、5%CO2培养箱中培养至融合态。加入1μg/mLLPS刺激24h,然后加入50μL已活化浓度为1×108CFU/mL的菌液培养3h,收集上清液。根据NO检测试剂盒说明书操作,计算NO质量浓度公式如下:
注:式中:c表示标准品(亚硝酸钠标准液,浓度20μmol/L);n为稀释倍数(n=4)。
从图2可以看出,随着乳酸菌浓度增大,对细胞生长促进作用逐渐增强。与正常组相比,当菌体菌体浓度为109CFU/mL时,SS-31对细胞存活率为111.61%,具有显著差异(P<0.05);当菌体浓度为108CFU/mL时,SS-31的细胞存活率97.05%虽有差异,但不显著(P>0.05);与正常组相比,菌体浓度105、106、107CFU/mL时细胞存活率均显著性降低(P<0.05),且细胞存活受到抑制。综上选择菌体浓度为108CFU/mL作为后续实验的最佳作用浓度。
从图3可以看出,发酵乳杆菌SS-31和LPS处理后的RAW264.7分泌NO的水平。LPS诱导RAW264.7细胞分泌大量NO(P<0.05),乳酸菌SS-31能够显著抑制NO分泌(P<0.05),抑制率为84.31%。
(5)炎症因子IL-1β、IL-6、IL-10的测定:将胰酶消化后的RAW264.7细胞悬液,调整为2.5×105cell/mL,均匀接种于24孔板,37℃、5%CO2培养箱中培养至融合态。加入浓度为1μg/mL的LPS刺激24h,然后加入50μL已活化浓度为1×108CFU/mL的菌液培养3h,收集上清液。根据ELISA试剂盒说明书操作步骤测定细胞上清液中炎症因子IL-1β、IL-6、IL-10的含量。
从图4可以看出,LPS诱导RAW264.7细胞分泌大量炎症因子IL-1β(P<0.05),乳酸菌SS-31能够显著抑制IL-1β分泌(P<0.05),抑制率为49.21%。
从图5可以看出,LPS诱导RAW264.7细胞分泌大量炎症因子IL-6(P<0.05),乳酸菌SS-31能够显著抑制IL-6分泌(P<0.05),抑制率为14.12%。
从图6可以看出,LPS诱导RAW264.7细胞降低促炎因子IL-10的分泌(P<0.05),乳酸菌SS-31能够显著促进IL-10分泌(P<0.05),促进率为86.80%。
以上研究结果表明发酵乳杆菌在体外抗炎上有明显的效果,可应用在抗炎药物、功能发酵食品上。
实施例2发酵培养基成分的优化
(1)发酵培养基碳源种类的影响
按照实施例1中的方法,在含如下组成的发酵培养基中培养:碳源20.00g/L、胰蛋白胨20.00g、磷酸氢二钾9.00g、硫酸锰0.50g,硫酸镁1.00g,吐温80 1.00g,调节pH至6.5,115℃高压灭菌20min。其中,碳源分别为葡萄糖、麦芽糖、蔗糖、乳糖。所得发酵液中活菌数如表1所示,表1中小写字母不同表示不同菌株之间存活率差异的显著性(P<0.05)。
表1不同碳源条件下对发酵乳杆菌SS-31生长的影响
(2)发酵培养基氮源种类的影响
按照实施例1中的方法,在含如下组成的发酵培养基中培养:麦芽糖20.00g/L、氮源20.00g、磷酸氢二钾9.00g、硫酸锰0.50g,硫酸镁1.00g,吐温80 1.00g,调节pH至6.5,115℃高压灭菌20min。其中,氮源分别为蛋白胨、酵母浸粉、牛肉胨、胰蛋白胨。所得发酵液中活菌数如表2所示,表2中小写字母不同表示不同菌株之间存活率差异的显著性(P<0.05)。
表2不同氮源条件下对发酵乳杆菌SS-31生长的影响
(3)发酵培养基无机盐种类的影响
按照实施例1中的方法,在含如下组成的发酵培养基中培养:麦芽糖20.00g/L、酵母浸粉20.00g、无机盐9.00g、硫酸锰0.50g,硫酸镁1.00g,吐温80 1.00g,调节pH至6.5,115℃高压灭菌20min。其中,无机盐分别为磷酸氢二钾、磷酸二氢钾、柠檬酸铵、醋酸钠。所得发酵液中活菌数如表3所示,表3中小写字母不同表示不同菌株之间存活率差异的显著性(P<0.05)。
表3不同无机盐条件下对发酵乳杆菌SS-31生长的影响
(4)发酵培养基麦芽糖、酵母浸粉、磷酸氢二钾添加量的影响
按照实施例1中的方法,在表4组成的各配方发酵培养基中培养。所得发酵液中活菌数如表5所示。
表4不同碳源、氮源、无机盐含量配比的培养基
种类 | 麦芽糖 | 酵母浸粉 | 磷酸氢二钾 | 硫酸锰 | 硫酸镁 | 吐温80 |
配方1 | 5 | 20 | 9 | 0.5 | 1 | 1 |
配方2 | 10 | 20 | 9 | 0.5 | 1 | 1 |
配方3 | 15 | 20 | 9 | 0.5 | 1 | 1 |
配方4 | 20 | 20 | 9 | 0.5 | 1 | 1 |
配方5 | 25 | 20 | 9 | 0.5 | 1 | 1 |
