CN108324731B - 提高抗氧化活性和增强抑菌作用的生物多糖颗粒的制备方法及其应用 - Google Patents
提高抗氧化活性和增强抑菌作用的生物多糖颗粒的制备方法及其应用 Download PDFInfo
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Abstract
本发明涉及一种提高抗氧化活性和增强抑菌作用的生物多糖颗粒的制备方法:取一定量的生物多糖,溶于去离子水,25℃恒温水浴锅中搅拌至生物活性多糖充分水合溶解,去除未溶解的活性多糖和杂质,制得浓度为1%~3%的生物活性多糖溶液,并调节其pH值为5~7;按照生物活性多糖溶液与95%乙醇的为1:3~1:20体积比,将95%乙醇逐滴滴加至生物活性多糖溶液,边搅拌边滴加,滴加和搅拌的温度均为25~50℃,滴加完乙醇后继续搅拌0.5~3h,3,000~1,0000rpm离心5~10min,95%乙醇洗涤,得到生物活性多糖纳米颗粒。本发明还提供该生物活性多糖纳米颗粒在清除自由基和抑菌方面的应用。该方法绿色安全、简单环保,制得的生物多糖颗粒非常好的抗氧化活性、抑菌作用和稳定性。
Description
技术领域
本发明属于食品药品技术领域,具体涉及一种提高抗氧化活性和增强抑菌作用的生物多糖颗粒的制备方法及其应用。
背景技术
生物多糖是由多个单糖分子脱水缩合而成,是一类分子机构复杂且庞大的糖类物质,不仅可作为一种能量物质和结构成分,还可参与细胞代谢及生理的调节特性。生物活性多糖作为一种活性组分,具有某种特殊生理活性的多糖化合物,广泛的存在于各种植物和真菌中,如茶叶、苦瓜、灵芝和香菇等。生物活性多糖具有各种各样生物功能,包括抗氧化、抑菌性、抗免疫调节、抗病毒和抗肿瘤等功效,以及具有降血糖和降血脂效果。生物活性多糖作为一种天然活性成分因其具有多种生物活性,且无毒、无害、无残留、无抗药性的优点,备受国内外研究学者青睐,成为医药、农业、食品和化妆品等领域的研究热点。当前,生物活性多糖的研究主要集中在多糖生物大分子的提取及其生物功能性的应用。生物活性多糖稳定性差,对温度和盐浓度的耐受性差,其抗氧化和抑菌性较弱,尤其是在敏感环境中无法有效发挥抗氧化和抑菌作用。此外,由于它们的亲水性和不带电荷性质,中性多糖表现出网状内皮系统的低生物利用度和清除率等缺点,限制了活性多糖应用范围。
发明内容
针对生物活性多糖稳定性差,对温度和盐浓度的耐受性差,其抗氧化和抑菌性较弱,尤其是在敏感环境中无法有效发挥抗氧化和抑菌作用,另外,中性多糖表现出网状内皮系统的低生物利用度和清除率的问题,本发明提供一种提高抗氧化活性和增强抑菌作用的生物活性多糖颗粒的制备方法,该方法绿色安全、简单环保,制得的生物多糖颗粒显示出非常好的抗氧化活性和抑菌作用,在pH、热、离子强度等敏感环境中的表现出更好的稳定性,还具有通过生物屏障的更高穿透率。
本发明是通过以下技术方案实现的:
本发明一方面提供一种提高抗氧化活性和增强抑菌作用的生物多糖颗粒的制备方法,包括以下步骤:
(1)生物活性多糖溶液的制备:取一定量的生物多糖粉末,溶于去离子水,25℃恒温水浴锅中搅拌至生物活性多糖充分水合溶解,去除未溶解的活性多糖和杂质,制得浓度为1%~3%的生物活性多糖溶液,并调节其pH值为5~7;
(2)生物活性多糖纳米颗粒的制备:按照生物活性多糖溶液与95%乙醇的为1:3~1:20体积比,将95%乙醇逐滴滴加至生物活性多糖溶液,边搅拌边滴加,滴加和搅拌的温度均为25~50℃,滴加完乙醇后继续搅拌0.5~3h,3,000~1,0000rpm离心5~10min,95%乙醇洗涤,得到生物活性多糖纳米颗粒,冻干。
