CN113424964B - 一种组合物在制备调节人乳营养和免疫成分的保健食品或食品中的应用 - Google Patents
一种组合物在制备调节人乳营养和免疫成分的保健食品或食品中的应用 Download PDFInfo
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- CN113424964B CN113424964B CN202110670703.5A CN202110670703A CN113424964B CN 113424964 B CN113424964 B CN 113424964B CN 202110670703 A CN202110670703 A CN 202110670703A CN 113424964 B CN113424964 B CN 113424964B
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Abstract
本发明公开了一种组合物在制备调节人乳营养和免疫成分的保健食品或食品中的应用，该组合物包含唾液酸和益生菌。通过实验研究表明，本发明组合物中唾液酸与益生菌具有协同增效作用，能够调节哺乳人群的乳汁营养和免疫成分，进而影响母乳喂养婴儿肠道有益菌群的建立和免疫***的发育成熟。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种组合物在制备调节人乳营养和免疫成分的保健食品或食品中的应用。
背景技术
母乳喂养对婴幼儿巨大的健康效应。由于母乳中含有丰富的营养物质（如蛋白质、脂肪和大量的低聚糖）、免疫因子以及其他活性物质，对婴儿的身体和智力发育、免疫成熟、有益肠道菌群建立和发展起到关键作用。
唾液酸在母乳中广泛存在，是人乳低聚糖（HMO）的重要组成单元，在母乳中大量存在（0.3-1.5 mg/ml）。在母乳中，唾液酸化的低聚糖约占HMO总量的30%。研究显示，HMO具有调节婴儿肠道健康、有利于益生菌生长与定植、防御呼***和泌尿***感染、刺激产生抗体、提高机体免疫力以及促进婴儿大脑发育等功能。
益生菌已被大量证明能够有效提高肠道有益菌群，改善肠道菌群结构，提高免疫力。研究显示，服用益生菌不仅能够促进消化道膜免疫***的激活, 在孕妇及产后服用益生菌，还能够影响对脐血及母乳的免疫指标。
目前，益生菌产品多用于改善胃肠道功能，尚未发现将益生菌和唾液酸组合应用来调节人乳营养和免疫成分，通过改善母乳营养进而促进婴幼儿健康状况的文献报道。
发明内容
鉴于此，本发明的首要目的在于提供一种组合物在制备调节人乳营养和免疫成分的保健食品或食品中的应用，该组合物中唾液酸和益生菌协同增效，能够提高人乳中功能性的唾液酸化低聚糖和调节免疫因子，进而促进子代肠道有益菌群增殖和免疫***的发育成熟。
本发明是通过以下技术方案实现：
一种组合物在制备调节人乳营养和免疫成分的保健食品或食品中的应用，所述组合物包括唾液酸和益生菌。
优选的，所述组合物中唾液酸的含量为10~500mg。
优选的，所述组合物中唾液酸的含量为50~300mg。
优选的，所述组合物中益生菌的含量为1×108～1×1011cfu。
优选的，所述益生菌包含细菌、酵母或其任何组合，所述细菌选自乳酸杆菌属、双歧杆菌属、芽孢杆菌属、丙酸杆菌属、链球菌属、乳球菌属、片球菌属、肠球菌属、葡萄球菌属或其任何组合；所述酵母选自酿酒酵母、布拉氏酵母、马克斯克鲁维酵母或其任何组合。
更优选的，所述益生菌选自发酵乳杆菌CECT 5716、鼠李糖乳杆菌HN001、短双歧杆菌M-16V、乳双歧杆菌HN019或植物乳杆菌Lp-155中的一种或几种的混合。
更优选的，所述益生菌选自发酵乳杆菌CECT 5716、鼠李糖乳杆菌HN001和短双歧杆菌M-16V按1:9:5的比例组合。
本发明所述的组合物中唾液酸和益生菌具有协同增效作用，能够提高乳汁中游离的唾液酸转化为具有功能性的唾液酸化低聚糖，且能够提高乳汁中的免疫因子水平。
本发明所述的保健食品或食品的剂型为粉剂、片剂、胶囊剂、颗粒剂、口服液、糖果、饮料或乳制品。
