【作者】 施悦；

【导师】 张丽娜；

【作者基本信息】 江南大学 ， 食品科学与工程， 2022， 硕士

【摘要】 食物过敏是婴幼儿常见的过敏性疾病之一,加强早期预防对保障婴幼儿健康生长具有重要意义。合理利用益生元因其安全有效的特点被认为是较为前瞻、具有潜力的食物过敏预防途径。乳寡糖是婴幼儿最初接触到的一类益生元,有研究证明其具有缓解过敏性疾病的潜力。动物乳中含有种类丰富的寡糖,并且具有与母乳寡糖相似的生物活性功能。合理利用天然动物乳源寡糖,不仅有利于动物乳源及其生物活性成分的开发利用,还可能为缓解婴幼儿过敏性疾病提供一种有潜力且有效的途径。为此,本研究首先利用糖组学的方法对不同物种的乳进行综合评价,探究不同物种及不同泌乳期内寡糖的差异,并通过动物实验对动物乳中具有优势的寡糖的降敏功效进行评价,同时对其作用机制进行初步解析,主要研究内容如下:(1)特种乳中寡糖的定性定量研究:本章以特种乳(山羊乳、绵羊乳、骆驼乳)为研究对象,以牛乳和母乳作为对照,分析了不同物种乳中寡糖在结构及含量上的差异。结果表明,动物乳中的寡糖与母乳中的寡糖在结构和含量上均存在较大差异。在结构上,动物乳中鉴定到的寡糖以非岩藻糖基化中性寡糖和唾液酸化寡糖为主,母乳中鉴定到的寡糖以岩藻糖基化中性寡糖为主。在含量上,母乳中8种目标寡糖的总含量是骆驼乳的15倍,是牛乳和山羊乳的50倍,是绵羊乳的188倍。在我们研究的4种动物乳中,骆驼乳表现出明显的优势,具体为骆驼乳中鉴定到的寡糖种类与母乳中鉴定到的相似度最高;同时骆驼乳中寡糖的总含量显著高于其他3种动物乳,这主要是由于骆驼乳中含有丰富的3’-唾液酸乳糖(3’-Sialyllactose,3’-SL)。(2)特种乳中寡糖随泌乳期的变化规律研究:本章以特种乳(山羊乳、骆驼乳)为研究对象,以牛乳和母乳作为对照,分析了不同物种乳中寡糖随泌乳期的变化规律。结果表明,不同物种乳中寡糖的结构随泌乳期的延长变化较小,不同物种乳中寡糖总含量随泌乳期的延长均呈现下降的趋势,但变化规律在物种间存在差异性。牛乳中寡糖的总含量在泌乳早期急剧下降,后趋于稳定,唾液酸化寡糖、岩藻糖基化中性寡糖和非岩藻糖基化中性寡糖的含量比例在不同阶段均以唾液酸化寡糖为优势。山羊乳中寡糖的总含量持续下降,其中唾液酸化寡糖的含量比例下降,非岩藻糖基化中性寡糖的含量比例上升。骆驼乳中寡糖总含量的下降趋势较缓,在过渡乳和成熟乳中均以唾液酸化寡糖为优势。母乳中寡糖的总含量持续下降,其中唾液酸化寡糖的含量比例先上升后下降,非岩藻糖基化中性寡糖的比例持续上升,岩藻糖基化中性寡糖的比例呈先下降后上升。在我们研究的3种动物乳中,骆驼乳表现出明显的优势,具体为在过渡乳和成熟乳中,均以骆驼乳中鉴定到的寡糖种类与母乳中鉴定到的相似度最高,同时骆驼过渡乳和成熟乳中寡糖的总含量均明显高于牛乳和山羊乳。(3)特种乳中优势寡糖3’-SL的降敏功效研究:本章将不同剂量的3’-SL和驼乳和母乳中寡糖的含量比例复配的2’-岩藻糖基乳糖(2’-Fucosyllactose,2’-FL)和3’-SL混合物灌胃给β-乳球蛋白(β-Lactoglobulin,β-Lg)过敏小鼠,通过比较不同干预方式下小鼠的过敏程度及症状,验证3’-SL及其与2’-FL复配混合物的降敏功效,并对其降敏机制进行初步解析。结果表明3’-SL及其与2’-FL复配的混合物干预可改善小鼠的特异性皮炎病变及过敏症状,并减轻特异性皮炎和过敏反应给小鼠生长发育带来的影响。其中,高剂量的3’-SL和中剂量的以母乳寡糖含量比例复配的2’-FL和3’-SL混合物的干预方式具有最优的降敏效果,说明3’-SL对过敏反应具有调节作用并呈剂量依赖性,2’-FL和3’-SL在降敏功效中存在协同效应。3’-SL及其与2’-FL复配一方面可通过抑制肥大细胞聚集、抑制肥大细胞β-氨基己糖苷酶(β-hexosaminidase,β-Hex)的分泌进而减轻过敏症状的产生;另一方面可作为免疫调节因子对机体炎症反应进行调控,通过抑制Toll样受体4(Toll like receptor 4,TLR4)/核因子κB(Nuclear factor-κ-gene binding,NF-κB)通路信号传导进而抑制白细胞介素4(Interleukin-4,IL-4)、白细胞介素6(Interleukin-6,IL-6)等促炎因子的表达,同时刺激抗炎因子白细胞介素10(Interleukin-10,IL-10)的分泌进而调节免疫应答,促使辅助型T细胞1(Helper T cell 1,Th1)和辅助型T细胞2(Helper T cell 2,Th2)恢复新的平衡。此外,3’-SL及其与2’-FL复配的混合物可通过调节肠道菌群的多样性及结构,进而起到减轻过敏症状的效果。更多还原

