CN114391652B - 一种具有降血脂和调节肠道菌群结构功能的人参不溶性膳食纤维组合物及其制备方法与应用 - Google Patents
一种具有降血脂和调节肠道菌群结构功能的人参不溶性膳食纤维组合物及其制备方法与应用 Download PDFInfo
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- Medicines Containing Plant Substances (AREA)
Abstract
本发明公开了一种具有降血脂和调节肠道菌群结构功能的人参不溶性膳食纤维组合物及其制备方法与应用,属于组合物技术领域。为了提供一种可以降血脂和调节肠道菌群结构的组合物,所述组合物包括人参提取物,是将人参经过水煎煮后,取其药渣再由热稳定α淀粉酶、碱性蛋白酶、淀粉葡萄糖苷酶按照质量比为1:1:(1‑3)处理后获得的人参提取物。本发明提供了人参不溶性膳食纤维组合物在制备降血脂保健品中的应用和在调节高血脂造成的肠道菌群结构紊乱中的应用。
Description
技术领域
本发明属于组合物领域,具体涉及一种具有降血脂和调节肠道菌群结构的人参不溶性膳食纤维组合物及其制备方法与应用。
背景技术
随着人们生活水平的不断提高,高血脂、脂肪肝等由饮食习惯而导致的血脂异常疾病发病率正逐年提高。药物治疗存在的不良反应和副作用,使得人们更加关注健康饮食对高血脂慢性病的治疗和缓解作用,开发具有降血脂功效的保健食品对于现代社会的健康需求具有重要意义。
发明内容
本发明的目的是为了提供一种可以降血脂和调节肠道菌群的组合物。
本发明提供一种人参不溶性膳食纤维组合物,所述组合物包括人参提取物。
进一步地限定,所述组合物还包括黄酮提取物。
进一步地限定,所述组合物是由以下质量分数的原料组成:黄酮提取物质量分数0.1-0.5%和质量分数人参提取物1-10%。
进一步地限定,所述黄酮提取物由山楂黄酮提取物和菊花黄酮提取物组成。
进一步地限定,所述山楂黄酮提取物和菊花黄酮提取物的质量比为1:1。
本发明提供一种上述组合物的制备方法,其特征在于,所述制备方法如下:
(1)将人参经过2次水煎提取后,取其药渣再由经过热稳定α淀粉酶、碱性蛋白酶、淀粉葡萄糖苷酶按照质量比为1:1:(1-3)处理后获得人参提取物;
(2)将步骤(1)获得的人参提取物和黄酮提取物进行混合。
进一步地限定,所述组合物还包括猪油、白糖、蛋黄粉、胆固醇和胆酸钠。
本发明提供上述的组合物在制备降血脂保健品中的应用。
本发明提供上述的组合物在制备调节肠道菌群结构的保健品中的应用。
有益效果:本发明将人参不溶性膳食纤维及其组合物以1%和10%的添加量加入高脂血症小鼠的高脂饲料中,研究人参IDF对小鼠高脂血症的影响。结果表明:与基础对照组相比,高脂模型组小鼠的体质量显著增加(P<0.05),血清中总胆固醇(totalcholesterol,TC)、甘油三酯(triglyceride,TG)、低密度脂蛋白胆固醇(low densitylipoprotein cholesterol,LDL-C)浓度、动脉粥样硬化指数(atherosclerosis index,AI)及脂/体比极显著增高(P<0.01),高密度脂蛋白胆固醇(high density lipoproteincholesterol,HDL-C)浓度极显著降低(P<0.01);血清及肝脏中超氧化物歧化酶(superoxide dismutase,SOD)活力降低,丙二醛(malondialdehyde,MDA)含量增加(P<0.05)。与高脂对照组对比,PPDF能极显著降低小鼠的体质量及血清中TC、TG、LDL-C浓度、AI值、脂/体比,极显著增加HDL-C浓度(P<0.01);血清及肝脏中的SOD活力增强,MDA含量显著降低(P<0.05)。
附图说明
图1为人参IDF对高脂血症小鼠体质量(A)、总增重(B)、总饲料采食量(C)和脏器指数(D)的影响;
图2为各组小鼠肝脏HE染色切片观察结果。A,正常组;B,HFD组;C,1%IDF组;D,10%IDF组;E,Simvastatin组;
图3为各组小鼠附睾脂肪组织HE染色切片观察结果。A,正常组;B,HFD组;C,1%IDF组;D,10%IDF组;E,Simvastatin组;F,各组脂肪细胞表面积柱状图;
