CN101184780B - β-葡聚糖和甘露聚糖的制备 - Google Patents

β-葡聚糖和甘露聚糖的制备 Download PDF

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CN101184780B
CN101184780B CN2006800150267A CN200680015026A CN101184780B CN 101184780 B CN101184780 B CN 101184780B CN 2006800150267 A CN2006800150267 A CN 2006800150267A CN 200680015026 A CN200680015026 A CN 200680015026A CN 101184780 B CN101184780 B CN 101184780B
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J·J·塞德马克
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Abstract

本发明公开了制备酵母β-葡聚糖和甘露聚糖制备物的方法。所述方法使用：自溶处理，然后用蛋白酶、葡聚糖酶或脂肪酶中的一种或多种进行酶处理。生产的制备物可用于食品增补剂、药品、化妆品、动物饲料和营养辅助品。

Description

β-葡聚糖和甘露聚糖的制备
交叉引用的相关申请
本申请要求于2005年5月5日递交的美国临时申请号60/677,973的优先权，通过引用将该申请的主题全文并入本文。
背景技术
本发明涉及β-葡聚糖/甘露聚糖制备物及其制备方法。具体而言，本发明涉及由微生物产生的制备物，包括β-(1，3/1，6)葡聚糖及甘露聚糖，所述微生物包括但不限于酵母。
“葡聚糖”是指主要或全部由D-葡萄糖构成的寡糖或多糖的通称。葡聚糖广泛地分布在自然界中，对于维持细菌、酵母及植物细胞的结构完整性尤其重要。举例来说，葡聚糖与其它多糖(例如甘露聚糖和几丁质)，共同决定着细胞壁的外形和机械强度。在这些细胞中葡聚糖通常占细胞壁重量的大约40％到50％。
作为D-葡萄糖的聚合物，D-葡萄糖单元可以通过各种方式连接在一起。例如，具有(1，3)、(1，4)、(1，6)和(1，2)键(糖苷键)的葡聚糖都是已知的。可能的键的多样化意味着葡聚糖通常是多支链化合物。在这种高度可变的方式下单个葡萄糖单体以及母体分子的所有立体形状都可以相连，结果是许多形式都是可能的。一种常见的葡聚糖是β-(1，3)-键连的吡喃葡萄糖(通常称为β-葡聚糖)。一些物种的细胞壁包含与β-(1，6)键连的吡喃葡萄糖偶联的β-(1，3)-键连的吡喃葡萄糖。例如，酿酒酵母(Saccharaomyces cerevisiae)的细胞壁主要是由β-键连的葡聚糖组成的，其主要是β-(1-3)-键连的葡萄糖单元构成的主链，而分子间和分子内的少量成分是通过β-(1-6)-键形成支链。
葡聚糖由于其化学特性而在化学、食品和制药工业中具有多种用途。例如，它们可以用作增稠剂(viscosity imparting agent)、乳化剂、纤维、膜、涂料、亲合层析法和凝胶电泳的支持物，可用于细胞培养基，用作滤板，和用于粘固剂。其还广泛地用作食品增稠剂和食用纤维的来源以及药品中的载体以及涂层剂。已经表明在人和动物体内葡聚糖具有免疫药理学活性。例如，已经显示在虾、鱼、家禽、猪、牛、兔、小鼠、大鼠和人中对致病微生物具有强烈的免疫刺激和保护作用。通过与Toll-样受体Dectin-1相互作用，酵母β-葡聚糖可以刺激脊椎动物和无脊椎动物的先天性(非特异性)免疫应答。这种结合刺激了巨噬细胞中活性氧种类的产生，增强它们的吞噬作用并杀死微生物。这些被刺激的免疫细胞还产生细胞因子(cytokin)，其能循环到动物全身并与其它免疫细胞相互作用以增强该动物的免疫状况。
来自酵母及其他生物体的β-葡聚糖的纯化已被广泛地研究，并且已知有多种方法。这些方法中大部分基于β-(1-3)-葡聚糖在碱或有机溶剂中的不可溶性。主要的已知方法是：(a)用浓氢氧化钠高温提取，然后用酸高温提取并用乙醇沉淀(参见，例如，Manners，D.J.等人，Biochem.J.13519-30(1973)，Jamas，S.等人，美国专利号4,810,646、5,028,703以及5,250,436)。这些方法中有许多需要预先匀浆化酵母细胞，还有许多需要多次重复各提取步骤；(b)用浓苯酚∶水(1∶1)提取由酵母自溶作用或酶降解而产生的酵母细胞壁制备物(参见，例如，Truscheit E.等人的美国专利号4,138,479)；以及(c)单独用或者在存在碱的情况下用有机溶剂(例如异丙醇、乙醇、丙酮或甲醇)提取(参见，例如欧洲专利申请号515216)。已知酸处理降低了葡聚糖材料中β-(1-6)-键的数目，从而增加了粘度。
甘露聚糖是一种由甘露糖单元组成的聚合物。在酵母中，甘露聚糖既与酵母细胞壁的外表面中的蛋白质相结合，作为muscigenous多糖，又与内细胞膜中的蛋白质相结合。通常其占细胞壁干重的大约20-50％。甘露聚糖与核心-肽链相连为寡聚物或聚合物。该复合物包含大约5-50％的蛋白质。寡聚甘露聚糖直接结合到丝氨酸和苏氨酸上，而聚合的甘露聚糖则通过N-乙酰葡糖胺结合到天冬酰胺上。在这种甘露糖-蛋白质复合物中，甘露糖单元是通过α-1，6、α-1，2和α-1，3-键相连的。
甘露聚糖-寡聚糖(MOS)能通过蛋白水解作用从酵母细胞壁中释放出来。释放出的MOS能有效地与肠道的细菌性病原体结合并妨碍其在肠道建群(colonize)的能力。例如，大肠杆菌(E.coli)、沙门氏菌(Salmonellaspp.)和霍乱弧菌(Vibrio cholera)在其表面具有特异性地结合到这种MOS甘露糖糖残基上的蛋白质(凝集素)。
考虑到葡聚糖的许多用途和应用，在本领域内显然需要有一种避免利用高浓度碱或酸和高温的提取β-葡聚糖/甘露聚糖的方法，这样才能提高葡聚糖和甘露聚糖的回收率，得到生物学有用的制备物。
附图说明
图1是本发明生产β-葡聚糖/甘露聚糖制备物的方法的一种实施方式的流程图。
图2是本发明生产β-葡聚糖/甘露聚糖制备物的方法的另一实施方式的流程图。
发明概述
