CN111466571A - 一种食药用猴头菇干粉的制备方法 - Google Patents
一种食药用猴头菇干粉的制备方法 Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
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Abstract
本发明公开了一种食药用猴头菇干粉的制备方法,涉及食药制品加工技术领域,本发明通过提取前对猴头菇的加热加压处理来软化和破坏细胞壁结构,从而利于后续提取时多糖成分的浸出;通过激活剂的添加来提高复合酶的酶解活性,从而在缩短提取时间的同时提高猴头菇多糖的提取率;利用醇沉法并结合DEAE纤维素柱层析来对提取物进行分离纯化,从而提高产品中猴头菇多糖的纯度。
Description
技术领域:
本发明涉及食药制品加工技术领域,具体涉及一种食药用猴头菇干粉的制备方法。
背景技术:
猴头菇,又叫猴头菌,只因外形酷似猴头而得名。猴头菌是鲜美无比的山珍,菌肉鲜嫩,香醇可口,有“素中荤”之称。作为食用菌王国的珍品,猴头菇属于药食两用真菌,尤其是其医药方面的药用价值,受到广大消费者的青睐,具有健脾养胃、安神、抗癌的功效。
猴头菇的主要成分是多糖类,具有助消化、利五脏的功能,从猴头菇子实体中提取得到的多糖是主链由β-1,3-糖苷键、支链为β-1,6-糖苷键连接的葡聚糖混合物。目前猴头菇多糖对的提取方法主要采用水提法、酸提法、碱提法、酶提法,其中水提法成本投入低,但提取时间长且提取率不高;酸提法和碱提法易使糖苷键发生断裂,部分多糖因发生水解而降低提取率,并且杂质含量不低;相对来说,酶提法的提取条件较为温和,但单独采用酶提法无法得到高提取率、高纯度的猴头菇多糖产品。因此,本发明以酶提法为基础,通过配合其他提取和纯化手段来保证猴头菇多糖产品的提取率和纯度。
发明内容:
本发明所要解决的技术问题在于提供一种食药用猴头菇干粉的制备方法,先经接种培养得到猴头菇,再经提取得到猴头菇提取物,并经纯化来提高猴头菇提取物中的多糖含量,最后制得食药用猴头菇干粉。
本发明所要解决的技术问题采用以下的技术方案来实现:
一种食药用猴头菇干粉的制备方法,包括以下制备步骤:
(1)菌种的斜面活化:在无菌环境下,向试管斜面中接入母种,并置于培养箱中恒温培养,直至试管斜面长满菌丝,得到活化菌种;
(2)菌种的扩大培养:在无菌环境下,向一级种子罐中接入活化菌种,恒温培养,得到一级种子罐菌种;
(3)发酵罐培养:向发酵罐中接入一级种子罐菌种,通入无菌空气进行恒温发酵,得到液体菌种;
(4)固体培养及出菇:向培养瓶中接入液体菌种,置于培养室中培养,当瓶口出现子实体后加大温差并给予散射光,即将产生孢子前应及时采收,干燥入库;
(5)预处理:将干燥的猴头菇置于高压釜中,通入惰性气体进行加热加压处理,并利用粉碎机制成粉体,得到猴头菇粉;
(6)酶法提取:将猴头菇粉加水浸泡,并调节pH值至6.0-7.0,再加入复合酶和激活剂,保温酶解,提取结束后升温灭酶,离心,上清液经浓缩后加入乙醇搅拌,静置,过滤,得到粗提物;
(7)纯化:将粗提物进行DEAE纤维素柱层析,依次用蒸馏水和碳酸氢钠溶液洗脱,浓缩,得到多糖提取物;
(8)制粉:向多糖提取物中加入药用辅料,混合均匀,真空干燥,并利用粉碎机制成粉体,分装,检验,得到猴头菇干粉。
所述加热加压处理的压力为2-5MPa,温度为45-55℃。
所述复合酶的添加量为猴头菇粉质量的0.1-0.5%。
