CN117586429A - 一种油黄口蘑多糖及其制备方法和应用 - Google Patents
一种油黄口蘑多糖及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种油黄口蘑多糖(TF‑P)及其制备方法和应用。所述TF‑P的组成包括葡萄糖和阿拉伯糖,其中葡萄糖和阿拉伯糖残基的摩尔比为7:3,TF‑P的化学结构中包含1,4‑连接的葡萄糖残基和1,4‑连接的阿拉伯糖残基。所述TF‑P经油黄口蘑子实体热水浸提、乙醇醇沉、除蛋白、离子交换柱层析、透析和浓缩得到。本发明的TF‑P具有显著的免疫调节活性和抗肿瘤活性,可应用在药品、保健品或食品中。
Description
技术领域
本发明涉及真菌多糖应用技术领域,特别涉及一种油黄多糖及其制备方法和应用。
背景技术
食用菌俗称蘑菇,是一种大型真菌,其子实体中富含蛋白质、维生素、矿质元素、氨基酸以及多糖等营养物质。食用菌多糖具有抗病毒、抗氧化、抗肿瘤、降血脂、促进免疫细胞的增殖分化和淋巴因子的分泌、激活补体等免疫调节等生物活性等特点,安全无毒,其在保健食品和生物医药等领域受到广泛的关注。而且食用菌多糖是一种非特异性免疫增强剂,它可通过多种途径提高机体免疫功能,而对机体没有副作用。
油黄口蘑(Tricholoma flavovirens)又名荞面菌、黄荞面菌、黄丝菌、油蘑、马口蘑、黄白蘑、荞面蘑、黄茅草以及松毛菇等,隶属担子菌亚门、层菌纲、伞菌目、白蘑科、口蘑属,其味道鲜美,营养丰富,属优良食菌。油黄口蘑的子实体黄色,菌盖宽5-10cm,扁半球形至平展,顶部稍凸起,淡黄色、柠檬黄色,具褐色鳞片,粘,边缘平滑易开裂。菌肉白色至带淡黄色,稍厚。菌褶淡黄色至柠檬黄色,稍密,弯生,不等长,边缘锯齿状。菌柄长4.5-7cm,粗0.8-2cm,圆柱形,淡黄色,具纤毛状小鳞片,内实至松软,基部稍膨大。孢子印白色。孢子无色,光滑,卵圆形至宽椭圆形,6-7.5μm×4-5μm。
张水花等在《微波辅助提取油黄口蘑多糖工艺及其体外抗氧化性研究》(北方园艺,2018,419(20):120-124.)报道了液料比、微波功率、微波时间、提取次数对云南野生油黄口蘑子实体多糖提取率的影响,在单因素试验基础上,采用正交实验优化了工艺条件,对最佳工艺条件下提取产物进行了DPPH、ABTS+清除研究。结果表明,对油黄口蘑多糖提取率影响最大的是微波时间,其次是液料比,提取次数影响最小;优化工艺条件为液料比15:1mL/g,微波功率320W,微波时间3min,提取3次,在此条件下多糖提取率达4.11%。抗氧化性研究表明,多糖具有良好的抗氧化能力。在一定浓度范围内,清除能力与浓度呈正相关,当多糖浓度分别为3.5、2.5g/L时对DPPH·及ABTS+·的清除率分别达到88%和90%。
Pessoa等在《Action of bioactive compounds in cellular oxidativeresponse.》(Energy Reports,2020,6(1):891-896.)报道了从油黄口蘑子实体中分离到的多糖具有抗氧化的特性。
可见,现有技术中缺少对油黄口蘑多糖的精细结构、油黄口蘑多糖在免疫调节活性中应用的研究。
发明内容
本发明克服了现有技术中存在的缺陷,提供了一种油黄口蘑多糖及其制备方法和应用。
本发明的第一方面提供了一种油黄口蘑多糖(TF-P),其为由葡萄糖和阿拉伯糖组成的杂多糖,其中葡萄糖和阿拉伯糖残基的摩尔比为7:3。
进一步地,所述多糖的化学结构中包含1,4-连接的葡萄糖残基和1,4-连接的阿拉伯糖残基。
进一步地,所述多糖的重均分子量为10000-20000Da(如10000Da、11000Da、12000Da、13000Da、14000Da、15000Da、15010Da、15020Da、15030Da、15040Da、15050Da、15060Da、15070Da、15080Da、15090Da、16000Da、17000Da、18000Da、19000Da、20000Da),优选地,为12000-18000Da,进一步优选地,为13000-17000Da。
