CN116083268B - 一种阿氏芽孢杆菌及其在改善雪茄香气中的应用 - Google Patents
一种阿氏芽孢杆菌及其在改善雪茄香气中的应用 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/18—Treatment of tobacco products or tobacco substitutes
- A24B15/20—Biochemical treatment
-
- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B3/00—Preparing tobacco in the factory
- A24B3/12—Steaming, curing, or flavouring tobacco
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- 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
- C12N1/20—Bacteria; Culture media therefor
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- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- General Health & Medical Sciences (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供了一种阿氏芽孢杆菌及其在改善雪茄香气中的应用,该阿氏芽孢杆菌命名为阿氏芽孢杆菌YNLTT1,于2022年06月24日保藏于中国典型培养物保藏中心,保藏编号为CCTCCNo:M2022956。本发明的阿氏芽孢杆菌YNLTT1具有产蛋白酶活性,可应用于雪茄烟叶中性香气成分的提升,对国产雪茄品质提高有较好的促进作用。
Description
技术领域
本发明属于生物技术领域,具体涉及一种阿氏芽孢杆菌及其在改善雪茄香气中的应用。
背景技术
雪茄作为烟草领域代表性产品之一,极具研究价值。烟草企业均致力于改善雪茄发酵条件进而提升产品质量,国产四川特色雪茄产品为提升优质雪茄比例,优化烟草发酵环节尤为重要。
雪茄是一种经晾干、发酵、陈化形成的全叶卷制特殊烟草制品,香气浓郁、焦油含量小、劲头大,主要由不同发酵方式的茄衣、茄套、茄芯组成。烟草的主要成分为糖类、含氮化合物、生物碱、香味物质、矿质元素等,其中含氮化合物蛋白质与烟草品质密切相关。烟草蛋白质含量较少将有助于烟气抽吸质量,可能是烟草中有害物质的前体物。因此适度降解国产雪茄烟叶的蛋白质对于优化雪茄特征风味具有重要意义。
发明内容
针对现有技术中存在的上述问题,本发明提供一种阿氏芽孢杆菌及其在改善雪茄香气中的应用,该阿氏芽孢杆菌具有产蛋白酶活性,可应用于雪茄烟叶中性香气成分的提升,对国产雪茄品质提高有较好的促进作用。
为实现上述目的,本发明解决其技术问题所采用的技术方案是:提供一种阿氏芽孢杆菌(Bacillus aryabhattai),命名为阿氏芽孢杆菌YNLTT1,于2022年06月24日保藏于中国典型培养物保藏中心,保藏编号为CCTCC No:M2022956。
上述阿氏芽孢杆菌YNLTT1在改善雪茄香气中的应用。
一种用于改善雪茄香气的酶制剂,该酶制剂的活性成分包括上述阿氏芽孢杆菌YNLTT1。
一种用于改善雪茄香气的复合微生物菌制剂,该复合微生物菌剂的活性成分包括上述阿氏芽孢杆菌YNLTT1。
综上所述,本发明具备以下优点:
1、阿氏芽孢杆菌YNLTT1具有产蛋白酶活性,可应用于雪茄烟叶中性香气成分的提升,能够提升四川雪茄(什邡)的品质,增加烟叶原本的香气成分,对于中烟工业生产优质国产雪茄有较大的研究意义。
