CN113440578B - Ethanol extract of co-fermentation product with memory improving effect - Google Patents
Ethanol extract of co-fermentation product with memory improving effect Download PDFInfo
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- CN113440578B CN113440578B CN202110531140.1A CN202110531140A CN113440578B CN 113440578 B CN113440578 B CN 113440578B CN 202110531140 A CN202110531140 A CN 202110531140A CN 113440578 B CN113440578 B CN 113440578B
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Abstract
The invention discloses an ethanol extract of a co-fermentation product with the functions of improving memory and a preparation method thereof, the extract is prepared by taking lucid ganoderma and cordyceps militaris as fermentation strains and taking gastrodia elata as a medicinal substrate to carry out liquid co-culture fermentation to obtain the fermentation product and extracting the fermentation product with ethanol, wherein the cordyceps militaris strain is B1528, and the strain preservation number is as follows: CCTCC NO: m2019789, deposited at 9.10.2019 in the center of China center for type culture Collection; the ganoderma lucidum strain is B1.4, and the preservation number of the strain is as follows: CCTCC NO: m2019790, deposited at the China center for type culture Collection on 2019, 10/9. The invention provides a brand-new product for fermenting gastrodia elata by utilizing lucid ganoderma and cordyceps militaris by utilizing a co-culture fermentation technology of traditional Chinese medicines, an ethanol extract of the product has an obvious memory improving effect, and can be used for preparing health-care products for improving memory and medicines for treating diseases with memory disorder symptoms, such as Alzheimer's disease and the like. Has good application prospect.
Description
Technical Field
The invention relates to a microbial fermentation conversion product of traditional Chinese medicine, a preparation method and application of effects thereof, in particular to an ethanol extract of a co-fermentation product of ganoderma lucidum and cordyceps militaris with the effects of improving memory and a preparation method thereof.
Background
Diseases with memory disorder as the main symptom have great harm to human health, such as Alzheimer's Disease (AD), whose clinical manifestations include memory impairment, dyskinesia and persistent cognitive decline. Alzheimer's disease is one of the leading causes of disability and death in contemporary humans, and its resulting learning and memory impairment has severely affected patients' quality of life. At present, no specific medicine is available for treating the Alzheimer disease, and the medicine treatment is mainly used, and non-medicine treatment and careful nursing are assisted to relieve symptoms or delay the development of the disease. With the increasing number of AD patients in the world, anti-AD drugs in the market cannot meet the requirements of the patients, so that the development of novel anti-AD drugs for improving the learning and memory disorders of the AD patients is necessary.
Disclosure of Invention
The invention aims to solve the technical problem of providing an ethanol extract of a co-fermentation product with the function of improving and improving memory, a preparation method and application thereof, and provides a new choice for the drug treatment of Alzheimer's disease.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
an ethanol extract of co-fermentation product with memory improving and improving effects is prepared by taking Ganoderma and Cordyceps militaris as fermentation strains, and rhizoma Gastrodiae as drug-property matrix, and performing bidirectional liquid co-culture fermentation; the cordyceps militaris strain is B1528, and the strain preservation number is as follows: CCTCC NO: m2019789, deposited at 9.10.2019 in the center of China center for type culture Collection; the ganoderma lucidum strain is B1.4, and the preservation number of the strain is as follows: CCTCC NO: m2019790, deposited at the China center for type culture Collection on 9/10/2019.
