CN116121146A - 一株产乳酸能力较强的戊糖片球菌及其应用 - Google Patents
一株产乳酸能力较强的戊糖片球菌及其应用 Download PDFInfo
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- CN116121146A CN116121146A CN202310222999.3A CN202310222999A CN116121146A CN 116121146 A CN116121146 A CN 116121146A CN 202310222999 A CN202310222999 A CN 202310222999A CN 116121146 A CN116121146 A CN 116121146A
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- pediococcus pentosaceus
- lactic acid
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- cholesterol
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Abstract
本发明公开了一株产乳酸能力较强的戊糖片球菌及其应用,涉及微生物技术领域。该戊糖片球菌为PediococcuspentosaceusTE0307,其已于2022年12月14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,中国科学院微生物研究所,保藏编号为CGMCCNo.26186。本发明提供的TE0307产乳酸能力强,对多种常见的抗生素敏感,对酸、胆盐、人工胃液、人工肠液有较好的耐受能力,对人结肠癌细胞HT‑29有较强黏附能力,对多种人畜肠道病原菌均具有较强的抑制能力,具有较好的降血糖、降胆固醇和抗氧化能力,在功能性食品领域中具有显著的应用价值。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一株产乳酸能力较强的戊糖片球菌及其应用。
背景技术
乳酸菌是一类能利用可发酵碳水化合物产生大量乳酸的细菌的统称,广泛存在于人体的肠道中,通过发酵产生的有机酸、特殊酶系、酸菌素等物质具有特殊生理功能。大量研究资料表明,乳酸菌能促进动物生长,调节胃肠道正常菌群、维持微生态平衡,从而改善胃肠道功能、提高食物消化率和生物效价、降低血清胆固醇、控制内毒素、抑制肠道内腐败菌生长、提高机体免疫力等。戊糖片球菌属于乳酸菌的一种,其益生功能也受到相当的关注。
乳酸菌的益生功能与其产酸能力密切相关,特别是产乳酸、乙酸等短链脂肪酸的能力直接影响其功能的发挥。产生的乳酸等有机酸能显著降低环境pH值和Eh(氧化还原电位)值,使肠内处于酸性环境,对于致病菌等有拮抗作用。最新研究表明,乳酸是一种存在于细胞与器官间的重要中间代谢物,在生物体的代谢网络中扮演着重要角色,它能通过调节丙酮酸的代谢流向,调节机体内NADH/NAD+的比值,调节机体的氧化还原和呼吸状态,调控全身能量代谢的平衡,在细胞器之间和细胞之间架起糖酵解和氧化磷酸化的桥梁,还通过各种细胞和组织中表达的受体发挥多功能信号分子的作用,在代谢调节、疾病预测、癌症治疗等方面发挥着越来越重要的作用(万娟,et al."乳酸作为信号分子作用的研究进展.";李媛,et al."乳酸在糖尿病认知功能障碍的作用及中医药研究进展.")。
现如今,对肠道中微生物的开发和研究重视程度越来越高,也获得了很多具有多种功能的微生物,对微生物的开发和应用作出了显著的贡献。中华蟾蜍是我国传统药用动物,分布广泛,资源丰富,研究发现蟾蜍体表和肠道都有丰富的乳酸菌,因此,蟾蜍肠道作为一种微生物资源地,具有广阔的开发出新的且具备有益特征的乳酸菌株的前景。
发明内容
本发明的目的是提供一株产乳酸能力较强的戊糖片球菌及其应用,开发了一种新的戊糖片球菌TE0307,其产乳酸能力强,对多种常见的抗生素敏感,对酸、胆盐、人工胃液、人工肠液有较好的耐受能力,对人结肠癌细胞HT-29有较强黏附能力,对多种人畜肠道病原菌均具有较强的抑制能力,具有较好的体外降血糖、降胆固醇和抗氧化能力,在功能性食品领域加工中具有显著的应用价值。
