CN111657501A - Oyster peptide cordyceps sinensis mycelium composition with function of enhancing immunity and application thereof - Google Patents
Oyster peptide cordyceps sinensis mycelium composition with function of enhancing immunity and application thereof Download PDFInfo
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- CN111657501A CN111657501A CN202010410352.XA CN202010410352A CN111657501A CN 111657501 A CN111657501 A CN 111657501A CN 202010410352 A CN202010410352 A CN 202010410352A CN 111657501 A CN111657501 A CN 111657501A
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- oyster peptide
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- cordyceps
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Abstract
The invention provides an oyster peptide cordyceps mycelium composition with an immunity enhancing function and application thereof, wherein the oyster peptide cordyceps mycelium composition comprises the following components in parts by weight: 25-48 parts of oyster peptide; 30-45 parts of cordyceps mycelia; 5-20 parts of astragalus extract; 2-10 parts of a wolfberry fruit extract. The invention adopts oyster peptide and cordyceps sinensis fermentation mycelium as main raw materials, and astragalus extract and medlar extract as auxiliary raw materials, and has scientific compatibility and synergistic effect. In addition, the invention adopts raw materials which are homologous in medicine and food, has the advantages of efficacy and safety, no toxic or side effect and can be widely applied to the development of various health-care foods and foods.
Description
Technical Field
The invention belongs to the technical field of food processing, and particularly relates to an oyster peptide cordyceps mycelium composition with an immunity enhancing function and application thereof.
Background
The oyster is the first cultured shellfish in the world and is also one of four cultured shellfish in China. The oyster contains rich nutrient substances, such as protein, glycogen, vitamins, and trace elements such as zinc, selenium, iron, copper, iodine, and the like, and particularly contains rich taurine, so that the oyster is a marine mollusk with extremely high nutritional value. Oyster meat is not only rich in nutrition, but also has unique physiological health care function and medicinal value, oyster is recorded in compendium of materia medica to have the efficacies of reducing phlegm and softening hard masses, clearing heat and removing dampness, relieving heart and spleen qi pain, dysentery, red and white turbidity, eliminating hernia and masses, goiter and tuberculosis; the Shen nong Ben Cao Jing records that oyster has the effects of astringing yin, suppressing yang, suppressing sweating, reducing phlegm and softening hard masses, and oyster is approved by the Ministry of health of China to be listed as one of health care products which are both medicines and foods. The preparation of oyster peptide by utilizing oyster proteolysis is an important way for high-value utilization of oysters, and the oyster peptide completely reserves the original nutritional ingredients of the oysters, such as vitamins, trace elements, taurine and the like, is more easily absorbed and utilized by the body and plays a biological function. Modern medical research proves that the oyster peptide has the physiological effects of enhancing immunity, resisting fatigue, resisting oxidation, resisting aging, resisting tumors, reducing blood sugar, inhibiting ACE activity and the like.
Cordyceps sinensis is a dry complex of stroma and larva corpse of Cordyceps sinensis parasitizing on larva of insect of Hepialidae, and is medicinal fungus of Cordyceps. Cordyceps sinensis is sweet in flavor and warm in nature. It enters lung and kidney meridians. The Chinese medicinal composition is mild and nourishing, has the effects of tonifying kidney, benefiting lung, stopping bleeding and reducing phlegm, is one of rare Chinese medicinal materials for nourishing and strengthening body, has various physiological pharmacology and active functions, and is used for treating diseases such as kidney deficiency and essence deficiency, impotence and spermatorrhea, soreness and pain of waist and knees, chronic cough and dyspnea due to deficiency of vital energy, chronic cough and hemoptysis. Because of the strict parasitic conditions and special geographical environment of the cordyceps sinensis, and the mining of wild cordyceps sinensis and the damage of the propagation environment of the cordyceps sinensis by people, the natural yield is sharply reduced, and the resources are gradually exhausted. Numerous studies have shown that: the fermented cordyceps sinensis mycelia and the natural cordyceps sinensis have similar chemical components, pharmacological effects and clinical effects, can be used as a substitute of the natural cordyceps sinensis to effectively make up for the deficiency of wild cordyceps sinensis resources, and becomes a hotspot for development of the cordyceps sinensis resources. According to the research on literature data and the database of the State administration of market supervision and management, the existing cordyceps mycelium health-care food is mostly prepared by directly and simply crushing fermented cordyceps mycelium and then preparing the crushed cordyceps mycelium into products such as capsules, tablets and the like, and because the cordyceps mycelium is high in price and large in dosage, the eating cost is high, and the cordyceps mycelium is unacceptable for common people; secondly, pure fungus powder products have single functions, are not compounded with effective functions, are difficult to exert the maximum efficacy of the cordyceps sinensis, cause the waste of nutrient components and seriously limit the high-valued development and utilization and economic benefits of the cordyceps sinensis mycelia.
