Construction method of animal model with stable intestinal flora in four bounds and application of animal model in evaluation of immunomodulatory function of infant formula powder

Abstract

The invention discloses a construction method of an animal model with stable intestinal tract four-bound flora and an intestinal tract four-bound core flora used for evaluating the immune regulation function of infant blending powder. According to the method, milk powder base powder containing milk fat globule membranes is used for intervening SD male young rats in the second day of birth in a certain mode, and four flora models with stable intestinal bacteria, fungi, archaea and viruses of the young rats in the lactation period are constructed through metagenome sequencing. The infant blending powder base powder containing the milk fat globule membrane remarkably increases immune related species, and intestinal tract four-boundary core florae remarkably related to immunity are obtained through Spearman correlation analysis. The obtained intestinal tract four-bound flora model of the baby rats in the lactation period can be used for evaluating the immune regulation function of baby blending powder.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and biotechnology, and specifically relates to a method for constructing an animal model with stable four-kingdom intestinal flora and an application of the model in evaluating the immunoregulatory function of infant formula. Background Art

[0002] Breast milk is the most perfect food for infants in their early growth, and has an important impact on the health and cognition of infants throughout their life cycle. However, when breastfeeding is not possible or breast milk is insufficient due to various reasons, infant formula is the best substitute for breast milk; it is a special formula designed according to the nutritional needs of infants, which can provide infants with comprehensive and balanced nutrition and has a vital impact on the early growth and development of infants and the formation of their intestinal microbial communities.

[0003] Infancy is a critical period for the development of the human immune system and a period when diseases are prone to occur. Therefore, good immunity is an important guarantee for the healthy growth of infants and young children. The intestinal microbial community is crucial to the development of the innate immune system, the formation of the adaptive immune system, and the acquired growth and development of infants and young children. It not only participates in the activation and maturation of the infant immune system, regulates immune responses, maintains intestinal epithelial homeostasis, regulates host metabolism, regulates the nervous system and brain development, but also has a good improvement effect on infections, allergies, and inflammation in infants and young children.

[0004] Although more and more studies have shown that infant formula containing functional components such as human milk-derived probiotics and human milk oligosaccharides can play a good regulatory role on the intestinal microbial community by promoting the growth of beneficial bacteria in the intestines of infants and young children, thereby improving the immune function of infants and young children; however, the evaluation of the regulation of immune function is mostly achieved through relevant immune indicators such as the serum and immune organs of model animals, and there is a lack of an evaluation model for the intestinal flora.

[0005] At present, most of the research on intestinal flora focuses only on bacteria; however, as the research continues to deepen, people have found that in addition to bacteria, the intestine also includes fungi, viruses, archaea and other microbial communities, which have co-evolved with the intestine to form a complex symbiosis and mutualistic relationship, thereby promoting the body's immunity and health; at the same time, there are few studies on the four-kingdom flora at the level of "bacteria-fungi-virus-archaea", especially the four-kingdom core flora for evaluating the immune regulation function of infant formula. Therefore, it is necessary to construct an animal evaluation model from the perspective of the four-kingdom flora of the intestine.

[0006] However, the microbial community in the intestine is easily affected by genetic, environmental, dietary and other factors, and has poor stability, making it difficult to form a stable intestinal microbial community. Summary of the invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for constructing an animal model with stable intestinal four-kingdom flora.

[0008] The technical problem that the present invention also aims to solve is to provide an application of the model construction method in evaluating the immunoregulatory function of infant formula.

[0009] Technical solution: In order to solve the above technical problems, the present invention provides a method for constructing an animal model with stable intestinal four-kingdom flora, the construction method comprising administering samples of milk powder-based milk containing milk fat globule membrane to lactating animals on the 2nd to 23rd day after birth in stages, the sample temperature is maintained at 36-38°C during gavage, the animal body temperature is maintained at 35-36°C during the experiment, and the dosage of the milk powder-based milk containing milk fat globule membrane is 0.1-0.8 mL / time, 2-4 times / d.

[0010] Wherein, the milk powder base powder includes but is not limited to infant formula milk powder base powder.

[0011] Among them, each kilogram of milk powder base contains 0.40kg desalted whey powder, 0.18kg food vegetable blending oil, 0.19kg lactose, 0.02kg whey protein powder, 0.005kg concentrated whey protein powder, 0.005kg compound nutritional enhancers (vitamins, minerals) and 0.2kg milk powder spray-dried from 1.6kg raw cow's milk.

[0012] The animals include but are not limited to young mice. Preferably, the animals of the present invention are male SD young mice in the lactation period on the second day after birth.

[0013] Among them, the frequency of the phased gavage intervention is: on the 2nd to 4th day after birth of the pups, 0.1-0.2 mL / time, 2 times / day; on the 5th to 9th day, 0.3-0.5 mL / time, 4 times / day; on the 10th to 23rd day: 0.5-0.8 mL / time, 4 times / day; after the 23rd day: 1 mL / time, 4 times / day.

[0014] The interval between each intervention on the young mice was 3 to 4 hours, and the abdomen of the young mice was gently massaged for 1 to 2 minutes after each intervention.

[0015] Wherein, the concentration of milk fat globule membrane is 500-1000 mg / kg (MFGM mass / mouse body weight).

[0016] Wherein, the milk powder concentration in the infant formula milk liquid is 130-160 g / kg of infant formula milk powder base powder, and the composition is the same as above.

[0017] The present invention also includes the application of the construction method in evaluating the immunomodulatory function of infant formula.

[0018] Among them, the application includes performing KEGG functional enrichment analysis on the four-kingdom intestinal flora in the constructed model, and predicting immune-related species, performing Spearman correlation analysis on the predicted species with the immune genes in the pathway and immune-related phenotypic indicators, and obtaining the four-kingdom flora significantly related to immunity, and comparing the flora corresponding to the infant formula to be tested with them. If the flora composition and abundance are similar or the same, it has high immune regulation ability, otherwise the immune regulation ability is lower than that of the model group.

[0019] The method for obtaining the intestinal four-kingdom flora for evaluating the immunomodulatory function of infant formula of the present invention comprises:

[0020] (1) Using the cloud platform of Shanghai Meiji Biopharmaceutical Technology Co., Ltd., KEGG functional enrichment analysis was performed on the stable four-kingdom intestinal flora obtained by the above construction method;

[0021] (2) Screening immune-related pathways and creating gene sets, then annotating the gene sets to species and predicting immune-related species;

[0022] (3) Spearman correlation analysis was performed on the predicted species with the immune genes in the pathway and the immune-related phenotypic indicators, and the four-kingdom bacterial community that was significantly correlated with immunity (P < 0.05) was obtained, which is the four-kingdom core bacterial community of the intestine that can be used to evaluate the immune regulatory function of infant formula.

