CN111560328A - 一种枯草芽孢杆菌fg7及其制备酵素饮料的方法 - Google Patents
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Abstract
本发明公开了一种枯草芽孢杆菌FG7及其制备酵素饮料的方法,从发酵椰子水中分离出内生菌,获得一株用于制备酵素饮料的芽孢杆菌菌株,命名为FG7;经过形态学和分子生物学方法鉴定为枯草芽孢杆菌(Bacillus subtilis);于2020年4月13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,保藏号为CGMCC No.19566。利用枯草芽孢杆菌FG7发酵得到的椰子酵素饮料风味独特,含糖量减少且有机酸含量提高。
Description
技术领域
本发明涉及微生物应用领域,具体涉及一种枯草芽孢杆菌FG7及其制备酵素饮料的方法,具体地,该酵素饮料为椰子水酵素。
背景技术
椰子是生长在热带和亚热带地区的植物,随着成熟度的增加,其蔗糖含量增加,而葡萄糖和果糖含量会减少。新鲜的椰子水含有多种营养物质,除了多种碳水化合物,还有矿物质、氨基酸、维生素、抗氧化物等,具有易于消化、甘甜解渴的价值。目前椰子水的利用率很低,大部分工厂加工椰子时只有少部分椰子水被利用为饮料外,大部分被丢弃,因其营养丰富,容易被环境中的微生物利用,造成了严重的资源浪费和环境污染,所以对椰子水的合理应用具有良好的生态效益和经济价值。
枯草芽孢杆菌(Bacillus subtilis)是自然界中存在较为丰富的一个类群,其可以在几种无脊椎动物和脊椎动物(包括人类)的胃肠道生长定居。枯草芽孢杆菌无致病性,细胞壁组成只有磷壁酸和肽聚糖,不含热源性脂多糖这种内毒素,是安全的微生物,发酵条件简单,成本较低,良好的生产技术和发酵基础,在简便的培养条件中也能生长。枯草芽孢杆菌可产生和分泌多种对动物肠道有益的物质,包括消化酶类、B族维生素和多种抑菌物质。此外,枯草芽孢杆菌刺激动物肠道,激活免疫系统,提高对疾病的抵抗能力。有资料表明枯草芽孢杆菌可以耐唾液和胆汁的攻击,有较好的耐酸碱特性,在酸性胃环境中能保持活性,是微生物中可直达大小肠的活菌。
发明内容
为了克服现有技术,本发明的目的在于提供一种枯草芽孢杆菌FG7及其制备酵素饮料的方法,在发酵椰子水中提取一种新的枯草芽孢杆菌菌株,命名为FG7,利用该FG7菌株能制备风味独特、低糖、高有机酸的椰子水酵素,在食品和日化领域具有良好的应用情景,能产生一定的经济效益和生态效益。
本发明的目的采用如下技术方案实现:
一种枯草芽孢杆菌FG7,其特征在于,从发酵椰子水中分离出内生菌,获得一株用于制备酵素饮料的芽孢杆菌菌株,命名为FG7;经过形态学和分子生物学方法鉴定为枯草芽孢杆菌(Bacillus subtilis);于2020年4月13 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,单位地址为北京市朝阳区北辰西路1号院3号,保藏号为CGMCC No.19566。
具体地,所述发酵椰子水为新鲜椰子水于20~30℃发酵7天所得。
枯草芽孢杆菌FG7筛选分离于自然发酵的椰子水,其菌落形态为:菌落表面粗糙不规则且边缘不整齐,菌落不透明,呈现灰白色;经过分子生物学鉴定为枯草芽孢杆菌。利用枯草芽孢杆菌FG7制备椰子水酵素,能产生较好的经济效益和生态效益。
枯草芽孢杆菌FG7制备酵素饮料的方法,包括以下步骤:
1)将FG7菌株接种到LB固体培养基上,25~35℃恒温培养;从平板上挑取单克隆接入LB液体培养基,30℃恒温培养,得到FG7菌株一级种子液;
2)将步骤1)所得的FG7菌株一级种子液以1~5%的接种量接入到LB 液体培养基中,25~35℃恒温培养,得到FG7菌株二级种子液;