The preparation method of the co-fermentation product ethanol extract with the memory improving and improving functions is characterized by comprising the following steps:
(1) preparing a culture medium:
strain activation medium: conventional PDA culture medium;
preparing seeds and a fermentation medium: taking 20g of potatoes, cleaning, peeling, cutting into small pieces, putting into a pot, adding 100mL of water, boiling for 10-15 minutes, filtering, adding 1.82g of cane sugar, 3.00g of peptone, 0.09g of monopotassium phosphate and 3.00g of rhizoma gastrodiae powder into filtrate, heating and stirring uniformly, cooling, keeping the pH natural, and sterilizing at 121 ℃ for 30min for later use;
preparation of initial liquid culture medium: taking 20g of potatoes, cleaning, peeling, cutting into small pieces, putting into a pot, adding 100mL of water, boiling for 10-15 minutes, filtering, adding 2g of glucose and 3g of rhizoma gastrodiae into filtrate, heating, stirring uniformly, cooling, naturally adjusting pH, and sterilizing at 121 ℃ for 30min for later use;
(2) activating strains: respectively transferring Ganoderma strain B1.4 and Cordyceps militaris strain B1528 to PDA plate, and culturing at 25 deg.C in dark for 7 days;
(3) preparing a seed solution: under the aseptic condition, punching an activated ganoderma lucidum strain B1.4 and a cordyceps militaris strain B1528 with the diameter of 5mm from the flat plate in the step (2) by using a puncher, inoculating the activated ganoderma lucidum strain B and the cordyceps militaris strain B1528 into a 500mL conical flask filled with 100mL of seed liquid culture medium, and performing shake culture for 5 days in a shaking incubator at 200r/min and 25 ℃ under the dark condition for later use;
(4) liquid fermentation: inoculating activated ganoderma lucidum strain B1.4 and cordyceps militaris strain B1528 into an initial liquid culture medium, wherein the cordyceps militaris-ganoderma lucidum inoculation ratio is 1:2, the inoculation amount is 3mL/100mL, after shaking up gently, putting into a constant-temperature culture shaking table, and carrying out dark culture at the temperature of 25 ℃ for 15d at the speed of 200rpm/min to obtain a gastrodia elata product obtained by co-culturing and fermenting ganoderma lucidum and cordyceps militaris.
(5) Preparing an ethanol extract of a fermentation product: centrifuging the obtained fermentation product (10000rpm/10min) to obtain solid, drying the solid at 60 deg.C (6889.11g), pulverizing into powder (791.85g), adding 10 times volume of 95% ethanol, soaking at 25 deg.C for 24 hr, filtering with Buchner funnel, leaching the residue once again, mixing the extractive solutions, and concentrating into extract (76.77g) with extraction rate of 9.7%.
The invention has the beneficial effects that: compared with the prior art, the invention provides a brand-new product for fermenting the gastrodia elata by utilizing the ganoderma lucidum and the cordyceps militaris by utilizing the co-culture fermentation technology of the traditional Chinese medicines, the product has an obvious memory improving effect, and can be used for preparing health-care products for improving the memory and medicines for treating diseases with memory disorder symptoms, such as Alzheimer's disease and the like. Has good application prospect.
The invention adopts a response surface design method to obtain the optimal fermentation process condition, and the ethanol extract of the fermentation product obtained by the optimal fermentation process condition is applied to a rat with learning and memory disorder model to research the influence of the ethanol extract of the fermentation product on the rat with learning and memory disorder. The ethanol extract is found to have a remarkable effect of improving memory. The invention uses the co-culture fermentation technology, not only improves the utilization of medicinal bacteria and traditional Chinese medicines, but also provides a new material basis for the research and development of the anti-dysmnesia medicine, and has important theoretical and practical significance.
The present invention will be further described with reference to the following embodiments.
Process optimization for co-culture fermentation of gastrodia elata by ganoderma and cordyceps militaris
1. Conditions of the experiment
1.1 strain: ganoderma Ganoderma Lucidum B1.4, the strain preservation number is: CCTCC m 2019790, preserved in the China center for type culture Collection in 2019, 10 and 9 months; cordyceps militaris Cordyceppsmilitaris B1528, and the strain preservation numbers are as follows: CCTCC m 2019789, which is preserved in the China center for type culture Collection in 2019, 10 and 9 months.
