CN112204130B - 具有抗病毒和免疫调节功效的植物乳杆菌cjlp17及包含其的组合物 - Google Patents
具有抗病毒和免疫调节功效的植物乳杆菌cjlp17及包含其的组合物 Download PDFInfo
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- CN112204130B CN112204130B CN201980028241.8A CN201980028241A CN112204130B CN 112204130 B CN112204130 B CN 112204130B CN 201980028241 A CN201980028241 A CN 201980028241A CN 112204130 B CN112204130 B CN 112204130B
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Abstract
本申请涉及具有耐酸性、耐胆汁性、和免疫增强活性的新型植物乳杆菌CJLP17菌株,以及包含其的组合物。
Description
技术领域
本公开涉及具有耐酸性、耐胆汁性、和免疫增强活性的植物乳杆菌(Lactobacillus plantarum)CJLP17菌株。具体地,本公开涉及包括植物乳杆菌CJLP17菌株及其培养物的组合物、饲料添加剂、和功能食品。
背景技术
在当前的畜牧业中,家畜由于高度传染性的病毒性疾病而被杀死,而且这常常导致养殖场的经济损害。特别地,在养猪业中,由病毒和病菌导致的传染病——如四种主要慢性消耗病(即猪呼吸道疾病复合征、断奶后多系统衰竭综合征(postweaningmultisystemic wasting syndrome)、猪繁殖与呼吸综合征、和猪流行性腹泻)——已导致巨大的经济损失。
其中,猪流行性腹泻是由猪流行性腹泻病毒(PEDV)(冠状病毒科成员)的感染导致的猪消化系统疾病。病毒在小肠和大肠的绒毛中增殖,并在所有年龄段的猪(尤其是在小猪)中导致急性肠炎、呕吐、和水样腹泻。特别地,主要在冬季(从11月至4月)危害严重,且已知出生1周内的断奶前小猪死亡率为约50%,且在严重的情况下,由于极度脱水,死亡率可达几乎100%。
1971年在欧洲首次发现了PED病毒(Oldham J.,Letter to the editor.PigFarming,1972,10:72-73),且1976年在比利时进一步检测和分离了G1a型PEDV CV777(Pensaert MB等人,A new coronavirus-like particle associated with diarrhea inswine.Arch Virol.,1978,58:243–247)。在20世纪80年代,该病毒在整个欧洲传播,且在20世纪90年代,包括中国、韩国、日本等的东亚国家发生爆发。此外,比G1a型病原性更强的G2b型PEDV在2010年首次在中国出现了。这种新型PEDV已传播到北美(美国和加拿大)以及东南亚和欧洲,导致严重损害(Song D.等人,Porcine epidemic diarrhoea virus:acomprehensive review of molecular epidemiology,diagnosis,and vaccines,VirusGenes,2012,44:167–175)。在2013年,由于美国养猪业生产力的损失,损害估计为约2.2万亿韩元。在韩国,据报道PEDV每年在20%到40%的猪养殖场发生爆发,导致全部猪的6%被杀死。另据报道,进入和离开屠宰场的车辆的感染率达到约60%(韩国农村经济研究院(Korea Rural Economic Institute),韩国猪兽医协会(Korea Swine VeterinaryAssociation))。
到目前为止,从根本上预防G2b型PED病毒的唯一方法是彻底灭菌。许多养殖场使用人造感染方法或接种现有的G1a型PEDV疫苗来预防病毒性疾病导致的损害,但在预防G2b型PEDV感染方面存在局限性。为了克服这样的问题,用于预防(疫苗等)和治疗(IgY、精油、有机酸、益生菌等)PED病毒的制剂的开发正在积极进行。特别地,最近研究了使用刺激体内免疫系统同时具有抗病毒作用的功能性材料来增强免疫力的方法。
免疫通常分为先天免疫和适应性免疫。先天免疫是从第一线立即防御病原体感染的系统,直接作用于侵入物(抗原)或诱导适应性免疫。适应性免疫是更复杂和精确的系统,它识别和去除侵入物,或作为相应侵入物的记忆,从而提供与先天免疫相比更永久的免疫功能。作为与先天免疫相关的抗原呈递细胞,树突细胞(DC)、巨噬细胞、和天然杀伤细胞直接发挥先天免疫功能,并具有辅助激活各种类型T细胞的受体,从而分泌细胞因子。适应性免疫是针对已进入身体的抗原的二级防御系统,且是由B淋巴细胞和T淋巴细胞进行的特异性免疫应答。由抗原激活的T细胞控制的免疫应答包括细胞毒性T细胞应答、辅助性T细胞应答等。作为这些T细胞的前体,幼稚(naive)CD4 T细胞分泌白细胞介素12(IL-12)并诱导根除细胞内病原体的细胞毒性T淋巴细胞(CTL)应答,或分泌白细胞介素4(IL-4)并从而诱导特异性根除细胞外病原体的应答(B细胞的抗体分泌)。此外,上面枚举的免疫细胞通过精细和复杂的过程——通过分泌TGF-β(β转化生长因子)以抑制过度的免疫应答(如炎症反应)、通过响应于TGF-β和白细胞介素6(IL-6)的分泌而将B细胞转化成浆细胞来刺激抗体的产生、或者诱导根除错误的自身免疫和细胞外病原体的免疫应答(Th17)等——对已经进入身体的侵入物以适当的方式作出响应。
发明内容
[技术问题]
在这些情况下,本发明人通过分离和鉴定能够激活免疫系统同时表现出针对上述病毒的抑制活性的新型微生物,并确认其活性,从而完成了本公开。
[技术方案]
