CN111410701A - 一种青稞β-葡聚糖及其纯化工艺 - Google Patents
一种青稞β-葡聚糖及其纯化工艺 Download PDFInfo
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Abstract
本发明公开了一种青稞β‑葡聚糖及其纯化工艺，其中采用为大孔树脂脱色，琼脂糖凝胶柱色谱分子筛纯化，干燥等步骤。本发明从青稞中分离出一种新的青稞β‑葡聚糖，采用高效凝胶色谱‑示差‑激光散射检测(HPSEC‑dRI‑LS)和核磁共振碳谱(13C‑NMR)为主的多种方法对该种β‑葡聚糖进行结构鉴定，并通过酶抑制实验，验证了通过该法纯化所得具有明确结构和序列的青稞β‑葡聚糖的降血糖活性，为开发血糖调节的功能食品打下基础。
Description
技术领域
本发明属于生物医药领域，具体涉及一种青稞β-葡聚糖及其纯化工艺。
背景技术
青稞，大麦属，是青藏高原地区人民的主食之一，也是家畜饲料和工业原料作物。据检测，青稞中β-葡聚糖的含量较高，达到4％-8％。该β-葡聚糖是由吡喃型葡萄糖通过β-(1→3)或者β-(1→4)糖苷键连接而成的线型多糖。
近年来，青稞β-葡聚糖在食品科学领域受到很大关注，β-葡聚糖的许多重要的生理学功能和生理活性逐渐被人认知。据报道，青稞β-葡聚糖可以作为固有免疫和获得性免疫的调节剂，具有良好的降血糖、降血脂功能，并在抗氧化、抗肿瘤等方面也显示出良好的应用前景。由于对青稞β-葡聚糖的结构、活性研究较为有限，因而限制了其应用，多数仅将青稞打粉后制作面食、面饼等普通食品。虽然目前已经发现青稞β-葡聚糖提取物具有降血糖活性，但由于其均一分子量多糖纯化困难，并且结构鉴定复杂，所以降血糖作用机理尚未明确，阻碍了它们被开发成防治糖尿病功能食品乃至新药的进程。
发明内容
本发明目的是提供一种均一分子量结构的青稞β-葡聚糖及其纯化工艺，并且检测其血糖相关酶的抑制活性。为解决基于青稞β-葡聚糖纯品的降血糖活性机理打下基础。
一种青稞中具有血糖相关酶抑制活性的新的β-葡聚糖，结构式为：
优选地，所述青稞β-葡聚糖的结构中含有重复单Glc1→3Glc1→4Glc/Glc1→3Glc1→4Glc1→4Glc。
优选地，所述青稞β-葡聚糖的结构中具有β-(1→3)和β-(1→4)两种连接方式的直链葡聚糖。
优选地，所述青稞β-葡聚糖中β-(1→3)与β-(1→4)糖苷键的比值为1:2至1:10。
优选地，所述青稞β-葡聚糖分子量的分布范围为190,000～220,000Da，分散系数(Mw/Mn)为1.415～1.611。
另一方面，本发明还提供了上述青稞β-葡聚糖的纯化工艺，包括如下步骤：
(1)去色素：将青稞提取物利用大孔树脂去除色素，制得青稞β-葡聚糖溶液；
(2)分子筛纯化：将前述去色素的青稞β-葡聚糖溶液利用琼脂糖凝胶柱色谱分子筛纯化，制得青稞β-葡聚糖纯品溶液；
(3)干燥：将所述青稞β-葡聚糖溶液进行干燥，得到青稞β-葡聚糖纯品。
优选地，步骤(1)中大孔树脂的用量为2％-4％，温度为40℃-60℃，pH为2.0-4.0，时间为70-90min。
优选地，步骤(3)中的干燥方式为喷雾、带式、烘箱或冷冻中的任意一种。
本发明还提供了上述β-葡聚糖在制备降糖药物中的应用。
本发明还提供了上述的β-葡聚糖在制备降糖食品中的应用。
利用本发明的纯化工艺得到的青稞β-葡聚糖是分子量明确的均一分子量多糖，使得β-葡聚糖有较高的纯度，纯度达到98％以上，且色泽较好；得率较高，每100g干燥青稞粉中可提取3-10gβ-葡聚糖；该青稞β-葡聚糖结构序列明确，具有良好的血糖相关酶抑制活性，为开发血糖调节的功能食品打下基础。