[0023] Among them, the stable four-kingdom intestinal flora includes 232 species:

[0024] (1) 118 bacteria: Lactobacillus, unclassified_f__Muribaculaceae, Prevotella, unclassified_f__Lachnospiraceae, Bacteroides, Ligilactobacillus, unclassified_o__Bacteroidales, unclassified_f__oscillospiraceae, Phocaeicola, Muribaculum, Ruminococcus, Duncaniella, unclassified_d__Bacteria, unclassified_f__Bacteroidaceae, Parabacteroides, Eubacterium, Dorea, unclassified_p__Bacillota, Helicobacter, Alistipes, Paramuribaculum, Acetatifactor, Oscillibacter, Bifidobacterium, Segatella, Escherichia, Schaedler ella, Lawsonibacter, Desulfovibrio, Hungatella, Adlercreutzia, Acutalibacter, Roseburia, Paraprevotella, unclassified_f__Clostridiaceae, unclassified_f__Porphyromonadaceae, Faecalibaculum, Pseudoflavonifractor, Streptococcus, Barnesiella, Allobaculum, Enterocloster, Flavonifractor, Thomasclavelia, Odoribacter, Lachnoclostridium, unclassified_f__Prevotellaceae, Parablautia, unclassified_f__Erysipelotrichaceae, Chlamydia, Clostridioides, Coprococcus, Anaerotruncus, Dysosmobacter, Turicibacter,Ruminiclostridium, Ruthenibacterium, Parasutterella, Candidatus_Amulumruptor, Butyricicoccus, Flintibacter, Intestinimonas, Lepagella, Eisenbergiella, unclassified_p__Bacteroidota, Sporofaciens, Otoolea, Emergencia, Leyella, Negativibacillus, unclassified_o__Lactobacillales, unclassified_f__Coriobacteriaceae, Butyrivibrio, Veillonella, Neglectibacter, Subdoligranulum, Klebsiella, Anaerostipes, Streptomyces, Colidextribacter, Candidatus_Homeothermus, Holdemania, Massiliimalia, Bacillus, Hemini philus, Collinsella, Fusicatenibacter, Agathobacter, Lacrimispora, Coprobacter, Paenibacillus, Porphyromonas, Vermiculatibacterium, unclassified_f__Christensenellaceae, Candidatus_Spyradocola, Vibrio, unclassified_f__Paludibacteraceae, Hominisplanchenecus, Turicimonas, Hespellia, Eggerthella, Provencibacterium, Angelakisella, Salmonella, Flavobacterium, unclassified_o__Candidatus_Gastranaerophilales, Bilophila, Listeria, Tannerella, Acinetobacter, Agathobaculum, Parvibacter, Ornithobacterium,Rikenella, Acetivibrio, Pseudomonas, unclassified_c__Deltaproteobacteria, Solobacterium;

[0026] (2) 77 fungi: Rattus, Lupinus, Trichuris, Mus, Tanacetum, Rhynchophorus, Plasmodium, Spinacia, Cricetulus, Heliosperma, Rhynchospora, Carnegiea, Capsicum, Zophobas, Schistosoma, Musa, Rhizopus, Elysia, Toxocara, Halteria, Rubroshorea, Aristolochia, Malus, Beta, Triticum, Solanum, Trypanosoma, Quillaja, Saitoella, Peromyscus, Phyllostomus, Trichoderma, Fusarium, Adiantum, Nymphon, Rousettus, Pisum, Anisakis, Aspergillum lus, Acacia, Ailuropoda, Pipistrellus, Trifolium, Jimgerdemannia, Colletotrichum, Arctia, Medicago, Apodemus, Arvicanthis, Abscondita, Camellia, Dipodomys, Ensete, Mastomys, Microtus, Helianthus, Homo, Marchantia, Chiloscyllium, Genlisea, Allacma, Cyprideis, Oryza, Onchocerca, Gossypium, Galeopterus, Botryococcus, Thalictrum, Phodopus, Myotis, Ditylenchus, Sphagnum, Pinus, Daucus, Brassica, Ictidomys, Neotoma;

[0027] (3) 14 viruses: unclassified c Caudoviricetes, unclassified d Viruses, unclassified_o__Crassvirales, unclassified_f__Microviridae, Junduvirus, Gammaretrovirus, unclassified_f__Ackermannviridae, Betaretrovirus, unclassified_p__Cressdnaviricota, Pseudotevenvirus, Wadgaonvirus, unclassified_f__Retroviridae, Skunavirus, Intracisternal_A-particles;

[0028] (4) 23 Archaea: Methanosphaera genus, unclassified_p__Candidatus_Bathyarchaeota genus, unclassified_d__Archaea genus, unclassified_f__Candidatus_Methanomethylophilaceae genus, unclassified_p__Thermoproteota genus, u nclassified_p__Candidatus_Woesearchaeota genus, unclassified_c__Thermoplasmata genus, unclassified_c__Candidatus_Nanoarchaeia genus, unclassified_p__Euryarchaeota genus, Methanomicrobium genus, Methanomassiliic occus genus, Methanoculleus genus, unclassified_f__Methanomassiliicoccaceae genus, Methanobacterium genus, unclassified_p__Nanoarchaeota genus, unclassified_p__Candidatus_Aenigmarchaeota genus, Thermococcus genus, Methano Lobus genus, unclassified_o__Methanomicrobiales genus, Methanothrix genus, unclassified_p__Candidatus_Lokiarchaeota genus, unclassified_o__Methanosarcinales genus, unclassified_p__Candidatus_Heimdallarchaeota genus.

[0035] The four-kingdom intestinal core flora used to evaluate the immunomodulatory function of infant formula includes 112 species:

[0036] (1) 66 bacteria: Acinetobacter, unclassified_c__Deltaproteobacteria, Pseudomonas, Listeria, Parvibacter, Agathobaculum, Fusicatenibacter, unclassified_o__Candidatus_Gastranaerophilales, Rikenella, Solobacterium, Streptomyces, Salmonella, Chlamydia, Hominisplanchenecus, Provencibacterium, Eggerthella, Holdemania, Angelakisella, Massil iimalia, Klebsiella, Lacrimispora, Leyella, unclassified_f__Christensenellaceae, Bacillus, Paenibacillus, Candidatus_Spyradocola, Vermiculatibacterium, Negativibacillus, Agathobacter, Colidextribacter, Veillonella, Neglectibacter, Sporofaciens, Butyricicoccus, unclassified_f__Coriobacteriaceae, Subdoligranulum, unclassified_p_

[0037] _Bacteroidota, Coprococcus, Eisenbergiella, Intestinimonas, Lachnoclostridium, Ruminiclostridium, Dysosmobacter, Thomasclavelia, Streptococcus, Parasutterella, Faecalibaculum, unclassified_f__Erysipelotrichaceae, Segatella, Pseudoflavonifractor, Candidatus_Amulumruptor , Odoribacter, Lawsonibacter, Schaedlerella, Bifidobacterium, Paramuribaculum, Oscillibacter, Parabacteroides, Escherichia, unclassified_d__Bacteria, Ruminococcus, unclassified_f__Bacteroidaceae, unclassified_o__Bacteroidales, Phocaeicola, Ligilactobacillus, Bacteroides;

[0038] (2) 36 fungi: Chiloscyllium, Ictidomys, Neotoma, Trifolium, Ditylenchus, Galeopterus, Genlisea, Phodopus, Brassica, Myotis, Microtus, Rousettus, Arctia, Pisum, Mastomys, Ailuropoda, Medicago, Pipistrellus, Apodemus, Triticum, Dipodomys, Arvicanthis, Fusarium, Homo, Phyllostomus, Trypanosoma, Elysia, Peromyscus, Musa, Schistosoma, Rhizopus, Plasmodium, Cricetulus, Mus, Trichuris, Rattus;

[0039] (3) 6 viruses: unclassified_o__Crassvirales, unclassified_c__Caudoviricetes, Betaretrovirus, Gammaretrovirus, unclassified_f__Retroviridae, and unclassified_d__Viruses;

[0040] (4) 4 archaea: unclassified_p__Candidatus_Bathyarchaeota, Methanosphaera, Methanomicrobium, unclassified_o__Methanomicrobiales

[0041] The present invention attempts to use the infant formula milk powder base powder containing breast milk-derived probiotics and human milk oligosaccharides, which has a good regulatory effect on the intestinal microbial community, to construct a lactating mouse model with stable intestinal flora, and explore the four-kingdom core flora in the intestine that is closely related to immunity; to provide theoretical support for the development and application of infant formula powder with functions such as immune regulation. However, through our experimental design and the results of three batches of experiments, it was found that the intestinal flora of lactating mice intervened by the infant formula milk powder base powder containing milk fat globule membrane, which has an unknown effect in the prior art, is not only bacteria, but also includes fungi, viruses, Archaea and other stable species. The number and abundance ratio, as well as the relative error of the abundance ratio are better than the infant formula milk powder base powder containing breast milk-derived probiotics and human milk oligosaccharides.

