CN116616348B - 含唾液酸的组合物及其在益生菌增殖和调节免疫中的应用 - Google Patents
含唾液酸的组合物及其在益生菌增殖和调节免疫中的应用 Download PDFInfo
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- CN116616348B CN116616348B CN202310896572.1A CN202310896572A CN116616348B CN 116616348 B CN116616348 B CN 116616348B CN 202310896572 A CN202310896572 A CN 202310896572A CN 116616348 B CN116616348 B CN 116616348B
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- sialic acid
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- lactoferrin
- sialyl
- milk
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Abstract
本发明公开了一种含唾液酸的组合物及其在益生菌增殖和调节免疫中的应用,所述组合物按照重量百分比计,包括唾液酸化乳铁蛋白5‑10%,和/或,唾液酸化乳脂球膜70‑90%,所述组合物中总唾液酸的含量大于0.1mg/g。所述组合物还可以包括益生菌菌粉。本发明还提供了所述组合物的应用,可用于制备食品、保健品、药品和营养补充剂。进一步地,可以用于制备奶粉、可以用于制备调节肠道内唾液酸水平的产品、用于制备调节肠道内益生菌水平的产品、以及用于制备调节免疫力的产品。本技术方案所提供的组合物可有效的调节肠道微生态,调节免疫,促进肠道具有唾液酸代谢能力的菌群生长,增强对炎症因子的抑制效果。
Description
技术领域
本发明涉及益生菌,尤其涉及一种含唾液酸的组合物及其在益生菌增殖和调节免疫中的应用。
背景技术
对于婴儿而言,母乳喂养是最为理想的,但是由于多种原因只能用牛乳或其他动物乳来替代母乳喂养。人们发现,母乳的具体组成随着哺乳期的时间推进而变化。母乳可以分为初乳(哺乳期0-5天)、早期乳(6-15天)、过渡乳(哺乳期16-30天)和成熟乳(31-360天)四个阶段,其蛋白质、脂肪、碳水化合物等成分呈现动态变化。母乳的这些变化,可能是为了更好地适应婴幼儿在不同阶段对营养物质和发育所需。婴幼儿营养组合物也在深入研究不同阶段中具有更加精细和贴近母乳的配方。
已知母乳中含有丰富的免疫成分,如乳铁蛋白、免疫球蛋白、乳脂球膜等,已有丰富的研究证实它们在调节肠道微生态、抑制病原菌定植、与免疫细胞相互作用促进免疫球蛋白IgG、IgA活化、促进中性粒细胞发育、防止上呼吸道病毒感染、降低过敏、促进婴幼儿免疫功能发育等方面的作用。
这些免疫分子多数为聚糖修饰的糖缀合物,包括糖蛋白、糖肽、糖脂等,还包括由单糖单元组成的游离母乳低聚糖等结构。
唾液酸(N-乙酰基神经氨酸)是一种对哺乳动物发育、细胞识别、细胞黏附和信号传导至关重要的链状糖分子,也是母乳中糖缀合物及母乳低聚糖末端常见的单糖单元。唾液酸可被唾液酸结合免疫球蛋白凝集素受体(Siglecs)识别,抑制或激活免疫细胞。唾液酸与蛋白或脂质连接形成的糖缀合物,是潜在的Siglecs反式配体,可能提高Siglecs的亲和力。
已知人类初乳(HC)、过渡期(HT)和成熟乳(HM)中N-聚糖呈现动态变化,从HC、HT、HM中分别鉴定了27、37和33种N-聚糖,其中唾液酸化N-聚糖的相对含量增加,并且发现在哺乳期间岩藻糖基化N-糖减少。
乳铁蛋白是一种由上皮细胞持续分泌的多功能免疫蛋白,存在母乳、泪液和中性粒细胞中,是母乳中含量丰富且具备重要生理功能的一种蛋白。乳铁蛋白具有多种功能,其抗菌特性最突出。
“唾液酸化乳铁蛋白”是指碳水化合物以寡糖形式在乳铁蛋白上的天冬酰胺的侧链氨基部分(-NH2)连接、且寡糖基末端为唾液酸的乳铁蛋白。从结构上来讲,乳铁蛋白是一个包含约700个氨基酸的多肽链,此肽链的两端是两个同源球状域,即N-环和C-环。N-环对应第1-333个氨基酸残基,而C-环对应第345-692个氨基酸残基,这两个域由一段短链α-螺旋相连接。N-环和C-环都包含金属离子(如铁、锌等)结合位点和糖基化位点(即可以和碳水化合物进行糖基化反应的氨基酸)。一般来说,牛乳铁蛋白的糖基化位点为第233位、第368位、第476位、以及第545位的天冬酰胺残基(Asparagine,Asn);人乳铁蛋白的糖基化位点为第138位和第479位天冬酰胺残基(Asn 138 和Asn 479)。
有研究认为母乳蛋白的糖基化可能会阻断或调节病原体与上皮表面细胞的结合,这可能是母乳喂养的婴儿胃肠道受到保护、免受感染的原因。
已有的母乳替代品和婴幼儿营养品主要考虑母乳中蛋白、脂质和碳水化合物的种类、数量和功能,但很少考虑糖缀合物在泌乳过程中的聚糖变化对肠道菌群的影响及其功能,使得婴幼儿无法得到与母乳喂养相同或相似的健康益处。
发明内容
为了克服现有技术的不足,本发明的目的之一在于提供一种组合物。
本发明的目的之二在于提供组合物的应用。
本发明的目的之一采用如下技术方案实现:
一种组合物,其特征在于,按照重量百分比计,包括唾液酸化乳铁蛋白5-10%,和/或,唾液酸化乳脂球膜70-90%,所述组合物中总唾液酸的含量大于0.1mg/g。
作为本发明可选的方案,所述组合物还包括益生菌菌粉。补充合适的益生菌有助于受试者的健康成长,特别地,补充的益生菌可以利用唾液酸化乳铁蛋白和/或唾液酸化乳脂球膜上的唾液酸为碳源,促进自身的生长,从而更好地助于受试者健康,例如有利于肠道健康、促进排便、提高免疫等。
所述受试者可以包括不同年龄段的人群,例如婴幼儿群体、青少年群体、成年人群体和中老年人群体。
作为本发明可选的方案,所述益生菌菌粉包括双歧杆菌菌粉和/或乳杆菌菌粉。双歧杆菌是人体肠道的一类益生菌,乳杆菌也是人体肠道中一种常见的菌,补充合适的双歧杆菌、乳杆菌,可以调节肠道菌群的平衡,改善肠道功能,提高免疫功能等。此外,双歧杆菌和乳杆菌具有唾液酸代谢能力,它们可以利用唾液酸化乳铁蛋白和/或唾液酸化乳脂球膜上的唾液酸为碳源,促进自身的生长,从而更好地助于受试者健康。
作为本发明可选的方案,所述双歧杆菌包括长双歧杆菌、短双歧杆菌、动物双歧杆菌、乳双歧杆菌、两歧双歧杆菌和婴儿双歧杆菌中的一种或任意组合;优选地,所述双歧杆菌包括动物双歧杆菌BB12和/或长双歧杆菌BB536。
作为本发明可选的方案,所述乳杆菌包括鼠李糖乳杆菌、植物乳杆菌和发酵乳杆菌中的一种或任意组合;优选地,所述鼠李糖乳杆菌包括鼠李糖乳杆菌LGG;所述发酵乳杆菌包括发酵乳杆菌LC40(CECT5716)。
作为本发明可选的方案,按照重量百分比计,所述组合物还包括益生菌菌粉0.5-20%;优选地,所述益生菌菌粉的占比为0.5-10%,更加优选地为1-5%,例如,所述组合物中益生菌菌粉的占比为0.5%,0.8%,1%,1.3%,2%,2.6%,3%,3.5%,4%,4.5%,5%,6%,7%,8%,9%,10%等。
作为本发明可选的方案,所述组合物中的唾液酸由唾液酸化乳铁蛋白和/或唾液酸化乳脂球膜提供,即所述组合物中的唾液酸仅由唾液酸化乳铁蛋白和/或唾液酸化乳脂球膜提供,不包含游离的唾液酸。例如,当所述组合物同时包含唾液酸化乳铁蛋白和唾液酸化乳脂球膜时,组合物中的唾液酸仅由唾液酸化乳铁蛋白和唾液酸化乳脂球膜提供。
作为本发明可选的方案,本发明所提供的组合物,可以包含游离的唾液酸,即所述组合物包含唾液酸化乳铁蛋白,和/或,唾液酸化乳脂球膜外,还可以包含游离的唾液酸。
作为本发明可选的方案,按照重量百分比计,所述组合物包括唾液酸化乳铁蛋白5-10%,唾液酸化乳脂球膜70-90%,益生菌菌粉0.5-20%;所述组合物中的总唾液酸含量大于0.5mg/g。
作为本发明可选的方案,按照重量百分比计,所述组合物包括唾液酸化乳铁蛋白5-10%,唾液酸化乳脂球膜70-90%,益生菌菌粉0.5-10%;所述组合物中的总唾液酸含量大于0.5mg/g。
作为本发明可选的方案,按照重量百分比计,所述组合物包括唾液酸化乳铁蛋白6-9%,唾液酸化乳脂球膜75-85%,益生菌菌粉1-5%;所述组合物中的总唾液酸含量大于0.8mg/g。
作为本发明可选的方案,按照重量百分比计,所述组合物包括唾液酸化乳铁蛋白8.5%,唾液酸化乳脂球膜90%,益生菌菌粉1.5%;所述组合物中的总唾液酸含量大于1mg/g。
作为本发明可选的方案,所述组合物中的总唾液酸含量为1~25mg/g,优选为1~20mg/g,也可以为1~2mg/g,更加具体地,可以为1mg/g、1.3mg/g、1.4mg/g、1.5mg/g、2mg/g、5mg/g、10mg/g、15mg/g、20mg/g等。
作为本发明可选的方案,所述唾液酸化乳铁蛋白中的唾液酸含量为20~50μg/g;所述唾液酸化乳脂球膜中的唾液酸含量为1300~1800μg/g。
作为本发明可选的方案,所述益生菌菌粉中益生菌的活菌数为1×106~1×1012 CFU/g(CFU/mL)。
作为本发明可选的方案,所述组合物用于调节肠道内的唾液酸水平;
和/或,所述组合物用于调节肠道内益生菌水平;
和/或,所述组合物用于调节免疫。
作为本发明可选的方案,所述组合物用于促进肠道内具有唾液酸代谢能力的益生菌生长,所述益生菌包括双歧杆菌和/或乳杆菌;
和/或,所述组合物用于调节细胞因子NO、TNF-α、IL-1β、IL-6、IL-10和IL-12的水平,以调节免疫系统的功能。
本发明的目的之二采用如下技术方案实现:
本发明还提供了将目的之一任一项所述的组合物在制备食品、保健品、药品和营养补充剂中的应用。
作为本发明可选的方案,所述食品包括乳粉和乳液。所述食品可以为婴幼儿食品、青少年食品、或中老年人食品。所述乳粉可以为婴幼儿乳粉、青少年乳粉、或中老年人乳粉。所述乳液可以为婴幼儿乳液、青少年乳液、或中老年人乳液。
本发明还提供了将目的之一任一项所述的组合物在制备调节肠道内唾液酸水平产品中的应用。
例如,一种用于调节肠道内唾液酸水平的产品,包括目的之一任一项所述的组合物。
本发明还提供了将目的之一任一项所述的组合物在制备调节肠道内益生菌水平产品中的应用。优选地,所述调节肠道内益生菌水平包括促进肠道内具有唾液酸代谢能力的益生菌生长,所述益生菌包括双歧杆菌和/或乳杆菌。
例如,一种用于调节肠道内益生菌水平的产品,包括目的之一任一项所述的组合物。
本发明还提供了将目的之一任一项所述的组合物在制备调节免疫力产品中的应用。