Oyster peptide is used as a high-quality marine functional protein peptide, cordyceps sinensis is used as a high-quality edible and medicinal fungus resource, the oyster peptide has unique physiological functions, and certain research and application are realized in the aspect of functional food development.
Disclosure of Invention
Aiming at the existing problems and product defects, the invention provides an oyster peptide cordyceps mycelium composition with the function of enhancing immunity and application thereof. The oyster peptide and the cordyceps sinensis fermented mycelia are used as main raw materials, the astragalus extract and the medlar extract are used as auxiliary raw materials, the compatibility is scientific, the synergy is realized, and the composition can remarkably enhance the immunity of the organism and also has certain functions of resisting viruses, tonifying the kidney and benefiting the lung.
In order to achieve the purpose, the invention adopts the following technical scheme:
the oyster peptide cordyceps mycelium composition with the function of enhancing immunity comprises the following components in parts by weight:
oyster peptide: 25-48 parts;
cordyceps mycelia: 30-45 parts of a stabilizer;
astragalus extract: 5-20 parts of a stabilizer;
wolfberry fruit extract: 2-10 parts.
Further, the oyster peptides are small molecular oligopeptides with molecular weight less than 1000Da and protein content more than 75%.
Further, the preparation method of the oyster peptide comprises the following steps:
(1) soaking fresh oysters for desalination, cleaning, shelling and removing internal organs to obtain desalted oyster meat, and homogenizing the desalted oyster meat to obtain oyster slurry;
(2) adding water into the oyster slurry, then adding chitosan immobilized compound protease, carrying out heat preservation and enzymolysis, and centrifuging to obtain supernatant, namely oyster enzymolysis liquid;
(3) ultrafiltering the Concha Ostreae enzymolysis solution with ultrafiltration membrane with cut-off molecular weight of 1000 daltons, collecting filtrate, and concentrating the filtrate to obtain concentrated solution;
(4) spray drying the concentrated solution to obtain oyster peptide.
Further, the weight ratio of the chitosan immobilized compound protease to the desalted oyster meat is 0.2-1%, wherein the mass ratio of the compound protease to the chitosan immobilized compound protease is 5-15%.
Further, the compound protease comprises pancreatin, papain and flavourzyme, and the weight ratio of the pancreatin to the papain to the flavourzyme is 3-5: 1-2.
Further, the cordyceps sinensis mycelia are prepared by submerged fermentation of cordyceps sinensis liquid, and the adenosine content is more than 0.8 per mill; the Cordyceps is hirsutella hepiali Chen et Shen or Paecilomyces hepiali Chen et Shen.
Furthermore, the content of protein in the oyster peptide cordyceps mycelium composition is more than 30%, and the content of adenosine is more than 0.3 per mill.
Further, the oyster peptide cordyceps mycelium composition comprises the following components in parts by weight: 45 parts of oyster peptide, 40 parts of hirsutella hepiali Chen et Shen mycelium, 10 parts of astragalus extract and 5 parts of wolfberry extract.
The invention provides application of the oyster peptide and cordyceps mycelia composition in preparation of health-care products or foods.
Furthermore, the dosage form of the health care product is oral liquid, capsules, tablets, pills, powder or granules.
Further, the chitosan immobilized compound protease is chitosan microparticle immobilized compound protease or magnetic Fe3O4-chitosan microparticles immobilized complex protease.
Further, the concentration method is vacuum concentration or nanofiltration concentration.
Compared with the prior art, the invention has the advantages and beneficial effects that:
(1) the invention takes oyster peptide and cordyceps mycelia as main materials and takes astragalus extract and medlar extract as auxiliary materials, and the oyster peptide and the cordyceps mycelia are scientifically matched and synergized to fully exert the efficacy and activity of nutrient active substances. The oyster peptide is a low molecular peptide prepared by precisely and efficiently carrying out enzymolysis on oysters by using chitosan immobilized compound protease, the process is simple and efficient, byproducts are few, the content of low molecular oyster peptide protein is high, the sensory flavor is excellent, nutritional and functional components such as vitamins, mineral substances, taurine and the like contained in the oysters are completely reserved, the absorption and utilization rate is high, and researches show that the oyster peptide has an excellent immunity enhancing function. The cordyceps sinensis fermentation mycelium contains active substances and physiological functions of cordyceps sinensis polysaccharide, adenosine and the like which are similar to wild cordyceps sinensis resources, and has a good immunity enhancing function. The oyster peptide and the cordyceps sinensis mycelia are scientifically compatible, so that the immunity enhancing function of the oyster protein peptide, the cordyceps sinensis polysaccharide and the cordyceps sinensis adenosine (nucleic acid) is jointly exerted, different nutrients can be mutually promoted to be absorbed, the absorption utilization rate of active nutrients is improved, the nutrient waste is avoided, the activity effect is better exerted, the oyster cordyceps sinensis mycelia simultaneously contain three nutrients of protein, polysaccharide and nucleic acid, the nutrition is balanced, the development of products with different functions is facilitated, and the application range is wider.
