CN110734880B - 源于广西巴马具有高产维生素B能力的植物乳杆菌Bama06及其用途 - Google Patents
源于广西巴马具有高产维生素B能力的植物乳杆菌Bama06及其用途 Download PDFInfo
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Abstract
源于广西巴马具有高产维生素B能力的植物乳杆菌Bama06及其用途,从广西巴马长寿老人肠道中分离得到的乳酸菌,测定不同乳酸菌在37℃下于MRS/M17培养基、牛乳培养基及豆浆培养基中发酵50 h发酵液的pH和活菌数,得到可以在不同培养基中生长产酸的乳酸菌,同时利用高效液相色谱法测定发酵液中维生素B的种类与产量,筛选出具有高产维生素B能力和较高产酸能力及活力的乳酸菌;经API试剂条及16s rDNA测序分析将筛选出的菌株Bama06鉴定为植物乳杆菌。本发明菌株的耐酸耐胆盐能力较强,可应用于制作富含维生素B的功能食品,利用本发明菌制得的产品,具有富含维生素B的特点。
Description
技术领域
本发明涉及生物技术领域,特别涉及源于广西巴马具有高产维生素B能力的植物乳杆菌Bama06及其用途。
技术背景
维生素B是机体必须的微量营养素之一,具有多种生理功能,常以辅酶的形式参与调节各种代谢反应,对人体健康十分重要。维生素B在人体含量很少,大部分不能由人体自身产生,很多人由于膳食营养不平衡、吸收效率低而导致维生素B缺乏进而引起疾病,须通过外源性的维生素B营养食品或者药物来补充。因此,开发科学、健康的维生素膳食补充产品成为食品行业关注的焦点。
乳酸菌作为公认的安全微生物,具有合成维生素B的能力。生物合成的维生素B具有安全性、经济性及环境友好性等优点,因此,使乳酸菌发酵食品成为人们日常维生素B营养强化的补充剂可进一步为人类健康事业添砖加瓦。
发明内容
本发明第一个目的在于提供一种具有高产维生素B能力和较高产酸能力及活力的乳酸菌,以期进一步开发利用。
源于广西巴马具有高产维生素B能力的植物乳杆菌(Lactobacillus plantarum)Bama06,于2019年10月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址: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,保藏编号为CGMCC NO.18694。
经实验验证,本发明菌株在同一批次广西巴马长寿老人肠道中分离的63株乳酸菌中产维生素B的种类与产量最高,表明本发明菌株具有良好的产维生素B的能力。经测定发现在MRS、牛乳及豆浆基质中37℃发酵50 h后,发酵液pH值分别为3.99、4.74和3.99,且活菌数均大于109 CFU/mL,有较高的产酸能力及活力,经37℃发酵50 h,在MRS发酵液中维生素B1、维生素B5及叶酸分别超过2.0 mg/L、41.5mg/L及0.5mg/L;在牛乳发酵液中维生素B2、维生素B3和维生素B5分别大于8.4mg/L、5.0mg/L和11.9mg/L;在豆浆发酵液中维生素B3的生物合成量大于1.0 mg/L。
通过API试剂条及16S rDNA序列比对分析将该菌株鉴定为植物乳杆菌。本发明菌株经耐酸耐胆盐实验发现,该菌株在pH为3.0的人工胃液中存活率为98.67%,在胆盐浓度为0.10%、0.30%、0.50%的MRS培养基中培养3h存活率分别大于71.9%、43.3%及11.2%。
本发明的第二个目的是提供高产维生素B和较高产酸能力及活力的植物乳杆菌Bama06的制备方法,即采用传统涂布平板分离法从广西巴马长寿村采集的样品中分离乳酸菌。
本发明的第三个目的是提供高产维生素B能力和较高产酸能力及活力乳酸菌的用途。
本发明菌株在牛乳基质中表现出良好的维生素B合成能力,可以利用其研发富含维生素B的发酵乳产品或乳酸菌饮料。
