CN116762867A

CN116762867A - Application of vitamin D3 in preparation of infant formula milk powder for relieving cow milk allergic reaction caused by beta-lactoglobulin

Info

Publication number: CN116762867A
Application number: CN202310776625.6A
Authority: CN
Inventors: 李欣; 陈红兵; 曾小玲; 鄢宇婷; 潘丽娜; 汪家琦; 叶望娟; 李威; 董玲; 梁琳; 胡一波
Original assignee: Ausnutria Dairy China Co ltd
Current assignee: Ausnutria Dairy China Co ltd
Priority date: 2023-06-28
Filing date: 2023-06-28
Publication date: 2023-09-19

Abstract

The application provides application of vitamin D3 in preparing infant formula milk powder for relieving infant cow milk allergy caused by beta-lactoglobulin. The application constructs a Caco-2 cell model to evaluate the biosafety of vitamin D2 and vitamin D3 and the transport and absorption capacity of the vitamin D2 and the vitamin D3 across cell membranes. The relief effect of vitamin D3 on milk allergy young mice is evaluated by adopting vitamin D3 intervention in different sensitization stages and combining the clinical pathological characteristics, the beta-lactoglobulin specificity IgE, igG, igG1 and IgA level in serum, jejunum HE staining, spleen cell secretion cytokines and other aspects. The experimental results show that: the transport and absorption amount of vitamin D3 is about 2 times that of vitamin D2. The long-term vitamin D3 intervention on the sensitized young mice can obviously reduce the specific antibody level in the serum of the young mice, repair intestinal barrier and regulate the Th1/Th2 balance in the young mice.

Description

Application of vitamin D3 in preparation of infant formula milk powder for relieving cow milk allergic reaction caused by beta-lactoglobulin

Technical Field

The application relates to the technical field of foods, in particular to application of vitamin D3 in preparing infant formula milk powder for relieving cow milk allergic reaction caused by beta-lactoglobulin.

Background

Food allergy is a phenomenon in which the body generates an adverse specific immune response when repeatedly exposed to a specific food. More than 90% of food allergy is caused by eight major types of foods including milk, eggs, peanuts, soybeans, wheat, nuts, fish and shellfish. The most common food allergens for infants are eggs, followed by milk, shrimp and fish. One research study in the united states estimated that 10.8% of people are allergic to food, while infants are more susceptible to food allergy than adults. Japanese investigation showed that the prevalence of food allergy in infants is 5-10% and that of infants is 13%. The nutrition and health monitoring project data of the chinese residents in 2013 show that the food allergy rate of the infants between 2 and 3 years old is 4.37%. In the 2017 national food allergy questionnaire, the incidence of food allergy for children was 5.83%. Recent investigation and research show that the incidence rate of food allergy of infants and infants with Chinese cow's milk allergy is 2.69%.

Food allergy is an immune disease, which is the result of an immune disorder and loss of normal oral tolerance. The immune mechanism of food allergy is the reaction to ingested antigen: first, immediate allergic reactions, mainly IgE-mediated allergic reactions, specific IgE antibodies bind to mast cells and basophils, triggering the release of mediators, leading to rapid physiological responses. The corresponding is delayed allergic reaction, and the delayed allergic inflammatory process is mainly limited to intestinal tracts, and is the basis of food protein-induced colorectal colitis, enterocolitis and eosinophilic esophagitis syndrome. Infant immune system development is imperfect and food allergy is more likely to occur than in adults.

Food allergy symptoms are mainly skin, gastrointestinal and respiratory symptoms. For infants, skin symptoms are mostly acute urticaria, atopic dermatitis and angioedema; gastrointestinal symptoms are diarrhea, vomiting, constipation, severe patients may have hematochezia or shock, and non-IgE mediated allergic patients may have eosinophilic esophagitis, colitis; respiratory symptoms include allergic rhinitis, conjunctivitis, asthma, etc.

Vitamin D nutrition intervention is the first to be focused on for infant allergy, as early as 2007, camargo et al reported for the first time that an automatic epinephrine injection needle has a latitude gradient in the United states, the prescription use rate in northern areas with less sunlight is 4 times higher than that in southern areas, and ultraviolet rays can promote vitamin D synthesis, and this report initiates an extensive study on vitamin D intervention effects, so that the food allergy nutrition intervention gradually goes into the field of vision of people, and more vitamin and fatty acid nutrition intervention effects for infants are studied nowadays. One study of asthma based on the sauter arabic population showed that: the dietary intake of quantitative dietary fiber, vitamin E, magnesium, calcium, sodium and potassium in the diet of allergic patients is obviously less than that of normal people, which also suggests that nutrient intervention has certain relevance to immune diseases. Vitamins play an important role in the immune system. Studies have shown that vitamins and their metabolites can exert important regulatory effects by affecting innate and adaptive immune responses. Among these, the most remarkable effects are fat-soluble vitamins A, D and E as important antioxidants.

