CN114350538B - 一种副干酪乳杆菌及其应用 - Google Patents
一种副干酪乳杆菌及其应用 Download PDFInfo
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Abstract
本发明提供了一种副干酪乳杆菌及其应用,所述副干酪乳杆菌为副干酪乳杆菌(Lactobacillusparacasei)L‑113,保藏于中国微生物菌种保藏管理委员会普通微生物中心,其保藏编号为CGMCCNo.23525。本发明的副干酪乳杆菌L‑113具有高效发酵产丁酸和耐酸性能,能够增强细胞免疫、体液免疫和NK细胞活性,具有显著增强免疫的功能。
Description
技术领域
本发明属于生物保健技术领域,尤其涉及一种副干酪乳杆菌及其应用。
背景技术
丁酸又名酪酸,是一种短链脂肪酸,它是人肠道上皮细胞的主要营养物质,人体内超过95%的丁酸在结肠内产生和吸收,一定水平的丁酸能够使结肠细胞保持稳定,从而可防止或抑制癌变,调节肠道菌群失调和治疗肠易激综合征、抗生素相关性肠炎、急慢性腹泻等疾病。
副干酪乳杆菌是一种有益人体健康的益生菌,从不同的副干酪乳杆菌菌株具有不同的功效。比如CN113355252A公开副干酪乳杆菌WG-7可有效地调节肠道菌群,促进机体微生态正向平衡,CN112534043A公开副干酪乳杆菌(LactobacillusParacasei)KBL382、副干酪乳杆菌KBL384或副干酪乳杆菌KBL385抑制肠炎引起的体重损失和结肠长度降低,从而表现出对肠炎的治疗作用,CN110643532B公开副干酪乳杆菌F3能够降解调和油、花生油和芝麻油,对芝麻油的降解效果最好,能够作为食品添加剂或者直接制成食品、保健品等。近年来,益生菌的研究由之前的针对肠道发展到针对人体微生态、免疫、脑肠轴、慢性疾病、代谢性疾病等前沿领域,在最近的保健食品用益生菌备案中,国内菌种资源备案的菌株也相对较少,需要拓宽该系列益生菌种类是现阶段急需解决的技术问题。
发明内容
有鉴于此,本发明的目的在于提供一种副干酪乳杆菌L-113,该菌具有发酵产丁酸能力,能够增强细胞免疫(包括脾淋巴细胞转化和迟发型变态反应)和NK细胞活性,具有增强免疫的功能。
为了实现上述发明目的,本发明提供了以下技术方案:
本发明提供了一种副干酪乳杆菌,所述副干酪乳杆菌为副干酪乳杆菌L-113,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.23525。
本发明提供了所述的副干酪乳杆菌在免疫调节中的应用。
优选的,所述应用为在制备免疫调节的药物中的应用。
优选的,所述药物包括药学上可接受的佐剂,所述药物和其药学上可接受的佐剂根据给药途径不同制备成散剂、片剂、颗粒剂、胶囊剂、溶液剂、乳剂或混悬剂。
优选的,所述药物中含有所述副干酪乳杆菌的活菌体和/或死菌体作为活性成分。
进一步优选的,所述药物中含有所述副干酪乳杆菌的活菌体,或活菌体和死菌体的混合菌体作为活性成分。
优选的,所述混合菌体中活菌体的数量高于死菌体的数量。
优选的,以每克或每毫升所述药物为基准,所述副干酪乳杆菌添加量为106-1011CFU/g或CFU/mL。
进一步优选的,以每克或每毫升所述药物为基准,所述副干酪乳杆菌添加量为108-1010CFU/g或CFU/mL。
与现有技术相比,本发明具有如下有益效果:
本发明首次获得副干酪乳杆菌L-113,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.23525。
本发明副干酪乳杆菌L-113具有高效发酵产丁酸和耐酸性能。
本发明副干酪乳杆菌L-113干预后,小鼠脾淋巴细胞转化、迟发型变态反应、抗体生成细胞检测、NK细胞活性结果为阳性,表明该菌株在细胞免疫功能、体液免疫功能、NK细胞活性三个方面结果均为阳性,可判定该菌株具有显著增强免疫力的功能。
附图说明
图1PCR筛选丁酸激酶(BUK)基因片段;
图2L-113发酵的丁酸产量结果;
图3小鼠脾淋巴细胞转化结果;
图4小鼠迟发型变态反应实验结果;
图5抗体生成细胞检测结果;
图6血清溶血素测定结果;
图7NK细胞活性测定结果。
生物保藏说明
副干酪乳杆菌L-113,拉丁文为Lactobacillusparacasei,保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),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.23525。
具体实施方式
