CN106755184B - 干巴菌菌丝体多糖及其制备方法和应用 - Google Patents
干巴菌菌丝体多糖及其制备方法和应用 Download PDFInfo
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- CN106755184B CN106755184B CN201611167404.5A CN201611167404A CN106755184B CN 106755184 B CN106755184 B CN 106755184B CN 201611167404 A CN201611167404 A CN 201611167404A CN 106755184 B CN106755184 B CN 106755184B
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
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- Life Sciences & Earth Sciences (AREA)
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- General Chemical & Material Sciences (AREA)
- Gerontology & Geriatric Medicine (AREA)
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- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Cosmetics (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及干巴菌多糖技术领域,特别涉及一种干巴菌菌丝体多糖,保藏编号为CGMCC No.12977干巴菌菌株TG‑1在液体培养基中发酵,菌丝体提取多糖,层析分离后用去离子水、0.05、0.1、0.3 mol/L的NaCl溶液洗脱,分别得到MPS‑1、MPS‑2、MPS‑3和MPS‑4。TG‑1在液体发酵培养时菌丝体生长旺盛,菌丝体多糖产量高,对干巴菌的开发利用及干巴菌菌丝体多糖的规模化生产具有非常重要的意义;MPS‑2的抗氧化能力最强,其次为MPS‑3,MPS‑4和MPS‑1,为多糖的活性分析以及多糖抗衰老功能食品的开发奠定了理论基础;干巴菌菌丝体多糖还具有优良的保湿能力。
Description
技术领域
本发明涉及干巴菌多糖技术领域,特别涉及一种干巴菌菌丝体多糖,还涉及所述干巴菌菌丝体多糖的制备方法及其应用。
背景技术
干巴菌(Thelephora ganbajun Zang)是我国特有的革菌属(Thelephora)珍稀野生食用菌,口感好营养价值高,仅仅分布在我国滇中800~2300 m高海拔地区的弱酸性红壤地面上。干巴菌与松树有外生菌根关系,人工栽培难以成功,因此导致干巴菌无序乱采现象严重,产量逐年下降,每年干巴菌上市時总是供不应求,价格逐年升高。由于其分布的局限性,国内外对干巴菌的研究极为有限,主要集中在干巴菌的分类、菌种分离、菌种鉴定和生态环境等方面。因此,干巴菌是一种具有极高经济价值及营养价值的亟待研究开发的菌种资源。
目前应用于化妆品的多糖类保湿剂主要有透明质酸、甲壳素及其衍生物、植物多糖提取物、肝素等。近年来将具有生物活性的食用真菌多糖作为保湿剂应用于化妆品,满足了人们对高品质化妆品的需求,成为保湿化妆品的发展趋势。干巴菌菌丝体多糖(Myceliapolysaccharide, MPS)良好的保湿和抗氧化特性是化妆品市场最重要的功能性诉求。
