CN111704679A - 一种猴头菌发酵菌丝体β-葡聚糖及其制备方法和应用 - Google Patents
一种猴头菌发酵菌丝体β-葡聚糖及其制备方法和应用 Download PDFInfo
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- CN111704679A CN111704679A CN202010655498.0A CN202010655498A CN111704679A CN 111704679 A CN111704679 A CN 111704679A CN 202010655498 A CN202010655498 A CN 202010655498A CN 111704679 A CN111704679 A CN 111704679A
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Abstract
本发明提供了一种猴头菌发酵菌丝体β‑葡聚糖及其制备方法和应用,涉及猴头菌应用技术领域。本发明利用饱和硫酸铵盐析沉淀法对猴头菌发酵菌丝体20%醇沉大分子量多糖进行简单有效的分离纯化,成功分离富集到诱变产生的新差异多糖组分β‑葡聚糖(X10‑H3P20)。该葡聚糖具有显著地的体外激活巨噬细胞释放NO的活性,具有广阔的应用前景。
Description
技术领域
本发明属于猴头菌应用技术领域,具体涉及一种猴头菌发酵菌丝体β-葡聚糖及其制备方法和应用。
背景技术
猴头菌[Hericium erinaceus(Bull.)Pers]属担子菌门(Basidiomycota),伞菌纲(Agaricomycetes),红菇目(Russulales),猴头科(Hericiaceae),广泛生长于亚洲东部山区,珍贵的食药兼用菌,在中医药领域有着悠久历史。猴头菌生物活性物质种类丰富,包括多糖、甾醇类、萜类、脂肪酸、酚类等化合物,猴头菌具有重要的生物反应调节剂能力,猴头菌多糖(β-葡聚糖)被公认为重要的活性物质,具有免疫调节、抗癌、降血脂、抗疲劳、抗胃炎、抗氧化和保护神经系统预防退行性脑疾病等功效。随着社会压力日益增大,人类癌症和慢性代谢疾病愈发严重,人们对生活质量要求不断提高,猴头菌日益成为功能性食品和药物研发的有力资源,近年来已被广泛应用于食品、医药、保健品和化妆品等领域,猴菇菌片、山珍精、猴头菇口服液、猴菇饮料、猴菇饼干等产品陆续面世,引起越来越多研究者的关注。
从猴头菌属真菌的子实体、菌丝体及菌丝发酵液中分离得到的多糖统称为猴头菌多糖。现代医学和药理学的很多研究表明,猴头菌多糖在提高机体免疫力、抗癌抑瘤、降血压、降血糖等多方面均具有一定的疗效。野生猴头菌天然资源有限,目前以人工栽培为主,但因其人工栽培也存在周期长、产量低的不足。而液体发酵可以在较短时间内获得大量的菌丝体及猴头菌多糖,具有生产周期短、产量高、成本低、工艺设备简单、可控性好等优点。深层发酵法还能够突破猴头菌生长的环境限制因素,能提供充足的养料供生物活性物质积累,更易于工业化持续大量生产,具有广泛的市场前景。
发明人前期通过ARTP诱变育种获得高产多糖的猴头菌株321,通过对高产多糖诱变菌株321菌丝体多糖和出发菌株(亲本菌株0605)菌丝体多糖理化特性和体外免疫活性比较,诱变前后多糖分子量分布差异主要集中在20%醇沉部分,诱变后菌丝体多糖产生一新的多糖组分,且相较亲本菌株具有更好的体外免疫活性,但菌丝体20%醇沉多糖组分分子量可高达千万级,通过凝胶柱层析和离子交换层析分离纯化困难。
发明内容
