CN116948916B - 一种干酪乳杆菌及其应用 - Google Patents
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Abstract
本发明公开了一种干酪乳杆菌及其应用，该干酪乳杆菌GS0605已于2023年5月22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.27393。该菌株安全性好，在哥伦比亚血平板上不产生溶血现象，对多种常见类型的抗生素敏感，对人工胃、肠液有较好的耐受能力，对人结肠癌细胞黏附能力强，其发酵上清液与结肠癌细胞HCT‑116共培后表现出较强的癌细胞生长活性抑制能力和促癌细胞凋亡能力，同时对大肠埃希氏菌、铜绿假单胞菌等多种致病菌有较强的抑制能力，产乙酸和乳酸能力强，对胆固醇具有较高的降解作用，抗氧化作用较强。因此，该菌株益生作用明显，可应用于人和动物功能性食品领域。
Description
技术领域
本发明涉及微生物技术领域，具体涉及一种具有结肠癌细胞生长抑制作用的干酪乳杆菌及其应用。
背景技术
微生物及其代谢物在药物开发和疾病的辅助治疗中扮演着越来越多的角色，其中，干酪乳杆菌(Lactobacillus casei)广泛存在于植物、发酵食品、人和动物类肠道等多种环境中，它以其将糖类发酵为乳酸的能力和健康益处而闻名。特别是干酪乳杆菌可以通过抑制有害细菌的生长和增加有益菌的数量，来调节肠道菌群平衡；可以通过减少肠道炎症、促进肠道黏膜健康和增强免疫功能等方式，改善肠道健康状况；可以降低人体中的胆固醇水平，有助于预防心血管疾病等疾病。
结直肠癌(Colorectal Cancer，CRC)是全球几大常见的恶性肿瘤之一，其发病率高、致死性强。随着饮食文化的多元化发展以及生活方式的改变，世界上多数国家结直肠癌的发病率都呈上升趋势，严重威胁着人类的健康。根据美国癌症协会的最新统计，新发结直肠癌的发病率和致死率仍居新发肿瘤的第三位，仅次于肺癌和乳腺癌。因此，开发具有结肠癌细胞生长抑制作用和促癌细胞凋亡作用的干酪乳杆菌具有很大应用价值。
发明内容
本发明的目的是提供一种干酪乳杆菌及其应用，该菌株对结肠癌细胞具有抑制作用，对多种致病菌具有较强的抑制能力，抗氧化作用较强，且对胆固醇具有较高的降解作用，该菌益生作用明显，在人和动物功能性食品领域具有显著的应用价值。
为了达到上述目的，本发明提供了一种干酪乳杆菌GS0605，该菌株GS0605于2023年5月23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，地址为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，保藏编号为CGMCC NO:27393。
本发明还提供了一种由上述干酪乳杆菌GS0605制备的菌剂，该菌剂可用于大肠埃希氏菌、铜绿假单胞菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、乙型副伤寒沙门氏菌或痢疾志贺氏菌的防治。
本发明提供的干酪乳杆菌GS0605可用于制备抗结肠癌药物或抗结肠癌辅助药物。
本发明还提供了一种抗结肠癌药物或抗结肠癌辅助药物，该药物包含上述干酪乳杆菌GS0605的发酵上清液。
本发明提供的干酪乳杆菌GS0605或其发酵上清液可用于制备降胆固醇药物，还可用于制备抗氧化功能药物。
本发明还提供了一种抗氧化药物或降胆固醇药物，该药物包含上述干酪乳杆菌GS0605的发酵上清液。
本发明提供的干酪乳杆菌GS0605可用于制备调节人或动物肠道菌群的功能性食品。
本发明还提供了一种包含上述干酪乳杆菌GS0605的功能性食品，其中，该功能性包含对胆固醇的降解或/和抗氧化。
本发明提供的干酪乳杆菌GS0605分离自酸菜，具有以下优点：
本发明提供的干酪乳杆菌GS0605在MRS琼脂培养基上生长良好，对酸和胆盐有较好的耐受能力。
本发明提供的干酪乳杆菌GS0605对人结肠癌细胞黏附能力好，并具有较强的癌细胞活性抑制能力和促癌细胞凋亡能力。
