Agilawood-containing composition for improving immunity and preparation method thereof

Abstract

The invention relates to an agilawood-containing composition for improving immunity and a preparation method of the agilawood-containing composition, and belongs to the technical field of biology, and the agilawood-containing composition contains soft-shelled turtle extract peptide, white poria peptide, agilawood extract, snake gourd seed extract, astragalus membranaceus, houttuynia cordata, honeysuckle, blueberries, sea cucumbers, zinc gluconate and the like. The soft-shelled turtle extract peptide, the white poria peptide and the snake gourd seed extract are obtained by adopting a special enzymolysis method, the agilawood extract is an ethanol extract, and the agilawood extract is matched with other components according to a specific proportion to have a better effect of quickly improving immunity, maintaining body health, resisting viruses, and realizing biological immune regulation without antibiotic drug dependence.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an agarwood-containing composition for improving immunity and a preparation method thereof. Background Art

[0002] In today's society, people's pace of life is accelerating, work pressure is increasing, and coupled with the influence of environmental pollution, bad living habits and other factors, the body's immunity is facing many challenges. As the key line of defense for the human body to resist the invasion of external pathogens and maintain physical health, immunity is becoming more and more important, which has also prompted the market demand for immunity-enhancing products to continue to grow.

[0003] In traditional medical concepts, many natural plants and animals can not only provide basic nutrients for the human body, but also have medicinal effects, and have unique advantages in regulating human functions and enhancing immunity. For example, wolfberry is rich in wolfberry polysaccharides, which can stimulate and regulate the activity of cells in the human immune system, promote the phagocytic ability of macrophages, and enhance the proliferation and differentiation of lymphocytes, thereby enhancing the body's immune defense function. Another example is astragalus. The active substances in astragalus, such as astragalus polysaccharides and flavonoid compounds, have antioxidant, anti-inflammatory and immune cytokine secretion regulating effects, which can effectively enhance the body's nonspecific immunity and specific immunity, and help the body resist the invasion of various diseases.

[0004] With the rapid development of modern science and technology, people have conducted more in-depth research on the mechanism of action of ingredients in improving immunity. Advanced extraction technologies, such as supercritical fluid extraction, ultrasound-assisted extraction, and enzymatic extraction, can more efficiently and accurately separate and enrich ingredients with immunomodulatory activity from medicinal materials and food ingredients to ensure the efficacy and stability of the product.

[0005] In the consumer market, consumers' awareness of health products is gradually improving, and they are more inclined to choose natural, safe, and side-effect-free medicinal and food products to improve immunity, rather than simply relying on Western medicine chemical synthetic drug treatment. However, there are also some problems in the current medicinal and food product market, such as uneven effects, no obvious short-term effects, long treatment courses, and dependence. This requires increasing scientific research efforts, in-depth exploration of the immunomodulatory potential of more medicinal and food ingredients, and optimization of product formulas and production processes, so as to provide consumers with better quality, efficient, and safe immunity-enhancing products to meet consumers' demand for health products. Summary of the invention

[0006] The existing products for improving immunity have different effects, are not obvious in the short term, have a long course of treatment, and are addictive. The present invention provides an agarwood composition for improving immunity and a preparation method thereof. The composition contains turtle extract peptides, white Poria peptides, agarwood extracts, snake melon seed extracts, astragalus, houttuynia cordata, honeysuckle, blueberry, sea cucumber, zinc gluconate and other ingredients; turtle extract peptides, white Poria peptides and snake melon seed extracts are obtained by a special enzymatic hydrolysis method, and the agarwood extract is an ethanol extract. When used in combination with other ingredients in a specific ratio, it has better and faster immunity improvement effects, maintains body health, is anti-viral, and has biological immune regulation without antibiotic drug dependence. The specific technical scheme is as follows:

[0007] An agarwood composition for improving immunity, the composition comprising the following raw materials in parts by weight: 5 to 8 parts of turtle extract peptide, 1 to 3 parts of white tuckahoe peptide, 0.5 to 1.5 parts of agarwood extract, 0.3 to 0.8 parts of snake gourd seed extract, 8 to 12 parts of astragalus, 1 to 3 parts of houttuynia cordata, 1 to 3 parts of honeysuckle, 0.5 to 2 parts of blueberry, 0.5 to 1 part of sea cucumber and 0.05 to 0.1 parts of zinc gluconate;

[0008] The turtle extracted peptide is an extracted peptide below 10 kDa obtained by sequentially enzymatically hydrolyzing turtle protein with serrapeptase, keratinase and nattokinase; the white poria peptide is an extracted peptide below 5 kDa obtained by co-enzymatically hydrolyzing white poria with hemicellulase, pectinase and ficin, followed by graded enzymatic hydrolysis with trypsin and bromelain; the agarwood extract is an ethanol-soaked extract of agarwood; the snake melon seed extract is a product obtained by co-enzymatically hydrolyzing the seed flesh of snake melon seeds with hemicellulase, pectinase and ficin, followed by enzymatic hydrolysis with papain, and then extracting with ethanol to obtain an extract below 5 kDa.

[0009] In the above composition, the preparation method of the turtle extracted peptide comprises the following steps: adding turtle protein to distilled water with a mass of 8 to 12 times that of turtle protein, stirring evenly to prepare a protein homogenate, adjusting the pH to 6.5 to 7.0, adding 0.5% to 1% of the mass of turtle protein serrapeptase, performing enzymolysis at a constant temperature of 45°C to 50°C for 60min to 90min, heating to inactivate the enzyme, reducing the temperature to 40°C to 60°C, adjusting the pH to 7.0 to 9.0, adding 0.5% to 1% of the mass of turtle protein keratinase, and performing enzymolysis at a constant temperature of 45°C to 55°C. The method comprises the following steps: enzymatically hydrolyze for 60 to 90 minutes, heat up to inactivate the enzyme, reduce the temperature to 40 to 55 degrees Celsius, adjust the pH value to 6.5 to 7.0, add 0.5% to 1% by weight of nattokinase of turtle protein, perform enzymatic hydrolysis at 50 to 55 degrees Celsius for 50 to 80 minutes, heat up to inactivate the enzyme, reduce the temperature to room temperature, centrifuge at 4000 to 6000 r / min for 15 to 20 minutes, take the supernatant, perform ultrafiltration using a 10 kDa ultrafiltration membrane, take the ultrafiltration component below 10 kDa, and freeze-dry it to obtain turtle extracted peptides.

[0010] In the preparation method of turtle extracted peptides, the preparation method of turtle protein comprises the following steps: taking fresh turtle meat of Chinese soft-shelled turtle, washing, draining, cutting into small pieces less than 2 cm, obtaining turtle meat fragments, adding 4°C to 6°C pre-cooled physiological saline of 4 to 6 times the mass of the turtle meat fragments to the turtle meat fragments, crushing and homogenizing, so that the turtle meat tissue is fully broken, releasing the protein components in the cells, and obtaining a homogenate; centrifuging the homogenate at 4000 r / min to 6000 r / min for 20 to 30 min, taking the supernatant, adding ammonium sulfate to the supernatant, stirring while adding, so that the protein gradually precipitates out, and then standing at 4°C to 6°C for 4h to 6h to allow the precipitate to fully form, and then centrifuging at 6000 r / min to 8000 r / min for 20min to 30min, collecting the precipitate, and washing and desalting the precipitate 2 to 3 times to obtain turtle protein.

[0011] In the above-mentioned composition, the preparation method of the white tuckahoe peptide comprises the following steps: taking white tuckahoe for crushing, adding distilled water of 8 to 12 times the mass of white tuckahoe, adjusting the pH to 5.5 to 6.5, adding hemicellulase of 0.5% to 1.5% by mass of white tuckahoe, pectinase of 0.5% to 1.5% by mass of white tuckahoe, and fig protease of 0.5% to 1.5% by mass of white tuckahoe in sequence, performing enzymolysis at 50°C to 55°C for 90min to 120min, heating to inactivate the enzyme, cooling to room temperature, adjusting the pH to 7.5 to 8.0, adding trypsin of 0.5% to 1.0% by mass of white tuckahoe, performing enzymolysis at 35°C to 38°C for 50min to 80min, heating to inactivate the enzyme, cooling to 50°C to 60°C, adjusting the pH to 6.0 to 7.0, adding bromelain of 0.5% to 1.0% by mass of white tuckahoe, and performing enzymolysis at 50°C to 60°C for 50min n~80min, raise the temperature to inactivate the enzyme, cool to room temperature, centrifuge at 4000r / min~6000r / min for 15min~20min, take the supernatant, use 5kDa ultrafiltration membrane for ultrafiltration, take the ultrafiltration component below 5kDa, freeze-dry, and obtain white Poria peptide.

