CN112979837B - 一种狗头枣阿拉伯半乳聚糖的制备方法和应用 - Google Patents
一种狗头枣阿拉伯半乳聚糖的制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种狗头枣阿拉伯半乳聚糖(SJP‑1b)的制备方法和应用。该方法是先运用乙醇(30%、50%、70%和80%(v/v))分级沉淀将狗头枣粗多糖进行分离纯化,相应得到四个多糖组分,将含有半乳糖醛酸的SJP‑1组分的多糖再次运用乙醇(10%和20%(v/v))分级沉淀得到了得率较高的阿拉伯半乳聚糖SJP‑1b。该阿拉伯半乳聚糖SJP‑1b的相对摩尔比为Ara:Gal=3.06:1,得率更高且为0.54%,具有较高的免疫调节活性。本发明通过两次分级沉淀,得到了更加全面、细分的多糖组分,整体操作步骤简单,耗时短,得率高,得到的阿拉伯半乳聚糖组分纯度高,适用于大规模生产。
Description
技术领域
本发明属于糖生物学技术领域,具体涉及一种狗头枣多糖组分的制备方法和应用。
背景技术
多糖是由十个以上的单糖通过糖苷键结合而成的天然生物大分子,广泛参与细胞的各项生命活动,如细胞识别、生长、分化、机体代谢、胚胎发育、免疫应答等[1]。多糖具有抗肿瘤、抗病毒、免疫调节等多种生物活性,且无毒副作用,成为天然药物及保健品研发的重要组成部分[2-4]。
多糖的活性与结构密切相关[5],多糖结构受分离纯化方法的影响,因此多糖的分离纯化方法至关重要[6]。传统的多糖分离方法主要是通过阴离子交换柱层析结合分子筛柱层析进行分离纯化,即通过水浸提样品,收集并浓缩上清液,加入乙醇,收集沉淀,得到粗多糖;然后将水提醇沉得到的粗多糖经离子交换柱层析及分子筛柱层析对多糖样品进行分离纯化。这种分离纯化方法操作复杂、耗时长、产率低,并会造成部分糖组分丢失。此外,多糖的分离纯化方法还包括乙醇分级沉淀法,即取粗多糖配制成一定浓度的水溶液,缓慢加入乙醇至终浓度为30%,进行第一级醇沉,收集得到30%醇沉的多糖组分;再取上清液,加入继续补加无水乙醇至终浓度50%,进行第二级淀,收集得到50%醇沉的多糖组分;再取上清液,加入无水乙醇……如此依次可继续获得30%、50%、70%醇沉的多糖组分等,并对这些多糖组分进行理化性质测定和活性研究。目前该方法已用于分离纯化枸杞子粗多糖、黄芪多糖等[5,7],其中枸杞子多糖经乙醇分级沉淀纯化得到的阿拉伯半乳聚糖具有强的免疫调节作用。
红枣,又名大枣,枣属,属于鼠李科。红枣主要分布在中国西北地区、黄河流域和东部地区,是传统药食两用植物。研究显示,红枣中含有许多丰富的化学成分,包括多糖、黄酮、三萜酸、氨基酸、多酚和维生素C等,其中多糖是红枣中主要的活性成分之一。据报道红枣多糖具有免疫调节[8]、抗氧化[9]、抗肿瘤[10]、降血糖[11]、保肝[12]、保护胃肠道[13]等生物活性。狗头枣作为一种重要的红枣品种,产于“红枣之乡”一带,是陕西省的一个重大产业,狗头枣含有丰富的多糖类物质而被广泛用作各种食品和保健品开发的原材料,在食品保健、生物医药等方面有很高的应用价值。
