CN116144523B - 一种可发酵大豆低聚糖的哈尔滨乳杆菌及其应用 - Google Patents
一种可发酵大豆低聚糖的哈尔滨乳杆菌及其应用 Download PDFInfo
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Abstract
一种可发酵大豆低聚糖的哈尔滨乳杆菌及其应用,该菌株为哈尔滨乳杆菌grx‑SOS05,是从传统发酵豆制品老北京酸豆汁样品中筛选得到,本发明菌株可发酵大豆低聚糖中蔗糖、棉子糖和水苏糖,尤其是不可消化的棉子糖和水苏糖,其在以棉子糖或水苏糖为唯一碳源的MRS培养基中生长情况良好,可以用来制备发酵豆乳,发酵豆乳酸度为56.7°T,贮藏14天后持水力为76%,活菌数为7.53log(CFU/ml),也可与其他乳酸菌复配制备发酵乳饮料等,更可将该哈尔滨乳杆菌的培养物干燥后制备益生菌粉。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种可发酵大豆低聚糖的哈尔滨乳杆菌及其应用。
背景技术
随着人们膳食营养观念的变化及对健康食品需求的日益增加,植物基发酵乳作为牛乳制品的健康替代品和营养补充源,受到越来越多消费者的关注。当前国内市场尚无有影响力的豆植物基发酵乳产品上市。
豆植物基发酵乳含有丰富的大豆蛋白、大豆异黄酮等功能物质,营养价值高,且其不含牛乳过敏原、乳糖,无胆固醇、饱和脂肪酸等动物源性成分,可以满足乳糖不耐症、减脂群体等特殊人群需求。但豆乳中蔗糖、棉子糖、水苏糖为主要碳源,由于人体内缺乏水解棉子糖和水苏糖的消化酶α-D-半乳糖苷酶,使豆植物基发酵乳中棉子糖和水苏糖在小肠内不被消化吸收,进入大肠后被结肠中肠道微生物发酵产生气体,容易引起人体消化不良、腹胀、肠鸣等不良反应,限制了豆植物基发酵乳的进一步开发利用。
发明内容
本部分的目的在于概述本发明的实施例的一些方面以及简要介绍一些较佳实施例。在本部分以及本申请的说明书摘要和发明名称中可能会做些简化或省略以避免使本部分、说明书摘要和发明名称的目的模糊,而这种简化或省略不能用于限制本发明的范围。
鉴于上述和/或现有技术中存在的问题,提出了本发明。
因此,本发明的目的是,克服现有技术中的不足,提供一种可发酵大豆低聚糖的哈尔滨乳杆菌。
为解决上述技术问题,本发明提供了如下技术方案:所述菌株是哈尔滨乳杆菌(Lactobacillus harbinensis),命名为grx-SOS05,保藏于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.:25445。
本发明的再一个目的是,解决现有技术中的不足,提供一种可发酵大豆低聚糖的哈尔滨乳杆菌的应用。
为解决上述技术问题,本发明提供了如下技术方案:一种可发酵大豆低聚糖的哈尔滨乳杆菌的应用,其特征在于,所述哈尔滨乳杆菌在发酵豆乳、乳酸菌饮料及功能食品中的应用;
为解决上述技术问题,本发明提供了如下技术方案:所述发酵豆乳在8h时的pH为4.57,酸度值为56.7°T,活菌数为7.74logCFU/ml,持水力为72.1%,黏度为1513mPa.s,硬度为0.436N,贮藏14d后,活菌数为7.53logCFU/ml。
本发明有益效果:
(1)提供一株可发酵大豆低聚糖的哈尔滨乳杆菌,以期进一步开发利用,该乳酸菌菌株为哈尔滨乳杆菌Lactobacillus harbinensis grx-SOS05,已于2022年8月1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CGMCCNO.:25445。
(2)初筛挑选了发酵豆乳凝乳时间短、产酸和质构特性优的菌株,复筛挑选可以高转化棉子糖、水苏糖的菌株。
(3)本发明菌株可用于制备发酵乳,特别是发酵豆乳、豆植物基发酵混合乳;也可用于制备益生菌粉,应用于食品中。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其它的附图。其中:
图1为本发明实施例1中菌株在正常葡萄糖MRS平板上的菌落形态图。
图2为本发明实施例1中菌株的普通光学显微镜观察形态图。
图3为本发明实施例1中氨基酸脱羧酶测定图,其中A为酪氨酸检测结果B为组氨酸检测结果。
图4为本发明实施例1中硝基还原酶检测图。
