CN107974424B - 空间罗伊氏乳杆菌Fullarton-9-25及应用 - Google Patents
空间罗伊氏乳杆菌Fullarton-9-25及应用 Download PDFInfo
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- CN107974424B CN107974424B CN201711421391.4A CN201711421391A CN107974424B CN 107974424 B CN107974424 B CN 107974424B CN 201711421391 A CN201711421391 A CN 201711421391A CN 107974424 B CN107974424 B CN 107974424B
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- lactobacillus reuteri
- fullarton
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- microbial inoculum
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一株空间罗伊氏乳杆菌Fullarton‑9‑25及应用。本发明所提供的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton‑9‑25,它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.14941。本发明所提供的罗伊氏乳杆菌Fullarton‑9‑25,与地面对照菌株相比,疏水性更高,对多种细菌的抑菌作用更强,产罗伊氏菌素的能力更高,且安全性评价实验的结果表明是安全的。因此,本发明所提供的罗伊氏乳杆菌Fullarton‑9‑25具有继续开发的潜力和价值。
Description
技术领域
本发明属于微生物领域,涉及一株空间罗伊氏乳杆菌Fullarton-9-25及应用。
背景技术
罗伊氏乳杆菌(Lactobacillus reuteri)是目前已报道的几乎可存在于所有脊椎动物和哺乳动物肠道内的乳酸杆菌,常栖息于人和动物的肠道系统中,对人和动物无害,具有良好的生物相容性,是具有改善过敏体质、预防过敏反复发作,以及调节肠道功能的益生菌。近年来,益生菌已经成为微生物学领域的研究热点,并在保健食品和乳品工业上得到广泛应用。目前,罗伊氏乳杆菌已经我国批准为可用于保健食品的益生菌菌株参考。
罗伊氏乳杆菌不仅具备乳酸菌主要的有益功效,而且还具备产生广谱抗菌物质的特效。它代谢甘油产生一种特殊的抑菌物质——罗伊氏素(reuterin)。罗伊氏素是一种广谱抗菌剂,可抑制革兰氏阳性菌、革兰氏阴性菌、酵母菌、霉菌、病原虫、原生动物等的生长,不仅可以有效作用于细菌,同样也可以作用于某些真菌和原生动物。罗伊氏菌素作为抗菌物质的优越性已引起人们越来越多的关注,也因其独特的生化特性及对人和动物安全无毒而具有十分广阔的应用前景。罗伊氏素的主要成分是3-羟基丙醛(3-HPA)的单体、水合物和环化二聚体。除了抗菌外,3-HPA单体还是一种潜在的重要化工原料,可作为多种新兴化学品如丙烯醛、丙烯酸、1,3-丙二醇等的前体,用于制备新型聚合物材料;可和蛋白质中的氨基反应形成交联,有望取代化学合成的戊二醛和环氧化合物作为新型生物交联剂。
发明内容
本发明的目的是提供一株新的空间罗伊氏乳杆菌(Lactobacillus reuteri)及应用。
本发明所提供的罗伊氏乳杆菌(Lactobacillus reuteri)具体为罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25,它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.14941。
本发明中的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25神州十一号飞船搭载罗伊氏乳杆菌SS23(即来自于中国工业微生物菌种保藏管理中心(CICC)保藏编号为CICC 6118的罗伊氏乳杆菌(Lactobacillus reuteri),该菌株在ATCC的编号为53608)进入太空,在太空中飞行31天18.5小时,飞船返回地球以后,采取一系列的筛选实验获得的。已经于2017年11月20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.14941。