图4为人参IDF对高脂血症小鼠肠道菌群多样性和分类组成的影响。A,Alpha多样性Chao1指数和Simpson指数;B,基于Bray-Crutis距离的Beta多样性主坐标分析(Principal coordinates analysis,PCoA);C,门水平分类学组成相对含量;D,属水平分类学组成相对含量;
图5为人参IDF对高脂血症小鼠肠道特征菌株构成的影响。A,OTU韦恩图;B,标志物种分类学LefSe(LDA Effect Size)分析。节点大小对应于该OTU的平均相对丰度;空心节点代表组间差异不显著的OTU,实心节点则代表丰度较高和组间差异显著的OTU。字母则标识了组间存在显著差异的分类单元的名称;C,种水平物种组成热图;D,标志物种随机森林模型分析。右侧Importance表示从上到下物种对模型的重要性依次递减,可认为这些重要性靠前的物种是组间差异的标志物种;
图6为人参IDF对高脂血症小鼠肠道菌群代谢功能的影响。A,各组KEGG信号通路功能基因表达相对丰度;B,各组功能基因表达丰度出现显著差异的几个信号通路。HFD vsNormal,ΔP<0.05;ΔΔP<0.01;HFD vs IDF,*P<0.05;**P<0.01。
具体实施例
动物与试剂:SPF级5周龄雄性C57BL/6J小鼠,体质量12~15g,由辽宁长生生物技术有限公司提供,动物生产许可证号:SCXK(辽)2020-0001。
总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)、低密度脂蛋白胆固醇(lowdensity lipoprotein cholesterol,LDL-C)、超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、丙氨酸转氨酶(alanine aminotransferase,ALT)、天冬氨酸转氨酶(Aspartate aminotransferase,AST)、碱性磷酸酶(alkalinephosphatase,ALP)试剂盒南京建成生物工程研究;血糖试纸,三诺生物传感股份有限公司;其余试剂均为分析纯,购于国药集团化学试剂有限公司。
仪器与设备:超微粉碎机济南健辰机械有限公司;Explorer EX125DZH型准微量电子天平美国奥豪斯仪器有限公司;Megafuge 8R高速低温离心机美国赛默飞世尔公司;超低温冰箱青岛海尔空调电子有限公司;BioTek-酶标仪美国伯腾公司;Sinocare安稳+血糖仪三诺生物传感股份有限公司;BX43荧光正置显微镜日本奥林巴斯公司。
实施例1.制备人参提取物和人参不溶性膳食纤维组合物
人参原料按照1:10(w/v)料液比沸水煎煮2次,每次1.5h,过滤后得到药渣,60℃烘干粉碎备用。
在60℃环境下,人参药渣按照20倍体积料液比与水混匀,经过热稳定经α-淀粉酶(质量分数为1%)、碱性蛋白酶(质量分数为1%)和淀粉葡萄糖苷酶(质量分数为3%)依次水解1.0h。反应结束后,离心所得残渣即为人参非水溶性膳食纤维(人参IDF)。
实施例2.添加人参组合物的高脂饲料配方
普通饲料77%、猪油10%、白糖5%、蛋黄粉5%、胆固醇2.5%、胆酸钠0.5%。
高脂饲料中普通饲料76%,人参IDF 1%,猪油10%、白糖5%、蛋黄粉5%、胆固醇2.5%、胆酸钠0.5%,制成1%IDF定制饲料。
高脂饲料中普通饲料67%,人参IDF 10%,猪油10%、白糖5%、蛋黄粉5%、胆固醇2.5%、胆酸钠0.5%,制成10%IDF定制饲料。
山楂/菊花黄酮提取物(购自西安圣青生物科技有限公司)以1:1比例混合,制成黄酮混合物。黄酮混合物0.3%,人参1%IDF定制饲料99.7%,制成HT+1%IDF定制饲料。
山楂/菊花黄酮提取物(购自西安圣青生物科技有限公司)以1:1比例混合,制成黄酮混合物。黄酮混合物0.3%,人参10%IDF定制饲料99.7%,制成HT+10%IDF定制饲料。
利用以下实验验证实验效果:
一、高脂血症模型的建立
