A composite nutrient supplement emulsion for improving immunity and preparation method thereof

Abstract

The present invention relates to the biological field, in particular, to a composite nutrient supplement emulsion for improving immunity and a preparation method thereof, wherein the composite nutrient supplement emulsion for improving immunity further improves walnut peptide, and finally screens out walnut peptides such as SEQ ID NO: 8 and SEQ ID NO: 15, which have higher DPPH and ABTS clearance rates; cell experiments have confirmed that walnut peptides can lower the content of IL-10 and increase the content of IL-17A, and walnut peptides can regulate inflammatory factors to achieve the purpose of improving body immunity, and mouse experiments have confirmed that spleen lymphocyte proliferation ability and mouse NK cell activity are significantly increased; and mouse experiments have confirmed that the composite nutrient supplement emulsion is better than simple walnut peptide, indicating that other components can play a synergistic effect with walnut peptide. The composite nutrient supplement emulsion of the present invention can improve immunity and has great economic value.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to a composite nutrient supplement emulsion for improving immunity and a preparation method thereof. Background Art

[0002] The immune system is a self-defense system that helps humans stay healthy and avoid various diseases. It has the ability to resist the invasion of bacteria, fungi, and viruses, remove damaged, degenerated, and aging cells, remove metabolic wastes, and maintain a stable internal environment. As we age, the body's immune system is the first and most vulnerable to damage, which has the greatest impact on the human body and is most closely related to diseases. As people's living standards improve, health products with immune-regulating effects are gaining more and more attention, and research and development of Chinese medicine immune regulators is urgent.

[0003] Studies have shown that walnut peptides have antioxidant, anti-fatigue, blood pressure lowering, and memory-enhancing effects. For example, the prior art "Research on the Immunomodulatory Effect of Walnut Peptides", Yang Shengjie et al., "Northern Pharmacy" 2019 Vol. 16 No. 7, discloses the study of the immunomodulatory effect of walnut peptides by in vitro detection of the effects of cellular inflammatory factors and in vivo zebrafish immunomodulatory models. The results showed that walnut peptides can regulate IL-10 and IL-17A cellular inflammatory factors, and have significant improvements and promotion effects on the inhibition of zebrafish macrophages and the number of phagocytic ink macrophages, with an improvement of up to 83.33% and a promotion multiple of 3.4 times. In summary, walnut peptides have good immunomodulatory effects and can be developed into drugs and foods for enhancing immunity.

[0004] For example, the prior art CN 108085356 B discloses a method for industrially producing high-purity walnut peptides using low-temperature pressed walnut meal as raw material. Using cold-pressed walnut meal as raw material, after dispersion and hydration, it is hydrolyzed by tannin, cellulase, and medium-temperature amylase, then denatured, and then hydrolyzed by alkaline protease and neutral protease step by step, enzyme inactivation, centrifugation, plate and frame filtration to obtain high-purity walnut peptides. Through the scientific optimization of pretreatment, enzymolysis, and refining process, it is possible to achieve very economical industrial preparation of high-purity walnut peptides, with a protein content of more than 90%, a peptide content of more than 85%, a relative molecular weight of less than 2000Dal peptides accounting for more than 85%, and a relative molecular weight of less than 1000Dal peptides accounting for more than 50%. The product has good quality, weak bitterness, is easy to match with other food ingredients, and has outstanding effects on brain health and intelligence and immunity enhancement. It can be widely used in various types of food and health products as a high-quality functional base material.

[0005] For example, the walnut peptide GDLASKHLE disclosed in the prior art CN 118452281 A has a DPPH free radical scavenging ability equivalent to that of Vc and a higher ABTS free radical scavenging ability. The prepared modulated milk powder containing walnut peptide can improve the proliferation ability of spleen lymphocytes and the activity of NK cells of immunocompromised mice. The modulated milk powder can effectively prevent and improve diseases, enhance disease resistance, and improve the body's immunity.