相应的,本发明还提供一种活性成分为所述罗伊氏乳杆菌(Lactobacillusreuteri)Fullarton-9-25的菌剂。
所述菌剂中除了含有作为活性成分的所述罗伊氏乳杆菌(Lactobacillusreuteri)Fullarton-9-25外,还含有辅料。所述辅料的作用可为赋形、充当载体、提高稳定性、增溶、助溶、缓释等。
所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25或所述菌剂在生产罗伊氏素中的应用也属于本发明的保护范围。
所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25或所述菌剂在制备抗菌药物中的应用也属于本发明的保护范围。
其中,所述菌可为细菌,如革兰氏阳性细菌或革兰氏阴性细菌,也可为真菌。
进一步地,所述细菌具体可为大肠杆菌、金黄色葡萄球菌、沙门氏菌和/或单增李斯特菌等。
所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25或所述菌剂在制备发酵乳制品中的应用也属于本发明的保护范围。
所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-35或所述菌剂在制备生产发酵乳制品所使用的发酵剂中的应用也属于本发明的保护范围。
进一步地,在制备所述发酵乳制品的过程中所使用的原料乳可为牛乳、羊乳、豆乳等,既可以是脱脂乳,也可以是非脱脂乳。
进一步地,所述发酵乳制品可为酸奶、开菲尔、发酵酪乳、酸奶酒、乳酒等。
所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25或所述菌剂在如下任一中的应用也属于本发明的保护范围:
(a1)调节人或动物胃肠道微生态平衡,或者制备用于调节人或动物胃肠道微生态平衡的产品;
(a2)缓解肠炎,或者制备用于缓解肠炎的产品;
(a3)辅助保护胃粘膜,或者制备用于辅助保护胃粘膜的产品;
(a4)通便,或者制备用于通便的产品;
(a5)增强人或动物免疫力,或者制备用于增强人或动物免疫力的产品。
其中,所述产品可为药品、营养品或者保健品等。
所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25或所述菌剂在如下任一中的应用也属于本发明的保护范围:
(b1)制备食品添加剂;
(b2)制备动物饲料添加剂。
本发明所提供的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25,与地面对照菌株相比,疏水性提高了0.77倍,发酵上清液(pH=6.5)对大肠杆菌的抑制率增加了0.40倍,对金黄色葡萄球菌的抑制率增加了0.12倍,对沙门氏菌的抑菌率提高11.79倍;产罗伊氏菌素的能力提高了0.90倍;溶血实验结果是γ-溶血,即不溶血,结果见表1。综上所述,Lactobacillus reuteri Fullarton-9-25具有继续开发的潜力和价值。
表1 Fullarton-9-25和地面菌株的性质
保藏说明
菌种名称:罗伊氏乳杆菌
拉丁名:Lactobacillus reuteri
参椐的生物材料(株):Fullarton-9-25
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称:CGMCC
地址:北京市朝阳区北辰西路1号院3号
保藏日期:2017年11月20日
保藏中心登记入册编号:CGMCC No.14941
附图说明
图1为编号为F-9-25的菌株和地面对照菌的镜检照片。A:编号为F-9-25的菌株;B:地面对照菌。
图2为地面对照菌及其经空间搭载所得的各诱变菌株对低pH和胆盐的耐受性。
图3为地面对照菌及其经空间搭载所得的各诱变菌株的细胞表面疏水性的测定。
图4为地面对照菌及其经空间搭载所得的各诱变菌株产罗伊氏素的能力测定。
图2-4中的wild type即表示地面对照菌株GS23。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