本公开的一个目的是提供具有耐酸性、耐胆汁性、和免疫增强活性的,以保藏号KCCM12249P保藏的植物乳杆菌CJLP17菌株。
本公开的另一目的是提供包含植物乳杆菌CJLP17菌株或其培养物的组合物。
本公开的又一目的是提供包含植物乳杆菌CJLP17菌株或其培养物的饲料添加剂。
本公开的再一目的是提供包含植物乳杆菌CJLP17菌株或其培养物的功能食品。
[有益效果]
本公开的植物乳杆菌CJLP17菌株具有优异的耐酸性和耐胆汁性,且因此可以作为益生菌提供。CJLP17菌株在体内激活免疫细胞,且从而允许调节免疫功能,并且特别地表现出针对猪流行性腹泻病毒的优异抑制活性。此外,可以发现,当家畜被口服给予本公开的植物乳杆菌CJLP17菌株时,不仅通过免疫调节作用显著降低腹泻发生率,而且显示出改善平均日增重(ADG)的效果。因此,本公开可以提供新型植物乳杆菌CJLP17菌株,其具有针对PEDV的抗病毒活性、免疫增强活性、以及改善家畜的平均日增重和降低腹泻发生率的作用,且因此,该菌株可以有效地用于药物组合物、食品组合物、或饲料组合物。
附图说明
图1显示了CJLP17的电子显微镜图像。
图2显示了确认菌株溶血活性的血琼脂平板图像。
图3是确认CJLP17细胞毒性的图表。
图4是显示培养猪的脾细胞(图表中表示为Spln)和外周血单核细胞(图表中表示为PBMC)后IL-12分泌的测量结果的图表。
图5是显示培养猪的脾细胞(图表中表示为Spln)和外周血单核细胞(图表中表示为PBMC)后IL-10分泌的测量结果的图表。
图6是培养肠系膜淋巴结(图表中表示为mLN)和派尔集合淋巴结(Peyer’s patch)细胞(图表中表示为PP)后,通过测量TGF-β的分泌,显示与阴性对照相比增加(pg/mL)的图表。
图7显示了示例根据植物乳杆菌CJLP17针对PED病毒感染的抑制作用的显微镜图像。
图8是显示口服给予CJLP17对断奶小猪ADG平均值的影响的图表。
图9是显示口服给予CJLP17对断奶小猪腹泻发生率的影响的图表。
具体实施方式
以下,将更详细地描述本公开。同时,本文公开的每个描述和实施方式可分别应用于其它描述和实施方式。也就是说,本文公开的各种要素的所有组合都落入本公开的范围内。进一步,本公开的范围不受以下具体描述的限制。
在本公开的一个方面,为了克服上述目的,提供了具有耐酸性、耐胆汁性、和免疫增强活性的植物乳杆菌CJLP17菌株,其以保藏号KCCM12249P保藏。
如本文所用,术语“乳杆菌(Lactobacillus)”是指属于自然界中广泛分布的好氧或兼性厌氧革兰氏阳性杆菌(bacillus,)的属的微生物。属于乳杆菌属的微生物包括植物乳杆菌等。本发明人提供了属于植物乳杆菌的新型菌株,其以保藏号KCCM12249P保藏。其对应于益生菌菌株,对人体无害,且可以无副作用地使用。
如本文所用,术语“益生菌”是指进入身体并提供保健益处的活细菌。目前已知的大部分益生菌一直以来通过诸如乳杆菌的乳酸菌制成的发酵乳制品来消耗(摄取,)。然而,近年来,益生菌在市场上可以除了乳杆菌以外,还含有诸如双歧杆菌属(Bifidobacterium)、肠球菌属(Enterococcus)一些菌种的发酵乳、颗粒剂、粉剂等形式获得。
如本文所用,术语“耐酸性”是指耐受高酸度的性质。如果益生菌是耐酸的,即使当通过口服给予消耗暴露于胃中的强酸性条件下时,它们也可以防止被降解或损坏。
如本文所用,术语“耐胆汁性”是指对胆汁中消化酶的抗性。胆汁在肝脏中制成并储存于胆囊中,并且是帮助在小肠的十二指肠中消化脂肪的弱碱性的棕绿色液体,并含有通过乳化脂肪帮助消化和吸收的各种酶。胆汁是减少益生菌给予效果的主要原因之一,因为它作用于通过口服给予摄取的益生菌。
具体地,本公开的植物乳杆菌CJLP17菌株是从已经发生过猪流行性腹泻病毒的国内(韩国)养殖场中的母猪和小猪的粪便和初乳中分离获得的。本公开的菌株的形态学特征是该菌株是革兰氏阳性杆菌,并且由SEQ ID NO:1的16s rDNA核苷酸序列表示。分析了核苷酸序列,并发现具有与植物乳杆菌约99%的同源性。因此,本发明人于2018年4月13日将新分离的植物乳杆菌CJLP17菌株保藏在韩国微生物保藏中心,保藏号为KCCM12249P。
为了长期稳定地维持(保存,)本公开的植物乳杆菌CJLP17菌株,该菌株可以通过将细胞溶解在保存溶液(通过将一定量的甘油在水中混合而制备)中在-70℃下保存,或者可以通过将细胞悬浮在灭菌的10%脱脂乳中来冷冻干燥,但不限于此。
植物乳杆菌CJLP17可能具有针对病原微生物的抗菌活性。
如本文所用,术语“抗菌”是指通过抑制包括病原微生物的细菌的生长或增殖来预防或治疗细菌感染的活性。
如本文所用,“病原微生物”是指通过寄生于人体而导致疾病的微生物。术语“病原性”是指病原体感染宿主并导致疾病的能力,且可以统称细菌或酵母的渗透性、增殖性、和毒素生产力。病原微生物的实例可以包括选自大肠杆菌(E.coli)、产肠毒素大肠杆菌(Enterotoxigenic E.coli)、金黄色葡萄球菌(Staphylococcus aureus)、鼠伤寒沙门氏菌(Salmonella typhimurium)、和副溶血弧菌(Vibrio parahaemolyticus)的至少一种病原微生物,但不限于此。
本公开的植物乳杆菌CJLP17菌株具有针对病原微生物的抗菌活性,但并非所有属于乳杆菌属的普通微生物都具有针对病原微生物的抗菌活性。
本公开的植物乳杆菌CJLP17菌株可以不表现出针对红细胞的溶血活性。溶血是指红细胞的破坏和血红蛋白向周围区域(外部,)的释放,并且是由从体内有害细菌产生的酶使红细胞溶血的作用。因此,CJLP17被认为是即使它被给予体内也不会导致血管中的溶血的稳定的微生物。
此外,本公开的植物乳杆菌CJLP17菌株可以对抗生素具有弱耐药性或不具有耐药性。抗生素可以具体地是但不限于氨苄西林、克林霉素、艮他霉素、卡那霉素、红霉素、氨苄西林/青霉烷砜、氯霉素、或链霉素。因此,即使当CJLP17用于药物、饲料添加剂等时,其对抗生素没有耐药性,因此导致相关药理作用或环境问题的概率低。
