Composition for promoting non-specific immunity of organism and application thereof

Abstract

The invention discloses a composition for promoting non-specific immunity of an organism and application of the composition, and belongs to the technical field of nutritional compositions. The composition is prepared from the following raw materials: 2 '-fucosyllactose and 6'-sialic acid lactose in a weight ratio of (1.15-2): (0.3-0.37). The addition amount of the 2 '-fucosyllactose can be greatly reduced only by adding a small amount of 6'-sialic acid lactose, and the same effect as that of high-content 2 '-fucosyllactose in the aspect of non-specific immunity is achieved, that is, the composition can greatly improve the non-specific immunity of an organism, and the non-specific immunity of the organism is improved. By enhancing the ability of mononuclear macrophages, regulating intestinal flora and increasing the concentration of short-chain fatty acid, the effect of enhancing the non-specific immunity of the organism is achieved. The preparation method is suitable for preparing formula milk powder or other food with the effect of improving non-specific immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of nutritional compositions, and relates to a human milk oligosaccharide composition, in particular to a composition for promoting nonspecific immunity of an organism and an application thereof. Background Art

[0002] The immune system of infants in the early stages of life is still in the development stage. Among them, the specific immune system develops slowly and needs to be "activated" through vaccines or related diseases; the nonspecific immune system (often referred to as the innate immune system), which is relatively mature at birth, provides important immune support for early infants. In addition to the skin and mucous membrane systems that first come into contact with pathogens, the nonspecific immune system also includes nonspecific immune cells such as phagocytes and killer cells, and immune molecules such as cytokines. Infants are very sensitive to pathogens in the environment after birth. Therefore, infants are a high-risk group for respiratory and gastrointestinal diseases. Activating the nonspecific immune system and enhancing the function of nonspecific immune cells are crucial to the health of infants.

[0003] Studies have shown that breast milk contains a large number of active ingredients, such as human milk oligosaccharides, which are believed to promote the maturation of the infant's immune system. Current studies have shown that supplementing with different human milk oligosaccharides has different degrees of effect on intestinal health, neurodevelopment and immune regulation. Even so, more research is still needed on the optimal amount and type of human milk oligosaccharides to explore the best combination for promoting infant immunity, especially non-specific immunity. Summary of the invention

[0004] The purpose of the present invention is to provide a composition for promoting nonspecific immunity of the body and its application, wherein the composition can promote nonspecific immunity of the body and improve the immunity of infants and young children.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A composition for promoting non-specific immunity of an organism is prepared from raw materials comprising 2'-fucosyllactose and 6'-sialyllactose in a weight ratio of 1.15-2:0.3-0.37.

[0007] As a limitation, the composition also includes skimmed milk powder.

[0008] As a limitation, the promotion of the body's nonspecific immunity is achieved by enhancing the capacity of immune cells, regulating the abundance of intestinal flora and increasing the concentration of short-chain fatty acids.

[0009] As a further limitation, the immune cells include mononuclear macrophages;

[0010] The regulation of intestinal flora abundance refers to increasing the abundance of beneficial bacteria and reducing the abundance of harmful bacteria;

[0011] The short-chain fatty acids include butyric acid, valeric acid and isovaleric acid.

[0012] Butyric acid is the most important short-chain fatty acid in colon cell metabolism. About 80% of butyric acid can be metabolized by intestinal epithelial cells, which can maintain the integrity of the intestinal barrier and enhance intestinal immunity. Valeric acid is relatively rare in nature, and its content in short-chain fatty acids is lower than that of acetic acid, propionic acid and butyric acid. It is produced by fermentation products of certain intestinal bacteria and helps to regulate and maintain intestinal flora. Valerate plays an important role in the treatment of inflammatory and autoimmune diseases. The composition of the present invention can significantly increase the content of butyric acid and valeric acid at the same time.

