Use of food, composition in the preparation of functional food and in the preparation of dairy products

Abstract

The present application relates to the application of food and composition in the preparation of functional food and dairy products. The food comprises a composition, which is composed of epigallocatechin gallate, ginseng powder and polydextrose; the mass ratio of the epigallocatechin gallate, the ginseng powder and the polydextrose is 1:(0.62-10):(0.6-46.16); the mass percentage of ginsenosides in the ginseng powder is 8% or more; the purity of the polydextrose solid is 90% or more; and the purity of the epigallocatechin gallate is 94% or more. In addition, the present application also provides the application of the above-mentioned composition in the preparation of functional food and dairy products. In the food provided by the present application, the components can effectively inhibit the M1 polarization of macrophages, promote the M2 polarization of macrophages, reduce the secretion level of pro-inflammatory cytokines, inhibit the inflammatory response, and realize the rapid improvement of the immune response ability of the body through synergistic effect at the cellular level.

Description

[0001] The present application is a divisional application of the parent application "Immune Regulation Composition and Application Thereof", the parent application has an application number of 202311473271.4 and a filing date of November 8, 2023. TECHNICAL FIELD

[0002] The present application relates to the field of food, in particular to a food, application of a composition in preparation of functional food and application of a composition in preparation of dairy products. BACKGROUND

[0003] With the acceleration of modern life pace, the increase of work pressure, irregular work and rest and unbalanced diet, combined with the damage of industrialized production to the environment and the generation of adverse weather such as haze, the incidence of human immune system dysfunction is on the rise, and the ability to resist pathogenic bacteria also decreases. In this case, the human immune system may have primary or secondary dysfunction, which may lead to primary immunodeficiency disease, secondary immunodeficiency disease, allergic disease, autoimmune disease, rheumatic immune disease, infectious disease and tumor. Therefore, strengthening immunity is of great significance for preventing diseases, coping with viral infections and promoting overall health.

[0004] At present, the immune regulation drugs used in daily life are mostly achieved by directly regulating the expression level of anti-inflammatory factors and pro-inflammatory factors to achieve anti-inflammatory and immune regulation effect. Such drugs need to be taken for a long time and have a relatively slow effect time. At the same time, the existing immune regulation drugs have complex composition and high production cost, which makes it difficult to achieve widespread application and popular use of the drugs.

[0005] M1 type macrophages and M2 type macrophages are two subtypes of macrophages, which play different roles and functions in the immune system. M1 type macrophages usually play a role in immune factors and cellular activation type immune response. M2 type macrophages usually play a role in tissue repair, immune inflammation and anti-inflammatory process. During the differentiation process of macrophages, the polarization ratio of M1 type macrophages and M2 type macrophages has a direct effect on the regulation of immunity. However, there is no composition in the existing immune regulation composition that can directly affect the polarization ratio of M1 type macrophages and M2 type macrophages from the level of cell tissue to achieve immune regulation effect. SUMMARY

[0006] In order to solve the above problems existing in the existing immune regulation drugs, the present application provides an immune regulation composition and application thereof.

[0007] In order to achieve the above purpose, the present application adopts the following technical scheme:

[0008] An immune-regulating composition comprising epigallocatechin-3-gallate (EGCG), ginseng powder and polydextrose (PDX);

[0009] The mass ratio of the epigallocatechin-3-gallate, the ginseng powder and the polydextrose is 1:(0.62-10):(0.6-46.16).

[0010] Preferably, the mass ratio of the epigallocatechin-3-gallate, the ginseng powder and the polydextrose is 1:(0.62-10):(13.3-46.16).

[0011] Preferably, the mass ratio of the epigallocatechin-3-gallate, the ginseng powder and the polydextrose is 1:(1.79-10):(13.3-46.16).

[0012] Preferably, the mass ratio of the epigallocatechin-3-gallate, the ginseng powder and the polydextrose is 1:1.79:46.16.

[0013] Preferably, the mass content of ginsenosides in the ginseng powder is 8% or above.

[0014] Preferably, the purity of the polydextrose solid is 90% or above.