[0042] The present invention specifically constructs a lactation mouse model with stable intestinal four-kingdom flora in the early stage, including:

[0043] (1) Three batches of animal experiments were conducted using male lactating mice born from the same pure line Sprague-Dawley (SD) pregnant mice. The lactating male mice on the second day of birth were intervened by four methods: infant formula milk powder (IF_Con group), infant formula milk powder containing mixed human milk oligosaccharides (HMOs) (IF_HM group), infant formula milk powder containing milk fat globule membrane (MFGM) (IF_MF group), and infant formula milk powder containing breast milk probiotics (IF_BL group); the body temperature of the mice was maintained at 35-36℃. During the model construction process, the mice were gradually separated from their mothers according to their weight;

[0044] (2) After the intervention, fresh fecal samples of the young mice treated with the four intervention methods were collected, DNA was extracted, and metagenomic sequencing was performed on the Illumina NovaSeq6000 platform;

[0045] (3) Using the cloud platform of Shanghai Meiji Biopharmaceutical Technology Co., Ltd. to perform bioinformatics analysis on the data obtained from sequencing. The species annotation analysis of this patent is performed at the genus level;

[0046] (4) The Kruskal-Wallis rank sum test was used to perform a differential analysis of the read counts and abundance ratios of the same species in three batches of the same intervention method. After removing species with significant differences (P < 0.05), the species with stable intestinal tract were further screened based on their read counts and abundance ratios.

[0047] The stable species in the present invention refers to stable species with a large abundance ratio, a large number, and a low relative error in abundance ratio obtained by performing a Kruskal-Wallis rank sum difference test on stable species obtained by different intervention methods.

[0048] The present invention obtains a stable intestinal four-kingdom flora for evaluating the immunoregulatory function of infant formula through the above construction method.

[0049] Beneficial effects: The present invention uses infant formula milk powder containing milk fat globule membrane to intervene in SD male mice on the second day of birth in a certain way to construct a stable four-kingdom flora model of intestinal bacteria, fungi, archaea and viruses in lactating mice. KEGG functional enrichment analysis was performed on the stable four-kingdom flora; immune-related species were screened; Spearman correlation analysis was performed on immune-related species and genes with immune phenotypic indicators to obtain the four-kingdom core flora of the intestine that is significantly related to immunity. The four-kingdom flora model of the intestinal tract of lactating mice obtained by the present invention can be used to evaluate the immunoregulatory function of infant formula. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Stable bacteria (genus level) in the intestines of lactating mice with different intervention methods;

[0051] Figure 2 Stable fungi (genus level) in the intestines of lactating rats with different intervention methods;

[0052] Figure 3 Stable viruses (genus level) in the intestines of lactating mice with different intervention methods;

[0053] Figure 4 Stable Archaea (genus level) in the intestines of lactating mice with different intervention methods;

[0054] Figure 5 Species annotations in immune-related pathways (genus level); A, bacteria; B, fungi; C, viruses; D, archaea;

[0055] Figure 6 Immune indicators in the serum of lactating mice (x±s, n=24); A, immunoglobulin; B, immune factor;

[0056] Figure 7 Heatmap of the correlation between species in immune-related pathways and immune-related genes;

[0057] Figure 8 Heatmap of the correlation between species in immune-related pathways and serum immune indicators; A, correlation with immune factors; B, correlation with immunoglobulins;

[0058] Fig. 9 Four kingdoms of bacteria in the intestines of lactating mice that are significantly correlated with serum immune indicators; A, bacteria; B, fungi; C, viruses; D, archaea. DETAILED DESCRIPTION

[0059] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0060] Example 1

[0061] The present invention utilizes infant formula milk powder base powder (each kilogram of milk powder base powder contains 0.40kg of desalted whey powder, 0.18kg of food vegetable blending oil, 0.19kg of lactose, 0.02kg of whey protein powder, 0.005kg of concentrated whey protein powder, 0.005kg of compound nutrient enhancer (vitamins, minerals) and 0.2kg of milk powder spray-dried from 1.6kg of raw cow's milk) and infant formula milk powder base powder containing mixed human milk oligosaccharides (human milk oligosaccharides, HMOs), milk fat globule membrane (Milk fatglobule membrane, MFGM) and breast milk-derived probiotics (the same composition as above), and constructs a stable four-kingdom flora model of intestinal bacteria, fungi, archaea and viruses of lactating mice through a certain feeding method and three batches of experiments; and screens out the four-kingdom core flora significantly associated with immunity.

[0062] 1. Preparation of intragastric samples

[0063] Control group (IF_Con): Infant formula milk powder base powder (same composition as above) was dissolved in 40°C water at 143 g / kg;

[0064] Human milk oligosaccharide group (IF_HM): 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose (DSM (China) Co., Ltd.) were mixed in the ratio of 53.50%, 12.40%, 19.58%, 3.15%, 2.95% and 8.43%, and the mixed human milk oligosaccharides were dissolved in the above-mentioned infant formula base powder (composition as above) milk at a final concentration of 1244 mg / kg (HMOs mass / rat weight);

[0065] Milk fat globule membrane group (IF_MF): MFGM (Lacprodan MFGM-10 from Arla Foods, Denmark) was dissolved in the above-mentioned infant formula milk powder (same composition as above) at a final concentration of 570 mg / kg (MFGM mass / mouse body weight);

[0066] Breast milk probiotic group (IF_BL): Bifidobacterium breve grx201 (Gu Ruixia, Hu Pengyu, Zhang Chenchen, et al. Bifidobacterium breve grx201 resistant to oxidative stress and its application [P], ZL 202011391809.3) and Lactobacillus plantarum M731 (Yujun Huang, Junjuan Yu, Ruixia Gu, et al. Screening of Lactobacillus from breast milk and infant feces and evaluation of their bile salt tolerance [J]. Quality Assurance and Safety of Crops&Foods, 2022, 14(4): 115-124) were activated and then incubated for 10 min at 4 °C. 9 CFU / mL was suspended in the above-mentioned infant formula milk powder base powder (composition as above) milk;

[0067] The samples were prepared and used immediately, and placed in a water bath at 36-38°C to keep warm during intragastric administration.

[0068] 2. Animal experiment grouping and intervention methods

[0069] Pregnant mice in animal experiments were from the same pure line of animals SD (Jinan Pengyue Experimental Animal Breeding Co., Ltd., certificate: SCXK (Lu) 20190003), fed with basic feed, free access to food and water during the experiment, controlled environmental temperature at 20-25 ° C, relative humidity at 50%-65%, and pups were born 19-22 days later; sterilized corn cob litter was replaced every day 1-3 days before delivery to avoid irritating the mother mice by replacing the litter on the first day after delivery. After the second day after birth, the corn cob litter was replaced 2-3 times a week, and the litter was replaced with corn cobs and sawdust after the 7th day. The care and use of pregnant mice and lactating pups in this study were approved by the Ethics Committee of Yangzhou University (No.: 202307006), and the breeding facility license number was SYXK (Su) 2022-0044.

