CN109053925B - 一种碱性黄秋葵多糖的分离提取方法及其用途 - Google Patents
一种碱性黄秋葵多糖的分离提取方法及其用途 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
本发明公开了一种碱性黄秋葵多糖的分离提取方法及其用途,以市售新鲜黄秋葵嫩果为原料,经热风干燥并粉碎后,称取适量的黄秋葵干粉,在一定的碱性pH值和适当的温度下进行多糖的提取、分离。黄秋葵作为一种种植广泛的蔬菜,相比较传统的中药,价格低廉,得到的黄秋葵多糖对人体无副作用。同时,相比较现有调节肠道益生菌群保健品,黄秋葵多糖不会产生腹泻,克服了来源于牛乳的低聚糖产品的缺陷。
Description
技术领域
本发明涉及一种碱性黄秋葵多糖的分离提取方法及其用途,属于农产品加工技术领域。
背景技术
黄秋葵Abelmoschus esculentus(L.)Moench,又名秋葵、羊角豆,是锦葵科秋葵属一年生草本植物。原本产于印度,现已引进我国广泛种植。黄秋葵通常是作为时令蔬菜,它的幼苗、叶、芽、花、果、种子均可食用。但由于其富含丰富的营养物质,如蛋白质、脂肪、碳水化合物、膳食纤维、维生素和矿物质等化合物,因此也作为保健品原料。黄秋葵的嫩果部分含有大量的粘液物质,有助于肠胃消化,具有开发成为调节肠道功能保健品的潜力。
目前关于黄秋葵多糖活性的研究,大多偏向于抗疲劳、降血糖、降血脂以及免疫调节等方面,关于黄秋葵多糖对调节肠道益生菌群功能的影响尚未见报道。而根据现有研究,已发现多种植物多糖具有调节肠道菌群稳定的功能,且不会产生如源于牛乳的低聚糖产品的腹泻副作用。因此分离出一种具有调节肠道功能的黄秋葵多糖组分,有助于开发利用黄秋葵资源,增加它的经济效益。
发明内容
本发明的目的是提供一种碱性黄秋葵多糖的分离提取方法及其用途。本发明碱性黄秋葵多糖具有调节肠道益生菌群的功能,本发明制备方法简单易行,原料利用率高,适合工业化生产。
本发明碱性黄秋葵多糖的分离提取方法,包括如下步骤:
步骤1:预处理
取新鲜的黄秋葵嫩果,洗净、去蒂、去籽,干燥后粉碎,过60目筛,得到黄秋葵干粉;取500g获得的黄秋葵干粉,浸泡于4~5倍体积的无水乙醇中,4℃静置24h,抽滤后收集滤饼;
步骤2:回流提取
将步骤1得到的滤饼置于pH值为8.5的氢氧化钠溶液中,加热提取两次,离心后合并上清液;
步骤3:浓缩醇沉
将步骤2获得的上清液浓缩至原体积的1/5~1/10,向浓缩液中加入4倍体积的无水乙醇,搅拌均匀后于4℃下静置8~12h使其充分沉淀,离心后收集沉淀物;
步骤4:除蛋白
用少量蒸馏水将沉淀物溶解,用Sevag法除去溶液中的蛋白质直至紫外检测无280nm处的蛋白吸收峰,对去除蛋白后的溶液进行浓缩和透析;
步骤5:分级
首先将步骤4得到的溶液5000g进行离心或者过滤处理,然后上DEAE cellulose-52柱,分别用水和0.3mol/L氯化钠溶液进行洗脱,至苯酚-硫酸法显色无黄色出现,收集洗脱液,将所得洗脱液浓缩至原体积的1/10~1/20;
步骤6:纯化
将步骤5得到的浓缩液5000~6000g离心,然后于葡聚糖凝胶G-150色谱柱中洗脱,洗脱液为水,用苯酚-硫酸法检测洗脱后的样品,收集多糖含量最高(吸光度值最大)的洗脱组分,经72h蒸馏水透析、浓缩、冷冻干燥后得到黄秋葵多糖。
步骤2中,滤饼与氢氧化钠溶液的料液比为1g:20ml(m:V)。
步骤2中,加热提取温度为80~90℃,每次提取时间为2~3h。
步骤2中,离心条件为3500~4000r/min,时间10~15min。
步骤3中,离心条件为3500~4000r/min,时间10~15min。
步骤4中,透析时透析袋的截留分子量为3500Da。
步骤6中,冷冻干燥条件为-40℃冷冻干燥15h。
本发明获得的黄秋葵多糖的重均分子量为9.79×104Da。
本发明获得的碱性黄秋葵多糖的用途,是在制备调节肠道益生菌群功能产品中的应用。
本发明与现有技术相比,其有益效果体现在:
1、黄秋葵多糖中的果胶含量较高,且具有分子量大、粘度大、水溶性差等不利于提取的特点,在碱性条件下提取可以减小果胶类多糖的分子量,而对多糖的其他品质、性质影响较小。
2、本发明操作简单易行,成本低廉却能大幅度提高多糖的提取率,能为企业带来经济效益,适合大规模推广。
3、相比较现有肠道菌群保健品,黄秋葵多糖克服了来源于牛乳的低聚糖产品带来的腹泻缺陷。
具体实施方式
下面结合具体的实施例对本发明技术方案做详细说明,这些实施例仅用来说明本发明,并不限制本发明的范围。在不脱离本发明构思的前提下,做出的相关调整和改进,均属于本发明的保护范围。
实施例1:
1、取黄秋葵干粉500g,用2500ml无水乙醇浸泡过夜(脱脂),过滤掉乙醇,用5000mlpH值8.5的氢氧化钠溶液在90℃下回流提取2次,每次2小时,将所得的水提液在4000r/min下离心15min,合并离心后的上清液;
2、将所得上清液于55℃旋转蒸发浓缩至500ml,用4倍体积的无水乙醇进行醇沉,4℃静置12小时,4000r/min离心10min,对得到的沉淀物进行-40℃冷冻干燥15h得到黄秋葵粗多糖;
3、将步骤2得到的黄秋葵粗多糖用1000ml蒸馏水复溶,用5倍体积的氯仿与1倍体积的正丁醇混匀后配制成Sevag试剂对溶液除蛋白,反复6次直至紫外检测无280nm处的蛋白吸收峰;将除蛋白后的溶液浓缩至200ml,用蒸馏水透析72h;
4、将步骤3所得透析液3500r/min离心10min,取上清液上DEAE cellulose-52柱进行分级纯化,分别用水和0.3mol/L氯化钠溶液进行洗脱,至苯酚-硫酸法显色无黄色出现,收集洗脱液,将其浓缩至原体积的1/10;
5、将步骤4得到的浓缩液进行透析除盐,4000r/min离心10分钟后,取上清液上葡聚糖凝胶G-150色谱柱,用水以流速0.5ml/min洗脱,收集洗脱液,透析后减压浓缩至20ml,-40℃冷冻干燥15h,得到黄秋葵多糖2g。
实施例2:
1、取黄秋葵干粉500g,用2000ml无水乙醇浸泡过夜(脱脂),过滤掉乙醇,用7500mlpH值8.5的氢氧化钠溶液在85℃下回流提取2次,每次3小时,将所得的水提液在4000r/min下离心15min,合并离心后的上清液;
2、将所得上清液于55℃旋转蒸发浓缩至750ml,用4倍体积的无水乙醇进行醇沉,4℃静置12小时,4000r/min离心10min,对得到的沉淀物进行-40℃冷冻干燥15h得到黄秋葵粗多糖;
3、将步骤2得到的黄秋葵粗多糖用1500ml蒸馏水复溶,用5倍体积的氯仿与1倍体积的正丁醇混匀后配制成Sevag试剂对溶液除蛋白,反复6次直至紫外检测无280nm处的蛋白吸收峰;将除蛋白后的溶液浓缩至500ml,用蒸馏水透析72h;
4、将步骤3所得透析液3500r/min离心10min,取上清液上DEAE cellulose-52柱进行分级纯化,分别用水和0.2mol/L氯化钠溶液进行洗脱,至苯酚-硫酸法显色无黄色出现,收集洗脱液,将其浓缩至原体积的1/10;
5、将步骤4得到的浓缩液进行透析除盐,4000r/min离心10分钟后,取上清液上葡聚糖凝胶G-150色谱柱,用水以流速1ml/min洗脱,收集洗脱液,透析后减压浓缩至50ml,-40℃冷冻干燥15h,得到黄秋葵多糖3.6g。
实施例3:
1、取黄秋葵干粉500g,用2500ml无水乙醇浸泡过夜(脱脂),过滤掉乙醇,用8000mlpH值8.5的氢氧化钠溶液在85℃下回流提取3次,每次3小时,将所得的水提液在4000r/min下离心15min,合并离心后的上清液;
2、将所得上清液于55℃旋转蒸发浓缩至800ml,用4倍体积的无水乙醇进行醇沉,4℃静置12小时,4000r/min离心10min,对得到的沉淀物进行-40℃冷冻干燥15h得到黄秋葵粗多糖;
3、将步骤2得到的黄秋葵粗多糖用1500ml蒸馏水复溶,用5倍体积的氯仿与1倍体积的正丁醇混匀后配制成Sevag试剂对溶液除蛋白,反复6次直至紫外检测无280nm处的蛋白吸收峰;将除蛋白后的溶液浓缩至300ml,用蒸馏水透析72h;
