Polyphenol-rich wild phellinus igniarius SH-27 fermentation liquor and application thereof
The PSO-BP neural network model optimizes the fermentation conditions of mulberry yellow liquid, solves the problem of insufficient polyphenol production in deep fermentation of mulberry yellow liquid, and achieves efficient production of polyphenol metabolites, and has broad medicinal potential.
Patent Information
- Application Number
- CN202510770511.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-10
- Publication Date
- 2025-08-15
AI Technical Summary
During the deep fermentation of mulberry yellow liquid, the impact of environmental factors on polyphenol synthesis has not been fully explained, and the fermentation and metabolism regulation process of high-quality and high-yield mulberry yellow polyphenol fermentation and control of polyphenols has been lacking, resulting in insufficient yield of polyphenol metabolites.
The PSO-BP neural network model is used to optimize the fermentation conditions of mulberry yellow liquid, and the liquid culture medium and fermentation parameters are optimized to improve the yield of polyphenol metabolites. The specific steps include mycelium activation, seed liquid culture, fermentation broth preparation and optimization of fermentation conditions.
The yield of mulberry polyphenol metabolites was significantly improved, with an average yield of 33.72 mg/L, an increase of 33.5%, and provides an effective optimization method for liquid fermentation of medicinal fungi.
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Abstract
Description
Technical Field
[0001] The invention relates to a polyphenol-rich wild phellinus igniarius SH-27 fermentation liquid and application thereof, belonging to the technical field of biological fermentation. Background Art
[0002] Thick hairy fibrous fungus ( Inonotus hispidus ), commonly known as the rough-haired yellow-brown inoculum or the rough-haired yellow-brown inoculum, is a mulberry-like fungus with important nutritional and medicinal value. Current studies have shown that mulberry-like fungi have good therapeutic effects in preventing or treating tumors, immune regulation, anti-inflammatory, anti-infection, and antioxidant effects, showing broad application prospects. The term mulberry-like fungus was first recorded in the "Medicinal Treatise" by Zhen Quan in the early Tang Dynasty. According to the "Medicinal Treatise", mulberry-like fungus is cold in nature and slightly bitter in taste. It is used in traditional Chinese medicine to treat night sweats, dysentery, hematuria, rectal prolapse and bleeding, umbilical and abdominal pain, amenorrhea, leucorrhea, spleen deficiency and diarrhea, etc.; the "Shennong Bencao Jing" also highly praised the medicinal effects of mulberry-like fungus, believing that it has the effects of prolonging life, improving the function of the digestive system, and detoxification.
[0003] Polyphenols are the main pigment components in the fruiting bodies of Phellinus igniarius. Their chemical structure primarily consists of an aromatic ring and multiple hydroxyl groups. Numerous studies on Phellinus igniarius have shown that the active substances contained in it, represented by polyphenols, have great medicinal potential in the fields of antioxidant, anti-inflammatory, anti-cancer, anti-tumor, and treatment and intervention of diabetes and hypertension.
[0004] As the concept of medicine and food sharing a common origin becomes more widely accepted, a wide range of beverages and foods rich in medicinally active ingredients are appearing in our daily lives. With increasing demands for quality of life and the growing demand for health services driven by an aging population, the healthcare industry faces a promising future. Medicinal fungi, as "green" health resources, have attracted significant attention from researchers both domestically and internationally, and Coriolus versicolor has been extensively studied.
[0005] my country has already conducted extensive research on polyphenols from the fruiting bodies of Phellinus igniarius. In recent years, researchers have begun studying the polyphenol metabolites of Phellinus igniarius mycelium, but this research lacks systematic and in-depth understanding. In particular, research on submerged fermentation of Phellinus igniarius is fragmented, leaving many unresolved questions. For example, the impact of environmental factors (temperature, rotational speed, pH, and dissolved oxygen) on polyphenol synthesis during submerged fermentation remains unclear, and further research is needed to determine the metabolic regulation of high-quality, high-yield Phellinus igniarius polyphenol fermentation.
[0006] In order to solve these problems, the present invention combines artificial intelligence algorithms and optimizes the liquid fermentation conditions of Phellinus igniarius through the PSO-BP neural network model, significantly improving the yield of polyphenol metabolites. Summary of the Invention
[0007] In view of the deficiencies in the prior art, the present invention provides a polyphenol-rich wild Phellinus igniarius SH-27 fermentation liquid and its application.
