CN116496426A - 一种螺旋藻多糖胶体及其制备方法和应用 - Google Patents
一种螺旋藻多糖胶体及其制备方法和应用 Download PDFInfo
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- 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
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
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- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
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- A—HUMAN NECESSITIES
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
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- A61L15/42—Use of materials characterised by their function or physical properties
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
本发明属于微藻生物技术领域，具体为一种螺旋藻多糖胶体及其制备方法和应用。本发明的螺旋藻多糖胶体从螺旋藻中提取得到，其主要成分为半乳糖及葡萄糖醛酸，并含有核糖、岩藻糖、木糖及葡萄糖；该螺旋藻多糖胶体具有良好的溶胀性及保水性，并且粘弹性与透明质酸相似，可以形成稳定凝胶状态，能够有效的清除自由基，具有良好的体外抗氧化能力，在医用敷料及美容材料等领域具有广泛的应用前景。
Description
技术领域
本发明属于微藻生物技术领域，具体涉及螺旋藻多糖胶体及其制备方法和应用。
背景技术
微藻固定大气中的二氧化碳，并通过光合作用将其转化为生物量。它储存了一些有价值的成分，如脂质、碳水化合物及一些天然色素，用于营养、动物饲料、营养补充剂以及健康产品的制造。其中，螺旋藻作为一种蓝藻，含有60％-70％的蛋白质，15％-20％的多糖以及一些其他的营养物质，不仅作为一些营养食品的补充剂，螺旋藻中含有的多糖还具有多种生物活性。
螺旋藻多糖的生物活性以及药理研究已经有大量报道，它可以降低血糖水平，提高胰岛素抵抗并抑制胰岛β细胞的凋亡，在糖尿病及其并发症的治疗中起着重要作用。此外，它可以通过清除体外自由基、增加超氧化物歧化酶、过氧化氢酶等方式改善氧化应激、抑制脂质过氧化等方式发挥抗衰老作用。螺旋藻多糖还能够在刺激巨噬细胞、T、B淋巴细胞的同时，引起依赖性免疫反应，起到预防肿瘤的作用。
目前，微藻多糖产品日益增多，其中，螺旋藻多糖在功能性食品和饮料中应用广泛，其应用形式主要有片剂、胶囊、冲剂等。充分发挥其“药食同源”功效，在大健康产业功能产品领域为人体健康助力。但是其作为凝胶形态的产品鲜有报到，凝胶产品由于其高含水量、良好的保水性、优良的生物相容性以及和细胞外基质相类似的微观结构，被广泛应用于组织工程支架、医用敷料及美容材料。因此，以螺旋藻提取物为原料，制备不同结构与功能应用的螺旋藻多糖胶体，有助于丰富功能型螺旋藻高附加值产品的综合性开发与应用。
发明内容
针对目前天然多糖胶体种类的不足以及活性的差异，本发明提供了一种螺旋藻多糖胶体(Spirulina sp.polysaccharide hydrogel，简称SPH)及其制备方法和应用。
本发明提供的螺旋藻多糖胶体，从螺旋藻中提取得到，提取的具体步骤如下：
(1)初步提取：称取干燥的螺旋藻冻干粉2～5g，将其研磨，加入100mL去离子水，于60～90℃水浴锅中，加热4～5小时后，于8000～10000rpm离心20～40min，得到沉淀和上清液，将上清液置于真空抽率装置进行抽滤，过0.45μm滤膜，将抽滤后收集得到的液体于25～60℃真空浓缩至原体积的1/10，得到浓缩液；