(2) Huang Qi, originally listed as the top grade in Shen nong Ben Cao Jing, is sweet in taste, slightly warm in nature, enters lung and spleen meridians, and has a variety of pharmacological actions such as immunoregulation, organ protection, blood sugar reduction, anti-tumor and anti-aging. The astragalus polysaccharide is used as the main active component of the astragalus extract, can promote the differentiation of immune cells, regulate immune response factors, components and enzyme activity, regulate and control the transcription expression of immune related genes, promote the development of immune organs and play a remarkable role in immune regulation; the astragalus polysaccharide has a remarkable immunity enhancing function and a remarkable antiviral effect, the cell survival rate of the astragalus polysaccharide under the same concentration is higher than that of acyclovir, after the astragalus polysaccharide acts on the type I herpes simplex virus, the cytopathology change is similar to that of acyclovir, and the antiviral effect of the astragalus polysaccharide is better than that of acyclovir. Astragalus membranaceus, a traditional Chinese medicine with the effect of remarkably enhancing immunity and antivirus, has been listed in 'diagnosis and treatment scheme of pneumonia infected by novel coronavirus' and applied to resisting novel coronavirus pneumonia epidemic situation. The medlar serving as a traditional Chinese medicine has the effects of tonifying kidney, producing sperm, nourishing liver, improving eyesight, enriching blood, soothing nerves, promoting fluid production, quenching thirst, moistening lung, relieving cough and the like. The fructus Lycii extract is mainly fructus Lycii polysaccharide, and has effects of regulating immunity, resisting oxidation, resisting aging, reducing blood sugar, reducing blood lipid, etc. According to the invention, by scientifically mixing the raw materials such as the oyster peptide, the cordyceps sinensis mycelium, the astragalus extract, the wolfberry extract and the like, the nutrition effect components are balanced, the sensory flavor is excellent, the active components can be mutually promoted to be absorbed, the nutrition function and the activity effect of different raw materials can be better exerted, the immunity enhancement function of the product is obviously enhanced, and the functions of resisting virus, tonifying kidney and benefiting lung and the like are achieved.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the following specific examples.
Example 1: oyster peptide cordyceps mycelium composition and product development
The oyster peptide cordyceps sinensis mycelium composition with the function of enhancing immunity provided by the invention comprises the following components in parts by weight: 25-48 parts of oyster peptide, 30-45 parts of cordyceps mycelia, 5-20 parts of astragalus extract and 2-10 parts of wolfberry extract.
The oyster peptide is a micromolecular oligopeptide with the molecular weight of less than 1000Da and the protein content of more than 75 percent, and the preparation method of the oyster peptide is as follows:
(1) adding 200kg of clear water into 100kg of fresh oysters, soaking for 1 hour, and then draining the water; soaking in clear water for 2 times to obtain desalted Concha Ostreae; removing shell and viscera of desalted Concha Ostreae, and homogenizing desalted Concha Ostreae with tissue triturator to obtain Concha Ostreae slurry.
(2) Adding 20L of water into 10kg of oyster slurry, uniformly mixing, adjusting the temperature of the oyster slurry to 50 ℃, adjusting the pH value to 7.0, and adding 50g of chitosan microparticle immobilized compound protease (the chitosan microparticle immobilized compound protease contains 5g of compound protease) under stirring, wherein the compound protease comprises pancreatin, papain and flavourzyme in a weight ratio of 3.5: 1: 1.5. Keeping the temperature for reaction for 5 hours, quickly cooling to room temperature after the reaction is finished, centrifuging at 10000rpm for 10 minutes to remove undegraded solid matters and immobilized enzymes, and obtaining the supernatant which is the oyster enzymolysis liquid. The hydrolysis degree of the enzymolysis liquid is 30.5 percent, and the recovery rate of the soluble protein is 74.2 percent.
(3) Filtering the oyster enzymolysis liquid by using a filter membrane with the aperture of 0.45 mu m, then performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 1000 daltons, and collecting filtrate; the filtrate was concentrated at 50 ℃ under reduced pressure to a concentrate with a soluble solids content of 20%.
(4) Spray drying the concentrated solution to obtain oyster peptide. The weight average molecular weight of the oyster peptide is 870Da, the protein content is 82.2%, and the ash content is 6.5%.
The content of adenosine in Cordyceps mycelium is 0.8 ‰, and the product is obtained by fermenting Cordyceps liquid. Weighing raw materials in different parts by weight, stirring and mixing uniformly to obtain the oyster peptide cordyceps mycelium composition. The prepared oyster peptide cordyceps mycelium composition has protein content higher than 30% and adenosine content higher than 0.3 ‰.