有鉴于此,本发明的目的在于提供一种猴头菌发酵菌丝体大分子量多糖及其制备方法和应用。本发明利用饱和硫酸铵盐析沉淀方法对大分子量多糖进行分步纯化,并成功分离富集到差异多糖组分,经鉴定为β-葡聚糖,实现了一种猴头菌发酵菌丝体β-葡聚糖及其制备方法和应用。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种猴头菌发酵菌丝体β-葡聚糖的制备方法,包括以下步骤:(1)将猴头菇发酵菌丝体20%醇沉多糖溶解于水中,向所述水中添加硫酸铵达30%饱和度,4℃静置8~12h后,离心收集沉淀;
(2)将所述沉淀复溶于蒸馏水后,流水透析48h后干燥,得X30-H3P20;
(3)将所述X30-H3P20溶解于水中,向所述水中添加硫酸铵达10%饱和度,4℃静置8~12h后,离心收集沉淀,得菌丝体β-葡聚糖X10-H3P20。
优选的,步骤(1)所述猴头菇发酵菌丝体20%醇沉多糖来源于猴头菇高产多糖菌丝体,所述猴头菇高产多糖菌丝体来源于菌株0605的ARTP诱变菌株321。
优选的,所述猴头菇发酵菌丝体20%醇沉多糖的制备方法,包括:(a)将所述ARTP诱变菌株321的干燥菌丝体与水混合后提取,将提取液浓缩;所述浓缩的浓缩比为1g:3ml;
(b)向所述浓缩后的提取液的上清液中添加无水乙醇,使乙醇在所述上清液中的体积浓度达到20%,4℃静置12h后,离心收集沉淀;
(c)将所述沉淀用体积浓度为20%的乙醇水溶液醇洗后,用蒸馏水溶解,冷冻干燥得所述20%醇沉多糖。
优选的,步骤(a)中所述提取时,所述干燥菌丝体和水的质量体积比为1g:20ml;所述提取的温度为100℃。
优选的,步骤(b)所述离心的转速为10000rpm。
优选的,步骤(c)所述溶解后,还包括将所述溶解后得到的溶解液90℃水浴挥醇2h,并离心除去不溶沉淀。
本发明还提供了一种利用上述制备方法得到的菌丝体β-葡聚糖X10-H3P20,所述菌丝体β-葡聚糖X10-H3P20的数均分子量1.1×106Da,重均分子量为1.3×106Da。
本发明还提供了利用上述制备方法制备得到的菌丝体β-葡聚糖X10-H3P20或所述菌丝体β-葡聚糖X10-H3P20在制备体外激活巨噬细胞释放NO药物中的应用。
本发明提供了一种猴头菌发酵菌丝体β-葡聚糖的富集纯化方法,利用饱和硫酸铵盐析沉淀方法对20%醇沉大分子量多糖进行简单有效的分步纯化,成功分离富集到诱变产生的新差异多糖组分-β-葡聚糖。本发明实施例中以诱变菌株321和出发菌株0605的菌丝体20%醇沉多糖为例进行猴头菌菌丝体β-葡聚糖的分步纯化,经30%饱和度硫酸铵盐析沉淀后,富集到的X30-H3P20组分差异多糖组分占比由15.9%增加到35.9%,经进一步10%饱和度硫酸铵盐析沉淀得到富集组分X10-H3P20,差异多糖组分在该多糖组分中占比达到了66%,实现了多糖差异组分的有效富集和纯化,提供了一种大分子量猴头菌菌丝体活性多糖的简单、快速、有效的纯化富集方法;且X10-H3P20较富集前具有明显的体外激活巨噬细胞释放NO的活性,可作为免疫调节剂/膳食营养补充剂应用于健康食品或药品中,服务于大健康产业。
附图说明
图1为饱和硫酸铵盐析法纯化猴头菌菌丝体多糖流程图;
图2为20%醇沉多糖组分HPSEC图谱;其中H1P20为猴头菌出发菌株0605发酵菌丝体20%醇沉多糖组分;H3P20为猴头菌ARTP诱变菌株321发酵菌丝体20%醇沉多糖组分;
图3为30%饱和度硫酸铵盐析分离多糖组分HPSEC图,其中S30-H3P20为H3P20多糖组分30%饱和度硫酸铵盐析上清组分;X30-H3P20为H3P20多糖组分30%饱和度硫酸铵盐析沉淀组分;
图4为10%饱和度硫酸铵盐析分离多糖组分HPSEC图,其中S10-H3P20为X30-H3P20经10%饱和度硫酸铵盐析上清组分;X10-H3P20为X30-H3P20多糖组分10%饱和度硫酸铵盐析沉淀组分;