一方面，本发明提供一种处理酵母细胞的方法，其使用以下步骤：使酵母细胞自溶以释放酵母细胞壁，用外源的蛋白酶温育该酵母细胞壁，将该酵母细胞壁分离成富含葡聚糖的组分和富含甘露聚糖的组分，并超滤富含甘露聚糖的组分，形成滤液和渗余物。
另一方面，本发明提供一种处理酵母细胞的方法，其使用以下步骤：使酵母细胞在40℃到65℃温度下自溶以释放酵母细胞壁，在pH 9到10用外源的蛋白酶温育该酵母细胞壁，并用酶(例如淀粉酶、脂肪酶或其组合)温育所述经蛋白酶处理过的细胞壁。
另一方面，本发明提供一种包含α-甘露聚糖的组合物，其中至少85％(w/w)的总α-甘露聚糖具有10,000Da或更高的分子量。
本发明的其它实施方式包括那些包含本发明方法制备的葡聚糖或甘露聚糖的动物饲料、食品增补剂、药品、化妆品以及营养辅助品(neutraceutical)。
发明简述
在一个实施方式中，本发明提供一种方法，其产生富含β(1，3)和β-(1，6)葡聚糖的不溶性细胞壁制备物以及富含甘露聚糖的可溶性部分。本发明的方法包括细胞壁来源(例如酵母，诸如啤酒酵母或面包酵母)的自溶步骤，继之以酶消化步骤。在一个方面，酶消化是使用一种高pH蛋白酶进行。在另一方面，酶消化是使用酶(例如，高pH蛋白酶、淀粉酶、葡糖淀粉酶和/或脂肪酶)的组合进行。在一个实施方式中，酶消化是使用一种高pH蛋白酶继之以一种或多种其它酶(例如，淀粉酶、葡糖淀粉酶和/或脂肪酶)进行。
在另一实施方式中，本发明提供一种富含β-(1，3)和β-(1，6)葡聚糖的细胞壁制备物，而在另一实施方式中，提供一种富含甘露聚糖的可溶组分。
结合详细的说明和附图，本发明的其它方面将变得清楚。
发明详述
在详细解释本发明的任何实施方式之前，应当理解本发明并非将其应用仅限制在下面说明书中列出或在附图中所呈现的组分的细节或组分的排列。本发明能够具备其它实施方式，并可以通过多种方式实施或进行。此外，还应当理解，本文所用措词和术语是用于说明目的，而不应该被认为是对于本发明的限制。本文中所用的“包括”、“包含”或“具有”及其变体意在包括其后所列的各项和其等同物以及补充项。
还应当理解，本文所述的任何数值范围包括从下限值到上限值的所有数值。例如，如果一种浓度范围陈述为1％到50％，其意指诸如2％到40％、10％到30％、或1％到3％等等的数值都已在该说明书中清楚地列举。这些仅仅是具体地意指的实例，列举的最低值和最高值之间的所有可能数值组合都被认为是在本申请中清楚地陈述了的。
除非另有陈述，说明书和权利要求所用的表达组分数量、反应条件等等的所有数字在一切情况下应被理解为被术语“约”修饰。因此，除非标明与此相反，在下面说明书和所附权利要求中所述的数字参数均为近似值，可依据本发明设法获得的期望特性而变化。最低限度，而不是试图将等同物的教条应用限制于权利要求的范围，各数字参数应该至少是按照报道的有效数字的数字，并通过应用普通四舍五入的方法(roundingtechniques)来解释。
尽管阐明本发明宽范围的数值范围和参数都是近似值，在特定实施例中所列的数值是尽可能精确的记录。但是，任何数值都自然地包含某些不可避免的误差，这些误差来自在其各自试验测定中得到的标准差。
β-葡聚糖/甘露聚糖制备物可，在微酸性的/接近中性的pH以及仅在适度升高的温度下使用简单的自溶方法从微生物(例如酵母)制备。自溶后是酶消化。在一个实施方式中，该酶促步骤使用通常约0.05％-1wt％的高pH蛋白酶(例如，获自Genencore International的Protex 6L或由发酵地衣芽孢杆菌(Bacillus lichenformis)获得)，在碱性pH和升高的温度下进行。
作为β-葡聚糖/甘露聚糖来源的适用酵母种包括，但不限于，酿酒酵母(Saccharomyces cerevisiae)(包括面包酵母菌株以及啤酒酵母菌株)、脆壁克鲁维氏酵母(Kluyveromyces fragilis)的酵母菌株和假丝酵母属(Candida)菌株(例如产朊假丝酵母(Candida utilis))及其组合。其它的β-葡聚糖/甘露聚糖适宜来源的酵母菌株包括，但不限于，戴耳克氏糖酵母(Saccharomyces delbruekii)、罗茜糖酵母(Saccharomyces rosei)、Saccharomyces microellipsodes、卡尔斯伯糖酵母(Saccharomycescarlsbergensis)、粟酒裂殖糖酵母(Schizosaccharomyces pombe)、乳克鲁维氏酵母(Kluyveromyces lactis)、多孢克鲁维氏酵母(Kluyveromycespolysporus)、白色假丝酵母(Candida albicans)、阴沟假丝酵母(Candidacloacae)、热带假丝酵母(Candida tropicalis)、季也蒙氏假丝酵母(Candidaguilliermondii)、温奇氏汉逊氏酵母(Hansenula wingei)、Hansenula arni、亨利氏汉逊氏酵母(Hansenula henricii)、美洲汉逊氏酵母(HansenulaAmericana)及其组合。这些酵母菌株可通过分批发酵或者连续发酵培养在食品等级营养物中的培养物中进行制备。
许多其它种的微生物，包括但不限于，细菌、真菌及植物(例如单细胞藻类)，已在本领域被报道作为一种β-葡聚糖/甘露聚糖的来源。可用于本发明作为β-葡聚糖和/或甘露聚糖来源的其它微生物包括但不限于：细菌，例如产碱菌属(Alkaligenes)，特别是粪产碱菌混合基因变种(Alkaligenes faecalis Var.mixogenes)(ATCC-21680)，土壤杆菌属(agrobacterium)，纤维单胞菌属(Cellulomonas)，例如ATCC 21399及产黄纤维单胞菌(Cellulomonas flavigena)(ATCC 53703)，和盘多毛孢属(Pestalotia)；真菌，例如短柄霉属(Aureobasidum)，如出芽短柄霉(Aureobasidum pullulans)菌株IFO446及短柄霉属K-1种(FERMP1289)，蘑菇属(Agaricus)，香菇属(Lentinus)，平菇(Pleurotus ostreatus)，Macrophomopsis，如菌株KOB55；灵芝属(Ganoderma)，裂褶菌属(Schizophylla)，Fachyma hoelen，Pestahlia，革盖菌属(Coriolus)，及其组合。非微生物，如植物，也可用于本发明作为β-葡聚糖和/或甘露聚糖的来源。