也可将该乳酸菌的培养物经冷冻干燥用于生产含有维生素B的膳食功能补充剂。
由于生物合成的维生素B更适合人体吸收,更安全,该乳酸菌发酵物可以用作一种食疗性产品。
本发明从广西巴马长寿老人肠道中分离得到的乳酸菌,测定不同乳酸菌在37℃下于MRS/M17培养基、牛乳培养基及豆浆培养基中发酵50 h发酵液的pH和活菌数,得到可以在不同培养基中生长产酸的乳酸菌,同时利用高效液相色谱法测定发酵液中维生素B的种类与产量,筛选出具有高产维生素B能力和较高产酸能力及活力的乳酸菌;经API试剂条及16srDNA测序分析将筛选出的菌株Bama06鉴定为植物乳杆菌(Lactobacillus plantarum)。通过对植物乳杆菌Bama06耐酸耐胆盐能力的研究发现,本发明菌株的耐酸耐胆盐能力较强,可应用于制作富含维生素B的功能食品,利用本发明菌制得的产品,具有富含维生素B的特点。
附图说明
图1为本发明菌株API鉴定结果;
图2为本发明菌株16S rDNA PCR扩增产物电泳图;
图3为本发明实验中维生素B标准品测定图谱;
图4为本发明菌株API试剂条鉴定图;
图5为本发明菌株16S rDNA序列。
具体实施方法
本发明采用传统涂布平板分离法从广西巴马长寿村采集的样品中分离乳酸菌,通过测定发酵液中的pH和活菌数确定活力好、产酸高的乳酸菌菌株;通过测定乳酸菌在MRS/M17、牛乳及豆浆培养基中的产维生素B的量与种类筛选出产维生素B能力较强的乳酸菌。
相关培养基及试剂配方
MRS培养基:蛋白胨10.0g,三水醋酸钠3.0g,牛肉膏8.0g,柠檬酸三铵2.0g,酵母浸膏4.0g,七水硫酸镁0.2g,葡萄糖10.0g,一水硫酸锰0.04g,吐温-80 1.0mL,磷酸氢二钾2.0g,硫乙醇酸钠2.0g,蒸馏水1000 mL。调pH至6.4,121℃灭菌15min,冷却备用。
M17培养基:植物蛋白胨5 g,聚蛋白胨5 g,牛肉膏2.5 g,酵母提取物5 g,抗坏血酸钠0.5 g,乳糖10 g,甘油10 g,磷酸氢二钠5 g,七水硫酸镁0.25 g,蒸馏水1000 mL。121℃灭菌15 min,冷却备用。
牛乳培养基:称取7%的蔗糖溶于热水中,将全脂乳粉按12%的比例溶于80℃的糖水中,95℃灭菌20 min,冷却备用。
豆浆培养基:称取大豆100 g,浸泡于1000 mL水中,24℃下浸泡12 h。按1:8体积比例加入蒸馏水,磨浆3 min后过筛,豆浆中加入7%的蔗糖,95℃灭菌20 min,冷却备用。
具体步骤:
1样品采集
采集广西巴马长寿村中长寿人群的粪便样品,加入1mL灭菌液体石蜡油密封后,迅速置于冰盒内,分离样品中的乳酸菌。
2乳酸菌的分离
将采集的样品经梯度稀释后,分别接种于MRS固体培养基、M17固体培养基中,37℃厌氧培养48h,挑取平板上的典型菌落,划线分离得到纯菌落。挑取各平板上的纯菌落于MRS液体培养基中,37℃厌氧培养24 h后,4℃冰箱冷藏备用或冻干保藏。
3乳酸菌的发酵特性
将分离得到的乳酸菌活化后按3%的比例分别接种到MRS/M17液体培养基、牛乳培养基及豆浆培养基中,于37℃下培养50 h后测定发酵液pH和活菌数。结果发现,Bama06在MRS、牛乳及豆浆基质中经过50 h的发酵后发酵液pH值分别为3.99、4.74和3.99,且活菌数均大于109 CFU/mL,表明该菌株在不同的基质中有较高的产酸能力及活力。
4筛选具有高产维生素B能力的乳酸菌
4.1发酵液制备:
将乳酸菌培养液在5000×g下离心5 min收集菌体,用盐水溶液(0.85% m/v NaCl)洗涤3次,将菌体重悬于盐水溶液中制成供试菌液备用。将供试菌液按3%的体积比加入到20mL的MRS/M17、牛乳及豆浆培养基中。在37℃的恒温培养箱中培养50 h后,取1 mL发酵液加入等体积超纯水稀释,于3000 rmp离心5 min后取上清。
4.2发酵液中维生素B的测定
