CN102497869A - 作为具有新功能益处的母乳等同性不可消化低聚糖的岩藻糖基乳糖 - Google Patents
作为具有新功能益处的母乳等同性不可消化低聚糖的岩藻糖基乳糖 Download PDFInfo
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- CN102497869A CN102497869A CN201080040930XA CN201080040930A CN102497869A CN 102497869 A CN102497869 A CN 102497869A CN 201080040930X A CN201080040930X A CN 201080040930XA CN 201080040930 A CN201080040930 A CN 201080040930A CN 102497869 A CN102497869 A CN 102497869A
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Abstract
本发明涉及用于刺激NK细胞的具有岩藻糖基乳糖的营养组合物。所述组合物适合用于婴儿。
Description
技术领域
本发明涉及具有不可消化的低聚糖的婴儿营养物，具体涉及其用于刺激免疫系统的用途。
背景技术
与婴儿配方喂养的婴儿相比，人乳喂养的婴儿具有较低的感染发生率，所述感染包括病毒感染。人乳中的许多组分，包括免疫球蛋白(例如IgA)、白细胞介素(IL)-1、IL-6、IL-8、IL-10、干扰素-γ(IFN-γ)、免疫活性细胞、转化生长因子-β(TGF-β)、乳铁蛋白、核苷酸和不可消化的低聚糖(NDO)，被认为参与对抗肠道或呼吸系统病原体感染的保护作用。发现TGF-β和膳食核苷酸是负责增加自然杀伤细胞活性的组分。
NDO是人乳的主要组分并且是人乳天然免疫系统的主要成分。人NDO促进以双歧杆菌(bifidobacteria)和乳酸杆菌(lactobacilli)为主的有益微生物丛的生长。还已知一些人NDO能够直接防止病原体和毒素的粘附。
人乳是优选的婴儿食物。但是，母乳喂养并不总是可行或合乎需要的。在所述情况下，婴儿配方或后续配方(follow on formulae)是一个良好的替代品。这些配方应该具有最佳的组成以便尽可能地模拟母乳的有益效果。
WO 2007/067053公开了包含植物来源的益生元菊粉和半乳糖醛酸寡糖以及由乳糖合成的益生元反式低聚半乳糖以减少感染的婴儿配方。
WO 2007/010084公开了用于进行免疫刺激的甘露聚糖多糖和甘露聚糖低聚糖。
US 6,576,251公开了用于通过肠内途径或肠胃外途径给予的饮食食物的碳水化合物混合物，所述混合物包含(a)单糖、(b)低聚糖(至多为己糖)和(c)多糖(至少为庚糖)，其中所述a、b、c的混合比例(按重量计)为：a＝1，b＝40至1000，c＝1至50，并且所述混合物包含至少1个重量百分比的岩藻糖，其以游离的形式并且/或者与低聚糖和/或多糖结合的形式存在。据报道所述碳水化合物混合物既具有营养作用也具有生物学作用，其比单独成分的相应作用明显更大。
EP 1 629 850提供了一种用于治疗和/或预防呼吸道感染和/或呼吸道感染疾病的方法和组合物，所述方法包括对哺乳动物口服给予组合物，所述组合物包含一种含不可消化低聚糖和至少5wt％可消化半乳糖的半乳糖。
EP 2 072 052涉及一种适合用于预防免疫缺陷个体中的机会性感染的组合物，所述组合物包含益生元和岩藻糖基化低聚糖，所述岩藻糖基化低聚糖选自2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、二岩藻糖基乳糖、乳糖基-N-岩藻戊糖、乳糖基-N-岩藻己糖、岩藻糖基乳糖基-N-己糖和岩藻糖基乳糖基-N-新己糖。所述文献进一步公开了所述组合物在预防免疫缺陷个体的机会性感染中的用途。
发明内容
