Nutritional composition for improving the intestinal microflora and uses thereof

Abstract

The present application belongs to the field of food, and particularly relates to a nutritional composition for improving intestinal microecology and use thereof. The nutritional composition provided by the present application comprises essential active ingredients shown in (I) and (II) as follows: (I) lacto-N-neotetraose, (II) 1,2-diacyl-sn-glycero-3-phosphocholine; and in the nutritional composition, the mass ratio of the lacto-N-neotetraose to the 1,2-diacyl-sn-glycero-3-phosphocholine is 1:(0.010-3.0). Studies show that both lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine can synergistically improve the intestinal microecological imbalance of offspring caused by maternal immune activation and provide benefits for the nervous system development of offspring.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food, in particular to a nutritional composition for improving the intestinal microecology and uses thereof. BACKGROUND

[0002] During pregnancy, the maternal immune system status will change dramatically to maintain the immune balance with the fetus, prevent rejection reaction, and maintain natural immunity and adaptive immune mechanisms against infection. Exposure to various factors such as environment during pregnancy will lead to an imbalance in the level of inflammatory factors in the body, resulting in a state of maternal immune activation (MIA). Maternal immune activation is considered a risk factor for the etiology of various mental and nervous system diseases in offspring, and the impact on offspring is heterogeneous, and the neurodevelopment of offspring under the background of maternal immune activation is affected by various factors.

[0003] In recent years, the role of maternal immune activation caused by pregnancy infection in neurodevelopmental disorders (NDDs) in offspring has become a research hotspot and has made some progress. Studies have found that the immature fetal brain is particularly susceptible to MIA damage. MIA can play an important role in the occurrence and development of neurodevelopmental disorders in offspring by changing the susceptibility of the genome, affecting epigenetic modification, microglial cell activation and inflammation in the brain.

[0004] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk, with a content second only to lactose and fat, and even higher than that of protein. Human milk oligosaccharides are a class of complex sugars with a variety of structures. According to the molecular structure characteristics, HMOs can be divided into neutral fucosylated HMOs, neutral non-fucosylated HMOs and acidic sialylated HMOs. Studies have shown that the total HMOs content in the early lactation period (i.e. 1 month), 2'-fucosyllactose (2'-FL), 6'-sialyllactose (6'-SL) and 3'-sialyllactose (3'-SL) are positively correlated with the development level of cognitive, language and motor abilities in later infants (i.e. 6-24 months old). Animal experiments have also shown that the intake of HMOs composition can significantly improve the spatial memory ability of rodents and piglets and accelerate the learning speed of operation tasks.

[0005] Studies have found that HMOs have a regulating effect on the intestinal health of the subject. For example, reference document 1 discloses that human milk oligosaccharides (including 2'-FL, 3-FL, DFL, LNT, LNnT, 3'-SL, 6'-SL, LNFP-I, or a mixture thereof, etc.) can be used to increase the abundance of Akkermansia in the human gastrointestinal tract, thereby helping to alleviate brain-gut disorders such as stress, anxiety and depression, etc.; reference document 2 discloses a nutritional composition comprising at least two human milk oligosaccharides, which is used to reduce the concentration of harmful proteolytic metabolites such as branched-chain short-chain fatty acids in the digestive tract of infants or young children, thereby helping to resist intestinal inflammation, intestinal infection, etc.

[0006] 1,2-diacyl-sn-glycero-3-phosphocholine (PC) is an asymmetric molecule with a glycerol skeleton, which naturally exists in egg yolk, soybean, sunflower seed, animal liver, peanut, etc. 1,2-diacyl-sn-glycero-3-phosphocholine is the precursor of choline, the memory factor in the brain, and is very critical to brain development. At present, 1,2-diacyl-sn-glycero-3-phosphocholine is mainly used for liposome preparation.

[0007] Reference documents:

[0008] Reference document 1: CN111683665A;

[0009] Reference document 2: CN110650635A. SUMMARY

[0010] Problems to be solved by the invention

[0011] Studies have shown that maternal immune activation plays an important role in the occurrence and development of neurodevelopmental disorders in offspring, and intestinal flora homeostasis imbalance can regulate gastrointestinal physiology, immune function, etc. through the gut-brain axis to participate in the occurrence and development of neurodevelopmental disorders.

[0012] For neurodevelopmental disorders, behavior intervention and drug intervention are currently used, but these methods have limited effect, large side effects, etc., especially for younger populations. And the younger the age, the more difficult it is to detect neurodevelopmental disorders, and the current judgment standard for neurodevelopmental disorders is often after 3 years old, which will miss the key intervention period of brain development in the early 1000 days of life.

[0013] In view of this, the present application has carried out a large amount of research, and found that maternal immune activation can cause intestinal microecological imbalance in offspring, including changes in intestinal flora and its metabolites, changes in intestinal structure and function, and the occurrence of intestinal inflammation. Based on this, the present application believes that how to reduce, improve or inhibit the adverse effects of maternal immune activation on the intestinal health of offspring, thereby providing help for the neurodevelopment of offspring through the gut-brain axis, has important research significance.

[0014] Although the effects of nutrients on intestinal health have been studied in, for example, the above-mentioned cited documents 1 and 2, such studies are still insufficient, and the disclosed intervention subjects are mainly healthy humans, and cited document 1 emphasizes that the intervention subjects are non-infant groups. The effects of nutrients on intestinal conditions in specific states (e.g., intestinal conditions of offspring reared by females who have experienced or are in a state of maternal immune activation), particularly on developmental damage (i.e., innate, systemic, and diffuse damage, rather than acquired, acquired, and localized damage), have not been studied in the prior art.

[0015] In this regard, the present application has been extensively studied and unexpectedly found that a combination of lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine can improve intestinal microecological imbalance of offspring caused by maternal immune activation, including improving intestinal microorganisms and their metabolic changes, intestinal structure and barrier function damage, and intestinal inflammation, based on the concept of the gut-brain axis, this improvement will be beneficial to the healthy development of the nervous system of offspring, and the present application unexpectedly found that when the mass ratio of lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine is within a certain range, there is a synergistic effect between the two.

[0016] Solution to the problem

[0017] [1]. A nutritional composition, wherein it is a nutritional composition having an effect of improving intestinal microecological imbalance of offspring caused by maternal immune activation, the nutritional composition comprising the following essential active ingredients shown as (I) and (II):

[0018] (I) lacto-N-neotetraose,

[0019] (II) 1,2-diacyl-sn-glycero-3-phosphocholine;

[0020] And in the nutritional composition, the mass ratio of the lacto-N-neotetraose to the 1,2-diacyl-sn-glycero-3-phosphocholine is 1:(0.010-3.0).

[0021] [2]. The nutritional composition according to [1], wherein the nutritional composition further comprises a human milk oligosaccharide other than lacto-N-neotetraose.

[0022] [3]. The nutritional composition according to [1] or [2], wherein the nutritional composition further comprises any one or more of 1,2-diacyl-sn-glycero-3-phosphoinositide, 1,2-diacyl-sn-glycero-3-phospho serine, 1,2-diacyl-sn-glycero-3-phosphoethanolamine, 1,2-diacyl-sn-glycero-3-phosphoglycerol, and N-acyl-D-sphingosine-1-phosphocholine.

[0023] [4]. Use of the nutritional composition according to any one of [1] to [3] in the manufacture of a food for improving intestinal microflora imbalance in an offspring caused by maternal immune activation.

[0024] [5]. The use according to [4], wherein the improving intestinal microflora imbalance in an offspring caused by maternal immune activation comprises improving intestinal microbiota changes in an offspring caused by maternal immune activation;

[0025] the improving intestinal microbiota changes in an offspring caused by maternal immune activation comprises any one or more of (al) to (a3) below:

[0026] (al) improving reduction in abundance of neurodevelopment-beneficial genera in an offspring's intestine caused by maternal immune activation, the neurodevelopment-beneficial genera comprising any one or more of Bifidobacterium, Allobaculum, and Gordonibacter;

[0027] (a2) improving reduction in abundance of neurodevelopment-beneficial species in an offspring's intestine caused by maternal immune activation, the neurodevelopment-beneficial species comprising any one or more of Bifidobacterium_pseudolongum and Akkermansia_muciniphila;

[0028] (a3) improving increase in abundance of neurodevelopment-harmful species in an offspring's intestine caused by maternal immune activation, the neurodevelopment-harmful species comprising Ruminococcus_gnavus_AGR2154.

[0029] [6]. The use according to [4] or [5], wherein the improving intestinal microflora imbalance in an offspring caused by maternal immune activation comprises improving short-chain fatty acid content changes in an offspring's intestine caused by maternal immune activation.

[0030] the improving short-chain fatty acid content changes in an offspring's intestine caused by maternal immune activation comprises (bl) below:

[0031] (b1) improving a decrease in the content of any one or more of acetic acid, propionic acid, butyric acid, and valeric acid in the offspring's intestine caused by maternal immune activation.

[0032] [7]. The use according to any one of [4] to [6], wherein the improvement in the intestinal dysbiosis in the offspring caused by maternal immune activation comprises an improvement in the damage to the intestinal structure and / or barrier function in the offspring caused by maternal immune activation.

[0033] The improvement in the damage to the intestinal structure and / or barrier function in the offspring caused by maternal immune activation comprises any one or more of the following (c1) to (c4):

[0034] (c1) improving a decrease in the thickness of the muscularis layer in the offspring's intestine caused by maternal immune activation;

[0035] (c2) improving a decrease in the crypt depth in the offspring's intestine caused by maternal immune activation;

[0036] (c3) improving a decrease in the secretion of mucin by goblet cells in the offspring's intestine caused by maternal immune activation;

[0037] (c4) improving a decrease in the expression level of intestinal barrier function-related proteins in the offspring's intestine caused by maternal immune activation, the intestinal barrier function-related proteins comprising any one or more of Claudin-1, Occludin, MUC2, and ZO-1.

[0038] [8]. The use according to any one of [4] to [7], wherein the improvement in the intestinal dysbiosis in the offspring caused by maternal immune activation comprises an improvement in the elevated level of inflammation in the offspring's intestine caused by maternal immune activation.

[0039] The improvement in the elevated level of inflammation in the offspring's intestine caused by maternal immune activation comprises the following (d1) and / or (d2):

[0040] (d1) improving an increase in the expression level of pro-inflammatory factors in the offspring's intestine caused by maternal immune activation, the pro-inflammatory factors comprising any one or more of TNF-a, IL-1b, IL-17A, and IL-6;

[0041] (d2) improving a decrease in the expression level of anti-inflammatory factors in the offspring's intestine caused by maternal immune activation, the anti-inflammatory factors comprising IL-10.

[0042] [9]. The use according to any one of [4] to [8], wherein the food product comprises any one or more of infant formula and infant complementary food.

[0043]

[10] . The use according to any one of [4] to [9], wherein the content of the lacto-N-neotetraose is 0.16 to 0.48 g / 100 g and the content of the 1,2-diacyl-sn-glycero-3-phosphocholine is 25 to 400 mg / 100 g, based on the total dry matter content of the food, in the food.

[0044]

[11] . Use of the nutritional composition according to any one of [1] to [3] in the manufacture of a food product for any one or more of the following effects (z1) to (z4):

[0045] (z1) improving offspring changes in gut microbiota caused by maternal immune activation,

[0046] (z2) improving offspring changes in short-chain fatty acid content in the gut caused by maternal immune activation,

[0047] (z3) improving offspring damage to gut structure and / or barrier function caused by maternal immune activation,

[0048] (z4) improving offspring increased levels of inflammation in the gut caused by maternal immune activation.