[0070] According to the principle that there was no difference in average body weight within and between groups, the newborn male mice were divided into 4 groups, with 2 cages for each intervention method, 1 mother mouse and 4 to 6 male mice in each cage; infant formula milk powder-based milk, infant formula milk powder-based milk containing HMOs, infant formula milk powder-based milk containing MFGM, and infant formula milk powder-based milk containing breast milk-derived probiotics were used to intervene in lactating mice on the second day after birth;

[0071] Four intervention methods: 0.1-0.2 mL / time, 2 times / day on the 2nd to 4th day after birth; 0.3-0.5 mL / time, 4 times / day on the 5th to 9th day; 0.5-0.8 mL / time, 4 times / day on the 10th to 23rd day; 1 mL / time, 4 times / day after the 23rd day; each intervention interval was 3-4 hours, and the abdomen of the puppies was gently massaged for 1-2 minutes after each intervention;

[0072] During the experiment, the body temperature of the pups was maintained at 35-36°C. When the pups weighed 20-25g, they were separated from their mothers during the day and put into cages at night. After their weight reached 30g, they were completely separated from their mothers. Each batch of the experiment lasted 28 days.

[0073] 3. Analysis of the four kingdoms of intestinal flora in young mice

[0074] After 28 days of intervention in each batch, fresh fecal samples were collected from the four groups of young mice, and DNA was extracted and then subjected to metagenomic sequencing on the Illumina NovaSeq 6000 platform. The average sequencing data volume of a single sample was 10G;

[0075] The Kruskal-Wallis rank sum test was used to analyze the abundance of the same species at the genus level in three batches of the same intervention method. After removing the species with significant differences (P<0.05), further screening was performed according to the following conditions: bacterial reads greater than 2970, and the abundance ratio greater than 0.01%; fungal reads greater than 17, and the abundance ratio greater than 0.04%; viral reads greater than 20, and the abundance ratio greater than 0.008%; archaea reads greater than 7, and the abundance ratio greater than 0.03%; The abundance ratios and numbers of stable species obtained by the four intervention methods are shown in Table 1, and the species are shown in Table 2. Figure 1 to Figure 4 .

[0076] Table 1 Stable species abundance ratio and number in the intestine of lactating mice with different intervention methods (x±s, n=24)

[0077]

[0078] Note: Abundance ratio refers to the ratio of the abundance of stable species at the same kingdom and genus level to the total abundance of species at the same kingdom and genus level.

[0079] As shown in Table 1, among the four intervention methods, the number of stable bacteria, fungi and viruses in the intestines of lactating mice obtained by milk fat globule membrane intervention was 118, 77 and 14 (genus level), respectively, which was higher than the number of species obtained by other intervention methods; their abundance in the intestines of young mice accounted for 83.38%, 88.74% and 97.67%, respectively, which were significantly higher than 31.61%, 12.32% and 1.30% in the control group.

[0080] (P<0.05), 46.52%, 7.30%, 1.98% in the human milk oligosaccharide group (P<0.05), and 24.91%, 6.66%, 0.84% ​​in the human milk probiotic group (P<0.05); the number of stable archaea (genus level) in the intestine of lactating mice obtained by milk fat globule membrane intervention was 23, which was higher than the 12 and 9 in human milk oligosaccharides and human milk probiotics, and its abundance accounted for 44.97%, which was higher than 23.68% in the control group, 4.46% in the human milk oligosaccharide group, and 24.49% in the human milk probiotic group;

[0081] At the same time, the relative error of the abundance of bacteria in the intestines of three batches of young mice intervened by milk fat globule membrane was 5.33%, which was 6.39% of the average abundance, while the other three intervention methods were all greater than 16.29%; similarly, the relative error of fungi obtained by milk fat globule membrane intervention was 7.56% of the average abundance, while the other three intervention methods were all greater than 44.16%; the relative error of viruses obtained by milk fat globule membrane intervention was 1.81% of the average abundance, while the other three intervention methods were all greater than 58.46%; the relative error of archaea obtained by milk fat globule membrane intervention was 59.48% of the average abundance, while the other three intervention methods were all greater than 65.02%; this shows that the abundance changes of the stable four-kingdom bacterial community obtained by milk fat globule membrane intervention in the intestines of three batches and 24 young mice were lower than those of other intervention methods, which further verifies that the four-kingdom bacterial community obtained by milk fat globule membrane intervention is relatively stable.

[0082] Depend on Figures 1 to 4 It can be seen that the stable intestinal four-kingdom flora obtained by intervention with milk fat globule membrane includes a total of 232 species:

[0083] (1) 118 bacteria: Lactobacillus, unclassified_f__Muribaculaceae, Prevotella, unclassified_f__Lachnospiraceae, Bacteroides, Ligilactobacillus, unclassified_o__Bacteroidales, unclassified_f__oscillospiraceae, Phocaeicola, Muribaculum, Ruminococcus, Duncaniella, unclassified_d__Bacteria, unclassified_f__Bacteroidaceae, Parabacteroides, Eubacterium, Dorea, unclassified_p__Bacillota, Helicobacter, Alistipes, Paramuribaculum, Acetatifactor, Oscillibacter, Bifidobacterium, Segatella, Escherichia, Schaedler ella, Lawsonibacter, Desulfovibrio, Hungatella, Adlercreutzia, Acutalibacter, Roseburia, Paraprevotella, unclassified_f__Clostridiaceae, unclassified_f__Porphyromonadaceae, Faecalibaculum, Pseudoflavonifractor, Streptococcus, Barnesiella, Allobaculum, Enterocloster, Flavonifractor, Thomasclavelia, Odoribacter, Lachnoclostridium, unclassified_f__Prevotellaceae, Parablautia, unclassified_f__Erysipelotrichaceae, Chlamydia, Clostridioides, Coprococcus, Anaerotruncus, Dysosmobacter, Turicibacter,Ruminiclostridium, Ruthenibacterium, Parasutterella, Candidatus_Amulumruptor, Butyricicoccus, Flintibacter, Intestinimonas, Lepagella, Eisenbergiella, unclassified_p__Bacteroidota, Sporofaciens, Otoolea, Emergencia, Leyella, Negativibacillus, unclassified_o__Lactobacillales, unclassified_f__Coriobacteriaceae, Butyrivibrio, Veillonella, Neglectibacter, Subdoligranulum, Klebsiella, Anaerostipes, Streptomyces, Colidextribacter, Candidatus_Homeothermus, Holdemania, Massiliimalia, Bacillus, Hemini philus, Collinsella, Fusicatenibacter, Agathobacter, Lacrimispora, Coprobacter, Paenibacillus, Porphyromonas, Vermiculatibacterium, unclassified_f__Christensenellaceae, Candidatus_Spyradocola, Vibrio, unclassified_f__Paludibacteraceae, Hominisplanchenecus, Turicimonas, Hespellia, Eggerthella, Provencibacterium, Angelakisella, Salmonella, Flavobacterium, unclassified_o__Candidatus_Gastranaerophilales, Bilophila, Listeria, Tannerella, Acinetobacter, Agathobaculum, Parvibacter, Ornithobacterium,Rikenella, Acetivibrio, Pseudomonas, unclassified_c__Deltaproteobacteria, Solobacterium;

