CN111447935A - 用于预防或治疗沙门氏菌病的组合物 - Google Patents
用于预防或治疗沙门氏菌病的组合物 Download PDFInfo
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- CN111447935A CN111447935A CN201780097437.3A CN201780097437A CN111447935A CN 111447935 A CN111447935 A CN 111447935A CN 201780097437 A CN201780097437 A CN 201780097437A CN 111447935 A CN111447935 A CN 111447935A
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Abstract
本发明涉及用于预防或治疗动物中的沙门氏菌病的用途的组合物,所述用途包括将所述组合物施用至所述动物,其中所述组合物包含至少0.01重量%的鼠李糖半乳糖醛酸聚糖I(RG‑I)多糖的干物质,所述鼠李糖半乳糖醛酸聚糖I(RG‑I)多糖具有大于2kDa的分子量并且具有由半乳糖醛酸残基和鼠李糖残基组成的主链,所述鼠李糖残基包含在α(1→4)‑半乳糖醛酸‑α(1→2)‑鼠李糖残基中,其中所述RG‑I多糖中的半乳糖醛酸残基与鼠李糖残基的摩尔比在50:1至1:1的范围内。RG‑I多糖在治疗或预防沙门氏菌病中的应用为抗生素的广泛应用提供了替代选择。此外,这些RG‑I多糖可以用于预防或治疗由对抗生素具有抗性的沙门氏菌菌株引起的沙门氏菌病。
Description
本发明的技术领域
本发明涉及动物中的沙门氏菌病的预防。更具体地,本发明涉及通过向这样的动物施用包含鼠李糖半乳糖醛酸聚糖I(rhamnogalacturonan I,RG-I)多糖的组合物来预防或治疗沙门氏菌病,所述鼠李糖半乳糖醛酸聚糖I(RG-I)多糖具有大于2kDa的分子量并且具有由半乳糖醛酸残基和鼠李糖残基组成的主链,所述鼠李糖残基包含在α(1→4)-半乳糖醛酸-α(1→2)-鼠李糖残基中,其中所述RG-I多糖中的半乳糖醛酸残基与鼠李糖残基的摩尔比在50:1至1:1的范围内。
上述RG-I多糖可以分离自水果和蔬菜,如苹果、菜椒、越桔、胡萝卜、柑橘、葡萄、甜菜、橄榄、豌豆或马铃薯。
发明背景
沙门氏菌病是由两种沙门氏菌(肠道沙门氏菌(Salmonella enterica)和邦戈尔沙门氏菌(Salmonella bongori))的有机体引起的人类和动物的感染性疾病。尽管沙门氏菌主要是肠道细菌,但它们在环境中广泛存在,并且通常存在于农场废水、人类污水和任何受到粪便污染的物质中。沙门氏菌有机体是人类腹泻和全身感染的病因,最常见的是作为源自动物和环境的食物的二级污染物,通常是由于食用动物的亚临床感染导致肉、蛋和乳的污染或者已经用粪便废弃物施肥或灌溉的水果和蔬菜的二级污染。
人类沙门氏菌病是最常见和经济上重要的人畜共患病之一。沙门氏菌有机体也可以存在于动物饲料中,引起动物,特别是家禽和猪的亚临床胃肠道运输或感染性疾病。所有国家都已经认识到沙门氏菌病,但该沙门氏菌病似乎在集约化畜牧业领域中是最普遍的,尤其是在猪和小牛以及在限制范围内饲养的一些类型的家禽中。几种血清型是宿主特异性的(例如绵羊中的流产沙门氏菌(S.abortusovis)或人类中的伤寒沙门氏菌(S.typhi))或宿主适应性的(例如猪中的猪霍乱沙门氏菌(S.choleraesuis)和牛中的都柏林沙门氏菌(S.dublin))。
该病可以影响所有种类的家畜;幼年动物以及怀孕和泌乳的动物是最易感的。肠道疾病是最常见的临床表现,但可以观察到广泛的临床症状,包括急性败血病、流产、关节炎和呼吸疾病。沙门氏菌感染可以不利地影响消化,并且可以损害幼年动物的生长。许多动物,尤其是猪和家禽也可以被感染,但没有显示出临床疾病。这样的动物可能对成群的牛羊(flocks and herds)之间的感染传播以及作为食物污染和人类感染的来源很重要。
WHO提供了关于开发用于预防和控制人类食源性疾病(包括沙门氏菌感染)的适当措施的信息。最常见的感染媒介物是蛋和蛋制品、家禽肉和来自其它食用动物的肉以及肉制品。在许多欧洲国家,肠炎沙门氏菌(Salmonella enteritidis)和鼠伤寒沙门氏菌(Salmonella.typhimurium)是最广泛的血清型。在北美,鼠伤寒沙门氏菌是主要的血清型。
