Ganoderma lucidum peptide for enhancing immunity as well as preparation method and application thereof

Abstract

The invention discloses a ganoderma lucidum peptide for enhancing immunity as well as a preparation method and application thereof, and belongs to the technical field of functional peptide preparation. The ganoderma lucidum peptide provided by the invention contains various active ingredients / nutritional ingredients, is high in utilization rate of ganoderma lucidum, can improve the health level of intestinal tracts, promotes absorption and utilization of nutrient substances by the intestinal tracts, and provides a fundamental material guarantee for improving the immunity of the organism; immune cells are activated, and related lymphatic tissues of intestinal tracts are stimulated, so that the effects of regulating cellular immunity and humoral immunity levels are achieved; the preparation method provided by the invention does not use high-temperature, high-pressure and toxic and harmful reagents, has mild conditions, and is simple, easy to operate and environment-friendly; no toxic and harmful substances are generated, and generated filter residues can be directly used as animal feed or plant fertilizer; the prepared ganoderma lucidum peptide is small in molecule, high in activity, rich in active substance variety, high in yield and easy to absorb and utilize by organisms.

Description

Technical Field

[0001] This invention relates to the field of functional peptide preparation technology, and in particular to a Ganoderma lucidum peptide that enhances immunity, its preparation method, and its application. Background Technology

[0002] In a broad sense, Ganoderma lucidum refers to all fungi in the genus Ganoderma, a large group of medicinal fungi, also known as auspicious herbs or "immortal herbs," with very low toxicity; "consumption in large quantities and for extended periods will not cause harm." In a narrow sense, Ganoderma lucidum refers specifically to the fungus Ganoderma sinense (also known as Reishi mushroom) of the Polyporaceae family. Ganoderma lucidum (Leyss. Ex Fr.) Karst. or Ganoderma lucidum Ganoderma sinense The dried fruiting bodies of Zhao, Xu, and Zhang are sweet in taste and neutral in nature, and enter the heart, lung, liver, and kidney meridians. Studies have shown that Ganoderma lucidum contains a variety of active ingredients: 1) polysaccharides, including glucan A and B, Ganoderma lucidum polysaccharides, β-glucan, etc.; 2) triterpenoids, including ganoderic acids A, B, C, C2, D, E, F, K, M, oleanolic acid, etc.; 3) sterols and alkaloids, including betaine, ganoderic acid A, ganoderic acid B, etc.; 4) proteins and amino acids, including essential amino acids (such as lysine and leucine); 5) trace elements and vitamins, including zinc, selenium, iron, etc.

[0003] Reishi mushrooms are hard and rich in lignin and other components. Consuming them directly or in powder form can irritate the stomach and intestines, potentially causing digestive discomfort such as abdominal pain and bloating. There is also a risk of microbial infection. Studies have shown that some people may experience allergic reactions to components of reishi, resulting in rashes, throat swelling, or difficulty breathing. Furthermore, most nutrients in reishi require specific conditions to dissolve and become absorbable by the body; direct consumption or ingestion of powder results in extremely low utilization of the effective components. Traditional methods include decocting or soaking reishi in alcohol, both of which are time-consuming, especially soaking in alcohol. The effective components are extremely limited; boiling denatures and deactivates proteins and small peptides, rendering them insoluble in water or turning them into residue, thus wasting them. Soaking in alcohol only yields components soluble in a certain concentration of alcohol, wasting a large amount of beneficial components. Summary of the Invention

[0004] The purpose of this invention is to provide a Ganoderma lucidum peptide that enhances immunity, its preparation method, and its application, in order to solve the problems of extremely low utilization rate of beneficial components in Ganoderma lucidum in traditional methods, resulting in a large waste of resources, or safety hazards and discomfort to the body.

[0005] To achieve the above objectives, the present invention provides a method for preparing Ganoderma lucidum peptides that enhance immunity, the steps of which are as follows: S1. After removing impurities from the Ganoderma lucidum, chop it, grind it into powder, and make it into a suspension; S2. Add live bacteria to the suspension obtained in S1 and react under stirring conditions for 24-48 hours to obtain fermentation broth; S3. After diluting the fermentation broth, ultrasonically break it up and filter to collect the filtrate; S4. Add protease to the filtrate for enzymatic hydrolysis, and freeze-dry the hydrolysate after sterilization.