[0049]

[12] . The use according to

[11] , wherein the improving offspring changes in gut microbiota caused by maternal immune activation comprises any one or more of the following (a1) to (a3):

[0050] (a1) improving offspring decreases in abundance of neurodevelopmentally beneficial genera in the gut caused by maternal immune activation, the neurodevelopmentally beneficial genera comprising any one or more of Bifidobacterium, Allobaculum, and Gordonibacter;

[0051] (a2) improving offspring decreases in abundance of neurodevelopmentally beneficial species in the gut caused by maternal immune activation, the neurodevelopmentally beneficial species comprising any one or more of Bifidobacterium_pseudolongum and Akkermansia_muciniphila;

[0052] (a3) improving offspring increases in abundance of neurodevelopmentally detrimental species in the gut caused by maternal immune activation, the neurodevelopmentally detrimental species comprising Ruminococcus_gnavus_AGR2154.

[0053]

[13] . The use according to

[11] or

[12] , wherein the improving offspring changes in short-chain fatty acid content in the gut caused by maternal immune activation comprises the following (b1):

[0054] (b1) improving a decrease in the content of any one or more of acetic acid, propionic acid, butyric acid, and valeric acid in the offspring's intestine caused by maternal immune activation.

[0055]

[14] . The use according to any one of

[11] to

[13] , wherein the improvement of the damage to the offspring's intestinal structure and / or barrier function caused by maternal immune activation comprises any one or more of the following (c1) to (c4):

[0056] (c1) improving a decrease in the offspring's intestinal muscle layer thickness caused by maternal immune activation;

[0057] (c2) improving a decrease in the offspring's intestinal crypt depth caused by maternal immune activation;

[0058] (c3) improving a decrease in the offspring's intestinal goblet cell secretion of mucin caused by maternal immune activation;

[0059] (c4) improving a decrease in the offspring's intestinal expression of barrier function-related proteins including any one or more of Claudin-1, Occludin, MUC2, and ZO-1 caused by maternal immune activation.

[0060]

[15] . The use according to any one of

[11] to

[14] , wherein the improvement of the increase in the level of inflammation in the offspring's intestine caused by maternal immune activation comprises the following (d1) and / or (d2):

[0061] (d1) improving an increase in the expression of pro-inflammatory factors including any one or more of TNF-a, IL-1b, IL-17A, and IL-6 in the offspring's intestine caused by maternal immune activation;

[0062] (d2) improving a decrease in the expression of anti-inflammatory factors including IL-10 in the offspring's intestine caused by maternal immune activation.

[0063]

[16] . The use according to any one of

[11] to

[15] , wherein the food product comprises any one or more of infant formula and infant complementary food.

[0064]

[17] . The use according to any one of

[11] to

[16] , wherein the content of the lacto-N-neotetraose is 0.16 to 0.48 g / 100 g and the content of the 1,2-diacyl-sn-glycero-3-phosphocholine is 25 to 400 mg / 100 g based on the total dry matter content of the food product in the food product.

[0065] Effects of the Invention

[0066] The present application proposes that the combination of lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine can improve the intestinal microecological imbalance of offspring caused by maternal immune activation, including improving the intestinal flora and its metabolism of offspring, especially having a promoting effect on the abundance of beneficial bacteria to neural development in the intestinal tract of offspring, such as Bifidobacterium, Allobaculum and Gordonibacter, and species such as Bifidobacterium_pseudolongum and Akkermansia_muciniphila, and having a reducing effect on the abundance of harmful bacteria to neural development in the intestinal tract of offspring, such as Ruminococcus_gnavus_AGR2154, and having a promoting effect on the content of main metabolic products of intestinal microbial fermentation, short-chain fatty acids (including acetic acid, propionic acid, butyric acid and valeric acid), and the improvement of the intestinal microecological imbalance of offspring caused by maternal immune activation by the combination of lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine also includes improving the damage of intestinal structure and barrier function of offspring, and the increase of intestinal inflammation level of offspring, that is, the combination of lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine can comprehensively improve the intestinal flora and the intestinal structure environment supporting the survival of flora, the function of intestinal flora is highly dependent on the intestinal environment in which it survives, and the integrity of intestinal function is regulated by flora, and this comprehensive improvement of intestinal microecology can provide benefits for the neural system development of offspring through the gut-brain axis. Alternatively, it can also be considered that the combination of lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine can improve the intestinal microecological imbalance accompanied by abnormal neural development of offspring born by female who has experienced or is in the state of maternal immune activation.

[0067] More importantly, the improvement proposed by the present application is for the intestinal microecological imbalance of offspring caused by maternal immune activation, that is, the improvement proposed by the present application is for the improvement of developmental period damage which occurs earlier, which provides help for the reversal of congenital developmental deficiency or abnormality.

[0068] And when the mass ratio of lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine is within a certain range, there is a synergistic effect between the two. The present application unexpectedly found that lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine can significantly amplify each other's improvement effect on the intestinal microecological imbalance of offspring caused by maternal immune activation.

[0069] Meanwhile, the nutritional composition containing the combination of lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine according to the present application can be used independently as a nutritional fortifier, or can be added to various foods, and can be ingested by eating, and can play a role in the early stage of life, i.e., the critical period of neural development of infants and young children. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 : Comparison results of the abundance of Bifidobacterium in the intestines of mice in the four example groups. DETAILED DESCRIPTION

[0071] The embodiments of the present application will be described below, but the present application is not limited thereto. The present application is not limited to each of the configurations described below, and various modifications can be made within the scope of the present application, and embodiments obtained by appropriately combining the technical means disclosed in each of the different embodiments and examples are also included in the technical scope of the present application.

[0072] In the present application, "comprise", "have", "include" or "contain" can mean inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. At the same time, "comprise", "have", "include" or "contain" can also mean closed, excluding additional, unrecited elements or method steps.

[0073] In the present application, "may" indicates both the meaning of performing a certain process and the meaning of not performing a certain process.

[0074] In the present application, "optional" or "optionally" means that a certain substance, component, execution step, applied condition, etc. is used or not used.

[0075] In the present application, "value A~value B", "value A-value B", "value A or more / less" means a value range including the end point values A and B.

[0076] In the present application, "about" is used to define the numerical range and parameters of the present application as approximate numerical values, and the relevant numerical values in the specific examples have been presented as accurately as possible. Unless otherwise explicitly stated, it should be understood that all ranges, numbers, values and percentages used in the present application are modified by "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1% or ±0.5% of a certain value or range. And the numerical values and value ranges appearing in the present application should be understood to include the systematic errors that are unavoidable in industrial production.

[0077] In the present application, "some specific / preferred embodiments", "further specific / preferred embodiments", "embodiments", and the like mean that the particular element (e.g., feature, structure, property, and / or characteristic) described in relation to the embodiment is included in at least one embodiment described herein, and can or can not be present in other embodiments. In addition, it should be understood that the described elements can be combined in any suitable manner in various embodiments.

[0078] In the present application, the unit names used are international standard unit names, and if not specifically stated, "%" used means weight or mass percentage.

[0079] In the present application, "infants" means the human group up to 3 years of age, which includes infants from 0 to 6 months of age, older infants from 6 to 12 months of age, and toddlers from 12 to 36 months of age.

[0080] In the present application, "animal milk" means a liquid obtained from mammary glands of mammals in the lactation period. The term "animal milk" should be interpreted broadly and encompasses both raw milk (i.e., a liquid obtained directly from mammary glands) and standardized dairy products such as, for example, skim milk or whole milk.

[0081] Unless otherwise defined, other technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0082] I. Nutritional composition

[0083] The present application provides a nutritional composition comprising essential active ingredients shown in (I) and (II) below: (I) lacto-N-neotetraose, (II) 1,2-diacyl-sn-glycero-3-phosphocholine; and, in the nutritional composition, the mass ratio of the lacto-N-neotetraose to the 1,2-diacyl-sn-glycero-3-phosphocholine is 1:(0.010-3.0).

[0084] Lacto-N-neotetraose (LNnT) is a linear tetrasaccharide consisting of D-galactose, N-acetylglucosamine, D-galactose, and D-glucose, arranged in the order of D-galactose linked to N-acetylglucosamine (GlcNAc) by a β-(1→4) bond, followed by D-galactose by a β-(1→3) bond, and then D-glucose by a β-(1→4) bond. Its molecular formula is C 26 H 45 NO 21 .

[0085] The source of LNnT is not particularly limited in the present application, and typically, it can be obtained by ordinary chemical synthesis method, microbial fermentation method, etc.

[0086] 1,2-diacyl-sn-glycero-3-phosphocholine (PC) is composed of a sn-glycerol backbone, a phosphocholine head and two fatty acid chain tails, and the fatty acid chain tails can be various fatty acids. Typically, one tail is a saturated fatty acid (e.g. palmitic acid, stearic acid), and the other is an unsaturated fatty acid (e.g. oleic acid, linoleic acid, arachidonic acid).

[0087] The source of 1,2-diacyl-sn-glycero-3-phosphocholine is not particularly limited in the present application, and typically, it is of animal origin, such as from cow milk, goat milk, cow liver and / or egg yolk.

[0088] The present application found that, compared with lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine alone, the nutritional composition provided by the present application can more effectively improve the intestinal microecological imbalance of offspring caused by maternal immune activation, that is, there is a synergistic effect between the two.

[0089] In some embodiments, in the nutritional composition described in the present application, the (I) lacto-N-neotetraose and (II) 1,2-diacyl-sn-glycero-3-phosphocholine are the main active ingredients, that is, the nutritional composition described in the present application mainly relies on the (I) lacto-N-neotetraose and (II) 1,2-diacyl-sn-glycero-3-phosphocholine contained therein to exert specific physiological activity functions, such as improving the intestinal microecological imbalance of offspring caused by maternal immune activation. In other words, in some embodiments, the active ingredients (ingredients for exerting specific physiological activity functions, i.e. ingredients for exerting the effect of improving the intestinal microecological imbalance of offspring caused by maternal immune activation) of the nutritional composition consist of the following (I) and (II): (I) lacto-N-neotetraose, (II) 1,2-diacyl-sn-glycero-3-phosphocholine.

[0090] In some embodiments, the nutritional composition comprises active ingredients (ingredients for exerting specific physiological activity functions, i.e. ingredients for exerting the effect of improving the intestinal microecological imbalance of offspring caused by maternal immune activation) and non-active ingredients (substances that do not exert the effect of improving the intestinal microecological imbalance of offspring caused by maternal immune activation). Exemplarily, the non-active ingredients can be other nutrients, any food acceptable adjuvant, and / or substances that are produced in the production or acquisition process of the active ingredients and cannot be effectively separated from the active ingredients or do not need to be separated. In some embodiments, the nutritional composition consists of the active ingredients and the non-active ingredients.

[0091] In some embodiments, the nutritional composition further comprises a human milk oligosaccharide other than lacto-N-neotetraose. Exemplary human milk oligosaccharides other than lacto-N-neotetraose include 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, and difucosyllactose, among others.

[0092] In some embodiments, the nutritional composition further comprises any one or more of 1,2-diacyl-sn-glycero-3-phosphoinositol, 1,2-diacyl-sn-glycero-3- phosphosermine, 1,2-diacyl-sn-glycero-3-phosphoethanolamine, 1,2-diacyl-sn-glycero-3- phosphoglycerol, and N-acyl-D-sphingosine-1-phosphocholine.

[0093] In some embodiments, the nutritional composition consists of components set forth in (I) and (II) (I) lacto-N-neotetraose, (II) 1,2-diacyl-sn-glycero-3- phosphocholine.