在猪中出现了鼠伤寒沙门氏菌的单相变体(S.4,5,12:i:-DT193,对氨苄青霉素、链霉素、磺酰胺和四环素具有抗性),并且在世界范围内的几个国家中引起了人类沙门氏菌病的爆发,并且存在具有不同抗性模式的几种其它新兴的单相S.4,5,12:i:-和S.4,12:i:-菌株,近年来,已经在许多国家的各种动物物种和人类中认识到所述菌株。
广泛的致病原内的抗微生物抗性是国家和多个部门广泛关注的日益增长的公共卫生威胁。世界各国政府越来越开始关注这一严重问题,以致于威胁着现代医学的成就。后抗生素时代-其中常见的感染和轻微的损伤可以杀人-在21世纪完全不是世界末日幻想,而是有非常现实的可能性。
在畜牧业-包括牲畜、家禽和鱼类养殖-中使用抗生素导致日益增加的认识到需要采取紧急措施以避免不适当的使用,并且减少在畜牧业和水产养殖以及在人类中的抗生素使用。
Petersen等人(Some putative prebiotics increase the severity ofSalmonella enterica serovar typhimurium infection in mice,BMC Microbiology(2009),9:245)描述了研究的结果,其中检查了小鼠中的鼠伤寒沙门氏菌SL1344感染是否可以通过施用具有不同结构和消化性分布的膳食碳水化合物来预防。在口服沙门氏菌攻击(107CFU)之前,给BALB/c小鼠喂食含有10%的以下碳水化合物中的任一种的饮食,持续三周,并与喂食基于玉米淀粉的对照饮食的小鼠进行比较:菊粉、低聚果糖、低聚木糖、低聚半乳糖、苹果果胶、聚葡萄糖或β-葡聚糖。与喂食基于玉米淀粉的对照饮食的小鼠相比,喂食含低聚果糖或低聚木糖的饮食的小鼠在肝、脾和肠系膜淋巴结中具有显著更高(P<0.01和P<0.05)数量的鼠伤寒沙门氏菌SL1344。在用补充有苹果果胶的饮食喂食的小鼠的回肠和粪便内容物中检测到显著增加的沙门氏菌的量(P<0.01),然而这些小鼠在肝、脾和淋巴结中没有显示出显著高于来自对照组的动物的鼠伤寒沙门氏菌的数量(P<0.20)。
Parsons等人(Dietary Supplementation with Soluble Plantain Non-StarchPolysaccharides Inhibits Intestinal Invasion of Salmonella typhimurium in theChicken,PLOS ONE,2014年2月,第9卷,第2期,e87658)报道了膳食车前草纤维防止鸡肠粘膜被沙门氏菌侵入。用来自孵化的喂食含有可溶性车前草NSP的颗粒料(pellet diet)(0至200mg/d)的小鸡进行体内实验,并在8日龄时口服感染鼠伤寒沙门氏菌4/74。用车前草NSP50mg/d的体内饮食补充将鼠伤寒沙门氏菌的侵入减少了98.9%(95%CI,98.1-99.7;p<0.0001),如通过来自脾组织的活细菌计数所反映的。体外研究证实了车前草NSP(5-10mg/ml)抑制鼠伤寒沙门氏菌4/74对猪上皮细胞系的粘附(73%的平均抑制(95%CI,64-81);P,0.001)和对初生鸡盲肠隐窝的粘附(82%的平均抑制(95%CI,75-90);P,0.001)。车前草NSP的抑制活性主要存在于酸性/果胶(富含同型半乳糖醛酸聚糖的)组分中。
Aziz(The effect of the Capsicum annuum in the diet of broilers on theisolation and shedding rate of Salmonella paratyphoid,Kufa Journal ForVeterinary Medical Sciences Vol.(1)No.(1)2010,28-38)描述了一项研究,其中研究了作为对抗肉鸡中鼠伤寒沙门氏菌攻击剂量的抗生素的可能替代选择的辣椒(Capsicumannuum)的抑制作用。结果显示,使用具有1%和2%的百分比的辣椒的混合饮食对鼠伤寒沙门氏菌感染是有效的。
WO 2011/136634描述了果胶降解产物用于预防和/或治疗患有沙门氏菌感染或易受沙门氏菌感染的对象的体重减轻中的用途。
US 2010/0047209描述了减轻农场动物中的沙门氏菌感染的益生菌组合物,所述益生菌组合物包含一种或多种微生物培养物,所述微生物培养物选自鼠乳杆菌(Lactobacillus murinus)、戊糖乳杆菌(L.pentosus)、唾液乳杆菌唾液亚种(L.salivarius sub-species salivarius)和戊糖片球菌(Pediococcus pentosaceus)。