植物乳杆菌CJLP17菌株可增强免疫细胞的激活以增加细胞因子的分泌量,或可以体内给予以促进免疫功能。
如本文所用,术语“免疫细胞”是指在体内起免疫功能作用的所有细胞,并且可以大体上()分为T细胞和B细胞。免疫细胞可包括但不限于Th1或Th2细胞。本公开的植物乳杆菌CJLP17菌株可以具有刺激免疫细胞的活性,从而增加细胞因子(如IL-12、IL-10、和/或TGF-β)的分泌量。
在通常显示高死亡率的病毒性疾病中,由病毒引起的细胞或组织自身坏死可导致其它菌株诱导的继发感染和败血症、由过度激活的免疫应答导致的炎症疾病、或食欲下降和脱水。因此,当抑制病毒感染的抗病毒功效(与Th1和Th2有关)和调节继发感染和过度炎症反应的免疫应答(Th2,抗炎)同时增强时,可以实现对病毒性疾病的有效预防和治疗的效果。由此,关于通过益生菌提供免疫增强效果同时增强Th1和Th2的菌株在本领域中是未知的,并且这样的菌株已由本发明人新鉴定。
如本文所用,术语“细胞因子”是指用作控制和刺激身体防御系统的信号物质的糖蛋白,并且可以是例如IL-12、IL-10或TGF-β,但不限于此。
当经由口服途径向家畜给予时,菌株可促进家畜的生长或降低腹泻发生率。
菌株可能具有针对猪流行性腹泻病毒(PEDV)的抗病毒活性。
猪流行性腹泻病毒是冠状病毒,其感染猪的肠细胞,导致猪流行性腹泻,猪流行性腹泻诱导严重腹泻和脱水。当本公开的植物乳杆菌CJLP17菌株或其培养物被处理时,可以显著抑制PEDV的活性和感染。因此,包括植物乳杆菌CJLP17菌株或其培养物的组合物可被用作针对PEDV的抗病毒组合物,或用于预防或治疗猪流行性腹泻的药物组合物,用于预防或改善猪流行性腹泻的医药外品、或饲料组合物。
在本公开的另一方面,提供了包括植物乳杆菌CJLP17菌株的组合物、其培养物、其浓缩物、或其干燥形式。关于菌株的进一步细节可通过参考上述描述来找到。
本公开的新分离的菌株可以通过用于培养乳杆菌属菌株的常规方法培养。作为培养基,可以使用天然培养基或合成培养基。作为培养基的碳源,可以使用例如葡萄糖、蔗糖、糊精、甘油、淀粉等。作为氮源,可以使用蛋白胨、肉提取物、酵母提取物、干酵母、大豆、铵盐、硝酸盐和其它有机或无机含氮化合物,但氮源不限于此。作为包含在培养基中的无机盐,可以使用镁、锰、钙、铁、钾等,但无机盐不限于此。除了碳源、氮源和无机盐的成分之外,还可以向培养基添加氨基酸、维生素、核酸、和相关化合物。本公开的新分离的菌株可在20℃至40℃的温度范围内培养12小时至4天。
具体地,新分离菌株的培养液可以是含有细胞的粗培养液,或也可以是从中移除了培养上清液的细胞、或浓缩细胞。培养液的组成不仅可以另外地含有常规培养乳杆菌所需的组分,而且还含有对乳杆菌的生长协同起作用的组分,并且其组成可以由本领域技术人员容易地选择。
此外,菌株可以处于液体状态或干燥状态,且干燥方法可以包括但不限于风干、自然干燥、喷雾干燥、和冻干。
在组合物中,植物乳杆菌CJLP17菌株的浓度可以是103CFU/mL至108CFU/mL、104CFU/mL至108CFU/mL、105CFU/mL至108CFU/mL、106CFU/mL至108CFU/mL、103CFU/mL至107CFU/mL、104CFU/mL至107CFU/mmL、105CFU/mL至107CFU/mL、106CFU/mL至107CFU/mL,但不限于此。
组合物可进一步包括冷冻保护剂或赋形剂。更具体地,组合物可进一步包括选自甘油、海藻糖、麦芽糊精、脱脂乳粉和淀粉中的至少一种冷冻保护剂;和/或选自葡萄糖、糊精和脱脂乳中的至少一种赋形剂。基于组合物的总重量,可以含有0.01wt%至20wt%或0.01wt%至10wt%的量的本公开的冷冻保护剂。具体地,基于组合物的总重量,组合物中可以含有5wt%至20wt%的量的甘油、2wt%至10wt%的量的海藻糖、2wt%至10wt%的量的麦芽糊精、0.5wt%至2wt%的量的脱脂乳粉、和0.1%至1%的量的淀粉。此外,基于组合物的总重量,可以含有75wt%至95wt%或85wt%至9wt5%的量的赋形剂。
此外,用于制备包括植物乳杆菌CJLP17菌株、其培养物、其浓缩物、或其干燥形式的组合物的方法可以包括将添加剂与植物乳杆菌CJLP17菌株、其培养物、其浓缩物、或其干燥形式混合的步骤。添加剂可以是上述冷冻保护剂或赋形剂。
在又一方面,提供了益生菌制剂,其包括作为活性成分的本公开的新分离的菌株或其培养物。
益生菌被固定在肠道中的消化道壁上,以防止有害细菌的定居(),并抑制病毒的增殖。此外,益生菌产生的有益消化酶通过有利于营养物的吸收和利用而促进生长。
组合物可选自食品、功能食品、饲料、饲料添加剂、化妆品组合物、药物组合物、和医药外品。
包含植物乳杆菌CJLP17菌株或其培养物的组合物可以具有针对病原微生物的抗菌活性。菌株、病原微生物、和抗菌活性如上所述。
具体地,病原微生物可以是选自大肠杆菌、产肠毒素大肠杆菌、金黄色葡萄球菌,鼠伤寒沙门氏菌、和副溶血弧菌中的至少一种。
包括植物乳杆菌CJLP17菌株或其培养物的组合物可以是用于增强免疫力的组合物。
在本公开的再一方面,提供了饲料添加剂或饲料组合物,其包括植物乳杆菌CJLP17菌株或其培养物。
上述植物乳杆菌CJLP17菌株如上所述。具体地,本公开的植物乳杆菌CJLP17菌株可以被添加到饲料添加剂或包含饲料添加剂的饲料组合物中,用于提供抗菌活性、促进生长、降低腹泻发生率、和抑制病毒活性的目的。
如本文所用,术语“饲料添加剂”是指为提供各种效果的目的(如补充营养物和防止体重下降、促进饲料中纤维素的消化率、改善乳品质、防止繁殖障碍和提高妊娠率、防止夏季高温应激等)而添加到饲料中的物质。根据牲畜和鱼类饲料控制法(Control ofLivestock and Fish Feed Act),本公开的饲料添加剂属于补充饲料,且可以进一步包括矿物质制剂,如碳酸氢钠、膨润土、氧化镁、复合矿物质、以及包括锌、铜、钴、硒等的痕量矿物质;维生素制剂,如胡萝卜素、维生素E、维生素A、维生素D、维生素E、烟酸、复合维生素B等;氨基酸保护制剂,如甲硫氨酸、赖氨酸等;脂肪酸保护制剂,如脂肪酸钙等;以及活细菌和酵母制剂,如益生菌(乳酸菌)、酵母培养物、真菌发酵产物等。
