CN113717875A - 一种格氏乳杆菌及其应用 - Google Patents
一种格氏乳杆菌及其应用 Download PDFInfo
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- CN113717875A CN113717875A CN202110288581.3A CN202110288581A CN113717875A CN 113717875 A CN113717875 A CN 113717875A CN 202110288581 A CN202110288581 A CN 202110288581A CN 113717875 A CN113717875 A CN 113717875A
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- lactobacillus gasseri
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- Tropical Medicine & Parasitology (AREA)
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- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及微生物技术领域,尤其是涉及一种格氏乳杆菌及其应用。所述菌株包括如SEQ ID NO:1所示的16s RNA序列。本发明筛选得到的格氏乳杆菌具有增强细胞免疫和体液免疫功能的效果,且效果稳定,相比较其他格氏乳杆菌株,本发明提高免疫功能的效果更佳,可以更好的治疗或预防免疫功能低下/紊乱性疾病。
Description
技术领域
本发明涉及微生物技术领域,尤其是涉及一种格氏乳杆菌及其应用。
背景技术
在现代的不良饮食和应激水平升高的环境下,人们的免疫功能极易受到影响,导致免疫功能低下/紊乱性疾病的发病率逐年升高,如肿瘤及其放化疗的损伤,辐射损伤,使用免疫抑制治疗(包括糖皮质激素以及其它免疫抑制剂),高龄,过敏性疾病如荨麻疹,湿疹,特应性皮炎,过敏性鼻炎,过敏性哮喘,过敏性紫癜,食物过敏相关消化道疾病,反复或慢性病毒感染(如流感,乙型肝炎等)。这些疾病的治疗往往很困难,短期多采用激素抑制症状,长期则难以治愈,复发缓解,预后不佳,因此,增强免疫力,纠正免疫紊乱需要新的方法。
益生菌作为微生态调节剂,能减少肠内有害菌、产生有益的营养物质、保护胃肠粘膜结构和功能的完整性,并通过微生物相关分子模式(MAMP)与肠道的抗原呈递细胞(APC)上的模式识别受体(PRR)的相互作用来调节免疫***,使人体达到免疫平衡的健康状态。近年来,益生菌已经成为新颖的、天然的治疗药物和食品。2010年我国***批准了可用于食品的益生菌种有15种。格氏乳杆菌(Lactobacillus gasseri)属于其中的一种,它是一种普遍存在的人体共生细菌,广泛存在于正常人的口腔、小肠、大肠、***粘膜上,且未发现任何的不安全性。该微生物已在健康的人类中确立了安全性,亦是美国食品药品监督管理局(FDA)表列安全菌种之。一般而言,格氏乳杆菌是耐酸和耐胆汁的(文献1-4),经研究发现:它能够维持人类肠道稳态、维护***健康、预防过敏反应、抑制幽门螺旋杆菌感染、减轻病毒感染引起的症状。然而,格氏乳杆菌CECT5714和鸡形杆状杆菌CECT5711的组合可刺激NK细胞并增加分泌的IgA水平,但对于格氏乳杆菌ATCC 33323或格氏乳杆菌TMC0356均未观察到这种作用(文献5-6),这说明不同的菌株可能功效相差较大,因此,亟需一种针对上述的免疫***疾病功效更好的新菌株。
文献1:Fernández MF Boris S&Barbés C(2003)Probiotic properties ofhuman lactobacilli strains to be used in the gastrointestinal tract.J ApplMicrobiol94:449–455.
文献2:Strahinic I Busarcevic M Pavlica D Milasin J Golic N&Topisirovic L(2007)Molecular and biochemical characterizations of human orallactobacilli as putative probiotic candidates.Oral Microbiol Immunol22:111–117.
文献3:Azcarate-Peril MA Altermann E Goh YJ et al.(2008)Analysis ofthe genome sequence of Lactobacillus Gasseri ATCC 33323reveals the molecularbasis of an autochthonous intestinal organism.Appl Environ Microbiol74:4610–4625.