4.2.1液相色谱分析条件:色谱柱:ZORBAX SB-C18柱(4.6 mm×250 mm,5μm);柱温:30℃;流动相:B磷酸二氢钾缓冲液(磷酸调节pH=2.5),D乙腈,洗脱梯度条件为:0-6 min100% B,随后20 min内B相体积比由100%线性下降至75%,保持5 min后,恢复至100% A平衡色谱柱10 min;流速:1.0 mL/min;进样体积:20 μL;采集波长分别设定为205、246、261、267、283、290 nm。
4.2.2标准液的配制:在黑暗条件下,将维生素B1、B3、B5、B6及B12标准品用0.01mol/L的盐酸溶液配制成2 mg/mL的标准储备溶液,于4℃冰箱内保存备用。维生素B7、叶酸与维生素B2标品先用0.25 mol/L NaOH溶液溶解,再用0.01 mol/L的盐酸溶液定容成2 mg/mL的标准储备溶液。取少量维生素标准储备液稀释、定容配制成维生素标准溶液,标准溶液浓度为500 μg/mL。过0.22 μm水系滤膜后上机测定,进样20 μL,记录每种维生素的出峰时间。取B族维生素的标准品储备液,利用0.01 mol/L的盐酸溶液稀释标准储备溶液,控制混合标品中维生素B1、B2、B3、B6、B7、B12及叶酸浓度为5 μg/mL,维生素B5浓度为50 μg/mL,过0.22 μm水系滤膜后上机测定,依次进样1、10、20、30、40和50 μL。根据峰面积和进样量制得标准回归方程,8种维生素B的标准回归方程相关系数均大于0.999。
4.2.3样品前处理:取1 mL上述4.1中上清发酵液,过0.22 μm水系滤膜后上机测定。
4.2.4数据的处理:根据标准液中各维生素的出峰时间,确定发酵液中所含维生素的种类,根据标准品回归方程计算各维生素B产量。对本实验中63株乳酸菌产B族维生素种类与产量的比较,发现乳酸菌Bama06具有产B族维生素量高,种类更全的优点。在MRS发酵液中检测到2.08 mg/L的维生素B1,41.86mg/L的维生素B5及0.53mg/L的叶酸,在牛乳发酵液中检测到8.43mg/L的维生素B2,5.04mg/L的维生素B3及11.90mg/L的维生素B5,在豆浆发酵液中检测到1.04 mg/L的维生素B3。
5乳酸菌的鉴定
综合以上试验结果发现,菌株Bama06具有高产维生素B的能力,同时具有较强的活力和产酸能力。因此选择菌株Bama06作为本发明菌株,并对其进行鉴定。
(1)API鉴定
利用API 50 CHL系列鉴定试剂条对菌株Bama06进行鉴定,结果表明,本发明菌株Bama06为植物乳杆菌,鉴定率为99.9%。
(2)16s rDNA序列测定进行菌株的鉴定:将制备的乳酸菌基因组DNA作为PCR扩增的模板,采用50 μL反应体系进行PCR扩增。模板DNA 2 uL,10 mmol上下游引物各1.5 μL,2×Taq酶25 μL,以ddH2O补至50 μL。PCR循环参数为:94℃预变性5 min;94℃变性1 min,64℃退火45 s,72℃延伸1 min,循环35次;72℃延伸8 min。吸取PCR产物5 μL与1 mL Loadingbuffer混合,用1.0%琼脂糖凝胶电泳,电压为80 V。电泳结束后用溴化乙锭EB染色20-30min,拍照。可约1500 bp的条带即为目标条带,见图1。电泳检测扩增产物后,送至上海生工生物工程技术服务有限公司进行测序。将测序得到的序列与GenBank数据库中的序列进行比对分析,结果表明,本发明菌株Bama06为植物乳杆菌,鉴定率为100%。
6植物乳杆菌Bama06耐酸耐胆盐能力的测定
将活化好的植物乳杆菌Bama06在5000×g下离心5 min收集菌体,用盐水溶液(0.85% m/v NaCl)洗涤3次,将菌体重悬于盐水溶液中制成菌悬液,取1.0mL的菌悬液接种至9.0mL的pH3.0人工胃液中,37℃培养,分别在0h和3h用平板计数法测定活菌数,计算其存活率(%)。
存活率(%)=3h的活菌数/0h的活菌数×100%
