CN116583193A - 作为抗菌剂和抗氧化剂益生菌的微生物菌株乳酸片球菌tak 589 coccobest - Google Patents
作为抗菌剂和抗氧化剂益生菌的微生物菌株乳酸片球菌tak 589 coccobest Download PDFInfo
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- CN116583193A CN116583193A CN202180077725.9A CN202180077725A CN116583193A CN 116583193 A CN116583193 A CN 116583193A CN 202180077725 A CN202180077725 A CN 202180077725A CN 116583193 A CN116583193 A CN 116583193A
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Abstract
微生物菌株乳酸片球菌TAK 589 CoccobEst是一种新的抗菌剂和抗氧化剂益生菌，CoccobEst抑制肠病原体单核细胞增生李斯特氏菌、大肠杆菌、肠炎沙门氏菌、肠沙门氏菌鼠伤寒血清变型、索氏志贺氏菌、金黄色葡萄球菌、粪肠球菌和阪崎肠杆菌的生长，但不抑制天然肠道微生物群的乳杆菌。具有蛋白水解活性的周质肽聚糖水解酶增强该菌株的抗菌特性。CoccobEst用作畜牧饲料添加剂，以防止农场动物和宠物的胃肠道中的细菌感染，并预防和减少腹泻。CoccobEst具有高度活跃的抗氧化剂防御系统，增加谷胱甘肽的氧化还原活性，减少细胞内的氧化应激。因此，CoccobEst用作人的食品补充剂或功能性食品，以预防胃肠道的细菌感染，并预防和减少氧化应激。此外，CoccobEst在发酵蔬菜中用作起子培养物。
Description
技术领域
本发明涉及生物技术的领域。本发明的益生菌乳酸细菌涉及用于增强宿主生物体的天然免疫力和用于预防和缓解多种疾病的用途。
背景技术
联合国粮食和农业组织(FAO)以及世界卫生组织(WHO)将益生菌定义为活的微生物，所述微生物当以足量作为膳食补充剂施用或掺入功能性食品时，对胃肠道的微生物平衡具有有利影响，并且因此有利于宿主的健康。功能性食品或组合物包含额外的组分或生物活性制剂，包括益生菌，其具有健康益处或降低疾病的风险。
物种和样本的显著不同的肠道微生物群以及与宿主的相互作用根据遗传和环境因素以及疾病而变化。当肠道微生物群的平衡在期望的方向受到影响时，可以增强对免疫、心理和代谢病况的抵抗力以及它们对治疗的反应。施用益生菌促进肠道微生物群的定殖抵抗力以及对应激和肠源性感染的抵抗力(Sassone-Corsi等人2015.How BeneficialMicrobes Cooperate with Immunity to Provide Colonization Resistance toPathogens.J Immunol.194：4081-4087)。
大多数益生菌是乳酸细菌或乳酸发酵细菌。这些包括形成同型发酵片球菌属的四联球菌，其已经适应广泛范围的pH水平、温度和渗透压，且因此能够定殖肠道。
当选择适合施用于人和动物的益生菌时，遵循安全性、功能性和技术特性的原则(FAO/WHO 2001.Health and Nutritional Properties of Probiotics in FoodIncluding Powder Milk with Live Lactic Acid Bacteria；EFSA Panel on Additivesand Products or Substances used in Animal Feed.2011.Technical guidance：Tolerance and efficacy studies in target animals.EFSA J.9：2175)。
关于安全性，必须排除对抗生素具有抗性或致病性或与感染性疾病相关的细菌菌株。在此方面，分离菌株的功能，即其定殖肠道的能力及其生物活性比其起源更重要(Saarela等人2000.Probiotic bacteria：safety，functional and technologicalproperties.J Biotechnol.84：197-215)。技术特性包括在生产和保存工艺的过程中保留益生菌菌株的生物活性和感官特性，包括确保益生菌在通过肠道后的活力。
向动物施用抗生素引起的问题
在野外出生的动物从它们的母亲获取肠道微生物群。现代畜牧业方法使母亲无法与其后代充分接触，且因此，年幼动物的肠道微生物群可能无法足够快速地发育或充分发育。这无法为新生动物提供针对病原体的足够保护；此外，来自身体和情绪环境变化的应激，即与其母亲分开或断奶，使年幼者特别容易受到感染的影响(Corcionivoschi等人2010.The Effect of Probiotics on Animal Health.A review.J Anim SciBiotechnol.43：1)。
为了防止感染并刺激生长，畜牧业几十年来一直在使用抗生素。因此，自然选择和基因突变显著增加抗生素抗性病原体的选择以及抗生素抗性基因在农场动物间以及从农田向地下水的水平转移(FAO.2016.Probiotics in animal nutrition-Production，impact and regulation，Y.S.Bajagai等人FAO Animal Production and Health PaperNo.179.Rome；Chee-Sanford等人2001.Occurrence and Diversity of TetracyclineResistance Genes in Lagoons and Groundwater Underlying Two Swine ProductionFacilities.Appl Environ Micro-biol.67：1494-1502)。
