Preparation method and application of marasmius albus fermentation product

Abstract

The invention belongs to the technical field of biology, and relates to a preparation method and application of Marasmilla sp leavening, and the preparation method mainly comprises the following steps: preparing strains, preparing primary liquid and secondary liquid strains, preparing a production culture medium, inoculating, culturing, collecting mycelia and exopolysaccharides, concentrating the fermentation liquor under reduced pressure, separating the polysaccharides by using an alcohol precipitation method, and obtaining the Marasmilla sp leavening. And freeze-drying the polysaccharide to prepare the polysaccharide freeze-dried powder. The optimum carbon source for preparing the fermentation product from the white marasmius is maltose, the optimum nitrogen source is yeast extract, the yield of mycelia in the fermentation product can reach 14.25 g / L, and exopolysaccharides are not lower than 0.75 g / L. The fermented product has the function of inhibiting the activity of alpha-glucosidase and can be used as a potential biological raw material for assisting in reducing blood sugar. The method for liquid culture has the advantages of rapidness, high efficiency, simplicity and convenience, realizes mass production of mycelia and exopolysaccharides of the white marasmius communis, and solves the problems that wild resources are difficult to collect and less in quantity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for preparing a fermentation product by liquid culture of Microcarpa serrata and application thereof. Background Art

[0002] Marasmiellus fungi are inextricably linked to human life and are the most common decomposers in ecosystems. Some species possess significant medicinal value. According to current classical taxonomy, there are over 400 species of Marasmiellus (Singer 1976, Antonín & Noordeloos 2010, etc.). Guizhou Province, located in southwestern my country, is one of the world's modern biodiversity hotspots, boasting exceptionally high biodiversity and numerous endemic species. Studies have reported the pharmacological effects of a unique species of Marasmiellus: compounds isolated from the fermentation broth exhibit weak cytotoxic activity against BEL-7702 (human liver cancer cells) and SGC-7901 (human gastric cancer cells), and exhibit high acetylcholinesterase inhibitory activity (Yang, 2014).

[0003] In the spring or summer and autumn forests, on the fallen branches and leaves, the microdermabranch fungi grow in scattered, grouped or sometimes nearly in clusters, but the fruiting bodies are small and not easily detected, and the strain separation is relatively difficult. The inventors obtained wild marasmiellus sp when conducting resource surveys and collections in Guizhou Province. After strain separation and identification, it was confirmed that the strain obtained by separation was white microdermabranch (Marasmiellus sp). The liquid culture method of mycelium and exopolysaccharide was carried out using this self-isolated strain to obtain pure mycelium and exopolysaccharide products of the species. After testing, the mycelium of white microdermabranch is rich in nutrients such as crude protein and amino acids, with an ash content of 10% and an active substance intracellular polysaccharide content of 3.3%. These early research and development data show that the white microdermabranch strain has good application potential. By optimizing the culture conditions, its mycelium biomass and exopolysaccharide yield can be significantly improved, providing valuable biological raw materials for research and development and application. Carrying out its artificial cultivation has great economic value and scientific significance. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies for utilizing the macrofungus Microdermabranchus. It provides a liquid culture method for the mycelium and exopolysaccharides of Microdermabranchus albus, establishes corresponding culture conditions and processes, and enables artificial mass production of this resource. This extraction method offers the advantages of high efficiency, simplicity, and ease of operation, resulting in a product with high cost-effectiveness and high purity. The detailed invention is as follows.

[0005] White Microdermabranch Species: The strain White Microdermabranch Marasmiellus sp.DCY6478 is isolated from the wild fruiting body of White Microdermabranch ( Figure 1This strain is currently deposited with the Guangdong Provincial Center for Microbiological Culture Collection under the accession number GDMCC No. 65883 and a date of February 14, 2025. Detailed information on this strain is as follows: Marasmiellus sp., deposited with the Guangdong Provincial Center for Microbiological Culture Collection at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, date of deposit: February 14, 2025, and GDMCC No. 65883. The viability of this biological material (strain) was tested by the collection center and confirmed to be viable.

