CN114886119A - Application of 3' -sialyllactose in preparation of functional food for relieving food allergy - Google Patents
Application of 3' -sialyllactose in preparation of functional food for relieving food allergy Download PDFInfo
- Publication number
- CN114886119A CN114886119A CN202210657070.9A CN202210657070A CN114886119A CN 114886119 A CN114886119 A CN 114886119A CN 202210657070 A CN202210657070 A CN 202210657070A CN 114886119 A CN114886119 A CN 114886119A
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- CN
- China
- Prior art keywords
- sialyllactose
- fucosyllactose
- mice
- allergy
- food
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- A23V2400/175—Rhamnosus
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Abstract
The invention discloses application of 3' -sialyllactose in preparation of functional food for relieving food allergy, and belongs to the technical field of functional food. 3 '-sialyllactose and the remitting effect of compounding the 3' -sialyllactose with 2 '-fucosyllactose on sensitized mice are further examined by adopting different doses of 3' -sialyllactose and different compounding ratios of the 3 '-sialyllactose to the 2' -fucosyllactose and combining clinical pathological characteristics, levels of beta-Lg specific IgE and IgG1 in serum, H & E staining of jejunal tissues, toluidine blue staining of intestinal permeability, intestinal flora and the like. Experimental results show that 3 ' -sialyllactose and the compound of 3 ' -sialyllactose and 2 ' -fucosyllactose can obviously reduce the content of sensitized antibodies in serum, improve colon tissue structure and enrich the diversity of intestinal microorganisms. Therefore, the 3 ' -sialyllactose and the compound of the 3 ' -sialyllactose and the 2 ' -fucosyllactose (1:6, w/w) can be used as functional food for well relieving food allergy.
Description
Technical Field
The invention relates to application of 3' -sialyllactose in preparation of functional food for relieving food allergy, and belongs to the technical field of functional food.
Background
Food allergy is a common allergic disease, and is characterized in that hypersensitivity is caused by immune disorder driven by allergen, and the major clinical manifestations are respiratory tract obstruction (asthma, chest distress, palpitation, dyspnea and the like), skin allergy (allergic dermatitis, urticaria and the like) and gastrointestinal tract diseases (stomachache, abdominal pain, diarrhea and the like), and severe patients may suffer from anaphylactic shock and even death. In recent years, the incidence of food allergy has shown a significant trend upward and has become a global public health problem. Infancy is a high-incidence stage of food allergies, while severe allergic reactions are common in infants and early-school children. There are data showing that in our country and some western countries, over 8% of children's health is affected by food allergies, and the prevalence of food allergies within 1 year of age is significantly higher than after 1 year of age. Food allergy in infants has serious effects on physical and mental health of the infant patient and child-care pressure of parents.
The current treatment method for the disease mainly comprises antihistamine drugs, allergic reaction medium drug-resistant drugs, antigen-antibody reaction inhibition drugs and the like, can relieve allergic symptoms, is mainly histamine H1 receptor antagonists clinically, such as diphenhydramine, promethazine, chlorpheniramine and the like, is the most widely applied nonspecific abnormal antiallergic drugs at present, has certain curative effect, but can generate side effects such as headache and somnolence caused by central inhibition and xerostomia, blurred vision, dysuria, constipation and the like caused by choline resistance, and can not only cause drug failure after long-term use, but also cause various adverse reactions and even toxic and side effects for severe people. Moreover, infants have high sensitivity to the drugs, and are not suitable for use. In the absence of effective treatment means, strengthening early prevention is of great significance in ensuring the healthy growth of infants. The current major prevention strategies for food allergy include diet avoidance, pure breast feeding, feeding with hydrolyzed formula milk powder, and rational utilization of prebiotics, among others. The rational utilization of prebiotics, by virtue of their safety and efficacy, is considered a relatively prospective and potential food allergy prevention approach. Related researches show that the incidence of allergic diseases of infants in two years can be remarkably reduced by supplementing prebiotics in the early life. The fecal microbiome composition and metabolites of infants fed with a hydrolyzed whey protein formula containing specific prebiotics (mixture of short chain neutral galacto-oligosaccharides (GOS) and long chain fructo-oligosaccharides (FOS) in a ratio of 9: 1) more closely resemble breast-fed infants and are effective in preventing the occurrence of infantile eczema. Lacto-oligosaccharides are a class of prebiotics initially contacted by infants, and related researches prove that 2 ' -fucosyllactose (Fuc (alpha 1-2) Gal (beta 1-4) Glc) has the potential of relieving food allergy, and 2 ' -fucosyllactose (2 ' -FL) can reduce the expression of proinflammatory factors and relieve allergic symptoms by inhibiting pathway signals of beta-Lg allergen activated TLR 4/NF-kB. However, 2 '-fucosyllactose is an oligosaccharide contained in relatively high amounts in breast milk, and is contained in very small amounts in animal milk, and it is not realistic to obtain a large amount of 2' -FL by separation and extraction from breast milk. At present, the method for producing 2' -fucose lactose mainly comprises a chemical synthesis method, an enzyme catalysis synthesis method and a microbial fermentation method. The chemical synthesis method requires high cost of raw materials, and generally, the production process is complicated and time-consuming. The enzyme-catalyzed synthesis needs specific fucosyltransferase, and although the reaction conditions for synthesizing 2' -fucosyllactose by the enzyme method are mild and controllable, the time is short, and the product is easy to purify, the cost of glycosyl donor needed for production is high, and the enzyme-catalyzed synthesis depends on the discovery of protein engineering for improving the efficiency of the glycosyltransferase to a great extent, and needs a certain time to separate from industrial production. The microbial fermentation production is the main mode for industrially producing the 2' -fucose lactose at present, the production mode has relatively low cost and can be produced in a large scale, but the production mode mainly utilizes escherichia coli and a transgenic technology, and the national safety regulation is not passed at present.
