CN111704678A - 一种平菇半乳甘露葡聚糖及其制备方法和用途 - Google Patents
一种平菇半乳甘露葡聚糖及其制备方法和用途 Download PDFInfo
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Abstract
本发明为一种平菇半乳甘露葡聚糖及其制备方法和用途,属于食用菌有效成分制备领域。其平菇半乳甘露葡聚糖是由平菇干燥子实体经过常温破壁提取,结合挤压分离、除蛋白、离子交换层析获得。破壁结合挤压过滤,在该制备方法中缩短了提取时间、减少了试剂用量、节约了能源、提高了多糖得率,并且采用的挤压过滤技术适用于粘稠溶液的分离,获得的平菇半乳甘露葡聚糖具有保护成肌细胞氧化损伤的功能活性,可用于制备保护成肌细胞氧化损伤的辅助药品、保健食品。
Description
技术领域
本发明涉及一种平菇半乳甘露葡聚糖及其制备方法和用途,属于天然产物领域。
背景技术
平菇,学名糙皮侧耳,在我国食用菌年产量中位居前三位。平菇富含蛋白质、多糖、维生素以及微量元素等营养成分,中医认为其具有治疗腰腿疼痛、四肢麻木和舒筋活络等功效。多糖是平菇发挥其药理作用的主要活性成分。受制于平菇多糖制备及药品或保健食品等产品的相关技术开发尚未成熟,尤其是平菇多糖功效的研究,如平菇多糖保肝活性、调节免疫活性仍处于实验室阶段,因此市场上缺少相关产品面世。
近几年,平菇多糖的提取条件的优化、结构分析和药理活性筛选已有一定的研究基础。目前,平菇多糖的提取方法主要有热水浸提法、酶解法、超声波辅助提取法、微波辅助提取法等。热水浸提法是提取多糖最常用的方法,工艺方法简单,但存在提取时间长、所用溶剂量大、高温易破坏多糖结构等缺点;酶解法专一性强,只能酶解特定糖苷键,酶制剂费用较高(杨勇,首坤秀,李立郎,等.复合酶法提取平菇多糖工艺优化[J].山地农业生物学报,2019,38(1):29-34);超声波辅助提取噪音较大(邓斌.超声联合酶法提取平菇多糖工艺优化[J].黑龙江八一农垦大学学报,2019,31(5):66-72);而微波辅助提取受设备限制,提取量较小(张晓,朱彩平,邓红,等.均匀设计优化超声协同酶法提取平菇多糖工艺[J].食品与机械,2016,32(9):166-171)。上述现有技术均不能满足该类产品产业化开发及生产的大量需求。
发明内容
本发明是针对现有研究及技术的不足,旨在提供一种平菇半乳甘露葡聚糖及其制备方法和用途。
本发明所述的一种平菇半乳甘露葡聚糖,是从平菇中提取的,由岩藻糖、阿拉伯糖、半乳糖、葡萄糖、果糖组成的α-吡喃聚糖,分子量范围2.75×105Da~8.70×105Da。
本发明所述一种平菇半乳甘露葡聚糖的制备方法为由平菇干燥子实体经过常温破壁提取,结合挤压分离、除蛋白、离子交换层析获得。具体步骤如下:
a、对平菇干燥子实体进行粉碎,过40目筛,形成平菇干燥子实体粉末,将平菇干燥子实体粉末装入破壁机中,按料液比1:10~1:80加入去离子水,破壁2~20min,挤压过滤获得原液,在70℃~90℃温度下浓缩提取的原液,浓缩液体积为原液体积的1/3~1/4,冷却至室温,加3~4倍浓缩液体积的无水乙醇,形成混合液1,在4℃环境中静置12h后,5000~10000r/min对混合液1进行离心,离心时间为5~10min,收集下层沉淀,挥发残余乙醇,冷冻干燥,干燥粉末即为平菇子实体粗多糖;