[0006] Molecular identification of the strain was conducted: The test strain was purified and molecular phylogenetic identification was performed using ITS rDNA sequence analysis. Genomic DNA was extracted, amplified, and sequenced using universal primers. BLAST sequence alignment of this sequence against the NCBI database revealed a 99.70% similarity with the Marasmiellus candidus sequence number OP364581 in the NCBI database, meeting the requirements for species identification. The isolated strain was ultimately confirmed to be Marasmiellus sp.

[0007] Based on the fermentation characteristics of the strain, the fermentation conditions and process were optimized, a method for preparing the fermentation product was established, and the application of the fermentation product was verified. The specific content and steps include S1-S6.

[0008] S1. Preparation of strains: Inoculate the white microdermabranch strain ( Figure 2 ) Culture in dark at 20-28°C on PDA tube medium for 10-15 days until the mycelium covers 2 / 3 or more of the slope.

[0009] S2. Preparation of primary liquid culture: The grown slant tube culture was inoculated into a primary liquid culture medium. The weight percentage of each component in the primary liquid culture medium is as follows: carbon source 1%-3%, nitrogen source 0.2%-1%, potassium dihydrogen phosphate 0.05%-0.1%, magnesium sulfate 0.05% and vitamin B1 0.002%-0.005%. Each component was dissolved in water and dispensed into a culture container, with the container filling 20%-60%. The pH value was 5.5-7.5. The culture was shaken at 20-32°C with a shaker speed of 120-200 r / min for 6-14 days until the mycelial balls were filled with liquid culture medium. The culture medium can then be used as a primary liquid culture medium. Figure 3 ); When the diameter of the mycelial ball of the first-level liquid culture is greater than 10mm, it can be broken up under sterile conditions using a cell disruptor to make it into a uniform mycelial slurry, thereby improving its propagation capacity.

[0010] S3. Preparation of secondary liquid culture: The primary liquid culture is inoculated into a secondary liquid culture medium at an inoculum volume of 5%-15% (V / V). The weight percentages of the components in the secondary liquid culture medium are as follows: carbon source 1%-3%, nitrogen source 0.2%-1%, potassium dihydrogen phosphate 0.05%-0.1%, magnesium sulfate 0.05% and vitamin B1 0.002%-0.005%. Each component is dissolved in water, the pH value is 5.5-7.5, the container filling volume and other culture conditions are the same as those of the primary liquid culture, and the culture is carried out for 4-10 days until the mycelial balls fill the entire culture medium, and the secondary liquid culture is used.

[0011] S4. Inoculation of production medium: Inoculate the above secondary liquid strains into the production liquid medium at a ratio of 5%-15% (V / V). The formula of the production liquid medium is determined according to the target product of liquid culture. The container is filled with 20%-60%. Culture at 20-32°C with shaking and a shaking speed of 120-200 r / min for 4-14 days, or use a fermenter as the culture equipment ( Figure 4 ), 0.01-0.1% defoamer must be added to the culture medium, the ventilation volume is 6-12L / min, the stirring speed is 150-200r / min, and the fermentation culture is carried out for 4-14 days. When the mycelial balls fill the entire culture medium, the culture can be terminated and the product can be collected and processed ( Figure 5 ).

[0012] S5. Collection and cleaning of mycelium: Use centrifugation or plate filter press to separate the mycelium and fermentation liquid. Rinse the mycelium with water 2-3 times to remove the residual culture medium. Figure 6 ), drying the mycelium at 45-80°C, with a mycelium yield of not less than 10 g / L.

[0013] S6. Extraction and purification of extracellular polysaccharides: The supernatant obtained by centrifugation in step S5 or the filtrate obtained by filtration, i.e., the fermentation broth, is concentrated under reduced pressure, and then the polysaccharides are separated by alcohol precipitation. Four volumes of anhydrous ethanol are added to make the final ethanol concentration 75% or above, and the mixture is allowed to stand overnight at 4°C. The polysaccharides are collected by centrifugation and freeze-dried to prepare a polysaccharide freeze-dried powder with a yield of not less than 0.75 g / L. According to production requirements, the fermentation concentrate can also be hydrolyzed with a protease (such as neutral protease, trypsin, papain) before the polysaccharide is separated by alcohol precipitation to hydrolyze the protein into small molecule peptides or amino acids to improve the purity of the white microdermabranch polysaccharide. The polysaccharide yield is not less than 0.55 g / L, and the polysaccharide content is not less than 55%.