[0006] Preferably, in S1, Ganoderma lucidum is the fruiting body of Ganoderma lucidum; impurity removal means removing impurities, including culture medium materials and dust; chopping means chopping to a diameter of no more than 3-5 mm; and grinding the powder and passing it through an 80-100 mesh sieve.

[0007] Preferably, in step S1, before preparing the suspension, sterile water is added to the Ganoderma lucidum powder until the water is saturated, and then 1 volume of sterile water is added and the mixture is stirred for 1-3 hours to obtain the suspension.

[0008] Preferably, the live bacteria in S2 are Bacillus subtilis, and the amount added is such that the number of live bacteria in the suspension is not less than 10. 7 -10 9 CFU / mL; stirring conditions: 35-37℃.

[0009] Preferably, the conditions for ultrasonic disruption in S3 are 100-120Hz ultrasound for 15-20 minutes, and the temperature of the ultrasonic mixture does not exceed 40℃.

[0010] Preferably, in S3, a large-aperture screen and a small-aperture screen are used sequentially during filtration; the large-aperture screen is 80-100 mesh, and the small-aperture screen is 200 mesh.

[0011] Preferably, after filtration in S3, the filter residue is rinsed 2-3 times with sterile water at 37-40℃, and the rinsing liquid is collected and added to the filtrate.

[0012] Preferably, the protease in S4 is pepsin or trypsin, and the pH needs to be adjusted to the optimal pH range of the enzyme using NaOH solution or HCl solution during enzymatic hydrolysis; sterilization is performed by passing the enzyme through a 0.22μm filter membrane.

[0013] The immune-enhancing Ganoderma lucidum peptides prepared by the method described above.

[0014] The application of Ganoderma lucidum peptides, which enhance immunity as described above, in the preparation of health foods that enhance immunity.

[0015] The preparation method provided by this invention uses Ganoderma lucidum as raw material. After grinding into powder, warm water is added to soak and break the soluble substances in the cells. Then, probiotics are used to decompose / enzymatically hydrolyze the components in the Ganoderma lucidum particles. The decomposed / enzymatically hydrolyzed Ganoderma lucidum particles and probiotics are then ultrasonically crushed, filtered, and the filtrate is obtained. After enzymatic hydrolysis, it is freeze-dried to obtain a mixture containing a variety of active ingredients, including beneficial components such as minerals, polypeptides, and vitamins derived from Ganoderma lucidum, as well as products of probiotic decomposition / enzymatic hydrolysis, metabolic products during the growth and reproduction of probiotics, and components contained in the probiotic cells themselves.

[0016] The Bacillus subtilis DE111 strain used in this invention is a probiotic approved by the National Health Commission of my country (2024), GRAS certified by the US FDA, and licensed by the Australian TGA. It has been validated through multiple clinical studies, demonstrating its safety and reliability. It is suitable for use in food fermentation and probiotic preparations. It can survive in the acidic environment of the stomach and colonize the small intestine, promoting intestinal flora diversity; it helps break down food and improve digestive function by secreting enzymes such as proteases; it enhances the body's immune response by stimulating gut-associated lymphoid tissue (GALT); it produces bacteriocins and antimicrobial compounds, significantly inhibiting pathogens; and it generates short-chain fatty acids (such as butyric acid) under aerobic conditions, among other beneficial effects.

