CN116948865B - 一株凝结芽孢杆菌及其应用 - Google Patents
一株凝结芽孢杆菌及其应用 Download PDFInfo
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- CN116948865B CN116948865B CN202310473617.4A CN202310473617A CN116948865B CN 116948865 B CN116948865 B CN 116948865B CN 202310473617 A CN202310473617 A CN 202310473617A CN 116948865 B CN116948865 B CN 116948865B
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- bacillus coagulans
- rice
- fermented
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- vermicelli
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- A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
本发明公开了一株凝结芽孢杆菌，所述菌株分类命名为凝结芽孢杆菌（Bacillus coagulans）PR06，已于2021年12月10日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO：M 20211589。本发明提供的凝结芽孢杆菌PR06具有较强的耐酸和耐胆盐能力，对大多数抗生素没有抗性。本发明还公开了所述凝结芽孢杆菌在制备发酵型米粉丝或发酵型米粉条产品中的应用。利用本发明提供的凝结芽孢杆菌PR06所制备的发酵型茶叶米粉丝无苦涩味、蒸煮和感官品质优、风味愉悦。因此，本发明提供的凝结芽孢杆菌PR06具有较高的社会应用价值。
Description
技术领域
本发明涉及微生物菌株及其应用技术领域，具体涉及一株凝结芽孢杆菌及其应用。
背景技术
凝结芽孢杆菌(Bacillus coagulans)为厚壁菌门芽孢杆菌属，胞体呈杆状、芽孢端生、无鞭毛，是一种兼性厌氧的革兰氏阳性菌。凝结芽孢杆菌在有氧及无氧的环境下都可生长，能适应低氧的肠道环境，对酸和胆汁有较高的耐受性，能够进行乳酸发酵，产生的L-乳酸能降低肠道pH值，抑制有害菌，并能促进双歧杆菌等有益菌的生长和繁殖。凝结芽孢杆菌能够形成芽孢，与其他不产乳酸的芽孢杆菌相比有利于恢复胃肠道的微生态平衡。凝结芽孢杆菌芽孢在人体中约4-6h便可萌发，其中85％的菌体可顺利通过消化系统，最终于肠道中萌发并繁殖。相比于胃肠道内固有的重要的乳杆菌和双歧杆菌，凝结芽孢杆菌不仅兼具二者的双重功效，同时还具备芽孢杆菌耐高温、易于培养与贮存稳定等独特优势。研究表明，凝结芽孢杆菌能够提高机体免疫力，增强抗病能力，促进机体消化代谢和营养物质的吸收。目前，凝结芽孢杆菌已被中国农业部列为可直接饲用于畜禽生产的新型饲料添加剂。凝结芽孢杆菌是我国批准的可用于普通食品的菌种之一，于中华人民共和国国家卫生和计划生育委员会2014年第6号公告。在食品领域，凝结芽孢杆菌主要应用于益生菌软糖和常温酸性饮品中，目前行业内尚未见到将凝结芽孢杆菌应用于发酵型茶叶米粉丝的相关报道。