图5为本发明实施例1中溶血活性检测图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合说明书实施例对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是本发明还可以采用其他不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似推广,因此本发明不受下面公开的具体实施例的限制。
其次,此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例,也不是单独的或选择性的与其他实施例互相排斥的实施例。
实施例1
哈尔滨乳杆菌的制备方法
本发明采用涂布平板分离法从传统发酵豆制品老北京酸豆汁样品中分离乳酸菌,通过测定乳酸菌发酵豆乳的凝乳时间、pH、活菌数、持水力、黏度和硬度等指标,筛选出产酸高、质构优的乳酸菌发酵豆乳菌株;通过测定乳酸菌在含有以棉子糖、水苏糖为唯一碳源的MRS培养基中600nm处吸光度值(OD600),筛选出可发酵大豆低聚糖的菌株;通过测定发酵前后豆乳中大豆低聚糖的含量,确定高转化大豆低聚糖的乳酸菌菌株。
1.乳酸菌的分离
本发明菌株是可发酵大豆低聚糖的哈尔滨乳杆菌,是发明人从传统发酵豆制品老北京酸豆汁中筛选得到的,经鉴定属于哈尔滨乳杆菌(lactobacilus harbinensis),命名为grx-SOS05,该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心。
2.乳酸菌在豆乳培养基中的产酸特性、生长特性、质构特性
豆乳培养基:挑选正常大豆,清洗干净并放入蒸馏水中浸泡过夜,再用80℃蒸馏水磨浆后去渣,调整豆水比为1:10制成豆浆,过100目筛后煮沸,分装在发酵瓶中,105℃灭菌15min,待冷却至室温后放置4℃冰箱,备用。
将筛选的菌株活化后按3%的添加量接种至100mL豆乳培养基发酵瓶,37℃培养箱发酵,每隔0.5h观察豆乳凝乳情况,记录凝乳时间。在发酵8h时取样对发酵样品进行pH、滴定酸度(称取5.0g左右的发酵样品,记录质量m,用40ml蒸馏水稀释并加入两滴酚酞指示剂,用0.1mol/L的NaOH滴定至微粉红色,30s内不变色,记录体积V)活菌数、持水力、黏度、硬度测定,所有实验均平行测定三次,取平均值。
结果表明,该发明菌株在豆乳培养基中凝乳时间为6h,其在8h时的pH为4.57,酸度值为56.7°T,活菌数为7.74log(CFU/ml),持水力为72.1%,黏度为1513mPa.s,硬度为0.436N,具有较好的产酸特性、生长特性及质构特性。
3.乳酸菌在棉子糖和水苏糖培养基中的生长情况
MRS液体培养基:葡萄糖20.00g,醋酸钠5.00g,磷酸氢二钾2.00g,蛋白胨10.00g,吐温-80 1.00mL,牛肉膏10.00g,四水硫酸锰0.05g,酵母提取物5.00g,柠檬酸二铵2.00g,七水硫酸镁0.20g,加蒸馏水定容至1000mL,121℃灭菌15min。
MRS-Glc+Raf培养基:将MRS培养基里的葡萄糖换成棉子糖,其余不变,121℃灭菌15min,备用。
MRS-Glc+Sta培养基:将MRS培养基里的葡萄糖换成水苏糖,其余不变,121℃灭菌15min,备用。
MRS-Glc培养基:将MRS培养基里的葡萄糖去掉,无碳源,其余不变,121℃灭菌15min,备用。
将菌株以3%的添加量接种至MRS-Glc+Raf和MRS-Glc+Sta液体培养基里,37℃培养,在0h、20h时取样,采用酶标仪测定发酵液在600nm处吸光度值(OD600),记录乳酸菌在MRS-Glc+Raf和MRS-Glc+Sta液体培养基的生长情况,以不含糖的空白培养基MRS-Glc培养基为阴性对照。
结果表明,该菌株比其他菌株在MRS-Glc+Raf培养基和MRS-Glc+Sta培养基的生长情况都要好,OD600棉子糖达到0.830,OD600水苏糖达到0.685。
4.乳酸菌发酵大豆低聚糖能力评价
将乳酸菌在MRS液体培养基中活化2代后,分别接入以棉子糖、水苏糖为唯一碳源的MRS-Glc+Raf培养基和MRS-Glc+Sta培养基,37℃培养箱发酵,在0h、12h、24h取样测定其棉子糖和水苏糖含量。取1ml的乳酸菌发酵液,8000r/min离心2min,取上清液经0.22μm水相滤膜过滤后用于HPLC检测,分析上清液中棉子糖和水苏糖的含量。
