Preparation method and application of composite polypeptide metagen solid preparation

Abstract

The invention provides a preparation method and application of a composite polypeptide metagen solid preparation, the composite polypeptide metagen solid preparation comprises a metagen and a peptide component, the metagen is prepared by fermenting lactobacillus plantarum nbk-MA2 and lactobacillus kefiranosus subsp.kefiranosus ZW3, the peptide component comprises albumin polypeptide, cordyceps sobolifera peptide, ginseng peptide, corn oligopeptide and collagen peptide, and the composite polypeptide metagen solid preparation is prepared by fermenting lactobacillus plantarum nbk-MA2, lactobacillus kefiranosus subsp.kefiranosus ZW3 and lactobacillus kefiranosus subsp.kefiranosus ZW3. The protein powder is prepared from protein powder raw materials through protease treatment. According to the composite polypeptide metagen solid preparation, the synergistic interaction effect can be achieved through compounding of the composite metagen and the peptide assembly, immunity is improved, tumor growth and migration are inhibited, tumor cell apoptosis is promoted, toxic and side effects are avoided, and the composite polypeptide metagen solid preparation is a food-borne functional product with good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method and application of a composite polypeptide postbiotic solid preparation. Background Art

[0002] Immunity is the body's own defense system, mainly against exogenous pathogenic microorganisms, including bacteria, viruses, tuberculosis, etc. invading the body, playing a defensive role. Another is the ability to target the body's own aging cells, cancer cells, or mutated cells and eliminate these cells. Almost all diseases are closely related to immunity. Immunity imbalance will lead to the occurrence of various diseases, such as respiratory diseases, gastrointestinal diseases, cardiovascular diseases, nervous system diseases, tumors, etc. There are many factors affecting human immunity, including age, sleep, exercise, dietary nutrition, mental factors, etc. Among them, nutritional factors are crucial. Sufficient nutrition can keep the human immune system in a good state, better resist the invasion of bacteria and viruses, reduce the risk of disease and improve the prognosis of diseases.

[0003] To meet the needs of different consumers, more products or services with strong pertinence and clear functions, such as enhancing immunity, have emerged in the market. Traditional such products mainly rely on Chinese herbal medicines. However, through various clinical applications, it has been found that these products not only cause various side effects, such as gastrointestinal discomfort, rashes, etc., but also greatly increase the burden on the liver and kidneys.

[0004] Therefore, developing food-derived, safe and highly effective functional products is the new direction of the future market. Summary of the Invention

[0005] The present invention provides a preparation method and application of a composite polypeptide postbiotic solid preparation. The preparation can enhance immunity by increasing the levels of IgM and IgG antibodies in the serum of mice, promoting the secretion of TNF-α and TNF-γ, and increasing the levels of IL-2, IL-9 and IL-10. At the same time, it can inhibit tumors by inhibiting the growth and migration of tumor cells and promoting the apoptosis of tumor cells.

[0006] In the first aspect, the present invention provides a composite polypeptide postbiotic solid preparation, and the composite polypeptide postbiotic solid preparation includes postbiotics and peptide components.

[0007] The postbiotics include the cells and fermentation products of Lactobacillus plantarum nbk-MA2 and Lactobacillus kefiranofaciens subsp. kefiranofaciens ZW3.

[0008] The preservation number of Lactobacillus plantarum nbk-MA2 is CGMCC NO.16872, and the preservation number of Lactobacillus kefiranofaciens subsp. kefiranofaciens ZW3 is CGMCC NO.2809.

[0009] The peptide component includes albumin polypeptide, cicada flower peptide, ginseng peptide, corn oligopeptide, and collagen peptide.

[0010] Preferably, the compound postbiotic solid preparation contains, by weight: 30 - 50 parts of postbiotics and 50 - 70 parts of the peptide component, and the total amount is preferably 100 parts.

[0011] Preferably, the preparation method of the postbiotics includes the following steps: (1) Inoculate Lactobacillus plantarum nbk - MA2 and Lactobacillus kefiranofaciens subsp. kefiranofaciens ZW3 into the seed medium for activation respectively to obtain the first - stage seed liquid; (2) Inoculate the first - stage seed liquid into the seed medium for anaerobic culture respectively to obtain the second - stage seed liquid; (3) Inoculate the second - stage seed liquid into the fermentation medium respectively for high - density fermentation; (4) Sterilize at 115 °C for 15 min after fermentation is completed respectively; (5) Concentrate the obtained fermentation liquid to a solid content of 50% respectively, then freeze - dry, pulverize, and configure into a postbiotic composition according to the ratio of Lactobacillus plantarum nbk - MA2:Lactobacillus kefiranofaciens subsp. kefiranofaciens ZW3 = 2.5:1 - 3.5:1.

