Screening method of ganoderma lucidum strain suitable for fermenting brown rice
By using solid-state fermentation to screen Ganoderma lucidum strain ACCC 51677 for fermenting brown rice, the problems of high cost of Ganoderma lucidum fruiting bodies and complexity of liquid deep fermentation were solved, achieving efficient fermentation of brown rice and improving its nutritional value.
Patent Information
- Application Number
- CN202511041180.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-28
- Publication Date
- 2025-10-28
AI Technical Summary
In the existing technology, the raw material cost of Ganoderma lucidum fruiting bodies is high and the quality is unstable. Liquid deep fermentation equipment requires large investment and is complicated to operate, making it difficult to efficiently utilize Ganoderma lucidum for brown rice fermentation. Furthermore, the existing microbial fermentation strains have poor adaptability and effectiveness to brown rice.
A suitable Ganoderma lucidum strain for fermenting brown rice was screened using a solid-state fermentation method. The Ganoderma lucidum strain was activated and a brown rice culture medium was prepared for fermentation. The content of Ganoderma lucidum triterpenes was measured, and strain ACCC 51677 was selected as the best strain, which has a short fermentation cycle, high peak content, and good stability.
This study achieved high efficiency and stability in Ganoderma lucidum brown rice fermentation, improved the nutritional value and processing characteristics of brown rice, and provided a scientific basis for the efficient utilization of Ganoderma lucidum resources.
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Figure CN120843642A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of Ganoderma lucidum strain screening technology, specifically a method for screening Ganoderma lucidum strains suitable for fermenting brown rice. Background Technology
[0002] Rice (Oryza sativa L.) is one of the most important food crops for humankind. China has the longest history of rice cultivation in the world, with Guizhou province having an annual rice planting area of over 10 million mu. Rice, processed from rice, is the staple food on Chinese tables, meeting the body's daily needs for protein, lipids, carbohydrates, and other nutrients. However, long-term consumption of refined rice and flour can easily lead to nutritional deficiencies and increase the risk of disease. As consumers' living standards improve, their awareness of health is gradually increasing, and their interest in developing whole grain foods is constantly growing.
[0003] Brown rice is a whole grain rice composed of the bran, germ, and endosperm remaining after rice has been hulled. It not only contains the nutrients of regular rice, but its bran layer also contains bioactive substances such as γ-aminobutyric acid (GABA), tocotrienols, and γ-oryzanol, making it rich in nutrients and potentially helping to prevent obesity, diabetes, and other diseases. However, the bran layer contains cellulose, hemicellulose, and lignin, which affect the taste and processing characteristics of brown rice, making it less appealing to consumers. Currently, the use of microbial fermentation to improve the nutritional value and processing characteristics of grains is widely studied. The fermentation strains used are mostly bacteria (lactic acid bacteria, Bacillus subtilis, etc.), fungi (yeasts, molds, etc.), or a mixture of multiple strains. Studies have shown that various enzymes produced by microorganisms can degrade macromolecules such as proteins, starches, and cellulose in grains, improving their swelling capacity. Grains fermented by microorganisms exhibit improved nutritional value and processing characteristics. However, research on directly using edible fungi to ferment grains is relatively limited.