在本发明的一个实施例中,所述多糖的重均分子量为15075Da。
进一步地,上述多糖包含如下结构式:
其中,n为1-20的整数(如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20),优选地,为2-15的整数,更优选地,为3-10的整数。
其中,Glcp为葡萄糖,Arap为阿拉伯糖。
本发明的第二方面提供了一种组合物,所述组合物包含第一方面所述的多糖。
本发明的第三方面提供了一种如第一方面所述的油黄口蘑多糖的制备方法,所述制备方法包括以油黄口蘑子实体为原料进行提取的步骤。
进一步优选地,所述制备方法包括通过水提醇沉法提取得到粗多糖的步骤。
进一步优选地,所述制备方法还包括(例如通过离子交换柱层析)对粗多糖进行纯化的步骤。
在本发明的一个实施方式中,所述制备方法包括如下步骤:
(1)取油黄口蘑子实体粉末,用热水浸提,将所得水提物依次经浓缩、醇沉、烘干得到粗多糖;
(2)将步骤(1)所得粗多糖通过离子交换柱层析,洗脱,收集洗脱液;
(3)将步骤(2)所得洗脱液用透析袋进行透析浓缩。
进一步地,所述制备方法还包括步骤(4),具体为将完成步骤(3)后的透析袋内液体冷冻干燥。
进一步地,步骤(1)中,所述浸提的温度可以为80-100℃(如80、85、90、95、100℃);在本发明的一个实施例中,该浸提温度为98℃。
进一步地,步骤(1)中,油黄口蘑子实体粉末与水的质量比(W/V,mg/mL)为1:1-10(如1:1、1:2、1:3、1:5、1:8、1:10);在本发明的一个实施例中,该质量比为1:3。
进一步地,步骤(1)中,所述浸提的次数为1-5次(如1、2、3、4、5次);在本发明的一个实施例中,该浸提次数为3次。
进一步地,步骤(1)中,每次浸提时间为1-10小时(如1、3、6、8、10小时);在本发明的一个实施例中,每次浸提时间为6小时。
在本发明的一个实施例中,步骤(1)中的浸提步骤可以包括:取油黄口蘑子实体粉末,与水混合,并置于水浴中煮沸。
进一步地,步骤(1)中,醇沉步骤中,醇与水提物浓缩液的体积比为1-10:1(如1:1、3:1、4:1、5:1、10:1);在本发明的一个实施例中,该体积比为3:1;
在本发明的一个实施例中,上述醇沉步骤中,醇为乙醇。
在本发明的一个实施方式中,步骤(1)包括:取油黄口蘑子实体粉末,热水浸提,收集上清液,浓缩,加入无水乙醇,收集沉淀,烘干,除去其中蛋白质,得到粗多糖。
进一步地,步骤(2)中,离子交换柱可以为纤维素柱,该纤维素柱的填料如DEAE纤维素。
进一步地,步骤(2)中,洗脱所用洗脱剂可以为NaCl溶液;具体地,该NaCl溶液浓度为0.01-1.0mol/L(如0.01、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、1.0)mol/L。
进一步地,步骤(2)中,洗脱可以为梯度洗脱,洗脱剂浓度可以为0.01-1.0mol/L(如0.01、0.1、0.2、0.3、0.4、0.5、0.6、0.8、1.0)mol/L。
在本发明的一个实施方式中,步骤(2)包括:将步骤(1)所得粗多糖的水溶液通过纤维素柱,梯度洗脱,收集洗脱液,浓缩。
进一步地,步骤(3)中,透析袋的截留分子量为5000-10000Da(如5000、6000、7000、8000、9000、10000Da);在本发明的一个实施例中,该截留分子量为7000Da。
在本发明的一个实施例中,步骤(3)包括:将步骤(2)所得洗脱液置于透析袋中进行透析,透析两天。
本发明的第四方面提供了一种如第一方面所述的油黄口蘑多糖或如第二方面所述的组合物或如第三方面所述制备方法制备得到的粗多糖的应用,所述的应用包括:
(1)在制备能够增强免疫力的产品中的应用;
(2)在制备具有抗肿瘤活性的产品中的应用。
进一步地,所述产品为食品、保健品或药品。
进一步地,所述应用中,所述多糖可单独使用,可也与其他活性成分联合使用。
进一步地,所述产品中油黄口蘑多糖的浓度为0.5-25μg/mL(如0.5μg/mL、0.625μg/mL、1.25μg/mL、2.5μg/mL、5μg/mL、10μg/mL、20μg/mL、25μg/mL),优选为0.6-20μg/mL。
进一步地,当为提升RAW 264.7和免疫T细胞的增殖效果时,油黄口蘑多糖的浓度优选为20μg/mL;当为提升免疫B细胞的增殖效果时,油黄口蘑多糖的浓度优选为5μg/mL。
进一步地,当为提升MFC和LLC细胞的抑制效果时,油黄口蘑多糖的浓度优选为10μg/mL;当为提升CT26.WT细胞的抑制效果时,油黄口蘑多糖的浓度优选为1.25μg/mL。
本发明的有益效果是:
本发明自油黄口蘑分离纯化得到多糖TF-P,并对其分子量、单糖组成、化学结构等进行了分析鉴定,确定了其重均分子量和结构组成。细胞实验表明,该多糖具有显著的免疫调节活性,特别在5μg/mL浓度时,B细胞的增殖率最高;特别在20μg/mL浓度时,T细胞和RAW264.7细胞促增殖率最高。该多糖还具有显著的抗肿瘤活性,特别在1.25μg/mL浓度时,CT26.WT细胞的增殖率最高;特别在10μg/mL浓度时,MFC细胞和LLC细胞抑制率最高。
附图说明
图1所示为TF-P的GPC谱图;
图2所示为TF-P的红外谱图;
图3所示为TF-P的HPLC谱图;
图4所示为TF-P的1H NMR谱图;
图5所示为TF-P的13C NMR谱图;
图6所示为TF-P的1H-1H-COSY谱图;
图7所示为TF-P的HMQC谱图;
图8所示为TF-P的HMQC谱图;
图9所示为TF-P对B细胞增殖的影响的实验结果;
图10所示为TF-P对T细胞增殖的影响的实验结果;
图11所示为TF-P对RAW264.7细胞增殖的影响的实验结果;
图12所示为TF-P对MFC细胞增殖的影响的实验结果;
图13所示为TF-P对CT26.WT细胞增殖的影响的实验结果;
图14所示为TF-P对LLC细胞增殖的影响的实验结果。
具体实施方式
除非另有定义,本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
在本发明中,“油黄口蘑(Tricholoma flavovirens)”是指担子菌亚门、层菌纲、伞菌目、白蘑科、口蘑属真菌,其包含子实体和菌丝体。
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1油黄口蘑多糖TF-P的分离提取
1、油黄口蘑多糖TF-P的分离提取
1.1水提醇沉法提取油黄口蘑粗多糖
称取干燥的油黄口蘑子实体200g粉碎,于烧杯中以料液1:3的比例加入粉碎后的油黄口蘑子实体与蒸馏水,在98℃水浴6小时,收集上清液浓缩,重复3次,最终将全部的上清液浓缩至200mL。加入三倍体积的无水乙醇,使其沉淀,收集沉淀并干燥,除去提取液中的蛋白质,从而获得油黄口蘑粗多糖。
1.2DEAE纤维素柱层析法分离纯化油黄口蘑粗多糖
精确称取50g DEAE纤维素,并将其溶解在1L的超纯水中,充分搅拌,若无肉眼可见的纤维素颗粒则停止搅拌。静置24h,弃去上清液,待用。配置0.5mol/L NaOH,浸泡纤维素6h,用超纯水洗涤至中性后,弃去上清,再加入0.5mol/L HCl浸泡6h,蒸馏水洗涤至中性,弃去上清;再加入0.5mol/LNaOH再次浸泡6h,用蒸馏水洗涤至中性,静置待用。
将活化的纤维素装柱后,经蒸馏水压柱平衡24h后即可进行粗多糖的分离纯化。将粗多糖稀释后的上清液(5mL)加入DEAE纤维素柱中,加入不同浓度的NaCl(0.01mol/L,0.05mol/L,0.1mol/L)进行洗脱。多糖用硫酸-苯酚法测定。将洗脱液浓缩至5mL,将样品在纤维素柱上纯化。用透析袋(Mw≥7kDa)透析,持续48h,冷冻干燥,得到油黄口蘑多糖,命名为TF-P。
