CN108546306B - 一种蛹虫草培养基多糖及其分离纯化方法和用途 - Google Patents
一种蛹虫草培养基多糖及其分离纯化方法和用途 Download PDFInfo
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- CN108546306B CN108546306B CN201810389081.7A CN201810389081A CN108546306B CN 108546306 B CN108546306 B CN 108546306B CN 201810389081 A CN201810389081 A CN 201810389081A CN 108546306 B CN108546306 B CN 108546306B
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- polysaccharide
- cordyceps militaris
- culture medium
- militaris culture
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Abstract
本发明公开了一种蛹虫草培养基多糖及其分离纯化方法和用途,该多糖含有以下摩尔百分比的单糖:0.11%核糖、0.11%鼠李糖、0.45%***糖、0.13%木糖、14.50%甘露糖、83.96%葡萄糖、0.73%半乳糖。采用本发明的提取方法不影响蛹虫草培养基多糖P1的生物活性,所得到的多糖纯品P1纯度高、性质稳定,在抗氧化、降尿酸、抑菌方面具有显著效果,有益于人体代谢;成本较低,多糖P1纯品可进一步用于保健品、药品、化妆品的开发。
Description
技术领域
本发明涉及一种蛹虫草培养基多糖及其分离纯化方法和用途。
背景技术
多糖(Polysaccharide)又称多聚糖,由醛糖或酮糖通过糖苷键相连接在一起的线性或分枝链状聚合物,是聚合度大于10的极性复杂大分子,分子量一般为数万以上,是构成生命活动的四大基本物质之一,生物体内的多糖除了以游离态存在外,也会与蛋白质或脂肪结合成蛋白多糖和脂多糖。
蛹虫草(C.militaris)又称北虫草,分类学上属于子囊菌亚门、核菌纲、球壳菌目,与北冬虫夏草同属麦角科虫草属,主要分布在我国东北、华北、西北等地区。蛹虫草可寄生于鳞翅目、鞘翅目、双翅目等昆虫的幼虫或蛹体上,能采用家蚕蛹、大米培养基等进行人工批量培育。
由于天然的蛹虫草资源有限,目前人工栽培的较多,主要有三种培养方法:(1)采集野生的蛹虫草,进行菌种的分离、纯化,然后将蛹虫草菌种接种在含蚕蛹粉的大米等固体培养基上,在一定的温度、湿度和光照条件下,培养35-45d获得蛹虫草子实体;(2)将蛹虫草菌种接种在家蚕幼虫或活蛹体内,在一定的温度、湿度和光照条件下,培养35-45d获得蛹虫草子实体;(3)以大豆粉蔗糖或玉米浆蔗糖为培养基,采用液体发酵培养蛹虫草。
我国采用人工方法培养蛹虫草子实体的生产已有相当规模,但在子实体收获的同时,也产生了大量的蛹虫草培养基下脚料,不仅造成环境的污染,也是不可忽视的资源浪费。
研究表明,蛹虫草培养基下脚料中含有虫草素、虫草多糖等生物活性物质。因此为了更好地开发利用蛹虫草资源,提高经济效益,完善蛹虫草产业链,对蛹虫草培养基下脚料的利用和研究开发已成为必要。
中国专利申请(申请号201110086808.2)公开了一种从蛹虫草培养基中提取多糖的方法,利用不同的酶液来去除蛹虫草培养基中的蛋白、淀粉,再经醇沉得到蛹虫草培养基粗多糖;其操作过程复杂,成本较高,且不能将多糖进一步分离纯化,无法得到纯度较高的多糖组分。