At present, most of domestic and foreign researches on the relationship between vitamin D and food allergy are epidemiological surveys, some vitamin D intake is estimated even through a questionnaire form, the real vitamin D state in the body cannot be accurately reflected, and only a small amount of clinical researches reflect the vitamin D level in the body by detecting 25- (OH) D, so that experimental researches on vitamin D and food allergy are lacking at present. Vitamin D also requires more research in relation to food allergy.

Disclosure of Invention

In view of the above, the technical problem to be solved by the application is to provide an application of vitamin D3 in preparing infant formula milk powder for relieving cow milk allergy caused by beta-lactoglobulin.

The application provides application of vitamin D3 in preparing infant formula milk powder for relieving infant cow milk allergy caused by beta-lactoglobulin.

Preferably, the allergic reaction comprises a gastrointestinal allergic reaction, a skin allergic reaction and/or a respiratory allergic reaction.

Preferably, the gastrointestinal allergic reaction of the infant cow milk comprises one or more of diarrhea, vomiting or constipation.

Preferably, the skin allergy comprises acute urticaria, atopic dermatitis or angioedema.

Preferably, the respiratory allergic reaction comprises one or more of allergic rhinitis, conjunctivitis or asthma.

Preferably, the dose of vitamin D3 is 0.55-5.54 mug/kg.bw.

Preferably, the alleviating comprises: decreasing secretion of Th2 type cytokines, increasing secretion of Th1 type cytokines or regulating Th1/Th2 balance in vivo is progressing to normal levels.

Preferably, the alleviating comprises: reduce histamine inflammation levels or reduce beta-lactoglobulin-specific antibody levels in serum.

Preferably, the β -lactoglobulin-specific antibodies include IgE, igG, igG1 and IgA.

Preferably, the alleviating comprises: improving intestinal villus damage.

Compared with the prior art, the application provides the application of vitamin D3 in preparing the infant formula milk powder for relieving the infant cow milk allergy caused by beta-lactoglobulin. The application constructs a Caco-2 cell model to evaluate the biosafety of vitamin D2 and vitamin D3 and the transport and absorption capacity of the vitamin D2 and the vitamin D3 across cell membranes. The relief effect of vitamin D3 on milk allergy young mice is evaluated by adopting vitamin D3 intervention in different sensitization stages and combining the clinical pathological characteristics, the beta-lactoglobulin specificity IgE, igG, igG1 and IgA level in serum, jejunum HE staining, spleen cell secretion cytokines and other aspects. The experimental results show that: the transport and absorption amount of vitamin D3 is about 2 times that of vitamin D2. The long-term vitamin D3 intervention on the sensitized young mice can obviously reduce the specific antibody level in the serum of the young mice, repair intestinal barrier and regulate the Th1/Th2 balance in the young mice. Therefore, the vitamin D3 can be used for preparing infant formula milk powder for relieving cow milk allergy.

Drawings

FIG. 1 is an evaluation of vitamin D for biosafety of Caco-2 cells in example 1;

FIG. 2 is the transport and absorption capacity of vitamin D across cell membranes in example 1;

FIG. 3 is a graph showing weight change during the priming and modeling of the young rats in example 1;

FIG. 4 is a graph showing the allergy symptoms score of the young mice in example 1;

FIG. 5 shows the temperature of the young mice in example 1 at various stages of their challenge;

FIG. 6 shows the levels of beta-lactoglobulin-specific IgE, igG, igG1 and IgA in the sera of young mice in example 1;

FIG. 7 is the level of histamine in the sera of young mice in example 1;

FIG. 8 shows the levels of cytokine secretion by the spleen cells of pups in example 1;

FIG. 9 shows the HE staining results of jejunal tissue of the pups in example 1;

FIG. 10 is a grouping of animals and treatment in example 1;

FIG. 11 shows the levels of beta-lactoglobulin-specific IgE, igG, igG and IgA in the serum of young mice of example 2;

FIG. 12 is the level of histamine in the sera of young mice in example 2;

FIG. 13 is the levels of cytokine secretion by the spleen cells of pups in example 2;

FIG. 14 is a grouping and treatment of animals according to example 2.

Detailed Description

The application provides application of vitamin D3 in preparing infant formula milk powder for relieving cow milk allergy caused by beta-lactoglobulin, which relates to the technical field of food, and can be realized by appropriately improving process parameters by a person skilled in the art by referring to the content of the application. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and they are intended to be within the scope of the present application. While the methods and applications of this application have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the application can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the application.