米粉是我国南方地区常见的主食类食品，食用人口达数亿之多，是市场规模最大的米制品。2019年，我国常德米粉和桂林米粉年产值分别超过20亿和25亿元。米粉可分为发酵米粉和非发酵米粉，研究表明发酵米粉在口感和风味上都有较大改善，蒸煮特性和复水性也更佳。由于普通米粉的营养成分较为单一(淀粉约75％、蛋白质约7％)，缺少保健功能成分。近年来随着人们保健意识的增强，普通米粉已经不能满足人们对营养和保健的要求。茶在我国被誉为“国饮”，茶叶的保健功效早已被大众所接受，其中的保健成分主要包括茶多酚和茶氨酸等。茶叶食品符合消费者们对保健的期望与要求，将茶叶应用到各类食品中加工成具茶叶特色和保健功效的食品越来越受到消费者们的青睐。由于米粉中的淀粉相对容易被人体消化吸收，食用后易引起人体血糖水平的升高，不大适宜糖尿病患者食用。研究表明多酚类物质能与淀粉大分子发生络合作用，改变淀粉分子的结晶形态，进而影响淀粉凝胶的老化行为和消化性质，起到抗消化的作用。因此，在米粉加工过程添加含有多酚类物质的茶粉，可以起到延缓米粉消化的效果，适合于高血糖的患者食用。然而，由于茶叶中的茶多酚和咖啡碱会导致米粉产生一定的苦涩味。
为解决上述问题，本申请人致力于筛选出用于制备无苦涩味的发酵型茶叶米粉丝或米粉条的菌株，为发酵型茶叶米粉丝或米粉条产品的开发生产提供新的菌源。
发明内容
本发明的目的是提供一株凝结芽孢杆菌及其应用，开发了一株能够用于制备无苦涩味的发酵型茶叶米粉丝(条)的菌株。
本发明的目的是通过以下技术方案实现的：
一株凝结芽孢杆菌，所述菌株分类命名为凝结芽孢杆菌(Bacillus coagulans)PR06，已于2021年12月10日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO：M20211589，保藏单位地址：湖北省武汉市武昌区八一路299号，中国典型培养物保藏中心，邮编：430072。
本发明提供的凝结芽孢杆菌PR06分离自人体粪便，菌落特征为：在MRS平板培养基上培养24～48h后，菌落形态表现为乳白色，圆形，表面光滑，湿润有光泽；扫描电镜观察显示菌体为杆状，大小为0.2～0.4×2.0～5.0μm；最适生长温度50℃。
本发明还提供了所述的凝结芽孢杆菌PR06在普通发酵型米粉丝或普通发酵型米粉条加工生产中的应用。
本发明还提供了所述的凝结芽孢杆菌PR06在发酵型茶叶米粉丝或发酵型茶叶米粉条加工生产中的应用。
本发明的显著优点在于：
本发明提供的凝结芽孢杆菌PR06具有较强的耐酸和耐胆盐能力，对大多数抗生素没有抗性。利用本发明提供的凝结芽孢杆菌PR06所制备的普通发酵型米粉丝感官品质优、风味独特且愉悦。利用本发明提供的凝结芽孢杆菌PR06所制备的发酵型茶叶米粉丝无苦涩味、蒸煮和感官品质优、风味愉悦。因此，本发明提供的凝结芽孢杆菌PR06具有较高的社会应用价值。
附图说明
图1为凝结芽孢杆菌PR06的菌落形态图。
图2为凝结芽孢杆菌PR06的扫描电镜图。
图3为凝结芽孢杆菌PR06在不同温度下的生长曲线图。
图4为凝结芽孢杆菌PR06发酵茶叶米粉丝成品图(蒸煮前)。
图5为凝结芽孢杆菌PR06发酵茶叶米粉丝成品图(蒸煮后)。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，若未特别指明，实施例中的培养基及实验条件均为常规培养基和实验条件。实施例中所用的试验材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1凝结芽孢杆菌(Bacillus coagulans)PR06的分离与鉴定
1、菌株分离
取新鲜人体粪便样品1.0g，装入装有9.0mL无菌水的无菌离心管中，震荡混匀，得到的菌悬液置于80℃沸水中水浴30min，取出；然后用10倍梯度稀释法依次稀释，选择适宜浓度梯度的稀释液100μL，加入MRS琼脂(胰蛋白胨10.0g/L、牛肉粉5.0g/L、酵母粉4.0g/L、葡萄糖20.0g/L、吐温80 1.0mL/L、K2HPO4·3H2O 1.5g/L、无水乙酸钠3.0g/L、柠檬酸铵2.0g/L、MgSO4·7H2O 0.2g/L、MnSO4·H2O 0.04g/L，琼脂粉20.0g/L，蒸馏水1L，pH值为6.2～6.4)平板中，用无菌涂布棒涂布均匀，以无菌水为空白对照，涂布均匀后置于45℃恒温培养箱倒置培养48～72h。挑取白色或米白色、有光泽、圆形、边缘整齐，菌落的菌株，在MRS琼脂平板培养基上进行反复划线纯化培养，直到得到单菌落，将该单菌落菌株命名为PR06。