[0015] Preferably, the purity of the epigallocatechin-3-gallate is 94% or above.

[0016] The present application also provides a food product comprising the immune-regulating composition.

[0017] The present application also provides a health product comprising the immune-regulating composition.

[0018] The present application also provides the use of the immune-regulating composition in a product for enhancing human immunity, the product being selected from one of a dairy product, a functional food, a health product and a medicine.

[0019] The present application also provides the use of the immune-regulating composition in a product for resisting inflammation, the product being selected from one of a dairy product, a functional food, a health product and a medicine.

[0020] The present application also provides the use of the immune-regulating composition in a product for inhibiting M1 macrophage polarization.

[0021] The present application also provides the use of the immune-regulating composition in a product for promoting M2 macrophage polarization.

[0022] M1 type macrophages secrete pro-inflammatory cytokines (TNF-a and IL-1β), which can promote the development of inflammation, accelerate the degradation of extracellular matrix and apoptosis, and regulate and promote Th1 type immune response. M2 type macrophages secrete anti-inflammatory cytokines, promote the proliferation and activation of T cells, regulate Th2 type immune response, and help tissue remodeling. In the immune regulation composition provided by the present application, through the synergistic effect of EGCG, ginseng powder and PDX, the M1 polarization of macrophages can be effectively inhibited at the cellular level, the M2 polarization of macrophages can be promoted, the pro-inflammatory cytokines can be effectively reduced, the inflammatory response can be inhibited, and the rapid improvement of the immune response ability of the body can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The graph is the cell viability influence curve of different test agents at different concentrations on macrophages in Example 1;

[0024] Figure 2 The column chart is the inhibition effect of EGCG, ginseng powder and PDX compound composition, EGCG and ginseng powder compound composition and PDX on M1 type macrophage polarization when the dosages of each component are shown in Table 1 in Example 2;

[0025] Figure 3 The column chart is the inhibition effect of EGCG, ginseng powder and PDX compound composition, EGCG and ginseng powder compound composition and PDX on M1 type macrophage polarization when the dosages of each component are shown in Table 2 in Example 2;

[0026] Figure 4 The column chart is the inhibition effect of EGCG, ginseng powder and PDX compound composition, EGCG and ginseng powder compound composition and PDX on M1 type macrophage polarization when the dosages of each component are shown in Table 3 in Example 2;

[0027] Figure 5 The column chart is the promotion effect of EGCG, ginseng powder and PDX compound composition, EGCG and ginseng powder compound composition and PDX on M2 type macrophage polarization when the dosages of each component are shown in Table 1 in Example 2;

[0028] Figure 6 The column chart is the promotion effect of EGCG, ginseng powder and PDX compound composition, EGCG and ginseng powder compound composition and PDX on M2 type macrophage polarization when the dosages of each component are shown in Table 2 in Example 2;

[0029] Figure 7Bar chart of the effect of the combination of EGCG, ginseng powder and PDX, the combination of EGCG and ginseng powder and PDX on the promotion of M2 macrophage polarization in the case of the dosages of each component shown in Table 3 in Example 2;

[0030] Figure 8 Graph of the effect of different PDX concentrations on the secretion level of proinflammatory factor TNF-α detected in the comparative example;

[0031] Figure 9 Graph of the effect of different yeast beta-glucan (YBG) concentrations on the secretion level of proinflammatory factor TNF-α detected in the comparative example;

[0032] Figure 10 Bar chart of the effect of different composition ratios in Table 4 and Table 5 on the secretion level of proinflammatory factor TNF-α in the comparative example.

[0033] Figure 11 Bar chart of the inhibitory effect of different composition ratios in Table 6 and Table 7 on M1 macrophage polarization in the comparative example.

[0034] Figure 12 Bar chart of the promoting effect of different composition ratios in Table 6 and Table 7 on M2 macrophage polarization in the comparative example. DETAILED DESCRIPTION

[0035] The specific embodiments of the present application will be further described in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. The components not explicitly shown in the present application can be obtained from the market.