对抗生素具有抗性的病原体在农场动物间的传播是对人类健康的威胁，因为它们沿着食物链传播并引起严重的暴发-在1990年代丹麦和美国的暴发与多药耐药肠沙门氏菌鼠伤寒血清变型DT104在猪肉中的传播以及喹诺酮抗性的空肠弯曲杆菌在家禽中的传播直接相关(Molbak等人1999.An outbreak of multidrug-resistant，quinolone-resistantS.enterica Typhimurium DT104.NEngl J Med.341：1420-1425；Smith等人1999.Quinolone-resistant Campylobacter jejuni infections in Minnesota，1992-1998Investigation team.N Engl J Med.340：1525-1532)。
益生菌在兽医学中的施用
根据欧洲议会和理事会的条例(EG)号1831/2003，自2006年起禁止在动物饲料中补充抗生素以预防疾病(包括消化道感染)和刺激生长。同一条例将益生菌分类为各种动物物种的畜牧学营养添加剂中的肠道菌群稳定剂。
益生菌添加剂减少小牛肠道中大肠菌的丰度及其引起的腹泻，以及增加小牛的摄食量，刺激生长和降低发病率(Wallace和Newbold.1995.Bacteriology in Animal Feedand Animal Feeding.VCH Verlagsgesellschaft mbH)。此外，益生菌促进肠道定殖抗性(Jatkauskas和Vrotniakien.2010.Effects of probiotic dietary supplementation ondiarrhoea patterns，fecal microbiota and performance of early weanedcalves.Vet Med Praha.55：494-503)。
益生菌在小牛出生后发育的早期(第7-10天)更有效，此时疾病的症状更频繁地显现并且并发症可能更严重(Kawakami等人2010.Feeding of Lactic Acid Bacteria andYeast on Growth and Diarrhea of Holstein Calves.J Anim Vet Adv.9：1112-1114)。因此，当务之急是在出生后尽可能早地抑制病原体在动物肠道中的传播。
将片球菌作为益生菌施用于农场动物
施用于仔猪和鸡以增加其重量的乳酸片球菌(Pediococcus acidilactici)CNCMMA 18/5M在欧盟被归类为肠道菌群稳定剂(2013年5月6日的委员会实施条例(EU)号413/2013)。
在出生后的前42天内向仔猪施用该菌株对其重量增加具有积极影响，并且保护小肠的粘膜，以及增加断奶期间的抵抗力定殖和应激(Di Giancamillo等人2008.Effects oforally administered probiotic Pediococcus acidilactici on the small and largeintestine of weaning piglets.A qualitative and quantitative micro-anatomicalstudy.Histol.Histopathol.23：651-664)。
当向鸡施用时，乳酸片球菌CNCM MA 18/5M对蛋的重量、蛋壳的厚度、饲料的效率、脂肪酸组成和蛋黄的胆固醇水平具有积极影响，并减少缺陷蛋的数量(Mikulski等人2012.Effects of dietary probiotic Pediococcus acidilactici supplementation onperformance，nutrient digestibility，egg traits，egg yolk cholesterol，and fattyacid profile in laying hens.Poultry Science，91：2691-2700)。
乳酸片球菌的一些益生菌菌株已经归因于调节蛋白信号传导分子的合成的能力，由此减少体内的炎性过程。
从韩国国菜中分离的乳酸片球菌LDTM 5201抑制在感染区域诱导炎性反应的IL-8细胞因子的转录。此外，当通过发酵乳制品或作为饲料添加剂施用时，同一菌株扩增体内抗炎反应介质或IL-10细胞因子的转录(KR 20180117761，Seoul National University R&DBFoundation，2018)。施用乳酸片球菌NRRL B-50517通过在体内增加IL-10细胞因子的转录和M2巨噬细胞并减少IL-6和IL-23细胞因子的转录，对炎性反应的发展具有类似的抑制作用(US 201916266397，Imagilin Technology LLC，2019)。
乳酸片球菌被用于几种由益生菌细菌物种组成的组合物中-其含有影响消化的额外组分，诸如菊粉、吸附剂和肠道蠕动的抑制剂-用于减少和预防小牛中腹泻的情况(EP20110711323，TechnischeMünchen，2011)。
片球菌在发酵植物物质中的用途
发酵是最简单的蔬菜保存方法之一。向切碎的蔬菜中添加食盐促进汁液的分离，所述汁液是发育中的微生物群的营养培养基。发酵需要厌氧条件以防止不期望的微生物群的生长，因此发酵蔬菜技术中的任何错误以及蔬菜的天然微生物群中存在的微生物的影响可能导致最终产品软或粘或产品变色(Pudnir和Jain.2010.Changes in microflora ofsauerkraut during fermentation and storage.World J Dairy Food Sci5：221-225)。几个因素限制了起子培养物在蔬菜发酵中的用途-蔬菜的固有微生物群、1-2％的食盐浓度、温度的波动。此外，起子培养物不得对最终产品的感官特性产生负面影响。