[0008] The technical solutions of the present invention are as follows: A polyphenol-rich wild mulberry fungus SH-27 fermentation liquid, the fermentation liquid is prepared by fermenting wild mulberry fungus SH-27 preserved in the Guangdong Provincial Microbial Culture Collection Center, the wild mulberry fungus SH-27 is a thick hair fibrous fungus ( Inonotus hispidus ), the deposit number is GDMCC No: 65865.
[0009] The method for preparing the polyphenol-rich wild Phellinus igniarius SH-27 fermentation broth comprises the following steps: 1) A circular moss with a diameter of 4-6 mm was taken from the mycelium of wild Phellinus igniarius SH-27 and inoculated onto a PDA solid medium plate. The culture was performed for 7-8 days to activate the strain. 2) Take fresh mycelial lawn from the edge of the activated strain and inoculate it into PDB liquid medium for 7-10 days to obtain seed liquid; 3) The seed liquid is inoculated into a fermentation medium at a volume ratio of 5-20%, and fermentation is performed to obtain a polyphenol-rich wild phellinus igniarius SH-27 fermentation liquid.
[0010] According to the preferred embodiment of the present invention, the culture conditions in step 1) are: 22-30°C.
[0011] According to the preferred embodiment of the present invention, the culture conditions in step 2) are: 100-200 rpm, 22-30°C.
[0012] According to the preferred embodiment of the present invention, the culturing time of the seed solution in step 2) is 168 hours.
[0013] According to the present invention, preferably, the components of the PDA solid culture medium in step 1) are: 12 g / L potato extract powder, 20 g / L glucose, 15 g / L agar, the solvent is water, and the pH is natural; the components of the PDB liquid culture medium in step 2) are: 12 g / L potato extract powder, 20 g / L glucose, the solvent is water, and the pH is natural.
[0014] According to a preferred embodiment of the present invention, the inoculation amount of step 2) is: 3 to 4 pieces of bacterial moss with a diameter of 4 to 6 mm are inoculated per 150 mL of culture medium.
[0015] According to the preferred embodiment of the present invention, the fermentation culture conditions in step 3) are: pH 6.5-7.5, temperature 22-30° C., ventilation volume 1-4 L / h, and stirring speed 100-200 rpm.
[0016] Further preferably, the fermentation culture conditions in step 3) are: pH 7, temperature 30°C, ventilation volume 2 L / h, and stirring speed 100 rpm.
[0017] According to the preferred embodiment of the present invention, the fermentation culture cycle in step 3) is 5 to 12 days.
[0018] Further preferably, the fermentation culture cycle in step 3) is 8 days.
[0019] According to the present invention, preferably, the culture medium for the fermentation culture in step 3) is PDB liquid culture medium.
[0020] The application of the polyphenol-rich wild mulberry fungus SH-27 fermentation liquid in the preparation of antioxidant and anti-inflammatory functional foods or medicines.
[0021] Beneficial effects: 1) The wild Morinda igniarius SH-27 screened out in the present invention is rich in active ingredients. The polyphenol content of its liquid cultured mycelium pellets is 29.74 mg / g, the triterpenoid content is 3.52 mg / g, the polysaccharide content is 88.61 mg / g, and the flavonoid content is 5.14 mg / g. By optimizing the liquid culture medium, the polyphenol content of its mycelium pellets can reach 46.98 mg / g. Its metabolites have great medicinal potential in the treatment and intervention of antioxidant, anti-inflammatory, anti-cancer, anti-tumor, diabetes and hypertension.
[0022] 2) The present invention uses the PSO-BP neural network model to optimize and increase the yield of mulberry ignia polyphenol metabolites, with an average yield of 33.72 mg / L, which is 33.5% higher than the fermentation broth obtained under the initial fermentation conditions and the single-factor experimental conditions.
[0023] 3) The PSO-BP neural network model used in the present invention optimizes fermentation conditions to increase the yield of mulberry ignia polyphenol metabolites, and also provides an excellent method for optimizing liquid fermentation conditions of medicinal fungi. It can effectively solve the problems of complex multi-factor coupling effects, low optimization efficiency, and insufficient optimization accuracy of traditional methods such as response surface methodology, and has good application potential in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 This is the morphological diagram of the wild Phellinus igniarius SH-27 strain; Figure 2 This is the morphological picture of the fruiting body of wild Phellinus igniarius SH-27; Figure 3 is the precise gradient parallel coordinate diagram; Figure 4 The scatter plot of the average yield of polyphenol metabolites of wild mulberry fungus SH-27; Figure 5This is the structure diagram of the PSO-BP neural network; Figure 6 is the mean square error curve of the model. DETAILED DESCRIPTION
[0025] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods. However, the following examples do not represent the scope of protection of the rights defined by the present invention. The scope of protection of the rights of the present invention shall be subject to the claims.
