Tricholoma matsutake milk tablet for preventing tumors and preparation method thereof

Abstract

The invention discloses a tricholoma matsutake milk tablet for preventing tumors and a preparation method thereof, and belongs to the technical field of medicine and health food. The tablet comprises dry milk powder, dry tricholoma matsutake extract powder, dry fructus broussonetiae powder and dry garlic powder. The tricholoma matsutake extract dry powder is prepared through the steps of enzymolysis pretreatment, ultrasonic-assisted extraction, decoloration purification and the like. The dry garlic powder is prepared by enzymolysis pretreatment, spray drying, microencapsulation treatment and coating of a protective layer. By optimizing the component proportion and innovating the preparation process, the problems of low bioavailability, insufficient synergistic effect, gastric mucosa irritation and the like of natural active components are solved. High-quality protein in the dry milk powder and the tricholoma matsutake polysaccharide form a nanoscale complex, so that the bioavailability of the polysaccharide is improved; the multi-layer protection mechanism of the dried garlic powder can retain the activity of allicin and reduce the stimulation of gastric mucosa; the fructus broussonetiae component and the tricholoma matsutake polysaccharide synergistically enhance immune response and relieve eye discomfort possibly caused by allicin. Milk lipid is used as a natural adhesive, so that the safety and taste of the tablet are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical and health food, and more specifically, relates to a Tricholoma matsutake milk tablet for preventing tumors and a preparation method thereof. Background Art

[0002] As a major global disease seriously threatening human health, the incidence and mortality of tumors are constantly rising. At present, there are many bottlenecks in functional foods for tumor prevention: natural active ingredients such as polysaccharides, polyphenols, and organosulfides generally face problems such as poor water solubility and low stability, and are easily degraded by gastric acid, digestive enzymes, and oxidative environment in the gastrointestinal tract, resulting in low bioavailability; the action targets of functional foods with single components are limited, and when multiple active ingredients are compounded, due to the unclear interaction mechanism between substances, the synergistic effect is insufficient or even antagonistic reactions occur; some anti-tumor active ingredients stimulate the gastric mucosa or cause discomfort reactions, affecting the product applicability and consumer acceptance; in traditional preparation processes, the extraction efficiency of active ingredients is low, the purification effect is poor, and tablet production often relies on additives such as artificial binders and stabilizers, which pose safety risks and affect the disintegration and taste of the product. The present invention aims to overcome the above defects and provide a Tricholoma matsutake milk tablet for preventing tumors with significantly improved bioavailability of natural active ingredients, enhanced synergistic effect, reduced gastric mucosa irritation, and advanced preparation process. Summary of the Invention

[0003] In view of the above situation, to overcome the defects of the prior art, the present invention optimizes the component ratio and innovates the preparation process, solves the problems of low bioavailability of natural active ingredients, insufficient synergistic effect, gastric mucosa irritation, and additive dependence in the prior art, and achieves a significant improvement in the immune regulation and tumor prevention effects.

[0004] To achieve the above object, the following technical solutions are adopted: On the one hand, the present invention provides a Tricholoma matsutake milk tablet for preventing tumors, comprising the following components in parts by weight: 80 - 90 parts of milk dry powder, 4 - 8 parts of Tricholoma matsutake extract dry powder, 3 - 7 parts of Broussonetia papyrifera dry powder, and 1.5 - 2.5 parts of garlic dry powder.

[0005] Milk is rich in various nutrients. After being made into milk powder, it can retain key substances such as high-quality protein, calcium, and fat-soluble vitamins to the greatest extent. Among them, high-quality proteins such as whey protein and casein are not only important components that make up human tissues and organs, provide essential amino acids for the body, and maintain normal physiological functions, but also can form nano-scale complexes with polysaccharides such as β-glucan in the matsutake extract. Utilizing the amphiphilic structure of proteins can effectively improve the water solubility and intestinal mucosal adsorption of polysaccharides, making the polysaccharides more stable in the gastrointestinal tract, significantly enhancing their bioavailability, and then more efficiently exerting immunomodulatory and tumor prevention effects. At the same time, the calcium in milk powder is the main component of human bones and teeth, helping to maintain bone health, while fat-soluble vitamins (such as vitamins A, D, E, K, etc.) play important roles in regulating the immune system, promoting cell growth and differentiation, etc., providing strong support for the body's immune defense mechanism. Matsutake is a precious medicinal and edible fungus. The extract powder of matsutake contains rich polysaccharide components such as β-glucan, which are the core active substances for exerting anti-tumor effects. β-glucan can stimulate the body's immune system, activate immune cells such as macrophages, T lymphocytes, and natural killer cells, enhance their phagocytic ability and cytotoxicity, and thus effectively identify and eliminate abnormal tumor cells in the body. In addition, matsutake also contains various amino acids, trace elements (such as zinc, selenium, etc.), and bioactive components (such as matsutake polypeptide, matsutake alcohol, etc.). Amino acids are the basis for synthesizing proteins and various bioactive substances, helping to maintain the body's normal metabolism and immune function; the trace element selenium is an important antioxidant that can scavenge free radicals in the body, reduce oxidative damage, and lower the risk of cell carcinogenesis; matsutake polypeptide has antioxidant, anti-inflammatory, and immunomodulatory effects, and can inhibit the growth and metastasis of tumor cells through various pathways. As a traditional tonic Chinese medicine, the dry powder of Broussonetia papyrifera contains various active ingredients such as flavonoids, terpenoids, and polysaccharides. Flavonoid components have strong antioxidant capabilities, can scavenge excess free radicals in the body, reduce oxidative stress damage to cells, and at the same time can regulate the immune system and enhance the body's anti-tumor immune response. Terpenoid components can play anti-tumor roles by inhibiting the proliferation of tumor cells, inducing apoptosis of tumor cells, and inhibiting tumor angiogenesis. Regarding the possible discomfort symptoms such as transient blurred vision caused by allicin, the active ingredients in Broussonetia papyrifera can relieve the relevant discomfort by inhibiting histamine release, reducing the permeability and inflammatory response of eye blood vessels, and at the same time regulating the microcirculation of the eyes and improving the nutritional supply of eye tissues. More importantly, the polysaccharide of Broussonetia papyrifera and the polysaccharide of matsutake have a synergistic effect in the immune regulation pathway. The two interact with each other, can regulate the body's tolerance to active ingredients, enhance the activity and function of immune cells, jointly strengthen the body's immune response, and improve the preventive effect against tumors. The main active ingredient in garlic dry powder is allicin, which has broad-spectrum biological activities and is outstanding in anti-tumor aspects. It can induce tumor cell cycle arrest, causing tumor cells to stagnate in the G2 / M phase or S phase, preventing their DNA replication and cell division, thereby inhibiting the proliferation of tumor cells. At the same time, allicin can also induce apoptosis of tumor cells by activating the apoptosis signaling pathway, reducing the number of tumor cells. However, allicin is vulnerable to destruction by light, heat, and gastrointestinal enzymatic hydrolysis. In addition, garlic also contains other components such as organic sulfides, and these components act synergistically with allicin to jointly exert antibacterial, anti-inflammatory, and anti-tumor effects.