(2)去除杂质：将制备得到的浓缩液，加入其体积1/3的Sevage试剂混合震荡后静置10～30min，6000～8000rpm离心5～10min，重复2～3次，取得水层去除交界处变性蛋白质；然后将水层过大孔树脂交换柱，收集水层溶液；
(3)乙醇诱导：将水层溶液加入乙醇至终浓度60～90％(v/v)进行诱导，将诱导后的混合溶液于0～4℃静置12～16小时，6000～8000rpm离心5～10min，收集得到螺旋藻多糖胶体SPH。
本发明制备的螺旋藻多糖胶体，其主要成分为半乳糖(55.37％)及葡萄糖醛酸(19.63％)，并含有的核糖(6.12％)、岩藻糖(4.06％)、木糖(4.63)及葡萄糖(6.56％)，还含有少量的阿拉伯糖(1.37％)、塔罗糖(0.91％)、鼠李糖(0.67％)及甘露糖(0.68％)。
本发明中，用于提取多糖胶体的螺旋藻为生长至稳定期的螺旋藻。
本发明中，步骤(1)中优选水浴温度为80℃，浸提时间为4小时。
本发明中，步骤(3)中优选加入乙醇至终浓度为60％(v/v)。
本发明中，所述螺旋藻多糖胶体(SPH)的溶胀性与透明质酸无显著性差异，并且显著高于海藻酸钠(p<0.05)。
通过流变学特性测定分析表明，所述螺旋藻多糖胶体(SPH)粘度较高，且粘弹性稳定，具有凝胶性行为，可以形成稳定的凝胶状态，具有良好的溶胀性及保水性；体外抗氧化试验表明，具有良好的自由基清除能力，体现出较高的体外抗氧化活性，可进一步进行水凝胶的开发与应用。具体如，生物医药方面，医用敷料(如伤口敷料)的制备，也可用于医美产品的制备。
附图说明
图1为本发明实施例提供的SPH的提取流程图。
图2为本发明实施例提供的SPH照片(图A为脱色前；图B为脱色后)。
图3为本发明实施例提供的SPH的单糖组成分析图。
图4为本发明实施例提供的SPH的溶胀性分析图。
图5为本发明实施例提供的SPH的保水性分析图。
图6为本发明实施例提供的SPH的静态流变学特性分析图。
图7为本发明实施例提供的SPH的动态流变学特性分析图。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
本发明提供了螺旋藻多糖胶体的制备方法，以及对制备的螺旋藻多糖胶体进行持水性试验(包括溶胀性测定分析及保水性测定分析)、流变学特性测定分析、体外抗氧化试验分析等。
1、SPH的制备，参见图1所示；
(1)初步提取：称取干燥的螺旋藻冻干粉2g，将其研磨，加入100mL去离子水，于80℃水浴锅中，加热4小时后，于10000rpm离心30min，得到沉淀和上清液，将沉淀加入100mL去离子水，继续加热2小时，混合两次上清液；将上清液置于真空抽率装置进行抽滤，过0.45μm滤膜，将收集得到的液体于50℃真空浓缩至原体积的1/10；
(2)去除杂质：将步骤(1)制备得到的浓缩液，加入其体积1/3的Sevage试剂(氯仿:正丁醇＝4:1)混合震荡后静置30min，8000rpm离心10min，取得水层去除交界处变性蛋白质；重复3次，直到离心后试剂层交界处没有白色沉淀出现；然后将水层溶液过D280大孔树脂交换柱，脱色1小时，经过紫外分光光度计检测，叶绿素基本除去，收集水层溶液；
(3)乙醇诱导：将步骤(2)得到的水层溶液，加入乙醇至终浓度60％(v/v)进行诱导，将诱导后的混合溶液于4℃静置12小时，8000rpm离心10min，收集得到SPH，如图2所示。
2、SPH的单糖组成分析
采用气质联用法，测定SPH的单糖组成；准确称取10mg螺旋藻多糖胶体冻干粉，加入1mL 4mol·L-1的三氟乙酸(TFA)，110℃水解3h，放入真空干燥箱中70℃减压干燥，将干燥后的样品进行硅烷化处理，振荡待其充分溶解后，于70℃恒温干燥箱中反应30min，随后使其温度逐渐降低，上样检测；
色谱条件：色谱柱为DB-17(30m×0.32mm×0.25μm)，进样口温度280℃，FID检测器温度300℃，载气为氮气，柱压73.0kPa，柱流量1.00mL·min-1，分流比10:1，程序升温从50℃保持3min，以10℃·min-1升至280℃，保留5min，进样量为1μL；结果如图3所示；