2、油黄口蘑多糖TF-P的结构鉴定
运用酸水解、甲基化分析、高效凝胶渗透色谱法、高效液相色谱、气相色谱和质谱联用技术、红外谱技术、核磁共振技术,对油黄口蘑多糖(TF-P)进行结构解析。
2.1分子量的测定
将10mg油黄口蘑多糖TF-P样品用1mL ddH2O溶解,超声5min,进行GPC分析。
2.2油黄口蘑多糖TF-P的红外谱分析
将2mg TF-P与KBr混合压片,通过红外分光光度计扫描4000cm-1-400cm-1范围。
2.3油黄口蘑多糖TF-P的单糖组成分析
将六种标准品和经TFA酸水解后的TF-P样品用流动相(75%乙腈)溶解后进行HPLC分析。
2.4油黄口蘑多糖TF-P的核磁共振分析
取50mg TF-P溶于0.6mL重水(D2O)中,装入核磁管中,在核磁共振仪上检测。
2.5油黄口蘑多糖TF-P的甲基化与硅烷化衍生后进行GC-MS分析
称取20mg TF-P样品,将烧杯密封,向密封的烧杯中加入2mL DMSO(二甲基亚砜),轻轻振荡烧杯,使TF-P充分溶解。然后加入200mg的NaOH,直至NaOH刚好不溶解为止并将其放置在摇床中室温振荡1h。振荡完成后,加入1.5mL碘甲烷,避光反应1h,反应后加水终止反应。用氯仿萃取产物,干燥后得甲基化多糖。甲基化多糖经TFA完全酸水解后,水洗三次,得甲基化完全酸水解产物。
将上述样品与2mL六甲基二硅烷胺、1mL三甲基氯硅烷以及2mL的无水吡啶充分反应,并在50℃条件下水浴20min,采用低温高速离心机在转速12000rpm/min,4℃条件下离心10min,弃去沉淀,用0.22μm过滤器过滤,取上层溶液用于GC-MS分析。
3、结果
3.1油黄口蘑多糖TF-P的基本性质结果
TF-P的GPC谱图如图1所示,其显示TF-P的重均分子量为15075Da。
3.2油黄口蘑多糖TF-P的FTIR谱分析
采用傅里叶红外光谱对TF-P进行初级结构表征,结果如图2所示,TF-P在4000~500cm-1范围内有明显信号峰。在特征峰区(4000~1250cm-1),3439.88cm-1处的强宽峰由糖分子的O-H伸缩振动引起,表明多糖分子内或分子间存在氢键;2927.11cm-1处为亚甲基的C-H伸缩振动峰;1639.10cm-1处尖峰由C=O非对称伸缩振动引起;1402.43cm-1处为C-H面内弯曲振动峰。在指纹图谱区(1250~500cm-1),1081.93cm-1处为吡喃糖环C-O伸缩振动峰。TF-P的FT-IR数据表明TF-P具有多糖类物质的特征吸收峰且结构中含有吡喃糖环。
3.3油黄口蘑多糖TF-P的单糖组成分析
将TF-P完全水解后,采用HPLC对其进行单糖组成分析,结果如图3所示,其中4.284min处为阿拉伯糖,5.063min处为葡萄糖。且阿拉伯糖和葡萄糖含量比为30.16:69.84,约为3:7。
3.4油黄口蘑多糖TF-P的NMR谱分析
TF-P的1H NMR结果如图4所示。结果显示,TF-P有两个异头氢信号,分别为:δ4.96和δ4.88,且积分面积比为7:3。δ3.0~4.2之间的信号归属为糖残基中C2-C6的氢信号。
TF-P的13C NMR结果如图5所示,TF-P在δ101.38和δ97.80处有两个异头碳信号。δ60~78之间的信号归属为糖残基中C2-C6的碳信号。
TF-P的1H-1H-COSY谱图如图6所示,据其可以鉴定相邻氢核间的耦合关系。信号A和B为TF-P异头氢与相邻碳上的氢之间的化学信号,以信号A(δ4.96/δ3.62)为例:其异头氢化学位移为δ4.96,对应的邻位氢化学位移为δ3.62,则位于δ4.96/δ3.62的信号峰归属为1,4-葡萄糖残基H1与H2的共振耦合信号。信号B(δ4.88/δ3.76)则归属于1,4-阿拉伯糖残基上H1与H2的共振耦合信号。