具体地，本发明的方法涉及产生富含(1，3)-和β-(1，6)-葡聚糖内容物和甘露聚糖内容物的细胞壁制备物的生产，由包括但不限于酵母的微生物产生。在一个示范性的实施方式中，该方法包括第一步酵母(例如啤酒酵母)的自溶，(通常7％到18％，优选10％到17％，更优选8％到12％或13％到16％的固态浆料)。自溶可适当地在至少pH 4、优选至少pH 4.5、更优选至少pH 5时进行。自溶可适当地在低于pH 8、优选低于pH 7、更优选低于pH 6时进行。进行自溶的温度可适当地为至少30℃，优选至少35℃，更优选至少40℃，甚至更优选至少45℃。进行自溶的温度可适当地为低于55℃，优选低于52℃，甚至更优选低于50℃。自溶可适当地进行至少10小时，优选至少16小时，更优选至少24小时。该自溶可适当地进行少于100小时，优选少于48小时，并且更优选少于36小时。然后适当地通过离心分离该酵母，产生一种提取物和低β-葡聚糖含量的细胞壁流质。另一步，在至少pH 8.5、优选至少pH 9、更优选至少pH9.2时用酶处理细胞壁流质，该酶包括但不限于蛋白酶，例如碱性蛋白酶。该pH还可适当地低于10.5，优选低于10，更优选低于9.8。该蛋白酶处理可适当地在至少45℃温度、优选至少50℃、更优选至少53℃下进行。该蛋白酶处理可适当地在低于70℃、优选低于65℃、更优选低于60℃、甚至更优选低于57℃的温度下。该蛋白酶处理可适当地进行至少5小时，优选至少8小时，更优选至少10小时，甚至更优选至少12小时。该蛋白酶处理可适当地进行少于48小时，优选少于36小时，更优选少于24小时，甚至更优选小于18小时。然后离心分离第二产物，产生一种富含甘露聚糖(α-甘露聚糖)的提取物和一种富含β-葡聚糖的细胞壁制备物。然后干燥(例如喷雾干燥)这种β-(1，3/1，6)细胞壁产品，这致使产品聚集成约100-300微米或更大的颗粒。然后将甘露聚糖提取物经过10,000分子量的超滤以产出富含甘露聚糖的高分子量的渗余物。
如上所述的这种示范性的方法如图1的流程图所示。活体酵母在以下过程中进行自溶，其间内源的酵母酶分解和溶解一些酵母高分子。通过离心从不溶的酵母细胞壁中分离可溶性提取物。然后用高pH蛋白酶处理细胞壁，进一步从细胞壁中去除蛋白质，随后同样去除依附于细胞壁蛋白质的甘露聚糖。然后通过离心从二次提取物中分离出富含β-葡聚糖的细胞壁。具有高分子量的甘露聚糖可通过10,000Da超滤该二次提取物进一步纯化和浓缩甘露聚糖。
在另一实施方式中，方法包括了第一步的酵母(例如啤酒酵母)的自溶，(通常8％-12％的固态浆料)。自溶可适当地在至少pH 4、优选至少pH 4.5，更优选至少pH 5时进行。该pH还可适当地低于8，优选低于7，更优选低于6。进行自溶的温度可适当地为至少30℃，优选至少40℃，更优选至少45℃。该温度还可适当地低于55℃，优选低于53℃，更优选低于50℃。自溶可适当地进行至少10小时，优选至少16小时，更优选至少24小时。该自溶可适当地进行少于100小时，优选少于48小时，并且更优选少于36小时。然后通过离心适当地分离酵母，产生一种提取物和一种低β-葡聚糖含量的细胞壁流质。另一步用酶处理该细胞壁流质。该酶步骤首先在碱性pH(例如，在至少pH 8.5，优选至少9，更优选至少9.2)时使用一种高pH蛋白酶。该pH还可适当地低于10.5，优选低于10，更优选低于9.8。该蛋白酶处理可适当地在至少45℃、优选至少50℃、更优选至少53℃的温度下进行。该蛋白酶处理可适当地在低于70℃、优选低于65℃、更优选低于60℃、甚至更优选低于57℃的温度下进行。该蛋白酶处理可适当地进行至少5小时，优选至少8小时，更优选至少10小时，甚至更优选至少12小时。该蛋白酶处理可适当地进行少于48小时，优选小于36小时，更优选小于24小时，更优选小于18小时。蛋白酶酶步骤后是用葡糖淀粉酶(例如来自曲霉属的种)、淀粉酶(例如来自枯草芽孢杆菌(Bacillus subtili)、米曲霉(Aspergillusoryzae)的α淀粉酶；来自黑曲霉(Aspergillus niger)或根霉属霉菌的淀粉葡萄糖苷酶)和/或脂肪酶(例如来自洋葱假单胞菌(Pseudomonascepacia)、皱褶假丝酵母(Candida rugosa)和爪哇毛霉(Mucor javanicus)的脂肪酶；通常约0.05％-1wt％)进行温育。用葡糖淀粉酶、淀粉酶和/或脂肪酶温育是在中性到微酸性的pH和高温条件下适当地进行的。例如，该pH可以适当地为至少为3.5，优选至少4，更优选至少4.5。该pH还可适当地为低于7，优选低于6，更优选低于5.5。与葡糖淀粉酶、淀粉酶和/或脂肪酶进行温育的温度可适当地为至少40℃，优选至少45℃，更优选至少50℃，甚至更优选至少53℃。该温度也可适当地从低于70℃，优选低于65℃，更优选低于60℃，甚至更优选低于58℃。尤其是如果该蛋白酶、淀粉酶或脂肪酶是耐热酶，可适当地采用至少60℃、至少65℃、至少70℃、至少75℃、至少80℃、至少85℃或至少90℃的温度。与碱性蛋白酶一起进行的温育还可以继之以与葡糖淀粉酶和脂肪酶的组合、淀粉酶和脂肪酶的组合或葡糖淀粉酶、淀粉酶和脂肪酶的组合一起的温育。
如上所述的这种示范方法如图2的流程图所示。在图2描绘的方法中，活体酵母在以下过程中进行自溶，其间内源的酵母酶会分解和溶解一些酵母高分子。来自自溶作用的细胞壁首先用高pH蛋白酶处理。与该高pH蛋白酶的温育适当地在50℃到65℃的温度进行大约10到16小时。然后用淀粉酶(或其它葡聚糖酶)或脂肪酶、或淀粉酶和脂肪酶的组合处理细胞壁。与该淀粉酶和/或脂肪酶的温育适当地在pH 4到7和50℃到65℃的温度进行大约4到10小时。淀粉酶可以溶解残留的α-葡聚糖，例如那些可能仍然残留在细胞壁上的糖原。脂肪酶可以降解富含脂类和脂肪的细胞壁膜。然后可以离心细胞壁流质，产生富含甘露聚糖的二次提取物，和富含β-葡聚糖的细胞壁产品。该细胞壁产品可进行干燥(例如喷雾干燥)。该二次甘露聚糖提取物可通过超滤膜，例如10,000Da的超滤膜、50,000Da的超滤膜或100,000Da的超滤膜，从而富集渗余物中的甘露聚糖。