[0012] Further preferably, the seed medium includes the following components by mass fraction: 1 - 3% glucose, 0.5 - 2% peptone, 0.5 - 2% yeast extract powder, 0.2 - 1% beef extract powder, 0.2 - 1% anhydrous sodium acetate, 0.1 - 0.5% ammonium citrate, 0.1 - 0.5% dipotassium hydrogen phosphate, 0.05 - 0.2% Tween 80 and the balance of water, and the pH value is 6.7 - 6.9; The fermentation medium includes the following components by mass fraction: 1 - 3% glucose, 0.5 - 2% peptone, 0.5 - 2% yeast extract powder, 0.2 - 1% beef extract powder, 0.2 - 1% anhydrous sodium acetate, 0.1 - 0.5% ammonium citrate, 0.1 - 0.5% dipotassium hydrogen phosphate, 0.05 - 0.2% Tween 80 and the balance of water, and the pH value is 6.3 - 6.5.

[0013] Further preferably, the high - density fermentation collects the fermentation liquid and performs subsequent sterilization when the cell density reaches 10 9 cfu / mL; The freeze - drying is to pre - freeze the fermentation liquid at - 38 °C for 2 h, vacuum - dry the pre - frozen fermentation liquid at 0.2 mba, and pulverize the vacuum - freeze - dried product to 20 mesh to obtain the postbiotic preparation.

[0014] The Lactobacillus plantarum nbk-MA2 was screened from kefir in Tibet Autonomous Region, and has antioxidant function and the function of enhancing immunity by reducing the secretion of inflammatory factors; Lactobacillus kefiranofaciens subsp. kefiranofaciens ZW3 is a new national food raw material strain, which can regulate the intestinal microecology, enhance the intestinal barrier function and improve the intestinal immunity.

[0015] Preferably, the preparation method of the peptide component comprises the following steps: (1) Mix egg white powder, Cordyceps cicadae fruit body powder, ginseng powder, corn protein powder and collagen powder with water respectively. The mixed slurry is subjected to high-temperature denaturation, the supernatant is removed, and then rinsed; (2) Mix the collected precipitate with water, and carry out enzymatic hydrolysis under the action of protease, then heat up to 95 °C to inactivate the enzyme for 15 min, filter and collect the filtrate, and carry out drying treatment to obtain dried albumin peptide, Cordyceps cicadae peptide, ginseng peptide, corn oligopeptide and collagen peptide; (3) Obtain the peptide component according to the ratio of dried albumin peptide:Cordyceps cicadae peptide:ginseng peptide:corn oligopeptide:collagen peptide of 7-13:0.5-1.5:0.5-1.5:0.5-1.5:15-25.

[0016] Further preferably, in the preparation method of the peptide component, the high-temperature denaturation temperature is 80-100 °C and the time is 10-60 min.

[0017] Further preferably, the protease is one or more of papain, alkaline protease and neutral protease; The conditions of the enzymatic hydrolysis treatment are: the enzymatic hydrolysis temperature is 40-60 °C, the time is 2-8 h, and the pH value is 8-9.

[0018] Further preferably, the polypeptide content of each peptide in the peptide component is 30-90%; the distribution ratio of polypeptides with a relative molecular weight of less than 1000 Da in the peptide component is not less than 80%, preferably 80-95%.

[0019] Preferably, the drying treatment includes: concentrating the filtrate and then carrying out spray drying. Further, the spray drying includes: the inlet air temperature is 160-200 °C, the outlet air temperature is 85-90 °C, the induced air rotor power is 30-40 Hz, and the nozzle rotor power is 16-20 Hz.

[0020] The source of the albumin polypeptide, ovalbumin, is a recognized "model protein". Its bioavailability is higher than that of ordinary proteins, the amino acid composition has an approximation rate of 99.8% with human serum albumin, it is absorbed quickly, has a reasonable amino acid pattern, high nutritional value, can provide a material basis for white blood cells and immune organs to secrete immune factors, and has various effects such as enhancing immunity, increasing muscle mass, anti-inflammatory, and increasing the amount of white blood cells. Albumin polypeptide resists the attack of various viruses and microorganisms on the human body and prevents various cancers caused by immune system imbalance by enhancing the cellular immune function and the phagocytosis or killing ability of macrophages and natural killer cells. The main components of ginseng peptide are proteins, ginsenosides, polysaccharides, etc., and it has biological activities such as enhancing immunity, anti-tumor, anti-inflammatory, delaying skin aging, anti-diabetes and its complications, and anti-depression. The polysaccharide in ginseng peptide can inhibit the activity of tumor cells and promote the proliferation of lymphocytes. Cordyceps cicada peptide can regulate the immune activity of T cells, inhibit the growth of melanoma, inhibit the proliferation of T-lymphocytes, inhibit the proliferation of Hela cells, block the cell cycle of S phase, and promote apoptosis, thereby inhibiting the growth of cancer cells. Corn peptide can easily scavenge superoxide anion free radicals and hydroxyl free radicals, and inhibit the occurrence of lipid peroxidation reactions in cells and tissues; by scavenging peroxidized lipids, oxygen free radicals and NO, and then inhibiting the production of key inflammatory cytokines such as IL1-β, it plays an important role in blocking the inflammatory cascade reaction to enhance immunity; collagen peptide can reduce the levels of pro-inflammatory cytokines such as interleukin-1β, interleukin-6 and tumor necrosis factor-α, and prevent and improve immune deficiency. The compound of probiotics and peptide components can achieve a synergistic effect.

[0021] In a second aspect, the present invention provides the use of the above-mentioned compound polypeptide probiotic solid preparation in the preparation of a drug for enhancing immunity, inhibiting the growth and migration of tumor cells, and promoting the apoptosis of tumor cells.

