CN111671765B - 一种褐藻膳食纤维在制备治疗或预防代谢综合症的药品、食品和/或保健品中的应用 - Google Patents
一种褐藻膳食纤维在制备治疗或预防代谢综合症的药品、食品和/或保健品中的应用 Download PDFInfo
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Abstract
本本发明公开了一种褐藻膳食纤维在制备治疗或预防代谢综合症的药品、食品和/或保健品中的应用。该褐藻膳食纤维中可溶性膳食纤维(SDF)和不溶性膳食纤维(IDF)的比例为1:0.7‑1.3,总膳食纤维含量高于80%。该褐藻膳食纤维在动物实验水平上已经证明具有显著糖脂代谢调节活性,降低代谢紊乱小鼠的血糖和血脂,改善糖尿病小鼠的胰岛素抵抗,降低代谢紊乱小鼠血清中各炎性因子的水平。该膳食纤维样品能有效的改善高脂饮食诱导的代谢紊乱小鼠的肠道菌群,增加肠道有益菌双歧杆菌的含量,促进肠道中短链脂肪酸的产生。综上,该褐藻膳食纤维具有良好糖脂紊乱调节活性,可用于制备治疗或预防代谢综合症的药品、食品和/或保健品的开发。
Description
技术领域
本发明属于食品保健领域,具体涉及一种褐藻膳食纤维在药品、食品和/或保健品方面的应用。
背景技术
代谢综合症包括人体蛋白质,脂肪,碳水化合物和其他物质的代谢紊乱。代谢综合症的症状包括葡萄糖耐量不全,内脏肥胖,血脂异常,高血糖和高血压以及胰岛素抵抗,这被证明是心血管疾病(CVD)和2型糖尿病(T2DM)的独立危险因素。除此之外,患有代谢综合症的人似乎易患其他疾病,特别是多囊卵巢综合症,脂肪肝,胆囊结石,哮喘,睡眠障碍和某些癌症,例如大肠癌和乳腺癌。与单因素代谢异常相比,由于多因素相互作用,糖脂代谢异常更为复杂。该综合症的病因在很大程度上尚不清楚,但涉及遗传,代谢和环境因素(包括饮食)之间的复杂相互作用。随着肥胖症和糖尿病的全球流行,全世界代谢综合症患者的数量急剧增加,不仅在成年人或老年人口,在儿童和年轻人中患病人数也逐渐增加。它已成为全球主要的公共卫生挑战之一,需要紧急的治疗和干预措施。
膳食纤维是植物中的一系列结构性多糖和木质素,无法被人胃和小肠中的酶消化。作为第七大类营养素,膳食纤维具有较好的吸水性和膨胀力,对于润肠通便具有积极的作用。褐藻是膳食纤维的重要来源,与陆生植物中膳食纤维主要是果胶和纤维素等成分不同,褐藻作为低等隐花植物,其细胞壁成分主要是由甘露糖醛酸、古罗糖醛酸、葡萄糖醛酸、甘露糖、木糖等组成的酸性多糖和半纤维素类成分。大型藻类的可溶性或粘性较高的纤维成分通常较高,结构上的特异性使得褐藻膳食纤维具有新颖的生理功能。本发明公开了褐藻膳食纤维在防治代谢综合症的药品、食品和/或保健品中的应用。
发明内容
本发明的目的是提供一种褐藻膳食纤维在制备代谢综合症防治药品、食品和和或保健品中的应用,褐藻膳食纤维提取原料为提取褐藻糖胶后的废弃的褐藻渣,极大的减少了资源的浪费。经动物实验表明此膳食纤维具有极好的调节代谢紊乱的活性。
上述褐藻膳食纤维的制备工艺为:
褐藻干燥粉碎(过100-300目筛)后用稀酸浸泡,弃去酸液余下藻体,藻体加碱和过氧化氢处理,提取液过滤、超滤、浓缩、在浓缩液中添加乙醇进行沉淀,经过过滤,沉淀干燥粉碎为可溶性膳食纤维;提取液过滤后的藻渣经蛋白酶处理后干燥得不溶性膳食纤维,两者混合得褐藻总膳食纤维。