[0006] Furthermore, the dried powder of matsutake extract is prepared by the following steps: (1) Enzymatic pretreatment: Wash and freeze-dry fresh matsutake, then crush it to 80 mesh, add it to a phosphate buffer solution containing calcium chloride, and add a composite enzyme preparation composed of cellulase, pectinase, and β-glucanase. The total enzyme addition amount is 2.5% of the substrate mass. Extract it by shaking in a water bath at 50 °C for 3 h to obtain an extract. (2) Ultrasonic-assisted extraction: Transfer the enzymolysis solution to an ultrasonic reactor, add 0.1% sodium bisulfite of the total mass of the extract as an antioxidant, and perform ultrasonic-assisted extraction. (3) Decolorization and purification: Adjust the pH of the extract to 7.0, add 3 - 5% activated carbon of the total mass of the extract and 0.5 - 2% diatomaceous earth of the total mass of the extract, stir and decolorize at 60 °C for 40 min, and obtain a clarified extract after suction filtration through a Buchner funnel. (4) Membrane separation and concentration: Perform fractional treatment using a hollow fiber ultrafiltration membrane, concentrate the retentate by rotary evaporation, slowly add food-grade ethanol to the concentrated solution to a final concentration of 75%, let it stand at 4 °C for 8 h, then centrifuge at 3000 rpm for 15 min, and collect the precipitate. (5) Column chromatography purification: Dissolve the precipitate in deionized water, load it onto a gel column for elution, collect the main peak component, desalt the eluate through a 10 kDa ultrafiltration membrane, and obtain the dried powder of matsutake extract by spray drying.

[0007] Furthermore, the garlic dry powder is prepared by the following steps: a. Enzymatic pretreatment: After crushing fresh garlic at low temperature, add 0.5% - 1.5% cellulase, and enzymolyze it at 40 - 45 °C for 30 - 60 min to obtain an enzymolysis solution. b. Spray drying: Spray-dry the enzymolysis solution to obtain garlic powder. c. Microencapsulation treatment: Mix the garlic powder with β-cyclodextrin coating solution, and form microencapsulated particles using a fluidized bed coating technology. The inlet air temperature of the fluidized bed is 70 °C, the outlet air temperature is 50 °C, the spray pressure is 0.2 MPa, the atomizing air is 20 m 3 / h, and the material fluidization speed is 0.55 m / s. d. Coating with a protective layer: Spray-coat a protective layer composed of milk lipids on the surface of the microencapsulated particles to form garlic dry powder with a double-layer embedding structure.

[0008] Preferably, the phosphate buffer solution is a 0.05 mol / L phosphate buffer solution containing 0.3 - 0.7% calcium chloride, the pH value of the phosphate buffer solution is controlled at 5.0 - 6.0, and the solid-liquid mass ratio of the Tricholoma matsutake raw material to the phosphate buffer solution is 1:10 - 1:20.

[0009] Preferably, the composite enzyme preparation is composed of cellulase, pectinase, and β-glucanase in a mass ratio of 2 - 4:1 - 3:1, and the total enzyme addition amount is 1.5 - 3.5% of the substrate mass.

[0010] Preferably, the power of the ultrasonic-assisted extraction is 350 - 450 W, the frequency is 25 - 30 kHz, it adopts a pulse mode of working for 3 s / interval for 2 s, the treatment temperature is controlled at 60 - 70 °C, and the treatment time is 30 - 50 min.

[0011] Preferably, the ultrafiltration membrane system uses a hollow fiber membrane with a molecular weight cut-off of 50 - 150 kDa, the operating pressure is 0.3 - 0.5 MPa, and the concentration end point is 1 / 6 - 1 / 10 of the original volume.

[0012] Preferably, the column chromatography purification is eluted with a 0.1 - 0.5 mol / L NaCl solution, and the flow rate is controlled at 0.5 - 1.5 mL / min.

[0013] Preferably, the garlic powder is mixed with the β-cyclodextrin coating solution in a mass ratio of 1:2 - 1:7, and the concentration of the β-cyclodextrin coating solution is 5% - 15%.