[0004] Ganoderma is a dark-colored fungus with a smooth appearance and woody texture. More than 240 compounds have been isolated from Ganoderma, including polysaccharides and triterpenes, which have been proven to possess antiviral, antitumor, and anti-aging pharmacological activities, making it a valuable health food with medicinal and edible properties. A variety of foods are processed from Ganoderma, such as instant tea, fiber biscuits, and functional beverages. Ganoderma fruiting bodies are commonly used in food processing, but these raw materials are generally expensive and product quality is inconsistent. Some studies have used submerged fermentation (SMF) to cultivate Ganoderma, utilizing the mycelium and crude enzyme solution produced during SMF for further processing. However, SMF for industrial production requires significant equipment investment and complex operation, necessitating strict sterilization. Besides SMF, another microbial fermentation method is solid-state fermentation. Solid-state fermentation (SSF) has been reported to produce excellent growth of filamentous fungi, with significantly higher levels of fungal metabolites compared to liquid deep fermentation. Furthermore, solid-state fermentation requires less investment, does not require strict aseptic techniques, and is suitable for industrial production. Summary of the Invention
[0005] In view of the problems existing in the prior art, the present invention discloses a method for screening Ganoderma lucidum strains suitable for fermenting brown rice. The technical solution adopted includes the following steps: Step 1: Prepare the experimental material Ganoderma lucidum spawn and activate it. In a clean bench, take 1-2 loops of Ganoderma lucidum spawn stored at 4℃ and inoculate it into sterile PDB liquid culture medium. Shake well and place it in a shaker at 28℃ and 180r / min for activation. Activate continuously for 72h. When the culture medium becomes turbid and a large number of mycelia appear, the activation is complete. Store at 4℃ for later use. Step 2: Preparation of Ganoderma lucidum brown rice fermentation: After screening and removing impurities from the brown rice raw material, soak it in a 5% sodium hypochlorite solution for 3 minutes to remove impurities. After cleaning, soak it in water for 24 hours until the brown rice softens. Add 3% glucose, 0.2% magnesium sulfate and 0.1% potassium dihydrogen phosphate by mass and stir well. Dispense 200g into 500ml Erlenmeyer flasks and sterilize at 121℃ for 2 hours. Cool to room temperature to obtain brown rice culture medium. In a clean bench, use a sterile pipette to inject 10ml of the activated bacteria from Step 1 into the brown rice culture medium. Shake well and incubate at a constant temperature of 25℃. Take samples after fermentation every 5 days, dry and grind them into powder, sieve and store for later use. Take samples 10 times in total, for a total of 50 days. Step 3: Prepare Ganoderma lucidum triterpenoid extract and determine its content. Weigh 25g of sample and add 100mL of 5% NaHCO3 solution. Extract by ultrasonication for 30min, filter, adjust the pH of the filtrate to 3.0 with 6mol / L hydrochloric acid solution, and then extract twice with 10mL of carbon tetrachloride-acetone solution (volume ratio 1:1). Concentrate the combined filtrate to 3mL using a vacuum concentrator and dry in an oven to obtain Ganoderma lucidum triterpenoid extract. Weigh 10mg of extract and dilute to 100mL with methanol. Measure the absorbance at 200nm wavelength and calculate the Ganoderma lucidum triterpenoid content.
[0006] As a preferred embodiment of the present invention, the Ganoderma lucidum strain is one or more of Ganoderma lucidum strain ACCC 51565, Ganoderma lucidum strain ACCC 52697, Ganoderma lucidum strain ACCC 51677, and Ganoderma lucidum strain ACCC 51673.
[0007] As a preferred technical solution of the present invention, the preparation method of PDB liquid culture medium in step one is as follows: weigh 35g of potato dextrose broth (PDB) culture medium, add 1000mL of distilled water, stir with a magnetic stirrer until completely dissolved, titrate with 4mol / L hydrochloric acid to pH 4.5, sterilize at 121℃ for 30min, and cool to room temperature for later use.
[0008] As a preferred technical solution of the present invention, step two, removing impurities from brown rice raw materials, involves removing moldy, broken grains, and stones.
[0009] As a preferred technical solution of the present invention, in step two, the sample is dried, ground into powder, sieved to 80 mesh, and stored at -80℃.
[0010] As a preferred embodiment of the present invention, step three, the determination of Ganoderma lucidum triterpenoid content, further includes the plotting of a Ganoderma lucidum triterpenoid standard curve. The method for plotting the Ganoderma lucidum triterpenoid standard curve is as follows: 10 mg of oleanolic acid standard is weighed into a 100 mL volumetric flask and diluted with methanol to obtain a 10 mg / 100 mL oleanolic acid standard stock solution; the stock solution is then diluted with methanol to prepare standard solutions of 0.08 mg / mL, 0.04 mg / 100 mL, 0.02 mg / 100 mL, 0.01 mg / 100 mL, and 0.005 mg / 100 mL for later use; a UV spectrophotometer is used to perform a full-wavelength scan, with the maximum absorption wavelength at 200 nm, to measure the oleanolic acid standard solutions of different concentrations, and standard curves are plotted with concentration and absorbance values on the x and y axes, respectively.