蔬菜发酵中的主要乳酸细菌物种是肠膜明串珠菌(Leuconostocmesenteroides)、短乳杆菌(Lactobacillus brevis)、戊糖片球菌(Pediococcuspentosaceus)和植物乳杆菌(Lactobacillus plantarum)(Applications ofBiotechnology to Traditional Fermented foods.1992.Report of an ad hocpanel ofthe board on science and technology for international development.NationalAcademy Press，Washington，D.C.；Plengvidhya等人2007.DNA Fingerprinting ofLactic Acid Bacteria in Sauerkraut Fermentations.Appl.Environ.Microbiol.73：7697-7702)。
在这方面，已知食用由大豆、姜黄和枳椇属树的果实与乳酸片球菌IOB701和其他益生菌菌株制成的发酵功能性食品减轻饮酒的后果，并支持肝脏在酒精分解中的活，性，以及降低肝损伤的风险(CN 201910930726，Tianjin Chuangyuan Biotechnology Co.，LTD，2019)。
以上表明，具有各种特性的乳酸片球菌菌株已经被研究和使用。然而，同一物种的不同菌株不具有相同的特性，这是因为由于遗传变异或个别菌株特异性的特性导致的物种内差异。
已经从各种环境(青贮饲料、粪便样品、牛奶以及发酵的蔬菜和果汁)中分离的片球菌对差异很大的抗生素具有菌株特异性的敏感性(Singla等人2018.Antibioticsusceptibility profile of Pediococcus spp.from diverse sources.3Biotech.8：489)。例如，当测试几种乳酸片球菌菌株时发现对常见抗生素的广泛抗性(Daniels等人2007.Susceptibility of Pediococcus spp.to antimicrobial agents.J ApplMicrobiol.102：384-389)。因此，密切相关的益生菌细菌菌株可以根据其基因型、表型和功能性而不同(Barros等人2001.Phenotypic and Genotypic Characterization ofPediococcus Strains Isolated from Human Clinical Sources.J.Clin.Microbiol.39：1241-1246)。
显现特定菌株特异性特性的主要先决条件是在微生物细胞中存在引起它的基因(Marteau.2011.Evidence of Probiotic Strain Specificity Makes Extrapolation ofResults Impossible from a Strain to Another，Even from the Same Species.Annalsof Gastroenterology&Hepatology)。
为此，需要一种乳酸片球菌菌株，其具有期望的特性并且适合用于人食品和动物饲料中以及用于发酵植物物质。
本发明的公开内容
本发明涉及分离的细菌菌株乳酸片球菌TAK 589 Coccobest、所述菌株的冻干形式、包含所述菌株的组合物和所述菌株作为食品或饮料中的益生菌添加剂、农场动物和宠物的畜牧饲料添加剂以及人的功能性食品或饮料的添加剂的用途。本发明的目的是提出一种具有抗微生物和抗氧化活性的新型益生菌细菌菌株，其适用于修正和稳定胃肠道微生物群，并由此用于在体内预防发生细菌感染以及预防和/或减少氧化应激。此外，乳酸片球菌TAK 589 Coccobest适合用作发酵植物物质的技术起子培养物。
乳酸片球菌TAK 589 Coccobest适合以微生物的冻干培养物的形式作为食品添加剂施用并为人和/或农场动物和宠物(包括鱼和鸟类)制备功能性食品。包含乳酸片球菌TAK589 Coccobest的组合物中的其他组分可包括完全代乳品、牛奶、起子发酵物、浓缩饲料、预混物和其他组分。
本发明的菌株可用于预防人、农场动物和宠物的胃肠道中的细菌感染，并因此用于预防和/或减少腹泻。引起上述细菌感染的微生物包括单核细胞增生李斯特氏菌(Listeria monocytogenes)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、肠炎沙门氏菌(Salmonella enteritidis)、肠沙门氏菌鼠伤寒血清变型(Salmonella enterica serovar Typhimurium)、索氏志贺氏菌(Shigella sonnei)、粪肠球菌(Enterococcus faecalis)和阪崎肠杆菌(Cronobacter sakazakii)。
乳酸片球菌TAK 589 Coccobest的抗细菌和抗氧化特性可安全地用于多种生物技术领域，包括植物物质的发酵或人益生菌膳食补充剂中。
乳酸片球菌TAK 589 Coccobest已根据国际承认为专利程序目的保藏微生物的布达佩斯条约以DSM编号32372保藏在Deutsche Sammlung für Mikroorganismen undZellkulturen GmbH(德国微生物和细胞培养物保藏中心GmbH)(2016年9月23日)。
形态学特性
乳酸片球菌TAK 589 Coccobest菌株在胃肠道微生物群的研究期间通过如下从健康小牛的粪便样品中分离出来：在盐水溶液(0.9％NaCl)中培养小牛的稀释粪便物质(1-2-10-7)并将其接种在deMan-Rogosa-Sharpe(MRS)琼脂(OXOID，UK)上，将其在IG 150培养箱(Jouan，France)中在微需氧环境(10/5/85CO2/O2/N2)中在37℃下孵育48小时。对培养的微生物菌落进行描述、计数并确定微生物的总量。乳酸片球菌TAK 589 Coccobest是一种革兰氏阳性球菌，其细胞成对或形成四联球菌。
生理和生化特性
乳酸片球菌TAK 589 Coccobest可以通过在微需氧环境中在MRS液体培养基中孵育24-48小时来培养，这导致一致混浊的生长。在MRS琼脂培养基中在微需氧环境(10/5/85CO2/O2/N2)中在37℃下培养48小时后，微生物菌落具有1.5-2.5mm的直径，规则的边缘，且为灰白色和凸起的。菌株的最佳生长温度为37℃至45℃；该菌株也可以在7℃和15℃下繁殖。培养基的最佳pH水平为6.5。