人乳与家畜乳的区别在于人乳包含更多的NDO并且区别在于所述NDO在结构上是不同的。人NDO组是非常复杂的，因为它代表超过130种具有多种糖组成的不同化合物的异质性组。由于其复杂的和多形态的结构，所以大规模合成是复杂的。因此，制备具有与人乳等同的NDO组成的婴儿营养物在技术上和经济上尚不可行。
最近，新技术已可用于化学合成与具体人NDO等同的具体类型的NDO，从而提供在体内和体外测定中测试具体人NDO的免疫调节能力的机会。
发明人意外地发现岩藻糖基乳糖(fucosyllactose；FL)——一种在人母乳中大量存在的并具有较简单结构的低聚糖——可特别地增加自然杀伤(NK)细胞的数目并从而增加自然杀伤细胞的活性。NK细胞在对抗病毒感染和肿瘤细胞的天然防御中起着重要作用。特定低聚糖负责增加NK细胞活性的这一发现使得现在能够设计含FL(更具体为2’-FL)的营养组合物用于增加NK细胞和/或NK细胞活性。
具体实施方式
因此，本发明涉及一种刺激受试者中的NK细胞活性和/或NK细胞增殖的方法，所述方法包括对所述受试者给予一种含岩藻糖基乳糖的组合物，所述组合物不是人乳。在本发明的一个实施方案中，本发明的方法是用于刺激受试者中的NK细胞活性和/或NK细胞增殖的非医疗方法。在一个实施方案中，本发明方法用于治疗和/或预防受试者中的病毒感染。
本发明还涉及一种治疗和/或预防受试者中的病毒感染的方法，所述方法包括对所述受试者给予含岩藻糖基乳糖的组合物，所述组合物不是人乳。
本发明还可被表述为岩藻糖基乳糖在制备用于刺激NK细胞活性和/或NK细胞增殖的组合物中的用途，所述组合物不是人乳。在一个实施方案中，所述组合物用于治疗和/或预防病毒感染。
本发明还可被表述为岩藻糖基乳糖在制备用于治疗和/或预防病毒感染的组合物中的用途，所述组合物不是人乳。
本发明还可被表述为一种含岩藻糖基乳糖的用于刺激NK细胞活性和/或NK细胞增殖、特别是用于在刺激NK细胞活性和/或NK细胞增殖中使用的组合物，所述组合物不是人乳。在一个实施方案中，所述组合物用于治疗和/或预防病毒感染。
本发明还可被表述为一种含岩藻糖基乳糖的用于治疗和/或预防病毒感染、特别是用于在治疗和/或预防病毒感染中使用的组合物，所述组合物不是人乳。
本发明还涉及一种通过刺激自然杀伤(NK)细胞活性和/或NK细胞增殖而治疗和/或预防感染的方法，所述方法包括对所述受试者给予一种含岩藻糖基乳糖的组合物，所述组合物不是人乳。
本发明还可被表述为岩藻糖基乳糖在制备用于通过刺激自然杀伤(NK)细胞活性和/或NK细胞增殖而治疗和/或预防感染的肠内组合物中的用途，所述组合物不是人乳。
本发明还可被表述为一种通过刺激自然杀伤(NK)细胞活性和/或NK细胞增殖而用于治疗和/或预防感染、特别是在治疗和/或预防感染中使用的含岩藻糖基乳糖的组合物，所述组合物不是人乳。
本发明还涉及一种用于增强接种应答的方法，所述方法包括对所述受试者给予一种含岩藻糖基乳糖的组合物，所述组合物不是人乳。在一个实施方案中，所述方法用于增强对病毒抗原接种的接种应答。
本发明还可被表述为岩藻糖基乳糖在制备用于增强接种应答的肠内组合物中的用途。在一个实施方案中，所述组合物用于增强对病毒抗原接种的接种应答。
本发明还可被表述为一种用于增强接种应答、特别是用于在增强接种应答中使用的含岩藻糖基乳糖的组合物。在一个实施方案中，所述组合物用于增强对病毒抗原接种的接种应答。
根据本发明方法给予的或根据本发明使用的组合物优选经肠给予，更优选口服给予。或换言之所述组合物优选用于经肠给予、优选用于口服给予；或换言之所述组合物是肠内组合物，优选口服组合物。
岩藻糖基乳糖
本发明组合物包含岩藻糖基乳糖。岩藻糖基乳糖(FL)是在人乳中存在的不可消化的低聚糖。它不存在于牛乳中。它由连接在一起三个单糖单元：岩藻糖、半乳糖和葡萄糖构成。经β1→4键(linkage)连接至葡萄糖单元的半乳糖单元称为乳糖。岩藻糖单元经α1，2键而连接至乳糖的半乳糖单元(2’-岩藻糖基乳糖，2’-FL)或经α1，3键而连接至乳糖的葡萄糖单元(3-岩藻糖基乳糖，3-FL)。本发明组合物优选包含2’-FL。