[0017] Therefore, the present invention provides a Ganoderma lucidum peptide that enhances immunity, its preparation method, and its application, with the following specific technical effects: (1) The Ganoderma lucidum peptide provided by the present invention contains a variety of active ingredients / nutrients, including polysaccharides, trace elements, vitamins and polypeptides contained in Ganoderma lucidum itself, as well as probiotic secretions and / or metabolites (such as bacteriocins, antibacterial compounds, short-chain fatty acids), and components contained in the probiotic cells themselves. Under the action of antibacterial compounds / bacteriocins and short-chain fatty acids, the growth and reproduction of harmful bacteria in the intestine are inhibited, and the intestinal mucosa is repaired. Under the action of beneficial components contained in Ganoderma lucidum (such as polysaccharides, alkaloids, small molecule peptides, trace elements, and vitamins), the level / risk of intestinal inflammation is reduced, the level of intestinal health is improved, and the absorption and utilization of nutrients in the intestine are promoted, providing a fundamental material guarantee for improving the body's immunity. Under the action of small molecule peptides, polysaccharides, probiotic components and short-chain fatty acids in Ganoderma lucidum, immune cells are activated and intestinal-associated lymphoid tissue is stimulated, thereby achieving the effect of regulating cellular immunity and humoral immunity. The experiments of this invention show that the Ganoderma lucidum peptides prepared in this invention have a good effect on improving / balancing the body's immune level. (2) The preparation method provided by the present invention does not use high temperature, high pressure and toxic and harmful reagents, the conditions are mild, the preparation method is simple and easy to operate, and the environment is friendly; no toxic and harmful substances are generated, and the filter residue produced can be directly used as animal feed or plant fertilizer; the prepared Ganoderma lucidum peptides have small molecules, strong activity, rich variety of active substances, high yield, high utilization rate of Ganoderma lucidum, and are easily absorbed and utilized by the body.

[0018] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0019] The technical solution of the present invention will be further described below through embodiments.

[0020] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention are clearly and completely described below through embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanations of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0022] Example 1 The specific steps for preparing a Ganoderma lucidum peptide are as follows: S1. After removing impurities (such as culture medium) from the surface of dried Ganoderma lucidum (fruiting body), cut the Ganoderma lucidum into 5mm slices, then chop and grind the slices into powder, pass through a 100-mesh sieve, take 500g of Ganoderma lucidum powder and add 40℃ sterile distilled water until the Ganoderma lucidum powder no longer absorbs water, then add sterile distilled water until the volume of the absorbed Ganoderma lucidum powder is 1 times, stir to form a suspension, and keep at 200rpm and 40℃ for 2h.

[0023] S2. After the designated time, add Bacillus subtilis DE111 to bring the viable count to approximately 10. 8 CFU / mL, kept at 200 rpm and 37°C for 24 h.

[0024] S3. Add one volume of pre-cooled sterile distilled water (0℃) to the fermentation broth obtained in S2, and sonicate in several batches at 120Hz for 15 minutes, keeping the temperature below 40℃ during the sonication process (when the temperature reaches 40℃, place it in ice water to cool it down).

[0025] S4. Filter the ultrasonic mixture multiple times to remove visible insoluble matter, then filter through a 200-mesh sieve. Take the filtrate and rinse the filter residue twice with one volume of sterile distilled water preheated at 37°C. Collect the rinsing liquid and add it to the filtrate. Adjust the pH of the filtrate to 7.5 with 1M NaOH, add 2g of trypsin (enzyme activity of 10000U), and enzymatically hydrolyze for 20 minutes at 37°C and 200rpm.

[0026] S5. Filter the enzymatically hydrolyzed solution through a 0.22 μm filter membrane, freeze-dry the filtrate, and then pulverize it. A total of 89.3 g was obtained.

[0027] Example 2 The specific steps for preparing a Ganoderma lucidum peptide are as follows: S1. After removing impurities (such as culture medium) from the surface of dried Ganoderma lucidum (fruiting body), cut the Ganoderma lucidum into 5mm slices, then chop and grind the slices into powder, pass through an 80-mesh sieve, take 500g of Ganoderma lucidum powder and add 40℃ sterile distilled water until the Ganoderma lucidum powder no longer absorbs water, then add sterile distilled water until the volume of the absorbed Ganoderma lucidum powder is 1 times, stir to form a suspension, and keep at 200rpm and 40℃ for 2h.

[0028] S2. After the designated time, add Bacillus subtilis DE111 to bring the viable count to approximately 10. 8 CFU / mL, kept at 200 rpm and 37°C for 24 h.

[0029] S3. Add one volume of pre-cooled sterile distilled water (0℃) to the fermentation broth obtained in S2, and sonicate in several batches at 100Hz for 15 minutes, keeping the temperature below 40℃ during the sonication process (when the temperature reaches 40℃, place it in ice water to cool it down).