图5为纯化富集前后猴头菌多糖的红外光谱图;
图6为纯化富集前后猴头菌菌丝体多糖对RAW264.7巨噬细胞释放NO的影响,其中PBS磷酸缓冲液为阴性对照;LPS细菌脂多糖为阳性对照(1μg/ml)。
具体实施方式
本发明提供了一种猴头菌发酵菌丝体β-葡聚糖的制备方法,包括以下步骤:(1)将猴头菇发酵菌丝体20%醇沉多糖溶解于水中,向所述水中添加硫酸铵达30%饱和度,4℃静置8~12h后,离心收集沉淀;
(2)将所述沉淀复溶于蒸馏水后,流水透析48h后干燥,得X30-H3P20;
(3)将所述X30-H3P20溶解于水中,向所述水中添加硫酸铵达10%饱和度,4℃静置8~12h后,离心收集沉淀,得菌丝体β-葡聚糖X10-H3P20。
本发明将猴头菇发酵菌丝体20%醇沉多糖溶解于水中,向所述水中添加硫酸铵达30%饱和度,4℃静置8~12h后,离心收集沉淀。本发明所述猴头菇发酵菌丝体20%醇沉多糖与水的质量体积比优选为20mg:50ml。本发明所述溶解优选为在70℃恒温震荡3h直至完全溶解。本发明所述猴头菇发酵菌丝体20%醇沉多糖优选来源于猴头菇高产多糖菌丝体,所述猴头菇高产多糖菌丝体优选来源于猴头菌菌株0605的ARTP诱变菌株321(a.杨珊,杨焱,李巧珍,等.常压室温等离子体诱变筛选高产多糖猴头菌株的研究.上海农业学报,2019,35(5):6~11;b.宋甜甜,吴迪,张赫男,等.ARTP诱变猴头菌株的发酵菌丝体多糖理化性质及体外免疫活性.菌物学报.2018,37(6):794~804)。
本发明所述猴头菇发酵菌丝体20%醇沉多糖的制备方法,优选包括:(a)将所述ARTP诱变菌株321的干燥菌丝体加水浸润后提取,将提取液浓缩、离心,收集上清提取液;所述浓缩的浓缩比为1:3(W/V,g/ml);
(b)向所述上清提取液中添加无水乙醇,使乙醇在所述上清提取液中的体积浓度达到20%,4℃静置12h后,离心收集沉淀;
(c)将所述沉淀用体积浓度为20%的乙醇水溶液醇洗后,用蒸馏水溶解,冷冻干燥得所述20%醇沉多糖。
本发明步骤(a)中所述提取时,所述干燥菌丝体和水的质量体积比优选为1g:20ml;所述提取的温度优选为100℃。本发明在所述提取时,优选提取2次,每次提取2h,并将每次提取得到的上清液混合。本发明步骤(b)所述离心的转速优选为10000rpm。本发明步骤(c)中所述醇洗的次数优选为2次,在进行所述醇洗时剧烈振荡,离心收集沉淀。本发明将经过所述醇洗后的沉淀用蒸馏水溶解,在所述溶解后,优选还包括将所述溶解后得到的溶解液90℃水浴挥醇2h,并离心除去不溶沉淀。本发明对所述冷冻干燥的具体参数并没有特殊限定,利用本领域的常规冷冻干燥方法即可。
本发明所述干燥菌丝体优选为利用ARTP诱变菌株321的菌丝块经斜面培养-平板活化-种子培养-液体发酵培养后得到。本发明对所述斜面培养、平板培养、种子培养和液体发酵培养的培养条件和培养基并没有特殊限定,利用本领域的常规培养方法即可。
得沉淀后,本发明将所述沉淀复溶于蒸馏水,流水透析48h后干燥,得X30-H3P20。本发明所述流水透析可以除去组分中的硫酸铵盐。本发明所述干燥优选为冷冻干燥。本发明为考察盐析分离效果,优选还包括对盐析处理上清液也进行相同的流水透析后干燥,得S30-H3P20。
得X30-H3P20后,本发明将所述X30-H3P20溶解于水中,向所述水中添加硫酸铵达10%饱和度,4℃静置8~12h后,离心收集沉淀,得菌丝体β-葡聚糖X10-H3P20。本发明所述溶解时X30-H3P20和水的质量体积比优选为1:1(W/V,g/ml)。