进一步的,如上所用稀酸为0.05-0.3mol/L柠檬酸、盐酸或硫酸,最优为0.1mol/L柠檬酸;10-20倍体积,其中最优为15倍;浸泡时间为0.5-5h,最优为2h;浸泡温度为20-60℃,最优为50℃。
进一步的,如上所述的作用碱为含有0.05%-2%过氧化氢的0.05%-3%Na2CO3溶液,最优为含有1%过氧化氢的1.5% Na2CO3溶液;10-30倍体积,其中最优为15倍;处理温度为40-90℃,最优为60℃;处理时间为1-5h,最优为3h。
进一步的,所述无水乙醇的添加量为浓缩液的0.67-3倍,最优为1.8倍。
进一步的,所述蛋白酶为碱性蛋白酶,用量为蛋白酶与藻渣的比例用量为0.5-2%,优选1%。
进一步的,所述的褐藻膳食纤维,其特征在于:此膳食纤维中可溶性膳食纤维(SDF)和不溶性膳食纤维(IDF)的比例为1:0.7-1.3,总膳食纤维含量高于80%。
该褐藻膳食纤维系从海洋褐藻包括海带、裙带菜、马尾藻、墨角藻、鹿角菜、泡叶藻、昆布、枝管藻、鼠尾藻、全缘马尾藻、羊栖菜、硇洲马尾藻、海黍子、海蒿子等褐藻中提取制备。
与现有的技术相比,本发明具有如下优点:
本发明的褐藻膳食纤维的制备方法提取率较高,去除了褐藻多糖硫酸酯和甘露醇等成分,对褐藻中可溶性膳食纤维和不溶性膳食纤维进行了充分的提取。
本发明的褐藻膳食纤维在动物模型上具有较好的降糖活性,能够调节代谢紊乱小鼠的血脂,改善胰岛素抵抗,对代谢紊乱小鼠的肝脏、肾脏具有很好的保护作用。
本发明的褐藻膳食纤维能够改善小鼠高脂饮食引起的肠道菌群失调,调节肠道有益菌的比例,尤其是增加肠道益生菌双歧杆菌的相对丰度。
本发明的褐藻膳食纤维在制备防治代谢综合症的药品、食品、保健品具有广泛的应用。
该褐藻膳食纤维在动物实验水平上已经证明具有显著糖脂代谢调节活性,降低代谢紊乱小鼠的血糖和血脂,改善糖尿病小鼠的胰岛素抵抗,降低代谢紊乱小鼠血清中各炎性因子的水平(TNF-α、IL-6、IL-β、MCP-1),有效保护小鼠的肝脏、肾脏等器官。该膳食纤维样品能有效的改善高脂饮食诱导的代谢紊乱小鼠的肠道菌群,增加肠道有益菌双歧杆菌的含量,促进肠道中短链脂肪酸的产生。综上,该褐藻膳食纤维具有良好糖脂紊乱调节活性,可用于制备治疗或预防代谢综合症的药品、食品和/或保健品的开发。
附图说明
图1.小鼠空腹血糖水平(*P<0.05或**P<0.01,vs Neg组;#P<0.05或##P<0.01,vsCon组)
图2.A:小鼠葡萄糖耐量测定变化曲线;B:小鼠葡萄糖耐量(*P<0.05或**P<0.01,vs Neg组;#P<0.05或##P<0.01,vs Con组)
图3.A:小鼠空腹胰岛素水平;B:小鼠胰岛素稳态模型(*P<0.05或**P<0.01,vsNeg组;#P<0.05或##P<0.01,vs Con组)
图4.A:小鼠肝脏指数;B:小鼠内脏脂肪指数(*P<0.05或**P<0.01,vs Neg组;#P<0.05或##P<0.01,vs Con组)
图5.小鼠血脂水平。A:血清甘油三酯;B:血清总胆固醇;C:血清低密度脂蛋白胆固醇;D:血清高密度脂蛋白胆固醇(*P<0.05或**P,<0.01,vs Neg组;#P<0.05或##P<0.01,vsCon组)