In view of the abundant varieties of oligosaccharides in animal milk, the animal milk oligosaccharide is not only a natural milk source of the milk oligosaccharide, but also has higher safety compared with the human milk oligosaccharide obtained by the existing biosynthesis method. Based on the research on the structure, content and change rule of oligosaccharide along with lactation period of animal milk by the inventor, the inventor finds that 3' -sialyllactose (Neu5Ac (alpha 2-3) Gal (beta 1-4) Glc) is a sialylated oligosaccharide with extremely high content in animal milk, but whether the sialylated oligosaccharide has a desensitization effect or not and whether the sialylated oligosaccharide has a synergistic effect with other oligosaccharides in desensitization are not reported. In 2016, the European Union has approved 3 '-sialyllactose for use as a "new food material", and 3' -sialyllactose has also passed the "generally accepted safety" material declaration in the United states. The 3' -sialyllactose-enriched formula milk powder produced in the European Union and the United states is already marketed.
Disclosure of Invention
[ problem ] to provide a method for producing a semiconductor device
Provides a new application of 3' -sialyllactose.
[ technical solution ] A
The first object of the present invention is to provide the use of 3' -sialyllactose for the preparation of an allergic reaction health food or an antiallergic drug for alleviating food allergy caused by β -Lg allergen.
The second object of the present invention is to provide the use of 3' -sialyllactose for the preparation of health foods or antiallergic drugs for alleviating atopic dermatitis caused by β -Lg allergens.
The third purpose of the invention is to provide the application of the 3' -sialyllactose in preparing the anaphylactic reaction health-care food or the antiallergic medicine for relieving the enteritis caused by the beta-Lg allergen.
The fourth purpose of the invention is to provide the application of the 3' -sialyllactose in the preparation of health-care food or anti-allergic medicine for relieving the allergic reaction caused by beta-Lg allergen and with the content of beta-Lg specific IgE and IgG1 being increased.
As a preferred embodiment of the present invention, in the aforementioned use, the dose of 3' -sialyllactose is 200-600. mu.g/kg.bw.
A fifth object of the present invention is to provide an allergy health food for alleviating food allergy caused by β -Lg allergen, in which anti-allergic components are 3 '-sialyllactose and 2' -fucosyllactose.
In a preferred embodiment of the present invention, the mass ratio of 3 '-sialyllactose to 2' -fucosyllactose is 1:6 or 1000: 1.
As a preferred embodiment of the present invention, the total dose of 3 '-sialyllactose and 2' -fucosyllactose is 400. mu.g/kg.bw.
The sixth purpose of the invention is to provide a composite prebiotic solid beverage for relieving food allergy caused by beta-Lg allergen, which comprises 3' -sialyllactose, fructo-oligosaccharide, resistant dextrin, mannose oligomer and lactobacillus rhamnosus LR 863; wherein the mass percentage of the 3' -sialyllactose is 2%.
The seventh object of the present invention is to provide a composite prebiotic solid beverage for alleviating food allergy caused by beta-Lg allergen, comprising 3 '-sialyllactose, 2' -fucosyllactose, fructo-oligosaccharide, resistant dextrin, mannose oligomer and lactobacillus rhamnosus LR 863; wherein the total mass percentage of the 3 '-sialyllactose and the 2' -fucosyllactose is 2 percent; the mass ratio of 3 '-sialyllactose to 2' -fucosyllactose is 1: 6.
