CN112852679B - 一株益生凝结芽孢杆菌及其应用 - Google Patents
一株益生凝结芽孢杆菌及其应用 Download PDFInfo
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- CN112852679B CN112852679B CN202110285874.6A CN202110285874A CN112852679B CN 112852679 B CN112852679 B CN 112852679B CN 202110285874 A CN202110285874 A CN 202110285874A CN 112852679 B CN112852679 B CN 112852679B
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Abstract
本发明公开了一株益生凝结芽孢杆菌及其应用，属于微生物技术领域。本发明的凝结芽孢杆菌菌株BC99的保藏编号为CGMCC No.21801，其分离来源安全，生长速度快，制备的菌粉芽孢数可达到3.9×1011CFU/g。本发明菌株同时具有较强的抑制病原菌的能力，对多种抗生素敏感，生物安全性高，具备潜在的益生功能。本发明菌株的耐热性能优异，可用于食品或饮品中，使食品或饮品具有益生功能。
Description
技术领域
本发明属于微生物技术领域，尤其是涉及一株益生凝结芽孢杆菌及其应用。
背景技术
凝结芽孢杆菌（最初命名芽孢乳杆菌）最先是由俄国科学家Horowitz和Wlassowa分离纯化得到的，是一种不仅能产乳酸，还能产芽孢的革兰氏阳性兼性厌氧细菌。凝结芽孢杆菌除了具有乳酸菌的所有特点外，还具有耐高温，抗逆性强，复活率高，代谢旺盛等优良特性。凝结芽孢杆菌在肠道中还能产生大量的有益代谢产物，如L-乳酸、凝固素、lactosporin、淀粉酶、蛋白酶、维生素、氨基酸和多种短链脂肪酸等，这些物质有助于机体消化吸收，抑制有害细菌生长，从而有效地调节肠道微生物菌群平衡，增加肠道上皮细胞的黏膜性，有利于有益菌在肠道中定植，提高上皮细胞的生存力，建立完整的生物屏障系统，提高提供机体的免疫力。如产Ｌ-乳酸特征，能降低肠道pH值，抑制有害菌，从而促进双歧杆菌等益生菌的生长与繁殖。凝固素是一种抗菌肽物质，在60℃、pH值4～8的条件下处理90min仍保持生物活性，且不受淀粉酶、酯酶和有机溶剂的影响，对李斯特菌、肠球菌和明串珠球菌等有抑制作用。研究发现，lactosporin能显著的杀灭阴道致病菌，而不会影响多数非致病性菌株的生长。
凝结芽孢杆菌作为人和动物用益生菌，已列入IDF名单，欧盟 QPS推荐生物制剂列表。凝结芽孢杆菌还通过美国 GRAS 认定，在加拿大、澳大利亚和新西兰等多个国家作为普通食品管理。近年来，凝结芽孢杆菌作为新型微生态制剂，由于其益生作用和高抗逆特性，在食品、医药、保健、畜牧和水产养殖等行业中被广泛应用。
发明内容
本发明的目的在于提供一种生长快速、芽孢数高、益生功能强的凝结芽孢杆菌及其应用，其能广泛应用于食品领域。
本发明的目的通过下述技术方案实现：
一种凝结芽孢杆菌菌株BC99，该菌株是从自然界样品分离到的200多株凝结芽孢杆菌细菌中反复比较筛选得到，其微生物保藏号为CGMCC No.21801；分类命名为：凝结芽孢杆菌Bacillus coagulans；保藏时间：2021年02月01日；保藏单位：中国微生物菌种保藏管理委员会普通微生物中心；保藏地址：北京市朝阳区北辰西路1号院3号。
所述凝结芽孢杆菌利用PCR扩增相应的16S rRNA基因序列，与NCBI 数据库Genbank中已登录的基因序列进行分析得知，该菌株与数据库中不同Bacillus coagulans菌株的16S rDNA序列性具有99%的相似性。
本发明所述的凝结芽孢杆菌菌株BC99具有如下特征：
（1）菌株来源于健康婴儿粪便样品中，分离样品来源安全。
（2）生长速度快，细胞密度高。
（3）芽孢数高，菌粉芽孢率可达到90%。制备的凝结芽孢杆菌菌粉的活菌数是4.3×1011CFU/g，芽孢数可达到3.9×1011CFU/g，芽孢率为90.7%。