5周龄雄性C57BL/6J小鼠在恒温(23±3)℃、12h/12h昼夜交替、相对湿度(50±10)%条件下适应性饲养1周。之后按体质量随机分为2组,其中1组饲喂基础饲料做正常对照组(Normal组,共8只),其余小鼠饲喂高脂饲料做高脂模型组(HFD组),4周后称量小鼠体质量,眼眶取血测TC、TG含量,判断高脂血症小鼠是否造模成功。
1.动物的分组及给药
造模成功的HFD组小鼠随机分成6组,每组7只:HFD组、1%IDF组、10%IDF组、HT+1%IDF组、HT+10%IDF组、阳性药组(辛伐他丁,Simvastatin)。HFD组和Simvastatin组继续饲喂高脂饲料,Normal组继续饲喂基础饲料。1%IDF组和10%IDF组饲喂相应1%IDF和10%IDF定制饲料,HT+1%IDF组和HT+10%IDF组饲喂相应的HT+IDF复合物定制饲料,Simvastatin组以10mg/kg剂量(现用现配)每天灌胃给药,Normal组及HFD组小鼠灌胃同等体积纯净水。连续灌胃12周,期间自由摄食、饮水。
给药期间每周观察记录小鼠的摄食和饮水状况、精神状态、毛发情况和体质量。给药结束前2天无菌收集小鼠粪便,于-80℃冰箱保存待测。给药结束后禁食不禁水12h,小鼠摘眼球取血,分离血清测定血液指标。全部小鼠脱臼处死迅速解剖取肝脏、脾脏、附睾脂肪组织等器官,观察并称质量。部分肝脏及附睾脂肪组织用10%福尔马林固定液固定,进行病理学组织切片分析。
2.小鼠脏器指数及脂肪组织系数测定
将小鼠的肝脏、脾脏、附睾脂肪等脏器组织用生理盐水冲洗干净,滤纸吸干水分后称质量。脏器指数按公式(1)计算,附睾脂肪系数计算公式相同。
结果:整个实验周期中小鼠生长状态良好,活动正常,未出现受伤或死亡现象。随着实验的进行,各组小鼠体重逐渐增加,从第6周开始HFD组体重显著高于正常组(P<0.01)和其他IDF组(P<0.01,图1A)。实验结束时,HFD组小鼠在采食量显著低于正常组的前提下(图1C),增重显著升高(图1B),且周身较肥胖,毛发油亮。在采食量相近的前提下,10%IDF组和HT+10%IDF组增重幅度最低,显著低于HFD组(P<0.01)和1%IDF组、HT+1%IDF和Simvastatin组(图1B)。IDF组普遍对肝脏指数影响不显著,但能够显著降低脂肪含量(P<0.05和P<0.01),同时增加脾脏指数(P<0.01,图1D)。总体来说,10%IDF组对高脂血症小鼠体重、采食和脏器指数等指标作用最显著,而HT+IDF组与IDF组的作用效果未见明显差异。
3.小鼠血脂指标的测定
冰上融化于-80℃冰箱保存的待测血清,根据试剂盒说明方法测定血清TC、TG、HDL-C、LDL-C水平。动脉粥样硬化指数(atherosclerosis index,AI)按以下公式(2)计算。
4.小鼠血清肝功指标及肝脂指标测定
取小鼠血清,根据试剂盒说明方法直接检测血清肝功三项ALT、AST、AKP指标。
取100mg肝组织,切碎置于离心管内,按照1:10(m:V)溶于无菌磷酸盐缓冲液中(pH7.4),自动匀浆仪低温快速匀浆,3 500r/min低温离心15min,取上清液于无菌离心管中4℃保存待测。根据试剂盒说明方法检测肝脏组织中TC、TG含量。
血糖、血脂、肝功三项和肝脂指标检测结果:血液检测发现(表1),IDF干预能改善高脂血症小鼠的血糖、血脂升高和肝功异常现象。1%IDF显著降低了空腹血糖(P<0.05)、血清TG(P<0.05)、肝脏TC、TG(P<0.05)以及肝功三项水平(P<0.05和P<0.01),显著提高了血清HDL-C水平;10%IDF显著降低了血清和肝脏TC(P<0.05)、TG(P<0.01)和血清AST(P<0.01)、ALP水平(P<0.05),显著提高了血清HDL-C水平(P<0.01)。IDF组、HT+IDF组和Simvastatin组均能显著降低HFD组的动脉硬化指数(P<0.01)。IDF组与HT+IDF组血糖、血脂、肝功和肝脂指标水平接近。
表1 人参IDF对高脂血症小鼠血糖、血脂、肝功和肝脂指标的影响
注:横向比较差异显著性,不同字母表示差异显著(p<0.05或p<0.01),相同字母或无标注表示差异不显著。
5.小鼠血清及肝脏SOD活力和MDA水平测定
取小鼠血清或肝脏组织上清,根据试剂盒说明方法检测其中SOD活力和MDA水平。