[0012] In the above composition, the preparation method of the agarwood extract comprises the following steps: taking agarwood medicinal materials, crushing them to obtain powder, adding ethanol 6 to 8 times the mass of the powder to the powder, mixing evenly, sealing and soaking for 7 to 10 days in a light-proof room temperature environment, centrifuging at 4000 r / min to 6000 r / min for 15 to 20 min, taking the supernatant, evaporating under reduced pressure at 40°C to 50°C to remove ethanol, and drying at 40°C to 45°C to constant weight to obtain the agarwood extract.

[0013] In the above-mentioned composition, the preparation method of the snake melon seed extract comprises the following steps: taking the seed flesh of the snake melon seed, crushing it to obtain a crushed material, adding distilled water 8 to 12 times the mass of the crushed material, adjusting the pH to 5.5 to 6.5, sequentially adding 0.5% to 1.5% of hemicellulase by weight of the crushed material, 0.5% to 1.5% of pectinase by weight of the crushed material, and 0.5% to 1.5% of fig protease by weight of the crushed material, performing enzymolysis at a constant temperature of 50°C to 55°C for 90min to 120min, heating to inactivate the enzyme, reducing the temperature to 55°C to 65°C, adding 0.5% to 1% of papain by weight of the crushed material, performing enzymolysis at a constant temperature of 55°C to 65°C for 60min to 90min, heating to inactivate the enzyme, reducing the temperature to room temperature, adding 1 to 3 times the mass of ethanol by weight of the crushed material for soaking for 1h to 2h, and then rotating the mixture at 4000r / min to 6000r / min. The mixture was centrifuged for 15 to 20 minutes, the supernatant was taken, and ultrafiltration was performed using a 5 kDa ultrafiltration membrane. The ultrafiltration fraction below 5 kDa was taken and freeze-dried to obtain the snake gourd seed extract.

[0014] In the above preparation method, the temperature for heating to inactivate the enzyme is 90° C. to 95° C., and the time for heating to inactivate the enzyme is 15 min to 40 min.

[0015] In the above composition, the astragalus is astragalus dry powder, the houttuynia cordata is houttuynia cordata dry powder, the honeysuckle is honeysuckle dry powder, the blueberry is blueberry dry powder, and the sea cucumber is sea cucumber dry powder; the astragalus dry powder, houttuynia cordata dry powder, honeysuckle dry powder, blueberry dry powder, sea cucumber dry powder and zinc gluconate are all sieved through 150-250 meshes.

[0016] The method for preparing the above-mentioned agarwood-containing composition for improving immunity comprises the following steps:

[0017] According to the mass proportions, astragalus, houttuynia, honeysuckle, blueberry, sea cucumber and zinc gluconate were mixed evenly, and then agarwood extract and snake melon seed extract were added, and the mixture was continued to be evenly mixed. Finally, turtle extract peptide and white Poria peptide were added, and the mixture was evenly mixed to obtain a composition.

[0018] In the above preparation method, the mixing speed is 10 rpm to 30 rpm; and the mixing temperature is below 20°C.

[0019] The present invention provides an agarwood-containing composition for improving immunity and a preparation method thereof, and the beneficial effects are as follows:

[0020] 1. The composition of the present invention contains turtle extract peptide, white tuckahoe peptide, agarwood extract, snake melon seed extract, astragalus, houttuynia, honeysuckle, blueberry, sea cucumber and zinc gluconate and other ingredients; turtle extract peptide as a bioactive peptide, has the effect of immunomodulation, and enhances the body's immunity by activating immune cells and regulating immune-related signal pathways. White tuckahoe peptide is also a peptide substance, which helps to regulate the body's physiological functions and plays a role in immune enhancement by enhancing the body's defense mechanism. Agarwood extract contains a variety of bioactive ingredients, which have a regulatory effect on the body's immune system and can help enhance the body's resistance. After special enzymatic hydrolysis and other treatments, snake melon seed extract contains ingredients that are beneficial to the immune system and participate in the immune regulation process. Astragalus is a traditional Chinese medicinal material with the effects of replenishing qi and strengthening the exterior, which can enhance the body's immune function and improve the body's disease resistance. Houttuynia has the effects of clearing heat and detoxifying, and indirectly improves the body's defense ability through anti-inflammatory and antibacterial effects. Honeysuckle has the effects of clearing heat and detoxifying, anti-inflammatory, etc., which helps to remove heat and toxins in the body and plays a certain regulatory role on the immune system. Blueberries are rich in antioxidants, such as vitamin C and anthocyanins, which can resist oxidation, scavenge free radicals, and reduce the damage of oxidative stress to the immune system, thereby indirectly enhancing immunity. Sea cucumbers are rich in various nutrients, such as sea cucumber polysaccharides, which have immune regulation and anti-fatigue effects, and help enhance the body's immune function. Zinc gluconate provides zinc, participates in the synthesis of various enzymes and the functional regulation of immune cells, and plays an important role in maintaining normal immune function.

[0021] Second, the present invention uses a variety of enzymes such as serrapeptase, keratinase, nattokinase, etc. to enzymolyze turtle protein step by step. Different enzymes have different action sites on proteins, and can gradually decompose turtle protein into peptide segments of characteristic sequences. After entering the human body, it can participate in the physiological process of immune regulation more quickly, and activate the receptors on the surface of immune cells as signal molecules, start the activation and proliferation process of immune cells, and thus enhance immunity. The 10kDa ultrafiltration membrane can remove macromolecular impurities greater than 10kDa. After ultrafiltration, the ultrafiltration components below 10kDa obtained have higher purity. These small molecule peptides can more effectively avoid the interference of impurities on their absorption and action, ensuring that they can accurately play the role of regulating immune cell metabolism, promoting the secretion of immune factors, and thus enhancing the body's immunity.

[0022] Three, the present invention is enzymatically hydrolyzed white tuckahoe by hemicellulase, pectinase, ficin, trypsin, bromelain successively.These enzymes can act on the different components in white tuckahoe respectively, hemicellulase and pectinase can decompose cell wall components, make the effective ingredients in the cell easier to release, and subsequent protease can decompose protein into peptides of specific sequence.This composite enzymatic hydrolysis method can fully extract the effective ingredients in white tuckahoe, and convert into small molecule peptides, these peptides can participate in the signaling pathway of regulating immune cells, promote the growth and differentiation of immune cells, thereby enhancing immunity.5kDa ultrafiltration membrane can effectively remove macromolecular impurities greater than 5kDa, so that the purity of the white tuckahoe peptide obtained is higher.Highly purified white tuckahoe peptide, after entering the human body, can more effectively bind to the receptors on the surface of immune cells, participate in the immune regulation metabolic process in the body, avoid the interference and adverse effects brought by impurities, and are more conducive to enhancing the immune function of the body.

[0023] Fourth, the present invention crushes the agarwood medicinal material and then seals and soaks it in ethanol in a dark and room temperature environment. This method can fully dissolve the terpenoids, flavonoids and other biologically active components in the agarwood in ethanol. These components have antioxidant and anti-inflammatory effects, and indirectly have a positive impact on the immune system by regulating the body's redox state and inflammatory response, reducing the damage of oxidative stress to immune cells, inhibiting the interference of inflammatory mediators on the immune system, and thus enhancing the body's immunity.

