CN112203519A - 纯化唾液酸乳糖的简单方法 - Google Patents
纯化唾液酸乳糖的简单方法 Download PDFInfo
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- CN112203519A CN112203519A CN201980036628.8A CN201980036628A CN112203519A CN 112203519 A CN112203519 A CN 112203519A CN 201980036628 A CN201980036628 A CN 201980036628A CN 112203519 A CN112203519 A CN 112203519A
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- sialyllactose
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- aqueous solution
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- A—HUMAN NECESSITIES
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Abstract
提供一种从其他碳水化合物中纯化唾液酸乳糖的方法,其特征在于,所述方法包括以下步骤:使包含唾液酸乳糖的水溶液使用不同的膜进行两个膜过滤步骤,所述不同的膜为截留分子量为约300道尔顿至约500道尔顿的膜以及截留分子量为约600道尔顿至约800道尔顿的膜。
Description
技术领域
本发明涉及一种纯化唾液酸乳糖(sialyllactose)的方法。更具体而言,本发明涉及一种简单且经济的方法,用于将唾液酸乳糖从其他碳水化合物例如乳糖和单糖以及更大的寡糖例如二唾液酸乳糖中分离,如果唾液酸乳糖通过微生物发酵产生,则所述二唾液酸乳糖可作为发酵液中的污染性碳水化合物存在。
背景技术
人乳被认为是婴儿发育的最佳饮食。它由脂肪、蛋白质、维生素、矿物质、微量元素和复杂寡糖组成。除乳糖外,人乳——以及其他哺乳动物的乳——还包含多种结构不同的寡糖,也称为人乳寡糖(HMO)(Usashima T.等人,(2011)Milk Oligosaccharides,NovaBiomedical Books,New York ISBN 978-1-61122-831-1)。在HMO中,观察到唾液酸化的HMO(SHMO)支持对肠道致病菌和病毒的抗性。有趣的是,最近的研究进一步证明了长链SHMO对坏死性小肠结肠炎的保护作用,坏死性小肠结肠炎是早产儿中最常见和致命的疾病之一。此外,SHMO被认为会支持婴儿的大脑发育及其认知能力。而且,唾液酸化的寡糖已显示出中和包括大肠杆菌(Escherichia coli)、霍乱弧菌(Vibrio cholerae)和沙门氏菌(Salmonella)的多种病原微生物的肠毒素。此外,发现唾液酸化的寡糖干扰幽门螺杆菌(Helicobacter pylori)对肠道的定殖,从而预防或抑制胃溃疡和十二指肠溃疡。
在唾液酸化的寡糖中,3’-唾液酸乳糖和6’-唾液酸乳糖是人乳中含量最丰富的成员。
由于唾液酸化的寡糖具有复杂的结构,因此其化学或(化学)酶促合成具有挑战性,并伴随着大量难题,例如立体化学的控制、特定连接的形成、原料的可获得性等。因此,可商购获得的唾液酸化的寡糖由于其天然来源的低量而非常昂贵。
因此,已在微生物代谢工程制备唾液酸化寡糖方面做出努力,因为这种方法是工业规模制备HMO的最有前景的方法。为了通过微生物发酵制备SHMO,通常在外源唾液酸的存在下培养微生物。