所述复合酶由蛋白酶、纤维素酶和半纤维素酶组成,质量比为1-5:1-5:0.1-2。
所述激活剂选自硫酸镁、磷酸二氢钙中的一种。
所述激活剂的添加量为猴头菇粉质量的0.1-0.5%。
所述蛋白酶优选木瓜蛋白酶。
所述酶解温度为55-65℃。
所述灭酶温度为80-90℃。
所述碳酸氢钠溶液的浓度为0.05-0.3mol/L。
硫酸镁和磷酸二氢钙是本领域已知的酶激活剂,除这两种物质以外本发明还采用了新型化合物作为酶激活剂,并取得了在相同提取时间下明显提高猴头菇多糖提取率的技术效果。
所述环丙基乳酸乙酰胺是由摩尔比1:1的乳酸和1-环丙基乙胺盐酸盐通过酰胺缩合反应制成。
本发明的有益效果是:
(1)本发明通过提取前对猴头菇的加热加压处理来软化和破坏细胞壁结构,从而利于后续提取时多糖成分的浸出;
(2)本发明通过激活剂的添加来提高复合酶的酶解活性,从而在缩短提取时间的同时提高猴头菇多糖的提取率;
(3)本发明利用醇沉法并结合DEAE纤维素柱层析来对提取物进行分离纯化,从而提高产品中猴头菇多糖的纯度。
具体实施方式:
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施例,进一步阐述本发明。
以下实施例和对比例中的提取多糖用干燥的猴头菇来自于同批培养的猴头菇且含水量在11%;药用辅料为羟丙基-β-环糊精和甘露醇,多糖提取物与羟丙基-β-环糊精、甘露醇的质量比为1:3:1;羟丙基-β-环糊精来源于山东滨州智源生物科技有限公司的药品级,甘露醇来源于寿光市华力糖醇有限公司;木瓜蛋白酶、纤维素酶和半纤维素酶来源于夏盛(北京)生物科技有限公司的木瓜蛋白酶FDY-4003、纤维素酶CEL-01FDG-2225和半纤维素酶FDG-2255。
实施例1
(1)菌种的斜面活化:在无菌环境下,向试管斜面中接入母种,并置于培养箱中25℃恒温培养15天,直至试管斜面长满菌丝,得到活化菌种;
(2)菌种的扩大培养:在无菌环境下,向一级种子罐中接入活化菌种,25℃恒温培养7天,得到一级种子罐菌种;
(3)发酵罐培养:向发酵罐中接入一级种子罐菌种,通入无菌空气进行25℃恒温发酵7天,得到液体菌种;
(4)固体培养及出菇:向培养瓶中接入液体菌种,置于温度25℃、湿度55%的培养室中培养,当瓶口出现子实体后加大温差并给予散射光,即将产生孢子前应及时采收,干燥入库;
(5)预处理:将干燥的猴头菇置于高压釜中,通入惰性气体进行加热加压处理15min,压力3MPa,温度55℃,并利用粉碎机制成100目的粉体,得到猴头菇粉;
(6)酶法提取:将200g猴头菇粉加1000g水浸泡3h,并调节pH值至6.0-7.0,再加入1g复合酶和1g硫酸镁,55℃保温酶解5h,提取结束后升温至85℃灭酶,离心,上清液经浓缩后加入乙醇搅拌,静置,过滤,得到粗提物;复合酶由木瓜蛋白酶、纤维素酶和半纤维素酶组成,质量比为2:2:0.5;
(7)纯化:将粗提物进行DEAE纤维素柱层析,依次用蒸馏水和0.15mol/L碳酸氢钠溶液洗脱,浓缩,得到多糖提取物;
(8)制粉:向多糖提取物中加入药用辅料,混合均匀,真空干燥,并利用粉碎机制成粉体,分装,检验,得到猴头菇干粉。
实施例2
实施例2与实施例1的区别之处在于将激活剂硫酸镁替换为磷酸二氢钙。
(1)菌种的斜面活化:在无菌环境下,向试管斜面中接入母种,并置于培养箱中25℃恒温培养15天,直至试管斜面长满菌丝,得到活化菌种;
(2)菌种的扩大培养:在无菌环境下,向一级种子罐中接入活化菌种,25℃恒温培养7天,得到一级种子罐菌种;