[0013] As a further limitation, the beneficial bacteria include Akkermansia, and the harmful bacteria include Lachnospiraceae_NK4A136_group and Ruminococcus.

[0014] The present invention also provides a formula milk powder, comprising the above-mentioned composition for promoting nonspecific immunity of an organism.

[0015] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0016] The present invention provides a composition for promoting nonspecific immunity of an organism. 2'-fucosyllactose can promote nonspecific immunity only when it is added in high content, and has no effect on improving the index when it is added in low content. However, only a small amount of 6'-sialyllactose is needed to greatly reduce the amount of 2'-fucosyllactose added, and achieve the same effect as that of high-content 2'-fucosyllactose, that is, the composition can greatly improve the nonspecific immunity of an organism, especially cellular immunity, and achieves the effect of enhancing the nonspecific immunity of an organism by enhancing the capacity of mononuclear macrophages, regulating the abundance of intestinal flora and increasing the concentration of short-chain fatty acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the carbon clearance ability of mice in Example 4;

[0018] Figure 2 This is a graph showing the results of NK cell activity in Example 4;

[0019] Figure 3 This is a PCA principal component analysis result diagram of the intestinal flora of mice treated with the HMOs composition based on unweighted_unifrac in Example 5;

[0020] Figure 4This is a PCA principal component analysis result diagram of the intestinal flora of mice treated with skim milk powder containing the HMOs composition based on unweighted_unifrac in Example 5;

[0021] Figure 5 This is a relative abundance distribution diagram of the intestinal flora of mice treated with the HMOs composition in Example 5;

[0022] Figure 6 This is a relative abundance distribution diagram of the intestinal flora of mice treated with skim milk powder containing the HMOs composition in Example 5;

[0023] Figure 7 The relative abundance differences of the intestinal flora genus level of mice treated with the HMOs composition in Example 5;

[0024] Figure 8 The relative abundance differences of the intestinal flora genus level of mice treated with skim milk powder containing HMOs composition in Example 5;

[0025] Fig. 9 This is a graph showing the effect of HMOs composition treatment on the SCFAs content in the cecum contents of mice in Example 5;

[0026] Fig.10 This is a graph showing the effect of skim milk powder treatment containing the HMOs composition on the SCFAs content in the cecum of mice in Example 5;

[0027] Fig.11 The Spearman study on the correlation between TOP50 bacterial genera and SCFAs in Example 5, wherein: Fig.11 A is the result of HMOs composition treatment, Fig.11 B is the result of treating skim milk powder with HMOs composition. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below by specific examples. It should be understood that the described examples are only used to explain the present invention, and are not intended to limit the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] Example 1

[0032] This example provides a human milk oligosaccharide composition that promotes the non-specific immunity of the body. The raw materials for making it include 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL) with a weight ratio of 1.15:0.3.

[0033] Example 2

[0034] This example provides a human milk oligosaccharide composition that promotes the non-specific immunity of the body. The raw materials for making it include 2'-FL and 6'-SL with a weight ratio of 2:0.37.

[0035] Example 3

[0036] This example provides an infant formula milk powder, which is added with 1.82 wt% of the human milk oligosaccharide composition. The raw materials for making the human milk oligosaccharide composition include 2'-FL and 6'-SL with a weight ratio of 1.5:0.35.

[0037] Example 4

[0038] This example discloses the application of the human milk oligosaccharide composition in improving the non-specific immunity of the body (especially cellular immunity), and verifies the effect through experiments. The specific steps are as follows:

[0039] S1. Experimental preparation

[0040] Experimental animals: Healthy male BALB / c mice, 4 weeks old, weighing 14 - 17 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the production license number SCXK(E)2022 - 0030. In the SPF-level animal laboratory of this center, the temperature is 20 - 26 °C, and the humidity is 40 - 70%. The use license number is SYXK(E)2023 - 0045.