配方6 | 15 | 5 | 9 | 0.5 | 1 | 1 |
配方7 | 15 | 10 | 9 | 0.5 | 1 | 1 |
配方8 | 15 | 15 | 9 | 0.5 | 1 | 1 |
配方9 | 15 | 20 | 9 | 0.5 | 1 | 1 |
配方10 | 15 | 25 | 9 | 0.5 | 1 | 1 |
配方11 | 15 | 20 | 3 | 0.5 | 1 | 1 |
配方12 | 15 | 20 | 5 | 0.5 | 1 | 1 |
配方13 | 15 | 20 | 7 | 0.5 | 1 | 1 |
配方14 | 15 | 20 | 9 | 0.5 | 1 | 1 |
配方15 | 15 | 20 | 11 | 0.5 | 1 | 1 |
表5不同配方下对发酵乳杆菌SS-31生长的影响
从表5可以看出,碳源是组成培养基的主要成分,碳源浓度对乳酸菌生长起到关键作用。通过研究选择合适发酵乳杆菌发酵的碳源为麦芽糖,含量15g/L。在糖浓度过高时,菌株前期大量增殖生长,发酵产生大量酸,导致培养基环境偏酸性化,抑制菌株的生长代谢。氮源为菌体生长代谢提供必要元素,富含氨基酸、无机盐和维生素。通过研究确定适合发酵乳杆菌的氮源是酵母浸粉,含量为20g/L。在氮源前期浓度过低时,培养基内营养物质不足菌体生长缓慢;当氮源浓度过高时,菌体生长过快导致菌体老化,自溶。无机盐也是菌体生长代谢的重要因素,能够构成细胞物质以及调节渗透压。通过研究确定适合发酵乳杆菌的无机盐是磷酸氢二钾,含量为9g/L。乳酸菌在生长代谢过程中分解糖类产生大量乳酸,随着时间培养,乳酸大量堆积导致培养基内pH下降,乳酸菌生长受到抑制。无机盐的存在,可以调节培养基的pH,中和过酸,从而促进菌体的生长。并且无机盐中的无机离子通过阳离子、阴离子的乳酸菌吸收进行一系列生物合成代谢、酶活性激活等,为其生长补充了微量元素。
实施例3发酵乳杆菌SS-31培养条件的影响
按照实施例1中的方法,在含如下组成的发酵培养基中培养:麦芽糖15.00g/L、酵母浸粉20.00g、磷酸氢二钾9.00g、硫酸锰0.50g,硫酸镁1.00g,吐温80 1.00g,调节pH至6.5,115℃高压灭菌20min。将已活化三代后的发酵乳杆菌SS-31以3%(v/v)接种量到增殖培养基中,调节初始pH为6.8,以流加中和剂氨水维持培养基pH 6.8±0.02,置于37℃恒温箱内培养24h,测定其活菌数。所得发酵液中活菌数如表6所示,表6中小写字母不同表示不同菌株之间存活率差异的显著性(P<0.05)。
表6发酵乳杆菌SS-31在流加氨水培养24h后的活菌数
中和剂名称 | 未流加中和剂 | 氨水 |
活菌数(×1010CFU/mL) | 0.80b | 1.19a |
由上述实施例结果可知:发酵乳杆菌SS-31发酵的碳源选择麦芽糖、氮源为酵母浸粉,无机盐为磷酸氢二钾,有利于发酵乳杆菌SS-31的高密度培养,用该培养基培养发酵乳杆菌SS-31,活菌数高于其它培养基类型。使用本发明的高密度发酵培养基,在发酵条件为:发酵温度为37℃、接种量为3%、初始pH值为pH 6.8,期间添加的氨水保持发酵液pH值为6.8±0.02,经37℃培养24h后,发酵乳杆菌SS-31发酵液中的活菌浓度可达到1.19×1010CFU/mL,较MRS培养基高出近23倍,与其它培养基类型相比,该培养基更适合培养发酵乳杆菌SS-31。
实施例4发酵乳杆菌SS-31的应用
(1)含该乳酸菌发酵乳的制作方法
将鲜牛乳在100℃加热15分钟或140℃,加热3-5s,冷却至35-37℃,接种该乳酸菌,接种量为3%-5%,使其发酵剂浓度达到108CFU/mL以上,35-37℃发酵至pH值为4.2-4.5,即制成含该乳酸菌的乳酸菌奶饮料。
(2)乳酸菌发酵粉剂的制备:
收集乳酸菌发酵液,4℃条件下4000r/min离心20分钟,弃上清,收集菌体沉淀,用冻干保护剂洗脱沉淀,保护剂:沉淀=8:1,保护剂的配方为:10%脱脂乳,3%海藻糖,1%L-谷氨酸钠,1%吐温80,收集保护剂和菌泥混合物,真空浓缩,喷雾干燥,即得到发酵乳杆菌SS-31发酵剂粉剂。
(3)含该乳酸菌生物饲料发酵剂
取10mL发酵乳杆菌SS-31发酵液,加入1g生物饲料。发酵培养液成分为:麦芽糖15.00g/L,蔗糖20.00g/L、酵母浸粉20.00g/L,磷酸氢二钾9.00g/L,硫酸锰0.50g/L,硫酸镁1.00g/L,吐温80 15.00g/L,纤维素酶20.00g/L,培养18-24h,真空浓缩,喷雾干燥,得到含发酵乳杆菌SS-31的生物饲料发酵剂。
序列表
<110> 广西大学