3)将步骤2)所得的FG7菌株二级种子液按0.25~0.5%的接种量接种至椰子水发酵培养基中,发酵后,得到粗产物;其中,发酵温度为25~35℃,发酵时间为120~288h。所用的椰子水发酵培养基为2~5wt%蔗糖、2~5wt%葡萄糖,其余为椰子水。粗产物的糖度小于5°Bx,粗产物的pH值为3-4。
4)将步骤3)所得的粗产物过滤,离心取上层清液,得到酵素饮料。
相比现有技术,本发明的有益效果在于:
本发明利用椰子水变质快的特性,在发酵椰子水中提取出枯草芽孢杆菌 FG7并应用于制备酵素饮料,由于枯草芽孢杆菌FG7具有耐受性强,能够耐受肠胃道低pH和高胆盐浓度,能够在肠道定植、生长繁殖,调节肠道菌群平衡,发挥功能,提高营养物质在肠道中的消化吸收的作用,利用枯草芽孢杆菌FG7发酵得到的椰子酵素饮料风味独特,含糖量减少且有机酸含量提高。
附图说明
图1为枯草芽孢杆菌FG7 16S rRNA扩增图;
图2为枯草芽孢杆菌FG716S rRNA系统发育进化树;
图3为实施例1的椰子水发酵pH值变化;
图4为实施例1的椰子水发酵糖度变化。
具体实施方式
下面,结合附图以及具体实施方式,对本发明做进一步描述,需要说明的是,在不相冲突的前提下,以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
枯草芽孢杆菌FG7的筛选和鉴定
新鲜椰子水于30℃放置自然发酵7天得到发酵椰子水,在12000r/min离心 5min收集菌体,稀释后涂布于LB固体培养基上,在30℃恒温培养至有菌落长出。
通过菌落形态学观察菌落表面粗糙不规则且边缘不整齐,菌落不透明,呈现灰白色,属于芽孢杆菌类;提取菌株基因组DNA,如图1所示,用16S rRNA通用引物进行PCR扩增,得到枯草芽孢杆菌FG7的16S rRNA,在生工生物(上海)有限公司测序后得到的序列信息在NCBI网站上进行提交,得到GenBank号为MT218353。利用NCBI进行Blast进行比对。系统发育分析利用Clustalx软件进行核酸多重序列排列,利用MEGA 7软件 Kimura2-parameter计算进化距离,使用Neighbor-joinjing方法构建如图2 所示的系统发育进化树。结果显示FG7和Bacillus subtilis亲缘关系近,可以确定为枯草芽孢杆菌,分别命名为Bacillussubtilis FG7。
实验例1
枯草芽孢杆菌FG7用于制备椰子水酵素,具体步骤如下:
1)将FG7菌株接种到LB固体培养基上,30℃恒温培养;从平板上挑取单克隆接入LB液体培养基,30℃恒温培养,得到FG7菌株一级种子液;
2)将步骤1)所得的FG7菌株一级种子液以1%的接种量接入到LB 液体培养基中,30℃恒温培养,得到FG7菌株二级种子液;
3)将步骤2)所得的FG7菌株二级种子液按0.25%的接种量接种至椰子水发酵培养基中,发酵后,得到粗产物;其中,发酵温度为30℃,发酵时间为288h。中途测量发酵液pH值和糖度,釆用手持糖度仪进行检测,结果以°Bx表示,当pH值或糖度不再降低时停止发酵。所用的椰子水发酵培养基为5wt%蔗糖、2wt%葡萄糖,其余为椰子水。在本实施例中,时刻记录椰子水发酵过程中的pH值变化和糖度变化,得到图3和图4。
4)将步骤3)所得的粗产物过滤,离心取上层清液,得到椰子水酵素。
实验例2
枯草芽孢杆菌FG7用于制备椰子水酵素,具体步骤如下:
1)将FG7菌株接种到LB固体培养基上,30℃恒温培养;从平板上挑取单克隆接入LB液体培养基,30℃恒温培养,得到FG7菌株一级种子液;
2)将步骤1)所得的FG7菌株一级种子液以1%的接种量接入到LB 液体培养基中,30℃恒温培养,得到FG7菌株二级种子液;
3)将步骤2)所得的FG7菌株二级种子液按0.5%的接种量接种至椰子水发酵培养基中,发酵后,得到粗产物;其中,发酵温度为25~35℃,发酵时间为120h。所用的椰子水发酵培养基为2wt%蔗糖、2wt%葡萄糖,其余为椰子水。