[0011] As a preferred embodiment of the present invention, the linear regression equation for plotting the Ganoderma lucidum triterpenoid standard curve is: y = 7.7985x + 0.054, R02 =0.9962.
[0012] The beneficial effects of this invention are as follows: By comprehensively evaluating the changes in Ganoderma lucidum triterpenoid content during the fermentation of brown rice by four Ganoderma lucidum strains, this invention found that Ganoderma lucidum ACCC 51677 strain is more suitable for brown rice fermentation; it has a short fermentation cycle, high peak content, and good stability, providing a scientific basis for the efficient utilization of Ganoderma lucidum resources. Attached Figure Description
[0013] Figure 1 This is a standard curve of Ganoderma lucidum triterpenes according to the present invention;
[0014] Figure 2 This is a graph showing the changes in the content of Ganoderma lucidum triterpenes in fermented brown rice (ACCC 51565) according to the present invention.
[0015] Figure 3 This is a graph showing the change in Ganoderma lucidum triterpenoid content in fermented brown rice (ACCC 52697) according to the present invention.
[0016] Figure 4 This is a graph showing the changes in the content of Ganoderma lucidum triterpenes in fermented brown rice (ACCC 51677) according to the present invention.
[0017] Figure 5 This is a graph showing the changes in the content of Ganoderma lucidum triterpenes in fermented brown rice (ACCC 51673) according to the present invention. Detailed Implementation
[0018] Example 1
[0019] This invention discloses a method for screening Ganoderma lucidum strains suitable for fermenting brown rice. The technical solution includes the following steps: Step 1: Prepare the experimental material, Ganoderma lucidum spawn, and activate the Ganoderma lucidum spawn. The preparation method of PDB liquid culture medium is as follows: Weigh 35g of potato dextrose broth (PDB) medium, add 1000mL of distilled water, stir with a magnetic stirrer until completely dissolved, titrate with 4mol / L hydrochloric acid to pH 4.5, sterilize at 121℃ for 30min, cool to room temperature for later use. In a clean bench, take 1-2 loops of Ganoderma lucidum spawn stored at 4℃ and inoculate it into the sterilized PDB liquid culture medium. Shake well, place in a shaker at 28℃ and 180r / min for activation. Activate continuously for 72h. When the culture medium becomes turbid and a large number of mycelia appear, the activation is complete. Store at 4℃ for later use. Step 2: Preparation of Ganoderma lucidum brown rice fermentation. After screening and removing impurities from the brown rice raw material, including removing mold, broken grains and stones, soak the raw material in a 5% sodium hypochlorite solution for 3 minutes to remove bacteria. After cleaning, soak the raw material in water for 24 hours until the brown rice softens. Add 3% glucose, 0.2% magnesium sulfate and 0.1% potassium dihydrogen phosphate by mass and stir well. Dispense 200g into 500ml Erlenmeyer flasks and sterilize at 121℃ for 2 hours. Cool to room temperature to obtain brown rice culture medium. In a clean bench, use a sterile pipette to inject 10ml of the activated bacteria from Step 1 into the brown rice culture medium into each flask. Shake well and incubate at a constant temperature of 25℃. Take samples after fermentation every 5 days, dry and grind them into powder, sieve and store for later use. The dried and ground powder is sieved to 80 mesh and stored at -80℃. Take samples 10 times for a total of 50 days. Step 3: Prepare Ganoderma lucidum triterpenoid extract and determine its content. Weigh 25g of sample and add 100mL of 5% NaHCO3 solution. Extract by ultrasonication for 30min, filter, adjust the pH of the filtrate to 3.0 with 6mol / L hydrochloric acid solution, and then extract twice with 10mL of carbon tetrachloride-acetone solution (volume ratio 1:1). Concentrate the combined filtrate to 3mL using a vacuum concentrator and dry in an oven to obtain Ganoderma lucidum triterpenoid extract. Weigh 10mg of extract and dilute to 100mL with methanol. Measure the absorbance at 200nm wavelength and calculate the Ganoderma lucidum triterpenoid content. like Figure 1 As shown, step three, the determination of Ganoderma lucidum triterpenoid content, also includes the plotting of a Ganoderma lucidum triterpenoid standard curve. The method for plotting the Ganoderma lucidum triterpenoid standard curve is as follows: Weigh 10 mg of oleanolic acid standard into a 100 mL volumetric flask and dilute to volume with methanol to obtain a 10 mg / 100 mL oleanolic acid standard stock solution; dilute the stock solution sequentially with methanol to obtain standard solutions of 0.08 mg / mL, 0.04 mg / 100 mL, 0.02 mg / 100 mL, 0.01 mg / 100 mL, and 0.005 mg / 100 mL for later use; perform a full-wavelength scan using an ultraviolet spectrophotometer, with the maximum absorption wavelength at 200 nm, and measure the oleanolic acid standard solutions of different concentrations, plotting the standard curve with concentration and absorbance values on the x and y axes respectively; the linear regression equation for the plotting of the Ganoderma lucidum triterpenoid standard curve is: y = 7.7985x + 0.054, R 2 =0.9962.