2’-FL，优选α-L-Fuc-(1→2)-β-D-Gal-(1→4)-D-Glc，和3-FL，优选α-L-Fuc-(1→3)-[β-D-Gal-(1→4)]-D-Glc)，可购自例如Sigma-Aldrich。或者，它们可自人乳中分离，例如如Andersson&Donald，1981，J Chromatogr.211：170-1744中所述；或由遗传修饰的微生物产生，例如如Albermann et al，2001，Carbohydrate Res.334：97-103中所述。
优选地，所述组合物包含1mg至3g岩藻糖基乳糖/100ml，更优选包含10mg至2g/100ml，甚至更优选20mg至100mg FL/100ml。以干重计，所述组合物优选地包含0.007wt％至20wt％的岩藻糖基乳糖，更优选0.07wt％至10wt％，甚至更优选0.15wt％至1wt％的岩藻糖基乳糖。更少量的岩藻糖基乳糖在增加NK细胞和/或增加NK细胞活性方面效果较低，而过高的量将导致该产品的不必要的高成本。
除FL之外的不可消化的低聚糖
本发明组合物优选地包含除FL之外的不可消化的低聚糖(NDO)。优选地，所述除FL之外的NDO刺激双歧杆菌和/或乳酸杆菌的生长，更优选双歧杆菌。双歧杆菌和/或乳酸杆菌的含量增加会刺激形成健康的肠内微生物丛。所述NDO优选地在肠中不被消化，或者仅被部分地消化，所述消化是通过存在于人上消化道(特别是小肠和胃)中的酸或消化酶作用进行的，并且所述NDO会被人肠内微生物丛发酵。例如，人们认为蔗糖、乳糖、麦芽糖和常见的麦芽糖糊精是可消化的。
本发明组合物优选地包含DP范围在2-250、更优选2-60的不可消化的低聚糖。所述不可消化低聚糖优选地为选自以下的至少一种、更优选为至少两种、优选为至少三种：低聚果糖、低聚半乳糖、低聚木糖、低聚阿拉伯糖、阿拉伯半乳寡糖、低聚葡萄糖、低聚壳糖(chito-oligosaccharides)、葡糖甘露寡糖、半乳甘露寡糖、甘露聚糖寡糖(mannan-oligosaccharide)、含唾液酸的低聚糖，以及糖醛酸低聚糖。低聚果糖组包括菊粉，低聚半乳糖组包括反式低聚半乳糖或β-低聚半乳糖，低聚葡萄糖组包括环糊精、龙胆寡糖和黑曲霉寡糖和不可消化的聚葡萄糖(polydextrose)，半乳甘露寡糖组包括部分水解的瓜尔胶，糖醛酸低聚糖组包括半乳糖醛酸寡糖和果胶降解产物。
更优选地，本发明组合物包含选自以下的至少一种、更优选为至少两种、最优选为三种：低聚果糖、β-低聚半乳糖和糖醛酸低聚糖。更优选地，所述组合物包含β-低聚半乳糖。
在一个优选实施方案中，所述组合物包含菊粉和短链低聚果糖的混合物。在一个优选实施方案中，所述组合物包含低聚半乳糖和低聚果糖的混合物，其中低聚果糖选自短链低聚果糖和菊粉，更优选菊粉。优选地，至少两种不同的不可消化低聚糖的混合物可更大程度地刺激肠内微生物丛的有益细菌。优选地，所述两种不同的不可消化低聚糖(优选低聚半乳糖和低聚果糖)的混合物中两种低聚糖的重量比在25-0.05之间，更优选在20-1之间。低聚半乳糖，优选β-低聚半乳糖，更能够刺激双歧杆菌。优选地，本发明组合物包含聚合度(DP)为2-10的低聚半乳糖(优选β-低聚半乳糖)和/或DP为2-60的低聚果糖。
低聚半乳糖优选地为β-低聚半乳糖。在一个特别优选的实施方案中，本发明组合物包含β-低聚半乳糖([半乳糖]n-葡萄糖；其中n为2-60的一个整数，即2、3、4、5、6、......、59、60；优选地n选自2、3、4、5、6、7、8、9和10)，其中半乳糖单元主要经β键而连接在一起。β-低聚半乳糖也称作反式低聚半乳糖(TOS)。β-低聚半乳糖以例如Vivinal(TM)的商标名(Borculo Domo Ingredients，Netherlands)市售。另一合适来源为Bi2Munno(Classado)。优选地，TOS包含以总键数计至少80％的β-1，4和β-1，6键，更优选至少90％。