2、菌株鉴定
(1)菌落及菌体的形态观察：将菌株PR06在MRS琼脂平板培养基上进行划线培养，在37℃恒温培养24～48h后，菌落形态(图1)显示为乳白色，圆形，表面光滑，湿润有光泽。在扫描电子显微镜下观察，菌体呈杆状，大小为0.2～0.4μm×2.0～5.0μm，其扫描电镜图见图2。
(2)菌株PR06的生长特征观察：将菌株PR06菌悬液按体积比3.0％的接种量分别接种于MRS液体培养基中，置于不同温度下培养24h，发现菌株在30℃～60℃环境下生长良好，最适生长温度为50℃(见图3)。
(3)菌株PR06的分子生物学鉴定：采用细菌全基因组快速抽提试剂盒，提取菌株PR06的全基因组，通过选用细菌16S rDNA通用引物27F(5’-AGAGTTTGATCCTGGCTCAG-3’)和1492R(5’-TACGGCTACCTTGTTACGACTT-3’)进行PCR，然后测序分析。测序结果经BLAST比对，鉴定该菌株为凝结芽孢杆菌，基因序列如SEQ ID NO.1所示，命名为：凝结芽孢杆菌(Bacillus coagulans)PR06，于2021年12月10日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO：M 20211589。保藏单位地址：湖北省武汉市武昌区八一路299号，中国典型培养物保藏中心，邮编：430072。
实施例2凝结芽孢杆菌(Bacillus coagulans)PR06对糖类的利用性应用API50CHL试剂盒(Biomerieux，法国)检测实施例1中分离的菌株PR06对糖类的利用性，结果(表1)显示：本发明凝结芽孢杆菌(Bacillus coagulans)PR06能够利用核糖、D-半乳糖、D-葡萄糖、D-果糖、D-甘露糖、甘露醇、山梨醇、N-乙酰葡萄糖胺、七叶苷柠檬酸铁、D-麦芽糖、D-乳糖、D-密二糖、D-蔗糖、海藻糖、D-棉子糖和淀粉。
表1应用API50CHL试剂盒检测菌株PR06对糖类的利用性结果
| 底物成分 | 48h反应结果 | 底物成分 | 48h反应结果 |
| 对照 | - | 七叶苷柠檬酸铁 | + |
| 甘油 | - | 水杨苷(柳醇) | - |
| 赤藓醇 | - | D-纤维二糖 | - |
| D-阿拉伯糖 | - | D-麦芽糖 | + |
| L-阿拉伯糖 | - | D-乳糖 | + |
| 核糖 | + | D-密二糖 | + |
| D-木糖 | - | D-蔗糖 | + |
| L-木糖 | - | 海藻糖 | + |
| 阿东糖醇 | - | 菊粉(菊糖) | - |
| β-甲基-D-木糖苷 | - | D-松三糖 | - |
| D-半乳糖 | + | D-棉子糖 | + |
| D-葡萄糖 | + | 淀粉 | + |
| D-果糖 | + | 糖原 | - |
| D-甘露糖 | + | 木糖醇 | - |
| L-山梨糖 | - | D-龙胆二糖(拢牛儿糖) | - |
| L-鼠李糖 | - | D-土伦糖(D-松二糖) | - |
| 卫矛醇 | - | D-来苏糖 | - |
| 肌醇 | - | D-塔格糖 | - |
| 甘露醇 | + | D-岩藻糖(D-岩糖) | - |
| 山梨醇 | + | L-岩藻糖 | - |
| α-甲基-D-吡喃甘露醇苷 | - | D-阿拉伯醇(D-阿拉伯糖醇) | - |
| α-甲基-D-吡喃葡萄糖苷 | - | L-阿拉伯醇(L-阿拉伯糖醇) | - |
