CN112538447B - 北工商节杆菌菌种及其应用 - Google Patents
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Abstract
本发明公开了北工商节杆菌新菌种及其应用,所述菌株为北工商节杆菌(Arthrobacter beigongshangensis),其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC NO.19210。试验证明,本发明提供的新菌种北工商节杆菌(Arthrobacter beigongshangensis),该菌株分离自白酒窖泥,其能够产生浓香风味物质,可用于发酵酿酒。
Description
技术领域
本发明涉及酿酒微生物技术领域,具体涉及一种北工商节杆菌菌种及其应用。
背景技术
白酒是世界三大蒸馏酒之一,采用独特的发酵技术生产,已有数千年的历史。窖池整个内壁都覆盖有预培养的窖泥。将发酵的原料蒸煮进行混合,粉碎和蒸馏。向蒸过的原料中添加糖化发酵剂,然后放入地窖进行发酵,将发酵的物料从地窖中取出并蒸馏以制成中国白酒。窖泥的微生物会产生各种风味成分,例如丁酸、己酸和己酸乙酯。己酸乙酯被认为是影响浓香型白酒风味和品质的关键成分。酿造过程中,窖泥微生物具有重要的生香增味作用,浓香型白酒质量随着酒窖年龄的增加而提高。浓香型白酒的高品质归因于窖泥的成熟过程,这导致了微生物群落结构的均衡和窖泥的多样性,从而产生了独特的风味。
浓香型白酒具有“窖香浓郁、绵甜醇厚、香味协调、回味悠长”的风味特征,深受广大消费者喜爱,年销售额约占我国白酒总量的70%。窖泥中的微生物会产生各种风味成分,例如丁酸,己酸和己酸乙酯,己酸乙酯被认为是影响浓香型白酒风味和品质的关键成分。窖泥微生物菌群是影响窖泥和白酒质量优劣的主要因素,同时也是白酒酿造过程中重要的微生物来源。窖泥中的微生物多种多样,具有非常丰富的物种多样性,但是大多数微生物还处于难培养状态。微生物在不同环境中的可培养性虽有不同,但都很低,如海水中为0.1%以下,在土壤中约为0.3%。使用传统的细菌培养方法仅能培养自然生境中的一小部分细菌。应用传统方法培养出的微生物都是已知微生物,尚未发现新的微生物物种。
发明内容
为此,本发明提供北工商节杆菌菌种及其应用。
为了实现上述目的,本发明提供如下技术方案:
本发明提供北工商节杆菌(Arthrobacter beigongshangensis)其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC NO.19210。
北工商节杆菌的16S rDNA序列如SEQ ID No.1所示。
本发明还提供北工商节杆菌或其发酵产物或其培养产物在酿酒过程中产生风味物质的应用。
本发明另一方面还提供含所述的北工商节杆菌的微生物菌剂。
本发明另一方面还提供北工商节杆菌的筛选培养方法,其包括将窖泥稀释后涂布于TSA培养基上,筛选得到所述北工商节杆菌。
本发明的一个实施例中,所述TSA培养基包括以下质量的原料:酪蛋白胰酶消化物15g、大豆粉木瓜蛋白酶消化物5g、氯化钠5g、琼脂15g。
本发明的一个实施例中,所述筛选培养的时间为20-28h。
本发明的一个实施例中,所述筛选培养温度为35-40℃。
本发明所述的菌株或所述的菌剂在如下(A1)或(A2)中的应用:(A1) 产生浓香风味物质;(A2)制备具有浓香风味物质的产品,也属于本发明保护的范围。
本发明的所述的菌株或所述的菌剂在如下(B1)或(B2)中的应用:(B1) 酿酒;(B2)制备酿酒产品,也属于本发明的保护范围
本发明具有如下优点:
试验证明,本发明提供一株菌种北工商节杆菌(Arthrobacterbeigongshangensis),该菌株分离自窖泥,其能够产生浓香风味物质,可用于发酵酿酒。
本发明分离菌种,先采用TSA培养基,常见的细菌繁殖较慢,同一培养皿上,难培养芽孢杆菌属细菌生长较快,受常规微生物影响小;同时,延长培养时间36h后挑取单菌落,难培养的微生物经过长时间充分的生长,直至可见的菌落。
菌种保藏说明:本发明的北工商节杆菌(Arthrobacter beigongshangensis),于2019年12月16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,保藏地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,其保藏编号为CGMCC NO.19210。