Mixing the oyster peptide-cordyceps sinensis mycelium composition with a proper amount of auxiliary materials, and preparing products of different formulations. The auxiliary materials are common edible and medicinal auxiliary materials such as lubricants, glidants, diluents and the like such as magnesium stearate, microcrystalline cellulose, silicon dioxide, starch, dextrin and the like, and the auxiliary processing is carried out to prepare different product formulations which are any one of oral liquid, capsules, tablets, pills, powder and granules, so that the requirements of different markets and customers are met.
The oyster peptide cordyceps mycelium compositions which are prepared from the raw materials in different parts by weight are shown in table 1.
TABLE 1 oyster peptides Cordyceps mycelium compositions
| Sample number | Oyster peptide (share) | Chinese caterpillar fungus mycelium (parts) | Radix astragali extract (parts) | Extract of Chinese wolfberry fruit (parts) |
| Sample 1 | 45 | 40 | 10 | 5 |
| Sample 2 | 25 | 45 | 20 | 10 |
| Sample 3 | 48 | 32 | 15 | 5 |
| Sample No. 4 | 43 | 35 | 5 | 7 |
| Sample No. 5 | 40 | 38 | 20 | 2 |
| Sample No. 6 | 40 | 38 | 12 | 10 |
| Sample 7 | 30 | 42 | 18 | 10 |
| Sample 8 | 36 | 40 | 20 | 4 |
| Sample 9 | 35 | 45 | 12 | 8 |
| Sample 10 | 42 | 38 | 15 | 5 |
The oyster peptide cordyceps sinensis mycelium composition prepared by the invention is yellow to faint yellow powder, and has no agglomeration, no impurity and no bad smell. The water soluble powder is quickly dissolved in water, and no visible impurities or precipitates are generated after the water soluble powder is dissolved in water. The protein content is more than 30 percent, the adenosine content is more than 0.3 per mill, and the water content is less than 10 percent.
Weighing the raw materials according to the weight part of the composition sample 1, uniformly mixing, adding 1% of magnesium stearate, and filling into capsules to obtain the oyster peptide cordyceps mycelium capsules.
Weighing the raw materials according to the weight part of the composition sample 2, uniformly mixing, adding 5% of dextrin and 1% of magnesium stearate, and granulating to obtain the oyster peptide cordyceps mycelia granules.
Weighing the raw materials according to the weight part of the composition sample 3, uniformly mixing, adding 1% of magnesium stearate and 1.5% of silicon dioxide, granulating, and tabletting to obtain the oyster peptide cordyceps mycelium tablet.
Weighing the raw materials according to the weight part of the composition sample 4, uniformly mixing, adding 10% of starch, 1% of magnesium stearate and 1.5% of silicon dioxide, and packaging to obtain the oyster peptide cordyceps mycelium powder.
Example 2: animal experiment of oyster peptide mycelium composition for enhancing immunity
1 Experimental materials and methods
1.1 Experimental materials
Oyster peptide, oyster peptide cordyceps mycelium composition sample 1;
positive control drug: levamisole hydrochloride.
1.2 Experimental animals and groups
135 female BALB/C mice (SPF grade) with age of 6-8 weeks and weight of 18-22 g are randomly divided into 9 groups of 15 mice each.
1.3 Experimental methods
After 3 days of adaptive feeding, 135 healthy female BALB/C mice were randomized into 9 groups: blank control group (NC), model control group (MC), positive control group (PC), oyster peptide low dose group (S1-L), oyster peptide medium dose group (S1-M), oyster peptide high dose group (S1-H), oyster peptide cordyceps mycelium composition low dose group (S2-L), oyster peptide cordyceps mycelium composition medium dose group (S2-M), oyster peptide cordyceps mycelium composition high dose group (S2-H), wherein the sample concentrations of the medium, low and high dose groups are respectively 0.5g/kg.bw, 1.0g/kg.bw and 2.0g/kg.bw, the positive control is 1.0g/kg.bw levamisole hydrochloride, and the immunosuppressant is 60mg/kg.bw cyclophosphamide. After grouping, the test substance and the immunosuppressant were administered according to table 2 to perform an experiment, and the test substance was continuously administered for 28 days with free diet and drinking water, fasting for 12 hours, and sampled to determine the relevant immunological index.
Table 2 animal experimental groups and embodiments
1.4 measurement indices and results
1.4.1 body weight and thymus index, spleen index determinations and results
Mice were weighed daily during the experiment, sacrificed 30 days later, the thymus, spleen removed and weighed, and thymus and spleen indices calculated using the following formulas: thymus index equals thymus weight/body weight (mg/g) and spleen index equals spleen weight/body weight (mg/g). Data processing was performed using SPSS 22 and results are expressed as mean ± standard deviation and are shown in table 3.
