Lactoferrin composition and application thereof for improving immunity by regulating macrophage immune activity

Abstract

The present invention discloses a lactoferrin composition and its application for improving immunity by regulating macrophage immune activity, and belongs to the technical field of functional compositions. The present invention obtains a lactoferrin composition with regulating macrophage immune activity by compounding lactoferrin, α-lactalbumin, N-acetylneuraminic acid and yeast β-glucan in a specific ratio, the raw material of the composition is easy to obtain, and four components work together to significantly enhance its phagocytic activity without changing the activity of macrophages, regulate the polarization state of macrophages in inflammatory response, inhibit macrophages from polarizing to pro-inflammatory M1 type, and promote macrophages to polarize to anti-inflammatory M2 type; At the same time, the composition can also enhance the antigen presentation ability of macrophages and the activation of immune T cells, and improve body immunity. Therefore, the composition provided by the present invention can be used to prepare health products, food or medicines, etc. that improve immunity.

Description

Technical Field

[0001] The invention relates to a composition, in particular to a lactoferrin composition and application thereof that improves immunity by regulating the immune activity of macrophages. Background Art

[0002] Since infants and young children's immune systems are not yet fully developed, their resistance and immunity are relatively weak, making them susceptible to infection by pathogenic bacteria and viruses. The most direct manifestation of physiological low immunity in infants and young children is that they are prone to illness, such as colds, fevers, coughs, asthma, vomiting, diarrhea or constipation and other acute and chronic diseases; in addition, physiological low immunity in infants and young children is also manifested in that it often takes a long time to recover after being sick, and the course of the disease is long and recurrent. In the long run, it will not only hinder the development and maturation of various organs in infants and young children's bodies, but also have varying degrees of negative impact on the intellectual development and cognitive level of infants and young children.

[0003] Macrophages are immune cells that play an important role in immune defense in the body. They are an important component and functional component of the second line of defense of the human immune system - the nonspecific immune system. They are also one of the basic guarantees for the normal functioning of the human immune system. When pathogens invade the body, macrophages help the body resist the invasion of foreign pathogens by engulfing, clearing pathogens, activating other immune cells, etc., and maintain the balance and stability of the immune system. Therefore, enhancing the immune response and phagocytic ability of macrophages is the key to enhancing the body's immunity. At present, there are many functional products on the market that claim to enhance the immunity of infants and young children, but there are generally some disadvantages. Therefore, it is necessary to develop safe, effective, and long-term edible functional products that enhance the immunity of infants and young children.

[0004] Lactoferrin (LF) is an important iron carrier protein that is widely present in the milk and other body fluids of mammals. In addition to playing an important role as an iron carrier protein in iron metabolism, lactoferrin also exhibits multiple physiological activities such as regulating inflammatory response, antibacterial and antioxidant functions. α-Lactalbumin exists in the milk of all mammals and is the main protein in human milk. α-Lactalbumin helps improve digestion, promote absorption, promote the conversion of lactose, and improve cognitive ability. Its nutritional value is higher than that of standard milk protein.

[0005] A variety of compositions capable of improving immunity are disclosed in the prior art, such as a Chinese patent document with publication number CN102551073A, which discloses a nutritional composition containing α-lactalbumin and lactoferrin and its application, the nutritional composition includes α-lactalbumin powder, lactoferrin powder, fruit powder and inulin; a Chinese patent document with publication number CN109007037A discloses a composition for enhancing infant immunity and its products and applications, the composition includes 30-60 parts of milk powder, 0.05-2 parts of lactoferrin, 5-30 parts of concentrated whey protein, 5-15 parts of prebiotics, 20-50 parts of desalted whey powder, 1-5 parts of lactalbumin, 0.1-2 parts of N-acetylneuraminic acid and 0.003-0.05 parts of folic acid compounds, by weight. In the above inventions, the effect of the composition was verified by experiments on phagocytosis of chicken red blood cells by mouse peritoneal macrophages, but the effect of the composition on other immune functions of macrophages was not involved. Therefore, it is necessary to develop a composition that can regulate multiple immune activities of macrophages. Summary of the invention

[0006] In view of the shortcomings in the prior art, the present invention provides a lactoferrin composition that has an immunity-improving effect and can regulate the immune activity of macrophages. The components of the composition are easy to obtain, and can significantly enhance the phagocytic activity of macrophages without changing the activity of macrophages, regulate the polarization state of macrophages in inflammatory responses, inhibit the polarization of macrophages to the pro-inflammatory M1 type, promote the polarization of macrophages to the anti-inflammatory M2 type, enhance the antigen presentation ability of macrophages and the activation ability of immune T cells, and improve the body's immunity.