In the present application, the term "and/or" describes an association relationship of an association object, which means that three relationships may exist, for example, a and/or B may mean that a exists alone, a and B exist together, and B exists alone. Wherein A, B may be singular or plural.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

The application provides application of vitamin D3 in preparing infant formula milk powder for relieving infant cow milk allergy caused by beta-lactoglobulin (BLG).

The allergic reaction of the present application includes gastrointestinal allergic reaction, skin allergic reaction and/or respiratory allergic reaction.

In one embodiment, the application provides the use of vitamin D3 for the preparation of an infant formula to alleviate gastrointestinal allergic reactions caused by beta-lactoglobulin (BLG).

In one embodiment, the application provides the use of vitamin D3 for the preparation of an infant formula to alleviate skin allergy caused by beta-lactoglobulin (BLG).

In one embodiment, the application provides the use of vitamin D3 for the preparation of an infant formula to alleviate respiratory allergic reactions caused by beta-lactoglobulin (BLG).

Food allergy symptoms are mainly skin, gastrointestinal and respiratory symptoms.

For infants, skin symptoms are mostly acute urticaria, atopic dermatitis and angioedema; gastrointestinal symptoms are diarrhea, vomiting, constipation, severe patients may have hematochezia or shock, and non-IgE mediated allergic patients may have eosinophilic esophagitis, colitis; respiratory symptoms include allergic rhinitis, conjunctivitis, asthma, etc.

As a preferred embodiment of the present application, in the aforementioned application, the vitamin D is vitamin D3 in an amount of 0.55 to 5.54. Mu.g/kg.bw, preferably specifically 0.55ug/kg, 2.77ug/kg or 5.54ug/kg; or a point value between any two of the above.

The above dosages of the present application are calculated as equivalent dose ratio values calculated as a function of body surface area between humans and animals, as known to those skilled in the art.

For example, the dose administered to mice is converted to human dose conversion ratio 387.9, and it is known that 20g mice daily ingest a dose of 0.1 μg, 0.5 μg, 1 μg, equivalent dose converted to human 0.1 x 389.7/70=0.55 ug/kg;0.5 x 387.9/70=2.77 ug/kg;1 x 387.9/70=5.54 ug/kg.

The application aims to provide a cow milk allergy model of a young rat of one week, wherein an allergen is beta-lactoglobulin. Four intraperitoneal injections of BLG were performed for sensitization and two lavage shots of BLG. The intraperitoneal injection dose of BLG was 25. Mu.g, and the lavage challenge dose of BLG was 5mg. The sensitization effect is judged by detecting the level of beta-lactoglobulin specific IgE by blood serum after the three-sensitization and the four-sensitization.

The application aims to provide a construction method for vitamin D3 intervention cow milk allergy young mice, which is characterized in that the vitamin D3 is used for stomach infusion intervention, and the construction method is four times a week for 28 times. The dose of vitamin D3 was 100. Mu.g/kg.bw.

The application adopts a mode of combining intraperitoneal injection sensitization and gastric lavage excitation to construct an animal sensitization model.

According to the application, the alleviating comprises: decreasing secretion of Th2 type cytokines, increasing secretion of Th1 type cytokines or regulating Th1/Th2 balance in vivo is progressing to normal levels.

According to the application, the alleviating comprises: reduce histamine inflammation levels or reduce beta-lactoglobulin-specific antibody levels in serum. The beta-lactoglobulin-specific antibodies include IgE, igG, igG1 and IgA.

According to the application, the alleviating comprises: improving intestinal villus damage.

The application provides application of vitamin D3 in preparing infant formula milk powder for relieving infant cow milk allergy caused by beta-lactoglobulin. The application constructs a Caco-2 cell model to evaluate the biosafety of vitamin D2 and vitamin D3 and the transport and absorption capacity of the vitamin D2 and the vitamin D3 across cell membranes. The relief effect of vitamin D3 on milk allergy young mice is evaluated by adopting vitamin D3 intervention in different sensitization stages and combining the clinical pathological characteristics, the beta-lactoglobulin specificity IgE, igG, igG1 and IgA level in serum, jejunum HE staining, spleen cell secretion cytokines and other aspects. The experimental results show that: the transport and absorption amount of vitamin D3 is about 2 times that of vitamin D2. The long-term vitamin D3 intervention on the sensitized young mice can obviously reduce the specific antibody level in the serum of the young mice, repair intestinal barrier and regulate the Th1/Th2 balance in the young mice. Therefore, the vitamin D3 can be used for preparing infant formula milk powder for relieving cow milk allergy.