结果:IDF干预对高脂血症小鼠血清和肝脂组织的氧化应激水平均有所改善。1%IDF和10%IDF显著提高了血清和肝脏SOD活力(P<0.01),显著降低了肝脏MDA水平(P<0.01),10%IDF还显著降低了血清MDA水平(P<0.01)。HT+1%IDF和HT+10%IDF同样显著提升了血清和肝脏SOD活力(P<0.05或P<0.01),显著降低了肝脏MDA水平(P<0.01)。IDF组和HT+IDF组的作用水平接近。
表2 人参IDF对高脂小鼠血清和肝脏组织氧化应激水平的影响
6.小鼠肝脏及脂肪组织病理学切片观察
将小鼠肝脏及附睾脂肪垫用10%中性福尔马林固定48h,流水冲洗,梯度乙醇脱水,二甲苯透明,透蜡,包埋,脱蜡切片,苏木精-伊红(hematoxylin-eosin,HE)染色,显微镜观察肝脏及脂肪组织结构。
结果:(1)肝脏组织HE染色观察结果
结合前面对小鼠外观特征、血清和肝脏组织生化指标检测结果,IDF组和HT+IDF组的整体作用水平接近,为深入揭示IDF对高脂血症小鼠的健康改善作用,选择1%IDF组和10%IDF组进行后续组织切片和肠道微生物多样性分析。
对高脂饮食可能影响较明显的肝脏和附睾脂肪组织进行了HE染色及切片观察。与正常组相比(图2A),HFD组小鼠肝组织HE染色切片出现些许空泡样脂肪组织堆积,导致中央门静脉周围的肝细胞结构不如正常组致密完整(图2B)。IDF各组小鼠肝组织空泡样脂肪结构减少。与HFD组相比,中央门静脉周围的肝细胞结构较致密(图2C和2D)。阳性药肝组织中仍可见少量空泡样脂肪组织存在(图2E)。
(2)附睾脂肪组织HE染色观察结果
IDF组显著改善了高脂血症小鼠的附睾脂肪细胞肥大。10%IDF对附睾脂肪细胞肥大的抑制作用最显著(P<0.01),这与IDF组小鼠体重显著减轻的特征相符。
7.数据统计分析
所有实验平行重复3次,数据采用Microsoft Excel 2007软件进行处理,结果均以平均值±标准差表示。采用SPSS 22.0软件进行数理统计分析,组间比较采用单因素方差分析(One Way ANOVA),P<0.05有显著差异,P<0.01有极显著差异。统计结果由GraphpadPrism 8.0软件作图。
二、粪便肠道菌群多样性分析
对人参IDF干预高脂血症小鼠粪便肠道菌群变化进行了分析,如图4所示。目前主要使用alpha多样性和beta多样性指数,分别表征物种在生境内和生境间的多样性,以综合评价各组样本微生物多样性水平。Alpha多样性中Chao 1指数代表样本物种丰富度,Simpson指数代表样本物种多样性。
与正常组相比,HFD组Chao1指数降低14.18%。与HFD组相比,1%IDF组Chao1指数提高了25.91%,10%IDF组Chao1指数提高了29.48%(图4A)。Beta多样性PcoA分析显示(图4B),HFD组与其余各组差异最大,IDF组多样性水平与正常组更为接近。与高脂血症组HFD相比,1%IDF和10%IDF的肠道菌群多样性都不同程度增加了。
通过门(图4C)和属(图4D)水平分类组成分析发现,高脂饮食导致HFD组放线菌门(Actinobacteria)、疣微菌门(Verrumcomicrobiota)丰度显著增加,拟杆菌门(Bacteroidota)丰度显著降低。IDF组使以上菌群丰度得到改变,但不同剂量效果不同。1%IDF主要增加了Firmicutes丰度,进一步降低了Bacteroidota丰度;10%IDF则显著增加了Bacteroidota和Proteobicteria丰度(图4C)。进一步属水平分析发现,IDF组显著降低了HFD组中的双歧杆菌属(Bifidobacterium)和阿克曼菌(Akkermansia1)丰度;1%IDF主要增加了乳杆菌属(Lactobacillus)丰度,10%IDF主要增加了苏黎世杆菌(Turicibacter)丰度(图4D)。
三、粪便肠道菌群组成及标志性物种分析
在QIIME2平台按照95%的一致性划分高质量序列(OTUs),正常组、HFD组、1%IDF组和10%IDF组的独有OTU分别为4843、4052、7456和6240(图5A)。相对于模型组,1%IDF的独有OTU增加了84%,10%IDF的独有OTU增加了53.99%,这与图4A中Chao1指数的升高相一致。LEfSe分析可以直接在所有分类水平的角度对各组样品同时进行差异分析,以寻找各组间稳健的标志物种。