进一步的,所述物活性多糖为茶多糖、苦瓜多糖、灵芝多糖、南瓜多糖、香菇多糖或海带多糖中的一种或几种。
进一步的,所述步骤(2)中的生物活性多糖溶液与95%乙醇的比例为1:4,1:5或者1:10。
进一步的,所述步骤(2)中冻干工艺为:真空度5~10Pa,温度-80~-60℃,时间48~72h。
本发明另一方面提供上述方法制备的生物活性多糖纳米颗粒在清除自由基和抑菌方面的应用。
进一步的,上述生物活性多糖纳米颗粒用于清除DPPH自由基、羟基自由基、超氧化物自由,抑制金黄色葡萄球菌、沙门氏菌、大肠杆菌和枯草芽孢杆菌。
本发明的有益效果:
(1)本方法采用纳米沉淀法,可提高抗氧化活性和增强抑菌作用的纳米化生物活性多糖颗粒及其制备方法具有操作简单、绿色环保、无毒、成本低等特点,
(2)本方法制备得到的生物活性多糖纳米颗粒具有尺寸小,比表面积大,生物相容性好、可降解、分布均匀等特点,
(3)本方法制备得到的纳米化的生物活性多糖颗粒增强了生物活性多糖的具有耐酸碱、耐盐和扩大了范围内优越的稳定性和生物活性;
(4)本方法制备得到的纳米化的生物活性多糖颗粒,增加了生物活性多糖对组织的附着力,减少对血液中对蛋白的吸附量,提高循环次数,延长生物活性多糖的在血液中的滞留时间,提高其生物利用率,有效减少生物活性成分的添加量和毒副作用。
(5)本方法制备得到的纳米化的生物活性多糖颗粒显著增强了生物活性多糖的抗氧化性、抑菌效果并延长其抑菌活性等特性。
附图说明
图1为实施例1不同乙醇倍数制备的茶多糖纳米颗粒的粒径分布、平均粒径、电位及分散指数图;
图2为实施例1不同乙醇倍数制备的茶多糖纳米颗粒的透射电镜图
图3为实施例1不同乙醇倍数制备的茶多糖纳米颗粒的傅里叶红外光谱图;
图4为实施例1温度对茶多糖纳米颗粒的颗粒粒径、电势和浊度的影响图;
图5为实施例1盐离子浓度对茶多糖纳米颗粒的颗粒粒径、电势和浊度的影响图;
图6为实施例1pH水平对茶多糖纳米颗粒的颗粒粒径、电势和浊度的影响图;
图7为实施例1~3生物活性多糖纳米颗粒在37℃(pH 7.4)对BSA不同时间的吸附量图;
图8为实施例2不同浓度的灵芝多糖和灵芝多糖纳米颗粒的抑菌效果图;
图9为实施例3不同浓度的苦瓜多糖和苦瓜多糖纳米颗粒的抑菌效果图;
图10为实施例2~3相同浓度的灵芝多糖和灵芝多糖纳米颗粒随时间变化的抑菌效果图;
图11为实施例2~3相同浓度的苦瓜多糖和苦瓜多糖纳米颗粒随时间变化的抑菌效果图;
图12为实施例2不同乙醇倍数制备的灵芝多糖纳米颗粒的平均粒径、电位及分散指数图;
图13为实施例2不同乙醇倍数制备的灵芝多糖纳米颗粒的透射电镜图;
图14为实施例3不同乙醇倍数制备的苦瓜多糖纳米颗粒的平均粒径、电位及分散指数图;
图15为实施例3不同乙醇倍数制备的苦瓜多糖纳米颗粒的透射电镜图;
图16~18为实施例1~3生物活性多糖纳米颗粒清除DPPH自由基图;
图19~21为实施例1~3生物活性多糖纳米颗粒清除羟基自由基图;
图22~24为实施例1~3生物活性多糖纳米颗粒清除超氧化物自由基图。
以上各图中大写的A-E分别代表乙醇倍数为1:3;1:4;1:5;1:10;1:20。
具体实施方式
下面结合具体实施例及附图对本发明做进一步详细说明。
实施例1制备茶多糖纳米颗粒(TP-NPs)
步骤:
(1)生物多糖溶液的制备:取1~3g的茶多糖,溶于100mL去离子水,25℃恒温水浴锅中搅拌2h,茶多糖充分水合溶解,3,000~5,000rpm离心5~10min,去除未溶解的活性多糖和杂质,制得浓度为%~3%茶多糖溶液,取一定量的茶多糖溶液,分别调节茶多糖pH值为5~7;