[0020] Example 2
[0021] This embodiment is basically the same as Embodiment 1, except that the Ganoderma lucidum strain used is Ganoderma lucidum strain ACCC51565; Figure 2As shown, when Ganoderma lucidum strain ACCC 51565 fermented brown rice, the content of Ganoderma lucidum triterpenes showed a trend of first increasing and then decreasing over time. The content of Ganoderma lucidum triterpenes was the highest at 30 days of fermentation, reaching 3.83 mg / g. As the fermentation time increased, the content of Ganoderma lucidum triterpenes gradually decreased and finally remained at 3.50 mg / g.
[0022] Example 3
[0023] This embodiment is basically the same as Embodiment 1, except that the Ganoderma lucidum strain used is Ganoderma lucidum strain ACCC52697; Figure 3 As shown, the content of Ganoderma triterpenes in fermented brown rice (ACCC 52697) showed a trend of first increasing and then decreasing over time. The content of Ganoderma triterpenes was the highest at 40 days of fermentation, reaching 4.07 mg / g. As the fermentation time increased, the content of Ganoderma triterpenes gradually decreased and finally remained at 2.36 mg / g.
[0024] Example 4
[0025] This embodiment is basically the same as Embodiment 1, except that the Ganoderma lucidum strain used is Ganoderma lucidum strain ACCC51677; Figure 4 As shown, when Ganoderma lucidum ACCC 51677 fermented brown rice, the content of Ganoderma lucidum triterpenes showed a trend of first increasing and then decreasing over time. The content of Ganoderma lucidum triterpenes was the highest at 30 days of fermentation, reaching 4.10 mg / g. As the fermentation time increased, the content of Ganoderma lucidum triterpenes gradually decreased and finally remained at 3.50 mg / g.
[0026] Example 5
[0027] This embodiment is basically the same as Embodiment 1, except that the Ganoderma lucidum strain used is Ganoderma lucidum strain ACCC51673; Figure 5 As shown, the content of Ganoderma lucidum triterpenes in fermented brown rice (ACCC 51673) showed a trend of first increasing and then decreasing over time. The content of Ganoderma lucidum triterpenes was the highest at 35 days of fermentation, reaching 4.47 mg / g. As the fermentation time increased, the content of Ganoderma lucidum triterpenes gradually decreased and finally remained at 2.90 mg / g.
[0028] In summary, based on the changes in Ganoderma lucidum triterpenoid content during the fermentation of brown rice by Ganoderma lucidum strains, it was found that Ganoderma lucidum strain ACCC51677 is more suitable for brown rice fermentation, with a shorter fermentation cycle, higher peak content, and better stability. Ganoderma lucidum strains ACCC51565, ACCC52697, ACCC51677, and ACCC51673 were all purchased from the Chinese Institute of Agricultural Resources and Regional Planning; the brown rice was purchased from Guizhou Qianba Rice Industry Co., Ltd.
[0029] Components not described in detail in this article are existing technologies.
[0030] While the specific embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention, and modifications or variations without creative effort are still within the protection scope of the present invention.