【Abstract】 Food allergy is one of the common allergic diseases in infants.Improving early prevention is of great significance to ensure the healthy growth of infants.Making rational use of prebiotics is considered as a prospective and potential approach for the prevention of food allergy due to its safety and effectiveness.Milk oligosaccharides are a kind of prebiotics that infants are exposed to initially,and some studies have proved that milk oligosaccharides have the potential to alleviate allergic diseases.Animal milk contains a variety of oligosaccharides,which have similar biological activity functions to human milk oligosaccharides.The rational use of natural animal milk oligosaccharides is not only conducive to the development and utilization of animal milk sources and their bioactive components,but also may provide a safe and feasible new idea for alleviating allergic diseases in infants.Therefore,in this study,the method of glycomics was used to comprehensively evaluate the milk of different species,to explore the differences of milk oligosaccharides in different species and different lactation periods,and the alleviation effect on allergy of the dominant oligosaccharides in animal milk was evaluated through animal experiments,and its mechanism was preliminarily analyzed.The main research contents are as follows:(1)Qualitative and quantitative research on oligosaccharides in the milk of special animal: This chapter takes the milk of special animal(caprine milk,ovine milk and camel milk)as the research object,and uses bovine milk and human milk as the control to analyze the differences in composition and concentration of oligosaccharides in milk of different species.The results showed that there were significant differences in structure and concentration of oligosaccharides in animal milk and human milk.In terms of structure,the main oligosaccharides identified in animal milk were non-fucosylated neutral oligosaccharides and sialylated oligosaccharides,while the main oligosaccharides identified in human milk were fucosylated neutral oligosaccharides.The concentration of eight oligosaccharides in human milk was approximately 15 times higher than that in camel milk,50 times higher than that in bovine and caprine milk,and 188 times higher than that in ovine milk.Among the four kinds of animal milk studied,camel milk showed obvious advantages,specifically,the similarity of oligosaccharides identified in camel milk and human milk was the highest.Meanwhile,the total concentration of oligosaccharides in camel milk is significantly higher than that in the other three animal milk,which is mainly due to the rich3’-sialyllactose(3’-SL)in camel milk.(2)Study on the change law of oligosaccharides in special milk with lactation period:This chapter takes the milk of the milk of special animal(caprine milk and camel milk)as the research object,and uses bovine milk and human milk as the control to analyze the changes of oligosaccharides in different species with lactation period.The results showed that the composition of oligosaccharides in milk of different species changed little with the prolongation of lactation period,and the total concentration of oligosaccharides in milk of different species showed a downward trend with the prolongation of lactation period,but the variation pattern was different among species.The total concentration of oligosaccharides in bovine milk decreased sharply in early lactation,and then tended to be stable.In different stages,the concentration ratio of sialylated oligosaccharides was the dominant when comparing with fucosylated neutral oligosaccharides and non-fucosylated neutral oligosaccharides.The total concentration of oligosaccharides in caprine milk continually decrease,among which the concentration proportion of sialylated oligosaccharides decreased,while the concentration proportion of non-fucosylated neutral oligosaccharides increased.The decrease trend of total oligosaccharides concentration in camel milk was slow,and sialylated oligosaccharides were dominant in transitional milk and mature milk.The total oligosaccharides concentration in human milk continually decrease,in which the proportion of sialylated oligosaccharides increased at first and then decreased,the proportion of non-fucosylated neutral oligosaccharides continued to increase,and the proportion of fucosylated neutral oligosaccharides first decreased and then increased.Among the three kinds of animal milk studied,camel milk showed obvious advantages,specifically,in transition milk and mature milk,the oligosaccharides identified in camel milk and human milk had the highest similarity,and the total concentration of oligosaccharides in camel transition milk and mature milk was significantly higher than that in bovine milk and caprine milk.(3)Study on alleviation effect on allergy of 3’-SL that is the dominant oligosaccharide in special animal milk.In this chapter,β-Lactoglobulin(β-Lg)allergic mice were given a gavage of different doses of 3’-SL and mixture of 2’-fucosyllactose(2’-FL)and 3’-SL prepared in the proportion of milk oligosaccharides in camel milk and human milk.By comparing the allergic degree and symptoms of mice under different intervention methods,the alleviation effect on allergy of milk oligosaccharides was verified,and its mechanism was preliminarily analyzed.The results showed that milk oligosaccharide intervention could improve the atopic dermatitis lesions and allergic symptoms of mice,and alleviate the effects of atopic dermatitis and allergic reaction on the growth and development of mice.Among different interventions,the intervention with high dose of 3’-SL and the mixture of 2’-FL and 3’-SL prepared in the proportion of oligosaccharides in human milk has the optimal efficacy of alleviating allergies,indicating that 3’-SL had a dose-dependent effect on alleviating allergy,and 2’-FL and 3’-SL had a synergistic effect on alleviating allergy.On the one hand,3’-SL and the mixture of2’-FL and 3’-SL can alleviate allergic symptoms by inhibiting the aggregation of mast cells and inhibiting the secretion of β-hexosaminidase(β-Hex)in mast cells;On the other hand,it can be used as an immunoregulatory factor to regulate the inflammatory response by inhibiting the signal transduction of toll like receptor 4(TLR4)/ nuclear factor-κ-gene binding(NF-κB)pathway,thereby inhibiting the expression of pro-inflammatory factors(such as interleukin-4(IL-4)and interleukin-6(IL-6)),and stimulating anti-inflammatory the secretion of the factor interleukin-10(IL-10)to regulate the immune response and restore the balance of helper T cell 1(Th1)and helper T cell 2(Th2).In addition,3’-SL and the mixture of 2’-FL and 3’-SL can reduce allergic symptoms by regulating the diversity and structure of intestinal flora.更多还原