[0022] In a third aspect, the present invention provides a product containing the above-mentioned compound polypeptide probiotic solid preparation; The product is a drug or a health food.

[0023] Preferably, the compound polypeptide probiotic solid preparation can be applied to powders, tablets, capsules and oral liquids.

[0024] The beneficial effect of the present invention is that the provided compound polypeptide probiotic solid preparation has no toxic and side effects, can enhance immunity by increasing the levels of IgM and IgG antibodies in the serum of mice, promoting the secretion of TNF-α and TNF-γ, and increasing the levels of IL-2, IL-9 and IL-10. At the same time, it can play a good role in inhibiting tumors by inhibiting the growth, proliferation, promoting the apoptosis of tumor cells and inhibiting the migration of tumor cells. Description of the Drawings

[0025] To more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0026] Figure 1 It is a graph showing the results of serum IgM in each group of mice after adding the postbiotic solid preparation with composite polypeptide in Example 3 of the present invention.

[0027] Figure 2 It is a graph showing the results of serum IgG in each group of mice after adding the postbiotic solid preparation with composite polypeptide in Example 3 of the present invention.

[0028] Figure 3 It is a graph showing the results of serum TNF-α in each group of mice after adding the postbiotic solid preparation with composite polypeptide in Example 3 of the present invention.

[0029] Figure 4 It is a graph showing the results of serum TNF-γ in each group of mice after adding the postbiotic solid preparation with composite polypeptide in Example 3 of the present invention.

[0030] Figure 5 It is a graph showing the results of serum IL-2 in each group of mice after adding the postbiotic solid preparation with composite polypeptide in Example 3 of the present invention.

[0031] Figure 6 It is a graph showing the results of serum IL-9 in each group of mice after adding the postbiotic solid preparation with composite polypeptide in Example 3 of the present invention.

[0032] Figure 7 It is a graph showing the results of serum IL-10 in each group of mice after adding the postbiotic solid preparation with composite polypeptide in Example 3 of the present invention.

[0033] Figure 8 It is the IC50 result of A549 cells after adding the postbiotic solid preparation with composite polypeptide in Example 4 of the present invention.

[0034] Figure 9 It is the IC50 result of 4T1 cells after adding the postbiotic solid preparation with composite polypeptide in Example 4 of the present invention.

[0035] Figure 10 It is the IC50 result of HuccT1 cells after adding the postbiotic solid preparation with composite polypeptide in Example 4 of the present invention.

[0036] Figure 11 It is the IC50 result of HepG2 cells after adding the postbiotic solid preparation with composite polypeptide in Example 4 of the present invention.

[0037] Figure 12 This is the tumor cell proliferation result graph of the postbiotic solid preparation and paclitaxel group after adding the composite polypeptide in Example 4 of the present invention.

[0038] Figure 13 This is the microscopic examination graph of 4T1 cell apoptosis staining in the postbiotic solid preparation and paclitaxel group after adding the composite polypeptide in Example 4 of the present invention.

[0039] Figure 14 This is the microscopic examination graph of A549 cell apoptosis staining in the postbiotic solid preparation and paclitaxel group after adding the composite polypeptide in Example 4 of the present invention.

[0040] Figure 15 This is the microscopic examination graph of HepG2 cell apoptosis staining in the postbiotic solid preparation and paclitaxel group after adding the composite polypeptide in Example 4 of the present invention.

[0041] Figure 16 This is the microscopic examination graph of HuccT1 cell apoptosis staining in the postbiotic solid preparation and paclitaxel group after adding the composite polypeptide in Example 4 of the present invention.

[0042] Figure 17 This is the result graph of the scratch experiment of each tumor cell in the postbiotic solid preparation and paclitaxel group after adding the composite polypeptide in Example 4 of the present invention. Detailed implementation manners

[0043] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the protection scope of the present invention.

[0044] Example 1 Preparation of postbiotic freeze-dried powder Preparation of seed medium: 2% glucose, 2% peptone, 0.5% yeast extract powder, 1% beef extract powder, 0.5% anhydrous sodium acetate, 0.2% ammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.1% Tween 80 and the balance of water, with a pH value of 6.8.

[0045] Preparation of fermentation medium: 2% glucose, 2% peptone, 0.5% yeast extract powder, 1% beef extract powder, 0.5% anhydrous sodium acetate, 0.2% ammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.1% Tween 80 and the balance of water, with a pH value of 6.4.

[0046] The preparation method of the postbiotic is as follows: (1) Inoculate the preserved Lactobacillus plantarum nbk-MA2 and the glycerol tube strain of Lactobacillus kefiranofaciens subsp. kefiranofaciens ZW3 into the seed medium at an inoculation amount of 3% (v / v) respectively, and culture them statically under anaerobic conditions at 37 °C for 18 h to obtain the primary seed liquid.

[0047] (2) Inoculate the primary seed liquid prepared in (1) into the seed medium at an inoculation amount of 3% (v / v), and culture it statically under anaerobic conditions at 36 °C for 15 h to obtain the secondary seed liquid.