[0007] The specific technical solutions adopted are as follows:

[0008] A lactoferrin composition for improving immunity by regulating the immune activity of macrophages comprises, by weight, 1 to 98 parts of lactoferrin, 1 to 98 parts of alpha-lactalbumin, 0.05 to 30 parts of N-acetylneuraminic acid and 0.05 to 12.5 parts of yeast beta-glucan.

[0009] The regulation of macrophage immune activity includes at least one of the following:

[0010] (1) Enhance the phagocytic activity of macrophages;

[0011] (2) Regulate the polarization state of macrophages in inflammatory responses, inhibiting the polarization of macrophages toward the pro-inflammatory M1 type and / or promoting the polarization of macrophages toward the anti-inflammatory M2 type;

[0012] (3) Enhance the antigen presentation ability of macrophages.

[0013] Optionally, the macrophages are RAW264.7 macrophages.

[0014] Preferably, the lactoferrin composition comprises, by weight, 1 to 70 parts of lactoferrin, 1 to 75 parts of α-lactalbumin, 0.1 to 30 parts of N-acetylneuraminic acid and 0.1 to 5 parts of yeast β-glucan.

[0015] Preferably, the lactoferrin composition comprises, by weight, 1 to 10 parts of lactoferrin, 65 to 75 parts of α-lactalbumin, 15 to 30 parts of N-acetylneuraminic acid and 1 to 5 parts of yeast β-glucan.

[0016] Most preferably, the lactoferrin composition comprises, by weight, 5.5 parts of lactoferrin, 69.2 parts of α-lactalbumin, 23 parts of N-acetylneuraminic acid and 2.3 parts of yeast β-glucan.

[0017] The present invention also provides the use of the lactoferrin composition in preparing a product having an immunity improving effect.

[0018] The present invention also provides a product with immunity improving effect, comprising the lactoferrin composition; the dosage form of the product includes but is not limited to tablets, capsules, granules, powders, oral liquids, beverages and the like.

[0019] Optionally, the product is a health product, food or medicine, and its ingredients also include pharmaceutically acceptable excipients, or food-acceptable matrices or additives.

[0020] Specifically, in the product, the mass percentage of the lactoferrin composition is 1% to 100%.

[0021] Preferably, in the product, the mass percentage of the lactoferrin composition is 10% to 90%.

[0022] More preferably, in the product, the mass percentage of the lactoferrin composition is 25% to 70%.

[0023] The product improves immunity by regulating the immune activity of macrophages, and regulating the immune activity of macrophages includes at least one of the following:

[0024] (1) Enhance the phagocytic activity of macrophages;

[0025] (2) Regulate the polarization state of macrophages in inflammatory responses, inhibiting the polarization of macrophages toward the pro-inflammatory M1 type and / or promoting the polarization of macrophages toward the anti-inflammatory M2 type;

[0026] (3) Enhance the antigen presentation ability of macrophages.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention obtains a composition capable of regulating the immune activity of macrophages by compounding lactoferrin, α-lactalbumin, N-acetylneuraminic acid and yeast β-glucan in a specific ratio. The raw materials of the composition are easy to obtain, and the composition can significantly enhance the phagocytic activity of macrophages without changing the activity of macrophages, regulate the polarization state of macrophages in inflammatory reactions, inhibit the polarization of macrophages to pro-inflammatory M1 type, and promote the polarization of macrophages to anti-inflammatory M2 type, thereby participating in the immune regulation process of the body. In addition, the composition can also enhance the antigen presentation ability of macrophages and the activation of immune T cells, thereby improving the immunity of the body.

[0029] (2) The composition for regulating macrophage immune activity provided by the present invention is highly safe and can be used to prepare products with immunity-improving effects and can be consumed for a long time as a common food, health product or medicine.