(2)生物活性多糖纳米颗粒的制备:按照茶多糖溶液与95%乙醇的为1:3,1:4,1:5,1:10和1:20体积比,将95%乙醇逐滴滴加至茶多糖溶液,边搅拌边滴加,滴加和搅拌的温度为25~50℃,滴加完乙醇后继续搅拌0.5~3h,3,000~6,000rpm离心5~10min,95%乙醇洗2~3次,得到茶多糖纳米颗粒,冻干得到茶多糖纳米颗粒粉末。
实施例2制备淀苦瓜多糖纳米颗粒(MCP-NPs)
步骤:
((1)苦瓜多糖溶液的制备:取1~3g的苦瓜多糖,溶于100mL去离子水,25℃恒温水浴锅中搅拌2h,苦瓜多糖充分水合溶解,3,000~5,000rpm离心5~10min,去除未溶解的活性多糖和杂质,得到浓度为1%~3%的苦瓜多糖溶液,取一定量的苦瓜多糖溶液,分别调节其pH值为5~7;
(2)苦瓜多糖纳米颗粒的制备:按照苦瓜多糖溶液与95%乙醇的为1:3,1:4,1:5,1:10和1:20体积比,将95%乙醇逐滴滴加至苦瓜多糖溶液,边搅拌边滴加,滴加完乙醇后继续搅拌0.5~3h,3,000~6,000rpm离心5~10min,95%乙醇洗2~3次,得到苦瓜纳米颗粒,冻干得到苦瓜多糖纳米颗粒粉末。
实施例3制备灵芝多糖纳米颗粒(GLP-NPs)
步骤:
(1)灵芝多糖溶液的制备:取1~3g的灵芝多糖,溶于100mL去离子水,25℃恒温水浴锅中搅拌2h,灵芝多糖充分水合溶解,3,000~5,000rpm离心5~10min,去除未溶解的灵芝多糖和杂质,制得浓度为1%~3%的灵芝多糖溶液;取一定量的灵芝多糖溶液,分别调节灵芝多糖pH值为5~7;
(2)灵芝多糖纳米颗粒的制备:按照灵芝多糖溶液与95%乙醇的为1:3,1:4,1:5,1:10和1:20体积比,将95%乙醇逐滴滴加至灵芝多糖溶液,边搅拌边滴加,滴加完乙醇后继续搅拌0.5~3h,3,000~6,000rpm离心5~10min,95%乙醇洗3次,得到灵芝纳米颗粒,冻干得到灵芝多糖纳米颗粒粉末。
实施例1~3所制备的纳米化生物活性多糖颗粒的性能检测:
(1)纳米化生物活性多糖颗粒的抗氧化活性测定:
DPPH(1,1-二苯基-2-三硝基苯肼)自由基清除能力的测定:10~2000μg/mL的生物活性多糖和活性多糖纳米颗粒溶解在去离子水,充分溶解后,2mL的DPPH乙醇溶液(0.2mM)分别与2mL的生物活性多糖和多糖纳米颗粒溶液混合均匀,暗处反应30min,相同体积的去离子水作为空白对照,Vc为阳性对照,测定517nm处的吸光度值,计算其半抑制浓度和自由基清除率。
羟基自由基清除能力的测定:将1.0mL FeSO4(9.0mM),1.0mL H2O2(8.8mM)和1.0mL水杨酸(9.0mM)与不同浓度的生物活性多糖或多糖纳米颗粒(10~2000μg/mL)分别混合、混合均匀,37℃保温处理1h,Vc为阳性对照,测定样品在510nm处的吸光值,计算其半抑制浓度和自由基清除率。
超氧化物自由基清除能力的测定:将5mL的50.0mM Tris-HCl缓冲液(pH 8.1)分别与4.0mL不同浓度的生物活性多糖或多糖纳米颗粒(10~2000μg/mL)充分混合。25℃处理20min,向混合物中加入1.0mL的3.0mM连苯三酚,并将混合物在25℃处理5min,然后加入1.0mL HCl(10.0mM)终止反应,并在320nm测定吸光值,计算其半抑制浓度和自由基清除率。
随后,测定盐离子(NaCl)和温度(65℃)处理的多糖纳米颗粒溶液对DPPH、羟基自由基(·OH)和超氧化物自由基清除能力,上述不同浓度的样品分别在0.8mM盐离子浓度和温度(65℃)条件下,放置2h后,再与DPPH乙醇溶液混合,测定其对DPPH自由基清除活力,不处理的多糖纳米颗粒作为对照样。DPPH自由基清除率计算公式如下:
自由基清除率(%)=[1-(样品的吸光度—对照的吸光度)/空白的吸光度]×100%
图16~24显示的为实例1~3制备得到的茶多糖纳米颗粒、灵芝多糖纳米颗粒和苦瓜多糖纳米颗粒的DPPH、羟基自由基(·OH)和超氧化物自由基清除能力测定结果,由图可得,与原生物活性多糖相比,生物活性多糖纳米颗粒显著增加了对DPPH、·OH和超氧化物自由基的清除能力(9%~25%),同时增加了生物活性多糖的耐受温度和盐离子浓度;由表1~表3可得,生物活性多糖纳米颗粒化后,其清除自由基的IC50值显著降低,即相同效率下,有效减少生物活性多糖的添加量。
表1生物活性多糖和生物活性多糖纳米颗粒清除DPPH的半抑制浓度(IC50)
表2生物活性多糖和生物活性多糖纳米颗粒清除羟基自由基的半抑制浓度(IC50)
表3生物活性多糖和生物活性多糖纳米颗粒清除超氧化物自由基的半抑制浓度(IC50)
(2)灵芝多糖和苦瓜多糖纳米颗粒抑菌性的测定:
牛肉膏蛋白胨液体培养基制备:准确称取蛋白胨10g、牛肉膏5g、氯化钠5g,倒入1000mL蒸馏水中水浴加热充分溶解。用2%NaOH溶液调节pH值至7.2,分装在三角瓶中,包扎,高压灭菌锅灭菌(121℃湿热灭菌15min),待用。
无菌生理盐水配制:称取9g NaCL溶于1000mL蒸馏水中,分装在三角瓶中,加塞,包扎,高压灭菌(121℃,15min),备用。
菌悬液制备:将菌落接种在液体培养基中,摇床培养(37℃培养24h)即可得到一定浓度的菌悬液。无菌生理盐水稀释菌悬液,使菌悬液的吸光值在0.7~0.8,封口置于4℃冰箱,备用。
不同时间灵芝多糖、苦瓜多糖、灵芝多糖纳米颗粒和苦瓜多糖纳米颗粒对四种类型细菌(金黄色葡萄球菌,沙门氏菌,大肠杆菌和枯草芽孢杆菌)的抑制作用:在无菌条件下,分别取稀释过的菌悬液18mL置于试管中,取400μL浓度为3000μg/mL生物活性多糖和多糖纳米颗粒溶液,旋涡混匀器混匀,37℃摇床中培养,以不加样品溶液的菌悬液作对照,以蒸馏水作为空白对照。分别测定0,2,8,12,24,36和48h测定样品菌悬液在600nm处的吸光度值,重复3次,取平均值,确定最适作用时间。
不同浓度灵芝多糖、苦瓜多糖、灵芝多糖纳米颗粒和苦瓜多糖纳米颗粒对四种菌的抑制作用:最适条件下,用无菌蒸馏水将生物活性多糖和多糖纳米颗粒溶液的浓度稀释成250;500;1000;1500;2000;3000和4000μg/mL。分别取上述菌的悬液18mL和400μL不同浓度的生物活性多糖和多糖纳米颗粒液,旋涡混合器混匀,37℃摇床培养24h,以不加样品液的菌悬溶液作对照,以蒸馏水作空白对照,测其600nm处的吸光度值,重复3次,取平均值。
图8~9为不同浓度的灵芝多糖或苦瓜多糖纳米颗粒对革兰氏阳性和阴性菌的抑菌效果图,由图可得浓度越高,抑菌效果越好;且相同浓度下,生物活性多糖纳米颗粒的抑菌效果强于生物活性多糖;
图10~11为相同浓度的灵芝多糖或苦瓜多糖纳米颗粒抑菌时间对革兰氏阳性和阴性菌的抑菌效果图,由图可得活性多糖纳米颗粒可显著延长抑菌效果。
(3)生物活性多糖纳米颗粒的大小及形态
图1,12,14为激光动态光散射、平均粒径、电位及分散指数图,当95%乙醇与溶液的比例为1:10时,茶多糖,灵芝多糖和苦瓜多糖纳米颗粒的平均粒径分别为99±15,95±7和141±9nm;纳米颗粒的分散指数均小于0.5,说明纳米颗粒在水溶液中具有良好的分散性,且多糖纳米颗粒均带负电荷。
图2,13,15为茶多糖,灵芝多糖和苦瓜多糖纳米颗粒的透射电镜图谱,由图看出,不同生物活性多糖纳米颗粒的具有良好的分散性,呈表面光滑的圆球形,而不同生物活性多糖纳米颗粒的大小不同,且都均在60~250nm。