[0014] Preferably, the protein content of the milk dry powder is ≥30%, and the fat content is ≥25%.

[0015] On the other hand, the present invention also provides a preparation method of Tricholoma matsutake milk tablets for preventing tumors, including the following steps: S1. Raw material mixing: Using a V-type mixer, put the milk dry powder, Tricholoma matsutake extract dry powder, Broussonetia papyrifera dry powder, and garlic dry powder into the mixer, and mix at a speed of 25 rpm for 40 min until each component is evenly dispersed; S2. Granulation and tabletting: Add purified water accounting for 7.5% of the total mass to the mixed material as a wetting agent, use an SWLP-∮15 type granulator to prepare 30-mesh granules, and dry them in an oven at 55 °C until the water content ≤5%; after drying, the granules are sieved, and tablets are pressed using a rotary tablet press. The rotation speed of the tablet press is controlled at 30 - 50 rpm, and the hardness of the tablets is controlled at 4 - 6 kgf / mm 2 .

[0016] The beneficial effects of the present invention are as follows: Compared with the prior art, through enzymatic hydrolysis pretreatment and ultrasonic-assisted extraction technology, the extraction rate of Tricholoma matsutake polysaccharide in the present invention is increased by more than 1 time compared with the traditional hot water extraction method, and the water solubility is increased by more than 2 times, significantly improving the stability and bioavailability of the polysaccharide in the gastrointestinal tract. At the same time, the retention rate of allicin in garlic is increased by 88%-90% compared with non-microencapsulated garlic powder, and the survival rate of gastric mucosal cells (AGS) is increased by 20%-27%. In addition, in animal experiments, the highest tumor inhibition rate in the example group reached 55.6%, the tumor weight was reduced by 55.6% compared with the model group, and the levels of immune factors IL-2, TNF-α, IFN-γ in the serum and the spleen index were all significantly increased, fully demonstrating the significant advantages of the present invention in immune regulation and tumor prevention.

[0017] (1) Through enzymatic hydrolysis pretreatment and ultrasonic-assisted extraction technology, the polysaccharide components such as β-glucan enriched from Tricholoma matsutake in the present invention are the core anti-tumor active substances. The high-purity Tricholoma matsutake polysaccharide obtained by gel chromatography refinement in the present invention can form a nano-level complex with whey protein in milk, and utilize the amphiphilic structure of the protein to improve the water solubility and intestinal mucosal adsorption of the polysaccharide, enhance the stability of the polysaccharide in the gastrointestinal tract, and significantly improve its bioavailability, so as to more effectively play the role of immune regulation and tumor prevention.

[0018] (2) Allicin in garlic has broad-spectrum antibacterial and tumor cell cycle arrest-inducing effects, but it is easily destroyed by light, heat and gastrointestinal enzymatic hydrolysis. The present invention realizes the retention of allicin activity through a multi-layer protection mechanism. First, low-temperature enzymatic hydrolysis pretreatment destroys the garlic cell wall under mild conditions and releases allicin precursor substances. Secondly, the molecular capsule formed by β-cyclodextrin coating encapsulates allicin through hydrophobic interaction, improving its light stability. Finally, the outer protective film formed by milk lipids utilizes the biocompatibility of liposomes and intestinal epithelial cells to achieve the controlled release of allicin in the alkaline environment of the small intestine, reducing its direct stimulation to the gastric mucosa.

[0019] (3) As a traditional tonic Chinese medicine, the flavonoids and terpenoids contained in Fructus Broussonetiae have antioxidant and optic nerve protection effects. Aiming at the transient blurred vision that may be caused by allicin, the extract of Fructus Broussonetiae can relieve the discomfort symptoms by inhibiting histamine release and regulating ocular microcirculation. Moreover, Fructus Broussonetiae polysaccharide and Tricholoma matsutake polysaccharide have a synergistic effect in the immune regulation pathway, regulating the body's tolerance to active ingredients and strengthening the immune response.

[0020] (4) Milk dry powder not only provides high-quality protein, calcium and fat-soluble vitamins. The whey protein in milk dry powder can form a gel layer in the stomach to adsorb irritating components such as allicin, reducing its direct contact with the gastric mucosa. The casein phosphopeptide in milk forms a coordination bond with the hydroxyl group of Tricholoma matsutake polysaccharide, promoting the intestinal absorption of minerals such as calcium and zinc. At the same time, milk fat acts as a natural binder during granulation, reducing the use of artificial additives and endowing the tablets with good disintegration and taste. Detailed implementation mode

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0022] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only and do not limit the content of this application.

[0023] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from commercial channels unless otherwise specified.

[0024] Example 1: A Tricholoma matsutake milk tablet for preventing tumors and its preparation method. The Tricholoma matsutake milk tablet comprises the following components in parts by weight: 80 parts of milk dry powder, 4 parts of Tricholoma matsutake extract dry powder, 4 parts of Broussonetia papyrifera dry powder, and 1.5 parts of garlic dry powder.