发明内容
本发明的首要目的在于提供一种蛹虫草培养基多糖。
本发明的另一目的是提供上述蛹虫草培养基多糖的分离纯化方法,利用超声波辅助水提醇沉法对蛹虫草废弃培养基进行多糖提取,并通过离子交换层析法对多糖进行分离纯化,由此提取分离出纯度较高、具有生物活性的多糖组分。
本发明的再一目的在于提供上述蛹虫草培养基多糖的用途。
本发明的目的通过下述技术方案实现:
一种蛹虫草培养基多糖,包含以下摩尔百分比的单糖:0.11%核糖、0.11%鼠李糖、0.45%***糖、0.13%木糖、14.50%甘露糖、83.96%葡萄糖、0.73%半乳糖;单糖的组成是由GC-MS面积归一化法计算出来的,是除溶剂峰外,把所有的出峰面积看作100%,计算主峰面积占总面积的百分数,可能所测的未知样品里包含了微量的标准品以外的单糖,或是由计算方法造成的偏差,导致该单糖的摩尔百分比组成不足100%,这属于正常情况;
所述蛹虫草培养基多糖的平均分子量为2.18k Da;
所述的蛹虫草培养基多糖中存在非常微量的硫酸基,还含有吡喃糖环和α-型糖苷键。
上述蛹虫草培养基多糖的分离纯化方法,包括以下步骤:
(1)提取蛹虫草培养基多糖:将蛹虫草大米培养基下脚料干燥后粉碎,过筛得下脚料干粉;称取干粉加入15-16倍质量的蒸馏水,超声波处理30min以上,在70℃下回流提取1.5-2.0h,提取若干次合并提取液,提取液过滤后浓缩,得到多糖浓缩液;在多糖浓缩液中加入3-4倍体积的95%(V/V)乙醇,搅拌后于4℃中静置过夜;离心后将沉淀烘干得到蛹虫草培养基多糖提取物;
步骤(1)所述的过筛优选过40目筛;
步骤(1)所述的浓缩优选在50-55℃下浓缩;
步骤(1)所述的离心优选5000r/min离心15min;
(2)蛹虫草培养基多糖的脱色:将蛹虫草培养基多糖提取物加蒸馏水溶解,调节pH值至8.0-8.5,滴加H2O2溶液至无色,50-55℃保温2h以上;
步骤(2)所述的H2O2溶液,浓度优选30%(V/V);
(3)酶法结合Sevage法脱蛋白:
3-1:将木瓜蛋白酶溶液与蛹虫草培养基多糖提取液混合,两者的体积比为1.0:1.5~1.0:1.7,60-70℃酶解2-3h;
所述的木瓜蛋白酶溶液用pH值6.0的PBS缓冲液配制而成,其中木瓜蛋白酶的浓度优选250U/ml;
3-2:在酶解液中加入1/5体积的Sevage试剂,振荡培养30min以上,然后多次离心直至无蛋白沉淀析出为止,取上清液,烘干得到蛹虫草培养基粗多糖;
步骤(3)所述的Sevage试剂,是氯仿和正丁醇按体积比5:1配制而成;
步骤(3)所述的振荡培养,摇床转速优选150r/min;
步骤(3)所述的离心优选4000r/min离心20-30min;
(4)蛹虫草培养基多糖的分离纯化:将蛹虫草培养基粗多糖用蒸馏水溶解后上DEAE琼脂糖凝胶FF(DEAE Sephrose Fast Flow)离子交换层析柱,用蒸馏水进行洗脱,得到所述的蛹虫草培养基多糖P1。
多糖的分离纯化不仅是一个除杂的过程,同时也是将混合多糖分离为单一组分的过程。柱层析法可分为离子交换层析和凝胶柱层析。离子交换层析是依据离子电荷密度的不同而进行分级分离,阴离子交换剂的电荷基团带正电,反离子带负电,因此这种交换剂可以与溶液中的负电荷化合物或阴离子进行交换反应,而阳离子交换剂则反之。
本发明的蛹虫草培养基多糖可以作为抗氧化剂或抑菌剂应用,也可以用于制备具有降尿酸功效的药物。
本发明通过水提醇沉法得到蛹虫草培养基粗多糖,利用离子交换层析柱进行粗多糖的分离纯化,相对于现有技术具有如下的优点及效果:
(1)本发明充分利用了培养蛹虫草而产生的培养基下脚料,符合变废为宝的绿色环保理念,建立了一条完整可行的蛹虫草培养基多糖提取、分离纯化、理化性质、结构和生物活性研究的技术路线,完善了人工培养蛹虫草产业的残渣处理工艺,为食用菌多糖的提取分离纯化提供了技术指导。
(2)本发明所用的水提醇沉法可以完成大通量的多糖提取操作,成本较低,重复性好,得率高,适用于工业化大规模生产。
(3)本发明所用的DEAE Sephrose Fast Flow理化稳定性和机械性能更好,交换容量大,可以在位清洗,床体积随pH的离子强度变化很小,由于流速和载量高,适合于进行大量粗产品的纯化工作。
(4)采用本发明的提取方法不影响蛹虫草培养基多糖P1的生物活性,所得到的多糖纯品P1纯度高、性质稳定,在抗氧化、降尿酸、抑菌方面具有显著效果,有益于人体代谢;成本较低,多糖P1纯品可进一步用于保健品、药品、化妆品的开发。
(5)本发明创造性地将多糖的水提醇沉提取方法与离子交换层析分离纯化方法结合起来用于蛹虫草培养基下脚料多糖的研究,比较并得到较优的工艺参数,并确定DEAESephrose Fast Flow作为层析柱填料,为蛹虫草培养基下脚料多糖的提取分离纯化提供技术指导和新思路。
附图说明
图1是蛹虫草培养基多糖的洗脱曲线图。
图2是多糖P1的紫外光谱图。
图3是多糖P1的红外光谱图。
图4是多糖P1的ABTS自由基清除能力。
图5是多糖P1的OH自由基清除能力。
图6是多糖P1的抑菌实验结果图;其中,Sa-对金黄色葡萄球菌的抑菌圈,Pa-对绿脓杆菌的抑菌圈,1-多糖P1产生的抑菌圈,0-对照组的抑菌圈。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
本发明中,蛹虫草培养基多糖的理化性质分析由中国广州分析测试中心进行,报告编号分别为2018001306-1b。
实施例1
一种从蛹虫草培养基下脚料中提取并分离纯化多糖的方法,包括以下步骤:
(1)提取蛹虫草培养基多糖:称取55g蛹虫草大米培养基下脚料充分干燥后粉碎,过40目筛,称取50g干粉加入800ml的蒸馏水,超声30min,在70℃下回流提取1.5h,提取3次合并滤液,真空抽滤后于55℃下浓缩至100ml,得到多糖浓缩液;在多糖浓缩液中加入400ml的95%乙醇,不断搅拌使多糖均匀沉淀,于4℃中静置过夜;5000r/min离心15min后将沉淀烘干得到蛹虫草培养基多糖提取物;
(2)蛹虫草培养基多糖的脱色:将蛹虫草培养基多糖提取物加蒸馏水溶解配成浓度为0.05g/ml的多糖提取液,加入NaOH调节pH值至8.0,滴加30%的H2O2至无色,50℃下保温2h;
(3)酶法结合Sevage法脱蛋白:
精确称取木瓜蛋白酶0.1g,用pH值6.0的PBS缓冲液溶解成终浓度为250U/ml的溶液,与蛹虫草培养基多糖提取液混合,酶液与蛹虫草培养基多糖提取液的体积比为1.0:1.5,64℃下酶解3h;
在酶解液中加入1/5体积的Sevage试剂(氯仿:正丁醇=5:1),置于摇床150r/min振荡30min后4000r/min离心20min,重复离心多次直到无蛋白沉淀析出,合并上清液,50℃下烘干得到蛹虫草培养基粗多糖;
(4)蛹虫草培养基多糖的分离纯化:
称取0.1g蛹虫草培养基粗多糖溶于10ml蒸馏水中,上DEAE Sephrose Fast Flow离子交换层析柱,用蒸馏水进行洗脱,流速为0.5ml/min,每10min收集1管,以苯酚-硫酸法逐管检测多糖含量,依据洗脱曲线(图1)合并,得到蛹虫草培养基多糖P1。