图6.小鼠血清肝脏生化指标。A:血清谷草转氨酶(AST)、谷丙转氨酶水平(ALT)及比值;B:血清白蛋白(ALB)、球蛋白(GLOB)水平及比值(*P,<0.05或**P,<0.01,vs Neg组;#P<0.05或##P<0.01,vs Con组)
图7.小鼠血清肾脏功能生化指标。A:血清尿酸水平;B:血清肌酐水平;C:血清尿素氮水平(*P<0.05或**P<0.01,vs Neg组;#P<0.05或##P<0.01,vs Con组)
图8.小鼠血清炎性因子水平。A:血清TNF-α水平;B:血清IL-6水平;C:血清IL-β水平;D:血清MCP-1水平(*P<0.05或**P,<0.01,vs Neg组;#P<0.05或##P<0.01,vs Con组)
图9.小鼠肝脏活性氧水平。A:肝脏SOD水平;B:肝脏MDA水平;C:肝脏GSH-Px水平;D:肝脏CAT水平(*P,<0.05或**P<0.01,vs Neg组;#P<0.05或##P<0.01,vs Con组)
图10.小鼠结肠内容物短链脂肪酸(SCFAs)含量。A:乙酸含量;B:丙酸含量;C:异丁酸含量;D:丁酸含量;E:异戊酸含量;F:戊酸含量(*P<0.05或**P,<0.01,vs Neg组;#P,<0.05或##P<0.01,vs Con组)
图11.小鼠肝脏石蜡切片H&E染色(200X和400X)
图12.膳食纤维改变了高脂饮食诱导的代谢紊乱小鼠肠道的菌群组成。A:肠道的微生物显示在门级分类堆叠图;B:PCoA主坐标分析,属水平;C:物种距离热图(每行和列都代表样本,颜色代表距离大小,颜色越接近红色距离越远,越接近绿色距离越近);LEFse分析鉴定出各组差异最丰富的分类单元,数据展示在LDA Score(D)中。
具体实施方式
通过以下具体实施例对本发明方案作进一步的具体说明,但本发明要求保护的范围并不局限于实例表述的范围。
实施例1:海带膳食纤维的制备、理化性质及成分分析
(1)海带膳食纤维的制备1
称取100g海带干粉,加入15倍体积的0.1M柠檬酸溶液置于50℃中浸泡2h,4000rpm转速离心15min,弃去酸液,收集固体成分,固体成分清洗至中性后加15倍体积含有1%过氧化氢的1.5% Na2CO3溶液于60℃水浴处理3h,提取液过滤,将固体成分与溶液分开,溶液部分超滤除盐、浓缩、使用终体积60%的乙醇沉淀,沉淀脱水烘干得可溶性膳食纤维14.70g。碱液处理之后的固体藻渣用1%的碱性蛋白酶40℃处理3h,离心弃去上清液,固体部分干燥得到不溶性膳食纤维16.28g。两者混合得总褐藻膳食纤维,总回收率30.98%。
(2)海带膳食纤维的制备2
同上述制备方法,但是将含1%过氧化氢的1.5% Na2CO3溶液,替代成为含1%过氧化氢的水溶液,不使用蛋白酶处理藻渣。经干燥粉碎得可溶性膳食纤维8.23g。碱液处理之后的固体藻渣直接干燥得到不溶性膳食纤维19.08g。两者混合得总褐藻膳食纤维,总回收率27.31%。
(3)褐藻膳食纤维成分分析
蛋白质的测定方法采用BCA法;水分的测定使用烘箱烘干法;脂肪含量的测定采用GB5009.8-85中索式抽提法;淀粉定性采用碘液;灰分采用GB5009.4-2016中的方法;膳食纤维的含量按GB5009.88-2008的方法测定;单糖组成测定按PMP衍生法测定