[0030] S4. Filter the ultrasonic mixture multiple times to remove visible insoluble matter, then filter through a 200-mesh sieve. Take the filtrate and rinse the filter residue twice with one volume of sterile distilled water preheated at 37°C. Collect the rinsing liquid and add it to the filtrate. Adjust the pH of the filtrate to 7.5 with 1M NaOH, add 2g of trypsin (enzyme activity of 10000U), and enzymatically hydrolyze for 20 minutes at 37°C and 200rpm.

[0031] S5. Filter the enzymatically hydrolyzed solution through a 0.22μm filter membrane, freeze-dry the filtrate, and then pulverize it. A total of 85.1g was obtained.

[0032] Example 3 The specific steps for preparing a Ganoderma lucidum peptide are as follows: S1. After removing impurities (such as culture medium) from the surface of dried Ganoderma lucidum (fruiting body), cut the Ganoderma lucidum into 5mm slices, then chop and grind the slices into powder, pass through a 100-mesh sieve, take 500g of Ganoderma lucidum powder and add 40℃ sterile distilled water until the Ganoderma lucidum powder no longer absorbs water, then add sterile distilled water until the volume of the absorbed Ganoderma lucidum powder is 1 times, stir to form a suspension, and keep at 200rpm and 40℃ for 2h.

[0033] S2. After the designated time, add Bacillus subtilis DE111 to bring the viable count to approximately 10. 8 CFU / mL, kept at 200 rpm and 37°C for 48 h.

[0034] S3. Add one volume of pre-cooled sterile distilled water (0℃) to the fermentation broth obtained in S2, and sonicate in several batches at 120Hz for 15 minutes, keeping the temperature below 40℃ during the sonication process (when the temperature reaches 40℃, place it in ice water to cool it down).

[0035] S4. Filter the ultrasonic mixture multiple times to remove visible insoluble matter, then filter through a 200-mesh sieve. Take the filtrate and rinse the filter residue twice with one volume of sterile distilled water preheated at 37°C. Collect the rinsing liquid and add it to the filtrate. Adjust the pH of the filtrate to 7.5 with 1M NaOH, add 2g of trypsin (enzyme activity of 10000U), and enzymatically hydrolyze for 20 minutes at 37°C and 200rpm.

[0036] S5. Filter the enzymatically hydrolyzed solution through a 0.22μm filter membrane, freeze-dry the filtrate, and then pulverize it. A total of 87.9g was obtained.

[0037] Effect test The Ganoderma lucidum peptides prepared in Examples 1-3 were subjected to mouse immunogenicity tests, as detailed below: Forty 6-week-old male Balb / c mice (22±2g) were randomly divided into four groups of 10 mice each. Each group was inoculated with an equal amount of influenza A virus (0.5mL virus suspension / 22g mouse) for three consecutive days. All mice were fed normally during the experiment, with free access to food and water.

[0038] One group of mice was randomly selected and inoculated with influenza A virus. Simultaneously, each day between 9 and 10 AM, 1g of the Ganoderma lucidum peptide prepared in Example 1 (dissolved in 1mL of sterile distilled water) was administered by gavage. This group was designated as Example 1 group. The treatment methods for mice in Example 2 and Example 3 groups were the same as for Example 1 group, except that they were administered the Ganoderma lucidum peptide prepared in Example 2 and Example 3 by gavage daily, respectively. This gavage treatment continued for 4 weeks. The remaining group served as the control group, receiving an equal volume of physiological saline by gavage at the same time.

[0039] After the experiment, the incidence of disease in the mice was detected, statistically analyzed, and recorded.

[0040] Mice were euthanized by cervical dislocation, and their spleen and thymus were removed, weighed, and their weights recorded. The spleen / body weight ratio and thymus / body weight ratio (mg / 10g) were calculated. The results are shown in Table 1.

[0041] Table 1 Statistical Results

[0042] It can be seen that the influenza A virus used in this invention caused disease in all mice, indicating that the immunocompromised mouse model was successfully constructed. The Ganoderma lucidum peptides prepared in Examples 1-3 all improved the spleen and thymus indices of immunocompromised mice, meaning that the Ganoderma lucidum peptides prepared in Examples 1-3 all improved the immunity of immunocompromised mice. The Ganoderma lucidum peptide in Example 1 showed the best effect and relatively high yield. In Example 2, the simultaneous increase in Ganoderma lucidum powder particle size and decrease in ultrasonic intensity may have resulted in a less effective fragmentation of the Ganoderma lucidum fruiting body compared to Example 1, thus affecting the dissolution of active substances. In Example 3, the excessively long fermentation time of Bacillus subtilis DE111 may have led to excessive consumption of beneficial components in the Ganoderma lucidum.