[0024] Five, the present invention uses hemicellulase, pectinase, ficin, papain to enzymolyze snake gourd seeds in sequence. These enzymes can decompose cell wall components and proteins in snake gourd seeds, so that the effective ingredients therein are fully released. The small molecule components obtained by enzymolysis contain substances that are beneficial to the immune system, including amino acids, specific peptides and other bioactive molecules, which can participate in the metabolic process of immune cells, promote the proliferation and activation of immune cells, and thus enhance the body's immunity. The 5kDa ultrafiltration membrane removes macromolecular impurities greater than 5kDa, thereby improving the purity of the snake gourd seed extract. The high-purity extract can reduce the interference of impurities on its role in the body, and is more conducive to the interaction of the active ingredients therein with immune cells and immune-related molecules, activating the signal pathways of immune cells, promoting the secretion of immune factors, and thus improving the body's immunity. After enzymolysis, soaking in ethanol can further extract and enrich the effective ingredients on the one hand, because some ingredients have better solubility in ethanol; on the other hand, centrifugation after ethanol soaking can remove some impurities with low solubility in ethanol, further improving the quality of the extract.

[0025] 6. Agarwood extract and snake melon seed extract each contain different types of immunomodulatory components, such as terpenoids in agarwood and bioactive peptides in snake melon seeds. These components act on the immune system through different pathways, promoting the proliferation of lymphocytes and the ability to produce antibodies. When agarwood extract and snake melon seed extract are combined, they can regulate the immune system from multiple aspects, enhance the activity and function of immune cells, produce a synergistic effect, and more effectively improve the body's immunity. Both extracts have anti-inflammatory effects, regulate the secretion balance of tumor necrosis factor-α, and avoid affecting the normal function of the immune system; their synergistic effect can more effectively inhibit inflammatory responses, provide a good environment for the normal function of the immune system, reduce the damage of inflammation to immune cells, and thus indirectly enhance the body's immunity. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0027] Example 1

[0028] A composition containing agarwood for improving immunity, the composition comprises the following raw materials in parts by weight: 6.5 parts of turtle extract peptide, 2 parts of white tuckahoe peptide, 1 part of agarwood extract, 0.5 parts of snake melon seed extract, 10 parts of astragalus, 2 parts of houttuynia, 2 parts of honeysuckle, 1.2 parts of blueberry, 0.8 parts of sea cucumber, and 0.08 parts of zinc gluconate. Among them, astragalus is astragalus dry powder, houttuynia is houttuynia dry powder, honeysuckle is honeysuckle dry powder, blueberry is blueberry dry powder, and sea cucumber is sea cucumber dry powder; astragalus dry powder, houttuynia dry powder, honeysuckle dry powder, blueberry dry powder, sea cucumber dry powder and zinc gluconate are all sieved through 200 meshes.

[0029] The preparation method of turtle extracted peptides includes the following steps: taking fresh turtle meat of Chinese soft-shelled turtle, washing, draining, cutting into small pieces less than 2 cm, obtaining turtle meat fragments, adding 4°C pre-cooled physiological saline of 5 times the mass of the turtle meat fragments to the turtle meat fragments, crushing and homogenizing, so that the turtle meat tissue is fully broken, releasing the protein components in the cells, and obtaining a homogenate; centrifuging the homogenate at 5000r / min for 25min, taking the supernatant, adding ammonium sulfate to the supernatant, stirring while adding, so that the protein gradually precipitates out, and then standing at 4°C for 5h to allow the precipitate to fully form, and then centrifuging at 6000r / min for 25min, collecting the precipitate, and washing and desalting the precipitate twice to obtain turtle protein. Add turtle protein to distilled water with a mass of 10 times that of turtle protein, stir evenly to prepare protein homogenate, adjust pH 6.8, add 0.8% of the mass of turtle protein serrapeptase, enzymolyze at 48°C for 80 minutes, heat to 90°C to inactivate the enzyme for 20 minutes, reduce to 50°C, adjust pH 8.0, add 0.8% of the mass of turtle protein keratinase, enzymolyze at 50°C for 75 minutes, heat to 90°C to inactivate the enzyme for 20 minutes, reduce to 50°C, adjust pH 6.6, add 0.6% of the mass of turtle protein nattokinase, enzymolyze at 50°C for 60 minutes, heat to 90°C to inactivate the enzyme for 20 minutes, reduce to room temperature, centrifuge at 5000r / min for 18 minutes, take the supernatant, ultrafilter with a 10kDa ultrafiltration membrane, take the ultrafiltration fraction below 10kDa, freeze-dry, and obtain turtle extracted peptide.

[0030] The preparation method of white tuckahoe peptide comprises the following steps: taking white tuckahoe for crushing, adding distilled water 10 times the mass of white tuckahoe, adjusting pH6.0, adding hemicellulase 1% by mass of white tuckahoe, pectinase 1% by mass of white tuckahoe, and fig protease 1% by mass of white tuckahoe in sequence, enzymolysis at 52°C for 100min, heating to 90°C for 20min to inactivate the enzyme, cooling to room temperature, adjusting pH7.7, adding trypsin 0.6% by mass of white tuckahoe, enzymolysis at 37°C for 65min, heating to 90°C for 20min to inactivate the enzyme, cooling to 55°C, adjusting pH6.6, adding bromelain 0.8% by mass of white tuckahoe, enzymolysis at 55°C for 60min, heating to 90°C for 20min to inactivate the enzyme, cooling to room temperature, centrifuging at 5000r / min for 18min, taking the supernatant, ultrafiltration with a 5kDa ultrafiltration membrane, taking the ultrafiltration fraction below 5kDa, and freeze-drying to obtain white tuckahoe peptide.

[0031] Among them, the preparation method of agarwood extract includes the following steps: taking agarwood medicinal materials, crushing them to obtain powder, adding ethanol 7 times the mass of the powder to the powder, mixing evenly, sealing and soaking for 8 days in a light-proof room temperature environment, centrifuging at 5000r / min for 16 minutes, taking the supernatant, evaporating under reduced pressure at 45°C to remove ethanol, and drying at 42°C to constant weight to obtain the agarwood extract.

[0032] The preparation method of the snake melon seed extract comprises the following steps: taking the seed flesh of the snake melon seeds, crushing it to obtain a crushed material, adding distilled water 10 times the mass of the crushed material, adjusting the pH to 6.1, sequentially adding hemicellulase 1.2% by mass of the crushed material, pectinase 1% by mass of the crushed material, and fig protease 1.2% by mass of the crushed material, performing enzymolysis at 52°C for 110 minutes, heating to 90°C to inactivate the enzyme for 20 minutes, reducing the temperature to 60°C, adding papain 0.8% by mass of the crushed material, performing enzymolysis at 60°C for 80 minutes, heating to 92°C to inactivate the enzyme for 40 minutes, reducing the temperature to room temperature, adding ethanol 2 times the mass of the crushed material for soaking for 1.5 hours, and then centrifuging at 5000r / min for 15 minutes, taking the supernatant, ultrafiltering with a 5kDa ultrafiltration membrane, taking the ultrafiltration component below 5kDa, and freeze-drying to obtain the snake melon seed extract.

[0033] The method for preparing the above-mentioned agarwood-containing composition for improving immunity comprises the following steps:

[0034] According to the mass proportions, astragalus, houttuynia, honeysuckle, blueberry, sea cucumber and zinc gluconate were mixed evenly at 15°C and 20 rpm, and then the agarwood extract and snake melon seed extract were added, and the mixing was continued at 15°C and 20 rpm, and finally the turtle extract peptide and white Poria peptide were added, and the mixture was mixed evenly at 15°C and 20 rpm to obtain a composition.

[0035] Example 2

[0036] A composition containing agarwood for improving immunity, the composition comprises the following raw materials by weight: 5 parts of turtle extract peptide, 1 part of white tuckahoe peptide, 0.5 parts of agarwood extract, 0.3 parts of snake gourd seed extract, 8 parts of astragalus, 1 part of houttuynia, 1 part of honeysuckle, 0.5 parts of blueberry, 0.5 parts of sea cucumber, and 0.05 parts of zinc gluconate. Among them, astragalus is astragalus dry powder, houttuynia is houttuynia dry powder, honeysuckle is honeysuckle dry powder, blueberry is blueberry dry powder, and sea cucumber is sea cucumber dry powder; astragalus dry powder, houttuynia dry powder, honeysuckle dry powder, blueberry dry powder, sea cucumber dry powder and zinc gluconate are all sieved through 150 mesh.