本发明提供的干酪乳杆菌GS0605对大肠埃希氏菌、铜绿假单胞菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、乙型副伤寒沙门氏菌和痢疾志贺氏菌等常见肠道病原细菌具有较强的抑菌效果。
本发明提供的干酪乳杆菌GS0605产乙酸和乳酸能力较强，对胆固醇降解能力强，抗氧化作用较强。
附图说明
图1为本发明中干酪乳杆菌GS0605的***进化关系图。
图2为本发明中不同浓度的干酪乳杆菌GS0605发酵上清液对结肠癌细胞生长的抑制作用结果。
图3为本发明中GC-MS检测干酪乳杆菌GS0605发酵上清液中短链脂肪酸的总离子流图谱。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅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干酪乳杆菌GS0605的分离及鉴定
1、材料准备
贵州酸菜样品：采自贵州省黎平县果利寨；
16S rDNA通用引物：27F和1492R通用引物由生工生物工程(上海)有限公司合成，序列如下：
27F(SEQ ID No.1)：5’-AGAGTTTGATCMTGGCTCAG-3’；
1492R(SEQ ID No.2)：5’-GGTTACCTTGTTACGACTT-3’。
MRS肉汤培养基的配方(每升)：酪蛋白酶消化物10.0g、牛肉膏粉10.0g、酵母膏粉4.0g、柠檬酸三铵2.0g、乙酸钠5.0g、硫酸镁0.2g、硫酸锰0.05g、磷酸氢二钾2.0g、葡萄糖20.0g、吐温-80，最终pH为5.7左右。
MRS琼脂培养基的配方(每升)：蛋白胨10.0g、牛肉膏粉5.0g、酵母膏粉4.0g、葡萄糖20.0g、吐温80 1.0mL、磷酸氢二钾2.0g、乙酸钠5.0g、柠檬酸三铵2.0g、硫酸镁0.2g、硫酸锰0.05g、琼脂15.0g，最终pH为6.2左右。
2、具体操作方式
取少许上述的酸菜样品于50mLMRS肉汤中，充分振荡混匀，置于37℃恒温摇床培养24h。采用10倍梯度稀释，涂布接种于MRS琼脂培养基，37℃培养24h后挑取单菌落，并连续纯化3次。将纯化后的菌株接种于600μL MRS肉汤培养基中，37℃摇床培养18h，加入400μL浓度50％(V/V)的无菌甘油，于-80℃超低温冰箱中冻存备用。
冻存菌株活化培养后用MRS肉汤培养基进行扩培，用天根细菌基因组DNA提取试剂盒提取菌株DNA，16S rRNA扩增采用菌落PCR技术完成，通过采用通用引物27F/1492R对其基因组总DNA为模板进行PCR扩增，PCR产物测序由生工生物工程(上海)有限公司完成，经测序可知，其16S rRNA序列如SEQ ID No.3所示。将序列与NCBI中BLAST进行比对，其中一株与标准菌株Lactobacillus casei strain JGB29的相似率达98.45％，可初步鉴定该菌株为干酪乳杆菌(Lactobacillus casei)，命名为GS0605，该菌株与其它菌株的***发育关系如附图1。
16S rRNA序列(SEQ ID No.3)：
TGGATTTGCGGCTGCTATACATGCAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTTCG GGGCAAATGACAGTGTGCATGGTGTCGTCAGCTCGTGTCGTGAGATGTGGTAGTCCCGCACGAGCGCACCCTATGACTAGTGCAGCATTAGTTGGCACTCTAGTAGACTGGCGTGACTATCGAGAGGTGGGATGACGTCAATCATCATGCCC。
将干酪乳杆菌GS0605接种于MRS琼脂培养基上，37℃培养24h后，观察并记录单菌落的形态。采用试剂盒法对干酪乳杆菌GS0605进行革兰氏染色，在显微镜下观察并记录染色后的细菌形态。干酪乳杆菌GS0605在MRS琼脂培养基上生长良好，菌落形态乳白色、圆形凸起、边缘平整、表面光滑，镜检可见菌体呈杆状、紫色，符合干酪乳杆菌的形态特征。结合16S rRNA序列判定该菌GS0605为干酪乳杆菌(Lactobacillus casei)，将该菌株于2023年5月23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，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，保藏编号为CGMCC NO:27393。