目前对于红枣多糖的研究主要集中于木枣、金丝小枣和新疆红枣等品种。红枣多糖的分离纯化主要采用传统的柱层析法,Lin[14]等人用碱提法得到红枣粗多糖,除蛋白脱色后经DEAE-Sepharose色谱柱和Sephacryl S-300凝胶层析法纯化红枣粗多糖,最终冻干得到纯化多糖SAZMP3,该多糖组分的分子量为9.73kDa,主链为1,4-α-D-GalA,支链为1,3-β-D-Galp,1,3,5-Araf,1,2,4-α-L-Rhap,支链末端连接着1-Araf,1-Rhap,1-Galp。Wang[15]等人用超声波辅助提取得到红枣粗多糖,然后经DEAE-Sepharose离子交换色谱分离纯化得到HJP1组分,理化性质分析显示HJP1主要由鼠李糖、半乳糖醛酸、半乳糖和阿拉伯糖组成,分子量为6.76kDa。HJP1的主要结构为(1→5)-Araf,(1→3)-Rhap,(1→4)-Galp。
目前对于狗头枣多糖的研究较少,且未见采用上述乙醇分级沉淀法分离纯化其粗多糖。发明人通过深入研究还认识到,目前对于红枣多糖的提取分离还不够充分,并存在一些技术偏见。
参考文献:
[1]Szymanski C M,Wren B W.Protein glycosylation in bacterial mucosalpathogens.Nature Reviews Microbiology,2005,3(3):225.
[2]Sheeja Varghese,Manu M.Joseph,Aravind S.R.,Unnikrishnan B.S.,T.TSreelekhaThe inhibitory effect of anti-tumor polysaccharide from Punicagranatum on metastasis,International Journal of Biological Macromolecules 103(2017)1000–1010.
[3]Feng-wei Ma,Si-yuan Kong,Hong-sheng Tan,Rong Wu,Bing Xia,Yan Zhou,Hong-xi Xu.Structural characterization and antiviral effect of a novelpolysaccharide PSP-2B from Prunellae Spica,Carbohydrate Polymers 152(2016)699–709.
[4]Chi,A.,Kang,C.Z.,Zhang,Y.,Tang,L.,Guo,H.H.,Li,H.,et al.(2015).Immunomodulatingand antioxidant effects of polysaccharide conjugates fromthe fruits of Ziziphus Jujube on chronic fatigue syndrome rats.CarbohydratePolymers,122,189–196.M.Jin,Q.Huang,K.Zhao,P.Shang,Biological activities andpotentia health benefit effects of polysaccharides isolated from Lyciumbarbarum L,Int.J.Biol.Macromol.54(2013)16–23.
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发明内容
本发明提供了一种狗头枣阿拉伯半乳聚糖(SJP-1b)的制备方法和应用。
本发明的构思和创新之处主要在于:
乙醇分级沉淀的原理是多糖在不同浓度的乙醇中溶解度不同。通常认为,低浓度的乙醇中醇沉得到的多糖得率也就更少,所以很少有人研究尝试将第一次醇沉得到的多糖(尤其是低浓度醇沉产物)进行二次醇沉,而且二次醇沉之后得到的多糖的糖组成也不明确。