低聚果糖是包含DP或平均DP为2-250、更优选2-100、甚至更优选10-60的β-连接的果糖单元链的NDO。低聚果糖包括菊粉、果聚糖和/或混合类型的多聚果糖(polyfructan)。尤其优选的低聚果糖是菊粉。适合用于所述组合物的低聚果糖也是市售可得的，例如(Orafti)。优选地，低聚果糖具有的平均DP大于20。
糖醛酸低聚糖优选地获自果胶降解产物。因此本发明组合物优选地包含DP为2-100的果胶降解产物。所述果胶降解产物优选地是由苹果果胶、甜菜果胶和/或柑橘果胶制备。优先地，糖醛酸低聚糖是半乳糖醛酸寡糖。优选地，所述组合物包含FL和选自低聚半乳糖和糖醛酸低聚糖中的一种。
除FL之外，最优选地，所述组合物包含β-低聚半乳糖、低聚果糖和糖醛酸低聚糖。发现所述结合物与岩藻糖基乳糖(特别是2’-岩藻糖基乳糖)起协同作用。β-低聚半乳糖∶低聚果糖∶糖醛酸低聚糖的重量比优选地为(20-2)∶1∶(1-20)，更优选地为(20-2)∶1∶(1-10)，甚至更优选地为(20-2)∶1∶(1-3)、甚至更优选地为(12-7)∶1∶(1-2)。最优选地，所述重量比为约9∶1∶1.1。优选地，FL与β-低聚半乳糖(优选TOS)的重量比为5-0.05，更优选地为5-0.1，更优选地为2-0.1。优选地，FL与低聚果糖(优选菊粉)的重量比为10-0.05，更优选地为10-0.1，更优选地为2-0.5。优选地，FL与糖醛酸低聚糖(优选衍生自果胶)的重量比为10-0.05，更优选地为10-0.1，更优选地为2-0.5。
优选地，所述组合物包含80mg至4g的不可消化低聚糖(包括岩藻糖基乳糖)/100ml，更优选地含150mg至2g/100ml，甚至更优选地包含300mg至1g的不可消化低聚糖/100ml。以干重计，所述组合物优选地包含0.25wt％至25wt％的不可消化低聚糖(包括岩藻糖基乳糖)，更优选为0.5wt％至10wt％，甚至更优选为1.5wt％至7.5wt％。更低量的不可消化低聚糖在刺激微生物丛中的有益细菌方面效果较小，而过高量将导致胃气胀和腹部不适的副作用。
营养组合物
优选地，所述含岩藻糖基乳糖的组合物是营养组合物。本发明组合物不是人乳。本发明组合物优选地经肠内给予，更优选地口服给予。
本发明组合物优选为营养配方，优选婴儿配方。本发明组合物可有利地用作婴儿的完全营养物。本发明组合物优选包含脂质组分、蛋白质组分和碳水化物组分，并且本发明组合物优选地以液态形式给予。本发明包括干燥食物，优选粉末，其附有关于将所述干燥食物混合物与合适液体(优选水)混合的说明书。
本发明有利地提供了一种组合物，其中脂质组分提供5-50％的总卡路里，蛋白质组分提供5-50％的总卡路里，可消化的碳水化物组分提供15-85％的总卡路里。本发明有利地提供了一种组合物，其中脂质组分提供20-50％的总卡路里，蛋白质组分提供5-30％的总卡路里，可消化的碳水化合物组分提供30-70％的总卡路里。优选地，在本发明组合物中，脂质组分提供35-50％的总卡路里，蛋白质组分提供7.5-12.5％的总卡路里，可消化的碳水化物组分提供40-55％的总卡路里。为计算蛋白质组分的％总卡路里，需要考虑由蛋白质、肽和氨基酸所提供的总能量。
本发明组合物优选地包含至少一种选自以下的脂质：动物脂质(除了人脂质以外)和植物脂质。优选本发明组合物包含植物脂质与至少一种选自以下的油的结合物：鱼油、动物油、藻油、真菌油和细菌油。本发明组合物优选地包含长链多不饱和脂肪酸(LC-PUFA)。LC-PUFA是含两个或更多个不饱和键的长度为20-24个碳原子、优选20或22个碳原子的脂肪酸和脂肪酰基链。更优选地，本发明组合物包含二十碳五烯酸(EPA，n-3)、二十二碳六烯酸(DHA，n-3)和/或花生四烯酸(ARA，n-6)。