[0094] In some embodiments, in the nutritional composition, the mass ratio of the lacto-N-neotetraose to the 1,2-diacyl-sn-glycero-3-phosphocholine is 1 : (0.010-3.0); for example, it can be 1 :0.010, 1 :0.020, 1 :0.030, 1 :0.040, 1 :0.050, 1 :0.060, 1 :0.070, 1 :0.080, 1 :0.090, 1 :0.10, 1 :0.11, 1 :0.12, 1 :0.13, 1 :0.14, 1 :0.15, 1 :0.16, 1 :0.17, 1 :0.18, 1 :0.19, 1 :0.20, 1 :0.21, 1 :0.22, 1 :0.23, 1 :0.24, 1 :0.25, 1 :0.26, 1 :0.27, 1 :0.28, 1 :0.29, 1 :0.30, 1 :0.31, 1 :0.32, 1 :0.33, 1 :0.34, 1 :0.35, 1 :0.36, 1 :0.37, 1 :0.38, 1 :0.39, 1 :0.40, 1 :0.41, 1 :0.42, 1 :0.43, 1 :0.44, 1 :0.45, 1 :0.46, 1 :0.47, 1 :0.48, 1 :0.49, 1 :0.50, 1 :0.51, 1 :0.52, 1 :0.53, 1 :0.54, 1 :0.55, 1 :0.56, 1 :0.57, 1 :0.58, 1 :0.59, 1 :0.60, 1 :0.61, 1 :0.62, 1 :0.63, 1 :0.64, 1 :0.65, 1 :0.66, 1 :0.67, 1 :0.68, 1 :0.69, 1 :0.70, 1 :0.71, 1 :0.72, 1 :0.73, 1 :0.74, 1 :0.75, 1 :0.76, 1 :0.77, 1 :0.78, 1 :0.79, 1 :0.80, 1 :0.81, 1 :0.82, 1 :0.83, 1 :0.84, 1 :0.85, 1 :0.86, 1 :0.87, 1 :0.88, 1 :0.89, 1 :0.90, 1 :0.91, 1 :0.92, 1 :0.93, 1 :0.94, 1 :0.95, 1 :0.96, 1 :0.97, 1 :0.98, 1 :0.99, 1 :1.0, 1 :1.2, 1 :1.3, 1 :1.4, 1 :1.5, 1 :1.6, 1 :1.7, 1 :1.8, 1 :1.9, 1 :2.0, 1 :2.1, 1 :2.2, 1 :2.3, 1 :2.4, 1 :2.5, 1 :2.6, 1 :2.7, 1 :2.8, 1 :2.9, or 1 :3.0, etc.; preferably, the mass ratio of the lacto-N-neotetraose to the 1,2-diacyl-sn-glycero-3-phosphocholine is 1 : (0.030-2.5); more preferably, the mass ratio of the lacto-N-neotetraose to the 1,2-diacyl-sn-glycero-3-phosphocholine is 1:(0.10-1.5); even more preferably, the mass ratio of the lacto-N-neotetraose to the 1,2-diacyl-sn-glycero-3-phosphocholine is 1:(0.80-1.5).

[0095] The present application does not make special limitation to the form of the nutritional composition, which can typically be a liquid or a solid, etc. From the perspective of production, transportation, storage and use convenience, the nutritional composition of the present application is preferably a powdered solid.

[0096] II. Use of the nutritional composition

[0097] The present application proposes that the combination of lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine in a certain ratio can improve the intestinal microecological imbalance of offspring caused by maternal immune activation (non-therapeutic purpose), be beneficial to the development of offspring's neural health through the gut-brain axis, and have a synergistic effect between the two substances.

[0098] The "maternal immune activation" of the present application mainly refers to the abnormal activation state of the immune system of a female during pregnancy, which can include immune homeostasis disorders such as inflammatory factor level disorders. Factors that can cause the abnormal activation of the immune system of a female during pregnancy include but are not limited to infection of bacteria, viruses, etc.

[0099] The "intestinal microecological imbalance" of the present application mainly refers to the imbalance state caused by changes in the composition of intestinal flora, changes in bacterial metabolic activity or changes in the distribution of flora, abnormal changes in normal intestinal structure (such as the thickness of the intestinal muscle layer, the crypt structure, etc.) and physiological function (such as barrier function, etc.), and the generation of inflammatory response.

[0100] The improvement of the intestinal microecological imbalance of offspring caused by maternal immune activation of the present application is not for the purpose of treating or preventing diseases, and at the same time, the intestinal microecological imbalance of the present application has not reached the extent that can be recognized as a disease.

[0101] Based on this, the present application provides the use of the above-mentioned nutritional composition in the preparation of a food for improving the intestinal microecological imbalance of offspring caused by maternal immune activation.

[0102] In some embodiments, the improvement of the intestinal microecological imbalance of offspring caused by maternal immune activation includes the improvement of the intestinal flora changes of offspring caused by maternal immune activation.

[0103] In some specific embodiments, the improvement of the intestinal flora changes of offspring caused by maternal immune activation includes any one or more of the following (a1)-(a3):

[0104] (a1) ameliorating a decrease in abundance of neurodevelopmentally-beneficial genera in the offspring gut caused by maternal immune activation, the neurodevelopmentally-beneficial genera comprising any one or more of Bifidobacterium, Allobaculum, and Gordonibacter;

[0105] (a2) ameliorating a decrease in abundance of neurodevelopmentally-beneficial species in the offspring gut caused by maternal immune activation, the neurodevelopmentally-beneficial species comprising any one or more of Bifidobacterium_pseudolongum and Akkermansia_muciniphila;

[0106] (a3) ameliorating an increase in abundance of neurodevelopmentally-detrimental species in the offspring gut caused by maternal immune activation, the neurodevelopmentally-detrimental species comprising Ruminococcus_gnavus_AGR2154.

[0107] In some particular embodiments, the ameliorating of offspring gut microbiome changes caused by maternal immune activation comprises the following (a1):

[0108] (a1) ameliorating a decrease in abundance of neurodevelopmentally-beneficial genera in the offspring gut caused by maternal immune activation, the neurodevelopmentally-beneficial genera comprising Bifidobacterium, Allobaculum, and Gordonibacter.

[0109] In some particular embodiments, the ameliorating of offspring gut microbiome changes caused by maternal immune activation comprises the following (a2):

[0110] (a2) ameliorating a decrease in abundance of neurodevelopmentally-beneficial species in the offspring gut caused by maternal immune activation, the neurodevelopmentally-beneficial species comprising Bifidobacterium_pseudolongum and Akkermansia_muciniphila.

[0111] In some particular embodiments, the ameliorating of offspring gut microbiome changes caused by maternal immune activation comprises the following (a3):

[0112] (a3) ameliorating an increase in abundance of neurodevelopmentally-detrimental species in the offspring gut caused by maternal immune activation, the neurodevelopmentally-detrimental species comprising Ruminococcus_gnavus_AGR2154.

[0113] In some specific embodiments, the improvement in the offspring gut microbiota changes caused by maternal immune activation comprises both (al) and (a2):

[0114] (al) improvement in the reduction of abundance of neurodevelopmentally beneficial genera in the offspring gut caused by maternal immune activation, the neurodevelopmentally beneficial genera comprising Bifidobacterium, Allobaculum, and Gordonibacter;

[0115] (a2) improvement in the reduction of abundance of neurodevelopmentally beneficial species in the offspring gut caused by maternal immune activation, the neurodevelopmentally beneficial species comprising Bifidobacterium_pseudolongum and Akkermansia_muciniphila.

[0116] In some embodiments, the improvement in the offspring gut dysbiosis caused by maternal immune activation comprises improvement in the offspring gut short-chain fatty acid content changes caused by maternal immune activation.

[0117] In some specific embodiments, the improvement in the offspring gut short-chain fatty acid content changes caused by maternal immune activation comprises (bl):

[0118] (bl) improvement in the reduction of content of any one or more of acetic acid, propionic acid, butyric acid, and valeric acid in the offspring gut caused by maternal immune activation.

[0119] In some specific embodiments, the improvement in the offspring gut short-chain fatty acid content changes caused by maternal immune activation comprises (bl):

[0120] (bl) improvement in the reduction of content of acetic acid and butyric acid in the offspring gut caused by maternal immune activation.

[0121] In some specific embodiments, the improvement in the offspring gut short-chain fatty acid content changes caused by maternal immune activation comprises (bl):

[0122] (bl) improvement in the reduction of content of acetic acid, propionic acid, butyric acid, and valeric acid in the offspring gut caused by maternal immune activation.

[0123] In some embodiments, the improvement in the offspring gut dysbiosis caused by maternal immune activation comprises improvement in the offspring gut structural and / or barrier function impairment caused by maternal immune activation.

[0124] In some specific embodiments, the improvement in the offspring gut structural and / or barrier function impairment caused by maternal immune activation comprises any one or more of (cl) - (c4):

[0125] (c1) ameliorating a decrease in the thickness of the muscularis layer of the offspring gut caused by maternal immune activation;

[0126] (c2) ameliorating a decrease in the crypt depth of the offspring gut caused by maternal immune activation;

[0127] (c3) ameliorating a decrease in the goblet cell mucin secretion of the offspring gut caused by maternal immune activation;

[0128] (c4) ameliorating a decrease in the expression of proteins related to barrier function of the offspring gut caused by maternal immune activation, including any one or more of Claudin-1, Occludin, MUC2, and ZO-1.

[0129] In some embodiments, the ameliorating of the damage to the structure and / or barrier function of the offspring gut caused by maternal immune activation comprises (c1):

[0130] (c1) ameliorating a decrease in the thickness of the muscularis layer of the offspring gut caused by maternal immune activation.

[0131] In some embodiments, the ameliorating of the damage to the structure and / or barrier function of the offspring gut caused by maternal immune activation comprises (c2):

[0132] (c2) ameliorating a decrease in the crypt depth of the offspring gut caused by maternal immune activation.

[0133] In some embodiments, the ameliorating of the damage to the structure and / or barrier function of the offspring gut caused by maternal immune activation comprises (c3):

[0134] (c3) ameliorating a decrease in the goblet cell mucin secretion of the offspring gut caused by maternal immune activation.

[0135] In some embodiments, the ameliorating of the damage to the structure and / or barrier function of the offspring gut caused by maternal immune activation comprises (c4):

[0136] (c4) ameliorating a decrease in the expression of proteins related to barrier function of the offspring gut caused by maternal immune activation, including any one or more of Claudin-1, Occludin, MUC2, and ZO-1.

[0137] In some embodiments, the ameliorating of the damage to the structure and / or barrier function of the offspring gut caused by maternal immune activation comprises (c2) and (c4):

[0138] (c2) ameliorating a decrease in the crypt depth of the offspring gut caused by maternal immune activation;

[0139] (c4) improving the decrease in the expression level of intestinal barrier function-related proteins, including Claudin-1, Occludin, MUC2 and ZO-1, in the offspring caused by maternal immune activation.

[0140] In some embodiments, the improving the intestinal microflora imbalance in the offspring caused by maternal immune activation comprises improving the increase in the level of intestinal inflammation in the offspring caused by maternal immune activation.

[0141] In some specific embodiments, the improving the increase in the level of intestinal inflammation in the offspring caused by maternal immune activation comprises the following (d1) and / or (d2):

[0142] (d1) improving the increase in the expression level of proinflammatory factors, including any one or more of TNF-a, IL-1 β, IL-17A and IL-6, in the intestine of the offspring caused by maternal immune activation;

[0143] (d2) improving the decrease in the expression level of anti-inflammatory factors, including IL-10, in the intestine of the offspring caused by maternal immune activation.

[0144] In some specific embodiments, the improving the increase in the level of intestinal inflammation in the offspring caused by maternal immune activation comprises the following (d1):

[0145] (d1) improving the increase in the expression level of proinflammatory factors, including TNF-a, IL-1 β, IL-17A and IL-6, in the intestine of the offspring caused by maternal immune activation.