附图说明
图1为本发明中青稞β-葡聚糖的高效凝胶色谱-示差检测；
图2为本发明中青稞β-葡聚糖的高效凝胶色谱-示差检测-激光散射检测；
图3为本发明中青稞β-葡聚糖的红外吸收光谱；
图4为本发明中青稞β-葡聚糖的核磁共振谱；
图5为青稞β-葡聚糖的血糖相关酶抑制活性示意图。
具体实施方式
本发明提供一种青稞β-葡聚糖的纯化工艺，包括以下步骤：
(1)去色素：将青稞提取物利用大孔树脂去除色素，制得青稞β-葡聚糖溶液；
(2)分子筛纯化：将前述去色素青稞β-葡聚糖溶液利用琼脂糖凝胶柱色谱分子筛纯化，制得青稞β-葡聚糖纯品溶液；
(3)干燥：将所述青稞β-葡聚糖溶液进行干燥，得到青稞β-葡聚糖纯品。
在一个实施例中，该步骤可以具体如下执行：将经过处理的溶液，以喷雾或带式或烘箱或冷冻等方式进行干燥，得到精制β-葡聚糖产品。
上述工艺所制得的青稞β-葡聚糖的结构特征为含有重复单元Glc1→3Glc1→4Glc/Glc1→3Glc1→4Glc1→4Glc，且他们之间存在多个结构序列均一但不同长度的β-(1→4)连接的葡萄糖片段。
根据核磁共振结果，在上述条件下提取纯化的样品为具有β-(1→3)和β-(1→4)两种连接方式的直链葡聚糖，并且β-(1→3)与β-(1→4)糖苷键的比值确定为1:2至1:10。根据HPSEC-dRI-LS分析结果，分子量的分布范围为190,000～220,000Da，分散系数(Mw/Mn)为1.415～1.611。
取上述工艺所得β-葡聚糖进行体外血糖相关酶抑制试验，具体实验条件以及实验结果如下：
步骤一：将精制青稞β-葡聚糖溶于纯净水或15％乙醇溶液中，检测对α-葡萄糖苷酶、α-淀粉酶和蔗糖酶的抑制活性。
在一个实施例中，该步骤可以具体如下执行：α-葡萄糖苷酶反应体系为β-葡聚糖纯净水或15％乙醇溶液，同2倍体积0.5U/mLα-葡萄糖苷酶(PBS,pH6.8)37℃结合5min，同1倍体积2.5mM PNPG(PBS,pH6.8)37℃反应30min，用4倍体积0.2M Na2CO3终止，405nm测OD值；α-淀粉酶反应体系为β-葡聚糖纯净水或15％乙醇溶液，同1倍体积0.8μg/mlα-淀粉酶(Tris-HCl,pH7.0)37℃结合10min，同2倍体积3％可溶性淀粉(Tris-HCl,pH7.0)37℃反应20min，用4倍体积DNS显色液终止，570nm测OD值；蔗糖酶反应体系为β-葡聚糖纯净水或15％乙醇溶液，同1倍体积6μg/ml蔗糖酶(Tris-HCl,pH4.5)37℃结合10min，同1倍体积150mM蔗糖(Tris-HCl,pH4.5)37℃反应15min，用3倍体积DNS显色液终止，570nm测OD值。
经上述检测后，青稞β-葡聚糖对α-葡萄糖苷酶的抑制率为89.5％(5mg/mL的水溶液)、93.1％(5mg/mL的15％乙醇溶液)，对α-淀粉酶的抑制率为13.4％(2.5mg/mL的水溶液)和18.7％(5mg/mL的15％乙醇溶液)，对蔗糖酶的抑制率为43.9％(3.33mg/mL的水溶液)和34.0％(6.66mg/mL的15％乙醇溶液)。由此可见，本发明的青稞β-葡聚糖有较好α-葡萄糖苷酶抑制性、温和的蔗糖酶抑制性、较弱的α-淀粉酶抑制性。
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图和实施例进一步说明本发明的技术方案。但是本发明不限于所列出的实施例，还应包括在本发明所要求的权利范围内其他任何公知的改变。