[0041] Dose design and grouping: After one week of adaptive feeding and observation of the mice, they were randomly divided into 5 groups, with 8 mice in each group, which were set as the blank control group (NC), low-dose 2'-FL group (LF), high 2'-FL group (HF), low-dose 2'-FL + 6'-SL group (LFS), and high-dose 2'-FL + 6'-SL group (HFS). Among them, the mice in the LF, HF, LFS, and HFS groups were respectively gavaged with 1.15 g / L of 2'-FL (the solvent is physiological saline), 2.0 g / L of 2'-FL (the solvent is physiological saline), a human milk oligosaccharide composition composed of 1.15 g / L of 2'-FL and 0.30 g / L of 6'-SL (the solvent is physiological saline), and a human milk oligosaccharide composition composed of 2.0 g / L of 2'-FL and 0.37 g / L of 6'-SL (the solvent is physiological saline) once a day. The NC group was gavaged with the same dose of physiological saline according to the body weight of the mice. After continuously gavaging the test substances for 30 days, the test was carried out.

[0042] S2. Mononuclear macrophage immune function-mouse carbon clearance experiment

[0043] When a certain dose of ink is injected into the blood, a large number of granular foreign bodies in the ink will be quickly phagocytosed by the macrophages present in the organs. The measured phagocytic index can not only reflect the rate at which carbon particles are cleared in the mouse body, but also indicate the phagocytic ability of mononuclear macrophages to a certain extent.

[0044] Diluted India ink (1:4, V / V) was injected into the tail vein of each mouse, and the time was immediately measured. At 2 and 10 minutes after the injection, 20 μL of blood was collected from the medial angular venous plexus and added to 2 mL of 0.1% Na 2 CO 3 The absorbance value (Na 2 CO 3 The solution was used as the control group). The mice were killed by dislocation and the liver and spleen were weighed. The vertical axis was the logarithm of the blood carbon concentration, and the horizontal axis was time. A straight line was drawn. The phagocytic rate of the measured mononuclear macrophages was represented by the slope of the straight line (k), and its carbon clearance ability was represented by the phagocytic index (a). The statistical results of the phagocytic index are shown in Figure 1 The following is the formula for calculating the phagocytic rate and phagocytic index:

[0045]

[0046] Where k is the phagocytosis rate, min -1 ; OD is absorbance, dimensionless; t is time, min; a is phagocytic index, dimensionless.

[0047] Depend on Figure 1 It can be seen that compared with the blank control group, the carbon clearance phagocytosis index of mice in the LF group, HF group, LFS group and HFS group increased by 2.67%, 10.44%, 12.67% and 12.67%, respectively. The HF group, LFS group and HFS group could significantly enhance the carbon clearance ability of mice (P<0.05), that is, low concentration of 2'-FL could not produce a significant effect on the carbon clearance ability, and only a small amount of 6'-SL was needed to greatly reduce the amount of 2'-FL added, achieving a better effect than high content of 2'-FL.

[0048] S3. Determination of NK cell activity

[0049] NK cells are lymphocytes with natural killing ability and are also an important type of immune regulatory cells. They do not require prior antigen stimulation or antibody participation, and regulate the body's immune function by releasing cytokines.

[0050] 24h before the experiment, the target cell YAC-1 was passaged, washed 3 times with Hank's solution, and the target cell concentration was adjusted to 4×105 / mL with RPMI1640 culture medium (containing 10% calf serum). The spleen of the mouse was taken out after dislocation and sacrificed, and the spleen cell suspension was prepared, washed 3 times with Hank's solution (10min, 1500r / min), and then the spleen cells were resuspended with 2mL RPMI1640 (containing 10% calf serum), and the number of spleen cells was counted after trypan blue staining, and the cell concentration was adjusted to 2×107 / mL to make the effector-target ratio 50:1. Take 100 μL of target cells and effector cells respectively into a U-shaped 96-well plate, add 100 μL of 2.5% Triton and 100 μL of target cells into the target cell maximum release well, then add 100 μL of culture medium and 100 μL of target cells into the target cell natural release well, set up three parallel wells above, put the well plate into a CO2 incubator and culture for 4 hours, centrifuge in a centrifuge (5 min, 1500 r / min), aspirate 100 μL of supernatant from each well and place it in an ELISA plate, add 100 μL of LDH matrix solution at the same time, time for 10 minutes, then add HCl solution (30 μL, 1 mol / L) into each well to terminate the reaction, finally measure OD490nm and calculate NK cell activity. When the NK cell activity of the sample group is significantly higher than that of the normal group, it can be judged as positive.