[0146] In some specific embodiments, the improving the increase in the level of intestinal inflammation in the offspring caused by maternal immune activation comprises the following (d2):

[0147] (d2) improving the decrease in the expression level of anti-inflammatory factor IL-10 in the intestine of the offspring caused by maternal immune activation.

[0148] Numerous studies have shown that gut microbes and their metabolites, gut structure and barrier function, and gut inflammation levels, can affect the central nervous system through endocrine, immune pathways, etc., and are directly related to the structural development of the nervous system, and affect brain function. Experimental data shows that lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine have an improving effect on the imbalance of the intestinal microecology of offspring caused by maternal immune activation. Based on this, the present application believes that by making the offspring produced by females who have experienced or are in a state of maternal immune activation consume the above-mentioned nutritional composition, not only is beneficial to the stable and healthy intestinal microecology, but also can achieve the effect of assisting in neural development through the gut-brain axis regulation. Alternatively, it can be considered that by making the offspring produced by females who have experienced or are in a state of maternal immune activation consume the above-mentioned nutritional composition, the imbalance of the intestinal microecology accompanied by abnormal neurological phenomena in the offspring can be improved.

[0149] The food described in the present application exerts the above-mentioned effects after being consumed by the offspring (offspring produced by females who have experienced or are in a state of maternal immune activation).

[0150] For specific foods containing or prepared using the above-mentioned nutritional composition, the present application does not make special limitations.

[0151] In some embodiments, the form of the food described in the present application is liquid or solid under normal temperature conditions.

[0152] In some embodiments, the food described in the present application includes any one or more of infant formula and infant complementary foods.

[0153] In some embodiments, the food described in the present application is a dairy product, such as milk powder, cheese, yogurt, liquid milk, etc.

[0154] In some embodiments, the food described in the present application is a confectionery, such as hard candy, gummy candy, crisp candy, pressed candy, and aerated candy, etc.

[0155] In some embodiments, the food described in the present application is a beverage, such as a carbonated beverage, a tea-based beverage, a coffee-based beverage, a fruit and vegetable juice beverage, and a lactic acid bacteria beverage, etc.

[0156] In some embodiments, the food described in the present application is a baked food, such as bread, cake, and biscuit, etc.

[0157] In some embodiments, the food described in the present application is a dietary supplement, such as a hard capsule, a soft capsule, a tablet, an oral liquid, a pill, a granule, and a powder, etc.

[0158] In some embodiments, in the food product according to the present application, the lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine are all derived from the nutritional composition. In other embodiments, in the food product according to the present application, the lacto-N-neotetraose and 1,2-diacyl-sn-glycero-3-phosphocholine are derived from the nutritional composition as well as from other food raw materials, such as animal milk and the like.

[0159] In some embodiments, in the food product according to the present application, the mass ratio of the lacto-N-neotetraose to the 1,2-diacyl-sn-glycero-3-phosphocholine is 1:(0.010-3.0); preferably, the mass ratio of the lacto-N-neotetraose to the 1,2-diacyl-sn-glycero-3-phosphocholine is 1:(0.030-2.5); more preferably, the mass ratio of the lacto-N-neotetraose to the 1,2-diacyl-sn-glycero-3-phosphocholine is 1:(0.10-1.5); even more preferably, the mass ratio of the lacto-N-neotetraose to the 1,2-diacyl-sn-glycero-3-phosphocholine is 1:(0.80-1.5).

[0160] The present application does not particularly limit the absolute content of the lacto-N-neotetraose and the 1,2-diacyl-sn-glycero-3-phosphocholine in the food product, which should meet the requirements of the local food-related laws and regulations.

[0161] In some embodiments, in the food product according to the present application, the content of the lacto-N-neotetraose is 0.16-0.48 g / 100 g, and the content of the 1,2-diacyl-sn-glycero-3-phosphocholine is 25-400 mg / 100 g, based on the total dry matter content of the food product.

[0162] In addition to the above-described components in the nutritional composition, the food product can further comprise other ingredients, such as common food ingredients of protein / amino acid, carbohydrate, fat, vitamin, mineral and the like.

[0163] In addition, according to the type of food product and the final needs of the target object, in some embodiments, the food product further comprises any one or more of the following ingredients: plant product ingredient, animal milk product ingredient, animal meat product ingredient, functional additive ingredient and any acceptable adjuvant.

[0164] Plant product ingredients, which can include, for example, fruits such as figs, pomegranates, kiwis, oranges, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, emblics, and mulberries, or extracts thereof; fruit and vegetable materials such as onions, cucumbers, tomatoes, cauliflowers, red beets, spinach, kohlrabi, Brussels sprouts, garlic, basil, Oregon grass, or extracts thereof; cereals such as rice (indica rice, japonica rice, waxy rice), wheat (wheat, barley, oat, rye), corn, sorghum, millet, foxtail millet, japonica, buckwheat, soybeans, beans, peas, mung beans, adzuki beans, kidney beans, or extracts thereof; nut materials such as walnuts, pistachios, cashews, hazelnuts, almonds, apricot kernels, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts, or extracts thereof; coffee or extracts thereof; and some medicinal and edible plant materials or extracts thereof.

[0165] Animal milk product ingredients, which can include, for example, fresh milk derived from cows, sheep, and the like, and reprocessed milk products such as whole milk powder, skim milk powder, concentrated whey protein powder, desalted whey powder, whey protein powder, hydrolyzed whey protein powder, casein powder, and the like.

[0166] Animal meat product ingredients, which can include, for example, meat product ingredients derived from pigs, cows, sheep, aquatic animals, or birds.

[0167] Functional additive ingredients, which can include, for example, vitamin supplements (e.g., vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, biotin, and the like), mineral supplements (e.g., iron, copper, manganese, zinc, cobalt, nickel, chromium, vanadium, fluorine, selenium, iodine, silicon, tin, and the like), nucleotide supplements (e.g., inosine), dietary fibers (e.g., inulin, konjac powder, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrins, resistant dextrins, soybean fibers, and the like), functional polyunsaturated fatty acid supplements (e.g., arachidonic acid oil powder, docosahexaenoic acid oil powder, and the like), and the like.

[0168] Any acceptable adjuvants, which can include, for example, solvents, antioxidants, antibacterial agents, thickening agents, diluents, co-solvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweetening agents, food flavors, food colorants, and the like.

[0169] Embodiments of the present application will be described in detail below with reference to Examples and Experimental Examples, but those skilled in the art will appreciate that the following Examples and Experimental Examples are for illustrative purposes only and should not be construed as limiting the scope of the present application. Unless otherwise specified, the specific conditions in the Examples and Experimental Examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The materials or instruments used are commercially available conventional products unless otherwise specified.

[0170] Experimental Example 1: Effect of nutrients on the basic growth indicators of MIA offspring

[0171] 1. Experimental method

[0172] 1.1. Construction of maternal immune homeostasis disorder model

[0173] Select 150 female mice and 30 male mice, and use the female and male mice mainly for offspring breeding. When caging, select mice with shiny hair and healthy bodies, and cage them according to a female to male ratio of 2:1. Weigh the female mice regularly in the following week, and when the body weight of the female mice increases significantly, separate them and inject 20 mg / kg poly(I:C) solution (Sigma-Aldrich, item number P9582, the injection form of poly(I:C) is a solution, the solvent is a PBS solution, and the concentration is 5 mg / mL) into the abdominal cavity to construct a maternal immune homeostasis disorder model.

[0174] 1.2. Offspring intervention

[0175] During the experiment, the male offspring were used as experimental objects. After the female parent mice gave birth for 3 weeks, the male offspring were weaned and raised separately. When the male offspring were 4 weeks old, they were respectively given intragastrically PBS solution or different proportions of nutrient solution, and the intragastric period was 6 weeks.

[0176] 1.3. Grouping of offspring animal intervention

[0177] The experiment was divided into 10 groups, with 12 mice in each group, namely a control group, a model group, and 8 nutrient intervention groups. Among them, “control group mice” refer to mice obtained by injecting PBS solution into the mother mouse during pregnancy, and “model group mice” refer to mice obtained by injecting poly(I:C) solution into the mother mouse during pregnancy. During the experiment, each mouse was raised in a standard cage and placed in an environment with a 12 h light / dark cycle, humidity of 50±15%, and temperature of 22±2℃, and the mice were free to eat and drink water.

[0178] Lacto-N-neotetraose (LNnT) (purchased from DSM Vitamin Trade (Shanghai) Co., Ltd.) and 1,2-diacyl-sn-glycero-3-phosphocholine (PC) (purchased from Beijing Libaoyi Trade Co., Ltd.) were selected for different single doses or different ratio combinations, and the improvement effect of different dose combinations on intestinal microecology was investigated. The experimental design is shown in Table 1. LNnT was selected for high and low dose intervention, with 133 mg / kg body weight / day and 400 mg / kg body weight / day, respectively; PC was also selected for high and low dose intervention, with 21 mg / kg body weight / day and 166 mg / kg body weight / day, respectively. Four single factor intervention doses of the two substances were used as comparative examples, and LNnT and PC were combined in different doses to form four different ratio combinations of LNnT:PC as four embodiments, i.e. 1:0.158, 1:1.25, 1:0.0525 and 1:0.415.

[0179] Table 1 Experimental intervention scheme design

[0180]

[0181] 1.4. Monitoring of basic growth indicators

[0182] During the 6-week intervention period, the body weight, food intake and water intake of the mice were recorded once a week.

[0183] 2. Experimental results

[0184] The body weight change data of the mice in the control group, model group and 8 intervention groups within 6 weeks of intervention are shown in Table 2. As can be seen from the table, the body weight of the mice in the control group was low at the time of intervention (0 days), with an average of about 14.38 g, the body weight of the mice in Example 4 group was the lowest, about 12.25 g, the body weight of the mice in Example 3 group was slightly higher than that of the control group, about 14.73 g, and the initial body weight of the mice in the remaining groups was greater than or equal to 15 g. After significant difference analysis, it was found that at the time of grouping, there was no significant difference between Example 3 group and the control group (p>0.05), the body weight of the control group was significantly higher than that of Example 4 group (p<0.0001), and the body weight of the mice in the remaining groups was significantly higher than that of the control group (p<0.01, p<0.001, p<0.0001). After 42 days of intervention, it was found that the body weight of the mice in Example 4 group was the lowest, about 20.47 g, while the body weight of the mice in the other groups was above 22 g. After significant difference analysis, it was found that the body weight of the mice in Example 4 group was significantly lower than that of the mice in the other groups (p<0.0001). Further comparison of the weight gain of the mice in each group showed that there was no significant difference in the weight gain of the mice in the control group, model group and 8 intervention groups within 42 days (p>0.05).

[0185] Meanwhile, the food intake and water intake of the 10 groups of mice were recorded during the intervention period, and the experimental results are shown in Table 3. As can be seen from the table, the food intake of the 10 groups of mice was about 3 g / day, and the water intake was in the range of 3.59-4.12 g / day during the entire intervention period. After analyzing the significant differences between groups, it was found that there was no significant difference in food intake and water intake of the 10 groups of mice during the 42-day intervention period (p>0.05).

[0186] The above analysis shows that maternal immune activation modeling has no effect on the weight gain of offspring mice, and nutritional intervention also does not change the food intake and water intake of mice.

[0187] Table 2 Changes in body weight of offspring mice in each group during the 6-week intervention period (g)

[0188]

[0189] Table 3 Comparison of average daily food intake and water intake of offspring mice in each group during the intervention period

[0190]

[0191] Experimental Example 2: Evaluation of the efficacy of nutritional intervention in regulating the intestinal flora of MIA offspring mice

[0192] 1. Experimental method

[0193] 1.1. Construction of maternal immune homeostasis disorder model

[0194] The same as Experimental Example 1.