[0030] (3) The composite composition of lactoferrin, α-lactalbumin, N-acetylneuraminic acid and yeast β-glucan has a significant synergistic effect on improving immunity. Removing any nutrient component will lead to a weakening or loss of immune regulation ability. In addition, the formula of the present invention reduces the use of high-cost lactoferrin raw materials, thereby reducing cost investment while ensuring the improvement of immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a graph showing the effect of the product made from the lactoferrin composition on the relative cell viability and relative phagocytic activity of RAW264.7 macrophages, wherein A is the relative cell viability of RAW264.7 macrophages, and B is the relative phagocytic activity of RAW264.7 macrophages; compared with the control group 0 μg / mL, *.p<0.05, **.p<0.01.

[0032] Figure 2 Relative mRNA expression of pro-inflammatory M1 macrophage markers; compared with the normal control group Con, **.p<0.01; compared with the model group LPS, #.p<0.05, ##.p<0.01.

[0033] Figure 3 Relative mRNA expression of anti-inflammatory M2 macrophage markers; compared with the normal control group Con, **.p<0.01; compared with the model group IL-4, #.p<0.05, ##.p<0.01.

[0034] Figure 4 Relative mRNA expression of antigen presentation-related markers in RAW264.7 macrophages; compared with the normal control group Con, *.p<0.05, **.p<0.01.

[0035] Figure 5 Relative mRNA expression levels of pro-inflammatory M1 macrophage markers under the action of different concentrations of lactoferrin composition; compared with the control group Con, **.p<0.01; compared with the LPS-treated group, #.p<0.05, ##.p<0.01.

[0036] Figure 6 The relative mRNA expression levels of anti-inflammatory M2 macrophage markers under the action of different concentrations of lactoferrin composition; compared with the control group Con, **.p<0.01; compared with the IL-4 treatment group, #.p<0.05, ##.p<0.01.

[0037] Figure 7 The relative mRNA expression of pro-inflammatory M1 macrophage markers under the action of different nutrient combinations; compared with the control group Con, **.p<0.01; compared with the LPS-treated group, #.p<0.05.

[0038] Figure 8 The relative mRNA expression of anti-inflammatory M2 macrophage markers under the action of different nutritional combinations; compared with the control group Con, **.p<0.01; compared with the IL-4 treatment group, #.p<0.05. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with the embodiments and drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0040] In the following examples and comparative examples, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in the field or the product instructions can be used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels or prepared by existing technologies.

[0041] Embodiment 1 (hard capsule)

[0042] According to weight proportions, 69.2 parts of α-lactalbumin, 23 parts of N-acetylneuraminic acid, 2.3 parts of yeast β-glucan and 5.5 parts of lactoferrin are uniformly mixed in proportion, and then filled with a capsule filling machine to obtain a hard capsule product for improving immunity.

[0043] Example 2 (granules)

[0044] According to weight proportions, 2.5 parts of α-lactalbumin, 2 parts of N-acetylneuraminic acid, 0.5 parts of yeast β-glucan, 65 parts of lactoferrin and 30 parts of lactose are uniformly mixed, granulated, dried and then packaged to obtain a granule product for improving immunity.

[0045] Embodiment 3 (powder)

[0046] According to weight proportions, 1.1 parts of α-lactalbumin, 4 parts of N-acetylneuraminic acid, 0.7 parts of yeast β-glucan, 55 parts of lactoferrin, 0.2 parts of vitamin C, 5 parts of fruit powder and 34 parts of oligofructose are uniformly mixed in proportion and packaged to obtain a powder product for improving immunity.

[0047] Example 4 (microcapsules)

[0048] According to weight proportions, 6 parts of α-lactalbumin, 0.75 parts of N-acetylneuraminic acid, 0.5 parts of yeast β-glucan, 65 parts of lactoferrin, 12.75 parts of maltodextrin and 15 parts of whey protein powder are taken; the α-lactalbumin, N-acetylneuraminic acid, yeast β-glucan and lactoferrin are uniformly mixed, then embedded with a mixed solution of maltodextrin and whey protein powder, dried at low temperature and then packaged to obtain a microcapsule product for improving immunity.

