CN101711112B - 粒状可溶的葡聚糖制备 - Google Patents

粒状可溶的葡聚糖制备 Download PDF

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CN101711112B
CN101711112B CN200880014957.4A CN200880014957A CN101711112B CN 101711112 B CN101711112 B CN 101711112B CN 200880014957 A CN200880014957 A CN 200880014957A CN 101711112 B CN101711112 B CN 101711112B
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D·J·考克斯
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Abstract

粒状β-葡聚糖在提高的压力和温度下溶解以形成粒状可溶的β-葡聚糖。粒状可溶的β-葡聚糖能够被干燥成粉末形式且随后再溶解。

Description

粒状可溶的葡聚糖制备
背景技术
本申请要求2007年5月8日申请的，发明名称为“可溶葡聚糖制备”的U.S.临时申请No.60/916,690的权益。
本发明涉及一种粒状可溶的β-葡聚糖。更具体而言，本发明涉及一种改良方法和粒状可溶的β-葡聚糖的组合物。
葡聚糖通常描述为葡萄糖的聚合物且来源于酵母、细菌、真菌和植物例如燕麦和大麦。已知含β(1-3)-连接的吡喃葡萄糖主链的葡聚糖具有生物活性，特别是，它们表现出调控免疫系统，并且近来表现出诱导造血干细胞和祖细胞(HSPC)活动。
之前，生产高质量的可溶β-葡聚糖很昂贵。例如，昂贵的成本抑制其作为营养增补剂或食品成分。因此，需要一种改良的可溶β-葡聚糖。
发明概述
粒状β-葡聚糖在升高的温度和压力下溶解在酸性溶液中。采用例如离心和/或色谱技术可将制得的可溶β-葡聚糖进一步澄清和纯化。制得的产品甚至在还含有高分子量葡聚糖时也可干燥成粉末且随后再溶解。
附图说明
图1-4是粒状可溶的β-葡聚糖样品的RI踪迹和数据转化图。
图5是各种β-葡聚糖诱导TNFa的生产的示意图。
发明内容
本发明所用的起始材料是由任何方法制备的粒状，或不可溶的β-葡聚糖。粒状β-葡聚糖起始材料的大小范围可从整个(whole)葡聚糖颗粒小至亚微米大小的颗粒。起始材料中的杂质水平也可变化。但是，随着起始材料中杂质水平的增加，回收的可溶葡聚糖数量会降低。适宜作为起始材料的一个例子是来自Biothera的WGP3-
许多来源的任意b-葡聚糖都可用于本发明。这些来源的例子为酵母、大麦、燕麦、蘑菇和其他真菌等。
在压力和提高温度下以酸处理粒状β-葡聚糖以产生可溶β-葡聚糖。开始时，将丸化的、粒状β-葡聚糖在缓冲液中再悬浮并混合于可密封反应容器中且调至pH3.6。加入缓冲试剂以使每升总体积的最终悬浮混合物含有约0.61g乙酸钠，5.24ml冰醋酸和430g丸化的，粒状β-葡聚糖。容器经氮气吹扫以去除氧气并增加反应容器内的压力。
通常地，将容器内部压力调至35PSI，将悬浮液加热至约135℃至多约5.5小时。发现在这样的条件下β-葡聚糖将溶解。随着温度从135℃下降，溶解量也下降。
应该注意的是这个温度和压力仅对上述实施方式是最优的。如果反应条件和/或试剂有任何改变则可能需要优化和调整温度和压力。
增加的压力和温度，通过实质上在工艺中消除使用有害化学物质，使其相对于现有技术中溶解β-葡聚糖的工艺具有优势。之前所用的有害化学物质包括例如可燃的VOC例如醚和乙醇，极强酸例如甲酸和硫酸和极高pH的苛性碱溶液。本工艺不仅更安全，而且通过减少所用的不同化学物质的量和所涉及的步骤数量，更为经济。