[0025] The Tricholoma matsutake extract dry powder is prepared through the following steps: (1) Enzymatic hydrolysis pretreatment: Wash and freeze-dry fresh Tricholoma matsutake and crush it to 80 meshes, add it to a phosphate buffer solution containing calcium chloride, add a composite enzyme preparation composed of cellulase, pectinase and β-glucanase, and the total enzyme addition amount is 1.5% of the substrate mass. Extract at 50°C in a water bath with shaking for 3 h to obtain an extract; wherein, the phosphate buffer solution is a 0.05 mol / L phosphate buffer solution containing 0.3% calcium chloride, the pH value is controlled at 5.0, the solid-liquid mass ratio of Tricholoma matsutake raw material to phosphate buffer solution is 1:10, and the mass ratio of cellulase, pectinase and β-glucanase in the composite enzyme preparation is 2:1:1; (2) Ultrasonic-assisted extraction: Transfer the enzymolysis solution into an ultrasonic reactor, add sodium bisulfite at 0.1% of the total mass of the extraction solution as an antioxidant, and perform ultrasonic-assisted extraction. The ultrasonic power is 350 W, the frequency is 25 kHz, and the pulsed mode of working for 3 s / interval for 2 s is adopted. The treatment temperature is controlled at 60 °C, and the treatment time is 30 min; (3) Decolorization and purification: Adjust the pH of the extraction solution to 7.0, add activated carbon at 3% of the total mass of the extraction solution and diatomaceous earth at 0.5% of the total mass of the extraction solution, stir and decolorize at 60 °C for 40 min, and obtain a clarified extraction solution after suction filtration through a Buchner funnel; (4) Membrane separation and concentration: Use a hollow fiber ultrafiltration membrane with a molecular weight cut-off of 50 kDa for fractional treatment. The operating pressure is 0.3 MPa, and the concentration end point is 1 / 10 of the original volume. Concentrate the retentate by rotary evaporation, slowly add food-grade ethanol to the concentrated solution to a final concentration of 75%, let it stand at 4 °C for 8 h, and then centrifuge at 3000 rpm for 15 min to collect the precipitate; (5) Column chromatography purification: Dissolve the precipitate in deionized water, use a Sepharose CL-6B gel chromatography column for elution, use a 0.1 mol / L NaCl solution for elution, control the flow rate at 0.5 mL / min, collect the main peak component, desalt the eluate through a 10 kDa ultrafiltration membrane, and obtain the dried powder of Tricholoma matsutake extract by spray drying.

[0026] The garlic dry powder is prepared by the following steps: a. Enzymolysis pretreatment: After crushing fresh garlic at low temperature, add 0.5% cellulase and enzymolyze at 40 °C for 30 min to obtain an enzymolysis solution; b. Spray drying: Spray dry the enzymolysis solution to obtain garlic powder; c. Microencapsulation treatment: Mix the garlic powder with β-cyclodextrin coating solution at a mass ratio of 1:2. The concentration of the β-cyclodextrin coating solution is 5%. Use fluidized bed coating technology to form microencapsulated particles. The inlet air temperature of the fluidized bed is 60 °C, the outlet air temperature is 40 °C, the spray pressure is 0.1 MPa, and the atomizing air is 10 m 3 / h, and the material fluidization speed is 0.3 m / s; d. Coating the protective layer: Spray-coat a protective layer composed of milk lipids on the surface of the microencapsulated particles to form garlic dry powder with a double-layer embedding structure The protein content of the milk dry powder is ≥30%, and the fat content is ≥25%.

[0027] The preparation method of the tumor-preventing Tricholoma matsutake milk tablets includes the following steps: S1. Raw material mixing: Put the milk dry powder, Tricholoma matsutake extract dry powder, Broussonetia papyrifera dry powder, and garlic dry powder into a V-type mixer, mix at a speed of 25 rpm for 40 min until each component is evenly dispersed; S2. Granulation and tabletting: Add purified water accounting for 7.5% of the total mass to the mixed materials as a wetting agent, prepare 30-mesh granules using a SWLP-∮15 type granulator, and dry them in an oven at 55°C until the water content ≤ 5%; after drying, the granules are sieved, and tablets are pressed using a Z-9 type rotary tablet press. The rotation speed of the tablet press is controlled at 30 - 50 rpm, and the hardness of the tablets is controlled at 4 - 6 kgf / mm 2 .

[0028] Example 2. A Tricholoma matsutake milk tablet for preventing tumors and its preparation method. The Tricholoma matsutake milk tablet comprises the following components in parts by weight: 90 parts of milk dry powder, 8 parts of Tricholoma matsutake extract dry powder, 7 parts of Broussonetia papyrifera dry powder, and 2.5 parts of garlic dry powder.

[0029] The Tricholoma matsutake extract dry powder is prepared through the following steps: (1) Enzymatic pretreatment: Wash and freeze-dry fresh Tricholoma matsutake, then crush it to 80 meshes, add it to a phosphate buffer solution containing calcium chloride, and add a composite enzyme preparation composed of cellulase, pectinase, and β-glucanase. The total enzyme addition amount is 3.5% of the substrate mass. Extract it by shaking in a water bath at 50°C for 3 h to obtain an extract; wherein, the phosphate buffer solution is a 0.05 mol / L phosphate buffer solution containing 0.7% calcium chloride, the pH value is controlled at 6.0, the solid-liquid mass ratio of Tricholoma matsutake raw material to the phosphate buffer solution is 1:20, and the mass ratio of cellulase, pectinase, and β-glucanase in the composite enzyme preparation is 4:3:1; (2) Ultrasonic-assisted extraction: Transfer the enzymatic hydrolysate to an ultrasonic reaction kettle, add 0.1% sodium bisulfite of the total mass of the extract as an antioxidant, and perform ultrasonic-assisted extraction. The ultrasonic power is 450 W, the frequency is 30 kHz, and the pulse mode of working for 3 s / interval for 2 s is adopted. The treatment temperature is controlled at 70°C, and the treatment time is 50 min; (3) Decolorization and purification: Adjust the pH of the extract to 7.0, add 5% activated carbon and 2% diatomaceous earth of the total mass of the extract, stir and decolorize at 60°C for 40 min, and obtain a clarified extract after suction filtration through a Buchner funnel; (4) Membrane separation and concentration: Perform fractional treatment using a hollow fiber ultrafiltration membrane with a cut-off molecular weight of 150 kDa, the operating pressure is 0.5 MPa, the concentration end point is 1 / 6 of the original volume. Concentrate the retentate by rotary evaporation, slowly add food-grade ethanol to the concentrated solution to a final concentration of 75%, let it stand at 4°C for 8 h, then centrifuge at 3000 rpm for 15 min, and collect the precipitate; (5) Column chromatography purification: The precipitate was redissolved in deionized water, eluted using a Sepharose CL-6B gel chromatography column, eluted with a 0.5 mol / L NaCl solution, the flow rate was controlled at 1.5 mL / min, the main peak component was collected, and after desalting the eluate through a 10 kDa ultrafiltration membrane, the matsutake extract dry powder was obtained by spray drying.