发明人克服了本领域技术人员存在的技术偏见,将第一次乙醇分级沉淀的30%醇沉多糖样品重新用水溶解,然后用乙醇再次分级沉淀,发现可以将半乳糖醛酸组分分离,并且得到了一种得率高、免疫活性较强的阿拉伯半乳聚糖组分。典型的实例是:
首先,对狗头枣粗多糖进行一次乙醇分级沉淀,得到对应于不同浓度醇沉(30%、50%、70%、80%)的多糖组分,分别记为30%组分(SJP-1)、50%组分(SJP-2)、70%组分(SJP-3)、80%组分(SJP-4);理化性质分析表明,30%多糖组分(SJP-1)得率最高,主要由半乳糖醛酸组成;
然后,对30%多糖组分(SJP-1)进行二次乙醇分级沉淀,得到对应于不同浓度醇沉(10%、20%)的多糖组分,分别记为SJP-1a、SJP-1b,最后的上清液记为SJP-1c。理化性质分析及实验表明,SJP-1b的得率高于SJP-1a和SJP-1c,且免疫调节作用强于SJP-1、SJP-1a和SJP-1c,与SJP-4和LBP-I-3(枸杞子阿拉伯半乳聚糖)大致相当。
基于此,本发明提出以下技术方案:
一种红枣阿拉伯半乳聚糖(SJP-1b)的制备方法,包括以下步骤:
对红枣粗多糖进行一次乙醇分级沉淀,得到对应于不同浓度醇沉的多糖组分;
取含有半乳糖醛酸的多糖组分(SJP-1)再次运用乙醇分级沉淀,其中对应于浓度20%醇沉的沉淀物即阿拉伯半乳聚糖(SJP-1b)。
可选地,所述一次乙醇分级沉淀的初始醇沉浓度为30%,其醇沉产物即所述含有半乳糖醛酸的多糖组分(SJP-1)。
进一步地,步骤中的参数优化如下:
步骤1)红枣粗多糖一次分级醇沉
配制1.5%的粗多糖水溶液,缓慢加入30%的无水乙醇在4℃下沉淀12h,10000r/10min离心,收集沉淀溶于双蒸水,冷冻干燥后得30%组分多糖;
步骤2)30%组分多糖二次分级醇沉
称取30%组分粗多糖约1g,加入50mL的双蒸水至完全溶解,缓慢加入10%的无水乙醇4℃沉淀12h,10000r/10min离心,收集沉淀溶解并冷冻干燥,得到10%多糖组分。同理收集上清补加20%无水乙醇沉淀12h,收集沉淀冻干,得到20%多糖组分,即阿拉伯半乳聚糖组分。
对于上述红枣粗多糖,可以采用现有的制备方法,例如水提醇沉法制备红枣粗多糖。
本发明也对水提醇沉法制备红枣粗多糖进行了优化,具体如下:
(i)将红枣(优选狗头枣)在60℃下烘干之后粉碎,称取100g枣干粉,料液比为1:10[v/v],水浴锅80℃-92℃提取,10000r/10min离心并收集残渣和上清,将残渣重复上述步骤进行复提一次;
(ii)合并两次水提上清液浓缩至150mL,将上清液:乙醇=1:4[v/v]于4℃醇沉12h,10000r/10min离心,并收集多糖沉淀复溶于双蒸水中;
(iii)用sevage法除蛋白,玻璃纸透析三天,冷冻干燥4-6天后获得狗头枣粗多糖。
除蛋白过程中应保证降蛋白除干净为止,所以透析时间至少三天,且透析过程中,透析袋装多糖溶液不能太满,留足够空间便于透析完全。
进一步地,步骤(i)中,提取温度为90℃,提取时间至少为2h,以保证糖醛酸组分被充分提取出来。
进一步地,步骤(ii)中所使用的乙醇的浓度为95%以上,醇沉时间为12h,醇沉温度为4℃。该环节所使用的乙醇的浓度影响沉淀的多糖的单糖组成,乙醇浓度应确保95%以上。