[0049] Embodiment 5 (soft capsule)

[0050] According to weight parts, 20 parts of alpha-lactalbumin, 1.5 parts of N-acetylneuraminic acid, 0.5 parts of yeast beta-glucan, 8 parts of lactoferrin, 4 parts of beeswax, 3 parts of lecithin and 63 parts of vegetable oil are uniformly mixed in proportion to obtain contents, gelatin, glycerol and water are mixed in a ratio of 1:0.4:1 to prepare a gelatin solution, and pellets are pressed by an automatic soft capsule pill pressing machine, and after drying, a soft capsule product for improving immunity is obtained.

[0051] Embodiment 6 (solid beverage)

[0052] According to weight proportions, 20 parts of α-lactalbumin, 0.75 parts of N-acetylneuraminic acid, 0.25 parts of yeast β-glucan, 1 part of lactoferrin, 13 parts of xylo-oligosaccharides, 15 parts of isomaltooligosaccharides, 30 parts of glucose and 20 parts of milk powder are uniformly mixed in proportion and packaged to obtain a solid beverage (powder) product for improving immunity.

[0053] Example 7 (Tablet)

[0054] According to weight proportions, 12 parts of α-lactalbumin, 2 parts of N-acetylneuraminic acid, 0.5 parts of yeast β-glucan, 1 part of lactoferrin, 34.5 parts of oligogalactose and 50 parts of maltodextrin are uniformly mixed in proportion, granulated and dried, and then tabletted to obtain a tablet product for improving immunity.

[0055] Example 8 (beverage)

[0056] According to weight proportions, 10 parts of α-lactalbumin, 1 part of N-acetylneuraminic acid, 0.5 part of yeast β-glucan, 2 parts of lactoferrin, 12 parts of whole milk powder and 74.5 parts of purified water are mixed evenly in proportion and packaged to obtain a formula dairy product for improving immunity.

[0057] Example 9 (beverage)

[0058] According to weight proportions, 1 part of α-lactalbumin, 0.2 parts of N-acetylneuraminic acid, 2 parts of yeast β-glucan, 1 part of lactoferrin, 0.4 parts of vitamin C, 10 parts of glucose, 10 parts of milk powder and 75.4 parts of purified water are mixed in proportion and packaged to obtain a beverage product for improving immunity.

[0059] Embodiment 10 (powder)

[0060] According to weight proportions, 20 parts of α-lactalbumin, 1 part of N-acetylneuraminic acid, 1.5 parts of yeast β-glucan, 30 parts of lactoferrin, 32.5 parts of fruit powder and 15 parts of inulin are uniformly mixed in proportion and packaged to obtain a powder product for improving immunity.

[0061] Sample analysis

[0062] 1. Experimental Materials

[0063] Experimental sample: lactoferrin composition (69.2 parts of α-lactalbumin, 23 parts of N-acetylneuraminic acid, 2.3 parts of yeast β-glucan, 5.5 parts of lactoferrin) + matrix (whole milk powder), the addition amount of the lactoferrin composition is 2.17% based on the total mass of the mixture.

[0064] Experimental cells: Mouse macrophage RAW264.7 cell line (Chuanqiu, Shanghai).

[0065] 2. Macrophage Culture

[0066] Mouse macrophage RAW264.7 cell line was used for the experiment. DMEM medium containing 10% newborn calf serum, 100 IU / mL penicillin and 100 μg / mL streptomycin was used and placed at 37°C and 5% CO 2 The cells were cultured under saturated humidity and the culture medium was changed every two days. When the cells reached 80% density, the cells were subcultured for the next experiment.

[0067] 3. Real-time fluorescence quantitative polymerase chain reaction (qPCR)

[0068] Total cellular RNA was extracted using Trizol reagent (Novizan, Nanjing). RNA concentration was determined using a Nano-300 spectrophotometer (Aosheng, Hangzhou), and cDNA was synthesized using a cDNA reverse transcriptase kit (Qingke, Beijing). Quantitative real-time PCR was performed using SYBR Green real-time PCR Master Mix (Qingke, Beijing) on ​​a CFX Connectreal-time PCR system (Bio-rad, USA). β-actin transcription was used as a housekeeping gene for data normalization.

[0069] 4. Determination of relative cell viability and relative phagocytic activity of macrophages

[0070] The relative viability and relative phagocytic activity of macrophages were detected by MTT colorimetric assay and neutral red staining, respectively.