本发明的制备物可通过任何适用的方法进行干燥，所述方法包括但不限于：冻干、滚筒式干燥、烤箱干燥、喷雾干燥、环形干燥(ring drying)及其组合和/或使用成膜设备进行干燥，和要么不采用进一步的处理，要么可使用任何适当的技术进行研磨。
适当地，该高pH蛋白酶可以在pH 7以上具有最优的蛋白水解活性。适宜的蛋白酶包括但不限于获自下述来源的那些蛋白酶：中华猕猴桃(Actinidia chinensis)、菠萝(Ananas comosus)、曲霉属的种(例如黑曲霉、黑曲霉泡盛曲霉变种(A.niger var.awamori)、米曲霉、酱油曲霉(A.sojae)、蜂蜜曲霉(A.melleus))、芽孢杆菌属的种(例如枯草芽孢杆菌、嗜碱芽孢杆菌(B.alcalophilus)、溶淀粉芽孢杆菌(B.amyloliquefaciens)、嗜碱芽孢杆菌(B.halodurans)、迟缓芽孢杆菌(B.lentus)、地衣芽孢杆菌(B.licheniformis)、嗜热脂肪芽孢杆菌(B.stearothermophilus)、嗜热芽孢杆菌(B.thermoproteolyticus))、番木瓜(Carica papya)、栗疫病菌(Cryphonectria parasitica)、栗疫菌(Endothiaparasitica)、脱毛榕木(Ficus glabrata)、乳克鲁维式酵母，柑桔青霉(Penicillum citrinum)、米赫根毛霉(Rhizomucor miehei)、雪白根霉(Rhizopus niveus)、来自小牛、山羊或牛胃或猪胰腺，及其组合。适宜的蛋白酶可包括但不限于，市售的酶例如枯草杆菌蛋白酶Carlsberg、枯草杆菌蛋白酶BPN′、枯草杆菌蛋白酶Novo、枯草杆菌蛋白酶309、枯草杆菌蛋白酶147及枯草杆菌蛋白酶168、AlcalaseTM、SavinaseTM、PrimaseTM DuralaseTM、DurazymTM、EsperaseTM、和KannaseTM(获自NovoNordisk A/S)；MaxataseTM、MaxacalTM、MaxapemTM、OptimaseTM、Properase TM、Purafect TM、Purafect OxPTM、FN2TM、和FN3TM(获自Genencor International Inc.)；和ValidaseTM AFP、ValidaseTM FP浓缩物、ValidaseTM FP 500、ValidaseTM FP II、ValidaseTM TSP浓缩物、碱性蛋白酶浓缩物、Bromelain(获自Valley Research，South Bend，IN)，及其组合。
适宜的淀粉酶包括植物、动物、细菌或真菌来源的那些淀粉酶，及其组合。淀粉酶包括但不限于，获自芽孢杆菌属的种(例如地衣芽孢杆菌、溶淀粉芽孢杆菌、枯草芽孢杆菌、嗜热脂肪芽孢杆菌)，米曲霉，黑曲霉，黑曲霉泡盛曲霉变种，蛾微杆菌(Microbacterium imperiale)，青绿色热单孢(Thermomonospora viridis)，大麦芽(大麦属的种)，猪胰腺(Sus种)，及其组合的葡糖淀粉酶或α-淀粉酶。有用的淀粉酶实例包括但不限于：市售淀粉酶，例如葡糖淀粉酶浓缩物(GlucoamylaseConcentrate)、DuramylTM、TermamylTM、FungamylTM和BANTM(获自Novo Nordisk A/S)；RapidaseTM和PurastarTM(获自Genencor InternationalInc.)；和ValidaseTM BAA，ValidaseTM HT340L，ValidaseTM FAA，ValidaseTM AGS，ValidaseTM GA，ValidaseTM RGA(获自Valley Research，SouthBend，IN)，及其组合。可以适当地采用淀粉酶的终浓度至少0.001％、优选至少0.01％、更优选至少0.02％。可以适当地采用淀粉酶的终浓度小于0.1％、优选小于0.05％、更优选小于0.1％。
可用于本发明的脂肪酶包括但不限于：来自腐质霉属(等同于嗜热真菌属(Thermomyces))的脂肪酶，例如来自疏棉状嗜热丝孢菌(H.lanuginosa(T.lanuginosus))、特异腐质霉(H.insolens))的脂肪酶；假单胞菌属脂肪酶，例如来自产碱假单胞菌(P.alcaligenes)或类产碱假单胞菌(P.pseudoalcaligenes)、洋葱假单胞菌(P.cepacia)、施氏假单胞菌(P.stutzeri)、荧光假单胞菌(P.fluorescens)、假单胞菌属种菌株SD 705、威州假单胞菌(P.wisconsinensis)的脂肪酶；芽孢杆菌脂肪酶，例如来自枯草芽孢杆菌、嗜热脂肪芽孢杆菌或短小芽孢杆菌(B.pumilus)的脂肪酶(WO 91/16422)；米曲霉，黑曲霉，解脂假丝酵母(Candidalipolytica)，皱褶假丝酵母，爪哇毛霉，娄格法尔特氏青霉(Penicillumroqueforti)，米赫根毛霉，德列马根霉(Rhizopus delemar)，雪白根霉(Rhizopus niveus)，米根霉，无根根霉(Rhizopus arrhizus)，及其组合。市售脂肪酶包括但不限于：LipolaseTM及Lipolase UltraTM(Novo NordiskA/S)，及真菌脂肪酶(Fungal Lipase)8000及胰脂肪酶(Pancreatic Lipase)250(获自Valley Research，South Bend，IN)。
来自酵母细胞自溶作用的产物适当地还包含以干燥固体计总产物的至少20％、优选至少23％、更优选至少25％的蛋白质。该产物同样适当地包含以干燥固体计小于总产物45％、优选小于40％、更优选小于35％的蛋白质。来自酵母细胞自溶作用的产物适当地还包含以干燥固体计总产物的至少20％、优选至少23％、要优选至少25％的葡聚糖。该产物同样适当地包含以干燥固体计小于总产物的45％、优选小于40％、更优选小于35％的葡聚糖。