例如,一种用于调节免疫力的产品,包括目的之一任一项所述的组合物。
本发明还提供了一种营养制品,所述营养制品包括目的之一任一项所述的组合物。所述营养制品除了包括奶粉外,还可以包括液体奶、酸奶、奶酪、婴幼儿米粉等。
本发明还提供了一种奶粉,所述奶粉包括目的之一任一项所述的组合物,所述奶粉中唾液酸的重量百分比为0.07-0.25%;更加优选地,所述奶粉中唾液酸的重量百分比为0.07-0.15%,即所述奶粉中,唾液酸在奶粉中的含量为70-150mg/100g奶粉。
本发明还提供了一种奶粉,所述奶粉包括目的之一任一项所述的组合物,所述组合物在所述奶粉中的重量百分比为3-10%,优选地为3-8%。更加优选地,所述组合物在所述奶粉中的重量百分比为3.7-4.3%,即在所述奶粉中,100g奶粉含有3.7-4.3g所述组合物(所述组合物可以为唾液酸化乳铁蛋白和唾液酸化乳脂球膜的组合,优选为唾液酸化乳铁蛋白、唾液酸化乳脂球膜和益生菌菌粉的组合)。
本发明所提供的奶粉,奶粉中的唾液酸除了可以由唾液酸化乳铁蛋白和唾液酸化乳脂球膜提供外,还可以由奶粉中的其它成分提供,例如游离的唾液酸、唾液酸化母乳低聚糖,神经节苷脂等。
本发明所提供的奶粉,所述奶粉包括婴幼儿奶粉、青少年奶粉、成年人奶粉和中老年人奶粉。
本发明所提供的奶粉,除了包含目的之一任一项所述的组合物外,还可以包括其他成分,例如,其他成分可以包括以下中的一种或多种:蛋白质、氨基酸、碳水化合物、低聚糖、脂质、膳食纤维、益生元、必需脂肪酸、核苷酸、维生素、矿物质和其它微量营养素。
其中,蛋白质可以包括但不限于:酪蛋白、α-乳白蛋白、乳清、大豆蛋白、大米蛋白、玉米蛋白、燕麦蛋白、大麦蛋白、小麦蛋白、黑麦蛋白、豌豆蛋白、菜籽(油菜籽)蛋白、卵蛋白、葵花籽蛋白、马铃薯蛋白、鱼蛋白、肉蛋白、血清白蛋白或免疫球蛋白,以及它们的任意组合。
氨基酸可以包括但不限于:亮氨酸、苏氨酸、酪氨酸、异亮氨酸、精氨酸、丙氨酸、组氨酸、脯氨酸、缬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、丝氨酸、赖氨酸、蛋氨酸、色氨酸、天冬酰胺或天冬氨酸,以及它们的任意组合。
碳水化合物可以包括但不限于:乳糖、蔗糖、麦芽糖糊精或淀粉,以及它们的任意组合。
低聚糖可以包括但不限于:低聚半乳糖(GOS)、低聚果糖(FOS)、异麦芽低聚糖(IMO)、低聚木糖(XOS)、低聚阿拉伯木糖(AXOS)、低聚甘露糖(MOS)、大豆低聚糖、2’-唾液酸乳糖、2’-唾液酸乳糖胺、3’-唾液酸乳糖、3’-唾液酸乳糖胺、6’-唾液酸乳糖、6’-唾液酸乳糖胺、2’-岩藻糖基乳糖或乳糖-N-新四糖,以及他们的任意组合。
脂质可以包括但不限于:棕榈油精、高油酸葵花油、高油酸红花油、亚麻籽油、核桃油、芥花油、鱼油、椰子油、牛乳脂或1,3-二油酸2-棕榈酸甘油三酯,以及它们的任意组合。
膳食纤维可以包括但不限于:聚葡萄糖、可溶性纤维、大豆纤维或菊粉,以及它们的任意组合。
益生元可以包括但不限于:聚葡萄糖(PDX)、半乳糖、低聚半乳糖(GOS)、低聚果糖(FOS)、异麦芽低聚糖(IMO)、低聚木糖(XOS)、低聚阿拉伯木糖(AXOS)、低聚甘露糖(MOS)、大豆低聚糖等。
必需脂肪酸可以包括但不限于:亚油酸(LA)、α-亚麻酸(ALA)和多不饱和脂肪酸(PUFA)、DHA、神经节苷脂(单唾液酸神经节苷脂3(GM3)和双唾液酸神经节苷脂3(GD3))、磷脂(诸如鞘磷脂、磷脂磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇、磷酯酰丝氨酸)等。
核苷酸可以包括但不限于:单磷酸胞苷(CMP)、单磷酸尿苷(UMP)、单磷酸腺苷(AMP)或单磷酸鸟苷(GMP),以及它们的组合。
维生素、矿物质和其它微量营养素可以包括但不限于:维生素A、维生素B1、维生素B2、维生素B6、维生素B9、维生素B12、维生素E、维生素K、维生素C、维生素D、叶酸、肌醇、烟酸、生物素、泛酸、胆碱、钙、磷、碘、铁、镁、铜、锌、锰、氯、钾、钠、硒、铬、钼、牛磺酸、L-肉碱以及它们的组合。矿物质通常以盐的形式添加。
相比现有技术,本发明的有益效果在于:
本技术方案所提供的组合物,可有效的调节肠道微生态,调节免疫,促进肠道具有唾液酸代谢能力的菌群生长,增强对炎症因子的抑制效果。
附图说明
图1为本发明实施例2所提供的动物双歧杆菌BB12和长双歧杆菌BB536的生长曲线图;
图2为本发明实施例3所提供的细胞因子NO的含量展示图;
图3为本发明实施例3所提供的细胞因子TNF-α的含量展示图;
图4为本发明实施例3所提供的细胞因子IL-1β的含量展示图;
图5为本发明实施例3所提供的细胞因子IL-6的含量展示图;
图6为本发明实施例3所提供的细胞因子IL-10的含量展示图;
图7为本发明实施例3所提供的细胞因子1L-12的含量展示图;
图8为本发明实施例4所提供的细胞因子NO的含量展示图;
图9为本发明实施例4所提供的细胞因子TNF-α的含量展示图;
图10为本发明实施例4所提供的细胞因子IL-1β的含量展示图;
图11为本发明实施例4所提供的细胞因子IL-6的含量展示图;
图12为本发明实施例4所提供的细胞因子IL-10的含量展示图;
图13为本发明实施例4所提供的细胞因子1L-12的含量展示图;
图14为本发明实施例5所提供的细胞因子NO的含量展示图;