[0071] RAW264.7 macrophages were cultured at 5 × 10 3 The cells were inoculated in a 96-well plate at a density of 100 μg / mL. After culturing for 24 h, the cells were treated with 0, 5, 10, 25, 50, and 100 μg / mL of the experimental samples for 24 h, rinsed once with PBS, and 10 μL of MTT (Seville, Wuhan) and 100 μL of serum-free DMEM medium were added to each well. After incubation at 37°C for 3 h, the supernatant was discarded, the cells were rinsed once with PBS, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the generated crystals. The cells were oscillated at room temperature for 15 min, and then the optical density (OD) value was measured at 570 nm using a microplate reader to evaluate the relative cell viability of the macrophages after treatment with the experimental samples.

[0072] Similarly, RAW264.7 macrophages were cultured at 5 × 10 3 The cells were inoculated in a 96-well plate at a density of 100 μg / mL. After culturing for 24 h, the cells were treated with 0, 5, 10, 25, 50, and 100 μg / mL of the experimental samples for 24 h, rinsed once with PBS, and 10 μL of neutral red staining solution (Biyuntian, Shanghai) and 100 μL of serum-free DMEM culture medium were added to each well. After incubation at 37°C for 3 h, the supernatant was discarded, the cells were rinsed once with PBS, and 200 μL of neutral red detection lysis solution was added to each well to lyse the cells. The cells were oscillated at room temperature for 10 min, and the optical density (OD) value was measured at 540 nm using a microplate reader to evaluate the relative phagocytic activity of the macrophages after treatment with the experimental samples.

[0073] Macrophages are involved in the recognition, phagocytosis and degradation of cell debris and pathogens and are the first responders of immune response. Therefore, the effects of experimental sample treatment on the relative cell viability and relative phagocytic activity of macrophages were first determined. After 24 hours of treatment with different concentrations of experimental samples, the relative cell viability and relative phagocytic activity of RAW264.7 macrophages were Figure 1 As shown in A and B in the figure. The results showed that compared with the 0 μg / mL group, the relative cell viability of macrophages did not change significantly after treatment with 5, 10, 25, 50 and 100 μg / mL product mixture for 24 hours, that is, the product mixture had no effect on the proliferation of RAW264.7 macrophages ( Figure 1 Neutral red staining results ( Figure 1 B) in the figure shows that after 24 hours of treatment, the product mixtures of 10, 25, 50 and 100 μg / mL can significantly promote the relative phagocytic activity of macrophages, among which the relative phagocytic activity of cells treated with the product mixture of 50 μg / mL is the highest. The above experimental results show that the product mixture can enhance the phagocytic activity of macrophages, which is achieved without changing the viability of macrophages. Subsequent experiments were carried out at three concentrations of 10, 25 and 50 μg / mL.

[0074] 5. Macrophage polarization treatment

[0075] RAW264.7 macrophages were cultured in 12-well plates (1×10 5 cells / well). In the treatment-induced pro-inflammatory M1 macrophage polarization experiment, RAW264.7 macrophages were co-treated with different concentrations of product mixtures (10, 25, 50 μg / mL) and lipopolysaccharide (LPS) (1 μg / mL, Sigma-Aldrich, USA) for 24 h. In the treatment-induced anti-inflammatory M2 macrophage polarization experiment, RAW264.7 macrophages were co-treated with different concentrations of product mixtures (10, 25, 50 μg / mL) and interleukin-4 (IL-4) (10 ng / mL, Sigma-Aldrich, USA) for 24 h. The relative mRNA expression levels of pro-inflammatory M1 macrophage markers (TNF-α, IL-1β, CCL5, MCP-1) and anti-inflammatory M2 macrophage markers (Arg1, CD206, Mgl2, Chi3l3) were analyzed by qPCR.