b、加入纯净水,配制成浓度为5mg/mL的平菇子实体粗多糖溶液后,加入氯仿正丁醇混合溶液,磁力搅拌,离心分层,收集上层溶液,重复加入氯仿正丁醇混合溶液1次,离心,取上清液进行减压浓缩至原体积1/3~1/4,加入3~4倍体积的无水乙醇,在4℃环境中静置12h后,形成混合溶液2,5000~10000r/min对混合液2进行离心,离心时间为5~10min,收集下层沉淀,挥发残余乙醇,冷冻干燥,干燥粉末即为平菇子实体多糖;
c、平菇子实体多糖加去离子水溶解,定容,上样DEAE 52纤维素柱层析,依次用纯净水及浓度为0.1mol/L的NaCl溶液洗脱,流速为5mL/3min,进行收集,每管收集5mL,苯酚硫酸法测定各管糖含量,根据各管糖含量收集0.1mol/L的NaCl溶液洗脱峰,冷冻干燥即为平菇半乳甘露葡聚糖。
步骤a所述的挤压过滤是采用单螺杆挤压机,单螺杆挤压机的模孔直径为8~16mm、挤压机套筒温度为40~90℃、螺杆转速为160~240r/min。
上述平菇半乳甘露葡聚糖具有制备保健食品、药品的用途。
一种可用于保护成肌细胞损伤的保健食品、药品组合物,所述保健食品、药品组合物中含有上述的平菇半乳甘露葡聚糖成分。
上述可用于保护成肌细胞损伤的保健食品、药品组合物的剂型可以为片剂、颗粒剂、胶囊剂或溶液剂。
本发明积极效果在于:首次从;并在平菇半乳甘露葡聚糖制备过程中采用了常温破壁提取结合挤压过滤的提取分离方法,缩短了提取时间、减少了试剂用量、节约了能源、提高了多糖得率,并且采用的挤压过滤方法适用于粘稠溶液的分离;同时公开了平菇半乳甘露葡聚糖具有保护成肌细胞氧化损伤的功能活性,可用于制备保护肌肉细胞辅助药品、保健食品,为平菇的深加工产品开发提供基础数据,具有重要的经济和市场价值。
附图说明
下面结合附图说明和具体实施方式对本发明作进一步说明:
图1为本发明平菇半乳甘露葡聚糖DEAE 52洗脱曲线;
图2为本发明平菇半乳甘露葡聚糖高效凝胶渗透层析;
图3为本发明平菇半乳甘露葡聚糖的红外光谱;
图4为本发明平菇半乳甘露葡聚糖单糖组成分析;
图5为本发明平菇半乳甘露葡聚糖对氧化损伤肝细胞抗氧化酶SOD的活性影响;
图6为本发明平菇半乳甘露葡聚糖对氧化损伤肝细胞抗氧化酶GSH-Px的活性影响;
图7为本发明平菇半乳甘露葡聚糖对氧化损伤肝细胞氧化产物MDA生成量的影响;
图8为本发明平菇半乳甘露葡聚糖对氧化损伤肝细胞活性氧ROS生成量的影响。
具体实施方式
下面将结合具体实施方式进一步说明本发明,但本发明要求保护的范围并不局限于下列实施方式。
实施例1:
1、对平菇干燥子实体进行粉碎,过40目筛,形成平菇干燥子实体粉末,将平菇干燥子实体粉末装入破壁机中,按料液比1:40加入去离子水,破壁10min,挤压过滤采用单螺杆挤压机,单螺杆挤压机的模孔直径16mm、挤压机套筒温度58℃、螺杆转速220r/min。提取液70℃浓缩至原有体积1/3,冷却至室温,加3倍体积无水乙醇,4℃静置12h,10000r/min,离心5min,挥发残余乙醇,冷冻干燥,干燥粉末即为平菇子实体粗多糖。
2、平菇子实体粗多糖配制成5mg/ml溶液,加入氯仿正丁醇混合溶液(体积比4:1),磁力搅拌,离心分层,提取上层溶液,重复加入氯仿正丁醇混合溶液(体积比4:1)1次,离心,取上清液进行减压浓缩至原体积1/3,加入3倍无水乙醇,4℃静置12h,10000r/min,离心5min,挥发残余乙醇,冷冻干燥,干燥粉末即为平菇子实体多糖。