结果HFD组的标志物种集中在Actinobacteria中的Bifidobacteriales菌,而1%IDF组的标志物种则转向了Firmicutes,并以Lactobacillus菌为主;10%IDF组的标志物种则偏离Firmicutes继续向Proteobicteria转变,并以Turicibacteraceae菌为主(图5B)。
通过种水平物种组成热图分析各组主要菌种(图5C)。HFD组中Bifidobacteriumpseudolongum、Bifidobacterium bifidum、Ruminococcus flavefaciens、Akkermansiamuciniphila、Lactobacillus salivarius菌丰度较高。1%IDF组中Clostridium colinum、Staphylococcus sciuri、Lactobacillus ruminis、Butyricicoccus pullicaecorum、Staphylococcus saprophyticus、Lactobacillus hamsteri等菌丰度较高。10%IDF组中Ruminococcus callidus、Desulfovibrio_C21_c20、Alistipes massiliensis、Alistipesindistinctus、Mucispirillum schaedleri、Parabacteroides distasonis、Helicobacterganmani、Butyricicoccus pullicaecorum等菌丰度较高。进一步通过随机森林模型分析丰度高的组间差异标志物种(图5D)。Bifidobacterium pseudolongum和Bifidobacteriumbifidum是HFD组中最能代表菌群结构特征的2个高丰度特征物种;Lactobacillushamsteri和Butyricicoccus pullicaecorum是1%IDF组中最能代表菌群结构特征的前两位高丰度物质;Butyricicoccus pullicaecorum是10%IDF组中最能代表菌群结构特征的第一位高丰度物质。从以上结果可以看出,IDF干预能有效改善HFD组的肠道菌群结构。1%IDF的剂量能使得乳酸菌、丁酸盐产生菌等有益菌株大量增殖,10%IDF的剂量在促进丁酸盐产生菌增殖的同时,也促进了Alistipes massiliensis、Alistipes indistinctus等菌的增殖,揭示了人参IDF在改善高脂血症小鼠肠道菌群结构中的独特作用。
四、粪便肠道菌群代谢功能分析
通过KEGG信号通路数据库(https://www.kegg.jp/)对各组肠道菌群功能基因表达情况进行了分析(图6)。一级信号通路下各组基因表达丰度总体水平较接近(图6A)。二级信号通路中,与HFD组相比,IDF组进一步提高了肠道菌群中细胞活性基因丰度,10%IDF显著降低了传染性疾病相关基因丰度(P<0.01),进一步提高了免疫系统基因丰度(P<0.01)(图6B)。而1%IDF则显著抑制了高脂血症小鼠免疫系统的高基因丰度(P<0.01)。以上结果说明了IDF能够纠正高脂血症小鼠异常表达的基因丰度,但1%IDF和10%IDF的效果不同。
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 (4)
1.一种人参非水溶性膳食纤维在制备降血脂和调节高脂血症肠道菌群结构的组合物中的应用,其特征在于,调节高脂血症肠道菌群结构为促进丁酸盐产生菌增殖。
2.根据权利要求1所述的应用,其特征在于,所述组合物还包括黄酮提取物。
3.根据权利要求2所述的应用,其特征在于,所述黄酮提取物由山楂黄酮提取物和菊花黄酮提取物组成。
4.根据权利要求3所述的应用,其特征在于,所述山楂黄酮提取物和菊花黄酮提取物的质量比为1:1。
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