CN117467032A - 一种金耳子实体提取物及其制备方法 - Google Patents
一种金耳子实体提取物及其制备方法 Download PDFInfo
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- CN117467032A CN117467032A CN202311597690.9A CN202311597690A CN117467032A CN 117467032 A CN117467032 A CN 117467032A CN 202311597690 A CN202311597690 A CN 202311597690A CN 117467032 A CN117467032 A CN 117467032A
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- Prior art keywords
- tremella aurantialba
- extract
- fruiting body
- tremella
- aurantialba
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
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Abstract
本发明提供了一种金耳子实体提取物的制备方法,包括如下步骤:将金耳子实体干燥粉碎,加水90‑100℃下水提1‑3h,然后采用高压纳米均质机均质处理,离心去除残渣,即得到金耳子实体提取液。本发明还提供了由上述方法制备得到的金耳子实体提取物。本发明提供的金耳子实体提取物,能够发挥益生元的功效,促进短链脂肪酸的生成。本发明提供的金耳提取物的制备方法具有工艺简便、生态环保、生产效率高等优点,采用该方法可促进金耳子实体多糖的释放,得率可达61.64%,金耳子实体提取物中多糖含量为53.11%,糖醛酸含量为30.64%。
Description
技术领域
本发明属于食用菌多糖制备领域,具体的说涉及一种金耳子实体提取物及其制备方法。
背景技术
金耳(学名:Naematelia aurantialba(Bandoni&M.Zang)Millanes&Wedin),是我国极具特色的一种稀有珍贵食药用菌,隶属于担子菌门(Basidiomycota),银耳纲(Tremellomycetes),银耳目(Tremellales),耳包革科(Naemateliaceae),耳包革属(Naematelia Fr)。在我国金耳主要集中分布于西南地区的云南、西藏、四川、贵州等地。金耳子实体整体韧胶质,形状呈不规则脑状至纯分叶状,基部狭窄,长度近10cm,厚达4-8cm,新鲜时颜色为黄色至橙红色,干时肉桂色或金黄色。据《中国药用真菌》介绍,金耳子实体性温带寒,具有化痰止咳、清心补脑、滋阴补肺、益气补血的功效。金耳中含有多糖、蛋白质、氨基酸、类胡萝卜素、维生素等多种活性成分,尤其是金耳多糖,含量丰富,且具有调节免疫、抗肿瘤、抗氧化、降血糖、保护肝脏、抗炎等药理作用,已成为我国功能性食品和药品领域开发的重要原料。
目前获得金耳子实体多糖的方法主要是热水提取法,金耳属于一种胶体食用菌,目前的提取方法中金耳提取液黏度大,使多糖的提取过程存在释放难、得率低等问题,从而限制了其进一步的研究及应用。因此,需要研究开发一种更为高效的金耳子实体提取方法。
发明内容
本发明首先提供了一种金耳子实体提取物的制备方法,包括如下步骤:
将金耳子实体干燥粉碎,加水90-100℃下煮沸水提1-3h,然后采用高压纳米均质机均质处理,离心去除残渣,即得到金耳子实体提取液;
所述水提料液比为1:40~60(w/v,g/mL);
所述高压纳米均质机均质处理条件为50HZ、800bar;
优选的,所述先将金耳子实体干燥粉碎成细粉(150目);