[0030] The garlic dry powder is prepared by the following steps: a. Enzymatic pretreatment: After freshly crushed garlic was broken at low temperature, 1.5% cellulase was added, and enzymatic hydrolysis was carried out at 45 °C for 60 min to obtain an enzymatic hydrolysate; b. Spray drying: The enzymatic hydrolysate was spray dried to obtain garlic powder; c. Microencapsulation treatment: The garlic powder was mixed with β-cyclodextrin coating solution at a mass ratio of 1:7, the concentration of the β-cyclodextrin coating solution was 15%, and microencapsulated particles were formed by fluidized bed coating technology. The inlet air temperature of the fluidized bed was 80 °C, the outlet air temperature was 60 °C, the spray pressure was 0.3 MPa, and the atomizing air was 30 m 3 / h, and the material fluidization speed was 0.8 m / s; d. Coating the protective layer: A protective layer composed of milk lipids was spray-coated on the surface of the microencapsulated particles to form garlic dry powder with a double-layer embedding structure.

[0031] The protein content of the milk dry powder is ≥30%, and the fat content is ≥25%.

[0032] The preparation method of the matsutake milk tablets for preventing tumors includes the following steps: S1. Raw material mixing: The milk dry powder, matsutake extract dry powder, Broussonetia papyrifera dry powder, and garlic dry powder were put into a V-type mixer and mixed at a speed of 25 rpm for 40 min until each component was evenly dispersed; S2. Granulation and tabletting: Purified water accounting for 7.5% of the total mass was added to the mixed material as a wetting agent, 30-mesh granules were prepared using a SWLP-∮15 type granulator, and dried in an oven at 55 °C until the water content ≤5%; after drying, the granules were sieved, and tablets were pressed using a Z-9 type rotary tablet press. The rotation speed of the tablet press was controlled at 30 - 50 rpm, and the hardness of the tablets was controlled at 4 - 6 kgf / mm 2 .

[0033] Example 3. A matsutake milk tablet for preventing tumors and its preparation method. The matsutake milk tablet comprises the following components in parts by weight: 86 parts of milk dry powder, 6 parts of matsutake extract dry powder, 6 parts of Broussonetia papyrifera dry powder, and 2 parts of garlic dry powder.

[0034] The matsutake extract dry powder is prepared by the following steps: (1)Enzymatic pretreatment: Wash and freeze-dry fresh Tricholoma matsutake, then crush it to 80 mesh. Add it to a phosphate buffer solution containing calcium chloride, and add a composite enzyme preparation composed of cellulase, pectinase, and β-glucanase. The total enzyme addition is 2.5% of the substrate mass. Extract it by shaking in a water bath at 50 °C for 3 h to obtain an extract. Among them, the phosphate buffer solution is a 0.05 mol / L phosphate buffer solution containing 0.5% calcium chloride, the pH value is controlled at 5.5, the solid-liquid mass ratio of Tricholoma matsutake raw material to the phosphate buffer solution is 1:15, and the mass ratio of cellulase, pectinase, and β-glucanase in the composite enzyme preparation is 3:2:1; (2)Ultrasound-assisted extraction: Transfer the enzymolysis solution to an ultrasonic reactor, add 0.1% sodium bisulfite of the total mass of the extract as an antioxidant, and perform ultrasonic-assisted extraction. The ultrasonic power is 400 W, the frequency is 27.5 kHz, and the pulse mode of working for 3 s / interval for 2 s is adopted. The treatment temperature is controlled at 65 °C, and the treatment time is 40 min; (3)Decolorization and purification: Adjust the pH of the extract to 7.0, add 4% activated carbon of the total mass of the extract and 1.25% diatomaceous earth of the total mass of the extract, stir and decolorize at 60 °C for 40 min, and obtain a clarified extract after suction filtration through a Buchner funnel; (4)Membrane separation and concentration: Use a hollow fiber ultrafiltration membrane with a molecular weight cut-off of 100 kDa for fractional treatment. The operating pressure is 0.4 MPa, and the concentration end point is 1 / 8 of the original volume. Concentrate the retentate by rotary evaporation, slowly add food-grade ethanol to the concentrated solution to a final concentration of 75%, let it stand at 4 °C for 8 h, and then centrifuge at 3000 rpm for 15 min to collect the precipitate; (5)Column chromatography refinement: Dissolve the precipitate in deionized water, elute it with a Sepharose CL - 6B gel chromatography column, use a 0.3 mol / L NaCl solution for elution, control the flow rate at 1.0 mL / min, collect the main peak component, desalt the eluate through a 10 kDa ultrafiltration membrane, and obtain the dry powder of Tricholoma matsutake extract by spray drying.