[ PROBLEMS ] the present invention
(1) The invention researches 3 '-sialyllactose, utilizes animal experiments, adopts a mouse model with early skin exposure allergen and food allergy, inspects the influence of the intervention of 3' -sialyllactose on the model mouse from the aspects of atopic dermatitis lesion degree, allergic symptoms, empty enteritis condition, immune response of organisms and the like, and finds out that: compared with a model group, the 3' -sialyllactose can improve the pathological change degree and the situation of the enteritis of the allergic mice to a certain degree, relieve the influence of allergic symptoms and allergic reaction on the growth and development of the mice, and reduce the level of specific antibodies in the serum of the allergic mice. Compared with a model group, the desensitization effect of 3 '-sialyllactose in different doses is found that both a high dose group (600 mu g/kg.bw) and a medium dose group (400 mu g/kg.bw) can obviously improve the pathological change degree and the situation of enteritis in allergic mice, reduce the influence of allergic symptoms and allergic reactions on the growth and development of the mice and reduce the level of specific antibodies in serum of the allergic mice, wherein the desensitization effect of the high dose 3' -sialyllactose group is optimal, and the levels of specific IgE and IgG1 in the serum of the mice are reduced to the highest degree compared with the level in the model group and respectively reach 70.60% and 49.41%. Therefore, the invention discovers that the 3' -sialyllactose has obvious relieving effect on the symptoms of the beta-Lg allergen combined food allergy for the first time;
(2) the research of the invention finds that the 3' -sialyllactose is oligosaccharide with the highest content in animal milk. The milk source is easier to obtain than breast milk when the animal milk is separated and prepared, and the existing research shows that the recovery rate of oligosaccharide can reach 95.6-98.3% by performing rough separation in a mode of combining microfiltration with ultrafiltration and nanofiltration, and the oligosaccharide is further enriched in a mode of performing ion exchange by QFF anion exchange resin (QFF), so that the method is suitable for industrial production. On one hand, the oligosaccharide prepared by separating animal milk has high safety, on the other hand, the structure of the separated oligosaccharide is more similar to the structure and the function of the natural oligosaccharide in milk, and a mixture of 3' -sialyllactose and other oligosaccharides can be prepared, which is favorable for better simulating the efficacy of a natural oligosaccharide compound; compared with 2' -fucose lactose, the method is more beneficial to large-scale production and industrial application;
(3) based on the relieving effect of 3 ' -sialyllactose on allergic symptoms, the invention further explores the interaction of 3 ' -sialyllactose and 2 ' -fucosyllactose on the effect of relieving food allergy, and further investigates the relieving effect of a mixture prepared by compounding 3 ' -sialyllactose and 2 ' -fucosyllactose in the ratio of oligosaccharide content in breast milk and oligosaccharide content in animal milk on the allergic symptoms of mice through clinical pathological characteristics. The experimental result shows that the 3 '-sialyllactose and 2' -fucosyllactose mixture compounded according to the oligosaccharide content proportion in breast milk can obviously improve the pathological change degree and the situation of the enteritis symptom of allergic mice, relieve the influence of the allergic symptoms and the allergic reaction on the growth and development of the mice and reduce the specific antibody level in the serum of the allergic mice through the synergistic action of the 3 '-sialyllactose and the 2' -fucosyllactose: in comparison of the single 3 ' -sialyllactose, the single 2 ' -fucosyllactose, and the different combination ratios (3 ' -sialyllactose: 2 ' -fucosyllactose ═ 1: 6; 3 ' -sialyllactose: 2 ' -fucosyllactose ═ 1000:1), the most potent desensitizing effect was found for the 1:6 combination of 3 ' -sialyllactose and 2 ' -fucosyllactose, and the reduction of specific IgE and IgG1 in mouse serum from the model group was 55.71% and 51.92%, respectively, better than the reduction of the single 3 ' -sialyllactose IgE and IgG1 from the model group (45.75% and 34.30%) and the reduction of the 2 ' -fucosyllactose IgE and IgG1 from the model group (36.73% and 30.50%), and better than the reduction of the combination of 3 ' -sialyllactose: the 2' -fucosyllactose group (animal milk oligosaccharide content ratio group) had a reduced level of IgE and IgG1 (54.34% and 36.10%) compared to the model group. Therefore, the 3 '-sialyllactose and the 2' -fucosyllactose compounded according to the content proportion of the oligosaccharides in the breast milk have a synergistic effect and can be used as functional food for well relieving food allergy.
Drawings
FIG. 1 is a graph showing the body weight change of a mouse during molding in example 1;
FIG. 2 is the lesion level of atopic dermatitis of ear in the mouse of example 1;
FIG. 3 is the allergic symptom score of the mouse in example 1;
FIG. 4 is a graph of the levels of β -Lg-specific IgE and IgG1 in the sera of mice in example 1;
FIG. 5 is the result of H & E staining of jejunal tissue of the mouse in example 1;
FIG. 6 shows toluidine blue staining results of jejunal tissues of mice in example 1;
FIG. 7 is the body weight change of the mouse during the molding in example 2;
FIG. 8 shows the degree of ear atopic dermatitis lesions of the mice in example 2;
FIG. 9 is the allergy symptom score of the mouse in example 2;
FIG. 10 is a graph of β -Lg-specific IgE and IgG1 levels in the sera of mice in example 2;
FIG. 11 is the result of H & E staining of jejunal tissue of mice in example 2;
FIG. 12 shows toluidine blue staining results of jejunal tissues of mice in example 2;
FIG. 13 is the Chaol index, Shanon index of the mouse intestinal microbiology assay in example 2.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.