实施例2GS0605对结肠癌细胞的作用研究
1、干酪乳杆菌GS0605对人结肠癌细胞的粘附性探究
将干酪乳杆菌GS0605冻存菌种复苏培养后接种于MRS肉汤培养基中，37℃恒温培养24h。培养完毕后置于-4℃、5000r/min条件下离心10min，并用无菌PBS缓冲液多次洗涤。调整菌悬液浓度至1×106CFU/mL后备用。复苏人结肠癌细胞HCT116，将其接种至六孔细胞培养皿，添加DMEM完全培养基并置于37℃、5％CO2中培养，两天更换一次培养液。当细胞贴壁状态达80％时，使用0.25％胰酶-EDTA进行消化，并传代培养。培养完毕后用细胞血球计数板对其进行计数，并将细胞浓度调整至5×106个/mL。将1mL细胞悬浮液加至六孔细胞培养皿的其中一培养孔中，置于培养箱中培养。待培养板中的细胞长至单层，弃掉DMEM培养液，用无菌PBS将每孔冲洗3次。将1mL已制备好的菌悬液加入细胞孔，轻微晃动细胞培养板，吸取孔中少量菌液用于平板计数，其结果作为菌悬液中的初始活菌数。将细胞板置于37℃孵育2h，弃去培养基并用无菌PBS缓冲液洗涤3次。使用0.7mL 0.25％胰蛋白酶-EDTA消化细胞10min，待细胞完全脱落后加入0.3mL DMEM培养液终止消化，收集黏附实验结束后的培养液进行平板计数，其结果作为黏附活菌数。粘附率(％)＝结束期乳酸菌数目/初始乳酸菌接种数目×100％，得到GS0605对HCT116细胞的粘附率情况见下表1，
表1干酪乳杆菌GS0605对人结肠癌细胞HCT116的粘附率(％)
可知，干酪乳杆菌GS0605对人结肠癌细胞具有较强的粘附能力，平均粘附率为41.06％，有利于发挥对结肠癌细胞生长和扩散的抑制作用。
2、GS0605发酵上清液对结肠癌细胞的生长抑制作用探究
制备干酪乳杆菌GS0605的发酵上清液：将干酪乳杆菌GS0605复苏后接种于MRS肉汤培养基中，37℃恒温培养24h，菌悬液浓度约为1×109CFU/mL。培养完毕后置于-4℃、5000r/min条件下离心10min，收集上清液，用细菌滤膜过滤后备用。
癌细胞复苏：在15mL离心管中准备好预热的含有10％FBS和1％双抗的DMEM培养基，置于无菌操作台上，取冻存的人结肠癌细胞(HCT116)于37℃水浴锅中快速解冻。待细胞解冻后，将其于无菌操作台上转移到准备好的含有培养基的15mL离心管中，1000r/min离心3min重悬细胞，吸到6cm的细胞培养皿中，在5％CO2培养箱中37℃培养24h后，更换新鲜的培养基。
细胞传代：待细胞生长状态良好，且密度达到80％左右时进行传代。将培养基和无菌的PBS在37℃水浴锅中预热之后置于无菌操作台上，吸掉细胞培养皿中旧的培养基，用预热的PBS洗细胞以除去死细胞和残留的培养基，再用0.25％的胰酶消化细胞，待细胞变成圆形之后用移液枪吸掉胰酶，培养基终止消化，将细胞收集到无菌的离心管中。1000r/min离心3min吸掉上清，用新鲜的培养基重悬细胞后将其吸到新的细胞培养皿中，置于细胞培养箱中培养。
细胞毒性实验：用CCK-8试剂盒(Cell Counting Kit-8细胞计数试剂)测定干酪乳杆菌GS0605发酵上清液对结肠癌细胞生长的影响。将对数生长期的HCT116细胞消化离心后均匀铺入96孔板中，计数保证每孔2000个细胞。培养12小时待细胞贴壁稳定后加入配置好的1％(发酵上清液：细胞培养液的体积比)GS0605发酵上清液，设置对照(1％的培养基)和三个处理(具体见下表2)，每个处理设置5个重复，培养48小时后进行CCK-8试剂盒检测，在细胞培养箱内继续孵育1小时，用酶标仪450nm测定吸光度，测定结果如下表2所示，
表2GS0605发酵上清液及联合几种抗癌药物对结肠癌细胞生长的影响(吸光度)