[0085] (2) 77 fungi: Rattus, Lupinus, Trichuris, Mus, Tanacetum, Rhynchophorus, Plasmodium, Spinacia, Cricetulus, Heliosperma, Rhynchospora, Carnegiea, Capsicum, Zophobas, Schistosoma, Musa, Rhizopus, Elysia, Toxocara, Halteria, Rubroshorea, Aristolochia, Malus, Beta, Triticum, Solanum, Trypanosoma, Quillaja, Saitoella, Peromyscus, Phyllostomus, Trichoderma, Fusarium, Adiantum, Nymphon, Rousettus, Pisum, Anisakis, Aspergillum lus, Acacia, Ailuropoda, Pipistrellus, Trifolium, Jimgerdemannia, Colletotrichum, Arctia, Medicago, Apodemus, Arvicanthis, Abscondita, Camellia, Dipodomys, Ensete, Mastomys, Microtus, Helianthus, Homo, Marchantia, Chiloscyllium, Genlisea, Allacma, Cyprideis, Oryza, Onchocerca, Gossypium, Galeopterus, Botryococcus, Thalictrum, Phodopus, Myotis, Ditylenchus, Sphagnum, Pinus, Daucus, Brassica, Ictidomys, Neotoma;

[0086] (3) 14 viruses: unclassified_c__Caudoviricetes, unclassified_d__Viruses, unclassified_o__Crassvirales, unclassified_f__Microviridae, Junduvirus, Gammaretrovirus, unclassified_f__Ackermannviridae, Betaretrovirus, unclassified_p__Cressdnaviricota, Pseudotevenvirus, Wadgaonvirus, unclassified_f__Retroviridae, Skunavirus, Intracisternal_A-particles;

[0087] (4) 23 Archaea: Methanosphaera genus, unclassified_p__Candidatus_Bathyarchaeota genus, unclassified_d__Archaea genus, unclassified_f__Candidatus_Methanomethylophilaceae genus, unclassified_p__Thermoproteota genus, u nclassified_p__Candidatus_Woesearchaeota genus, unclassified_c__Thermoplasmata genus, unclassified_c__Candidatus_Nanoarchaeia genus, unclassified_p__Euryarchaeota genus, Methanomicrobium genus, Methanomassiliic occus genus, Methanoculleus genus, unclassified_f__Methanomassiliicoccaceae genus, Methanobacterium genus, unclassified_p__Nanoarchaeota genus, unclassified_p__Candidatus_Aenigmarchaeota genus, Thermococcus genus, Methano Lobus genus, unclassified_o__Methanomicrobiales genus, Methanothrix genus, unclassified_p__Candidatus_Lokiarchaeota genus, unclassified_o__Methanosarcinales genus, unclassified_p__Candidatus_Heimdallarchaeota genus.

[0091] 4. Immune-related pathway analysis

[0092] The cloud platform of Shanghai Meiji Biotechnology Co., Ltd. was used to perform KEGG functional enrichment analysis on 232 species, including 118 stable bacteria, 77 fungi, 14 viruses and 23 archaea in the intestines of the above-mentioned lactating mice obtained by intervention with milk fat globule membrane; then, immune-related pathways were screened, and the results are shown in Table 2.

[0093] Table 2 KEGG functional enrichment analysis of species

[0094]

[0095]

[0096] As shown in Table 2, among the enriched KEGG pathways, Allograft rejection, Asthma, Primary immunodeficiency, Rheumatoidy_arthritis, Natural killer cell mediatedcytotoxicity, RIG-I-like receptor signaling pathway, Toll like receptorsignaling pathway were screened. pathway; these pathways are enriched with IGH (IgE, IgM, IgG, IgD, IgA), IgA (3.4.24.13), IFNA (IFN-α), EDA2R, TNFRSF27, XEDAR (EDA2R tumor necrosis factor receptor superfamily member 27), IL16 (IL16), IL4R, CD124 (IL-4R interleukin 4 receptor), IL2RB, CD122 (IL2R interleukin 2 receptor β) and other immune-related genes, and involve core cellular immune signaling pathways such as TCR signaling pathway, intestinal epithelial cell immunity, short-chain fatty acid absorption and other signaling pathways.

[0097] 5. Immune-related species annotation

[0098] The cloud platform of Shanghai Meiji Biopharmaceutical Technology Co., Ltd. was used to annotate species in 68 immune-related pathways, and a total of 166 species were annotated at the genus level. The results are shown in Figure 5 .

[0099] Depend on Figure 5It can be seen that the 166 annotated species include 114 bacteria such as Acinetobacter, Pseudomonas, Listeria, Parvibacter, Agathobaculum, Fusicatenibacter, Negativibacillus, Agathobacter, Colidextribacter, Lachnoclostridium, Segatella, Pseudoflavonifractor, Odoribacter, Lawsonibacter, Schaedlerella, and Bifidobacterium; Rattus, Chiloscyllium, Ictidomys, Neotoma, Trifolium, and Dityle nchus, Galeopterus, Genlisea, Phodopus, Brassica, Myotis, Microtus, Rousettus, 38 fungi; 6 viruses, including unclassified_o__Crassvirales, unclassified_c__Caudoviricetes, Betaretrovirus, Gammaretrovirus, and 8 archaea, including unclassified_p__Candidatus_Bathyarchaeota, Methanosphaera, Methanomicrobium, unclassified_o__Methanomicrobiales, and unclassified_d__Archaea;

[0102] 6. Serum immune indexes of young mice

[0103] After each batch of experiments using milk fat globule membrane intervention, the young mice were fasted overnight, blood was collected from the eyeball and transferred to anticoagulant tubes, incubated in a 37°C incubator for 30 min, and centrifuged at 3000×g and 4°C for 10 min to separate serum;

[0104] The levels of immune factors such as immunoglobulin A (IgA), immunoglobulin M (IgM), secretory immunoglobulin A (SIgA), immunoglobulin G (IgG), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), immunoglobulin G2a (IgG2a), interferon-γ (INF-γ) and necrosis factor-α (TNF-α) in serum were determined according to the instructions of the ELISA kit. The results are shown in Table 1. Figure 6 .

[0105] Depend on Figure 6 It can be seen that the levels of IgA, IgM, sIgA, IgG and IgG2a in the serum of lactating mice treated with milk fat globule membrane at 28 days were 16.69, 1518.05, 17.46, 703.26 and 1374.67 μg / mL respectively;

[0106] The contents of IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α were 290.74, 103.001, 30.38, 50.37, 1418.76 and 252.65 pg / mL, respectively.

[0107] 7. Correlation analysis of the four kingdoms of intestinal flora related to immune regulation

[0108] In order to understand the relationship between microorganisms enriched in immune signaling pathways and immune factors and immune genes, we conducted a Spearman correlation study and drew a heat map based on the correlation coefficient matrix; first, the original variables X and Y were converted into ranks RX and RY through rank transformation, and then the Pearson correlation coefficient was used to perform a correlation analysis on RX and RY, thereby obtaining the Spearman rank correlation coefficient to quantify the consistency or difference between the two variables in the rank order. (Reference: Tang D, Wang Y, Kang W, et al. Chitosan attenuates obesity by modifying the intestinal microbiota and increasing serum leptin levels in mice [J]. Journal of Functional Foods 64 (2020): 103659.), the results are shown in Figure 7 and Figure 8 .