TABLE 3 Effect of oyster peptide and oyster peptide Cordyceps mycelium composition on body weight, thymus index, spleen index of mice
| Grouped/(g/kg.bw) | Initial body weight/(g) | Final body weight/(g) | Spleen index/(mg/g) | Thymus index/(mg/g) |
| NC | 19.01±1.04 | 26.54±1.32 | 5.10±0.61 | 2.95±0.58 |
| MC | 19.27±0.98 | 25.95±1.46 | 4.36±0.56 | 2.46±0.56 |
| PC | 19.11±1.03 | 26.32±1.52 | 4.94±0.49 | 2.84±0.71 |
| S1-L | 19.14±0.99 | 26.07±1.44 | 4.75±0.59 | 2.55±0.49 |
| S1-M | 19.17±1.03 | 26.21±1.63 | 4.81±0.64 | 2.58±0.62 |
| S1-H | 19.32±1.04 | 26.15±1.99 | 4.79±0.69 | 2.62±0.56 |
| S2-L | 19.16±0.99 | 27.07±1.44 | 4.76±0.69 | 2.86±0.53 |
| S2-M | 19.25±1.05 | 27.16±1.51 | 4.92±0.65 | 2.98±0.59 |
| S2-H | 19.21±1.01 | 27.25±1.68 | 5.01±0.70 | 3.06±0.63 |
As can be seen from the table, the weight of the mice was significantly reduced after the mice were subjected to immunosuppressive treatment, and in the subsequent experiments, the weights of the mice using the composition of oyster peptide and oyster peptide cordyceps mycelia were recovered, indicating that the composition of oyster peptide and oyster peptide cordyceps mycelia was able to reduce the weight loss of the mice caused by CTX. And the thymus index and the spleen index are detected simultaneously to evaluate the effect of the oyster peptide and the oyster peptide composition on immune organs, compared with an NC group, the spleen index and the thymus index of other groups have no obvious difference (P is more than 0.05), compared with an MC group, the oyster peptide sample group and the oyster peptide cordyceps mycelium composition sample group are increased, and the experimental sample can improve the damage of cyclophosphamide on the immune organs of mice and play a role in enhancing the immunity.
1.4.2 Effect of oyster peptide and oyster peptide Cordyceps mycelium composition on mouse cellular immune function
Measurement of delayed allergic reaction in mice, serum hemolysin level, antibody production number, and phagocyte function. The mouse spleen lymphocyte transformation experiment and the NK cell activity determination are all determined by using the kit. Data processing was performed using SPSS 22 and results are expressed as mean ± standard deviation and are shown in table 4.
TABLE 4 influence of oyster peptide and oyster peptide Cordyceps mycelium composition on immune function of mouse cells
Note: p < 0.05 and P < 0.01 in the blank group, and P < 0.05 and P < 0.01 in the model group
As shown in Table 4, in the mouse delayed type allergy test, S1-M, S1-H, S2-L has a significant difference (P is less than 0.05) in toe swelling degree compared with NC and MC, PC, S2-M and S2-H have a significant difference (P is less than 0.01) in toe swelling degree, and the oyster peptide and cordyceps sinensis mycelium composition has a more significant effect than the oyster peptide at the same concentration. In the ConA-induced spleen lymphocyte transformation experiment, compared with NC and MC, the S1-M group and the S1-H group have significant differences (P < 0.05) with the S2-M group and the S2-H group. The results show that the oyster peptide and oyster peptide cordyceps sinensis mycelium composition can promote the immune function of mouse cells, and the oyster peptide cordyceps sinensis mycelium composition has a more remarkable effect in promoting the immune function of mouse cells under the same concentration.
The results of the effect of oyster peptide and oyster cordyceps mycelia peptide composition on the humoral immune function of mice are shown in table 5.
TABLE 5 Effect of oyster peptide and oyster peptide Cordyceps mycelium composition on humoral immune function in mice
Note: p < 0.05 and P < 0.01 in the blank group, and P < 0.05 and P < 0.01 in the model group
In mouse antibody production experiments, the PC, S1-M, S1-H, S2-M and S2-H groups have significant difference (P is less than 0.01) compared with the NC group, the S2-L group has significant difference (P is less than 0.05) compared with the NC group, all sample groups and positive control groups have significant improvement (P is less than 0.01) compared with the MC group, and the effect of the oyster peptide and cordyceps mycelium composition is better than that of oyster peptide under the same concentration. In an experiment for measuring the level of the mouse serum hemolysin, compared with a blank group and a model group, each experimental sample group is improved, an S2-M group has significant difference (P is less than 0.05) compared with an NC group, and S2-H has very significant difference (P is less than 0.01); compared with the MC group, the S1-H and S2-L groups have significant difference (P < 0.05), and the S2-M and S2-H groups have very significant difference (P < 0.01). The results show that the oyster cordyceps mycelium composition can activate the immune function of a mouse body better than oyster peptide.
