CN113652461A - 一种黄精植物甾醇多糖蛋白的制备方法及其活性制品 - Google Patents
一种黄精植物甾醇多糖蛋白的制备方法及其活性制品 Download PDFInfo
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Abstract
本发明公开一种黄精植物甾醇多糖蛋白的制备方法，以黄精植物发酵物进入肠胃道后的肝肠循环吸收后，由下腔动脉路径进入泌尿道系统血管循环引发的局部作用。为此，本发明所要解决的技术问题在于现有发酵技术中从发酵物分离多糖蛋白，并解决提取率较低，且生物活性不高的问题，同时，本发明还公开一种黄精植物甾醇多糖蛋白的活性制品，其具有促骨盆底骨骼肌强度，及抗血管收缩功效。
Description
技术领域
本发明涉及天然植物提取物的技术领域，尤其涉及一种黄精植物甾醇多糖蛋白的制备方法及其活性制品。
背景技术
黄精(Polygonatum sibiricum Red.)为百合科植物的干燥根茎。黄精以根茎入药。具有补气養陰，健脾，润肺，益肾功能。用于治疗脾胃虚弱，体倦乏力，口干食少，肺虚燥咳，精血不足，内热消渴等症。性味甘,平，归肺，脾，肾经。用于阴虚肺燥，干咳少痰，及肺肾阴虚的劳嗽久咳，用于脾胃虚弱，既补脾阴，又益脾气，用于肾虚精亏得头晕，腰膝酸软，须发早白及消渴等。
发明内容
本发明的一个目的在于：提供一种黄精植物甾醇多糖蛋白的制备方法及其活性制品，用以解决现有技术存在的问题。
为达此目的，本发明采用以下技术方案：
一种黄精植物甾醇多糖蛋白的制备方法，包括以下步骤：
(1)预处理：取新鲜黄精根部，去除黄精表面的附着物，称重(湿重)并作为计量依据，将黄精切成丁状，粉碎后的黄精中加入4-8倍重量的水，使用匀浆机或胶体磨将粉碎的黄精顶制作成匀浆状；
(2)一次提取：采用过滤的方法将步骤(1)获得的黄精匀浆进行过滤，分别收集滤饼和滤液，滤液为第一次提取液；
(3)二次提取：将步骤(2)获得的黄精滤饼重新悬浮在3-5倍重量的水中，调节溶液pH至5.0-7.0，过滤后收集滤液，为第二次提取液；
(4)酶解：合并两次提取的滤液，加入0.1-5.0％黄精重量的复合酶解物，在30-50℃范围内搅拌6-12h；复合酶解物是由质量比为1-3：0.5-2的纤维素酶、木瓜蛋白酶组成。
(5)沉淀处理：将步骤(4)获得的酶解液调节pH至3.0-4.5并加入硫酸盐溶液，搅拌均匀，静置后使用离心法将滤液进行处理，收集滤液；
(6)干燥：将步骤(5)获得的滤液进行超滤处理，收集截留液，喷雾干燥，获得的粉末即为粗黄精多糖蛋白组合物。
作为一种优选的技术方案，在所述步骤(4)中，所述复合酶解物发酵产物的制备方法为：取经过清洗的菠萝原料，设定酶解条件为料液比1∶10(m/V)、纤维素酶CellulaseACCF-4740添加量2％、温度55℃、pH4.5、反应时间2.5h，进行酶解，纤维素酶水解结束后，沸水浴灭酶15min，9000r/min离心，收集上清液得到菠萝水解液；菠萝酶解液灭酶活后，调节pH值至6.2-6.6，高压蒸汽灭菌温度为121℃，时间为20min，然后接种体积分数1％的MRS培养基活化的戊糖片菌液到菠萝酶解液，37℃恒温培养箱静置，进行发酵；发酵结束后，9000r/min离心，收集上清液得到复合酶解物。