同时,将乳酸菌接入豆乳培养基中,37℃培养箱发酵,取发酵8h的豆乳样品。样品处理:取2mL发酵豆乳样品,加入1.5mL无菌去离子水溶解,60℃水浴10min;加入0.25mLCarrez I溶液(0.5mol/L亚铁氰化钾水溶液aqueous potassium ferrocyanide),0.25mLCarrez II溶液(0.5mol/L醋酸锌水溶液aqueous zinc acetate)和1mL乙腈,混匀,室温静置1h,10000r/min离心8min,取上清液经0.22μm水相滤膜过滤用于HPLC检测。
结果表明:该发明菌株在以棉子糖、水苏糖为唯一碳源的MRS-Glc+Raf培养基和MRS-Glc+Sta培养基中,发酵24h后,棉子糖和水苏糖含量从20g/L分别降低到4.2g/L和4.67g/L;在发明菌株发酵的豆乳中,蔗糖含量降低到0.19g/L,水苏糖含量降低到0.25g/L,棉子糖的含量降低到检测不到的水平。
5.菌株安全性检测
a)抗生素耐药性敏感测定
采用药敏纸片琼脂扩散法检验乳酸菌对抗生素的敏感性。选用10种抗生素包括氨苄西林、万古霉素、头孢唑林、复方新诺明、链霉素、利福平、克林霉素、四环素、氯霉素、青霉素。将该发明菌株活化两代后,取1ml菌液于1.5ml离心管中,8000r/min、5min离心,离心后的菌液用无菌生理盐水稀释至OD600=0.5,取200μl稀释液于MRS固体培养基中涂布,在无菌操作下将药敏纸片取出并贴在平板内,每个平板内贴3个药敏纸片。室内放置30min,然后放置于37℃培养箱培养24h后测量并记录抑菌圈直径。结果参照表1,根据美国CLSI制定的抗微生物药物敏感试验执行标准来评价乳酸菌对各种抗生素的耐药性。
表1乳酸菌对抗生素耐药性判断标准
表2发明菌株对抗生素耐药性结果
结果表明:该发明菌株对头孢唑林、利福平、链霉素、氯霉素、青霉素、四环素6种抗生素敏感,对氨苄西林-舒巴坦、复方新诺明、万古霉素、克林霉素4种抗生素耐药。
b)有毒物质产生测定
(1)生物胺的生成,将活化2代的乳酸菌用生理盐水稀释后,涂布于添加酪氨酸和组氨酸的氨基酸脱羧酶检测培养基中,37℃培养3d后,观察培养基颜色变化情况,颜色变紫则为阳性,颜色变黄则为阴性,同时用金黄色葡萄球菌做对照实验。
(2)亚硝酸盐的生成,将活化2代的乳酸菌按照3%的添加量接种于硝酸盐培养基中,37℃培养4d后,向培养液中依次滴加3-5滴α-奈胺溶液和对氨基苯磺酸溶液,同时用金黄色葡萄球菌做对照实验,观察实验结果。
(3)溶血素的生成,将待测乳酸菌和对照阳性菌金黄色葡萄球菌活化2代后划线于哥伦比亚血琼脂板内,37℃培养箱培养48h后观察菌落周围是否有溶血圈出现。
结果如图3、4、5所示,该菌株氨基酸脱羧酶测定、硝基还原酶测定、溶血活性测定均为阴性,表明其是安全菌株。
6.乳酸菌的鉴定
6.1形态观察和革兰氏染色
肉眼观察MRS固体平板上菌落特征,并挑取单菌落于载玻片上,经革兰氏染色后,通过生物学显微镜观察菌株形态。
如图1、2所示,发明菌株的菌落形态为乳白色,近圆形凸起且光滑,边缘整齐。经革兰氏染色后,革兰氏染色呈阳性,长杆状,无芽孢。
6.2菌株16S rDNA鉴定
(1)提取基因组:菌株活化后,取1ml菌液离心,选用细菌基因组DNA提取试剂盒提取DNA;
(2)PCR扩增:以菌株基因组DNA为模板进行扩增,引物为(27F:AGAGTTTGATCCTGGCTCAG;1492R:GGTTACCTTGTTACGACTT),PCR扩增程序:95℃、5min;95℃、30s;55℃、15s;72℃、2min,30个循环后继续72℃延伸5min。
将PCR产物电泳检测后送至上海生工生物工程股份有限公司进行测序。测序结果:其核苷酸长度在1500bp附近,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为哈尔滨乳杆菌,16S rDNA序列一致性达86%。16S rDNA序列如下所示,