2、筛选所得的阿氏芽孢杆菌YNLTT1可用于后续相关产品的开发,比如酶制剂和复合微生物菌制剂。
3、采用气相色谱串联质谱仪器分析添加菌制剂与未添加菌制剂的中性香气成分,这对于添加降解蛋白质微生物后雪茄产品品质提高的机制有一定贡献,具有学术研究意义。
4、在传统分离纯化微生物的基础上,选用烟草浸提物培养基作为分离培养基,更好的模拟微生物生长环境,除此之外,选择筛选微生物的材料为优质发酵雪茄一级品更具有代表性,筛选所得阿氏芽孢杆菌培养简便快捷,成本低,不需要专门的商品酶处理,生物友好。实验验证菌株具有一定的耐碱特性及优良发酵特性,结合气相色谱质谱串联分析发酵结果,分析结果更加具有科学代表性,雪茄烟叶中性致香成分解析中该菌株发酵效果增香显著。
附图说明
图1为烟叶细菌分离纯化示意图;
图2为单菌落点板酪蛋白培养基水解实验图;
图3为阿氏芽孢杆菌YNLTT1菌株水解酪蛋白实验结果图;
图4为酪氨酸标准曲线;
图5为不同菌株蛋白酶活力测定图;
图6为阿氏芽孢杆菌YNLTT1菌株菌落形态;
图7为阿氏芽孢杆菌YNLTT1菌株生长曲线;
图8为阿氏芽孢杆菌YNLTT1生物扫描电镜图;
图9为阿氏芽孢杆菌YNLTT1耐碱性验证实验结果;
图10为比对结果示意图;
图11为阿氏芽孢杆菌YNLTT1系统发育树。
具体实施方式
实施例1阿氏芽孢杆菌YNLTT1的分离纯化、筛选和鉴定
通过传统分离方法从优质发酵雪茄烟叶分离纯化可培养的微生物,筛选出降解蛋白质的功能微生物,应用到国内雪茄,提升香气及品质,具体步骤如下:
一、雪茄烟叶微生物分离纯化
(1)筛选原料:优质发酵雪茄烟叶茄芯、茄套、茄衣;
(2)雪茄烟叶表面微生物培养基:10%烟草浸提物培养基(1L):100ml烟草浸提液+9g胰蛋白胨+9g氯化钠+4.5g酵母浸提物+20g琼脂,pH自然,温度室温;
其中,烟草浸提液制备:15g雪茄烟叶+400ml蒸馏水,煮沸提取30min,室温冷却,两层纱布过滤,得烟叶沸水浸提液;
(3)菌悬液制备:将烟叶于40℃烘箱中平衡水分3小时后,取1g中部烟叶在无菌条件下,利用灭菌的剪刀剪碎后,浸泡于100mL无菌水中,于37℃,200r/min振荡培养30min以上,无菌单层纱布过滤培养液,9000r/min离心15min后,弃上清,沉淀用5mL无菌水重悬,得原始菌悬液;
(4)分离纯化:将原始菌液稀释0、10、100、1000、10000和100000倍后,分别吸取100μL在烟叶水浸提物固体培养基涂布均匀,37℃培养48h;培养所获得的菌落,应用平板划线法纯化多次,直至获得单菌落。
分离纯化得到4株不同形态的菌株,其菌株如图1所示。
二、降解蛋白质功能细菌的筛选及蛋白酶活力测定
(1)烟叶中分离纯化的4株菌的功能性筛选:因烟叶中大分子物质蛋白质的存在对于雪茄制品的抽吸特性有较为消极的影响,故选择降解大分子物质蛋白质为筛选标准,确定筛选培养基为酪蛋白降解培养基,将4株菌接种于酪蛋白培养基进行点板实验,观察菌株生长情况以及蛋白质水解透明圈大小
(2)蛋白质降解功能菌的降解特性及蛋白酶活力测定
1、蛋白酶筛选固体培养基:酪蛋白10.0g/L,酵母提取物5.0g/L,NaCl 4.0g/L,琼脂粉20g/L,pH 7.2
2、烟叶浸提物液体培养基(1L):100ml烟草浸提液+9g胰蛋白胨+9g氯化钠+4.5g酵母浸提物
3、菌株蛋白质降解能力测定:挑取单菌落接种于蛋白酶筛选固体培养基中,在37℃的条件下倒置培养48h,用直尺测量菌落直径(d)及降解圈直径(D),计算菌株降解蛋白质的降解指数I
4、粗酶液制备:将上述蛋白质降解能力较强的菌株接种于烟叶浸提物液体培养基中,37℃培养至待菌株OD660为0.8-1.2之间时,用于后续测定蛋白酶活力
5、测定蛋白酶活力:将5ml粗酶液于4℃8000r/min离心5min,弃沉淀,取上清加入50ml磷酸盐缓冲液摇匀后适当稀释后作为蛋白酶待测液,于波长660nm处测定吸光度。