本发明还明确了:一种阿拉伯半乳聚糖(SJP-1b)可用于制备辅助增强免疫功能的产品;所述阿拉伯半乳聚糖(SJP-1b)来源于狗头枣,阿拉伯半乳聚糖(SJP-1b)的单糖组成主要为62.2%的Ara、20.3%的Gal、5.9%的Glc、4.0%的Xyl。
上述辅助增强免疫功能的产品例如食品、药物等。
与现有技术相比,本发明具有以下优点:
(1)本发明针对红枣粗多糖,通过两次分级沉淀,得到了更加全面、细分的多糖组分,避免了经传统柱层析法分离纯化后造成的多糖组分损失,样品浪费等。
(2)本发明中两次分级沉淀都使用的是无水乙醇,该试剂来源丰富、价格低廉,整体操作步骤简单,耗时短,得率高,得到的阿拉伯半乳聚糖组分纯度高,适用于大规模生产。
(3)本发明得到的阿拉伯半乳聚糖可以增强巨噬细胞的免疫活性,可用于制备辅助增强免疫功能的食品、药品等。
(4)本发明能够使多糖组分得到最大化的利用,对红枣多糖(尤其是狗头枣多糖)的高值化开发具有深远的意义。
附图说明
图1为SJP-1及其二次醇沉产物的气相色谱图。
图2为SJP-1及其二次醇沉产物的分子量图谱
图3为SJP-1、SJP-1a、SJP-1b和SJP-1c的免疫活性比较的统计图;其中:(A)细胞活力;(B)细胞酸性磷酸酶活力;(C)细胞吞噬能力;(D)细胞NO分泌量;实验结果表示为平均值±标准差(n=5);与空白组相比,*p<0.05,**p<0.01***p<0.001。
图4为SJP-4、SJP-1b和LBP-I-3免疫活性比较的统计图;其中:(A)细胞活力;(B)细胞酸性磷酸酶活力;(C)细胞吞噬能力;(D)细胞NO分泌量;实验结果表示为平均值±标准差(n=5);与空白组相比,*p<0.05,**p<0.01***p<0.01。
具体实施方式
以下结合附图和实施例进一步详述本发明。
本实施例介绍狗头枣多糖(组分)的制备方法,具体包括以下步骤:
1)狗头枣粗多糖的制备:
将狗头枣剪碎后放置在60℃烘箱中干燥48h,粉碎机将其碎成狗头枣干粉,称取100g枣干粉进行预实验,料液比蒸馏水:红枣=10:1,在90℃条件下提取2h并重复提取两次,收集两次上清液浓缩至150mL后,以浓缩液:乙醇=1:4的比例在4℃醇沉12h,10000rpm离心10min,收集沉淀之后savage法除蛋白,玻璃纸透析三天,冻干得狗头枣粗多糖。
2)对狗头枣粗多糖进行一次分级醇沉:
将狗头枣粗多糖经乙醇(30%、50%、70%和80%(v/v))分级沉淀得到四个多糖组分,具体步骤如下:配制1.5%的粗多糖(SJP)水溶液。缓慢加入30%的无水乙醇在4℃下沉淀12h,10000rpm/10min离心,收集沉淀复溶于双蒸水,冷冻干燥后得30%组分(SJP-1),收集上清均匀搅拌并补加无水乙醇至浓度为50%,4℃沉淀12h,10000rpm/10min离心后收集沉淀并重复上述步骤,得到50%组分(SJP-2);同理继续收集上清补加无水乙醇至浓度为70%,在4℃沉淀12h,得到70%组分(SJP-3);收集最后的上清补加无水乙醇至80%,在4℃沉淀12h,得到80%组分(SJP-4);理化性质分析,30%多糖组分(SJP-1)得率最高,主要由半乳糖醛酸组成,但30%多糖组分的分子量较大溶解性较差;
此前的研究只是将枸杞子粗多糖、黄芪粗多糖等经该一次乙醇分级沉淀后得到的多糖直接用于理化性质和活性研究,且本领域普遍认为低浓度的乙醇中醇沉得到的多糖得率也就更少,二次沉淀没有意义(得不到高产率且纯度较高的多糖),二次醇沉之后得到的多糖的糖组成也不明确。所以,此前还是多采用柱层析法进行第二次的分离纯化,但是这种方法会造成部分糖组分的丢失,且得率较低。现有技术未见对红枣粗多糖进行二次乙醇分级沉淀并得到阿拉伯半乳聚糖组分。