1.2 medicinal materials and reagents:
gastrodia elata-Guizhou Daling medicine industry Co., Ltd (batch 181201), cordycepin reference substance-Shanghai source leaf biotechnology Co., Ltd (batch Y02M6K1), gastrodin reference substance-SIGMA (batch SLBN0006V), oleanolic acid reference substance-Chinese food and drug verification research institute (batch 308D024), acetonitrile chromatogram purity-America Tiandi (batch 18075173), absolute ethanol-Chongqing Chuandong chemical industry Co., Ltd (batch 20181101), methanol chromatogram purity-Tianjin Konmi chemical reagent Co., Ltd (batch 20180506), sulfuric acid-Chongqing Chuandong chemical industry Co., Ltd (batch 20160501), ethyl acetate-Chongqing Chuanwa chemical industry Co., Ltd (batch 20170919), glacial acetic acid-Taicang Hahuhui reagent Hajin reagent Co., Ltd (batch 2018425), vanillin-Tianmi chemical reagent Co., Ltd (batch 20170110), Gangzhou Dazui Kanji chemical reagent Co., Ltd (batch 82923), and pure water purification research institute (batch 20180721).
2. Experimental methods
2.1 preparation of culture Medium: activating strains: conventional PDA culture medium; seed liquid culture medium: 100mL of water, 20g of potatoes and 2g of glucose, wherein the pH is natural, and the mixture is sterilized at 121 ℃ for 30min for later use; initial liquid medium: 100mL of water, 20g of potatoes, 2g of glucose and 3g of gastrodia elata, wherein the pH is natural, and the mixture is sterilized at 121 ℃ for 30min for later use.
2.2 activating the strains: transferring Ganoderma strain B1.4 and Cordyceps militaris strain B1528 to PDA plate, and culturing at 25 deg.C in dark for 7 days.
2.3 preparing seed liquid: under the aseptic condition, activated strains B1.4 and B1528 with the diameter of 5mm are beaten from a plate of 2.2 by a puncher, are inoculated into a 500mL conical flask filled with 100mL seed liquid culture medium, and are shake-cultured for 5 days in a shaking incubator at the temperature of 25 ℃ at 200r/min under the dark condition for later use.
2.4 liquid fermentation: and (3) taking 1.5mL of each of the B1.4 seed solution and the B1528 seed solution, inoculating the seed solutions into an initial liquid culture medium, shaking the seed solutions lightly and uniformly, putting the seed solutions into a constant-temperature culture shaking table, and carrying out dark culture at the temperature of 25 ℃ at 200 rpm/min. 3 parallel samples were set for each condition.
2.5 index determination
2.5.1 measurement of Biomass: taking 250mL of fermentation product, centrifuging for 10min at 10000r/min, discarding supernatant, collecting mycelium, and weighing by an electronic balance.
2.5.2 determination of Ganoderma triterpene (Total triterpene-sterol)
2.5.2.1 method for extracting Ganoderma triterpene: adding 2mL of fermentation product into a centrifuge tube, adding 50mL of absolute ethyl alcohol, shaking to mix thoroughly, performing ultrasonic treatment for 45min, centrifuging at 4000rpm/min for 5min, taking 200 μ L of supernatant into a test tube, and drying at 70 deg.C to obtain the final product.
2.5.2.2 determination of Ganoderma triterpene content: precisely adding 200 mu L of vanillin glacial acetic acid solution (vanillin is 0.5g, glacial acetic acid constant volume is 10mL) into the sample obtained from 2.5.2.1, shaking up, immediately cooling in ice bath for 5min after water bath at 70 ℃ is carried out for 15min, precisely adding 4mL ethyl acetate, and shaking up. The absorbance was measured at a wavelength of 546nm by UV-visible spectrophotometry, and the measurement was carried out using absolute ethanol as a blank.