[0026] Example 1 Isolation, Screening and Identification of Wild Phellinus igniarius (1) Wild Phellinus igniarius fruiting bodies were collected from the century-old ancient mulberry garden in Xiajin, Shandong Province, China. Under sterile conditions, the surface was disinfected by wiping with 75% (v / v) alcohol cotton. The alcohol cotton should not be too wet, and no alcohol or cotton wool should remain on the fruiting bodies after wiping. The blade was burned with an alcohol lamp to disinfect it, and then the blade was used to cut along the fiber of the fruiting body. After cutting, the blade was burned again, and a square was cut in the center of the cut surface with the blade to facilitate picking up with tweezers. The cut fruiting body tissue was picked up with pointed tweezers burned with an alcohol lamp; (2) Preparation of mother culture medium: After sterilizing the plate at 121°C under high temperature and high pressure, pour the prepared and sterilized PDA solid culture medium onto the clean bench and wait for it to solidify; The PDA solid culture medium comprises the following components: 12 g / L potato extract powder, 20 g / L glucose, 15 g / L agar, water as solvent, and natural pH.
[0027] (3) Preparation of mother culture: Transfer the fruiting body tissue separated in step (1) to the PDA solid culture medium in step (2) and culture in a 30°C constant temperature box in the dark; (4) Preparation of original seed: When the mycelium has grown but other bacteria have not yet grown, pick the tip of the mycelium and place it on the PDA solid culture medium, and culture it in a 30℃ constant temperature box in the dark.
[0028] This strain grows slowly. After about three weeks of culture, the colony diameter is about 7~9cm. In the early stage of growth, white velvety hyphae grow. As the hyphae grow, they gradually change from white to yellow-brown, but the fresh hyphae at the edge of the colony are still white. Figure 1 As shown, this is the daily storage form of the strain. Mycelium or fungus moss can be picked from this strain in subsequent experiments.
[0029] (5) Expansion culture: Pick the hyphae from the edge of the colony of the original culture for liquid culture, culture in a shake flask at 24°C and 100 rpm for 10-14 days to obtain mycelial balls; The liquid culture medium is PDB liquid culture medium, which comprises 12 g / L potato extract powder, 20 g / L glucose, water as solvent, and natural pH.
[0030] (6) Identification of fungal species: The mycelial pellets obtained in step (5) were ground with liquid nitrogen, and the sample genome was extracted using a plant genomic DNA extraction kit. ITS-PCR amplification was performed using universal primers for the fungal internal transcribed spacer region, ITS1 / ITS4, synthesized by Shanghai Sangon Biotechnology Co., Ltd.; The primers are as follows: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', ITS4:5'-TCCTCCGCTTATTGATATGC-3', The ITS-PCR experiment was performed with a system of 50 μL, as shown in Table 1.
[0031] Table 1
[0032] The reaction conditions were as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, with a final extension at 72°C for 5 min. The amplified products were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.
[0033] The above ITS sequences were BLASTed using the NCBI database and found to be Inonotus hispidus The clone SH1 similarity was as high as 99%, and it was identified as a strain of Porifera scalypifolia ( Inonotus hispidus ) and named SH-27. It was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on February 10, 2025. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65865.
[0034] The wild mulberry fungus SH-27 screened by the present invention belongs to the family of Coriolus, the genus of Microporus, and has different shapes and colors, black or yellow-brown, with a cap in the shape of a horseshoe, and solitary fruiting bodies. The surface of some sample fruiting bodies is smooth, while the surface of some sample fruiting bodies has rough objects. The mycelium morphology is as follows: Figure 1 As shown, the fruiting body morphology is as follows Figure 2 The ITS sequence of the wild mulberry fungus SH-27 is shown in SEQ ID NO.1.
[0035] Example 2 Liquid Culture of Wild Phellinus Igneus SH-27 An alcohol lamp was lit on a sterile clean bench to create a local aseptic operating environment. Mycelial moss was picked from the mycelium of wild Phellinus igneus SH-27 and inoculated into the optimized culture medium for liquid culture. The culture was shaken at 24°C and 100 rpm for 10-14 days to obtain mycelial balls.