为了达到上述目的,本发明提供了一种戊糖片球菌TE0307,该戊糖片球菌为Pediococcuspentosaceus TE0307,该菌已于2022年12月14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,中国科学院微生物研究所,保藏编号为CGMCC No.26186。
本发明提供的戊糖片球菌TE0307可用于抑制大肠埃希氏菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、铜绿假单胞菌、单增李斯特菌、溶血性葡萄球菌、多杀性巴氏杆菌或/和小肠结肠炎耶尔氏菌。
本发明还提供了一种由戊糖片球菌TE0307所制备的生防菌剂,该生防菌剂可用于大肠埃希氏菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、铜绿假单胞菌、单增李斯特菌、溶血性葡萄球菌、多杀性巴氏杆菌或/和小肠结肠炎耶尔氏菌的防治。
本发明还提供了一种由戊糖片球菌TE0307所制备的生物制剂。
本发明提供的戊糖片球菌TE0307可应用于功能性食品领域中,尤其可用于制备成具有降低血液血糖水平或降低胆固醇的功能性食品等。
本发明提供的戊糖片球菌TE0307及其应用,具有以下优点:
本发明提供的戊糖片球菌TE0307分离筛选自自然保护区中华蟾蜍肠道,在MRS琼脂培养基上生长良好,对人肠道模拟环境适应性强,细胞黏附能力强,丰富了乳酸菌的资源库。
本发明提供的戊糖片球菌TE0307产乳酸能力强,抗氧化能力强,能产多种短链脂肪酸,具有较好的体外降血糖、降胆固醇能力。
本发明提供的戊糖片球菌TE0307对大肠埃希氏菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、单增李斯特菌、溶血性葡萄球菌、多杀性巴氏杆菌、小肠结肠炎耶尔氏菌等常见病原细菌抑菌能力较强,应用范围广,潜力大。
附图说明
图1为本发明中的菌株TE0307的系统发育关系图。
图2为本发明中的戊糖片球菌TE0307在MRS琼脂培养基上的菌落形态图。
图3为本发明中的戊糖片球菌TE0307在MRS琼脂培养基上的革兰氏染色结果图。
图4为本发明中戊糖片球菌TE0307菌株产乳酸能力测定标准曲线图。
图5为本发明中戊糖片球菌TE0307菌株在血平板上的培养结果。
图6为本发明中戊糖片球菌TE0307菌株发酵液短链脂肪酸GC-MS测定总离子流色谱图。
图7为本发明中戊糖片球菌TE0307菌株发酵液DPPH自由基清除率标准曲线图。
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。
说明:实施例中所用到的方法,未见特殊说明的均为常规方法,未见特殊说明的试剂均为常规试剂。
本实施例中用到的标准菌株LGG是鼠李糖乳杆菌Lactobacillus rhamnosus ATCC53103。
实验例1戊糖片球菌的分离和鉴定
1.1材料准备
中华蟾蜍采自四川自然保护区;
16s rRNA通用测序引物‘27F和1492R’由生工生物工程(上海)有限公司合成,序列如下(5’→3’):
27F(SEQ ID NO.1):AGAGTTTGATCCTGGCTCAG
1492R(SEQ ID NO.2):GGTTACCTTGTTACGACTT
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。按上述配比称取各组分,加入蒸馏水或去离子水1L,分装于锥形瓶,于121℃高压条件灭菌15min即得。
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。按上述配比称取各组分,加入蒸馏水或去离子水1L,分装于锥形瓶,于121℃高压条件灭菌15min即得。
1.2菌株的筛选分离
将在无菌条件下取中华蟾蜍肠道,挤出肠道内容物少许,接种于50mL MRS肉汤中,充分振荡混匀,置于37℃恒温摇床培养24h。吸取培养液1mL,采用10倍梯度稀释,分别取20μL,涂布接种于含碳酸钙的MRS琼脂培养基上(碳酸钙2g/100mL,在倒平板前加入混匀),37℃培养24h后,挑取具有较大溶钙圈的单菌落,同样方法连续分离纯化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)的灭菌甘油,于-80℃超低温冰箱中冻存备用。