文献4:Jensen H Grimmer S Naterstad K&Axelsson L(2012)In vitro testingof commercial and potential probiotic lactic acid bacteria.Int J FoodMicrobiol153:216–222.
文献5:Olivares M Díaz-Ropero MAP Gómez N Lara-Villoslada F SierraSMaldonado JA Martín R López-Huertas E Rodríguez JM&Xaus J(2006a)Oraladministration of two probiotic strains,Lactobacillus gasseri CECT5714 andLactobacillus coryniformis CECT5711,enhances the intestinal function ofhealthy adults.Int J Food Microbiol107:104–111.
文献6:Stoeker L Nordone S Gunderson S Zhang L Kajikawa A LaVoy AMiller M Klaenhammer TR&Dean GA(2011)Assessment of Lactobacillus gasseri as acandidate oral vaccine vector.Clin Vaccine Immunol18:1834–1844.
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种格氏乳杆菌及其应用,本发明筛选得到的格氏乳杆菌具有增强细胞免疫和体液免疫功能的效果,且效果稳定,相比较其他格氏乳杆菌株,本发明提高免疫功能的效果更佳,可以更好的治疗或辅助治疗或预防免疫功能低下/紊乱性疾病。
为实现上述目的,本发明采取的技术方案为:
本发明的第一方面提供了一种菌株,所述菌株包括如SEQ ID NO:1所示的16s RNA序列。
作为本发明所述菌株的优选实施方式,所述菌株为格氏乳杆菌(Lactobacillusgasseri)TTYS-839,所述格氏乳杆菌(Lactobacillus gasseri)TTYS-839已于2020年07月02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,保藏地址为:北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.20178。
本发明从健康人体肠道菌群中筛选得到一株具有增强细胞免疫和体液免疫功能的菌株,该菌株具有如SEQ ID NO:1所示的16s RNA序列,该序列长度为1510bp,经过基因比对,该菌株被鉴定为格氏乳杆菌(Lactobacillus gasseri)。本发明人将其命名为格氏乳杆菌(Lactobacillus gasseri)TTYS-839。相比于其他现有的格氏乳杆菌,本发明增强细胞免疫和体液免疫功能的效果更佳,可以更好的治疗或辅助治疗或预防免疫功能低下/紊乱性疾病。
其中疾病包括但不限于以下疾病:肿瘤及其放化疗的损伤,辐射损伤,使用免疫抑制治疗(包括糖皮质激素以及其它免疫抑制剂);过敏性疾病如荨麻疹,湿疹,特应性皮炎,过敏性鼻炎,过敏性哮喘,过敏性紫癜,食物过敏相关消化道疾病,反复或慢性病毒感染(如流感,乙型肝炎等)。
本发明的第二方面提供了上述菌株在制备用于调节免疫的药物中的应用。
本发明的第三方面提供了一种药物组合物,所述药物组合物包括上述的菌株及在药学上可接受的载体。
作为本发明所述药物组合物的优选实施方式,所述药物组合物的剂型包括片剂、胶囊剂、口服剂或冻干粉剂。
作为本发明所述药物组合物的优选实施方式,所述药学上可接受的载体为脱脂奶、乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖、海藻糖、淀粉、***胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁、矿物油中的至少一种。
本发明的第四方面提供了上述的药物组合物在制备用于调节免疫的药物中的应用。
本发明的第五方面提供了上述的菌株在制备用于调节免疫的食品、保健品中的应用。
作为本发明所述应用的优选实施方式,所述食品包括特殊医学用途配方食品。
本发明的第六方面提供了一种培养液,包括上述的菌株。
本发明还提供了一种用于调节免疫的食品、保健品,包括上述的菌株。更优选的是,所述食品、保健品包括上述菌株、含有上述菌株的裂解物、含有上述菌株的培养上清液、含有上述菌株的代谢产物中的任意一种。
与现有技术相比,本发明具有以下有益效果:
本发明提供了一种格氏乳杆菌(Lactobacillus gasseri)TTYS-839,该菌株具有增强细胞免疫和体液免疫功能的效果,且效果稳定,相比较其他格氏乳杆菌株,本发明提高免疫功能的效果更佳,可以更好的治疗或辅助治疗或预防免疫功能低下/紊乱性疾病。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。
实施例1、本发明的格氏乳杆菌(Lactobacillus gasseri)TTYS-839的保藏信息
本发明的格氏乳杆菌来源于2岁左右的健康幼儿的肠道菌群,幼儿身高体重与同龄人相比有明显优势,且聪明活泼,日常活动显示其智商与情商优于同龄儿童。利用乳酸菌培养基(MRS肉汤培养基)对该儿童的粪便进行培养,获得一批生长旺盛的乳酸菌类培养物。经过MRS平板的划线分离、菌落纯培养及16SRNA测序鉴定,鉴定了多种有益的乳酸菌类型,最终获得一株格氏乳杆菌(Lactobacillus gasseri),将其命名为格氏乳杆菌(Lactobacillus gasseri)TTYS-839。
格氏乳杆菌(Lactobacillus gasseri)TTYS-839已于2020年07月02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,保藏地址为:北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.20178。
实施例2、本发明的格氏乳杆菌(Lactobacillus gasseri)TTYS-839对免疫功能的影响
一、实验动物
昆明种小鼠,雄性18-22g,120只,购于沈阳药科大学实验动物中心。(饲养环境:22±2℃,湿度为45~65%,12h昼夜交替);自由进食与饮水。
二、实验方法
分组:
将小鼠适应性饲养7天后,按照体重随机分为对照组、含有格氏乳杆菌(Lactobacillus gasseri)TTYS-839的培养液10mL/kg、20mL/kg、30mL/kg、40mL/kg剂量组、阳性药组(匹多莫德0.2g/kg),共6组,每组20只。
给药:
灌胃给予匹多莫德、含有格氏乳杆菌(Lactobacillus gasseri)TTYS-839的培养液10mL/kg、20mL/kg、30mL/kg、40mL/kg剂量组灌胃给予相应剂量的药,对照组给予相应体积的生理盐水,连续给药28天。末次给药禁食12小时,每组取10只进行DNFB诱导小鼠耳肿胀实验,余下10只进行血清溶血素测定。
1、DNFB诱导小鼠耳肿胀实验:
每鼠腹部皮肤用硫化钡脱毛,范围约3cmx3cm、用DNFB溶液50uL均匀涂抹致敏。5天后,用DNFB溶液10uL均匀涂抹于右耳(两面)进行攻击。攻击后24h颈椎脱臼处死小鼠,剪下相同面积的左右耳片,称重,计算耳肿胀双侧耳重差值。
2、血清溶血素测定
取羊血(SRBC),用生理盐水洗涤3次,每次离心(2000r/min)10min。将压积SRBC用生理盐水配成2%(v/v)的细胞悬液,每只鼠腹腔注射0.2mL进行免疫。5d后,开腹、下腔静脉取血。2000r/min离心10min,收集血清。将血清稀释100倍待测。取试管,依次加入稀释的血清0.25mL,10%的绵羊红细胞悬液0.25mL,补体0.25mL,同样方法做样品空白对照,以0.25mL生理盐水代替血清样品,将试管摇匀后置37℃水浴中保温30min,然后移至冰浴中终止反应,2000rpm离心取上清液于540nm测量吸光度值。
HC50=(样品吸光度/绵羊红细胞半数溶血时吸光度)*稀释倍数。
三、检测指标
1、DNFB诱导小鼠耳肿胀双侧耳重差值;
2、免疫器官脾脏及胸腺的脏器指数;
3、小鼠血清溶血素水平。
四、统计学分析
数据采用SPSS 21.0分析软件进行分析,实验结果以平均值±标准差(mean±SD)表示。多组均数之间的比较采用单因素方差分析(One-Way ANOVA)评价整体方差差异,并用LSD进行多重比较。对于两组间比较,采用非配对t检验进行比较。P<0.05认为有显著性差异。
五、实验结果
表1含有格氏乳杆菌(Lactobacillus gasseri)TTYS-839的培养液对KM小鼠DNFB诱导的耳肿胀及脏器指数的影响(mean±SD)
#P<0.05,##P<0.01,与对照组相比;
如表1所示:与对照组相比,含有(20mL/kg、30mL/kg、40mL/kg)格氏乳杆菌(Lactobacillus gasseri)TTYS-839的培养液各剂量组耳肿胀程度均有不同程度上升,其中含有30mL/kg格氏乳杆菌(Lactobacillus gasseri)TTYS-839的培养液组和匹多莫德组具有显著性差别,以上结果表明含有格氏乳杆菌(Lactobacillus gasseri)TTYS-839的培养液对小鼠细胞免疫有增强作用。含有(30mL/kg、40mL/kg)格氏乳杆菌(Lactobacillusgasseri)TTYS-839的培养液剂量组的脾脏及胸腺脏器指数均有不同程度上升,其中含有40mL/kg格氏乳杆菌(Lactobacillus gasseri)TTYS-839的培养液组脾脏脏器指数显著性增加。以上结果表明含有格氏乳杆菌(Lactobacillus gasseri)TTYS-839的培养液对免疫***具有增强作用。
