CN110408664A - 一种具有高抗氧化活性新琼六八糖的制备方法 - Google Patents
一种具有高抗氧化活性新琼六八糖的制备方法 Download PDFInfo
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Abstract
本发明公开了一种具有高抗氧化活性新琼六八糖的制备方法,属于海洋生物技术领域。本发明以琼胶寡糖为原料,利用酶降解法制备得到琼六八糖,通过测定新琼寡糖的清除羟自由基能力的测定和新琼寡糖的清除超氧阴离子能力,实现高抗氧化活性新琼六八糖的均一性制备。本发明采用酶法降解,使得制备过程简单快捷、产物的率高、质量稳定,并且该产品具有较高的抗氧化活性,开发具有抗氧化功能的保健食品前景广阔。
Description
技术领域
本发明涉及新琼寡糖领域,具体地说是一种具有高抗氧化活性的新琼六糖和新琼八糖制备方法。
背景技术
作为海洋多糖,琼胶具有高粘度度特性。它水溶性低,不易被吸收,因此在应用方面受到很大限制。琼胶降解得到的琼胶寡糖水溶性好,有利于人体吸收,它不仅具有功能性低聚糖的一般特性,还具有许多常见寡糖无法替代的生理功能。琼胶寡糖具有对微生物的抑菌、增殖肠道益生菌作以及抗病毒的作用,此外还具有抗肿瘤和免疫增强作用、抗炎作用、抗氧化作用以及吸湿保湿性和美白作用等生物活性,是一种极具开发潜力的低聚糖。
相关研究初步表明,琼胶寡糖具有较好的抗氧化性能。薛长湖等通过研究不同聚合度的琼胶低聚糖对DPPH, 超氧阴离子和羟自由基的清除效果,结果表明了琼胶寡糖具有很好的抗氧化活性。人的体内会产生自由基,过多的自由基会损坏肝脏功能,降低细胞的抵抗力和减弱人体免疫力,还会阻碍细胞的正常发育,引起细胞和个体的衰老。而琼胶寡糖具有较强的抗氧化能力,能够清除多种自由基,可作为一种天然,无毒,高效的抗氧化剂,将来可运用于药品、保健品、食品添加剂、饲料添加剂和化妆品等行业,具有明显经济效益和市场前景。
研究显示酶解的新琼六八糖具有较强的抗氧化功能。新琼六八糖的制备,以发酵制备的琼胶酶为工具酶,琼脂为底物,降解产物滤去除蛋白,薄层分析显示得到主产物。在琼胶寡糖制备方面,酶解法较传统的化学法具有底物专一性强、产物特异性高、反应条件温和等优点,能够保证酶解产物结构不被破坏,从而保证寡糖产品的稳定性,使高纯度寡糖的大量制备成为可能。
发明内容
为克服上述现有技术中的不足,本发明提供一种具有高抗氧化活性新琼六八糖的制备方法,以琼胶寡糖为原料,利用酶降解法制备得到琼六八糖,通过测定新琼寡糖的清除羟自由基能力的测定和新琼寡糖的清除超氧阴离子能力,实现高抗氧化活性新琼六八糖的均一性制备。本发明采用酶法降解,使得制备过程简单快捷、产物的率高、质量稳定,并且该产品具有较高的抗氧化活性,开发具有抗氧化功能的保健食品前景广阔。
本发明通过下述技术方案实现上述技术效果:
一种具有高抗氧化活性新琼六八糖的制备方法,具体包括以下步骤:
1)准确配制质量浓度0.25%琼脂糖溶液,90~110℃加热使其完全溶解;
2)冷却至35~45℃,每100mL底物加入5~10mLß-琼胶酶的粗酶液,混匀,40~60℃,100~200rpm振荡反应2~8h;
3)沸水浴加热5~15min灭活酶蛋白,0~10℃完全冷却沉降;
4) 0~10℃,5000~15000r/min离心5~15min除去不溶物;
5)使用旋转蒸发仪浓缩样品体积,置0~10℃放置过夜;
6) 0~10℃, 5000~15000r/min离心2~8min,上清液用0.22um滤膜过滤去除杂质;
7)冷冻干燥至粉末状,即得新琼六八糖,于干燥处保存。
优选的,步骤2)中粗酶液的加入量为8mL。
优选的,步骤4)和6)中离心转速为10000r/min。
上述ß-琼胶酶的粗酶液通过如下步骤制备:分别配制斜面培养基、种子培养基和发酵培养基,于灭菌锅中115℃~130℃灭菌20~40min;将类芽抱杆菌菌株Paenibacillus sp.WL-15接于斜面培养基,于恒温生化培养箱内15~35℃条件下培养12~36 h后接于种子培养基中,置于恒温培养摇床中15~35℃ , 100~300r/min 条件下扩大培养12~36h,再以0.5:15( w/w)的接种量的接入发酵培养基中,于恒温培养摇床中以同样的条件发酵培养12~36h;然后将发酵培养液在1~7℃ ,5000-15000r/min条件下离心5~15min,取其上清液,即为所需的胞外酶ß-琼胶酶的粗酶液,于0~10℃冰箱中保存备用。
优选的,上述斜面培养基和活化培养基的成分组成为:琼胶5%、NaCl 3.0%、尿素0.1%、酵母粉0.1%、MgSO4 0.05%、K2HPO4 0.1%、FeSO4 0.002%、Fe2(SO4)3 0.001%、CaCl20.02%。