本工艺的另一个优势是上述处理的长度控制制得的粒状可溶的β-葡聚糖分子量。如全文所用的术语，“粒状可溶的β-葡聚糖”可以是任何可溶的，粒状状或再溶解形式。处理时间长度与制得的分子量大小负相关。换而言之，随着处理时间的增长，制得的粒状可溶的β-葡聚糖的平均分子量降低。该原理如下文所示。
在上述工艺期间的多个时间点(60，90，120和240分钟)，从一批粒状可溶的β-葡聚糖悬浮液中收集四个样品。通过尺寸排阻色谱对比右旋糖苷分子量标记测定样品的分子量。结果如表1所汇总。
表1
取样时间 Mn* Mw** Mz*** 己糖浓度
60分钟 5,500 352,000 4,250,000 5.16mg/mL
90分钟 8,500 1,180,000 5,430,000 12.33mg/mL
120分钟 6,600 354,000 2,730,000 11.26mg/mL
240分钟 5,100 56,500 362,000 ≈25mg/mL
*由 M ‾ n = Σ i N i M i Σ i N i 计算的数均分子量
**由 M ‾ w = Σ i N i M i 2 Σ i N i M i 计算的重均分子量
其中Ni是分子量Mi的分子数量
***z-平均分子量，其是基于聚合物链的体积。
由上述数据可证明，在约90分钟时最大平均尺寸的粒状β-葡聚糖慢慢开始发生降解。90分钟的时间点之后，平均尺寸减小。时间点的特定数据如图1-4所示。
图1包括了组A和B。组A是60分钟样品的折射率(RI)踪迹描述。组B中，将组A中产生的数据部分转化并点绘在所示图表中。曲线10表示转化的原始数据。曲线12表示特定分子量的累计百分率。通过由已知大小的右旋糖苷生成的标准曲线测定分子量(数据未显示)。
90分钟样品生成的数据如图2所示。具有曲线14和16的组A和组B，对应图1的组A和组B。
120分钟样品生成的数据如图3所示。具有曲线18和20的组A和组B对应图1的组A和组B。
240分钟样品生成的数据如图4所示。具有曲线22和24的组A和组B对应图1的组A和组B。
特定大小的粒状可溶的葡聚糖可更适用于特定用途。例如，当局部应用时，较小分子量的分子可更好地渗透皮肤。另一方面，更大分子量分子可更好地适宜口服施用。因此，易于控制葡聚糖分子平均大小的能力提供了明显有竞争力的优势。
通过采样反应器内容物而实验性地测定热处理精确时间。可实施各种试验例如凝胶渗透色谱(GPC)分析法。该工艺可使符合特定规格特性和杂质水平的粒状可溶的材料的收率最大化。一旦β-葡聚糖被溶解，则冷却混合物以终止反应。
可在该点清洗和利用粒状可溶的β-葡聚糖，但是，对于药物用途则通常要进行进一步纯化。可采用以下步骤中的一个或多个的任意组合以进一步纯化和/或澄清粒状可溶的β-葡聚糖。如有需要也可采用本领域已知的其他手段。适宜的手段包括例如离心或连续流离心，其消除了大量的脂质杂质。
在一些情况中，粒状可溶的β-葡聚糖内部的脂质包含体可增强其对某些传染病的效力或增强免疫系统的效力。此处所述的工艺提供一种有效控制和改变粒状可溶的β-葡聚糖组合物中脂质数量的方式。
可选地或额外地，可过滤粒状可溶的β-葡聚糖。在一个实施方式中，以0.2μm的过滤器过滤材料。
也可采用色谱进行纯化。粒状可溶的β-葡聚糖可在制备中的某些点针对色谱进行调节。例如，若色谱步骤包括疏水作用色谱(HIC)，可以硫酸铵和醋酸钠溶液将粒状可溶的β-葡聚糖调节为适宜的传导率和pH。适宜溶液是3.0M硫酸铵，0.1M醋酸钠，其被用于将pH调整至5.5。
可利用的其他色谱步骤是凝胶渗透色谱(GPC)。可能需要多重色谱循环以确保负载不超出柱容量。
在该点上，将粒状可溶的β-葡聚糖纯化备用。通常收集的粒状可溶的β-葡聚糖具有＞1,000,000Da的平均分子量和高多分散性。这种粒状可溶的β-葡聚糖产品特别是高分子量范围如超过1000000道尔顿(Da)的优势在于产品可被喷雾干燥成粉末形式然后在pH约5和更高尤其是弱酸至强碱的溶液中随后再溶解。此外，再溶解发生在低至约室温的温度下(～25℃)。依据再溶解之后的最终浓度和所用溶剂，再溶解的时间和温度要求会变化。