自由基是机体细胞代谢过程中产生的活性物质,如果自由基在体内过多积累就会引起生物膜的不饱和脂类发生氧化反应,形成脂质过氧化物,从而引起细胞结构和功能的改变,造成对机体的损害,引起各种疾病,这就是引起机体衰老的主要原因。干巴菌MPS可以清除体内的自由基,进而起到保护生物膜和延缓衰老的作用。
因此,干巴菌MPS可以作为天然、营养、安全的化妆品保湿剂和抗衰老功能食品,从而为干巴菌资源开发提供有效的途径。
陆文娟等在《响应面法优化提取干巴菌多糖的工艺研究》《南京师范大学学报(工程技术版)》,2015,15(3):84-92,以干巴菌子实体为原料,采用超声细胞破碎法提取其多糖,在单因素实验的基础上,采用响应面法对提取工艺进行优化,通过Box-Behnken设计,建立并分析了各因素与多糖得率关系的数学模型,结果显示,最佳工艺条件为:液料比为38:1,提取时间为3 h,提取温度为88℃,超声功率为603 W,重复2次,测定干巴菌多糖的得率为5.96%。但并没有涉及干巴菌的液体发酵培养、干巴菌多糖的分离纯化以及具有清除自由基、抗氧化功效的多糖组分。
发明内容
为了解决以上现有技术中干巴菌多糖在提取和应用中存在的利用不充分的问题,本申请提供了由干巴菌菌株(Thelephoraganbajun Zang)TG-1发酵得到的菌丝体多糖。本课题组从云南地区分离纯化得到了一株干巴菌菌株(TG-1),并且研究发现干巴菌在液体发酵培养时菌丝体生长旺盛,多糖产量高,并且通过抗氧化实验和保湿实验证明干巴菌菌丝体多糖(MPS)具有良好的保湿和抗氧化特性。
本申请还提供了所述干巴菌菌丝体多糖的制备方法。
本申请还提供了所述干巴菌菌丝体多糖的应用。
本发明是通过以下步骤得到的:
一种干巴菌菌丝体多糖,是通过以下步骤得到的:
保藏编号为CGMCC No.12977干巴菌菌株TG-1在液体培养基中发酵,菌丝体经过分离,提取干巴菌菌丝体多糖MPS,然后经过阴离子交换柱层析分离,依次用去离子水、0.05、0.1、0.3 mol/L的NaCl溶液进行洗脱,分别得到干巴菌多糖组分MPS-1、MPS-2、MPS-3和MPS-4。
所述的干巴菌菌丝体多糖,优选液体培养基中含有马铃薯150~250 g/L,葡萄糖15~25 g/L,蛋白胨1~3 g/L,硫酸镁0.5~2.5 g/L,磷酸二氢钾0.5~2.5 g/L,pH为4.5~7,于15~30 ℃发酵培养。
所述的干巴菌菌丝体多糖,菌丝体经过分离,提取干巴菌菌丝体多糖的优选操作如下:
(1)将菌丝体分离后烘干或冻干,并粉碎得到干巴菌菌丝体粉末;
(2)干巴菌菌丝体粉末与去离子水以1﹕10~1﹕30的比例混合,调节pH值为7~9,超声波破碎,超声功率为200~800 w,超声时间为5~20 min,然后置于50~100 ℃水浴中,提取1~3 h,离心取上清液;
(3)离心后的菌丝体残渣按步骤(2)再提取2~4次,合并上清液;
(4)将上清液浓缩至原体积的3/4~1/2,加入1~3倍体积乙醇,静置至沉淀完全析出,3000~15000 r/min离心5~15 min,弃上清并于40~60 ℃条件下烘干,烘干后再次溶解,离心,上清液去蛋白至少3次,冻干后即为干巴菌菌丝体多糖。
所述的干巴菌菌丝体多糖,优选干巴菌菌丝体多糖经过阴离子交换柱层析分离操作如下:
将干巴菌菌丝体多糖溶于去离子水中,得到上样溶液,将上样溶液用规格为1.6× 30 cm的DEAE-52纤维素阴离子交换柱进行层析分离,依次用去离子水和0.05、0.1、0.3mol/L的NaCl溶液进行洗脱,洗脱液的流速为1 mL/min,分别得到的洗脱组分冻干后即为干巴菌多糖组分MPS-1,MPS-2,MPS-3和MPS-4。
所述的干巴菌菌丝体多糖在制备抗衰老保健品和化妆品中的应用。
优选干巴菌菌丝体多糖为干巴菌多糖MPS及其组分MPS-1、MPS-2、MPS-3和MPS-4。