所有的氢的化学位移都总结于表1中。
TF-P的HMQC光谱图如图7所示,据其可以鉴定近程相关的1H和13C间的耦合关系。TF-P中信号A(H1/C1:δ4.96/δ101.38)和信号B(H1/C1:δ4.88/δ97.80)分别归属为1,4-葡萄糖残基和1,4-阿拉伯糖残基的H1与C1的耦合信号,与1H-1H COSY谱中信号A和B相对应。
TF-P的HMBC光谱图如图8所示,据其可以鉴定远程相关的1H和13C间的耦合关系。信号A(H1/C3:δ3.62/δ69.60)归属为1,4-葡萄糖残基的H2与C4的共振信号,信号B(H2/C4:δ4.88/δ69.19)归属为1,4-阿拉伯糖残基的H1与C3的耦合信号。
所有的碳的化学位移都总结于表2中。
表1 TF-P的1H的化学位移
表2 TF-P中13C的化学位移
3.5油黄口蘑多糖TF-P的气相色谱与质谱分析
甲基化结果如表3所示,表明TF-P的主要重复结构单元由1,4-连接的葡萄糖和1,4-连接的阿拉伯糖残基交替连接构成。
表3 TF-P甲基化结果分析
实施例2油黄口蘑多糖TF-P的免疫调节活性测定(在体外采用两种CCK-8法测定油黄口蘑多糖TF-P的免疫调节及抗肿瘤活性)
1、试剂
CCK-8试剂盒、RPIM1640、FBS、DMSO、双抗等,均为市售产品。
2、仪器
酶标仪;细胞培养箱。
3、方法
3.1TF-P对免疫细胞(B细胞、T细胞和RAW264.7细胞)的增殖影响
通过细胞计数试剂盒(CCK-8)法测定油黄口蘑多糖(TF-P)对T细胞、B细胞和RAW264.7细胞增殖的影响。体外培养T细胞、B细胞和RAW264.7细胞至对数生长期,细胞计数板计数后,用新的培养液将细胞悬液稀释至1×105个/mL,将细胞悬液加入96孔板,每孔100μL,将96孔板放入CO2培养箱中培养24h。24h后,实验组分别加入不同质量浓度的TF-P溶液(最终质量浓度为0.625、1.25、2.5、5、10、20μg/mL),阳性对照加入100μL LPS溶液(最终质量浓度5μg/mL),空白组加入100μL细胞培养液。在CO2培养箱中培养24h后,分别向每孔加入5μl的CCK-8,放入CO2培养箱中孵育3h后在酶标仪上检测(450nm)吸光度值并拍摄图像。
3.2TF-P对肿瘤细胞(MFC细胞、CT26.WT细胞和LLC细胞)的增殖影响
采用CCK-8法检测多糖对MFC、CT26.WT和LLC细胞的抑制作用。除阳性对照组(MAN组)加入等体积终浓度为5μg/mL的甘露聚糖肽溶液(MAN)外,其余同3.1。
4、结果
4.1TF-P对B细胞的增殖影响
结果如图9所示,与空白组相比,LPS组能显著(P<0.05)促进B细胞增殖,增殖率为50.50%;当TF-P的终浓度为0.625~20μg/mL范围时,能显著(P<0.05)促进B细胞的增殖;且TF-P终浓度为5μg/mL时,TF-P对B细胞的增殖效果最为明显,最大增殖率达到42.65%。
4.2TF-P对T细胞的增殖影响
结果如图10所示,与空白组相比,LPS组能显著(P<0.05)促进T细胞增殖,增殖率为60.86%;当TF-P的终浓度为0.625~20μg/mL范围时,能显著(P<0.05)促进T细胞的增殖;当TF-P终浓度为20μg/mL时,TF-P对T细胞的增殖效果最为明显,最大增殖率达到65.44%。
4.3TF-P对RAW264.7细胞的增殖影响
结果如图11所示,与空白组相比,LPS组能显著(P<0.05)促进RAW264.7细胞增殖,增殖率为24.53%;当TF-P的终浓度为1.25~20μg/mL范围时,均能显著(P<0.05)促进RAW264.7细胞的增殖;当TF-P终浓度为20μg/mL时,TF-P对RAW264.7细胞的增殖效果最为明显,最大增殖率达到28.75%。
4.4TF-P对MFC细胞的增殖影响