上述实验结果如图2-3所示。由图2可知,阿氏芽孢杆菌YNLTT1菌株(H-1-3)降解能力最好,降解指数D/d达3.75。
且酪氨酸标准曲线如图4所示,由此得到不同菌株蛋白酶活力测定图如图5所示。由图4-5可知,阿氏芽孢杆菌YNLTT1菌株(即H-1-3)的活力最好。
三、降解蛋白质功能菌的生理生化实验及分子生物学鉴定
(1)生理生化形态
经过平板划线分离后,菌落颜色整体呈不透明乳白色,单菌落呈光滑状,中央凸起为白色粗糙状,边缘光滑整齐。
镜检观察为中长杆菌,呈正圆柱形,菌体两端多钝圆,结果如图6所示。经革兰氏染色后镜检观察是紫色,为革兰氏阳性菌。
将该功能菌株于37℃培养箱中静置培养48h,间隔一定时间测其菌液吸光度值变化情况,其结果如图7所示。
由图7可知,菌株在25h生长速率达到最大值,随后呈现衰退并保持稳定状态。
生理生化鉴定实验项目包括革兰氏染色、甲基红实验、吲哚实验、V-P实验、糖发酵实验、好氧生长实验。
结果表明染色呈紫色为革兰氏阳性菌;甲基红实验、糖发酵实验、好氧生长呈阳性;吲哚实验、V-P实验呈阴性,如表1所示。
表1阿氏芽孢杆菌YNLTT1生理生化实验
注:“+”表示阳性,“-”表示阴性
(2)细菌扫描电镜观察
细菌样品前处理:将菌液以3000r/min转速离心10min后,弃上清液。注入2.5%戊二醛,静置2h以上固定。固定后离心,将细菌沉积,弃上清,以蒸馏水重悬浮,重复3次,最后以蒸馏水重悬。分别用30%、50%、70%、80%、90%梯度浓度酒精脱水,每个梯度中静置10min,脱水完毕,用纯酒精脱水3次,每次30min,然后用纯叔丁醇置换酒精3次,每次30min,最后吸取混匀的细菌-叔丁醇悬浮液滴在覆有盖玻片的样品台上,置冷冻干燥机内真空干燥,结果如图8所示。
(3)为测试该菌株耐碱性能,本实验设置额外添加不同浓度的NaOH的三种培养基作为供试生长培养基,然后测菌株不同生长时间的吸光度值,如图9所示。由图9可知,初步确定该菌株具有耐碱性,实验结果表明6g/L NaOH的生长状况较良好。
(4)分子生物学鉴定
实验步骤:
1、DNA提取
使用TSINGKE植物DNA提取试剂盒(通用型),具体步骤如下:
(1)将Spin Column置于Collection Tube中,加入250μl Buffer BL,12000rpm/min离心1min活化硅胶膜;
(2)取样本干燥组织(不大于20mg),加入液氮充分研磨。研磨后置于1.5ml离心管中,加入400μl Buffer gP1,涡旋振荡1min,65℃水浴10-30min,期间可取出颠倒混匀以充分裂解;
(3)加入150μl Buffer gP2,涡旋振荡1min,冰浴5min;
(4)12000rpm/min离心5min,将上清转移至新的离心管中;
(5)加入上清等体积的无水乙醇,立即充分振荡混匀,液体全部转入Spin Column中,12,000rpm/min离心30s,弃废液;
(6)向Spin Column中加入500μl Buffer Pw(使用前已加入无水乙醇),12000rpm/min离心30s,弃废液;
(7)向Spin Column中加入500μl Wash Buffer(使用前已加入无水乙醇),12000rpm/min离心30s,弃废液;
(8)重复操作步骤7;
(9)将Spin Column放回Collection Tube中,12,000rpm/min离心2min,开盖晾干1min;
(10)取出Spin Column,放入一个干净的离心管中,在吸附膜的中央处加50-100μlTE Buffer(65℃预热TE Buffer),20-25℃放置2min,12000rpm/min离心2min。
2、PCR扩增及电泳检测