1.4.3 Effect of oyster peptides and oyster peptide compositions on NK cell Activity in mice
The results of the effect of oyster peptides and oyster peptide compositions on the activity of mouse NK cells are shown in table 6.
TABLE 6 Effect of oyster peptide and oyster peptide Cordyceps mycelium composition on NK cell Activity in mice
| Dosage/(g/kg. bw) | NK cell Activity/(%) |
| NC | 37.46±4.36 |
| MC | 35.67±3.57 |
| PC | 43.46±4.23*# |
| S1-L | 40.56±3.88 |
| S1-M | 41.44±3.97 |
| S1-H | 43.37±4.10*# |
| S2-L | 41.36±4.02# |
| S2-M | 42.15±3.99*## |
| S2-H | 43.59±4.35*## |
Note: p < 0.05 and P < 0.01 in the blank group, and P < 0.05 and P < 0.01 in the model group
The samples S1-H, S2-M, S2-H can improve the activity of NK cells of mice, but under the same concentration, the oyster peptide and cordyceps sinensis mycelium composition has more obvious effect than the oyster peptide and has obvious difference (P is less than 0.05) compared with an NC group and an MC group.
In conclusion, compared with a blank control group, the oyster peptide and oyster peptide cordyceps mycelium composition has activity in promoting the cellular immune function, the humoral immune function and the NK cell activity of a mouse, the effect is obvious in a medium-high dose group, and the oyster peptide and oyster peptide composition in the same dose group are compared and analyzed, so that the oyster peptide composition has more remarkable capability of activating and promoting the immune activity of the mouse under the same dose. Provides technical support for the development of health food and food of the oyster peptide-cordyceps mycelium composition.
Example 3: cell experiment for improving immunity function of oyster peptide mycelium composition
1 Experimental materials and methods
1.1 Experimental materials
Oyster peptide solution, oyster peptide Cordyceps mycelium composition solution, macrophage RAW264.7, cultured with Gibco Eagle supplemented with 10% Fetal Bovine Serum (FBS) at 37 deg.C and 5% CO2And (5) culturing in an incubator, and culturing the cells for 36-48 h to reach a logarithmic growth phase for subsequent experiments.
1.2 experimental grouping:
macrophage RAW264.7 was divided into a blank control group (NC), a positive control group (PC), an oyster peptide low dose group (S1-L), an oyster peptide medium dose group (S1-M), an oyster peptide high dose group (S1-H), an oyster peptide cordyceps mycelium composition low dose group (S2-L), an oyster peptide cordyceps mycelium composition medium dose group (S2-M), and an oyster peptide cordyceps mycelium high dose group (S2-H).
Physiological saline is added into the blank control group, Lipopolysaccharide (LPS) with the concentration of 2 mug/mL is added into the positive control group, and the low, medium and high dosage groups of the oyster peptide and oyster peptide cordyceps mycelia composition have the addition concentrations of 50, 100 and 200 ug/mL.
1.2.1 Effect of oyster peptides and oyster peptide compositions on the proliferation and phagocytic Capacity of RAW264.7 macrophages
RAW264.7 macrophages in 96-well plates at 5 × 103The cells/well (100. mu.L/well) were cultured overnight at a density, 100. mu.L of a sample solution at a concentration of 50, 100, 200ug/mL and 2. mu.g/mL of LPS were added, respectively, to each set of 10 duplicate wells, and cultured at 37 ℃ for 24 hours. Cell proliferation assay: 10 μ LMTT (5mg/mL) was added to each well and the cells were incubated at 37 ℃ for an additional 4h, the supernatant was discarded and the cells were lysed with 150 μ L DMSO solution. The absorbance of the solution at 570nm was measured using a microplate reader. Cell phagocytosis assay: 100 μ L of 0.1% neutral red solution was added to each well and incubated at 37 ℃ for an additional 1 hour. Cells were washed 3 times with PBS and then 100 μ L of cell lysis buffer (ethanol: glacial acetic acid ═ 1: 1) was added to each well. The cell culture plate was allowed to stand at 4 ℃ overnight. The absorbance at 570nm was measured with a microplate reader.
The results of testing the effect of oyster peptide and oyster peptide cordyceps mycelium composition on the proliferation of RAW264.7 macrophages by MTT are shown in table 7, and the results are expressed as the mean ± standard deviation by data processing using SPSS 22.