According to the application, by constructing a Caco-2 cell model, the biological safety of vitamin D and the transport and absorption capacity of vitamin D across cell membranes are evaluated, and the result shows that: the transport and absorption amount of vitamin D3 is about 2 times that of vitamin D2. Based on the above, the application researches the cow milk allergy intervention effect of vitamin D3, utilizes animal experiments, adopts a mode of combining intraperitoneal injection sensitization and gastric lavage excitation to construct an animal sensitization model, and evaluates the influence of the intervention of vitamin D3 on cow milk allergy young mice in terms of clinical pathological characteristics such as allergy-related symptoms, anal temperature change conditions, histamine levels in serum, beta-lactoglobulin specificity IgE, igG, igG1 and IgA levels in serum, jejunum inflammation conditions, spleen cell secretion Th1 and Th2 type cytokines levels and the like. The experimental results show that: compared with a model group, the vitamin D3 can reduce symptoms of allergic mice and inflammatory levels such as histamine to a certain extent, reduce the beta-lactoglobulin specific antibody level in serum of the allergic mice, reduce secretion of Th2 type cytokines in spleen, increase secretion of Th1 type cytokines, and regulate and control the development of Th1/Th2 balance in the bodies of the mice to normal level.

The application compares the allergy relieving effect of vitamin D3 in different intervention stages, and further discovers that compared with a model group, the effect of improving cow milk allergy in a long-term intervention group which intervenes in the early stage of sensitization is better than that in a middle-term intervention group which intervenes in the middle stage of sensitization and a short-term intervention group which intervenes in the later stage of sensitization. The long-term intervention group can relieve anaphylactic symptoms, reduce the level of histamine in serum, reduce the level of specific antibodies in serum of the allergic mice, obviously improve the damage condition of intestinal villus of the mice, and furthest develop the balance of Th1/Th2 of the mice to the normal level. Wherein the histamine content in the serum of the mice in the model group is up to 36.59ng/mL, and the histamine level in the serum of the mice is obviously reduced to 20ng/mL to 23ng/mL after the intervention of vitamin D. The spleen of the young mice in the model group secreted Th2 type cytokines IL-4 and IL-13 to be obviously increased, and the content of IL-4 and IL-13 is averagely reduced by 18 percent and 32 percent by the intervention of vitamin D, which is also the most obvious reduction of the long-term intervention group.

The application creates a method for establishing a food allergy model by utilizing a young mouse, which is a technical difficulty for establishing a relevant food allergy model due to the immature immune system of the young mouse, the imperfect weight, the light volume, the feeding of the young mouse and the like, and the application researches the food allergy model of the young mouse, breaks through the problems of difficult feeding and difficult administration of the young mouse and has great guiding significance for the establishment of the young mouse model.

To further illustrate the present application, the present application provided below in connection with the examples relates to the field of food technology, and in particular to the use of vitamin D3 for preparing infant formulas for alleviating cow's milk allergy caused by beta-lactoglobulin.

Example 1: transport and absorption capacity of vitamin D in Caco-2 cells and application of vitamin D3 in young mice relieving cow milk allergy

The Caco-2 cells of 30-35 generations can be used for constructing a single-layer intestinal epithelial cell model, and after washing and digesting the Caco-2 cell suspension with higher cell fusion degree, counting the cell suspension and adjusting the concentration of the cell suspension to 2 multiplied by 10 ⁶ And each mL. 500. Mu.L of complete medium was added dropwise to the blank upper well (AP side) of the 12-well Transwell plate, 500. Mu.L of cell suspension was added dropwise to the experimental upper well, and 1500. Mu.L of complete medium was added to the lower well (BL side) of all wells.

The proliferation toxicity of vitamin D to Caco-2 cell monolayer model was determined using CCK-8 kit, and the specific experimental procedure was as follows.

(1) Cell culture: after washing and digestion of the Caco-2 cell suspension in logarithmic growth phase in the same "2.3.1.3 passage", the cell suspension was counted and its concentration was adjusted to 2X 10 ⁵ Taking 96-hole cell culture plates per mL, adding 100 mu LHank's solution into the outer ring, arranging vitamin D sample holes, control holes and blank holes in the outer ring, and arranging 3 parallel holes; control wells, vitamin D sample wells were added with 100 μl of cell suspension per well, and blank wells were added with DMEM complete medium without cells. Culturing for about 36h until the cells are successfully attached.

(2) Sample adding: after culturing, the cells were observed with a microscope until the confluency reached 90%, the old medium in each well was discarded, 100. Mu.L of incomplete medium was added to the control well, 50mg of vitamin D3 and vitamin D2 standard were dissolved in 1mL of DMSO, 1. Mu.L of vitamin D/DMSO solution was added to 1mL of incomplete medium to a final concentration of 50. Mu.g/mL, and the incomplete medium was sequentially diluted to 40. Mu.g/mL, 30. Mu.g/mL, 20. Mu.g/mL, and 10. Mu.g/mL in a gradient manner. 100. Mu.L of each sample was added to each well of the sample well, and 100. Mu.L of incomplete medium was added to the blank wells. Culturing for 24h.