[0006] However, the walnut peptides in the above-mentioned prior art are either a mixture or a single polypeptide, and the effect of improving immunity needs to be improved. Therefore, it is urgent to explore more walnut peptides and composite nutrient supplement lotions containing walnut peptides for improving immunity. Summary of the invention

[0007] In order to solve the above problems, the present invention first provides a composite nutrient supplement emulsion for improving immunity, which comprises walnut peptide, calcium carbonate, vitamin K2, vitamin D3, colloid and sugar alcohol; the walnut peptide is shown in SEQ ID NO: 8 or SEQ ID NO: 15.

[0008] Preferably, the colloid comprises one or more of gum arabic, xanthan gum and pectin.

[0009] Preferably, the colloid comprises gum arabic, xanthan gum and pectin.

[0010] Preferably, the sugar alcohol includes one or more of maltitol liquid, xylitol, and sorbitol liquid.

[0011] Preferably, the sugar alcohol includes maltitol liquid, xylitol and sorbitol liquid.

[0012] Preferably, it comprises walnut peptide, vitamin D3, vitamin K2, calcium carbonate, Tween-80, Span-80, soy lecithin, gum arabic, xanthan gum, pectin, maltitol liquid, xylitol, sorbitol liquid and purified water.

[0013] Preferably, it contains 1% by mass of walnut peptide, 3% by mass of vitamin D3, 3.5% by mass of vitamin K2, 1% by mass of calcium carbonate, 5% by mass of Tween-80, 2% by mass of Span-80, 1.5% by mass of soy lecithin, 1% by mass of gum arabic, 1% by mass of xanthan gum, 0.5% by mass of pectin, 1% by mass of maltitol liquid, 1% by mass of xylitol, 1% by mass of sorbitol liquid, and 0.2% by mass of the rest being purified water.

[0014] The present invention also provides a method for preparing the above-mentioned composite nutrient supplement emulsion for improving immunity, comprising the following steps:

[0015] The preparation method of the composite nutrient supplement emulsion comprises the following steps:

[0016] S1: Mix vitamin D3, vitamin K2 and calcium carbonate in proportion, add soybean lecithin, Span-80, gum arabic, xanthan gum, pectin, maltitol liquid, xylitol and sorbitol liquid, and stir and heat at 30° C. and 700 rpm for 40 min to dissolve all of the soybean lecithin, gum arabic, xanthan gum, pectin, maltitol liquid, xylitol and sorbitol liquid to obtain an oil phase;

[0017] S2: Tween-80 and purified water were fully mixed in proportion at room temperature and a rotation speed of 650 rpm, and stirred to dissolve to obtain an aqueous phase;

[0018] S3: dispersing the oil phase in step S1 dropwise into the water phase in step S2, stirring while adding, until the dispersion is complete, stirring is continued for 30 minutes to obtain a crude emulsion, and pasteurizing;

[0019] S4: The crude emulsion in step S3 is homogenized at 45 MPa high pressure for 3 times, the first time for 6 minutes, the second time for 4 minutes, and the third time for 3 minutes, and then packaged to obtain the composite nutrient supplement emulsion for improving immunity.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects:

[0021] On the basis of previous research, the present invention further improved walnut peptides, and finally screened out walnut peptides such as SEQ ID NO: 8 and SEQ ID NO: 15, which have higher DPPH and ABTS clearance rates; cell experiments confirmed that walnut peptides can downregulate the content of IL-10 and increase the content of IL-17A, which shows that walnut peptides can regulate inflammatory factors to achieve the purpose of improving the body's immunity, and mouse experiments confirmed that the proliferation capacity of spleen lymphocytes and the activity of mouse NK cells are significantly increased; and mouse experiments confirmed that the effect of preparing a composite nutrient supplement emulsion is better than that of a simple walnut peptide, indicating that other components can have a synergistic effect with walnut peptides. The composite nutrient supplement emulsion of the present invention can improve immunity and has great economic value. DETAILED DESCRIPTION

[0022] In order to make the technical problems to be solved, technical solutions and advantages of the present invention more clear, they are described in detail below in conjunction with specific embodiments.