TABLE 7 Effect of oyster peptides and oyster peptides Cordyceps mycelium compositions on macrophage proliferation and phagocytosis
| grouping/(ug/mL) | Cell viability/% | Phagocytic power/(%) |
| NC | 100.00±5.46 | 36.12±3.25 |
| PC | 114.68±5.28** | 42.31±4.20** |
| S1-L | 106.35±6.19 | 37.46±4.59 |
| S1-M | 103.45±6.24 | 39.28±4.68 |
| S1-H | 101.21±5.98 | 41.58±4.53** |
| S2-L | 110.35±5.12* | 39.85±4.22 |
| S2-M | 109.58±5.28 | 41.64±4.65 |
| S2-H | 106.21±5.98** | 43.88±4.11** |
Note: p < 0.05, P < 0.01, compared to blank
Research shows that the cell survival rate is in negative correlation with the concentrations of the oyster peptide and the oyster cordyceps sinensis mycelium composition, and the influence of the concentrations of different oyster peptide solutions on the survival rate of macrophages has no statistical significance (P is more than 0.05). The oyster peptide cordyceps mycelium composition has the highest cell survival rate at 50ug/mL, has a significant difference (P is less than 0.01) compared with a blank group, and the 200ug/mL complex inhibits the growth of macrophages but is still higher than the blank group, so that the high-concentration complex does not influence the proliferation activity of the macrophages.
Phagocytosis is one of the response characteristics of macrophages to pathogens and cancer cells, the immune function of the cells is reflected by measuring the phagocytosis function of the phagocytosis cells, the influence of oyster peptide and oyster peptide cordyceps mycelium composition on the phagocytosis capability of RAW264.7 macrophages is measured by adopting a neutral red phagocytosis experiment in the experiment, the result is shown in table 6, and the research shows that, compared with an NC group, 200ug/mL of oyster peptide remarkably improves the phagocytosis capability (P is less than 0.01) of the macrophages, the phagocytosis capability reaches the maximum, and the phagocytosis capability of the oyster peptide is close to that of a positive control group. The oyster peptide cordyceps mycelium composition has a phagocytic index of 43.88 +/-4.11% at a concentration of 200ug/ml, is close to that of a positive control group, has a very significant difference (P is less than 0.01) compared with a blank control group, and has a more significant effect than that of oyster peptide at the same concentration. Therefore, the oyster peptide cordyceps mycelium composition can activate and promote the phagocytic capacity of macrophages and improve the immunological activity of cells.
1.2.2 Effect of oyster peptide and oyster peptide Cordyceps mycelium composition on RAW264.7 macrophage immune factor
RAW264.7 macrophages in log phase growth in 96 well plates at 5 × 105The cells/well (100 uL/well) were cultured overnight at density, and then treated with 100uL of 50, 100, 200ug/mL sample solution and 2ug/mL LPS at 37 deg.C, 5% C02Culturing in incubator for 24 hr, mixing 50uL cell supernatant with 50uL Griess solution, reacting in dark for 10min, measuring absorption intensity at 540nm with microplate reader, calculating NO release amount, detecting TNF- α, IL-6 and IL-1 β with ELISA kit, calculating TNF- α, IL-6 and IL-1 β according to standard curveConcentration of IL-1 β.
The results of the effect of oyster peptides and oyster peptide compositions on RAW264.7 macrophage immune factor are shown in table 8, and are presented as mean ± standard deviation using SPSS 22 for data processing.
TABLE 8 Effect of oyster peptide and oyster peptide Cordyceps mycelium compositions on RAW264.7 macrophage immune factor
| grouping/(ug/mL) | NO release amount/(μmoL/L) | IL-1β/(ng/L) | IL-6/(ng/L) | TNF-α/(ng/L) |
| NC | 2.95±0.11 | 61.25±4.58 | 45.46±3.58 | 64.46±6.15 |
| PC | 5.28±0.22* | 96.36±5.27* | 93.64±4.55* | 95.28±5.28* |
| S1-L | 3.59±0.18* | 85.27±5.96 | 53.25±5.64 | 76.74±5.69 |
| S1-M | 4.28±0.11* | 92.65±6.18* | 76.85±5.85* | 85.82±5.48* |
| S1-H | 4.96±0.20* | 97.29±5.94* | 89.36±5.35* | 93.64±5.86* |
| S2-L | 4.25±0.25* | 87.37±5.24 | 55.69±5.69 | 80.56±5.49 |
| S2-M | 4.96±0.62* | 93.89±6.15* | 75.25±5.26* | 89.37±5.16* |
| S2-H | 5.36±0.52* | 98.56±5.64* | 91.86±5.48* | 95.18±4.55* |
Note: p < 0.05, P < 0.01, compared to blank
NO can promote apoptosis and phagocytosis of macrophage, and can be used as index for detecting macrophage immunocompetence. The influence of oyster peptide and oyster peptide cordyceps mycelia composition on NO release of RAW264.7 macrophages is shown in Table 8, and experimental results show that the NO release amount increases with the increase of concentration, the oyster peptide cordyceps mycelia composition sample has stronger capability of releasing NO of the macrophages, and the NO release amounts of different oyster peptide and oyster peptide cordyceps mycelia sample concentrations are significantly different (P is less than 0.05) compared with NC groups, so that the oyster peptide and oyster peptide cordyceps mycelia composition can promote the release of NO of the macrophages and improve the immunocompetence of cells.