如本文所用,术语“饲料”是指动物进食、摄食和消化或适合于进食、摄食和消化的任何天然或人造饮食、单餐等,或单餐的组分。可以将包括根据本公开用于预防或治疗代谢疾病的组合物作为活性成分的饲料制备成本领域已知的各种形式的饲料,并且可以优选地包括浓缩饲料、粗饲料、和/或特种饲料。
出于本公开的目的,待饲养的受试者可以包括可以摄取本公开的饲料的任何生物,并且可以包括猪。
根据本公开的饲料组合物中植物乳杆菌CJLP17菌株的含量可以根据待施用的家畜的种类和年龄、施用形式、期望效果等适当控制,并且可以是例如以0.01wt%至20wt%的量使用,但不限于此。
为了给予,本公开的饲料组合物可以进一步包括下列一种或多种的混合物:有机酸,如柠檬酸、富马酸、己二酸、乳酸等;磷酸盐,如磷酸钾、磷酸钠、多磷酸盐等;和天然抗氧化剂,如多酚、儿茶素、生育酚、维生素C、绿茶提取物、壳聚糖、鞣酸等。如果需要的话,可以添加其它常规添加剂,如抗流感剂、缓冲剂、抑菌剂等。进一步,可以另外添加稀释剂、分散剂、表面活性剂、粘合剂、或润滑剂以将该组合物配制成可注射制剂(如水溶液、悬浮液、乳剂等)、胶囊、颗粒剂、或片剂。
另外,除了如作为辅助组分的氨基酸、无机盐、维生素、抗氧化剂、抗真菌剂、抗菌物剂等的各种助剂,以及包括植物蛋白饲料(如粉碎的或破碎的小麦、大麦、玉米等)、动物蛋白饲料(如血粉、肉粉、鱼粉等)、动物脂肪、和植物脂肪的主要成分之外,本公开的饲料组合物还可以与营养补充剂、生长促进剂、消化吸收促进剂、和预防剂一起使用。
当本公开的饲料组合物被用作饲料添加剂时,饲料组合物可以原样添加或与其它组分一起使用,或者可以根据常规方法适当地使用。饲料组合物可以与无毒的、药学上可接受的载体组合而以速释或缓释剂型的给予形式制备。可食用载体可以是玉米淀粉、乳糖、蔗糖、或丙二醇。固体载体可以是片剂、粉剂、锭剂等的给予形式,并且液体载体可以是糖浆剂、液体悬浮液、乳剂、溶液等的给予形式。进一步,给予剂可以包括防腐剂、润滑剂、溶液促进剂、或稳定剂,并且还可以包括用于改善其它炎症疾病的其它试剂和对于预防病毒有用的物质。
基于家畜饲料的干重,根据本公开的饲料组合物可以以每1kg约10g至500g,具体地10g至100g的量混合。在完全混合之后,饲料组合物可以提供为糊状物(),或可以进一步经历粒化、膨胀化(/>)、或挤出工艺,但不限于此。
在本公开的又一方面,提供了食品组合物或功能食品,其包含植物乳杆菌CJLP17菌株或其培养物。
具体地,本公开的组合物可以被添加到食品组合物,用于提供抗菌活性、促进生长、促进免疫、降低腹泻发生率、和抑制病毒活性的目的。植物乳杆菌CJLP17菌株如上所述。食品组合物可以包括饮食学上可接受的载体。
本公开的食品组合物包括所有形式的功能食品、营养补充剂、保健食品、和食品添加剂,并且这些类型的食品组合物可以根据常规方法制备成各种形式。
当植物乳杆菌CJLP17菌株被用作食品添加剂时,植物乳杆菌CJLP17菌株可以原样添加或与其它食品或食品成分组合使用,或者可以根据常规方法适当使用。混合活性成分的量可根据使用目的(预防、保健、或治疗处理)适当确定。通常,在制备食品或饮料时,基于包含植物乳杆菌CJLP17菌株的原料组合物,植物乳杆菌CJLP17菌株以0.0001wt%至1wt%,具体地按重量计0.001wt%至0.1wt%的量添加。然而,在为了保健和卫生目的或为了控制健康目的而长期给予的情况下,量可以小于上述范围。
对食品的类型没有特别限制。可以将上述物质添加到其中的食品的实例包括肉类、香肠、面包、巧克力、糖果、小吃、甜食、比萨饼、方便面、其它面条、口香糖、乳制品(包括冰淇淋)、各种汤、饮品、茶、饮料、酒精饮料、复合维生素等,并且包括在普通意义上的所有保健功能食品。
本公开的保健饮料组合物可以如在常规饮料一样进一步含有各种调味剂或天然碳水化合物作为另外的组分。前述天然碳水化合物可以包括单糖,如葡萄糖、果糖等;二糖,如麦芽糖、蔗糖等;多糖,如糊精、环糊精等;和糖醇,如木糖醇、山梨糖醇、赤藓糖醇等。天然甜味剂,如奇异果甜蛋白、甜叶菊提取物等;和合成甜味剂,如糖精、阿斯巴甜等可以用作甜味剂。基于按重量计100份的本公开的组合物,另外的组分的比例可以在按重量计0.01至0.04份,具体地按重量计0.02至0.03份的范围内。
除了前述组分以外,本公开的组合物可以含有各种营养补充剂、维生素、电解质、调味剂、着色剂、果胶酸和其盐、藻酸和其盐、有机酸、保护性胶体增稠剂、pH控制剂、稳定剂、防腐剂、甘油、醇、碳酸饮料中使用的碳酸化剂等。这种添加剂的比例并不重要,但是基于按重量计100份的本公开的组合物,通常在按重量计0.01至0.1份的范围内选择。而且,本公开的组合物可以含有用于制备天然果汁、果汁饮料、或蔬菜饮料的果肉。这种果肉的比例并不重要,但是基于按重量计100份的本公开的组合物,通常在按重量计0.01至10份的范围内选择。这种组分可以单独使用或组合使用。
可以向受试者给予本公开的组合物,其包含植物乳杆菌CJLP17菌株、其培养物、其浓缩物、或其干燥形式,。通过上述给予,对于本公开受试者,组合物可以表现出组合物的效果(即,针对病原微生物的抗菌活性、免疫增强活性等)。
受试者可以包括除人类之外或包括人类在内的动物。动物不仅可以包括人类,而且可以包括需要发挥组合物的上述效果的所有动物。具体地,动物可以是哺乳动物,如牛、马、绵羊、猪、山羊、骆驼、羚羊、狗、猫等。作为另一个实例,动物可以是宠物。
用于给予的剂量不受限制,但是组合物可以以基于植物乳杆菌CJLP17菌株的106CFU/天或更多、107CFU/天或更多、108CFU/天或更多、109CFU/天或更多、或1010CFU/天或更多的量给予。