取冻存菌种20μL,接种于MRS液体培养基进行增殖培养,采用天根细菌基因组DNA提取试剂盒提取菌株DNA,16S rRNA扩增采用菌落PCR技术完成,PCR产物测序由生工生物工程(上海)有限公司完成。将16S rRNA测序结果与GenBank序列进行NCBIBLAST比对,有6株被鉴定为戊糖片球菌(Pediococcuspentosaceus),编号分别为TS0202、TE0302、TE0307、TE3704、TE3724、TE3904。其中TE0307菌株的16SrRNA序列如SEQ ID NO.3所示,将序列进行NCBIBLAST比对,与标准菌株Pediococcuspentosaceus ATCC25745相似率达99.70%,菌株TE0307与其它菌株的系统发育关系如图1所示。将该菌于2022年12月14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,中国科学院微生物研究所,保藏编号为CGMCC No.26186。
将戊糖片球菌TE0307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,戊糖片球菌TE0307在MRS琼脂培养基上的菌落形态如图2所示,可见,该菌株生长良好,菌落形态乳白色、圆形凸起、边缘平整、表面光滑。采用试剂盒法对戊糖片球菌TE0307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,其中,戊糖片球菌TE0307在MRS琼脂培养基上的革兰氏染色结果如图3所示,可知,镜检可见菌体呈紫色,符合革兰氏阳性菌的染色特征。
实验例2戊糖片球菌TE0307的产乳酸能力测定
乳酸具有重要的生理功能,能调节胃肠道正常菌群,从而改善胃肠道功能、提高食物消化率和生物效价、降低血清胆固醇、控制内毒素、抑制肠道内腐败菌生长、提高机体免疫力等。乳酸在乳酸脱氢酶的作用下生成丙酮酸,同时使NAD+还原生成NADH和H+,H+传递给PMS生成的PMSH2还原MTT生成紫色物质,在570nm处有特征吸收峰,据此可以定量测定乳酸含量。
2.1材料准备
仪器设备:天平、研钵/匀浆器、离心机、可见分光光度计、微量玻璃比色皿、恒温水浴锅、乙醇和蒸馏水。
试剂及溶液配制:采用乳酸含量测定试剂盒(北京索莱宝科技有限公司),各试剂如表1所示。试剂二:液体置于试剂瓶内EP管中。临用前按试剂二(V):蒸馏水(V)=10μL:450μL的比例配制试剂二溶液,现用现配;试剂四:临用前每瓶加入3mL蒸馏水混匀,可分装后-20℃保存,避免反复冻融;标准品:临用前加入1.04mL蒸馏水配成100μmol/mL的标准溶液。
表1试剂名称、规格和保存条件
2.2标准曲线的绘制
仪器准备:分光光度计预热30min以上,波长调至570nm,分光光度计用乙醇调零。
标准液的稀释:将100μmol/mL的标准溶液用蒸馏水稀释为2.5、1.25、0.625、0.3125、0.15625、0.078μmol/mL的标准溶液待测。各试剂使用如表2所示。
表2测定管及试剂用量
标准曲线的绘制:以各标准溶液浓度为X轴,以其对应的吸光值(ΔA标准)为Y轴,绘制标准曲线,得到标准方程:Y=KX+b,将ΔA测定带入公式中得到X(μmol/mL),得出,戊糖片球菌TE0307菌株产乳酸能力测定标准曲线如图4所示。
2.3发酵液乳酸含量测定
将实验例1中鉴定保存的6株戊糖片球菌TS0202、TE0302、TE0307、TE3704、TE3724、TE3904菌种活化培养后,接种于MRS肉汤培养基,37℃培养24h,4℃、12000g离心10min后取上清液保存备用。取100μL发酵上清液,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。
其中,乳酸含量的计算公式如下:
乳酸含量(μmol/mL)=X×(V上清+V提取液二)÷[V液体×V上清÷(V提取液一+V液体)]。
式中的V上清:加入的上清液体积,0.8mL;V提取液二:加入提取液二的体积,0.15mL;V提取液一:加入的提取液一,1mL;V液体:液体样本体积,0.1mL。
戊糖片球菌TE0307发酵液乳酸含量见测定结果如表3所示,可知,所测的六株戊糖片球菌发酵液中,乳酸含量最低的为TE3904,浓度为12.30±0.64μmol/mL,而TE0307的产乳酸能力最强,发酵液中乳酸浓度达到82.45±5.25μmol/mL,乳酸含量为TE3904菌株的6.7倍,结果表明戊糖片球菌TE0307作为一种乳酸菌具有很好的开发利用价值。
表3戊糖片球菌TE0307发酵液乳酸含量(μmol/mL)
实验例3戊糖片球菌TE0307的溶血性和抗生素抗性测定