[0014] Preferably, the carbon source in step S2 and step S3 is one of maltose, glucose, dextrin, and soluble starch.

[0015] Preferably, the carbon source in step S2 and step S3 is maltose.

[0016] Preferably, the nitrogen source in step S2 and step S3 is one of yeast extract, peptone, beef extract powder, or soy peptone.

[0017] Preferably, the nitrogen source in step S2 and step S3 is yeast extract.

[0018] Preferably, the formula of the production liquid culture medium in step S4 is the same as the formula of the first-level liquid culture medium; wherein, when the main target product of the production culture is extracellular polysaccharide, the carbon source is a non-starch substance.

[0019] Preferably, the inoculation amount in steps S2, S3 and S4 is 10%.

[0020] Preferably, the culture temperature in steps S2, S3, and S4 is 28°C.

[0021] Preferably, the shaking speed in steps S2, S3 and S4 is 120-150 r / min, 0.05% defoaming agent is added to the culture medium during fermentation tank production, the ventilation volume is 10 L / min, and the stirring speed is 180 r / min.

[0022] The invention relates to the application of fermentation products of Marasmiellus sp. DCY6478, i.e. mycelium and fermentation liquid, in the preparation of auxiliary blood sugar lowering products.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention adopts a liquid culture production method for Microcarpa spp., which has the advantages of being fast, efficient, and simple. The product obtained by liquid culture is relatively simple and easier to extract, separate, and purify. It can achieve the mass production of Microcarpa spp. fermentation products, mainly mycelium and extracellular polysaccharides, to meet market demand, solve the problems of difficult collection and small quantity of wild resources, and avoid the trouble of destroying wild resources due to excessive manual picking. Microcarpa spp. mycelium contains rich nutrients and active ingredients, including proteins, amino acids, polysaccharides, flavonoids, etc., and can be used as a biological raw material for the development of auxiliary blood sugar lowering products, with great economic value and certain scientific significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Other features, objects and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings.

[0026] Figure 1 Wild fruiting bodies of Microcarpa leucophylla

[0027] Figure 2 Microdermabranch slant fungus

[0028] Figure 3 Microdermabranchus first-grade liquid fungus

[0029] Figure 4 Preparation of Microcarpa alba fermentation products in fermentation tank

[0030] Figure 5 Fermentation collection

[0031] Figure 6 Fermented mycelium DETAILED DESCRIPTION

[0032] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific implementation methods.

[0033] Example 1

[0034] Taking a 500 ml Erlenmeyer flask as a fermentation container as an example, liquid culture of the mycelium of Microdermabrasion edulis was carried out.

[0035] The slant strain was inoculated on PDA culture medium and cultured in the dark at 25°C for 15 days. The grown slant strain was then transferred to a primary liquid culture medium with the following formula: 20 g glucose, 5 g peptone, 0.5 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, and 0.02 g vitamin B1. The above raw materials were dissolved in water and the volume was made up to 1 L with water. The pH was adjusted to 6.5 with hydrochloric acid or sodium hydroxide. The culture was divided into 500 mL Erlenmeyer flasks with 250 mL per bottle. The culture was shaken at 25°C with a shaker speed of 120 r / min. After culturing for 8 days, the mycelial balls filled the liquid culture medium with an average diameter of about 2-3 mm, which was used as the primary strain.

[0036] The above primary strains were inoculated into a secondary liquid culture medium (formula: 2% carbon source, 0.4% peptone, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.002% vitamin B1) at an inoculum rate of 10%. The above raw materials were dissolved in water and the pH was adjusted to 6.5 with hydrochloric acid and sodium hydroxide. The carbon sources were glucose, maltose, and soluble starch, respectively. The culture was divided into 500 ml Erlenmeyer flasks and the volume of the liquid culture medium was 250 ml. The culture was shaken at 25°C with a shaker speed of 120 r / min for 6 days until the mycelial balls filled the entire culture medium. The mycelial balls were collected by filtration, the mycelium was dried at 50°C, and the mycelium was weighed.