苯酚-硫酸法检测多糖含量具体操作:精密称取105℃干燥至恒重的无水葡萄糖标准品0.1g,置于100ml容量瓶中加蒸馏水溶解并定容,摇匀,配成1mg/ml的标准品溶液备用。取该溶液分别稀释成10、20、40、60、80、100μg/ml不同浓度的标准溶液。分别吸取上述溶液各1ml置于试管中,加入6%苯酚溶液0.5ml并混匀,再加入2.5ml浓硫酸混匀,室温静置20min,以蒸馏水为空白对照,于490nm处测定吸光度,以葡萄糖浓度为横坐标,OD值为纵坐标,绘制标准曲线。未知样品用标准曲线法测定多糖含量。
将洗脱得到的多糖组分浓缩、透析后冷冻干燥,得到蛹虫草培养基多糖纯品,命名为P1。
实施例2
对实施例1得到的蛹虫草培养基多糖P1进行紫外光谱分析,各称取1mg多糖样品,配制1mg/mL多糖溶液,扫描在200-800nm范围内紫外图谱。
图2为P1的紫外光谱图,结果显示,P1在260nm、280nm处有微弱的吸收峰,表明P1含有微量的蛋白质和核酸。
实施例3
对实施例1得到的蛹虫草培养基多糖P1进行多糖分子量分析,具体实验方法如下:
采用凝胶渗透色谱法(GPC)测定分子量。称取冷冻干燥的多糖样品2mg,加0.02M磷酸缓冲液溶解,配制成2.0mg/mL溶液,用0.22μm无菌滤膜过滤,取过滤清液待用。
色谱条件:柱温35℃;0.02mol/L磷酸盐缓冲液(pH值7.0)为流动相,流速0.6ml/min,进样量20μL;TSK凝胶保护柱(40mm×6.0mm),TSKG-4000K凝胶柱(300mm×7.8mm)和TSKG-2500K凝胶柱(300mm×7.8mm);Waters 2414示差折光检测器检测。配制一系列不同分子量的葡聚糖溶液(700,400,200,100,50,30,10,5kD)作为标样,做标准曲线。样品的分子量根据其相应的洗脱体积对照标准曲线进行计算。
结果表明,蛹虫草培养基多糖P1的平均分子量为2.18k Da。
实施例4
对实施例1得到的蛹虫草培养基多糖P1进行单糖组成分析,具体方法如下:
称取多糖样品10mg,加入5mL三氟乙酸(4M),110℃水解2h。水解液于50℃真空旋转蒸发至干,用色谱纯甲醇清洗3次(加入色谱纯甲醇,再进行旋干,反复3次,直至旋干物质再无三氟乙酸味道),得多糖水解物。
在多糖水解物中依次加入10mg盐酸羟胺、1mg内标肌醇和2mL吡啶,密封,90℃水浴30min后加入2mL醋酸酐90℃水浴30min,加入2mL水终止反应。加入2mL二氯甲烷萃取,重复2次,合并二氯甲烷相,加入无水硫酸钠干燥,过0.22μm有机微孔滤膜,备用。
采用气相色谱仪分析,分析柱为HP-5MS石英毛细管柱(30m×0.25mm×0.25μm)。升温程序如下:进样口温度为250℃,初始柱温100℃,保持0.5min;然后以20℃/min升至140℃,保持5min;以3℃/min速度升至160℃;再以10℃/min速度升到250℃,保持5min。进样体积为1μL;分流比为10:1;流动相为氦气;流速为1mL/min。
各种单糖标准品(鼠李糖、***糖、核糖、木糖、甘露糖、葡萄糖和半乳糖)按照相同步骤进行实验,按照相同的检测程度,将处理后的标准品单糖进行气相色谱分析。
测得蛹虫草培养基多糖的单糖组成结果如下表:
表1蛹虫草培养基多糖P1的单糖组成
实施例5
对实施例1得到的蛹虫草培养基多糖P1进行傅里叶红外光谱分析:
称取2mg多糖样品,将其与干燥后的KBr(溴化钾)于研钵中混匀研磨,经压片机压成片,采用傅里叶变换红外光谱仪,在400-4000cm-1的波数范围内扫描。
图3为P1的红外光谱图,在3402、3401cm-1处的峰分别是P1的O-H伸缩振动产生的,而2929cm-1处的峰是由C-H振动产生的,1622、1642cm-1处的峰分别是P1的C=O伸缩振动产生的,这些都是多糖的特征峰,说明P1属于多糖类物质。