本发明提供了一种副干酪乳杆菌,所述副干酪乳杆菌为副干酪乳杆菌L-113,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.23525。
在本发明中,所述副干酪乳杆菌具有高效产丁酸和耐酸性能。所述副干酪乳杆菌的筛选方法可选为筛选体外PCR扩增具有丁酸激酶(BUK)基因片段的菌株。
本发明提供所述副干酪乳杆菌在免疫调节中的应用。本发明中所述副干酪乳杆菌可发酵产丁酸。本发明中所述副干酪乳杆菌能够增强细胞免疫和NK细胞活性,所述细胞免疫包括脾淋巴细胞转化和迟发型变态反应。
在本发明中,所述的副干酪乳杆菌在制备免疫调节的药物中的应用。
本发明中药物优选包括药学上可接受的佐剂,所述药物和其药学上可接受的佐剂根据给药途径不同制备成散剂、片剂、颗粒剂、胶囊剂、溶液剂、乳剂或混悬剂。本发明中所述佐剂既可以是各种制剂中常规使用的,如:但不仅限于等渗剂、缓冲液、矫味剂、赋形剂、填充剂、粘合剂、崩解剂和润滑剂等;也可以是为了与所述物质相适应而选择使用的,如:乳化剂、增溶剂、抑菌剂、止痛剂和抗氧剂等,这类佐剂能有效提高组合物所含化合物的稳定性和溶解性或改变化合物的释放速率和吸收速率等,从而改善各种化合物在生物体内的代谢,进而增强组合物的给药效果。此外,还可以为实现特定的给药目的或方式,如:缓释给药、控释给药和脉冲给药等,而使用的佐剂,如:但不仅限于明胶、白蛋白、壳聚糖、聚醚和聚酯类高分子材料,如:但不仅限于聚乙二醇、聚氨酯、聚碳酸酯及其共聚物等。所称的“有利于给药”的主要表现有:但不仅限于提高治疗效果、提高生物利用度、降低毒副作用和提高患者顺应性等。
在水溶液注射剂中,佐剂一般包括等渗剂和缓冲液,以及必要的乳化剂(如:Tweeen-80、Pluronic和Poloxamer等)、增溶剂和抑菌剂等。此外,还包括含有药学上可接受的其它药用佐剂,如:抗氧剂、pH调节剂和止痛剂等。用于制取口服液体制剂的佐剂一般包括溶剂,以及必要的矫味剂、抑菌剂、乳化剂和着色剂等。用于制取片剂的佐剂一般包括填充剂(如:淀粉、糖粉、乳糖、可压性淀粉、微晶纤维素、硫酸钙、磷酸氢钙和甘露醇等)、粘合剂(如:乙醇、淀粉浆、羧甲基纤维素钠、羟丙基纤维素、甲基纤维素、乙基纤维素、羟丙基甲基纤维素、明胶溶液、蔗糖溶液和聚乙烯吡咯烷酮的水溶液或醇溶液等)、崩解剂(如:干淀粉、羧甲基淀粉钠、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮和交联羧甲基纤维素钠)和润滑剂(如:硬脂酸镁、微粉硅胶、滑石粉、氢化植物油、聚乙二醇4000、聚乙二醇6000和月桂醇硫酸镁等)等。用于制取乳剂的佐剂一般为水、油(如:脂肪酸)、乳化剂,以及必要的防腐剂和矫味剂等。用于制取颗粒剂的佐剂与片剂类似,但造粒过程不同。根据需要,将制得的颗粒剂与助流剂混合后装入胶囊即得胶囊剂。
在本发明中,所述药物中优选含有所述副干酪乳杆菌的活菌体和/或死菌体作为活性成分,更优选含有所述副干酪乳杆菌的活菌体,或活菌体和死菌体的混合菌体作为活性成分。本发明中所述混合菌体中活菌体的数量高于死菌体的数量。
在本发明中,以每克或每毫升所述药物为基准,所述副干酪乳杆菌添加量优选为为106-1011CFU/g或CFU/mL,更优选为108-1010CFU/g或CFU/mL。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1体外筛选
1.乳酸菌菌种活化
在无菌操作条件下,取来源于中国农业大学营养与健康系的40种乳酸菌冻干粉菌株,分别接种于10mL脱脂乳中,充分振荡后,37℃静置培养,凝固后以5%的接种量于脱脂乳中继续培养传代。传代3次后,分别取第3代菌液4℃保存备用。
2.乳杆菌耐酸能力测定
37℃活化40种待测乳酸菌冻干粉菌株12h后,以2%的接种量分别接种于pH 2.0的MRS培养基中。37℃下培养2h,分别在0和2h时进行平板计数。每组试验至少有3个重复。表1结果显示,在pH 2.0的MRS培养基中,培养2h后能够存活的菌有14株;M023-D-13显示出最强的耐酸性,存活性达到96.32%;菌株人工胃液耐受能力从高到低依次为M023-D-13、L-113、LCS、G021-A-10、LR-F、17-14-1、D061-A-3、17-12、D061-A-04、17-9、E30-B-10、17-13、Lduo、植物(F)。
表1菌株耐酸能力
3.菌体DNA提取
采用天根细菌DNA提取试剂盒分别对步骤1获得的40株第3代菌液进行DNA提取,按照内附说明书进行操作,提取后的DNA在-20℃可长期保存使用。
4.PCR扩增