[0023] Example 1 Synthesis of walnut peptide

[0024] Referring to CN 118452281 A, single-point and multi-point mutants were designed based on GDLASKHLE (SEQ ID NO: 1). The results are shown in Table 1. The underlined amino acids are mutation sites.

[0025] Table 1 Mutants of walnut peptides

[0026] Mutants Peptide sequence SEQ ID NO: 1 GDLASKHLE SEQ ID NO: 2 <![CDATA[ S DLASKHLE]]> SEQ ID NO: 3 <![CDATA[G L LASKHLE]]> SEQ ID NO: 4 <![CDATA[GD A ASKHLE]]> SEQ ID NO: 5 <![CDATA[GDL K SKHLE]]> SEQ ID NO: 6 <![CDATA[GDLA Q KHLE]]> SEQ ID NO: 7 <![CDATA[GDLAS R PLEASE]]> SEQ ID NO: 8 <![CDATA[GDLASK S THE]]> SEQ ID NO: 9 <![CDATA[GDLASKH Q And]]> SEQ ID NO: 10 <![CDATA[GDLASKHL R ]]> SEQ ID NO: 11 <![CDATA[ K DLAS Q HERE]]> SEQ ID NO: 12 <![CDATA[G K LA Q GOOD]]> SEQ ID NO: 13 <![CDATA[GD S ASK A THE]]> SEQ ID NO: 14 <![CDATA[GDL L SKH G E]]> SEQ ID NO: 15 <![CDATA[ S DL K SKH R E]]> SEQ ID NO: 16 <![CDATA[G K THERE K K S THE]]> SEQ ID NO: 17 <h2 style=";text-align:left;direction:ltr"><![CDATA[GDL <h2 style=";text-align:left;direction:ltr"> Q <h2 style=";text-align:left;direction:ltr"> S<h2 style=";text-align:left;direction:ltr"> S <h2 style=";text-align:left;direction:ltr"> HL<h2 style=";text-align:left;direction:ltr"> R <h2 style=";text-align:left;direction:ltr"> ]]><h2 style=";text-align:left;direction:ltr">

[0027] The walnut peptides in Table 1 were synthesized by solid phase synthesis and purified by HPLC to obtain the walnut peptides in Table 1 with a purity of 95%.

[0028] Example 2 Effect detection of walnut peptide mutants

[0029] DPPH free radical scavenging ability test:

[0030] An equal amount of sterile distilled water was added to prepare the walnut peptides of SEQ ID NO: 1-SEQ ID NO: 17 to obtain 0.6 mg / mL walnut peptides. 1 mL of the walnut peptide sample solutions of different groups were added to 2 mL of DPPH (0.1 mM, methanol solution) solution, mixed and placed at room temperature in the dark for 30 min, and the absorbance was measured at 517 nm. Distilled water was used as the blank group, ascorbic acid (Vc) was used as the positive control, and all samples were measured in three groups in parallel and the average value was calculated. The scavenging ability is represented by the scavenging rate, and the calculation formula is as follows:

[0031] Clearance rate (%) = [A0-(Ai-A1)] / A0 × 100%

[0032] A0——absorbance of blank group (1 mL distilled water instead of peptide sample);

[0033] Ai——absorbance of sample solution;

[0034] A1 – absorbance of 2 mL methanol instead of DPPH.