[0036] Example 1

[0037] Cell activity detection

[0038] 1. Materials and reagents

[0039] RAW264.7 macrophages, lipopolysaccharide (LPS), epigallocatechin gallate (EGCG), polydextrose (PDX), ginseng powder, TNF-α ELISA Kit (Jiangsu Enzyme Biotechnology Co., Ltd., kit model number MB2868A), cell culture medium, PBS buffer. The purity of the polydextrose solid is 90%, the purity of the epigallocatechin gallate is 94%, and the mass percentage content of ginsenosides in the ginseng powder is 8%.

[0040] 2. Cell activity

[0041] CCK-8 assay was used to determine the cell proliferation ability after the intervention of the compound composition. Cells were inoculated and 8 cell concentration gradients (1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL) were set. 10 μL of the test agent (EGCG, ginseng powder or PDX) was added to 100 μL of culture medium. After the addition of CCK-8, the cells were incubated in an incubator until the OD value was about equal to 1, and the cell viability of each test group was calculated.

[0042] The effects of EGCG, ginseng powder and PDX on the survival rate of macrophages at different concentrations are shown in Figure 1

[0043] After treating the cells with different concentrations of the test agent, the results showed that ginseng powder and PDX had no obvious cytotoxicity, and ginseng powder had a promoting effect on cell proliferation. EGCG showed a certain cytotoxicity at a concentration of 20 μg / mL.

[0044] Example 2

[0045] The following three groups of EGCG, ginseng powder and PDX compound mass ratios and doses were used for subsequent anti-inflammatory efficacy verification experiments.

[0046] Ratio I: the mass ratio of the test composition was EGCG: ginseng powder: PDX = 1:0.62:0.6.

[0047] The dosage of the test composition is shown in Table 1.

[0048] Table 1 Dosage of test composition

[0049]

[0050] Ratio II: the mass ratio of the test composition was EGCG: ginseng powder: PDX = 1:1.79:46.16.

[0051] The dosage of the test composition is shown in Table 2.

[0052] Table 2 Dosage of test composition

[0053]

[0054] Ratio III: the mass ratio of the test composition was EGCG: ginseng powder: PDX = 1:10:13.3.

[0055] The dosage of the test composition is shown in Table 3.

[0056] Table 3 Dosage of test composition

[0057] ​

[0058] Macrophage polarization test was performed using the above three groups of test compositions respectively.

[0059] Logarithmic growth phase RAW264.7 cells were inoculated in a 24-well plate at a concentration of 2x10 5 After overnight culture at 37°C, 5% CO2, the cells were washed with PBS, and 100 ng / mL LPS and 20 ng / mL IFN-γ were added to induce M1 polarization, 20 ng / mL IL-4 was added to induce M2 polarization, and different proportions of the composition were added (the control group was added with an equal amount of PBS). After 24 h of culture, the M1 and M2 cells were collected for polarization test.

[0060] Polarization test method:

[0061] M1 macrophage polarization test method:

[0062] 1. The cells were collected in a flow tube, washed twice with pre-cooled PBS to remove the culture medium, and centrifuged at 300xg, 4°C for 5 min each time.

[0063] 2. Incubate CD16 / 32 at 4°C for 10 min.

[0064] 3. Wash three times with pre-cooled PBS, centrifuge at 300xg, 4°C for 5 min each time.

[0065] 4. Incubate F4 / 80 and CD86 antibodies at 4°C for 30 min.

[0066] 5. Wash three times with pre-cooled PBS, centrifuge at 300xg, 4°C for 5 min each time. Resuspend the cells in 300 uL PBS and detect on a cell screen.

[0067] M2 macrophage polarization test method:

[0068] 1. The cells were collected in a flow tube, washed twice with pre-cooled PBS to remove the culture medium, and centrifuged at 300xg, 4°C for 5 min each time.

[0069] 2. Incubate CD16 / 32 at 4°C for 10 min.

[0070] 3. Wash three times with pre-cooled PBS, centrifuge at 300xg, 4°C for 5 min each time.

[0071] 4. Incubate F4 / 80 antibody at 4°C for 30 min.

[0072] 5. Wash three times with pre-cooled PBS, centrifuge at 300xg, 4°C for 5 min each time.