3、平菇子实体多糖加去离子水溶解,定容,上样DEAE 52纤维素柱层析(4.0×30cm),依次用纯净水及浓度为0.1mol/L的NaCl溶液洗脱,流速5mL/3min,每管收集5mL。苯酚硫酸法测定各管糖含量,根据各管糖含量收集洗脱峰,获得多糖组分POP-1、POP-2和POP-3,结果如图1所示。继续将POP-2进行凝胶渗透色谱分析(TSK-gel G-3000PWXL色谱柱(7.8×300mm)),色谱条件:色谱柱:DionexTMCarboPacTMPA20。流动相:A相:ddH2O;B相:200mMNaOH;C相:200mMNaOH/500mMNaAC。流速:0.5mL/min。分子量以lgMw=-0.228t+7.831计算,获得平均分子量为3.98×105Da的平菇半乳甘露葡聚糖,结果如图2所示。红外光谱检测平菇半乳甘露葡聚糖为α-吡喃糖,结果如图3所示。取2mg平菇半乳甘露葡聚糖通过含有1M HCl的甲醇水解,水解产物通过2MTFA水解后,水解产物通过1-苯基-3-甲基-5-吡唑啉酮(PMP)衍生化后,产物经Compass C18 column(250×4.6mm)色谱柱分离,HPLC分析,UV245nm检测洗脱产物,结果如图4所示。结果表明,平菇半乳甘露葡聚糖由岩藻糖、阿拉伯糖、半乳糖、葡萄糖、果糖组成。
实施例2:
1、对平菇干燥子实体进行粉碎,过40目筛,形成平菇干燥子实体粉末,将平菇干燥子实体粉末装入破壁机中,按料液比1:10加入去离子水,破壁20min,挤压过滤采用单螺杆挤压机,单螺杆挤压机的模孔直径为8mm、挤压机套筒温度90℃、螺杆转速240r/min。提取液90℃浓缩至原有体积1/4,冷却至室温,加4倍体积无水乙醇,4℃静置12h,5000r/min,离心10min,挥发残余乙醇,冷冻干燥,干燥粉末即为平菇子实体粗多糖。
2、平菇子实体粗多糖配制成5mg/ml溶液,加入氯仿正丁醇混合溶液(体积比4:1),磁力搅拌,离心分层,提取上层溶液,重复加入氯仿正丁醇混合溶液(体积比4:1)1次,离心,取上清液进行减压浓缩至原体积1/3,加入3倍无水乙醇,4℃静置12h,5000r/min,离心10min,挥发残余乙醇,冷冻干燥,干燥粉末即为平菇子实体多糖。
3、平菇子实体多糖加去离子水溶解,定容,上样DEAE 52纤维素柱层析(4.0×30cm),依次用纯净水及浓度为0.1mol/LNaCl溶液洗脱,流速5mL/3min,每管收集5mL。苯酚硫酸法测定各管糖含量,根据各管糖含量收集洗脱峰,获得多糖组分POP-1、POP-2和POP-3,结果如图1所示。继续将POP-2进行凝胶渗透色谱分析(TSK-gel G-3000PWXL色谱柱(7.8×300mm)),色谱条件:色谱柱:DionexTMCarboPacTMPA20。流动相:A相:ddH2O;B相:200mMNaOH;C相:200mMNaOH/500mMNaAC。流速:0.5mL/min。分子量以lgMw=-0.228t+7.831计算,获得平均分子量为2.75×105Da的平菇半乳甘露葡聚糖,结果如图2所示。红外光谱检测平菇半乳甘露葡聚糖为α-吡喃糖,结果如图3所示。取2mg平菇半乳甘露葡聚糖通过含有1M HCl的甲醇水解,水解产物通过2M TFA水解后,水解产物通过1-苯基-3-甲基-5-吡唑啉酮(PMP)衍生化后,产物经Compass C18 column(250×4.6mm)色谱柱分离,HPLC分析,UV245nm检测洗脱产物,结果如图4所示。结果表明,平菇半乳甘露葡聚糖由岩藻糖、阿拉伯糖、半乳糖、葡萄糖、果糖组成。