PMP法测定单糖组成的具体步骤:取20mg膳食纤维置于水解瓶中,加入1mL水,1mL4mol/L的三氟乙酸,封口后105℃水解4h,冷却至室温之后,2mol/L的氢氧化钠中和。取100μL上述溶液,加入120μL 0.5mol/L的PMP(甲醇溶解),100μL 0.3mol/L的氢氧化钠,封口70℃反应30min。取出反应液加100μL 0.3mol/L的盐酸中和,加入500μL氯仿萃取三次。标准品做同样处理,高效液相测定单糖组成。
C-18色谱柱(安捷伦,4.6x150mm,5μm),柱温30℃,流动相为PBS缓冲液:乙腈=81:19,流速0.8mL/min,在波长254nm检测。
(4)褐藻膳食纤维的理化性质
膨胀力测定:称取1g膳食纤维的粉末放入量筒中,测干粉体积,加入20℃水,使总体积达到50mL,摇匀,使其充分吸收水分,在20℃下静置24h,读取吸水饱胀后的膳食纤维的体积,计算膨胀力。
膨胀力(%)=(膨胀后体积(mL)–干粉体积(mL))/样品干质量(g)×100%
持水力测定:称取1g膳食纤维粉质放入量筒中,加入20℃水,饱和1h,将膳食纤维移至滤筛布上沥干后,把保留在滤布上结合了水的膳食纤维移到表面皿中称重。
持水力(%)=[样品湿质量(g)–样品干质量(g)]/样品干质量(g)×100%
表1:海带膳食纤维的成分及理化性质
表2:海带膳食纤维的单糖组成
表1表明,海带膳食纤维中含有较少的蛋白质、脂肪,淀粉检测为阴性,总膳食纤维的含量高达82.79%,可溶性膳食纤维的比例占总膳食纤维的44.72%。与已经公开的褐藻膳食纤维相比,如(CN 102787519 B)具有较好的持水性和溶胀性。
经比较,不经Na2CO3的转化作用的膳食纤维样品可溶性膳食纤维的产率减少。蛋白质含量增加,持水力和膨胀力较小。
表2表明海带膳食纤维是一种以鼠李糖和甘露糖醛酸为主的多糖。
实施例2:马尾藻膳食纤维的制备、理化性质及成分分析
(1)称取100g马尾藻干粉,加入12倍体积0.15M柠檬酸溶液置于45℃水浴中浸泡2h,4000rpm转速离心15min,弃去酸液,收集固体成分,流动水清洗固体成分至中性后加15倍体积含有2%过氧化氢的1% Na2CO3溶液与70℃水浴处理2h,提取液过滤,将固体成分与溶液分开,溶液超滤除盐、浓缩、使用终体积75%的乙醇沉淀,沉淀脱水烘干得可溶性膳食纤维13.77g。碱液处理之后的固体藻渣用碱性蛋白酶40℃处理3h,离心弃去上清液,干燥得到不溶性膳食纤维15.02g。两者混合得总褐藻膳食纤维,总回收率28.79%。
(2)马尾藻膳食纤维成分及性质研究
参考实施例1的方法
表3:马尾藻膳食纤维的成分及理化性质
表4:马尾藻膳食纤维的单糖组成
表3表明,马尾藻膳食纤维中蛋白质、脂肪含量较少,无淀粉总膳食纤维的含量高达84.19%,可溶性膳食纤维的比例占总膳食纤维的46.72%。同时具有较好的持水性和溶胀性。
表4表明马尾藻膳食纤维中鼠李糖、甘露糖醛酸和葡萄糖的含量较高。
实施例3:动物实验
选择实施例1中的海带膳食纤维样品在动物模型上验证其功能。
选择四周龄C57BL/6J雄性小鼠,体重20±2g,分为4组(空白组、阴性组、膳食纤维组、二甲双胍组)),每组12只。空白组饲喂10%脂肪供能的普通饲料。其余3组饲喂60%脂肪供能的高脂饲料7周后开始给药。阴性对照组灌胃生理盐水,膳食纤维组灌胃海带膳食纤维500mg/kg/天,阳性药选择二甲双胍(西格玛奥德里奇上海贸易有限公司),灌胃剂量为200mg/kg/天。用药8周,灌胃期间持续饲喂高脂饲料并监测体重、饮食、血糖变化。8周后,无菌代谢笼收集小鼠粪便用于肠道微生物的分析。小鼠解剖取血清、肝脏、白色脂肪、棕色脂肪,结肠内容物等液氮处理之后置于-80℃冰箱备用,用于生化测定及组织形态观察。