将活化好的乳酸菌按3%的接种量分别接种至含0.00%(即空白)、0.10%、0.30%及0.50%胆盐的MRS培养基中,37℃培养,分别在0h和3h用平板计数法测定活菌数,计算其存活率(%)。
存活率(%)=3h的活菌数/0h的活菌数×100%
结果表明,植物乳杆菌Bama06在pH为3.0的人工胃液中存活率为98.67%,在胆盐浓度为0.10%、0.30%、0.50%的MRS培养基中培养3h存活率分别为71.97%、43.33%及11.25%。
应用实施例
1含该乳酸菌发酵乳的制作方法:
将鲜牛乳加热到50℃以上,加入6%的白砂糖搅拌至完全溶解,预热到60~65℃,20Mpa压力均质,90℃左右热处理5-8分钟,冷却至42~45℃,接种该乳酸菌,接种量为2%~3%。40~42℃发酵至pH值为4.2~4.5,冷却,冷藏即制成含该乳酸菌的酸乳。经测定,发酵乳中活菌数高于109 CFU/mL,且含有丰富的维生素B2、B3和B5。
2含该乳酸菌饮料的制作方法:
将应用示例1中的发酵乳,用杀菌并冷却的水、糖浆、乳化稳定剂等混合物稀释2-4倍,制成可直接饮用的乳酸菌饮料。该乳酸菌饮料不仅口感酸甜,风味良好,还含有较高的维生素B2和B3含量。
3含B族维生素的功能补充剂制作方法:
将该乳酸菌冷冻至-40℃~-60℃,经干燥,至含水量为3%~5%,压碎成粉末状,可将粉末状产品加入奶粉、低聚糖、麦芽糊精、风味剂等制成粉状或片状产品,用于日常食用。该食品中富含多种维生素B,且为菌株自身发酵产生,更具有安全性。
以上利用本发明菌制得的产品,具有富含B族维生素的特点,日常食用可补充人体B族维生素,适合熬夜,睡眠不足,精神压力大的人群。
序列表
<110> 扬州大学 江苏宇航食品科技有限公司
<120> 源于广西巴马具有高产维生素B能力的植物乳杆菌Bama06及其用途
<160> 1
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 人工序列(Artificial Sequence)
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cctaaaaggt taccccaccg actttgggtg ttacaaactc tcatggtgtg acgggcggtg 60
tgtacaaggc ccgggaacgt attcaccgcg gcatgctgat ccgcgattac tagcgattcc 120
gacttcatgt aggcgagttg cagcctacaa tccgaactga gaatggcttt aagagattag 180
cttactctcg cgagttcgca actcgttgta ccatccattg tagcacgtgt gtagcccagg 240
tcataagggg catgatgatt tgacgtcatc cccaccttcc tccggtttgt caccggcagt 300
ctcaccagag tgcccaactt aatgctggca actgataata agggttgcgc tcgttgcggg 360
acttaaccca acatctcacg acacgagctg acgacaacca tgcaccacct gtatccatgt 420
ccccgaaggg aacgtctaat ctcttagatt tgcatagtat gtcaagacct ggtaaggttc 480
ttcgcgtagc ttcgaattaa accacatgct ccaccgcttg tgcgggcccc cgtcaattcc 540
tttgagtttc agccttgcgg ccgtactccc caggcggaat gcttaatgcg ttagctgcag 600
cactgaaggg cggaaaccct ccaacactta gcattcatcg tttacggtat ggactaccag 660