罗伊氏乳杆菌富乐顿亚种(Lactobacillus reuteri FSMCC)由富乐顿生物工程科技(北京)有限公司空间微生物菌种库提供,涉及菌种为地面对照菌GS23(Lactobacillusreuteri FSMCC GS23)和空间搭载菌SS23(Lactobacillus reuteri FSMCC SS23),两者为同一株菌,为来自于中国工业微生物菌种保藏管理中心(CICC)保藏编号为CICC6118的罗伊氏乳杆菌(Lactobacillus reuteri),该菌株在ATCC的编号为53608。其中空间搭载菌SS23进入太空后的搭载时间为31天18.5小时。
下述实施例的各试验中所测各项指标样品均为三个平行,所得数据以平均值±标准偏差的形式表达。数据的统计分析采用GraphPad Prism 6,采用t-检验分析各诱变菌株与地面菌株的差异显著性,当p<0.05时即表示差异显著,显著水平标记为*;当p<0.01时,显著水平标记为**,当p<0.001时,显著水平标记为***。。
实施例1、罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25的分离与鉴定
一、太空诱变菌株的分离
将空间搭载菌SS23的甘油保藏管用MRS培养基活化三代后,在MRS固体平板上采用四区划线法分离,37℃倒置培养24h,挑取菌落形态与地面对照菌GS23有差异的单菌落或者随机挑选菌落,共挑选100个菌落。采用平板划线法进行分离纯化三次,进行形态学观察和革兰氏染色,并且使用甘油冷冻法进行保存。
结果显示:对分离纯化的菌落进行革兰氏染色,均为革兰氏阳性菌,菌体形态呈短杆或长杆。图1为编号为F-9-25的菌株和地面对照菌的镜检照片,两株菌形态无明显差异。
二、发酵性能的测定
将分离纯化到的100个菌株接于灭菌的12%(m/v)脱脂乳培养基中,37℃发酵,观察凝乳快慢进行初筛。培养至凝乳后,置于4℃冰箱进行过夜后熟,测定脱脂乳培养基pH值和粘度。以地面对照菌GS23为对照,选择与对照菌株差异大的太空诱变菌株进行下一步实验。将初筛出的诱变菌株传代5次,再次验证菌株的发酵性能,以防止诱变菌株回复突变的发生。粘度的测定采用DV-III型粘度计进行,使用LV3探头,转速200rpm,测定时间为2min。
结果显示:对挑选出的100个菌落进行发酵性能,检测的菌株均能够凝乳,但是凝乳时间具有较大差异。选择出了与对照菌株发酵性能具有明显差异的太空诱变菌株11株,实验结果见表2。从凝乳时间来看,编号为F-9-20、F-9-35、F-9-79和F-9-87的菌株凝乳时间显著短于对照菌株的,其余菌株反之。从发酵脱脂乳的pH来看,这11株诱变菌株均与对照菌株的pH差异不显著。从发酵脱脂乳的粘度来看,编号为F-9-17、F-9-18、F-9-35、F-9-40、F-9-58、F-9-71、F-9-79和F-9-87的菌株的粘度与对照菌株具有显著差异。
表2罗伊氏乳杆菌凝乳时间和产酸产粘性质
菌株 | 凝乳时间/h | pH | 粘度 |
地面对照菌GS23 | 26.17±0.29 | 4.92±0.033 | 263.00±20.30 |
F-9-04 | 34.50±0.50<sup>***</sup> | 4.97±0.02 | 244.33±7.57 |
F-9-17 | 114.67±0.77<sup>***</sup> | 5.03±0.03 | 429.00±14.73<sup>***</sup> |
F-9-18 | 132.33±1.04<sup>***</sup> | 5.05±0.03 | 488.67±12.50<sup>***</sup> |
F-9-20 | 22.67±0.58<sup>***</sup> | 4.90±0.02 | 273.33±9.45 |
F-9-25 | 46.50±0.05<sup>***</sup> | 4.83±0.03 | 257.00±35.03 |
F-9-35 | 20.67±0.58<sup>***</sup> | 4.89±0.01 | 426±22.27<sup>***</sup> |
F-9-40 | 67.17±0.29<sup>***</sup> | 4.84±0.03 | 310±13.05<sup>*</sup> |
F-9-58 | 52.33±0.58<sup>***</sup> | 4.85±0.03 | 359.00±40.95<sup>*</sup> |