图15为本发明实施例5所提供的细胞因子TNF-α的含量展示图;
图16为本发明实施例5所提供的细胞因子IL-1β的含量展示图;
图17为本发明实施例5所提供的细胞因子IL-6的含量展示图;
图18为本发明实施例5所提供的细胞因子IL-10的含量展示图;
图19为本发明实施例5所提供的细胞因子1L-12的含量展示图。
具体实施方式
下面,结合附图以及具体实施方式,对本发明做进一步描述,需要说明的是,在不相冲突的前提下,以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。在下述实施例中所采用的原材料、设备等除特殊限定外均可以通过购买方式获得。
本发明发现唾液酸化乳铁蛋白(SA-LF)、唾液酸化乳脂球膜(SA-MFGM)和双歧杆菌具有协同作用,唾液酸化乳铁蛋白和唾液酸化乳脂球膜可在双歧杆菌分泌的糖苷酶和唾液酸酶等作用下释放唾液酸,双歧杆菌可利用和代谢唾液酸,进而促进双歧杆菌的增殖。唾液酸化乳铁蛋白、唾液酸化乳脂球膜和双歧杆菌三者进行组合,通过调节肠道内唾液酸的水平,可有效的调节肠道微生态,调节免疫,促进肠道具有唾液酸代谢能力的菌群生长,更进一步增强了对炎症因子的抑制效果。
以下实施例中,唾液酸化乳铁蛋白和唾液酸化乳脂球膜样品从Sigma公司购买,双歧杆菌及乳杆菌由市场采购。
实施例1
唾液酸化乳铁蛋白和唾液酸化乳脂球膜中的唾液酸含量测定
实验目的:通过液质联用建立唾液酸化乳铁蛋白和唾液酸化乳脂球膜上的唾液酸定性定量分析方法。
实验设计:唾液酸化乳铁蛋白和唾液酸化乳脂球膜样品在盐酸溶液中加热水解,释放出唾液酸,提取液经固相萃取柱净化后,样品试液用反相色谱柱分离,采用超高效液相色谱-串联质谱(UPLC-MS/MS)进行测定,外标法定量。
乳铁蛋白样品处理:
(1)试样提取。称取唾液酸化乳铁蛋白1g,加入3 mL水,涡旋振荡30s,转至25 mL具塞比色管中,沿管壁缓慢加入10mL盐酸溶液,一边加一边振荡,盖上玻璃塞,涡旋混匀,置于80℃水浴中水解40 min,在水浴过程中每5 min振荡一次。取出比色管,冷却至室温,用乙腈-水溶液(1+9)定容至25 mL, 混匀,静置5min。取10 mL上层溶液放入离心管中,加入5 mL三氯甲烷,涡旋混匀后,9000 r/min离心5 min,取上清液待净化。
(2)试样净化。固相萃取柱依次用3mL甲醇和3mL水活化,负压抽干,样液以小于1mL/min 的流速通过固相萃取柱,收集流出液。准确吸取100μL样液,加入950μL乙腈-水溶液(1+9),混匀,过0.22μm微孔滤膜后,待测。
乳脂球膜样品处理:
试样提取。称取唾液酸化乳脂球膜1g,加入3 mL水,涡旋振荡30s,转至25 mL具塞比色管中,沿管壁缓慢加入10mL盐酸溶液,一边加一边振荡,盖上玻璃塞,涡旋混匀,置于80℃水浴中水解40 min,在水浴过程中每5 min振荡一次。取出比色管,冷却至室温,用乙腈-水溶液(1+9) 定容至100mL, 混匀,静置5min。取10 mL上层溶液放入离心管中,加入5 mL三氯甲烷,涡旋混匀后,9000 r/min离心5 min,取上清液待净化。
试样净化。固相萃取柱依次用3mL甲醇和3mL水活化,负压抽干,样液以小于1mL/min 的流速通过固相萃取柱,收集流出液。准确吸取50μL样液,加入950μL乙腈-水溶液(1+9),混匀,过0.22μm微孔滤膜后,待测。
标准品溶液制备:精确称取唾液酸标准品10mg于10mL容量瓶中,用乙腈-水(80:20,v/v)定容至刻度,得到浓度1mg/mL的母液,置于-20℃保存备用;准确移取母液至容量瓶中,用流动相定容至刻度,逐级稀释为0.1、0.2、0.4、0.8、1.0、2.0mg/mL的标准品溶液,4℃下储存备用。
检测条件:
色谱柱: C18柱,3.0mm x 150mm,1.8μm;流速: 0.3mL/min;柱温: 30℃;
进样量:2μL;流动相: A相: 0.1% 甲酸;B相: 乙腈 (梯度洗脱);洗脱条件见下表1。
表1 洗脱条件记录表
时间/min | A(%) | B(%) |
0.0 | 80 | 20 |
4.0 | 10 | 90 |
4.1 | 10 | 90 |
6.0 | 10 | 90 |
6.1 | 80 | 20 |
10.0 | 80 | 20 |
电离方式:
电喷雾电离(ESI),负离子模式。
扫描方式:多反应监测(MRM)。
毛细管电压:3500 V,裂解电压:120 V。
干燥气温度:300 ℃,干燥气流速:8 L/min。
鞘气温度:350 ℃;鞘气流速:10 L/min。
定性离子对及碰撞能量/eV: 308/87.0 (-17), 308/170.0 (-13);定量离子对及碰撞能量/eV: 308/87.0 (-17)。
方法学考察:
线性关系:在已确定的仪器条件下,分别对0.1、0.2、0.4、0.8、1.0、2.0mg/mL的标准工作液进样分析,以唾液酸的定量离子峰面积(Y)与唾液酸的质量浓度(x,μg/mL)进行线性回归,绘制标准曲线。在0.1~1.0μg/mL 范围内,唾液酸的线性回归方程为y=3301.5x-80.23,R2=0.9988。
精密度试验:取1.0、0.4、0.1μg/mL 唾液酸标准品溶液,重复进样6次测定,其相对标准偏差(RSD)为2.5%,表明方法的精密度良好。