4、将步骤3所得透析液3500r/min离心10min,取上清液上DEAE cellulose-52柱进行分级纯化,分别用水和0.5mol/L氯化钠溶液进行洗脱,至苯酚-硫酸法显色无黄色出现,收集洗脱液,将其浓缩至原体积的1/10;
5、将步骤4得到的浓缩液进行透析除盐,4000r/min离心10分钟后,取上清液上葡聚糖凝胶G-150色谱柱,用水以流速1ml/min洗脱,收集洗脱液,透析后减压浓缩至30ml,-40℃冷冻干燥15h,得到黄秋葵多糖4.2g。
以下通过试验例进一步说明本发明所述黄秋葵多糖的物理化学性质和对肠道的调节功能。
试验例1:黄秋葵多糖组分的纯度及分子量的测定
仪器:Agilent 1260高校液相色谱,Agilent 1260 Infinity蒸发光检测器;色谱柱:TSK-GEL G4000 PWXL(300×7.8mm);流动相:纯水;流速:1ml/min;柱温:30℃;漂移管温度:110℃;标准品:葡聚糖T系列T-3,T-10,T-70,T-100,T-200。以保留时间t为横坐标,标准葡聚糖分子量的对数lgMw为纵坐标,得到标准曲线方程为:lgMw=-0.5539t+9.474。
分别将实施例1、2和3得到的黄秋葵多糖样品进行分子量测定,保留时间分别为8.075,8.116和8.091min,均为单一对称峰,计算可得黄秋葵多糖的重均分子量平均为9.79×104Da。
试验例2:黄秋葵多糖组分的单糖组成分析
精密称取实施例3得到的黄秋葵多糖10mg,加入5ml 2mol/L的三氟乙酸,充氮气排尽空气后,封管,110℃水解8h。将水解液在50℃下浓缩,用甲醇除去三氟乙酸,蒸干后水解产物用蒸馏水复溶至1ml。
向复溶后的1ml样品中加入1ml的0.6mol/L的氢氧化钠溶液,充分混匀后,加入2ml0.5mol/L的PMP甲醇溶液,封管混匀后,70℃水浴100min,取出冷却后,加入2ml 0.3mol/L的盐酸溶液中和氢氧化钠,加入14ml去离子水以及20ml氯仿萃取,除去有几层,对得到的上层水相再萃取2次,得到PMP衍生化样品。单糖标准品则为:鼠李糖,甘露糖,葡萄糖,半乳糖,果糖,阿拉伯糖,木糖,葡萄糖醛酸,半乳糖醛酸。衍生化处理方法与样品处理方法相同。
HPLC检测条件:流动相A为磷酸盐缓冲液(pH 6.7),流动相B为乙腈(A:B=80:20,V/V),柱温为20℃,色谱柱为C18柱,检测器波长254nm,进样量20μL。
根据液相结果,得到实施例3中的黄秋葵多糖单糖组成及摩尔比为甘露糖:鼠李糖:半乳糖醛酸:半乳糖=0.84:3.12:2.15:5.89。
实验例3:黄秋葵多糖组分对正常小鼠肠道菌群的作用
实验动物:雄性昆明小鼠,体重18~22g,由安徽中医药大学提供。
实验方法:将40只小鼠平均分成4组:空白对照组,黄秋葵粗多糖组,黄秋葵多糖组,阳性对照组。饲喂1周基础饲料后,空白对照组灌胃的是生理盐水,阳性对照组灌胃的是金百通的低聚果糖口服液,黄秋葵粗多糖组灌胃的是提取的粗多糖,黄秋葵多糖组灌胃的是纯化后的多糖。实验组灌胃的剂量为200mg/kg。灌胃20天,每3天收集一次小鼠粪便,称量一次小鼠体重和摄食量。小鼠粪便用来检测短链脂肪酸含量和主要营养物质的表观吸收率。实验结束时于无菌条件下取小鼠盲肠内容物,采用高通量测序对盲肠内容物进行菌群组成分析。表1为小鼠灌胃期间的体重,表2为小鼠摄食量和主要营养物质表观吸收率,表3为小鼠短链脂肪酸含量,表4为基因水平下的物种丰度。
由表1结果可知,与空白对照组相比,灌胃前后小鼠的体重没有明显变化(P>0.05)。说明饲喂黄秋葵多糖不会显著促进或抑制小鼠体重的增长。同时灌胃期间未见小鼠出现腹泻现象,未有副作用出现。
注:与空白对照组比较*P<0.05,**P<0.01。
由表2结果可知,与空白对照组相比,实验组碳水化合物和蛋白质的表观吸收率没有显著提高(P>0.05),但是脂肪的表观吸收率有明显降低(P<0.01),说明黄秋葵多糖能够减少脂肪的吸收。实验组的摄食量相比较空白组,也有明显提高(P<0.05),说明多糖能够促进小鼠摄食量。同时相比较粗多糖组,纯化后的多糖组的实验结果更趋近于阳性组。