ggtatctaat cctgtttgct acccatactt tcgagcctca gcgtcagtta cagaccagac 720
agccgccttc gccactggtg ttcttccata tatctacgca tttcaccgct acacatggag 780
ttccactgtc ctcttctgca ctcaagtttc ccagtttccg atgcacttct tcggttgagc 840
cgaaggcttt cacatcagac ttaaaaaacc gcctgcgctc gctttacgcc caataaatcc 900
ggacaacgct tgccacctac gtattaccgc ggctgctggc acgtagttag ccgtggcttt 960
ctggttaaat accgtcaata cctgaacagt tactctcaga tatgttcttc tttaacaaca 1020
gagttttacg agccgaaacc cttcttcact cacgcggcgt tgctccatca gactttcgtc 1080
cattgtggaa gattccctac tgctgcctcc cgtaggagtt tgggccgtgt ctcagtccca 1140
atgtggccga ttaccctctc aggtcggcta cgtatcattg ccatggtgag ccgttacccc 1200
accatctagc taatacgccg cgggaccatc caaaagtgat agccgaagcc atctttcaaa 1260
ctcggaccat gcggtccaag ttgttatgcg gtattagcat ctgtttccag gtgttatccc 1320
ccgcttctgg gcaggtttcc cacgtgttac tcaccagttc gccactcact caaatgtaaa 1380
tcatgatgca agcaccaatc aataccagag ttcgttcgac 1420
Claims (6)
1.源于广西巴马具有生产维生素B能力的植物乳杆菌(Lactobacillus plantarum)Bama06,保藏编号为CGMCC NO.18694,具有生产维生素B的能力,同时具有耐酸耐胆盐能力。
2.如权利要求1所述源于广西巴马具有生产维生素B能力的植物乳杆菌Bama06的发酵性能检测方法,其特征是,经37℃发酵50h,在MRS发酵液中维生素B1、维生素B5及叶酸分别超过2.0mg/L、41.5mg/L及0.5mg/L;在牛乳发酵液中维生素B2、维生素B3和维生素B5分别大于8.4mg/L、5.0mg/L和11.9mg/L;在豆浆发酵液中维生素B3的生物合成量大于1.0mg/L。
3.如权利要求1所述源于广西巴马具有生产维生素B能力的植物乳杆菌Bama06的耐酸耐胆盐性能检测方法,其特征是,在pH为3.0的人工胃液中存活率大于98.5%,在胆盐浓度为0.10%、0.30%、0.50%的MRS培养基中培养3h存活率分别大于71.9%、43.3%及11.2%。
4.如权利要求1所述源于广西巴马具有生产维生素B能力的植物乳杆菌Bama06用于制备富含维生素B的发酵乳的应用,所述维生素B为维生素B1、维生素B2、维生素B3、维生素B5。
5.如权利要求1所述源于广西巴马具有生产维生素B能力的植物乳杆菌Bama06用于制备富含维生素B的发酵乳饮料的应用,所述维生素B为维生素B1、维生素B2、维生素B3、维生素B5。
6.如权利要求1所述源于广西巴马具有生产维生素B能力的植物乳杆菌Bama06用于制备富含维生素B的活菌制剂的应用,所述维生素B为维生素B1、维生素B2、维生素B3、维生素B5。
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