[0035] ABTS free radical scavenging ability test:

[0036] Preparation of ABTS free radical stock solution: Take 10 mL of ABTS solution with a concentration of 7.4 mmol / L and 10 mL of K2S2O8 solution with a concentration of 2.6 mmol / L, mix them, and oxidize them at room temperature in the dark for 12 h to obtain a stock solution. The ABTS stock solution was diluted with PBS (0.2 M, pH = 7.4) before use, and its absorbance value was 0.80 ± 0.02 at 734 nm. Add an equal amount of sterile distilled water to prepare walnut peptides of SEQ ID NO: 1-SEQ ID NO: 17 to obtain 0.6 mg / mL walnut peptides. Take 2 mL of ABTS solution, add different groups of walnut peptide sample solutions, mix, let stand at room temperature for 6 min, and measure the absorbance at 734 nm. Ascorbic acid (Vc) was used as a positive control, and all samples were measured in three groups in parallel and the average value was calculated. The ABTS scavenging ability is represented by the scavenging rate, and the calculation formula is as follows:

[0037] Clearance rate (%) = [A0-(Ai-A1)] / A0 × 100%

[0038] Where, A0——blank control (1 mL distilled water instead of peptide sample);

[0039] Ai——absorbance of sample solution;

[0040] A1 - sample control (2 mL 0.2 M buffer instead of ABTS).

[0041] The results are shown in Table 2.

[0042] Table 2 Effects of walnut peptide mutants

[0043] Mutants DPPH clearance rate (%) ABTS clearance rate (%) Positive control (Vc) 99.4 97.8 SEQ ID NO: 1 98.6 95.3 SEQ ID NO: 2 74.4 85.3 SEQ ID NO: 3 63.1 75.2 SEQ ID NO: 4 52.3 62.1 SEQ ID NO: 5 41.7 51.4 SEQ ID NO: 6 82.1 83.1 SEQ ID NO: 7 55.5 61.3 SEQ ID NO: 8 99.5 98.1 SEQ ID NO: 9 90.1 89.1 SEQ ID NO: 10 86.2 79.7 SEQ ID NO: 11 64.6 65.9 SEQ ID NO: 12 77.4 79.6 SEQ ID NO: 13 86.5 79.8 SEQ ID NO: 14 78.2 69.7 SEQ ID NO: 15 99.9 99.9 SEQ ID NO: 16 68.9 70.9 SEQ ID NO: 17 68.6 81.3

[0044] From the results in Table 2, it can be seen that the free radical scavenging rates of mutants SEQ ID NO: 8 and SEQ ID NO: 15 were significantly improved compared with SEQ ID NO: 1 (P<0.05), and SEQ ID NO: 8 and SEQ ID NO: 15 were selected for subsequent experiments.

[0045] Example 3 Inflammatory Factor Cell Experiment

[0046] Human peripheral blood mononuclear cells (HPBMC, purchased from LONZA, Switzerland) were used to evaluate the effect of walnut peptides on the IL-10 and IL-17A inflammatory factors of cells. The measurement results are shown in Table 3.

[0047] Method: 10 μg / mL anti-CD3 was coated on a 6-well plate (200 μL / well) and incubated at 4°C for 18-24 hours. HPBMC were activated and diluted to 5×10 5 cells / mL, and the final volume was 36mL, which contained anti-CD28 (2μg / mL), rhIL-2 (10ng / mL), and rhIL-4 (50ng / mL). The 6-well plate coated with anti-CD3 was rinsed with culture medium, and the HPBMC dilution was transferred to this 6-well plate and cultured in a carbon dioxide incubator. After 2 days, the HPBMC dilution in this 6-well plate was collected and centrifuged, the supernatant was removed, and the culture medium containing rhIL-2 (10ng / mL) and rhIL-4 (50ng / mL) was added to disperse and mix, so that the concentration of cells was 5×10 5cells / mL, and then transferred to a cell culture bottle and cultured in a carbon dioxide incubator. After 2 days, HPBMCs were collected, centrifuged to remove the supernatant, washed with culture medium, and centrifuged again to remove the supernatant. Culture medium containing 5ng / mL PMA was added to disperse and mix to make the cell concentration 5×10 5 cells / mL, the final volume was 36mL, and placed in a carbon dioxide incubator. After 4 hours, centrifugation was performed, and the supernatant was analyzed by CBA. The operation method was carried out according to the BD CBA Human Th1 / Th2 / Th17 Cytokine Kit (purchased from BD Company, USA) Instruction Manual to analyze the changes of IL-10 and IL-17A. Statistical analysis was performed using analysis of variance and Dunnett's T-test, and P<0.05 indicated significant differences.