不同处理的GS0605发酵上清液对结肠癌细胞生长的抑制情况如图2所示，其中，图中的A:对照；B:发酵上清液(1％)；C:发酵上清液(1％)+奥沙利铂(Oxaliplatin)20μM)；D:发酵上清液(1％)+5-氟尿嘧啶(5fu)20μM。结合表2可知，1％干酪乳杆菌GS0605发酵上清液对人结肠癌细胞的生长表现出明显的抑制作用，培养48h后的结肠癌细胞密度为对照的76.61％。当1％的发酵上清液与抗癌药物5-氟尿嘧啶(5fu，20μM)或奥沙利铂(Oxaliplatin，20μM)联合使用时效果更为明显，培养48h后的结肠癌细胞密度分别为对照的36.78％和48.66％。说明干酪乳杆菌GS0605发酵上清液单独使用或与抗癌药物联合使用，对人结肠癌细胞的生长都有明显的抑制作用，提示干酪乳杆菌GS0605发酵上清液可用于制备开发抗结肠癌药物或抗结肠癌辅助药物。
3、GS0605发酵上清液对人结肠癌细胞的促凋亡作用
3.1材料与方法
细胞培养：将对数生长期的HCT116细胞消化离心后均匀铺入96孔板中，计数保证每孔2000个细胞。培养12小时待细胞贴壁稳定后加入GS0605发酵上清液1％，每个处理设置5个重复，培养48小时后提取蛋白质。
蛋白提取：将培养好的细胞用PBS洗两遍后，尽可能地吸掉细胞表面所有残余的PBS。将细胞置于冰浴中，加入含有蛋白酶抑制剂Cocktail的RIPA裂解液(蛋白酶抑制剂按照1：100的比例稀释)，将细胞全部刮到1.5mL无菌离心管中，置于冰浴中裂解30min。12000r/min4℃离心10min，收集上清液到新的1.5mL无菌离心管中，即为所提取的蛋白样品。该蛋白样品可以直接进行蛋白定量分析或者保存在-80℃冰箱中备用。对于定量分析之后的蛋白样品，可加入蛋白缓冲液，置于金属浴中98℃煮10min，使蛋白变性，瞬时离心后保存于-20℃冰箱备用。
蛋白定量：用碧云天蛋白定量试剂盒进行定量分析。将蛋白样品稀释20倍加入到标记好的96孔板中。按照BCA试剂A与B的比例为50:1配制BCA工作液，充分混匀，每孔加入200μL BCA工作液后，摇晃混匀30s，盖住各样品孔，放置在37℃的恒温培养箱中30min。在562nm处测定吸光值，根据各蛋白样品的校正吸光值，在标准曲线的线性范围内读出各样品的蛋白浓度，根据样品体积和稀释度计算原来样品中的蛋白量。
3.2实验结果
(1)GS0605发酵上清液对细胞粘附因子β-连环蛋白的影响
癌细胞扩散的特征是细胞间无序的相互作用和细胞粘附。β-连环蛋白(β-catenin)是一种多功能蛋白，是重要的细胞粘附分子，参与细胞生长与修复，在肿瘤发生与转移的过程中起着重要作用。结直肠癌的手术后复发率较高，患者的生存时间较短，几乎所有结直肠癌中Wnt/β-catenin信号通路的突变最终都会导致β-catenin的积累。因此，β-连环蛋白的含量可以作为临床上评判结直肠癌发展及预后的一个辅助因素，β-连环蛋白的核积累可能是结直肠癌侵袭、转移和预后不良等恶性相关的标志物。实验结果显示，在结肠癌细胞(HCT116)培养液中加入1％的GS0605发酵上清液，培养48h后，β-连环蛋白(β-catenin)含量大大降低，仅为不加发酵液的对照的42％。与此同时，磷酸化β-连环蛋白(P-β-catenin)的含量也有明显下降，蛋白含量仅为对照的71％(见下表3)。磷酸化β-连环蛋白(P-β-catenin)从粘附复合物中分离并转移到细胞质中，被降解或转位到细胞核，触发Wnt通路激活。Wnt/β-catenin信号通路是一条多功能的通路，在多种癌症中发生着β-catenin信号的紊乱，参与调控结直肠癌、***癌、乳腺癌和恶性血液病等癌症的癌变。
表3GS0605发酵上清液对结肠癌细胞β-连环蛋白含量的影响(吸光度)
由表3的结果可知，GS0605发酵上清液可以明显降低结肠癌细胞的β-连环蛋白和磷酸化β-连环蛋白的表达量，抑制癌细胞间的粘连性，从而抑制癌细胞的迁移扩散。
(2)GS0605发酵上清液对细胞凋亡因子的影响
髓样细胞白血病1(Mcl-1)的过度表达或扩增常见于多种癌症类型，因此被认为MCL-1是最相关的癌蛋白之一，具有抑制癌细胞凋亡的作用。研究表明，细胞培养基中加入1％的GS0605发酵上清液，能使结肠癌细胞MCL-1蛋白总量明显降低(见下表4)，说明发酵液具有促癌细胞凋亡活性。研究表明，mcl-1基因亚型1在结肠肿瘤组织及相应旁组织中的mRNA和蛋白质水平表达均高于正常组，mcl-1亚型1和亚型2对结肠癌肿瘤恶性程度呈正相关，可以作为临床评估结肠癌恶性程度有效的诊断指标之一。