[0048] (3) Inoculate the secondary seed liquid cultured in (2) into the fermentation medium at an inoculation amount of 5% (v / v), and culture it statically under anaerobic conditions at 37 °C for 8 h to obtain the fermentation broth.

[0049] Treat the fermentation broth at 115 °C for 15 min, pre-freeze it at -38 °C for 2 h, and then perform vacuum freeze-drying on the pre-frozen fermentation broth with a vacuum degree of 0.2 mba; after vacuum freeze-drying, crush it to 20 mesh to prepare the freeze-dried powder.

[0050] Example 2 Preparation of Composite Polypeptide Postbiotics Solid Preparation Preparation of composite polypeptide: (1) Mix egg white powder, Cordyceps cicadae fruit body powder, ginseng powder, corn protein powder, and collagen powder with water respectively. The material-liquid ratio of the protein powder to water is 1:50. Perform high-temperature denaturation on the mixed slurry at 80 - 100 °C for 10 - 60 min, remove the supernatant, rinse, and let it stand for precipitation.

[0051] (2) Mix the collected precipitate with water, perform enzymatic hydrolysis under the action of papain, inactivate the enzyme at 95 °C for 15 min, filter, and collect the filtrate.

[0052] (3) After concentrating the filtrate at 65 °C, perform spray drying. The inlet air temperature is 180 °C, the outlet air temperature is 87 °C, the induced draft rotor power is 35 Hz, and the nozzle rotor power is 18 Hz.

[0053] The polypeptide content of albumin peptide in the proteolytic polypeptide is 80%; the polypeptide content of Cordyceps cicadae peptide is 30%; the polypeptide content of ginseng peptide is 35%; the polypeptide content of corn oligopeptide is 85%; the polypeptide content of collagen peptide is 70%. The distribution ratio of the proteolytic polypeptide of the present invention with a relative molecular weight below 1000 Da is 85%.

[0054] First, compound the polypeptide prepared by spray drying in the ratio of albumin peptide:Cordyceps cicadae peptide:ginseng peptide:corn oligopeptide:collagen peptide = 10:1:1:1:20, and then compound it with the postbiotic freeze-dried powder in Example 1 in the ratio of 3:2 to obtain the composite polypeptide postbiotics solid preparation.

[0055] Effect of Compound Polypeptide Postbiotics Solid Preparation in Example 3 on the Levels of IgM, IgG, TNF-α, TNF-γ, IL-2, IL-9, and IL-10 in Mouse Serum After 3 days of adaptive feeding, ICR mice were randomly divided into 4 groups: blank control group, model group, low-dose group of compound polypeptide postbiotics solid preparation, and high-dose group of compound polypeptide postbiotics solid preparation, with 15 mice in each group. The model group and the two experimental groups were intraperitoneally injected with cyclophosphamide [80 mg / (kg·bw)] for 3 consecutive days, and the blank control group was simultaneously intraperitoneally injected with an equal volume of normal saline. Administration started 1 day after the last intraperitoneal injection. The two experimental groups were respectively gavaged with compound polypeptide postbiotics at concentrations of 0.9 g / (kg·bw) and 1.8 g / (kg·bw) for 30 consecutive days. At the same time, the blank control group and the model group were gavaged with normal saline. During the experiment, the mice were allowed to eat and drink freely. After the last administration, the mice were fasted overnight for 12 h, sacrificed in the early morning the next day, and biological samples were collected for detection of relevant indicators.

[0056] After anesthesia, the mice were sacrificed by eye enucleation to collect blood. The collected mouse blood was centrifuged at 24 °C, with a centrifugal radius of 6 cm and 4000 r / min for 8 min. After centrifugation, the upper serum part was aspirated with a pipette for standby.

[0057] According to the instructions of the ELISA kit, the levels of IgM, IgG, TNF-α, TNF-γ, IL-2, IL-9, and IL-10 in mouse serum were measured using the corresponding ELISA kits for mice to evaluate the immune level of the mice. The results are shown in Figures 1 - 7 .

[0058] In the figure, # represents P < 0.05 compared with the model group, ## represents P < 0.01 compared with the model group, and represents P < 0.001 compared with the model group; * represents P < 0.05 compared with the model group, ** represents P < 0.01 compared with the model group, and *** represents P < 0.001 compared with the model group.

[0059] (I) Changes in the Levels of IgM and IgG in Mouse Serum Immunity generally refers to the body's ability to fight against infections and pathogens, relying on the coordinated action of various immune cells and molecular systems. IgM is the earliest antibody to respond in the immune system and is usually rapidly produced during the body's primary infection. It can recognize and neutralize foreign pathogens (such as bacteria and viruses), helping the immune system to eliminate these pathogens. An increase in IgM levels usually indicates that the body is dealing with an acute or recent infection. In a body with stronger immunity, IgM will rapidly increase and participate in the fight against pathogens when facing an infection. This reflects that the body's immune system can effectively recognize and respond to new infections; if the immune system is weak, the response of IgM may be slower or the production may be insufficient, resulting in a weaker ability to fight against infections.