实施例
在下文中,将通过实施例的方式更详细地描述本公开。然而,给出这些实施例仅仅是为了示例目的,并且本公开的范围不意图受这些实施例的限制。
实施例1:菌株的分离和选择
1-1.样品收集和菌株的分离
从已发生PEDV慢性爆发的国内(韩国)养殖场收集了母猪和小猪的粪便和初乳样品。将由此收集的样品进行连续稀释,涂抹在固体MRS和BHI培养基上,并在37℃下温育48小时。通过将从每种样品中分离出的菌株转移到新鲜培养基中而将它们纯分离,并且在-70℃或更低温度下将纯分离和温育的菌株保存在补充有20%甘油的营养培养基中。结果,总共收集了1,552种菌株,并且通过以下实施例选择了具有优异抗病毒活性的菌株。
1-2.菌株的耐酸性和耐胆汁性的评估
为了选择可用作益生菌的菌株,评估了所收集的菌株的耐酸性和耐胆汁性。
制备了用于评估耐酸性的人造胃液培养基。更具体地,通过向液体MRS培养基中添加胃蛋白酶以调节pH至2.5,随后灭菌来制备人造胃液培养基。
第二次继代培养后,在37℃下将实施例1-1的菌株在液体MRS培养基中进行静态培养18小时。将1%的预温育的菌株接种到人造胃液培养基,并在37℃下进行静态培养,并且在0小时和3小时对培养液进行取样。将取样的培养液连续稀释并涂抹在固体MRS培养基上,并在37℃下温育48小时以测量活细胞数。
制备了人造胆汁培养基用于评估耐胆汁性。更具体地,通过将0.5%牛胆汁(即,公牛胆汁)添加到液体MRS培养基,随后灭菌来制备人造胆汁培养基。
第二次继代培养后,在37℃下将实施例1-1的菌株在液体MRS培养基中进行静态培养18小时。将1%的预温育的菌株接种到人造胆汁培养基,并在37℃下进行静态培养,并且在0小时和24小时对培养液取样。将取样的培养液连续稀释并涂抹在固体MRS培养基上,并在37℃下温育48小时以测量活细胞数。
通过以上评估,选择了具有最高耐酸性和耐胆汁性的菌株CJLP17。如通过下述实施例确认的,由此选择的菌株被鉴定为植物乳杆菌。为了将CJLP17菌株的耐酸性和耐胆汁性与常规已知菌株的耐酸性和耐胆汁性比较,以与上述方法相同的方式评估了从韩国微生物保藏中心获得的植物乳杆菌标准菌株(KCCM12116)的耐酸性和耐胆汁性。
[表1]耐酸性评估(单位:CFU/mL)
0小时 | 3小时 | |
植物乳杆菌CJLP17 | 1.1×106 | 4.0×106 |
植物乳杆菌(KCCM12116) | 2.3×107 | 1.3×107 |
[表2]耐胆汁性评估(单位:CFU/mL)
0小时 | 24小时 | |
植物乳杆菌CJLP17 | 1.3×106 | 1.1×107 |
植物乳杆菌(KCCM12116) | 2.1×107 | 1.6×106 |
根据上述表1和表2,在人造胃液培养基和人造胆汁培养基中的植物乳杆菌标准菌株(KCCM12116)的细胞数量减少。结果,可以看出并非所有一般已知的植物乳杆菌都具有耐酸性和耐胆汁性。
同时,植物乳杆菌CJLP17菌株在两种培养基中的细胞数均实际增加,表明该菌株具有优异的耐酸性和耐胆汁性。
1-3:菌株的形态学和生化特征的研究
为了鉴定CJLP17菌株,首先研究了菌株的形态学和生化特征。对于形态学特征,作为革兰氏染色的结果,该菌株是革兰氏阳性的,并且通过电子显微镜确认,发现是杆菌(图1)。为了分析生化特征,用API 50CHL系统(biomerieux Vitek,Inc.,法国)分析了菌株的糖发酵模式(表3)。
[表3]分离菌株的糖发酵模式分析
+:阳性,-:阴性
1-4.菌种的鉴定
为了更准确地鉴定菌株,进行了基于DNA序列的分子系统发育方法( )。如下进行了序列分析:使用PCR预混合液(Bioneer,韩国)与通用引物27F(5′AGAGTTTGATCMTGGCTCAG 3′)和1492R(5′GGTTACCTTGTTACGACTT 3′)扩增16s rDNA的基因。在基因扩增时,反应溶液的总体积被调节到20μL,并在94℃下进行反应,持续1分钟,在56℃下持续1分钟,和在72℃下持续1分钟,并将该循环总共重复30次以分析扩增的DNA序列。分离菌株的分析的16srDNA核苷酸序列由SEQ ID NO:1表示。
作为分析的结果,CJLP17被鉴定为与植物乳杆菌具有99%同源性的微生物。因此,将分离的菌株命名为“植物乳杆菌CJLP17”,并于2018年4月13日将新鉴定的微生物以植物乳杆菌CJLP17保藏在韩国微生物保藏中心,保藏号为KCCM12249P。
实施例2:CJLP17抗菌活性特征的分析
将大肠杆菌、产肠毒素大肠杆菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、和副溶血弧菌制备为病原微生物,且评估了CJLP17针对这些病原微生物的抗菌活性。
将CJLP17菌株接种到液体MRS培养基中,并在37℃下进行静态培养24小时。在病原微生物中,大肠杆菌、产肠毒素大肠杆菌、鼠伤寒沙门氏菌、和金黄色葡萄球菌接种在液体BHI培养基中,并在37℃下以200rpm温育了18小时;且副溶血弧菌被接种在补充有3% NaCl的液体LB培养基中,并在30℃下以200rpm温育了18小时。
将10μL的CJLP17的培养液等分到固体MRS培养基并干燥,然后将其在37℃下预温育18小时。将1%的病原微生物的培养液各自接种到10mL的固体BHI培养基中,然后将所得产物倒入其中预培养了CJLP17的固体MRS培养基上,并使其完全凝固。然后,测量了在37℃下温育24小时形成的环的尺寸。随着环的尺寸增加,发现了抗菌活性是高的。为了比较抗菌活性,以与上述方法相同的方式评估了植物乳杆菌标准菌株(KCCM12116)的抗菌活性。
[表4]
(单位:mm)
通过上述方法测量环的尺寸的结果,如表4中所示,确认了与植物乳杆菌标准菌株相比,CJLP17对所有病原微生物表现出优异的抗菌活性。
实施例3:菌株稳定性的评估
3-1.菌株溶血活性的确认