注:与空白对照组比较**P<0.01。
由表3结果可知,小鼠在未灌胃多糖的时候,各组之间总短链脂肪酸含量无显著差异。当灌胃了多糖以后,相比较空白对照组,实验组小鼠的总短链脂肪酸含量开始提高,当灌胃结束后,实验组的含量相比较空白组,得到显著提高(P<0.01)。这是因为当机体进食多糖以后,部分不能被消化的多糖会经微生物进行发酵生成短链脂肪酸。在肠道功效方面,短链脂肪酸能降低肠道pH,使肠道中的游离氨浓度下降,有利于粪便中有害物质的排出,从而保持肠道健康。
注:与空白对照组比较**P<0.01。
由表4结果可知,相比较空白组,实验组的乳酸菌(Lactobacillus)丰度有显著提高(P<0.01),拟杆菌(Bacteroides)的丰度则显著下降(P<0.01)。双歧杆菌(Bifidobacterium)、肠球菌(Enterococcus)和大肠杆菌(Escherichia)则无明显差异(P>0.05)。根据《保健食品检测与评价技术标准》中有关调节肠道菌群实验的规定,可以发现黄秋葵多糖具有调节肠道菌群的功能,且在实验过程中未发现副作用。说明以天然植物为原料的保健品,安全度更高。
以上显示和描述了本发明的基本原理、具体实施方式及实验。从事本行业的人员应该了解,这些实施例和说明书中描述的仅用来说明本发明,并不限制本发明的范围,在不脱离本发明实质的构思的前提条件下,还可以做出若干调整或改进,均属于本发明的保护范围。
Claims (8)
1.一种碱性黄秋葵多糖的用途,其特征在于:所述碱性黄秋葵多糖在制备调节肠道益生菌群功能产品中的应用;
所述碱性黄秋葵多糖是通过包括如下步骤的方法分离提取获得:
步骤1:预处理
取新鲜的黄秋葵嫩果,洗净、去蒂、去籽,干燥后粉碎,过60目筛,得到黄秋葵干粉;取500 g获得的黄秋葵干粉,浸泡于4~5倍体积的无水乙醇中,4℃静置24h,抽滤后收集滤饼;
步骤2:回流提取
将步骤1得到的滤饼置于pH值为8.5的氢氧化钠溶液中,加热提取两次,离心后合并上清液;
步骤3:浓缩醇沉
将步骤2获得的上清液浓缩至原体积的1/5~1/10,向浓缩液中加入4倍体积的无水乙醇,搅拌均匀后于4℃下静置8~12h使其充分沉淀,离心后收集沉淀物;
步骤4:除蛋白
用少量蒸馏水将沉淀物溶解,用Sevag法除去溶液中的蛋白质直至紫外检测无280nm处的蛋白吸收峰,对去除蛋白后的溶液进行浓缩和透析;
步骤5:分级
首先将步骤4得到的溶液5000g进行离心或者过滤处理,然后上DEAE cellulose-52柱,分别用水和0.5mol/L氯化钠溶液进行洗脱,至苯酚-硫酸法显色无黄色出现,收集洗脱液,将所得洗脱液浓缩至原体积的1/10~ 1/20;
步骤6:纯化
将步骤5得到的浓缩液5000~ 6000g 离心,然后于葡聚糖凝胶G-150色谱柱中洗脱,洗脱液为水,用苯酚-硫酸法检测洗脱后的样品,收集多糖含量最高的洗脱组分,经72h蒸馏水透析、浓缩、冷冻干燥后得到黄秋葵多糖。
2.根据权利要求1所述的用途,其特征在于:
步骤2中,滤饼与氢氧化钠溶液的料液比为1g:20ml。
3.根据权利要求1所述的用途,其特征在于:
步骤2中,加热提取温度为80~90℃,每次提取时间为2~3h。
4.根据权利要求1所述的用途,其特征在于:
步骤2中,离心条件为3500~4000 r/min,时间10~15 min。
5.根据权利要求1所述的用途,其特征在于:
步骤3中,离心条件为3500~4000 r/min,时间10~15 min。
6.根据权利要求1所述的用途,其特征在于:
步骤4中,透析时透析袋的截留分子量为3500 Da。
7.根据权利要求1所述的用途,其特征在于:
步骤6中,冷冻干燥条件为-40℃冷冻干燥15h。
8.根据权利要求1所述的用途,其特征在于:
所述黄秋葵多糖的重均分子量为9.79×104 Da。
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