(3) Adding CCK-8 working solution: after 24 hours of culture, the liquid in the wells was discarded, 100. Mu.L of complete medium and 10. Mu.L of CK-8 working solution were added to each well, and the mixture was stirred gently and then placed in an incubator for 2 hours of culture.

(4) And (3) detection and calculation: the plates were removed and the absorbance at 450nm was measured for each well to calculate cell activity as follows:

cell activity (%) = (sample well OD value-blank well OD value)/(control well OD value-blank well OD value) ×100%

The established Caco-2 cell monolayer model can be used for carrying out a transmembrane transport experiment of vitamin D2 and vitamin D3, and the specific steps are as follows:

(1) sample adding: wells with similar resistance values were used as sample wells, 500 μl of vitamin D at appropriate concentration was added to the upper chamber, and 1500 μl of LHank's buffer was added to the lower chamber.

(2) And (3) transferring: incubate in incubator for 4h. After the end, the liquid in the lower chamber is sucked.

(3) Cracking: after the sample was aspirated, the sample was washed 2 times with PBS for 1 minute and 5 minutes, 200. Mu.LWESTERn and IP cell lysate were added to the upper chamber, reacted on ice for 5 minutes, after the completion of which the cell lysate was blown off, collected, centrifuged at 10000 Xg for 5 minutes, and the supernatant was taken to determine the vitamin D2 and vitamin D3 contents.

(4) And (3) measuring: the content of vitamin D in the lower chamber liquid and lysate was detected using a human vitamin D2 ELISA commercial kit and a human vitamin D3 ELISA commercial kit.

One week old young mice were sensitized by a combination of intraperitoneal injection and lavage for stimulation of beta-lactoglobulin. For consideration of the day-old young mice, 25 pregnant mice are selected from about 15 pregnant mice, a proper quiet environment is provided for production, 125 mice are produced, 60 female mice are selected for random grouping, 10 mice are selected for each group, the mice are sensitized by intraperitoneal injection 1 week after production, and development indexes such as weight are monitored every 2 days; the mice were grouped as follows: group A: negative control group (PBS); group B: adjuvant control group (Al); group C: positive control group (BLG); group D: long-term intervention group (VD-L); group E, middle intervention group (VD-M); group F short-term intervention group (VD-S). Of these B, C, D, E, F groups, BLG injections (25. Mu.g of BLG in PBS per 100. Mu.L) were performed weekly. Group D (VD-L group) was intervened four times a week by lavage 100. Mu.L with 1. Mu. gVD3 in from day 0 of sensitization; group E (VD-M group) was intervened four times a week by lavage 100. Mu.L containing 1. Mu. gVD3 from 10 days of sensitization; group F (VD-S) was intervened four times a week by lavage 100. Mu.L with 1. Mu. gVD3 in from day 21 of sensitization.

Figure 10 is an animal grouping and treatment.

Serum was isolated weekly from week 3 (d 14) by venous blood sampling from the eyeball and IgE levels were determined by indirect ELISA to see if there was a significant change. The method comprises the following specific steps:

(1) Coating: 100. Mu.L of 2.5. Mu.g/mL BLG was coated on each 96-well ELISA plate and incubated at 4℃for 16h.

(2) And (3) blocking: the next day, the microplate was removed, returned to room temperature, the coating was decanted, washed three times with PBST (0.1% Tween), 5min each time. mu.L of 3% fish gelatin was added for blocking and incubated at 37℃for 1.5h.

(3) An anti-reaction: plates were washed three times with PBST and 100. Mu.L of diluted baby mouse serum was added to each well of the ELISA plate at a serum dilution of 1:200 (IgE), respectively. Incubate in an oven at 37℃for 1h.

(4) Secondary reaction: plates were washed three times with PBST and 100. Mu.L of 1:5000 dilution of the corresponding goat anti-mouse secondary IgE was added per well of the ELISA plate. Incubate in an oven at 37℃for 1h.

(5) Biotin-avidin reaction: plates were washed three times with PBST and 100 μl1 was added: 60 dilution of HRP-labeled avidin. Incubate in an oven at 37℃for 1h.

(6) Color development and termination: the plates were washed three times with PBST, 100. Mu.L of TMB chromogenic solution was added to each well of the ELISA plate, incubated at 37℃for 13-15min, and the reaction was stopped by adding 50. Mu.L of 2M H2SO4 and detecting OD at 450nm in 3 min.

Two shots (30 min apart) were performed on day 28, and the lavage BLG was 5mg in 100 μlpbs. After 20min of the second excitation, the symptoms of the young mice are observed and scored according to the scoring standard of table 1, and meanwhile, the change of the anus temperature of the young mice after 20min of the first excitation, 30min of the second excitation and 50min of the second excitation is detected, analyzed and compared. Serum was collected by taking blood from the eyeball 18h after the first excitation, sacrificed and spleens were removed.