(3)发酵罐培养:向发酵罐中接入一级种子罐菌种,通入无菌空气进行25℃恒温发酵7天,得到液体菌种;
(4)固体培养及出菇:向培养瓶中接入液体菌种,置于温度25℃、湿度55%的培养室中培养,当瓶口出现子实体后加大温差并给予散射光,即将产生孢子前应及时采收,干燥入库;
(5)预处理:将干燥的猴头菇置于高压釜中,通入惰性气体进行加热加压处理15min,压力3MPa,温度55℃,并利用粉碎机制成100目的粉体,得到猴头菇粉;
(6)酶法提取:将200g猴头菇粉加1000g水浸泡3h,并调节pH值至6.0-7.0,再加入1g复合酶和1g磷酸二氢钙,55℃保温酶解5h,提取结束后升温至85℃灭酶,离心,上清液经浓缩后加入乙醇搅拌,静置,过滤,得到粗提物;复合酶由木瓜蛋白酶、纤维素酶和半纤维素酶组成,质量比为2:2:0.5;
(7)纯化:将粗提物进行DEAE纤维素柱层析,依次用蒸馏水和0.15mol/L碳酸氢钠溶液洗脱,浓缩,得到多糖提取物;
(8)制粉:向多糖提取物中加入药用辅料,混合均匀,真空干燥,并利用粉碎机制成粉体,分装,检验,得到猴头菇干粉。
实施例3
实施例3与实施例1的区别之处在于将激活剂替换为环丙基乳酸乙酰胺。
环丙基乳酸乙酰胺的制备:先分别将10mol乳酸、10mol 1-环丙基乙胺盐酸盐、11mol EDCI和11mol HOBt用水溶解制成溶液,再将这四种溶液混合,并加热至65℃保温反应3h,反应液浓缩后柱层析,得到环丙基乳酸乙酰胺。元素分析:理论值C,61.12;H,9.62;N,8.91;O,20.35.实测值C,61.13;H,9.61;N,8.90;O,20.36.
(1)菌种的斜面活化:在无菌环境下,向试管斜面中接入母种,并置于培养箱中25℃恒温培养15天,直至试管斜面长满菌丝,得到活化菌种;
(2)菌种的扩大培养:在无菌环境下,向一级种子罐中接入活化菌种,25℃恒温培养7天,得到一级种子罐菌种;
(3)发酵罐培养:向发酵罐中接入一级种子罐菌种,通入无菌空气进行25℃恒温发酵7天,得到液体菌种;
(4)固体培养及出菇:向培养瓶中接入液体菌种,置于温度25℃、湿度55%的培养室中培养,当瓶口出现子实体后加大温差并给予散射光,即将产生孢子前应及时采收,干燥入库;
(5)预处理:将干燥的猴头菇置于高压釜中,通入惰性气体进行加热加压处理15min,压力3MPa,温度55℃,并利用粉碎机制成100目的粉体,得到猴头菇粉;
(6)酶法提取:将200g猴头菇粉加1000g水浸泡3h,并调节pH值至6.0-7.0,再加入1g复合酶和1g环丙基乳酸乙酰胺,55℃保温酶解5h,提取结束后升温至85℃灭酶,离心,上清液经浓缩后加入乙醇搅拌,静置,过滤,得到粗提物;复合酶由木瓜蛋白酶、纤维素酶和半纤维素酶组成,质量比为2:2:0.5;
(7)纯化:将粗提物进行DEAE纤维素柱层析,依次用蒸馏水和0.15mol/L碳酸氢钠溶液洗脱,浓缩,得到多糖提取物;
(8)制粉:向多糖提取物中加入药用辅料,混合均匀,真空干燥,并利用粉碎机制成粉体,分装,检验,得到猴头菇干粉。
对比例1
对比例1与实施例1的区别之处在于未添加激活剂。
(1)菌种的斜面活化:在无菌环境下,向试管斜面中接入母种,并置于培养箱中25℃恒温培养15天,直至试管斜面长满菌丝,得到活化菌种;
(2)菌种的扩大培养:在无菌环境下,向一级种子罐中接入活化菌种,25℃恒温培养7天,得到一级种子罐菌种;
(3)发酵罐培养:向发酵罐中接入一级种子罐菌种,通入无菌空气进行25℃恒温发酵7天,得到液体菌种;