来自酵母细胞自溶作用的产物适当地同样包含以干燥固体计总产物的至少5％、优选至少7％、更优选至少10％的α-葡聚糖。该产物同样适当地包含以干燥固体计小于总产物的20％、优选小于18％、更优选小于15％的α-葡聚糖。来自酵母细胞自溶作用的该产物适当地包含以干燥固体计总产物的至少7％、优选至少10％、更优选至少12％的β-葡聚糖。该产物同样适当地包含以燥固体计小于总产物的22％、优选小于20％，更优选小于18％的β-葡聚糖。来自酵母细胞自溶作用的该产物适当地包含以干燥固体计总产物的至少5％、优选至少7％、更优选至少10％的甘露聚糖。该产物同样适当地包含以干燥固体计小于总产物的20％、优选小于18％、更优选小于15％的甘露聚糖。
富含β-(1，3/1，6)葡聚糖产物的细胞壁产品的特征在于，例如，至少50％、至少55％、至少60％或至少65％的β-(1，3/1，6)葡聚糖，而蛋白质含量小于20％、小于15％或小于10％。富含甘露聚糖的产物(二次甘露聚糖提取物)的特征在于包含至少50％、优选至少55％、更优选至少57％的甘露聚糖。该富含甘露聚糖的产物的特征还可以在于包含小于70％、优选小于68％、更优选小于65％的甘露聚糖。该富含甘露聚糖的产物(二次甘露聚糖提取物)的特征还在于包含至少25％、优选至少27％、更优选至少29％的蛋白质。该富含甘露聚糖的产物的特征还可以在于包含小于35％、优选小于32％、更优选小于30％的蛋白质。
超滤步骤可以这样进行：迫使根据本文所述方法产生的提取物(例如二次甘露聚糖提取物)在压力下穿过超滤膜。适当地，该超滤膜包括一种或多种半透膜。该半透膜或超滤膜可以具有一定的截流分子量，例如至少8,000Da、优选至少10,000Da、更优选至少25,000Da、更优选至少50,000Da、更优选至少100,000Da、更优选至少150,000Da。应当理解该超滤膜可以具有本文所述的那些数值之间的任一数值的截流分子量，包括但不限于至少15,000Da、20,000Da、30,000Da、40,000Da、60,000Da、70,000Da、80,000Da、90,000Da、110,000Da、120,000Da、130,000Da及140,000Da的截流分子量。适宜的超滤膜包括但不限于，获自A/G Technology Corp，Needham，MA的空心纤维膜。
二次甘露聚糖提取物经过滤之后在渗余物中的总二次甘露聚糖的至少80％(w/w)、优选至少85％(w/w)、更优选至少90％(w/w)的分子量可高于所用过滤膜的截流分子量。例如，如果二次甘露聚糖提取物应用了10,000Da的截流分子量，那么在渗余物中通常至少80％(w/w)、优选至少85％(w/w)、更优选至少90％(w/w)的总甘露聚糖的分子量高于10,000Da。如果二次甘露聚糖提取物应用了50,000Da的截流分子量，那么在渗余物中通常至少80％(w/w)、优选至少85％(w/w)、更优选至少90％(w/w)的总甘露聚糖的分子量高于50,000Da。如果二次甘露聚糖提取物应用了100,000Da的截流分子量，那么在渗余物中通常至少80％(w/w)、优选至少85％(w/w)、更优选至少90％(w/w)的总甘露聚糖的分子量高于100,000Da。如果二次甘露聚糖提取物应用了150,000Da的截流分子量，那么在渗余物中通常至少80％(w/w)、优选至少85％(w/w)、更优选至少90％(w/w)的总甘露聚糖的分子量高于150,000Da。
该超滤步骤可任选地包括使甘露聚糖提取物通过两个或多个不同截流分子量的超滤膜。最后的渗余物包含一种富含甘露聚糖的产物，其中大多数甘露聚糖的分子量介于所述超滤器的截流分子量之间。在该实施方式中，在最后的渗余物中至少80％(w/w)、优选至少85％(w/w)、更优选至少90％(w/w)的总甘露聚糖的分子量介于超滤膜的截流分子量之间。
自溶细胞壁经酶处理之后从富含葡聚糖产物中分离的二次甘露聚糖提取物的特征在于，例如，15％到50％的甘露聚糖，20％到30％的蛋白质及20％到25％的其它组分。当根据本发明方法超滤二次甘露聚糖提取物时，渗余物可包含至少50％、优选至少52％、更优选至少55％、更优选至少60％的甘露聚糖。该渗余物可包含低于70％、优选低于65％、更优选小于62％的甘露聚糖。该渗余物可进一步包含至少10％、优选至少12％、更优选至少15％、甚至更优选至少17％的蛋白质。该渗余物可进一步包含低于33％、优选低于30％、更优选低于22％的蛋白质。
可以预见本发明的制备物在例如食品增补剂、药品(例如改善免疫应答)、化妆品、动物饲料及营养辅助品中具有重要性。例如，一种动物饲料可以适当地包含1到10g制备物/kg饲料。适当地，以重量/重量计，该制备物可以包含该饲料总重的至少0.01％、优选至少0.02％、更优选至少0.05％、甚至更优选至少0.1％，并低于该饲料总重的5％、优选低于2％、更优选低于0.5％、甚至更优选低于0.3％。适合的动物饲料包括，但不限于，牛、马、猪、家禽、鱼(例如，甲壳动物、有壳的水生动物)、鸟类和宠物(例如，猫、狗)饲料。液体组合物可以包含0.1％-1wt％的本发明的制备物。本发明的制备物还可与在农业上可接受的载体一起，以及任选的在农业上可接受的营养物、除草剂或杀虫剂一起，用于植物保护组合物。
例如，根据本发明制备的富含β-葡聚糖的部分可适当地在动物及人食品、药品中用作免疫刺激物或软化剂、降胆固醇剂、以及食品和饮料中的增稠剂。如果加入到一种软化剂、洗液或乳膏剂中并用于治疗病症(condition)，该β-葡聚糖可以适当地以至少0.05％、优选至少0.1％、更优选至少0.5％并小于10％、优选小于5％、更优选小于2％的浓度(w/w)存在。适当地，根据本发明制备的β-葡聚糖部分可以用于例如通过掺入乳膏剂、洗液或软化剂中治疗湿疹。湿疹包括各种发炎的皮肤病症，包括遗传过敏性皮炎(“遗传过敏性湿疹(atopic eczema”)，并会在儿童期影响世界人口的约10％到约20％。湿疹似乎是身体免疫系统的一种异常应答。
根据本发明制备的富含甘露聚糖的产品还有许多用途。例如，甘露聚糖产品可以用于动物饲料工业，能够有利地结合霉菌毒素以及病原菌，防止细菌在肠道建群。
总之，除了别的之外本发明应用相对温和的工艺操作条件，提供了富含β-葡聚糖及甘露聚糖的制备物。
在下面实施例中阐述了本发明的各种特征及方面。