[0037] Under the same culture conditions, the culture medium without adding carbon source was used as the blank control group in the secondary liquid culture medium. The culture medium with different carbon sources produced different mycelium yields of Microdermabrasion spp. When maltose was used as the carbon source, the mycelium yield of Microdermabrasion spp. was the highest, at 14.25 g / L; followed by glucose, at 13.01 g / L; when dextrin and soluble starch were used as carbon sources, the yields were both 10.88 g / L; when no carbon source was added to the secondary liquid culture medium, the mycelium yield of Microdermabrasion spp. was 2.08 g / L.

[0038] Example 2

[0039] Taking a 500 ml Erlenmeyer flask as a fermentation container as an example, liquid culture of the mycelium of Microdermabrasion edulis was carried out.

[0040] Inoculate the slant onto PDA medium and incubate in the dark at 25°C for 15 days. The grown slant was then transferred to a primary liquid culture medium with the following formulation: 20g glucose, 5g peptone, 0.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, and 0.02g vitamin B1. Dissolve these ingredients in water and dilute to 1L. Adjust the pH to 6.5 with hydrochloric acid or sodium hydroxide. Aliquot into 500mL Erlenmeyer flasks, each containing 250mL. Incubate at 25°C with shaking at 120 rpm. After 8 days, mycelial pellets will fill the liquid culture medium and have an average diameter of approximately 2-3 mm. These pellets are then used as the primary culture medium.

[0041] The above primary strain was inoculated into a liquid culture medium (formula: 2% glucose, 0.4% nitrogen source, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, and 0.002% vitamin B1) at a 10% (v / v) inoculum ratio. These ingredients were dissolved in water and the pH was adjusted to 6.5 with hydrochloric acid and sodium hydroxide. The nitrogen source was selected from yeast extract, peptone, beef extract powder, soy peptone, etc. The culture medium was divided into 500 mL Erlenmeyer flasks, each filled with 250 mL of liquid culture medium. The culture was shaken at 25°C with a shaker speed of 120 rpm for 4 days until mycelial pellets filled the entire culture medium. The mycelial pellets were collected by filtration, dried at 50°C, and weighed.

[0042] Under the same culture conditions, the culture medium without nitrogen source added in the secondary liquid culture medium was used as the blank control group. The culture medium with different nitrogen sources produced different mycelium yields of Microdermabrasion spp. When yeast extract was used as the nitrogen source, the mycelium yield was the highest, at 13.5 g / L; followed by peptone, with a yield of 12.24 g / L; when beef extract powder was used as the nitrogen source, the yield was 10.81 g / L; when soy peptone was used as the nitrogen source, the yield was 10.75 g / L; when the culture medium without nitrogen source added was used, the yield was 2.52 g / L.

[0043] Example 3

[0044] Taking a 500 ml Erlenmeyer flask and a 5 liter small fermentation tank as fermentation containers as examples, liquid culture of the mycelium of Microdermabrasion edulis was carried out.

[0045] The slant culture, the primary liquid culture and the secondary liquid culture were cultured using the same method steps, culture medium formulation and culture conditions as in Example 1.

[0046] The secondary liquid spawn is inoculated at a 10% (v / v) ratio into a production liquid culture medium (1.5% maltose, 0.5% yeast extract, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.002% vitamin B1, 0.02% defoamer, pH 7.0). The fermenter is filled to a volume of 2.5 L. The culture is maintained at 25°C with a stirring rate of 150 r / min and an aeration rate of 10 L / min. The culture is continued for 72 hours until the mycelial pellets fill the entire medium. The pellets are then collected by centrifugation. The mycelium is then dried at 50°C, yielding a mycelial yield of 13 g / L. This completes the cultivation and collection of the mycelium for the white sedge, ready for product processing.