从图3可见，经气质联用测定分析，SPH多糖的主要由半乳糖Gal(55.37％)及葡萄糖醛酸GlcUA(19.63％)组成，含有少量的核糖Rib(6.12％)、岩藻糖Fuc(4.06％)、木糖Xly(4.63)及葡萄糖Glc(6.56％)，还含有少量的阿拉伯糖Ara(1.37％)、塔罗糖Gal(0.91％)、鼠李糖Rha(0.67％)及甘露糖Man(0.68％)。
3、SPH的溶胀性分析
将SPH冻干粉放入模具中，并用紫外光照射30s，从模具中取出样品，在37℃的PBS缓冲液中孵育2小时，使其达到平衡状态，并称取膨胀后的SPH的质量记为Ms；随后将其进行冻干，其重量记为MI，其中透明质酸及海藻酸钠作为对照组，试验平行重复三次，如图4所示；
溶胀率＝(Ms-MI)/MI；
结合图4分析可见，SPH的溶胀性显著高于海藻酸钠(p<0.05)且与透明质酸并无显著性差异，其中，SPH溶胀性为50.10％。
4、SPH的保水性分析
将上述在PBS缓冲液中达到溶胀平衡的样品取出称重记为Mp，然后将其暴露于室温条件下72h，期间每隔一定时间取出称量记为Mk；采用公式计算保水率，其中透明质酸及海藻酸钠作为对照组，试验平行重复三次，如图5所示；
保水率＝(Mk-MI)/(Mp-MI)×100％
结合图5分析可见，三种多糖的保水性均随着时间延长而呈现下降的趋势，其中SPH的保水性与透明质酸无显著性差异，透明质分子能携带500倍以上的水分子，是当今公认的最佳保湿成分，由此可见，SPH的保水性能比较突出，可应用于医用敷料和美容材料等生物医药领域。
5、SPH的流变学特性分析
利用DHR-1流变仪(TA.美国)进行流变学特性测定；流变仪基板选择平行板转子(直径40mm)，进行静态流变学试验，试验前溶液需静止2min；温度控制在25±0.1℃下，剪切速率从0.01-100s-1，其中样品浓度均为20mg/mL，观察剪切速率对粘度的影响，且试验平行重复三次；进行动态粘弹性行为，将样品进行频率扫描，利用弹性模量G’和粘性模量G”作为频率的函数来观察SPH的粘弹性变化，如图6所示。
结合图6分析可见，不同样品都表现出典型的非牛顿流体行为和明显的剪切稀化现象，可能是由于大分子沿流线的方向打开的纠结缠绕结构不能及时更好的恢复。随着剪切速率的升高，表观粘度降低，对于SPH，当剪切速率从0.01s-1增加到100s-1时，粘度从2.26pa.s降低到0.14pa.s。相同浓度下的透明质酸的表观粘度最高，其次是SPH，海藻酸钠最低。
从动态粘弹性分析可以看出，低频率下，G’<G”，SPH和透明质酸的多糖分子进行充分的纠缠后断裂，之后溶液表现出稀化现象，此时，溶液体系主要以弹性为主。随着震荡频率的升高，形成凝胶的接枝区域，此时，G’>G”，溶液主要以粘性为主，说明由于多糖分子间链接形成的网络结构影响了水分子的迁移率，分子间形成紧密的分子交联体。当频率在50Hz前后，SPH和透明质酸的多糖的tanδ接近于1，说明螺旋藻多糖胶体具有凝胶性行为，可以形成稳定的凝胶状态，可应用于水凝胶的开发与应用。
6、螺旋藻多糖胶体的体外抗氧化活性分析
(1)DPPH自由基清除能力的测定：取SPH、透明质酸以及海藻酸钠配为样品溶液(0.0、0.2、0.4、0.6、0.8、1.0mg/mL)，将50μL样品与150μL 0.2mmol/L DPPH溶液混合，设置为Ai组；将50μL样品与150μL无水乙醇溶液混合，设置为A0组；将0.2mmol/L DPPH与H2O混合，设置为Aj组。静置30min，测定吸光度(517nm)。以VC为阳性对照，试验重复三次。
DPPH自由基清除率(％)＝[1-(Ai-Aj)/A0]×100