The effect of oyster peptide and oyster peptide cordyceps mycelium composition on the secretion capacity of RAW264.7 macrophage to secrete IL-1 beta, IL-6 and TNF-alpha is shown in table 8, and the research shows that, after the oyster peptide solution and the oyster peptide cordyceps mycelium composition solution are treated for 24 hours, 100-200 ug/mL of oyster peptide and oyster peptide cordyceps mycelium composition can promote macrophages to secrete IL-1 beta, IL-6 and TNF-alpha, the oyster peptide cordyceps sinensis mycelium composition has more obvious effect and obvious difference (P is less than 0.05) compared with a blank control group, therefore, the oyster peptide and oyster peptide cordyceps mycelium composition can activate and promote macrophages to secrete IL-1 beta, IL-6 and TNF-alpha, and the oyster peptide composition can better activate cell secretion and improve the cell immunocompetence compared with oyster peptide.
By carrying out immunological research on cells, the oyster peptide and the oyster peptide cordyceps mycelium composition can improve the survival rate of macrophage cells, the phagocytic capacity of the cells, the NO release amount and the secretion amounts of IL-1 beta, IL-6 and TNF-alpha, and have particularly obvious medium and high dose groups and stronger cell immunoregulation activity. The influence of the oyster peptide and the oyster peptide cordyceps mycelium composition on the cellular immunity under the same dosage is comparatively analyzed, and the oyster peptide cordyceps mycelium composition is more remarkable in the aspect of promoting the cellular immune activity compared with the oyster peptide under the same dosage. The oyster peptide cordyceps mycelium composition has a stronger activation and promotion effect on the cellular immune function, and lays a foundation for the development of health-care food and food of the oyster peptide cordyceps mycelium composition.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. An oyster peptide cordyceps mycelium composition with the function of enhancing immunity is characterized in that: the oyster peptide cordyceps mycelium composition comprises the following components in parts by weight:
oyster peptide: 25-48 parts;
cordyceps mycelia: 30-45 parts of a solvent;
astragalus extract: 5-20 parts of a solvent;
wolfberry fruit extract: 2-10 parts.
2. The oyster peptide cordyceps mycelium composition according to claim 1, wherein: the oyster peptides are small molecular oligopeptides with molecular weight less than 1000Da and protein content more than 75%.
3. The oyster peptide cordyceps mycelium composition according to claim 2, wherein: the preparation method of the oyster peptide comprises the following steps:
(1) soaking fresh oysters for desalination, cleaning, shelling and removing internal organs to obtain desalted oyster meat, and homogenizing the desalted oyster meat to obtain oyster slurry;
(2) adding water into the oyster slurry, then adding chitosan immobilized compound protease, carrying out heat preservation and enzymolysis, and centrifuging to obtain supernatant, namely oyster enzymolysis liquid;
(3) ultrafiltering the Concha Ostreae enzymolysis solution with ultrafiltration membrane with cut-off molecular weight of 1000 daltons, collecting filtrate, and concentrating the filtrate to obtain concentrated solution;
(4) spray drying the concentrated solution to obtain oyster peptide.
4. The oyster peptide cordyceps mycelium composition according to claim 3, wherein: the weight ratio of the chitosan immobilized compound protease to the desalted oyster meat is 0.2-1%, and the mass ratio of the compound protease in the chitosan immobilized compound protease to the chitosan immobilized compound protease is 5-15%.
5. The oyster peptide cordyceps mycelium composition according to claim 4, wherein: the compound protease comprises pancreatin, papain and flavourzyme, wherein the pancreatin: papain: the weight ratio of the flavourzyme is 3-5: 1: 1-2.
6. The oyster peptide cordyceps mycelium composition according to claim 1, wherein the cordyceps mycelium is prepared by submerged fermentation of cordyceps sinensis liquid, wherein the cordyceps sinensis is hirsutella hepiali Chen et Shen or paecilomyces hepiali Chen et Shen; the content of adenosine in the cordyceps sinensis mycelia is more than 0.8 per mill.
7. The oyster peptide cordyceps mycelium composition according to claim 1, wherein the oyster peptide cordyceps mycelium composition has a protein content of more than 30% and an adenosine content of more than 0.3 ‰.
8. The oyster peptide cordyceps mycelium composition according to claim 1, wherein the oyster peptide cordyceps mycelium composition comprises the following components in parts by weight: 45 parts of oyster peptide, 40 parts of hirsutella hepiali Chen et Shen mycelium, 10 parts of astragalus extract and 5 parts of wolfberry extract.
9. The oyster peptide-cordyceps mycelium composition according to claim 1 to 8, wherein the oyster peptide-cordyceps mycelium composition is used for preparing health products or food.
10. The use of the oyster peptide cordyceps sinensis mycelium composition according to claim 9 in the preparation of health products or foods, wherein the composition comprises: the health product is in the form of oral liquid, capsule, tablet, pill, powder or granule.
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