重复性试验:对同一种样品,分别取6份,按照样品处理方法处理后进样分析,RSD为4.2%,表明该方法重复性较好。
加样回收试验:测定本发明实施例的唾液酸化乳铁蛋白和唾液酸化乳脂球膜中唾液酸含量分别约为30.2μg/g和1603.8μg/g。取乳铁蛋白样品,加入唾液酸浓度为样品100%的标准品,取5份进样分析,计算唾液酸回收率;另取乳脂球膜样品,加入唾液酸浓度为样品100%的标准品,取5份进样分析,计算唾液酸回收率。
如表2和表3结果显示,乳铁蛋白回收率为102.3-108.6%;乳脂球膜加样回收率为99.5-101.1%。
表2 唾液酸化乳铁蛋白样品的加标回收实验结果记录表
未添加标准品含量(μg/g) | 测定含量(μg/g) | 回收率(%) |
30.2 | 61.1 | 102.3 |
30.2 | 63.0 | 108.6 |
30.2 | 61.5 | 103.6 |
30.2 | 61.4 | 103.3 |
30.2 | 62.1 | 105.6 |
表3 唾液酸化乳脂球膜样品的加标回收实验结果记录表
未添加标准品含量(μg/g) | 测定含量(μg/g) | 回收率(%) |
1603.8 | 3199.6 | 99.5 |
1603.8 | 3186.7 | 98.7 |
1603.8 | 3153.1 | 96.6 |
1603.8 | 3186.7 | 98.7 |
1603.8 | 3225.2 | 101.1 |
实施例2
本实施例旨在研究唾液酸化乳铁蛋白、唾液酸化乳脂球膜对动物双歧杆菌BB12和长双歧杆菌BB536生长促进作用的影响。
实验方法:
在无碳源改良MRS培养基中加入唾液酸化乳铁蛋白、或唾液酸化乳脂球膜作为唯一碳源,测定双歧杆菌的生长情况。
改良MRS培养基配制:
配制1L的MRS基础培养基需称量如下试剂溶解于1L去离子水中。胰蛋白胨:10 g;酵母提取物:2.5 g;L-半胱氨酸:1 g;柠檬酸二胺 2 mL;乙酸钠:0.9 g;硫酸锰:0.2 g ;KH2PO4:2.0 g;K2HPO4:0.45 g;硫酸镁:0.5 g;吐温-80:1mL。溶解后,加入刃天青溶液1 mL后,煮沸至培养基变色(红色转黄色)后,马上充氮使培养基液面保持无氧,蠕动泵分装至10ml西林瓶中,压盖密封,高压蒸汽灭菌后使用。
细菌培养:
分别选取目的动物双歧杆菌BB12和长双歧杆菌BB536进行鉴定和活化。划线分离培养提取单菌落到MRS液体培养液培养至对数期。接种菌液至新的MRS培养液再次培养至对数期。移取10μl生长至对数期的新鲜菌液,接种到含改良MRS培养液的西林瓶,厌氧培养48h,通过生长曲线测定仪来检测益生菌对碳源利用情况和生长情况。待细菌长到平稳期,将菌液进行离心(4℃,12000 rpm,10分钟),取上清液进行乳铁蛋白和乳脂球膜及其代谢产物含量检测。每个组做3个平行重复。实验分组情况见下表4。
表4 实验分组情况记录表
序号 | 分组 | 说明 |
1 | 空白对照 | 1% SA-LF(唾液酸化乳铁蛋白)改良MRS培养基(无双歧杆菌接种) |
2 | 双歧杆菌对照组 | 空白改良MRS培养基(接种BB12和BB536) |
3 | SA-LF实验组 | 1% SA-LF(唾液酸化乳铁蛋白)改良MRS培养基(接种BB12和BB536) |
4 | SA-MFGM实验组 | 1% SA-MFGM(唾液酸化乳脂球膜)改良MRS培养基(接种BB12和BB536) |
5 | SA-LF+SA-MFGM实验组 | 0.5% SA-LF+0.5% SA-MFGM改良MRS培养基(接种BB12和BB536) |
双歧杆菌生长曲线
双歧杆菌的生长曲线实验结果如图1所示,从图中可得:
(1)空白对照组无读数,说明实验用培养液无污染。
(2)双歧杆菌对照组有微弱生长,说明即使不添加SA-LF以及不添加SA-MFGM,培养基中仍残余少量可供细菌生长的营养物质,供细菌生长。
(3)SA-LF实验组相对于双歧杆菌对照组有明显生长,说明添加的SA-LF可作为双歧杆菌的碳源,促进其生长。说明动物双歧杆菌BB12和长双歧杆菌BB536能够降解SA-LF的糖基结构,并利用其作为碳源生长。
(4)SA-MFGM实验组相对于双歧杆菌对照组有明显生长,说明添加的SA-MFGM可作为双歧杆菌的碳源,促进其生长。说明动物双歧杆菌BB12和长双歧杆菌BB536能够降解SA-MFGM的糖基结构,并利用其作为碳源生长。并且,SA-MFGM相比SA-LF更加利于双歧杆菌的生长。
(5)SA-LF+SA-MFGM实验组中,双歧杆菌的生长最为旺盛,双歧杆菌的生长优于SA-LF实验组、以及优于SA-MFGM实验组,说明了SA-LF和SA-MFGM起到了协同增效的作用,能够共同促进动物双歧杆菌BB12和长双歧杆菌BB536的生长。
综上所述,动物双歧杆菌BB12和长双歧杆菌BB536能够降解唾液酸化乳铁蛋白(SA-LF)的糖基结构,也能够降解唾液酸化乳脂球膜(SA-MFGM)的糖基结构,并利用其作为碳源进行生长。并且,唾液酸化乳铁蛋白和唾液酸化乳脂球膜能够协同促进动物双歧杆菌BB12和长双歧杆菌BB536的生长。
2、培养液中唾液酸的含量测定
实施例2中采用的唾液酸化乳铁蛋白,其唾液酸的含量为30.2μg/g,唾液酸化乳脂球膜中唾液酸的含量为1603.8μg/g。