[0035] The garlic dry powder is prepared by the following steps: a. Enzymatic pretreatment: After low-temperature crushing of fresh garlic, add 1.0% cellulase and enzymolyze at 42.5 °C for 45 min to obtain an enzymolysis solution; b. Spray drying: Spray-dry the enzymolysis solution to obtain garlic powder; c. Microencapsulation treatment: Mix the garlic powder with β-cyclodextrin coating solution at a mass ratio of 1:4.5. The concentration of the β-cyclodextrin coating solution is 10%. Use fluidized bed coating technology to form microencapsulated particles. The inlet air temperature of the fluidized bed is 70 °C, the outlet air temperature is 50 °C, the spray pressure is 0.2 MPa, and the atomizing air is 20 m 3 / h, and the material fluidization speed is 0.55 m / s; d. Coating with a protective layer: Spray-coat a protective layer composed of milk lipids on the surface of the microencapsulated particles to form garlic dry powder with a double-layer embedding structure.

[0036] The protein content of the milk dry powder is ≥ 30%, and the fat content is ≥ 25%.

[0037] The preparation method of the Tricholoma matsutake milk tablets for preventing tumors includes the following steps: S1. Raw material mixing: Put the milk dry powder, Tricholoma matsutake extract dry powder, Broussonetia papyrifera seed dry powder, and garlic dry powder into a V-type mixer and mix at a speed of 25 rpm for 40 min until each component is evenly dispersed; S2. Granulation and tabletting: Add purified water accounting for 7.5% of the total mass to the mixed materials as a wetting agent, use a SWLP-∮15 type granulator to prepare 30-mesh granules, and dry them in an oven at 55 °C until the water content is ≤ 5%; after drying, the granules are sieved, and a Z-9 type rotary tabletting machine is used to press them into tablets. The rotation speed of the tabletting machine is controlled at 30 - 50 rpm, and the hardness of the tablets is controlled at 4 - 6 kgf / mm 2 .

[0038] Comparative Example 1: The difference between this comparative example and Example 3 is that the Broussonetia papyrifera seed dry powder component is removed, and the rest is the same as in Example 3.

[0039] Comparative Example 2: The difference between this comparative example and Example 3 is that there is no enzymatic hydrolysis and ultrasonic-assisted process, but a traditional Tricholoma matsutake extraction process is used, that is, direct hot water extraction (100 °C, 2 h) is used without enzymatic hydrolysis pretreatment and ultrasonic-assisted extraction, and the rest is the same as in Example 3.

[0040] Comparative Example 3: The difference between this comparative example and Example 3 is that the garlic powder is not microencapsulated, that is, only steps a (enzymatic hydrolysis pretreatment) and b (spray drying) are carried out during the preparation of the garlic dry powder, steps c (microencapsulation) and d (protective layer coating) are omitted, and the garlic powder is directly used instead of the garlic dry powder in Example 3, and the rest is the same as in Example 3.

[0041] Test Example 1: Detection of the extraction rate and water solubility of Tricholoma matsutake polysaccharide, and the measurement results are shown in Table 1.

[0042] (1) Weigh 500 g of fresh Tricholoma matsutake raw materials (about 50 g after freeze-drying), prepare Tricholoma matsutake extract dry powder according to the methods of Examples 1 - 3 and Comparative Example 2 (traditional hot water extraction), and calculate the extraction rate according to the following formula: Extraction rate (%) = (mass of Tricholoma matsutake extract dry powder / mass of fresh Tricholoma matsutake raw materials) × 100%.

[0043] (2)Weigh 100 mg of each Tricholoma matsutake polysaccharide sample, add 10 mL of deionized water, stir magnetically at 25 °C for 30 min, and centrifuge at 3000 rpm for 10 min.

[0044] Take the supernatant, determine the polysaccharide content by the phenol-sulfuric acid method, and calculate the solubility (g / 100 mL of water).

[0045] As can be seen from Table 1, through enzymatic pretreatment and ultrasonic-assisted extraction technology, the extraction rate of Tricholoma matsutake polysaccharide in the present invention is greatly improved compared with the traditional hot water extraction method, and the water solubility is also greatly enhanced. This indicates that this process effectively breaks the cell wall structure of Tricholoma matsutake, promotes the release and dissolution of polysaccharides such as β-glucan, and forms a nano-complex combined with milk protein, which can significantly improve the stability and bioavailability of polysaccharides in the gastrointestinal tract.

[0046] Table 1 Results of the determination of the extraction rate and water solubility of Tricholoma matsutake polysaccharide in Test Example 1 Among them, + It means that compared with Comparative Example 2, P < 0.05, and the extraction rate and water solubility are significantly improved.

[0047] Test Example 2 Detection of the retention rate of allicin in garlic dry powder and the irritation to gastric mucosa, and the determination results are shown in Table 2.

[0048] (1)Calculate the allicin retention rate according to the following method: Chromatographic conditions: C18 column (4.6 × 250 mm, 5 μm), mobile phase methanol: water = 70:30, flow rate 1.0 mL / min, detection wavelength 254 nm, injection volume 10 μL.

[0049] Determine the allicin content in the fresh garlic enzymatic hydrolysate (after step a) and the microencapsulated garlic dry powder (Examples 1-3, Comparative Example 3) respectively, and calculate the retention rate: Retention rate (%) = (Allicin content in the sample / Allicin content in the enzymatic hydrolysate) × 100%.

[0050] (2)Detect the irritation to gastric mucosa of the milk tablets according to the following method.