[0109] Depend on Figure 7 and Figure 8 It can be seen that the four-kingdom core flora of the intestine that are significantly associated with immune factors and immune genes include 112 species:

[0110] (1) 66 bacteria: Acinetobacter, unclassified_c__Deltaproteobacteria, Pseudomonas, Listeria, Parvibacter, Agathobaculum, Fusicatenibacter, unclassified_o__Candidatus_Gastranaerophilales, Rikenella, Solobacterium, Streptomyces, Salmonella, Chlamydia, Hominisplanchenecus, Provencibacterium, Eggerthella, Holdemania, Angelakisella, Massiliimalia, Klebsiella, Lacrimispora, Leyella, unclassified_f__Christensenellaceae, Bacillus, Paenibacillus, Candidatus_Spyradocola, Vermiculatibacterium, Negativibacill lus, Agathobacter, Colidextribacter, Veillonella, Neglectibacter, Sporofaciens, Butyricicoccus, unclassified_f__Coriobacteriaceae, Subdoligranulum, unclassified_p_Bacteroidota, Coprococcus, Eisenbergiella, Intestinimonas, Lachnoclostridium, Ruminiclostridium, Dysosmobacter, Thomasclavelia, Streptococcus, Parasutterella, Faecalibaculum, unclassified_f__Erysipelotrichaceae, Segatella, Pseudoflavonifractor, Candidatus_Amulumruptor, Odoribacter, Lawsonibacter, Schaedlerella, Bifidobacterium,Paramuribaculum, Oscillibacter, Parabacteroides, Escherichia, unclassified_d__Bacteria, Ruminococcus, unclassified_f__Bacteroidaceae, unclassified_o__Bacteroidales, Phocaeicola, Ligilactobacillus, Bacteroides;

[0111] (2) 36 fungi: Chiloscyllium, Ictidomys, Neotoma, Trifolium, Ditylenchus, Galeopterus, Genlisea, Phodopus, Brassica, Myotis, Microtus, Rousettus, Arctia, Pisum, Mastomys, Ailuropoda, Medicago, Pipistrellus, Apodemus, Triticum, Dipodomys, Arvicanthis, Fusarium, Homo, Phyllostomus, Trypanosoma, Elysia, Peromyscus, Musa, Schistosoma, Rhizopus, Plasmodium, Cricetulus, Mus, Trichuris, Rattus;

[0112] (3) 6 viruses: unclassified o Crassvirales, unclassified c Caudoviricetes, Betaretrovirus, Gammaretrovirus, unclassified_f__Retroviridae, and unclassified_d__Viruses;

[0113] (4) 4 archaea: unclassified_p__Candidatus_Bathyarchaeota, Methanosphaera, Methanomicrobium, and unclassified_o__Methanomicrobiales.

[0114] Among them, the genera of Colidextribacter, Veillonella, Agathobaculum, Bifidobacterium, Parabacteroides, Agathobaculum, Thomasclavelia, Fusarium, Phocaeicola, and Escherichia were significantly positively correlated with serum IgA, IgG, IgM, sIgA, IgG2a, IFN-γ, IL-2, IL-4, IL-6, and IL-10, respectively. (P<0.05); Listeria, Faecalibaculum, Ligilactobacillus, Agathobacter, Eggerthella, Medicagos, Fusarium, Agathobaculum, Eggerthella, Trifolium, etc. were significantly negatively correlated with serum IgA, IgG, IgM, sIgA, IgG2a, IFN-γ, IL-2, IL-4, IL-6, and IL-10, respectively (P<0.05). Acinetobacter, Brassica, Chlamydia, Homo, Mus and other genera were significantly positively correlated with IGH, IgA, IFNA, EDA2R, TNFRSF27, XEDAR, IL16, IL4R, CD124, IL2RB, CD122 and other immune genes annotated in the pathway (P<0.05); Butyricicoccus, Parasutterella, Segatella, unclassified_o__Methanomicrobiales, unclassified_o__Crassvirales were significantly negatively correlated with IgA, IFNA, EDA2R, TNFRSF27, XEDAR, IL4R, CD124 (P<0.05).

[0115] In summary: After treating young mice with milk fat globule membrane intervention, there was a relatively stable four-kingdom bacterial community in the intestinal microorganisms, and the stable four-kingdom bacterial community was significantly correlated with immune factors and immune genes in the serum.

[0116] Example 2

[0117] 1. Use 130-160g / kg of commercially available conventional stage 1 infant formula (each kilogram of infant formula contains 0.39kg of desalted whey powder, 0.18kg of food vegetable blending oil, 0.19kg of lactose, 0.02kg of whey protein powder, 0.005kg of concentrated whey protein powder, 0.005kg of compound nutritional enhancers (vitamins, minerals), 0.0006kg of lactoferrin, 0.00015kg of maltodextrin (containing 3.0×10 10 The intervention was carried out with 0.1-0.2 mL / time of milk (infant formula weight / water weight) containing 0.16 kg of eicosapentaenoic acid (CFU) animal Bifidobacterium bb-12, 0.016 kg of eicosapentaenoic acid, 0.012 kg of docosahexaenoic acid, and 1.45 kg of raw cow's milk spray-dried into 0.1813 kg of milk powder) from the same purebred SD pregnant mice (Jinan Pengyue Experimental Animal Breeding Co., Ltd., certificate: SCXK (Lu) 20190003) during lactation. The intervention was carried out on the 2nd to 4th day after the birth of the pups. , 2 times / d; 5th to 9th day, 0.3-0.5mL / time, 4 times / d; 10th to 23rd day: 0.5-0.8mL / time, 4 times / d; after 23rd day: 1mL / time, 4 times / d; each intervention interval was 3-4h, and the abdomen of the young mice was gently massaged for 1-2min after each intervention; during the experiment, the body temperature of the young mice was maintained at 35-36℃. When the weight of the young mice reached 20-25g, they were separated from the mother mice during the day and caged together at night; after the weight reached 30g, they were completely separated from the mother mice;

[0118] Sterilized corn cob litter was replaced every day 1-3 days before delivery to avoid irritating the mother rat by changing the litter on the first day after delivery. After the second day after birth, the corn cob litter was replaced 2-3 times a week, and the litter was replaced with corn cobs and sawdust after the seventh day. The care and use of pregnant and lactating rats in this experiment were approved by the Ethics Committee of Yangzhou University (No. 202307006), and the breeding facility license number was SYXK (Su) 2022-0044.

[0119] After 28 days of intervention, fresh fecal samples were collected from the pups, and DNA was extracted and then subjected to metagenomic sequencing on the Illumina NovaSeq 6000 platform. The average sequencing data volume for a single sample was 10G.

[0120] 2. The intestinal flora of the four kingdoms of young mice was analyzed using the cloud platform of Shanghai Meiji Biomedical Technology Co., Ltd., and the intestinal flora of the young mice with milk fat globule membrane intervention (hereinafter referred to as model young mice) constructed in Example 1 was used to evaluate the immunomodulatory function of infant formula. The results are shown in Fig. 9 .