The food allergy model for mouse skin sensitization used in the present invention is a method for constructing a food allergy model for mouse skin sensitization in patent publication No. CN114010766A, and is modified appropriately.
Example 1: application of 3 '-sialyllactose (3' -SL) in relieving food allergy of mice
Modeling by a method of early exposure of beta-Lg allergen in combination with food allergy to the skin. The 60 mice were divided on average into 6 groups of 10 mice each, blank, MC903, model, low dose of 3 ' -sialyllactose (200 μ g/kg.bw), medium dose of 3 ' -sialyllactose (400 μ g/kg.bw) and high dose of 3 ' -sialyllactose (600 μ g/kg.bw), respectively. All mice were acclimatized for 3 days, starting on day 4 with the corresponding gavage of normal saline or the corresponding dose of 3' -sialyllactose. The molding process included a sensitization phase (first 14 days) and a challenge phase (last 4 days), for 18 days. In the sensitization stage: except for a blank group and an MC903 group, coating an MC903 ethanol solution (the concentration is 0.1mmol/L, the coating amount is 20 mu L of total double ears) on double ears of each mouse in the morning every day, and coating a PBS solution (the concentration is 10g/L, the coating amount is 10 mu L of total double ears) containing beta-Lg on the double ears after the ethanol on the double ears of the mouse is naturally air-dried; for the blank group of mice, each mouse was applied daily to both ears only with the same dose of PBS solution without β -Lg every morning; coating MC903 ethanol solution on ears of each mouse in the morning every day for MC903 group mice, and coating PBS solution without beta-Lg on the ears after the ethanol is naturally dried; after 14 days of continuous application, the priming phase was entered. And (3) an excitation stage: in the morning of the day after the end of the sensitization phase, each mouse of the other groups was first challenged by gavage with a PBS solution containing β -Lg, except for the blank group and the MC903 group; in the afternoon of the fifth day after the end of the sensitization phase, each mouse of the other groups was subjected to a second challenge by gavage with a PBS solution containing β -Lg, except for the blank group and the MC903 group; the gavage beta-Lg is dissolved in a PBS (pH 7.4) solution, the concentration is 250g/L, and the gavage amount of each mouse is 200 mu L; for the blank group and the MC903 group, both excitations were gavaged with PBS solution without beta-Lg; after the second challenge, mice were scored for clinical symptoms of allergy with specific scoring criteria as shown in the table below.
TABLE 3 mouse allergy symptom scoring criteria Table
The experimental method comprises the following steps: during the animal experiment, the body weight of the mice was weighed daily, a body weight change curve was plotted, and the degree of ear atopic dermatitis lesions of the mice was observed. After a second challenge with a high dose of allergen, mice were scored for clinical symptoms of allergy according to the allergy symptom scoring table. The day after the second challenge, the mice were sacrificed and blood was collected and the level of specific IgE/IgG1 in the mouse serum was determined by ELISA. Meanwhile, the jejunum tissue of the mouse is fixed in 4% neutral formaldehyde, tissue sections are carried out, H & E staining and toluidine blue staining are respectively used, and the jejunum tissue structure and the aggregation condition of the mast cells of the mouse are observed under a biological microscope.
The results obtained in this example were analyzed and shown in fig. 1-6:
(1) the body weight change of the mice during molding is shown in fig. 1: during the sensitization phase (0-14d), the body weight of the mice in the blank group steadily increased, and the growth rate of the mice in other groups is obviously reduced, wherein the average body weight of the mice in the MC903 group and the model group is obviously reduced, and the body weight of the mice with low, medium and high daily gavage dosage of 3' -sialyllactose is increased to a certain extent, although the growth rate is reduced compared with the blank group. In the challenge period (14-18d), the average body weight of the model group mice is still continuously reduced, and the body weight of the mice intragastrically administered with 3' -sialyllactose is not obviously reduced every day, although the growth rate is reduced compared with that of the blank group; the growth rate was significantly increased in the high dose (600 μ g/kg.bw)3 ' -sialyllactose group compared to the low dose (200 μ g/kg.bw)3 ' -sialyllactose group and the medium dose (400 μ g/kg.bw)3 ' -sialyllactose group. The 3 '-sialyllactose can reduce the influence of the anaphylactic reaction on the growth and development of the mice to a certain extent, and particularly the high-dose (600 mu g/kg. bw) 3' -sialyllactose group can reduce the influence of the anaphylactic reaction on the growth and development of the mice to the greatest extent.