优选的,上述发酵培养基的成分组成为:乳糖0.5%、琼胶5%、NaCl 3.0%、尿素0.1%、酵母粉0.1%、MgSO4 0.05%、K2HPO4 0.1%、FeSO4 0.002%、Fe2(SO4)3 0.001%、CaCl2 0.02%。
本发明提供了一种具有高抗氧化活性的新琼六八糖的制备方法和应用,与现有技术相比具有如下优势:
一是本申请以琼胶寡糖为原料,利用酶降解法制备得到新琼六八糖,产品中新琼六八糖含量高,具有较强的抗氧化能力,对羟自由基和超氧阴离子的清除能力较强,实现高抗氧化活性新琼六八糖的均一性制备;
二是本发明制备的新琼寡糖除了具有产物专一性好、反应条件温和、无污染等众多优点,还能保证酶解产物的结构及其活性不被破坏,从而保证了制备出的琼新琼寡糖品质,为研究新琼寡糖的各种活性奠定了基础;本发明减少了酶的分离纯化步骤,大大简化了制备工艺,降低了生产成本,从而提高新琼寡糖的利用度,并且该产品具有抗氧化活性,可用于开发具有抗氧化功能的保健食品等。;
三是试验结果表明新琼六八糖降解酶发酵培养基中添加乳糖对发酵产酶具有一定的抑制作用;但是研究结果表明在发酵培养基中添加乳糖有利于提高通过粗酶液制备的新琼六八糖的抗氧化能力;相同条件下不添加乳糖发酵培养的粗酶液酶解后的新琼六八糖粗酶液抗氧化能力较实施例3下降34.7%。
附图说明:
图1:新琼六八糖的TLC图。
具体实施方式
实施例1 高抗氧化活性新琼六八糖的制备
一种具有高抗氧化活性新琼六八糖的制备方法,具体包括以下步骤:
1)准确配制质量浓度0.25%琼脂糖溶液,90~110℃加热使其完全溶解;
2)冷却至35~45℃,每100mL底物加入8mLß-琼胶酶的粗酶液,混匀,40~60℃,100~200rpm振荡反应5h;
3)沸水浴加热10min灭活酶蛋白,0~10℃完全冷却沉降;
4) 0~10℃, 10000r/min离心10min除去不溶物;
5)使用旋转蒸发仪浓缩样品体积,置0~10℃放置过夜;
6) 0~10℃, 10000r/min离心5min,上清液用0.22um滤膜过滤去除杂质;
7)冷冻干燥至粉末状,即得新琼六八糖,于干燥处保存;
上述ß-琼胶酶的粗酶液通过如下步骤制备:分别配制斜面培养基、种子培养基和发酵培养基,于灭菌锅中115℃~130℃灭菌30min;将类芽抱杆菌菌株Paenibacillus sp.WL-15接于斜面培养基,于恒温生化培养箱内15~35℃条件下培养24 h后接于种子培养基中,置于恒温培养摇床中15~35℃ , 200r/min 条件下扩大培养24h,再以0.5:15( w/w)的接种量的接入发酵培养基中,于恒温培养摇床中以同样的条件发酵培养24h;然后将发酵培养液在1~7℃ , 10000r/min条件下离心10min,取其上清液,即为所需的胞外酶ß-琼胶酶的粗酶液,于0~10℃冰箱中保存备用。
实施例2 新琼六八糖的检测
对实施例1制备的新琼六八糖进行薄层层析:
薄层板:德国Merck公司TLC Silica gel 60 F254 plate;正丁醇/甲酸/水=4/6/1(V/V);显色剂:苯胺/二苯胺;上样:毛细管点样,0.5 µL;
点样后,将薄层硅胶板置于层析缸中展层。待前沿到达硅胶板顶端时取出,热风吹干。喷显色剂加热显色;
结果如图1所示。
实施例3 高抗氧化活性的新琼六八糖的抗氧化能力测定
1、新琼六八糖糖清除超氧阴离子能力的测定
试剂:Xanthine(黄嘌呤): 0.4mmol/l、Xanthine oxidase(黄嘌呤氧化酶)贮液: 1unit/mL、0.05 unit/mL、NBT: (Nitro blue tetrazolium chloride氯化硝基四氮唑蓝),0.24mmol、PBS(0.01mol/L,pH=8.0)、PBS(0.01mol/L,pH=7.4)、Ascorbic acid: 1mg/mL 、HCl:1mol/l、NaOH:1mol/l;
测定方法:
在96孔板孔中依次加入100μL 黄嘌呤(0.4mmol/l)和NBT(0.24mmol/l)的混合液(每种各50μL,溶于0.01mol/l的PBS,pH=8.0),100μL黄嘌呤氧化酶(0.049units/ml), 50μL不同浓度的κ-卡拉胶寡糖溶液(浓度分别为1mg/ml、2mg/ml、4mg/ml、 8mg/ml)。混匀后37℃孵育30min, 酶标仪测OD560值(扣除OD800值)。不加样品溶液作为空白对照,Vc(维生素C)为阳性对照,每个浓度的样品平行测定三次,分别计算清除率及样品的IC50;
样品清除率=(OD空白-OD样品)/OD空白×100%.