B细胞淋巴瘤/白血病-2基因(bcl-2)，具有明显抑制细胞凋亡的作用，可以抑制由多种细胞毒素所引起的细胞死亡，与结肠癌、卵巢癌等多种癌症密切相关，该基因的过度表达能增强细胞对大多数细胞毒素的抵抗性。实验表明，细胞培养基中加入1％的GS0605发酵上清液，能使结肠癌细胞Bcl-2蛋白总量下降(见下表4)，再次证明发酵液具有促癌细胞凋亡活性。
P62蛋白为一种癌基因蛋白，转录调控的靶基因参与细胞周期的调控、细胞增殖、细胞凋亡及永生化等过程，在诸多肿瘤的发生中起着重要作用。P62基因可以帮助人体更好地识别和抵御外源抗原，从而减轻感染带来的危害。p62蛋白还可以通过促进选择性自噬，来防止遗传毒性和致癌突变的积累，从而发挥抑癌作用。当P62基因与抗原相结合时，它会引发抗原特异性的T细胞和B细胞功能。T细胞会分泌特异性Cytokine来杀死外来病原体，而B细胞会分泌特异性抗体来抑制病原体的复制。因此，P62基因能够结合抗原，促进免疫应答，帮助人体抵御外来的抗原，从而减少病毒感染或细菌侵染所造成的伤害。实验表明，细胞培养基中加入1％的益生菌发酵上清液，能使结肠癌细胞P62蛋白总量明显增加(见下表4)，说明发酵上清液的促凋亡活性。
表4GS0605发酵上清液对细胞凋亡因子的影响
由表4的结果可知，GS0605发酵上清液能够降低抗细胞凋亡因子基因mcl-1和bcl-2的表达，同时使具有促癌细胞凋亡和选择性自噬的p62基因上调，从多个方面证实GS0605发酵上清液具有诱导和促进癌细胞凋亡的作用，进一步证明GS0605发酵上清液可用于制备抗结肠癌药物或辅助药物，用于治疗结肠癌。
实施例3干酪乳杆菌GS0605对人肠道的适应能力及安全性探究
1、模拟胃液、肠液耐受性
模拟胃液和肠液购自上海源叶生物科技有限公司。人工胃液模拟液成分为稀盐酸、胃蛋白酶和氯化钠，最终pH＝2.5；人工肠液模拟液成分为磷酸二氢钾和胰蛋白酶，最终pH＝6.8。将待测菌株干酪乳杆菌GS0605在MRS琼脂平板上进行复苏并活化3代，并调节菌液初始浓度达到1×106CFU/mL。将1mL待测菌株干酪乳杆菌GS0605菌液接种于模拟胃液中，37℃培养3h。培养完毕，取20μL菌液涂布于MRS琼脂平板培养基，设置3个重复，于37℃培养24h，观察培养后MRS平板表面是否有菌落生长。同样，将1mL待测菌株干酪乳杆菌GS0605菌液接种于模拟肠液中，37℃培养6h后，涂平板，37℃培养24h，查看菌落生长情况。
胃模拟液耐受性试验结果表明，干酪乳杆菌GS0605在模拟胃液中处理3h后仍可在MRS琼脂平板上长出正常菌落；肠模拟液耐受性试验结果表明，干酪乳杆菌GS0605在模拟肠液中耐受6h后仍可在MRS琼脂平板上长出正常菌落；说明干酪乳杆菌GS0605具有较强的耐酸和耐胆盐特性。
2、干酪乳杆菌GS0605菌株溶血性及抗生素抗性
将干酪乳杆菌GS0605菌株复苏并活化3代后，划线接种于哥伦比亚血平板上，37℃培养24h后观测待测菌落周围是否出现溶血环，并以溶血性葡萄球菌作为阳性对照。采用纸片琼脂扩散法测试菌株对常见大类抗生素的药敏性。将干酪乳杆菌GS0605菌株复苏并活化3代，菌液浓度调整至1×106CFU/mL，用无菌棉签将菌液均匀地涂抹于MRS培养基平板表面，室温10min后放入药敏纸片，37℃培养24h后，用游标卡尺测量各药敏纸片周围的抑菌圈直径，每种抗生素重复3次，试验结果参照美国临床实验室标准委员会(NCCLS)标准判断菌株的药物敏感性，结果以敏感(S)、中介(I)和耐药(R)表示。
溶血性实验结果表明，在菌落周围没有出现溶血环现象；对测试的5种抗生素表现为敏感(S)或中介(I)，说明所述干酪乳杆菌GS0605菌株的安全性良好，结果下表5。
表5干酪乳杆菌GS0605菌株对5种抗生素的敏感性
实施例4干酪乳杆菌GS0605的其他益生性能
1、短链脂肪酸和乳酸产生能力
发酵液的制备：将所述干酪乳杆菌GS0605保存菌株活化培养24h后，吸取4μL菌液加入到4mL肉汤培养基中，37℃培养24h备用。