[0037] The preparation method of turtle extracted peptides includes the following steps: taking fresh turtle meat of Chinese soft-shelled turtle, washing, draining, cutting into small pieces less than 2 cm, obtaining turtle meat fragments, adding 5°C pre-cooled physiological saline of 5 times the mass of the turtle meat fragments to the turtle meat fragments, crushing and homogenizing, so that the turtle meat tissue is fully broken, releasing the protein components in the cells, and obtaining a homogenate; centrifuging the homogenate at 4000r / min for 20min, taking the supernatant, adding ammonium sulfate to the supernatant, stirring while adding, so that the protein gradually precipitates out, and then standing at 5°C for 4h to allow the precipitate to fully form, and then centrifuging at 7000r / min for 30min, collecting the precipitate, and washing and desalting the precipitate three times to obtain turtle protein. Add turtle protein to distilled water with 9 times the mass of turtle protein, stir evenly to make protein homogenate, adjust pH6.7, add 0.6% of the mass of turtle protein serrapeptase, enzymolyze at 45°C for 70 minutes, heat to 95°C to inactivate the enzyme for 15 minutes, reduce to 40°C, adjust pH8.2, add 0.9% of the mass of turtle protein keratinase, enzymolyze at 45°C for 85 minutes, heat to 95°C to inactivate the enzyme for 15 minutes, reduce to 40°C, adjust pH6.9, add 0.8% of the mass of turtle protein nattokinase, enzymolyze at 50°C for 75 minutes, heat to 95°C to inactivate the enzyme for 15 minutes, reduce to room temperature, centrifuge at 6000r / min for 20 minutes, take the supernatant, use 10kDa ultrafiltration membrane for ultrafiltration, take the ultrafiltration component below 10kDa, freeze-dry, and obtain turtle extracted peptide.

[0038] The preparation method of white tuckahoe peptide comprises the following steps: crushing white tuckahoe, adding distilled water 11 times the weight of white tuckahoe, adjusting the pH to 6.2, adding hemicellulase 1.3% by weight of white tuckahoe, pectinase 1.2% by weight of white tuckahoe, and fig protease 0.8% by weight of white tuckahoe in sequence, performing enzymolysis at 50°C for 110 minutes, heating to 95°C to inactivate the enzyme for 15 minutes, cooling to room temperature, adjusting the pH to 7.6, adding trypsin 0.7% by weight of white tuckahoe, and performing enzymolysis at 50°C for 110 minutes. White tuckahoe peptide was prepared by enzymolysis at 35°C for 65 min, then heated to 95°C for 15 min to inactivate the enzyme, then cooled to 50°C, adjusted to pH 6.7, added with 0.8% bromelain by weight of white tuckahoe, and enzymolyzed at 50°C for 75 min, then heated to 95°C for 15 min to inactivate the enzyme, cooled to room temperature, and centrifuged at 4000 r / min for 20 min. The supernatant was taken and ultrafiltered using a 5 kDa ultrafiltration membrane. The ultrafiltration fraction below 5 kDa was taken and freeze-dried to obtain white tuckahoe peptide.

[0039] Among them, the preparation method of agarwood extract includes the following steps: taking agarwood medicinal materials, crushing them to obtain powder, adding ethanol 7.5 times the mass of the powder to the powder, mixing evenly, sealing and soaking for 9 days in a light-proof room temperature environment, centrifuging at 6000r / min for 15min, taking the supernatant, evaporating under reduced pressure at 48°C to remove ethanol, and drying at 44°C to constant weight to obtain agarwood extract.

[0040] The preparation method of the snake melon seed extract comprises the following steps: taking the seed flesh of the snake melon seeds, crushing it to obtain a crushed material, adding distilled water 11 times the mass of the crushed material, adjusting the pH to 6.2, sequentially adding hemicellulase 1.4% by mass of the crushed material, pectinase 0.9% by mass of the crushed material, and fig protease 1.3% by mass of the crushed material, performing enzymolysis at a constant temperature of 50°C for 110 minutes, heating to 95°C to inactivate the enzyme for 15 minutes, reducing the temperature to 55°C, adding papain 0.7% by mass of the crushed material, performing enzymolysis at a constant temperature of 55°C for 85 minutes, heating to 95°C to inactivate the enzyme for 30 minutes, reducing the temperature to room temperature, adding ethanol 2.5 times the mass of the crushed material to soak for 2 hours, and then centrifuging at 6000r / min for 15 minutes, taking the supernatant, ultrafiltration using a 5kDa ultrafiltration membrane, taking the ultrafiltration component below 5kDa, and freeze-drying to obtain the snake melon seed extract.

[0041] The method for preparing the above-mentioned agarwood-containing composition for improving immunity comprises the following steps:

[0042] According to the mass proportions, astragalus, houttuynia, honeysuckle, blueberry, sea cucumber and zinc gluconate were mixed evenly at 10 ℃ and 10 rpm, and then the agarwood extract and snake melon seed extract were added, and the mixture was further mixed at 10 ℃ and 10 rpm, and finally the turtle extract peptide and white Poria peptide were added, and the mixture was mixed evenly at 10 ℃ and 10 rpm to obtain a composition.

[0043] Example 3

[0044] A composition containing agarwood for improving immunity, the composition comprises the following raw materials in parts by weight: 5 parts of turtle extract peptide, 3 parts of white tuckahoe peptide, 0.5 parts of agarwood extract, 0.8 parts of snake gourd seed extract, 8 parts of astragalus, 3 parts of houttuynia, 1 part of honeysuckle, 2 parts of blueberry, 0.5 parts of sea cucumber, and 0.1 parts of zinc gluconate. Among them, astragalus is astragalus dry powder, houttuynia is houttuynia dry powder, honeysuckle is honeysuckle dry powder, blueberry is blueberry dry powder, and sea cucumber is sea cucumber dry powder; astragalus dry powder, houttuynia dry powder, honeysuckle dry powder, blueberry dry powder, sea cucumber dry powder and zinc gluconate are all sieved through 250 mesh.

[0045] The preparation method of turtle extracted peptides includes the following steps: taking fresh turtle meat of Chinese soft-shelled turtle, washing, draining, cutting into small pieces less than 2 cm, obtaining turtle meat fragments, adding 4°C pre-cooled physiological saline of 4 times the mass of the turtle meat fragments to the turtle meat fragments, crushing and homogenizing, so that the turtle meat tissue is fully broken, releasing the protein components in the cells, and obtaining a homogenate; centrifuging the homogenate at 4000r / min for 20min, taking the supernatant, adding ammonium sulfate to the supernatant, stirring while adding, so that the protein gradually precipitates out, and then standing at 4°C for 4h to allow the precipitate to fully form, and then centrifuging at 6000r / min for 20min, collecting the precipitate, and washing and desalting the precipitate twice to obtain turtle protein. Add turtle protein to distilled water 8 times the mass of turtle protein, stir evenly to make protein homogenate, adjust pH6.5, add 0.5% of the mass of turtle protein serrapeptase, enzymolyze at 45°C for 60 minutes, heat to 90°C to inactivate the enzyme for 40 minutes, reduce to 60°C, adjust pH7.0, add 0.5% of the mass of turtle protein keratinase, enzymolyze at 55°C for 60 minutes, heat to 90°C to inactivate the enzyme for 40 minutes, reduce to 40°C, adjust pH6.5, add 0.5% of the mass of turtle protein nattokinase, enzymolyze at 50°C for 50 minutes, heat to 90°C to inactivate the enzyme for 40 minutes, reduce to room temperature, centrifuge at 4000r / min for 15 minutes, take the supernatant, ultrafilter with 10kDa ultrafiltration membrane, take the ultrafiltration fraction below 10kDa, freeze-dry, and obtain turtle extracted peptide.