实施例1：使用高pH蛋白酶处理酵母
在夹层式不锈钢容器中将来自商品级的自溶啤酒酵母(酿酒酵母)的31.1kg的细胞壁部分加热到55℃。总固相为10.7％，在该固相中蛋白质总比例为24.5％。用氢氧化钠将pH提高到9.5，并加入0.1％(基于总重)的Protex 6L(一种碱性蛋白酶，获自Genencor，Palo Alto，CA)。在55℃搅拌细胞壁16小时。在85℃加热30分钟使Protex 6L失活，使用连续倾析法，用Alpha Laval Gyro型转筒离心机分离细胞壁。用与除去的提取物等体积的水洗涤不溶的细胞壁部分三次。将洗涤的细胞壁部分浓缩到固相15.4％，盐酸调整pH到7.0，喷雾干燥该部分。将一部分来自Protex 6L处理的提取物(相当于图1所示2°提取物)浓缩到固相28.3％，调整pH为7.0并喷雾干燥该提取物。残余的2°提取物用UFP-10-C-6A10,000NMWC空心纤维膜(获自A/G Technology Corp，Needham，MA)超滤。将富含甘露聚糖的高分子量渗余物的pH调整为7.0并喷雾干燥。调整该3°提取物(滤液)的pH为7.0，浓缩并喷雾干燥。
利用下列技术分析由此方法产生的产品的组成：利用LECO蛋白质测定仪(LECO Corp.，St.Joseph，MI)测定蛋白质；利用MegazymeInternational Mushroom及酵母β-葡聚糖试剂盒(获自MegazymeInternational，Wicklow，爱尔兰)测量总葡聚糖、α-葡聚糖和β-葡聚糖；利用己糖激酶、葡糖-6-磷酸脱氢酶、磷酸葡糖异构酶及磷酸甘露糖异构酶，通过碳水化合物的酸性水解测定甘露聚糖，结合分光光度分析测定游离甘露糖；使用Blich，E.G.和Dyer，W.J.Can.J.Biochem.Physiol.(1959)37，911的甲醇-氯仿提取法测定脂肪；使用Yellow Springs InstrumentsBiochemistry分析器(获自YSI Incorporated，Yellow Springs，OH)测定游离葡萄糖。这些分析结果示于表1。
表1
Figure 2006800150267A00800011
ND指未确定的。
实施例2：使用高pH蛋白酶和葡糖淀粉酶处理酵母
将16,000加仑的由原啤酒酵母提取物产生的细胞壁膏状物加热到55℃，用氢氧化钠调整pH为9.5。加入0.1％(v/v)的Protex 6L，混合物在55℃放置14小时。用HCl将pH降到pH 5.0。Protex 6L在pH 5下没有活性，不会破坏加入的酶。加入0.0175％(重量∶总重)的葡糖淀粉酶浓缩物(获自Valley Research，South Bend，IN)。在55℃放置4小时，然后升温到88℃使该酶失活。用Westfalia转筒分离器(获自WestfaliaSeparator，Inc.，Northvale，NJ)分离加热的物料。浓缩大部分提取物(图2中所示的2°提取物)并喷雾干燥。一部分2°提取物用UFP-10-C-6A10,000NMWC空心纤维膜(获自A/G Technology Corp，Needham，MA)超滤。浓缩渗余物及滤液并喷雾干燥。根据在实施例1所述的技术分析该喷雾干燥产品。结果列于表2。通过离心用水洗涤该细胞壁部分，浓缩并喷雾干燥。
表2
Figure 2006800150267A00800021
ND指未确定的。
比较表1和2的数据可以看出在实施例2处理过程中加入的葡糖淀粉酶的效果。在实施例2的过程中，超滤之后在渗余物和滤液中并没有检测到α-葡聚糖。同样，来自实施例2处理过程的2°和3°提取物中的游离葡萄糖水平(如表2所示)比来自实施例1的2°和3°提取物(如表1所示)高出很多。
实施例3：使用以不同顺序加入到自溶的酵母细胞壁中的葡糖淀粉酶和高pH蛋白酶处理酵母
向两个夹层式不锈钢容器中分别加入25Kg来自商业级原啤酒酵母提取物的细胞壁，其中酵母细胞已经经过自溶作用。固相为11.8％。将两个容器加热到55℃。容器1的pH调整为5.0并加入0.1％(重量∶总重)的葡糖淀粉酶浓缩物(获自Valley Research，South Bend，IN)。连续温育14小时，将pH升高到9.5。然后加入0.10％的Protex 6L，继续温育4小时。在不同的时间点取样并测定在葡糖淀粉酶作用下释放的游离葡萄糖。
开始时容器2的pH升高到9.5并加入0.1％(重量∶总重量)的Protex6L。在55℃温育混合物14小时。然后将pH降到5.0并加入0.1％的葡糖淀粉酶浓缩物。继续温育4小时。在不同的时间点取样并测定在葡糖淀粉酶作用下释放的游离葡萄糖。表3表明在不同时间两个容器中游离葡萄糖的水平。
表3
Figure 2006800150267A00800031
表3的数据表明在Protex 6L之前加入葡糖淀粉酶时，如在容器1中，则细胞壁的变化不足以使葡糖淀粉酶进入并消化在啤酒酵母自溶作用之后嵌入细胞壁内部的大分子α-葡聚糖(糖原)。相反，在容器2中，在葡糖淀粉酶之前加入蛋白酶，使得葡糖淀粉酶能够进入并消化α-葡聚糖，释放出多得多的葡萄糖。在pH 5.0下，尽管容器1中的葡糖淀粉酶作用时间(14小时)要比在容器2中的葡糖淀粉酶作用时间(4小时)长，但仍是这样的情况。因此，为了最优地去除来自啤酒酵母细胞壁的糖原/α-葡聚糖，应在葡糖淀粉酶之前加入碱性蛋白酶Protex 6L。
实施例4：根据图2所示处理过程处理啤酒酵母和面包酵母。
将220g来自商业级的自溶的初级培养面包酵母(固相15％)或啤酒酵母(固相11.8％)的细胞壁加热到55℃，调整pH为9.5。然后用0.1％(重量：总重)Protex 6L处理该细胞壁14小时。14小时后，将pH降到5.0，每个容器中加入0.0175％的葡糖淀粉酶浓缩物。在55℃下再温育该培养瓶4小时。用YSI生化分析器(Biochemistry Analyzer)监测游离葡萄糖。结果如表4所示。
表4
Figure 2006800150267A00800041
来自面包酵母的自溶作用的细胞壁所含的糖原低于来自啤酒酵母的细胞壁，主要是因为好氧培养的面包酵母累积的β-葡聚糖往往少于厌氧培养的啤酒酵母。用葡糖淀粉酶温育之后的啤酒酵母细胞壁释放出的葡萄糖多于面包酵母细胞壁释放出的葡萄糖。因此图2的处理过程对于处理来自啤酒酵母细胞壁的β-葡聚糖非常有效。