此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。
实施例
本实施例提供了一种青稞β-葡聚糖的纯化工艺，包括如下步骤：利用AB-8大孔树脂吸附青稞提取物去除色素，树脂用量2％-4％、温度40℃-60℃、pH2.0-4.0、时间70-90min。在6000r/min的转速下离心分离15min，去除溶液中的树脂，收集上清液。上清液适当浓缩，过Sepharose CL-4B琼脂糖凝胶柱，用蒽酮-硫酸法在620nm隔管检测多糖，刚果红法检测是否为β-葡聚糖。收集含纯β-葡聚糖的组分合并。冷冻干燥，得到0.3gβ-葡聚糖产品。
1、青稞β-葡聚糖的结构表征
对实施例1中所得的产品分别进行单糖组成和分子量分析、红外分析、核磁共振分析。
(1)单糖组成和分子量分析
单糖组成分析：
多糖水解：称取10mg精制的青稞多糖样品于安瓿瓶中，加入2mL2 mol/L的三氟乙酸，封管后置于120℃干燥箱中水解，2h后取出冷却，离心后取上清液，真空浓缩至干，得单糖样品置于干燥箱中备用；标准单糖的衍生化：称取葡萄糖、木糖、甘露糖、半乳糖、阿拉伯糖、鼠李糖各约10mg，分别加入10mg盐酸羟胺及1mL无水吡啶，充分振荡溶解，于90℃水浴反应30min，取出，冷却到室温，加入1mL无水醋酸酐，于90℃水浴酰化反应30min，取出冷却后真空浓缩蒸干，残渣加入1mL氯仿溶解，进行气相色谱分析；样品单糖的衍生化：将上述备用的青稞多糖水解产物中，加入10mg盐酸羟胺及1mL无水吡啶，加入内标溶液0.1mL。充分振荡溶解，于90℃水浴反应30min，取出，冷却到室温，加入1mL无水醋酸酐，于90℃水浴酰化反应30min，取出冷却后真空浓缩蒸干，残渣加入1mL氯仿溶解，自动进样器进行样1ul，进行气相色谱分析。气相色谱分析方法：色谱条件：进样口温度250℃，分流比20：1；恒流模式，柱流量1.5mL/min；柱温：初温190℃，保持5min，以8℃/min升到260℃；通过HPSEC-dRI-LS的方式对实施例1中纯化所得青稞β-葡聚糖进行单糖组成分析。在分析之前，采用2M三氟乙酸(TFA)对实施例1中纯化所得样品进行降解，降解12h后，通过旋转蒸发的方式，将具有强腐蚀作用的TFA去除。分别配制阿拉伯糖、半乳糖、葡萄糖以木糖标准品溶液及其混合液，采用HPEAC-PAD对降解样品以及标准品溶液进行分离分析，分析柱为CarboPac PA-1column(4×250mm，Dionex，Sunnyvale，CA)，并选取15mM NaOH溶液为流动相，以1mL/min进行等度洗脱。根据降解后的样品和标准单糖的PAD色谱图对比，样品降解后只有葡萄糖被检测到，葡萄糖色谱峰的纯度超过98％。因此可确定纯化所得β-葡聚糖纯度较高(>98％)，如图1所示。
分子量分析：
采用HPSEC-dRI-LS的方式，对实施例1β-葡聚糖进行分子量分析。方法：色谱柱(Shodex SB-803HQ，Showa Denko K.K.，日本)。通过注射器过滤器(0.45μm孔)过滤样品，并将20μl滤液注入HPSEC色谱柱。用流速为0.6ml/min的0.15M NaNO3洗脱柱，使用激光散射检测器(Wyattawn heleos-II，Wyatt DAWN Technology，美国)(LS)和折射率检测器(OptilabT-rEX，Wyatt DAWN)洗脱美国，技术(dRI)。结果如图2所示：所述青稞β-葡聚糖分子量的分布范围为190,000～220,000Da，分散系数(Mw/Mn)为1.415～1.611。