| N-乙酰葡萄糖胺 | + | 葡萄糖酸钾(盐) | - |
| 苦杏仁苷 | - | 2酮基葡萄糖酸钾(盐) | - |
| ARBULIN(熊果苷) | - | 5酮基葡萄糖酸钾(盐) | - |
备注：+表示阳性结果，说明菌株能够利用受试底物；-表示阴性结果，说明菌株不能利用受试底物。
实施例3凝结芽孢杆菌(Bacillus coagulans)PR06的耐酸及耐胆盐能力
将活化好的菌株PR06制成悬液，取1.0mL菌悬液(浓度为2.0×107cfu/mL)分别接种至9.0mL的pH为2.5、3.0、3.5或胆盐浓度为1.0、2.0、3.0和4.0g/L的MRS液体培养基中，45℃培养4h，分别在培养后0h和培养后4h平板计数法测定活菌数，计算存活率(％)。
存活率(％)＝(4h的活菌数/0h的活菌数)×100。
表2凝结芽孢杆菌PR06的耐酸和耐胆盐能力
表2结果显示，凝结芽孢杆菌PR06在pH为3.0、3.5或胆盐浓度为1.0、2.0、3.0和4.0g/L的环境下具有较高的存活率，说明该菌株具有较强的耐酸和耐胆盐能力，能够顺利通过胃肠道进入机体大肠发挥益生作用。
实施例4凝结芽孢杆菌(Bacillus coagulans)PR06的抗生素抗性将PR06菌液接种至MRS液体培养基中，于45℃生化培养箱中培养24h，取100μL菌液于MRS琼脂平板中涂布，在其上放置药片，所属药片包含红霉素、青霉素、庆大霉素，卡那霉素，链霉素，杆菌肽，氯霉素，万古霉素，四环素，氨苄青霉素，头孢西丁，利福平，新霉素，萘啶酮酸，环丙沙星，多粘菌素，于45℃生化培养箱中倒置培养24h，观察并记录抑菌圈大小。实验结果如表3所示。
表3抗生素对凝结芽孢杆菌PR06的抑菌效果
表3结果显示，凝结芽孢杆菌PR06仅对杆菌肽和多粘菌素这两种抗生素有抗性，对其他大多数抗生素均没有抗性，说明PR06具有较好的应用安全性。
实施例5凝结芽孢杆菌PR06普通发酵型米粉丝的制备和感官品评将直链淀粉含量超过20％且陈化1年～1.5年的早籼米进行磨粉并过80目筛得到大米粉，取800g大米粉样品，加入150g玉米淀粉和50g马铃薯淀粉，充分混匀后，平铺在紫外灯下杀菌24h，加入适量纯水使米浆水分含量为55％～60％。于超净工作台上为米浆接种活化好的凝结芽孢杆菌PR06(浓度：2.0×107cfu/mL，接种量2％v/v)，于30～50℃下发酵8～18h后，将发酵米浆倒入自熟挤丝成型米粉机，将获得的粉丝蒸制2～10min，冷却，于4～8℃下老化3～10h，最后烘干至水分含量低于14％，获得成品。对成品粉丝进行感官品评，结果表明，凝结芽孢杆菌PR06普通发酵型米粉丝的感官品质优，咀嚼时具有令人愉悦的酸甜感。
实施例6凝结芽孢杆菌PR06发酵型茶叶米粉丝的制备
将直链淀粉含量超过20％且陈化1年～1.5年的早籼米进行磨粉并过80目筛得到大米粉，取800g大米粉样品，加入150g玉米淀粉和50g马铃薯淀粉，充分混匀后，平铺在紫外灯下杀菌24h，加入适量茶水(取50g岩茶茶粉，加入1L蒸馏水，121℃灭菌15min，离心，取上清液)，使米浆水分含量为55％～60％。于超净工作台上为米浆接种活化好的凝结芽孢杆菌PR06(浓度：2.0×107cfu/mL，接种量2％v/v)，于30～50℃下发酵8～18h后，将发酵米浆倒入自熟挤丝成型米粉机，将获得的粉丝蒸制2～10min，冷却，于4～8℃下老化3～10h，最后烘干至水分含量低于14％，获得成品。图4和图5显示了凝结芽孢杆菌PR06发酵茶叶米粉丝蒸煮前后的状态图。
实施例7凝结芽孢杆菌PR06发酵茶叶米粉丝蒸煮品质的检测对实施例6中的米粉丝样品的蒸煮品质进行检测，具体参照标准(SN/T 0395-2018)测定米粉粉丝的断条率、蒸煮损失率和复水时间，参照标准(QB/T 2652-2004)测定米粉丝的复水率。测定结果如表4所示。