(1)小鼠空腹血糖水平
小鼠空腹血糖测定使用罗氏卓越金采血糖仪测定。
图1显示阴性组小鼠与空白组小鼠空腹血糖水平有显著性差异,与高脂饮食小鼠相比,二甲双胍作为临床上广泛使用的降糖药,能显著降低小鼠的空腹血糖血糖,海带膳食纤维也能显著降低小鼠空腹血糖水平。
(2)小鼠血清胰岛素水平
小鼠血清胰岛素水平采用酶联免疫吸附试剂盒按照说明书操作测定。
海带膳食纤维和二甲双胍显著降低高脂饮食小鼠的血清胰岛素水平,使之接近于正常水平,增加了胰岛素的敏感性(图3A)。
(3)口服葡萄糖耐受实验(OGTT)
小鼠禁食10-12h,灌胃D-葡萄糖溶液(2g/kg),灌胃后0、30、90、120min测定小鼠的血糖水平。
药时曲线下面积(AUC)=0.5(G0h+G0.5 h)×0.5+0.5(G2h+G0.5h)×1.5
稳态模型评估(HOMA-IR)=G0*I0/22.5
其中,G0空腹血糖水平mmoL/L;I0空腹胰岛素水平mU/L
药时曲线下面积越小,说明小鼠对糖的耐受能力越强,对糖的敏感度和调节能力越强。结果显示服用膳食纤维和二甲双胍能够有效的改善代谢紊乱小鼠的葡萄糖耐量,增强胰岛素的敏感性(图2);据胰岛素稳态评价模型(图3B),海带膳食纤维显著的降低了胰岛素稳态评估模型的系数,减轻了高脂饮食导致的胰岛素抵抗。
(4)肝脏指数和内脏脂肪指数的测定
取小鼠肝脏,内脏脂肪(附睾脂肪、肾周脂肪、腹膜后脂肪),生理盐水清洗两次,吸水纸吸干水分立即称重,小鼠肝脏,脂肪重量与体重的比值即肝脏指数和内脏脂肪指数。
图4显示海带膳食纤维显著降低了代谢紊乱小鼠的肝脏指数和内脏脂肪指数,降低了脂肪在肝脏及内脏部位的堆积,具有一定的减脂效果。
(5)小鼠血清生化
取小鼠血清,全自动生化分析仪(Beckman Coulter AU5800)分析小鼠血脂、肝脏功能、肾脏功能的生化指标。
高脂血症是一种脂质代谢疾病,它是代谢综合症的一个重要症状。全自动生化分析仪测定小鼠血清中CHOL、TG、LDL-C、HDL-C的含量。由图5可见,高脂饮食组小鼠血清的CHOL、TG、LDL-C、HDL-C含量均显著高于空白组。高脂饮食导致小鼠血脂异常。补充膳食纤维能非常显著降低血清TG、HDL-C的水平,对LDL-C和CHOL水平显著降低。膳食纤维能有效改善高脂饮食诱导的血脂异常。
海带膳食纤维的补充显著降低了谷草转氨酶(AST)水平和小鼠AST/ALT的比值(图6A),该比值能够表明肝脏细胞损伤情况,结果表明褐藻膳食纤维和二甲双胍能够有效的减少高脂饮食对肝脏的损伤。图6B显示高脂饮食组小鼠肝脏白蛋白与球蛋白的比值降低,表明高脂饮食影响了小鼠肝脏合成蛋白的能力,海带膳食纤维及二甲双胍对小鼠肝脏白蛋白与球蛋白的比值有一定的恢复作用,但不显著。
图7测定了小鼠血清中尿酸(UA)、肌酐(CRE)尿素氮(UREA)水平。高脂饮食加重了小鼠肾脏的负担。与空白组相比高脂饮食组小鼠血清UA、CRE、UREA水平明显的升高,提示小鼠肾脏功能损伤及高尿酸血症。而海带膳食纤维使UA、CRE、UREA显著的降低至正常水平,说明该膳食纤维减少肾脏的损伤,对代谢紊乱小鼠肾脏有较好的保护作用。