优选地，本发明组合物包含以总脂肪含量计至少0.1wt.％，优选至少0.25wt.％，更优选至少0.6wt.％，甚至更优选至少0.75wt.％的具有20和22个碳原子的LC-PUFA。
LC-PUFA(特别是具有20和22个碳原子的LC-PUFA)的含量优选地不超过总脂肪含量的6wt％，更优选地不超过3wt.％，因为需要尽可能接近地模拟人乳。LC-PUFA可作为游离脂肪酸，以甘油三酯形式、甘油二酯形式、甘油单酯形式、磷脂形式，或作为上述一种或多种的混合物来提供。本发明组合物优选地包含以总脂肪计5-75wt％、优选10-50wt％的多不饱和脂肪酸。
在营养组合物中所使用的蛋白质优选地选自：非人的动物蛋白质(优选乳蛋白质)、植物蛋白质(优选大豆蛋白质和/或稻蛋白质)、其水解产物、其游离氨基酸以及它们的混合物。本发明组合物优选地包含酪蛋白、乳清、水解的酪蛋白和/或水解的乳清蛋白。优选地，所述蛋白质包含完整蛋白质，更优选完整牛乳清蛋白和/或完整牛酪蛋白蛋白质。
本发明组合物优选地包含选自以下的可消化的碳水化合物：蔗糖、乳糖、葡萄糖、果糖、玉米糖浆固体、淀粉和麦芽糖糊精，更优选地为乳糖。
从以上可以看出，同样重要的是所述液体食物不具有过度的卡路里密度，但仍提供足够的卡路里以喂养受试者。因此，所述液体食物优选地具有在0.1-2.5kcal/ml之间的卡路里密度、甚至更优选在0.5-1.5kcal/ml之间、最优选在0.6-0.8kcal/ml之间的卡路里密度。
优选地，本发明组合物包含核苷酸和/或核苷，更优选核苷酸。优选地，所述组合物包含5’-单磷酸胞苷、5’-单磷酸尿苷、5’-单磷酸腺苷、5’-单磷酸鸟苷和/或5’-单磷酸肌苷，更优选5’-单磷酸胞苷、5’-单磷酸尿苷、5’-单磷酸腺苷、5’-单磷酸鸟苷和5’-单磷酸肌苷。优选地，所述组合物包含5-100mg、更优选5-50mg、最优选10-50mg核苷酸和/或核苷/100克所述组合物干重。核苷酸和/或核苷的存在有利地刺激NK细胞活性。人们认为核苷酸和/或核苷与本发明组合物的岩藻糖基乳糖起协同作用。
应用
在一个实施方案中，本发明组合物用于刺激自然杀伤细胞活性和/或自然杀伤细胞增殖。在一个实施方案中，本发明组合物用于治疗和/或预防病毒感染。在一个实施方案中，用于刺激自然杀伤细胞活性和/或自然杀伤细胞感染，并且/或者用于治疗和/或预防病毒感染的所述组合物是用于给予HIV患者、年长患者和/或肿瘤患者。
NK细胞是一类细胞毒性淋巴细胞，其构成先天性免疫系统的主要组分。NK细胞在抵御细胞内感染的防御中起着重要作用。NK细胞被定义为在人体中不表达T-细胞抗原受体(TCR)或Pan T标记CD3或表面免疫球蛋白(Ig)B细胞受体，但通常表达表面标记CD16(FcγRIII)和CD56的大颗粒状淋巴细胞。它们被称作“自然杀手(nature killer)”，因为最初的观点是它们不需要活化以便杀伤不含I型主要组织相容性复合体(MHC)的“自身”标记的细胞。NK细胞具有两种主要类型的效应子作用：细胞杀伤和分泌细胞因子。增加NK细胞活性(通过增加NK细胞的数目和/或增加NK细胞的具体活性)导致对抗病毒感染的抵抗力增加。因此，含岩藻糖基乳糖的营养组合物优选地用于预防和/或治疗病毒感染，更优选由以下病毒引起的病毒感染：正粘病毒科尤其是流感病毒、疱疹病毒科、轮状病毒、巨细胞病毒、杯状病毒科、呼吸道合胞病毒、人免疫缺陷病毒和/或鼻病毒。因此，含岩藻糖基乳糖的营养组合物优选地用于预防和/或治疗病毒感染，更优选地所述病毒感染为普通感冒、流行性感冒、麻疹、水痘、病毒性腹泻、病毒性胃肠炎、HIV感染和/或病毒性呼吸道感染。在一个优选实施方案中，本发明用于HIV患者。在一个实施方案中，本发明涉及对HIV患者提供营养物。本发明组合物用于HIV患者是有利的，因为HIV患者具有降低的自然杀伤细胞活性。
因此，含岩藻糖基乳糖的营养组合物用于婴儿配方是特别有益的。在一个实施方案中，本发明涉及对婴儿提供营养物。配方喂养的婴儿与成人相比具有发育不全的免疫系统，并且与人乳喂养的婴儿相比更易于发生病毒感染。优选地，所述婴儿为0-36个月大，更优选为0-18个月大，甚至更优选为0-12个月，最优选为0-6个月大。婴儿越小，免疫系统发育越不健全。
