CN116590205B - 一种动物双歧杆菌亚种组合菌剂在减肥制剂制备中的应用 - Google Patents
一种动物双歧杆菌亚种组合菌剂在减肥制剂制备中的应用 Download PDFInfo
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Abstract
本发明属于微生物新应用技术领域,具体涉及一种动物双歧杆菌亚种组合菌剂在制备减肥制剂中的应用。主要包括将动物双歧杆菌亚种TY‑S01+U9制备成组合菌剂,应用在减肥制剂中,其可以有效降低体脂率、BMI指数、血脂水平和炎症因子含量,还能够有效促进有益菌的增殖,如双歧杆菌属和布劳特氏菌属,从而有效改善肥胖情况。
Description
技术领域
本发明属于益生菌技术领域,特别涉及一种动物双歧杆菌亚种TY-S01+U9组合菌剂在减肥制剂制备中的应用。
背景技术
肥胖会导致多种疾病,如心血管疾病等。此外,肥胖还会导致脂肪细胞释放炎症因子,如肿瘤坏死因子α(TNF-α)和C反应蛋白(CRP)等,从而引发机体低度炎症,抑制脂肪细胞氧化供能,进一步促进肥胖的发展,对人体健康造成危害。
目前,肥胖的临床管理主要通过以下三个方式——生活方式干预、减肥药物干预和减重手术干预,但这些方式较难坚持或会产生一定副作用,因此有必要开发新型的药物作为现有抗肥胖的直接手段或辅助手段。益生菌因其安全、无副作用等优点,能够通过多种方式改善肥胖症状,如抑制肠道有害菌增殖或缓解炎症等方式。
基于此,挖掘一种可以应用在减肥制剂中的益生菌具有重大意义。
发明内容
针对以上问题,本发明目的在于提供一种益生菌在减肥制剂制备中的应用,该益生菌为动物双歧杆菌亚种TY-S01+U9(保藏编号为CGMCC No.21255和CGMCC No.11560),其可以通过降低体脂率、BMI指数、血脂水平和炎症因子含量以及促进有益菌增殖达到改善肥胖的目的。
为了达到上述目的,本发明可以采用以下技术方案:
本发明一方面提供一种动物双歧杆菌亚种TY-S01+U9在减肥制剂制备中的应用,其中,动物双歧杆菌乳亚种TY-S01的保藏编号为CGMCC No.21255,动物双歧杆菌乳亚种U9的保藏编号为CGMCC No.11560。
保藏菌种信息:
动物双歧杆菌乳亚种TY-S01,保藏机构:中国微生物菌种保藏管理委员会普通微生物中心(CGMCC);地址:北京市朝阳区北辰西路1号院3号;保藏日期:2020年11月27日;保藏编号:CGMCCNo.21255;分类命名:动物双歧杆菌乳亚种Bifidobacterium animalis subsp.lactis。该菌株已经在CN116270758A专利文献中公开。
动物双歧杆菌乳亚种U9,保藏机构:中国微生物菌种保藏管理委员会普通微生物中心(CGMCC);地址:北京市朝阳区北辰西路1号院3号;保藏日期:2015年11月04日,保藏编号为CGMCC NO. 11560;分类命名:动物双歧杆菌乳亚种Bifidobacteriumanimalis subsp.lactis。该菌株已经在CN113583923B专利文献中公开。
本发明用到的对照分离菌株:
动物双歧杆菌乳亚种A6,(Bifidobacterium animalis subsp. Lactis,A6),保藏机构:中国微生物菌种保藏管理委员会普通微生物中心(CGMCC);地址:北京市朝阳区北辰西路1号院3号;保藏日期:2014年06月05日,保藏编号为CGMCC NO. 9273。该菌株已经在CN104224851B专利文献中公开。
在一些具体实施例中,上述应用可以包括以下一种或多种应用组合:
(1)动物双歧杆菌亚种TY-S01+U9在制备降低体脂率的制剂中的应用;
(2)动物双歧杆菌亚种TY-S01+U9在制备降低BMI指数的制剂中的应用;
(3)动物双歧杆菌亚种TY-S01+U9在制备降低血脂水平的制剂中的应用;