综上,褐藻膳食纤维不仅降低了高脂血症的风险,对肥胖小鼠的内脏器官,如肝脏、肾脏,都有极好的保护作用,在一定程度上改善了高脂饮食诱导的小鼠糖脂代谢紊乱。其中,膳食纤维对肝脏的保护作用可通过肝脏病理切片分析进一步解释。
(6)小鼠血清炎性因子水平
小鼠血清炎性因子(TNF-α;IL-6;IL-β;MCP-1)采用酶联免疫吸附试剂盒按照说明书操作测定。
发明人比较了血浆中各种炎性标记物。图8高脂饮食小鼠炎性标记物的水平,如TNF-α、MCP-1、IL-6、IL-β,明显高于空白组。值得关注的是,膳食纤维的补充显著降低了血清中TNF-α、IL-6、IL-β的水平,但对炎性因子MCP-1水平未有显著性影响。以上研究表明:补充膳食纤维能够改善高脂饮食引起的炎症反应,减慢代谢综合症的发展进程。
(7)小鼠肝脏氧化应激水平
取适量小鼠肝脏,称重,加入适当生理盐水,使用手持自动研磨器在冰浴条件下制成匀浆,添加生理盐水至终浓度为1mg/ml,取肝脏匀浆按照说明使用酶联免疫吸附试剂盒测定肝脏中MDA、CAT、SOD、GSH-Px水平。
为了进一步确定褐藻膳食纤维对代谢紊乱小鼠的保护作用,我们对参与氧化应激相关参数进行了研究。肝组织丙二醛(MDA)的生成是衡量脂质过氧化的可靠方法。此外,我们还评估了包括CAT、SOD、GSH-Px在内的抗氧化酶的活性,以评估补充海带膳食纤维对肝脏氧化应激水平的影响。
图9显示与高脂饮食组小鼠相比,组肝脏中MDA的水平极显著降低,即海带膳食纤维减少了MDA的产生,抑制了肝脏脂质过氧化。其次,肝脏中抗氧化酶的产生也显著的升高,对氧化物的清除能力大大的增加。海带食纤维增加了代谢紊乱小鼠的抗氧化能力,减少了氧自由基对肝脏的损伤。
(8)结肠内容物短链脂肪酸含量的测定
短链脂肪酸(SCFAs)是膳食纤维通过一些细菌的消化产生的含小于6个碳原子的短链有机酸。乙酸、丙酸和丁酸是肠道中主要的SCFAs,作为大肠细胞的主要能量来源,SCFAs调控着肠道内多种营养物质的吸收及激素产生,广泛参与能量代谢。
短链脂肪酸使用气相色谱法进行测定。结肠内容物(50mg)加入2.5mL水做成混悬液,加入1mol/L HCl溶液调节pH=2-3,冰浴超声提取10min,2次,12000rpm/min离心15min,取上清液加入1mL乙酸乙酯(乙醚)萃取,离心后取上清液使用气相色谱测定。柱子:HP-INNOWAX Polyethylene Glycol column(30m×320μm×0.5μm),烘箱温度初始保持90℃,0min,升至150℃,5℃/min升至180℃,维持1min,入口温度为250℃,载气流速为2mL/min。以含乙酸、丙酸、丁酸、异丁酸、戊酸、异戊酸(西格玛奥德里奇上海贸易有限公司)的混标为标准品。
实验结果显示结肠内容物中含量最丰富的SCFAs是乙酸,其次是丙酸和丁酸。高脂饮食使肠道中SCFAs的产生显著降低。海带膳食纤维的补充非常显著增加了小鼠结肠内容物中乙酸、丙酸、异丁酸、戊酸的产生,对丁酸和异戊酸也有显著性影响。二甲双胍组乙酸和戊酸的产生与高脂饮食组小鼠有显著性差异,说明二甲双胍对肠道微生物组成也有一定的影响。
(9)肝脏组织学分析(H&E染色)
分别从各组小鼠中分离肝组织和脂肪组织。组织用4%多聚甲醛组织固定液固定,脱水,包埋,切片。4μm的切片使用苏木精-伊红染色。使用Aperio AT2数字切片扫描仪扫描图像,拍照获得图像,观察肝脏的病理状态。