本发明所述制备方法的流程(饱和硫酸铵盐析分离纯化猴头菌菌丝体多糖)优选如图1所示,诱变菌株321较出发菌株0605的菌丝体20%醇沉多糖的差异组分在H3P20多糖组分中质量比仅有15.9%,经30%饱和度硫酸铵盐析沉淀后,富集到的X30-H3P20组分差异多糖组分占比增加到35.9%,该组分又经10%饱和度硫酸铵盐析沉淀得到富集组分X10-H3P20,差异多糖组分在该多糖组分中占比达到了66%。通过硫酸铵盐析沉淀方法我们对差异多糖组分实现了有效的富集。
本发明还提供了一种利用上述制备方法得到的菌丝体β-葡聚糖X10-H3P20,所述菌丝体β-葡聚糖X10-H3P20的数均分子量1.1×106Da,重均分子量为1.3×106Da。本发明对所述菌丝体β-葡聚糖X10-H3P20进行红外光谱分析,3420cm-1处有强的O-H伸缩振动吸收峰,2900cm-1处有明显的C-H伸缩振动吸收峰,这两组吸收峰是糖类的特征峰;在1650cm-1处有明显的C=O的伸缩振动峰;1160cm-1、1072cm-1和1038cm-1之间的一组吸收峰是C-O-H侧基的伸缩振动和吡喃糖环的C-O-H糖苷带振动造成的。890cm-1附近是吡喃糖β型碳氢键变角振动的特征峰,糖环中存在β-吡喃糖苷类。
本发明还提供了利用上述制备方法制备得到的菌丝体β-葡聚糖X10-H3P20或所述菌丝体β-葡聚糖X10-H3P20在制备体外激活巨噬细胞释放NO药物中的应用。本发明实施例中进行了体外刺激巨噬细胞释放NO的活性试验,结果表明所述菌丝体β-葡聚糖X10-H3P20具有较明显的体外激活巨噬细胞释放NO的活性,在浓度为500μg/ml时释放量最高可达12.68μmol/L,与阳性对照LPS效果相当。本发明对所述药物的剂型并没有特殊限定。本发明所述药物中优选还包括药学上可接受的辅料。
下面结合实施例对本发明提供的猴头菌发酵菌丝体β-葡聚糖及其制备方法和应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
1、高产多糖猴头菌菌株321发酵菌丝体的培养
1)斜面培养
取一块生长活力较旺盛的菌丝块(约5×5cm)接种于PDA试管斜面后置于26℃恒温培养箱中静置培养12~15d左右,直至菌丝体将铺满斜面,用报纸包裹避光保存至4℃冰箱中备用。
2)平板培养
从试管斜面上取一块生长活力较旺盛的菌丝块(约5×5cm)接种于PDA平板中央,用封口膜封口后置于26℃恒温培养箱中静置培养12~15d,当菌丝体长满平板约80%时,用报纸包裹避光保存至4℃冰箱中备用。
3)种子培养
接种前将平板放入26℃恒温培养箱中活化1~2h。用已灭菌的打孔器(直径8mm)取平板培养的猴头菌块10~12块接种于容量为250ml含有100ml的种子培养基(马铃薯葡萄糖肉汤24g/L,KH2PO4 2g/L和MgSO4·7H2O 1g/L,于灭菌锅中121℃,灭菌30min)三角摇瓶中。在26℃、150r/min恒温震荡摇床中发酵培养10d后即得种子液。
4)液体发酵培养
将培养好的种子液以10%(V/V)的接种量接入发酵培养基(葡萄糖20g/L,酵母自溶粉10g/L,KH2PO4 2g/L和MgSO4·7H2O 1g/L)中。在26℃、150r/min培养8d,纱布过滤收集菌丝体,并蒸馏水洗涤3次,冷冻干燥,得干燥菌丝体。
2、菌丝体多糖的分离纯化
1)猴头菌发酵菌丝体多糖组分制备
分别准确称取猴头菌液体发酵干燥菌丝体20.00g,按料液比1:20(W/V,g/ml)加入蒸馏水,100℃提取2h,取上清,提取两次,合并提取液,按浓缩比1:3(W/V,g/ml)浓缩。上清添加无水乙醇,使乙醇体积终浓度达到20%,4℃冰箱中静置12h,10000r/min离心后沉淀用20%乙醇醇洗两次,醇洗时剧烈振荡,收集沉淀,蒸馏水充分溶解后,90℃水浴挥醇2h,离心除去不溶沉淀,冷冻干燥,获得20%醇沉多糖组分。