[0047] Example 4

[0048] Taking a 500 ml Erlenmeyer flask as a fermentation container as an example, liquid culture of the mycelium and extracellular polysaccharide of Microdermabrasion alba was carried out simultaneously.

[0049] Inoculate the slant onto PDA medium and incubate in the dark at 25°C for 15 days. Transfer the grown slant to primary liquid culture medium with the following formulation: 20g glucose, 5g peptone, 0.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, and 0.02g vitamin B1. Dissolve these ingredients in water and dilute to 1L. Adjust the pH to 6.0 with hydrochloric acid or sodium hydroxide. Aliquot into 500mL Erlenmeyer flasks, each containing 250mL. Incubate at 25°C with shaking at 150 rpm for 7 days. Mycelial pellets will fill the liquid culture medium and serve as the primary culture medium.

[0050] The above primary strains were inoculated into liquid culture medium (formula: glucose 2%, nitrogen source 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, vitamin B1) at an inoculum rate of 8% (V / V). 0.002%, the above raw materials were dissolved in water, and then the pH was adjusted to 6.5 with hydrochloric acid and sodium hydroxide. Among them, the nitrogen source was selected from yeast extract, peptone, beef extract, soy peptone, etc., and was divided into 500 ml Erlenmeyer flasks and filled with liquid culture medium with a volume of 250 mL. The culture was shaken at 25°C with a shaker speed of 150 r / min for 4 days until the mycelial balls filled the entire culture medium. The mycelial balls and supernatant were collected by centrifugation, and the mycelium was dried at 50°C and weighed. The mycelium yield was 12 g / L. 4 times the volume of anhydrous ethanol was added to the supernatant to make the final ethanol concentration of 75% or above, and the mixture was allowed to stand at 4°C overnight. The polysaccharide was collected by centrifugation and freeze-dried to prepare polysaccharide freeze-dried powder. The yield of extracellular polysaccharide was 0.89 g / L.

[0051] Fermentation application verification.

[0052] After the fermentation product of the present invention is prepared, two products, the mycelium of white microdermabranch mushroom and the extracellular sugar, can be obtained, which contain nutrients and multiple effective ingredients and can be used as food. The following are the results of the auxiliary hypoglycemic activity analysis: α-glucosidase is an important enzyme for maintaining human sugar metabolism. α-glucosidase inhibitors can effectively control the activity of α-glucosidase and inhibit α-glucosidase from catalyzing the decomposition of polysaccharides into monosaccharides, thereby reducing the incidence of type II diabetes. The in vitro hypoglycemic activity of the two fermentation products was determined by α-glucosidase experiments, using acarbose as a positive control. The test results showed that the inhibition rates of the aqueous extract of white microdermabranch mushroom mycelium and the fermentation broth on α-glucosidase were 38.20% and 33.72%, respectively, and had good in vitro hypoglycemic activity.

[0053] The results of activity test showed that the mycelium and fermentation liquid of Microcarpa leucophylla contained rich active ingredients and had the function of inhibiting the activity of α-glucosidase. Microcarpa leucophylla can be used as a biological raw material to assist in lowering blood sugar, which has great economic value and certain scientific significance.

Claims

1. Based on the fermentation characteristics of the strain, the fermentation conditions and process were optimized, and a method for preparing the fermentation product was established. The specific contents and steps include: S1. Preparation of strains: Inoculate the white spores of Microdermabrasion on PDA test tube culture medium and culture in the dark at 20-28°C for 10-15 days. It can be used when the mycelium has grown to 2 / 3 or more of the slope. S2. Preparation of a primary liquid culture: The grown slant tube culture is inoculated into a primary liquid culture medium. The weight percentages of the components in the primary liquid culture medium are as follows: 1%-3% carbon source, 0.2%-1% nitrogen source, 0.05%-0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, and 0.002%-0.005% vitamin B1. Each component is dissolved in water and dispensed into a culture container at 20%-60% of the container volume. The pH is 5.5-7.