GAGGGGGGGGGGGGTCCCTACACGTTTCAAGCCGAACGAGGCTGGTTCAGTTTGCGATGGTGCTTGCATCACCAATTACCAATCACACCGAGCGTCGGACAGGTGATCACATATGCGCAACCTGCCCTTCTACAGGGGATAACATTTGGAAACTGATGCTTATACCGCATAATCACGGAGACCGCTTGGTCTCCGGGTAAAAGATGGTGCCAGCTATCTCTGACCGATGGACCCGCGCCGTATTATCCAGTTGGTGGGTTAACGGGCTACCATACCGATGATACCTTACGACCTGTCAGGGTTTCGGCCACTTTGGGACTGACACACGGCCCAAACTCCTACGGGAGGCAGAATTATGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCATCACCGCATGAGTGTTTAAGGCTTTCGGGTCCTATTACTCTGTTATTGAAAAAGAACGTGTGTGACAGTAACTGGTCATGCAGTGACGGTATTCAATTCAAAAGTCACTGCTAACTACCTGCCACCATACGCGGTAATACGTAAGTGGTATCCGTATTCCTGATTTATTGGGCGTAAAGCGAGTGCAGGCGGTCTTTTAATTCTGATGTGAAAGCCTTCGGCTTAACAGTAGAACGGCATCTGACATTCTTAAACTTGAATGCAGAAAATGAAAGTGGATCTCCATGTGTAGCGGTGAAATGTGTATATATAAGGAAGAACACCCGAGGGCGAAGGCTGCTCTCTGGTACTGTAACTGAAGCCTTAGCTCCATAGCTAGCGTACCAAACATGGATAACATACCCTGGTATTCCCACCGTAAACAATGATAACTATTTTGTTGCATGGCTTCTCCTCCTTTCCCGCCGAAACTAACTTAATTACTATTCCTG
表3乳酸菌16S rDNA基因序列同源性比对结果
实施例2
1.本发明菌株可制备发酵豆乳。
挑选正常大豆,清洗干净并放入蒸馏水中浸泡过夜,再用80℃蒸馏水磨浆后去渣,调整豆水比为1:10制成豆浆,过100目筛后煮沸,可添加蔗糖,分装在发酵瓶中,105℃灭菌15min,备用。取发明菌株,活化后接种至豆乳培养基发酵,接种量为3%,37℃发酵8h后可冷藏食用。
2.本发明菌株可制备成益生菌粉,应用于食品中。
将该乳酸菌冷冻至-40℃~-60℃,冻干后制成粉末状,可将该粉末加入各种饮品或者食物中做为补充剂。
本发明提供一株可发酵大豆低聚糖的乳酸菌,以期进一步开发利用。该乳酸菌菌株为哈尔滨乳杆菌grx-SOS05,已于本发明申请日前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。
该菌株是从传统发酵豆制品老北京酸豆汁中分离得到的,经实验验证,该菌株在以棉子糖为唯一碳源的MRS培养基中生长OD600达到0.830,以水苏糖为唯一碳源的MRS培养基中生长OD600达到0.685,其发酵豆乳8h时的pH为4.57,酸度值为56.7°T,活菌数为7.74log(CFU/ml),持水力为72.1%,黏度为1513mPa.s,硬度为0.436N,贮藏14d后,活菌数为7.53log(CFU/ml)。
本发明菌株可用于制备发酵乳,特别是发酵豆乳、豆植物基发酵混合乳;也可用于制备益生菌粉,应用于食品中。
应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (3)
1.一种可发酵大豆低聚糖的哈尔滨乳杆菌,其特征在于:所述菌株是哈尔滨乳杆菌(Lactobacillus harbinensis),命名为grx-SOS05,保藏于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.:25445。
2.如权利要求1所述的可发酵大豆低聚糖的哈尔滨乳杆菌的应用,其特征在于,所述哈尔滨乳杆菌在发酵豆乳、乳酸菌饮料及功能食品中的应用。
3.根据权利要求2所述的应用,其特征在于,包括:所述发酵豆乳在8h时的pH为4.57,酸度值为56.7°T,活菌数为7.74logCFU/ml,持水力为72.1%,黏度为1513mPa.s,硬度为0.436N,贮藏14d后,活菌数为7.53logCFU/ml。
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