[0046] The preparation method of white tuckahoe peptide comprises the following steps: crushing white tuckahoe, adding distilled water 8 times the weight of white tuckahoe, adjusting the pH to 5.5, sequentially adding hemicellulase 0.5% by weight of white tuckahoe, pectinase 0.5% by weight of white tuckahoe, and fig protease 0.5% by weight of white tuckahoe, performing enzymolysis at 50°C for 90 minutes, heating to 90°C to inactivate the enzyme for 40 minutes, cooling to room temperature, adjusting the pH to 7.5, adding trypsin 0.5% by weight of white tuckahoe, and performing enzymolysis at 50°C for 90 minutes. Enzyme, constant temperature enzymolysis at 37 ° C for 50 minutes, heated to 90 ° C to inactivate the enzyme for 40 minutes, lowered to 55 ° C, adjusted to pH 6.0, added with 0.5% bromelain by weight of white Poria cocos, constant temperature enzymolysis at 55 ° C for 50 minutes, heated to 90 ° C to inactivate the enzyme for 40 minutes, lowered to room temperature, centrifuged at 4000 r / min for 15 minutes, took the supernatant, ultrafiltered with a 5 kDa ultrafiltration membrane, took the ultrafiltration component below 5 kDa, freeze-dried, and obtained white Poria cocos peptide.

[0047] Among them, the preparation method of agarwood extract includes the following steps: taking agarwood medicinal materials, crushing them to obtain powder, adding ethanol 6 times the mass of the powder to the powder, mixing evenly, sealing and soaking for 7 days in a light-proof room temperature environment, centrifuging at 4000r / min for 15min, taking the supernatant, evaporating under reduced pressure at 40°C to remove ethanol, and drying at 40°C to constant weight to obtain the agarwood extract.

[0048] The preparation method of the snake melon seed extract comprises the following steps: taking the seed flesh of the snake melon seeds, crushing it to obtain a crushed material, adding 8 times the amount of distilled water to the crushed material, adjusting the pH to 5.5, sequentially adding 0.5% of the weight of hemicellulase, 0.5% of the weight of pectinase, and 0.5% of the weight of fig protease to the crushed material, performing enzymolysis at 55°C for 90 minutes, heating to 90°C to inactivate the enzyme for 40 minutes, reducing the temperature to 55°C, adding 0.5% of the weight of papain to the crushed material, performing enzymolysis at 55°C for 60 minutes, heating to 94°C to inactivate the enzyme for 40 minutes, reducing the temperature to room temperature, adding 1 times the weight of ethanol to the crushed material for soaking for 1 hour, and then centrifuging at 4000r / min for 15 minutes, taking the supernatant, ultrafiltering with a 5kDa ultrafiltration membrane, taking the ultrafiltration component below 5kDa, and freeze-drying to obtain the snake melon seed extract.

[0049] The method for preparing the above-mentioned agarwood-containing composition for improving immunity comprises the following steps:

[0050] According to the mass proportions, astragalus, houttuynia, honeysuckle, blueberry, sea cucumber and zinc gluconate were mixed evenly at 18°C ​​and 15 rpm, and then the agarwood extract and snake melon seed extract were added, and the mixture was further mixed at 18°C ​​and 15 rpm, and finally the turtle extract peptide and white Poria peptide were added, and the mixture was mixed evenly at 18°C ​​and 15 rpm to obtain a composition.

[0051] Example 4

[0052] A composition containing agarwood for improving immunity, the composition comprises the following raw materials in parts by weight: 8 parts of turtle extract peptide, 3 parts of white tuckahoe peptide, 1.5 parts of agarwood extract, 0.8 parts of snake gourd seed extract, 12 parts of astragalus, 3 parts of houttuynia, 3 parts of honeysuckle, 2 parts of blueberry, 1 part of sea cucumber, and 0.1 parts of zinc gluconate. Among them, astragalus is astragalus dry powder, houttuynia is houttuynia dry powder, honeysuckle is honeysuckle dry powder, blueberry is blueberry dry powder, and sea cucumber is sea cucumber dry powder; astragalus dry powder, houttuynia dry powder, honeysuckle dry powder, blueberry dry powder, sea cucumber dry powder and zinc gluconate are all sieved through 200 meshes.

[0053] The preparation method of turtle extracted peptides includes the following steps: taking fresh turtle meat of Chinese soft-shelled turtle, washing, draining, cutting into small pieces less than 2 cm, obtaining turtle meat fragments, adding 6°C pre-cooled physiological saline of 4 times the mass of the turtle meat fragments to the turtle meat fragments, crushing and homogenizing, so that the turtle meat tissue is fully broken, releasing the protein components in the cells, and obtaining a homogenate; centrifuging the homogenate at 4000r / min for 30min, taking the supernatant, adding ammonium sulfate to the supernatant, stirring while adding, so that the protein gradually precipitates out, and then standing at 4°C for 6h to allow the precipitate to fully form, and then centrifuging at 6000r / min for 30min, collecting the precipitate, and washing and desalting the precipitate twice to obtain turtle protein. Add turtle protein to distilled water with a mass of 12 times that of turtle protein, stir evenly to make a protein homogenate, adjust the pH to 6.5, add 1% of the mass of turtle protein serrapeptase, enzymolyze at 50°C for 60 minutes, heat to 95°C to inactivate the enzyme for 25 minutes, reduce the temperature to 60°C, adjust the pH to 8.6, add 0.5% of the mass of turtle protein keratinase, enzymolyze at 55°C for 90 minutes, heat to 90°C to inactivate the enzyme for 25 minutes, reduce the temperature to 55°C, adjust the pH to 6.5, add 1% of the mass of turtle protein nattokinase, enzymolyze at 55°C for 50 minutes, heat to 90°C to inactivate the enzyme for 20 minutes, reduce the temperature to room temperature, centrifuge at 6000r / min for 15 minutes, take the supernatant, use a 10kDa ultrafiltration membrane for ultrafiltration, take the ultrafiltration component below 10kDa, freeze-dry, and obtain turtle extracted peptide.

[0054] The preparation method of white tuckahoe peptide comprises the following steps: crushing white tuckahoe, adding distilled water 12 times the weight of white tuckahoe, adjusting the pH to 5.5, sequentially adding hemicellulase 1.5% by weight of white tuckahoe, pectinase 0.5% by weight of white tuckahoe, and fig protease 1.5% by weight of white tuckahoe, performing enzymolysis at 55°C for 90 minutes, heating to 95°C to inactivate the enzyme for 25 minutes, cooling to room temperature, adjusting the pH to 8.0, adding trypsin 0.5% by weight of white tuckahoe, and performing enzymolysis at 55°C for 90 minutes. Enzyme, constant temperature enzymolysis at 38 ° C for 80 minutes, heated to 90 ° C to inactivate the enzyme for 20 minutes, lowered to 60 ° C, adjusted to pH 6.0, added bromelain (1.0% by weight of white Poria cocos), constant temperature enzymolysis at 60 ° C for 50 minutes, heated to 90 ° C to inactivate the enzyme for 20 minutes, lowered to room temperature, centrifuged at 6000 r / min for 15 minutes, took the supernatant, ultrafiltered with a 5 kDa ultrafiltration membrane, took the ultrafiltration component below 5 kDa, freeze-dried, and obtained white Poria cocos peptide.

[0055] Among them, the preparation method of agarwood extract includes the following steps: taking agarwood medicinal materials, crushing them to obtain powder, adding ethanol 8 times the mass of the powder to the powder, mixing evenly, sealing and soaking for 9 days in a light-proof and room temperature environment, centrifuging at 6000r / min for 15min, taking the supernatant, evaporating under reduced pressure at 50°C to remove ethanol, and drying at 40°C to constant weight to obtain the agarwood extract.

[0056] The preparation method of the snake melon seed extract comprises the following steps: taking the seed flesh of the snake melon seeds, crushing it to obtain a crushed material, adding distilled water 12 times the mass of the crushed material, adjusting the pH to 5.5, sequentially adding hemicellulase 1.5% by mass of the crushed material, pectinase 0.5% by mass of the crushed material, and fig protease 1.5% by mass of the crushed material, performing enzymolysis at 55°C for 90 minutes, heating to 95°C to inactivate the enzyme for 25 minutes, reducing the temperature to 65°C, adding papain 1% by mass of the crushed material, performing enzymolysis at 65°C for 60 minutes, heating to 95°C to inactivate the enzyme for 35 minutes, reducing the temperature to room temperature, adding ethanol 3 times the mass of the crushed material to soak for 1 hour, and then centrifuging at 6000r / min for 15 minutes, taking the supernatant, ultrafiltering with a 5kDa ultrafiltration membrane, taking the ultrafiltration component below 5kDa, and freeze-drying to obtain the snake melon seed extract.