含岩藻糖基乳糖的组合物甚至更有利地用于早产婴儿和/或出生体重非常低或低的婴儿，因为这些婴儿甚至更容易受到和/或易于发生病毒感染。
含岩藻糖基乳糖的组合物甚至更有利地用于经由剖腹产术分娩的婴儿。剖腹产术出生的婴儿是在医院中具有更多病原体的环境中出生的，所述病原体是由母亲给予婴儿的抗体所无法有效抵抗的。剖腹产术出生的婴儿具有延迟的和次优的大肠肠道定殖并因此还更易于发生肠内感染。
含岩藻糖基乳糖的组合物可有利地用于年长者的营养物。在一个实施方案中，本发明涉及对年长者提供营养。年长者是年龄为55岁或更高的人，特别是年龄为65岁或更高的人。已证实年长者具有比健康年青成年个体更低的自然杀伤细胞活性。年长者尤其易发生病毒感染并发症。在一个优选的实施方案中，本发明用于治疗和/或预防年长者中的免疫衰老。年长者更易于发生肿瘤。NK细胞活性抑制肿瘤细胞增殖。在一个优选实施方案中，本发明用于癌症患者的营养物。肿瘤患者具有比健康年青成年个体更低的自然杀伤细胞活性。
实施例
实施例1：
材料和方法
对6-8周龄雌性C57BL/6小鼠(Charles River)给予半纯化的基于AIN-93G的膳食(Research Diet Service，Wijk bij Duurstede，theNetherlands)，其包含
1)比例为9∶1∶1.1的2wt％的β-低聚半乳糖(GOS；source VivinalGOS，Borculo Domo)、低聚果糖(FOS；source RaftilineHP，Orafti)和半乳糖醛酸寡糖(源自AOS)。AOS是果胶(Siidzucker AG，Mannheim，Germany)制备的，DP为1-20。其包含以总重量计约75％半乳糖醛酸低聚物；
2)1wt％乳糖新四糖(lactoneotetraose，LNnT)，
3)1wt％3’-唾液酸基乳糖(3’-SL)，或
4)1wt％2’-岩藻糖基乳糖(2’-FL)。
所有组均与未补充的对照膳食进行比较。在第一次接种之前14天开始膳食补充，持续至试验结束，即第一次接种之后31天。
接种试验是使用Influvac(Solvay Pharmaceuticals，Weesp，theNetherlands)从2005/2006季进行。小鼠接受初次接种和加强接种，其包括皮下注射(sc)总体积为100μl的1∶1的疫苗和佐剂的混合物。所述加强接种是在初次接种后第21天进行。所述试验在加强接种后第10天结束。在试验结束时采集血液样本。所包括的阴性对照组接受总体积为100μl的1∶1的PBS和佐剂的混合物注射。为测定NK细胞的百分比，将细胞用FITC-标记的抗小鼠CD3mAb结合PE-标记的抗小鼠NK1.1mAb进行标记。脾细胞悬液中的NK细胞细胞毒性是使用标准的51Cr释放测定法进行测定的。简言之，NK细胞细胞毒性是使用YAC-1靶细胞进行测试。特异性51Cr释放百分比计算为：特异性裂解的百分比＝(试验释放-自发释放)/(总洗涤剂释放-自发释放)×100。自发释放数值总是低于总裂解的15％。
调节T细胞(Treg)的百分比是通过使用别藻蓝蛋白(APC-)标记的抗小鼠CD3mAb、Pe-Cy5-标记的抗小鼠CD4mAb和藻红蛋白(PE)-标记的抗小鼠CD25mAb的流式细胞术(FACSCalibur)和异硫氰酸荧光素(FITC)-标记的Foxp3mAb的细胞内染色，根据制造商(eBiosciences，San Diego，CA)提供的说明书进行测定。
统计学分析是使用GraphPadPrism软件进行。测试组和对照组之间的统计学差异是通过ANOVA进行分析，如果多组与单一(对照)组进行比较时则用事后邓尼特检验(post hoc Dunnett’s test)。在所有试验中P-值＜0.05被认为是显著的。
结果