通过使用用于测定碳水化合物的发酵概况的API CHL50(bioMérieux，France)培养基和Maldi Biotyper质谱仪(Bruker Daltonik，Germany)，TAK 589 Coccobest菌株基于其生化活性被鉴定为乳酸片球菌。该菌株发酵：L-阿拉伯糖、核糖、D-木糖、D-半乳糖、D-葡萄糖、D-果糖、D-甘露糖、L-鼠李糖、N-乙酰氨基葡萄糖、苦杏仁苷、熊果苷、七叶苷、水杨苷、纤维二糖、D-海藻糖、龙胆二糖、塔格糖、葡萄糖酸钠。
基因组和分子特性
通过使用PRINSEQ网络软件(Schmieder和Edwards.2011.Quality control andpreprocessing of metagenomic datasets.Bioinformatics，27∶863-864.)，根据Phred质量评分(＜20)和长度(＜40bp)分选通过乳酸片球菌TAK 589 Coccobest的全基因组测序(Omega Bioservices，USA)获得的来自样品的DNA片段。基于RAST服务器的PATRIC软件用于从通过质量控制的样品完全组装和注释基因组(Brettin等人2015.RASTtk：amodular andextensible implementation of the RAST algorithm for building customannotation pipelines and annotating batches of genomes.Sci Rep.5：8365)。
基于结果，确定乳酸片球菌TAK 589 Coccobest基因组的核苷酸结构，并鉴定1,947个蛋白编码DNA序列(表1)。
表1.TAK 589 Coccobest的核苷酸特征
将在基因组注释期间鉴定的1,947个蛋白编码序列针对基于COG服务器(Tatusov.2000.The COG database：a tool for genome-scale analysis of proteinfunctions and evolution.Nucleic Acids Res.28：33-36)的数据库中的直向同源基因进行比对；1,677个序列根据功能分为19个更大类别(表2)。
表2.TAK 589 Coccobest的编码序列的分类
在乳酸片球菌TAK 589 Coccobest的基因组中鉴定周质肽聚糖水解酶(PGH)的基因序列，其酶位点具有蛋白水解活性并在体外抑制广泛范围的微生物的生长(Sharma等人2016.Prediction of peptidoglycan hydrolases-a new class of antibacterialproteins.BMC Genomics.17：1；García-Cano等人2011.Detection，cellularlocalization and antibacterial activity of two lytic enzymes of Pediococcusacidilactici ATCC 8042.J Appl Microbiol.111：607-615)。
将鉴定的PGH序列(GenBank AKD44141.1)针对NCB1保守结构域数据库进行比对，由此确定在早期研究的过程中确定的两个活性位点。第一个属于识别肽聚糖层的蛋白家族，即它能够通过破坏细胞膜的酰胺之间的键在细胞外水解肽聚糖。第二个活性位点属于具有类似作用的氨基葡萄糖苷酶超家族(Marchler-Bauer等人2016.CDD/SPARCLE：functional classification of proteins via subfamily domainarchitectures.Nucleic Acids Res.45：D200-D203)。
因此，乳酸片球菌TAK 589 Coccobest含有至少两种裂解酶，其活性位点促进讨论中的菌株的抗细菌活性。归因于上述PGH酶的对微生物生长的抑制作用已在体外得到证明，并且本发明的抗微生物特性在体内得到证明。在两种情况下，已经证明针对几种广泛传播的病原体(包括针对单核细胞增生李斯特氏菌、肠沙门氏菌鼠伤寒血清变型、金黄色葡萄球菌、粪肠球菌和大肠杆菌)的抗细菌作用的传播。
对抗生素的敏感性
根据ISO 10932标准使用VetMICTMpanels(SVE，Sweden)测试乳酸片球菌TAK 589Coccobest对抗生素的抗菌剂敏感性。根据欧洲食品安全局(EFSA)推荐的流行病学截止值确定最小抑制浓度并与BioCC微生物培养物保藏中心中的其他动物来源的片球菌进行比较(表3)。当微生物菌株在等于或低于特定抗菌化合物的截止值的浓度下受到抑制时，该微生物菌株被认为是敏感的(S≤×mg/L)。当微生物菌株在高于特定抗菌化合物的截止值的浓度下受到抑制时，该微生物菌株被认为是抗性的(R＞×mg/L)。
乳酸片球菌TAK 589 Coccobest的菌株对研究中使用的抗生素没有抗性。因此，与已经测试并发现对常见抗生素有抗性的几种其他乳酸片球菌菌株相比，它可以被认为是高度安全的标准(Daniels等人2007.Susceptibility of Pediococcus spp.toantimicrobial agents.J Appl Microbiol.102：384-389)。
表3.与两种片球菌相比，TAK 589 Coccobest的抗菌剂敏感性
a Guidance on the assessment of bacterial susceptibility toantimicrobials of human and veterinary importance.EFSA Journal.2012.10(6)：2740
抗细菌特性