综上所述，实施例1中纯化所得的青稞β-葡聚糖具有如下结构特征：存在重复单元Glc1→3Glc1→4Glc/Glc1→3Glc1→4Glc1→4Glc，且存在多个β-(1→4)连接的片段。根据核磁共振结果，在上述条件下提取纯化的样品为具有β-(1→3)和β-(1→4)两种连接方式的直链葡聚糖。根据HPSEC-dRI–LS分析结果，组成该β-葡聚糖的片段存在一定重复规律，即糖链存在两种序列连接，它们是分别为[Glc1→4Glc1→4Glc]n和[Glc1→3Glc1→4Glc]n；[Glc1→3Glc1→4Glc]n→4Glc是每一个周期中丰度最高的寡糖；均一的β-(1→4)连接的葡萄糖片段含量较少。此外，纯化所得葡聚糖中β-(1→3)与β-(1→4)糖苷键的比值确定为1:2-1:10。分子量的分布范围为190,000～220,000Da，分散系数(Mw/Mn)为1.415～1.611。
(2)红外分析：
红外光谱分析如图3所示，3419,1645，1120,1139cm-1表示羟基吸收峰；2923，1345cm-1表示次甲基吸收峰；1425cm-1表示亚甲基吸收峰。
(3)核磁共振分析
13C-NMR分析结果所得谱图如图4所示，根据已有知识，异头碳的化学位移大于103ppm则糖苷键类型为β型，可初步确定该糖链为β型。除此之外，根据文献中已有的凝胶多糖的常规13C-NMR峰位归属以及纤维素的固态13C-NMR峰位归属特征，可对青稞β-葡聚糖的谱图进行有效地归属。其中，104.04ppm与103.40ppm处的共振峰分别归属为β-(1→3)-D-Glc的异头碳C-1以及β-(1→4)-D-Glc的异头碳C-1，β-(1→3)-D-Glc的C-3共振峰出现在87.75ppm处，将73.40，71.06，76.73以及61.25ppm的共振峰分别归属为β-(1→3)-D-Glc的C-2，C-4，C-5and C-6，68.74，将72.66，80.77，75.38以及60.71ppm处的共振峰分别归属为C-2，C-3，C-4，C-5以及C-6，如表1。并且根据异头碳碳谱丰度推测上述青稞β-葡聚糖中β-(1→3)与β-(1→4)糖苷键的比值为1:2至1:10。
表1、青稞β-葡聚糖13C-NMR化学位移
2、青稞β-葡聚糖的血糖相关酶抑制活性评价
采用抑制目标酶降解底物的能力，对实施例1β-葡聚糖进行体外血糖相关酶的抑制活性分析。所采用方法如下：β-葡聚糖用纯净水或15％乙醇溶解，①在α-葡萄糖苷酶检测中，加入2倍体积0.5U/mLα-葡萄糖苷酶(PBS,pH6.8)，37℃孵育5min；加入1倍体积2.5mMPNPG(PBS,pH6.8)，37℃孵育30min；加入4倍体积0.2M Na2CO3；405nm测OD值。②在α-淀粉酶检测中，加入1倍体积0.8μg/mlα-淀粉酶(Tris-HCl,pH7.0),37℃孵育10min；加入2倍体积3％可溶性淀粉(Tris-HCl,pH7.0)，37℃孵育20min；加入4倍体积DNS显色液3，570nm测OD值。③加入1倍体积6μg/ml蔗糖酶(Tris-HCl,pH4.5)，37℃孵育10min；加入1倍体积150mM蔗糖(Tris-HCl,pH4.5)，37℃孵育15min；加入3倍体积DNS显色液，570nm测OD值。结果如图5和表2所示：所述青稞β-葡聚糖对三个酶均有抑制作用，有较强α-葡萄糖苷酶抑制性，抑制率为89.5％(5mg/mL的水溶液)、93.1％(5mg/mL的15％乙醇溶液)；有温和的蔗糖酶抑制性，抑制率为43.9％(3.33mg/mL的水溶液)和34.0％(6.66mg/mL的15％乙醇溶液)；有较弱的α-淀粉酶抑制性，抑制率为13.4％(2.5mg/mL的水溶液)和18.7％(5mg/mL的15％乙醇溶液)。