附图说明
为了更清楚地说明本发明的实施方式或现有技术中的技术方案,下面将对实施方式或现有技术描述中所需要使用的附图作简单地介绍。显而易见地,下面描述中的附图仅仅是示例性的,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图引伸获得其它的实施附图。
图1为本为本发明提供的北工商节杆菌(Arthrobacter beigongshangensis) 16SrDNA序列及进化树图;
图2为本发明提供的北工商节杆菌(Arthrobacter beigongshangensis)的呼吸醌甲基萘醌呼吸醌MK-8:MK-7(H2):MK-7:MK-6(H2)=69:14:5: 12检测结果图;
图3为本发明提供的北工商节杆菌(Arthrobacter beigongshangensis)极性酯检测结果图。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1、北工商节杆菌(Arthrobacter beigongshangensis)的分离与鉴定
一、北工商节杆菌(Arthrobacter beigongshangensis)菌种的分离
步骤一、配置TSA培养基:酪蛋白胰酶消化物15g、大豆粉木瓜蛋白酶消化物5g、氯化钠5g、琼脂15g。
步骤二、取窖泥10g,该窖泥来源于四川浓香型酒厂,用无菌蒸馏水稀释至10-5,涂布于配置的TSA培养基培养皿上28℃好氧培养;
步骤三、28℃好氧培养36h后挑取单菌落,三区划线纯化3次,得到纯培养细菌;
步骤四、提取细菌DNA,利用PCR技术扩增16SrDNA序列,通过序列比对,初步确定北工商节杆菌(Arthrobacter beigongshangensis)菌种。
二、北工商节杆菌(Arthrobacter beigongshangensis)的鉴定
从窖泥中分离筛选的菌为革兰氏阳性菌,节杆菌属,命名为:北工商节杆菌(Arthrobacter beigongshangensis)
1、北工商节杆菌(Arthrobacter beigongshangensis)对菌种的进行形态学、生理生化、细胞化学及基因水平研究。北工商节杆菌REN12T(Arthrobacterbeigongshangensis)生理生化特性
北工商节杆菌(Arthrobacter beigongshangensis)菌落形态为北工商节杆菌菌落均呈淡黄色,圆形,表面突起,边缘光滑,菌体为杆状,宽约0.5μm,长约2.0μm;革兰氏反应阳性,为革兰氏阳性菌,最适温度为30℃,最适pH值8,最适氯化钠浓度1%,可以水解淀粉,过氧化氢酶阳性,碱性磷酸盐酶,酯酶(C4),类脂酯酶(C8),白氨酸芳氨酶,缬氨酸芳氨酶,胰蛋白酶,酸性磷酸酶,萘酚-AS-BI-磷酸水解酶,β-半乳糖苷酶,α-葡萄糖苷酶,β-葡萄糖苷酶,α-甘露糖苷酶呈阳性反应。硝酸还原,水解葡萄糖苷酶-七叶灵。
2、北工商节杆菌(Arthrobacter beigongshangensis)细胞化学特征检测
通过GC气相色谱、HPLC液相色谱和TLC薄层色谱检测北工商节杆菌(Brevibacterium renqingensis)的脂肪酸、醌类型、极性脂等细胞化学组分(SasserM.Identification of bacteria by gas ghromatography of cellular fattyacids, MIDI Technical Note 101.Newark,DE:MIDIinc;1990.Minnikin DE,O’DonnellAG,Goodfellow M,Alderson G,Athalye M et al.Anintegrated procedure for theextraction of bacterial isoprenoid quinones andpolar lipids.J MicrobiolMethods 1984;2:233–241)。
北工商节杆菌(Arthrobacter beigongshangensis)细胞脂肪酸成分:饱和脂肪酸:C16:0,Antesio-C15:0,Antesio-C17:0,Iso-C14:0,Iso-C15:0,Iso-C16:0,Iso-C17:0;不饱和脂肪酸:C16:0N alcohol,细胞脂肪酸成分如表1所示。
表1