图11为四组肝细胞H&E染色切片。结果显示代谢紊乱小鼠肝细胞排列散乱,肝脂肪变性沉积,形成白色的空泡,并存在一定量的炎症细胞浸润。海带膳食纤维组的小鼠肝脏仍存在一定量的炎性细胞浸润,但是肝细胞排列明显较高脂饮食组小鼠整齐,肝小叶边缘清晰,存在脂肪变性沉积,但沉积量明显少于高脂饮食组。二甲双胍组肝脏接近空白组,肝脏无明显损伤。结合肝脏生化指标、肝脏氧化应激水平及组织病理分析可得海带膳食纤维能很好的保护肥胖小鼠的肝脏,减少高脂饮食对小鼠肝脏的损伤。
(10)肠道微生物测定
从小鼠粪便提取总基因组DNA。利用引物341F(5’-CCTAYGGGRBGCASCAG-3’)和806R(5’-GGACTACNNGGGTATC TAAT-3’)扩增细菌16s rDNA的V3+V4区(341~806)。然后用荧光仪Qubit3.0对扩增子进行汇集、纯化和定量。下一代测序由Illumina Hiseq2500PE250完成,该测序由广州基迪奥生物科技有限公司进行。
图12A为不同处理组肠道的微生物群落显示在门级分类堆叠图。图中我们可以看出各组在门级分类水平上主要由厚壁菌门、拟杆菌门、疣微菌门、变形菌门构成。在正常组饮食小鼠肠道中含量最高的是拟杆菌门,而高脂饮食改变了小鼠肠道微生物的组成,使厚壁菌门显著增多,而拟杆菌门水平降低。已有研究表明代谢综合症的发生与拟杆菌门与厚壁菌门的比例降低相关,这与我们观察到的一致。与高脂饮食组相比,膳食纤维的补充改变了肠道菌群的组成,提高了拟杆菌门与厚壁菌门的比率,减慢了代谢综合症发生的进程。二甲双胍也对肠道菌群组成产生了较大的影响,疣微菌门的比例大大增加。
通过PCoA主坐标图分析得到在属水平上各组在主坐标图上分离开形成独立的群体。在属水平(图12B)上,膳食纤维组与模型组和空白组的距离相当,说明食用膳食纤维之后小鼠的肠道微生物组成趋于正常水平发展。二甲双胍组属水平上距离空白组和模型组都相距较远,表现出特异的肠道微生物组成。PCoA主坐标分析的结果可通过物种距离热图(图12C)进一步说明。通过物种距离热图可以得到与PCoA主坐标图类似的结果。空白组与高脂饮食组肠道菌群物种距离较远,膳食纤维组与空白组和高脂饮食组在热图上偏向绿色,证明在膳食纤维的补充有效的逆转了高脂饮食导致的肠道菌群失调。
通过LEFse分析(图12D)组间菌群差异,可以找出各组间特异的指示菌群。
图12D中模型组中同属厚壁菌门梭菌目的毛螺菌科(Lachnospiraceae)、梭菌科(Clostridiaceae)、真杆菌属(Eubacterium)、消化球菌属(Peptococcus)、Ruminococcaceae、柔嫩梭菌(Clostridium,leptum)是指示物种。其中,消化球菌属与短链脂肪酸(SCFAs)的产生呈负相关。已有报道表明:二甲双胍的降糖机制与调节肠道菌群相关。研究表明:二甲双胍组中Akkermansia是一种益生菌,帮助维持消化道健康,降低肥胖、糖尿病、炎症等疾病的风险,它的数量增多会让宿主出现肥胖、炎症、II型糖尿病的概率降低。