实施例5：提取物在动物饲料中的用途
通过将两个组分干混在一起来配制自溶啤酒酵母细胞：来自图2处理的2°提取物为50∶50(基于干燥固体)混合物，其中2°提取物根据实施例2制备(即前后用蛋白酶和淀粉酶处理获得的甘露聚糖)。该混合物用来补充断乳后28天的幼猪的饲料。该混合物在第1阶段(0-7天)以3磅(lbs)/吨饲料加入，在第2阶段(7-14天)为2磅/吨饵料，第3阶段(14-28天)为2磅/吨饵料。对照组和处理组饲料均含有抗生素。根据体重将断乳后的猪(17-22天龄)随机地分配到对照组饲料或处理组饲料。每个饲料组有13只猪，分6个围栏。结果如表5所示。
表5
Figure 2006800150267A00800051
a，b是指差异显著，P＜0.10。
处理组饵料饲养的猪在14天明显地更重，并到28天猪重量有增加的趋势。
实施例6：酵母提取物在动物饲料中作为增味剂的用途。
犬食涂以油，然后将1.0％的来自图2所示处理过程的干燥的3°提取物(根据实施例2制备(即超滤之后的滤液))或者1.0％的已经被接受的犬增味剂喷涂到涂有油的犬食表面上。以1000g的定额在两天内提供给一组20只犬。食盆的位置每天互换，防止“左-右”偏爱。
每只犬在两天时间内食用的食物量列于表6。表6显示：根据实施例2制备的图2处理过程的3°提取物即使不高于标准增味剂(palatant)，但至少能够跟标准增味剂一样增强干燥犬食的可口性。
表6
Figure 2006800150267A00800061
实施例7(预示性)：酵母细胞壁-喷雾干燥粉的特性
根据实施例2所述方法制备高度纯化的酿酒酵母细胞壁产品。其具有高浓度的(β-1，3/1，6)葡聚糖。该产品通过了FDA的G.R.A.S.(通常被认为是安全的)。该产品具有高质量天然来源的(β-1，3/1，6)葡聚糖，可以用于各式各样的食品中的增补剂。已经表明该生物学活性物质刺激许多动物的免疫系统。该组合物和该产品的特性列于表7。
表7
Figure 2006800150267A00800071
实施例8(预示性)
啤酒酵母细胞壁膏状物被加热到131℉(55℃)。用50％氢氧化钠(每Kg细胞壁膏状物约5ml)将pH升到9.5。加入0.1％(体积：细胞壁膏状物总重)的Protex 6L(Genencore)。在131℉放置混合物14小时。用28％的HCl(盐酸)将pH降到5.0并加入0.0175％(重量：总重)的葡糖淀粉酶浓缩物(Valley Research)。在55℃放置混合物4小时，然后加热到185-195℉加热灭活该酶。分离各部分。在喷雾干燥富含β-葡聚糖的不溶部分之前，调整pH为6.5。喷雾干燥富含β-葡聚糖的不溶部分。
制备高度纯化的酿酒酵母细胞壁产品。其具有高浓度的(β-1，3/1，6)葡聚糖。该产品通过FDA的G.R.A.S.。该产品具有高质量的天然来源的(β-1，3/1，6)葡聚糖，可用于各式各样的食品中增补剂。已经表明该生物学活性物质能够刺激许多动物的免疫系统。该组合物和该产品的特性示于表7。
实施例9：使用高pH蛋白酶和脂肪酶处理酵母。
将220g来自商业级面包酵母自溶作用的细胞壁(固相15％)置于玻璃培养瓶中并搅拌。将温度升高到55℃，并用HCl将pH升到9.5。加入0.1％的Protex 6L，温育样品14小时。此时，将30g的等分试样装入适用于Sorvall SS34离心机转子的50ml离心管(获自Nalgene)中。各管中加入磁搅拌棒。向离心管加入下列A、B或C：
A.0.0175％的葡糖淀粉酶浓缩物(获自Valley Research)
B.0.1％的脂肪酶CR(一种获自Valley Research的三酰基甘油脂肪酶)
C.0.0175％的葡糖淀粉酶浓缩物+0.1％的脂肪酶CR。
各管在55℃搅拌温育四小时。85℃加热15分钟灭活所述酶，使用带有SS34转子的SorvallTM离心机沉淀(pellet)细胞壁(12,000r.p.m.10分钟)。然后用与除去的可溶性提取物等体积的水洗涤沉淀三次。细胞壁重悬浮到约固相15％并用Buchi Mini喷雾干燥器B-191喷雾干燥。分析干燥细胞壁的蛋白质(氮X 6.25；LECO蛋白质测定仪，获自LECO Corp.，St.Joseph，MI)，并利用Megazyme International Mushroom和酵母β-葡聚糖试剂盒(获自Megazyme International，Wicklow，Ireland)测量β-葡聚糖。结果如表8所示。
表8
Figure 2006800150267A00800081
实施例10(预示性)：实施例2的富含β-葡聚糖的产品在肉用仔鸡饲料中的应用
每天给来自1天龄的肉用仔鸡饲喂含1g/Kg实施例2的富含β-葡聚糖产品的标准鸡饲料(无抗生素)或者不含β-葡聚糖的标准鸡饲料(无抗生素)(对照)。7天后用雏鸡病原性大肠杆菌菌株对对照和β-葡聚糖饲养的两组雏鸡进行呼吸免疫攻击。持续给这些小鸡喂其各自的饲料，并记录一个月内的死亡率。
预计β-葡聚糖饲养的雏鸡的死亡率会明显低于标准饲料组。对于降低呼吸道感染引发的生产损失来说，β-葡聚糖对雏鸡免疫系统的刺激作用是有价值的。
实施例11(预示性)：实施例1的富含甘露聚糖的超滤液渗余物在雏鸡饲料中的用途
两星期内每天给肉用仔鸡饲喂含有1g/Kg实施例1的富含甘露聚糖超滤渗余物的标准鸡饲料(无抗生素)或者不含富集的甘露聚糖的标准鸡饲料(无抗生素)(对照)。然后给肉用仔鸡(对照和甘露聚糖饲养组)经口接种雏鸡病原性沙门氏菌属的菌株。持续给这些雏鸡喂其各自的饲料，监测一个月内的死亡率和发病率。
甘露聚糖会与沙门氏菌属结合并防止其与甘露聚糖饲料组雏鸡的肠道结合。预计这会显著降低甘露聚糖饲养雏鸡组的发病率及死亡率。
实施例12(预示性)：实施例1或2的富含β-葡聚糖的产品在虎虾(TigerShrimp)培养中的用途。
将一组虎虾(斑节对虾(Penaeus monodon)浸于不含富集的β-葡聚糖的溶液中(对照组)。在研究期间该组饲以不含富集的β-葡聚糖的商品虾食小球(commercial pellet)。第二组虎虾浸于含0.1％来自实施例1的富含β-葡聚糖的溶液中，然后饲以含0.1％来自实施例1的富集的β-葡聚糖的商品虾食颗粒。第三组虎虾浸于含0.1％来自实施例2的富含β-葡聚糖的溶液中，然后饲以含0.1％来自实施例2的富集的β-葡聚糖的商品虾食颗粒。监测几个月内各组的死亡率。