(4)动物双歧杆菌亚种TY-S01+U9在制备降低炎症因子含量的制剂中的应用。
需要说明的是,体脂率、BMI指数、血脂以及炎症因子为本领域所已知的术语,不特指某种意思,比如体脂率为人体内脂肪重量在人体总体重中所占的比例;BMI指数是国际上常用的衡量人体胖瘦程度以及是否健康的一个标准,计算公式为:BMI=体重÷身高²;血脂是血浆中的中性脂肪(甘油三酯)和类脂(磷脂、糖脂、固醇、类固醇)的总称;炎症因子是指参与炎症反应的各种细胞因子,主要有肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、白介素-6(IL-6)、转化生长因子-β(TGF-β)或致炎因子CRP等。将动物双歧杆菌亚种TY-S01应用在减肥制剂中,可以有效降低体脂率、BMI指数、血脂水平以及炎症因子含量。
在一些具体实施例中,动物双歧杆菌亚种TY-S01+U9在减肥制剂制备中的应用主要包括:
(1)在制备减少脂肪面积的制剂中的应用或者动物双歧杆菌亚种TY-S01+U9在制备提高肌肉率的制剂中的应用;
(2)在制备降低低密度脂蛋白胆固醇水平和/或高密度脂蛋白胆固醇水平和/或总胆固醇水平和/或甘油三酯水平的制剂的应用;
(3)在制备降低TNF-α含量和/或CRP含量的制剂中的应用。
需要说明的是,动物双歧杆菌亚种TY-S01+U9可以通过减少脂肪面积降低体脂率,降低低密度脂蛋白胆固醇水平和/或高密度脂蛋白胆固醇水平和/或总胆固醇水平和/或甘油三酯水平从而降低血脂水水平,以及通过降低TNF-α含量和/或CRP含量从而降低炎症因子含量。
在一些具体实施例中,动物双歧杆菌亚种TY-S01+U9在减肥制剂制备中的应用主要包括在促进肠道菌群中双歧杆菌属(Bifidobacterium)和/或布劳特氏菌属(Blautia)增殖的制剂中的应用。
需要说明的是,动物双歧杆菌亚种TY-S01+U9可以通过促进与肥胖相关有益菌的增殖,从而管控体重。
在一些具体实施例中,上述应用中,减肥制剂包括药物药剂或食物制剂。需要说明的是,可以将动物双歧杆菌亚种TY-S01+U9制备成食物和药物。另外,食物可药物的剂型可以是固体剂型,比如益生菌片、益生菌粉、益生菌胶囊以及益生菌丸子;也可以是液体剂型,比如益生菌饮料和益生菌搅拌型酸奶等;还可以是膏状剂型,比如凝固型酸奶等。还需要说明的是,将动物双歧杆菌亚种TY-S01+U9制备成制剂需要添加辅料,添加的辅料是根据所需要选择的剂型进行选择的,辅料为本领域所已知的。
本发明相比现有技术的有益效果为:将动物双歧杆菌亚种TY-S01+U9应用在减肥制剂中,可以有效降低体脂率、BMI指数、血脂水平和炎症因子含量,而且可以有效促进有益菌(双歧杆菌属和/或布劳特氏菌属)的增殖,从而有效改善肥胖情况。
附图说明
图1为TY-S01+U9对单纯性肥胖人群炎症因子(血清肿瘤坏死因子α(TNF-α))含量影响。
图2为TY-S01+U9对单纯性肥胖人群炎症因子(血清C反应蛋白(CRP))含量影响。
图3为TY-S01+U9对两组肠道菌群门水平群落组成的影响。
图4为TY-S01+U9对两组肠道菌群属水平群落组成的影响。
图5为TY-S01+U9对两组肠道菌群中双歧杆菌属丰度的影响。
图6为TY-S01+U9对两组肠道菌群中布劳特氏菌属丰度的影响。
具体实施方式
所举实施例是为了更好地对本发明进行说明,但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整,仍属于本发明的保护范围。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
以下将通过实施例对本发明进行详细描述。应当能够理解的是,以下实施例仅用于示例性地进一步详细解释和说明本发明的内容,而不用于限制本发明。