果胶是存在于陆生植物的初生细胞壁中的结构杂多糖。
果胶多糖是包含不同量的以下多糖组分的异质多糖组:
(i)同型半乳糖醛酸聚糖(HG),
(ii)木糖半乳糖醛酸聚糖(XG)
(iii)芹半乳糖醛酸聚糖(AG)
(iv)鼠李糖半乳糖醛酸聚糖-1(RG-I)和
(v)鼠李糖半乳糖醛酸聚糖-II(RG-II)。
图1提供了果胶多糖(包括上述5种多糖组分)的结构的示意图。注意多糖组分AG、XG和RG-II通常仅代表一小部分的果胶多糖。
多糖组分HG、AG、XG和RG-II各自包含由α-(1-4)-连接的D-半乳糖醛酸单糖单元的直链组成的主链。
只有RG-I包含由重复二糖单元的直链组成的主链:4)-α-D-半乳糖醛酸-(1,2)-α-L-鼠李糖-(1.RG-I的结构的示意图显示在图2中。
果胶多糖的组成和精细结构根据植物来源和应用的提取条件而变化很大。同型半乳糖醛酸聚糖结构域可以具有高达约100个连续D-GalA残基的长度。含有侧链的RG-I结构域通常被称为‘分枝区’或‘毛状区’,而同型半乳糖醛酸聚糖结构域(与RG-I结构域连接)通常不被糖苷/或糖苷侧链取代。
RG-I中的GalA残基经由第1位和第4位与Rha残基连接,而Rha残基经由端基(anomeric)和O-2位与GalA残基连接。通常,约20-80%的Rha残基在O-4位支化(取决于植物来源和分离方法),具有中性和酸性侧链。这些侧链主要由以各种方式连接的Ara和Gal残基组成,构成称为阿拉伯聚糖(arabinans)、阿拉伯半乳聚糖I(AG-I)和/或AG-II的聚合物。AGI由在α-L-阿拉伯糖基的O-3处具有取代的β-(1,4)-连接的D-Gal主链组成;Gal主链可以具有中间的(interspacing)α(1,5)-L-Ara单元。AG-II由高度分枝的半乳聚糖组成,其中主要为内部β(1,3)-连接的D-Gal在外部具有短的(1,6)-连接的链的取代。后者还具有(1,3)-和/或α(1,5)-连接的L-Ara的连接。侧链可以是直链或支链的,并且这些侧链中的一些可以用α-L-岩藻糖苷、β-D-葡糖苷酸和4-O-甲基β-D-葡糖苷酸残基终止。
WO2011/069781描述了能够调节免疫应答的多糖,所述多糖获自茶树(Camelliasinensis)物种,其中所述多糖的主链包含交替的鼠李糖半乳糖醛酸聚糖-1结构域和α(1,4)-连接的聚半乳糖醛酸或α(1,4)-连接的寡聚半乳糖醛酸结构域,其中所述多糖的主链中的半乳糖醛酸残基与鼠李糖基残基的摩尔比范围为2.5:1至1:1,并且其中所述多糖具有至少70kDa的分子量。
发明内容
发明人已经发现,通过向动物施用组合物可以非常有效地预防或治疗该动物中的沙门氏菌病,所述组合物包含至少0.01重量%的鼠李糖半乳糖醛酸聚糖I(RG-I)多糖的干物质,所述鼠李糖半乳糖醛酸聚糖I(RG-I)多糖具有大于2kDa的分子量并且具有由半乳糖醛酸残基和鼠李糖残基组成的主链,所述鼠李糖残基包含在α(1→4)-半乳糖醛酸-α(1→2)-鼠李糖残基中,其中所述RG-I多糖中的半乳糖醛酸残基与鼠李糖残基的摩尔比在50:1至1:1的范围内。
因此,本发明的一个方面涉及含有至少0.01重量%的RG-I多糖的干物质的组合物在预防或治疗动物中的沙门氏菌病中的用途,所述用途包括向动物施用所述组合物。
RG-I多糖在预防或治疗沙门氏菌病中的应用为抗生素的广泛应用提供了替代选择。此外,这些RG-I多糖可以用于预防或治疗由对抗生素具有抗性的沙门氏菌菌株引起的沙门氏菌病。
根据本发明使用的RG-I多糖可以合适地从水果和蔬菜中分离,如苹果、菜椒、越桔、胡萝卜、柑橘、葡萄、甜菜、橄榄、豌豆和马铃薯。WO 2012/148277描述了可以用于分离RG-I多糖的分离方法。
本发明的另一方面涉及制备可食用组合物的方法,所述方法包括将一种或多种可食用成分与多糖组分混合,所述多糖组分包含至少5重量%的RG-I多糖的干物质。
发明详述
本发明的第一方面涉及用于预防或治疗动物中的沙门氏菌病的用途的组合物,所述用途包括向所述动物施用所述组合物,其中所述组合物包含至少0.01重量%的鼠李糖半乳糖醛酸聚糖I(RG-I)多糖的干物质,所述鼠李糖半乳糖醛酸聚糖I(RG-I)多糖具有大于2kDa的分子量并且具有由半乳糖醛酸残基和鼠李糖残基组成的主链,所述鼠李糖残基包含在α(1→4)-半乳糖醛酸-α(1→2)-鼠李糖残基中,其中所述RG-I多糖中的半乳糖醛酸残基与鼠李糖残基的摩尔比在50:1至1:1的范围内。
本文所用的术语“沙门氏菌病”是指由沙门氏菌属细菌引起的感染。