将三种化学合成的人低聚糖的免疫调节作用与GOS/FOS/AOS进行比较。与对照喂养的动物相比，补充GOS/FOS/AOS、LNnT、2’-FL或3’-SL导致DTH反应——TH1依赖性参数——的显著增加。
值得注意的是，与对照和补充GOS/FOS/AOS的小鼠相比，NK细胞在脾脏中的百分比在补充人低聚糖的小鼠中显著增加，参见表1。使用2’-FL时该作用最高。为检测NK细胞百分比在补充人低聚糖的组中的增加是否还与功能性活性相关，在小鼠脾细胞中测量NK细胞活性。与对照相比，在补充2’-FL的小鼠脾细胞中检测到显著增加的NK细胞活性，参见表1。
表1：与人乳NDO类似的膳食NDO对NK细胞和NK细胞活性的作用
＊表示与对照组相比ρ＜0.05
＊＊表示与对照组相比ρ＜0.01
最后，从表2中可以看出，对于有FL的饮食，调节T细胞(Treg)的量降低至最大程度。
表2：与人乳NDO类似的膳食NDO对调节T细胞(Treg)百分比的作用
＊表示与对照组相比ρ＜0.05
调节T细胞的减少表示对免疫应答的抑制降低，并因此使得能够增加对病毒病原体的接种应答。在将进行接种时，调节T细胞的短暂减少是特别有益的。
总体而言，这些结果支持了，口服补充2’-FL刺激NK细胞的生长和/或活性。这些结果表示膳食2’-FL用于增强接种应答，尤其是对病毒抗原接种的接种应答的作用。这些结果表示膳食2’-FL用于治疗和/或预防病毒感染的作用。
实施例2
用于刺激NK细胞活性的婴儿配方包含：每100ml(13.9干重)
1.4g蛋白质(乳清和酪蛋白)
7.3g可消化糖类(包括乳糖)
3.6g脂肪(植物脂肪、鱼油)
0.8g不可消化的低聚糖，其中含80mg 2’-岩藻糖基乳糖、640mg β-低聚半乳糖和80mg低聚果糖
另外还包含本领域中已知的：胆碱、肌醇、牛磺酸、矿物质、微量元素和维生素。
实施例3
可用于刺激HIV患者中自然杀伤细胞活性的优选组合物包含：每100g干重
| 膳食纤维 | 5-50g |
| 低聚果糖 | 总膳食纤维的5％ |
| 低聚半乳糖 | 总膳食纤维的40％ |
| 果胶水解产物 | 总膳食纤维的50％ |
| 2’-FL | 总膳食纤维的5％ |
| N-乙酰半胱氨酸 | 0.5-5g |
| 碳水化合物(非膳食纤维) | 2-20g |
| 脂肪 | 4-20g |
Claims (11)
1.岩藻糖基乳糖在制备用于刺激自然杀伤(NK)细胞活性和/或NK细胞增殖的肠内组合物中的用途，所述组合物不是人乳。
2.岩藻糖基乳糖在制备用于治疗和/或预防病毒感染的肠内组合物中的用途，所述组合物不是人乳。
3.岩藻糖基乳糖在制备用于通过刺激自然杀伤(NK)细胞活性和/或NK细胞增殖而治疗和/或预防感染的肠内组合物中的用途，所述组合物不是人乳。
4.岩藻糖基乳糖在制备用于增强接种应答，优选地用于增强对病毒抗原接种的接种应答的肠内组合物中的用途。
5.前述权利要求任一项的用途，其中所述组合物用于给予婴儿、HIV患者、年长者和/或肿瘤患者。
6.前述权利要求任一项的用途，用于向婴儿提供营养。
7.前述权利要求任一项的用途，用于向HIV患者、年长者和/或肿瘤患者提供营养。
8.前述权利要求任一项的用途，其中所述组合物另外包含至少一种选自以下的物质：β-低聚半乳糖、低聚果糖和糖醛酸低聚糖。
9.前述权利要求任一项的用途，其中岩藻糖基乳糖为2’-岩藻糖基乳糖。
10.前述权利要求任一项的用途，其中所述组合物包含以所述组合物干重计0.07-1wt％的岩藻糖基乳糖。
11.前述权利要求任一项的用途，其中所述组合物包含以总能量计5-50％的蛋白质、15-85％的可消化碳水化合物和5-50％的脂肪。
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107405354A (zh) * | 2015-03-05 | 2017-11-28 | 格礼卡姆股份公司 | 治疗急性呼吸道感染的组合物和方法 |