非常值得关注的是膳食纤维组肠道中双歧杆菌(Bifidobacteriaceae)为指示菌群,双歧杆菌是一种益生菌,它可以通过产生乙酸、乳酸等短链脂肪酸来抑制肠道腐败菌的生长和有毒代谢产物的形成,刺激肠蠕动,从而减少水分的过度吸收而缓解便秘症状;双歧杆菌细胞能够吸附食物中的致癌和致突变物质,从而保护机体细胞免受这些致癌物质的损害;肠道的有害菌产生并释放内毒素进入血液到中,对肝脏会造成损伤,双歧杆菌制剂可以抑制产生内毒素的有害菌数量,从而对肝脏患者起到良好的治疗作用。除此之外,肠道放线菌(Actinobacteria)含量增加。空白组中拟杆菌(Bacteroidales)、Muribaculaceae、瘤胃球菌属(Ruminococcus)、脱硫弧菌属(Desulfovibiro)、毛形杆菌属(Lachnobacterium)、Gordonibacter等丰度增加。其中,拟杆菌(Bacteroidales)是肠道主要菌群之一,它在瘦鼠内的含量较高。Muribaculaceae与乙酸、丙酸、丁酸等短链脂肪酸(SCFAs)的产生呈正相关。综上,高脂饮食使肠道微生物的优势物种发生变化,而海带膳食纤维能改变高脂饮食小鼠肠道微环境,调节小鼠肠道微生物组成,进而改善高脂饮食诱导产生的代谢综合症的一系列症状。
补充内容
以上实施例仅用以说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案精神和范围。
Claims (8)
1.一种褐藻膳食纤维在制备改善代谢综合症的药品、食品和/或保健品中的应用;所述的褐藻膳食纤维由以下方法制备:褐藻干燥粉碎过100-300目筛后用稀酸浸泡,弃去酸液余下藻体,藻体加碱和过氧化氢处理,提取液过滤、超滤、浓缩、在浓缩液中添加浓缩液的0.67-3倍乙醇进行沉淀,经过过滤,沉淀干燥粉碎为可溶性膳食纤维;提取液过滤后的藻渣经蛋白酶处理后干燥得不溶性膳食纤维,两者混合得褐藻总膳食纤维;此膳食纤维中可溶性膳食纤维(SDF)和不溶性膳食纤维(IDF)的比例为1:0.7-1.3,总膳食纤维的含量高于80%:所述褐藻选自海带、裙带菜、马尾藻、墨角藻、鹿角菜、泡叶藻、昆布、枝管藻、鼠尾藻、全缘马尾藻、羊栖菜、硇洲马尾藻、海黍子或海蒿子。
2. 根据权利要求1所述的应用,其特征在于:其中,所用稀酸为0.05-0.3 mol/L柠檬酸、盐酸或硫酸;稀酸加入量为褐藻的10-20倍体积;浸泡时间为0.5-5h;浸泡温度为20-60℃。
3.根据权利要求2所述的应用,其特征在于:其中,所用稀酸为0.1mol/L柠檬酸;稀酸加入量为褐藻的15倍体积;浸泡时间为2h;浸泡温度为50℃。
4. 根据权利要求1所述的应用,其特征在于:其中,所述的碱为含有0.05%-2%过氧化氢的0.05%-3% Na2CO3溶液;所述的碱的加入量为藻体的10-30倍体积;处理温度为40-90℃;处理时间为1-5h。
5.根据权利要求4所述的应用,其特征在于:其中,所述的碱为含有1%过氧化氢的1.5%Na2CO3溶液;所述的碱的加入量为藻体的15倍体积;处理温度为60℃;处理时间为3h。
6.根据权利要求1所述的应用,其特征在于:其中,无水乙醇的添加量为浓缩液的0.67-3倍。
7.根据权利要求1所述的应用,其特征在于:所述的蛋白酶为碱性蛋白酶,用量为蛋白酶与藻渣的比例用量为0.5-2%。
8.根据权利要求1所述的应用,其特征在于:所述改善代谢综合症相关的药品、食品、保健品中添加药学及生物学上可接受的载体或辅料。
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