[0060] IgG is a major component of humoral immunity and can recognize and neutralize pathogenic microorganisms in the body. It clears these foreign substances by directly binding to the pathogens or by activating the complement system. The IgG antibody response is usually slower, but it can provide protection for long-term immunity. Especially after an infection, IgG can remain in the body for several months or even years. A lower IgG level may indicate a functional defect in the immune system and an inability to produce sufficient antibody responses to resist infections.

[0061] Figure 1 Results of mouse serum IgM Figure 2 Results of mouse serum IgG. Compared with the blank control group, the levels of IgM and IgG in the serum of mice in the model group were significantly decreased, indicating that the immune level of the model mice was inhibited after cyclophosphamide-induced modeling; compared with the model group, the levels of IgM and IgG in the low-dose and high-dose groups of the compound polypeptide postbiotic solid preparation increased to varying degrees. Among them, the IgM level in the high-dose group was 4.2 mg / mL, which was 75% higher than that in the model group; the IgG level in the high-dose group was 82.1 mg / mL, which was 55.5% higher than that in the model group. The increase in the two high-dose groups was the most significant, indicating that their ability to restore the humoral immune level of mice was significantly enhanced.

[0062] Changes in the levels of TNF-α and TNF-γ in mouse serum Tumor necrosis factor-α (TNF-α) is an important cytokine that plays a key role in immune responses. It is secreted by various cells in the immune system (such as macrophages, T cells, etc.) and participates in various physiological processes, including inflammatory responses, immune regulation, apoptosis, and tumor surveillance. TNF-α is involved in acute and chronic inflammatory responses and is an important regulator of the immune system. It can promote the secretion of inflammatory factors such as IL-1 and IL-6 and enhance the activity of immune cells.

[0063] TNF-γ is a cytokine secreted by immune cells such as T cells, natural killer cells (NK cells), and macrophages, and plays an important role mainly in immune responses and inflammatory processes. TNF-γ is one of the important factors that promote cell-mediated immune responses. In the case of infection or immune challenge, TNF-γ helps initiate the immune response by activating macrophages, enhancing their phagocytosis, and promoting antigen presentation.

[0064] Figure 3 Results of TNF-α in mouse serum Figure 4 Results of TNF-γ in mouse serum. Compared with the blank control group, the levels of TNF-α and TNF-γ in the model group were significantly decreased, and its immune level was inhibited; compared with the model group, the TNF-α and TNF-γ indexes in the low-dose and high-dose groups of the compound polypeptide postbiotic solid preparation showed significant changes to varying degrees under the regulation of the test substances administered. Generally speaking, the levels of TNF-α and TNF-γ in the serum of mice in the high-dose group were 161.2 pg / mL and 2.07 pg / mL respectively, which were increased by 79% and 70% respectively compared with the model group, and the elevation effects in the two high-dose groups were the most significant (P < 0.001). It shows that the high-dose group can better restore the reduction of TNF-α and TNF-γ secretion in the serum of immunocompromised mice, suggesting that the compound polypeptide postbiotic solid preparation has strong immune-enhancing activity.

[0065] Changes in the levels of IL-2, IL-9, and IL-10 in mouse serum IL-2 (Interleukin-2) is an important cytokine secreted by T cells, and its main function is to promote the proliferation and activation of T cells, thereby regulating immune responses. It plays a key role in maintaining the function of the immune system, promoting immune tolerance, and preventing immune dysregulation. When the body is infected or challenged by other immune factors, T cells will be activated and secrete IL-2. IL-2 promotes the proliferation of T cells and enhances the immune response. Therefore, the level of IL-2 is closely related to the activity of the body's immune system. Higher IL-2 levels usually reflect a stronger immune response. IL-2 not only promotes immune responses but also plays a role in immune tolerance. It helps the immune system distinguish "self" from "non-self" by regulating the function of regulatory T cells (Tregs), thereby preventing the immune system from attacking its own tissues. Therefore, too low an IL-2 level may lead to insufficient immune tolerance and increase the risk of autoimmune diseases.

[0066] IL-9 is a cytokine secreted by a variety of immune cells, mainly produced by T cells, mast cells and certain types of dendritic cells, etc. IL-9 plays an important role in immune responses and allergic reactions, and it has a certain impact on the regulation of the immune system. IL-9 helps to promote the proliferation of Th2 cells, and further enhances the production of cytokines such as IL-4, IL-5, IL-13, etc., which are very important for allergic reactions and anti-parasitic immunity.

[0067] IL-10 is an anti-inflammatory cytokine, mainly secreted by a variety of cells in the immune system (such as T cells, B cells, macrophages, etc.). Its main role is to inhibit inflammatory reactions and regulate the balance of the immune system. IL-10 can reduce excessive immune responses by inhibiting the secretion of pro-inflammatory cytokines (such as TNF-α, IL-6, IL-1, etc.), thereby avoiding tissue damage.