| CN107567588A (zh) * | 2015-05-19 | 2018-01-09 | 雀巢产品技术援助有限公司 | 用于鉴定母乳缺失岩藻糖基转移酶‑2相关聚糖以及喂食一定剂量所述聚糖的成套试剂盒 |
| CN104940925A (zh) * | 2015-07-17 | 2015-09-30 | 宁波荣安生物药业有限公司 | 一种人用狂犬病疫苗及其制备方法 |
| CN111050851A (zh) * | 2017-08-11 | 2020-04-21 | N·V·努特里奇亚 | 用于提高免疫适应性的人乳低聚糖 |
| CN107373674A (zh) * | 2017-08-25 | 2017-11-24 | 西宝生物科技（上海）股份有限公司 | 一种肿瘤治疗后的营养组合物及其应用 |
| CN112384079A (zh) * | 2018-07-12 | 2021-02-19 | N·V·努特里奇亚 | 用于治疗腹泻的营养组合物、其制备和治疗方法 |
| CN112911947A (zh) * | 2019-06-04 | 2021-06-04 | N·V·努特里奇亚 | 包含2’岩藻糖基乳糖和膳食丁酸的营养组合物 |
| CN116115623A (zh) * | 2022-12-12 | 2023-05-16 | 天津市肿瘤医院（天津医科大学肿瘤医院） | 2'-岩藻糖基乳糖作为一种抗肿瘤辅助药物的应用 |
| CN116115623B (zh) * | 2022-12-12 | 2024-02-23 | 天津市肿瘤医院（天津医科大学肿瘤医院） | 2'-岩藻糖基乳糖作为一种抗肿瘤辅助药物的应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| US10588965B2 (en) | 2020-03-17 |
| EP3342413B1 (en) | 2021-10-20 |
| US20160158346A1 (en) | 2016-06-09 |
| WO2011008087A1 (en) | 2011-01-20 |
| PL3342413T3 (pl) | 2022-03-28 |
| US20220023418A1 (en) | 2022-01-27 |
| ES2904608T3 (es) | 2022-04-05 |
| RU2530641C2 (ru) | 2014-10-10 |
| EP2813230A1 (en) | 2014-12-17 |
| BR112012000859B1 (pt) | 2021-08-03 |
| CN102497869B (zh) | 2013-11-27 |
| US11135290B2 (en) | 2021-10-05 |
| BR112012000859A2 (pt) | 2018-03-13 |
| US20180264104A1 (en) | 2018-09-20 |
| EP2453901A1 (en) | 2012-05-23 |
| ES2525565T3 (es) | 2014-12-26 |
| RU2012105310A (ru) | 2013-08-20 |
| PL2453901T3 (pl) | 2015-03-31 |
| MY195771A (en) | 2023-02-10 |
| EP3342413A1 (en) | 2018-07-04 |
| EP2453901B1 (en) | 2014-09-10 |
| EP4197542A1 (en) | 2023-06-21 |
| MY164674A (en) | 2018-01-30 |
| US20120177691A1 (en) | 2012-07-12 |
| US20200155672A1 (en) | 2020-05-21 |
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