TABLE 1 allergy symptom scoring criteria for young rats

The experimental method comprises the following steps: during the animal experiment, the body weight of the young mice was weighed every two days, and a body weight detection curve was drawn. The allergic clinical symptoms of the pups were scored according to the scoring table after the second challenge, the pups were sacrificed 18h after the second challenge, and the levels of histamine, beta-lactoglobulin-specific IgE, igG, igG1 and IgA in the serum of the pups were determined by ELISA. Meanwhile, taking jejunal tissues of the young mice, fixing in 4% paraformaldehyde, carrying out paraffin tissue section and HE staining, and observing jejunal tissue structures of the young mice by using a microscope. Separating spleen of young mouse under aseptic condition, grinding with 70 μm cell sieve and grinding rod in 6cm culture dish, adding 5mL erythrocyte lysate during grinding, washing cell sieve after grinding, adding 7mL 1640 culture medium (Cellmax RPMI1640, product number: CGM 112.05) 1200rpm, centrifuging for 5min, removing supernatant again, adding 2mL complete culture medium, counting, and adjusting cell concentration to 5×10 for each group ⁶ Plating after each mL, adding 1mL of cell suspension to each well of a 48-well sterile cell culture plate, and stimulating cells with 40. Mu.L of 10mg/mL of elicitor BLG, and placing the cells at 37℃with 5% CO ₂ Culturing in a sterile incubator for 72 hours. After the culture is finished, 1000g is centrifuged for 20min, cell supernatant is taken, split charging is carried out, and the cell supernatant is stored at the temperature of minus 80 ℃ for standby. IL-4, IL-13, IFN-gamma were detected using commercial ELISA kits.

The results obtained in this example were analyzed and are shown in FIGS. 1-9:

(1) Sample well concentrations of 50. Mu.g/mL, 40. Mu.g/mL, 25. Mu.g/mL, 20. Mu.g/mL, 15. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL and 1. Mu.g/mL were set for DMSO, vitamin D ₂ Vitamin D ₃ Vitamin D ₂ : vitamin D ₃ =1: 1 toxicity test, the results of which are shown in figure 1, A, B, C, D. All concentrations of DMSO have no toxic effects on cells when the vitamin D concentration is less thanAt 25 mug/mL, the cell activity is above 95%. The post-transfer concentrations were therefore selected to be 25. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL.

(2) Use of 25 μg/mL, 10 μg/mL, 5 μg/mL vitamin D ₂ Vitamin D ₃ The results of the transmembrane transport experiments are shown in FIG. 2. From the figure, it can be seen that in the Caco-2 cell monolayer model, the vitamin D content in the cell lysate is generally higher than that in the BL side ₃ The transport and absorption capacity is obviously higher than that of vitamin D ₂ When vitamin D ₂ Vitamin D in cell lysate at a concentration of 10. Mu.g/mL ₂ The concentration is highest and is obviously higher than that of vitamin D after transportation ₂ Concentration; when vitamin D ₃ Vitamin D in cell lysate at a concentration of 5. Mu.g/mL ₃ The highest concentration is also significantly higher than vitamin D after transportation ₃ Concentration.

(3) In the sensitization period, the weight change of the young mice is shown in figure 3, and the weight of all the young mice steadily rises in 0 to 28 days, which indicates that the method can ensure the normal growth and development of the young mice to the greatest extent so as to achieve the corresponding sensitization effect.

(4) The allergic symptom score of the mice after the large-dose excitation is shown as figure 4, the clinical symptom score of most of the mice in the model group reaches 3, and the mice show multiple scratching, nose scratching, activity and hair puffiness reduction, breathing acceleration and loose stool; the vitamin D3-interfered group of young mice more show the accelerated and reduced activities of breathing, the condition of loose stool disappears, and the symptoms are relieved.

(5) The body temperature of the young mice at various stages after the large dose challenge is shown in fig. 5, and several studies indicate that the mice will respond to the challenge for a period of time, with a consequent change in rectal temperature, which is generally manifested as a decrease in anal temperature. The anus temperature of the model group of young mice is obviously different from that of the other groups, and the body temperature of the young mice is obviously reduced. The anal temperature is at normal level after vitamin D3 intervention.