结果如图12所示,与空白组相比,MAN组能显著(P<0.05)抑制MFC细胞增殖,抑制率为72.43%;当TF-P的终浓度为0.625~20μg/mL范围时,能显著(P<0.05)抑制MFC细胞的增殖;且TF-P终浓度为10μg/mL时,TF-P对MFC细胞的抑制效果最为明显,最大抑制率达到69.88%。
4.5TF-P对CT26.WT细胞的增殖影响
结果如图13所示,与空白组相比,MAN组能显著(P<0.05)抑制CT26.WT细胞增殖,抑制率为39.58%;当TF-P的终浓度为0.625和1.25μg/mL时,能显著(P<0.05)抑制CT26.WT细胞的增殖;且TF-P终浓度为1.25μg/mL时,TF-P对CT26.WT细胞的抑制效果最为明显,最大抑制率达到40.99%。
4.6TF-P对LLC细胞的增殖影响
结果如图14所示,与空白组相比,MAN组能显著(P<0.05)抑制LLC细胞增殖,抑制率为39.19%;当TF-P的终浓度为1.25~20μg/mL范围时,能显著(P<0.05)抑制LLC细胞的增殖;且TF-P终浓度为10μg/mL时,TF-P对LLC细胞的抑制效果最为明显,最大抑制率达到67.91%。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换等,均应包含在本发明的保护范围之内。
本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。
本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。
Claims (10)
1.一种油黄口蘑多糖,其特征在于,所述多糖为由葡萄糖和阿拉伯糖组成的杂多糖,葡萄糖和阿拉伯糖残基的摩尔比为7:3;所述多糖的化学结构中包含1,4-连接的葡萄糖残基和1,4-连接的阿拉伯糖残基;
所述多糖包含如下结构:
其中,n为1-20的整数。
2.如权利要求1所述的多糖,其特征在于,所述多糖的重均分子量为10000-20000Da,优选地,为12000-18000Da,进一步优选地,为13000-17000Da,特别优选地,为15075Da。
3.如权利要求1所述的多糖,其特征在于,所述n为2-15的整数,优选地,为3-10的整数。
4.一种组合物,其特征在于,所述组合物包含权利要求1-3任一项所述的多糖。
5.如权利要求1所述的多糖的制备方法,其特征在于,包括以油黄口蘑子实体为原料进行提取的步骤:
优选地,所述制备方法包括通过水提醇沉法提取得到粗多糖的步骤;
进一步优选地,所述制备方法还包括对粗多糖进行纯化的步骤。
6.如权利要求5所述的制备方法,其特征在于,所述制备方法包括如下步骤:
(1)取油黄口蘑子实体粉末,用热水浸提,将所得水提物依次经浓缩、醇沉、烘干得到粗多糖;
(2)将步骤(1)所得粗多糖通过离子交换柱层析,洗脱,收集洗脱液;
(3)将步骤(2)所得洗脱液用透析袋进行透析浓缩。
7.如权利要求6所述的制备方法,其特征在于,步骤(1)中,所述浸提温度为80-100℃;
所述油黄口蘑子实体粉末与水的质量比为1:1-10;
所述醇与水提物浓缩液的体积比为1-10:1;
所述醇为乙醇。
8.如权利要求6所述的制备方法,其特征在于,步骤(2)中,所述离子交换柱为纤维素柱,其填料为DEAE纤维素;
所述洗脱所用的洗脱剂为NaCl溶液;
所述洗脱为梯度洗脱。
9.根据权利要求1-3任一项所述的多糖或权利要求4所述的组合物或权利要求4-9任一项所述制备方法制备的多糖的应用,其特征在于,所述应用包括:
(1)在制备能够增强免疫力的产品中的应用;
(2)在制备具有抗肿瘤活性的产品中的应用。
10.根据权利要求9所述的应用,其特征在于,所述产品中油黄口蘑多糖的浓度为0.5-25μg/mL。
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