[0068] Figure 5 For the results of mouse serum IL-2, Figure 6 For the results of mouse serum IL-9, Figure 7 For the results of mouse serum IL-10, compared with the blank control group, the levels of IL-2, IL-9 and IL-10 in the model group were significantly decreased, and its immune level was inhibited; compared with the model group, the concentrations of IL-2, IL-9 and IL-10 in the low-dose and high-dose groups of the compound polypeptide postbiotic solid preparation were significantly increased. Among them, the concentrations of IL-2, IL-9 and IL-10 in the high-dose group were 107.7 pg / mL, 24.7 pg / mL, 0.87 pg / mL respectively, which were increased by 137%, 115% and 112% compared with the model group. The results showed that the compound polypeptide postbiotic solid preparation could significantly up-regulate the levels of IL-2, IL-9 and IL-10 in immunosuppressed mice, and the high-dose group was closer to the level of normal mice, suggesting that in the immunosuppression model established in this experiment, the compound polypeptide postbiotic solid preparation has the effect of improving immune function, and the effect is more significant after increasing the dosage.

[0069] Example 4 Inhibitory effect of the compound polypeptide postbiotic solid preparation on tumor cells 1. Experimental reagents CCK-8 apoptosis staining kit; basal medium, 25% trypsin, fetal bovine serum, double antibody, phosphate buffer solution (PBS); crystal violet.

[0070] 2. Experimental cells Breast cancer cells (4T1), human lung adenocarcinoma epithelial cells (A549), human liver cancer cells (HepG2) and human cholangiocarcinoma cells (HuccT1).

[0071] Experimental method (1)Cell resuscitation Take out the cryopreserved breast cancer cells (4T1), human lung adenocarcinoma epithelial cells (A549), human hepatoma cells (HepG2) and human cholangiocarcinoma cells (HuccT1) from liquid nitrogen and thaw them rapidly. Add 4 mL of culture medium into a centrifuge tube, transfer the cell suspension into the centrifuge tube containing the culture medium, centrifuge at 1000 r / min for 5 min at room temperature, and pour out the upper-layer culture medium. Aspirate 3 - 5 mL of culture medium into the centrifuge tube containing cell clumps, then mix well, and then add the cell suspension into a cell culture dish and place it in an incubator for culture.

[0072] (2) Cell culture, subculture and cryopreservation Cell culture: Take out the 4T1, A549, HepG2 and HuccT1 cells after culturing for 24 h, discard the culture medium, then add 5 mL of PBS buffer, gently shake and wash, discard the PBS, and then add 10 mL of culture medium, and place the cells in an incubator at 37°C for culture. When the monolayer cell density of HepG2 and L02 cells in the cell culture dish reaches about 80%, cell subculture is carried out.

[0073] Cell subculture: Discard the culture medium in the culture dish, then add 5 mL of PBS buffer and gently shake and wash, discard the PBS, and repeat twice; add 1 mL of trypsin solution, incubate at 37°C for 1 min, gently pat, observe under a microscope, if most cells fall off, the cell digestion is complete, add 3 mL of culture medium and gently pipette to terminate the digestion; collect the cell suspension, centrifuge at 1000 r / min for 5 min at room temperature, pour out the upper-layer liquid; aspirate 6 mL of culture medium into the centrifuge tube, mix well, and then add the cell suspension into 2 cell culture dishes and place them in an incubator for culture. Select cells in the logarithmic growth phase for experiments.

[0074] Cell cryopreservation: When the monolayer cell density of 4T1, A549, HepG2 and HuccT1 cells reaches 90%, obtain the cell suspension according to the "cell subculture" operation; centrifuge the cell suspension at 1000 r / min for 5 min at room temperature, pour out the upper-layer supernatant, add cell cryopreservation solution and mix well, then place the cell cryopreservation solution containing cells in a cryopreservation tube, seal it, then place it at -80°C for cryopreservation, and finally store the cells in liquid nitrogen.

[0075] (I) Detection of IC50 of cells by CCK-8 method Take 4T1, A549, HepG2, and HuccT1 cells in the logarithmic growth phase. Obtain cell suspensions according to the method in "Cell Passage". After dilution, inoculate the cells into a 96-well plate with an inoculation volume of 100 μL per well and approximately 1000 cells per well. Then place them in an incubator and culture for 24 h. Divide the cells into a blank group, a control group, and a drug administration group. The blank group does not add cells. The cells in the control group are cultured with medium. The drug administration group is cultured with a medicated medium containing compound polypeptide postbiotics solid preparation at a concentration of 80 μg / mL to 40.96 mg / mL. After culturing the 96-well plate in an incubator for 24 h, aspirate the medium. Subsequently, add 90 μL of medium and 10 μL of CCK-8 reagent to each well, and then incubate in the incubator for 30 min. Measure the absorbance (A) using an enzyme-linked immunosorbent assay (ELISA) reader with a maximum absorption wavelength of 450 nm, and calculate the effect of the compound polypeptide postbiotics solid preparation on the IC50 values of A549, 4T1, HuccT1, and HepG2 cells.

[0076] After treating the cells with compound polypeptide postbiotics solid preparations at different concentrations (80 μg / mL to 40.96 mg / mL) for 24 h, detect the changes in cell viability, and use GraphPad Prism 9 to calculate the IC50 values of the cells. The results are as Figures 8 - 11 shown, where Figure 8 are the IC50 results of A549 cells, Figure 9 are the IC50 results of 4T1 cells, Figure 10 are the IC50 results of HuccT1 cells, Figure 11 are the IC50 results of HepG2 cells. It can be seen from the figure that the IC50 values of A549, 4T1, HuccT1, and HepG2 cells are 2849 μg / mL, 2124 μg / mL, 976.6 μg / mL, and 4582 μg / mL, respectively, indicating that the compound polypeptide postbiotics solid preparation has a certain inhibitory effect on tumor cells.