(2) The pathological degree of the mouse ear atopic dermatitis is shown in figure 2: in the sensitization stage, the ear of the allergic mouse has atopic dermatitis pathological symptoms such as dry cochlea, thickened keratinization, obvious dermatoglyph and the like. Wherein the cochlea of the model group mice is severely keratinized and is accompanied by obvious scab; the symptoms of atopic dermatitis lesions were reduced in mice perfused daily with 3 '-sialyllactose compared to the model group, with mice perfused with high doses of 3' -sialyllactose having the least degree of atopic dermatitis lesions and the ears exhibiting only mild keratosis. The 3 '-sialyllactose can reduce the pathological degree of the atopic dermatitis of the mice to a certain degree, and particularly, the 3' -sialyllactose group with high dose (600 mu g/kg. bw) can reduce the pathological degree of the atopic dermatitis of the mice to the maximum degree.
(3) The allergy symptom scores of the mice are shown in figure 3: allergic mice develop overt allergic symptoms during the challenge phase, including scratching of the head, slowed movement, diarrhea, a decrease in anal temperature, and the like. The allergy symptom score of the model group mice was significantly higher than that of the blank group and the MC903 group. The 3 '-sialyllactose intragastric mice score per day was significantly lower than the model group, and the mice with intragastric high dose of 3' -sialyllactose had the lowest allergic symptoms score.
(4) The levels of β -Lg specific IgE and IgG1 in mouse sera are shown in figure 4: the serum levels of specific IgE and IgG1 were significantly higher in the allergic mice than in the blank group and MC903 group. Wherein the specific IgE level in the serum of the mice with low, medium and high dose of 3' -sialyllactose after daily intragastric administration is respectively 41.93 percent, 45.75 percent and 70.60 percent lower than that of the model group. The level of specific IgG1 in serum of mice with low, medium and high doses of 3' -sialyllactose after daily intragastric administration is also significantly lower than that of the model group, and the reduction degrees are respectively 30.44%, 34.30% and 49.41%. In the intervention group, the serum levels of specific IgE and IgG1 were lowest in mice perfused with high doses of 3' -sialyllactose.
(5) The H & E staining results of mouse jejunal tissues are shown in fig. 5: the morphological structure of the jejunum tissue of the mice in the blank group and the MC903 group is complete, and the jejunum tissue of the mice in the model group has serious inflammation conditions such as villous cell necrosis, desquamation and the like. The jejunal inflammation condition of the mice intragastrically perfused with the 3 '-sialyllactose is improved to a different degree compared with that of the model group, wherein the jejunal inflammation condition of the mice intragastrically perfused with the 3' -sialyllactose group is the lightest, the villus structure of the small intestine is complete, and only the condition that a few villus cells are exfoliated is observed.
(6) Toluidine blue staining of mouse jejunal tissue is shown in fig. 6: mast cells in jejunum of mice in the blank group and MC903 group did not aggregate significantly. The number of jejunal mast cells of the mice in the model group is obviously increased. The aggregation phenomenon of mast cells in the jejunum of mice perfused with 3 '-sialyllactose daily is improved to a different extent than that of the model group, wherein the aggregation phenomenon of mast cells in the jejunum of mice perfused with high doses of 3' -sialyllactose is less.
The result shows that the high-dose 3' -sialyllactose has the effect of effectively relieving allergy.
Example 2: application of compound mixture of 3 '-sialyllactose and 2' -fucosyllactose in relieving food allergy of mice
The method of early exposing beta-Lg allergen to food allergy in skin is used for molding. The 60 mice were divided into 7 groups on average, and each group was 10 mice, which were a blank group, an MC903 group, a model group, a 3 '-sialyllactose group (400 μ g/kg. bw. 3' -sialyllactose), a 2 '-fucosyllactose group (400 μ g/kg. bw. 2' -fucosyllactose group), a breast milk oligosaccharide content ratio group (400 μ g/kg. bw. a mixture of 3 '-sialyllactose and 2' -fucosyllactose compounded, 2 '-fucosyllactose: 3' -sialyllactose ═ 6:1, w/w), an animal lactooligosaccharide content ratio group (400 μ g/kg. bw. 3 '-sialyllactose and 2' -fucosyllactose compounded mixture, 2 '-fucosyllactose: 3' -sialyllactose ═ 1:1000, w/w), respectively. All mice are adaptively fed for 3 days, corresponding gavage physiological saline or corresponding oligosaccharides are applied to the mice from the 4 th day, except for a blank group and an MC903 group, an MC903 ethanol solution (the concentration is 0.1mmol/L, the application amount is 20 mu L in total) is applied to double ears of each mouse every morning in a sensitization stage, and after the ethanol of the double ears of the mice is naturally air-dried, a PBS solution (the concentration is 10g/L, the application amount is 10 mu L in total) containing beta-Lg is applied to the double ears; for the blank group of mice, only PBS solution without β -Lg was applied to both ears of each mouse every morning in the sensitization phase; for MC903 group mice, applying MC903 ethanol solution to ears of each mouse every morning in the sensitization stage, and after ethanol is naturally dried, applying PBS solution without beta-Lg to ears. After two weeks of continuous application, the priming phase was entered. In the morning of the day after the end of the sensitization phase, each mouse of the other groups was first challenged by gavage with a PBS solution containing β -Lg, except for the blank group and the MC903 group; in the afternoon of the fifth day after the end of the sensitization phase, each mouse of the other groups was subjected to a second challenge by gavage with a PBS solution containing β -Lg, except for the blank group and the MC903 group; the gavage β -Lg was dissolved in PBS (pH 7.4) at a concentration of 250g/L and a gavage volume of 200 μ L per mouse. For the blank and MC903 groups, both challenges were gavaged with PBS solution without β -Lg. The mice with the second challenge were scored for clinical symptoms of allergy as shown in table 1. The body weight of the mice was recorded daily and the degree of ear skin atopic dermatitis lesions was observed, the mice were sacrificed the next day after the second challenge, blood and tissues were collected, and the allergic immunological index was analyzed.
