CN111875715B - 一种脱色枝瑚菌多糖的制备及应用 - Google Patents
一种脱色枝瑚菌多糖的制备及应用 Download PDFInfo
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- CN111875715B CN111875715B CN202010909599.6A CN202010909599A CN111875715B CN 111875715 B CN111875715 B CN 111875715B CN 202010909599 A CN202010909599 A CN 202010909599A CN 111875715 B CN111875715 B CN 111875715B
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
一种脱色枝瑚菌多糖的制备及应用。本发明的脱色枝瑚菌多糖是将含蛋白质、色素及其他杂质的枝瑚菌多糖水提液经W/O胶团萃取分离、透析冻干得到。所述脱色枝瑚菌多糖为淡黄色粉末,质量百分比组成为岩藻糖7.20%,鼠李糖0.10%,阿拉伯糖0.05%,半乳糖27.39%,葡萄糖41.24%,木糖5.34%,甘露糖18.19%和葡萄糖醛酸0.49%,具有较强的α‑淀粉酶抑制能力和DPPH自由基、ABTS自由基清除效果。本发明利用W/O胶团分离枝瑚菌多糖,制备方法快速高效、成本较低,所得脱色枝瑚菌多糖颜色浅、得率较高,利于后续纯化、结构分析和活性鉴定工作,为其后续在日化用品、功能食品及保健药品等领域的应用提供了可能性,社会经济效益显著。
Description
技术领域
本发明涉及食品资源开发与应用以及生物、医药技术领域,具体涉及一种脱色枝瑚菌多糖的制备及应用。
背景技术
枝瑚菌是一种常见的食药用真菌,中医文献记载枝瑚菌具有和胃现气、祛风、破血缓中的作用。此外,部分枝瑚菌对于艾氏癌(EC)和小鼠肉瘤(S-l80)有很强抑制作用。因此,枝瑚菌是我国野生食药用真菌资源中极其宝贵的组成部分,具有较大研究和应用价值。目前对枝瑚菌的研究主要集中在营养价值测定及多酚、黄酮、胡萝卜素等功能成分的分析上,对其大分子活性成分的报道较少。而多糖是食药用真菌中能够起到抗氧化、降血糖、抗肿瘤、抗病毒、抗衰老、免疫调节等功能的重要成分。
虽然已有研究者通过热水浸提法提取并研究枝瑚菌多糖的功能,如王丽娟等发现红顶枝瑚菌(Ramaria botrytoides)多糖具有羟基自由基清除活性(食品工业,2012,33(10):43-46),Li Hua纯化葡萄状顶枝瑚菌(Ramaria botrytis)多糖,并发现其多糖组分RBP-3表现出较强的羟基自由基清除活性,RBP-4具有较强的DPPH自由基清除活性和还原能力(Chemistry Central Journal,2017,11:24)。但由热水浸提法得到的多糖呈深棕褐色,含有较多的蛋白质、多酚等杂质,色素含量较高,不仅干扰其分离纯化、结构分析和活性鉴定,也影响多糖作为日化及食品添加剂、药物或保健品的外观。
目前常用的多糖分离方法有盐析法、柱层析法、制备性区域电泳法、超滤法等。但是此类方法存在步骤繁琐、操作周期长、成本高、产率低、易导致多糖活性降低或变质,不利于产业化等缺点,因此,开发新的多糖分离技术对多糖产业化具有重要意义。
发明内容
本发明目的是克服现有技术存在的上述不足,提供黄枝瑚菌中一种具有降血糖和抗氧化活性的脱色枝瑚菌多糖提取物及其制备方法和应用,该制备方法克服了传统分离方法操作步骤繁琐、周期长、成本高且易使多糖活性降低的缺陷。