[0076] To evaluate the immunomodulatory effect of the product mixture, LPS treatment was used to induce M1 macrophage polarization and IL-4 treatment was used to induce M2 macrophage polarization. The results were as follows: Figure 2 and Figure 3The results showed that the product mixture treatment significantly reduced the mRNA expression of pro-inflammatory M1 macrophage markers induced by LPS in a dose-dependent manner ( Figure 2 ), including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), CC chemokine ligand 5 (CCL5), and monocyte chemoattractant protein-1 (MCP-1). In addition, the product mixture dose-dependently increased the expression of IL-4-induced anti-inflammatory M2 macrophage markers in RAW264.7 cells ( Figure 3 ), such as arginase 1 (Arg1), macrophage mannose receptor (CD206) and macrophage galectin 2 (Mgl2), as well as chitinase 3-like protein 3 (Chi3l3). That is, the product mixture can regulate the polarization state of macrophages, reduce the polarization of unactivated macrophages toward M1 type, increase the polarization of unactivated macrophages toward M2 type, and regulate the immune function of macrophages.

[0077] 6. Evaluation of macrophage antigen delivery ability

[0078] In addition to participating in the innate immune response, macrophages can also present antigens to T cells and induce antigen-presenting cells to express co-stimulatory molecules, thereby initiating adaptive immune responses. Therefore, in order to evaluate the effect of the product mixture on the antigen presentation ability of macrophages, the mRNA expression of macrophage antigen presentation-related molecules and co-stimulatory molecules was detected without antigen stimulation. RAW264.7 macrophages were plated in 12-well plates (1×10 5 Cells / well) were cultured and treated with different concentrations of product mixture (10, 25, 50 μg / mL) for 24 h. The relative mRNA expression levels of antigen presentation-related molecules MHCⅡ and co-stimulatory molecules CD86 and CD80 were analyzed by qPCR. Figure 4 As shown, the experimental samples significantly increased the expression of antigen presentation-related molecules MHCⅡ and co-stimulatory molecules CD86 and CD80 of macrophages in a dose-dependent manner, indicating that the product mixture can promote the improvement of antigen presentation ability and the enhancement of T cell activation ability.

[0079] 7. Effects of different concentrations of lactoferrin combinations on macrophage immune activity

[0080] The above study shows that the experimental sample with a concentration of 50 μg / mL can effectively regulate the polarization of M1 / M2 macrophages and participate in immune regulation. Therefore, the concentration of the lactoferrin composition corresponding to "50 μg / mL" is defined as "1× concentration" to explore the effects of different concentrations of lactoferrin compositions on the immune activity of macrophages.

[0081] RAW264.7 macrophages were cultured in 12-well plates (1×105 cells / well). In LPS treatment, RAW264.7 cells were treated with different concentrations of lactoferrin compositions (1.085, 5.425, 10.85, 27.125, 54.25, 108.5 μg / mL, corresponding to 1×, 5×, 10×, 25×, 50×, 100× the total content of four nutrients of lactoferrin) and LPS (1 μg / mL, Sigma-Aldrich, USA) for 24 h. In IL-4 treatment, Raw264.7 cells were treated with different concentrations of lactoferrin compositions (1.085, 5.425, 10.85, 27.125, 54.25, and 108.5 μg / mL, corresponding to 1×, 5×, 10×, 25×, 50×, and 100× the total content of four nutrients of lactoferrin, respectively) and IL-4 (10 ng / mL, Sigma-Aldrich, USA) for 24 h.

[0082] The results are as follows Figure 5-6 As shown, the lactoferrin composition treatment significantly reduced the mRNA expression of LPS-induced pro-inflammatory M1 macrophage markers, including tumor necrosis factor-a (TNF-a), interleukin-1β (IL-Iβ), CC chemokine ligand 5 (CCL5), and monocyte chemoattractant protein-1 (MCP-1) in a dose-dependent manner within a certain range. In addition, the administration of the lactoferrin composition increased the expression of IL-4-induced anti-inflammatory M2 macrophage markers in RAW264.7 cells, such as arginase 1 (Argl), macrophage mannose receptor c-type 1 (CD206), and macrophage galectin 2 (Mgl2), as well as chitinase 3-like 3 (Chi3l3). It can be seen that the lactoferrin composition can reduce the activation of pro-inflammatory M1 macrophages and increase the proportion of anti-inflammatory M2 macrophages, thereby participating in the immune regulation process.