本发明所述的保健食品或食品包含有效量的上述组合物和食品学上可接受的辅料。作为优选，所述食品学上可接受的辅料包含但不限于：麦芽糊精、抗性糊精、乳糖、淀粉、膳食纤维、微晶纤维素、糖醇（如甘露糖醇）、助流剂（如硬脂酸镁）、甜味剂、香精、色素等。
本发明所述的保健食品或食品以本领域常规方法制备成常规形式进行包装使用。
本发明与现有技术相比，具有如下有益效果：
本发明提供一种组合物在制备调节人乳营养和免疫成分的保健食品或食品中的新应用，该组合物包含唾液酸和益生菌。通过研究表明，唾液酸和益生菌具有协同增效作用，制备得到的组合物能够提高乳汁中游离的唾液酸转化为具有功能性的唾液酸化低聚糖，如6’-唾液乳糖（6’-sialyllactose, 6’-SL），提高哺乳期女性血清和乳汁中免疫因子水平。进一步研究显示，这种调节作用能进而影响子代肠道有益菌群的建立和肠道免疫***的发育成熟。
附图说明
图1为WT和KO大鼠T淋巴细胞比例；
图2为BSA 免疫后大鼠产生IgG 能力；
图3为WT组和KO组大鼠肠道菌群分布；
图4为各组干预后的乳汁中总唾液酸低聚糖含量图；
图5为各组干预后的乳汁中6’-SL含量图；
图6各组干预后的乳汁中游离唾液酸含量图；
图7为NMDS分析各组仔鼠肠道菌群分布图；
图8为各组仔鼠肠道菌群分布和丰度图。
具体实施方式
下面通过具体实施方式来进一步说明本发明，以下实施例为本发明具体的实施方式，但本发明的实施方式并不受下述实施例的限制。
本发明所涉及唾液酸、发酵乳杆菌（Lactobacillus fermentum）CECT 5716、鼠李糖乳杆菌（Lactobacillus rhamnosus）HN001、短双歧杆菌（Bifidobacterium breve）M-16V、乳双歧杆菌(Bifidobacteriumlactis) HN019、植物乳杆菌（Lactobacillusplantarum Lp-155）均为商业化原料。
实施例1：
包含本发明所述组合物的粉剂配方和制备方法：
粉剂的制备方法包括：将原辅料按照表1进行准确称量备料；并根据原辅料性状以及用量选择是否粉碎、过筛、预干燥或者逐级混合。将唾液酸、益生菌以及辅料成分置于混合机中，混合至混合原料形状以及水分指标达到质量标准后，使用粉剂包装机进行袋包，并定期检测袋包密封性及其装量差异。
表1本发明粉剂配方[设计剂量：按人服用剂量1.5g/60kg/天]
实施例2：
实验目的：利用唾液酸基转移酶1（ST6GAL1）基因敲除大鼠模型分析唾液酸化聚糖缺乏对母乳成分及肠道微生态的影响。
实验设计：ST6Gal-1 (α-2,6 唾液酰基转移酶) 是一种重要的糖基转移酶，它以α-2,6-键连接的方式将唾液酸残基添加到聚糖底物的末端位置，从而形成唾液酸化的聚糖。ST6Gal-1基因是体内唾液酸转移相关功能的重要基因之一。本实验首先利用CRISPR/Cas9 ***建立了ST6Gal-1 基因敲除SD 大鼠模型（ST6Gal-1-/-，KO）。ST6 Gal-1 基因一共由6 个外显子组成，本实验所用模型是从2 号外显子开始切一直到6 号外显子，共敲除掉了51 kb 的碱基对。
利用杂合雄性大鼠（ST6 Gal-1+/-，♂）与杂合雌性大鼠（ST6 Gal-1+/-，♀）交配，对生成的新生子鼠进行PCR 鉴定野生（WT）、杂合、和敲除鼠（KO）。对选取纯合敲除鼠（ST6Gal-1-/-）进行饲养观察。
实验结果：
1. ST6 Gal-1-/-（KO）大鼠的免疫水平显著低于野生型（WT）大鼠
通过流式细胞术，发现总的T 淋巴细胞及其亚群CD4+ T 淋巴细胞和CD8+ T 淋巴细胞的比例和数量均减少（图1）。
利用牛血清蛋白（BSA）分别对WT 和KO 大鼠进行免疫，结果显示免疫后KO 大鼠血清IgG 水平明显低于WT 大鼠（图2）。
免疫实验结果显示，KO大鼠在细胞免疫和体液免疫方面都有明显缺陷。
2.ST6 Gal-1-/- (KO)大鼠肠道菌群发生明显改变
通过16S rRNA高通量测序对WT 大鼠、KO 大鼠肠道微生物群特征进行了分析。结果显示，在门水平上，KO 组的厚壁菌门（Firmicutes）和放线菌门(Actinobacteriota)相对丰度显著降低（图3A）。在属水平上，KO 组较WT 组乳酸杆菌 (Lactobacillus) 的相对丰度却降低（图3B）。