5. The culture is shaken at 20-32°C with a shaker speed of 120-200 r / min for 6-14 days until the mycelial pellets are filled with liquid culture medium. The culture can then be used as a primary liquid culture medium. When the diameter of the mycelial pellets of the primary liquid culture is greater than 10 mm, they can be aseptically broken using a cell disruptor to form a uniform mycelial homogenate, thereby improving their propagation capacity. S3. Preparation of a secondary liquid culture: inoculate the primary liquid culture at an inoculum ratio of 5%-15% (v / v) into a secondary liquid culture medium. The weight percentages of the components in the secondary liquid culture medium are as follows: carbon source 1%-3%, nitrogen source 0.2%-1%, potassium dihydrogen phosphate 0.05%-0.1%, magnesium sulfate 0.05%, and vitamin B1 0.002%-0.005%. Each component is dissolved in water, the pH is 5.5-7.5, the container filling amount, and other culture conditions are the same as those of the primary liquid culture medium. Cultivate for 4-10 days until the mycelial pellets fill the entire culture medium, and use this as the secondary liquid culture medium. S4. Inoculation and culture of production medium: Inoculate the above secondary liquid strain into the production liquid medium at an inoculum ratio of 5%-15% (V / V). The formula of the production liquid medium is determined according to the target product of liquid culture. The container filling volume is 20%-60%. Incubate at 20-32°C with shaking at a shaker speed of 120-200 r / min for 4-14 days. Alternatively, use a fermenter as the culture equipment. 0.01%-0.1% defoaming agent must be added to the culture medium. The ventilation volume is 6-12 L / min and the stirring speed is 150-200 r / min. Fermentation culture is carried out for 4-14 days until the mycelial balls fill the entire culture medium. Then the culture can be terminated and the product can be collected and processed; S5. Collection and cleaning of mycelium: Separate the mycelium and fermentation broth by centrifugation or filtration using a plate filter press. Rinse the mycelium 2-3 times with water to remove any residual culture medium. Different nitrogen sources may slightly vary the appearance of the mycelium. Dry the mycelium at 45-80°C. The mycelium yield should be no less than 10 g / L. S6. Extraction and purification of extracellular polysaccharides: The supernatant obtained by centrifugation in step S5 or the filtrate obtained by filtration, i.e., the fermentation broth, is concentrated under reduced pressure, and then the polysaccharides are separated by alcohol precipitation. Four volumes of anhydrous ethanol are added to make the final ethanol concentration 75% or above, and the mixture is allowed to stand overnight at 4°C. The polysaccharides are collected by centrifugation and freeze-dried to prepare a polysaccharide freeze-dried powder with a yield of not less than 0.75 g / L. According to production requirements, the fermentation concentrate can also be hydrolyzed with a protease (such as neutral protease, trypsin, papain) before the polysaccharide is separated by alcohol precipitation to hydrolyze the protein into small molecule peptides or amino acids to improve the purity of the white microcarpa polysaccharide. The polysaccharide yield is not less than 0.55 g / L, and the polysaccharide content is not less than 55%.

2. Preferably, the carbon source in step S2 and step S3 is one of maltose, glucose, dextrin, and soluble starch.

3. Preferably, the carbon source in step S2 and step S3 is maltose, glucose or maltose.

4. Preferably, the nitrogen source in step S2 and step S3 is one of yeast extract, peptone, beef extract powder, or soy peptone.

5. Preferably, the nitrogen source in step S2 and step S3 is yeast extract.

6. Preferably, the formula of the production liquid culture medium in step S4 is the same as the formula of the first-grade liquid culture medium; wherein, When the main target product of production and culture is extracellular polysaccharide, the carbon source is non-starch substance.

7. Preferably, the inoculation amount in steps S2, S3, and S4 is 10%.

8. Preferably, the culture temperature in steps S2, S3, and S4 is 28°C.

9. Preferably, the shaking speed in steps S2, S3, and S4 is 120-150 r / min, 0.05% defoaming agent is added to the culture medium during fermentation tank production, the ventilation volume is 10 L / min, and the stirring speed is 180 r / min.

10. White Micro-skin Umbrella ( Marasmiellus sp . ) Application of the fermentation products of DCY6478, i.e. mycelium and fermentation broth, in the preparation of auxiliary blood sugar lowering products.

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