所述的保健品是具有清除自由基、抗氧化作用的保健品。
所述的化妆品是具有清除自由基、抗氧化和保湿作用的化妆品。
有益效果:
1)利用液体发酵途径,制备干巴菌菌丝体多糖,为干巴菌的资源化利用提供了一条有效途径;
2)干巴菌菌株TG-1在液体发酵培养时菌丝体生长旺盛,菌丝体多糖产量高,因此,干巴菌菌株TG-1对干巴菌的开发利用及干巴菌菌丝体多糖的规模化生产具有非常重要的意义;
3)抗氧化实验表明,干巴菌菌丝体多糖及其组分具有优良的抗氧化活性,清除羟基自由基能力的强弱顺序为MPS-2 >MPS > MPS-3 > MPS-4 > MPS-1,清除DPPH自由基能力的强弱顺序为MPS-2 >MPS > MPS-3 > MPS-4 > MPS-1,清除ABTS自由基能力的强弱顺序为MPS-2 > MPS-3 > MPS-4 > MPS > MPS-1,因此,MPS的三个组分中MPS-2的抗氧化能力最强,其次为MPS-3,MPS-4和MPS-1,为干巴菌胞外多糖构效关系的研究、多糖的改造、多糖的活性分析以及多糖抗衰老功能食品的开发奠定了理论基础;
4)干巴菌菌丝体多糖MPS具有优良的保湿能力,在某些条件下,MPS的保湿能力要强于透明质酸(HA)和甘油(Gl),可以用于护肤保健。
菌种保藏信息
保藏时间:2016年 9月 23日,
保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,
保藏编号:CGMCC No. 12977,
保藏单位地址:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,邮政编码:100101分类命名:干巴菌 Thelephora ganbajun。
附图说明
图1 干巴菌菌丝体多糖MPS及其组分(MPS-1、MPS-2、MPS-3和MPS-4)清除羟基自由基的能力,
图2 干巴菌菌丝体多糖MPS及其组分(MPS-1、MPS-2、MPS-3和MPS-4)清除DPPH自由基的能力,
图3 干巴菌菌丝体多糖MPS及其组分(MPS-1、MPS-2、MPS-3和MPS-4)清除ABTS自由基的能力,
图4干巴菌菌丝体多糖MPS保湿率随时间的变化曲线(RH60%),
图5干巴菌菌丝体多糖MPS保湿率随时间的变化曲线(RH43%),
图6干巴菌菌丝体多糖MPS保湿率随时间的变化曲线(RH0%)。
具体实施方式
下面通过具体的实施例对本发明进行详细说明,但这些例举性实施方式的用途和目的仅用来例举本发明,并非对本发明的实际保护范围构成任何形式的任何限定,更非将本发明的保护范围局限于此。
实施例1菌株的获得
分别于云南省不同地区采集4个新鲜干巴菌子实体,用75%酒精进行表面消毒,再用无菌水冲洗数次后,用灭菌镊子撕取干巴菌内部组织小块接种到含氯霉素的马铃薯葡萄糖(PDA)培养基平板上,移入24 ℃培养箱中培养,待长出菌丝后,反复纯化,直至获得干巴菌纯培养物。于4个干巴菌子实体中分别筛选获得4株干巴菌菌种,并分别命名为TG-1、TG-2、TG-3、TG-4。
实施例2干巴菌的液体发酵
进行发酵罐培养实验,① 菌种的活化。将干巴菌菌株TG-1、TG-2、TG-3、TG-4分别接种于活化培养基平板(马铃薯 200 g/L,葡萄糖 20 g/L,硫酸镁 1 g/L,磷酸二氢钾 1.5g/L)上,24 ℃培养7 d。取0.5 cm2干巴菌活化菌种接入液体培养基中(马铃薯 200 g/L,葡萄糖 20 g/L,蛋白胨2 g/L,硫酸镁 1 g/L,磷酸二氢钾 1.5 g/L,pH自然),500 mL三角瓶装液量为300 mL,然后于摇床24 ℃振荡培养10 d。② 发酵罐培养条件。装量:7.5升;接种量:10 %;发酵温度:24 ℃;搅拌速度:350 r/min;泡沫控制:接种后加两滴消泡剂。③ 发酵过程中pH值及DO值的测定。利用pH电极及溶氧电极自动在线测定并记录数据。④ 菌丝体的分离。将干巴菌培养物离心(10000 r/min,10 min),获得淡黄色的菌丝体,反复离心洗涤菌丝体3遍后,将菌丝体置于烘箱中,55 ℃烘干。