[0083] 8. Effects of different nutritional combinations on macrophage polarization ability

[0084] A lactoferrin composition concentration of 10× (10.85 μg / mL) was selected for the nutrient combination experiment. At the same time, RAW264.7 cells were treated with four combinations of three ingredients (combination 1: N-acetylneuraminic acid, yeast-β-glucan and lactoferrin; combination 2: α-lactalbumin, yeast-β-glucan and lactoferrin; combination 3: α-lactalbumin, N-acetylneuraminic acid and lactoferrin; combination 4: α-lactalbumin, N-acetylneuraminic acid and yeast-β-glucan; the ratio of each component in the above combinations is the same as the above experiment) and LPS (1 μg / mL) or IL-4 (10 ng / mL) for 24 h.

[0085] To evaluate whether different combinations of nutrients have different effects on immune regulation, a 10× concentration of a combination containing four nutrients was compared with a combination containing three nutrients, and LPS was used to induce M1 macrophage polarization or IL-4 was used to induce M2 macrophage polarization. Figure 7-8 As shown, the results showed that the combination containing four nutrients significantly reduced the mRNA expression of LPS-induced pro-inflammatory M1 macrophage markers, including TNF-a, IL-Iβ, CCL5 and MCP-1, and any combination containing three ingredients had no improvement effect. In addition, the combination of four nutrients increased the expression of IL-4-induced anti-inflammatory M2 macrophage markers in RAW264.7 cells, such as Argl, CD206, Mgl2 and Chi313, and any combination containing three ingredients had no improvement effect. In summary, the combination of four nutrients can lead the transformation of macrophages to the anti-inflammatory M2 type, thereby participating in immune regulation, and the removal of any nutrient will lead to the loss of immune regulation ability, that is, the combination of four nutrients has a synergistic effect in exerting immune efficacy.

[0086] In summary, the in vitro experimental results of RAW264.7 macrophages show that the product mixture can significantly enhance the phagocytic activity of macrophages without changing the viability of macrophages, and it is dose-dependent within a certain concentration range. Secondly, the product mixture can regulate the polarization state of macrophages in inflammatory responses, inhibit the polarization of macrophages to the pro-inflammatory M1 type, and promote the polarization of macrophages to the anti-inflammatory M2 type. At the same time, the product mixture can significantly enhance the antigen presentation ability of macrophages and the activation ability of T cells. The lactoferrin composition can regulate the polarization state of macrophages, reduce the activation of M1 macrophages, and increase the proportion of M2 macrophages. The lack of any component will affect its technical effect. It shows that the composition has the activity of regulating the immune function of macrophages and can be used to prepare related products with immunity improvement effects.

[0087] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or substitutions in a similar manner made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A lactoferrin composition for improving immunity by regulating macrophage immune activity, characterized in that: According to weight, it is composed of 1-10 parts of lactoferrin, 65-75 parts of α-lactalbumin, 15-30 parts of N-acetylneuraminic acid and 1-5 parts of yeast β-glucan; The regulation of macrophage immune activity includes at least one of the following: (1) Enhance the phagocytic activity of macrophages; (2) Regulate the polarization state of macrophages in inflammatory responses, inhibiting the polarization of macrophages to the pro-inflammatory M1 type and / or promoting the polarization of macrophages to the anti-inflammatory M2 type; (3) Enhance the antigen presentation ability of macrophages.

2. The lactoferrin composition according to claim 1, characterized in that According to weight, the composition comprises 5.5 parts of lactoferrin, 69.2 parts of α-lactalbumin, 23 parts of N-acetylneuraminic acid and 2.3 parts of yeast β-glucan.

3. Use of the lactoferrin composition according to claim 1 or 2 in the preparation of a product having an immunity improving effect.

4. A product with an immunity-improving effect, characterized in that: The invention comprises the lactoferrin composition according to claim 1 or 2.

5. The product according to claim 4, characterized in that The product is a food or a medicine; the ingredients also include pharmaceutically acceptable excipients, or food-acceptable bases or additives.

6. The product according to claim 5, characterized in that The food is a health product.

7. The product according to claim 4, characterized in that The dosage forms of the product include tablets, capsules, granules, powders, oral liquids or beverages.

8. The product according to claim 4, characterized in that In the product, the mass percentage of the lactoferrin composition is 1% to 100%.

9. The product according to claim 4, characterized in that In the product, the mass percentage of the lactoferrin composition is 25% to 70%.

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