实施例3:
1、对平菇干燥子实体进行粉碎,过40目筛,形成平菇干燥子实体粉末,将平菇干燥子实体粉末装入破壁机中,按料液比1:80加入去离子水,破壁2min,挤压过滤采用单螺杆挤压机,单螺杆挤压机的模孔直径16mm、挤压机套筒温度40℃、螺杆转速160r/min。提取液80℃浓缩至原有体积1/3,冷却至室温,加3倍体积无水乙醇,4℃静置12h,7000r/min,离心8min,挥发残余乙醇,冷冻干燥,干燥粉末即为平菇子实体粗多糖。
2、平菇子实体粗多糖配制成5mg/ml溶液,加入氯仿正丁醇混合溶液(体积比4:1),磁力搅拌,离心分层,提取上层溶液,重复加入氯仿正丁醇混合溶液(体积比4:1)1次,离心,取上清液进行减压浓缩至原体积1/4,加入4倍无水乙醇,4℃静置12h,7000r/min,离心8min,挥发残余乙醇,冷冻干燥,干燥粉末即为平菇子实体多糖。
3、平菇子实体多糖加去离子水溶解,定容,上样DEAE 52纤维素柱层析(4.0×30cm),依次用纯净水及浓度为0.1mol/L的NaCl溶液洗脱,流速5mL/3min,每管收集5mL。苯酚硫酸法测定各管糖含量,根据各管糖含量收集洗脱峰,获得多糖组分POP-1、POP-2和POP-3,结果如图1所示。继续将POP-2进行凝胶渗透色谱分析(TSK-gel G-3000PWXL色谱柱(7.8×300mm)),色谱条件:色谱柱:DionexTMCarboPacTMPA20。流动相:A相:ddH2O;B相:200mMNaOH;C相:200mM NaOH/500mM NaAC。流速:0.5mL/min。分子量以lgMw=-0.228t+7.831计算,获得平均分子量为8.70×105Da的平菇半乳甘露葡聚糖,结果如图2所示。红外光谱检测平菇半乳甘露葡聚糖为α-吡喃糖,结果如图3所示。取2mg平菇半乳甘露葡聚糖通过含有1MHCl的甲醇水解,水解产物通过2M TFA水解后,水解产物通过1-苯基-3-甲基-5-吡唑啉酮(PMP)衍生化后,产物经CompassC18column(250×4.6mm)色谱柱分离,HPLC分析,UV245nm检测洗脱产物,结果如图4所示。结果表明,平菇半乳甘露葡聚糖由岩藻糖、阿拉伯糖、半乳糖、葡萄糖、果糖组成。
通过以下试验证明本发明一种可保护成肌细胞氧化损伤的平菇半乳甘露葡聚糖在药品、保健食品中的用途:
试验例1:
采用抗氧化试剂盒检测平菇半乳甘露葡聚糖对氧化损伤成肌细胞抗氧化酶酶活性的影响。将对数生长期的成肌细胞L6细胞以6×104个/mL接种于96孔板,每孔100μL。分组:①对照组;②阳性(香菇多糖1mg/mL)组;③平菇半乳甘露葡聚糖(2mg/mL、1.5mg/mL、1mg/mL、0.5mg/mL、0.25mg/mL、0.125mg/mL)组,每组设6个重复。待细胞生长24h后,贴壁状态良好,加入H2O2(400μM)继续培养4h。收集培养液并重悬细胞,按照SOD、GSH-Px和MDA试剂盒说明书操作,每组设置6个重复。
试验例2:
采用DCFH-DA荧光探针检测平菇半乳甘露葡聚糖对氧化损伤成肌细胞活性氧生成量的影响。将对数生长期的成肌细胞L6细胞以6×104个/mL接种于96孔板,每孔100μL。分组:①对照组;②阳性(香菇多糖1mg/mL)组;③平菇半乳甘露葡聚糖(2mg/mL、1.5mg/mL、1mg/mL、0.5mg/mL、0.25mg/mL、0.125mg/mL)组,每组设6个重复。待细胞生长24h后,贴壁状态良好,加入H2O2(400μM)继续培养4h。每孔加入含有10μLDCFH-DA荧光探针的无血清DMEM培养基,37℃培养箱中孵育30min,PBS缓慢清洗三次,置于多功能荧光酶标仪中检测荧光强度并绘制柱形图(激发波长为488nm,发射波长为525nm)。