本文所用的术语“沙门氏菌属”是指肠杆菌科的杆状(芽孢杆菌)革兰氏阴性细菌的属。沙门氏菌属的两种物种是肠道沙门氏菌和邦戈尔沙门氏菌。在温血动物和环境中发现了肠道沙门氏菌亚种。邦戈尔沙门氏菌限于冷血动物。
本文所用的术语“动物”是指脊椎动物,包括人。
本文所用的术语“支链多糖”是指包含通过糖苷键结合在一起的单糖单元的线性主链的多糖,其中主链内的至少一个单糖单元携带一个或多个糖苷连接的单糖单元的侧链。
如本文所用,术语“主链(backbone chain)”和“主链(backbone)”是同义词。
本文所用的术语“果胶多糖”是指分子量大于2kDa并且包含由半乳糖醛酸残基和鼠李糖残基组成的主链的任选分支的多糖,所述鼠李糖残基包含在α(1→4)-半乳糖醛酸-α(1→2)-鼠李糖残基中。
本文所用的术语“区段”是指多糖主链内两个糖苷键连接的单糖单元的序列,不包括与其连接的任何侧链。
本文所用的术语“结构域”是指区段加上与所述区段连接的任何侧链。
术语“鼠李糖半乳糖醛酸聚糖-I区段”或“RG-I区段”是指由半乳糖醛酸(GalA)和鼠李糖(Rha)对组成的区段,其中GalA残基经由第1位和第4位与Rha残基连接,而Rha残基经由端基和O-2位与GalA残基连接,即交替的α(1→4)-半乳糖醛酸-α(1→2)-鼠李糖残基。RG-I结构域可以包含侧链,如例如半乳聚糖、阿拉伯聚糖和阿拉伯半乳聚糖侧链。
术语“鼠李糖半乳糖醛酸聚糖-I多糖”或“RG-I多糖”是指任选分支的果胶多糖,其包含含有一个或多个鼠李糖半乳糖醛酸聚糖-I区段的主链。
术语“α(1,4)-连接的半乳糖醛酸区段”是指由α(1→4)-半乳糖醛酸残基组成的区段。
除了RG-I结构域之外,本发明的RG-I多糖还可以包含一个或多个以下结构域:
·同型半乳糖醛酸聚糖(HG),
·木糖半乳糖醛酸聚糖(XG),
·芹半乳糖醛酸聚糖(AG),
·鼠李糖半乳糖醛酸聚糖-II(RG-II)。
结构域XG、AG和RG-II通常仅占RG-I多糖的一小部分。
任选地存在于本发明的RG-I多糖中的HG结构域、XG结构域、AG和RG-II结构域包含由两个或更多个α-(1-4)-连接的D-半乳糖醛酸的线性链组成的主链。
HG结构域不包含任何侧链。HG结构域的主链内的半乳糖醛酸残基的羧基可以被酯化。半乳糖醛酸可以被甲基或乙酰基酯化,分别形成甲酯或乙酰酯。
XG结构域的主链包含一个或多个D-木糖形式的侧链。
AG结构域的主链包含由一个或多个D-芹菜糖残基组成的一个或多个侧链。
RG-II的主链包含仅由D-木糖或D-芹菜塘组成的一个或多个侧链。RG-II结构域的主链内的半乳糖醛酸残基的羧基可以被酯化。半乳糖醛酸可以被甲基或乙酰基酯化,分别形成甲酯或乙酰酯。
术语“乙酰化程度”是指每个半乳糖醛酸残基的乙酰基残基的数目,以百分比表示。
术语“甲基化程度”是指每个半乳糖醛酸残基的甲基残基的数目,以百分比表示。
可以通过本领域技术人员已知的分析技术来确定不同多糖的浓度及其单糖组成。酸水解(甲醇分解)后,中性糖的单糖组成可以通过高效阴离子交换色谱结合脉冲安培检测(HPAEC-PAD)适当地确定。
通过使用比色间羟基二苯基测定来确定糖醛酸(半乳糖醛酸是糖醛酸的主要成分)。
可以使用折射率(RI)检测(浓度),通过高效尺寸排阻色谱法来确定分子大小分布。
以上提及的分析方法描述于:Analytical Biochemistry,第207卷,第1期,1992,pg 176(用于中性糖分析)和Mol.Nutr.Food Res.,第61卷,第1期,2017,1600243(用于糖醛酸分析和分子大小分布)。
除非另有说明,否则本文提及的所有百分比是指重量百分比。
本发明涵盖RG-I多糖组合物用于预防或治疗沙门氏菌病的用途。在本发明的特别优选的实施方案中,将含有RG-I多糖的组合物施用至动物以预防沙门氏菌病。
当将RG-1多糖组合物施用至尚未感染沙门氏菌病的动物时,本发明的益处尤其明显。因此,可以预防由沙门氏菌病引起的临床疾病,或者至少可以降低临床疾病的严重程度。
根据本发明的沙门氏菌病的预防或治疗可以有利地用于农场动物以优化饲料利用和生长。
含有RG-I多糖的组合物优选包含至少0.03重量%,更优选0.1-10重量%,最优选0.2-5重量%的本文所限定的RG-I多糖的干物质。
如果RG-I多糖不再缠在细胞壁材料的基质中,则认为它在预防或治疗沙门氏菌病中是特别有效的。因此,在特别优选的实施方案中,本发明的含有RG-I多糖的组合物包含至少0.01重量%的干物质,更优选至少0.03重量%的干物质,甚至更优选至少0.1重量%的干物质,并且最优选至少0.2重量%的易溶于水的RG-I多糖的干物质。可以通过将100ml去矿物质水(20℃)与足够量的含有RG-I多糖的组合物组合以提供2.5克干物质,随后搅拌5分钟以及在100μm过滤器上过滤来确定易溶于水的RG-I多糖在含有RG-I多糖的组合物中的浓度。滤液中的RG多糖是易溶于水的RG-I多糖。