如图2所示,在菌株北工商节杆菌(Arthrobacter beigongshangensis)细胞呼吸醌组份检测,该菌株的主要优势醌甲基萘醌呼吸醌MK-8:MK-7(H2): MK-7:MK-6(H2)=69:14:5:12,醌类化合物是一类广泛存在于自然产物、抗肿瘤药物、体内生化代谢物、环境污染物或多环芳烃代谢产生的氧化活性物质。每种细菌都有一个主要的醌类组分,一个微生物群体中的醌类种类和数量的不同反映了群体组成的多样性,醌类谱已经作为细菌群体组成多样性的一个指标,在环境微生物中广泛使用。
北工商节杆菌(Arthrobacter beigongshangensis)极性酯的检测,具体步骤为:极性脂提取:取100mg北工商节杆菌冻干菌体,悬浮于9.5mL氯仿:甲醇:0.3%NaCl(2.5:5:2)溶液中。将上述菌体溶液放置80℃水浴,15min。冷却后,滤纸过滤至50mL离心管中,加入2.5mL氯仿和2.5mL 0.3%NaCl, 4000rpm离心5min。小心分取下层氯仿相至干净的旋转蒸发仪专用烧瓶中,在旋转蒸发仪上减压旋转蒸发除去氯仿,水浴温度不超过40℃。加入250μL 氯防-甲醇(2:1,v/v)转至棕色螺口样品瓶中,放置4℃冰箱中保存待测。
极性脂的TLC分析:将10cm×10cm的硅胶板(Merck公司25 TLC aluminiumnsheets 20cm×20cm Silica gel 60 F254)在110℃烘箱中活化1小时, 取出冷却。吸取2μL总脂样品点样于TLC板上,复点样3次。
将TLC薄板置于第一个层析缸内展层,第一向展层剂为氯仿:甲醇:水 (65:25:4,v/v),溶媒展至顶部后取出薄板吹干、将薄板放入第二个层析缸,第二向展层剂为氯仿:甲醇:乙酸:水(80:12:15:4,V/V),按与第一向垂直的方向上行,溶媒展至顶部取出薄板吹干,将磷钼酸显色剂喷洒至TLC板至完全湿润,100℃加热5-8min,将清晰斑点显出,立即在扫措仪上扫描TLC 板,记录结果。如图3所示,北工商节杆菌中含有3种极性酯,DPG:双磷脂酰甘油,diphosphatidylglycerol;PG:磷脂酰甘油,phosphatidylglycerol,
Glycophospholipid;PE:磷脂酰乙醇胺,phosphatidyl ethanolamin。与蛋白质、糖等构成细胞膜,对于物质运输、代谢及维持正常的渗透压都有重要作用。不同属菌的磷酸类脂组分是不同的,它是鉴别属的重要特征之一,是化学分类项目中不可缺少的分类指征。
3、北工商节杆菌(Arthrobacter beigongshangensis)16S rDNA序列及进化树
北工商节杆菌(Arthrobacter beigongshangensis),菌株由上海美吉生物医药科技有限公司进行全基因组测序,得到全基因组序列,其中,系统进化树如图1所示。
将测得的REN12T 16S rRNA序列在NCBI(https://www.ncbi.nlm.nih.gov/) 中与模式菌株进行比对,根据相似度选取16个模式菌株16S rRNA序列进行系统进化树的构建,如图1所示REN12T与节杆菌属的亲缘距离相近,且在系统进化树中为独立分支,确定其为节杆菌属,同时根据相似度(99.07%)得知其相似株为Arthrobacter stackebrandtii CCM2783。接着将REN12T菌株送往上海美吉生物医药科技有限公司进行全基因组测序,得到全基因组序列。同时从 NCBI中下载相似株全基因组,将REN12T全基因组与相似株Arthrobacter stackebrandtii CCM 2783在DSMZ(http://ggdc.dsmz.de/home.php)进行全基因组比对,基因组DNA-DNA同源性杂交(DDH):DNA同源性(DDH)为21.90% [19.7-24.4%],DDH小于70%。
虽然,上文中已经用一般性说明及具体实施例对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
SEQUENCE LISTING
<110> 北京工商大学
<120> 北工商节杆菌菌种及其应用
<130> GG19719810A
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1395
<212> DNA
<213> Artificial Sequence
<400> 1
gtcgaacgat gaaccccgct tgcgggggga ttagtggcga acgggtgagt aacacgtgag 60