2.5.3 determination of Cordycepin and Gastrodin
2.5.3.1 method for extracting cordycepin and gastrodin: adding 5mL of fermentation product into a centrifuge tube, adding 50% methanol, diluting to a constant volume of 10mL, shaking to mix well, performing ultrasonic treatment for 40min, adding 1mL of sample into a 1.5mLEP tube, centrifuging at 15000rpm/min for 5min, taking supernatant, and filtering with 0.22 μm microporous membrane.
2.5.3.2 determination of cordycepin and gastrodin content
Detecting the content of cordycepin and gastrodin in the fermentation product by High Performance Liquid Chromatography (HPLC). With the increase of the fermentation time, gastrodin cannot be detected in the fermentation product extract, which may be that during the fermentation process, gastrodin is converted or decomposed, and only the content of cordycepin in the fermentation product is detected finally.
Chromatographic conditions are as follows: a chromatographic column: agilent Poaris C18-A (250 mm. times.4.6 mm, 5 μm); column temperature: 25 ℃; detection wavelength: 220 nm; flow rate: 1.0 mL/min; mobile phase: 0.05% phosphoric acid solution-acetonitrile (97: 3); sample introduction amount: 10 μ L.
2.6 Single factor experiment
2.6.1 incubation time: the fermentation time is 10, 12, 14, 16, 18 and 20 days, the content of rhizoma gastrodiae is 3g, the content of glucose is 2g, the content of potato is 20g, and the content of water is 100 mL. The inoculation ratio of the cordyceps militaris to the lucid ganoderma is 1:1 (the inoculation amount is 3mL), the cordyceps militaris and the lucid ganoderma are cultured under dark conditions of 200rpm/min and 25 ℃, and 3 parallel samples are set in each condition. The biomass (wet weight), ganoderma triterpene and cordycepin in the fermentation product are measured at 10, 12, 14, 16, 18 and 20d of the fermentation.
2.6.2 inoculation ratio of cordyceps militaris to ganoderma lucidum: fermentation time is 15d, gastrodia elata is 3g, glucose is 2g, potatoes are 20g, and water is 100 mL. The inoculation amount is 3mL, and the inoculation ratio of the cordyceps militaris to the lucid ganoderma is set to be 4:1, 3:1, 2:1, 1:2, 1:3 and 1: 4. 200rpm/min, 25 ℃ dark conditions, each condition set up 3 parallel samples. Detecting the contents of biomass (wet weight), ganoderma triterpene and cordycepin in the fermentation product.
2.6.3 adding amount of rhizoma Gastrodiae: the fermentation time is 15 days, the addition amount of rhizoma Gastrodiae is 1g, 2g, 3g, 4g, potato is 20g, and water is 100 mL. The cordyceps militaris-ganoderma lucidum inoculation ratio is 1:2 (the inoculation amount is 3 mL). 200rpm/min, 25 ℃ dark conditions, each condition set up 3 parallel samples. Detecting the contents of biomass (wet weight), ganoderma triterpene and cordycepin in the fermentation product.
2.6.4 addition of carbon sources: fermentation time is 15 days, gastrodia elata is 3g, potatoes are 20g, and water is 100 mL. Four factors of malto-oligosaccharide, soluble starch, glucose and sucrose are set as carbon sources, and the addition amount is 2 g. The cordyceps militaris-ganoderma lucidum inoculation ratio is 1:2 (the inoculation amount is 3 mL). 200rpm/min, 25 ℃ dark conditions, each condition set up 3 parallel samples. Detecting the contents of biomass (wet weight), ganoderma triterpene and cordycepin in the fermentation product.
2.6.5 addition of Nitrogen sources: fermentation time is 15d, gastrodia elata is 3g, cane sugar is 2g, potatoes are 20g, and water is 100 mL. Six factors of peptone, yeast, ammonium chloride, ammonium sulfate, ammonium nitrate and sodium nitrate are set as carbon sources, and the addition amount is 2 g. The cordyceps militaris-ganoderma lucidum inoculation ratio is 1:2 (the inoculation amount is 3 mL). 200rpm/min, 25 ℃ dark conditions, each condition set up 3 parallel samples. Detecting the contents of biomass (wet weight), ganoderma triterpene and cordycepin in the fermentation product.