[0195] 1.2. Offspring intervention

[0196] The same as Experimental Example 1.

[0197] 1.3. Intervention grouping of offspring animals

[0198] The same as Experimental Example 1.

[0199] 1.4. Intestinal flora detection

[0200] Intestinal flora detection used 16S rRNA amplicon sequencing method. First, the DNA extraction kit was used to extract the intestinal contents DNA of mice in each group, and the extracted genomic DNA was detected by 1% agarose gel electrophoresis to determine the concentration and purity; the 3rd to 4th regions (V3-V4 region) in the 16S rDNA hypervariable region were used as the target sequencing fragments, and the corresponding primers were designed to amplify the target fragments by PCR. The PCR products were quantified using QuantiFluor-ST fluorescence meter, and then mixed in the corresponding proportion according to the sequencing requirement of each sample. After the quantitative and uniformization of the PCR amplification products, the samples were sequenced using the Illumina Miseq platform, and the sequencing analysis was entrusted to the platform of Guangzhou Kidio Biological Technology Co., Ltd. The raw data of sequencing was analyzed by R language tools and related analysis software for biological information analysis and data statistical analysis.

[0201] 2. Experimental results

[0202] First, we analyzed the intestinal flora composition of offspring ASD mice after maternal immune activation. The results showed that the alpha diversity of intestinal flora of ASD mice in the model group was significantly different from that in the control group, such as Shannon index (Shannon) and Simpson index (Simpson). On the β diversity, through principal coordinate analysis (PCoA), non-metric multidimensional scaling analysis (NMDS) and partial least squares discriminant analysis (PLS-DA), it was found that the flora of mice in the model group and the control group showed significant independence. It can be seen that maternal immune activation has a significant effect on the intestinal flora of offspring mice. Further analysis found that the intestinal flora of offspring mice after maternal immune activation showed significant differences in genus and species levels.

[0203] Table 4 shows the abundance changes of three probiotics related to neural development at genus level in the intestinal flora of mice in each group after modeling and intervention. First, g_Bifidobacterium is one of the most important and most studied beneficial bacteria in human intestinal flora, especially in infancy. This bacterium has multiple health benefits for the human body, such as regulating intestinal flora balance, regulating immune balance, enhancing intestinal barrier integrity, and promoting neural development. It is crucial for early life neural development editing: it can produce neuroactive metabolites (SCFAs, regulate tryptophan / serotonin), reduce systemic and neural inflammation, regulate microglia, activate the vagus nerve, regulate the HPA axis response to stress, and has great potential in assisting the improvement of neural development disorders, emotional disorders (anxiety, depression), and maintaining cognitive function. The present application found that the g_Bifidobacterium in the intestinal flora of offspring mice after maternal immune activation decreased to about 1 / 20 of the control group, which indicates that maternal immune activation caused a significant decrease in the abundance of Bifidobacterium in offspring, and the One-Way ANOVA difference significance analysis found that the model group and the control group produced a very significant difference (p<0.0001). After LNnT and PC intervention at low and high doses (Comparative Examples 1-4), the abundance of g_Bifidobacterium increased to some extent, and the difference significance analysis found that Comparative Examples 1-4 and the model group produced a significant difference (p<0.0001); Examples 1-4 showed that the LNnT and PC were compounded at different ratios to form compositions, which further improved the abundance of g_Bifidobacterium in the intestinal flora of mice compared with the model group and Comparative Examples 1-4, and the difference significance analysis found that the four examples were significantly higher than the four comparative examples, as shown in Table 5. This shows that the composition formed by compounding LNnT and PC at a ratio of 1:0.0525-1:1.25 has a better effect than the two substances alone in promoting the colonization of Bifidobacterium in the intestinal flora of offspring, especially Examples 1, 2, and 4 have obvious synergistic effect. And among the four examples, Example 2 can reach the level of the control group, and it is significantly higher than Examples 1, 3, and 4, as shown in Table 5. Figure 1

[0204] ​g_Allobaculum is a genus of Lachnospiraceae, which is an extremely important family in the gut microbiota, with a large number of members and plays an important role in host health. The most core potential function of g_Allobaculum is to ferment dietary fiber to produce short-chain fatty acids (SCFA) (especially acetic acid and butyric acid), which can enhance the intestinal barrier, reduce inflammation, stimulate the secretion of intestinal hormones, and thus help to improve obesity, insulin resistance and blood glucose control. Human observational studies have also found that it is positively correlated with lean body mass and metabolic health. Its efficacy in neurodevelopment is also mainly through the production of SCFAs, such as butyric acid, which can pass through the blood-brain barrier, promote the expression of brain-derived neurotrophic factor (BDNF) to support the survival of neurons and synaptic plasticity; butyric acid can also inhibit histone deacetylase (HDAC) to regulate neural gene expression and thus affect learning and memory. Currently, it has been found in clinical practice that the abundance of g_Allobaculum in the feces of people with neurodevelopmental disorders is significantly reduced, and the level of butyric acid is also reduced. In the present research, it was found that the abundance of the bacteria in the intestinal tract of normal control pups was 0.427, while the abundance of the bacteria in the intestinal tract of pups with neurodevelopmental disorders after maternal immune activation was reduced to 0.0911 (as shown in Table 4), and a significant difference was generated with the control group (p<0.0001); Comparative Examples 1-4 show the abundance of g_Allobaculum in the intestinal tract of pups after intervention with low and high doses of LNnT and PC alone, and from Table 4 it can be seen that the abundance of g_Allobaculum is 0.145-0.215, which is significantly higher than that of the model group (p<0.01, p<0.0001), which indicates that after the intervention of the two nutrients, the abundance of g_Allobaculum in the intestinal tract of pups can be significantly restored; but the present inventors have also surprisingly found that when LNnT and PC are compounded to form a composition to intervene in pups, the abundance of g_Allobaculum can be further increased to 0.338-0.447, and after significant difference analysis, it was found that the four examples were significantly higher than the model group and the four comparative examples, and the significant difference analysis results are shown in Table 6. This indicates that the composition formed by compounding LNnT and PC at a ratio of 1:0.0525-1:1.25 has a better effect than the two substances alone in promoting the colonization of g_Allobaculum in the intestinal tract of pups, especially Example 1, Example 2 and Example 4 have obvious synergistic effects. And in the four examples, the abundance of g_Allobaculum in Example 2 can reach the level of the control group, and it is significantly higher than Example 1, Example 3 and Example 4.

[0205] Gordonibacter belongs to the phylum Actinobacteria, class Roseibacteria, order Eggtithiaceae, and is a probiotic that can degrade plant polyphenols and produce urolithin, a high-value metabolite. The physiological activity of urolithin determines the important role of g_Gordonibacter in anti-aging and cell regeneration, metabolism and immune regulation, and estrogen metabolism intervention. In terms of neural development, g_Gordonibacter can indirectly affect brain health through the metabolite urolithin: 1) urolithin has strong antioxidant capacity and can reduce oxidative damage to neurons; 2) it can inhibit neuroinflammation, reduce microglial activation, and delay the progression of neurodegeneration. Currently, the abundance of g_Gordonibacter has been found to decrease in the feces of some Parkinson's patients, and is positively correlated with the level of urolithin. In the present invention, it was also found that the abundance of g_Gordonibacter in the offspring gut decreased significantly after maternal immune activation, as shown in Table 4. It can be seen that the relative abundance of g_Gordonibacter in the model group has decreased to about 0.0005, while the relative abundance of g_Gordonibacter in the normal control group is about 0.033, and a significant difference (p<0.0001) is generated between the two; Comparative Examples 1-4 show the relative abundance of the bacteria in the offspring gut after intervention with low and high doses of LNnT and PC, respectively. As can be seen from Table 4, the relative abundance of the bacteria in the offspring gut in the four comparative examples is increased to 0.003-0.01, which is higher than that in the model group, but no significant difference (p>0.05) is found between the four comparative examples and the model group after significant difference analysis, which indicates that the intervention of the two nutrients alone has no significant effect on the colonization of g_Gordonibacter; Examples 1-4 show the colonization of the bacteria in the offspring gut after intervention with the complex formed by the two nutrients in different ratios. As can be seen from Table 4, the relative abundance of g_Gordonibacter is increased to the range of 0.0195-0.0411, especially in Example 2, the relative abundance of the bacteria can reach 0.0411, and after significant difference analysis, it is found that Examples 1-4 are significantly higher than the model group and the four comparative examples, as shown in Table 7. Through analysis, we found that Examples 1, 2 and 4 have obvious synergistic effect. This indicates that the composition formed by LNnT and PC in the ratio of 1:0.0525-1:1.25 has a better effect on promoting the colonization of g_Gordonibacter in the offspring gut than the two nutrients alone.

[0206] Table 4 Comparison of the abundance of the differential bacteria related to neural development at the genus level in the gut of mice in each group

[0207]

[0208] Table 5 Significance analysis of the difference in the abundance of g_Bifidobacterium in the intestines of mice in each group

[0209]

[0210] Table 6 Significance analysis of the difference in the abundance of g_Allobaculum in the intestines of mice in each group

[0211]

[0212] Table 7 Significance analysis of the difference in the abundance of g_Gordonibacter in the intestines of mice in each group

[0213]

[0214] Further, at the species level, we found that the relative abundance of three species s_Bifidobacterium_pseudolongum, s_Akkermansia_muciniphila and s_Ruminococcus_gnavus_AGR2154 related to neural development differed under different nutritional interventions in mice in each group (results shown in Table 8). Among the three species, the first two belong to species beneficial to neural development, and the third species is a species detrimental to neural development.

[0215] First is s_Bifidobacterium_pseudolongum, which belongs to the genus Bifidobacterium. Its main functions for host health are metabolic regulation, immune regulation, and pathogen inhibition. In terms of neural development, it produces neuroactive metabolites, in addition to acetic acid and butyric acid. It is one of the strongest tryptophan metabolizing bacteria: indole-3-lactic acid produced after tryptophan metabolism can protect neurons from oxidative damage, promote intestinal barrier repair, and reduce brain inflammation. Another metabolite, a serotonin precursor, can increase peripheral serotonin levels and affect the brain's emotional center through the vagus nerve. In studies related to neurological disorders, high s_Bifidobacterium_pseudolongum abundance in the gut of infants has been associated with higher cognitive scores at 1 year of age in cohort studies. In patients with depression, s_Bifidobacterium_pseudolongum abundance was 3-5 times lower in the gut than in healthy controls. The present invention found that the abundance of this bacterium in the offspring's gut after maternal immune activation was about 29 times lower than in the control group, with a significant difference (p<0.0001) between the two groups. After separate intervention with low and high doses of nutrients LNnT and PC, the relative abundance of s_Bifidobacterium_pseudolongum increased to varying degrees compared to the model group, but after significant difference analysis, it was found that except for Comparative Example 1, which had no significant difference (p>0.05) with the model group, Comparative Examples 2-4 were significantly higher than the model group (p<0.05). Examples 1-4 showed that the relative abundance of s_Bifidobacterium_pseudolongum in the offspring's gut after intervention with compositions formed by compounding LNnT and PC at different ratios. As shown in Table 8, the relative abundance of this bacterium in the four examples was further increased, with a relative abundance of 0.0531 in Example 2, reaching the level of the control group. After significant difference analysis, it was found that the four examples were significantly higher than the four comparative examples, as shown in Table 9. This indicates that compositions formed by compounding LNnT and PC at a ratio of 1:0.0525-1:1.25 have a better effect than the two substances alone in promoting the colonization of s_Bifidobacterium_pseudolongum in the offspring's gut. Through analysis, we found that Examples 1, 2, and 4 had a clear synergistic effect.