国际公开物WO 2007/101862 A1和WO 2014/153252 A1公开了工程设计细菌来产生唾液酸化寡糖的方法和组合物。然而,已知6'-唾液酸乳糖的发酵制备与其他碳水化合物例如二唾液酸乳糖的生物合成有关(Drouillard,S.等人,(2010)通过代谢工程改变的大肠杆菌表达发光杆菌属种(Photobacterium sp.)JT-ISH-224中的多功能唾液酸转移酶有效合成6'-唾液酸乳糖、6,6'-二唾液酸乳糖和6'-KDO-乳糖,Carbohydrate Research 345:1394-1399),由于在基因工程细菌中表达了唾液酸转移酶的活性而由6'-唾液酸乳糖产生所述二唾液酸乳糖。此外,高压灭菌(热处理)碳水化合物(例如乳糖)引起不希望的副产物例如羟醛产物或美拉德产物的形成,或重排反应(例如乳糖转化为乳果糖)。最终产物中特别是以较大的量存在这类污染物是不希望的,甚至是不可接受的。
此外,使用基因工程细菌制备寡糖导致发酵液被微生物产生的重组材料例如核酸分子或多肽污染。然而,当今的监管机构或消费者都无法接受重组DNA或蛋白质对人类消费产品的污染。因此,需要将重组微生物产生的任何核酸和蛋白质从所需的人乳寡糖中去除。当今重组DNA分子的检测限非常低,这需要彻底纯化方案。在基于qPCR的DNA检测中,甚至可在样品中检测到一个DNA分子。
从发酵液中纯化各寡糖的“现有技术”方法在技术上是复杂的,并且通常不经济,特别是当所述寡糖旨在用于食品应用时。为了从复杂混合物例如乳清或糖蜜中纯化二糖乳糖或蔗糖,已开发了涉及多个结晶步骤的工业规模的方法。然而,所述方法是复杂的,并且仅产生低收率。而且,这些方法不适于使通过微生物发酵获得的寡糖适用于食品工业,因为这种发酵液——不同于乳清和糖蜜——还不是食品。
然而,如果所有工艺参数都是已知的且优化的,则诸如超滤、微滤和纳滤的过滤方法在技术上容易实施。渗滤是另一种合适的方法,其涉及向溶液中添加淡水以除去膜可渗透的组分。超滤和微滤通常用于将更大的分子例如蛋白质或细胞从发酵液或水溶液中分离。此外,通过纳滤除去水、矿物质和非常小的分子是众所周知的,并且在乳制品行业中用于乳清的浓缩和去矿化。在大多数情况下,用于纳滤和超滤的膜材料基于材料例如哌嗪、聚酰胺、聚醚砜、聚乙二醇和陶瓷。膜可以组装为例如中空纤维组件、螺旋卷式组件和平板膜,并且该组装会产生不同的分离性能。通常,纳滤膜具有的截留分子量为150-300道尔顿(Dalton)。截留分子量为400-600道尔顿的膜是非常罕见的,特别是对于大规模工业制备而言。这使得寡糖的分离仍然复杂,因为需要其他分离技术,例如分批或连续的色谱法。
尽管凝胶过滤色谱法是一种方便的实验室规模方法,但只有模拟移动床色谱法代表一种适合食品生产规模的6-SL纯化的合适方法。然而,所有其他已公布的方法的问题是不能有效地将污染性碳水化合物从所需产物中去除。
因此,本发明的目的是提供一种用于从微生物发酵中纯化唾液酸乳糖的简单且具有成本效益的方法,其中可将源自重组微生物的核酸和多肽从所需的寡糖以及所述其他碳水化合物中除去。
发明内容
提供一种纯化唾液酸乳糖的方法,所述方法简单、具有成本效益且可扩展。纯化唾液酸乳糖的方法可在不使用用于唾液酸乳糖结晶的有机溶剂的情况下进行。此外,纯化唾液酸乳糖的方法可在没有不连续的色谱分离步骤的情况下进行。
唾液酸乳糖代表人乳寡糖,非常需要将其包含在婴儿配方物和医学食品中。唾液酸乳糖的高成本阻碍了它的广泛使用,特别是用于婴儿配方物中。特别地,从微生物发酵中纯化产物通常是复杂且昂贵的。而且,有机溶剂和不连续的色谱分离步骤的使用使6'-唾液酸乳糖和其他中性寡糖价格过高。而且,乙醇的使用不被某些宗教食品标准(例如清真(Halal))所接受。因此,本发明涉及一种简单的具有成本效益的方法,以使用重组加工助剂从微生物发酵中纯化唾液酸乳糖,产生适合于人类食用的唾液酸乳糖产物,特别是适合于婴儿食品和医学营养产品的唾液酸乳糖产物。
纯化方法是基于使用两种不同的膜通过过滤从污染性碳水化合物中纯化/分离唾液酸乳糖,并且包括两个膜过滤步骤,其中一个膜的截留分子量为约300至约500道尔顿,并且其中另一个膜的截留分子量为约600至约800道尔顿。
具体实施方式