（4）对大肠杆菌、金黄色葡萄球菌和沙门氏菌致病菌均具有显著的抑制作用。
（5）对青霉素类（青霉素、氨苄西林、苯唑西林和阿莫西林）、头孢菌素类（头孢克洛、头孢曲松和头孢噻吩）、氨基糖苷类（链霉素、庆大霉素和卡那霉素）、大环内脂类（新霉素、红霉素和阿奇霉素）、四环素类（四环素）、糖肽类（万古霉素）、磺胺类（甲氧嘧啶）、喹诺酮类（环丙沙星）、林克胺类（克林霉素）、利福霉素类（利福平）和其他类（新生霉素）10类的20种常见抗生素敏感，安全性高。
本发明提供含有所述凝结芽孢杆菌BC99益生菌菌粉，菌粉的芽孢数可达到3.9×1011CFU/g。
所述的凝结芽孢杆菌BC99益生菌菌粉是采用下述方法制备的：凝结芽孢杆菌BC99菌种从甘油管出发，接入试管斜面活化，经一级种子摇瓶培养、二级种子摇瓶培养，进行5L发酵罐发酵，得到的发酵液经12000rmp离心后收集菌泥，通过喷雾干燥工艺得到菌粉，菌粉进行活菌计数检测，菌粉检测芽孢数可达到3.9×1011CFU/g。
所述的凝结芽孢杆菌BC99耐热性能优异，80℃水浴处理2h后，芽孢存活率98%；100℃高温处理10min后芽孢存活率96%。其菌粉可用于食品或饮品中，特别是热饮品，使食品或饮品具有益生功能。
所述的凝结芽孢杆菌BC99或其菌粉在咖啡中的应用。
所述的凝结芽孢杆菌BC99或其菌粉在茶饮料中的应用。
所述的凝结芽孢杆菌BC99或其菌粉在豆奶粉中的应用。
本发明凝结芽孢杆菌BC99菌株分离来源安全，生长速度快，制备的菌粉芽孢数可达到3.9×1011CFU/g。同时该菌株具有较强的抑制病原菌的能力，对多种抗生素敏感，生物安全性高，具备潜在的益生功能。该菌株制备的菌粉在咖啡、茶饮料和豆奶粉中应用，表现出热稳定性好，芽孢存活率高。
附图说明
图1是生长较快的前5株凝结芽孢杆菌的生长曲线图。
具体实施方式
下面结合实施例对本发明做进一步详细的描述，但本发明的实施方式不限于此。
实施例1
（1）凝结芽孢杆菌菌株的筛选
从全国各地采集传统的发酵食品、泡菜、发酵乳制品、健康人的口腔、健康婴幼儿的粪便和健康成年人的粪便样品，经过分离筛选，利用PCR扩增相应的16S rRNA基因序列，鉴定得到不同来源的凝结芽孢杆菌菌株200多株，菌株超低温甘油管保藏于武汉微康益生菌研究院有限公司实验室。
（2）凝结芽孢杆菌菌株生长速度比较实验
从甘油管出发，接种一环于PCA试管斜面培养基上，42℃培养48-72h，待大部分形成芽孢后，加入3-5mL无菌水，洗脱试管斜面菌苔，制备菌悬液。取菌悬液按照10-20%接种量接入到装有种子培养基的摇瓶中培养，摇瓶装液量6mL/50mL，42℃、200r/min培养4-6h得一级种子液；将一级种子液按照10-20%接种量接入到装有种子培养基的摇瓶中培养，摇瓶装液量30mL/250mL，42℃、200r/min培养2-4h得二级种子液；二级种子液以5%接种量转接到装有发酵培养基的摇瓶中培养，摇瓶装液量30mL/250mL，42℃、200r/min培养，进行生长曲线的测定。
所述的种子培养基配方：以重量百分比计，葡萄糖2.0%、酵母浸粉1.0%、蛋白胨1.0%、MgSO4·7H2O 0.03%、MnSO4 0.01%、NaCl 0.05%、K2HPO4 0.1%、FeSO4·7H2O 0.001%，CaCO3 0.05%，余量为水，调节初始pH值为7.0±0.1，115℃灭菌30min备用。
所述的发酵培养基配方：以重量百分比计，葡萄糖3.0%、酵母浸粉3.5%、蛋白胨4.0%、MgSO4·7H2O 0.06%、MnSO4 0.01%、NaCl 0.1%、K2HPO4 0.2%、FeSO4·7H2O 0.002%，CaCO3 0.1%，余量为水，调节初始pH值为7.0±0.1，115℃灭菌30min备用。
对200株菌株通过生长速度的比较实验，经过反复筛选、综合比较等实验，优选出凝结芽孢杆菌菌株3298，将其定名为凝结芽孢杆菌BC99，该菌株来源于健康婴儿粪便样品中，其生长OD值显著高于其它菌株，生长较快的前5株凝结芽孢杆菌的生长曲线图见图1。