F-9-71 | 31.33±0.29<sup>***</sup> | 4.83±0.02 | 415.67±16.04<sup>***</sup> |
F-9-79 | 18.00±0.50<sup>***</sup> | 4.73±0.01 | 378.00±19.08<sup>**</sup> |
F-9-87 | 14.17±0.29<sup>***</sup> | 4.83±0.03 | 330.67±24.83<sup>*</sup> |
三、模拟人消化道的耐受性实验
益生菌在人体内发挥益生特性的前提条件是保证其在胃液的酸性环境和肠道的胆盐环境中存活下来。本发明通过体外实验模拟人胃肠道的低pH,高胆盐环境,评价诱变菌株对模拟消化液的耐受能力。
1、低pH耐受性测定
将菌液接种于pH 2.5的MRS培养基中,置于37℃培养箱培养3h。采用生理盐水10倍稀释平板计数法对0h和3h的活菌数进行测定。
2、胆盐耐受性测定
将菌液接种于胆盐浓度为0.5%(m/v)的MRS培养基中,置于37℃培养箱培养4h。采用生理盐水10倍稀释,平板计数法对0h和4h的活菌数进行测定。菌株耐受性的结果用活菌数的变化来表示,计算公式如下:
RI=log N0/Nf;
式中,N0表示初始菌落总数;Nf表示最终菌落总数。
3、结果
实验结果见图2。研究发现,罗伊氏乳杆菌对低pH均有良好的耐受性,编号为F-9-87的菌株的耐酸能力是最好的;但是对胆盐的耐受性稍差,其中编号为F-9-17、F-9-18、F-9-35和F-9-58的菌株具有相对较好的耐胆盐能力,活菌数下降不到2个数量级。
四、菌体细胞表面疏水性的测定
罗伊氏乳杆菌在MRS培养基中过夜培养,离心,3mL PBS洗两次,调整至OD600值于0.8-1.0(A0)。1mL二甲苯添加到3ml菌悬液中,涡旋振荡120s,37℃静置1h,检测水相的OD值(A),以缓冲液为对照。H%=[(A0-A)/A0],认为大于50%也许具有较好的疏水性,即黏附性也可能较高。
从疏水性结果来看,编号为F-9-25、F-9-35、F-9-79、F-9-87的菌株的疏水性超过了50%,且显著高于对照菌株,见图3。
五、产胞外多糖(EPS)的检测
乳酸菌胞外多糖是指乳酸菌在生长代谢过程中分泌到细胞壁外的黏液多糖或荚膜多糖的总称。EPS具有多种生理功能,包括保护菌体、促进菌体黏附、降血压、降胆固醇,抗氧化、抗肿瘤、抗溃疡、抗病毒、改善肠道微生态环境和增强人体免疫力等。此外,EPS作为新型天然的食品添加剂,可改善食品的质构、口感、流变学特性和风味等指标,还可进一步提高产品的营养保健作用。本研究选取了前面研究得出与对照菌株性质具有较大差异的7株诱变菌株进行EPS的检测。
1、EPS的提取
将罗伊氏乳杆菌接种于10%(m/v)脱脂乳培养基中,在37℃培养至凝乳后,将凝乳破碎并搅拌均匀,分别吸取5mL样品于离心管中,并向发酵液中加入等体积的5%(m/v)的三氯乙酸(TCA)溶液,室温下静置30min沉淀蛋白,在4℃、10000r/min离心30min,用0.45μm滤膜滤得上清液,用蒸馏水稀释80倍,吸取每组滤液1mL于具塞试管中,分别加入1mL 6%(v/v)苯酚溶液,5mL浓硫酸,混合均匀,用蒸馏水作为空白试剂调零,于沸水浴中保持15min,然后迅速冰水浴冷却终止反应。在波长490nm处测定吸光度值,计算胞外多糖含量。
2、EPS的测定
采用苯酚-硫酸法测定EPS含量,以葡萄糖为标准品制作标准曲线。取适量分析纯葡萄糖置于鼓风干燥箱中80℃干燥至恒重,冷却后准确称取100mg葡萄糖于500mL容量瓶中,加蒸馏水至刻度。反应体系各溶液体积按表2添加,先将标准葡萄糖溶液加入到具塞刻度试管中,再加入5%(v/v)的苯酚溶液,最后加入10mL浓硫酸混匀静置,待其冷却后于490nm处测定吸光度,每组做3个平行。以葡萄糖含量(mg/L)为横坐标,吸光度(A490)为纵坐标绘制标准曲线。得线性回归方程为:y=1.405x-0.426,R2=0.987。同法测得EPS水溶液于波长490nm处的吸光度,通过回归方程计算菌株的EPS产量。
样品中多糖含量以质量分数ω计,单位以克每百克(g/100g)表示,按如下公式计算:
式中:
m1——从标准曲线上查得样品测定液中含糖量,单位为微克(μg);
V1——样品定容体积,单位为毫升(mL)
V2——比色测定时所移取样品测定液的体积,单位为毫升(mL);
m2——样品质量,单位为克(g);
0.9——葡萄糖换算成葡萄糖的校正系数。