PCR反应体系(25μL):模板DNA 1μL、上下引物各1μL、Taq Mix12.5μL、无菌水9.5μL。PCR反应程序:94℃预变性3min、94℃变性30s、55℃退火30s、72℃延伸1min、72℃保持5min,PCR 30个循环。
表2引物序列
5.琼脂糖凝胶电泳
将扩增后的PCR产物点入琼脂糖凝胶,在缓冲液中220V通电30min,得到的胶体在多功能成像仪中观察是否产生条带。利用PCR的方法筛选菌株的丁酸激酶(BUK)基因片段,结果如图1所示,只有副干酪乳杆菌L-113存在BUK基因片段,表明该菌具有产丁酸的能力。
6.产丁酸含量的测定
副干酪乳杆菌L-113菌株活化完成后,在无菌条件下,按照5%的接种量接种于MRS培养基中厌氧培养24h后,分别利用GC-MS对培养12h、18h、24h发酵液中丁酸含量进行测定,结果如图2所示,在24h丁酸产量最高,可达5.8g/L。
实施例2动物实验
1.菌株培养:在无菌操作条件下,取副干酪乳杆菌L-113冻干粉菌株,接种于10mL脱脂乳中,充分振荡后,37℃静置培养,凝固后以5%的接种量继续培养传代。传代3次后,取第3代菌液4℃保存备用。
2.实验动物分组及处理
购入健康6-8周balb/c雄性小鼠,18-22g,适应性喂养一周后,随机分成3组,空白对照组、L-113低剂量组(1×109cfu/kg)和L-113高剂量组(1×109cfu/kg),每组8只,灌胃45天,结束后戊巴比妥麻醉后解刨。
3.检测指标(方法参考《允许保健食品声称的保健功能目录非营养素补充剂(2020年版)(征求意见稿)》)
①体重
灌胃前后小鼠体重。
②脏器/体重比值测定
③细胞免疫功能测定:小鼠脾淋巴细胞转化实验,迟发型变态反应实验。
L-113对小鼠脾淋巴细胞转化的情况如图3所示,结果表明,L-113低剂量组和高剂量组均可提高脾淋巴细胞转化值,且高剂量组效果显著;对小鼠迟发型变态反应实验结果如图4所示,结果表明,L-113低剂量组和高剂量组的足趾变态反应差值显著高于对照组,且高剂量组效果显著,结果呈现阳性。
④体液免疫功能测定:抗体生成细胞检测,血清溶血素测定。
小鼠的抗体生成细胞检测的情况如图5所示,结果表明,L-113低剂量组和高剂量组均可提高抗体生成,且高剂量组效果显著,结果呈现阳性;小鼠血清溶血素测定实验结果如图6所示,与对照组相比,没有呈现阳性结果的组别。
⑤NK细胞活性测定
小鼠的NK细胞活性测定结果如图7,与对照组相比,L-113各剂量组的NK细胞活性均有明显增加,故判定为阳性。
4.结果判定
有助于增强免疫力功能判定:在细胞免疫功能、体液免疫功能、NK细胞活性三个方面任两个方面结果阳性,可判定该受试样品具有有助于增强免疫力功能作用。
其中细胞免疫功能测定项目中的两个实验结果均为阳性,或任一个实验的两个剂量组结果阳性,可判定细胞免疫功能测定结果阳性。体液免疫功能测定项目中的两个实验结果均为阳性,或任一个实验的两个剂量组结果阳性,可判定体液免疫功能测定结果阳性。NK细胞活性测定实验的一个以上剂量组结果阳性,可判定NK细胞活性结果阳性。
综上所述,副干酪乳杆菌L-113干预后,小鼠细胞免疫(包括脾淋巴细胞转化和迟发型变态反应)、NK细胞活性结果阳性,表明副干酪乳杆菌L-113具有增强小鼠免疫的功能。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.一种副干酪乳杆菌,其特征在于,所述副干酪乳杆菌为副干酪乳杆菌L-113,保藏编号为CGMCCNo.23525。
2.权利要求1所述的副干酪乳杆菌在制备免疫调节药物中的应用。
3.根据权利要求2所述的应用,其特征在于,所述药物包括药学上可接受的佐剂,所述药物和其药学上可接受的佐剂根据给药途径不同制备成散剂、片剂、颗粒剂、胶囊剂、溶液剂、乳剂或混悬剂。
4.根据权利要求2所述的应用,其特征在于,所述药物中含有所述副干酪乳杆菌的活菌体和/或死菌体作为活性成分。
5.根据权利要求4所述的应用,其特征在于,所述药物中含有所述副干酪乳杆菌的活菌体,或活菌体和死菌体的混合菌体作为活性成分。
6.根据权利要求5所述的应用,其特征在于,所述混合菌体中活菌体的数量高于死菌体的数量。
7.根据权利要求2-6任一项所述的应用,其特征在于,以每克或每毫升所述药物为基准,所述副干酪乳杆菌添加量为106-1011CFU/g或CFU/mL。
8.根据权利要求7所述的应用,其特征在于,以每克或每毫升所述药物为基准,所述副干酪乳杆菌添加量为108-1010CFU/g或CFU/mL。
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