提供一种从其他碳水化合物中纯化唾液酸乳糖的方法,其中所述方法包括以下步骤:使包含唾液酸乳糖和所述其他碳水化合物的水溶液使用不同的膜进行两个膜过滤步骤,其中一个膜的截留分子量为约300至约500道尔顿,并且其中另一个膜的截留分子量为约600至约800道尔顿。
截留分子量为约300至约500道尔顿的膜能够除去分子量小于唾液酸乳糖分子量的大部分碳水化合物。过滤后,SL和分子量大于SL分子量的碳水化合物被保留在渗余物中。
在一个另外的和/或替代的实施方案中,截留分子量为约300至约500道尔顿的膜的孔径为1至2nm。
合适的截留分子量小于SL分子量的膜为——例如——TriSep XN-45(TriSepCorporation,USA)、Dairy DK(Suez Water Technologies,前称为GE)和Filmtech NF270(Dow)。
截留分子量为约600至约800道尔顿的膜对唾液酸乳糖和分子量小于SL分子量的碳水化合物具有渗透性。过滤后,SL存在于滤液中,而分子量大于SL分子量的碳水化合物则保留在渗余物中。
在一个另外的和/或替代的实施方案中,截留分子量为约600至约800道尔顿的膜的孔径为2.5至3nm。
合适的对6-SL具有渗透性并在渗余物中保留分子量大于6-SL分子量的碳水化合物的膜为——例如——TangenX SIUS TFF 0.65kDa膜(Repligen Corporation)、Zirkonia组件3nm(Pervatech BV)和S-CUT YSNF-YS600(CUT/Bürkert)。
所述方法避免使用昂贵的不连续的色谱分离步骤,并且也不需要使用有机溶剂的沉淀或结晶步骤。因此,在一个另外的和/或替代的实施方案中,所述方法不包括一个或多个不连续的色谱分离步骤和/或一个或多个通过使用有机溶剂使6-SL沉淀和/或结晶的步骤。
在一个另外的和/或替代的实施方案中,所述水溶液由用于制备唾液酸乳糖的发酵或酶促方法获得。
本文中所述的方法适合于从微生物发酵或生物催化反应中以多吨量纯化人乳寡糖3'-唾液酸乳糖或6'-唾液酸乳糖,因为其在经济上可行且可扩展。
在一个另外的和/或替代的实施方案中,所述水溶液由发酵通过将生物质从发酵液中分离获得,即培养能够在培养基(发酵液)中并在允许微生物细胞产生唾液酸乳糖的条件下产生唾液酸乳糖的微生物细胞。优选地,将生物质从发酵液中分离包括至少一个超滤步骤,优选两个超滤步骤,更优选使用截留分子量为约500kDa的膜的第一超滤和使用截留分子量为约150kDa的膜的第二超滤。
在一个另外的和/或替代的实施方案中,所述水溶液用优选为H+形式的阳离子交换树脂和优选为Cl-形式的阴离子交换树脂处理。优选地,包含唾液酸乳糖和其他碳水化合物的水溶液在进行用于除去其他碳水化合物的膜过滤步骤之前用离子交换树脂处理。
在一个另外的和/或替代的实施方案中,所述方法还包括渗析步骤,优选电渗析步骤,以除去离子。优选地,在已从水溶液中除去所述其他碳水化合物之后,使包含唾液酸乳糖的水溶液进行渗析和/或电渗析。
产物可以最方便地以无菌浓缩物或喷雾干燥产物的形式提供。
所述方法能够纯化3’-唾液酸乳糖或6’-唾液酸乳糖,即将3-SL或6-SL从其他碳水化合物中分离,其中所述水溶液中唾液酸乳糖的纯度在纯化之前为≤70%、≤60%、≤50%、≤40%、≤30%、≤20%、≤10%或≤5%,和/或所述水溶液在纯化之后包含纯度为≥80%、优选≥85%或更优选≥90%的唾液酸乳糖。
在一个另外的和/或替代的实施方案中,纯化包括以下步骤:
i.)将生物质从发酵液中分离;
ii.)使无细胞发酵液进行至少一种离子交换树脂处理以除去带电荷材料,优选进行阴离子交换树脂处理和阳离子交换树脂处理;
iii.)使步骤ii.中获得的水溶液进行使用截留分子量为约300至约500道尔顿的膜的膜过滤步骤以除去分子量小于唾液酸乳糖分子量的碳水化合物;
iv.)使步骤iii.中获得的渗余物进行使用截留分子量为约600至约800道尔顿的膜的膜过滤步骤以除去分子量大于唾液酸乳糖分子量的碳水化合物;和——任选地——
v.)增加存在于步骤iv.的滤液中的唾液酸乳糖的浓度。