作为一种优选的技术方案，在所述步骤(4)中，酶解的原料是经过一次和二次提取后滤液的合并物。
作为一种优选的技术方案，在所述步骤(4)中，黄精多糖蛋白的制备方法为：提取液，4℃下提取44-50h，过滤，按0.5-1.5g/mL的添加量向滤液中缓慢添加硫酸铵，添加硫酸铵过程中不断搅拌，防止硫酸铵局部过饱和，然后静置45-50h，4℃条件下25000g离心5-10min，沉淀用酸性缓冲液复溶，4℃下分子量5000Da的透析袋透析，透析液经20000g离心10min，取上清液即为制备的扩张蛋白溶液；
其中，匀浆缓冲液组分为：25mmol/L 4-羟乙基哌嗪乙磺酸，1.5mmol/L焦亚硫酸钠，2mmol/L EDTA，0.1wt％TritonX-100，pH7.0；提取液组分为：15mmol/L 4-羟乙基哌嗪乙磺酸，1.0mmol/L乙二胺四乙酸，1.5mmol/L焦亚硫酸钠，0.5mol/L NaCl，pH6.0；酸性缓冲液配制是：将2.5g醋酸钠溶于水中，用冰醋酸调节pH至4.0，水定容至1L。
作为一种优选的技术方案，在所述步骤(5)中，向溶液中加入的硫酸盐，可以是硫酸铁、硫酸锌或硫酸钠或混合使用。
一种黄精植物甾醇多糖蛋白的活性制品，包括以下重量份数计组分：马卡酵素提取物10-20份、黄精酵素提取物20-48份、枸杞酵素提取物15-25份、山药酵素提取物15-20份、啤酒酵母提取物3-8份、海藻糖提取物3-8份、南瓜子提取物15-30份、牛磺酸提取物4-8份、L-精氨酸提取物5-10份、麦芽糊精13-18份、硬脂酸镁0.2-1份。
作为一种优选的技术方案，所述活性制品的浓度为65％-88％。
本发明的有益效果为：公开一种黄精植物甾醇多糖蛋白的制备方法，以黄精植物发酵物进入肠胃道后的肝肠循环吸收后，由下腔动脉路径进入泌尿道系统血管循环引发的局部作用。为此，本发明所要解决的技术问题在于现有发酵技术中从发酵物分离多糖蛋白，并解决提取率较低，且生物活性不高的问题，同时，本发明还公开一种黄精植物甾醇多糖蛋白的活性制品，其具有促骨盆底骨骼肌强度，及抗血管收缩功效。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。
一种黄精植物甾醇多糖蛋白的制备方法，包括以下步骤：
(1)预处理：取新鲜黄精根部，去除黄精表面的附着物，称重(湿重)并作为计量依据，将黄精切成丁状，粉碎后的黄精中加入4-8倍重量的水，使用匀浆机或胶体磨将粉碎的黄精顶制作成匀浆状；
(2)一次提取：采用过滤的方法将步骤(1)获得的黄精匀浆进行过滤，分别收集滤饼和滤液，滤液为第一次提取液；
(3)二次提取：将步骤(2)获得的黄精滤饼重新悬浮在3-5倍重量的水中，调节溶液pH至5.0-7.0，过滤后收集滤液，为第二次提取液；
(4)酶解：合并两次提取的滤液，加入0.1-5.0％黄精重量的复合酶解物，在30-50℃范围内搅拌6-12h；复合酶解物是由质量比为1-3：0.5-2的纤维素酶、木瓜蛋白酶组成。
(5)沉淀处理：将步骤(4)获得的酶解液调节pH至3.0-4.5并加入硫酸盐溶液，搅拌均匀，静置后使用离心法将滤液进行处理，收集滤液；
(6)干燥：将步骤(5)获得的滤液进行超滤处理，收集截留液，喷雾干燥，获得的粉末即为粗黄精多糖蛋白组合物。