β-溶血是其中由红细胞提供的磷脂被由有害细菌产生的磷脂酶水解,导致红细胞溶血的现象。为了确定CJLP17菌株的溶血活性,使用了血琼脂平板(绵羊血5%琼脂,Hanilkomed,韩国)。将菌株划线接种(streaking)至制备的血琼脂平板中,并在37℃下温育24小时以确认溶血。
结果,如图2中所示,确认了植物乳杆菌CJLP17菌株没有显示溶血活性。
3-2.抗生素敏感性的评估
将CJLP17菌株接种到液体MRS培养基中,并在37℃下进行静态培养24小时。将由此培养的细菌吸入灭菌棉拭子中,并涂抹在固体Mueller Hinton II培养基(Difco)上,然后将抗生素片置于该培养基上,并在37℃下温育24小时。将氨苄西林、克林霉素、艮他霉素、卡那霉素、红霉素、氨苄西林/青霉烷砜、氯霉素、和链霉素片(Oxoid,UK)用作用于抗生素测试的抗生素片。
作为CJLP17菌株的抗生素敏感性测试的结果,鉴定出CJLP17菌株对上述抗生素不具有耐性(表5)。因此,可以发现,即使将CJLP17菌株用于药品、饲料添加剂等中,考虑到它们对抗生素没有耐药性,也不太可能发生可引起关于耐药性和环境问题的问题。
[表5]根据抗生素的细菌生长的抑制
实施例4:细胞毒性的评估
为了研究菌株对细胞存活的影响,使用(3-(4,5-二甲基-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四氮唑(promega,USA)进行MTS试验以评估对IPEC-J2细胞(猪肠道上皮细胞(intestinal pig epithelium cells))的细胞毒性水平。将各细胞在96孔细胞培养板上温育,并且将CJLP17以103CFU/孔至108CFU/孔的不同浓度处理。24小时之后,将MTS溶液添加到细胞培养液中,并将细胞温育2小时,并且用酶标仪通过在490nm处测量吸光度来计算细胞存活率(%)。
结果,如图3中所示,当将细胞在6种不同浓度下处理时,确认了在107CFU/mL或更低浓度下几乎观察不到细胞死亡,且在108CFU/mL浓度下细胞显示出约80%的存活率。因此,可以确认CJLP17在107CFU/mL或更低浓度下基本上显示无细胞毒性。
实施例5:免疫增强活性的评估
为了确认CJLP17菌株的免疫增强作用,将IPEC-J2细胞在DMEM/F-12培养基(Dulbecco的改良Eagle培养基(Dulbecco’s Modified Eagle Medium):营养混合物(Nutrient Mixture)F-12)中温育。另外,从21日龄断奶小猪收集肠系膜淋巴结(mLN)中的淋巴细胞、外周血单核细胞(PBMC)、派尔集合淋巴结中的细胞、和脾细胞(Spln),并通过下列方法从各组织和血清中获得免疫细胞。
为了从脾细胞和肠系膜淋巴结中的淋巴细胞获得免疫细胞,使用注射器的活塞和70微米的细胞过滤器(BD Falcon)粉碎了切碎的组织,然后使用RPMI-1640培养基(RoswellPark Memorial Institute-1640,Gibco BRL,Grand Island,NY,USA)洗涤细胞。在最后的洗涤步骤之前,使用RBC裂解缓冲液(eBioscience,USA)移除了剩余的RBC(红细胞)。最后的洗涤步骤后收集的免疫细胞用台盼蓝染色,并用待用的血细胞计数器计数。
为了获得派尔集合淋巴结中的细胞,从小肠中分离出相应的组织,然后使用外科剪刀将其切碎并粉碎。随后,将细胞在含有Dnase I(Roche,德国)、Dispase II(Sigmaaldrich,USA)、抗生素、FBS、和15mM 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)的RPMI-1640培养基(pH 7.2)中反应1小时以获得单一细胞。此后,以与上述相同的方式获得免疫细胞。
将收集的血液以1:1的比例与PBS混合,并在Ficoll-Paque Plus(GE HealthcareLife Sciences,NJ,USA)上离心以分离WBC(白细胞)聚集层,并从分离层获得外周血单核细胞。
为了评价免疫增强作用,将CJLP17菌株与使用上述方法获得的每种类型的免疫细胞以10:1的比例混合,并且将混合物在含有5% CO2的细胞培养箱中在37℃下温育20小时。温育完成后,离心细胞,并使用细胞因子ELISA(酶联免疫吸附试验)评估了培养上清液的免疫激活作用。更具体地,对IL-12(白细胞介素-12)进行ELISA(R&D Systems,USA),IL-12(白细胞介素-12)激活防御外部侵入因子(病原体)的Th1(T辅助(T helper)1)相关的免疫机制。为了评估系统免疫中的Th1和Th2免疫应答,对于IL-12和IL-10,使用脾细胞和外周血单核细胞,以及为了更准确地测量肠道中的抗炎应答,使用派尔集合淋巴结中的细胞和肠系膜淋巴细胞进行了TGF-βELISA试验。
显示IL-12、IL-10和TGF-β的测量结果的图,分别显示在图4至图6中。在各图中,对照组使用没有菌株处理的阴性对照。
结果,如图4至图6中所示,确认了CJLP17菌株显示通过刺激免疫细胞增加IL-12、IL-10、和TGF-β的分泌量的活性。因此,可见CJLP17菌株表现出同时增强Th1免疫机制和Th2免疫机制的作用。
实施例6:针对病毒感染的抑制作用
为了测量CJLP17菌株针对病毒感染的抑制作用,制备了猪流行性腹泻病毒(PEDV、SM98、或KPEDV9菌株)。具体地,将病毒在Vero细胞(CCL-81,来源于绿猴属(Chlorocebus)的肾上皮细胞)中增殖,并且使用了MEM(Eagle's最小必需培养基(Minimum EssentialMedium),Gibco BRL,Grand Island,NY,USA)、热灭活的10% FBS(胎牛血清,v/v)和1%(v/v)青霉素/链霉素作为用于培养Vero细胞的培养基。将Vero细胞作为单层温育,用培养基洗涤两次,然后移除所有溶液。将病毒以0.1MOI(感染复数)的水平在含有用5μg/mL TPCK(N-甲苯磺酰基-L-苯丙氨酸氯甲基酮)处理的胰蛋白酶,不含FBS的MEM中混合,用最小体积的制备的培养细胞处理,然后在含有5% CO2的37℃细胞培养箱中温育2至3天。