2.6.6 addition of inorganic salts: fermentation time is 15d, gastrodia elata is 3g, cane sugar is 2g, peptone is 2g, potato is 20g, and water is 100 mL. Six factors of calcium chloride, ferric chloride, potassium dihydrogen phosphate, magnesium sulfate, sodium nitrate and zinc sulfate are set as carbon sources, and the addition amount is 0.1 g. The cordyceps militaris-ganoderma lucidum inoculation ratio is 1:2 (the inoculation amount is 3 mL). 200rpm/min, 25 ℃ dark conditions, each condition set up 3 parallel samples. Detecting the contents of biomass (wet weight), ganoderma triterpene and cordycepin in the fermentation product.
2.7Box-Behnken experiment: the addition amounts of the screened sucrose, peptone and potassium dihydrogen phosphate are considered factors, according to the Box-Behenken center experiment design principle, on the basis of a single-factor experiment, an optimal value is selected as a center point, one level value is selected from top to bottom as a response surface experiment design level, comprehensive scores obtained by calculating the contents of biomass, cordycepin and ganoderma triterpene are used as indexes, and three-factor three-level response surface analysis is designed (see table 1-2). And response surface regression analysis is carried out on the experimental data by adopting Design expert.V8.0.6 software.
TABLE 1-2 Box-Behnken Experimental factors and levels
2.8 validation experiment: and (4) carrying out a verification experiment by using the optimized formula of the fermentation medium, verifying whether the theoretical predicted value is consistent with the experimental detection value, and then carrying out reliability analysis to obtain the final optimized formula.
3. Results of the experiment
3.1 incubation time: under the basic conditions that the culture temperature is 25 ℃, the shaking table rotates at 200rpm/min, the inoculation ratio is 1:1 (the inoculation amount is 3mL), when the culture is carried out until the 14 th day, the content of cordycepin in the sample reaches a high value, and the content of ganoderma triterpene in the sample reaches a high value when the culture is carried out at the 16 th day (tables 1-3). The biomass of the sample reached a high value at 16d of cultivation. In order to obtain more cordycepin, ganoderma triterpene and biomass, the culture time is determined to be 15 days for carrying out experiments of other factors.
TABLE 1-3 Effect of time factors on Cordycepin content, Ganoderma triterpene content and biomass in fermentation product
3.2 inoculation ratio of cordyceps militaris and lucid ganoderma: the influence of the cordyceps militaris-ganoderma lucidum inoculation ratio on cordycepin content, ganoderma lucidum triterpene content and biomass in fermentation products is shown in tables 1-4 under the basic conditions that the culture temperature is 25 ℃, the shaking table rotates at 200rpm/min, the fermentation time is 15d, and the inoculation amount is 3 mL. When the inoculation ratio of the two bacteria is changed from 4:1 to 1:2 (the inoculation amount of the ganoderma lucidum is gradually increased), the biomass obtained by fermentation and the content of ganoderma lucidum triterpene are obviously increased. When the inoculation ratio is 4:1 or 3:1, the content of cordycepin and ganoderma triterpene is high, but the growth of mycelium is obviously influenced. When the inoculation amount of the ganoderma lucidum is excessive, the growth of two bacteria is not facilitated, and the generation of biomass is reduced. Comprehensive investigation shows that the cordyceps militaris-ganoderma lucidum inoculation ratio is 1:2, and the cordyceps militaris-ganoderma lucidum inoculation ratio is suitable for growth of mycelium and synthesis and accumulation of metabolites.