优选的,将金耳粉按照料液比1:50(w/v,g/mL)加水100℃提取2h;
优选的,将金耳粉水提液通过高压纳米均质机均质处理,高压纳米均质机参数如下:50HZ、800bar、均质7次;
优选的,将均质后金耳提取液离心处理,离心机参数如下:9000rpm、时间20min,去除沉淀,得到金耳均质提取液;
优选的,将金耳均质提取液真空冷冻干燥48h,得到金耳子实体提取物。
本发明的一种金耳子实体提取物的制备方法,包括如下步骤:金耳子实体干燥粉碎成150目,按照料液比1:50(w/v,g/mL)加水100℃提取2h;然后采用高压纳米均质机均质处理,高压纳米均质机参数如下:50HZ、800bar、均质7次;然后进行离心处理,离心机参数如下:9000rpm、时间20min,去除沉淀,得到金耳提取液;将金耳提取液真空冷冻干燥48h,得到金耳子实体提取物。
本发明还提供了上述制备方法制备得到的金耳子实体提取物。
本发明提供的制备方法可以降低金耳提取溶液的粘稠度,解决金耳多糖在提取制备过程中存在的释放量不高、得率较低等问题,具有工艺简单、生态环保、生产效率高等优点;制备得到的金耳子实体提取物富含多糖,提取物得率为61.64%。相较于金耳子实体的常规热水提取方法提高了42.78%,其中多糖含量为53.11%,糖醛酸含量为30.64%。提高了金耳在后续研究开发和应用中的可行性,提高其应用价值。
附图说明
图1实施例1金耳均质提取液
图2对比例1金耳提取液
图3对比例1和实施例1金耳提取液多糖分子量图谱对比图
图4对比例1和实施例1金耳提取物单糖组成对比图
其中11个标准品名称:1.岩藻糖;2.鼠李糖;3.阿拉伯糖;4.葡萄糖胺;5.半乳糖;6.葡萄糖;7.木糖;8.甘露糖;9.果糖;10.半乳糖醛酸;11.葡萄糖醛酸
图5体外模拟发酵过程中还原糖含量变化
图6体外模拟发酵过程中pH值变化
具体实施方式
为了进一步说明本发明,下面结合附图和实施例对本发明提供的技术方案进行详细地描述,但不能将它们理解为对本发明保护范围的限定。
材料
金耳子实体,云南菌视界生物科技有限公司工厂化栽培,栽培菌株编号为JSJ-X9,栽培周期为45d。
下列实施例中所用试剂如无特殊说明,均为普通市售产品。
实施例1
金耳子实体提取液的制备方法,步骤如下:
将金耳子实体烘干粉碎成细粉(150目),按照料液比1:50(w/v,g/mL)加水,100℃提取2h;然后经过GA-10H高压纳米均质机(朗灏孚纳米科技(上海)有限公司)均质处理:50HZ、800bar下处理7次,9000rpm下高速离心20min,去除沉淀,得到金耳子实体提取液。该提取液呈均一的流体状,粘度显著降低,提取液相较均质前澄清透明(图1)。
对比例1
采用实施例1中的水提法制备金耳提取液,区别在于,在金耳热水提取后未进行均质处理。步骤如下:
将金耳子实体烘干粉碎成细粉(150目),按照料液比1:50(w/v,g/mL)加水,100℃提取2h,9000rpm下高速离心20min,去除沉淀,得到金耳子实体提取液。该提取液粘度较高,流动性差,呈凝胶状(图2)。
实施例2
(一)金耳子实体提取物得率的比较
取一定量的实施例1和对比例1的金耳子实体提取液(图1与图2)分别进行真空冷冻干燥48h,分别得到金耳子实体提取物,计算得到实施例1的提取物得率为61.64%,对比例1的提取物得率为43.17%,相对于对比例1,实施例1的提取物得率提高了42.78%。
(二)多糖与糖醛酸含量的测定和比较
对实施例1、对比例1得到的金耳子实体提取物进行多糖与糖醛酸含量测定。
(1)总糖含量参考苯酚硫酸法进行测定。具体方法为:配置5%的苯酚溶液、不同浓度(0.00、0.01、0.02、0.04、0.06、0.08和0.10mg/mL)的葡萄糖标准品溶液。分别取标准品溶液与样品测定液1mL于长试管中,加5%的苯酚溶液与2.5mL浓硫酸,混匀,置于100℃水浴15min,取出,冷却至室温,490nm下测定吸光度,绘制标准曲线,计算总糖含量。