在所述步骤(4)中，所述复合酶解物发酵产物的制备方法为：取经过清洗的菠萝原料，设定酶解条件为料液比1∶10(m/V)、纤维素酶CellulaseACCF-4740添加量2％、温度55℃、pH4.5、反应时间2.5h，进行酶解，纤维素酶水解结束后，沸水浴灭酶15min，9000r/min离心，收集上清液得到菠萝水解液；菠萝酶解液灭酶活后，调节pH值至6.2-6.6，高压蒸汽灭菌温度为121℃，时间为20min，然后接种体积分数1％的MRS培养基活化的戊糖片菌液到菠萝酶解液，37℃恒温培养箱静置，进行发酵；发酵结束后，9000r/min离心，收集上清液得到复合酶解物。
在所述步骤(4)中，酶解的原料是经过一次和二次提取后滤液的合并物。
在所述步骤(4)中，黄精多糖蛋白的制备方法为：提取液，4℃下提取44-50h，过滤，按0.5-1.5g/mL的添加量向滤液中缓慢添加硫酸铵，添加硫酸铵过程中不断搅拌，防止硫酸铵局部过饱和，然后静置45-50h，4℃条件下25000g离心5-10min，沉淀用酸性缓冲液复溶，4℃下分子量5000Da的透析袋透析，透析液经20000g离心10min，取上清液即为制备的扩张蛋白溶液；
其中，匀浆缓冲液组分为：25mmol/L 4-羟乙基哌嗪乙磺酸，1.5mmol/L焦亚硫酸钠，2mmol/L EDTA，0.1wt％TritonX-100，pH7.0；提取液组分为：15mmol/L 4-羟乙基哌嗪乙磺酸，1.0mmol/L乙二胺四乙酸，1.5mmol/L焦亚硫酸钠，0.5mol/L NaCl，pH6.0；酸性缓冲液配制是：将2.5g醋酸钠溶于水中，用冰醋酸调节pH至4.0，水定容至1L。
在所述步骤(5)中，向溶液中加入的硫酸盐，可以是硫酸铁、硫酸锌或硫酸钠或混合使用。
一种黄精植物甾醇多糖蛋白的活性制品，包括以下重量份数计组分：马卡酵素提取物10-20份、黄精酵素提取物20-48份、枸杞酵素提取物15-25份、山药酵素提取物15-20份、啤酒酵母提取物3-8份、海藻糖提取物3-8份、南瓜子提取物15-30份、牛磺酸提取物4-8份、L-精氨酸提取物5-10份、麦芽糊精13-18份、硬脂酸镁0.2-1份。
所述活性制品的浓度为65％-88％。
黄精的根状茎含甾体皂甙，已分离出2个呋甾烯醇型皂甙和2个螺甾烯醇型皂甙。另外，含有黄精多糖A、B、C，三者的相对分子质量均大于20万Dalton，均由葡萄糖(glucose)、甘露糖(mannose)和半乳糖醛酸(galacturonicacid)按照摩尔比6：26：1缩合而成；又含黄精低聚糖A、B、C，相对分子质量分别为1630、862和472，系由果糖(fructose)与葡萄糖按摩尔比8：1、4：1和2：1缩合而成。这些有效成分作用于生理上可影响人体抗病原微生物作用，体外试验表明、黄精水提出液(1：320)对伤寒杆菌、金黄色葡萄球菌、抗酸杆菌有抑制作用，2％黄精在沙氏培养基内对常见致病真菌有不同程度的抑制作用。对肾上腺素引起的血糖过高呈显著抑制作用，增强精力，具有抗疲劳作用。其成分有抗氧化作用，实验动物中使小鼠肝脏超氧化物歧化酶(SOD)活性升高，心肌脂褐质含量降低。对心血管有调节作用，水浸膏明显增加麻醉犬冠脈流量，对垂体后叶素引起的兔心肌缺血有对抗作用，对抗垂体后叶素引起的T波增高，促进T波异常提前恢复，也可增强小鼠对缺氧的耐受力。以口服液方式进入肠胃道连续10天，可提高小鼠紅細胞膜Na+，K+-ATP酶活性。40％黄精醇提液腹腔注射(0.3ml/只)，能提高小鼠心肌、肝、脾组织細胞DNA对3H-TdR的掺入率。黄精煎剂灌胃(0.5g/只)使小鼠血浆cAMP，及cGMP含量均下降，而以cGMP下降更多，因此cAMP/cGMP比值大于对照组，对脾组织cAMP，cGMP含量均上升，但cAMP/cGMP比值无明显变化。