(6) The levels of beta-lactoglobulin-specific IgE, igG, igG and IgA in the sera of pups are shown in FIG. 6, and we found that IgG, igE, igG and IgA for BLG specificity in the sera of pups of the BLG group were elevated compared to the blank group, and there were significant differences, while the average levels of IgE and IgG antibodies in the sera of the pups of the VD intervention group were lower than that of the BLG group. Wherein the IgG, igG1 antibody levels of the long intervention group were significantly lower than those of the medium-dry pre-group and the short intervention group. The experimental results show that the intraperitoneal injection of the BLG sensitized young mouse model is successful, and the allergic mechanism of the BLG group young mouse serving as a positive control group is mainly Th2 type antibody. And the effect of the vitamin D3 long intervention group on relieving cow milk allergy is obviously better than that of the middle-dry pre-group and the short intervention group

(7) Histamine levels in the serum of the pups as shown in fig. 7, the levels in the plasma of the pups are closely related to food allergy.

FIG. 5 is a study of the level of histamine in the sera of the pups of example 1, which shows that the level of histamine in the serum of the pups of the BLG sensitized group was as high as 36.59ng/mL, and that the level of histamine in the serum of the pups was significantly reduced to 20ng/mL to 23ng/mL after the vitamin D3 was dried. This also demonstrates that vitamin D3 intervention can reduce histamine release in the body.

(8) As shown in fig. 8, the spleen cells of the pups secreted cytokine levels, and the spleen of the BLG sensitized group of pups secreted Th2 cytokines (IL-4, IL-13) were significantly increased compared to the PBS control group, the IL-4 level of the BLG group was 3 times that of the PBS group and the Al adjuvant, and the IL-13 level of the BLG group was 20 times that of the PBS group and the Al adjuvant, which showed that the pups of the BLG group were in a Th1/Th2 imbalance state, and the body balance was shifted toward the Th2 direction. While vitamin D3 intervention reduced IL-4, IL-13 levels by an average of 18% and 32%. It is evident that vitamin D3 can inhibit secretion of Th2 type cytokines by spleen cells of BLG sensitized pups. Whereas the vitamin D3 long-intervention group inhibited Th 2-type cytokine secretion to the greatest extent. In contrast, for Th1 cytokines, i.e., IFN-gamma, the vitamin D3-interfered group (VD-L, VD-M, VD-S group) had an increase in IFN-gamma of 144.67%, 1063.91% and 586.64%, respectively, compared to the positive control group BLG group. The amplification is very obvious, which indicates that the BLG immunized young mice after vitamin D3 is dry, the body immunity is biased towards the Th1 direction, and the body Th1/Th2 is regulated to develop towards the normal level.

(9) The HE staining results of the jejunal tissue of the pups are shown in FIG. 9, and the complete structure of the jejunum with PBS and Al groups can be seen, and the wool Mao Pai is orderly and orderly arranged; the jejunum of the model group, namely the BLG sensitization group, has obvious rupture, the intestinal villus has serious rupture and disturbance, and the structure is difficult to distinguish, which indicates that the allergic reaction of the mice in the BLG group involves serious gastrointestinal reaction; the structure of the long and medium stem pre-groups of VD is relatively similar to that of PBS, and the short intervention groups of VD also have intestinal villus rupture condition, but are far lighter than that of the BLG group. This also demonstrates that the long-term intervention group of vitamin D can alleviate intestinal allergy symptoms, and has a great relieving effect on allergy symptoms.

The results show that the long-term vitamin D3 intervention has the effect of effectively relieving allergy.

Example 2

Based on the construction of the early-stage method, the young mice of one week age are sensitized by the combination of intraperitoneal injection and gastric lavage excitation of beta-lactoglobulin, and the intervention experiment is carried out by adopting a long-term intervention method. Selecting 23 pregnant mice about 15 days of gestation, giving a suitable quiet environment for production, and selecting 121 mice from the pregnant mice, randomly grouping 50 female mice, wherein 10 mice in each group, and grouping the mice as follows: group A: negative control group (PBS); group B: adjuvant control group (Al); group C: positive control group (BLG); group D: low dose intervention group (0.1 μg VD3 group); group E: high dose intervention group (0.5 μg VD3 group). Wherein B, C, D, E groups were given once a week for BLG injections (100 μl of each injection contained 25 μg BLG). Group D was intervened by gavage 100 μl containing 0.1 μ gVD four times a week from day 0 of sensitization; group E was intervened by gavage 100. Mu.L containing 0.5. Mu. gVD3 four times a week from 10 days of sensitization.