各种乳酸片球菌菌株针对病原体的抗菌作用可能变化很大。例如，已经证明菌株QC38针对大肠杆菌和肠沙门氏菌鼠伤寒血清变型具有低抑制作用，并且针对金黄色葡萄球菌的生长具有中等抑制作用(Morales-Estrada等人2016.Partial Characterization ofBacteriocin Produced by Halotolerant Pediococcus acidilactici StrainQC38Isolated from Traditional Cotija Cheese.Pol J Microbiol.65：279-285)。同时，本发明的菌株针对这些病原体的抑制作用强。
基于体内研究结果，乳酸片球菌TAK 589 Coccobest抑制致病微生物。此类肠病原体包括单核细胞增生李斯特氏菌、大肠杆菌、肠炎沙门氏菌、肠沙门氏菌鼠伤寒血清变型、索氏志贺氏菌、金黄色葡萄球菌、粪肠球菌和阪崎肠杆菌(实施例1)。
为了评价乳酸片球菌TAK 589 Coccobest针对病原体的抗菌特性，使用划线法(Hütt等人2006.Antagonistic activity of probiotic lactobacilli and bifidobacteriaagainst entero-and uropathogens.J AppL Microbiol.100：1324-32)。为了测量针对靶标微生物的拮抗活性，测量以毫米计的抑制区的宽度，并且基于所用样品的结果计算算术平均值和标准偏差，并且基于此，评价菌株的拮抗活性。
乳酸片球菌TAK 589 Coccobest菌株在孵育24至48小时后针对选定的病原体具有强烈的拮抗特性；然而，该菌株不抑制属于天然肠道微生物群的乳杆菌(表4和5)。
表4.TAK 589 Coccobest的抗菌活性a
a在微需氧(10％CO2)和厌氧(5/90/5CO2/O2/N2)环境中在孵育24小时后的改良的MRS琼脂。
微需氧环境中的生长抑制区：低＜9.9；中等10.0-12.9；高＞13(mm)。厌氧环境中的生长抑制区：低＜4.9；中等5.0-5.9；高＞6(mm)。
表5.TAK 589 Coccobest的抗菌活性b
b在微需氧(10％CO2)和厌氧(5/90/5CO2/O2/N2)环境中在孵育48小时后的改良的MRS琼脂上。
微需氧环境中的生长抑制区：低＜9.9；中等10.0-12.9；高＞13(mm)。厌氧环境中的生长抑制区：低＜7.9；中等8.0-12.9；高＞13(mm)。
抗氧化特性
体内氧化应激由促进自由基的连锁反应的因素(即促氧化剂)引起。促氧化剂的连续不受控制的形成和作用引起对脂质、蛋白、核酸和碳水化合物的氧化损伤，这是许多疾病(心脏病发作、中风、动脉粥样硬化、癌症)的原因之一。其中，促氧化剂包括活性氧物质，其积累是细胞损伤的主要原因之一。
抗氧化剂是可以抑制或防止促氧化剂和连锁反应从非常低的浓度开始形成的酶或物质。特定的抗氧化酶和抗氧化物质两者均影响身体(例如谷胱甘肽)。此外，抗氧化剂从食物中吸收。在污染和应激性环境中，抗氧化保护需要额外的支持。其中，由于肠道益生菌的功能，抗氧化剂被释放至血流中。
在乳酸片球菌TAK 589 Coccobest的基因组中鉴定了抗氧化酶和转录因子的基因序列。这些包括NADH过氧化物酶(EC 1.11.1.1)、过氧化氢应激反应调节子(PerP)、硫氧还蛋白(Trx)、硫氧还蛋白还原酶(EC 1.8.1，9)、谷胱甘肽还原酶(EC 1.8.1.7)、硫醇过氧化物酶(EC 1.11.1.15)、甲硫氨酸亚砜还原酶A和B(EC 1.8.4.11和EC 1.8.4.12)、辅酶A二硫化物还原酶(EC 1.8.1.1)、锰过氧化氢酶(EC 1.11.1.6)等。
为了评价鉴定的抗氧化酶的作用，对它们进行体外测试，并与BioCC微生物培养物保藏中心中的来自动物来源的其他片球菌进行比较(表6)。
乳酸片球菌TAK 589 Coccobest菌株在MRS液体培养基中培养24小时，并在4℃下以10000×G离心5分钟；随后，除去上清液并将收集物(result)悬浮在1ml MQ水中。悬浮液的密度为每毫升109个细菌细胞，OD600为1.1。为了获得裂解物，将含有SDS(CAS#151-21-3)的裂解缓冲液(Qiagen，Holland)添加至悬浮液中，并将细菌细胞在室温下机械破碎15分钟。
用商业试剂盒(Cayman Chemicals，USA)测定总抗氧化活性(TAA)。该方法基于高铁肌红蛋白对ABTS(CAS#28752-68-3)的氧化以及样品中抑制性抗氧化剂的定量。用Synergy HTX分光光度计(BioTek，USA)以750nm的波长测量样品和标准品的吸光度。
用比色试剂盒(Invitrogen，USA)测定氧化型(GSSG)和还原型(GSH)谷胱甘肽。样品用5％苯甲酸(CAS#97-05-2)处理以从裂解物中除去蛋白。游离谷胱甘肽和其他硫醇用2-乙烯基吡啶(CAS#100-69-6)封闭，用于氧化型谷胱甘肽的定量。用Synergy HTX分光光度计以405nm的波长测量样品和标准品的吸光度。
用比色试剂盒(Thermo Scientific，USA)测定过氧化氢。该方法基于过氧化氢对莫尔盐(CAS#24389-93-3)的氧化同时通过山梨糖醇(CAS#50-70-4)进行增强。氧化的莫尔盐结合样品中的二甲苯酚(CAS#1611-35-4)。用Synergy HTX分光光度计以595nm的波长测量样品和标准品的吸光度。
结果表明，在含有活细胞的环境中和无细胞环境中，含有乳酸片球菌TAK 589Coccobest菌株的样品中的过氧化氢含量低(表6)。H2O2是体内形成活性自由基的前体，其扩散通过细胞结构，并且在酸性环境中是强氧化剂。
在无细胞环境中测量的还原型谷胱甘肽水平在乳酸片球菌TAK 589 Coccobest菌株的情况下最高，其是活性氧物质最重要的陷阱之一。谷胱甘肽的氧化还原活性、其抑制自氧化和保留还原形式的能力是细胞中最重要的氧化还原缓冲。谷胱甘肽形式的变化直接影响细胞的抗氧化活性(Smirnova和Oktyabrsky.2005.Glutathione inBacteria.Biokhimiya70：1199-211)。就此而言，与BioCC微生物培养物保藏中心中的来自动物来源的其他片球菌相比，GSSG/GSH的比率(细胞中氧化应激的指标)在乳酸片球菌TAK589 Coccobest菌株的情况下最低。