[0121] Depend on Fig. 9It can be seen that the abundance of species such as Veillonella, Agathobaculum, Chiloscyllium, Trifolium, Bifidobacterium, Eggerthella, Parvibacter, unclassified_c__Caudoviricetes, unclassified_d__Bacteria, unclassified_o__Candidatus_Gastranaerophilales, Lachnoclostridium, Ditylenchus, Agathobaculum, Hominisplanchenecus and Negativibacillus in the intestines of model rats was higher than that in the intestines of lactating rats intervened by commercial infant formula;

[0124] Depend on Fig. 9 It also showed that the abundance of species such as Listeria, Neglectibacter, Trichuris, unclassified_p__Candidatus_Bathyarchaeota, unclassified_f__Erysipelotrichaceae, Ligilactobacillus, Agathobacter, Brassica, Intestinimonas, unclassified_f__Christensenellaceae, Phocaeicola, Medicago, Fusarium, and Pisum in the intestines of model rat pups was lower than that in the intestines of lactating rat pups intervened with commercial infant formula;

[0125] The correlation analysis in the intestine of the model mice showed that Veillonella was significantly positively correlated with the IgG content in the serum of the mice (P<0.05); Agathobaculum, Chiloscyllium and Trifolium were significantly positively correlated with the IgM content in the serum of the mice (P<0.05); Bifidobacterium was significantly positively correlated with the sIgA content in the serum of the mice (P<0.05);

[0126] The genera of Eggerthella, Parvibacter, nclassified_c__Caudoviricetes, unclassified_d__Bacteria and unclassified_o__Candida_tus Gastranaerophilales were significantly negatively correlated with the content of IgG2a in the serum of young mice (P<0.05); the genera of Lachnoclostridium were significantly positively correlated with the content of IFN-γ in the serum of young mice (P<0.05); the genera of Ditylenchus were significantly positively correlated with the content of IL-2 in the serum of young mice (P<0.05); the genera of Agathobaculum, Chiloscyllium, Hominisplanchenecus and Negativibacillus were significantly negatively correlated with the content of IL-4 in the serum of young mice (P<0.05); the genera of Trifolium were significantly negatively correlated with the content of IL-10 in the serum of young mice (P<0.05).

[0129] Listeria, Neglectibacter, Trichuris, unclassified_p__Candidatus

[0130] The genus Bathyarchaeota was significantly negatively correlated with the content of IgA in the serum of young mice (P<0.05); the genus unclassified Erysipelotrichaceae was significantly negatively correlated with the content of IgG in the serum of young mice (P<0.05); the genus Ligilactobacillus was significantly negatively correlated with the content of IgM in the serum of young mice (P<0.05); the genus Agathobacter, Brassica, Intestinimonas, Trichuris, and unclassified Christensenellaceae were significantly negatively correlated with the content of sIgA in the serum of young mice (P<0.05); The genus A was significantly positively correlated with the content of IgG2a in the serum of young mice (P<0.05); the genus Medicago was significantly negatively correlated with the content of IgG2a in the serum of young mice (P<0.05); the genus Medicago was significantly negatively correlated with the content of IFN-γ in the serum of young mice (P<0.05); the genera Fusarium, Ligilactobacillus and Pisum were significantly negatively correlated with the content of IL-2 in the serum of young mice (P<0.05); the genera Ligilactobacillus were significantly positively correlated with the content of IL-4 in the serum of young mice (P<0.05); the genera Escherichia, Fusarium and Medicago were significantly positively correlated with the content of IL-10 in the serum of young mice (P<0.05).

[0131] Therefore, the levels of IgA, IgG, IgM, sIgA, IFN-γ, and IL-2 in the serum of the model mice were higher than those in the mice intervened with commercially available infant formula (this embodiment), while the levels of IgG2a, IL-4, and IL-10 in the serum were lower than those in the mice intervened with commercially available infant formula.

[0132] 3. According to the instructions of the ELISA kit, the levels of IgA, IgG, IgM, SIgA, IgG2a, IL-2, IL-4, IL-10 and INF-γ in the serum of young mice treated with commercially available infant formula were determined. The comparison with the relevant serum indicators of model young mice is shown in Table 3.

[0133] Table 3 Relevant immune indexes of serum of lactating mice (x±s, n=24)

[0134]

[0135] As can be seen from Table 3, the levels of IgA, IgG, IgM, sIgA, IFN-γ, and IL-2 in the serum of the model mice were significantly higher than those of the mice treated with commercial infant formula (P<0.05), while the levels of IgG2a and IL-4 in the serum were significantly lower than those of the mice treated with commercial infant formula (P<0.05). Although the content of IL-10 in the serum of the mice treated with commercial infant formula was not significantly different from that in the serum of the model mice of the present invention (P<0.05), it was higher than that of the model mice. The results showed that the serum-related immune indicators predicted by the four-kingdom flora of the intestine of lactating mice were consistent with the trends of the measured serum-related immune indicators. Therefore, the four-kingdom flora of the model mice can be better used to evaluate the immune ability of lactating mice.

Claims

1. A method for constructing an animal model with stable intestinal four-kingdom flora, characterized in that: The construction method comprises the steps of gavage intervention of samples of milk powder-based milk solution containing milk fat globule membrane to lactating animals on the 2nd to 23rd day after birth, wherein the sample temperature is maintained at 35 to 38° C. during gavage, and the body temperature of the animals is maintained at 35 to 36° C. during the experiment, and the dosage of the milk powder-based milk solution containing milk fat globule membrane is 0.1 to 0.8 mL / time, 2 to 4 times / day.

2. The method for constructing an animal model with stable intestinal four-kingdom flora according to claim 1, characterized in that: The animals are young mice.

3. The method for constructing an animal model with stable intestinal four-kingdom flora according to claim 2, characterized in that: The frequency of the phased gavage intervention is: on the 2nd to 4th day after birth, 0.1-0.2 mL / time, 2 times / day; on the 5th to 9th day, 0.3-0.5 mL / time, 4 times / day; on the 10th to 23rd day: 0.5-0.8 mL / time, 4 times / day; after the 23rd day: 1 mL / time, 4 times / day.

4. The method for constructing an animal model with stable intestinal four-kingdom flora according to claim 2, characterized in that: The interval between each intervention on the young mice was 3 to 4 hours, and the abdomen of the young mice was gently massaged for 1 to 2 minutes after each intervention.

5. The method for constructing an animal model with stable intestinal four-kingdom flora according to claim 2, characterized in that: The milk fat globule membrane concentration is 500-1000 mg / kg (MFGM mass / mouse body weight).

6. The method for constructing an animal model with stable intestinal four-kingdom flora according to claim 2, characterized in that: The milk powder concentration in the milk liquid is 130-160 g / kg of infant formula milk powder base powder.