[0077] (2) Detection of cell colony formation ability by plate cloning experiment Take tumor cells in the logarithmic growth phase and seed 1000 cells per well in a 6-well plate, making the cells evenly distributed singly in the wells. Change the medium every 2 days, and set up a control group, a paclitaxel group, and a compound polypeptide postbiotics solid preparation group. Culture the cells until white dots can be seen growing at the bottom of the plate with the naked eye, and when cell colonies are formed in clusters under the microscope and the number of cells in most of the wells in the blank group is ≥50, it is regarded as colony formation. Wash twice with PBS, fix with 4% paraformaldehyde for 15 min, then add 1 mL of crystal violet staining solution to each well and stain for 15 min. Place it in clean water to wash away the staining solution remaining in the 6-well plate, and drain the water. Use a microscope to photograph and record the general situation of each well's colonies, and count 1 cm under the microscope2 The number of cell colonies in the square grid.

[0078] The number of clone formations is an important indicator reflecting the proliferation ability of tumor cells. As Figure 12 shown, compared with the control group, there were almost no clone numbers in the paclitaxel positive drug group and the composite polypeptide postbiotics group, indicating that paclitaxel and the experimental group inhibited cell proliferation and had a strong inhibitory ability.

[0079] (III) Cell apoptosis experiment Inoculate 1.5×10 5 cells into a 6-well plate and culture overnight. Culture with the medium containing paclitaxel and composite polypeptide postbiotics for 24 h. Wash the cells 2 times with pre-cooled PBS, fill in the staining buffer, FITC staining solution and PI staining solution for staining, incubate on ice for 25 min in a light-proof environment, wash once with PBS, observe the staining of cells in different groups with a fluorescence inverted microscope, and take pictures for record.

[0080] Cell apoptosis is usually called programmed cell death, which is an active cell death process controlled by genes. It involves the activation, expression and regulation of a series of genes and is a death process that cells actively strive for to better adapt to the environment. Tumor cells have the ability of continuous proliferation and resistance to cell apoptosis. By inducing tumor cell apoptosis, the number of tumor cells can be effectively controlled, thus achieving the purpose of anti-tumor.

[0081] The Annexin V-FITC and PI double staining method is based on the principle that phosphatidylserine (PS) on the cell membrane flips from the inner side of the cell membrane to the surface of the cell membrane in the early stage of apoptosis. When both Annexin V-FITC and PI are negative (Annexin V- / PI-), it is a normal living cell; when both Annexin V-FITC and PI are positive (Annexin V+ / PI+), it is a late apoptotic cell, also called a necrotic cell; when Annexin V-FITC is positive and PI is negative (Annexin V+ / PI-), it is an early apoptotic cell. Figure 13 It is the microscopic examination picture of 4T1 cell apoptosis staining, Figure 14 It is the microscopic examination picture of A549 cell apoptosis staining, Figure 15 It is the microscopic examination picture of HepG2 cell apoptosis staining, Figure 16 It is the microscopic examination picture of HuccT1 cell apoptosis staining. As Figures 13 - 16 can be seen, paclitaxel and the experimental group can promote tumor cell apoptosis, and the effect is better than that of the single peptide component and the postbiotics group.

[0082] (IV) Cell scratch test Tumor cells 4T1, A549, HepG2, and HuccT1 with strong migratory ability during the logarithmic growth phase were inoculated with 3×10 5 cells into 6-well plates, with 3 replicates per group and 2.5 mL of culture medium in each well until the cells were evenly distributed. The 6-well plates were cultured in an incubator for 24 h (at this time, the cell density should be greater than 90%). After 24 h, the supernatant was discarded, and the cells were washed twice. A 200 μL pipette tip was used to quickly draw a blank scratch zone perpendicular to the bottom along the cross mark at the bottom of the 6-well plate. Try to use the same pipette tip for each well. The cells scraped off were washed away with PBS three times, and it was checked under an inverted microscope whether the scratch area was cleaned. A control group, a paclitaxel group, and a compound polypeptide postbiotics solid preparation group were set up. 2.5 mL of drug-containing and drug-free blank culture medium was added to each well and placed in an incubator for culture. After 48 h, observations and photographs were taken under an inverted microscope.

[0083] Metastasis, that is, the growth of tumor cells in organs far from their origin sites, is the ultimate and most fatal manifestation of cancer. The vast majority of cancer patients die from metastatic diseases rather than primary tumors. Tumor metastasis is an important link in the malignant development of tumors and the main reason for the failure of tumor treatment. The cell scratch assay can directly reflect the migratory ability of tumor cells.