2)诱变菌株32120%多糖组分中差异组分的分离纯化
将20mg20%醇沉多糖样品溶解于50ml蒸馏水中,70℃恒温震荡3h直至完全溶解,室温条件下缓慢向溶液中加入硫酸铵,不断搅拌使其溶解,直至硫酸铵饱和度达30%,4℃静置过夜后,高速离心得到上清和沉淀两部分,将沉淀复溶于蒸馏水中,对该部分溶液和离心后的上清组分流水透析48h以除去组分中的硫酸铵盐,离心,浓缩,冻干,分别命名为“S30-H3P20”和“X30-H3P20”。按上述操作步骤,对“X30-H3P20”差异组分进一步10%饱和度硫酸铵盐析分离富集,最终纯化富集到一种猴头菌发酵菌丝新的β-葡聚糖“X10-H3P20”。
饱和硫酸铵盐析沉淀分离纯化流程如图1,诱变菌株321较出发菌株0605的菌丝体20%醇沉多糖的差异组分在H3P20组分中占比仅有15.9%,经30%饱和度硫酸铵盐析沉淀后,富集到的X30-H3P20组分差异多糖组分占比增加到35.9%,该组分又经10%饱和度硫酸铵盐析沉淀得到富集组分X10-H3P20,差异多糖组分在该多糖组分中占比达到了66%。通过硫酸铵盐析沉淀方法对差异多糖组分实现了有效的富集。
对出发菌株0605、ARTP诱变菌株321的发酵菌丝体20%醇沉多糖进行反复醇洗后,进行流水透析,冻干,考察分子量分布情况,结果如图2所示,ARTP诱变对猴头菌发酵菌丝体大分子量多糖结构产生影响,多糖差异组分主要为保留时间36min左右的部分。因此接下来对该差异组分进行分离纯化。
猴头菌诱变菌株321的20%醇沉多糖组分经30%饱和度的硫酸铵盐析后,得到上清液和沉淀两个组分,分别为S30-H3P20和X30-H3P2,HPSEC图谱(图3)显示两部分的色谱峰在保留时间为36min处有较大差别,其中X30-H3P20组分将差异组分富集,但所占比例仅为35.9%,还需要针对保留时间为36min左右的差异组分多糖进行富集。
经30%饱和度硫酸铵盐析,得到已富集部分差异多糖组分的X30-H3P20,但是HPSEC图谱显示还有一个较大的多糖组分峰未除去,为了将该组分分离,将透析冻干后的X30-H3P20溶解后加入固体硫酸铵盐使其最终饱和度为10%,低温过夜后得到上清和沉淀两个组分,经过溶解透析冻干后得到S10-H3P20和X10-H3P20两个多糖组分。X10-H3P20液相色谱分析结果如图4所示,X30-H3P20经过10%饱和度硫酸铵盐析后,第一个分子量较大的峰明显减小,差异多糖组分相对富集,所占比例提升至66%。
实施例2
富集纯化的β-葡聚糖X10-H3P20结构和活性的分析
2.1分子量分析
样品的制备和处理
分别称取5mg待测样品,溶解于1ml流动相中,流动相含0.05mol/L的NaH2PO4和pH70.02%叠氮钠,配制成浓度5mg/ml的溶液。用12000×g离心20min后取上清液,经0.22μm的水相微孔膜过滤后进行HPSEC-MALLS-RI分析。
色谱分析条件:分析柱选TSKPWXL6000和TSKPWXL4000凝胶色谱柱串联分析,流速为0.5ml/min,色谱柱温用柱温箱恒定在35℃;激光检测器光源波长623.8nm。多糖在溶液中的折光指数增量(dn/dc)按照0.146ml/g计算。
数据处理
使用Astra(version 6.1.1,Wyatt Technology,Santa Barbara,CA)数据分析软件对光散射数据进行采集和分析,计算分子量。