[0051]

[0052] Depend on Figure 2 It can be seen that compared with the blank control group, the NK cell activities of mice in the LF group, HF group, LFS group and HFS group increased by 38.32%, 103.21%, 110.92% and 127.69%, respectively. The HF group, LFS group and HFS group can significantly enhance the NK cell activity of mice (P<0.05). That is, adding a small amount of 6'-SL can greatly reduce the amount of 2'-FL added and achieve the same effect as a high-content 2'-FL.

[0053] Example 5

[0054] This embodiment discloses the role of a human milk oligosaccharide composition and skim milk powder added with the human milk oligosaccharide composition in regulating the abundance of intestinal flora and increasing the concentration of short-chain fatty acids, thereby improving intestinal immunity, and verifies the effect through experiments, specifically comprising the following steps:

[0055] S1. Experimental Preparation

[0056] Experimental animals: Same as in Example 4;

[0057] Dosage design and grouping:

[0058] In the experiment of human milk oligosaccharide composition, the dosage design and grouping were the same as in Example 4;

[0059] In the experiment of adding skim milk powder with human milk oligosaccharide composition, the mice were observed for adaptive feeding for one week and then randomly divided into 6 groups, 8 mice in each group, which were set as blank control group (CT), skim milk powder group (SMP), skim milk powder + low dose 2'-FL group (LMF), skim milk powder + high dose 2'-FL group (HMF), skim milk powder + low dose 2'-FL + 6'-SL group (LMFS) and skim milk powder + high dose 2'-FL + 6'-SL group (HMFS);

[0060] The mice in SMP, LMF, HMF, LMFS and HMFS groups were gavaged with 0.588 g / mL skim milk powder, 0.472 g / mL skim milk powder + 1.15 g / L 2'-FL, 0.386 g / mL skim milk powder + 2.0 g / L 2'-FL, 0.444 g / mL skim milk powder + 1.15 g / L 2'-FL + 0.30 g / L 6'-SL, and 0.352 g / mL skim milk powder + 2.0 g / L 2'-FL + 0.37 g / L 6'-SL every day, respectively; the mice in CT group were gavaged with the same dose of normal saline according to their body weight, once a day, and the test was conducted after 30 days of continuous gavage.

[0061] S2. Effects of human milk oligosaccharide composition on intestinal microbiota of mice

[0062] Mouse fecal samples were collected and stored in a -80°C refrigerator for testing. According to the instructions of the bacterial DNA kit, the bacterial microbial genomic DNA in the intestinal contents was extracted, and the bacterial genome V3-V4 variable region was amplified using bacterial primer 27F and reverse primer 1492R. The PCR amplification products were sent to Beijing Biomike Biotechnology Co., Ltd. for high-throughput sequencing.

[0063] Chromatographic peak extraction and quantitative analysis were performed using Tracefinder software (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing data were analyzed on the cloud platform (https: / / international.biocloud.net / zh / dashboard) of Beijing Biomike Biotechnology Co., Ltd. The spliced ​​raw data were filtered using lima v1.7.0, cutadapt 1.9.1, and UCHIME v4.2 software. Reads were clustered at a similarity level of 97.0% using Usearch software to obtain OTUs. The Beta diversity index of the samples was evaluated using QIIME2 software. The results are shown in Figure 2. Figure 2-Figure 5 shown.