[0073] 6. Resuspend the cells in 250 μL of fixative and incubate at 4 °C for 20 min.

[0074] 7. Wash the cells twice with the cell membrane rupture solution, centrifuging at 400×g, 4℃, for 5 min each time.

[0075] 8. Incubate CD206 antibody in 100 μL of membrane permeabilization solution at 4 °C for 30 min.

[0076] 9. Wash three times with pre-cooled PBS, centrifuging at 400×g each time at 4℃ for 5 min. Resuspend the cells in 300uL PBS, pass through a cell mesh for analysis.

[0077] The inhibitory effects on M1 macrophage polarization under different ratios of EGCG, ginseng powder, and PDX are as follows: Figures 2-4 As shown.

[0078] Depend on Figures 2-4 It can be seen that, compared with the control group, the compound of EGCG, ginseng powder and PDX can inhibit the polarization of M1 macrophages and exhibit anti-inflammatory effects. In addition, in the above three different compound combinations, the proportion of M1 macrophages in the "EGCG + ginseng powder + PDX" group was lower than that in the "EGCG + ginseng powder" group and the "PDX" group, that is, the compound has a synergistic inhibitory effect on the polarization of M1 macrophages.

[0079] The promoting effect on M2 macrophage polarization under different ratios of EGCG, ginseng powder, and PDX is as follows: Figures 5-7 As shown.

[0080] Depend on Figures 5-7 It can be seen that, compared with the control group, the EGCG, ginseng powder and PDX compound can promote the polarization of M2 macrophages and exhibit anti-inflammatory effects. In addition, in the above three different compound combinations, the proportion of M2 macrophages in the "EGCG + ginseng powder + PDX" group was higher than that in the "EGCG + ginseng powder" group and the "PDX" group, that is, the compound has a synergistic effect on promoting the polarization of M2 macrophages.

[0081] Comparative Example

[0082] Like PDX, yeast β-glucan (YBG) is a prebiotic, an active substance found in yeast cells. It consists of a linear backbone of D-glucose linked by β-1,3 bonds and branched structures linked by β-1,6 bonds. Due to its various health-beneficial biological activities, it is widely used in the food and pharmaceutical industries. Cellular, animal, and human studies have all found that yeast β-glucan exhibits regulatory effects on immune cells, receptors, and cytokines.

[0083] In this comparative example, TNF-α was used as a reference index to compare the anti-inflammatory effects of "EGCG + ginseng powder + PDX" and "EGCG + ginseng powder + YBG" complex formulations at specific complex ratios and doses. The specific method is as follows:

[0084] The ratio of PDX and YBG was determined. Based on the aforementioned cytotoxicity experiment, the intervention concentration gradient was set for the intervention effect experiment, and TNF-α was used as an index to determine the half-maximal effective concentration (concentration for 50% of maximum effect, EC 50 ) for the complex ratio determination. The effect curve was formed by nonlinear fitting, and the EC 50 of PDX and YBG were calculated. Figure 8 Figure 9 As shown in , when at their respective EC 50 , the YBG EC 50 = 8.3 μg / mL, and the PDX EC 50 = 5.1 μg / mL, the inhibitory effects on TNF-α were equivalent, so the mass ratio of YBG to PDX was set to YBG:PDX = 1.63:1 in the comparative experiment.

[0085] The experiments were performed according to the following complex mass ratios and doses, respectively:

[0086] 1) EGCG: ginseng powder: PDX = 1:0.62:0.6;

[0087] The doses of each component are shown in Table 4.

[0088] Table 4 Doses of each component

[0089]

[0090] 2) EGCG: ginseng powder: YBG = 1:0.62:0.98;

[0091] The doses of each component are shown in Table 5.

[0092] Table 5 Doses of each component

[0093]

[0094] The effects of the above two complex formulations on reducing the secretion level of the pro-inflammatory factor TNF-α are shown in Figure 10 . As shown in Figure 10It can be seen that the "EGCG + ginseng powder + PDX" group has a significantly better effect on reducing the two cytokines than the "EGCG + ginseng powder + YBG" group, and different doses show a stable effect. However, the intervention effect of the "EGCG + ginseng powder + YBG" group shows different effects at different doses, that is, the "EGCG + ginseng powder + PDX" combination is better than the "EGCG + ginseng powder + YBG" combination in terms of anti-inflammatory effect.