(2)还原糖含量按照二硝基水杨酸法(DNS)进行测定。具体方法为:配制DNS试剂、不同浓度(0、0.1、0.2、0.4、0.6、0.8、1mg/mL)的葡萄糖标准品溶液。分别取葡萄糖标准品溶液与样品测定液2mL于25mL容量瓶中,加入5mL DNS试剂,100℃水浴5min,冷却,定容至25mL,摇匀,在540nm下测定吸光值,绘制标准曲线,计算还原糖含量。
(3)多糖含量计算公式如下:
多糖含量(%)=总糖含量(%)-还原糖含量(%)
(4)金耳子实体提取物糖醛酸含量参考间羟基联苯法进行测定。具体方法为:配置间羟基联苯工作液、四硼酸钠-硫酸溶液、1mg/mL半乳糖醛酸标准品溶液。将标准品溶液稀释成不同浓度的工作液与样品测定液1mL于长试管中,加入6mL四硼酸钠-硫酸溶液,在冰水中冷却后混匀,沸水浴加热5min,在冰水中冷却后再加入100μL间羟基联苯溶液,充分混匀后在525nm处测定吸光值,绘制标准曲线,计算糖醛酸含量。
通过上述方法对实施例1与对比例1中的组分含量进行了测定,实施例1与对比例1中的还原糖含量较少,均小于1%;实施例1与对比例1中的多糖含量分别为53.11%与50.84%,糖醛酸含量分别为30.64%与28.08%;结果表明,实施例1制备得到的提取物在提高得率的同时,多糖含量与糖醛酸也略有所提升。具体比较结果见如下表1。
表1实施例1和对比例1提取物组分含量
(三)金耳子实体提取物分子量测定
对实施例1、对比例1中金耳子实体提取物进行分子量测定。金耳子实体提取物分子量分布参考高效凝胶尺寸排阻色谱-多角度激光仪散射-示差折光检测仪联用法(HPSEC-MALLS-RID)进行测定。
仪器:Waters2695 HPLC泵、Waters2414示差检测器(美国Waters公司)、氦-氖激光光源的DAWN8+激光光散射检测器(美国Wyatt公司)。色谱柱:TSK-GEL系列G6000PWXL和G4000PWXL(7.8mm×300mm,日本TOSOH公司)串联。流动相:0.05mol/L硝酸钠、0.02%叠氮钠。柱温30℃,流速0.5mL/min,上样量100μL。使用Astra(version6.1.1)数据分析软件对光散射数据进行采集和分析,计算多糖分子量(Mw)。
如图3,金耳子实体提取物中以大分子量多糖为主,且组分单一。如表2,实施例1中金耳提取物大分子量多糖组分重均分子量Mw为5.812×105g/mol,对比例1中金耳提取物大分子量多糖组分Mw为1.511×106g/mol。实施例1分子量明显右移,表明实施例1制备得到的金耳子实体提取物中的多糖分子量明显降低,从而使其溶解度得到提升,释放量增加,因此提取得率显著增加。
表2对比例1和实施例1提取物中多糖分子量分布
(四)金耳子实体提取物单糖组成分析
对实施例1、对比例1金耳提取物中的多糖组分进行单糖组成分析。分别取2mg的实施例1与对比例1的提取物加入2mL 2.0mol/L的三氟乙酸(TFA),在110℃下水解4h,冷却后通过N2除去TFA。加入3mL甲醇,并继续用N2吹干(重复上述操作4-5次)。水解产物用超纯水完全溶解后定容至50mL。通过高效阴离子交换色谱仪(ICS-5000,美国Dionex公司)测定水解产物中单糖的组成和相应的摩尔比。
如图4,实施例1与对比例1提取物中的多糖单糖组成由葡萄糖、木糖、甘露糖、半乳糖醛酸和葡萄糖醛酸组成,其中甘露糖所占的比例最高,葡萄糖比例最低。具体比较数据见如下表3,可以看出实施例1和对比例1的提取物中的多糖单糖组成基本相似。
表3对比例1和实施例1金耳提取物单糖组成
(五)金耳子实体提取液的粘度比较
对实施例1和对比例1中制备得到的金耳提取液、蒸馏水的粘度进行检测,检测方法为:采用RVA-TM快速粘度计,取25mL样品,25℃,600r/min条件下持续3min。