[0057] The method for preparing the above-mentioned agarwood-containing composition for improving immunity comprises the following steps:

[0058] According to the mass proportions, astragalus, houttuynia, honeysuckle, blueberry, sea cucumber and zinc gluconate were mixed evenly at 20°C and 30 rpm, and then the agarwood extract and snake melon seed extract were added, and the mixing was continued at 20°C and 30 rpm, and finally the turtle extract peptide and white Poria peptide were added, and the mixing was evenly performed at 20°C and 30 rpm to obtain a composition.

[0059] Example 5

[0060] A composition containing agarwood for improving immunity, the composition comprises the following raw materials in parts by weight: 8 parts of turtle extract peptide, 1 part of white tuckahoe peptide, 1.5 parts of agarwood extract, 0.3 parts of snake gourd seed extract, 12 parts of astragalus, 1 part of houttuynia, 3 parts of honeysuckle, 0.5 parts of blueberry, 1 part of sea cucumber, and 0.05 parts of zinc gluconate. Among them, astragalus is astragalus dry powder, houttuynia is houttuynia dry powder, honeysuckle is honeysuckle dry powder, blueberry is blueberry dry powder, and sea cucumber is sea cucumber dry powder; astragalus dry powder, houttuynia dry powder, honeysuckle dry powder, blueberry dry powder, sea cucumber dry powder and zinc gluconate are all sieved through 150 mesh.

[0061] The preparation method of turtle extracted peptides includes the following steps: taking fresh turtle meat of Chinese soft-shelled turtle, washing, draining, cutting into small pieces less than 2 cm, obtaining turtle meat fragments, adding 6 times the mass of 6°C pre-cooled physiological saline to the turtle meat fragments, crushing and homogenizing, so that the turtle meat tissue is fully broken, releasing the protein components in the cells, and obtaining a homogenate; centrifuging the homogenate at 6000r / min for 30min, taking the supernatant, adding ammonium sulfate to the supernatant, stirring while adding, so that the protein gradually precipitates out, and then standing at 6°C for 6h to allow the precipitate to fully form, and then centrifuging at 8000r / min for 30min, collecting the precipitate, and washing and desalting the precipitate three times to obtain turtle protein. Add turtle protein to distilled water with a mass of 12 times that of turtle protein, stir evenly to prepare protein homogenate, adjust pH7.0, add 1% of turtle protein by mass of serrapeptase, enzymolyze at 50°C for 90 minutes, heat to 95°C to inactivate the enzyme for 30 minutes, reduce to 45°C, adjust pH9.0, add 1% of turtle protein by mass of keratinase, enzymolyze at 45°C for 90 minutes, heat to 95°C to inactivate the enzyme for 25 minutes, reduce to 52°C, adjust pH7.0, add 1% of turtle protein by mass of nattokinase, enzymolyze at 52°C for 80 minutes, heat to 90°C to inactivate the enzyme for 25 minutes, reduce to room temperature, centrifuge at 6000r / min for 20 minutes, take the supernatant, ultrafilter with a 10kDa ultrafiltration membrane, take the ultrafiltration fraction below 10kDa, freeze-dry, and obtain turtle extracted peptide.

[0062] The preparation method of white tuckahoe peptide comprises the following steps: crushing white tuckahoe, adding distilled water 12 times the weight of white tuckahoe, adjusting the pH to 6.5, adding hemicellulase 1.5% by weight of white tuckahoe, pectinase 1.5% by weight of white tuckahoe, and fig protease 1.5% by weight of white tuckahoe in sequence, performing enzymolysis at 55°C for 120 minutes, heating to 90°C to inactivate the enzyme for 15 minutes, cooling to room temperature, adjusting the pH to 8.0, adding trypsin 1.0% by weight of white tuckahoe, and performing enzymolysis at 55°C for 120 minutes. White tuckahoe peptide was prepared by enzymolysis at 36°C for 80 min, then heated to 90°C for 15 min to inactivate the enzyme, then cooled to 58°C, adjusted to pH 7.0, added with bromelain (1.0% by weight of white tuckahoe), and enzymolysis was carried out at 58°C for 80 min, then heated to 90°C for 15 min to inactivate the enzyme, cooled to room temperature, and centrifuged at 6000 r / min for 20 min. The supernatant was taken and ultrafiltered with a 5 kDa ultrafiltration membrane. The ultrafiltration fraction below 5 kDa was taken and freeze-dried to obtain white tuckahoe peptide.

[0063] Among them, the preparation method of agarwood extract includes the following steps: taking agarwood medicinal materials, crushing them to obtain powder, adding ethanol 8 times the mass of the powder to the powder, mixing evenly, sealing and soaking for 10 days in a light-proof room temperature environment, centrifuging at 6000r / min for 20min, taking the supernatant, evaporating under reduced pressure at 50°C to remove ethanol, and drying at 45°C to constant weight to obtain the agarwood extract.

[0064] The preparation method of the snake melon seed extract comprises the following steps: taking the seed flesh of the snake melon seeds, crushing it to obtain a crushed material, adding distilled water 12 times the mass of the crushed material, adjusting the pH to 6.5, sequentially adding hemicellulase 1.5% by mass of the crushed material, pectinase 1.5% by mass of the crushed material, and fig protease 1.5% by mass of the crushed material, performing enzymolysis at 53°C for 120 minutes, heating to 90°C to inactivate the enzyme for 30 minutes, reducing the temperature to 62°C, adding papain 1% by mass of the crushed material, performing enzymolysis at 62°C for 90 minutes, heating to 95°C to inactivate the enzyme for 30 minutes, reducing the temperature to room temperature, adding ethanol 3 times the mass of the crushed material to soak for 2 hours, and then centrifuging at 6000r / min for 20 minutes, taking the supernatant, ultrafiltering with a 5kDa ultrafiltration membrane, taking the ultrafiltration component below 5kDa, and freeze-drying to obtain the snake melon seed extract.

[0065] The method for preparing the above-mentioned agarwood-containing composition for improving immunity comprises the following steps:

[0066] According to the mass proportions, astragalus, houttuynia, honeysuckle, blueberry, sea cucumber and zinc gluconate were mixed evenly at 16°C and 20 rpm, and then the agarwood extract and snake melon seed extract were added, and the mixing was continued at 16°C and 20 rpm, and finally the turtle extract peptide and white Poria peptide were added, and the mixing was continued at 16°C and 20 rpm to obtain a composition.

[0067] In the above embodiments, the pH value was adjusted using 0.2 mol / L hydrochloric acid aqueous solution and 0.2 mol / L sodium hydroxide aqueous solution.

[0068] In the above-mentioned embodiments, in the method for preparing turtle protein, water washing and desalting is to resuspend the precipitate with distilled water, centrifuge and take the precipitate, which is one water washing and desalting.

[0069] The agarwood raw materials used in the above embodiments are sourced from Maoming Hetai Agarwood Planting Co., Ltd.

[0070] The enzyme sources used in the above embodiments are as follows: the enzyme activity of serrapeptase is 100,000 U / g, which is derived from Jiangsu Aofu Biotechnology Co., Ltd.; the enzyme activity of keratinase is 200,000 U / g, which is derived from Suzhou Zhuoxin Biotechnology Co., Ltd.; the enzyme activity of nattokinase is 40,000 U / g, which is derived from Shaanxi Langde Biotechnology Co., Ltd.; the enzyme activity of hemicellulase is 200,000 U / g, which is derived from Jiangsu Yuanshengtong Bioengineering Co., Ltd.; the enzyme activity of pectinase is 100,000 U / g, which is derived from Shandong Zheque Bioengineering Co., Ltd.; the enzyme activity of figin is 100,000 U / g, which is derived from Nanjing Shengshi Fanhua Biotechnology Co., Ltd.; the enzyme activity of trypsin is 4,000 U / g, which is derived from Xi'an Muguo Biotechnology Co., Ltd., porcine trypsin; the enzyme activity of bromelain is 200,000 U / g, which is derived from Shanxi Xinyu Biotechnology Co., Ltd.; the enzyme activity of papain is 100,000 U / g, which is derived from Shandong Jinchengda Biotechnology Co., Ltd.