上述抗氧化酶酶活性检测及活性氧生成实验结果如图5、图6、图7以及图8所示,当平菇半乳甘露葡聚糖的浓度为0.25~1.5mg/mL时,对可增强抗氧化酶酶活性、降低活性氧生成量,起着降低细胞氧化损伤水平的作用,对成肌细胞的氧化损伤具有保护作用。
本发明实施方式说明到此结束。
Claims (7)
1.一种平菇半乳甘露葡聚糖,其特征在于,该平菇半乳甘露葡聚糖是从平菇中提取的,由岩藻糖、阿拉伯糖、半乳糖、葡萄糖、果糖组成的α-吡喃聚糖,分子量范围2.75×105Da~8.70×105Da。
2.根据权利要求1所述的一种平菇半乳甘露葡聚糖的制备方法,其特征在于:该平菇半乳甘露葡聚糖由平菇干燥子实体经常温破壁提取,结合挤压分离,除蛋白,离子交换层析获得。
3.根据权利要求2所述的一种平菇半乳甘露葡聚糖的制备方法,其特征在于:上述制备方法具体步骤如下:
a、对平菇干燥子实体进行粉碎,过40目筛,形成平菇干燥子实体粉末,将平菇干燥子实体粉末装入破壁机中,按料液比1:10~1:80加入去离子水,破壁2~20min,挤压过滤获得原液,在70℃~90℃温度下浓缩提取的原液,浓缩液体积为原液体积的1/3~1/4,冷却至室温,加3~4倍浓缩液体积的无水乙醇,形成混合液1,在4℃环境中静置12h后,5000~10000r/min对混合液1进行离心,离心时间为5~10min,收集下层沉淀,挥发残余乙醇,冷冻干燥,干燥粉末即为平菇子实体粗多糖;
b、加入纯净水,配制成浓度为5mg/mL的平菇子实体粗多糖溶液后,加入氯仿正丁醇混合溶液,磁力搅拌,离心分层,收集上层溶液,重复加入氯仿正丁醇混合溶液1次,离心,取上清液进行减压浓缩至原体积1/3~1/4,加入3~4倍体积的无水乙醇,在4℃环境中静置12h后,形成混合溶液2,5000~10000r/min对混合液2进行离心,离心时间为5~10min,收集下层沉淀,挥发残余乙醇,冷冻干燥,干燥粉末即为平菇子实体多糖;
c、平菇子实体多糖加去离子水溶解,定容,上样DEAE 52纤维素柱层析,依次用纯净水及浓度为0.1mol/L的NaCl溶液洗脱,流速为5mL/3min,进行收集,每管收集5mL,苯酚硫酸法测定各管糖含量,根据各管糖含量收集0.1mol/L的NaCl溶液洗脱峰,冷冻干燥即为平菇半乳甘露葡聚糖。
4.根据权利要求3所述的一种平菇半乳甘露葡聚糖的制备方法,其特征在于:步骤a所述的挤压过滤采用单螺杆挤压机,单螺杆挤压机的模孔直径为8~16mm、挤压机套筒温度为40~90℃、螺杆转速为160~240r/min。
5.根据权利要求1~3所述的一种平菇半乳甘露葡聚糖在制备保健食品、药品中的用途。
6.一种可用于保护成肌细胞损伤的保健食品、药品组合物,其特征在于:所述保健食品、药品组合物中含有权利要求1~3所述的平菇半乳甘露葡聚糖成分。
7.根据权利要求6所述的一种可用于保护成肌细胞损伤的保健食品、药品组合物,其特征在于:所述保健食品、药品组合物的剂型可以为片剂、颗粒剂、胶囊剂或溶液剂。
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