[0216] Akkermansia muciniphila is a highly specialized bacterium in the gut that utilizes mucin, the main component of the intestinal mucus layer, as its main carbon and nitrogen source. It plays a very important role in human health, such as enhancing the intestinal barrier, regulating metabolism to improve obesity and diabetes, regulating the immune system, reducing inflammation, protecting the cardiovascular system, etc. In terms of neurodevelopment, a number of clinical studies have observed that the abundance of s_Akkermansia muciniphila in the gut of children with neurodevelopmental disorders is significantly lower than that of healthy children. The main mechanisms of action include: 1) stimulating goblet cells to produce more mucin and enhancing intestinal barrier function; 2) interacting with intestinal immune cells to regulate immune balance and inhibit microglial overactivation to protect the nervous system; 3) producing active metabolites related to neurodevelopment, such as short-chain fatty acids (acetic acid and propionic acid), bile acid metabolism, and tryptophan metabolism; 4) activating vagus nerve endings to accelerate neural signal transmission; and 5) promoting the production of neuroprotective factors. In the present invention, it was found that the relative abundance of such important probiotics in the gut of offspring after maternal immune activation decreased significantly (p<0.0001). Comparative Examples 1-4 showed that the relative abundance of s_Akkermansia muciniphila increased to some extent after LNnT and PC monomers were intervened at low and high doses, respectively, but after significant difference analysis, it was found that Comparative Example 1 and Comparative Example 4 had no significant difference (p>0.05) with the model group, and Comparative Example 2 and Comparative Example 3 had significant difference (p<0.01 and p<0.05) with the model group. Examples 1-4 showed that the abundance of the bacteria in the gut of offspring mice after combined intervention of the composition formed by compounding LNnT and PC at different ratios. As can be seen from Table 8, the relative abundance of the bacteria in the gut of offspring mice in the example group increased by an order of magnitude compared with the model group, and after significant difference analysis, it was found that the four examples were significantly higher than the four comparative examples, and the results are shown in Table 10. This shows that the composition formed by compounding LNnT and PC at a ratio of 1:0.0525-1:1.25 has a better effect than the two substances alone in promoting the colonization of s_Akkermansia muciniphila in the gut of offspring mice, and through analysis, we found that especially Example 1, Example 2 and Example 4 have obvious synergistic effect.

[0217] The s_Ruminococcus_gnavus_AGR2154 belongs to the genus g_Ruminococcus. The bacteria in this genus can have different effects on the host, such as producing butyric acid bacteria (R.bromii) and harmful (R.gnavus). The s_Ruminococcus_gnavus_AGR2154 detected in the present application is a genus-level rumenococcus that is harmful to the human body. Studies have found that R.gnavus, as a conditional pathogen, can activate the TLR4 signaling pathway by producing glycan envelopes, exacerbate intestinal inflammation by releasing TNF-α / IL-6, and further promote brain immune system disorders through the gut-brain axis. Clinical studies have found that the abundance of this bacterium in the intestines of patients with inflammatory bowel disease (IBD) increases by 300%, and its polysaccharide induces Th17 cell differentiation. In the present application, the abundance of this bacterium in the intestines of healthy control pups was found to be 0, while the abundance of this bacterium in the intestines of model pups was 0.00241. In Comparative Examples 1-4, the abundance of this bacterium in the intestines of pups after intervention with low and high doses of LNnT and PC, respectively, was significantly lower than that in the model group (p<0.0001). Further, after intervention with pups using compositions formed by compounding LNnT and PC at different ratios, it was found that the abundance of this bacterium in the intestines decreased exponentially, and in particular in Example 2, the average relative abundance of this bacterium was 0, reaching the level of the control group. Further, significant difference analysis of the examples and comparative examples found that the four examples were significantly lower than the four comparative examples (as shown in Table 11), and analysis found that in particular, Example 1, Example 2 and Example 4 had a clear synergistic effect. The above shows that the composition formed by compounding LNnT and PC at a ratio of 1:0.0525-1:1.25 has a better effect than the two substances alone in reducing the colonization of s_Ruminococcus_gnavus_AGR2154 in the intestines of pups. In particular, in Example 2, the composition formed by compounding LNnT and PC at a ratio of 1:1.25 had almost no colonization of s_Ruminococcus_gnavus_AGR2154 in the intestines of the offspring.

[0218] From the perspective of the composition of the flora, it was found that at the genus level, the composition formed by LNnT and PC at 1:0.0525-1:1.25 had better promoted the colonization of g_Bifidobacterium (Bifidobacterium), g_Allobaculum (Allobaculum) and g_Gordonibacter (Gordonibacter) beneficial to neural development than the two substances alone; at the species level, the composition formed by LNnT and PC at 1:0.0525-1:1.25 had better promoted the colonization of s_Bifidobacterium_pseudolongum and s_Akkermansia_muciniphila beneficial to neural development than the two substances alone, and could reduce the colonization of s_Ruminococcus_gnavus_AGR2154 harmful to neural development.

[0219] Table 8 Comparison of the abundance of different flora at the species level in the intestines of mice in each group

[0220]

[0221] Table 9 Significance analysis of the difference in the abundance of s_Bifidobacterium_pseudolongum in the intestines of mice in each group

[0222]

[0223] Table 10 Significance analysis of the difference in the abundance of s_Akkermansia_muciniphila in the intestines of mice in each group

[0224]

[0225] Table 11 Significance analysis of the difference in the abundance of s_Ruminococcus_gnavus_AGR2154 in the intestines of mice in each group

[0226]

[0227] Experimental Example 3: Nutritional substance intervention regulates the formation of short-chain fatty acids (SFCAs) in the intestines of offspring of MIA mice

[0228] 1. Experimental method

[0229] 1.1. Construction of maternal immune homeostasis disorder model

[0230] The same as Experimental Example 1.

[0231] 1.2. Offspring intervention

[0232] The same as Experimental Example 1.

[0233] 1.3. Offspring animal intervention grouping

[0234] The same as Experimental Example 1.

[0235] 1.4. Sample collection and index detection

[0236] 1.4.1. Fecal sample collection

[0237] One day before the mice were sacrificed, the mice were placed individually in a high-pressure sterilized beaker and waited for them to defecate freely. After the mice defecated, the feces were clamped with a high-pressure sterilized forceps and transferred to a sterile EP tube, and immediately transferred to a -80°C refrigerator for detection.

[0238] 1.4.2. Determination of short-chain fatty acids in the intestinal tract

[0239] Non-targeted metabolomics was used to determine short-chain fatty acids in mouse feces. Fecal non-targeted metabolomics testing was mainly tested by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. The mouse fecal sample was frozen with liquid nitrogen, then ground into powder, and an extractant was added for extraction. The pretreated sample was injected into the mass spectrometer instrument for positive and negative ion scanning twice. The original data file was obtained, including total ion flow chart (TIC), metabolite identification results, etc. The original data file was subjected to quality control and pretreatment, including peak alignment, denoising, normalization, etc. Appropriate statistical methods (such as PCA, PLS-DA, etc.) were used to compare and analyze the metabolite data of the experimental group and the control group, and to find the difference metabolites.

[0240] 2. Experimental results

[0241] From the analysis of changes in the structure of the flora, it can be seen that maternal immune activation has a significant impact on the intestinal flora of the offspring. Intestinal flora metabolites are short-chain fatty acids (SCFAs), which are fatty acids with less than 6 carbon atoms in the carbon chain, mainly including acetic acid, propionic acid, butyric acid, valeric acid, etc., among which acetic acid, propionic acid and butyric acid are the main ones, accounting for more than 95% of the entire SCFAs. These metabolites are considered to be the main energy source for intestinal epithelial cells and play an important role in maintaining intestinal environmental homeostasis. SCFAs can pass through the blood-brain barrier and have an impact on the central nervous system.

[0242] Among the SCFAs that have a positive effect on neural development, it is believed that acetic acid can promote normal neural development by supplying energy to the brain, inhibiting neural inflammation, promoting myelination, and strengthening the blood-brain barrier. There are clinical findings that the acetic acid level in the feces of children with neurodevelopmental disorders is generally low, and the number of acetic acid-producing bifidobacteria is reduced. The results of Example 2 have shown that bifidobacteria are significantly reduced in the model group, and from Table 12 it can be seen that the production of acetic acid in the intestinal tract of the offspring mice after maternal immune activation is significantly reduced (p<0.0001). The production of acetic acid in the intestinal tract of the offspring mice after LNnT and PC are separately intervened at low and high doses in Comparative Examples 1-4, respectively, can be seen from Table 12, which has improved to some extent compared with the model group, but after significant difference analysis, it is found that Comparative Example 1 has no significant difference (p>0.05) with the model group, and Comparative Examples 2-4 are significantly higher than the model group (p<0.0001). Examples 1-4 show the production of acetic acid in the intestinal tract of mice after LNnT and PC are compounded at different ratios to form compositions, and from the table it can be seen that the four examples not only have improved compared with the model group, but the value is higher than the four comparative examples, and after significant difference analysis, it is found that the four examples are significantly higher than the four comparative examples (as shown in Table 13), and through analysis we find that especially Example 1, Example 2 and Example 4 have obvious synergistic effect. This shows that the composition formed after LNnT and PC are compounded at a ratio of 1:0.0525-1:1.25 has better promotion of the production of acetic acid in the intestinal tract than the two substances alone, which is consistent with the change in the abundance of bifidobacteria. Further comparison of the four examples found that the content of acetic acid in Example 2 reached 8.86 μg / g, reaching the level of the normal control group. Another SCFA extremely important for neural development is butyric acid, which plays a neuroprotective role by promoting neuronal activity and synaptic plasticity, enhancing blood-brain barrier integrity, strong anti-inflammatory, and stimulating the expression of BDNF. In the present application, it is found that the content of butyric acid in the control group is 2.86 μg / g, while that in the model group is reduced to 0.46 μg / g, and there is a significant difference between the two (p<0.0001), which shows that the significant reduction of butyrate content in the intestinal tract of the offspring after maternal immune activation.The butyric acid content in the intestines of the offspring mice after LNnT and PC intervention at low and high doses, respectively, is shown in Comparative Examples 1-4. It can be seen that the butyrate content is increased to different degrees after the intervention of the two nutrients, but after significant difference analysis, it is found that the two nutrient interventions have no significant effect on the increase of butyric acid at low doses (Comparative Examples 1 and 3) (p>0.05), but at high doses (Comparative Examples 2 and 4), they are significantly higher than the model group (p<0.001 and p<0.0001); it is further found that the butyric acid content in the intestines of the mice in Examples 1-4 is further increased, which can reach or exceed the level of the control group, and after significant difference analysis, it is found that the butyric acid content in the four examples is significantly higher than that in the four comparative examples, as shown in Table 13, and through analysis, it is found that especially Examples 1, 2 and 4 have obvious synergistic effect. This shows that the composition formed by LNnT and PC at a ratio of 1:0.0525-1:1.25 has a better effect on promoting the production of butyric acid in the intestines than the two substances alone, especially Example 2 (LNnT:PC is 1:1.25), the content of butyric acid reaches 3.82 μg / g.