[0071] Comparative Example 1

[0072] In the method for preparing turtle extracted peptides, the turtle protein is not enzymatically hydrolyzed by keratinase and nattokinase; other parameters and methods are the same as in Example 1.

[0073] Comparative Example 2

[0074] In the method for preparing turtle extracted peptides, the turtle protein is not enzymatically hydrolyzed by serrapeptase and keratinase; other parameters and methods are the same as those in Example 1.

[0075] Comparative Example 3

[0076] In the method for preparing turtle extracted peptides, the turtle protein is not enzymatically hydrolyzed by nattokinase; other parameters and methods are the same as those in Example 1.

[0077] Comparative Example 4

[0078] In the preparation method of white Poria cocos peptide, white Poria cocos is not enzymatically hydrolyzed by ficin; other parameters and methods are the same as those in Example 1.

[0079] Comparative Example 5

[0080] In the preparation method of white Poria cocos peptide, trypsin and bromelain are not used for enzymolysis of white Poria cocos; other parameters and methods are the same as those in Example 1.

[0081] Comparative Example 6

[0082] In the preparation method of white Poria cocos peptide, white Poria cocos is not enzymatically hydrolyzed by bromelain; other parameters and methods are the same as those in Example 1.

[0083] Comparative Example 7

[0084] No agarwood extract is added to the composition; other parameters and methods are the same as in Example 1.

[0085] Comparative Example 8

[0086] No snake gourd seed extract was added to the composition; other parameters and methods were the same as in Example 1.

[0087] Comparative Example 9

[0088] Neither agarwood extract nor snake melon seed extract is added to the composition; other parameters and methods are the same as in Example 1.

[0089] Comparative Example 10

[0090] In the preparation method of the snake gourd seed extract, ficin is not used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0091] The compositions prepared in the above-mentioned embodiments and comparative examples were tested for safety and efficacy.

[0092] 1. Safety Testing

[0093] (1) Acute toxicity test: 160 healthy adult SPF Kunming mice were selected, half of which were male and half were female, and randomly divided into 16 groups (10 mice in each group), of which one group was a blank control group, which was given an equal amount of physiological saline by gavage. The remaining example groups and comparative example groups were given 4 mg / 20 g body weight of the agarwood composition by oral gavage to the mice. The mice were observed for 14 consecutive days, and their diet, drinking water, activities, fur and death were observed. The results showed that no poisoning symptoms or deaths occurred in any group, and each composition was safe and non-toxic.

[0094] (2) Long-term toxicity test: 160 healthy adult SPF Kunming mice were selected, half of which were male and half were female, and randomly divided into 16 groups (10 mice in each group), of which one group was a blank control group, which was given an equal amount of physiological saline by gavage. The remaining example groups and comparative example groups were given 0.5 mg / 20 g body weight of the agarwood-containing composition by oral gavage for 90 consecutive days, once a day. During the administration period, the general condition of the mice, including body weight, food intake, etc., was observed. After the experiment, the mice were killed and pathological histological examinations were performed on the main organs (including heart, liver, spleen, lung, and kidney). The results showed that the daily behavior of the mice was normal, and no obvious specific lesions were found in the histopathological examinations of the organs, indicating that the compositions were safe.

[0095] 2. Efficacy Testing

[0096] 340 SPF-grade healthy adult Kunming mice, half male and half female, were selected and randomly divided into 17 groups (20 mice in each group), of which 1 group was a blank control group, 1 group was an immune model group, and the rest were test groups (corresponding to the compositions of each comparative example and each embodiment, respectively). The mice in the immune model group and each test group were intraperitoneally injected with 80 mg / kg of cyclophosphamide to establish an immunosuppression model; the blank control group was only injected with normal saline. After the model was successfully established (white blood cell count ≤ 50% of the white blood cell count of the blank control group), the mice in each test group were gavaged with the composition at 0.3 mg / 20 g body weight every day, and the blank control group and the immune model group were gavaged with an equal amount of normal saline for 14 consecutive days. 24 hours after the last administration: 10 mice were randomly selected from each group for orbital blood sampling, serum separation, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of immunoglobulin G (IgG), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) in the serum. At the same time, the spleen of the mice was taken, and spleen cell suspension was prepared. The MTT method (detection wavelength was 570 nm) was used to detect the OD value of the proliferation ability of spleen lymphocytes. The higher the OD value, the stronger the metabolic activity of the cells. For the remaining 10 mice in each group, 1 ml of the concentration of 10 5 PFU / mL of influenza A virus H1N1, observe and record the number of mice that died within 14 days after infection, and calculate the mortality rate and survival protection rate. Mortality rate = number of mice surviving / total number of mice, survival protection rate = (number of mice surviving in the test group / total number of mice in the test group) × 100% - (number of mice surviving in the immune model group / total number of mice in the immune model group) × 100%. The test results are shown in Table 1 below.

[0097] Table 1 Efficacy index test results (average value)

[0098]

[0099] From the above experimental results, it can be seen that the agarwood-containing compositions prepared in Examples 1 to 5 have safe ingredients and no toxic effects. Compared with the immune model group, they can significantly increase the levels of immunoglobulin G (IgG) and interleukin-2 (IL-2) in human blood, and well regulate the content of tumor necrosis factor-α (TNF-α), so that the body maintains a healthy state. It can also increase the proliferation capacity of spleen lymphocytes, enhance the body's immune defense function, and enhance the body's resistance to infection.

[0100] The turtle extracted peptides of Example 1 contain small molecule peptides with special efficacy ingredients for improving immunity. These small molecule peptides are more easily absorbed and utilized by the human body, thereby having a better effect of improving immunity. It can be seen from the results of Comparative Examples 1 to 3 that in the preparation method of turtle extracted peptides, the turtle protein is not enzymatically hydrolyzed by keratinase and nattokinase, or is not enzymatically hydrolyzed by serrapeptase and keratinase, or is not enzymatically hydrolyzed by nattokinase, the composition of the extracted product is changed, and the peptide composition is different, resulting in a decrease in the effect of the composition in improving the body's immunity and enhancing resistance to infection.

[0101] The white Poria cocos peptide of Example 1 has a peptide segment with specific biological activity, which has a positive effect on regulating the immune system, enhancing the body's resistance, etc.; from the results of Comparative Examples 4 to 6, it can be seen that in the preparation method of white Poria cocos peptide, white Poria cocos is not enzymatically hydrolyzed by ficin, or is not enzymatically hydrolyzed by trypsin and bromelain, or is not enzymatically hydrolyzed by bromelain, and the composition of the extracted product changes, especially the structure of the small molecule peptide changes, resulting in a decrease in the effect of the composition on improving the body's immunity and enhancing resistance to infection.

[0102] From the results of Comparative Example 7, it can be seen that agarwood extract contains anti-inflammatory and antioxidant components, which indirectly regulate the normal functions of immune cells and improve the body's immune defense capabilities. If agarwood extract is not added to the composition, the effect of improving the body's immunity and enhancing resistance to infection is reduced.

[0103] From the results of Comparative Example 8, it can be seen that the snake melon seed extract helps to regulate the body's immune balance. Without adding the snake melon seed extract to the composition, the functions of regulating immune cell activity and promoting the secretion of immune factors are weakened.

[0104] From the results of Comparative Example 9, it can be seen that if agarwood extract and snake melon seed extract are not added to the composition at the same time, the effects of improving the body's immunity and enhancing infection resistance are significantly reduced, and the two have a good synergistic effect.

[0105] From the results of Comparative Example 10, it can be seen that in the preparation method of the snake gourd seed extract, ficin is not used for enzymatic hydrolysis, and the composition of the extracted product changes, resulting in a certain decrease in the effect of the composition on improving the body's immunity and enhancing resistance to infection.