[0051] AGS cells are cultured to the logarithmic phase and inoculated into a 96-well plate (1 × 10 4 cells / well) and cultured for 24 h.

[0052] Add the garlic powder suspension (concentration 1 mg / mL) treated differently in Examples 1-3 and Comparative Example 3, add an equal amount of PBS to the control group, discard the supernatant after culturing for 24 h, and detect the cell viability by the MTT method: Cell viability (%) = (OD value of the experimental group / OD value of the control group) × 100%.

[0053] As can be seen from Table 2, through the double embedding technology of β-cyclodextrin coating and milk lipid protective layer in the present invention, the retention rate of allicin in the examples is increased by 88%-90% compared with Comparative Example 3 without microencapsulation treatment, and the survival rate of gastric mucosal cells (AGS) is increased by 20%-27%. This confirms that the process effectively protects allicin from light, heat and enzymatic degradation, while reducing its direct stimulation to the gastric mucosa, not only retaining the activity of allicin inducing tumor cell cycle arrest, but also improving the safety and applicability of the product.

[0054] Table 2 Detection results of allicin retention rate and gastric mucosal irritation of garlic dry powder Among them, + It indicates that compared with Comparative Example 3, P<0.05, and the retention rate and cell survival rate are significantly improved.

[0055] Test Example 3: Performance test of immunomodulatory and anti-tumor activities of milk tablets, and the measurement results are shown in Table 3.

[0056] (1) The influence of milk tablets on the proliferation of T lymphocytes was measured according to the following method: Mouse spleen lymphocytes were isolated, adjusted to a concentration of 2×106 cells / mL, and inoculated into 96-well plates (100 μL / well).

[0057] The sample solutions of each example and comparative example (concentration 1 mg / mL) were added, and ConA (5 μg / mL) was added as the positive control. After culturing for 72 h, MTT (5 mg / mL, 20 μL / well) was added, and DMSO was added after 4 h to measure OD 570 nm.

[0058] (2) The influence of milk tablets on the phagocytic ability of macrophages was measured according to the following method: RAW264.7 cells were inoculated into 96-well plates (1×10 5 cells / well), the sample solution (1 mg / mL) was added, and after culturing for 24 h, neutral red staining solution (0.05%) was added, and OD 570 nm was measured after 1 h.

[0059] (2) The anti-tumor activity performance of milk tablets was measured according to the following method: HepG2 cells and A549 cells were respectively inoculated into 96-well plates (5×10 3 cells / well), the sample solution (concentration 0.5 / 1 / 2 mg / mL) was added, and after culturing for 48 h, the OD value was measured by the MTT method, and the inhibition rate was calculated: Inhibition rate (%) = (1 - OD value of experimental group / OD value of control group) × 100%.

[0060] As can be seen from Table 3, the T lymphocyte proliferation rate, macrophage phagocytosis rate, and inhibition rates of liver cancer and lung cancer cells in each example group were significantly better than those in the comparative example group. Among them, in Comparative Example 1 without Fructus Broussonetiae, all indicators decreased, indicating that the flavonoid and terpene components in Fructus Broussonetiae have a synergistic effect with Tricholoma matsutake polysaccharide, and can jointly enhance the anti-tumor effect by regulating the immune pathway (such as activating T cells and enhancing macrophage activity) and directly inhibiting tumor cell proliferation; while the traditional extraction process and the insufficient activity of unembedded garlic further verified the necessity of the component ratio and preparation process.

[0061] Table 3 Test results of immunomodulation and anti-tumor activity in Test Example 3 Among them, + Indicates that compared with all comparative example groups, P < 0.05, and the activity is significantly enhanced.

[0062] Test Example 4: In vivo efficacy verification experiment of milk tablets, and the measurement results are shown in Table 4.

[0063] (2) Animal grouping and treatment: Balb / c mice (6 weeks old, female, n = 48, 6 mice in each group): Normal group: Intragastric administration with normal saline; Model group: Intragastric administration with normal saline, and subcutaneous inoculation with S180 cells (1×10 6 cells / mouse); Example 1-3 and Comparative Example 1-3 groups: Intragastric administration with corresponding samples (0.5 g / kg / d); Continuous intragastric administration for 14 days, record the body weight every day, inoculate tumor cells on the 7th day, sacrifice the mice on the 14th day, weigh the tumor, and calculate the tumor inhibition rate: Tumor inhibition rate (%) = (tumor weight in the model group - tumor weight in the experimental group) / tumor weight in the model group × 100%.

[0064] (2) Detection of immune indexes: Levels of serum IL-2, TNF-α, and IFN-γ: ELISA method; Spleen index = spleen weight (mg) / body weight (g).

[0065] The animal experiment showed that the highest tumor inhibition rate in the example group reached 55.6%, the tumor weight in the example group was reduced by 55.6% compared with the model group, and the levels of immune factors IL-2, TNF-α, and IFN-γ in the serum and the spleen index were significantly increased, indicating that the product can play an anti-tumor role by enhancing the body's immune response (such as promoting cytokine secretion and improving the function of the spleen immune organ). In contrast, the tumor inhibition rate and immune indexes of Comparative Example -3 were lower than those in the example group, further corroborating that the optic nerve protection and immune synergy of Fructus Broussonetiae, the efficient extraction of Tricholoma matsutake polysaccharide, and the stable protection of the active ingredients of garlic are the key factors for enhancing the in vivo efficacy.

[0066] Table 4 Verification Results of In Vivo Efficacy for Test Example 4 Among them, + indicates P < 0.05 compared with the model group.