病毒感染是由病毒合胞体的形成确定的。当形成病毒合胞体时,在3至6小时内收集病毒培养液,并将细胞使用离心机移除并在-80℃下保存。为了计算病毒的感染滴度,将Vero细胞以2×104个细胞/0.1mL的密度(水平,)在96孔板中温育,并用PBS洗涤细胞。随后,向细胞中加入培养液(其中使病毒进行2倍连续稀释),并温育24至48小时以确认病毒感染,并通过Reed&Muench法计算了病毒的感染滴度。
为了测量CJLP17菌株针对病毒感染的抑制作用,将CJLP17菌株和在实施例5中提取的四种类型的免疫细胞反应20至24小时,以获得培养液。然后,将培养液各自在96孔细胞培养板(其中温育了IPEC-J2)上处理,并在含有5% CO2的37℃细胞培养箱中温育2至4小时。将剂量为100TCID50/mL(50%的组织细胞感染剂量)的PED病毒(SM98或KPEDV9)等分到每个平板并温育48小时。为了确认病毒感染,在培养完成后,细胞培养板用甲醇固定,用结晶紫染色,然后用显微镜检查其中细胞变性的孔,从而确认病毒感染。
[表6]植物乳杆菌CJLP17通过激活的免疫细胞针对PED病毒(SM98/KPEDV9菌株)的抑制作用
++:完全抑制,+:部分抑制,-:感染的
结果,如表6和图7中所示,确认了当猪的免疫细胞与植物乳杆菌CJLP17一起处理时,免疫细胞被菌株激活,并且病毒感染被显著抑制。因此,可以发现当免疫细胞与CJLP17组合处理时,它显示出针对PED病毒的优异的抗病毒活性。
实施例7:对家畜生长和腹泻的作用的确认
为了确认植物乳杆菌CJLP17菌株当经由口服途径给予时对家畜生长性能和腹泻发生率的作用,进行了实验如下:
购买了32只21日龄的断奶小猪,并在其中维持31±1℃的温度和50%的湿度的培养箱养殖场中饲养14天。饲料以不含抗生素的普通碎屑()饲料形式给予,且无限制地给予水。将植物乳杆菌CJLP17菌株以冻干粉的形式生产并保存在冰箱中,并将其彻底混合到碎屑饲料用于口服给予,使得每只小猪每天可以以1010CFU的量的摄取饲料。在共14天的断奶小猪试验中,菌株对小猪生产性(/>)和腹泻/软便(/>)发生率的作用示于图8和图9中。
结果,确认了与对照组相比,饲喂含有CJLP17的饲料的断奶小猪组的日增重(ADG)的平均值更优(图8)。另外,确认了与对照组相比,饲喂含有CJLP17的饲料的断奶小猪组显示出了48%的腹泻发生率改善作用(图9)。
因此,可以发现CJLP17不仅增加了家畜的生长,即生产性,而且表现出显著降低腹泻发生率的作用。
虽然已经参考特定的示例性实施方式描述了本公开,但是本公开所涉及领域的技术人员将理解,在不脱离本公开的技术精神或必要特征的情况下,本公开可以以其它具体形式体现。因此,上述实施方式被认为在所有方面都是示例性的,而不是限制性的。此外,本公开的范围由所附权利要求书而不是由具体实施方式限定,并且应当理解,从本公开的含义和范围及其等同概念得出的所有修改或变化都包括在所附权利要求书的范围内。
/>
<110> CJ第一制糖株式会社
<120> 具有抗病毒和免疫调节功效的植物乳杆菌CJLP17及包含其的组合物
<130> OPA19035-PCT
<150> KR 10-2018-0051380
<151> 2018-05-03
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 1333
<212> DNA
<213> 植物乳杆菌
<400> 1
gacgggcggt gtgtacaagg cccgggaacg tattcaccgc ggcatgctga tccgcgatta 60
ctagcgattc cgacttcatg taggcgagtt gcagcctaca atccgaactg agaatggctt 120
taagagatta gcttactctc gcgagttcgc aactcgttgt accatccatt gtagcacgtg 180
tgtagcccag gtcataaggg gcatgatgat ttgacgtcat ccccaccttc ctccggtttg 240
tcaccggcag tctcaccaga gtgcccaact taatgctggc aactgataat aagggttgcg 300
ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacaacc atgcaccacc 360
tgtatccatg tccccgaagg gaacgtctaa tctcttagat ttgcatagta tgtcaagacc 420
tggtaaggtt cttcgcgtag cttcgaatta aaccacatgc tccaccgctt gtgcgggccc 480
ccgtcaattc ctttgagttt cagccttgcg gccgtactcc ccaggcggaa tgcttaatgc 540
gttagctgca gcactgaagg gcggaaaccc tccaacactt agcattcatc gtttacggta 600
tggactacca gggtatctaa tcctgtttgc tacccatact ttcgagcctc agcgtcagtt 660
acagaccaga cagccgcctt cgccactggt gttcttccat atatctacgc atttcaccgc 720
tacacatgga gttccactgt cctcttctgc actcaagttt cccagtttcc gatgcacttc 780