3)对SJP-1进行二次分级醇沉
将SJP-1经乙醇(10%、20%(v/v))分级沉淀之后得到三个多糖组分,具体步骤如下:称取SJP-1约1g,加入50mL的双蒸水至完全溶解,缓慢加入10%的无水乙醇4℃沉淀12h,10000r/10min离心,分离并收集沉淀;
将以上经过10%醇沉后收集的沉淀溶解于双蒸水中,收集上清(旨在去除水不溶性的部分,即沉淀复溶水后形成的新的沉淀,可能是不溶于水的糖、蛋白和/或杂质),冷冻干燥,得到10%多糖组分(SJP-1a);
将以上经过10%醇沉后收集的上清补加20%无水乙醇沉淀12h,分别收集沉淀和上清,其中,沉淀冷冻干燥得到20%多糖组分(SJP-1b),也就是阿拉伯半乳聚糖组分,上清冷冻干燥得到的组分记为SJP-1c。
本实施例先采用乙醇(30%、50%、70%和80%(v/v))分级沉淀将狗头枣粗多糖进行分离纯化,得到了SJP-1,SJP-2,SJP-3和SJP-4四个多糖组分;然后将含有半乳糖醛酸的SJP-1组分的多糖再次运用乙醇(10%和20%(v/v))分级沉淀得到了得率较高的阿拉伯半乳聚糖SJP-1b。该阿拉伯半乳聚糖SJP-1b的相对摩尔比为Ara:Gal=3.06:1,得率更高且为0.54%,具有免疫调节活性。
具体实验结果如下:
1、得率:SJP-1a组分得率为0.33%,SJP-1b组分得率为0.54%,SJP-1c组分得率为0.19%。
2、单糖组成
参见图1和表1,可以看出,基于SJP-1进行二次醇沉得到的产物中:
SJP-1a组分主要由Ara(17.4%)和GalA(66.4%)组成,且半乳糖醛酸所占比较大;
SJP-1b组分为阿拉伯半乳聚糖组分,主要由Ara(62.2%)和Gal(20.3%)组成;
SJP-1c组分的单糖分布较为杂乱,主要由Ara(14.9%)、Man(10.9%)、Glc(35.0%)和GalA(27.0%)组成。
表1 SJP-1a~SJP-1c的单糖组成摩尔百分比
3、分子量
SJP-1组分分级沉淀的分子量如图2所示,首先SJP-1、SJP-1a和SJP-1b的分子量分布范围都较宽,SJP-1组分的分子量分布在9.2×104Da,SJP-1a组分的分子量分布在1.1×105Da,SJP-1b的分子量分布在1.2×105Da,分级沉淀得到的SJP-1a和SJP-1b相比于SJP-1组分的平均分子量较大,而SJP-1c组分可以明显的看到具有两个主峰,且分布都较宽,分别分布在9.2×104Da和0.86×104Da。
另外,经实验,基于以上二次分级醇沉的方法,对其中的粗多糖水溶液浓度、醇沉具体参数以及粗多糖本身的制备步骤参数等略作调整,最终也都可以获得上述产物,其得率、单糖组成以及分子量近似。
4、SJP-1、SJP-1a、SJP-1b和SJP-1c免疫活性比较
SJP-1、SJP-1a、SJP-1b和SJP-1c的免疫活性比较如图3所示。其中:
(A)表示SJP-1、SJP-1a、SJP-1b和SJP-1c多糖样品对RAW264.7细胞的细胞活力的影响,与空白组相比,当浓度小于等于100μg/mL时,SJP-1、SJP-1a、SJP-1b和SJP-1c多糖样品组的细胞活力呈浓度依赖性,四组多糖样品均对RAW264.7细胞活力无显著性影响,因此可选择100μg/mL浓度进行后续实验。