计算结果保留至小数点后两位。
3、结果
结果如表3。其中编号为F-9-17、F-9-35、F-9-58、F-9-71和F-9-87的诱变菌株的EPS产量要显著高于对照菌株。
表3罗伊氏乳杆菌产胞外多糖的含量
六、抑菌试验
1、无细胞上清液的制备
罗伊氏乳杆菌在MRS或MRS-glycerol(MRS-g,丙三醇浓度为400mM)培养基中37℃培养24h,离心,得上清液。为了排除有机酸的抑菌作用,用1M NaOH调节上清液的pH至6.5。将处理过的无细胞上清液经0.22μm滤膜过滤,4℃保存备用。
2、抑菌活性的检测
抑菌活性的检测采用96孔板共培养法。本实验采用的指示菌为大肠杆菌(Escherichia coli)ATCC 8739,金黄色葡萄球菌(Staphylococcus aureus)ATCC 25923,沙门氏菌(Salmonella enterica serovar Typhimurium)ATCC 14028,单增李斯特菌(Listeria monocytogenes)ATCC 19115,这些指示菌均在TSB培养基中37℃摇床培养。
96孔板共培养法:指示菌过夜培养,取0.1ml浓度为105cfu/ml的指示菌培养液加入96孔板中,再加入0.1ml无细胞培养液,37℃培养24h,酶标仪检测OD600。以加入TSB培养基的孔为对照,利用如下公式进行抑菌率的计算:
结果如表4所示:所有测试菌株的MRS上清液都具有抑菌作用,但是调节pH至6.5时都不具有抑菌效果,这说明抑菌成分主要是有机酸。然而,将菌株发酵MRS-g的上清液(pH=6.5)进行抑菌实验发现,很多菌株仍然具有抑菌活性,这说明具有抑菌作用的菌株可以代谢甘油,生成了某些抑菌成分。整体看来,编号为F-9-25、F-9-35、F-9-71对4种致病菌的抑制率均接近100%,表现出强大的抑菌作用。
表4 96孔板法测定罗伊氏乳杆菌MRS-g对病原菌的抑制率(%)
菌株编号 | E.coli | S.aureus | S.enterica | L.monocytogenes |
地面对照菌株GS23 | 70.9±0.06 | 89.2±0.87 | 7.8±0.14 | 100.1±0.97 |
F-9-04 | 69.6±1.85 | 82.3±0.92 | 4.0±0.42 | 100.1±0.37 |
F-9-17 | 65.6±0.96 | 34.3±1.12 | 7.6±0.43 | 0.3±0.04 |
F-9-18 | 99.1±0.03 | 100.3±0.04 | 46.6±0.48 | 101.3±0.64 |
F-9-20 | 99.0±0.23 | 99.8±0.10 | 46.9±8.36 | 101.3±0.85 |
F-9-25 | 98.9±0.10 | 100.1±0.17 | 99.9±0.10 | 99.4±0.85 |
F-9-35 | 98.9±0.10 | 99.9±0.28 | 99.9±0.05 | 100.0±0.32 |
F-9-40 | 54.0±0.52 | 44.1±0.45 | 4.4±0.16 | 94.3±0.32 |
F-9-58 | 73.1±2.52 | 100.3±0.11 | 58.8±1.16 | 100.4±0.21 |
F-9-71 | 99.0±0.03 | 100.2±0.11 | 100.1±0.09 | 100.4±0.37 |
F-9-79 | 46.8±0.03 | 8.4±0.07 | 4.0±0.10 | 87.6±0.53 |
F-9-87 | 27.7±1.10 | 8.4±2.33 | 2.5±0.10 | 30.6±0.37 |
3、罗伊氏菌素产生能力的检测
(1)待测上清液的制备
罗伊氏乳杆菌在MRS培养基中培养24h,离心,PBS洗一次,重新悬浮在甘油-水溶液,37℃孵育3h,离心获得上清液,置于4℃冷藏待测。
(2)标准曲线的制作
丙烯醛标准曲线的制作:用95%的乙醇稀释储备液,分别向10ml容量瓶中加入0-2ml储备液,不足2ml的用95%补足,加入1.2ml水。然后加入0.5ml的0.01M的色氨酸溶液和6.3ml浓HCl,置于60℃水浴5min,达到最深颜色。水浴后,测定OD560,用试剂对照做空白。最后将15μg、30μg、45μg、60μg、75μg、90μg丙烯醛与其对应的吸光度值建立标准曲线。
结果如图4所示,与地面对照菌株相比,编号为F-9-25菌株的罗伊氏素产量提高了0.90倍。