利用饱和硫酸铵盐析沉淀方法对ARTP诱变菌株321菌丝体20%醇沉多糖中较出发菌株的差异多糖组分实现了有效富集,对富集的差异多糖组分进行分子量分布分析,结果见表1,在分离纯化过程中差异多糖组分占比由15.9%增加至66%,该差异组分的重均分子量在一百万道尔顿以上。
表1分子量分布情况
2.2红外光谱
取2mg左右的样品与KBr粉末研磨混合均匀,压片后在400~4000cm-1范围内进行红外光谱分析
对差异多糖组分的基本结构进行分析,红外光谱如图5所示,“H3P20”经纯化富集后多糖特征吸收峰未发生明显改变。对差异多糖组分“X10-H3P20”红外光谱分析可知,3420cm-1处有强的O-H伸缩振动吸收峰,2900cm-1处有明显的C-H伸缩振动吸收峰,这两组吸收峰是糖类的特征峰;在1650cm-1处有明显的C=O的伸缩振动峰;1160cm-1、1072cm-1和1038cm-1之间的一组吸收峰是C-O-H侧基的伸缩振动和吡喃糖环的C-O-H糖苷带振动造成的。890cm-1附近是吡喃糖β型碳氢键变角振动的特征峰,糖环中存在β-吡喃糖苷类。
2.3单糖组成分析
采用高效阴离子交换色谱法测定不同菌株多糖组分的单糖组成:准确称取2mg样品于V型反应瓶中,加入2mol/L的3ml三氟乙酸(TFA),110℃,水解3h,冷却至室温后用氮气在45℃下吹干,加入3ml甲醇,吹干,重复4~5次操作以除去残留TFA。用超纯水将反应后的样品完全溶解,并定容至50ml,按合适浓度稀释后,吸取1ml,11000g离心15min,吸取上清液,上样检测。
色谱条件:Dionex公司的ICS2500离子色谱仪,CarboPacTMPA20预处理柱,CarboPacPA-20阴离子交换柱(150mm×3mmi.d.),脉冲安培检测器,柱温30℃流动相为超纯水和0.25mol/L的NaOH,流速为0.45ml/min,上样量25μL。
“H3P20”和“X10-H3P20”多糖组分单糖组成分析结果如表2所示,H3P20多糖组分主要含有半乳糖、葡萄糖、甘露糖,所占比例大约为1:1.5:1,经过纯化富集后得到的差异多糖组分X10-H3P20主要由葡萄糖构成,说明APTP诱变后产生的差异组分为β-葡聚糖。
表2单糖组成分析
注:表中数值单位为μg/ml。
实施例3
体外刺激巨噬细胞释放NO的活性试验
称取一定量透析过(分子量3.5kDa,3d)的猴头菌多糖,于灭过菌的Eppendof离心管中,在无菌条件下,用PBS溶液配成5mg/ml的样品母液,18000×g离心30min,在无菌操作台将上清转入灭菌管中,用PBS将样品稀释至50μg/ml、100μg/ml、200μg/ml、500μg/ml备用。
细胞培养:取对数生长期的RAW264.7巨噬细胞株(购自中科院细胞所),用DMEM完全培养基(购自Gibco公司)在37℃、含5%CO2条件下传代培养,用0.05%胰酶或5%EDTA溶液消化,混悬液1000rpm离心3min后收集细胞,计数备用。
Griess试剂的配制:在烧杯中加入6.25ml H3PO3,加入蒸馏水250ml,分别加入2.5g的sulfanilamide(4-氨基苯磺酰胺,Sigma公司)和0.25g的naphthylethylenediaminedihydrochloride(盐酸萘乙二胺,Sigma公司)用磁力搅拌器至全部溶解,棕色试剂瓶4℃冰箱保存。
标准曲线绘制:配成不同浓度的亚硝酸钠溶液,浓度梯度为0、5、10、15、20、25、30、35、40μM共九个;取100μL于96孔板孔,加入50μL Griess试剂,测定543nm吸光值,标准曲线每个浓度3个重复,根据吸光值绘制标准曲线,标准曲线公式为Y=13.2X+0.055,其中Y为吸光值,X为根据标准曲线计算出的产生NO的量,单位为μmol/L。