本发明利用W/O胶团首次从黄枝瑚菌(Ramaria flava)子实体中提取得到具有良好色泽和透明度的活性多糖,操作简便、耗时较短、得率高、活性保持率较高,对黄枝瑚菌在日化用品、功能食品和保健药品等领域的开发和应用具有重要意义。
本发明的技术方案为:
脱色枝瑚菌多糖,从枝瑚菌子实体中提取得到,为淡黄色粉末,质量百分比组成为岩藻糖7.20%、鼠李糖0.10%、阿拉伯糖0.05%、半乳糖27.39%、葡萄糖41.24%、木糖5.34%、甘露糖18.19%和葡萄糖醛酸0.49%。
所述的枝瑚菌为黄枝瑚菌(Ramaria flava),属于真菌界(Eumycota),担子菌亚门(Basidiomycotina),担子菌纲(Basidiomycetes),非褶菌目(Aphyllophorales),珊瑚菌科(Clavariaceae),枝瑚菌属(Ramaria)。
上述脱色枝瑚菌多糖的制备方法,将含蛋白质、色素及其他杂质的枝瑚菌多糖水提液经W/O胶团萃取分离后得到,分离过程包括如下步骤:
向枝瑚菌多糖水提液中加入离子强度调节剂得到多糖处理液;将多糖处理液与W/O胶团混合,静置2.5h后离心取下层溶液,最后将所得下层溶液透析、冷冻干燥后获得脱色枝瑚菌多糖。
所述离子强度调节剂为NaCl粉末,所加量与多糖水提液中多糖的质量比为3:1~1:1。
所述W/O胶团为表面活性剂在助溶剂的作用下连续分布于有机相中自发形成的纳米级别的微胶团;所述表面活性剂为十六烷基三甲基溴化铵,浓度为100-300mmol/L,助溶剂为正己醇,有机相为异辛烷,二者的体积比为1:2~1:5;多糖处理液与W/O胶团的体积比为3:1~7:1。
其中,枝瑚菌多糖水提液的制备过程如下:
(1)枝瑚菌子实体烘干粉碎后,400目过筛,蒸馏水(质量体积比为1:32~1:35)75~85℃浸提4~6h,抽滤后将所得滤渣在相同条件下继续浸提1次,合并两次滤液,即得枝瑚菌子实体粗提液;
(2)将(1)中枝瑚菌子实体粗提液蒸发浓缩,加入4倍体积的无水乙醇,边加边搅拌,4℃静置过夜后离心收集沉淀,沉淀用蒸馏水复溶后即得枝瑚菌多糖水提液。
本发明中脱色枝瑚菌多糖采用液相色谱法进行单糖组成检测,根据单糖及其衍生物标准品确定脱色枝瑚菌多糖的单糖组成及其质量百分比。
.本发明中脱色枝瑚菌多糖对α-淀粉酶具有较强的抑制作用,可用于制备具有降血糖作用的食品、保健品或药品。
本发明中脱色枝瑚菌多糖对DPPH、ABTS自由基具有一定的清除作用,能显著降低DPPH自由基和ABTS自由基水平,可用于制备具有抗氧化作用的日化用品、食品、保健品或药品。
本发明的优点和有益效果:
(1)本发明中脱色枝瑚菌多糖为天然提取物,具有良好的安全性;
(2)本发明中脱色枝瑚菌多糖有良好的抗氧化和降血糖效果;
(3)本发明中脱色枝瑚菌多糖呈现淡黄色,不含明显的多酚类色素;
(4)本发明采用W/O胶团对枝瑚菌多糖粗提物进行提取分离,不仅分离得到了脱色枝瑚菌多糖,提高了多糖糖含量(82.15%),利于后续纯化、结构分析和活性鉴定工作且脱除大量多酚类色素,脱色率达70.52%,改善了多糖的色泽及透明度,也进一步降低蛋白质含量,清除率达35%。
(5)本发明的提取分离方法富集能力强,准确率高,脱色枝瑚菌多糖得率为0.99%,且制备简单,成本低,适于大规模生产,为其后续在日化用品、功能食品及保健药品等领域的应用提供了可能性,其社会经济效益显著。
附图说明