7. The construction method according to claim 1, wherein, in the application, the stable intestinal four-kingdom flora in the animal model includes 232 species: (1) 118 bacteria: Lactobacillus, unclassified_f__Muribaculaceae, Prevotella, unclassified_f__Lachnospiraceae, Bacteroides, Ligilactobacillus, unclassified_o__Bacteroidales, unclassified_f__oscillospiraceae, Phocaeicola, Muribaculum, Ruminococcus, Duncaniella, unclassified_d__Bacteria, unclassified_f__Bacteroidaceae, Parabacteroides, Eubacterium, Dorea, unclassified_p__Bacillota, Helicobacter, Alistipes, Paramuribaculum, Acetatifactor, Oscillibacter, Bifidobacterium, Segatella, Escherichia, Schaedler ella, Lawsonibacter, Desulfovibrio, Hungatella, Adlercreutzia, Acutalibacter, Roseburia, Paraprevotella, unclassified_f__Clostridiaceae, unclassified_f__Porphyromonadaceae, Faecalibaculum, Pseudoflavonifractor, Streptococcus, Barnesiella, Allobaculum, Enterocloster, Flavonifractor, Thomasclavelia, Odoribacter, Lachnoclostridium, unclassified_f__Prevotellaceae, Parablautia, unclassified_f__Erysipelotrichaceae, Chlamydia, Clostridioides, Coprococcus, Anaerotruncus, Dysosmobacter, Turicibacter,Ruminiclostridium, Ruthenibacterium, Parasutterella, Candidatus_Amulumruptor, Butyricicoccus, Flintibacter, Intestinimonas, Lepagella, Eisenbergiella, unclassified_p__Bacteroidota, Sporofaciens, Otoolea, Emergencia, Leyella, Negativibacillus, unclassified_o__Lactobacillales, unclassified_f__Coriobacteriaceae, Butyrivibrio, Veillonella, Neglectibacter, Subdoligranulum, Klebsiella, Anaerostipes, Streptomyces, Colidextribacter, Candidatus_Homeothermus, Holdemania, Massiliimalia, Bacillus, Hemini philus, Collinsella, Fusicatenibacter, Agathobacter, Lacrimispora, Coprobacter, Paenibacillus, Porphyromonas, Vermiculatibacterium, unclassified_f__Christensenellaceae, Candidatus_Spyradocola, Vibrio, unclassified_f__Paludibacteraceae, Hominisplanchenecus, Turicimonas, Hespellia, Eggerthella, Provencibacterium, Angelakisella, Salmonella, Flavobacterium, unclassified_o__Candidatus_Gastranaerophilales, Bilophila, Listeria, Tannerella, Acinetobacter, Agathobaculum, Parvibacter, Ornithobacterium,Rikenella, Acetivibrio, Pseudomonas, unclassified_c__Deltaproteobacteria, Solobacterium; (2) 77 fungi: Rattus, Lupinus, Trichuris, Mus, Tanacetum, Rhynchophorus, Plasmodium, Spinacia, Cricetulus, Heliosperma, Rhynchospora, Carnegiea, Capsicum, Zophobas, Schistosoma, Musa, Rhizopus, Elysia, Toxocara, Halteria, Rubroshorea, Aristolochia, Malus, Beta, Triticum, Solanum, Trypanosoma, Quillaja, Saitoella, Peromyscus, Phyllostomus, Trichoderma, Fusarium, Adiantum, Nymphon, Rousettus, Pisum, Anisakis, Aspergillum lus, Acacia, Ailuropoda, Pipistrellus, Trifolium, Jimgerdemannia, Colletotrichum, Arctia, Medicago, Apodemus, Arvicanthis, Abscondita, Camellia, Dipodomys, Ensete, Mastomys, Microtus, Helianthus, Homo, Marchantia, Chiloscyllium, Genlisea, Allacma, Cyprideis, Oryza, Onchocerca, Gossypium, Galeopterus, Botryococcus, Thalictrum, Phodopus, Myotis, Ditylenchus, Sphagnum, Pinus, Daucus, Brassica, Ictidomys, Neotoma; (3) 14 viruses: unclassified_c__Caudoviricetes, unclassified_d__Viruses, unclassified_o__Crassvirales, unclassified_f__Microviridae, Junduvirus, Gammaretrovirus, unclassified_f__Ackermannviridae, Betaretrovirus, unclassified_p__Cressdnaviricota, Pseudotevenvirus, Wadgaonvirus, unclassified_f__Retroviridae, Skunavirus, Intracisternal_A-particles; (4) 23 Archaea: Methanosphaera genus, unclassified_p__Candidatus_Bathyarchaeota genus, unclassified_d__Archaea genus, unclassified_f__Candidatus_Methanomethylophilaceae genus, unclassified_p__Thermoproteota genus, u nclassified_p__Candidatus_Woesearchaeota genus, unclassified_c__Thermoplasmata genus, unclassified_c__Candidatus_Nanoarchaeia genus, unclassified_p__Euryarchaeota genus, Methanomicrobium genus, Methanomassiliic occus genus, Methanoculleus genus, unclassified_f__Methanomassiliicoccaceae genus, Methanobacterium genus, unclassified_p__Nanoarchaeota genus, unclassified_p__Candidatus_Aenigmarchaeota genus, Thermococcus genus, Methano Lobus genus, unclassified_o__Methanomicrobiales genus, Methanothrix genus, unclassified_p__Candidatus_Lokiarchaeota genus, unclassified_o__Methanosarcinales genus, unclassified_p__Candidatus_Heimdallarchaeota genus.

8. Use of the construction method according to any one of claims 1 to 7 in evaluating the immunomodulatory function of infant formula.

9. The use according to claim 8, characterized in that: The application includes performing KEGG functional enrichment analysis on the four-kingdom intestinal flora in the constructed model, screening pathways related to immunity, and creating a gene set, and then annotating the gene set to predict species related to immunity; performing Spearman correlation analysis on the predicted species with the immune genes in the pathway and the phenotypic indicators related to immunity, respectively, to obtain the four-kingdom flora significantly related to immunity, and comparing the flora corresponding to the infant formula to be tested with them; if the flora composition and abundance are similar or the same, it is a high immune regulation ability, otherwise the immune regulation ability is lower than that of the model group.

10. The use according to claim 8, characterized in that: The four-kingdom core flora of the intestine used to evaluate the immunomodulatory function of infant formula includes 112 species: (1) 66 bacteria: Acinetobacter, unclassified_c__Deltaproteobacteria, Pseudomonas, Listeria, Parvibacter, Agathobaculum, Fusicatenibacter, unclassified_o__Candidatus_Gastranaerophilales, Rikenella, Solobacterium, Streptomyces, Salmonella, Chlamydia, Hominisplanchenecus, Provencibacterium, Eggerthella, Holdemania, Angelakisella, Massiliimalia, Klebsiella, Lacrimispora, Leyella, unclassified_f__Christensenellaceae, Bacillus, Paenibacillus, Candidatus_Spyradocola, Vermiculatibacterium, Negativibacill us, Agathobacter, Colidextribacter, Veillonella, Neglectibacter, Sporofaciens, Butyricicoccus, unclassified_f__Coriobacteriaceae, Subdoligranulum, unclassified_p__Bacteroidota, Coprococcus, Eisenbergiella, Intestinimonas, Lachnoclostridium, Ruminiclostridium, Dysosmobacter, Thomasclavelia, Streptococcus, Parasutterella, Faecalibaculum, unclassified_f__Erysipelotrichaceae, Segatella, Pseudoflavonifractor, Candidatus_Amulumruptor, Odoribacter, Lawsonibacter, Schaedlerella, Bifidobacterium,Paramuribaculum, Oscillibacter, Parabacteroides, Escherichia, unclassified_d__Bacteria, Ruminococcus, unclassified_f__Bacteroidaceae, unclassified_o__Bacteroidales, Phocaeicola, Ligilactobacillus, Bacteroides; (2) 36 fungi: Chiloscyllium, Ictidomys, Neotoma, Trifolium, Ditylenchus, Galeopterus, Genlisea, Phodopus, Brassica, Myotis, Microtus, Rousettus, Arctia, Pisum, Mastomys, Ailuropoda, Medicago, Pipistrellus, Apodemus, Triticum, Dipodomys, Arvicanthis, Fusarium, Homo, Phyllostomus, Trypanosoma, Elysia, Peromyscus, Musa, Schistosoma, Rhizopus, Plasmodium, Cricetulus, Mus, Trichuris, Rattus; (3) 6 viruses: unclassified_o__Crassvirales, unclassified_c__Caudoviricetes, Betaretrovirus, Gammaretrovirus, unclassified_f__Retroviridae, and unclassified_d__Viruses; (4) 4 archaea: unclassified_p__Candidatus_Bathyarchaeota, Methanosphaera, Methanomicrobium, and unclassified_o__Methanomicrobiales.

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