4)将步骤3)所得的粗产物过滤,离心取上层清液,得到椰子水酵素。
实验例3
枯草芽孢杆菌FG7用于制备椰子水酵素,具体步骤如下:
1)将FG7菌株接种到LB固体培养基上,30℃恒温培养;从平板上挑取单克隆接入LB液体培养基,30℃恒温培养,得到FG7菌株一级种子液;
2)将步骤1)所得的FG7菌株一级种子液以1%的接种量接入到LB 液体培养基中,30℃恒温培养,得到FG7菌株二级种子液;
3)将步骤2)所得的FG7菌株二级种子液按0.5%的接种量接种至椰子水发酵培养基中,发酵后,得到粗产物;其中,发酵温度为25~35℃,发酵时间为168h。所用的椰子水发酵培养基为5wt%蔗糖、2wt%葡萄糖,其余为椰子水。
4)将步骤3)所得的粗产物过滤,离心取上层清液,得到椰子水酵素。
如图3-4所示,实施例1利用枯草芽孢杆菌FG7生产得到的椰子水酵素用于未发酵前相比,其pH值下降,产生了较多的有机酸,包括柠檬酸、乙酸、丁二酸和草酸等,是发酵椰子水主要的风味物质,具有可软化血管,促进钙、铁元素的吸收,能刺激消化腺的分泌活动,有增进食欲、帮助消化吸收及止渴解暑的功能;另外糖度的下降,符合现今减糖的生活方式,有利于人们的身体健康。这些都为发酵椰子水在食品和日化领域的应用提供理论基础。将枯草芽孢杆菌FG7应用于椰子水发酵解决了大量椰子水被废弃而污染环境的问题,同时产生了经济价值。
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
附录 枯草芽孢杆菌FG7 16S rRNA序列
TAGGCGGGTGCTATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCTCA
Claims (7)
1.一种枯草芽孢杆菌FG7,其特征在于,从发酵椰子水中分离出内生菌,获得一株用于制备酵素饮料的芽孢杆菌菌株,命名为FG7;经过形态学和分子生物学方法鉴定为枯草芽孢杆菌(Bacillus subtilis);于2020年4月13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,保藏号为CGMCC No.19566。
2.如权利要求1所述的枯草芽孢杆菌FG7,其特征在于,所述发酵椰子水为新鲜椰子水于20~30℃发酵7天所得。
3.一种采用如权利要求1所述的枯草芽孢杆菌FG7制备酵素饮料的方法,其特征在于,包括以下步骤:
1)将FG7菌株接种到LB固体培养基上,25~35℃恒温培养;从平板上挑取单克隆接入液体培养基,30℃恒温培养,得到FG7菌株一级种子液;
2)将步骤1)所得的FG7菌株一级种子液以1~5%的接种量接入到液体培养基中,25~35℃恒温培养,得到FG7菌株二级种子液;
3)将步骤2)所得的FG7菌株二级种子液按0.25~0.5%的接种量接种至椰子水发酵培养基中,发酵后,得到粗产物;
4)将步骤3)所得的粗产物过滤,离心取上层清液,得到酵素饮料。
4.如权利要求1所述的所述的枯草芽孢杆菌FG7制备酵素饮料的方法,其特征在于,步骤1)和步骤2)所用的液体培养基为LB液体培养基。
5.如权利要求1所述的枯草芽孢杆菌FG7制备酵素饮料的方法,其特征在于,步骤3)中所用的椰子水发酵培养基为2~5wt%蔗糖、2~5wt%葡萄糖,其余为椰子水。
6.如权利要求1或5所述的枯草芽孢杆菌FG7制备酵素饮料的方法,其特征在于,步骤3)得到的粗产物的糖度小于5°Bx,粗产物的pH值为3-4。
7.如权利要求1所述的枯草芽孢杆菌FG7制备酵素饮料的方法,其特征在于,步骤3)中,发酵温度为25~35℃,发酵时间为120~288h。
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