[0036] The optimized culture medium components are: glucose 30.1 g / L, yeast extract 3.15 g / L, MgSO4 3.1 g / L, KH2PO4 1.1 g / L, vitamin B1 0.1 g / L, solvent is water, pH 7.2.
[0037] Example 3 Determination method of active ingredient content of wild Phellinus igniarius SH-27 1. Determination of polyphenol content (1) Preparation of standard curve Accurately pipette 1 mL of the gallic acid standard stock solution (200 mg / L) into a 10 mL volumetric flask and dilute to volume with 70% ethanol to obtain the gallic acid working solution. Then, pipette 0, 0.2, 0.4, 0.6, 1.0, and 1.5 mL of the gallic acid working solution into separate 10 mL volumetric flasks. Add 2.5 mL of Folin-phenol reagent and shake well. Add 2.5 mL of 15% Na₂CO₃ solution, dilute to the mark with water, and shake well. Incubate in a 40°C water bath for 60 minutes, then allow to cool for 20 minutes. Measure the absorbance at 760 nm. Plot a standard curve with concentration as the horizontal axis and absorbance as the vertical axis.
[0038] (2) Detection of polyphenols in samples The mycelial pellets obtained in step (5) of Example 1 or the liquid culture of Example 2 were dried at 60°C and then ground into powder. 0.4 g of mycelial powder was accurately weighed and added with 4 mL of 70% ethanol. After ultrasonic extraction at 55°C for 1 h, the mixture was centrifuged at 6000 rpm for 10 min, and the supernatant was collected. The residue was extracted twice with 3 mL of 70% ethanol solution under the same extraction conditions as above. The supernatants from the three extractions were combined and diluted to 10 mL with 70% ethanol to obtain the ethanol extract sample solution for the determination of polyphenols, triterpenes, and flavonoids.
[0039] (3) Sample testing Pipette 1.0 mL of the alcohol extract into a 10 mL stoppered colorimetric tube. Add 2.5 mL of Folin-phenol reagent and shake well. Add 2.5 mL of 15% Na₂CO₃ solution, bring to volume with water, and shake well. Incubate in a 40°C water bath for 60 minutes, allow to cool for 20 minutes, and measure the absorbance at 760 nm. Calculate the total polyphenol concentration in each sample using the standard curve.
[0040] 2. Determination of polysaccharide content The extraction of polysaccharides was carried out according to the method of GB / T 15672-2009 for the determination of total sugar content in edible fungi. (1) Preparation of glucose standard curve To create a glucose standard curve: Pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of a glucose standard solution (100 mg / L) into a 10 mL stoppered colorimetric tube. Fill to 1.0 mL with distilled water. Add 1.0 mL of 5% (v / v) phenol solution to each solution. Quickly add 5.0 mL of concentrated sulfuric acid and allow to react for 10 minutes. Then, place the colorimetric tube in a 30°C water bath for 20 minutes. Measure the absorbance of a certain amount of the reaction solution at 490 nm. Based on the measured values, plot a standard curve with glucose concentration as the horizontal axis and absorbance as the vertical axis.
[0041] (2) Detection of polysaccharides in samples The mycelial pellets obtained from the liquid culture in step (5) of Example 1 were dried at 60°C and ground into powder. 0.25 g of the crushed mycelial powder was accurately weighed and added to 50 mL of distilled water. The mixture was then boiled in a water bath for 3 h. A group containing only distilled water and no mycelial powder was also boiled in a water bath for 3 h as a blank control. After the boiling water bath, the mixture was filtered, and the filtrate obtained was used as the sample for the intracellular polysaccharide content test.
[0042] (3) Sample testing Sample polysaccharide content determination: Accurately pipette 0.1 mL of each diluted sample (to ensure accurate experimental results, dilute the liquid to an absorbance between 0.2 and 0.8) into a 10 mL stoppered colorimetric tube. Fill to 1.0 mL with distilled water. Add 1.0 mL of 5% (v / v) phenol solution to the tube. Quickly add 5 mL of concentrated sulfuric acid, let the reaction stand for 10 minutes, and place the tube in a 30°C water bath for 20 minutes. Measure the absorbance using the same method as the standard curve. Perform three replicates for each sample. Use a blank control sample to zero the spectrophotometer. Calculate the polysaccharide content of each sample based on the standard curve.