[0243] Propionic acid and valeric acid are considered to have a dose-dependent effect on neuroprotection, and a suitable dose promotes neuroprotection and neuroplasticity, but when excessive, both are found to induce neuroinflammation. In the present application, it was found that the contents of propionic acid and valeric acid in the intestines of normal healthy control mice were 3.22 μg / g and 0.59 μg / g, respectively, and when the maternal immune activation occurred, both indicators in the intestines of the offspring were significantly reduced (propionic acid decreased to 0.85 μg / g, valeric acid decreased to 0.14 μg / g) (p<0.0001). As can be seen from Table 12, after the intervention of LNnT and PC at different doses (Comparative Examples 1-4) or different proportions of compositions (Examples 1-4), both SCFAs were improved to different degrees, but the highest value did not exceed the normal control group level. Further detailed comparison found that the propionic acid and valeric acid in the four comparative examples were higher than those in the model group, and after difference significance analysis, it was found that only high-dose (Comparative Examples 2 and 4) LNnT and PC intervention could significantly improve the propionic acid in the intestines of the offspring (p<0.0001), and there was no significant difference (p>0.05) between low-dose intervention (Comparative Examples 1 and 3) and the model group; but for valeric acid, both substances were significantly improved (p<0.05 or p<0.0001) when intervened at two doses alone; further found that Examples 1-4 were further improved than Comparative Examples 1-4, and after difference significance analysis, it was found that the propionic acid and valeric acid in the four examples were significantly higher than those in the four comparative examples, as shown in Table 13, and through analysis, it was found that especially Examples 1, 2 and 4 had a synergistic effect. This shows that the composition formed by compounding LNnT and PC at a ratio of 1:0.0525-1:1.25 has a better effect than the two substances alone in promoting the production of propionic acid and valeric acid in the intestines, especially Example 2 (LNnT:PC is 1:1.25), the contents of propionic acid and valeric acid are close to the control group.

[0244] Table 12 Short-chain fatty acid production in the intestines of mice in each group (μg / g)

[0245]

[0246] Table 13 Difference significance analysis of short-chain fatty acids in the intestines of mice in examples and comparative examples after correction

[0247]

[0248] Experimental Example 4: Nutritional substance intervention regulates the intestinal tissue and barrier function of MIA offspring

[0249] 1. Experimental method

[0250] 1.1. Construction of maternal immune homeostasis disorder model

[0251] The same as Experimental Example 1.

[0252] 1.2. Offspring intervention

[0253] The same as Experimental Example 1.

[0254] 1.3. Intervention grouping of offspring animals

[0255] The same as Experimental Example 1.

[0256] 1.4. Tissue sample collection and index detection

[0257] 1.4.1. Tissue sample collection

[0258] The mice were fasted for 12 h before sacrifice, and were anesthetized by intraperitoneal injection of a 1.25% tribromoethanol solution at a dose of 0.2 mL / 10 g bw. After eye blood collection, the mice were sacrificed by cervical dislocation. The plasma was obtained by centrifugation at 25°C, 3000 r / min for 15 min, and was stored at -80°C. The intestinal tissue samples were then collected by dissection on ice, wrapped in tin foil, quickly frozen in liquid nitrogen, and moved to a -80°C refrigerator. The tissues for pathological sectioning were placed in a 4% (v / v) paraformaldehyde solution for subsequent experiments.

[0259] 1.4.2. Quantitative analysis of intestinal morphology

[0260] Hematoxylin-eosin staining was used to analyze the thickness of the intestinal muscle layer and the depth of the crypt. The specific experimental steps were as follows:

[0261] (1) Preparation of paraffin sections

[0262] Immediately after sacrifice, the intestinal tissue was taken, cut in half, and placed in a 4% (v / v) paraformaldehyde / PBS fixing solution for 24 h. After dehydration, the tissue was embedded in paraffin, and the wax block was cut into 5 μm thin sections using a microtome. The sections were placed on the water surface at 42°C to flatten, and were then coated with APES-coated glass slides and inserted into a section holder for drying in a 37°C incubator.

[0263] (2) Hematoxylin-eosin (H&E) staining

[0264] The tissue sections were dewaxed and rehydrated in xylene I for 10 min, xylene II for 10 min, 100% ethanol I for 5 min, 100% ethanol II for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then washed with PBS for 3 times, each for 5 min. The sections were then stained with hematoxylin for 5 min, differentiated with 1% hydrochloric acid alcohol, and then washed with tap water for 15 min. The sections were counterstained with 1% diluted ammonia water, and then stained with eosin for 3 min. The sections were then dehydrated with ethanol, cleared with xylene, and mounted with neutral resin. Air-dried sections were observed and photographed under an optical microscope.

[0265] 1.4.3. Quantitative analysis of intestinal goblet cells

[0266] Alcian blue staining is commonly used to observe the secretion of mucin by goblet cells. The intestinal tissue sections were dewaxed in xylene for 5 min, rehydrated in 100%, 90%, and 80% gradient alcohol for 5 min each, and then in distilled water for 5 min. The sections were soaked in acidified solution for 3 min, and then the liquid on the sections was gently shaken off. The sections were stained with staining solution for 30 min, and then washed with running water for 5 min. The sections were counterstained with nuclear fast red staining solution for 10 min, and then washed with running water for 3 min. The sections were dehydrated in 80%, 90%, and 100% gradient ethanol for 5 min each, cleared in xylene for 5 min, mounted with neutral resin, and then air-dried. The colon tissue sections were observed under an optical microscope. The alcian blue staining was used for quantitative analysis of the secretion of mucin by goblet cells using Image J.

[0267] 1.4.4. Quantitative Real-time polymerase chain reaction

[0268] Quantitative Real-time polymerase chain reaction (qRT-PCR) is mainly used to analyze the gene transcription level changes of specific molecules in the colon, and the specific operation is as follows:

[0269] (1) Extraction of tissue RNA

[0270] TRIzol was used to extract mRNA from tissues, and the steps were referred to the instructions. A certain amount of mouse colon tissue was taken, grinding beads were added, and tissue homogenate was prepared. Then, 1 mL of TRIzol was added for homogenate lysis. Then, according to the instructions, the RNA was obtained by layering (chloroform extraction), precipitation (isopropanol), and washing (75% ethanol). Then, the RNA was dissolved in DEPC water and stored at -80°C.

[0271] (2) Reverse transcription (cDNA synthesis)

[0272] The extracted mRNA was reversely transcribed using PrimeScript™ RT Master Mix Reagent Kit (TaKaRa PrimeScript RT Master Mix, Dalian) according to the instructions.

[0273] (3) Index determination

[0274] According to the operation instructions of Takara Premix Ex Taq TM II RR036A kit, the PCR amplification system was configured as follows: 1 μL of upstream primer, 1 μL of downstream primer, 6 μL of ddH2O, 10 μL of Premix Ex Taq II, and 2 μL of cDNA template. The primers for each detection index are shown in Table 14.

[0275] Two-step PCR amplification standard procedure: 95°C for 30 s, 1 cycle; 95°C for 3 s, 60°C for 30 s, 40 cycles; enter the melting curve stage. Finally, the mRNA of GAPDH was used as an internal reference, and the relative expression amount of the gene was calculated according to 2 -△△Ct

[0276] Table 14 Gene primer sequences

[0277]

[0278] 2. Experimental results

[0279] In the above study, significant differences in intestinal microecology were found between the model group and the normal control group. The intestinal tissue state and barrier function were further analyzed. Table 15 shows the tissue state and barrier function of mice in each group. As can be seen from the table, the muscle layer thickness and crypt depth of the offspring decreased significantly after maternal immune activation (p<0.0001). This indicates that maternal immune activation not only has a serious impact on the neural development of the offspring, but also damages the intestinal tissue state to a certain extent. Comparative Examples 1-4 show that the muscle thickness and crypt depth have improved after intervention with low and high doses of LNnT and PC, but after significant difference analysis, it is found that there is no significant difference between the two indicators and the model group (as shown in Tables 16 and 17); the muscle layer thickness and crypt depth of the offspring in the four examples are significantly higher than those in the model group and the four comparative examples, and the significant difference is shown in Tables 16 and 17. This indicates that LNnT and PC alone have no significant effect on improving the muscle layer thickness and crypt depth of the offspring, but when they are compounded in different proportions, the muscle layer thickness and crypt depth of the offspring can be further improved, especially in Example 2, the muscle layer thickness can reach about 104.2 μm, and the crypt depth reaches 82.14 μm, both reaching the level of the control group.

[0280] ​Further, Alcian blue staining was used to investigate the relative amount of mucus secreted by goblet cells in the intestines of the pups in each group. Goblet cells and the mucus secreted by them are said to be the first line of defense of the gastrointestinal barrier function, and can interact with the intestinal immune system, playing an important role in the establishment of the human immune system. The relative amount of mucus secreted by goblet cells in the intestinal tissues of the pups in each group was quantitatively analyzed. As can be seen from Table 15, maternal immune activation significantly reduced the relative amount of mucus secreted by goblet cells in the intestines of the offspring. However, after intervention with LNnT and PC monomers at low and high different doses (Comparative Examples 1-4), the content was improved to a certain extent, and after difference significance analysis, it was found to be significantly higher than that of the model group (as shown in Table 18); it was further found that when they were compounded at different ratios (Examples 1-4), the content was further improved, and was significantly higher than that of the model group and the four comparative example groups. This shows that the composition formed by compounding LNnT and PC at a ratio of 1:0.0525-1:1.25 has a better effect than the two substances alone in promoting the secretion of mucus by goblet cells in the intestines, and has a synergistic effect between them. In particular, Example 2 is significantly higher than Example 1, Example 3 and Example 4 (p<0.001 or p<0.01).

[0281] Then, the expression of intestinal tissue barrier integrity related proteins was analyzed by qRT-PCR technology to reflect the influence of maternal immune activation on intestinal barrier function. Intestinal barrier integrity is closely related to the occurrence and development of various diseases. The tight junction between epithelial cells is an important part of the intestinal mechanical barrier. This connection is mainly composed of occludin, claudin, mucin (MUC), and ZO protein adhesion molecules. The mRNA expression of occludin, claudin-1, MUC2 and ZO-1 was used to illustrate the influence of maternal immune activation and nutritional intervention on the intestinal barrier function of the offspring. As can be seen from Table 19, the mRNA expression of the four proteins representing the intestinal barrier function was significantly reduced after maternal immune activation (p<0.0001), and after LNnT and PC were intervened with low and high doses respectively (Comparative Examples 1-4), the expression of the four barrier related protein mRNAs was significantly higher than that of the model group (p<0.05 or p<0.01 or p<0.001 or p<0.0001). Further found that when LNnT and PC were compounded in different proportions (Examples 1-4) to form a composition, the mRNA expression of the four barrier related proteins in the intestinal tract of the offspring was further improved, and the difference significance analysis found that the four examples were significantly higher than the model group (p<0.0001) and the four comparative examples (as shown in Tables 20-23), and the two had a synergistic effect. This shows that the composition formed by compounding LNnT and PC in the ratio of 1:0.0525-1:1.25 has a better effect than the two substances alone in promoting the mRNA expression of the barrier function related proteins in the intestine. Especially, Example 2 is significantly higher than Example 1, Example 3 and Example 4 (p<0.0001 or p<0.001 or p<0.01).

[0282] From these results, we can see that LNnT and PC can significantly promote the intestinal tissue state and barrier function of the offspring after maternal immune activation when they are compounded in the ratio of 1:0.0525, 1:0.158, 1:0.415 and 1:1.25, and the two have a synergistic effect.