Claims

1. An agarwood composition for improving immunity, characterized in that: The composition comprises the following raw materials in parts by weight: 5 to 8 parts of turtle extract peptide, 1 to 3 parts of white tuckahoe peptide, 0.5 to 1.5 parts of agarwood extract, 0.3 to 0.8 parts of snake gourd seed extract, 8 to 12 parts of astragalus, 1 to 3 parts of houttuynia cordata, 1 to 3 parts of honeysuckle, 0.5 to 2 parts of blueberry, 0.5 to 1 part of sea cucumber and 0.05 to 0.1 parts of zinc gluconate; The turtle extracted peptide is an extracted peptide below 10 kDa obtained by sequentially enzymatically hydrolyzing turtle protein with serrapeptase, keratinase and nattokinase; the white poria peptide is an extracted peptide below 5 kDa obtained by co-enzymatically hydrolyzing white poria with hemicellulase, pectinase and ficin, followed by graded enzymatic hydrolysis with trypsin and bromelain; the agarwood extract is an ethanol-soaked extract of agarwood; the snake melon seed extract is a product obtained by co-enzymatically hydrolyzing the seed flesh of snake melon seeds with hemicellulase, pectinase and ficin, followed by enzymatic hydrolysis with papain, and then extracting with ethanol to obtain an extract below 5 kDa.

2. The agarwood-containing composition for improving immunity according to claim 1, characterized in that: The preparation method of turtle extracted peptide comprises the following steps: adding turtle protein to distilled water with a mass of 8 to 12 times that of turtle protein, stirring evenly to prepare protein homogenate, adjusting pH to 6.5 to 7.0, adding 0.5% to 1% by mass of serrapeptase of turtle protein, performing enzymolysis at 45 to 50° C. for 60 to 90 minutes, heating to inactivate enzyme, cooling to 40 to 60° C., adjusting pH to 7.0 to 9.0, adding 0.5% to 1% by mass of keratinase of turtle protein, performing enzymolysis at 45 to 55° C. for 60 minutes, min~90min, heat up to inactivate enzyme, reduce to 40℃~55℃, adjust pH6.5~7.0, add 0.5%~1% by weight of nattokinase of turtle protein, perform enzymolysis at 50℃~55℃ for 50min~80min, heat up to inactivate enzyme, reduce to room temperature, centrifuge at 4000r / min~6000r / min for 15min~20min, take supernatant, perform ultrafiltration with 10kDa ultrafiltration membrane, take ultrafiltration component below 10kDa, freeze-dry, and obtain turtle extracted peptide.

3. The agarwood-containing composition for improving immunity according to claim 2, characterized in that: The method for preparing turtle protein comprises the following steps: taking fresh turtle meat of Chinese soft-shelled turtle, washing, draining, cutting into small pieces less than 2 cm, obtaining turtle meat fragments, adding 4°C to 6°C pre-cooled physiological saline of 4 to 6 times the mass of the turtle meat fragments to the turtle meat fragments, crushing and homogenizing, so that the turtle meat tissue is fully broken, and the protein components in the cells are released to obtain a homogenate; centrifuging the homogenate at 4000 r / min to 6000 r / min for 20 to 30 min, taking the supernatant, adding ammonium sulfate to the supernatant, stirring while adding, so that the protein is gradually precipitated, and then standing at 4°C to 6°C for 4h to 6h to allow the precipitate to be fully formed, and then centrifuging at 6000 r / min to 8000 r / min for 20min to 30min, collecting the precipitate, and washing and desalting the precipitate 2 to 3 times to obtain turtle protein.

4. The agarwood-containing composition for improving immunity according to claim 1, characterized in that: The preparation method of white tuckahoe peptide comprises the following steps: taking white tuckahoe and crushing it, adding distilled water of 8 to 12 times the mass of white tuckahoe, adjusting the pH to 5.5 to 6.5, sequentially adding hemicellulase of 0.5% to 1.5% by mass of white tuckahoe, pectinase of 0.5% to 1.5% by mass of white tuckahoe, and fig proteinase of 0.5% to 1.5% by mass of white tuckahoe, performing enzymolysis at a constant temperature of 50°C to 55°C for 90min to 120min, heating up to inactivate the enzyme, cooling down to room temperature, adjusting the pH to 7.5 to 8.0, adding trypsin of 0.5% to 1.0% by mass of white tuckahoe, and performing enzymolysis at a constant temperature of 50°C to 55°C for 90min to 120min, heating up to inactivate the enzyme, cooling down to room temperature, adjusting the pH to 7.5 to 8.0, and White tuckahoe peptide was prepared by enzymolysis at 35-38°C for 50-80 min, heating to inactivate the enzyme, cooling to 50-60°C, adjusting the pH to 6.0-7.0, adding bromelain (0.5%-1.0% by weight of white tuckahoe) and enzymolysis at 50-60°C for 50-80 min, heating to inactivate the enzyme, cooling to room temperature, centrifuging at 4000-6000 r / min for 15-20 min, taking the supernatant, ultrafiltration with a 5 kDa ultrafiltration membrane, taking the ultrafiltration fraction below 5 kDa, freeze-drying, and obtaining white tuckahoe peptide.

5. The agarwood-containing composition for improving immunity according to claim 1, characterized in that: The preparation method of the agarwood extract comprises the following steps: taking agarwood medicinal materials, crushing them to obtain powder, adding ethanol 6 to 8 times the mass of the powder to the powder, mixing them evenly, sealing and soaking them in a light-proof room temperature environment for 7 to 10 days, centrifuging at 4000 r / min to 6000 r / min for 15 to 20 minutes, taking the supernatant, evaporating under reduced pressure at 40°C to 50°C to remove ethanol, and drying at 40°C to 45°C to constant weight to obtain the agarwood extract.

6. The agarwood-containing composition for improving immunity according to claim 1, characterized in that: The preparation method of the snake melon seed extract comprises the following steps: taking the seed flesh of the snake melon seed, crushing it to obtain a crushed product, adding distilled water 8 to 12 times the crushed product, adjusting the pH to 5.5 to 6.5, sequentially adding hemicellulase 0.5 to 1.5% by weight of the crushed product, pectinase 0.5 to 1.5% by weight of the crushed product, and fig protease 0.5 to 1.5% by weight of the crushed product, performing enzymolysis at a constant temperature of 50 to 55° C. for 90 to 120 minutes, heating to inactivate the enzyme, and then cooling to 55° C. ~65℃, add 0.5%~1% papain by weight of the crushed material, perform enzymolysis at 55℃~65℃ for 60min~90min, raise the temperature to inactivate the enzyme, cool to room temperature, add 1 to 3 times the weight of ethanol by weight of the crushed material to soak for 1h~2h, then centrifuge at 4000r / min~6000r / min for 15min~20min, take the supernatant, perform ultrafiltration with a 5kDa ultrafiltration membrane, take the ultrafiltration component below 5kDa, freeze-dry, and obtain the snake melon seed extract.

7. The agarwood-containing composition for improving immunity according to claim 2, 4 or 6, characterized in that: The temperature for heating the enzyme to be inactivated is 90° C. to 95° C., and the time for heating the enzyme to be inactivated is 15 min to 40 min.

8. The agarwood-containing composition for improving immunity according to claim 1, characterized in that: The astragalus is astragalus dry powder, the houttuynia cordata is houttuynia cordata dry powder, the honeysuckle is honeysuckle dry powder, the blueberry is blueberry dry powder, and the sea cucumber is sea cucumber dry powder; the astragalus dry powder, houttuynia cordata dry powder, honeysuckle dry powder, blueberry dry powder, sea cucumber dry powder and zinc gluconate are all sieved through 150-250 meshes.

9. A method for preparing an agarwood composition for improving immunity, for preparing the agarwood composition for improving immunity as claimed in claim 1, characterized in that: The preparation method comprises the following steps: According to the mass proportions, astragalus, houttuynia, honeysuckle, blueberry, sea cucumber and zinc gluconate were mixed evenly, and then agarwood extract and snake melon seed extract were added, and the mixture was continued to be evenly mixed. Finally, turtle extract peptide and white Poria peptide were added, and the mixture was evenly mixed to obtain a composition.

10. The method for preparing the agarwood composition for improving immunity according to claim 9, characterized in that: The mixing speed is 10 rpm to 30 rpm; the mixing temperature is below 20°C.

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