本发明经验证，食用后4-12小时可达到抗局部微血管收缩和增强内生性贺尔蒙的效果以及增强骨盆底肌强度，基于本发明中主要活性组分为黄精植物多糖蛋白，配合从植物甾醇多糖蛋白提取出的活性成分以及其它配合植化素，以及植物中的微量元素钠、钾、钙、镁、磷、锌、铜、铁元素，在进入循环后，一方面可以作用在细胞酶系统，维持细胞内外信息及核酸生合成的平衡；另一方面是山药多糖、枸杞多糖与南瓜子提取物共同作用于宿主的腺体细胞，诱发内分泌调控，配合补充配方中的牛磺酸、L-精氨酸，可刺激摄护腺细胞，副肾脏腺体组织细胞等腺体器官，促进腺体细胞活性，通过增强骨盆底肌动作电位的反应性，从而增强机体的脑肠轴内分泌调控功能，易于被人体吸收，条件98％低龄人群长期服用无不适症状。
食疗价值如下：
①壮筋骨，益精髓，变白发：黄精、蒼朮各四斤，枸杞根、柏叶各五斤，天門冬三斤。煮汁一石，同曲十斤，糯米一石，如常酿酒钦。(《綱目》)
②补精气：枸杞子(冬采者佳)、黄精等分。为细末，二味招和，捣成块，捏作饼子，干复捣为末，炼蜜为丸，如梧桐子大。每服五十丸，空心温水送下。(《奇效良方》枸杞丸)
③治脾胃虚弱，体倦无力：黄精、党參、淮山药各一两，蒸鸡食。(《湖南农村常用中草药手册》)
④治肺劳咳血，赤白带：鲜黄精根头二两，冰糖一两。开水炖服。(《闽东本草》)
⑤治肺結核，病后体虚：黄精五钱至一两。水煎服或炖猪肉食。(《湖南农村常用中草药手册》)
⑥治小儿下肢痿软：黄精一两，冬蜜一两。开水炖服。(《闽东本草》)
⑦治胃热口渴：黄精六钱，熟地、山药各五钱，天花粉、麦门冬各四钱。水煎服。(《山东中草药手册》)
⑧治眼，補肝气，明目：蔓菁子一斤(以水淘净)，黄精二斤(和蔓菁子水蒸九次，曝干)。上药，捣细罗为散。每服，空心以粥饮调下二钱，日午晚食后，以温水再调服。(《圣惠方》蔓菁子散)
⑨治荣气不清，久风入脉，因而成癞，鼻坏色败，皮肤痒溃：黄精根(去皮洗净)二斤。日中曝令软，纳粟米饭甑中同蒸之，二斗米熟为度，不拘时服。(《圣济总录》)
⑩治蛲虫病：黄精八钱，加冰糖一两，炖服。〔《福建中医药》(6)：44，1965)。
需要声明的是，上述具体实施方式仅仅为本发明的较佳实施例及所运用技术原理，在本发明所公开的技术范围内，任何熟悉本技术领域的技术人员所容易想到的变化或替换，都应涵盖在本发明的保护范围内。
Claims (7)
1.一种黄精植物甾醇多糖蛋白的制备方法，其特征在于，包括以下步骤：
(1)预处理：取新鲜黄精根部，去除黄精表面的附着物，称重(湿重)并作为计量依据，将黄精切成丁状，粉碎后的黄精中加入4-8倍重量的水，使用匀浆机或胶体磨将粉碎的黄精顶制作成匀浆状；
(2)一次提取：采用过滤的方法将步骤(1)获得的黄精匀浆进行过滤，分别收集滤饼和滤液，滤液为第一次提取液；
(3)二次提取：将步骤(2)获得的黄精滤饼重新悬浮在3-5倍重量的水中，调节溶液pH至5.0-7.0，过滤后收集滤液，为第二次提取液；