(B)表示四组多糖样品对RAW264.7细胞酸性磷酸酶活力的影响,当浓度大于等于25μg/mL时,SJP-1、SJP-1b和SJP-1c能显著增强细胞酸性磷酸酶活力,其中当浓度为50μg/mL时,SJP-1b和SJP-1c处理组的酸性磷酸酶活力最强,分别为1.235和1.206。
(C)表示SJP-1、SJP-1a、SJP-1b和SJP-1c对RAW264.7细胞的吞噬能力的影响,SJP-1a组对RAW264.7细胞的吞噬能力无显著性影响,SJP-1c仅在浓度为50μg/mL时能显著性增强细胞的吞噬能力;当浓度大于等于25μg/mL时,SJP-1和SJP-1b能显著增加细胞的吞噬能力,且呈浓度依赖性,当浓度为100μg/mL时,细胞吞噬指数分别是1.34和1.43。
(D)表示四组多糖样品对RAW264.7细胞NO释放量的影响,当浓度大于等于25μg/mL时,SJP-1和SJP-1b能显著增加细胞的NO释放量,且与浓度呈正相关,当浓度等于100μg/mL时,四个样品均能显著增加细胞的NO释放量,NO释放量分别为18.818mol/L、15.702mol/L、20.753mol/L和17.545mol/L。综上所述,SJP-1b的免疫调节作用强于其它三个多糖组分。
5、SJP-4、SJP-1b和LBP-I-3免疫活性比较
SJP-4、SJP-1b和LBP-I-3的免疫活性比较如图4所示。其中:
(A)表示SJP-4、SJP-1b和LBP-I-3多糖样品对RAW264.7细胞的细胞活力的影响,与空白组相比,SJP-4、SJP-1b和LBP-I-3多糖样品组的细胞活力呈浓度依赖性,多糖浓度越高,细胞活力显著性增大,当多糖样品浓度为800μg/mL时,SJP-4、SJP-1b和LBP-I-3组的细胞活力分别是131.4%、126.7%和120.4%,SJP-4的细胞活力最大,但三组之间无显著性差异,说明都能三组多糖样品增强RAW264.7细胞活力作用相当,然而多糖样品浓度小于等于100μg/mL时,三组多糖样品均对RAW264.7细胞活力无显著性影响,因此可选择100μg/mL浓度进行后续实验。
(B)表示SJP-4、SJP-1b和LBP-I-3多糖样品对RAW264.7细胞酸性磷酸酶活力的影响,与空白组相比,三种多糖样品组的酸性磷酸酶活力呈浓度依赖性,当浓度为6.25μg/mL时,SJP-1b和LBP-I-3组的细胞酸性磷酸酶活力无显著性影响,当浓度为100μg/mL时,SJP-4、SJP-1b和LBP-I-3多糖样品组的细胞酸性磷酸酶活性指数分别是1.11、1.08和1.09,且各组之间无显著性差异。
(C)表示SJP-4、SJP-1b和LBP-I-3多糖样品对RAW264.7细胞的吞噬能力的影响,各组分多糖样品处理的细胞吞噬能力呈浓度依赖性,多糖浓度越高,细胞吞噬能力越强,其中SJP-4组的细胞吞噬能力的增长趋势较为明显,与空白组相比,当浓度为6.25μg/mL,SJP-1b组无显著性差异,当浓度为100μg/mL时,SJP-4、SJP-1b和LBP-I-3多糖样品的细胞吞噬指数分别是1.32、1.14和1.28,各组分间无显著性差异。
(D)表示SJP-4、SJP-1b和LBP-I-3对RAW264.7细胞NO分泌含量的影响,与空白组相比,当多糖样品浓度小于等于12.5μg/mL时,三组多糖样品均对RAW264.7细胞分泌NO含量无显著性影响当浓度为100μg/mL时,SJP-4、SJP-1b和LBP-I-3多糖样品组的细胞NO分泌量分别是25.5mol/L、22.39mol/L和23.38mol/L,三组之间无显著性差异,综上所述,SJP-4、SJP-1b和LBP-I-3多糖样品的免疫活性相当。