表2含有格氏乳杆菌(Lactobacillus gasseri)TTYS-839的培养液对KM小鼠血清溶血素抗体水平影响(mean±SD)
#p<0.05,##p<0.01,与对照组相比;
如表2所示:与对照组相比,含有(20mL/kg、30mL/kg、40mL/kg)格氏乳杆菌(Lactobacillus gasseri)TTYS-839的培养液剂量组均有不同程度上升,其中含有40mL/kg格氏乳杆菌(Lactobacillus gasseri)TTYS-839的培养液剂量组差异具有显著性,以上结果表明含有格氏乳杆菌(Lactobacillus gasseri)TTYS-839对小鼠体液免疫有增强作用。
本发明筛选得到的格氏乳杆菌(Lactobacillus gasseri)TTYS-839可以有效提高昆明种小鼠的细胞免疫和体液免疫功能,效果稳定,相比较其他格氏乳杆菌株,本发明提高免疫功能的效果更佳,可以更好的治疗或辅助治疗或预防免疫功能低下/紊乱性疾病。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 孙长春
<120> 一种格氏乳杆菌及其应用
<130> 2021.01.27
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1510
<212> DNA
<213> 人工合成
<400> 1
ggctcaggac gaacgctggc ggcgtgccta atacatgcaa gtcgagcgag cttgcctaga 60
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cacgtgggta acctgcccaa gagactggga taacacctgg aaacagatgc taataccgga 180
taacaacact agacgcatgt ctagagttta aaagatggtt ctgctatcac tcttggatgg 240
acctgcggtg cattagctag ttggtaaggt aacggcttac caaggcaatg atgcatagcc 300
gagttgagag actgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg 360
cagcagtagg gaatcttcca caatggacgc aagtctgatg gagcaacgcc gcgtgagtga 420
agaagggttt cggctcgtaa agctctgttg gtagtgaaga aagatagagg tagtaactgg 480
cctttatttg acggtaatta cttagaaagt cacggctaac tacgtgccag cagccgcggt 540
aatacgtagg tggcaagcgt tgtccggatt tattgggcgt aaagcgagtg caggcggttc 600
aataagtctg atgtgaaagc cttcggctca accggagaat tgcatcagaa actgttgaac 660
ttgagtgcag aagaggagag tggaactcca tgtgtagcgg tggaatgcgt agatatatgg 720
aagaacacca gtggcgaagg cggctctctg gtctgcaact gacgctgagg ctcgaaagca 780
tgggtagcga acaggattag ataccctggt agtccatgcc gtaaacgatg agtgctaagt 840
gttgggaggt ttccgcctct cagtgctgca gctaacgcat taagcactcc gcctggggag 900
tacgaccgca aggttgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 960
gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcca gtgcaaacct 1020
aagagattag gtgttccctt cggggacgct gagacaggtg gtgcatggct gtcgtcagct 1080