样品刺激巨噬细胞释放NO量的测定:收集RAW264.7细胞,用无色RPMI1640培养基(购自Gibco公司)(10%胎牛血清+1%抗生素液体)将细胞稀释至5×105/ml,加入96孔板,每孔加入180μL,然后再加入20μL待测样品,阴性对照为PBS,37℃培养48小时。取100μL上清于96孔板,加入50μl Griess试剂,室温孵浴10分钟,测定543nm吸光值。根据标准曲线计算细胞NO的释放量。
结果如图6所示,在浓度50μg/ml时,H3P20、X10-H3P20刺激巨噬细胞产生NO的量(单位:μmoL/L)分别为:4.15±0.15,7.77±0.28;在浓度100μg/ml时,产生NO量分别为4.32±0.35,9.19±0.24;在浓度200μg/ml时,产生NO量分别为5.21±0.27,12.03±0.36;在浓度500μg/ml时,产生NO量分别为7.68±0.12,12.68±0.22,阴性对照4.01±0.18,阳性对照(浓度1μg/ml)12.53±0.3μmoL/L,样品组显著高于阴性对照活性,差异多糖组分“X10-H3P20”较“H3P20”具有较明显的体外激活巨噬细胞释放NO的活性,显示出良好的应用前景。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.一种猴头菌发酵菌丝体β-葡聚糖的制备方法,其特征在于,包括以下步骤:
(1)将猴头菇发酵菌丝体20%醇沉多糖溶解于水中,向所述水中添加硫酸铵达30%饱和度,4℃静置8~12h后,离心收集沉淀;
(2)将所述沉淀复溶于蒸馏水后,流水透析48h后干燥,得X30-H3P20;
(3)将所述X30-H3P20溶解于水中,向所述水中添加硫酸铵达10%饱和度,4℃静置8~12h后,离心收集沉淀,得菌丝体β-葡聚糖X10-H3P20。
2.根据权利要求1所述制备方法,其特征在于,步骤(1)所述猴头菇发酵菌丝体20%醇沉多糖来源于猴头菇高产多糖发酵菌丝体,所述猴头菇高产多糖菌丝体来源于菌株0605的ARTP诱变菌株321。
3.根据权利要求2所述制备方法,其特征在于,所述猴头菇发酵菌丝体20%醇沉多糖的制备方法,包括:(a)将所述ARTP诱变菌株321的干燥菌丝体与水混合后提取,将提取液浓缩;所述浓缩的浓缩比为1g:3ml;
(b)向所述浓缩后的提取液的上清液中添加无水乙醇,使乙醇在所述上清液中的体积浓度达到20%,4℃静置12h后,离心收集沉淀;
(c)将所述沉淀用体积浓度为20%的乙醇水溶液醇洗后,用蒸馏水溶解,冷冻干燥得所述20%醇沉多糖。
4.根据权利要求3所述制备方法,其特征在于,步骤(a)中所述提取时,所述干燥菌丝体和水的质量体积比为1g:20ml;所述提取的温度为100℃。
5.根据权利要求3所述制备方法,其特征在于,步骤(b)所述离心的转速为10000rpm。
6.根据权利要求3所述制备方法,其特征在于,步骤(c)所述溶解后,还包括将所述溶解后得到的溶解液90℃水浴挥醇2h,并离心除去不溶沉淀。
7.一种利用权利要求1~6任一项所述制备方法制备得到的菌丝体β-葡聚糖X10-H3P20,其特征在于,所述菌丝体β-葡聚糖X10-H3P20的数均分子量1.1×106Da,重均分子量为1.3×106Da。
8.利用权利要求1~6任一项所述制备方法得到的菌丝体β-葡聚糖X10-H3P20或权利要求7所述菌丝体β-葡聚糖X10-H3P20在制备体外激活巨噬细胞释放NO药物中的应用。
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