根据本发明的特别优选的实施方案,本发明的组合物在至少3天的时间段内以提供至少1mg RG-I多糖/kg体重/天的量每天至少一次施用至动物。更优选地,通过每天至少一次施用含有RG-I多糖的组合物,在至少7天的时间段内,最优选在至少14天的时间段内提供至少10mgRG-I多糖/kg体重/天,甚至更优选至少100mg/kg体重/天,最优选至少250mgRG-I多糖/kg体重/天的量。
根据另一个优选的实施方案,将含有RG-I多糖的组合物在至少21天的时间段内施用至动物,以提供至少1mg RG-I多糖/kg体重/天,更优选50-500mg RG-I多糖/kg体重/天的量的RG-I多糖。
施用至动物的含有RG-I多糖的组合物优选是含有蛋白质、脂肪和矿物质的食物或饲料组合物。通常,对于动物,如猪、小牛和家禽,组合物含有10-60en%的蛋白质和5-50en%的脂肪以及15-90en%的碳水化合物和0-10wt.%的灰分。
含有RG-I多糖的组合物优选包含维生素的组合。更具体地,所述组合物包含:
·1,000-2,500IU/kg的维生素A;
·0.1-10mcg/kg的维生素B12;
·100-350IU/kg的维生素D;
·2-20IU/kg的维生素E;和
·0.25-0.60mg/kg维生素K。
根据另一个优选的实施方案,将含有RG-I多糖的组合物施用至尚未患有沙门氏菌病的动物。如实施例中所示,当在用沙门氏菌感染之前施用含有RG-I多糖的组合物时,所述组合物在增加动物从沙门氏菌病快速恢复的能力方面是特别有效的。
本发明的含有RG-I多糖的组合物可以有效地用于预防或治疗由肠道沙门氏菌引起的沙门氏菌病。甚至更优选地,所述组合物用于预防或治疗由肠炎沙门氏菌或鼠伤寒沙门氏菌引起的沙门氏菌病。因此,本发明的一个有利的实施方案涉及预防或治疗由肠炎沙门氏菌引起的沙门氏菌病。根据另一个有利的实施方案,本发明涉及预防或治疗由鼠伤寒沙门氏菌引起的沙门氏菌病。
根据优选的实施方案,含有RG-I多糖的组合物用于预防或治疗农场动物中的沙门氏菌病。根据特别优选的实施方案,所述组合物用于预防或治疗家禽,例如鸡、鸭、鹅或火鸡的沙门氏菌病。根据另一个特别优选的实施方案,所述组合物用于预防或治疗猪的沙门氏菌病。
根据另一个优选的实施方案,含有RG-I多糖的组合物用于预防或治疗人的沙门氏菌病。优选地,含有RG-I多糖的组合物用于治疗人的沙门氏菌病。
根据本发明使用的RG-I多糖优选获自植物来源,其选自苹果、菜椒、越桔、胡萝卜、柑橘、葡萄、马铃薯、甜菜和橄榄以及以上的组合。更优选地,RG-I多糖获自植物来源,其选自苹果(例如苹果渣)、菜椒、胡萝卜(例如胡萝卜浆)、柑橘(例如皮)、葡萄、甜菜(例如甜菜浆)、橄榄(例如橄榄浆)、豌豆(例如豌豆浆)、马铃薯(例如马铃薯浆)以及以上的组合。甚至更优选地,RG-I多糖获自植物来源,其选自苹果、胡萝卜、橄榄以及以上的组合。最优选地,RG-I多糖获自苹果和/或胡萝卜。
RG-I多糖具有包含鼠李糖半乳糖醛酸聚糖-I区段和任选地α(1,4)-连接的同型半乳糖醛酸区段的主链。RG-I多糖中的半乳糖醛酸残基与鼠李糖残基的摩尔比在20:1至1:1的范围内。优选地,RG-I多糖中的半乳糖醛酸残基与鼠李糖残基的摩尔比范围为15:1至1:1,更优选12:1至1:1,甚至更优选10:1至1:1,最优选9:1至1:1。
优选地,鼠李糖残基占RG-I多糖主链中的单糖残基的3-50%,更优选5-50%,并且最优选10-50%。
鼠李糖残基通常占RG-I多糖中包含的所有单糖残基(即包括侧链中包含的单糖残基)的3-50%,更优选3.5-40%,并且最优选4-35%。
半乳糖醛酸残基通常占RG-I多糖主链中的单糖残基的50-97%,更优选50-95%,并且最优选50-90%。
半乳糖醛酸残基通常占RG-I多糖中包含的所有单糖残基(即包括侧链中包含的单糖残基)的10-80%,更优选15-70%,并且最优选20-65%。
阿拉伯糖残基通常占胡萝卜RG-I多糖中包含的所有单糖残基的4-38%,更优选6-36%,并且最优选8-34%。
半乳糖残基通常占胡萝卜RG-I多糖中包含的所有单糖残基的4-42%,更优选8-40%,并且最优选10-38%。