提取的DNA样品适量稀释后作为PCR模板,以擎科1×TSE101金牌mix进行扩增16SrDNA,扩增长度为1500bp左右。细菌通用引物为5’-3’(AGTTTGATCMTGGCTCAGGGTTACCTTGTTACGACTT)。将扩增好的PCR产物进行琼脂糖凝胶电泳(2ul样品+6ul溴酚蓝),300V电压下12分钟。
3、比对鉴定
(1)用ContigExpress拼接测序结果,并去除两端不准的部分。
(2)将拼接好的序列在NCBI数据库(blast.ncbi.nlm.nih.gov)中进行比对,比对结果如图10所示。
(3)选择同源度最高的物种
经过鉴定为杆菌属,阿氏芽孢杆菌,命名为阿氏芽孢杆菌YNLTT1。
利用MEGA 11软件构建系统发育树如图11所示,其序列如SEQ ID No.1所示。
实施例2阿氏芽孢杆菌性能检测实验
气相色谱质谱分析对照组与实验组雪茄烟叶中性挥发性成分
1、样品来源:未发酵什邡雪茄茄套烟叶
2、样品前处理:对照组什邡雪茄茄套与实验组雪茄茄套于40℃烘干后,磨成粉末,过40目筛。准确称取烟末0.5g,于20mL顶空瓶中,对照组添加1ml灭菌后烟叶浸提物液体培养基,实验组添加1ml培养至OD660为0.8-1.2的阿氏芽孢杆菌菌悬液,28℃培养12d,将10ul乙酸苯乙酯内标滴在顶空瓶内侧,密封后置于固相微萃取加热台上70摄氏度平衡20min;将老化后的萃取头插入顶空瓶中,顶空采样(萃取温度为70℃萃取时间为30min),从顶空瓶拔出萃取头立即插入气相色谱仪进样口,在250℃解析5min,进行GC-MS/MS检测。
3、色谱分析条件:色谱柱:DB-5ms(30m×0.25mm×0.25μm)、载气:高纯氦气,流速1.5mL/min、进样量:1μL、进样口温度:250℃、进样方式:分流比10:1、升温程序:初始温度60℃,保持2min,以4℃/min升至240℃,保持5min。
4、质谱分析条件:离子源:EI源、电离电压70eV、离子源温度300℃、传输线温度:250℃、溶剂延迟时间3min、扫描方式:全扫描扫描范围:33-350amu。
5、仪器:生产厂家ThermoFisher赛默飞,美国,型号DSQ9000,三重四级杆气相-质谱联用仪。
对照组与实验组雪茄烟叶中性挥发性成分结果见表2。
表2菌悬液强化前后什邡雪茄茄套挥发性物质含量对比分析
由表2可知,实验组烟草生物碱含量高于对照组,但整体含量仍低于自然发酵烟草生物碱含量。其中,对照组共检出35种挥发性成分,实验组检出50种挥发性成分,经阿氏芽孢杆菌发酵后雪茄烟叶致香物质明显增加,比如新植二烯、异丁酸丙酯、2-哌嗪酮、茄酮、3-甲基丁酸、4-甲基戊酸等成分。
同时,萜类、酯类、醛酮类、酸类整体都有增加趋势。新增降烟碱成分尼可他因;酯类异戊酸异戊酯、反油酸乙酯;醛酮类香叶基丙酮、植酮、苯甲醛、六氢假紫罗酮;酸类中的氢化肉桂酸。其中异戊酸异戊酯具有苹果香气味;六氢假紫罗酮具有致香作用,赋予烟叶花果香及柏木香特征;氢化肉桂酸具有增香作用可作为香料的定香剂,具有协调烟叶抽吸特性的潜在功效。
虽然结合附图对本发明的具体实施方式进行了详细地描述,但不应理解为对本专利的保护范围的限定。在权利要求书所描述的范围内,本领域技术人员不经创造性劳动即可作出的各种修改和变形仍属本专利的保护范围。
Claims (3)
1. 一种阿氏芽孢杆菌(Bacillus aryabhattai),其特征在于,命名为阿氏芽孢杆菌YNLTT1,于2022年06月24日保藏于中国典型培养物保藏中心,保藏编号为CCTCC No:M2022956。
2. 权利要求1所述的阿氏芽孢杆菌 YNLTT1在改善雪茄香气中的应用。
3. 一种用于改善雪茄香气的复合微生物菌制剂,其特征在于,所述复合微生物菌剂的活性成分包括权利要求1所述的阿氏芽孢杆菌 YNLTT1。
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