然而,用于纯化唾液酸乳糖的其他方法相当复杂并因此价格昂贵,本文中前述的方法主要依赖于使用两个膜过滤步骤。
某些实施方案包括一个或多个其他步骤,例如渗析步骤(用于去除盐)、电渗析(用于去除带电荷分子)、活性炭处理(用于产物溶液的脱色)和/或其他过滤过程(例如内毒素去除和/或无菌过滤)。尽管不是必需的,但是所述方法可包括用有机溶剂(例如短链醇,如甲醇)处理包含唾液酸乳糖的水溶液以使污染性寡糖沉淀,或以在吸附至活性炭后用短链醇和水混合物洗脱,和/或以使唾液酸乳糖结晶。
由所述方法得到的水溶液包含唾液酸乳糖,但不含微生物细胞的核酸和多肽。此外,所述水溶液几乎不——如果有的话——包含单糖和/或二唾液酸乳糖。
将通过特定实施方案来描述本发明,但是本发明不限于此,而是仅由权利要求书来限定。此外,说明书和权利要求书中的术语“第一”、“第二”等用于区分相似的元素,而不一定用于描述时间、空间、等级或任何其他方面的顺序。应当理解,如此使用的术语在适当的情况下是可互换的,并且本文描述的本发明的实施方案能够以不同于本文描述或说明的其他顺序来操作。
应当注意,权利要求书中使用的术语“包含”不应解释为限于其后列出的方式;其不排除其他元素或步骤。因此,应将其解释为具体指明所提及的所述特征、整体、步骤或组件的存在,但并不排除存在或增加一个或多个其他特征、整体、步骤或组件或其组合。因此,表述“包含设备A和B的装置”的范围不应限于仅由组件A和B组成的装置。这意指就本发明而言,所述装置的相关组件仅为A和B。
在整个说明书中,提及“一个(one或an)实施方案”意指关于该实施方案描述的特定特征、结构或特性包括在本发明的至少一个实施方案中。因此,在遍及本说明书的不同位置中出现的短语“在一个实施方案中”并不一定都是指相同的实施方案,但是也可以指相同的实施方案。此外,在一个或多个实施方案中,特定特征、结构或特性可以以任何合适的方式组合,这对于本领域普通技术人员而言从本公开内容是容易得知的。
类似地,应当理解,在本发明的示例性实施方案的描述中,有时将本发明的各特征在单个实施方案、附图或其描述中组合在一起,以简化公开内容并有助于对各个发明方面中的一个或多个的理解。然而,本公开内容的方法不应解释为反映了以下意图:与各权利要求中明确列出的特征相比,所要求保护的发明需要更多的特征。而是,如以下权利要求所反映的,本发明的各方面在于比单个前述公开的实施方案的所有特征更少。因此,在详述的说明书之后随附的权利要求在此被明确地纳入该详述的说明书中,其中各权利要求独立地作为本发明的单独实施方案。
此外,尽管本文所述的一些实施方案包括一些特征,而不包括其他实施方案中所包括的其他特征,但是不同实施方案的特征的组合意欲落入本发明的范围内,并且形成不同的实施方案,如本领域技术人员所理解的。例如,在下文的权利要求中,任何所要求保护的实施方案可以任意组合使用。
此外,一些实施方案在本文中被描述为可由计算机系统的处理器或通过执行该功能的其他工具来实现的方法或方法的元素的组合。因此,具有用于执行所述方法或方法元素的必要指令的处理器形成用于执行所述方法或方法元素的工具。此外,本文描述的设备实施方案的元件为用于执行这样的功能的工具的示例,所述功能由用于实现本发明的目的的元件执行。
在本文提供的描述和附图中,阐述了许多具体细节。然而,应当理解,可在没有这些具体细节的情况下进行本发明的实施方案。在其他情况下,未详细示出众所周知的方法、结构和技术,以免混淆对本说明书的理解。
现将通过对本发明的几个实施方案的详细描述来描述本发明。显然,在不脱离本发明的真实精神或技术教导的情况下,可根据本领域技术人员的知识来构建本发明的其他实施方案,本发明仅由所附权利要求书的条款限定。
实施例
实施例1:6'-唾液酸乳糖的发酵制备