图1是分离前枝瑚菌多糖水相及W/O胶团分离后枝瑚菌多糖溶液对比图。
图2是标准单糖及其衍生物的液相色谱分析图。
图3是本发明脱色枝瑚菌多糖的液相色谱检测图。
图4是本发明脱色枝瑚菌多糖的傅里叶红外光谱图。
图5是本发明脱色枝瑚菌多糖对α-淀粉酶的抑制作用图。
图6是本发明脱色枝瑚菌多糖对DPPH自由基的清除作用图。
图7是本发明脱色枝瑚菌多糖对ABTS自由基的清除作用图。
具体实施方式
下面结合具体实施例对本发明做进一步的说明,但本发明并不限于此。
实施例1:
脱色枝瑚菌多糖的制备
(1)枝瑚菌子实体50℃烘干,粉碎后400目过筛,用蒸馏水(质量体积比为1:34)83℃浸提5h,抽滤后将所得滤渣在相同条件下继续浸提1次,合并两次滤液,即得枝瑚菌子实体粗提液;
(2)步骤(1)得到的枝瑚菌子实体粗提液经旋转蒸发浓缩后,向其中缓慢加入4倍体积的无水乙醇,边加边搅拌,4℃静置过夜后10000rpm离心10min收集沉淀,用最少体积的蒸馏水溶解沉淀后即得枝瑚菌多糖水提物。
(3)以硫酸-蒽酮法测定枝瑚菌多糖水提物中多糖含量后,将按与枝瑚菌多糖水提物中多糖质量比为1.8:1的比例向步骤(2)得到的枝瑚菌多糖水提物中加入NaCl粉末得到多糖处理液;
(4)按照体积比为3:7的比例混合正己醇和异辛烷,再向其中加入200mmol/L十六烷基三甲基溴化铵,制得W/O胶团,将步骤(3)得到的多糖处理液与W/O胶团按5:1体积比剧烈混合,静置2.5h后5000rpm离心10min收集下层溶液,分离效果如图1,然后将溶液转入截留分子量为3500Da的透析袋中4℃透析48h,冷冻干燥处理后即为本发明所述的脱色枝瑚菌多糖。
实施例2
脱色枝瑚菌多糖的制备
本实施例与实施例1相比,步骤(1)中枝瑚菌子实体粉末与蒸馏水的质量体积比为1:32,75℃热水浸提4h;步骤(3)中NaCl粉末与枝瑚菌多糖的质量比为1:1;步骤(4)中W/O胶团中正己醇和异辛烷体积比为1:2,十六烷基三甲基溴化铵浓度为100mmol/L,多糖水相与W/O胶团按3:1体积比剧烈混合。
其他条件同实施例1。
实施例3
脱色枝瑚菌多糖的制备
本实施例与实施例1相比,步骤(1)中枝瑚菌子实体粉末与蒸馏水的质量体积比为1:35,85℃热水浸提6h;步骤(3)中NaCl粉末与枝瑚菌多糖的质量比为3:1;步骤(4)中W/O胶团中正己醇和异辛烷体积比为1:5,十六烷基三甲基溴化铵浓度为300mmol/L,多糖水相与W/O胶团按7:1体积比剧烈混合。
其他条件同实施例1。
实施例4
脱色枝瑚菌多糖的制备
本实施例与实施例1相比,步骤(1)中枝瑚菌子实体粉末与蒸馏水的质量体积比为1:33,80℃热水浸提5h;步骤(3)中NaCl粉末与枝瑚菌多糖的质量比为2:1;步骤(4)中W/O胶团中正己醇和异辛烷体积比为1:4,,十六烷基三甲基溴化铵浓度为200mmol/L,多糖水相与W/O胶团按5:1体积比剧烈混合。
其他条件同实施例1。
实施例5
脱色枝瑚菌多糖的成分组成测定
取干净的15mLEP管,加入8mL无菌水,依次加入岩藻糖、鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖、半乳糖醛酸、葡萄糖醛酸、甘露糖醛酸各100mg,溶解后定容至10mL,配制成10mg/mL的标准液母液。将上述混标母液先稀释100倍,制备成100μg/mL工作液,将工作液梯度稀释后进行液相色谱检测,其中的色谱参数为:色谱系统采用的是Thermo ICS5000+离子色谱系统(Thermo Fisher Scientific,USA),采用DionexTMCarboPacTM PA20液相色谱柱,进样量为20μL。流动相为97.5%H2O–2.5%100mM NaOH溶液,流速为0.5mL/min,柱温为30℃,利用电化学检测器对单糖组分进行分析检测。以单糖标准品的量为横坐标,以单糖及其衍生物标准品的峰面积为纵坐标绘制标准曲线,获得拟合方程,标准单糖及其衍生物的出峰时间及拟合方程见表1,液相色谱分析见图2。