此外，测量的总抗氧化活性在乳酸片球菌TAK 589 Coccobest的细胞裂解物中最高(表6)。
表6.与其他片球菌相比，TAK 589 Coccobest的抗氧化活性
aTAA-总抗氧化活性
bGSH-还原型谷胱甘肽
cGSSG-氧化型谷胱甘肽
dGSSG/GSH-谷胱甘肽的氧化还原比
生物活性的保留
当使用粉末制备小牛的完全代乳品饲料(MRF)时，将其在40-50℃的水中稀释，这是益生菌菌株在更高温度下维持其生物活性的能力至关重要的原因。
为了证明这一点，用冻干的乳酸片球菌TAK 58 9 Coccobest培养物进行实验，将其搅拌至稀释的酪乳中并在85℃下加热20分钟。加热后，将其立即接种至MRS琼脂上，在37℃孵育48小时，其后计数微生物孔(microbial wells)。在实验的过程中，发现乳酸片球菌TAK 589 Coccobest的数量在60℃加热10-20分钟时没有变化，并且在70℃减少一个对数(图1)。
除了食品加工中使用的温度以外，在各种温度下在较长时段期间维持生物活性对于储存益生菌乳酸细菌的菌株是重要的。对于持续36个月的保留挑战测试，使用乳酸片球菌TAK 589 Coccobest培养物作为冻干粉末(表7)。
表7.TAK 589 Coccobest的生物活性保留挑战测试a
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a根据(改良的)ISO 15214：1998标准。
此外，在胃的酸性环境中和与胆汁酸接触下测试乳酸片球菌TAK 589 Coccobest菌株的生物活性。菌株在微需氧环境(10/5/85CO2/O2/N2)中在37℃下在MRS液体培养基中培养24小时。再制备三种MRS液体培养基用于测定对胆汁酸和胃酸的抗性：将3g/L胆汁提取物(Sigma，USA)添加至第一种MRS液体培养基中，将1M HCl溶液添加至第二种MRS液体培养基中，直到环境的pH水平为3，并且最后，除了1M HCl溶液以外，还将3g/L胃蛋白酶(EC3.4.23.1；Sigma，USA)添加至第三种MRS液体培养基中，以达到环境中2的pH水平。将先前培养的细菌培养物添加至改良的MRS液体培养基中，随后在37℃下在微需氧环境中孵育。
乳酸片球菌TAK 589 Coccobest菌株的存活能力用每小时接种进行证明(表8)。
表8.TAK 589 Coccobest对胆汁酸和酸性环境的抗性测试
定殖肠道的能力
证明乳酸片球菌TAK 589 Coccobest通过胃肠道的能力，作为在爱沙尼亚用32头荷斯坦雌性小牛进行的实验的一部分(实施例3)。新生小牛被分为测试和对照组，并从出生后第4天直至第16天饲喂6升来自当地农场的牛奶和用粉末制备的完全代乳品饲料(MRF)的混合物。将乳酸片球菌TAK 589 Coccobest益生菌细菌菌株添加至饲喂给测试组中的小牛的MRF中。在第一周和第二周结束时，用戴手套的手从小牛的直肠收集粪便样品至无菌杯中，并储存在-20℃且后来储存在-80℃。
通过使用菌株特异性引物和基因探针，用qPCR方法测量从小牛收集的粪便样品中的乳酸片球菌TAK 589 Coccobest的量。根据QIAamp DNA Stool Mini Kit(Qiagen，Germany)的方案从粪便样品中提取DNA。使用Synergy HTX分光光度计(BioTek，USA)检查DNA的浓度。为了确定乳酸片球菌TAK 589 Coccobest的菌株，制备每只小牛的DNA样品的等分试样，其中浓度为10ng/μL且最终体积为35μL。为了扩增DNA，对于细菌活性所必需的基因序列区域-BioY(WP_002832434.1)(一种属于生物素生物合成链的转运蛋白，其对于乳酸片球菌TAK 589 Coccobest是菌株特异性的)设计菌株特异性引物和TaqMan诊断基因探针(表9)。
表9.菌株特异性引物和TaqMan基因探针的分子特性
用PrimerQuest工具软件(Integrated DNA TechnoLogies，USA)设计寡核苷酸。
a反映核酸双链体的稳定性的解链温度
b与其自身紧密配合的内部互补区段
c形成二聚体的内部互补区段
将用于qPCR反应的2μl DNA等分试样(～20ng)干燥，即将DNA移液至微孔板上并在无菌条件下干燥过夜。然后，将20μL预先制备的反应混合物添加至每个样品中。将两个空白添加至板中以控制测定的纯度和可能的假阳性。每个样品的反应混合物包含以下组分：
4μL PrimeMini qPCR Assay(Integrated DNA Technologies，USA)
4μL 5x HOTProbe qPCR Mix(Solis BioDyne，Estonia)
12μL MQ去离子H20
QuantStudio热循环仪(Thermo Fisher Scientific，USA)用于qPCR反应。QuantStudio Design and Analysis Software(Thermo Fisher Scientific，USA)用于读取PCR板并分析结果。qPCR反应的条件如下：
在由TaqMan菌株特异性基因探针产生的荧光的帮助下，测试从小牛收集的粪便样品中的乳酸片球菌TAK 589 Coccobest。在PCR期间，引物和基因探针通过在特定序列中紧密配合或错配以菌株特异性方式结合。基因探针在5′末端与荧光团结合，并且在3′末端与淬灭剂结合。淬灭剂仅在荧光团附近时才有活性。当一条新的DNA链被合成时，基因探针的5′末端的荧光团就被释放。随着每个新的循环，荧光团以与扩增子的数量成比例进行释放。这导致越来越强的荧光，其使得能够定量反应混合物中的菌株特异性扩增子。当超过荧光的阈值时，记录循环阈值。用含有基因组DNA和反应混合物的对照样品评价诊断基因探针对乳酸片球菌TAK 589 Coccobest菌株的特异性。
分别地，在出生后第I周和第II周，在10个样品中的9个样品中和11个样品中的7个样品中，在测试组小牛的粪便样品中测试到乳酸片球菌TAK 589 Coccobest菌株的存在(表10)。在第I周，在13个样品中的1个样品中，在第II周，在7个样品中的1个样品中，在对照组小牛的粪便样品中测试到菌株的存在。因此，可以说乳酸片球菌TAK 589Coccobest能够成功定殖在小牛的肠道中。