RG-I多糖通常具有至少10kDa的分子量。优选地,RG-I多糖具有15kDa至2,000kDa,更优选20kDa至1,500kDa,甚至更优选35kDa至1,200kDa,最优选40kDa至1,000kDa的分子量。
包含在本发明组合物中的RG-I多糖的平均分子量优选超过30kDa,更优选超过40kDa,并且最优选超过60kDa。
优选地,RG-I多糖中少于85%的半乳糖醛酸残基以甲酯的形式酯化。更优选地,RG-I多糖具有0%至70%,更优选0%至60%,甚至更优选0%至55%,最优选0%至50%的酯化度。
优选地,RG-I多糖中0-95%的半乳糖醛酸残基以乙酰酯的形式酯化。更优选地,RG-I多糖具有在5%至90%,更优选7%至50%,最优选8%至30%的酯化度。
RG-I多糖的主链可以包含一个或多个侧链。这些侧链可以包含阿拉伯糖和/或半乳糖的残基,以及少量的单体岩藻糖、葡萄糖、葡糖醛酸、木糖和/或糖醛酸的残基。一个或多个侧链优选选自半乳聚糖侧链、阿拉伯聚糖侧链和阿拉伯半乳聚糖侧链。
阿拉伯糖聚糖侧链包含至少一个或多个α(1,5)-连接的阿拉伯糖残基,并且在RG-I结构域中的鼠李糖残基的O-4位是取代的。阿拉伯聚糖侧链可以是直链或支链的。如果侧链是直链的,则侧链由α(1,5)-连接的阿拉伯糖残基组成。如果阿拉伯聚糖侧链是分支的侧链,则一个或多个α-阿拉伯糖残基与α(1,5)-连接的阿拉伯糖的O-2和/或O-3连接。
半乳聚糖侧链包含至少一个或多个β(1,4)-连接的半乳糖残基并且在RG-I结构域中的鼠李糖残基的O-4位是取代的。
阿拉伯半乳聚糖侧链在RG-I结构域的鼠李糖残基的O-4位是取代的,并且可以是I型阿拉伯半乳聚糖(AGI)或II型阿拉伯半乳聚糖(AGII)。AGI由(1→4)-β-D-Galp主链组成,在该主链上可以在O-6位或在O-3位发生单体Galp单元的取代。AGI进一步被α-L-Araf-p残基和/或(1→5)-α-L-Araf短的侧链取代。AGII由用(1→6)-β-D-Galp二级链修饰的α(1→3)-β-D-Galp主链组成,所述二级链被阿拉伯糖基化(arabinosylated)。
优选地,阿拉伯糖残基和鼠李糖残基以小于30:1,更优选小于15:1,甚至更优选小于8:1,并且最优选小于5:1的摩尔比存在于RG-I多糖中。
半乳糖残基和鼠李糖残基优选以小于30:1,更优选小于15:1,甚至更优选小于8:1,并且最优选小于5:1的摩尔比存在于RG-I多糖中。
一方面阿拉伯糖残基和半乳糖残基与另一方面鼠李糖残基的组合通常以小于40:1,更优选小于20:1,并且最优选小于10:1的摩尔比存在于RG-I多糖中。
RG-I多糖优选以富含RG-I多糖的果胶多糖分离物的形式掺入含有RG-I多糖的组合物中。因此,在特别优选的实施方案中,RG-I多糖占含有RG-I多糖的组合物中存在的果胶多糖的至少10wt.%,更优选至少20wt.%,甚至更优选至少30wt.%,并且最优选至少40wt.%。
通常,半乳糖醛酸残基、鼠李糖残基、阿拉伯糖残基和半乳糖残基的组合占糖中存在的单糖残基的至少30mol.%,所述糖包含在含有RG-I多糖的组合物中。更优选地,该组合占所述糖中的单糖残基的至少40mol.%,甚至更优选至少45mol.%,更优选至少50mol.%,并且最优选至少70mol.%。
在特别优选的含有RG-I多糖的组合物中,半乳糖醛酸残基和鼠李糖残基的组合占糖中存在的单糖残基的至少25mol.%,更优选30-90mol.%,最优选35-70mol.%,所述糖包含在含有RG-I多糖的组合物中。
本发明的另一方面涉及制备可食用组合物的方法,所述方法包括将一种或多种可食用成分与多糖组分混合,所述多糖组分包含至少5重量%,更优选至少10重量%,并且最优选至少15重量%的上文所述的RG-I多糖的干物质。
根据特别优选的实施方案,RG-I多糖占多糖组分中存在的果胶多糖的至少20wt.%,更优选至少40wt.%,甚至更优选至少50wt.%并且最优选至少60wt.%。
多糖组分优选是获自植物来源的分离物,所述植物来源选自苹果、菜椒、越桔、胡萝卜、柑橘、葡萄、甜菜、橄榄、豌豆、马铃薯以及以上的组合。
根据另一个优选的实施方案,多糖组分的水含量小于6wt.%,更优选小于4wt.%,并且最优选小于2wt.%。
多糖组分优选是颗粒材料,例如粉末、粒状或球状材料。
多糖组分优选包含小于50重量%,更优选小于40重量%,并且最优选小于25重量%的糖的干物质,所述糖选自葡萄糖、果糖、蔗糖以及以上的组合。
本发明的又一方面涉及通过本发明方法制备的可食用组合物。
优选地,可食用组合物包含10-60en.%的蛋白质和5-50en.%的脂肪及15-90en.%的碳水化合物。