从理论上计算,当培养基中还有1%SA-LF时,则SA-LF在10mL培养基的含量为0.1g,此时SA的含量为3.02μg;当培养基中含有0.5%SA-LF时,则SA-LF在10mL培养基的含量为0.05g,此时SA的含量为1.51μg。
当培养基中还有1%SA-MFGM时,则SA-MFGM在10mL培养基的含量为0.1g,此时SA的含量为160.38μg;当培养基中含有0.5%SA-MFGM时,则SA-MFGM在10mL培养基的含量为0.05g,此时SA的含量为80.19μg。
对于0.5% SA-LF+0.5% SA-MFGM改良MRS培养基,SA的含量为81.7μg。
按照实施例1的方法对培养液中唾液酸(SA)的含量进行测定。实验结果如下表5所示。
表5 唾液酸含量测定结果记录表
T0/μg | T48/μg | |
空白对照 | 3.02 | 3.00 |
双歧杆菌对照组 | 0 | 0 |
SA-LF实验组 | 3.02 | 0 |
SA-MFGM实验组 | 160.38 | 0.05 |
SA-LF+SA-MFGM实验组 | 81.7 | 0.03 |
从表5中的结果可得,空白对照组中,唾液酸的含量在T0和T48时基本一致,说明了培养基没有受到污染。双歧杆菌对照组中,培养基中没有添加唾液酸,在T0和T48时唾液酸的检测结果均为0。而另外3个实验组中,唾液酸的含量在实验结束时均大大下降了,48小时后底物中残留的唾液酸几乎接近0,说明了动物双歧杆菌BB12和长双歧杆菌BB536能够利用SA-LF以及SA-MFGM上的SA进行生长。
实施例3
实施例3的目的在于研究唾液酸化乳铁蛋白(SA-LF)、唾液酸化乳脂球膜(SA-MFGM)与双歧杆菌的体外协同抗炎及免疫调节功能。具体地,实施例3研究了长双歧杆菌BB536、唾液酸化乳铁蛋白、唾液酸化乳脂球膜组合、以及它们三者的组合对细胞因子NO、TNF-α、IL-1β、IL-6、IL-10和IL-12的影响。
NO、TNF-α、IL-1β、IL-6均为促炎因子,可以诱导炎症的产生。IL-10、1L-12均为抗炎因子。其中,IL-10是一种具有重要免疫调节功能的多效细胞因子,通过活化的巨噬细胞来抑制炎症因子如TNF、IL-6、IL-1的表达以平衡炎症;IL-12是一种具有多种生物学活性的免疫细胞生长刺激因子,能促进T淋巴细胞和NK细胞的分化与增殖,调控细胞免疫,提高NK/LAK细胞的杀伤功能和特异性CTL细胞的应答能力,发挥免疫调节作用。
具体的实验分组如下表6所示。
表6 实施例3的实验分组记录表
添加成分 | 空白对照组 | 模型组 | 单独使用组1 | 单独使用组2 | 单独使用组3 | 联合干预组 |
LPS | \ | 100ng/ml | 100ng/ml | 100ng/ml | 100ng/ml | 100ng/ml |
长双歧杆菌BB536 | \ | \ | 1.5mg/g | \ | \ | 1.5mg/g |
SA-LF | \ | \ | \ | 8.5mg/g | \ | 8.5mg/g |
SA-MFGM | \ | \ | \ | \ | 90mg/g | 90mg/g |
实验步骤:
(1)取生长良好的对数期Raw264.7细胞,细胞消化离心后用新鲜含血清的培养基制成1×106个细胞/mL的细胞悬液,接种24孔板中,每孔500μL,至于37℃、95%湿度的二氧化碳培养相中培养;
(2)待细胞完全贴壁后,弃掉细胞培养基并加入新鲜无血清DMEM培养基继续培养12h;
(3)空白对照组换无血清培养基,LPS诱导组(模型组)换含有LPS终浓度为100ng/ml的无血清培养基培养;单独使用组分别含有LPS、以及不同浓度的长双歧杆菌BB536/唾液酸化乳铁蛋白/唾液酸化乳脂球膜的无血清培养基培养;联合干预组含有LPS、长双歧杆菌BB536、唾液酸化乳铁蛋白和唾液酸化乳脂球膜组合的无血清培养基培养;
(4)继续培养24h后,收集细胞培养上清液,用检测试剂盒依次测定细胞因子NO、TNF-α、IL-1β、IL-6、IL-10、1L-12的含量,并对其检测数据进行差异性分析。
实验结果:
实验结果如图2-7所示,图中,ns代表无统计学差异;*与对照组相比,有统计学差异(P<0.05);**与对照组相比,有显著性差异(P<0.01);***与对照组相比,有极显著性差异(P<0.001)。
与空白对照组相比,LPS诱导组中的促炎因子NO、TNF-α、IL-1β、IL-6的表达均具有显著性增高,说明各组抗炎细胞模型成功建立。
与模型组相比较,单独使用双歧杆菌可显著性降低NO、IL-1β、IL-6的表达,对TNF-α因子、IL-10和IL-12因子的表达无影响。
单独使用唾液酸化乳铁蛋白均能降低NO、TNF-α、IL-1β、IL-6的表达,对IL-12、IL-10因子的表达无影响。
单独使用唾液酸化乳脂球膜可显著性降低NO、TNF-α、IL-1β、IL-6的表达,提高IL-10、IL-12因子的表达。
联合干预组均能显著性降低NO、TNF-α、IL-1β、IL-6的表达,均能显著性升高IL-10和IL-12因子的表达,说明唾液酸化乳铁蛋白、唾液酸化乳脂球膜及双歧杆菌在抗炎作用及免疫调节方面具有显著的相互协同作用。
实验结果也表明,仅有联合干预组能够同时降低促炎因子NO、TNF-α、IL-1β、IL-6的含量,并同时提高抗炎因子IL-10、1L-12的含量,且与对照组相比,有极显著性差异。
实施例4