The experimental method comprises the following steps: during the animal experiment, the body weight of the mice was weighed daily, a body weight change curve was plotted, and the degree of ear atopic dermatitis lesions of the mice was observed. After a second challenge with a high dose of allergen, mice were scored for clinical symptoms of allergy according to the allergy symptom scoring table. The day after the second challenge, mice were sacrificed, blood was collected,ELISA method for specific IgE/IgG in mouse serum 1 Is analyzed by determination. At the same time, jejunum tissue of mice was fixed in 4% neutral formaldehyde and subjected to tissue sectioning using H, respectively&E staining and toluidine blue staining, and observing the jejunum tissue structure and the aggregation of mast cells of the mouse under a biological microscope. Collecting colon contents of mice, quickly freezing the colon contents by liquid nitrogen, storing the colon contents at the temperature of-80 ℃, and performing subsequent DNA extraction and detection, PCR amplification, product purification, library preparation and detection and Miseq computer high-throughput sequencing.
The results obtained in this example were analyzed and shown in fig. 7-13:
(1) the body weight change of the mice during molding is shown in fig. 7: during the sensitization phase, the body weight of the mice in the blank group steadily increased, and the growth rate of the mice in other groups was significantly reduced, wherein the average body weight of the mice in the MC903 group, the model group and the daily gavage of single 2' -fucosyllactose was decreased, and the body weight of the mice in other groups was still increased, although the growth rate was reduced compared with the blank group. In the challenge phase, the average body weights of the model group and the mice gazed with single 2 '-fucosyllactose were still continuously decreased, and the average body weights of the mice gazed with 3' -sialyllactose and the mixture of 3 '-sialyllactose and 2' -fucosyllactose per day were slightly increased, although the growth rate was significantly lower than that of the blank group; wherein, the breast milk oligosaccharide content proportion group is obviously superior to the animal milk oligosaccharide content proportion group in the aspect of weight gain.
(2) The pathological degree of the mouse ear atopic dermatitis is shown in fig. 8: in the sensitization stage, the ear of the allergic mouse has atopic dermatitis pathological symptoms such as dry cochlea, thickened keratinization, obvious dermatoglyph and the like. In which the cochlea of the model group mice was severely keratinized with significant crusting. Mice gavaged daily with 3 '-sialyllactose, 2' -fucosyllactose and a mixture of 3 '-sialyllactose and 2' -fucosyllactose had reduced symptoms of atopic dermatitis lesions. Compared with 3 ' -sialyllactose only for intragastric administration and 2 ' -fucosyllactose only for intragastric administration, mice with 3 ' -sialyllactose only for intragastric administration had less atopic dermatitis lesion symptoms. Compared with single intervention and compound intervention, the compound intervention has the best improvement effect, wherein the mouse compounded with the intragastric breast milk oligosaccharide content proportion has the lowest degree of atopic dermatitis lesion, and the ear part only shows slight keratinization.
(3) The allergy symptom scores of the mice are shown in fig. 9: allergic mice develop overt allergic symptoms during the challenge phase, including scratching of the head, slowed movement, diarrhea, a decrease in anal temperature, and the like. The allergy symptom score of the model group mice was significantly higher than that of the blank group and the MC903 group. The mice that were gavaged daily for 3 '-sialyllactose, 2' -fucosyllactose and the mixture of 3 '-sialyllactose and 2' -fucosyllactose scored significantly lower than the model group. Compared with 3 '-sialyllactose only for intragastric administration and 2' -fucosyllactose only for intragastric administration, the average value of the allergic symptom scores of the mice with 2 '-fucosyllactose only for intragastric administration is lower, but the final score is not obviously different from the allergic symptom scores of the mice with 3' -sialyllactose only for intragastric administration due to larger individual difference. Compared with single intervention of 3 '-sialyllactose and 2' -fucosyllactose and compound intervention of 3 '-sialyllactose and 2' -fucosyllactose, the compound intervention has better improvement effect, wherein the allergy symptom score of the mouse of the compound compounded by the intragastric breast milk oligosaccharide content ratio is lowest. Because the allergic symptom score is somewhat subjective, the effect of sensitization should also be assessed in conjunction with the levels of IgE and IgG1 in the blood.