此外,在P1的红外光谱中,1240cm-1处的弱峰是由S=O的伸缩振动所产生的,这说明P1中存在非常微量的硫酸基,1154cm-1处的峰是环上C-O的吸收峰,1081、1023cm-1的峰是由醇羟基的变角振动产生的,这三个峰说明P1中存在吡喃糖环,850cm-1处的峰表示P1中存在α-型糖苷键。
实施例6
对实施例1得到的蛹虫草培养基多糖P1进行抗氧化能力测定:
(1)ABTS自由基清除能力测定:
取7mmol/L ABTS水溶液和2.45mmol/L过硫酸钾水溶液各5mL混合,置于暗处反应12h,产生ABTS自由基,将ABTS自由基溶液稀释并使其在734nm波长条件下的吸光值为0.70±0.02。将1mL不同质量浓度的蛹虫草培养基多糖P1溶液与2mL ABTS自由基溶液混合均匀,10min后测其在734nm处的吸光值,记作A1;2mL ABTS自由基溶液与1mL蒸馏水混合后测定734nm处的吸光值,记作A0;测定2mL蒸馏水与1mL多糖溶液在734nm处的吸光值,记作A2。
清除率(%):Y=[1-(A1-A2)/A0]×100%
图4为蛹虫草培养基多糖P1的ABTS自由基清除能力。由图4可知,在浓度1.0~5.0mg/ml范围内,蛹虫草培养基多糖组分P1具有显著的清除ABTS自由基能力,且与多糖浓度呈正相关。当多糖浓度为5.0mg/ml时,P1的ABTS自由基清除率为85.4%。
(2)OH自由基清除能力测定:
分别在试管中加入不同浓度的蛹虫草培养基多糖P1溶液1mL,再加入1mL6mmol/LFeSO4溶液,1mL 6mmol/L H2O2溶液和1mL 6mmol/L水杨酸-乙醇溶液,37℃水浴1h,510nm处测定其吸光度Ai。用蒸馏水代替样品溶液测得A0,用蒸馏水代替H2O2溶液测得Aj。
清除率(%)=[1-(Ai-Aj)/A0]×100%
图5为蛹虫草培养基多糖P1的OH自由基清除能力。由图5可知,蛹虫草培养基多糖组分P1具有显著的清除OH自由基能力,在质量浓度1.0~5.0mg/ml范围内,P1随着溶液浓度的增加,对OH自由基的清除能力逐渐增强,在浓度为5.0mg/ml时,清除率可达81.03%。
实施例7
对实施例1得到的蛹虫草培养基多糖P1进行降尿酸研究:
(1)高尿酸血症模型的建立
雄性小鼠60只,饲养一周后,随机分为6组,每组10只,依次为空白组、模型组、多糖P1高(400mg/kg)、P1中(200mg/kg)、P1低(100mg/kg)剂量组以及阳性药物(50mg/kg)对照组。每天上午模型组、阳性对照组、多糖P1高中低剂量组灌胃给予黄嘌呤600mg/kg(ig)+腹腔注射氧嗪酸钾100mg/kg(ip.)造模前一个小时禁食不禁水,空白组给予同等剂量生理盐水。下午多糖P1高、中、低剂量组采用多糖混悬液进行灌胃给药,阳性对照组灌胃给予别嘌醇混悬液50mg/kg,模型组和空白组则灌胃给予同等体积的生理盐水,连续七天(2)体内生化指标的测定
第七天下午给药1h后称体重,断头取血,室温放置40min后,以3000r/min离心10min,吸取上层血清,采用试剂盒检测血清中尿酸(UA)、血清肌酐(CREA)和血清尿素氮(BUN)值,考察多糖P1对高尿酸血症模型小鼠UA、CREA和BUN及肾功能的影响。
测得蛹虫草培养基多糖P1的降尿酸作用结果如下表:
表2蛹虫草培养基多糖P1的降尿酸作用
由表2可以看出,与正常组比,模型组小鼠的血清肌酐、血清尿酸和血清尿素氮水平均显著升高了,表明造模成功。