表10.用菌株特异性引物的qPCR反应的结果
a反应的荧光信号超过阈值时的循环阈值
b组中的扩增样品/所有样品的数量
c，d结果的算术平均值和中值
附图简要说明
图1.当在高温下孵育10至20分钟时，乳酸片球菌TAK 589Coccobest在稀释酪乳中在log10系统中的存活率。
图2.在厌氧环境中的抑制。与从肠道分离的片球菌属物种的对照菌株相比，乳酸片球菌TAK 589 Coccobest对病原体的抗菌作用。在改良的MRS琼脂上，在孵育24小时后，在厌氧(5/90/5 CO2/O2/N2)环境中使用划线法(靶标微生物的生长抑制，以mm计)。
图3.微需氧环境中的抑制。与从肠道分离的片球菌属物种的对照菌株相比，乳酸片球菌TAK 589 Coccobest对病原体的抗菌作用。在改良的MRS琼脂上，在孵育24小时后，在微需氧(10％CO2)环境中使用划线法(靶标微生物的生长抑制，以mm计)。
实施方案的描述
实施例1.对致病性微生物的拮抗作用
目的：测试乳酸片球菌TAK 589 Coccobest针对最常见致病性微生物的拮抗特性。
方法：为了评价乳酸片球菌TAK 589 Coccobest针对病原体的拮抗特性，使用划线法；以毫米计测量由TAK 589 Coccobest和片球菌属物种对照菌株引起的病原体的生长抑制区(Hütt等人2006.Antagonistic activity of probiotic lactobacilli andbifidobacteria against entero-and uropathogens.J Appl Microbiol.100：1324-1332)。
在不同环境中针对八种病原体菌株评价片球菌属物种菌株的拮抗活性：单核细胞增生李斯特氏菌ATCC 51774；大肠杆菌ATCC 25922；肠炎沙门氏菌ATCC 13076；肠沙门氏菌鼠伤寒血清变型；索氏志贺氏菌ATCC 25931；金黄色葡萄球菌ATCC 25923；和粪肠球菌ATCC29212。首先，将乳酸片球菌TAK 589 Coccobest在IG 150培养箱(Jouan，France)中在MRS(Oxoid，UK)中在微需氧(10％CO2)环境在37℃下培养24小时。在有氧环境中在37℃下，使致病菌株在补充有血液的琼脂上生长24小时。为了评价抗菌特性，将20μL培养24小时的五种不同的片球菌菌株沿中线培养至改良的MRS琼脂培养基(不含硫酸三铵C6H17O7N3和醋酸钠C2H9O5Na)上并同时在37℃下在微需氧和厌氧(5/90/5 CO2/O2/N2)环境中培养。将要测试的病原体菌株以相反方向的两条垂直线接种，并在微需氧和厌氧环境中在37℃下孵育24小时。测量无生长区以确定对病原体的抑制。从肠道分离和属于BioCC微生物培养物保藏中心的片球菌属物种的对照菌株用作比较的基础。
结果：乳酸片球菌TAK 589 Coccobest在微需氧和厌氧环境中针对病原体的生长具有最强的抑制特性；其他菌株的影响是中等或低的(图2，图3)。
实施例2.对小牛的施用的作用(实验1)
目的：研究乳酸片球菌TAK 589 Coccobest对小牛肠道微生物群和腹泻发病率的作用。
方法：实验包括8头荷斯坦品种的爱沙尼亚雄性小牛。新生小牛被分为测试和对照组(两组：n＝4)。测试组中的小牛从第一天以1×1010CFU的每日剂量接受作为用冻干粉末制备的水溶液的乳酸片球菌TAK 589 Coccobest，直至小牛为5天龄。在小牛生命的第2天和第5天以及另外在出生后第8天从小牛收集粪便样品，以评价后效应。用戴手套的手从直肠收集粪便样品至无菌杯中，所述杯储存在-20℃，且后来储存在-80℃。测量样品中的乳杆菌的总量以及大肠杆菌和大肠菌微生物的量。此外，根据健康评分标准(威斯康星大学麦迪逊分校，兽医学院，小牛健康评分表和小牛健康评分标准(University of Wisconsin-Madison，School of Veterinary Medicine，Calf Health Scoring Chart and Calf HealthScoring Criteria))评价腹泻发病率和总体健康指标。
结果：对照组16个样品中的7个样品为腹泻阳性(44％)，且测试组16个样品中3个样品为腹泻阳性(19％)。施用乳酸片球菌TAK 589 Coccobest使测试组的粪便样品中大肠杆菌的量到出生后第5天减少1.6个对数，且到出生后第8天减少2.5个对数。测试组粪便样品中大肠菌微生物的量到第5天减少0.3个对数，且到第8天减少0.8个对数。同时，测试组的粪便样品中的乳杆菌的量并没有减少。
实施例3.对小牛的施用的作用(实验2)
目的：研究乳酸片球菌TAK 589 Coccobest对小牛肠道肠杆菌微生物群和腹泻发病率的作用。
方法：实验包括32头荷斯坦品种的爱沙尼亚雌性小牛。新生小牛被分为测试和对照组(两组：n＝16)，并在其生命的前三天给予初乳。在第3天后直至第16天，向小牛给予来自当地农场的牛奶和完全代乳品饲料(MRF)的混合物，所述完全代乳品饲料(MRF)用MRF粉末(20％脂肪、22％蛋白质、47.6％乳糖和16.4％酪蛋白)与温水(40℃)制备，其中比率为140g：890mL。从第17天开始，小牛仅给予MRF以饮用。从第3天开始，将乳酸片球菌TAK 589Coccobest(1.2-3.2×107CFU/mL)添加到至测试组中的小牛的MRF中。
在第4天和第10天之间，小牛每天接受6升MRF，且之后每天接受8升，直至断奶(近似在第70天)。在实验期间，兽医每天根据粪便评价标准(威斯康星大学麦迪逊分校，兽医学院，小牛健康评分表和小牛健康评分标准(University of Wisconsin-Madison，School ofVeterinary Medicine，Calf Health Scoring Chart and Calf Health ScoringCriteria))记录小牛粪便的黏稠度。测试组和对照组小牛之间的出生体重和Ig水平没有差异(表11)。出生后第2、7、18和30天用戴手套的手将小牛粪便样品从直肠收集至无菌杯中，所述杯储存在-20℃且后来储存在-80℃。根据ISO标准测定样品中的大肠杆菌、大肠菌微生物和肠杆菌的一般量。