(4) The levels of β -Lg specific IgE and IgG1 in mouse sera are shown in figure 10: the serum levels of specific IgE and IgG1 in the allergic mice were significantly higher than those in the blank group and the MC903 group, and the serum levels of specific IgE and IgG1 in the mice perfused daily with 3 '-sialyllactose, 2' -fucosyllactose and a mixture of 3 '-sialyllactose and 2' -fucosyllactose were significantly lower than those in the model group. The reduction degree (45.75%) of the specific IgE level in the serum of the mice perfused with single 3 '-sialyllactose is also obviously higher than that in the serum of the mice perfused with single 2' -fucosyllactose (36.73%). The serum level of specific IgG1 in mice with gavage alone of 3 '-sialyllactose was reduced to a slightly higher degree (34.30%) than in the model group (30.50%) than in the gavage alone of 2' -fucosyllactose. Comparison of 2 '-fucosyllactose, 3' -sialyllactose single intervention and complex intervention findings: compounding 3' -sialyllactose: the degree of reduction of specific IgE and IgG1 in serum of mice in the 2 '-fucosyllactose ═ 1:6 group (breast milk oligosaccharide content ratio group) was 55.71% and 51.92% respectively, better than that of 3' -sialyllactose alone IgE and IgG1 (45.75% and 34.30%) and that of 2 '-fucosyllactose IgE and IgG1 (36.73% and 30.50%) than those of the compound 3' -sialyllactose: the 2' -fucosyllactose group (animal milk oligosaccharide content ratio group) had a reduced level of IgE and IgG1 (54.34% and 36.10%) compared to the model group. The most reduced degree of specific IgE and IgG1 was found in the serum of mice formulated with the gavage breast milk oligosaccharide content ratio (3 '-sialyllactose: 2' -fucosyllactose ═ 1:6) compound.
The H & E staining results of mouse jejunal tissues are shown in fig. 11: the morphological structure of the jejunum tissue of the mice in the blank group and the MC903 group is complete, and the jejunum tissue of the mice in the model group has serious inflammation conditions such as villous cell necrosis, desquamation and the like. The conditions of the mice with 3 '-sialyllactose, 2' -fucosyllactose and the mixture of 3 '-sialyllactose and 2' -fucosyllactose which are intragastrically administered daily are improved to different degrees compared with the model group. Compared with single 3 '-sialyllactose for intragastric administration and single 2' -fucosyllactose for intragastric administration, the inflammation of jejunum tissue of mice with single 3 '-sialyllactose for intragastric administration is obviously better than that of single 2' -fucosyllactose for intragastric administration. Compared with single intervention and compound intervention, the compound intervention has better improvement effect, wherein the situation of the mouse jejunitis disease of the mixture compounded by the content of the breast milk oligosaccharide through gastric lavage is the lightest, the villus structure of the small intestine is complete, and only the situation of the shedding of a few villus cells is observed.
Toluidine blue staining of mouse jejunal tissue is shown in fig. 12: mast cells in jejunum of mice in the blank group and MC903 group did not aggregate significantly. The number of jejunal mast cells of the mice in the model group is obviously increased. The phenomena of mast cell aggregation in the jejunum of mice gavaged daily with 3 '-sialyllactose, 2' -fucosyllactose and a mixture of 3 '-sialyllactose and 2' -fucosyllactose were improved to a different extent than in the model group. Compared with single 3 '-sialyllactose for intragastric administration and single 2' -fucosyllactose for intragastric administration, the aggregation phenomenon of mast cells in jejunum tissue of mice with single 3 '-sialyllactose for intragastric administration is obviously better than that of single 2' -fucosyllactose for intragastric administration. Compared with single intervention and compound intervention, the compound intervention has better improvement effect, wherein the aggregation phenomenon of mast cells in jejunum of a mouse is less by a mixture compounded by per-day gavage according to the content proportion of breast milk oligosaccharide.
TABLE 1 relative abundance of the phylum of gut flora for the different treatment groups
The mouse intestinal microbiology analysis is shown in figure 13 and table 1: from the Chaol index and the Shanon index, the alpha-diversity of the intestinal flora of allergic mice is obviously reduced compared with that of blank mice, while the Chaol index and the Shanon index of the intestinal flora of mice perfused with 3 '-sialyllactose, 2' -fucosyllactose and a mixture of 3 '-sialyllactose and 2' -fucosyllactose are both increased compared with that of a model group, which shows that the intervention of 3 '-sialyllactose and a mixture compounded with the 2' -fucosyllactose can improve the abundance and diversity of the intestinal flora of the mice.