实验结果表明,在干巴菌的发酵过程中,中期由于菌体的代谢产物增多,pH相对减少较快,而后期相对平稳。从30 h开始,由于菌体的大量繁殖以及胞外多糖的大量产生,培养物逐渐变得粘稠,溶氧率降低较快,而后期,发酵体系粘稠度变化不大,溶氧相对稳定。干巴菌TG-1、TG-2、TG-3、TG-4的菌丝体生物量及MPS产量见表1。
表1 干巴菌TG-1、TG-2、TG-3、TG-4的菌丝体生物量及MPS产量
由表1可见,干巴菌菌株TG-1的菌丝体生物量及MPS产量显著高于菌株TG-2、TG-3、TG-4。因此,干巴菌TG-1是生产干巴菌MPS的理想菌株。
实施例3干巴菌菌丝体多糖的提取
1)干巴菌菌丝体的分离:
将干巴菌培养物离心(10000 r/min,10 min),收集菌丝体,并洗涤菌丝体3次。将菌丝体于55 ℃条件下烘干、粉碎,获得干巴菌菌丝体粉末。
2)干巴菌菌丝体多糖的提取:
取干巴菌菌丝体粉末,将菌丝体粉末与去离子水以1﹕20比例混合,调节pH值为8,超声波粉碎,超声功率为600 w,超声时间为10 min,然后置于90 ℃水浴中,提取2 h,离心取上清液。离心后的菌丝体残渣按上述步骤再提取2次,合并上清液。
3)干巴菌菌丝体多糖的纯化:
将多糖提取液浓缩至原体积1/2。加入2倍体积乙醇,静置至沉淀完全析出,15000r/min离心10 min,弃上清液并于55 ℃条件下烘干,粉碎,将其溶于去离子水中,除蛋白至少6次,冻干后即为MPS。将MPS溶于去离子水中,用0.45 μm的微孔滤膜过滤,得到上样溶液。将上样溶液用规格为1.6 × 30 cm的DEAE-52纤维素阴离子交换柱进行层析分离,依次用去离子水和0.05、0.1、0.3 mol/L的NaCl溶液进行洗脱,分别得到干巴菌多糖组分MPS-1,MPS-2,MPS-3,MPS-4,洗脱液的流速为1 mL/min。用全自动部分收集器收集洗脱液(2 mL/管),用苯酚-硫酸法逐管检测,将各洗脱组分透析除盐,冻干后即为干巴菌多糖组分MPS-1,MPS-2,MPS-3,MPS-4。
实施例4体外抗氧化实验
(1)羟基自由基清除能力的测定
采用Fenton法进行羟基自由基清除能力的测定。配置一系列不同浓度的干巴菌菌丝体多糖水溶液。将1 mL硫酸亚铁溶液(9 mmol/L),1 mL水杨酸乙醇溶液(9 mmol/L),1 mL多糖样品溶液和1 mL过氧化氢溶液(8.8 mmol/L)于试管中混合均匀,37 ℃水浴30 min。离心(5000 r/min,10 min)后取上清液于510 nm处测定吸光度,并用维生素C(Vitamin C)作为阳性对照。按照以下公式计算羟基自由基清除率(Scavenging rate, SR),SR (%) =(A0–A)/A0×100。其中,A0为空白对照的吸光度,A为多糖样品的吸光度。羟基自由基的清除能力以EC50值表示,EC50值代表羟基自由基清除50%時所需要的样品浓度。EC50值越小则代表羟基自由基的清除能力越强。
(2)DPPH自由基清除能力的测定
将干巴菌菌丝体多糖进行梯度稀释,配置一系列不同浓度的多糖水溶液。取2 mL多糖样品溶液于试管中,加入2 mL DPPH乙醇溶液(0.2 mmol/L),混合均匀后避光反应30min,然后在517 nm处测定其吸光度,用维生素C作为为阳性对照。根据吸光度计算DPPH自由基的清除能力,清除能力(SR)的计算公式为:SR (%) = [1–(A–A0)/A1]×100。其中,A为2 mL多糖样品溶液与2 mL DPPH乙醇溶液混合液的吸光度,A0为2 mL乙醇与2 mL多糖样品溶液混合液的吸光度,A1为2 mL去离子水与2 mL DPPH乙醇溶液混合液的吸光度。DPPH自由基的清除能力以EC50值表示,EC50值代表清除50% DPPH自由基的样品浓度。DPPH自由基的清除能力用BHT作为为阳性对照。