(4)固体培养及出菇:向培养瓶中接入液体菌种,置于温度25℃、湿度55%的培养室中培养,当瓶口出现子实体后加大温差并给予散射光,即将产生孢子前应及时采收,干燥入库;
(5)预处理:将干燥的猴头菇置于高压釜中,通入惰性气体进行加热加压处理15min,压力3MPa,温度55℃,并利用粉碎机制成100目的粉体,得到猴头菇粉;
(6)酶法提取:将200g猴头菇粉加1000g水浸泡3h,并调节pH值至6.0-7.0,再加入1g复合酶,55℃保温酶解5h,提取结束后升温至85℃灭酶,离心,上清液经浓缩后加入乙醇搅拌,静置,过滤,得到粗提物;复合酶由木瓜蛋白酶、纤维素酶和半纤维素酶组成,质量比为2:2:0.5;
(7)纯化:将粗提物进行DEAE纤维素柱层析,依次用蒸馏水和0.15mol/L碳酸氢钠溶液洗脱,浓缩,得到多糖提取物;
(8)制粉:向多糖提取物中加入药用辅料,混合均匀,真空干燥,并利用粉碎机制成粉体,分装,检验,得到猴头菇干粉。
对比例2
对比例2与实施例1的区别之处在于未对猴头菇进行高温高压处理。
(1)菌种的斜面活化:在无菌环境下,向试管斜面中接入母种,并置于培养箱中25℃恒温培养15天,直至试管斜面长满菌丝,得到活化菌种;
(2)菌种的扩大培养:在无菌环境下,向一级种子罐中接入活化菌种,25℃恒温培养7天,得到一级种子罐菌种;
(3)发酵罐培养:向发酵罐中接入一级种子罐菌种,通入无菌空气进行25℃恒温发酵7天,得到液体菌种;
(4)固体培养及出菇:向培养瓶中接入液体菌种,置于温度25℃、湿度55%的培养室中培养,当瓶口出现子实体后加大温差并给予散射光,即将产生孢子前应及时采收,干燥入库;
(5)预处理:将干燥的猴头菇利用粉碎机制成100目的粉体,得到猴头菇粉;
(6)酶法提取:将200g猴头菇粉加1000g水浸泡3h,并调节pH值至6.0-7.0,再加入1g复合酶和1g硫酸镁,55℃保温酶解5h,提取结束后升温至85℃灭酶,离心,上清液经浓缩后加入乙醇搅拌,静置,过滤,得到粗提物;复合酶由木瓜蛋白酶、纤维素酶和半纤维素酶组成,质量比为2:2:0.5;
(7)纯化:将粗提物进行DEAE纤维素柱层析,依次用蒸馏水和0.15mol/L碳酸氢钠溶液洗脱,浓缩,得到多糖提取物;
(8)制粉:向多糖提取物中加入药用辅料,混合均匀,真空干燥,并利用粉碎机制成粉体,分装,检验,得到猴头菇干粉。
分别取10mg上述实施例和对比例所制多糖提取物,加80mL蒸馏水煮沸1h后冷却至室温,再加蒸馏水定容至100mL。移取1.0mL到比色皿中,加2.0mL5.0%苯酚和5.0mL98%浓硫酸,摇匀后室温放置20min,在波长490nm处测定吸光度。根据标准品曲线,计算所制多糖提取物中猴头菇多糖的含量,并计算猴头菇多糖的提取率,如表1所示。
表1
项目 | 实施例1 | 实施例2 | 实施例3 | 对比例1 | 对比例2 |
提取率/% | 88.6 | 89.2 | 93.5 | 82.4 | 78.3 |
含量/% | 75.5 | 75.8 | 76.3 | 74.1 | 72.6 |
由表1可知,对猴头菇的高温高压处理、激活剂的添加都有利于提高猴头菇多糖的提取率和纯度。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (10)
1.一种食药用猴头菇干粉的制备方法,其特征在于,包括以下制备步骤:
(1)菌种的斜面活化:在无菌环境下,向试管斜面中接入母种,并置于培养箱中恒温培养,直至试管斜面长满菌丝,得到活化菌种;