3.3 addition amount of rhizoma gastrodiae: the influence of different rhizoma Gastrodiae addition amounts on fermentation was studied under the basic conditions of a culture temperature of 25 deg.C, a shaker rotation of 200rpm/min, a fermentation time of 15d, and a Cordyceps militaris-Ganoderma inoculation ratio of 1:2 (inoculum size of 3mL), and the results are shown in tables 1-5. When the gastrodia elata is not added for fermentation, the content of cordycepin and ganoderma triterpene in the fermentation product is the lowest, when the addition amount of the gastrodia elata is increased from 1g to 3g, the content of cordycepin, the content of ganoderma triterpene and biomass in the fermentation product are obviously increased, and the highest content is achieved when the addition amount of the gastrodia elata is 3 g. When the addition amount of the gastrodia elata is continuously increased from 3g to 4g, the cordycepin content, the ganoderma triterpene content and the biomass tend to decrease. Therefore, the optimum amount of rhizoma Gastrodiae is 3g/100 mL.
TABLE 1-4 influence of Cordyceps militaris-Ganoderma lucidum inoculation ratio on cordycepin content, Ganoderma lucidum triterpene content and biomass in fermentation product
Tables 1-5 influence of the addition of Gastrodia elata on cordycepin content, Ganoderma triterpene content and biomass in fermentation product
3.4 addition of carbon source: the influence of different carbon sources on fermentation products is analyzed under the basic conditions that the culture temperature is 25 ℃, the shaking table rotates at 200rpm/min, the fermentation time is 15d, the cordyceps militaris-lucid ganoderma inoculation ratio is 1:2 (the inoculation amount is 3mL), and the addition amount of gastrodia elata is 3g/100mL, and the results are shown in tables 1-6. When sucrose is used as an added carbon source, the content of ganoderma triterpene and biomass are the highest. When the low-polymer maltose or glucose is used as an added carbon source, the cordycepin content is higher, but the ganoderma triterpene content and the biomass are less; when soluble starch is used as an added carbon source, the cordycepin content, the ganoderma lucidum triterpene content and the biomass are low. Thus, the selection of sucrose as the carbon source for addition is contemplated.
Tables 1-6 influence of different carbon sources on cordycepin content, ganoderma triterpene content and biomass in fermentation product
3.5 addition of Nitrogen sources: the influence of different nitrogen sources on fermentation products is researched under the basic conditions that the culture temperature is 25 ℃, the shaking table rotates at 200rpm/min, the fermentation time is 15d, the cordyceps militaris-ganoderma lucidum inoculation ratio is 1:2 (the inoculation amount is 3mL), the addition amount of gastrodia elata is 3g/100mL, and the sucrose is 2g/100mL, and the results are shown in tables 1-7. When peptone is used as an added nitrogen source, the cordycepin content and biomass in the fermentation product reach the highest level, and the ganoderma triterpene content is higher. When ammonium chloride is used as an added nitrogen source, the content of ganoderma triterpene in a fermentation product is highest, but the content and biomass of cordycepin are lower. Peptone was therefore selected as the nitrogen source for addition.
Tables 1-7 influence of different nitrogen additions on cordycepin content, ganoderma triterpene content and biomass in fermentation product
3.6 addition of inorganic salts: the culture temperature is 25 ℃, the shaking table is rotated at 200rpm/min, the fermentation time is 15d, the cordyceps militaris-ganoderma lucidum inoculation ratio is 1:2 (the inoculation amount is 3mL), the addition amount of gastrodia elata is 3g/100mL, the sucrose is 2g/100mL, and the peptone is 2g/100mL, and when potassium dihydrogen phosphate is used as an added inorganic salt, the biomass in the fermentation product is highest, and the cordycepin content and the ganoderma lucidum triterpene content are higher (tables 1-8). When magnesium sulfate is used as an added inorganic salt, the cordycepin content in the fermentation product is highest, but the ganoderma triterpene content and biomass are lower; when zinc sulfate is used as the added inorganic salt, the content of ganoderma triterpene in the fermentation product reaches the highest value, but the content of cordycepin is the lowest value, and the biomass is less. Comprehensively considering, potassium dihydrogen phosphate is used as an added inorganic salt.