[0283] Table 15 Comparison of intestinal tissue states of mice in each group

[0284]

[0285] Table 16 Difference significance analysis of muscle layer thickness of intestinal tissue state of mice in each group

[0286]

[0287] Table 17 Analysis of the significance of the difference in the depth of the crypts of the intestinal tissue of the mice in each group

[0288]

[0289] Table 18 Analysis of the significance of the difference in the relative amount of the goblet cells of the intestinal tissue of the mice in each group

[0290]

[0291] Table 19 Comparative analysis of the mRNA expression amount of the intestinal barrier function proteins of the mice in each group

[0292]

[0293] Table 20 Comparative analysis of the difference in the mRNA expression amount of the intestinal barrier function protein Claudin-1 of the mice in the comparative and example groups

[0294]

[0295] Table 21 Comparative analysis of the difference in the mRNA expression amount of the intestinal barrier function protein Occludin of the mice in the comparative and example groups

[0296]

[0297] Table 22 Comparative analysis of the difference in the mRNA expression amount of the intestinal barrier function protein MUC2 of the mice in the comparative and example groups

[0298]

[0299] Table 23 Comparative analysis of the difference in the mRNA expression amount of the intestinal barrier function protein ZO-1 of the mice in the comparative and example groups

[0300]

[0301] Experimental Example 5: Nutritional substance intervention regulates the intestinal inflammation level of the offspring of MIA mice

[0302] 1. Experimental method

[0303] 1.1. Construction of a maternal immune homeostasis disorder model

[0304] The same as Experimental Example 1.

[0305] 1.2. Offspring intervention

[0306] The same as Experimental Example 1.

[0307] 1.3. Grouping of the intervention of the offspring animals

[0308] The same as Experimental Example 1.

[0309] 1.4. Tissue sample collection and index detection

[0310] 1.4.1. Tissue sample collection

[0311] The same as Experimental Example 3.

[0312] 1.4.2. Real-time fluorescent quantitative polymerase chain reaction

[0313] The experimental method is the same as that in Experimental Example 4, and the relevant gene primer sequences are shown in Table 24.

[0314] Table 24 Gene primer sequences

[0315]

[0316] 2. Experimental results

[0317] The present application also investigates the influence of maternal immune disorders on the intestinal immune level of offspring. The qRT-PCR technique is used to analyze the changes in pro-inflammatory factors and anti-inflammatory factors after maternal immune activation and nutritional intervention. Table 25 shows the mRNA expression of five immune factors IL-17A, TNF-α, IL-1β, IL-6 and IL-10.

[0318] 1) Inflammation initiation: IL-1β is produced by activated immune cells (such as macrophages, monocytes, dendritic cells) and some non-immune cells (such as epithelial cells). It acts as a core pro-inflammatory factor, involved in innate immunity and adaptive immunity. IL-1β and TNF-α are the initiators of early inflammation. TNF-α is the core initiator of the inflammatory cascade, a potent pro-inflammatory and induces apoptosis, playing a key role in autoimmune diseases, sepsis and metabolic diseases. In this invention, we found that the expression of IL-1β and TNF-α mRNA in the offspring colon was significantly increased (p<0.0001) after maternal immune activation, but after LNnT and PC were intervened alone at different doses (Comparative Examples 1-4), the mRNA expression of IL-1β and TNF-α was lower than that of the model group, and after significant difference analysis, it was found to be significantly lower than the model group (p<0.0001); Examples 1-4 showed that after LNnT and PC were compounded at different ratios to form compositions, the mRNA levels of pro-inflammatory factors IL-1β and TNF-α were further reduced, and after significant difference analysis, it was found to be significantly lower than the model group (p<0.0001), and the four examples were significantly lower than the four comparative examples, as shown in Tables 26 and 27. This shows that the composition formed by compounding LNnT and PC at a ratio of 1:0.0525-1:1.25 has a better effect than the two substances alone in reducing the initiation and amplification of inflammation in the intestine, and through analysis we found that especially Examples 1, 2 and 4 have obvious synergistic effect.

[0319] 2) Inflammation amplification: IL-6 will continuously amplify inflammation after inflammation initiation. In this invention, we found that the pro-inflammatory factor IL-17A also changed significantly after maternal immune activation. Table 25 shows that the expression of IL-6 and IL-17A mRNA in the offspring colon was significantly increased (p<0.0001) after maternal immune activation, but after LNnT and PC were intervened alone at different doses (Comparative Examples 1-4), the mRNA expression of IL-6 and IL-17A was lower than that of the model group, and after significant difference analysis, it was found to be significantly lower than the model group (p<0.0001); Examples 1-4 showed that after LNnT and PC were compounded at different ratios to form compositions, the mRNA levels of pro-inflammatory factors IL-6 and IL-17 were further reduced to 4 or less, and after significant difference analysis, it was found to be significantly lower than the model group (p<0.0001), and the four examples were significantly lower than the four comparative examples, as shown in Tables 28 and 29. This shows that the composition formed by compounding LNnT and PC at a ratio of 1:0.0525-1:1.25 has a better effect than the two substances alone in reducing the expression of pro-inflammatory factors IL-17A and IL-6 mRNA in the intestine, and the two have a synergistic effect.

[0320] 3) Decreased levels of anti-inflammatory factors: IL-10 is mainly secreted by regulatory T cells (Treg), macrophages, B cells and dendritic cells. It is an anti-inflammatory cytokine that inhibits excessive immune responses and maintains immune homeostasis. In this invention, it was found that the expression of the anti-inflammatory factor IL-10 mRNA in the offspring colon was significantly reduced after maternal immune activation (p<0.0001), but after intervention with LNnT and PC at different doses alone (Comparative Examples 1-4), the mRNA expression of IL-10 was higher than that of the model group, and after significant difference analysis, it was found that Comparative Examples 2-4 were significantly higher than the model group (p<0.01 or p<0.0001) except for Comparative Example 1; Examples 1-4 showed that the mRNA level of the anti-inflammatory factor IL-10 was further improved after intervention with the composition formed by LNnT and PC at different ratios, and after significant difference analysis, it was found that it was significantly higher than the model group (p<0.0001), and the four examples were significantly higher than the four comparative examples, as shown in Table 30. This shows that the composition formed by LNnT and PC at a ratio of 1:0.0525-1:1.25 has a better effect than the two substances alone in promoting the expression of anti-inflammatory factor IL-10 mRNA in the intestine. And through the analysis we found that especially Example 1, Example 2 and Example 4 have obvious synergistic effect.

[0321] The above results show that LNnT and PC can significantly regulate the inflammatory state of MIA offspring in the intestine at a ratio of 1:0.0525, 1:0.158, 1:0.415 and 1:1.25, and can significantly inhibit the start and amplification of the inflammatory response, and can promote the production of anti-inflammatory factors.

[0322] Table 25 Comparison and analysis of mRNA expression of inflammatory factors in the intestines of mice in each group

[0323]

[0324] Table 26 Difference comparison and analysis of TNF-α mRNA expression of inflammatory factors in the intestines of offspring mice in the comparative examples and examples

[0325]

[0326] Table 27 Difference comparison and analysis of IL-1β mRNA expression of inflammatory factors in the intestines of offspring mice in the comparative examples and examples

[0327]

[0328] Table 28 Difference comparison and analysis of IL-6 mRNA expression of inflammatory factors in the intestines of offspring mice in the comparative examples and examples

[0329]

[0330] Table 29 Comparative analysis of differences in intestinal inflammatory factor IL-17A mRNA expression in subgroups of Examples and Comparative Examples

[0331]

[0332] Table 30 Comparative analysis of differences in intestinal inflammatory factor IL-10 mRNA expression in subgroups of Examples and Comparative Examples

[0333]

Claims

1. A nutritional composition, characterized in that, It is a nutritional composition that improves the intestinal microecological imbalance in offspring caused by maternal immune activation, and the nutritional composition contains the essential active ingredients shown in (I) and (II) below: (I) Lactose-N-neotetrasaccharide, (II) 1,2-Diayl-sn-glycerol-3-phosphate choline; Furthermore, in the nutritional composition, the mass ratio of lactose-N-neotetrasaccharide to 1,2-diacyl-sn-glycerol-3-phosphocholine is 1:(0.80-1.5).

2. The nutritional composition according to claim 1, characterized in that, The nutritional composition also contains human milk oligosaccharides other than lactose-N-neotetrasaccharide.

3. The nutritional composition according to claim 1 or 2, characterized in that, The nutritional composition further comprises any one or more of 1,2-diacyl-sn-glycerol-3-phosphoinositol, 1,2-diacyl-sn-glycerol-3-phosphoserine, 1,2-diacyl-sn-glycerol-3-phosphoethanolamine, 1,2-diacyl-sn-glycerol-3-phosphoglycerol, and N-acyl-D-sphingosine-1-phosphocholine.

4. Use of the nutritional composition according to any one of claims 1-3 in the preparation of foods that improve the intestinal microecological imbalance in offspring caused by maternal immune activation.

5. The use according to claim 4, characterized in that, The improvement of offspring gut microbiota imbalance caused by maternal immune activation includes improving changes in offspring gut microbiota caused by maternal immune activation. The improvement in offspring gut microbiota changes resulting from maternal immune activation includes any one or more of the following (a1)-(a3): (a1) Improves the reduced abundance of beneficial bacteria genera in the offspring gut caused by maternal immune activation, wherein the beneficial bacteria genera include Bifidobacterium , Allobaculum and Gordonibacter Any one or more of the following; (a2) Improve the reduced abundance of beneficial bacteria for neural development in the offspring gut caused by maternal immune activation, wherein the beneficial bacteria for neural development include Bifidobacterium_pseudolongum and Akkermansia_ muciniphila Any one or more of the following; (a3) Improves the abundance of neurodevelopmentally harmful bacteria in the offspring gut caused by maternal immune activation, including... Ruminococcus_gnavus_AGR2154 .

6. The use according to claim 4, characterized in that, The improvement of offspring gut microbiota imbalance caused by maternal immune activation includes improving changes in short-chain fatty acid content in the offspring gut caused by maternal immune activation. The improvement in the changes in short-chain fatty acid content in the offspring gut caused by maternal immune activation includes the following (b1): (b1) Improves the reduction of one or more of acetic acid, propionic acid, butyric acid and valerate in the offspring gut caused by maternal immune activation.

7. The use according to claim 4, characterized in that, The improvement of offspring gut microbiota imbalance caused by maternal immune activation includes improving offspring gut structure and / or barrier function damage caused by maternal immune activation. The improvement of offspring intestinal structure and / or barrier function impairment caused by maternal immune activation includes any one or more of the following (c1)-(c4): (c1) Improves the reduction in intestinal muscle layer thickness in offspring caused by maternal immune activation; (c2) Improves the reduction in intestinal crypt depth in offspring caused by maternal immune activation; (c3) Improves the reduction of mucin secretion by goblet cells in offspring caused by maternal immune activation; (c4) Improve the reduced expression of intestinal barrier function-related proteins in offspring caused by maternal immune activation, wherein the intestinal barrier function-related proteins include any one or more of Claudin-1, Occludin, MUC2 and ZO-1.

8. The use according to claim 4, characterized in that, The improvement of offspring gut microbiota imbalance caused by maternal immune activation includes improving the elevated level of offspring gut inflammation caused by maternal immune activation; The improvement in offspring gut inflammation levels caused by maternal immune activation includes the following (d1) and / or (d2): (d1) Improves the increase in the expression of pro-inflammatory factors in the offspring gut caused by maternal immune activation, said pro-inflammatory factors including any one or more of TNF-α, IL-1β, IL-17A and IL-6; (d2) Improves the reduction in the expression of anti-inflammatory factors in the offspring gut caused by maternal immune activation, including IL-10.

9. The use according to any one of claims 4-8, characterized in that, The food includes any one or more of infant formula and infant complementary foods.

10. The use according to any one of claims 4-8, characterized in that, In the food, based on the total dry matter content of the food, the content of lactose-N-neotetrasaccharide is 0.16-0.48 g / 100 g, and the content of 1,2-diacyl-sn-glycerol-3-phosphocholine is 25-400 mg / 100 g.

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