实施例4的目的在于研究唾液酸化乳铁蛋白、唾液酸化乳脂球膜与鼠李糖乳杆菌LGG的体外协同抗炎及免疫调节功能。实施例4的实验步骤与实施例3相同,在此不赘述。实施例4的实验分组如下表7所示。
表7 实施例4的实验分组记录表
添加成分 | 空白对照组 | 模型组 | 单独使用组1 | 单独使用组2 | 联合干预组(结合态) | 联合干预组(游离态) |
LPS | \ | 100ng/ml | 100ng/ml | 100ng/ml | 100ng/ml | 100ng/ml |
鼠李糖乳杆菌LGG | \ | \ | 5mg/g | 5mg/g | 5mg/g | 5mg/g |
SA-LF | \ | \ | 5mg/g | \ | 5mg/g | \ |
SA-MFGM | \ | \ | \ | 90mg/g | 90mg/g | \ |
LF(经脱唾液酸基处理) | \ | \ | \ | \ | \ | 5mg/g |
MFGM(经脱唾液酸基处理) | \ | \ | \ | \ | \ | 90mg/g |
游离唾液酸 | \ | \ | \ | \ | \ | 144.49μg/g |
备注:结合态组中,用的原料为唾液酸化乳铁蛋白(SA-LF)和唾液酸化乳脂球膜(MFGM)。唾液酸化乳铁蛋白中唾液酸的含量为30.2μg/g;唾液酸化乳脂球膜中唾液酸的含量为1603.8μg/g。当SA-LF为5mg/g,SA为0.151μg/g;当SA-MFGM为90mg/g时,SA为144.342μg/g。
游离态组中,所用的乳铁蛋白经脱唾液酸基处理;所用的乳脂球膜经脱唾液酸基处理。游离态组中使用的唾液酸是单独的成分,其用量是根据结合态组中唾液酸总含量进行等量添加的。
具体的实验结果如图8-13所示。
从图8-13可得,“SA-LF+鼠李糖乳杆菌LGG组”和“SA-MFGM+鼠李糖乳杆菌LGG组”均能降低促炎因子NO、TNF-α、IL-1β、IL-6的含量,提高抗炎因子IL-10、1L-12的含量。
当SA-LF、SA-MFGM和鼠李糖乳杆菌组LGG进行组合时,能够起到协同增效的作用。结合态联合组对炎症因子的调节效果要优于游离态组的效果。
实施例5
实施例5的目的在于研究唾液酸化乳铁蛋白(SA-LF)、唾液酸化乳脂球膜(SA-MFGM)与长双歧杆菌的体内协同抗炎及免疫调节功能。实施例5利用小鼠抗炎模型,研究单独使用及协同使用三种成分对小鼠血清中细胞因子NO、TNF-α、IL-1β、IL-6、IL-10和IL-12的影响。
实验分组如下表8所示。
表8 实施例5的实验分组记录表
添加成分 | 空白对照组 | 模型组 | 单独使用组1 | 单独使用组2 | 单独使用组3 | 联合干预组 |
LPS | \ | 30mg/kg | 30mg/kg | 30mg/kg | 30mg/kg | 30mg/kg |
长双歧杆菌BB536 | \ | \ | 1.5mg/g | \ | \ | 1.5mg/g |
SA-LF | \ | \ | \ | 8.5mg/g | \ | 8.5mg/g |
SA-MFGM | \ | \ | \ | \ | 90mg/g | 90mg/g |
实验步骤:
采用体重范围在15-23g的SPF级ICR小鼠进行研究,将60只小鼠随机分为6组,每组10只,分别为空白对照组、模型组、单独使用组、联合干预组。
空白对照组和LPS诱导组(模型组)给予无菌生理盐水灌胃2周,每天1次;单纯使用组按照实验设计剂量灌胃2周,每天1次;联合对照组给与长双歧杆菌BB536+25mg/gSA-LF+50mg/gSA-MFGM灌胃2周,每天1次。
各组末次给药1h后,除空白对照组,其余各组小鼠腹腔注射30mg/kg的脂多糖LPS进行处理,空白对照组注射等量生理盐水。
12h后,摘小鼠眼球取血,将获得的血清在2-8℃、3500rpm条件下离心20min,取血清并按照试剂盒说明书检测血清中的NO、TNF-α、IL-1β、IL-6、IL-10和IL-12的含量,并对其检测数据进行差异性分析。
实验结果如图14-19所示,图中,ns代表无统计学差异;*与对照组相比,有统计学差异(P<0.05);**与对照组相比,有显著性差异(P<0.01);***与对照组相比,有极显著性差异(P<0.001)。
与空白对照组相比,LPS诱导组小鼠血清中的中的NO、TNF-α、IL-1β、IL-6含量均显著升高,表明诱导组、双歧杆菌组和联合干预组小鼠出现全身性炎症反应,提示造模成功。
与模型组相比较,单独使用双歧杆菌可显著性降低小鼠血清中的NO、IL-1β、IL-6、TNF-α的表达,对IL-10和IL-12因子的表达无影响。
单独使用唾液酸化乳铁蛋白均能降低NO、TNF-α、IL-1β的表达,对IL-6、IL-10、IL-12因子的表达无显著影响。
单独使用唾液酸化乳脂球膜可显著性降低NO、TNF-α、IL-1β、IL-6的表达,提高IL-10、IL-12因子的表达。
而联合干预组均能显著性降低NO、TNF-α、IL-1β、IL-6的表达,均能显著性升高IL-10和IL-12因子的表达,说明唾液酸化乳铁蛋白、唾液酸化乳脂球膜及双歧杆菌在抗炎作用及免疫调节方面具有显著的相互协同作用,从而减轻模型小鼠的炎症损伤和调节小鼠的免疫功能。
实施例6