以下实施例中,益生菌TY-S01来源于天友菌种库,其在中国微生物菌种保藏管理委员普通微生物中心(CGMC)保藏,保藏编号:CGMCC No.21255;保藏日期:2020年11月27日,分类命名:动物双歧杆菌亚种TY-S01(Bifidobacterium animalis subsp.lactisTY-S01)。
动物双歧杆菌乳亚种U9(Bifidobacterium animalis subsp. Lactis,U9),于2015年11月04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.11560。
动物双歧杆菌乳亚种A6(Bifidobacterium animalis subsp. Lactis,A6)于2014年06月05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CGMCCNo.9273。
以下实施例中,选择受试者的标准为:年龄在18~45岁之间;成人BMI ≥ 28kg/m²,或总脂肪百分率达到男>25%,女>30%,血脂指标无异常;同意在研究期间饮用受试产品;同意签署知情同意书。排除标准:患有心、肝、肾和造血系统等严重疾病,精神病患者;患有影响体重的疾病,如甲状腺功能减退和库欣综合征;短期内服用与受试功能有关的物品,影响到对结果的判断者;未按规定食用受试样品,无法判定功效或资料不全影响功效或安全性判断者;在过去一个月内参加了另一项临床试验。
以下实施例中,使用校准的身体成分仪(Inbody770)记录BMI、体脂率、内脏脂肪面积、肌肉率等信息,在脐下两个手指处测量腰围,在臀部最宽处测量臀围。
以下实施例中,肠道菌群多样性测序工作由上海美吉生物医药科技有限公司完成,测序利用Illumina.PE300测序平台,对16SrDNA进行高通量测序。
高通量测序的具体流程为:采集受试者的粪便保存至-80℃中,进行16srDNA高通量测序,包括DNA抽提、PCR扩增与产物纯化、PCR产物定量与均一化、构建PE文库以及测序,具体如下:
DNA抽提:完成基因组DNA抽提后,利用1%琼脂糖凝胶电泳检测抽提的基因组DNA;
PCR扩增与产物纯化:使用NanoDrop2000测定DNA浓度和纯度;使用338F (5’-ACTCCTACGGGAGGCAGCAG-3’)和806R (5'-GGACTACHVGGGTWTCTAAT-3’)对16S rRNA基因V3-V4 区进行PCR扩增。将同一样本的PCR产物混合后使用2%琼脂糖凝胶回收PCR产物,利用AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA)进行回收产物纯化,2%琼脂糖凝胶电泳检测,并用Quantus™ Fluorometer (Promega, USA) 对回收产物进行检测、定量。
构建PE文库包括:使用NEXTflex TMRapidDNA-SeqKit(BiooScientific,美国)进行建库:(1)接头链接;(2)使用磁珠筛选去除接头自连片段;(3)利用PCR扩增进行文库模板的富集;(4)磁珠回收PCR产物得到最终的文库;
测序包括:1)DNA片段的一端与引物碱基互补,固定在芯片上;2)以DNA片段为模板,芯片上固定的碱基序列为引物进行PCR合成,在芯片上合成目标待测DNA片段;3)变性、退火后,芯片上DNA片段的另一端随机与附近的另外一个引物互补,也被固定住,形成“桥”;4)PCR扩增,产生DNA簇;5)DNA扩增子线性化成为单链;6)加入改造过的DNA聚合酶和带有4种荧光标记的dNTP,每次循环只合成一个碱基;7)用激光扫描反应板表面,读取每条模板序列第一轮反应所聚合上去的核苷酸种类;8)将“荧光基团”和“终止基团”化学切割,恢复3'端粘性,继续聚合第二个核苷酸;9)统计每轮收集到的荧光信号结果,获知模板DNA片段的序列。