[0048] Table 3 Effects of walnut peptides on IL-10 and IL-17A inflammatory factors in cells

[0049] Walnut peptide Concentration (μg / mL) IL-10 (%) IL-17A (%) Negative control group / 100.0±1.34 100.0±2.33 SEQ ID NO: 1 100 76.1±2.13*** 145.3±3.11*** SEQ ID NO: 8 100 69.7±2.19*** 163.0±1.99*** SEQ ID NO: 15 100 63.5±1.52*** 178.0±3.37***

[0050] Compared with the negative control group: **P<0.01, ***P<0.001.

[0051] As shown in Table 3, walnut peptide can downregulate the content of IL-10 and increase the content of IL-17A. It can be seen that walnut peptide can regulate inflammatory factors to achieve the purpose of improving the body's immunity. Among them, the mutants SEQ ID NO: 8 and SEQ ID NO: 15 have better effects than SEQ ID NO: 1 (P<0.05).

[0052] Example 4 Preparation of a composite nutrient supplement emulsion for improving immunity

[0053] The composite nutrient supplement lotion 1 for improving immunity is composed of the following raw materials in percentage by weight:

[0054] Walnut peptide GDLASKHLE (SEQ ID NO: 1) 1%, vitamin D3 3%, vitamin K2 3.5%, calcium carbonate 1%, Tween-80 5%, Span-80 2%, soy lecithin 1.5%, gum arabic 1%, xanthan gum 1%, pectin 0.5%, maltitol liquid 1%, xylitol 1%, sorbitol liquid 1%, and the balance is purified water.

[0055] The preparation method of the composite nutrient supplement emulsion 1 for improving immunity comprises the following steps:

[0056] S1: Mix vitamin D3, vitamin K2 and calcium carbonate in proportion, add soybean lecithin, Span-80, gum arabic, xanthan gum, pectin, maltitol liquid, xylitol and sorbitol liquid, and stir and heat at 30° C. and 700 rpm for 40 min to dissolve all of the soybean lecithin, gum arabic, xanthan gum, pectin, maltitol liquid, xylitol and sorbitol liquid to obtain an oil phase;

[0057] S2: Tween-80 and purified water were fully mixed in proportion at room temperature and a rotation speed of 650 rpm, and stirred to dissolve to obtain an aqueous phase;

[0058] S3: Disperse the oil phase in step S1 dropwise into the water phase in step S2, stirring while adding until the dispersion is complete.

[0059] The stirring was continued for 30 min to obtain a crude emulsion, which was pasteurized;

[0060] S4: The crude emulsion in step S3 is homogenized at 45 MPa for 3 times, the first time for 6 minutes, the second time for 4 minutes, and the third time for 3 minutes, and then packaged to obtain the composite nutrient supplement emulsion 1 for improving immunity. Subsequently, the emulsion can be sterile filled and sealed for storage;

[0061] The compound nutrient supplement lotion 2 for improving immunity is different from the compound nutrient supplement lotion 1 for improving immunity in that the walnut peptide is SEQ ID NO: 8 replacing SEQ ID NO: 1.

[0062] The compound nutrient supplement lotion 3 for improving immunity is different from the compound nutrient supplement lotion 1 for improving immunity in that the walnut peptide is SEQ ID NO: 15 replacing SEQ ID NO: 1.