表1标准单糖及其衍生物的出峰时间及拟合方程
同时,称取5mg脱色枝瑚菌多糖于色谱瓶中,加入1mL浓度为2mol/L的三氟乙酸溶液,于121℃加热2小时;向色谱瓶中通入氮气,吹干溶液后向瓶中加入0.5mL甲醇溶解固形物,再用氮气吹干;甲醇重复清洗2-3次后将反应后的产物用1mL无菌水溶解,并用0.22μm滤膜过滤后进行液相色谱检测,根据出峰时间确定脱色枝瑚菌多糖的单糖组成类型,再根据峰面积通过拟合方程计算其浓度。脱色枝瑚菌多糖的单糖及其组成结果见表2,液相色谱检测见图3,实验结果表明,脱色枝瑚菌多糖的质量百分比组成为岩藻糖7.20%,鼠李糖0.10%,阿拉伯糖0.05%,半乳糖27.39%,葡萄糖41.24%,木糖5.34%,甘露糖18.19%和葡萄糖醛酸0.49%。
表2脱色枝瑚菌多糖的单糖组成及其浓度
实施例6
脱色枝瑚菌多糖的红外光谱分析
称取脱色枝瑚菌多糖粉末1~2mg与干燥溴化钾粉末混合,研细后,用压片机压成1mm薄片用于红外光谱分析,红外扫描范围为4000cm-1-500cm-1。如图4所示,脱色枝瑚菌多糖在3440cm-1附近出现强O-H伸缩振动吸收峰,2931cm-1附近出现强C-H伸缩振动吸收峰,是典型的多糖特征吸收峰。1650cm-1附近是C=O的伸缩振动吸收峰,1573cm-1附近是N-H的缩振动吸收峰,这两个特征吸收峰说明脱色枝瑚菌多糖中可能含有酰胺键。1070cm-1附近是的C-O-C糖化键振动和C-O-H拉伸振动重叠的吡喃糖环振动,说明脱色枝瑚菌多糖中含有吡喃糖环。
实施例7
脱色枝瑚菌多糖的降血糖活性鉴定
将不同浓度梯度(0.5mg/mL~2mg/mL)的脱色枝瑚菌多糖溶液与0.25mg/mL的α-淀粉酶溶液(2.5U/mL)等体积混匀,37℃孵育10min;加入等体积1%的淀粉溶液并混匀,37℃再次孵育30min;加入2倍淀粉溶液体积的DNS试剂,混匀后100℃水浴10min,检测反应体系OD540值,根据如下公式计算脱色枝瑚菌多糖对α-淀粉酶的抑制率,α-淀粉酶抑制率(%)==[1-(A3-A4)/(A1-A2)]×100%。其中A1为无抑制剂的正对照组,A2为无抑制剂无α-淀粉酶的空白对照组,A3为实验组,A4为无α-淀粉酶的阴性对照组。该反应体系以阿卡波糖为阳性对照。如图5所示,脱色枝瑚菌多糖对α-淀粉酶的抑制作用呈明显的剂量效应,当脱色枝瑚菌多糖作用浓度为2mg/mL时,对α-淀粉酶的抑制率为31.85±6.89%,是正对照阿卡波糖的39.08%。
实施例8
脱色枝瑚菌多糖的抗氧化活性鉴定
(1)脱色枝瑚菌多糖的DPPH自由基清除活性
将不同浓度梯度(125μg/mL~1000μg/mL)的脱色枝瑚菌多糖溶液与0.1mmol/L的DPPH-甲醇溶液等体积混匀,避光静置30min后检测反应体系OD570值,根据如下公式计算脱色枝瑚菌多糖对DPPH自由基的清除率,DPPH自由基清除率%=[1-(Ai-Ai0)/A0]×100%。其中A0为蒸馏水空白组,Ai0为无DPPH的对照组,Ai为实验组。该反应体系以维生素C为阳性对照。如图6所示,脱色枝瑚菌多糖对DPPH自由基的清除效果呈明显的剂量效应,当脱色枝瑚菌多糖作用浓度为1000μg/mL时,对DPPH自由基的清除率为76.39±2.04%,其IC50值为451.76±0.43μg/mL。