(4)茶多糖纳米颗粒的傅里叶红外光谱
如图3所示为茶多糖纳米颗粒的红外光谱图,随着乙醇与溶液比例的增加,3,600–3,200cm-1逐渐增强,且峰值向短波长移动,说明生物活性多糖纳米化后,氢键作用加强。
(5)茶多糖纳米颗粒敏感环境变化(温度、盐离子、pH水平)测试:
0.1%(w/v)的茶多糖纳米颗粒被分散在NaCl溶液中(0;100;200;300;400和500mM)。样品在室温放置60min,分别测定茶多糖纳米颗粒平均粒径、分散性指数、电荷和浊度的变化。
0.1%(w/v)的茶多糖纳米颗粒被分散在水溶液中。用盐酸和氢氧化钠溶液调节pH为2.0、5.1、7.4和9.2。样品被放置在室温放置60min,然后分别测定茶多糖纳米颗粒的平均粒径、分散性指数、电荷和浊度的变化。
0.1%(w/v)的茶多糖纳米颗粒分散在水溶液中。样品分别被放置在25(室温)、37和70℃放置60min,然后分别测定测定茶多糖纳米颗粒的平均粒径、分散性指数、电荷和浊度的变化。
如图4~6所示,茶多糖纳米颗粒具有良好的抵温度、抗盐和pH水平变化的能力。
(6)生物活性多糖纳米颗粒的蛋白吸附试验
以牛血清蛋白(BSA)为模板测定多糖纳米颗粒对蛋白吸附量的测定。0.15mg/mL的多糖纳米颗粒与0.25mg/mL的BSA溶液在pH为7.4,37℃下培养不同的时间。每隔一定的时间,取1mL样品,10,000g离心20min,水洗两次,取上清液,考马斯亮蓝法测定上清液中BSA含量,根据BSA标准曲线,计算得到上清液中BSA具体含量,从而计算得到多糖纳米颗粒吸附BSA含量。
图7为活性多糖纳米颗粒对BSA吸附量的测定。活性多糖纳米颗粒与BSA培养2h后,多糖纳米颗粒吸附少量的BSA,且36h后,多糖纳米颗粒仍对BSA呈现较小的吸附。然而,与茶多糖和苦瓜多糖纳米颗粒吸附量相比,灵芝多糖纳米颗粒吸附较小的BSA,可以有效的逃避体内蛋白对多糖纳米颗粒的吸附和包埋,表明了多糖纳米颗粒有利于在血液中增加循环次数,尤其是灵芝多糖纳米颗粒。因此,多糖纳米颗粒在改善血液的稳定和提高生物利用率呈现前景市场空间。
Claims (4)
1.一种提高抗氧化活性和增强抑菌作用的生物活性多糖颗粒的制备方法,其特征在于,包括以下步骤:
(1)生物活性多糖溶液的制备:取一定量的生物活性多糖粉末,溶于去离子水,25℃恒温水浴锅中搅拌至生物活性多糖充分水合溶解,去除未溶解的活性多糖和杂质,制得浓度为1%~3%的生物活性多糖溶液,并调节其pH值为5~7;
(2)生物活性多糖纳米颗粒的制备:按照生物活性多糖溶液与95%乙醇的为1:3~1:20体积比,将95%乙醇逐滴滴加至生物活性多糖溶液,边搅拌边滴加,滴加和搅拌的温度均为25~50℃,滴加完乙醇后继续搅拌0.5~3h,3,000~1,0000rpm离心5~10min,95%乙醇洗涤,得到生物活性多糖纳米颗粒,冻干;
所述生物活性多糖为茶多糖、苦瓜多糖、灵芝多糖中的一种或几种。
2.根据权利要求1所述的制备方法,其特征在于,所述步骤(2)中的生物活性多糖溶液与95%乙醇的比例为1:4,1:5或者1:10。
3.根据权利要求1所述的制备方法,其特征在于,所述步骤(2)中冻干工艺为:真空度5~10Pa,温度-80~-60℃,时间48~72h。
4.根据权利要求1所述的制备方法,其特征在于,所述方法制备的生物活性多糖纳米颗粒用于清除DPPH自由基、羟基自由基、超氧化物自由基,抑制金黄色葡萄球菌、沙门氏菌、大肠杆菌和枯草芽孢杆菌。
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