与模型组相比,多糖P1的低中高剂量组可使小鼠血清肌酐水平分别下降5.03%、20.19%、32.76%,可使小鼠血清尿酸水平分别下降39.58%、46.46%、53.72%,可使小鼠血清尿素氮水平分别下降22.77%、40.05%、51.59%。说明在该实验模型下,蛹虫草培养基多糖P1能够降低高尿酸血症的血清肌酐、血清尿酸和血清尿素氮水平。
实施例8
对实施例1得到的蛹虫草培养基多糖P1进行抑菌研究:
对常见的致病菌金黄色葡萄球菌(Sa)、绿脓杆菌(Pa)采用打孔法测定抑菌活性。主要操作过程如下:取直径为90mm的培养皿,倒平板后用移液枪取150μl的菌悬液,涂布均匀后每一平板用8mm打孔器打4孔,分别加入40μl备用的1mg/ml蛹虫草培养基多糖P1,对照组则每孔加入40μl生理盐水。置于37℃恒温培养箱内培养24h,用十字交叉法分别测量实验平板及对照平板上各抑菌圈的直径,并计算其平均值。
多糖P1的抑菌实验结果如图6所示,分别是P1对金黄色葡萄球菌的抑菌结果、金黄色葡萄球菌对照组、P1对绿脓杆菌的抑菌结果、绿脓杆菌对照组,测得抑菌圈的直径如表3。
表3抑菌圈直径(cm)
由表3可知,蛹虫草培养基多糖P1对金黄色葡萄球菌、绿脓杆菌均有不同程度的抑制作用,其中对金黄色葡萄球菌的抑制作用比绿脓杆菌的更强。
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上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (5)
1.一种蛹虫草培养基多糖在制备抗氧化剂和绿脓杆菌抑制剂中的应用,其特征在于:
所述的蛹虫草培养基多糖含有以下摩尔百分比的单糖:0.11%核糖、0.11%鼠李糖、0.45%***糖、0.13%木糖、14.50%甘露糖、83.96%葡萄糖、0.73%半乳糖;所述的蛹虫草培养基多糖含有硫酸基、吡喃糖环和α-型糖苷键,平均分子量为2.18k Da;
所述的蛹虫草培养基多糖的分离纯化方法包括以下步骤:
(1)提取蛹虫草培养基多糖:将蛹虫草大米培养基下脚料干燥后粉碎,过筛得下脚料干粉;称取干粉加入15-16倍质量的蒸馏水,超声波处理30min以上,在70℃下回流提取1.5-2.0h,提取若干次合并提取液,提取液过滤后浓缩,得到多糖浓缩液;在多糖浓缩液中加入3-4倍体积的95%(V/V)乙醇,搅拌后于4℃中静置过夜;离心后将沉淀烘干得到蛹虫草培养基多糖提取物;
(2)蛹虫草培养基多糖的脱色:将蛹虫草培养基多糖提取物加蒸馏水溶解,调节pH值至8.0-8.5,滴加H2O2溶液至无色,50-55℃保温2h以上;
(3)酶法结合Sevage法脱蛋白:
3-1:将木瓜蛋白酶溶液与蛹虫草培养基多糖提取液混合,两者的体积比为1.0:1.5~1.0:1.7,60-70℃酶解2-3h;
3-2:在酶解液中加入1/5体积的Sevage试剂,振荡培养30min以上,然后多次离心直至无蛋白沉淀析出为止,取上清液,烘干得到蛹虫草培养基粗多糖;
(4)蛹虫草培养基多糖的分离纯化:将蛹虫草培养基粗多糖用蒸馏水溶解后上DEAE琼脂糖凝胶FF离子交换层析柱,用蒸馏水进行洗脱,得到多糖。
2.根据权利要求1所述的应用,其特征在于:步骤(1)所述的浓缩是在50-55℃下浓缩。
3.根据权利要求1所述的应用,其特征在于:步骤(2)所述的H2O2溶液,浓度为30%(V/V)。
4.根据权利要求1所述的应用,其特征在于:步骤(3)所述的木瓜蛋白酶溶液用pH值6.0的PBS缓冲液配制而成,其中木瓜蛋白酶的浓度为250U/ml。
5.根据权利要求1所述的应用,其特征在于:步骤(3)所述的Sevage试剂,是氯仿和正丁醇按体积比5:1配制而成。
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