采用纸片琼脂扩散法测试菌株对常见抗生素的敏感性。将戊糖片球菌TE0307菌株活化培养,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,室温放置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。戊糖片球菌TE0307对6种抗生素的敏感性测定结果见下表4,可知,戊糖片球菌TE0307对测试的四环素、氨苄西林、头孢曲松、克林霉素、克拉霉素和氯霉素6种抗生素均表现为敏感(S)。
表4戊糖片球菌TE0307对6种抗生素的敏感性
有的细菌在生长过程中,可能产生溶血素,使红细胞破裂溶解,影响人体健康。在血平板上生长时,菌落周围可观察到透明或半透明的溶血环,许多细菌的致病性都与溶血特性相关。由于不同细菌产生的溶血素不同,溶血能力也不相同,在血平板上的溶血现象也不相同,因此,溶血试验常用来进行细菌的安全鉴定。将TE0307待测菌株使用接种环在MRS琼脂平板划线后,挑选单菌落,在血平板培养基上划线,培养24h后,观察溶血情况。溶血情况主要分为α溶血(菌落周围出现草绿色的溶血环)、β溶血(菌落周围出现清晰透明的溶血环)、γ溶血(菌落周围无溶血环),三次重复。戊糖片球菌TE0307菌株在血平板上的培养结果如图5所示,可见,在菌落周围没有出现溶血现象,表明其具备安全性。
实验例4戊糖片球菌TE0307对人体肠道环境的适应能力评估
4.1耐酸性和耐胆盐性评估:
将待测菌株戊糖片球菌TE0307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。使用盐酸和猪胆盐分别配制酸度(pH=3.0)及胆盐浓度(0.3%)的MRS肉汤培养基。将1mL待测菌株植物乳杆菌GZ2406菌液接种于pH=3.0的肉汤培养基中,37℃培养18h。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18h。观察培养后MRS平板表面是否有菌落生长。同样,将1mL待测菌株戊糖片球菌TE0307菌液接种于胆盐浓度为0.3%的MRS肉汤培养基,37℃培养18h后,涂平板,37℃培养24h,查看菌落生长情况。
根据耐酸性试验结果可知,戊糖片球菌TE0307在pH=3.0的MRS肉汤培养基中处理18h后仍可在MRS琼脂平板上长出正常菌落;耐胆盐性试验结果表明,戊糖片球菌TE0307在胆盐浓度为0.3%MRS肉汤培养基中耐受18h后仍可在MRS琼脂平板上长出正常菌落;说明戊糖片球菌TE0307具有较强的耐酸和耐胆盐特性,对人和动物的肠道环境有较强的适应能力。
4.2细胞粘附性试验:
将戊糖片球菌TE0307复苏后接种于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HT-29,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。并用标准菌株LGG作为对照,选择的标准菌株LGG是鼠李糖乳杆菌Lactobacillus rhamnosus ATCC 53103。
粘附率(%)=结束期乳酸菌数目/初始乳酸菌接种数目×100%
戊糖片球菌TE0307对人结肠癌细胞HT-29的粘附率结果见下表5。可知,戊糖片球菌TE0307对人结肠癌细胞HT-29的粘附率为78.07±8.54%,高于标准菌株LGG的细胞粘附性。
粘附率(%)=结束期乳酸菌数目/初始乳酸菌接种数目×100%
高黏附性的乳酸菌能够黏附于肠黏液层及上皮细胞,不易随着肠内容物流动被清除,有利于自身菌群的长期定植。同时,黏附的乳酸菌能够促进黏液层中黏蛋白的分泌,增加黏液层厚度,稳固黏液屏障功能。此外,乳酸菌能够通过竞争、排斥等作用,与致病菌竞争黏附位点,维持肠道菌群平衡。因此,乳酸菌的黏附性是评价其作为益生菌的重要标准。对肠上皮细胞的黏附特性评价方法通常采用易于体外培养的人结肠癌细胞HT-29作为细胞模型。戊糖片球菌TE0307对人结肠癌细胞HT-29的粘附率测定结果见下表5,可知,戊糖片球菌TE0307对人结肠癌细胞HT-29的粘附率高,为78.07±8.54%,高于标准菌株LGG的细胞粘附性。
表5戊糖片球菌TE0307对人结肠癌细胞HT-29的粘附率
4.3模拟胃液、肠液耐受实验:
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。将戊糖片球菌TE0307菌株复苏并活化,取1mL浓度为1×108CFU/mL菌液加人9mL模拟人工胃液中,并进行10倍梯度稀释,吸取20μL涂平板计活菌数,作为耐受人工胃液的初始活菌值;接菌的模拟胃液于37℃培养3h后,再次涂平板计活菌数,作为耐受人工胃液的结束活菌值。同理,将1mL浓度为1×108CFU/mL戊糖片球菌TE0307发酵液加入9mL模拟肠液中,并进行活菌计数,37℃培养6h后再次计活菌数,计算存活率。存活率=结束时活菌数/初始活菌数*100%。
戊糖片球菌TE0307菌株对于人工模拟胃、肠液的耐受能力见下表6所示,可知,戊糖片球菌TE0307菌株对于人工模拟胃、肠液有较好的耐受能力,在人工胃液中3h后存活率为75.76±4.43%,在人工肠液6h后存活率为66.67±2.29%。
表6戊糖片球菌TE0307菌株对于人工模拟胃、肠液的耐受能力(%)
实验例5戊糖片球菌TE0307发酵液短链脂肪酸含量测定
通常把碳原子数小于6的脂肪酸称为短链脂肪酸(short-chain fatty acids,SCFAs)。短链脂肪酸包括甲酸,乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸,被后肠迅速吸收后,既储存了能量又降低了渗透压,并且短链脂肪酸对于维持大肠的正常功能和结肠上皮细胞的形态和功能具有重要作用。短链脂肪酸是肠道菌群的重要效应因子,对人体健康非常重要,是益生菌评价的重要指标之一。
发酵液的制备:将戊糖片球菌TE0307保存菌株活化培养24h后,调整浓度为1×108CFU/mL,吸取4μL菌液加入到4mL肉汤培养基中,37℃培养24h,离心过滤后,取上清液保存备用。
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-QP2010 Plus),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/z 33-500。取发酵液4mL,加入浓度为200μg/mL2-乙基丁酸内标液10ul,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℃。5种短链脂肪酸(乙酸、正丁酸、异丁酸、异戊酸、异己酸)的浓度采用内标法计算。
戊糖片球菌TE0307菌株发酵液短链脂肪酸GC-MS测定总离子流色谱图如图6所示,其发酵液短链脂肪酸含量检测结果见下表7,可知,戊糖片球菌TE0307发酵液中乙酸含量为4.359μg/mL,还含有异丁酸、异戊酸和2-甲基丁酸三种三种短链脂肪酸。
表7戊糖片球菌TE0307发酵液短链脂肪酸含量(μg/mL)
实验例6戊糖片球菌TE0307菌株的人体益生性能评估
6.1戊糖片球菌TE0307菌株对常见肠道致病菌的抑制能力:
将大肠埃希氏菌(Escherichia coli CMCCB 44102)、金黄色葡萄球菌(Staphylococcus aureus CMCCB 50094)、鼠伤寒沙门氏菌(Salmonella typhimuriumATCC14028)、铜绿假单胞菌(Pseudomonas aeruginosa CMCCB10104)、空肠弯曲杆菌(CampylobacterjejuniATCC 33291)、大肠弯曲杆菌(Campylobacter coliATCC 43478)、单增李斯特菌、溶血性葡萄球菌、多杀性巴氏杆菌和小肠结肠炎耶尔氏菌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。
戊糖片球菌TE0307的抑菌活性评估测定结果见下表8所示,可知,戊糖片球菌TE0307菌株的发酵液对大肠埃希氏菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、铜绿假单胞菌、单增李斯特菌、溶血性葡萄球菌、多杀性巴氏杆菌、小肠结肠炎耶尔氏菌等致病菌的生长具有较强的抑制活性,优于LGG标准菌株的抑菌效果,由戊糖片球菌TE0307制备的生防菌剂可用于上述8种致病菌的防治。
表8戊糖片球菌TE0307菌株的抑菌活性评估(直径:mm)
6.2戊糖片球菌TE0307对α-葡萄糖苷酶的抑制作用:
α-葡萄糖苷酶参与碳水化合物的分解利用,研究表明抑制α-葡萄糖苷酶的活性可以降低人体血液中的血糖水平。通过将戊糖片球菌TE0307保存菌种复苏培养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)备用;取上清液25μL,加入PBS缓冲液(pH=6.8)50μL、20mmol/L的PNPG溶液50μL,37℃水浴10min;加入20U/mLα-葡萄糖苷酶溶液30μL,37℃继续反应10min;加入50μL的1mol/LNa2CO3溶液终止反应;在96孔板(365μL)中进行,每组设定3个重复;在酶标仪405nm处测定吸光值,计算得到α-葡萄糖苷酶抑制率(%)。计算公式为:
其中,A组为α-葡萄糖苷酶;B组为PBS缓冲液;C为待测样品组,含α-葡萄糖苷酶和待测样品;D组仅含待测样品。
戊糖片球菌TE0307发酵液对α-葡萄糖苷酶的抑制率检测结果见下表9,可知,戊糖片球菌TE0307发酵上清液具有抑制α-葡萄糖苷酶活性的作用,抑制率为10.74%,与标准菌株鼠李糖乳杆菌LGG的抑制活性相当,表明用戊糖片球菌TE0307制备的发酵液可能具有降低动物和人血液血糖水平的潜力,可以作为一种生物制剂,或制备成功能性食品,用于降低血液血糖水平,在功能性食品领域中具有潜在的应用价值。
表9戊糖片球菌TE0307发酵液对α-葡萄糖苷酶的抑制率(%)
6.3戊糖片球菌TE0307对胆固醇的降解作用:
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XX mL、6mol/LNaOH溶液12mL;
接种培养:将戊糖片球菌TE0307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。
胆固醇标准曲线的绘制:
混合酸配制:浓硫酸和冰乙酸按1:1比例混合摇匀;
邻苯二甲醛溶液(1mg/mL):25mL无水乙醇与25mg邻苯二甲醛混溶至棕色容量瓶中,冰箱保存;
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;
按下表10进行加样,共6种配比,震荡摇匀后静置30min。在550nm波长下测定吸光度(OD值),以胆固醇浓度为横坐标,吸光度为纵坐标,绘制标准曲线;
表10标准曲线绘制中各试剂的用量(mL)
胆固醇含量测定:分别取菌悬液和空白对照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;
戊糖片球菌TE0307对胆固醇的降解能力见下表11,可知,戊糖片球菌TE0307对胆固醇的降解率为41.26%,优于标准菌株鼠李糖乳杆菌-LGG的21.25%,说明戊糖片球菌TE0307在动物和人体内具有降低胆固醇的潜力,可以作为一种生物制剂,或制备成功能性食品,用于降低胆固醇,在功能性食品领域中具有潜在的应用价值。
表11戊糖片球菌TE0307对胆固醇的降解能力
6.4戊糖片球菌TE0307发酵液的抗氧化能力:
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。
6.4.1发酵液的制备:
将戊糖片球菌TE0307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。
6.4.2试剂与仪器:
总抗氧化能力(T-AOC)检测试剂盒(A015-1-2)、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。
6.4.3总抗氧化能力测定:
总抗氧化能力(Total antioxidant capacity,T-AOC)是指各种抗氧化物质构成的总抗氧化水平。研究抗氧化可以有效克服其所带来的危害,所以抗氧化被保健品、化妆品企业列为主要的研发方向之一,也是市场最重要的功能性诉求之一。许多抗氧化物质能使Fe3+还原成Fe2+,后者可与菲啉类物质形成稳固的络合物,通过比色可测出其抗氧化能力的高低。使用波长520nm,1cm光径,双蒸水调零,测定吸光度值。具体步骤按照总抗氧化能力(T-AOC)检测试剂盒说明书进行。戊糖片球菌TE0307发酵液的抗氧化能力测定结果见下表12,可知,戊糖片球菌TE0307发酵液的总抗氧化能力为104.63±2.53U/mL,明显高于对照菌株TE0302,说明TE0307发酵液的总抗氧化能力较强。
6.4.4DPPH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,得到DPPH自由基清除率标准曲线如图7所示。
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。戊糖片球菌TE0307发酵液的DPPH自由基清除能力测定结果见下表12,可知,戊糖片球菌TE0307发酵液的DPPH自由基清除能力701.62±7.16μgTrolox/mL,明显高于对照菌株TE0302,表明TE0307发酵液对DPPH自由基清除能力较强。