使用重组的合成6'-唾液酸乳糖的大肠杆菌菌株(E.coli BL21(DE3)ΔlacZ)进行6'-唾液酸乳糖补料分批发酵,所述大肠杆菌菌株表达来自鳆发光杆菌(Photobacteriumleiognathi)JT-SHIZ-119的α-2,6-唾液酸转移酶基因plsT6。为了增强CMP-唾液酸的生物合成,将编码来自大肠杆菌的葡糖胺-6-磷酸合酶GlmS、来自集胞藻属种(Synechocystissp.)的N-乙酰葡糖胺2-差向异构酶Slr1975、来自酿酒酵母(Saccharomyces cerevisiae)的葡糖胺6-磷酸N-乙酰基转移酶Gna1、来自大肠杆菌的磷酸烯醇丙酮酸合酶PpsA、均来自空肠弯曲杆菌(Campylobacter jejuni)的N-乙酰神经氨酸合酶NeuB和CMP-唾液酸合酶NeuA的基因都通过染色体整合到大肠杆菌BL21(DE3)宿主中。此外,将编码来自大肠杆菌的乳糖通透酶LacY的基因和来自大肠杆菌W的基因cscB(蔗糖通透酶)、cscK(果糖激酶)、cscA(蔗糖水解酶)和cscR(转录调控因子)整合到BL21基因组中,使得整合基因的转录从组成型启动子(四环素启动子Ptet或PT5启动子)开始。将由基因galE(UDP-葡萄糖-4-差向异构酶)、galT(半乳糖-1-磷酸尿苷基转化酶(galactose-1-phosphate uridylyltransferase))、galK(半乳糖激酶)和galM(半乳糖-1-差向异构酶)组成并表达这些基因的功能性半乳糖操纵子(gal-operon)从大肠杆菌K12转移至BL21菌株的基因组。为了防止N-乙酰葡糖胺-6-磷酸的降解,将编码N-乙酰葡糖胺-6-磷酸脱乙酰酶(NagA)、葡糖胺-6-磷酸脱氨酶(NagB)和N-乙酰葡糖胺特异性PTS蛋白IIABC(NagE)的基因从细菌染色体中删除。此外,分别将编码大肠杆菌PTS系统中用于甘露糖、葡萄糖、葡糖胺和N-乙酰葡糖胺的糖转运蛋白的操纵子manXYZ,以及编码N-乙酰神经氨酸裂解酶、N-乙酰甘露糖胺激酶、N-乙酰甘露糖胺-6-磷酸差向异构酶和唾液酸转运蛋白的基因nanA、nanK、nanE和nanT删除。还将编码N-乙酰半乳糖胺-6-磷酸脱乙酰酶(AgaA)的基因删除。
将产生6'-唾液酸乳糖的大肠杆菌菌株在定义的矿物盐培养基中培养,所述培养基包含7g·L-1 NH4H2PO4、7g·L-1 K2HPO4、2g·L-1 KOH、0.3g·L-1柠檬酸、5g·L-1 NH4Cl、1mL·L-1消泡剂(Struktol J673,Schill+Seilacher)、0.1mM CaCl2、8mM MgSO4、微量元素和2%蔗糖作为碳源。微量元素由0.101g·L-1次氮基三乙酸(pH 6.5)、0.056g·L-1柠檬酸铁铵、0.01g·L-1MnCl2x4H2O、0.002g·L-1 CoCl2x6H2O、0.001g·L-1 CuCl2x2H2O、0.002g·L-1硼酸、0.009g·L-1 ZnSO4x7H2O、0.001g·L-1 Na2MoO4x2H2O、0.002g·L-1 Na2SeO3、0.002g·L-1 NiSO4x6H2O组成。
蔗糖进料(500g·L-1)补充有8mM MgSO4、0.1mM CaCl2、微量元素和5g·L-1 NH4Cl。为了形成6'-唾液酸乳糖,使用216g·L-1的乳糖进料。通过使用氨溶液(25%v/v)控制培养基的pH。补料分批发酵在30℃下在恒定通气和搅拌下通过采用5.5-7mL·L-1·h-1(参照起始体积计)的蔗糖进料速率进行72小时。在发酵开始后72小时,大部分加入的乳糖转化为6'-唾液酸乳糖。为了去除发酵上清液中的残留乳糖,将β-半乳糖苷酶加入到发酵容器中。
实施例2:无细胞发酵液的制备
将细胞团块(发酵液的约10%)通过如下步骤从培养基中分离出:进行超滤(截留值为0.05μm)(CUT膜技术,Erkrath,Germany),然后使用MWCO为150kDa的过滤器(Microdyn-Nadir,Wiesbaden,Germany)进行错流过滤。
实施例3:离子交换树脂处理包含6'-唾液酸乳糖的无细胞发酵液