6、狗头枣阿拉伯半乳聚糖(SJP-1b)和枸杞子阿拉伯半乳聚糖(LBP-I-3)的得率及单糖组成对比
表2 SJP-1b和LBP-I-3的得率及单糖组成对比
从表2可以看出,狗头枣阿拉伯半乳聚糖(SJP-1b)比枸杞子阿拉伯半乳聚糖(LBP-I-3)的得率高一倍以上。
基于此,可以预期该红枣阿拉伯半乳聚糖(SJP-1b)可用于制备调节免疫功能(辅助增强免疫功能)的食品、药物等,其中药物可以是口服制剂,也可以是注射制剂。
另外,可以预期,按照上述多糖(组分)的制备方法,从木枣、金丝小枣和新疆红枣等其他品种的红枣中有可能也会得到阿拉伯半乳聚糖,只是不确定是否能够达到本实施例中SJP-1b同等的免疫活性。
Claims (6)
1.一种狗头枣阿拉伯半乳聚糖的制备方法,其特征在于,包括以下步骤:
对狗头枣粗多糖进行一次乙醇分级沉淀,一次乙醇分级沉淀的初始醇沉浓度为30%,得到含有半乳糖醛酸的多糖组分(SJP-1);
取含有半乳糖醛酸的多糖组分(SJP-1)再次运用乙醇分级沉淀,其中对应于浓度20%醇沉的沉淀物即阿拉伯半乳聚糖(SJP-1b);具体包括以下步骤:
0)将狗头枣烘干之后粉碎,取狗头枣干粉采用水提醇沉法得到红枣粗多糖,具体包括以下步骤:
(i)将狗头枣60℃烘干之后粉碎成粉,取枣干粉:双蒸水=1:10,温度控制在80℃-92℃之间;10000rpm离心10min并收集残渣和上清;将残渣溶于双蒸水重复以上步骤,复提一次;
(ii)合并两次上清液并浓缩,按照上清液:乙醇=1:4的比例进行醇沉,10000r/10min离心,并收集多糖沉淀溶于双蒸水中;
(iii)用Sevage法除蛋白,玻璃纸透析三天,冷冻干燥4-6天后获得狗头枣粗多糖;
1)取狗头枣粗多糖,配制质量分数为1.5%的粗多糖水溶液;加入30%的无水乙醇进行醇沉,然后离心,收集沉淀溶于双蒸水,冷冻干燥后得30%醇沉组分多糖;
2)称取30%醇沉组分多糖,加入双蒸水至完全溶解形成浓度为2mg/mL的多糖溶液;再加入终浓度10%的无水乙醇进行醇沉,然后离心,去除沉淀,收集上清液;对上清液补加终浓度20%的无水乙醇进行醇沉,然后离心,收集沉淀、冻干,得到阿拉伯半乳聚糖(SJP-1b)。
2.根据权利要求1所述的一种狗头枣阿拉伯半乳聚糖的制备方法,其特征在于,步骤1)和步骤2)中,每次醇沉均是在4℃下沉淀12h,然后10000r/10min离心。
3.根据权利要求2所述的一种狗头枣阿拉伯半乳聚糖的制备方法,其特征在于,步骤(i)中,提取温度为90℃,提取时间至少为2h,以保证糖醛酸组分被充分提取出来。
4.根据权利要求2所述的一种狗头枣阿拉伯半乳聚糖的制备方法,其特征在于,步骤(ii)中所使用的乙醇的浓度为95%以上,醇沉时间为12h,醇沉温度为4℃。
5.一种通过权利要求1-4任一所述制备方法制备的阿拉伯半乳聚糖(SJP-1b)在制备辅助增强免疫功能的产品中的应用;该阿拉伯半乳聚糖(SJP-1b)来源于狗头枣,该阿拉伯半乳聚糖(SJP-1b)的单糖组成主要为62.2%的Ara、20.3%的Gal、5.9%的Glc、4.0%的Xyl。
6.根据权利要求5所述的应用,所述辅助增强免疫功能的产品为食品或药物。
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