表1 新琼六八糖对超氧阴离子清除率
由表 1 可见,新琼六八糖对超氧阴离子能力有很好的抑制作用,而且随糖浓度的增加抑制作用提高。
2、新琼六八糖清除羟自由基能力的测定
试剂:1.865 mmol/L邻二氮菲、无水乙醇溶液、0.2 M的pH 7.4磷酸盐缓冲液、1.865mmol/L的FeSO4·7H2O溶液、0.03% (v/v) 的H2O2、1mg/ml抗坏血酸;
测定方法:
取1.0 mL浓度为1.865 mmol/L邻二氮菲的无水乙醇溶液于带塞试管中,分别加入浓度为0.2 M的pH 7.4磷酸盐缓冲液2 mL和1 mL不同浓度的κ-卡拉胶寡糖(浓度分别为1mg/ml、2mg/ml、4mg/ml、8mg/ml),充分混匀后加入1.0 mL浓度为1.865 mmol/L的FeSO4·7H2O溶液,再次混匀后加入1.0 mL 0.03% (v/v) 的H2O2,于37℃恒温水浴,60 min后,在536 nm下分别测量各组混合溶液的吸光度值得AS ,以蒸馏水代替样品作为空白组测吸光度值得Ab,以蒸馏水替代H2O2作为损伤组,测其吸光度值An,以抗坏血酸代替样品作为阳性对照,抗氧化剂的羟自由基清除率按以下公式计算:
羟自由基清除率(%)=[(As-An)/(Ab-An)] × 100
表2 新琼六八糖对羟基自由基清除率
由表2可以看出,新琼六八糖对羟自由基有一定的清除作用,而且清除作用随糖浓度的增加而提高。
以上实施例仅用于说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对被发明进行了详细的说明,但对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而对这些修改或者替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (7)
1.一种具有高抗氧化活性新琼六八糖的制备方法,其特征在于具体包括以下步骤:
1)准确配制质量浓度0.25%琼脂糖溶液,90~110℃加热使其完全溶解;
2)冷却至35~45℃,每100mL底物加入5~10mLß-琼胶酶的粗酶液,混匀,40~60℃,100~200rpm振荡反应2~8h;
3)沸水浴加热5~15min灭活酶蛋白,0~10℃完全冷却沉降;
4) 0~10℃,5000~15000r/min离心5~15min除去不溶物;
5)使用旋转蒸发仪浓缩样品体积,置0~10℃放置过夜;
6) 0~10℃, 5000~15000r/min离心2~8min,上清液用0.22um滤膜过滤去除杂质;
7)冷冻干燥至粉末状,即得新琼六八糖,于干燥处保存。
2.根据权利要求1所述的一种具有高抗氧化活性新琼六八糖的制备方法,其特征在于所述步骤2)中粗酶液的加入量为8mL。
3.根据权利要求1所述的一种具有高抗氧化活性新琼六八糖的制备方法,其特征在于所述步骤4)和6)中离心转速为10000r/min。
4.根据权利要求1所述的一种具有高抗氧化活性新琼六八糖的制备方法,其特征在于所述ß-琼胶酶的粗酶液通过如下步骤制备:分别配制斜面培养基、种子培养基和发酵培养基,于灭菌锅中115℃~130℃灭菌20~40min;将类芽抱杆菌菌株Paenibacillus sp.WL-15接于斜面培养基,于恒温生化培养箱内15~35℃条件下培养12~36 h后接于种子培养基中,置于恒温培养摇床中15~35℃ , 100~300r/min 条件下扩大培养12~36h,再以0.5:15( w/w)的接种量的接入发酵培养基中,于恒温培养摇床中以同样的条件发酵培养12~36h;然后将发酵培养液在1~7℃ ,5000-15000r/min条件下离心5~15min,取其上清液,即为所需的胞外酶ß-琼胶酶的粗酶液,于0~10℃冰箱中保存备用。
5.根据权利要求4所述的一种具有高抗氧化活性新琼六八糖的制备方法,其特征在于所述斜面培养基和活化培养基的成分组成为:琼胶5%、NaCl 3.0%、尿素0.1%、酵母粉0.1%、MgSO4 0.05%、K2HPO4 0.1%、FeSO4 0.002%、Fe2(SO4)3 0.001%、CaCl2 0.02%。
6.根据权利要求4所述的一种具有高抗氧化活性新琼六八糖的制备方法,其特征在于所述发酵培养基的成分组成为:乳糖0.5%、琼胶5%、NaCl 3.0%、尿素0.1%、酵母粉0.1%、MgSO4 0.05%、K2HPO4 0.1%、FeSO4 0.002%、Fe2(SO4)3 0.001%、CaCl2 0.02%。
7.按权利要求1所述方法制备的新琼六八糖。
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