菌群研究结果显示，KO组乳酸杆菌和其所属的厚壁菌门显著下降，双歧杆菌所在的放线菌门也显著下降，而乳酸杆菌和双歧杆菌是体内重要的有益菌和应用于益生菌的菌种之一。因此该结果提示，肠道有益菌，尤其是有益的乳酸菌，包括乳酸杆菌和双歧杆菌，与唾液酸转移酶活性以及功能性唾液酸化的聚糖的合成具有重要的正相关性。该结果为通过益生菌干预来改善唾液酸转移酶活性，进而提高功能性低聚糖的合成提供了重要的理论依据。
因此，本研究选取了具有典型代表性的乳酸菌益生菌，来自人乳的发酵乳杆菌CECT 5716，来自乳制品的鼠李糖乳杆菌HN001和乳双歧杆菌HN019、来自婴儿肠道短双歧杆菌M-16V等作为对象，研究补充益生菌和唾液酸对于母乳成分并进而对子代健康的影响。
实施例3：
实验目的：本实验利用动物实验研究单独使用唾液酸以及唾液酸复配益生菌对母乳成分以及子代的影响。
实验设计：本实验采用SPF级野生型SD大鼠及其子代进行研究。野生型母鼠和公鼠进行交配，待分娩后选取子代进行哺乳。将母鼠随机分为8组，其中，一组在子鼠出生14天后（第三周）开始进行单独唾液酸干预（SA组），3组进行单独益生菌干预（益生菌的种类和配比分别按照表1中三个配方中配比，编号为Pro1组、Pro2组、 Pro3组），3组进行唾液酸与益生菌组合干预（分别按照表1中三个配方中配比，编号为SA+Pro1组、SA+Pro2组、 SA+Pro3组），最后一组服用盐水作为对照(Con组)，持续2周（干预分组见表2）。哺乳期过后（四周）分别处死母鼠及子鼠。利用质谱法检测母鼠乳汁中低聚糖；利用ELISA检测母鼠血清、乳汁中以及免疫相关指标，以及仔鼠粪便中sIgA水平；利用16SrRNA测序法分析仔鼠肠道菌群。
表2动物实验分组
* Con为生理盐水对照组；
实验结果：
1. 母乳低聚糖组成
利用在线固相萃取-亲水作用色谱-质谱联用（SPE-HILIC-MS）方法对母鼠乳汁中的寡糖及游离唾液酸进行了profiling 分析。结果显示，SA组和益生菌单独干预组（Pro1、Pro2、Pro3）鼠乳唾液酸化寡糖的水平与对照组Con接近，无明显差别（p＞0.05）；相比之下，SA和益生菌组合的3个组（SA+Pro1、SA+Pro2、SA+Pro3）干预都明显上调了鼠乳总的唾液酸化低聚糖的水平（P<0.05）（图4），其中SA+Pro1组的效果比SA+Pro2和SA+Pro3组更好。
进一步对各组母鼠乳汁当中的主要HMO结构，6’-SL, 进行相对含量的分析。如图5所示，与对照组Con相比较，SA和益生菌复配的3个组（SA+Pro1、SA+Pro2、SA+Pro3）干预都显著提高了乳汁中6’-SL水平（P<0.05），同样可以看到SA+Pro1组比SA+Pro2和SA+Pro3组更高。而SA和益生菌单独组（Pro1、Pro2、Pro3）乳汁中主要HMO成分没有显著影响（P＞0.05）。
此外，针对乳汁中游离唾液酸的分析显示，相对于Con组，SA干预组的游离唾液酸水平上调（P<0.05）；益生菌单独干预（Pro1、Pro2和Pro3）组无显著变化（P＞0.05）；而SA和益生菌组合组（SA+Pro1、SA+Pro2、SA+Pro3）中乳汁中游离唾液酸降低（P<0.05）(图6)，其中SA+Pro1组和SA+Pro2组下降程度高于SA+Pro3组。结果说明，单独补充唾液酸，有助于提高乳汁中游离唾液酸的含量，但没有改变功能性低聚糖的组成。而唾液酸复配益生菌，两者能发挥协同作用，使更多的游离唾液酸转化为功能性低聚糖。且配方1和2中益生菌的菌种组合优于配方3。
HMO是母乳中重要的碳水化合物，唾液酸化的HMO是人乳中重要的组成部分。大量研究显示，HMO具有调节肠道菌群，预防呼吸和泌尿***感染，刺激产生抗体，提高免疫力和促进婴儿认知发育等功能。
综合以上结果，唾液酸联合益生菌能够起到协同增效作用，益生菌促进游离唾液酸合成功能性低聚糖，从而调节母乳HMO构成。
2. 母乳免疫因子