[0043] 3. Determination of triterpenoid content (1) Standard curve: Weigh the oleanolic acid standard and add anhydrous ethanol to prepare a 0.2 mg / mL standard solution. Accurately measure 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the standard solution into centrifuge tubes. Evaporate the solvent. Accurately add 0.2 mL of freshly prepared 0.05 g / mL vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid. Shake well. Heat in a 70°C water bath for 15 minutes, cool in an ice bath for 5 minutes, add 4 mL of ethyl acetate, shake well, and use the vanillin-glacial acetic acid and ethyl acetate mixture as a blank. Measure the absorbance of the standard at 560 nm to construct a standard curve.
[0044] (2) Sample testing Take 1 mL of the ethanol extract sample, add 0.2 mL of freshly prepared 0.05 g / mL vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid, shake well, heat in a 70°C water bath for 15 minutes, cool in an ice bath for 5 minutes, add 4 mL of ethyl acetate, shake well, and measure the absorbance. Calculate the triterpenoid content in each sample using the standard curve.
[0045] 4. Determination of flavonoid content (1) Standard curve: Accurately weigh 10 mg of rutin standard, dried to a constant weight, dissolve in anhydrous ethanol, and adjust to volume in a 50 mL volumetric flask. Pipette 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL of the 0.2 g / L rutin standard solution into a test tube and add anhydrous ethanol to make the volume 1.5 mL. Sequentially add 0.1 mL of 100 g / L aluminum nitrate solution and 0.1 mL of 98 g / L potassium acetate solution, and make up to 5 mL with water. Let the solution stand for 1 hour. Measure the absorbance at 420 nm using 30% (v / v) ethanol as a blank to construct a standard curve.
[0046] (2) Sample testing Take 1.5 mL of the alcohol extract sample and add 0.1 mL of 100 g / L aluminum nitrate solution and 0.1 mL of 98 g / L potassium acetate solution, respectively. Make up to 5 mL with water. Prepare three replicates for each sample and let it stand for 1 hour. Measure the absorbance using the same method as the standard curve. Calculate the flavonoid content in each sample using the regression equation of the standard curve.
[0047] The wild mulberry fungus SH-27 screened by the present invention has a liquid culture mycelial pellet with a polyphenol content of 29.74 mg / g, a polysaccharide content of 88.61 mg / g, a triterpene content of 3.52 mg / g, and a flavonoid content of 5.14 mg / g. By optimizing the liquid culture medium, the polyphenol content of the mycelial pellet can reach 46.98 mg / g.
[0048] Example 4 Determination of the optimal tolerance range of fermentation conditions by step-by-step single-factor experiment A single-factor optimization experiment method was used, with the polyphenol metabolite production of wild Phellinus igniarius SH-27 as the measurement indicator. The six-dimensional parameter ranges of pH, temperature, ventilation, inoculation age, fermentation cycle, and stirring speed were screened through ultra-wide tolerance range determination. Precision gradient design and fixed gradient were used to conduct single-factor experiments to obtain the fermentation results under various conditions and create a data matrix. The specific implementation steps are as follows: (1) Strain activation and seed liquid preparation: On a clean bench, use a 5 mm diameter and high-temperature sterilized puncher to take the mycelial lawn of wild mulberry fungus SH-27 and place it on a solidified PDA solid culture medium plate, and evenly inoculate 4 pieces on each plate. After sealing with a sealing film, place it in a 30℃ constant temperature incubator and culture for 7~8 days to activate the strain; use a sterile 5 mm diameter puncher to take three fresh mycelial lawns from the edge of the activated strain mycelium and inoculate them into PDB liquid culture medium (add 150 mL culture medium to a 250 mL conical flask), and culture in a constant temperature shaker at 30℃ and 200 rpm for 7~10 days to obtain seed liquids of different inoculation ages; The PDB liquid culture medium components are (g / L): 12 g / L potato extract powder, 20 g / L glucose, the solvent is water, and the pH is natural.
[0049] (2) Preparation of fermentation broth: Seed liquid of different inoculation ages was inoculated into a 5 L fermentation tank containing 3 L PDB liquid medium at a volume ratio of 10%. The pH, temperature, ventilation volume, stirring speed, and fermentation period were controlled by a single factor method, and the fermentation broth was collected for use. Among them, the 5L fermentation tank was provided by Shanghai Bailun Biotechnology Co., Ltd.