产品再溶解的能力使其可理想地生成适宜于例如速溶饮料粉末的速食形式的β-葡聚糖。简言之，干燥形式的粒状可溶的β-葡聚糖被再润湿以达到约9-12％的水分含量，其形成团聚体。通过双流体喷嘴将表面活性剂例如卵磷脂添加到团聚体中。依据所用表面活性剂和条件和将最终溶解产品的溶剂而改变表面活性剂的浓度。然后将团聚物通过流化床干燥器的三个阶段。第一阶段加热产品，第二阶段调节产品而第三阶段冷却产品。制得的产品筛分后形成速食产品。
形成速食产品的另一方式是通过添加第二干燥形式可溶成分例如蔗糖或食盐(NaCl)。在一个实施例中，200mg粉末状的，粒状可溶的β-葡聚糖和约0.5-约1.0g蔗糖晶体被混入并在mortor碗中研磨约2-3分钟。该混合物加入500ml室温水并剧烈摇晃。粒状可溶的β-葡聚糖再溶解。以NaCl进行相同实验得到一致的结果。
粒状可溶的β-葡聚糖产品，不论是以其溶解，粉末或再溶解形式，都可用于许多用途。粒状可溶的β-葡聚糖理想地用作人和农业/伴侣/外来动物的营养增补剂，食品成分，化妆品成分和药物产品。
可添加的干燥或润湿形式的粒状可溶的β-葡聚糖的典型食物包括例如谷物和谷物产品，焙烤商品和烘焙混合物，饮料和饮料基料，乳制品类似物例如豆乳，乳和乳产品，植物蛋白产品，加工水果和果汁，软糖，汤和汤混合物，酸奶，瓶装水和饮料，营养棒等。如果需要可添加多种掩蔽剂和其他添加剂以改善风味和质地。
以其液体形式，粒状可溶的β-葡聚糖可用于制备可食膜。通过任何本领域已知的很多手段可干燥液体产品薄层。可在液体产品中添加添加剂以形成多种质地的膜或使它们或多或少的柔韧。膜可用作药物或补充产品的载体。此处同样，可添加多种掩蔽剂以改善膜的风味。
粒状可溶的β-葡聚糖与现有技术的粒状β-葡聚糖相比也更有效。例如，该产品可对传染病或癌症更有效。效力增强可以是在吸收更好，更好的免疫系统增强，给药方案减少等方面。
为此，体外研究显示高达300μg/mL浓度的粒状可溶的β-葡聚糖不会由人类外周血单核细胞(PBMC)诱导TNFα。特别是，在含5％热灭活的FCS的cRPMI培养基中培养人PBMC。以各种β-葡聚糖(所有都不含内毒素)，TLR7/8激动剂，或媒介刺激细胞。使用各种浓度的β-葡聚糖。在5％CO2，37℃下过夜培育后，收集无细胞上清液，并在-20℃储存直至以ELISA进行细胞因子分析。图5中显示了三次重复的结果。如图所证明，整个(whole)葡聚糖颗粒刺激了TNFα的产生，而粒状可溶的产品则不会。因为TNFα是参与炎症反应的主要细胞因子，所以预期粒状可溶的产品不会诱导炎症反应。
尽管参照特定实施方式表现和描述本发明，本领域技术人员会理解可以在形式和细节上进行多种改变而不会使其偏离所附权利要求涵盖的本发明的精神和范围。

Claims (1)

1.组合物，含有能够被干燥成粉末形式且然后在pH5或更高的溶液中被再溶解的粒状可溶的β-葡聚糖，并且该β-葡聚糖具有超过1,000,000Da的平均分子量，其中制备该粒状可溶的β-葡聚糖的方法包括在135℃和35PSI溶解粒状β-葡聚糖至多90分钟。
2.如权利要求1所述的组合物，其中粒状可溶的β-葡聚糖来源于酵母。
3.如权利要求1所述的组合物，其中粒状可溶的β-葡聚糖是速食的。
4.如权利要求3所述的组合物，进一步含有：第二干燥形式的可溶成分。
5.如权利要求1所述的组合物，其中该溶液的温度为室温或更高。
6.组合物，含有能够被干燥成粉末形式且然后在25℃或更高温度的溶液中被再溶解的粒状可溶的β-葡聚糖，并且该β-葡聚糖具有超过1,000,000Da的平均分子量，其中制备该粒状可溶的β-葡聚糖的方法包括在135℃和35PSI溶解粒状β-葡聚糖至多90分钟。
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