根据另一个优选的实施方案,可食用组合物包含:
·1000-2500IU/kg的维生素A;
·0.1-10mcg/kg的维生素B12;
·100-350IU/kg的维生素D;
·2-20IU/kg of的维生素E;和
·0.25-0.60mg/kg的维生素K。
通过以下非限制性实例进一步说明本发明。
实施例
实施例1
使用以下描述的程序,从中试规模的干菜椒粉(Paprika Mild 80-100AtsaSteamtr-Felix Reverte S.A.)中分离RG-I多糖级分。
将菜椒材料(100kg)在温和转向下用80%的乙醇水溶液洗涤三次,即在80℃洗涤两次,持续2小时,然后在室温洗涤过夜;每次使用12.5%(w/v),以去除乙醇可溶材料。每次通过离心(1000G持续10min)回收乙醇不溶性残余物。将在3个洗涤循环之后获得的乙醇不溶性残余物干燥,并且将90kg用具有95℃的温度的1000L热水萃取两次,持续90分钟。每次,在1000G离心10分钟后保留上清液。随后将收集的上清液通过布过滤,并使用2KDa分子量截留膜进行超滤以去除小分子量材料。通过干燥截留物获得干燥的富含RG-I的提取物,产生约5kg干燥的富含RG-I的多糖提取物。
富集RG-I多糖的提取物的表征
在酸水解(甲醇分解)后测定提取物的单糖组成。使用高效阴离子交换色谱结合脉冲安培检测(HPAEC-PAD)分析中性单糖。使用比色间羟基二苯基测定来确定糖醛酸(半乳糖醛酸是糖醛酸的主要成分)。
乙酰化和甲基化的程度如下测定:
用氢氧化钠(0.1M,过夜,20℃)处理多糖样品(2-5mg)。通过使用配备有DB-WAXETR柱,Cryo Focus-4冷阱和FID检测的顶空GC测量释放的甲醇(改编自Huisman等人,FoodHydrocolloids,18,4,2004,665-668)。将样品中和(1M HCl),然后通过使用配备有带保护柱和RI检测的Aminex HPX 87H柱的HPLC定量释放的乙酰基(改编自Voragen等人,FoodHydrocolloids,1,1,1986,65-70)。将具有已知的甲基化和乙酰化程度的甜菜果胶用作标准。酯化度表示为释放的甲醇和乙酸的摩尔量占糖醛酸的量的百分比。
提取物的分子特征显示在表1中。
表1
实施例2
6周龄的无特定病原体的雌性C57BL/6小鼠随意接受无菌饮用水和含有30mmol/kg钙(未感染组和感染组)的半合成辐照的AIN-93G饮食(Research Diet Services,Wijk bijDuurstede,The Netherlands)或者补充有1%(w/w)的来自菜椒的富含RG-I的提取物的相同食物。
将小鼠随机分配到处理组中,并且每笼饲养3只小鼠,持续2周,然后再单独饲养一周。Reeves等人描述了AIN-93G饮食的组成(AIN-93 purified diets for laboratoryrodents:final report of the American Institute of Nutrition ad hoc writingcommittee on the reformulation of the AIN-76A rodent diet.J Nutr(1993)123:1939-1951)。
3周饮食处理后,用含有108CFU肠炎沙门氏菌(临床分离物,噬菌体4型;菌株NIZO1241,NIZO乳品研究所(NIZO food research),Ede,the Netherlands)的0.2mL盐水通过管饲法感染小鼠。将一组小鼠进行假处理(未感染,N=6),将另外两组进行感染(感染;N=12,测试组感染;N=12)。
从感染当天每天收集粪便。使小鼠安乐死,并感染后4天收集回肠样品。为了定量活的肠炎沙门氏菌的数量,从回肠中部切下大约1cm,纵向打开,并用盐水短暂冲洗。将粪便和回肠样品在无菌盐水中均质化,并将稀释液铺板在Brilliant Green改良板(BectonDickinson Diagnostics)上进行菌落形成单位(CFU)计数。细菌计数表示为每克湿重的logCFU。结果总结在表2和表3中。
表2
表3
实施例3
在人结肠的短期模拟中,测试了来自苹果的RG-I多糖针对肠炎沙门氏菌的抗病原活性。
由干燥的苹果渣(苹果汁生产的残余物,GreendFields,Poland)通过使用果胶酶(Ultra Mash,Novozymes)的水提取(10%w/w 2hr 45℃),热灭活(90℃,10min),通过倾析去除不溶性残余物,超滤(40kDa截留)和最后干燥来生产苹果RG-I多糖。