[0050] (3) Determination of the production of polyphenolic metabolites of wild SH-27 fungus: 100 μL of completely uniform fermentation broth was taken and analyzed by high performance liquid chromatography (HPLC) to determine the production of polyphenolic metabolites in the corresponding fermentation broth in mg / L.
[0051] The instrument used for the high performance liquid chromatography is an Agilent 1260 high performance liquid chromatography system.
[0052] (4) Ultra-wide tolerance range determination: Taking the production of polyphenolic metabolites of wild mulberry fungus SH-27 as the determination index and PDB liquid medium as the basic culture medium, the effects of multidimensional parameters pH, temperature, ventilation, inoculation age, fermentation cycle, and stirring speed on the production of polyphenolic metabolites of wild mulberry fungus SH-27 were evaluated. The basic fermentation conditions were pH 7, temperature 28℃, ventilation 2L / h, inoculation age 168h, fermentation cycle 7d, and stirring speed 200rpm. On this basis, single-factor experiments were conducted on the six parameters of pH, temperature, ventilation, inoculation age, fermentation cycle, and stirring speed. That is, under the premise of controlling other factors at the initial level, the level of a single factor was changed, and the production of polyphenolic metabolites of mulberry fungus in the fermentation broth at different levels of a single factor was measured to obtain the optimal tolerance range of the factor to be explored. The tolerance ranges are: pH 6.5~7.5, temperature 22~30℃, ventilation volume 1~4L / h, inoculation age 168~240h, fermentation cycle 5~12d, and stirring speed 100~200rpm.
[0053] (5) Precision gradient design: Within the tolerance range determined in step (4), each factor was transformed with a fixed gradient and randomly combined. The gradients of each factor were: pH value with a gradient of 0.5 units, temperature with a gradient of 2°C, ventilation with a gradient of 0.5 L / h, inoculation age with a gradient of 12 hours, fermentation cycle with a gradient of 1 day, and stirring speed with a gradient of 25 rpm. According to the randomly combined parameters, the fermentation of wild mulberry fungus SH-27 was carried out in a 5L fermenter. Each group of experiments was carried out three times, and the average yield of polyphenol metabolites in the fermentation broth was determined. The experimental results are shown in Figure 2. Figure 3 、 Figure 4 As shown, the results showed that the average yield of mulberry ignia polyphenol metabolites was 25.26 mg / L.
[0054] Example 5 PSO-BP neural network model optimizes fermentation conditions to increase the yield of polyphenol metabolites Based on the step-by-step single-factor experiment, the experimental data were used to take the six-dimensional parameters of pH, temperature, ventilation, inoculation age, fermentation cycle, and stirring speed as the input of the model. The yield of polyphenol metabolites of wild Phellinus igniarius SH-27 was taken as the target. The optimal fermentation parameters of wild Phellinus igniarius SH-27 were obtained by using the PSO (particle swarm optimization) algorithm. The accuracy and effectiveness of the PSO-BP neural network model were verified through experiments. The steps are as follows: (1) Data preprocessing: Based on the step-wise single-factor experiment, the experimental data were statistically analyzed and shuffled, and then normalized using the mapminmax function to eliminate the influence of dimensional differences. The statistical and normalized results are shown in Tables 2 and 3.
[0055] The mapminmax function formula is as follows:
[0056] Where: Y is the normalized value, X is the original value, X min is the minimum value in the original data, X max is the maximum value in the original data, and A and B are the start and end values of the normalized range.
[0057] Table 2. Statistical results
[0058] Table 3 Normalization results
[0059] (2) Construction and training of PSO-BP neural network model: The PSO-BP neural network was constructed using the mathematical software MATLAB R2023b. The relevant parameters were set as follows: the six-dimensional parameters pH, temperature, ventilation, inoculation age, fermentation cycle, and stirring speed were used as the input layer, with a node number of 6; the number of hidden layers was 1, and the number of nodes was set to 10 by trial and error; the yield of polyphenol metabolites of wild Morinda ignia SH-27 was used as the only output layer, with a node number of 1; the number of model iterations was set to 100. The statistical data obtained in step (1) was randomly divided, with 80% divided into the training set and 20% divided into the test set, to train the model. The mean square error (MSE) is used to measure the degree of difference between the model prediction value and the true value. The smaller the MSE value, the better the model prediction effect, and the preset threshold is 0.04g 2 / L 2 Using the test set MSE as the model training indicator, the final MSE was reduced to 0.038g 2 / L 2 , which is lower than the preset threshold. The PSO-BP neural network structure is as follows Figure 5 As shown in , the MSE value drops sharply in the second iteration of the model and reaches the lowest value after 18 iterations, as shown in Figure 6 As shown in Figure 2, the model prediction effect is the best at this time.