七、安全性评价(溶血试验)
溶血试验是将过夜培养的罗伊氏乳杆菌培养液划线至血平皿表面,37℃培养48h。观察是否出现溶血现象,即β-溶血(菌落周围出现大面积的清晰溶血圈)、α-溶血(菌落周围出现浅褐色或草绿色溶血圈)和γ-溶血(菌落周围没有溶血圈)。
结果显示,对11株诱变菌株和1株地面对照菌株进行溶血实验。研究发现,所有测试菌株均没有出现溶血现象,即γ-溶血。初步表明,经过太空诱变的候选益生菌株是安全的。
八、菌种的鉴定
通过DNA试剂盒对地面对照菌和各空间搭载诱变菌株分别进行DNA提取,提取完成后,则分别对上述的DNA提取物进行PCR扩增,扩增体系总体积为20μL,包括2个单位的TaqDNA聚合酶,PCR buffer,2.5mM MgCl2,500μM dNTP,100ng DNA模板和10pmol的通用引物1、2,P1:5’-AGTTTGATCMTGGCTCAG-3’;和P2:5’-GGTTACCTTGTTACGACTT-3’。扩增条件为:95℃预变性2min,95℃变性30s,51℃退火(复性)30s,72℃延伸1min,循环30次,最后72℃延伸2min,得最终产物,然后将产物送交上海生工测序。
通过16S rDNA测序的结果,并结合前文鉴定的形态学特征,可知地面对照菌各诱变菌株均为罗伊氏乳杆菌(Lactobacillus reuteri),编号为F-9-25的菌株的16S rDNA序列如SEQ ID No.1所示。
编号为F-9-25的菌株已经于2017年11月20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.14941,参椐的生物材料(株)为Fullarton-9-25。
罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25的培养温度为37℃;自然pH;培养基成分:酪蛋白胨10.0g,牛肉膏10.0g,酵母粉5.0g,葡萄糖5.0g,乙酸钠5.0g,柠檬酸二铵2.0g,Tween 80 1.0g,K2HPO4 2.0g,MgSO4.7H2O 0.2g,MnSO4.H2O 0.05g,琼脂15.0g,蒸馏水1.0L。
<110> 富乐顿生物工程科技(北京)有限公司
<120> 空间罗伊氏乳杆菌Fullarton-9-25及应用
<130> GNCLN172067
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1411
<212> DNA
<213> 罗伊氏乳杆菌(Lactobacillus reuteri)
<400> 1
ttttgcagtc gtacgcactg gcccaactga ttgatggtgc ttgcacctga ttgacgatgg 60
atcaccagtg agtggcggac gggtgagtaa cacgtaggta acctgccccg gagcggggga 120
taacatttgg aaacagatgc taataccgca taacaacaaa agccacatgg cttttgtttg 180
aaagatggct ttggctatca ctctgggatg gacctgcggt gcattagcta gttggtaagg 240
taacggctta ccaaggcgat gatgcatagc cgagttgaga gactgatcgg ccacaatgga 300
actgagacac ggtccatact cctacgggag gcagcagtag ggaatcttcc acaatgggcg 360
caagcctgat ggagcaacac cgcgtgagtg aagaagggtt tcggctcgta aagctctgtt 420
gttggagaag aacgtgcgtg agagtaactg ttcacgcagt gacggtatcc aaccagaaag 480
tcacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggat 540
ttattgggcg taaagcgagc gcaggcggtt gcttaggtct gatgtgaaag ccttcggctt 600
aaccgaagaa gtgcatcgga aaccgggcga cttgagtgca gaagaggaca gtggaactcc 660
