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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