[0084] The results are as Figure 17 shown. Compared with the control group, paclitaxel and the three experimental groups had a certain inhibitory effect on the migration of tumor cells. The inhibitory effect of the compound polypeptide postbiotics group on the migration of A549, HepG2, and HuccT1 cells was comparable to that of the paclitaxel group. It shows that the compound polypeptide postbiotics can significantly inhibit the migration of tumor cells, and the inhibitory effect is better than that of the single peptide component and the postbiotics group.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite polypeptide postbiotic solid preparation, characterized in that, Comprising postbiotics and peptide components; The postbiotics include the cells and fermentation products of Lactobacillus plantarum nbk-MA2 and Lactobacillus kefiranofaciens subsp. kefiranofaciens ZW3; The preservation number of Lactobacillus plantarum nbk-MA2 is CGMCC NO.16872, and the preservation number of Lactobacillus kefiranofaciens subsp. kefiranofaciens ZW3 is CGMCC NO.2809. The peptide components include albumin polypeptide, cicada flower peptide, ginseng peptide, corn oligopeptide, and collagen peptide.

2. The composite polypeptide postbiotic solid preparation according to claim 1, characterized in that the composite polypeptide postbiotic solid preparation contains, by weight: 30-50 parts of postbiotics and 50-70 parts of peptide components.

3. The composite polypeptide postbiotic solid preparation according to any one of claims 1 or 2, characterized in that The preparation method of the postbiotics includes the following steps: (1) Inoculate Lactobacillus plantarum nbk-MA2 and Lactobacillus kefiranofaciens subsp. kefiranofaciens ZW3 into the seed medium for activation respectively to obtain the primary seed liquid; (2) Inoculate the primary seed liquid into the seed medium for anaerobic culture respectively to obtain the secondary seed liquid; (3) Inoculate the secondary seed liquid into the fermentation medium for high-density fermentation respectively; (4) Sterilize at 115°C for 15 min after fermentation is completed respectively; (5) Concentrate the obtained fermentation broth to a solid content of 50% respectively, then freeze-dry, pulverize, and configure into a postbiotic composition according to the ratio of Lactobacillus plantarum nbk-MA2: Lactobacillus kefiranofaciens subsp. kefiranofaciens ZW3 = 2.5:1 to 3.5:

1.

4. The composite polypeptide postbiotic solid preparation according to claim 3, characterized in that, The seed medium includes the following components by mass fraction: 1-3% glucose, 0.5-2% peptone, 0.5-2% yeast extract powder, 0.2-1% beef extract powder, 0.2-1% anhydrous sodium acetate, 0.1-0.5% ammonium citrate, 0.1-0.5% dipotassium hydrogen phosphate, 0.05-0.2% Tween 80 and the balance of water, with a pH value of 6.7-6.9; The fermentation medium includes the following components by mass fraction: 1-3% glucose, 0.5-2% peptone, 0.5-2% yeast extract powder, 0.2-1% beef extract powder, 0.2-1% anhydrous sodium acetate, 0.1-0.5% ammonium citrate, 0.1-0.5% dipotassium hydrogen phosphate, 0.05-0.2% Tween 80 and the balance of water, with a pH value of 6.3-6.

5.

5. The probiotic solid preparation of the composite polypeptide according to claim 3, wherein The high-density fermentation collects the fermentation broth and performs subsequent sterilization when the cell density reaches 10 9 cfu / mL; The freeze-drying is to pre-freeze the fermentation broth at -38°C for 2 h, vacuum-dry the pre-frozen fermentation broth at 0.2 mba, and pulverize it to 20 meshes after vacuum freeze-drying to obtain the postbiotic preparation.

6. The compound polypeptide postbiotic solid preparation according to any one of claims 1 or 2, characterized in that, The preparation method of the peptide components includes the following steps: (1) Mix egg white powder, cicada flower fruiting body powder, ginseng powder, corn protein powder, and collagen powder with water respectively. The mixed slurry is subjected to high-temperature denaturation, the supernatant is removed, and rinsed; (2) Mix the collected precipitate with water, carry out enzymatic hydrolysis under the action of protease, then raise the temperature to 95°C to inactivate the enzyme for 15 min, collect the filtrate, and carry out drying treatment to obtain dried albumin peptide, cicada flower peptide, ginseng peptide, corn oligopeptide, and collagen peptide; (3) The dried albumin peptide: cicada flower peptide: ginseng peptide: corn oligopeptide: collagen peptide is used to obtain a peptide component according to a ratio of 7-13:0.5-1.5:0.5-1.5:0.5-1.5:15-25.

7. The composite polypeptide postbiotic solid preparation according to claim 6, wherein The high-temperature denaturation temperature is 80-100 °C and the time is 10-60 min; The protease is one or more of papain, alkaline protease, and neutral protease; The conditions for the enzymatic hydrolysis treatment are: the enzymatic hydrolysis temperature is 40-60 °C, the time is 2-8 h, and the pH value is 8-9.

8. The composite polypeptide postbiotics solid preparation according to claim 6, characterized in that, The polypeptide content of each peptide in the peptide component is 30-90%; the distribution ratio of polypeptides with a relative molecular weight of less than 1000 Da in the peptide component is not less than 80%, preferably 80-95%.

9. Use of the composite polypeptide postbiotic solid preparation according to any one of claims 1-8 in the preparation of drugs related to enhancing immunity, inhibiting the growth and migration of tumor cells, and promoting the apoptosis of tumor cells.

10. A product, characterized in that, Containing the composite polypeptide postbiotic solid preparation according to any one of claims 1-8; The product is a drug or a health food.

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