6.4.5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系统,产生超氧阴离子自由基,加入电子传递物质及Gress氏显色剂,使反应体系呈现紫红色,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
在反应系统中,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。戊糖片球菌TE0307发酵液的抗超氧阴离子能力测定结果见下表12,可知,戊糖片球菌TE0307发酵液对超氧阴离子自由基的抑制能力为553.37±8.27(U/L),明显高于对照菌株TE0302,表明TE0307发酵液对超氧阴离子自由基的抑制能力较强。
表12戊糖片球菌TE0307发酵液的抗氧化能力
综上可知,TE0307发酵液的抗氧化能力和自由基清除能力较强,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。
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 (7)
1.一种戊糖片球菌TE0307,其特征在于,该菌已于2022年12月14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,中国科学院微生物研究所,保藏编号为CGMCCNo.26186。
2.如权利要求1所述的戊糖片球菌TE0307在抑制大肠埃希氏菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、铜绿假单胞菌、单增李斯特菌、溶血性葡萄球菌、多杀性巴氏杆菌或/和小肠结肠炎耶尔氏菌中的应用。
3.一种由权利要求1所述戊糖片球菌TE0307所制备的生防菌剂。
4.如权利要求3所述的生防菌剂在大肠埃希氏菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、铜绿假单胞菌、单增李斯特菌、溶血性葡萄球菌、多杀性巴氏杆菌或/和小肠结肠炎耶尔氏菌防治中的应用。
5.一种由权利要求1所述戊糖片球菌TE0307所制备的生物制剂。
6.如权利要求1所述的戊糖片球菌TE0307在功能性食品领域中的应用。
7.根据权利要求6所述的应用,其特征在于,该应用包含制备成具有降低血液血糖水平或降低胆固醇的功能性食品。
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CN115747111A (zh) * | 2022-11-25 | 2023-03-07 | 四川大学 | 一株戊糖片球菌及其应用 |
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CN117586909A (zh) * | 2023-10-27 | 2024-02-23 | 青岛农业大学 | 一株戊糖片球菌lwq1及其应用 |
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CN115747111A (zh) * | 2022-11-25 | 2023-03-07 | 四川大学 | 一株戊糖片球菌及其应用 |
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CN117586909A (zh) * | 2023-10-27 | 2024-02-23 | 青岛农业大学 | 一株戊糖片球菌lwq1及其应用 |
CN117586909B (zh) * | 2023-10-27 | 2024-06-11 | 青岛农业大学 | 一株戊糖片球菌lwq1及其应用 |
CN117264850A (zh) * | 2023-11-09 | 2023-12-22 | 潍坊君薇生物科技有限责任公司 | 一种具有辅助治疗阴道炎和增强免疫力功能的戊糖片球菌sw006及其应用 |
CN117264850B (zh) * | 2023-11-09 | 2024-05-14 | 潍坊君薇生物科技有限责任公司 | 一种具有辅助治疗阴道炎和增强免疫力功能的戊糖片球菌sw006及其应用 |
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