As can be seen from Table 1, Bacteroidota (Bacteroidota) and Firmicutes (Firmicutes) are the major microorganisms in the intestinal tract of mice. Compared with a blank group of mice, the intestinal tract Bacteroidota of the model group of allergic mice is higher in abundance, and the intestinal tract Bacteroidute of the model group of allergic mice is lower in abundance. After a single dry prediction of 2' -fucosyllactose (400 μ g/kg. bw), the abundance of bacteroidata in the mouse gut was further increased than that of the model group and that of Firmicute was further decreased than that of the model group. Under the intervention of single 3' -sialyllactose (400 mu g/kg. bw), the abundance of Bacteroidota in the intestinal tract of the mouse is slightly lower than that of the intestinal tract of the mouse in the model group, and the abundance of Firmicute is slightly increased, so that the callback of the intestinal flora of the allergic mouse to the intestinal flora of the normal mouse is realized to a certain extent. Similarly, the breast milk oligosaccharide content ratio is compounded (3 '-sialyllactose and 2' -fucosyllactose are 1:6, 400 mu g/kg. bw) for gastric perfusion intervention, the abundance of bacteroideta in the intestinal tract of the mouse is more obviously reduced than that of the allergic mouse, the abundance of Firmicute is more obviously increased, and the composition of the intestinal flora of the mouse is more similar to that of a blank group of mice.
The result is combined to show that the mixture of 3 '-sialyllactose and 2' -fucosyllactose compounded according to the content proportion of breast milk oligosaccharide has excellent effect of relieving allergy.
Example 3: 3' -sialyllactose-enriched composite prebiotics solid beverage
A composite prebiotic solid beverage (10g) comprises 0.2g of 3' -sialyllactose, 0.5g of fructo-oligosaccharide, 8.6g of resistant dextrin, 0.5g of oligomannose and 0.2g of Lactobacillus rhamnosus LR863, and has the effect of relieving food allergy.
Example 4: 3 '-sialyllactose and 2' -fucosyllactose-enriched composite prebiotics solid beverage
A composite prebiotic solid beverage (10g) comprises 3 '-sialyllactose, 2' -fucosyllactose, 0.5g fructo-oligosaccharide, 8.6g resistant dextrin, 0.5g oligomannose and 0.2g lactobacillus rhamnosus LR863, wherein the total mass of the 3 '-sialyllactose and the 2' -fucosyllactose is 0.2g, and the mass ratio of the two is 1:6, and the prebiotic solid beverage has the effect of relieving food allergy.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
- Use of 3' -sialyllactose in the preparation of an allergic reaction health food or an antiallergic drug for alleviating food allergy caused by β -Lg allergen.
- Application of 3' -sialyllactose in preparing health food or antiallergic medicine for relieving atopic dermatitis caused by beta-Lg allergen is provided.
- Application of 3' -sialyllactose in preparation of health food or antiallergic medicine for relieving anaphylaxis caused by beta-Lg allergen.
- Application of 3' -sialyllactose in preparing health food or antiallergic medicine for relieving allergic reaction caused by beta-Lg allergen and with raised beta-Lg specific IgE and IgG1 content.
- 5. The use according to any one of claims 1 to 4, characterized in that the dose of 3' -sialyllactose is 200 μ g/kg.
- 6. An allergy-free health food for alleviating food allergy caused by β -Lg allergen, wherein the anti-allergy ingredient in the allergy-free health food is 3 '-sialyllactose and 2' -fucosyllactose.
- 7. The allergy free health food according to claim 6, wherein the mass ratio of 3 '-sialyllactose to 2' -fucosyllactose is 1:6 or 1000: 1.
- 8. The allergy free health food according to claim 6, wherein the total dose of 3 '-sialyllactose and 2' -fucosyllactose is 400 μ g/kg.bw.
- 9. A composite prebiotic solid beverage for alleviating food allergy caused by beta-Lg allergen comprises 3' -sialyllactose, fructo-oligosaccharide, resistant dextrin, mannose oligomer and Lactobacillus rhamnosus LR 863; wherein the mass percentage of the 3' -sialyllactose is 2%.
- 10. A composite prebiotic solid beverage for alleviating food allergy caused by beta-Lg allergen comprises 3 '-sialyllactose, 2' -fucosyllactose, fructo-oligosaccharide, resistant dextrin, mannose oligomer and Lactobacillus rhamnosus LR 863; wherein the total mass percentage of the 3 '-sialyllactose and the 2' -fucosyllactose is 2 percent; the mass ratio of 3 '-sialyllactose to 2' -fucosyllactose is 1: 6.
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