ttcggttgag ccgaaggctt tcacatcaga cttaaaaaac cgcctgcgct cgctttacgc 840
ccaataaatc cggacaacgc ttgccaccta cgtattaccg cggctgctgg cacgtagtta 900
gccgtggctt tctggttaaa taccgtcaat acctgaacag ttactcttaa atatgttctt 960
ctttaacaac agagttttac gagccgaaac ccttcttcac tcacggggcg ttgctccatc 1020
agactttcgt ccattgtgga agattcccta ctgctgcctc ccgtaggagt ttgggccgtg 1080
tctcagtccc aatgtggccg attaccctct caggttggct acgtatcatt gccatggtga 1140
gccgttacct caccatctag ctaatacgcc gcgggaccat ccaaaagtga tagccgaagc 1200
catctttcaa actcggacca tgcggtccaa gttgttatgc ggtattagca tctgtttcca 1260
ggtgttatcc cccgcttctg ggcaggtttc ccacgtgtta ctcaccagtt cgccactcac 1320
tcaaatgtaa atc 1333
Claims (14)
1.植物乳杆菌(Lactobacillus plantarum)CJLP17菌株,其具有耐酸性、耐胆汁性、和免疫增强活性,其以保藏号KCCM12249P保藏。
2.权利要求1所述的菌株,其中所述菌株具有针对病原微生物的抗菌活性。
3.权利要求2所述的菌株,其中所述病原微生物是选自大肠杆菌(E.coli)、金黄色葡萄球菌(Staphylococcus aureus)、鼠伤寒沙门氏菌(Salmonella typhimurium)、和副溶血弧菌(Vibrio parahaemolyticus)中的至少一种。
4.权利要求1所述的菌株,其中所述菌株通过促进免疫细胞的激活来增加细胞因子的分泌量。
5.权利要求4所述的菌株,其中所述细胞因子是IL-12、IL-10、或TGF-β。
6.权利要求1所述的菌株,其中所述菌株具有针对猪流行性腹泻病毒(PEDV)的抗病毒活性。
7.权利要求1所述的菌株,其中所述菌株当经由口服途径向家畜给予时,促进家畜的生长或降低腹泻发生率。
8.组合物,其包含权利要求1所述的植物乳杆菌CJLP17菌株或其培养物。
9.权利要求8所述的组合物,其中所述组合物进一步包含冷冻保护剂或赋形剂。
10.权利要求9所述的组合物,其中所述冷冻保护剂是选自甘油、海藻糖、麦芽糊精、脱脂乳粉、和淀粉中的至少一种,并且所述赋形剂是选自葡萄糖、糊精、和脱脂乳中的至少一种。
11.权利要求8所述的组合物,其中所述组合物具有针对病原微生物的抗菌活性。
12.权利要求11所述的组合物,其中所述病原微生物是选自大肠杆菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、和副溶血弧菌中的至少一种。
13.权利要求8所述的组合物,其中所述组合物增强免疫力。
14.饲料添加剂,其包含权利要求1所述的植物乳杆菌CJLP17菌株或其培养物。
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PCT/KR2019/005351 WO2019212299A1 (ko) | 2018-05-03 | 2019-05-03 | 항바이러스 및 면역조절 효능을 가지는 락토바실러스 플란타럼 cjlp17 및 이를 포함하는 조성물 |
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2018
- 2018-05-03 KR KR1020180051380A patent/KR20190127156A/ko not_active Application Discontinuation
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2019
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EP3747989A4 (en) | 2021-06-30 |
SG11202009008PA (en) | 2020-10-29 |
BR112020022085A2 (pt) | 2021-02-02 |
KR20190127156A (ko) | 2019-11-13 |
PL3747989T3 (pl) | 2023-02-06 |
PT3747989T (pt) | 2022-12-22 |
WO2019212299A1 (ko) | 2019-11-07 |
US11344588B2 (en) | 2022-05-31 |
JP2021516069A (ja) | 2021-07-01 |
DK3747989T3 (da) | 2022-10-17 |
PH12020551375A1 (en) | 2021-09-01 |
EP3747989B1 (en) | 2022-09-14 |
ES2929354T3 (es) | 2022-11-28 |
JP7052092B2 (ja) | 2022-04-11 |
EP3747989A1 (en) | 2020-12-09 |
CN112204130A (zh) | 2021-01-08 |
US20210038657A1 (en) | 2021-02-11 |
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