[0064] Depend on Figure 3It can be seen that NC and HF are completely separated, and the difference in community composition is quite obvious; NC partially overlaps with LF, LFS and HFS, and the composition of mouse intestinal flora is somewhat different; LF and HFS are close in distance, and the fecal microbial community composition is similar;

[0065] Depend on Figure 4 It can be seen that CT and SMP are close to each other, indicating that SMP has no significant effect on the structure of intestinal flora; CT partially overlaps with LMF, HMF, LMFS and HMFS, indicating that supplementation of skim milk powder containing HMOs will have a certain effect on the composition of the intestinal microbiota of mice;

[0066] Depend on Figure 5 It can be seen that at the phylum level, compared with the NC group, the relative abundance of Verrucomicrobia in the HF group was significantly increased, the relative abundance of Actinobacteria in the LFS group was significantly increased, and the relative abundance of Cyanobacteria in the LF, HF and HFS groups was significantly decreased;

[0067] Depend on Figure 6 It can be seen that at the phylum level, compared with the CT group, the relative abundance of Bacteroidetes in the SMP, LMF, HMF, LMFS and HMFS groups of mice increased, while the relative abundance of Desulfobacterota, Actinobacteriota and Patescibacteria decreased. Bacteroidetes can degrade carbohydrates to provide energy for the body, and can also resist the adhesion of pathogens on the surface of the intestinal mucosa and enhance the intestinal immune function of mice; Desulfobacterota usually has the ability to metabolize sulfuric acid, which will lead to the production of hydrogen sulfide. Studies have determined that Desulfobacterota is related to various diseases, including intestinal inflammation, Parkinson's disease, joint inflammation and diabetes.

[0068] At the genus level, the top 20 bacterial species in relative abundance were screened to analyze the effects of HMOs on specific intestinal microbiota. Figure 7 , Figure 8 shown.

[0069] Depend on Figure 7It can be seen that compared with the NC group, the relative abundance of Lachnospiraceae_NK4A136_group (harmful bacteria) in the LFS and HFS groups was significantly reduced, the relative abundance of unclassified_Lachnospiraceae (beneficial bacteria) in the LF group was significantly increased, the relative abundance of unclassified_Clostridia_UCG_014 (beneficial bacteria) and Bacteroides (beneficial bacteria) in the HFS group were significantly increased, the HMOs treatment significantly reduced uncultured_rumen_bacterium, the HF, LFS, and HFS groups all significantly reduced the relative abundance of Ruminococcus (harmful bacteria), and increased the relative abundance of Akkermansia (beneficial bacteria);

[0070] Depend on Figure 8 It can be seen that compared with the CT group, the relative abundance of unclassified_Lachnospiraceae (beneficial bacteria) in the SMP group was significantly increased; the relative abundance of Bacteroides (beneficial bacteria) and unclassified_Oscillospiraceae (beneficial bacteria) in each group increased, and the relative abundance of uncultured_Bacteroidales_bacterium and uncultured_rumen_bacterium decreased significantly; skim milk powder containing HMOs significantly increased Rikenellaceae_RC9_gut_group (beneficial bacteria) in the intestine.

[0071] S3. Determination of short-chain fatty acids

[0072] Take the contents of the cecum and put them in ultrapure water, vortex and oscillate to mix; adjust the pH of the suspension to acidic with dilute HCl, shake at room temperature and centrifuge at 14000r / min for 30min, take 200μL of the supernatant into a new centrifuge tube, add the internal standard 2-ethylbutyric acid (TEBA), pass through the membrane and wait for Shimadzu gas chromatograph analysis. The results are as follows: Figure 8 , Fig. 9 As shown;

[0073] Chromatographic conditions: Chromatographic column: Agilent DB-FFAP 125-3237 column (30m×0.52μm×0.50mm); hydrogen, air and nitrogen as supplementary gas flow rates were 30mL / min, 300mL / min and 20mL / min respectively; nitrogen was used as carrier gas with a flow rate of 6.0mL / min;

[0074] Column heating program: initial temperature 80°C, hold for 0.5 min; increase to 180°C at a heating rate of 8°C / min, hold for 1 min; increase to 200°C at a heating rate of 20°C / min, hold for 15 min.