（3）凝结芽孢杆菌菌株发酵罐放大比较实验
按照上述（2）制备5株凝结芽孢杆菌菌株BC99、1916、3702、611和2的二级种子液，再按照5%接种量转接到装有发酵培养基的发酵罐中进行发酵培养，42℃ 培养28-30h，得到发酵液。发酵液中的活菌数和芽孢数见下表1。
表1 不同凝结芽孢杆菌发酵液活菌数和芽孢数
| 菌株编号 | 发酵液活菌数（*10<sup>10</sup>CFU/g） | 发酵液芽孢数（*10<sup>10</sup>CFU/g） | 芽孢转化率（%） |
| BC99 | 1.5±0.06 | 1.32±0.03 | 88% |
| 1916 | 1.0±0.1 | 0.8±0.13 | 80% |
| 3702 | 0.6±0.28 | 0.45±0.32 | 75% |
| 611 | 0.9±0.11 | 0.7±0.09 | 77.8% |
| 2 | 0.6±0.38 | 0.42±0.58 | 70% |
凝结芽孢杆菌BC99发酵液活菌数为1.5×1010CFU/mL，芽孢数为1.32×1010CFU/g，芽孢转化率可达88%，高于其它筛选菌株。
（4）凝结芽孢杆菌益生菌菌粉活菌数和芽孢数检测
由（3）得到的发酵液经12000rmp离心后收集菌泥，通过喷雾干燥工艺得到菌粉。喷雾干燥塔进风温度为158-162℃，出风温度为 80-84℃，喷雾干燥塔内真空度为-0.01Mpa～-0.02MPa。
将凝结芽孢杆菌菌粉样品分别取0.1g加入到9.9g含有0.6%吐温80的带玻璃珠的无菌生理盐水中，在25℃温度下250rpm摇床震荡均质30min，便得到初始菌悬液。将均质后的初始菌悬液汲取2mL于离心管中浸入水浴锅进行 80℃水浴处理10min，取出水浴后的菌液冰水冷却至常温得到样品待测液。待测液用无菌生理盐水十倍梯度稀释，选择合适的稀释度用于计数，计算得到菌粉的芽孢数，结果见表2。
表2不同凝结芽孢杆菌菌粉的芽孢数
| 菌株编号 | 菌粉活菌数（*10<sup>11</sup>CFU/g） | 菌粉芽孢数（*10<sup>11</sup>CFU/g） | 存活率（%） |
| BC99 | 4.3±0.08 | 3.9±0.03 | 90.7% |
| 1916 | 4.1±0.12 | 3.2±0.29 | 78% |
| 3702 | 4.1±0.35 | 3.0±0.42 | 73.2% |
| 611 | 3.7±0.21 | 2.8±0.21 | 75.7% |
| 2 | 3.2±0.58 | 2.2±0.58 | 68.8% |
凝结芽孢杆菌BC99菌粉活菌数为4.3×1011CFU/mL，芽孢数为3.9×1011CFU/g，芽孢率可达90.7%，明显高于其它筛选菌株。
凝结芽孢杆菌BC99于2021年02月01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，保藏编号为CGMCC No.21801。
实施例2 凝结芽孢杆菌菌株BC99的抑菌实验
指示菌株菌悬液准备：将大肠杆菌、金黄色葡萄球菌和沙门氏菌分别在LB固体平板培养基上活化，37℃培养24h，挑取菌苔至生理盐水制备菌悬液，调整菌悬液浓度至108CFU/mL。
拮抗菌株菌液制备：将凝结芽孢杆菌按一定比例接种至液体MRS培养基中，42℃培养24h，得到培养液。
抑菌实验：将含有1.5%琼脂的肉汤培养基冷却至55℃左右，与指示菌株菌悬液按一定比例混匀，使得最终指示菌浓度在106CFU/mL，迅速倾注于预先放置牛津杯的平板中，待培养基冷却凝固后，取出牛津杯，在每孔中加入200μL拮抗菌液（108CFU/mL），以200μL无菌MRS液体培养基作为空白对照，静置数分钟后，放入37℃恒温培养箱中，培养适宜时间后观察，用游标卡尺测量抑菌圈直径，每个菌三个重复，结果见表3。
表3对病原菌的拮抗能力
表3结果表明，凝结芽孢杆菌BC99对大肠杆菌、金黄色葡萄球菌和沙门氏菌都有较强的抑制作用，其中对大肠杆菌的抑制作用最为显著。
实施例3 凝结芽孢杆菌BC99抗生素抗性实验