由实施例2获得的无细胞发酵液以1000升的体积包含6'-唾液酸乳糖(纯度为9%,以干重计),并使其通过H+形式的强阳离子交换树脂(购自公司Lanxess,Germany的S2568)以除去带正电荷的污染物(离子交换剂床体积大小为200L)。用去离子水继续从离子交换树脂中洗脱6-SL。然后通过添加氢氧化钠溶液将所得溶液的pH设定为pH7。然后使溶液(无延迟)通过阴离子交换剂柱(离子交换剂的床体积为200L)。所使用的强阴离子交换剂S6368A(Lanxess,Germany)为氯化物(Cl-)形式。用去离子水继续洗脱6-SL。所得溶液再次中和至pH 7。
实施例4:第一过滤以除去较小的分子/碳水化合物
使用TriSep XN-45(TriSep Corporation,USA)纳滤膜和500L去离子水对实施例3中所述的通过离子交换树脂处理而获得的溶液进行渗滤。使用纳滤膜(RE 8040-BE,CSM-Saehan)进一步浓缩所得的溶液,其中获得纯度为31%(以干重计)且电导率为8mS/cm的6'-唾液酸乳糖溶液。
实施例5:第二过滤以除去较大的分子/碳水化合物
为了将6'-唾液酸乳糖与发酵产生的副产物二唾液酸乳糖分离,使用第二膜处理。使用标称截留值为0.65kDa的TangenX SIUS TFF膜(Repligen Corporation)进行过滤。将包含94.4%6'-唾液酸乳糖和6.6%二唾液酸乳糖(比例15:1)的溶液用所述系统进行错流过滤,并确定渗透液和渗余物中6'-唾液酸乳糖和二唾液酸乳糖的浓度。结果表明,渗透液中二唾液酸乳糖的量从6.6%显著降低至0.2%(6'-唾液酸乳糖与二唾液酸乳糖的比例为55:1),而渗余物中6'-唾液酸乳糖与二唾液酸乳糖的比例为14:1。该结果表明,通过使用额外的过滤步骤,可以将6'-唾液酸乳糖与二唾液酸乳糖分离。该观察结果也可通过使用第二种来自不同供应商的具有相似截留值的膜来证明。通过使用标称截留值为600-800Da的S-CUT YSNF-YS600-2540-M46D-ATD膜(CUT Membrane Technology GmbH),还可通过错流过滤将6'-唾液酸乳糖与二唾液酸乳糖分离。
实施例6:电渗析处理
使用装配有PC-Cell ED 1000H膜堆的PC-Cell 15电渗析装置(PC-Cell,Heusweiler,Germany)将实施例5中获得的渗透液电渗析至2mS/cm。该膜堆装配有以下膜:阳离子交换膜CEM:PK SK和阴离子交换膜AEM:PcAcid60,其中尺寸排阻限为60Da。在电渗析过程中,将0.025M氨基磺酸(胺基磺酸)溶液用作电解质。电渗析后,获得包含纯度为83%(以干重计)的6'-唾液酸乳糖的溶液。除电渗析处理外,还可以使用渗滤方法。
实施例7:浓缩和活性炭处理
使用反渗透过滤器从水溶液中除去一些水,并在45℃下旋转蒸发,以获得25%(m/v)的6'-唾液酸乳糖溶液。然后将所得的水溶液用活性炭(Norit SA2)处理。将125g活性炭用于1L 25%(m/v)的6'-唾液酸乳糖。
实施例8:无菌过滤和内毒素去除
随后,通过使实施例7中获得的溶液通过6kDa过滤器(Pall Microza超滤中空纤维组件SEP-2013,Pall Corporation,Dreieich,Germany)将所述溶液进行无菌过滤。然后将无菌溶液在室温下保存,直至喷雾干燥。
实施例9:喷雾干燥6'-唾液酸乳糖
然后使用NUBILOSA LTC-GMP喷雾干燥器(NUBILOSA,Konstanz,Germany)将如此获得(实施例8)的无菌6'-唾液酸乳糖喷雾干燥。为了喷雾干燥6'-唾液酸乳糖,使溶液在3.5巴的压力下通过设定为130℃的喷雾干燥器喷嘴,并调节流量以使排气温度保持在67℃至69℃。使用这些设置,可获得湿度低于9%的喷雾干燥的粉末。湿度含量通过Karl-Fischer滴定法测定。
实施例10:6'-唾液酸乳糖的LC-MS/MS分析