(3)ABTS自由基的清除能力
将多糖分别用去离子水进行梯度稀释,配置一系列不同浓度的多糖水溶液。将7mmol/L ABTS溶液和4.9 mmol/L过硫酸钾溶液等体积混合,避光静置20 h,用磷酸盐缓冲液稀释,使其在734 nm处的吸光度为0.7,即为ABTS工作液。取0.8 mL ABTS工作液和0.2 mL样品溶液于试管中,摇匀,避光30 min,于734 nm处测定吸光度。SR (%) = [1–(A样品–A对照)/A空白]×100。空白管用去离子水替代样品,对照管用磷酸盐缓冲液替代ABTS工作液。
由图1可见,MPS及其组分MPS-1,MPS-2,MPS-3,MPS-4都具备羟基自由基的清除能力,并且随着浓度的增加表现出明显的量效关系。MPS及其组分MPS-1,MPS-2,MPS-3,MPS-4清除羟基自由基的EC50值分别为698.09 mg/L,1552.05 mg/L,391.85 mg/L,755.43 mg/L和1019.06 mg/L。因此,清除羟基自由基能力的强弱顺序为MPS-2 >MPS > MPS-3 > MPS-4> MPS-1。
由图2可见,MPS及其组分MPS-1,MPS-2,MPS-3,MPS-4都具备DPPH自由基的清除能力,并且,随着浓度的增加都表现出了明显的量效关系。MPS及其组分MPS-1,MPS-2,MPS-3,MPS-4清除DPPH自由基的EC50值分别为596.42 mg/L,1769.55 mg/L,475.31 mg/L,768.12mg/L和869.75 mg/L。因此,清除DPPH自由基能力的强弱顺序为MPS-2 >MPS > MPS-3 >MPS-4 > MPS-1。
由图3可见,MPS及其组分MPS-1,MPS-2,MPS-3,MPS-4都具备ABTS自由基的清除能力,并且,随着浓度的增加都表现出了明显的量效关系。MPS及其组分MPS-1,MPS-2,MPS-3,MPS-4清除ABTS自由基的EC50值分别为229.07 mg/L,315.67 mg/L,166.27 mg/L,177.46mg/L和190.04 mg/L。因此,清除ABTS自由基能力的强弱顺序为MPS-2 > MPS-3 > MPS-4 >MPS > MPS-1。抗氧化实验结果表明,MPS的三个组分中MPS-2的抗氧化能力最强,其次为MPS-3,MPS-4和MPS-1。
实施例5保湿实验
准确称取干巴菌菌丝体多糖(MPS)、高分子量透明质酸(HA-H)、低分子量透明质酸(HA-L)和甘油(Gl)样品0.2000 g于称量瓶(25×25)中,并配制成质量分数为10%的溶液,分别置于装有饱和醋酸钾(RH 60%)、饱和碳酸钠(RH 43%)和变色硅胶(RH 0%)的干燥器中,用甘油、低分子量及高分子量的透明质酸钠作为对照。将两个干燥器置于25 ℃的恒温箱中,分别在4,8,16,24,32,40,48,60,72,84,96,108,120 h后称重,计算保湿率。保湿率计算公式:保湿率%=(mn﹣m0)/mn×100%,式中:mn为放置之前样品质量,m0为放置之后样品质量。
由图4可以看出,在相对湿度60%的条件下,在120 h时,4种物质的保湿顺序为MPS>HA-L>HA-H>Gl,并且MPS、HA-H、HA-L和Gl的保湿率分别为64.44%,53.87%,56.96%和50.18%。因此,在该相对湿度条件下,干巴菌菌丝体多糖(MPS)的保湿能力要强于透明质酸(HA)和甘油(Gl)。