一种奶粉,包括以下组分:含有唾液酸的组合物3.7%,以及,其它成分96.3%。奶粉中唾液酸的占比为0.07%,奶粉中的唾液酸除了由唾液酸化乳铁蛋白和唾液酸化乳脂球膜提供外,还由奶粉中的其它成分提供。
按照在含有唾液酸的组合物中的重量百分比计,含有唾液酸的组合物由以下组分组成:唾液酸化乳铁蛋白10%,唾液酸化乳脂球膜87.4%,益生菌2.6%。
其它成分包括:蛋白质、氨基酸、碳水化合物、低聚糖、脂质、益生元、必需脂肪酸、核苷酸、维生素、矿物质和其它微量营养素。
实施例7
一种奶粉,包括以下组分:含有唾液酸的组合物4.3%,以及,其它成分95.7%。奶粉中唾液酸的占比为0.09%,奶粉中的唾液酸除了由唾液酸化乳铁蛋白和唾液酸化乳脂球膜提供外,还由奶粉中的其它成分提供。
按照在含有唾液酸的组合物中的重量百分比计,含有唾液酸的组合物由以下组分组成:唾液酸化乳铁蛋白8.5%,唾液酸化乳脂球膜90%,益生菌1.5%。
其它成分包括:蛋白质、氨基酸、碳水化合物、低聚糖、脂质、益生元、必需脂肪酸、核苷酸、维生素、矿物质和其它微量营养素。
实施例8
一种奶粉,包括以下组分:含有唾液酸的组合物3%,以及,其它成分97%。奶粉中唾液酸的占比为0.15%,奶粉中的唾液酸除了由唾液酸化乳铁蛋白和唾液酸化乳脂球膜提供外,还由奶粉中的其它成分提供。
按照在含有唾液酸的组合物中的重量百分比计,含有唾液酸的组合物由以下组分组成:唾液酸化乳铁蛋白9%,唾液酸化乳脂球膜89.7%,益生菌1.3%。
其它成分包括:蛋白质、氨基酸、碳水化合物、低聚糖、脂质、益生元、必需脂肪酸、核苷酸、维生素、矿物质和其它微量营养素。
实施例9
一种奶粉,包括以下组分:含有唾液酸的组合物5%,以及,其它成分95%。奶粉中唾液酸的占比为0.12%,奶粉中的唾液酸除了由唾液酸化乳铁蛋白和唾液酸化乳脂球膜提供外,还由奶粉中的其它成分提供。
按照在含有唾液酸的组合物中的重量百分比计,含有唾液酸的组合物由以下组分组成:唾液酸化乳铁蛋白5%,唾液酸化乳脂球膜90%,益生菌5%。
其它成分包括:蛋白质、氨基酸、碳水化合物、低聚糖、脂质、益生元、必需脂肪酸、核苷酸、维生素、矿物质和其它微量营养素。
实施例10
一种奶粉,包括以下组分:含有唾液酸的组合物10%,以及,其它成分90%。奶粉中唾液酸的占比为0.25%,奶粉中的唾液酸除了由唾液酸化乳铁蛋白和唾液酸化乳脂球膜提供外,还由奶粉中的其它成分提供。
按照在含有唾液酸的组合物中的重量百分比计,含有唾液酸的组合物由以下组分组成:唾液酸化乳铁蛋白8%,唾液酸化乳脂球膜82%,益生菌10%。
其它成分包括:蛋白质、氨基酸、碳水化合物、低聚糖、脂质、益生元、必需脂肪酸、核苷酸、维生素、矿物质和其它微量营养素。
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (11)
1.一种组合物,其特征在于,由按照重量百分比计的以下组分组成:唾液酸化乳铁蛋白5-10%,唾液酸化乳脂球膜70-90%,益生菌菌粉0.5-20%,所述益生菌菌粉包括双歧杆菌菌粉和/或乳杆菌菌粉,所述双歧杆菌包括动物双歧杆菌BB12和/或长双歧杆菌BB536;所述乳杆菌包括鼠李糖乳杆菌LGG;
所述组合物中总唾液酸的含量大于0.1mg/g,所述唾液酸化乳铁蛋白中的唾液酸含量为20~50μg/g;所述唾液酸化乳脂球膜中的唾液酸含量为1300~1800μg/g;
所述组合物不包含游离的唾液酸,所述组合物中的唾液酸由唾液酸化乳铁蛋白和唾液酸化乳脂球膜提供;
所述组合物用于调节肠道内的唾液酸水平;
和/或,调节肠道内益生菌水平;
和/或,调节免疫。
2.如权利要求1所述的组合物,其特征在于,按照重量百分比计,由以下组分组成:唾液酸化乳铁蛋白8.5%,唾液酸化乳脂球膜90%,益生菌菌粉1.5%;所述组合物中的总唾液酸含量大于1mg/g。
3. 如权利要求1所述的组合物,其特征在于,所述益生菌菌粉中益生菌的活菌数为1×106~1×1012 CFU/g。
4.如权利要求1所述的组合物,其特征在于,所述组合物用于促进肠道内具有唾液酸代谢能力的益生菌生长,所述益生菌包括双歧杆菌和/或乳杆菌;
和/或,所述组合物用于调节细胞因子NO、TNF-α、IL-1β、IL-6、IL-10和IL-12的水平,以调节免疫系统的功能。
5.权利要求1-4任一项所述的组合物在制备食品、保健品和药品中的应用。
6.如权利要求5所述的应用,其特征在于,所述食品包括乳粉和乳液。
7.权利要求1-4任一项所述的组合物在以下(Ⅰ)~(Ⅲ)中至少一种中的应用:
(Ⅰ)在制备调节肠道内唾液酸水平产品中的应用;
(Ⅱ)在制备调节肠道内益生菌水平产品中的应用;
(Ⅲ)在制备调节免疫力产品中的应用。
8.一种营养制品,其特征在于,包括权利要求1-4任一项所述的组合物。
9.一种奶粉,其特征在于,包括权利要求1-4任一项所述的组合物,所述奶粉中唾液酸的重量百分比为0.07-0.25%。
10.如权利要求9所述的奶粉,其特征在于,所述奶粉中的唾液酸由唾液酸化乳铁蛋白和唾液酸化乳脂球膜提供;
或者,所述奶粉中的唾液酸除了由唾液酸化乳铁蛋白和唾液酸化乳脂球膜提供外,还由以下中的一种或多种提供:游离的唾液酸、唾液酸化母乳低聚糖、神经节苷脂。
11.一种奶粉,其特征在于,包括权利要求1-4任一项所述的组合物,所述组合物在所述奶粉中的重量百分比为3-10%。
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