taacctgccc ttaactctgg gataagcctt ggaaacgggg tctaatactg gatattgact 120
tttcaccgca tggtggttgg ttgaaagatt tattggtttt ggatggactc gcggcctatc 180
agcttgttgg tgaggtaatg gctcaccaag gcgacgacgg gtagccggcc tgagagggtg 240
accggccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 300
attgcacaat gggcgaaagc ctgatgcagc gacgccgcgt gagggatgac ggccttcggg 360
ttgtaaacct ctttcagtag ggaacaaggc cagacgttgt ctggttgagg gtacttgcag 420
aagaagcgcc ggctaactac gtgccagcag ccgcggtaat acgtagggcg caagcgttat 480
ccggaattat tgggcgtaaa gagctcgtag gcggtttgtc gcgtctgccg tgaaagtccg 540
gggctcaacc ccggatctgc ggtgggtacg ggcagactag agtgatgtag gggagactgg 600
aattcctggt gtagcggtga aatgcgcaga tatcaggagg aacaccgatg gcgaaggcag 660
gtctctgggc attaactgac gctgaggagc gaaagcatgg ggagcgaaca ggattagata 720
ccctggtagt ccatgccgta aacgttgggc actaggtgtg ggggacattc cacgttttcc 780
gcgccgtagc taacgcatta agtgccccgc ctggggagta cggccgcaag gctaaaactc 840
aaaggaattg acgggggccc gcacaagcgg cggagcatgc ggattaattc gatgcaacgc 900
gaagaacctt accaaggctt gacatgaact ggaaacgcct ggaaacaggt gccccgcttg 960
cggtcggttt acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1020
gtcccgcaac gagcgcaacc ctcgttctat gttgccagca cgtgatggtg gggactcata 1080
ggagactgcc ggggtcaact cggaggaagg tgaggacgac gtcaaatcat catgcccctt 1140
atgtcttggg cttcacgcat gctacaatgg ccggtacaat gggttgcgat actgtgaggt 1200
ggagctaatc ccaaaaagcc ggtctcagtt cggattgggg tctgcaactc gaccccatga 1260
agtcggagtc gctagtaatc gcagatcagc aacgctgcgg tgaatacgtt cccgggcctt 1320
gtacacaccg cccgtcaagt cacgaaagtt ggtaacaccc gaagcccatg gcccaacccg 1380
caagggaggg agtgt 1395
Claims (4)
1.北工商节杆菌(Arthrobacter beigongshangensis),其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.19210。
2.含权利要求1所述的北工商节杆菌的微生物菌剂。
3.权利要求1所述的菌株或权利要求2所述的菌剂在如下(A1)或(A2)中的应用:
(A1)产生浓香风味物质;
(A2)制备具有浓香风味物质的产品。
4.权利要求1所述的菌株或权利要求2所述的菌剂在如下(B1)或(B2)中的应用:
(B1)酿酒;
(B2)制备酿酒产品。
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