其中，短链脂肪酸的检测：检测仪器为日本岛津公司气质联用仪(GCMS-QP2010Plus)，色谱柱为美国RESTEK(瑞斯泰克)公司Rtx-5熔融石英毛细管柱(30m×0.25mm×0.25μm)。GC升温程序为初始温度40℃保持5min，每分钟5℃升至150℃，以每分钟10℃升到280℃，并维持2min。载气为高纯氦气(纯度＞99.999％)，流速：1.0mL/min。MS条件：电离方式为EI；温度为200℃，接口温度220℃，质量扫描范围m/z33-500。取发酵液4mL，加入浓度为200μg/mL 2-乙基丁酸内标液10μL，样品以分流模式1:3的方式进样1μL，溶剂延迟时间设定为0.1min，进样口温度270℃。测定短链脂肪酸乙酸、异戊酸的含量，其浓度采用内标法计算，测定结果如下表6。
乳酸含量的测定：乳酸与糖代谢、酯类代谢、蛋白质代谢以及细胞内能量的代谢密切相关，乳酸含量是评估糖元代谢的和有氧代谢的重要指标。乳酸含量测定严格按照乳酸测定试剂盒(北京索莱宝科技有限公司)说明书进行。将菌种活化培养后，接种于MRS肉汤培养基，37℃培养24h，取100μL培养液加入1mL提取液一，4℃、12000g离心10min，取0.8mL上清液，再加入0.15mL提取液二，12000g离心10min后取上清液，在酶标仪570nm处测定OD值，测定结果如下表6。
表6干酪乳杆菌GS0605发酵液短链脂肪酸及乳酸含量(μg/mL)
通过GC-MS检测了5种短链脂肪酸，分别是乙酸、正丁酸、异丁酸、异戊酸、异己酸，总离子流图谱如图3所示。计算结果表明，干酪乳杆菌GS0605发酵液中乙酸含量最高，为76.317μg/mL，此外还检测到异戊酸，其它短链脂肪酸如正丁酸、异丁酸和2-乙基丁酸含量甚微。最新研究发现乙酸不仅增加了结肠中IgA(免疫球蛋白A是哺乳动物中最丰富的免疫球蛋白，维持粘膜表面稳态的一种成分)产生，而且还改变了IgA与特定微生物(包括肠杆菌)结合的能力。因此，认为肠道微生物产生的乙酸具有调节IgA产生以维持粘膜稳态的作用。丁酸和异戊酸也有非常重要的生理功能。
干酪乳杆菌GS0605菌株具有较强的产乳酸能力，发酵液中乳酸浓度为45.09μmol/mL。乳酸与糖代谢、酯类代谢、蛋白质代谢以及细胞内能量的代谢密切相关，乳酸含量是评估糖元代谢的和有氧代谢的重要指标。
2、对常见肠道致病菌的抑菌活性
将大肠埃希氏菌(Escherichia coli CMCCB 44102)、铜绿假单胞菌(Pseudomonasaeruginosa CMCCB 10104)、金黄色葡萄球菌(Staphylococcus aureus CMCCB 50094)、鼠伤寒沙门氏菌(Salmonella typhimuriumATCC14028)、乙型副伤寒沙门氏菌(SalmonellaparatyphiB CMCCB 50094)和痢疾志贺氏菌(Shigella dysenteriae CMCCB51105)分别接种于营养琼脂培养基，复苏并活化3次。吸取适量胰酪大豆胨液体培养基至离心管，将活化好的致病菌接种于肉汤培养基中，并调节菌液浓度达到1×108CFU/mL。吸取上述致病菌和肉汤的混合液1mL加至500mL灭菌后暂未凝固的营养琼脂培养基内(温度冷却至40℃左右)，充分混匀后按每皿20mL的量分装至培养皿中。待培养基冷却凝固后，使用直径6mm的打孔器在平板上打孔，制成致病菌琼脂板，每板对应一株菌，并设置三孔作为重复。将待测菌株进行复苏并活化，调节培养后的待测菌菌液浓度达到1×108CFU/mL。吸取50μL的待测菌菌液加至上述致病菌琼脂板孔内，于37℃培养24h。培养后，使用游标卡尺测量打孔点周围的抑菌圈直径大小并记录。将标准菌株LGG作为对照菌株，与待测菌株同时进行以上实验操作。
发酵液对大肠埃希氏菌、铜绿假单胞菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、乙型副伤寒沙门氏菌和痢疾志贺氏菌等致病菌的生长具有较强的抑制活性，与LGG标准菌株的效果基本相当(结果见下表7)，说明干酪乳杆菌GS0605可用于制备菌剂，该菌剂可用于上述大肠埃希氏菌、铜绿假单胞菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、乙型副伤寒沙门氏菌或痢疾志贺氏菌的防治，还可用于制备治疗由大肠埃希氏菌、铜绿假单胞菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、乙型副伤寒沙门氏菌或痢疾志贺氏菌所导致疾病的菌剂。