IL-10是乳汁中重要的免疫调节和抗炎细胞因子，在母乳喂养新生儿和婴儿肠道屏障动态平衡以及异常免疫反应的调节中发挥关键作用。TGF-β是影响细胞生长和分化的细胞因子，能促进乳导管生长和腺泡发育。研究显示，母乳中的TGF-β可以预防纯母乳喂养期间婴儿过敏性疾病的发生，并有助于启动新生儿IgA的产生。母乳免疫球蛋白中sIgA含量最高，在保护新生儿和婴儿黏膜，包括呼吸道、肠道和泌尿生殖道黏膜等，防御***中发挥重要作用，能帮助维持长期稳定平衡的肠道内环境，抵抗微生物的感染病并预防特应性皮炎。
通过酶联免疫吸附实验（ELISA）的检测了母鼠乳汁相关细胞因子及生长因子的水平，结果显示（表3）：相对于Con组，SA组在各项指标都没有显著变化（P＞0.05）；Pro1和Pro2组显著提高了乳汁中抗炎因子白介素10（IL-10）和分泌型免疫球蛋白A (sIgA)；Pro3组无显著影响。而对于SA和益生菌复配的3个组（SA+Pro1、SA+Pro2、SA+Pro3），乳汁中除IL-10和sIgA显著升高以外，转化生长因子TGF-β也显著升高，而且对IL-10和sIgA的提高程度比单独使用益生菌更高。
结果表明SA和益生菌的复配组合，在调节母乳免疫因子方面，相对于单独使用SA或益生菌，具有更好的效果。结果也显示，Pro1和Pro2配方中益生菌的菌种组合效果优于Pro3, 进一步的，Pro1中菌种的配比优于Pro2中的配比。
表3母鼠乳汁免疫因子含量
*与对照组(Con)对比，差异显著（P<0.05）。
综上结果表明，唾液酸联合益生菌能够更有效提高乳汁中免疫成分，有助于其母乳喂养的婴儿的肠道健康和免疫力。
3. 仔鼠血清和粪便免疫指标
ELISA 法检测了子鼠血清炎症相关细胞因子。结果显示，促炎因子IL-6 在3个SA和益生菌复配组都有所降低（P<0.05），在其他组中无显著变化。
进一步检测了各组子鼠血清和粪便中的sIgA的水平，结果显示3个SA和益生菌复配组（SA+Pro1、SA+Pro2、SA+Pro3）的血清和粪便中sIgA水平都显著高于Con组（P<0.05）。
同时可以看到，SA组和益生菌单独使用（Pro1、Pro2、Pro3）组粪便中sIgA也有提高趋势，但没有达到统计学上显著水平（表4）。结果说明，SA和益生菌的复配组合相对于单独干预，提高免疫力的能力更高。
表4仔鼠血清和粪便免疫指标
*与对照组(Con)对比，差异显著（P<0.05）。
4. 仔鼠肠道菌群
基于以上结果，选取Con组、SA组和SA+Pro1组，通过16S rRNA高通量测序研究子鼠肠道微生物群特征。通过NMDS 分析各组仔鼠菌群β多样性发现，Con组和SA 组及SA+Pro1组的菌群发生明显聚类，有统计学差异（p＜0.05），SA 组及SA+Pro1组的菌群结构更接近(图7)。说明母乳成分的改变，能够影响婴儿肠道菌群的组成结构。
在属水平上分析了各组子鼠肠道菌群中相对丰度（如图8），发现SA 组和SA+Pro1组菌群中有益的乳酸杆菌属（Lactobacillus）相对丰度均增高，其中SA+Pro1组升高的程度显著高于SA组，说明益生菌和SA组合对肠道有益菌群的调节作用优于单独使用SA。
Claims (4)
1.一种组合物在制备调节人乳营养和免疫成分的食品中的应用，其特征在于，所述组合物包括唾液酸和益生菌；所述调节人乳营养是指组合物能够促进乳汁中游离的唾液酸转化为具有功能性的唾液酸化低聚糖；所述组合物中唾液酸的含量为10~500mg，益生菌的含量为1×108～1×1011cfu；所述益生菌为发酵乳杆菌CECT 5716、鼠李糖乳杆菌HN001、短双歧杆菌M-16V按1:9:5或5:2:8的比例组合或所述益生菌为短双歧杆菌M16V、乳双歧杆菌HN019、植物乳杆菌Lp-115按5:4:6的比例组合。
2.根据权利要求1所述的应用，其特征在于，所述组合物中唾液酸的含量为50~300mg。
3.根据权利要求1所述的应用，其特征在于，所述组合物能够提高乳汁中的免疫因子水平。
4.根据权利要求1所述的应用，其特征在于，所述食品为粉剂、片剂、胶囊剂、颗粒剂、口服液、糖果、饮料或乳制品。
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