凝结芽孢杆菌划线接种于MRS琼脂平板上，42℃培养48h后，得到单菌落。挑取单菌落至生理盐水中制备菌悬液，调整菌悬液浓度为108CFU/mL。取100μL菌悬液，用无菌棉签均匀涂布于整个MRS固体平板上，将抗生素药敏试纸片按照一定间距有序置于平板表面。37℃培养适宜时间后观察，用游标卡尺测量抑菌圈直径，结果见表4。
表4 凝结芽孢杆菌BC99对抗生素的敏感性
供试菌株和20种常用抗生素进行对峙实验，表4结果表明，该菌株对20种抗生素都是敏感的，充分表明所筛选到的凝结芽孢杆菌BC99菌株是具有开发应用潜力的安全益生菌。
实施例4 凝结芽孢杆菌菌株BC99的耐热性实验
称取凝结芽孢杆菌菌粉（芽孢数在3.9×1011CFU/g）1g添加到1000mL无菌水中，充分混匀。各取凝结芽孢杆菌溶液1mL，分别进行80℃水浴处理、100℃高温处理，其中80℃条件下分别处理2h、5h，100℃高温条件下处理10min、30min。吸取处理液适度稀释，测定活菌数，以未处理的凝结芽孢杆菌溶液作为空白对照组，计算凝结芽孢杆菌的芽孢存活率，每次实验三次重复，结果见表5。
表5 BC99的耐热性评价
凝结芽孢杆菌BC99菌粉的耐热性实验结果表明，80℃水浴处理2h，芽孢存活率影响不大，处理5h后，仍还有80%芽孢存活率。100℃高温处理10min，芽孢存活率影响不大，处理30min后，仍还有82%芽孢存活率。
实施例5 凝结芽孢杆菌BC99菌粉在咖啡中的应用
称取凝结芽孢杆菌菌粉（芽孢数在3.9×1011CFU/g）0.5g添加到9.5g烘焙咖啡粉中，充分混匀。取1.15g含有凝结芽孢杆菌的咖啡粉末添加到200mL沸水中进行冲泡，在室温下搅拌均匀。冲泡处理后，分别在0、10min不同冷却时间下，取样测定芽孢数，每次实验三次重复，结果见表6。
表6 BC99在咖啡中冲泡后的芽孢活性评价
BC99在咖啡中的应用，对BC的热稳定性和芽孢存活率进行评价，结果显示其稳定性好，存活率高。
实施例6 凝结芽孢杆菌BC99菌粉在茶饮料中的应用
称取凝结芽孢杆菌菌粉（芽孢数在3.9×1011CFU/g）0.5g添加到9.5g红茶、绿茶或乌龙茶中，充分混匀。取1.15g含有凝结芽孢杆菌的茶叶添加到200mL沸水中进行冲泡，在室温下搅拌均匀，冲泡处理后，分别在0、10min不同冷却时间下，取样测定芽孢数，每次实验三次重复。以下表7提供BC在红茶中冲泡后的芽孢活性评价。
表7 BC99在红茶中冲泡后的芽孢活性评价
BC99在茶饮料中的应用，对BC的热稳定性和芽孢存活率进行评价，结果显示其稳定性好，对芽孢存活率完全不受影响。
实施例7 凝结芽孢杆菌BC99菌粉在豆奶粉中的应用
称取凝结芽孢杆菌菌粉（芽孢数在3.5×1011CFU/g）0.5g添加到9.5g豆奶粉中，充分混匀。取1.15g含有凝结芽孢杆菌的豆奶粉添加到200mL沸水中进行冲泡，在室温下搅拌均匀，冲泡处理后，分别在0、10min不同冷却时间下，取样测定芽孢数，每次实验三次重复，结果见表8。
表8 BC99在豆奶粉中冲泡后的芽孢活性评价
BC99在豆奶粉中的应用，对BC的热稳定性和芽孢存活率进行评价，结果显示其稳定性好，存活率高。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (8)
1.一种凝结芽孢杆菌（Bacillus coagulans）BC99，其特征在于：保藏编号为CGMCCNo.21801。
2.权利要求1所述的凝结芽孢杆菌BC99的菌粉的制备方法，其特征在于：包括如下步骤：凝结芽孢杆菌BC99发酵得到的发酵液经离心后收集菌泥，菌泥通过喷雾干燥得到菌粉。
3.权利要求1所述的凝结芽孢杆菌BC99在食品中的应用。
4.权利要求1所述的凝结芽孢杆菌BC99在饮品中的应用。
5.权利要求1所述的凝结芽孢杆菌BC99在热饮品中的应用。
6.权利要求1所述的凝结芽孢杆菌BC99在咖啡中的应用。
7.权利要求1所述的凝结芽孢杆菌BC99在茶饮料中的应用。
8.权利要求1所述的凝结芽孢杆菌BC99在豆奶粉中的应用。
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