atgtgtagcg gtggaatgcg tagatatatg ggaagacaca gtgcgagcgg ctgtctggtc 720
tgcaactgac gctgagctcg aagcatgggt agcgaacagg attagatacc ctggtagtcc 780
atgccgtaaa cgatgagtgc taggtgttgg agggtttccg cccttcagtg ccggagctaa 840
cgcattaagc actccgcctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg 900
ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc 960
aggtcttgac atcttgcgct aaccttagag ataaggcgtt cccttcgggg acgcaatgac 1020
aggtggtgca tggtcgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080
gcgcaaccct tgttactagt tgccagcatt aagttgggca ctctagtgag actgccggtg 1140
acaaaccgga ggaaggtggg gacgacgtca gatcatcatg ccccttatga cctgggctac 1200
acacgtgcta caatggacgg tacaacgagt cgcaagctcg cgagagtaag ctaatctctt 1260
aaagccgttc tcagttcgga ctgtaggctg caactcgcct acacgaagtc ggaatcgcta 1320
gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380
cacaccatgg gagtttgtaa cgctccaaag t 1411
Claims (11)
1.罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25,它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. 14941。
2.一种菌剂,它的活性成分为权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25。
3.权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25或权利要求2所述的菌剂在生产罗伊氏素中的应用。
4.权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25或权利要求2所述的菌剂在制备抗菌药物中的应用。
5.根据权利要求4所述的应用,其特征在于:所述菌为细菌或真菌。
6.根据权利要求5所述的应用,其特征在于:所述细菌为革兰氏阳性细菌或革兰氏阴性细菌。
7.根据权利要求6所述的应用,其特征在于:所述细菌为大肠杆菌、金黄色葡萄球菌、沙门氏菌和/或单增李斯特菌。
8.权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25或权利要求2所述的菌剂在制备发酵乳制品中的应用。
9.权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25或权利要求2所述的菌剂在制备用于生产发酵乳制备所使用的发酵剂中的应用。
10.权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25或权利要求2所述的菌剂在如下任一中的应用:
(a1)制备用于调节人或动物胃肠道微生态平衡的产品;
(a2)制备用于缓解肠炎的产品;
(a3)制备用于辅助保护胃粘膜的产品;
(a4)制备用于通便的产品;
(a5)制备用于增强人或动物免疫力的产品。
11.权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-9-25或权利要求2所述的菌剂在如下任一中的应用:
(b1)制备食品添加剂;
(b2)制备动物饲料添加剂。
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