Claims
1. A method for screening Ganoderma lucidum strains suitable for fermenting brown rice, characterized in that, Includes the following steps: Step 1: Prepare the experimental material Ganoderma lucidum spawn and activate it. In a clean bench, take 1-2 loops of Ganoderma lucidum spawn stored at 4℃ and inoculate it into sterile PDB liquid culture medium. Shake well and place it in a shaker at 28℃ and 180r / min for activation. Activate continuously for 72h. When the culture medium becomes turbid and a large number of mycelia appear, the activation is complete. Store at 4℃ for later use. Step 2: Preparation of Ganoderma lucidum brown rice fermentation: After screening and removing impurities from the brown rice raw material, soak it in a 5% sodium hypochlorite solution for 3 minutes to remove impurities. After cleaning, soak it in water for 24 hours until the brown rice softens. Add 3% glucose, 0.2% magnesium sulfate and 0.1% potassium dihydrogen phosphate by mass and stir well. Dispense 200g into 500ml Erlenmeyer flasks and sterilize at 121℃ for 2 hours. Cool to room temperature to obtain brown rice culture medium. In a clean bench, use a sterile pipette to inject 10ml of the activated bacteria from Step 1 into the brown rice culture medium. Shake well and incubate at a constant temperature of 25℃. Take samples after fermentation every 5 days, dry and grind them into powder, sieve and store for later use. Take samples 10 times in total, for a total of 50 days. Step 3: Prepare Ganoderma lucidum triterpenoid extract and determine its content. Weigh 25g of sample and add 100mL of 5% NaHCO3 solution. Extract by ultrasonication for 30min, filter, adjust the pH of the filtrate to 3.0 with 6mol / L hydrochloric acid solution, and then extract twice with 10mL of carbon tetrachloride-acetone solution (volume ratio 1:1). Concentrate the combined filtrate to 3mL using a vacuum concentrator and dry in an oven to obtain Ganoderma lucidum triterpenoid extract. Weigh 10mg of extract and dilute to 100mL with methanol. Measure the absorbance at 200nm wavelength and calculate the Ganoderma lucidum triterpenoid content.
2. The method for screening Ganoderma lucidum strains suitable for fermenting brown rice according to claim 1, characterized in that: The Ganoderma lucidum strain is one or more of Ganoderma lucidum strain ACCC 51565, Ganoderma lucidum strain ACCC 52697, Ganoderma lucidum strain ACCC 51677, and Ganoderma lucidum strain ACCC51673.
3. The method for screening Ganoderma lucidum strains suitable for fermenting brown rice according to claim 1, characterized in that: The preparation method of PDB liquid culture medium in step one is as follows: Weigh 35g of potato dextrose broth (PDB) culture medium, add 1000mL of distilled water, stir with a magnetic stirrer until completely dissolved, titrate with 4mol / L hydrochloric acid to pH 4.5, sterilize at 121℃ for 30min, and cool to room temperature for later use.
4. The method for screening Ganoderma lucidum strains suitable for fermenting brown rice according to claim 1, characterized in that: Step two, the removal of impurities from the brown rice raw material, involves removing moldy, broken grains, and stones.
5. The method for screening Ganoderma lucidum strains suitable for fermenting brown rice according to claim 1, characterized in that: After drying and grinding the sample in step two, it is sieved to 80 mesh and stored at -80℃ for later use.
6. The method for screening Ganoderma lucidum strains suitable for fermenting brown rice according to claim 1, characterized in that: The third step of the determination of Ganoderma lucidum triterpenoid content also includes the construction of a Ganoderma lucidum triterpenoid standard curve. The method for constructing the Ganoderma lucidum triterpenoid standard curve is as follows: Weigh 10 mg of oleanolic acid standard into a 100 mL volumetric flask and dilute to volume with methanol to obtain a 10 mg / 100 mL oleanolic acid standard stock solution; dilute the stock solution with methanol sequentially to prepare standard solutions of 0.08 mg / mL, 0.04 mg / 100 mL, 0.02 mg / 100 mL, 0.01 mg / 100 mL, and 0.005 mg / 100 mL for later use; perform a full wavelength scan using an ultraviolet spectrophotometer, with the maximum absorption wavelength at 200 nm, and measure the oleanolic acid standard solutions of different concentrations, and plot the standard curve with concentration and absorbance values on the x and y axes respectively.
7. The method for screening Ganoderma lucidum strains suitable for fermenting brown rice according to claim 6, characterized in that: The linear regression equation for plotting the standard curve of Ganoderma lucidum triterpenes is: y = 7.7985x + 0.054, R0 2 =0.9962.