[0060] (3) PSO algorithm optimization: With the goal of maximizing the production of polyphenolic metabolites from wild SH-27 fungus, the PSO algorithm was used to optimize the model trained in step (2). The PSO algorithm parameters were set as follows: population size 50, number of iterations 100, learning factor c1 = c2 = 1.5, and a linear decreasing strategy for the inertia weight (initial 0.9, final value 0.6). The optimal input parameters were: pH = 6.68, temperature = 30.36°C, ventilation volume = 1.81 L / h, inoculation age = 168.50 h, fermentation cycle = 7.53 d, and stirring speed = 100 rpm.
[0061] (4) Experimental verification: Based on the optimization results of step (3), the pH was set to 7, the temperature to 30℃, the ventilation volume to 2L / h, the inoculation age to 168h, the fermentation period to 8d, and the stirring speed to 100rpm. Experimental verification was carried out in a 5L fermenter. 100μL of completely uniform fermentation broth was taken and analyzed by high performance liquid chromatography (HPLC). The yield of polyphenol metabolites of wild mulberry fungus SH-27 in the corresponding fermentation broth was determined to be 33.72mg / L, which was 33.5% higher than the average yield of 25.26mg / L in the step-by-step single factor experiment. The results showed that the established PSO-BP neural network model was used to optimize the optimal fermentation conditions suitable for the production of polyphenol metabolites of wild mulberry fungus. This shows that the model is reliable and that the PSO-BP neural network model optimization can indeed increase the yield of polyphenol metabolites of wild mulberry fungus SH-27 compared with the original fermentation conditions.
[0062] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. A polyphenol-rich wild Phellinus igniarius SH-27 fermentation broth, characterized in that: The fermentation liquid is prepared by fermenting wild mulberry fungus SH-27 preserved in Guangdong Province Microbial Culture Collection Center. The wild mulberry fungus SH-27 is a thick hair fibrous fungus ( Inonotus hispidus ), the deposit number is GDMCC No: 65865.
2. The method for preparing the polyphenol-rich wild Phellinus igniarius SH-27 fermentation broth according to claim 1, characterized in that: The steps include: 1) A 4-6 mm diameter circular moss of wild Phellinus igniarius SH-27 mycelium was inoculated onto a PDA solid medium plate and cultured for 7-8 days to activate the strain. 2) Take the edge of the mycelium of the activated strain and inoculate it into PDB liquid medium for 7-10 days to obtain seed liquid; 3) The seed liquid is inoculated into a fermentation medium at a volume ratio of 5-20%, and fermentation is performed to obtain a polyphenol-rich wild phellinus igniarius SH-27 fermentation liquid.
3. The preparation method according to claim 2, wherein Step 1) The culture conditions are: 22-30°C.
4. The preparation method according to claim 2, wherein Step 2) The culture conditions are: 100-200 rpm, 22-30°C.
5. The preparation method according to claim 2, wherein Step 2) The seed solution is cultured for 168 h.
6. The preparation method according to claim 2, wherein Step 2) The inoculation amount is: 3 to 4 pieces of bacterial moss with a diameter of 4 to 6 mm are inoculated per 150 mL of culture medium.
7. The preparation method according to claim 2, wherein Step 3) The fermentation culture conditions are: pH 6.5-7.5, temperature 22-30°C, ventilation volume 1-4 L / h, and stirring speed 100-200 rpm; Further preferably, the fermentation culture conditions in step 3) are: pH 7, temperature 30°C, ventilation volume 2 L / h, and stirring speed 100 rpm.
8. The preparation method according to claim 2, wherein Step 3) The fermentation culture cycle is 5 to 12 days; Further preferably, the fermentation culture cycle in step 3) is 8 days.
9. The preparation method according to claim 2, wherein Step 3) The fermentation culture medium is PDB liquid culture medium.
10. Use of the polyphenol-rich wild Phellinus igniarius SH-27 fermentation broth according to claim 1 in the preparation of antioxidant and anti-inflammatory functional foods or medicines.