<120> 发酵乳杆菌及其培养和应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1063
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aaggcggggg gggtgctact acatgcaagt cgaacgcgtt ggcccaattg attgatggtg 60
cttgcacctg attgattttg gtcgccaacg agtggcggac gggtgagtaa cacgtacgta 120
acctgcccag aagcggggga caacatttgg aaacagatgc taataccgca taacaacgtt 180
gttcgcatga acaacgctta aaagatggct tctcgctatc acttctggat ggacctgcgg 240
tgcattagct tgttggtggg gtaacggcct accaaggcga tgatgcatag ccgagttgag 300
agactgatcg gccacaatgg gactgagaca cggcccatac tcctacggga ggcagcagta 360
gggaatcttc cacaatgggc gcaagcctga tggagcaaca ccgcgtgagt gaagaagggt 420
ttcggctcgt aaagctctgt tgttaaagaa gaacacgtat gagagtaact gttcatacgt 480
tgacggtatt taaccagaaa gtcacggcta actacgtgcc agcagccgcg gtaatacgta 540
ggtggcaagc gttatccgga tttattgggc gtaaagagag tgcaggcggt tttctaagtc 600
tgatgtgaaa gccttcggct taaccggaga agtgcatcgg aaactggata acttgagtgc 660
agaagagggt agtggaactc catgtgtagc ggtggaatgc gtagatatat ggaagaacac 720
cagtggcgaa ggcggctacc tggtctgcaa ctgacgctga gactcgaaag catgggtagc 780
gaacaggatt agataccctg gtagtccatg ccgtaacgat gagtgctagg tgttggaggg 840
tttccgccct tcagtgccgg agctaacgca ttaagcactc cgcctggggg agtacgaccg 900
caaggttgaa actcaaggaa ttgacggggg ccccgcacaa gcggtggagc atgtggttta 960
attcgaagct acgcgaagaa ccttaccagg tcttgacatc ttgcgccaat cctagagata 1020
gggcgttcct tcggaacgca atgacagggt ggtgccatgg tcc 1063
<210> 2
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
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agagtttgat ttgatcctgg ctag 24
<210> 3
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggttaccttg ttacgactt 19
Claims (3)
1.一株发酵乳杆菌,其特征在于为发酵乳杆菌(Lactobacillus fermentum)SS-31,保藏编号为CGMCC NO: 24925 。
2.权利要求1所述发酵乳杆菌的高密度发酵培养方法,其特征在于将发酵乳杆菌种子液接种到高密度发酵培养基中,接种量1%-5%,调整初始发酵pH值为5.8-7.8,发酵后采用15%-25%的氨水溶液控制发酵液pH值6.8±0.02,32-42℃培养12-24h;所述高密度发酵培养基每1L由以下含量成分组成:麦芽糖15.00g、酵母浸粉20.00g、磷酸氢二钾9.00g、硫酸锰0.50g,硫酸镁1.00g,吐温80 1.00g,水1000mL,调节pH至6.5,115℃高压灭菌20min。
3.一种乳酸菌饮料,其特征在于含有权利要求1所述发酵乳杆菌,其活菌数达到10 8CFU/mL以上。
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