[0063] Example 5 Mouse Immunity Test

[0064] Grouping:

[0065] Blank control group

[0066] Model control group

[0067] Experimental group 1: GDLASKHLE (SEQ ID NO: 1);

[0068] Experimental group 2: GDLASKSLE (SEQ ID NO: 8);

[0069] Experimental group 3: SDLKSKHRE (SEQ ID NO: 15);

[0070] Experimental group 4: Complex nutrient supplement lotion 1 for improving immunity;

[0071] Experimental group 5: Complex nutrient supplement lotion 2 to improve immunity;

[0072] Experimental group 6: Complex nutrient supplement lotion 3 to improve immunity;

[0073] method:

[0074] After SPF-grade C57BL / 6 mice were raised and stabilized for a period of time, the experimental mice were randomly divided into 8 groups according to their body weight, with 5 mice in each group.

[0075] The mice in the model control group and experimental group were intraperitoneally injected with 80 mg / kg cyclophosphamide for 3 consecutive days from the day of grouping. After that, the model was reinforced with injection once a week. The blank control group was intraperitoneally injected with the same amount of 0.9% sodium chloride injection during the same period. The modeling period was 30 days.

[0076] During the modeling period, mice in experimental groups 1-6 were gavaged with 0.4 mL / kg of the corresponding dose of walnut peptide or 40 mL / kg of the composite nutrient supplement emulsion to improve immunity, once a day for 30 consecutive days; the blank control group and the model control group were gavaged with the corresponding volume of pure water every day. Subsequently, all mice in the groups were used for ConA (concanavalin A)-induced mouse spleen lymphocyte transformation and mouse spleen NK cell activity assay tests, and the results are shown in Table 4.

[0077] Table 4 Mouse experimental results

[0078] Grouping <![CDATA[Lymphocyte proliferation ability (10 -2 )]]> NK cell activity (%) Blank control group 30 61 Model control group 17 25 Experimental Group 1 20 42 Experimental Group 2 23 29 Experimental Group 3 27 35 Experimental Group 4 25 56 Experimental Group 5 27 57 Experimental Group 6 29 59

[0079] As shown in Table 4, compared with experimental group 1, the proliferation ability of mouse spleen lymphocytes and the activity of mouse NK cells in experimental groups 2 and 3 were significantly increased (P<0.05); and compared with experimental group 1, experimental group 4, experimental group 5, experimental group 2, and experimental group 6, the proliferation ability of mouse spleen lymphocytes and the activity of mouse NK cells were significantly increased (P<0.05), which indicates that other components can have a synergistic effect with walnut peptide.

[0080] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite nutrient supplement lotion for improving immunity, characterized in that: The invention comprises walnut peptide, calcium carbonate, vitamin K2, vitamin D3, colloid and sugar alcohol; the walnut peptide is shown as SEQ ID NO: 8 or SEQ ID NO:

15.

2. The composite nutrient supplement emulsion for improving immunity according to claim 1, characterized in that: The colloid includes one or more of gum arabic, xanthan gum, pectin and carrageenan.

3. The composite nutrient supplement emulsion for improving immunity according to claim 1, characterized in that: The colloids include gum arabic, xanthan gum and pectin.

4. The composite nutrient supplement emulsion for improving immunity according to claim 1, characterized in that: The sugar alcohol includes one or more of maltitol liquid, xylitol, sorbitol liquid, and isomalt.

5. The composite nutrient supplement emulsion for improving immunity according to claim 1, characterized in that: The sugar alcohols include maltitol liquid, xylitol and sorbitol liquid.

6. The composite nutrient supplement emulsion for improving immunity according to claim 1, characterized in that: It contains walnut peptide, vitamin D3, vitamin K2, calcium carbonate, Tween-80, Span-80, soy lecithin, gum arabic, xanthan gum, pectin, maltitol liquid, xylitol, sorbitol liquid and purified water.

7. The composite nutrient supplement emulsion for improving immunity according to claim 1, characterized in that: It contains 1% by mass of walnut peptide, 3% of vitamin D3, 3.5% of vitamin K2, 1% of calcium carbonate, 5% of Tween-80, 2% of Span-80, 1.5% of soybean lecithin, 1% of gum arabic, 1% of xanthan gum, 0.5% of pectin, 1% of maltitol liquid, 1% of xylitol, 1% of sorbitol liquid, and the balance is purified water.