在虾的养殖历史上曾有过高的死亡率。与对照组相比，当虾浸于含β-葡聚糖的溶液中时，以及当该虾随后用含β-葡聚糖的饲料养殖时，预期来自实施例1和2的各酵母β-1，3-1，6-葡聚糖刺激虾的免疫应答。由于酵母β-葡聚糖对先天免疫系统的刺激作用，预计浸于和用酵母β-葡聚糖饲料饲养的虎虾组生长得更快，并预计其死亡率低于对照组。
实施例13(预示性)：实施例2富含β-葡聚糖的产品在湿疹治疗中的用途
选定一组患有湿疹且对当前接受的皮肤洗液治疗不响应的儿童，用1％含实施例2的富含β-葡聚糖的产品的悬浮液进行治疗。每天施用洗液两次。皮肤科医生每周评价皮肤的病变及疼痛的改良。预计β-葡聚糖洗液降低病变引发的疼痛并加快病变痊愈的速度。
实施例14(预示性)：实施例2富含β-葡聚糖的产品在健康快餐食品生产中的用途
将来自实施例2的酵母β-葡聚糖提取物按1％(w/w)加到冰淇淋中以部分替换脂肪。β-葡聚糖加固了冰淇淋的硬度和外形，但并不影响其质地。添加了β-葡聚糖的冰淇淋所含卡路里少于不含β-葡聚糖的冰淇淋。摄取这种添加后的冰淇淋，预计β-葡聚糖会刺激肠道的先天免疫系统从而有益于消费者的免疫状况。
将来自实施例2的酵母β-葡聚糖提取物按0.5％(w/w)及1％(w/w)加到饼干、快餐店及面包店项目(item)中。添加了β-葡聚糖的饼干、快餐店及面包店项目所含卡路里少于不含β-葡聚糖的饼干、快餐店及面包店项目。摄取这种添加后的饼干、快餐店及面包店项目后，预计β-葡聚糖刺激肠道的先天免疫系统从而有益于消费者的免疫状况。
尽管已经说明并例证了本发明的一些特性，本领域技术人员将理解能够在本发明描述的基础上进行各种改变，包括变化、填加及省略。所以，意指这些改变同样包括在本发明中，本发明的范围仅由所附权利要求在法律上的最广泛解释所限制。
本文引用的所有专利、出版物及参考文献均全文并入本文作为参考。在本公开文本与并入的专利、出版物及参考文献互相冲突的情况下，应该参照本公开文本。

Claims (30)

1.一种处理微生物细胞的方法，其包括：
(a)使所述微生物细胞自溶，以释放微生物细胞壁；
(b)用外源的蛋白酶温育所述微生物细胞壁；
(c)将所述微生物细胞壁分离为富含葡聚糖的组分和富含甘露聚糖的组分，所述富含葡聚糖的组分包括至少50％的β-(1，3/1，6)葡聚糖；以及
(d)超滤步骤(c)的富含甘露聚糖组分，以形成滤液和渗余物，所述渗余物包括至少50％的甘露聚糖和至少10％的蛋白质。
2.权利要求1的方法，其中所述微生物包括酵母、真菌或细菌中的至少一种。
3.权利要求1的方法，其中所述微生物细胞包括酵母细胞。
4.权利要求1的方法，其中步骤(b)的蛋白酶在步骤(c)之前被灭活。
5.权利要求1的方法，其中步骤(b)的温育在pH 9到10、温度50℃到65℃时进行。
6.权利要求1的方法，其中所述渗余物包含甘露聚糖，并且其中至少85％(w/w)的所述甘露聚糖的分子量至少为10,000Da。
7.权利要求1的方法，其中步骤(a)在pH 4到8进行。
8.权利要求1的方法，其中步骤(a)进行24到36小时。
9.权利要求1的方法，其中步骤(a)在35℃到55℃的温度下进行。
10.权利要求1的方法，进一步包括在动物饲料中使用步骤(c)的富含葡聚糖的组分。
11.权利要求1的方法，进一步包括在动物饲料中使用步骤(b)的经蛋白酶处理的细胞壁。
12.权利要求1的方法，进一步包括在动物饲料中使用步骤(d)的滤液。
13.权利要求1的方法，进一步包括在选自食品增补剂、药品、化妆品和营养辅助品的产品中使用步骤(c)的富含葡聚糖的组分。
14.权利要求1的方法，进一步包括在选自食品增补剂、药品、化妆品和营养辅助品的产品中使用步骤(b)的经蛋白酶处理的细胞壁。
15.权利要求1的方法，进一步包括在选自食品增补剂、药品、化妆品和营养辅助品的产品中使用步骤(c)的富含葡聚糖的组分中使用步骤(d)的滤液。
16.一种处理酵母细胞的方法，其包括：
(a)使酵母细胞在50℃到65℃的温度自溶以释放酵母细胞壁；
(b)在pH 9到10用外源的蛋白酶温育该酵母细胞壁；以及
(c)用包含选自淀粉酶、脂肪酶及其组合的至少一种的酶温育步骤(b)的经蛋白酶处理的细胞壁；以及
(d)将步骤(c)的经酶处理的细胞壁分离成富含葡聚糖的组分和富含甘露聚糖的组分，其中所述富含葡聚糖的组分包括至少50％的β-(1，3/1，6)葡聚糖以及所述富含甘露聚糖的组分包括至少50％的甘露聚糖和至少25％的蛋白质。
17.权利要求16的方法，其中所述酵母细胞包括啤酒酵母细胞。
18.权利要求16的方法，进一步包括在动物饲料中使用步骤(c)的经酶处理的细胞壁。
19.权利要求16的方法，进一步包括在选自食品增补剂、药品、化妆品和营养辅助品的产品中使用步骤(c)的经酶处理的细胞壁。
20.权利要求16的方法，其中步骤(c)在pH 4到6进行。
21.权利要求20的方法，进一步包括在动物饲料中使用步骤(d)的富含葡聚糖组分。
22.权利要求20的方法，进一步包括在选自食品增补剂、药品、化妆品和营养辅助品的产品中使用步骤(d)的富含葡聚糖的组分。
23.权利要求20的方法，进一步包括
(e)超滤步骤(d)的富含甘露聚糖的组分，以形成滤液和渗余物。
24.权利要求23的方法，其中所述渗余物包含甘露聚糖，并且其中至少85％(w/w)的所述甘露聚糖的分子量至少为10,000Da。
25.权利要求23的方法，进一步包括在动物饲料中使用步骤(e)的滤液。
26.权利要求23的方法，进一步包括在选自食品增补剂、药品、化妆品和营养辅助品的产品中使用步骤(e)的滤液。
27.一种包含通过权利要求1或16的方法获得的α-甘露聚糖的组合物，其中总α-甘露聚糖的至少85％(w/w)具有10,000Da或更高的分子量。
28.包含权利要求27的组合物的食品增补剂、药品、化妆品或营养辅助品。
29.包含权利要求27的组合物的动物饲料。
30.权利要求29的动物饲料，其中所述动物饲料为狗、猫、猪、鱼或牛饲料。
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