表7干酪乳杆菌GS0605菌株的抑菌活性评估(直径：mm)
3、干酪乳杆菌GS0605发酵上清液的抗氧化活性
抗氧化剂就是任何以低浓度存在就能有效抑制自由基的氧化反应的物质，以抵消自由基对人体细胞的氧化攻击。其作用机理可以是直接作用在自由基，或是间接消耗掉容易生成自由基的物质，防止发生进一步反应，机体抗氧化的能力越强，就越健康，生命也越长。越来越多的研究显示抗氧化是预防衰老的重要步骤，因为自由基或氧化剂会将细胞和组织分解，影响代谢功能，并会引起不同的健康问题。如果能够消除过多的氧化自由基，对于许多自由基引起的及老化相关疾病都能够预防。例如常见的癌症、动脉硬化、糖尿病、白内障、心血管病、老年痴呆、关节炎等，这些疾病都被认为与自由基相关。
发酵上清液的制备：将干酪乳杆菌GS0605冻存菌种复苏培养24h后，按2％接种量接种于MRS液体培养基中，在37℃条件下培养24h，菌液在4℃、4000r/min条件下离心15min，上清液经0.22μm滤膜过滤得到发酵上清液(CFS)冻存备用。
试剂与仪器：羟自由基测定试剂盒(A018-1-1)、DPPH自由基清除能力试剂盒(A153-1-1)和抑制与产生超氧阴离子自由基测定试剂盒(比色法A052-1-1)均由南京建成生物工程研究所生产。紫外可见分光光度计(UV752N型)，上海佑科仪器仪表有限公司生产；发酵液抗氧化能力测定由成都里来生物科技有限公司完成。
抑制羟自由基能力：羟自由基系活性氧的一种，具有极强的得电子能力，也就是氧化能力很强。羟自由基能杀死红细胞，降解DNA、细胞膜和多糖化合物。芬顿(Fenton)反应是最常见的产生羟自由基的化学反应，H2O2的量和Fenton反应产生的羟自由基量成正比，当给予电子受体后，用Griess试剂显色，形成红色物质，其呈色与羟自由基的多少成正比关系。严格按照说明书操作，在550nm处测吸光值。计算公式如下：
抑制羟自由基能力(U/mL)＝(A对照-A测定)/(A标准-A空白)×C标准×(1/V样)×N；
式中，C标准：标准品浓度，8.824mmol/L；V样：取样量，0.2mL；N：样本测试前稀释倍数。
测定结果如下表8，可知干酪乳杆菌GS0605发酵液具有较强的抑制羟自由基能力，为5378.08±143.38U/mL。
DPPH自由基清除能力：DPPH又称1,1-二苯基-2-三硝基苯肼，是一种很稳定的氮中心的自由基。由于DPPH自由基有单电子，在517nm处有一强吸收，其醇溶液呈紫色，当有自由基清除剂存在时，由于与其单电子配对而使其吸收逐渐消失，呈现的颜色越浅，因此，可以对样本中DPPH清除能力进行定量分析。按照试剂盒说明书将标准品粉剂一支加无水甲醇2mL溶解，即为0.5mg/mL(Trolox)标准应用液，再用无水甲醇分别稀释成5μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL，使用波长517nm，1cm光径，无水乙醇调零，测定各管吸光度，制作标准曲线。
DPPH自由基清除率(％)＝(1-(A测定－A对照)÷A空白)×100％
用从标准曲线算得的相当于抗氧化剂Trolox的量来表示样本的DPPH自由基清除能力。发酵液样品DPPH自由基清除能力(μg Trolox/mL)＝代入标准曲线得相当于Trolox的浓度×稀释倍数。干酪乳杆菌GS0605发酵液的DPPH自由基清除能力测定结果见表8，可知，干酪乳杆菌GS0605发酵液的DPPH自由基清除能力165.55±1.69μg Trolox/mL，表明干酪乳杆菌GS0605发酵液对DPPH自由基有一定的清除能力。
抗超氧阴离子能力：超氧阴离子自由基作为生物体代谢过程中产生的一种自由基，可攻击生物大分子，如脂质、蛋白质、核酸和聚不饱和脂肪酸等，使其交链或者断裂，引起细胞结构和功能的破坏，与机体衰老和病变有很密切的关系。本实验模拟机体中黄嘌呤与黄嘌呤氧化酶反应***，产生超氧阴离子自由基，加入电子传递物质及Gress氏显色剂，使反应体系呈现***，用分光光度计测其吸光度，当被测样本中含有超氧阴离子自由基抑制剂时，则比色时测定管的吸光度低于对照管的吸光度，通过以维生素C做标准，可计算出被检物品对超氧阴离子自由基的抑制能力。测定时用1cm光径比色杯，双蒸水调零，波长550nm处比色。