表2、青稞β-葡聚糖血糖相关酶的抑制活性
综上所述，本发明公开了一种青稞β-葡聚糖的纯化工艺，通过去色素、分子筛等关键纯化工艺，使得β-葡聚糖有较高的纯度，纯度达到98％以上，且色泽较好；得率较高，每100g干燥青稞粉中可提取3-10gβ-葡聚糖；结构序列明确；以及具有良好的血糖相关酶抑制活性，为开发血糖调节的功能食品打下基础。
以上所述实施例仅表达了本发明的实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种青稞中具有血糖相关酶抑制活性的β-葡聚糖，其特征在于，结构式为：
2.根据权利要求1所述的β-葡聚糖，其特征在于，所述青稞β-葡聚糖的结构中含有重复单Glc1→3Glc1→4Glc/Glc1→3Glc1→4Glc1→4Glc。
3.根据权利要求1所述的β-葡聚糖，其特征在于，所述青稞β-葡聚糖的结构中具有β-(1→3)和β-(1→4)两种连接方式的直链葡聚糖。
4.根据权利要求1所述的β-葡聚糖，其特征在于，所述青稞β-葡聚糖中β-(1→3)与β-(1→4)糖苷键的比值为1:2至1:10。
5.根据权利要求1所述的β-葡聚糖，其特征在于，所述青稞β-葡聚糖分子量的分布范围为190,000～220,000Da，分散系数(Mw/Mn)为1.415～1.611。
6.权利要求1-5中任一项所述的β-葡聚糖的纯化工艺，其特征在于，包括如下步骤：
(1)去色素：将青稞提取物利用大孔树脂去除色素，制得青稞β-葡聚糖溶液。
(2)分子筛纯化：将前述去色素的青稞β-葡聚糖溶液利用琼脂糖凝胶柱色谱分子筛纯化，制得青稞β-葡聚糖纯品溶液。
(3)干燥：将所述青稞β-葡聚糖溶液进行干燥，得到青稞β-葡聚糖纯品。
7.根据权利要求6所述的纯化工艺，其特征在于，步骤(1)中大孔树脂的用量为2％-4％，温度为40℃-60℃，pH为2.0-4.0，时间为70-90min，离心分离。
8.根据权利要求6所述的纯化工艺，其特征在于，步骤(3)中的干燥方式为喷雾、带式、烘箱或冷冻中的任意一种。
9.权利要求1-5中任一项所述的β-葡聚糖在制备降糖药物中的应用。
10.权利要求1-5中任一项所述的β-葡聚糖在制备降糖食品中的应用。
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| RONG, YINXIU ET AL: "Sequencing analysis of beta-glucan from highland barley with high performance anion exchange chromatography coupled to quadrupole time - Of - Flight mass spectrometry", 《FOOD HYDROCOLLOIDS》 * |
| 谢昊宇等: "青稞β-葡聚糖的分离纯化及理化特性研究", 《食品科技》 * |
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