以下实施例中,是基于I-Sanger云平台(https://www.i-sanger.com/)进行生物信息学及数据统计分析,生物信息分析方法包括:Miseq测序得到的PE.reads首先根据overlap关系进行拼接,同时对序列质量进行质控和过滤,区分样本后进行OTU聚类分析和物种分类学分析,基于分类学信息,在各个分类水平上进行群落结构的统计分析。在上述分析的基础上,对多样本的群落组成和系统发育信息进行多元分析和差异显著性检验等一系列深入的统计学和可视化分析。
实施例1益生菌组合对肥胖病症的群体实验
按照上述受试者标准招募200名受试者,随机分成两组,其中安慰剂组50人,试食期间每天饮用一袋麦芽糊精饮料;TY-S01+A6组50人,TY-S01+U9组50人,A6+U9组50人,每天饮用一袋益生菌固体饮料(活菌含量约为5×1010CFU/袋,其中TY-S01、A6、U9菌数根据不同组合分别按照1:1进行配比,其余部分用麦芽糊精补充; 安慰剂组为麦芽糊精)。一共干预12周,干预前和干预后各测量一次与肥胖指数相关的身体参数,采集一次血液和粪便样本,且共有190人完成试验(其中10人因个人原因无法进行干预后采样而退组)。受试者均为单纯性肥胖患者,各人口学和基线指标均无显著差异;受试期间,受试者的餐饮情况无显著差异,具体如下表1所示。
表1 人口基线数据
注:P表示安慰剂组和益生菌组组间差异;BMI、体脂率使用连续变量正态或近似正态分布的用平均数±标准差描述;年龄使用偏态分布,用中位数和四分位间距描述。
实施例2 动物双歧杆菌亚种TY-S01+U9对单纯性肥胖人群身体肥胖相关参数的调节效果
如表2所示,受试者干预12周后,安慰剂组、TY-S01+A6组、A6+U9组BMI和体脂率较干预前无显著变化,下降幅度大于安慰剂组,TY-S01+U9组干预后较干预前显著下降(p< 0.05);干预后安慰剂组和A6+U9组内脏脂肪面积较干预前无显著变化,TY-S01+A6组和TY-S01+U9内脏脂肪面积较干预前显著下降(p<0.05)。表明TY-S01+A6组和TY-S01+U9组有降低肥胖人群体脂的潜力,且TY-S01+U9的效果更好。
表2 干预前后肥胖人群身体肥胖相关参数
注:P指组内干预前后差异,BMI和体脂率属于正态或近似正态分布的连续变量,用平均数±标准差描述;内脏脂肪面积属于偏态分布数据,用中位数和四分位间距描述。
实施例3 动物双歧杆菌亚种TY-S01+U9对单纯性肥胖人群血脂代谢的调节效果
抽取受试者的空腹静脉血检测总胆固醇(TC)、总甘油三酯(TG)、低密度脂蛋白胆固醇(LDL_C)、高密度脂蛋白胆固醇(HDL_C),结果如表3所示,各组的甘油三酯水平在干预前后都没有显著差异;干预12周后,安慰剂组、TY-S01+A6组、A6+U9组TC和LDL_C较干预前无显著变化,TY-S01+U9组TC和LDL_C显著下降(p<0.05);干预后安慰剂组、TY-S01+A6组、TY-S01+U9组的HDL_C较干预前均显著降低(p<0.05)。表明TY-S01+U9有较好降血脂效果。
表3 干预前后人群血脂指标信息
注:P指组内干预前后差异,连续变量正态或近似正态分布的用平均数±标准差描述,偏态分布的用中位数和四分位间距描述。
实施例4 动物双歧杆菌亚种TY-S01+U9对炎症指标的调节效果
肥胖状态下,脂肪组织会分泌炎症因子,会引起全身的慢性炎症。在脂肪组织内,TNF-α是主要由脂肪细胞和基质血管细胞表达的一种炎症因子,能够抑制脂肪酸氧化的基因表达,增加参与胆固醇、脂肪酸再合成基因表达。CRP也是一种炎症标志物,是肥胖相关心血管疾病最相关的危险指标。
本发明实施例中抽取受试者血清,使用高速离心机分离血清(转速3500rpm,时间15min),使用ELISA试剂盒检测血清样本中肿瘤坏死因子α(TNF-α)、C反应蛋白(CRP)的含量。TNF-α含量如图1所示。安慰剂组和U9+A6组干预前后TNF-α无显著差异,TY-S01+U9组TNF-α显著下降;CRP含量如图2所示(左边为安慰剂组,中间为TY-S01+U9组,右边为U9+A6组),三组CRP含量都显著下降。以上结果表明TY-S01+U9组缓解炎症的效果最好。