抗超氧阴离子能力(U/L)＝(A对照-A测定)/(A对照-A标准)×C标准×1000×N
在反应***中，每升发酵液在37℃反应40分钟所抑制的超氧阴离子自由基相当于1mg的维生素C所抑制的超氧阴离子自由基的变化值为一个活力单位。干酪乳杆菌GS0605发酵液的抗超氧阴离子能力测定结果见下表8，可知，干酪乳杆菌GS0605发酵液对超氧阴离子自由基的抑制能力为722.77±7.42(U/L)，表明干酪乳杆菌GS0605发酵液对超氧阴离子自由基的抑制能力较强。
表8干酪乳杆菌GS0605发酵液的抗氧化能力
综上可知，GS0605发酵上清液的抗氧化能力较强，在功能性食品领域具有潜在的应用价值，包括用于改善人体健康水平等。
4、干酪乳杆菌GS0605对胆固醇的降解作用
现有研究表明，一些乳酸菌能够吸附或吸收胆固醇，通过排出体外达到降低动物体内胆固醇的目的。因此，在胆固醇-MRS培养基(MRS-CHOL培养基)中加入一定量的胆盐和胆固醇，培养后测定胆固醇浓度变化即可计算对胆固醇的降解能力。具体操作方法如下：
胆固醇溶液的制备：胆固醇0.06g、牛胆盐0.12g、蔗糖脂肪酸脂0.06g、冰乙酸5mL、吐温0.6mL，超声震荡至完全溶解，无菌条件下用0.22μm滤膜过滤后备用；
胆固醇-MRS培养基(MRS-CHOL培养基)制备：在300mL MRS液体培养基中加入胆固醇溶液5mL、6mol/LNaOH溶液12mL；
接种培养：将干酪乳杆菌GS0605菌株菌种活化培养24h,以2％的接种量接种于MRS-CHOL液体培养基中，37℃培养48h，另一份不接菌的为空白对照。
标准胆固醇工作液：0.05g胆固醇，使用冰乙酸定容至50mL，配置成质量浓度为1mg/mL的胆固醇标准溶液；使用冰乙酸稀释10倍，制成标准胆固醇工作液。
胆固醇含量测定：分别取菌悬液(实验组)和空白对照(对照组)500μL于5mL试管中，缓缓加入4.5mL无水乙醇，静置10min，3000r/min离心15min；取上清0.5mL于试管中加入1mg/mL邻苯二甲醛0.2mL、混合酸4.3mL，震荡摇匀，静置30min；在550nm波长下测定吸光值；由标准曲线计算样品中胆固醇含量：
其中C0为空白对照组的OD值；C1为实验组OD值。
实验结果表明，干酪乳杆菌GS0605对胆固醇的降解能力较强，平均降解率为40.12％，高于LGG标准菌株的36.82％，说明干酪乳杆菌GS0605具有降低胆固醇的潜力(见下表9)。
表9干酪乳杆菌GS0605对胆固醇的降解能力(％)
尽管本发明的内容已经通过上述优选实施例作了详细介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，本发明的保护范围应由所附的权利要求来限定。
Claims (4)
1.一种干酪乳杆菌(Lactobacillus casei)GS0605，其特征在于，该菌株GS0605于2023年5月23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，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，保藏编号为CGMCC NO.27393。
2.由权利要求1所述的干酪乳杆菌GS0605制备的菌剂，其特征在于，所述菌剂可用于大肠埃希氏菌、铜绿假单胞菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、乙型副伤寒沙门氏菌或痢疾志贺氏菌的防治。
3.如权利要求1所述的干酪乳杆菌GS0605在制备抗结肠癌药物或抗结肠癌辅助药物中的应用。
4.如权利要求1所述的干酪乳杆菌GS0605在制备乙酸、乳酸或/和异戊酸中的应用。
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