[0075] Depend on Fig. 9 It can be seen that compared with the NC group, the LF group significantly increased the content of n-butyric acid, the LFS group significantly increased the content of acetic acid, propionic acid, n-butyric acid and isovaleric acid, and the HFS group significantly increased the content of acetic acid, propionic acid, n-butyric acid, isovaleric acid and n-valeric acid. The total SCFAs concentrations of each group increased significantly.

[0076] Depend on Fig.10 It can be seen that compared with the CT group, the HMF, LMFS and HMFS groups all increased the SCFAs content. Among them, HMF intervention significantly promoted the content of isobutyric acid in cecal contents; LMFS intervention significantly promoted the growth of acetic acid, isobutyric acid and butyric acid in cecal contents; the content of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid in feces increased significantly under the intervention of HMFS; LMFS and HMFS treatments significantly increased the total SCFAs content in cecal contents;

[0077] Acetic acid can be absorbed by the intestinal wall and transported to the liver, providing energy for the liver, muscles and peripheral tissues. In addition, acetic acid can also be oxidized by peripheral tissues and play a role in their metabolism. Propionic acid can reduce fatty acid levels in serum and liver, increase satiety, reduce food intake, and prevent diet-induced obesity. Butyric acid, as the most important SCFAs in colon cell metabolism, is an important energy source for intestinal epithelial cells. About 80% of butyric acid can be metabolized by intestinal epithelial cells. Butyric acid can also maintain the integrity of the intestinal barrier and enhance intestinal immunity. Valeric acid is relatively rare in nature, and its content in short-chain fatty acids is lower than acetic acid, propionic acid and butyric acid. It is produced by fermentation products of certain intestinal bacteria and helps regulate and maintain intestinal flora. Valerate plays an important role in the treatment of inflammatory and autoimmune diseases.

[0078] The correlation between SCFAs and the abundance of the top 50 intestinal flora genera was analyzed. Fig.10 shown.

[0079] Depend on Fig.11 It can be seen that intervention with HMOs or skim milk powder containing HMOs can significantly promote the proliferation of beneficial bacteria that produce SCFAs in the intestine, thereby increasing the level of SCFAs, thereby regulating the homeostasis in the intestine and enhancing intestinal immunity.

Claims

1. A composition for promoting nonspecific immunity of an organism, characterized in that: The raw materials for making it include 2'-fucosyllactose and 6'-sialyllactose in a weight ratio of 1.15-2:0.3-0.

37.

2. The composition for promoting nonspecific immunity of an organism according to claim 1, characterized in that: The composition also includes skimmed milk powder.

3. The composition for promoting nonspecific immunity of an organism according to claim 1 or 2, characterized in that: The promotion of the body's nonspecific immunity is achieved by enhancing the capacity of immune cells, regulating the abundance of intestinal flora and increasing the concentration of short-chain fatty acids.

4. The composition for promoting nonspecific immunity of an organism according to claim 3, characterized in that: The immune cells include mononuclear macrophages; The regulation of intestinal flora abundance refers to increasing the abundance of beneficial bacteria and reducing the abundance of harmful bacteria; The short-chain fatty acids include butyric acid, valeric acid and isovaleric acid.

5. The composition for promoting nonspecific immunity of an organism according to claim 4, characterized in that: The beneficial bacteria include Akkermansia , harmful bacteria include Lachnospiraceae_NK4A136_group and Ruminococcus .

6. A formula milk powder, characterized in that: A composition for promoting nonspecific immunity of an organism comprising any one of claims 1 to 5.

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