(2)ABTS+自由基清除能力的测定：储备液：取40mg ABTS+与6.88mg过硫酸钾，溶于10mL蒸馏水，黑暗条件下保存12～16h(常温)。工作液：向以上溶液中加入适量磷酸缓冲液(pH 7.4)直至其在734nm下的吸光度为0.70±0.02。取SPH、透明质酸以及海藻酸钠配为样品溶液(0.0、0.2、0.4、0.6、0.8、1.0mg/mL)，将180μL工作液与20μL样品混合，设置为As组；将180μL工作液与20μL H2O混合，设置为Ab组。静置5min，测吸光度(734nm)，以VC为阳性对照，试验重复三次。
ABTS+自由基清除率(％)＝[1-(Ab-As)/Ab]×100
(3)羟自由基清除能力的测定：取SPH、透明质酸以及海藻酸钠配为样品溶液(0.0、0.2、0.4、0.6、0.8、1.0mg/mL)，取300μL 3mmol/L FeSO4溶液与300μL 6mmol/L水杨酸-乙醇溶液混合，加入100μL样品，最后加入300μL 9mmol/L H2O2溶液，为Ai组；以蒸馏水代替H2O2作为Aj组；以蒸馏水代替样品作为A0组。水浴30min(37℃)，测吸光度(510nm)。以VC为阳性对照，试验重复三次。
羟自由基清除率(％)＝[1-(Ai-Aj)/A0]×100
由表1分析可知，通过SPH对DPPH、ABTS+以及羟自由基清除率的IC50值发现，与透明质酸及海藻酸钠相比，SPH具有良好的抗氧化性作用及自由基清除率能力。自由基会引起脂质过氧化，导致人体衰老，造成组织损伤等。SPH可以通过清除自由基，防止细胞损伤，从而延缓衰老，可应用于医用敷料及医美材料等。
表1.体外抗氧化活性分析
以上所述仅为本发明的优选实施例而已，并不用于限制本发明。
对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (3)
1.一种螺旋藻多糖胶体，其特征在于，从螺旋藻中提取得到，其主要成分为55.37%半乳糖及19.63%葡萄糖醛酸，并含有6.12%核糖、4.06%岩藻糖、4.63%木糖及6.56%葡萄糖，还含有少量的1.37%阿拉伯糖、0.91%塔罗糖、0.67%鼠李糖及0.68%甘露糖；提取的具体步骤为：
（1）初步提取：称取干燥的螺旋藻冻干粉2~5 g，将其研磨，加入100 mL去离子水，于60~90 ℃水浴锅中，加热4~5小时后，于8000~10000 rpm离心20~40 min，得到沉淀和上清液，将上清液置于真空抽率装置进行抽滤，过0.45 μm滤膜，将抽滤后收集得到的液体于25~60 ℃真空浓缩至原体积的1/10，得到浓缩液；
（2）去除杂质：将制备得到的浓缩液，加入其体积1/3的Sevage试剂混合震荡后静置10~30 min，6000~8000 rpm离心5~10 min，重复2~3次，取得水层去除交界处变性蛋白质；然后将水层过大孔树脂交换柱，收集水层溶液；
（3）乙醇诱导：将水层溶液加入乙醇至终浓度60~90% （v/v）进行诱导，将诱导后的混合溶液于0~4 ℃静置12~16小时，6000~8000 rpm离心5~10 min，收集得到螺旋藻多糖胶体SPH。
2.一种如权利要求1所述的螺旋藻多糖胶体的制备方法，其特征在于，具体步骤为：
（1）初步提取：称取干燥的螺旋藻冻干粉2~5 g，将其研磨，加入100 mL去离子水，于60~90 ℃水浴锅中，加热4~5小时后，于8000~10000 rpm离心20~40 min，得到沉淀和上清液，将上清液置于真空抽率装置进行抽滤，过0.45 μm滤膜，将抽滤后收集得到的液体于25~60 ℃真空浓缩至原体积的1/10，得到浓缩液；
（2）去除杂质：将制备得到的浓缩液，加入其体积1/3的Sevage试剂混合震荡后静置10~30 min，6000~8000 rpm离心5~10 min，重复2~3次，取得水层去除交界处变性蛋白质；然后将水层过大孔树脂交换柱，收集水层溶液；
（3）乙醇诱导：将水层溶液加入乙醇至终浓度60~90% （v/v）进行诱导，将诱导后的混合溶液于0~4 ℃静置12~16小时，6000~8000 rpm离心5~10 min，收集得到螺旋藻多糖胶体SPH。
3.如权利要求1所述螺旋藻多糖胶体在制备医用敷料或者医美产品中的用途。
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