(2)菌种的扩大培养:在无菌环境下,向一级种子罐中接入活化菌种,恒温培养,得到一级种子罐菌种;
(3)发酵罐培养:向发酵罐中接入一级种子罐菌种,通入无菌空气进行恒温发酵,得到液体菌种;
(4)固体培养及出菇:向培养瓶中接入液体菌种,置于培养室中培养,当瓶口出现子实体后加大温差并给予散射光,即将产生孢子前应及时采收,干燥入库;
(5)预处理:将干燥的猴头菇置于高压釜中,通入惰性气体进行加热加压处理,并利用粉碎机制成粉体,得到猴头菇粉;
(6)酶法提取:将猴头菇粉加水浸泡,并调节pH值至6.0-7.0,再加入复合酶和激活剂,保温酶解,提取结束后升温灭酶,离心,上清液经浓缩后加入乙醇搅拌,静置,过滤,得到粗提物;
(7)纯化:将粗提物进行DEAE纤维素柱层析,依次用蒸馏水和碳酸氢钠溶液洗脱,浓缩,得到多糖提取物;
(8)制粉:向多糖提取物中加入药用辅料,混合均匀,真空干燥,并利用粉碎机制成粉体,分装,检验,得到猴头菇干粉。
2.根据权利要求1所述的食药用猴头菇干粉的制备方法,其特征在于:所述加热加压处理的压力为2-5MPa,温度为45-55℃。
3.根据权利要求1所述的食药用猴头菇干粉的制备方法,其特征在于:所述复合酶的添加量为猴头菇粉质量的0.1-0.5%。
4.根据权利要求1所述的食药用猴头菇干粉的制备方法,其特征在于:所述复合酶由蛋白酶、纤维素酶和半纤维素酶组成,质量比为1-5:1-5:0.1-2。
5.根据权利要求1所述的食药用猴头菇干粉的制备方法,其特征在于:所述激活剂选自硫酸镁、磷酸二氢钙中的一种。
6.根据权利要求1所述的食药用猴头菇干粉的制备方法,其特征在于:所述激活剂的添加量为猴头菇粉质量的0.1-0.5%。
7.根据权利要求4所述的食药用猴头菇干粉的制备方法,其特征在于:所述蛋白酶优选木瓜蛋白酶。
8.根据权利要求1所述的食药用猴头菇干粉的制备方法,其特征在于:所述酶解温度为55-65℃。
9.根据权利要求1所述的食药用猴头菇干粉的制备方法,其特征在于:所述灭酶温度为80-90℃。
10.根据权利要求1所述的食药用猴头菇干粉的制备方法,其特征在于:所述碳酸氢钠溶液的浓度为0.05-0.3mol/L。
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CN104231110A (zh) * | 2014-10-21 | 2014-12-24 | 哈尔滨艾博雅食品科技开发有限公司 | 一种猴头菇粗多糖的提取方法 |
CN108341891A (zh) * | 2018-05-09 | 2018-07-31 | 四川岚晟生物科技有限公司 | 一种猴头菇多糖的提取方法 |
CN110922494A (zh) * | 2019-11-01 | 2020-03-27 | 福州福德恩生物科技有限公司 | 一种猴头菇多糖提取分离方法 |
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CN112522344B (zh) * | 2020-12-23 | 2022-09-13 | 上海市农业科学院 | 一种猴头菇子实体粉固态酶解的方法 |
CN114907495A (zh) * | 2022-05-30 | 2022-08-16 | 王春艳 | 一种猴头菇多糖的高效提取工艺 |
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