(4)酶解：合并两次提取的滤液，加入0.1-5.0％黄精重量的复合酶解物，在30-50℃范围内搅拌6-12h；复合酶解物是由质量比为1-3：0.5-2的纤维素酶、木瓜蛋白酶组成。
(5)沉淀处理：将步骤(4)获得的酶解液调节pH至3.0-4.5并加入硫酸盐溶液，搅拌均匀，静置后使用离心法将滤液进行处理，收集滤液；
(6)干燥：将步骤(5)获得的滤液进行超滤处理，收集截留液，喷雾干燥，获得的粉末即为粗黄精多糖蛋白组合物。
2.根据权利要求1所述的一种黄精植物甾醇多糖蛋白的制备方法，其特征在于，在所述步骤(4)中，所述复合酶解物发酵产物的制备方法为：取经过清洗的菠萝原料，设定酶解条件为料液比1∶10(m/V)、纤维素酶CellulaseACCF-4740添加量2％、温度55℃、pH4.5、反应时间2.5h，进行酶解，纤维素酶水解结束后，沸水浴灭酶15min，9000r/min离心，收集上清液得到菠萝水解液；菠萝酶解液灭酶活后，调节pH值至6.2-6.6，高压蒸汽灭菌温度为121℃，时间为20min，然后接种体积分数1％的MRS培养基活化的戊糖片菌液到菠萝酶解液，37℃恒温培养箱静置，进行发酵；发酵结束后，9000r/min离心，收集上清液得到复合酶解物。
3.根据权利要求1所述的一种黄精植物甾醇多糖蛋白的制备方法，其特征在于，在所述步骤(4)中，酶解的原料是经过一次和二次提取后滤液的合并物。
4.根据权利要求1所述的一种黄精植物甾醇多糖蛋白的制备方法，其特征在于，在所述步骤(4)中，黄精多糖蛋白的制备方法为：提取液，4℃下提取44-50h，过滤，按0.5-1.5g/mL的添加量向滤液中缓慢添加硫酸铵，添加硫酸铵过程中不断搅拌，防止硫酸铵局部过饱和，然后静置45-50h，4℃条件下25000g离心5-10min，沉淀用酸性缓冲液复溶，4℃下分子量5000Da的透析袋透析，透析液经20000g离心10min，取上清液即为制备的扩张蛋白溶液；
其中，匀浆缓冲液组分为：25mmol/L 4-羟乙基哌嗪乙磺酸，1.5mmol/L焦亚硫酸钠，2mmol/L EDTA，0.1wt％TritonX-100，pH7.0；提取液组分为：15mmol/L 4-羟乙基哌嗪乙磺酸，1.0mmol/L乙二胺四乙酸，1.5mmol/L焦亚硫酸钠，0.5mol/L NaCl，pH6.0；酸性缓冲液配制是：将2.5g醋酸钠溶于水中，用冰醋酸调节pH至4.0，水定容至1L。
5.根据权利要求1所述的一种黄精植物甾醇多糖蛋白的制备方法，其特征在于，在所述步骤(5)中，向溶液中加入的硫酸盐，可以是硫酸铁、硫酸锌或硫酸钠或混合使用。
6.一种黄精植物甾醇多糖蛋白的活性制品，其特征在于，包括以下重量份数计组分：马卡酵素提取物10-20份、黄精酵素提取物20-48份、枸杞酵素提取物15-25份、山药酵素提取物15-20份、啤酒酵母提取物3-8份、海藻糖提取物3-8份、南瓜子提取物15-30份、牛磺酸提取物4-8份、L-精氨酸提取物5-10份、麦芽糊精13-18份、硬脂酸镁0.2-1份。
7.根据权利要求6所述的一种黄精植物甾醇多糖蛋白的活性制品，其特征在于，所述活性制品的浓度为65％-88％。
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