[0095] Meanwhile, the effects of the combination of "EGCG + ginseng powder + PDX" and "EGCG + ginseng powder + YBG" on the polarization of M1 and M2 macrophages were compared according to the following ratios and dosages.

[0096] 1) EGCG: Ginseng powder: PDX = 1:0.62:0.6;

[0097] The dosages of each component are shown in Table 6.

[0098] Table 6 Dosage of each component

[0099]

[0100] 2) EGCG: Ginseng powder: YBG = 1:0.62:0.98;

[0101] The dosages of each component are shown in Table 7.

[0102] Table 7 Dosage of each component

[0103]

[0104] The effects of the two compounds mentioned above on inhibiting M1 macrophage polarization are as follows: Figure 11 As shown, the effects of the two compounds mentioned above on promoting the polarization of M2 macrophages are as follows: Figure 12 As shown. By Figure 11 It can be seen that the inhibitory effect of the "EGCG + ginseng powder + PDX" group on M1 macrophage polarization is significantly better than that of the "EGCG + ginseng powder + YBG" group. Figure 12 It can be seen that the "EGCG + ginseng powder + PDX" group has a significantly better promoting effect on the polarization of M2 macrophages than the "EGCG + ginseng powder + YBG" group, proving that the "EGCG + ginseng powder + PDX" combination is superior to the "EGCG + ginseng powder + YBG" combination in anti-inflammatory effect.

Claims

1. A food product, characterized by, The food contains a composition consisting of epigallocatechin gallate, ginseng powder and polydextrose; the mass ratio of the epigallocatechin gallate, the ginseng powder and the polydextrose is 1: (0.62-10): (0.6-46.16); the mass percentage of ginsenosides in the ginseng powder is 8% or above; the purity of the polydextrose solid is 90% or above; and the purity of the epigallocatechin gallate is 94% or above.

2. The food product of claim 1, wherein: The mass ratio of the epigallocatechin gallate, the ginseng powder and the polydextrose is 1: (0.62-10): (13.3-46.16).

3. The food product of claim 1, wherein: The mass ratio of the epigallocatechin gallate, the ginseng powder and the polydextrose is 1: (1.79-10): (13.3-46.16).

4. The food product of claim 1, wherein: The mass ratio of the epigallocatechin gallate, the ginseng powder and the polydextrose is 1:1.79:46.

16.

5. Use of a composition for the manufacture of a functional food, characterized in that: The food contains a composition consisting of epigallocatechin gallate, ginseng powder and polydextrose; the mass ratio of the epigallocatechin gallate, the ginseng powder and the polydextrose is 1: (0.62-10): (0.6-46.16); the mass percentage of ginsenosides in the ginseng powder is 8% or above; the purity of the polydextrose solid is 90% or above; and the purity of the epigallocatechin gallate is 94% or above.

6. Use according to claim 5, wherein: The mass ratio of the epigallocatechin gallate, the ginseng powder and the polydextrose is 1: (0.62-10): (13.3-46.16).

7. The use according to claim 5, characterized in that: The mass ratio of the epigallocatechin gallate, the ginseng powder and the polydextrose is 1: (1.79-10): (13.3-46.16).

8. The use according to claim 5, characterized in that: The mass ratio of the epigallocatechin gallate, the ginseng powder and the polydextrose is 1:1.79:46.

16.

9. Use of a composition for the preparation of a dairy product, characterized in that: The food contains a composition consisting of epigallocatechin gallate, ginseng powder and polydextrose; the mass ratio of the epigallocatechin gallate, the ginseng powder and the polydextrose is 1: (0.62-10): (0.6-46.16); the mass percentage of ginsenosides in the ginseng powder is 8% or above; the purity of the polydextrose solid is 90% or above; and the purity of the epigallocatechin gallate is 94% or above.

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