如实施例1所述进行单糖组成的测定。结果显示在表4中。
表4
将含有结肠中存在的营养物(例如宿主来源的聚糖,如粘蛋白)的糖贫化的基础结肠培养基引入已经含有测试提取物(5g/L最终浓度)的70mL青霉素瓶子中。用橡胶塞密封瓶子,并通过用N2冲洗实现厌氧。随后,从冷冻的粪便样品制备人粪便接种物。在解冻,与厌氧的磷酸盐缓冲液混合,均质化,以及经由离心(2min,500G)去除颗粒之后,将粪便接种物加入到不同的瓶子中。粪便接种物以五倍稀释作为有害(dysbiotic)群落的技术模拟进行给药。另外,将在BHI肉汤中过夜生长的肠炎沙门氏菌培养物的2%接种物加入到温育中(3.62107CFU/mL最终浓度)。此时,开始温育48小时,在此期间将温度控制在37℃,并通过振荡器(90rpm)确保连续混合。24h和48h温育后取样。
使用利用100ppm链霉素(沙门氏菌菌株对其具有抗性)给药的选择性McConckey琼脂,使用选择性计数来评估肠炎沙门氏菌定殖。沙门氏菌物种由于不能利用乳糖而产生非粉红色菌落导致与其它细菌区分开。
糖贫化的结肠培养基和菊粉,一种众所周知的益生元(Beneo,DP 23,100%菊粉),分别用作阴性参照和阳性参照。
温育实验的结果显示在表5中。
表5
Claims (15)
1.用于预防或治疗动物中的沙门氏菌病的用途的组合物,所述用途包括将所述组合物施用至所述动物,其中所述组合物包含至少0.01重量%的鼠李糖半乳糖醛酸聚糖I(RG-I)多糖的干物质,所述鼠李糖半乳糖醛酸聚糖I(RG-I)多糖具有大于2kDa的分子量并且具有由半乳糖醛酸残基和鼠李糖残基组成的主链,所述鼠李糖残基包含在α(1→4)-半乳糖醛酸-α(1→2)-鼠李糖残基中,其中所述RG-I多糖中的半乳糖醛酸残基与鼠李糖残基的摩尔比在50:1至1:1的范围内。
2.根据权利要求1所述的用于预防或治疗动物中的沙门氏菌病的用途的组合物,其中将所述组合物在至少3天的时间段内以提供至少1mg RG-I多糖/kg体重/天的量每天至少一次施用至所述动物。
3.根据权利要求1或2所述的用于预防或治疗动物中的沙门氏菌病的用途的组合物,其中将所述组合物施用至尚未患有沙门氏菌病的动物。
4.根据前述权利要求中任一项所述的用于预防或治疗动物中的沙门氏菌病的用途的组合物,其中所述沙门氏菌病是由肠炎沙门氏菌(Salmonella enterica Serovarenteritidis)引起的。
5.根据前述权利要求中任一项所述的用于预防或治疗动物中的沙门氏菌病的用途的组合物,其中所述沙门氏菌病是由鼠伤寒沙门氏菌(Salmonella enterica Serovartyphimurium)引起的。
6.根据前述权利要求中任一项所述的用于预防或治疗动物中的沙门氏菌病的用途的组合物,其中所述动物是家禽。
7.根据权利要求1-5中任一项所述的用于预防或治疗动物中的沙门氏菌病的用途的组合物,其中所述动物是猪。
8.根据权利要求1-5中任一项所述的用于预防或治疗动物中的沙门氏菌病的用途的组合物,其中所述动物是人。
9.根据前述权利要求中任一项所述的用于预防或治疗动物中的沙门氏菌病的用途的组合物,其中所述组合物包含:
·1,000-2,500IU/kg的维生素A;
·0.1-10mcg/kg的维生素B12;
·100-350IU/kg的维生素D;
·2-20IU/kg的维生素E;和
·0.25-0.60mg/kg的维生素K。
10.根据前述权利要求中任一项所述的用于预防或治疗动物中的沙门氏菌病的用途的组合物,其中所述RG-I多糖的主链中的半乳糖醛酸残基与鼠李糖基残基的摩尔比不超过20:1。
11.根据前述权利要求中任一项所述的用于预防或治疗动物中的沙门氏菌病的用途的组合物,其中RG-I多糖占所述组合物中存在的果胶多糖的至少10wt.%。
12.根据前述权利要求中任一项所述的用于预防或治疗动物中的沙门氏菌病的用途的组合物,其中鼠李糖占所述RG-I多糖中包含的所有单糖残基的3-50%。
13.根据前述权利要求中任一项所述的用于预防或治疗动物中的沙门氏菌病的用途的组合物,其中所述RG-I多糖获自植物来源,所述植物来源选自:苹果、菜椒、越桔、胡萝卜、柑橘、葡萄、马铃薯、甜菜、橄榄、豌豆及以上的组合。
14.制备可食用组合物的方法,所述方法包括将一种或多种可食用成分与多糖组分混合,所述多糖组分包含至少5重量%的权利要求1所述的RG-I多糖的干物质。
15.通过权利要求14所述的方法获得的可食用组合物。
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