表11.测试组和对照组小牛在出生后的健康指标
结果：在它们生命的第15-65天之间，16头测试组小牛中头仅有一头具有腹泻(6.3％)，相比之下，16头对照组小牛中有7头具有腹泻(43.8％)。此外，两头对照组小牛死亡。到第30天，施用乳酸片球菌TAK 589 Coccobest使测试组的粪便样品中的大肠杆菌的量减少一个对数。到第30天，测试组的粪便样品中的大肠菌细菌和肠杆菌的量分别减少1.1和1.3个对数。同时，到第30天，对照组的粪便样品中的大肠杆菌的量增加0.5个对数。到第30天，对照组的粪便样品中的大肠菌细菌和肠杆菌的量增加0.4个对数。
实施例4.用TAK 589 Coccobest菌株发酵蔬菜(实验1)
目的：测试乳酸片球菌TAK 589 Coccobest对于发酵蔬菜(例如白色卷心菜(whitecabbage)或甘蓝变种capitata f. alba(Brassica oleracea var.capitataf.alba))的适用性。
方法：用食品加工机将卷心菜磨碎成细条，向其中加入食盐(1.7％)并小心混合。然后，将培养24小时且最终菌株密度为107CFU/g的乳酸片球菌TAK 589 Coccobest与卷心菜混合。卷心菜在18℃下发酵9天。自发发酵的卷心菜用作对照。在第2、5和9天测量卷心菜汁的pH水平。
结果：添加乳酸片球菌TAK 589 Coccobest有助于加速卷心菜的发酵。磨碎的卷心菜的基础pH水平为5.60。与自发发酵相比，磨碎的卷心菜的pH水平下降更快(表12)。pH水平的快速下降有助于抑制卷心菜的不期望的微生物群落，包括产生粘液的乳酸细菌(例如肠膜明串珠菌(Leuconostoc mesenteroides))。
表12.磨碎的卷心菜在发酵时段期间的pH下降
在两次测试期间，发酵卷心菜没有缺损，味道酸，且质地硬脆。乳酸片球菌TAK 589Coccobest适合作为技术起子培养物用于发酵中，以引导卷心菜的发酵。
实施例5.用TAK 589 Coccobest菌株发酵蔬菜(实验2)
目的：测试乳酸片球菌TAK 589 Coccobest对于发酵户外生长的蔬菜(例如黄瓜(cucumbers)或黄瓜(Cucumis sativus L))的适用性。
方法：制备由每1升水1汤匙食盐和1汤匙糖组成的溶液，用于发酵户外生长的爱沙尼亚黄瓜。将培养24小时的乳酸片球菌TAK 589Coccobest的培养物添加至溶液中，最终密度为107CFU/g。黄瓜(4-5根黄瓜，400mL液体)在30℃发酵24小时。在发酵结束时，测量pH水平并将黄瓜冷却至4℃。
结果：在发酵结束时，自发发酵黄瓜的pH水平为4.34。用乳酸片球菌TAK 589Coccobest发酵的黄瓜的pH水平为3.85。对照黄瓜和用乳酸片球菌TAK 589 Coccobest发酵的黄瓜都没有缺损。他们尝起来酸。用乳酸片球菌TAK 589 Coccobest发酵的黄瓜没有自发发酵黄瓜的苦的余味。因此，乳酸片球菌TAK 589 Coccobest适合作为技术起子培养物在发酵黄瓜时引导发酵。
Claims (8)
1.分离的益生菌微生物乳酸片球菌(Pediococcus acidilactici)TAK 589CoccobestDSM 32372。
2.权利要求1的微生物，其呈冻干形式。
3.包含权利要求1或2的微生物的组合物。
4.权利要求1或2中任一项的微生物作为益生菌食品和饮料添加剂的用途。
5.权利要求1或2中任一项的微生物作为畜牧饲料添加剂以在农场动物和宠物中预防胃肠道的细菌感染以及预防和/或减少腹泻的用途。
6.权利要求1或2中任一项的微生物作为功能性食品或饮料添加剂以在人中预防胃肠道的细菌感染以及预防和/或减少氧化应激的用途。
7.根据权利要求5或6所述的权利要求1或2中任一项的微生物的用途，其中引起细菌感染的微生物为单核细胞增生李斯特氏菌(Listeria monocytogenes)、大肠杆菌(Escherichia coli、)、肠炎沙门氏菌(Salmonella enteritidis)、肠沙门氏菌鼠伤寒血清变型(Salmonella enterica serovar Typhimurium)、索氏志贺氏菌(Shigella sonnei)、金黄色葡萄球菌(Staphylococcus aureus)、粪肠球菌(Enterococcus faecalis)和阪崎肠杆菌(Cronobacter sakazakii)。
8.权利要求1或2中任一项的微生物作为用于蔬菜发酵的技术起子培养物的用途。
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| EEP202000014 | 2020-09-18 | ||
| PCT/IB2021/000739 WO2022058798A2 (en) | 2020-09-18 | 2021-09-17 | Microorganism strain pediococcus acidilactici tak 589 coccobest as an antimicrobial and antioxidant probiotic |
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| JP7121867B1 (ja) * | 2022-03-28 | 2022-08-18 | エバラ食品工業株式会社 | 乳酸菌 |
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| KR102036275B1 (ko) * | 2018-10-02 | 2019-10-24 | 서울대학교 산학협력단 | 항-염증 활성이 우수한 페디오코커스 악시딜락티시 ldtm 5201 신균주(kctc 13563bp) |
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