实施例5 动物双歧杆菌亚种TY-S01+U9对肠道菌群的影响
(1)肠道菌群组成
为得知两组干预在各门水平和属水平上的群落结构组成情况并得知各组中微生物的相对丰度,对受试者肠道菌群门和属水平(丰度>1%)的物质组成进行分析。分析结果如图3~4所示。(其中,V0_A指安慰剂组干预前,V2_A指安慰剂组干预后,V0_B指U9+A6组干预前,V2_B指U9+A6组干预后。V0_D指TY-S01+U9组干预前,V2_D指TY-S01+U9组干预后),肥胖人群肠道菌群优势菌为厚壁菌门(Firmicutes),拟杆菌门(Bacteroidota),放线菌门(Actinobacteriota)和变形菌门(Proteobacteria),肥胖人群肠道菌群优势菌属为拟杆菌属(Bacteroides)、粪杆菌属(Faecalibacterium)、布劳特氏菌(Blautia)等。
(2)有益菌丰度
实验干预后,TY-S01+U9组双歧杆菌属(Bifidobacterium)和布劳特氏菌属(Blautia)丰富显著增加(p<0.05),安慰剂组和U9+A6组双歧杆菌属和布劳特氏菌属丰度干预前后无显著差异(图5~6)。可见TY-S01+U9能够促进肠道有益菌的增殖。
综上可见,益生菌TY-S01+U9的组合物干预能够降低单纯性肥胖人群的体脂和炎症水平,有应用在减脂产品中的潜质。
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 (9)
1.一种动物双歧杆菌乳亚种(Bifidobacterium animalis subsp. Lactis)益生菌组合物,其特征在于,由两种动物双歧杆菌乳亚种菌株TY-S01和U9组成,两株菌活菌含量1:1配比混合制备成菌剂;
动物双歧杆菌乳亚种TY-S01,保藏编号CGMCC No. 21255;
动物双歧杆菌乳亚种U9,保藏编号 CGMCC NO. 11560。
2.由动物双歧杆菌乳亚种TY-S01和动物双歧杆菌乳亚种U9制备的益生菌发酵物,其特征在于,两株菌活菌含量1:1 配比混合制备成益生菌发酵物,
动物双歧杆菌乳亚种TY-S01的保藏编号为CGMCC No. 21255;
动物双歧杆菌乳亚种U9的保藏编号为CGMCC NO. 11560。
3.由权利要求1所述的益生菌组合物或权利要求2所述的益生菌发酵物制备的产品,其特征在于,产品为食品、保健品、药品中的任意一种。
4.如权利要求3所述的产品,其特征在于,产品为益生菌片、益生菌粉、益生菌胶囊或益生菌乳品。
5.如权利要求4所述的产品,其特征在于,所述益生菌乳品为益生菌搅拌型酸奶或凝固型酸奶。
6.如权利要求1所述的益生菌组合物或权利要求2所述的益生菌发酵物在制备减肥制剂中的应用。
7.如权利要求6所述的应用,其特征在于,所述应用为以下的一种或多种,
(1)动物双歧杆菌亚种TY-S01+U9在制备降低体脂率的制剂中的应用;
(2)动物双歧杆菌亚种TY-S01+U9在制备降低BMI指数的制剂中的应用;
(3)动物双歧杆菌亚种TY-S01+U9在制备降低血脂水平的制剂中的应用;
(4)动物双歧杆菌亚种TY-S01+U9在制备降低炎症因子含量的制剂中的应用,所述炎症因子为TNF-α和CRP。
8.如权利要求6所述的应用,其特征在于,所述应用为以下的一种或多种,
(1)动物双歧杆菌亚种TY-S01+U9在制备减少脂肪面积的制剂中的应用;
(2)动物双歧杆菌亚种TY-S01+U9在制备降低低密度脂蛋白胆固醇水平和/或高密度脂蛋白胆固醇水平和/或总胆固醇水平和/或甘油三酯水平的制剂的应用。
9.如权利要求6所述的应用,其特征在于,所述应用为促进肠道菌群中双歧杆菌属和/或布劳特氏菌属增殖的应用。
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