由图5可以看出,在相对湿度43%的条件下,在120 h时,4种物质的保湿顺序为MPS>HA-H> HA-L>Gl,并且MPS、HA-H、HA-L和Gl的保湿率分别为89.17%,88.12%,86.63%和84.19%。因此,在该相对湿度条件下,干巴菌菌丝体多糖(MPS)的保湿能力要优于透明质酸(HA)和甘油(Gl)。
由图6可以看出,在相对湿度0%的条件下,在120 h时,4种物质的保湿顺序为HA-H>MPS>HA-L>Gl,并且MPS、HA-H、HA-L和Gl的保湿率分别为36.16%,38.34%,34.72%和18.71%。因此,在相对湿度0%的条件下,干巴菌菌丝体多糖(MPS)的保湿能力要强于低分子量透明质酸(HA-L)和甘油(Gl),而略低于高分子量透明质酸(HA-H)。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受实施例的限制,其它任何未背离本发明的精神实质与原理下所做的改变、修饰、组合、替代、简化均应为等效替换方式,都包含在本发明的保护范围之内。
Claims (4)
1.一种干巴菌菌丝体多糖,其特征在于是通过以下步骤得到的:
保藏编号为CGMCC No.12977干巴菌(Thelephora ganbajun)菌株TG-1在液体培养基中发酵,菌丝体经过分离,提取干巴菌菌丝体多糖MPS,然后经过阴离子交换柱层析分离,依次用去离子水和0.05、0.1、0.3 mol/L的NaCl溶液进行洗脱,分别得到干巴菌菌丝体多糖组分MPS-1、MPS-2、MPS-3和MPS-4;
所述的菌丝体经过分离,提取干巴菌菌丝体多糖MPS操作如下:
(1)将菌丝体分离后烘干或冻干,并粉碎得到干巴菌菌丝体粉末;
(2)干巴菌菌丝体粉末与去离子水以1﹕10~1﹕30的比例混合,调节pH值为7~9,超声波破碎,超声功率为200~800 w,超声时间为5~20 min,然后置于50~100 ℃水浴中,提取1~3 h,离心取上清液;
(3)离心后的菌丝体残渣按步骤(2)再提取2~4次,合并上清液;
(4)将上清液浓缩至原体积的2/3~1/4,加入1~3倍体积乙醇,静置至沉淀完全析出,3000~15000 r/min离心5~15 min,弃上清并于40~60 ℃条件下烘干,烘干后再次溶解,离心,上清液去蛋白至少3次,冻干后即为干巴菌菌丝体多糖MPS;
所述的干巴菌菌丝体多糖为干巴菌菌丝体多糖MPS、干巴菌菌丝体多糖组分MPS-1、MPS-2、MPS-3或MPS-4。
2.根据权利要求1所述的干巴菌菌丝体多糖,其特征在于液体培养基中含有马铃薯150~250 g/L,葡萄糖15~25 g/L,蛋白胨1~3 g/L,硫酸镁0.5~2.5 g/L,磷酸二氢钾0.5~2.5 g/L,pH为4.5~7,于15~30 ℃发酵培养。
3.根据权利要求1所述的干巴菌菌丝体多糖,其特征在于干巴菌菌丝体多糖MPS经过阴离子交换柱层析分离操作如下:
将干巴菌菌丝体多糖MPS溶于去离子水中,用0.45 μm的微孔滤膜过滤,得到上样溶液,将上样溶液用规格为1.6 × 30 cm的DEAE-52纤维素阴离子交换柱进行层析分离,依次用去离子水和0.05、0.1、0.3 mol/L的NaCl溶液进行洗脱,洗脱液的流速为1 mL/min,分别得到的洗脱组分冻干后即为干巴菌菌丝体多糖组分MPS-1,MPS-2,MPS-3和MPS-4。
4.一种权利要求1-3中任一项所述的干巴菌菌丝体多糖在制备具有清除自由基、抗氧化作用的保健品和具有清除自由基、抗氧化和保湿作用的化妆品中的应用。
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