实施例1和对比例1中制备得到的金耳提取液的粘度分别为93cP和226cP,蒸馏水的粘度为33cP,由此可见,实施例1中的金耳提取液粘度相较对比例1降低了58.85%,实施例1制备得到的金耳提取液的粘度得到了很大程度的改善。
(六)金耳子实体提取物益生元功效评价
建立体外粪便发酵模型对比实施例1、对比例1中金耳子实体提取物体外调节肠道菌群发酵产短链脂肪酸(SCFAs)的作用效果。
1.新鲜粪便样本来自4名健康志愿者(上海地区2名女性和2名男性,年龄20-30岁),配置粪便悬浮液:称取氯化钠8.0g,氯化钾0.2g,无水磷酸氢二钠1.15g,磷酸二氢钾0.2g于1L的蒸馏水中。高温灭菌后与粪便混合配置成10%(w/v)的粪便悬浮液。
2.体外发酵基础培养基配置:称取蛋白胨2.0g,酵母提取物2.0g,半胱氨酸盐酸盐0.5g,氯化钠0.1g,碳酸氢钠2.0g,磷酸二氢钾0.04g,无水磷酸氢二钾0.04g,七水硫酸镁0.01g,六水氯化钙0.01g,氯化血红素0.005g,胆盐0.5g,刃天青0.1g,吐温80 2.0mL,维生素K1 10μL于1L的蒸馏水中,pH调为7.0。
3.将一定比例对比例1和实施例1的金耳提取物分别与淀粉混合,作为体外粪便发酵所需的碳源。具体配置方法如下:向体外发酵基础培养基中添加0.2%的对比例1金耳提取物(w/v)与0.8%的淀粉(w/v)作为对比例1碳源组,命名为NAW-1;向体外发酵基础培养基中添加0.2%的实施例1金耳均质提取物(w/v)与0.8%的淀粉(w/v)作为实施例1碳源组,命名为NAW-2;向体外发酵基础培养基中添加1%无菌水(v/v)作为空白对照组,命名为CON;向体外发酵基础培养基中添加1%淀粉(w/v)作为阳性对照组,命名为AmY。将1.0mL的10%粪便悬浮液(w/v)分别加入上述四组不同碳源的培养基(9.0mL)中,于37℃厌氧条件下分别培养0、6、12、24和48h后,收集发酵液。
4.将收集到的发酵液以8000rpm离心10min,取1mL上清液,采用DNS法测定不同时间发酵液中还原糖含量的变化,以及采用pH计测定发酵过程中pH值的变化。
5.将收集到的发酵液以8000rpm离心10min,取1mL上清液用100μL H2SO4-H2O溶液(10%,v/v)酸化,充分涡旋混合2min,然后加入800μL无水乙醚进行液-液萃取,混匀后以14000rpm离心15min,待分层后取最上层有机相,加入0.25g无水硫酸钠,14000rpm离心15min,0.22μm滤膜过滤,用于进一步的气相色谱(GC-2010Plus)检测。
6.色谱条件:SHIMADZU GC-2010Plus系统配有自动进样器和氢离子火焰检测器(flame ionizationdetector,FID),使用SH-Rtx-Wax毛细管柱(30m×0.25mm×0.25μm)进行组分分离,进样量1μL,进样口温度230℃,FID检测器温度260℃,柱温箱升温程序为:初始柱温70℃保持1min,随后以10℃/min速率梯度升温至100℃,然后以2.5℃/min的速率梯度升温至120℃保持2min,最后以30℃/min的速率升温至230℃保持5min。
7.标准品配置:分别精密称取乙酸、丙酸、丁酸、戊酸和异戊酸0.1g溶于5mL超纯水中,制成标准贮备母液。分别配置浓度为1000、500、250、125、62.5、31.25μg/mL的系列混标溶液,将系列混标溶液经0.22μm微孔滤膜过滤后,通过气相色谱检测进行标准曲线的绘制。
结果及讨论:
CON、AmY、NAW-1与NAW-2组在发酵过程中还原糖含量与pH值的变化如图5、图6所示:
多糖可以作为人体肠道微生物群的碳源,并产生有益的代谢产物。发酵过程中,还原糖含量呈先增加后降低的趋势,是由于发酵前期(0-6h),肠道微生物中酶作用于多糖,使其糖苷键迅速断裂,糖苷键的断裂速度高于肠道微生物利用速度,从而使还原糖迅速积累。在6-48h的发酵时间内,菌群快速生长代谢,碳源被降解利用消耗,还原糖含量降低,在6-24h内,NAW-2组发酵液中的还原糖含量的变化趋势大于NAW-1组与AmY组,这表明在此阶段NAW-2组的利用率高于NAW-1组与AmY组。由此可见,实施例1与对比例1的金耳提取物均可以作为肠道微生物生长所需的碳源,且实施例1的利用率要好于对比例1。
体外发酵初期,各组间pH值在8-9之间。随着发酵的进行,AmY、NAW-1与NAW-2组的pH值均逐渐下降,发酵48h后,pH值分别从8.4左右降低至4.4左右。CON组的pH值在6h发酵后无明显变化,这可能是由于培养基中缺乏可利用的碳水化合物。上述结果表明,在被肠道微生物群降解和利用后,金耳子实体提取物与淀粉均可以产生酸性终产物,例如SCFAs,从而显著降低结肠环境的pH值。
CON、AmY、NAW-1与NAW-2组在发酵过程中短链脂肪酸(SCFAs)含量的变化如下表4所示。
SCFAs是肠道菌群发酵产生的重要代谢产物之一,其水平和类型取决于底物来源和肠道菌群的组成。肠道菌群发酵所产生的SCFAs包括乙酸、丙酸、丁酸、戊酸、异戊酸等,SCFAs在肠道中主要发挥抑制有害病原体生长、调节上皮屏障功能、降低结肠pH值和预防结直肠癌等重要作用。12h-48h,NAW-1组与NAW-2组中的总SCFAs含量均高于AmY组与CON组,这表明在碳源中添加实施例1与对比例1的金耳提取物,相较只添加淀粉,能够更好地促进粪便中部分肠道微生物的代谢,从而提高SCFAs的含量,金耳子实体提取物主要促进乙酸与丙酸的产生,乙酸是一种能源代谢物质,可以在肝脏、心脏和周围组织中代谢和吸收,丙酸可以降低肝脏和血浆中脂肪酸的含量,抑制胆固醇的合成。
NAW-1组与NAW-2组的总SCFAs含量相当,表明高压均质处理不会影响金耳子实体多糖的调节肠道菌群活性,金耳子实体多糖可作为益生元促进肠道菌群短链脂肪酸的产生。
表4体外模拟发酵过程中的短链脂肪酸含量变化
由以上实施例2可以得出,采用实施例1的提取方法得到的金耳子实体提取液可以解决金耳多糖在水提过程中粘度太高导致提取过程中存在的固液分离难、得率低等一系列问题;同时,实施例1的金耳子实体提取过程简便成本低,为后续的研究开发和应用提供更加完善的提取方法,并通过测定发酵过程中的SCFAs含量评估了实施例1的益生元效果,为后续金耳子实体多糖在益生元产品开发中的应用提供理论依据。
尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例,而不是全部实施例,可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
Claims (4)
1.一种金耳子实体提取物的制备方法,其特征在于包括如下步骤:
将金耳子实体干燥粉碎,加水90-100℃下水提1-3h,然后采用高压纳米均质机均质处理,离心去除残渣,即得到金耳子实体提取液;
其中水提的料液比为1:40~60,g/mL;
其中高压纳米均质机均质处理条件为50HZ、800bar。
2.根据权利要求1所述的金耳子实体提取物的制备方法,其中金耳子实体干燥粉碎成150目。
3.根据权利要求1所述的金耳子实体提取物的制备方法,其中金耳子实体干燥粉碎,按照料液比1:50加水100℃提取2h;然后采用高压纳米均质机均质处理,高压纳米均质机参数如下:50HZ、800bar、均质7次;然后进行离心处理,离心机参数如下:9000rpm、时间20min,去除沉淀,得到金耳子实体提取液;将金耳提取液真空冷冻干燥48h,得到金耳子实体提取物。
4.权利要求1-3任一项所述制备方法制备得到的金耳子实体提取物。
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