表4凝结芽孢杆菌PR06发酵茶叶米粉丝的蒸煮品质测定结果
| 样品 | 断条率％ | 蒸煮损失率％ | 复水率％ | 复水时间s |
| 实施例6发酵型茶叶米粉丝 | 3.7±0.3 | 4.3±0.1 | 203.6±3.3 | 223±7 |
| 未接种PR06的对照品 | 6.1±0.5 | 5.0±0.1 | 235.6±8.2 | 202±6 |
表4结果显示，利用本发明凝结芽孢杆菌PR06制备的发酵茶叶米粉丝蒸煮品质佳，断条率和蒸煮损失率均低于5.0％，复水率大于200％，复水时间在4min(240s)之内。
实施例8凝结芽孢杆菌PR06发酵茶叶米粉丝感官品质的检测
对实施例6中的米粉丝的感官品质进行评价，结果如表5所示。
表5凝结芽孢杆菌PR06发酵茶叶米粉丝感官评定标准及结果
表5结果显示，利用本发明凝结芽孢杆菌PR06制备的发酵型茶叶米粉丝感官品质优，总感官评分达到92分。
实施例9凝结芽孢杆菌PR06发酵茶叶米粉丝的风味物质检测
对实施例6中的发酵型茶叶米粉丝样品中的风味物质进行检测。
检测方法：取2g样品移入到15mL萃取瓶中，然后快速密封。将SPME萃取纤维头在GC-MS进样口于250℃老化至无杂峰。将样品瓶置于固相微萃取装置上，设定温度为85℃；将样品瓶放在萃取装置充分水浴加热5min；将SPME萃取头通过瓶盖插入样品的顶空部分，推出纤维头，萃取头高于样品上表面约1.0cm，顶空萃取45min；抽回纤维头，从样品瓶中拔出萃取头；再将萃取头插入GC-MS进样口，推出纤维头，于250℃解析5min，进样分析。GC检测条件如下：色谱柱：HP-5MS柱(30m×0.25mm,0.25μm)；载气：氦气；流量1mL/min；样品注入温度保持在250℃；气相色谱烘箱温度保持在40℃下3min，然后以4℃/min升高至150℃，然后以10℃/min升高至250℃，而后保持不变。MS条件如下：EI离子源，电子能量70eV，质量范围35～350AMU/s，GC-MS接口温度250℃，离子源温度230℃，四极杆温度为150℃。
凝结芽孢杆菌PR06发酵茶叶米粉丝风味物质的检测结果如表6所示。
表6凝结芽孢杆菌PR06发酵茶叶米粉丝的风味物质检测结果
表6结果显示，利用本发明凝结芽孢杆菌PR06制备的发酵型茶叶米粉丝的主要风味物质为壬醛、反式-2-庚醛、癸醛、香叶醇、十三醛、香叶基丙酮、2,4-二叔丁基苯酚、水杨酸辛酯和邻苯二甲酸二异丁酯等。
综上所述，本发明提供的凝结芽孢杆菌PR06具有较强的耐酸和耐胆盐能力，对大部分抗生素均有抗性，并且能够用于制备普通发酵型米粉丝或普通发酵型米粉条，也能够用于制备无苦涩味且蒸煮和感官品质俱佳的发酵型茶叶米粉丝或发酵型茶叶米粉条。鉴于凝结芽孢杆菌是我国批准的可用于普通食品的菌种之一，是属于行业内公认的安全性很高的菌株，因此本发明提供的凝结芽孢杆菌PR06具有很高的实际应用价值。
以上仅是本发明的若干个具体实施例。本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
Claims (3)
1.一株凝结芽孢杆菌（Bacillus coagulans）PR06，其特征在于：已于2021年12月10日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO：M 20211589，保藏地址为：湖北省武汉市武昌区八一路299号。
2. 如权利要求1所述的凝结芽孢杆菌（Bacillus coagulans）PR06在制备普通发酵型米粉丝或普通发酵型米粉条产品中的应用。
3. 如权利要求1所述的凝结芽孢杆菌（Bacillus coagulans）PR06在制备发酵型茶叶米粉丝或发酵型茶叶米粉条产品中的应用。
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