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgtca ttagttgcca 1140
tcattaagtt gggcactcta atgagactgc cggtgacaaa ccggaggaag gtggggatga 1200
cgtcaagtca tcatgcccct tatgacctgg gctacacacg tgctacaatg gacggtacaa 1260
cgagaagcga acctgcgaag gtaagcggat ctctgaaagc cgttctcagt tcgaactgta 1320
ggctgcaact cgcctacacg aagctggaat cgctagtaat cgcggatcag cacgccgcgg 1380
tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgagagtc tgtaacaccc 1440
aaagccggtg ggataacctt tataggagtc agccgtctaa ggtaggacag atgattaggg 1500
tgaagtcgta 1510
Claims (10)
1.一种菌株,其特征在于,所述菌株包括如SEQ ID NO:1所示的16s RNA序列。
2.如权利要求1所述的菌株,其特征在于,所述菌株为格氏乳杆菌(Lactobacillusgasseri),所述格氏乳杆菌(Lactobacillus gasseri)已于2020年07月02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.20178。
3.如权利要求1或2所述的菌株在制备用于调节免疫的药物中的应用。
4.一种药物组合物,其特征在于,所述药物组合物包括权利要求1或2所述的菌株及在药学上可接受的载体。
5.如权利要求4所述的药物组合物,其特征在于,所述药物组合物的剂型包括片剂、胶囊剂、口服剂或冻干粉剂。
6.如权利要求4所述的药物组合物,其特征在于,所述药学上可接受的载体为脱脂奶、乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖、海藻糖、淀粉、***胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁、矿物油中的至少一种。
7.如权利要求4所述的药物组合物在制备用于调节免疫的药物中的应用。
8.如权利要求1或2所述的菌株在制备用于调节免疫的食品、保健品中的应用。
9.如权利要求8所述的应用,其特征在于,所述食品包括特殊医学用途配方食品。
10.一种培养液,其特征在于,包括权利要求1或2所述的菌株。
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US20010051609A1 (en) * | 1999-03-19 | 2001-12-13 | Kousaku Murata | Immunomodulator, immunomodulator food and immunomodulator feed |
CN109161494A (zh) * | 2018-08-09 | 2019-01-08 | 北京市理化分析测试中心 | 具有调节免疫功能的复合菌剂及其制备方法和应用 |
CN111518724A (zh) * | 2020-01-19 | 2020-08-11 | 河北医科大学 | 格氏乳杆菌hmv18及其外泌蛋白和应用 |
CN112469812A (zh) * | 2018-05-23 | 2021-03-09 | Ko生物技术有限公司 | 格氏乳杆菌kbl697菌株及其用途 |
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US20010051609A1 (en) * | 1999-03-19 | 2001-12-13 | Kousaku Murata | Immunomodulator, immunomodulator food and immunomodulator feed |
CN112469812A (zh) * | 2018-05-23 | 2021-03-09 | Ko生物技术有限公司 | 格氏乳杆菌kbl697菌株及其用途 |
CN109161494A (zh) * | 2018-08-09 | 2019-01-08 | 北京市理化分析测试中心 | 具有调节免疫功能的复合菌剂及其制备方法和应用 |
CN111518724A (zh) * | 2020-01-19 | 2020-08-11 | 河北医科大学 | 格氏乳杆菌hmv18及其外泌蛋白和应用 |
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