为了定量分析,使用LC三重四极MS检测系统通过MRM(多重反应监测)进行质谱分析。在四极1中选择并分析前体离子,在碰撞池中使用氩气作为CID气体进行裂解,在四极3中进行碎片离子的选择。在带有XBridge Amide保护盒(guard cartridge)(3.5μm,2.1x10mm)(Waters,USA)的XBridge Amide HPLC柱(3.5μm,2.1x50mm(Waters,USA)上进行糖类的色谱分离。HPLC系统的柱箱温度为50℃。流动相由乙腈/H2O与10mM乙酸铵组成。将1μL样品注入仪器;以400μL/min的流速运行3.60min。以ESI阳离子化模式通过MRM分析6'-唾液酸乳糖。质谱仪以单位分辨率运行。唾液酸乳糖形成m/z 656.2[M+Na]的离子。唾液酸乳糖的前体离子在碰撞池中进一步破碎成碎片离子m/z 612.15、m/z 365.15和m/z 314.15。碰撞能量、Q1和Q3预偏置(Pre Bias)分别针对每种分析物进行优化。使用市售的标准品(Carbosynth,Compton,UK)建立定量方法。
Claims (8)
1.一种从其他碳水化合物中纯化唾液酸乳糖的方法,其特征在于,所述方法包括以下步骤:使包含唾液酸乳糖和其他碳水化合物的水溶液使用不同的膜进行两个膜过滤步骤,其中
-一个膜的截留分子量为约300至约500道尔顿,并且
-另一个膜的截留分子量为约600至约800道尔顿。
2.根据权利要求1所述的方法,其中所述水溶液由用于制备唾液酸乳糖的发酵或酶促方法获得。
3.根据权利要求1或2所述的方法,其中所述水溶液由发酵通过将生物质从发酵液中分离而获得。
4.根据权利要求3所述的方法,其中将生物质从发酵液中分离包括至少一个超滤步骤,优选两个超滤步骤,更优选使用截留分子量为约500kDa的膜的第一超滤和使用截留分子量为约150kDa的膜的第二超滤。
5.根据权利要求1至5中任一项所述的方法,其中包含唾液酸乳糖的水溶液用优选为H+形式的阳离子交换树脂和优选为Cl-形式的阴离子交换树脂处理,更优选在所述水溶液进行膜过滤步骤之前处理。
6.根据权利要求1至6中任一项所述的方法,其中所述方法还包括渗析步骤,优选电渗析步骤。
7.根据权利要求1至6中任一项所述的方法,其中在纯化之前所述水溶液中唾液酸乳糖的纯度为≤70%、≤60%、≤50%、≤40%、≤30%、≤20%、≤10%或≤5%,和/或在纯化之后所述水溶液包含纯度为≥80%、优选≥85%或更优选≥90%的唾液酸乳糖。
8.根据权利要求1至7中任一项所述的方法,其中所述唾液酸乳糖为3’-唾液酸乳糖或6’-唾液酸乳糖。
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KR20210025007A (ko) | 2021-03-08 |
CN112203519B (zh) | 2023-09-15 |
ES2933959T3 (es) | 2023-02-15 |
US20210212335A1 (en) | 2021-07-15 |
AU2019276095A1 (en) | 2020-12-24 |
JP2021526024A (ja) | 2021-09-30 |
SG11202011734QA (en) | 2020-12-30 |
EP3801037A1 (en) | 2021-04-14 |
MX2020012855A (es) | 2021-02-17 |
FI3801037T3 (fi) | 2023-01-13 |
WO2019229118A1 (en) | 2019-12-05 |
PH12020552067A1 (en) | 2021-07-05 |
PL3801037T3 (pl) | 2023-01-23 |
JP7356460B2 (ja) | 2023-10-04 |
AU2019276095B2 (en) | 2024-05-02 |
BR112020024463A2 (pt) | 2021-03-16 |
EP3801037B1 (en) | 2022-10-19 |
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