(2)脱色枝瑚菌多糖的ABTS自由基清除活性
将7.4mmol/L的ABTS液与2.6mmol/L的K2S2O8液等体积混合,摇匀后于室温、黑暗环境静置12h,将溶液稀释至OD734=0.7制成ABTS+·工作液;将ABTS+·工作液与不同浓度梯度(125μg/mL~1000μg/mL)的脱色枝瑚菌多糖溶液以4:1的体积比混合摇匀后,检测反应体系OD700值,并根据公式ABTS自由基清除率(%)=[1-(An-An0)/A0]×100%。计算脱色枝瑚菌多糖对ABTS自由基的清除活性,其中,A0为蒸馏水空白组,An0为无ABTS+·工作液的对照组,An为实验组。该反应体系以维生素C为阳性对照。如图7所示,脱色枝瑚菌多糖样品浓度在125μg/mL至1000μg/mL范围内,对ABTS自由基的清除活性与多糖浓度呈正相关,当作用浓度为1000μg/mL时,对ABTS自由基的清除率为98.60±0.36%,其IC50值为378.56±0.05μg/mL。
Claims (3)
1.一种脱色枝瑚菌多糖在制备具有α-淀粉酶抑制作用的食品、保健品或药品中的用途,其特征在于,所述脱色枝瑚菌多糖从枝瑚菌子实体中提取得到,为淡黄色粉末,由岩藻糖,鼠李糖,阿拉伯糖,半乳糖,葡萄糖,木糖,甘露糖和葡萄糖醛酸组成;所述的枝瑚菌为黄枝瑚菌(Ramaria flava),属于真菌界(Eumycota),担子菌亚门(Basidiomycotina),担子菌纲(Basidiomycetes),非褶菌目(Aphyllophorales),珊瑚菌科(Clavariaceae),枝瑚菌属(Ramaria);
所述脱色枝瑚菌多糖是将含蛋白质、色素及其他杂质的枝瑚菌多糖水提液经W/O胶团萃取分离后得到,所述W/O胶团为表面活性剂在助溶剂的作用下连续分布于有机相中自发形成的纳米级别的微胶团;分离过程包括如下步骤:
向枝瑚菌多糖水提液中加入离子强度调节剂得到多糖处理液;将多糖处理液与W/O胶团混合,静置后离心取下层溶液,最后将所得下层溶液透析、冷冻干燥后获得脱色枝瑚菌多糖;
其中,枝瑚菌多糖水提液的制备过程如下:
(1)枝瑚菌子实体烘干粉碎后,400目过筛,用蒸馏水75~85 ℃浸提4~6 h,抽滤后将所得滤渣在相同条件下继续浸提1次,合并两次滤液,即得枝瑚菌子实体粗提液;
(2)将(1)中枝瑚菌子实体粗提液蒸发浓缩,加入4倍体积的无水乙醇,边加边搅拌,4℃静置过夜后离心收集沉淀,沉淀用蒸馏水复溶后即得枝瑚菌多糖水提液。
2.根据权利要求1所述的用途,其特征在于,所述脱色枝瑚菌多糖的质量百分比组成为岩藻糖7.20%,鼠李糖0.10%,阿拉伯糖0.05%,半乳糖27.39%,葡萄糖41.24%,木糖5.34%,甘露糖18.19%和葡萄糖醛酸0.49%。
3.根据权利要求1至2任一项所述的用途,其特征在于,所述离子强度调节剂为NaCl粉末,所加量与多糖水提液中多糖的质量比为3:1~1:1;
所述表面活性剂为十六烷基三甲基溴化铵,浓度为100-300 mmol/L,助溶剂为正己醇,有机相为异辛烷,二者的体积比为1:2~1:5;多糖处理液与W/O胶团的体积比为3:1~7:1。
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