[0043] Therefore, the Ganoderma lucidum peptides provided by this invention contain a variety of active ingredients / nutrients, have a high utilization rate of Ganoderma lucidum, can improve intestinal health, promote the absorption and utilization of nutrients in the intestine, and provide a fundamental material guarantee for improving the body's immunity; they activate immune cells and stimulate intestinal-associated lymphoid tissue, thereby achieving the effect of regulating cellular immunity and humoral immunity; the preparation method provided does not use high temperature, high pressure and toxic and harmful reagents, the conditions are mild, the preparation method is simple and easy to operate, and it is environmentally friendly; no toxic and harmful substances are generated, and the filter residue can be directly used as animal feed or plant fertilizer; the prepared Ganoderma lucidum peptides have small molecules, strong activity, rich variety of active substances, high yield, and are easily absorbed and utilized by the body.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing Ganoderma lucidum peptides that enhance immunity, characterized in that, The steps are as follows: S1. After removing impurities from the Ganoderma lucidum, chop it, grind it into powder, and make it into a suspension; S2. Add live bacteria to the suspension obtained in S1 and react under stirring conditions for 24-48 hours to obtain fermentation broth; S3. After diluting the fermentation broth, ultrasonically break it up and filter to collect the filtrate; S4. Add protease to the filtrate for enzymatic hydrolysis, and freeze-dry the hydrolysate after sterilization.

2. The method for preparing Ganoderma lucidum peptides that enhance immunity according to claim 1, characterized in that: In S1, Ganoderma lucidum refers to the fruiting body of Ganoderma lucidum; impurity removal means removing impurities, including culture medium materials and dust; chopping means chopping to a diameter of no more than 3-5 mm; and grinding the powder and then passing it through an 80-100 mesh sieve.

3. The method for preparing Ganoderma lucidum peptides that enhance immunity according to claim 1, characterized in that: Before preparing the suspension in S1, add sterile water to the Ganoderma lucidum powder until the water is saturated, and then add 1 volume of sterile water and continue stirring for 1-3 hours to obtain the suspension.

4. The method for preparing Ganoderma lucidum peptides that enhance immunity according to claim 1, characterized in that: The live bacteria in S2 are Bacillus subtilis, and the amount added is such that the number of live bacteria in the suspension is not less than 10. 7 -10 9 CFU / mL; stirring conditions: 35-37℃.

5. The method for preparing Ganoderma lucidum peptides that enhance immunity according to claim 1, characterized in that: The conditions for ultrasonic disruption in S3 are: 100-120Hz ultrasound for 15-20 minutes, and the temperature of the ultrasonic mixture does not exceed 40℃.

6. The method for preparing Ganoderma lucidum peptides that enhance immunity according to claim 1, characterized in that: When filtering in S3, use a large-aperture screen followed by a small-aperture screen in sequence; the large-aperture screen is 80-100 mesh, and the small-aperture screen is 200 mesh.

7. The method for preparing Ganoderma lucidum peptides that enhance immunity according to claim 1, characterized in that: After filtration in S3, rinse the filter residue 2-3 times with sterile water at 37-40℃, collect the rinsing liquid and add it to the filtrate.

8. The method for preparing Ganoderma lucidum peptides that enhance immunity according to claim 1, characterized in that: The protease in S4 is either pepsin or trypsin. During enzymatic hydrolysis, the pH needs to be adjusted to the optimal pH range of the enzyme using NaOH or HCl solution. Sterilization is achieved by passing the enzyme through a 0.22μm filter membrane.

9. The Ganoderma lucidum peptide for enhancing immunity prepared by the preparation method according to any one of claims 1-8.

10. The application of the Ganoderma lucidum peptide as described in claim 9 in the preparation of health foods that enhance immunity.

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