[0067] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0068] All in all, if those of ordinary skill in the art are inspired by it and, without departing from the purpose of the present invention, design in a non-creative way similar methods and embodiments to this technical solution, they shall fall within the protection scope of the present invention.

Claims

1. A pine mushroom milk tablet for preventing tumors, characterized in that: The invention comprises the following components in parts by weight: 80-90 parts of dry milk powder, 4-8 parts of dry pine mushroom extract powder, 3-7 parts of mulberry seed powder, and 1.5-2.5 parts of dry garlic powder; The Tricholoma matsutake extract dry powder is prepared by the following steps: (1) Enzymatic pretreatment: Wash and freeze-dry fresh matsutake mushrooms, crush them into 80 mesh, add them into phosphate buffer containing calcium chloride, add a composite enzyme preparation consisting of cellulase, pectinase and β-glucanase, and add the total amount of enzyme added to 2.5% of the substrate mass. Extract them in a water bath at 50°C with shaking for 3 h to obtain an extract. (2) Ultrasonic-assisted extraction: The enzymatic hydrolysate was transferred into an ultrasonic reactor, and 0.1% sodium bisulfite as an antioxidant was added to the total mass of the extract for ultrasonic-assisted extraction; (3) Decolorization and purification: adjust the pH of the extract to 7.0, add 3-5% activated carbon and 0.5-2% diatomaceous earth, stir and decolorize at 60°C for 40 min, and obtain a clear extract after filtration through a Buchner funnel; (4) Membrane separation and concentration: hollow fiber ultrafiltration membrane was used for graded treatment, and the retentate was concentrated by rotary evaporation. Food-grade ethanol was slowly added to the concentrate to a final concentration of 75%. After standing at 4°C for 8 h, it was centrifuged at 3000 rpm for 15 min to collect the precipitate. (5) Column chromatography purification: The precipitate was redissolved in deionized water and eluted using a Sepharose CL-6B gel chromatography column to collect the main peak components. The eluate was desalted by a 10 kDa ultrafiltration membrane and then spray-dried to obtain a dry powder of the pine mushroom extract. The dried garlic powder is prepared by the following steps: a. Enzymatic pretreatment: After crushing the fresh garlic at low temperature, add 0.5%-1.5% cellulase, and perform enzymatic hydrolysis at 40-45℃ for 30-60min to obtain enzymatic solution; b. Spray drying: spray drying the enzymatic hydrolysate to obtain garlic powder; c. Microencapsulation treatment: Mix garlic powder with β-cyclodextrin coating liquid and use fluidized bed coating technology to form microencapsulated particles. The inlet air temperature of the fluidized bed is 70°C, the outlet air temperature is 50°C, the spray pressure is 0.2MPa, and the atomizing wind is 20m 3 / h, the material fluidization speed is 0.55m / s; d. Coating protective layer: spray a protective layer composed of milk lipids on the surface of the microencapsulated particles to form a double-layer encapsulated structure of garlic dry powder.

2. The pine mushroom milk tablet for preventing tumors according to claim 1, characterized in that: The phosphate buffer is a 0.05 mol / L phosphate buffer containing 0.3-0.7% calcium chloride, the pH value of the phosphate buffer is controlled at 5.0-6.0, and the solid-liquid mass ratio of the matsutake raw material to the phosphate buffer is 1:10-1:

20.

3. The pine mushroom milk tablet for preventing tumors according to claim 1, characterized in that: The composite enzyme preparation is composed of cellulase, pectinase and beta-glucanase in a mass ratio of 2-4:1-3:1, and the total amount of enzyme added is 1.5-3.5% of the mass of the substrate.

4. The pine mushroom milk tablet for preventing tumors according to claim 1, characterized in that: The power of the ultrasonic assisted extraction is 350-450W, the frequency is 25-30kHz, a pulse mode of working for 3s / resting for 2s is adopted, the processing temperature is controlled at 60-70°C, and the processing time is 30-50min.

5. The pine mushroom milk tablet for preventing tumors according to claim 1, characterized in that: The ultrafiltration membrane system adopts a hollow fiber membrane with a molecular weight cutoff of 50-150 kDa, an operating pressure of 0.3-0.5 MPa, and a concentration end point of 1 / 6-1 / 10 of the original volume.

6. The pine mushroom milk tablet for preventing tumors according to claim 1, characterized in that: The column chromatography purification is eluted with a 0.1-0.5 mol / L NaCl solution, and the flow rate is controlled at 0.5-1.5 mL / min.

7. The pine mushroom milk tablet for preventing tumors according to claim 1, characterized in that: The garlic powder and the beta-cyclodextrin coating solution are mixed in a mass ratio of 1:2-1:7, and the concentration of the beta-cyclodextrin coating solution is 5%-15%.

8. The pine mushroom milk tablet for preventing tumors according to claim 1, characterized in that: The protein content of the milk dry powder is ≥30%, and the fat content is ≥25%.

9. The method for preparing the tumor-preventing Matsutake milk tablet according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1. Raw material mixing: Using a V-type mixer, the milk powder, pine mushroom extract powder, paper mulberry powder, and garlic powder were put into the mixer and mixed at 25 rpm for 40 min until the components were evenly dispersed; S2. Granulation and tableting: Add 7.5% of the total mass of purified water as a wetting agent to the mixed material, use a SWLP-∮15 granulator to prepare 30 mesh granules, and dry them in a 55°C oven to a moisture content of ≤5%. After drying, the granules are sized and pressed into tablets using a rotary tablet press. The speed of the tablet press is controlled at 30-50rpm, and the hardness of the tablets is controlled at 4-6kgf / mm 2 .