The experimental method comprises the following steps: mice were sensitized by intraperitoneal injection 1 week after production, and two gastric lavage shots (30 min intervals) were performed on day 28, with 5mg of gastric lavage BLG in 100 μLPBS. Mice were sacrificed 18h after the second challenge and the serum levels of histamine, beta-lactoglobulin-specific IgE, igG, igG1 and IgA were determined by ELISA. Separating spleen of young mouse under aseptic condition, grinding with 70 μm cell sieve and grinding rod in 6cm culture dish, adding 5mL erythrocyte lysate during grinding, washing cell sieve after grinding, adding 7mL 1640 culture medium (Cellmax RPMI1640, product number: CGM 112.05) 1200rpm, centrifuging for 5min, removing supernatant again, adding 2mL complete culture medium, counting, and adjusting cell concentration to 5×10 for each group ⁶ Post-plating at one/mL in 48 well sterile cell culture plates1mL of cell suspension was added to each well, and cells were stimulated with 40. Mu.L of 10mg/mL of the elicitor BLG, and the cells were placed at 37℃with 5% CO ₂ Culturing in a sterile incubator for 72 hours. After the culture is finished, 1000g is centrifuged for 20min, cell supernatant is taken, split charging is carried out, and the cell supernatant is stored at the temperature of minus 80 ℃ for standby. IL-4, IL-13, IL-10, IFN-gamma were detected using commercial ELISA kits.

The results obtained were analyzed and are shown in fig. 11-13:

the levels of beta-lactoglobulin-specific IgE, igG, igG and IgA in the serum of the pups are shown in FIG. 11, and IgG, igE, igG and IgA for BLG specificity in the serum of the pups of the BLG group are elevated compared with the serum of the PBS group, and have significant differences, which indicates that the intraperitoneal injection of the BLG sensitized pups model is successful, and the allergic mechanism of the pups of the BLG group serving as a positive control group is mainly Th2 type antibody. The serum IgE and IgG antibodies of the VD intervention group mice are obviously lower than those of the BLG group, which indicates that the vitamin D3 intervention group has obvious effect of relieving cow milk allergy.

Histamine levels in the serum of the pups as shown in fig. 12, the levels in the plasma of the pups are closely related to food allergy.

Fig. 12 shows that in example 2, there is no significant difference in histamine release capacity between PBS and Al groups and between vitamin D3 intervention groups, and that in BLG sensitized groups, the histamine content in the young mouse serum is as high as 37.56ng/mL, and after low and high dose vitamin D3 intervention, the histamine level in the young mouse serum is significantly reduced to 20.08ng/mL to 22.58ng/mL, which proves that both vitamin D3 interventions can reduce the release of body histamine.

As shown in FIG. 13, the levels of cytokine secretion from spleen cells of pups were significantly increased in the BLG sensitized group, the levels of IL-4 in the BLG group were 3 times that in the PBS group and the Al group, and the levels of IL-13 in the BLG group were 20 times that in the PBS group and the Al group, compared with the PBS control group, indicating that the pups in the BLG group were in a Th1/Th2 imbalance state, and that the immune expression of the organism was shifted in the Th2 direction. While vitamin D3 intervenes in the Th2 type cytokines of mice in the group to a different degree, indicating that vitamin D3 can inhibit the secretion of Th2 type cytokines by spleen cells of BLG sensitized pups. Whereas for Th1 and Treg type cytokines (IL-10), vitamin D3 intervention groups were compared to the BLG groups, which had significantly higher levels of IFN-gamma, IL-10 than the PBS and Al groups. The VD intervention obviously increases IFN-gamma and IL-10, increases the level of Th1 and Treg type cytokines, and the intake of vitamin D can induce the expression of the Treg cytokines. Shows that the BLG immunized young mice after vitamin D3 is dry, the body immunity is biased towards the Th1 direction, and the body Th1/Th2 is regulated to develop towards the normal level.

The results show that the long-term intervention of vitamin D3 with the dose of 0.1-1.0 mug has the effect of effectively relieving allergy.

The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.

Claims

1. Use of vitamin D3 for the preparation of an infant formula for alleviating infant cow's milk allergy caused by beta-lactoglobulin.

2. The use according to claim 1, wherein the allergic reaction comprises a gastrointestinal allergic reaction, a skin allergic reaction and/or a respiratory allergic reaction.

3. The use according to claim 2, wherein the infant cow's milk gastrointestinal allergic reaction comprises one or more of diarrhea, vomiting or constipation.

4. The use according to claim 2, wherein the skin allergy comprises acute urticaria, atopic dermatitis or angioedema.

5. The use according to claim 2, wherein the respiratory allergic reaction comprises one or more of allergic rhinitis, conjunctivitis or asthma.

6. The use according to claim 1, wherein the dose of vitamin D3 is 0.55-5.54 μg/kg-bw.

7. The use of claim 1, wherein the mitigating comprises: decreasing secretion of Th2 type cytokines, increasing secretion of Th1 type cytokines or regulating Th1/Th2 balance in vivo is progressing to normal levels.

8. The use of claim 1, wherein the mitigating comprises: reduce histamine inflammation levels or reduce beta-lactoglobulin-specific antibody levels in serum.

9. The use of claim 8, wherein the β -lactoglobulin-specific antibody comprises IgE, igG, igG and IgA.

10. The use of claim 1, wherein the mitigating comprises: improving intestinal villus damage.