Tables 1-8 influence of different inorganic salts on cordycepin content, ganoderma triterpene content and biomass in fermentation product
3.7Box-Behnken experiment: on the basis of a single-factor experiment, the addition amounts of sucrose, peptone and potassium dihydrogen phosphate are selected as influencing factors, the cordycepin content, the ganoderma triterpene content and the biomass are respectively used as evaluation indexes, a three-factor three-level response surface analysis experiment is carried out, and the results are shown in tables 1-9.
TABLE 1-9 Box-Behnken Experimental factors and levels
TABLE 1-10 regression model ANOVA results (Biomass)
Note: R-Squared 0.9389, Adj R-Squared 0.8604, Adeq Precision 11.917.
TABLE 1-11 regression model analysis of variance results (Cordycepin)
Note: R-Squared 0.9281, Adj R-Squared 0.8356, Adeq Precision 9.782.
TABLE 1-12 regression model ANOVA results (Ganoderma triterpene)
Note: R-Squared 0.9107, Adj R-Squared 0.7960, Adeq Precision 9.392.
Three factors of sucrose (A), peptone (B) and potassium dihydrogen phosphate (C) were applied to biomass (Y) using Design expert.V8.0.6 software 1 ) Cordycepin (Y) 2 ) Ganoderma triterpene (Y) 3 ) And (3) carrying out response surface analysis, and establishing a ternary quadratic regression equation as follows:
Y 1 (biomass) ═ 914.3+135.08A-41.77B-1066.18C +1.32AB +64.73AC-22.68BC-8.99A 2 +2.23B 2 -289.84C 2 ；
Y 2 (cordycepin) ═ 17017.92+815.42A +557.92B-19626.34C +53.06AB +465.63AC +107.58BC-100.23A 2 -108.28B 2 -5375.81C 2 ；Y 3 (Ganoderma triterpene) ═ 3074.65+450.34A-139.30B-3583.93C +4.24AB +215.78AC-75.61BC-29.88A 2 +7.51B 2 -974.46C 2 。
From the analysis of variance shown in tables 1-10, 1-11, 1-12Discovery, regression model P<0.05, indicating that the regression model reaches a significant level, and the mismatching term P (biomass) is 0.2594>0.05, P (Cordycepin) ═ 0.5207>0.05, P (Ganoderma triterpene) 0.2693>0.05, the difference is not significant, the unknown factor is shown to have small interference on the test result, and the residual errors are all caused by random errors. Correlation coefficient R of model 2 (biomass) ═ 0.9389, R 2 (cordycepin) ═ 0.9281, R 2 The result shows that the model has better fitting degree and smaller experimental error when the ganoderma triterpene is 0.9107.
Based on the model, in order to determine that the partial derivatives of the optimal values Y of all factors to A, B, C are zero, the fitted quadratic equations are derived to obtain an equation set, the equation set is solved, the results are rounded and the integers are taken to obtain sucrose (A) ═ 1.82, peptone (B) ═ 3.00 and potassium dihydrogen phosphate (C) ═ 0.09, and the optimal culture conditions and the optimal culture medium composition are obtained by combining the results of single-factor experiments: 18.2g/L of sucrose, 30.0g/L of peptone, 0.9g/L of potassium dihydrogen phosphate, 1:2 of cordyceps militaris-lucid ganoderma inoculation ratio (inoculation amount is 30mL/L), 30.0g/L of gastrodia elata addition amount and 15d of culture time, wherein the biomass, the cordycepin and lucid ganoderma triterpene yields predicted under the optimized condition are 215.37g/L, 1142mg/L and 10.84mg/L respectively.
3.8 verification of experimental results: in order to test the reliability of an experimental model, the fermentation culture is carried out by adopting the process conditions, considering the feasibility of actual operation, the experimental conditions are determined as 18.2g/L of sucrose, 30.0g/L of peptone, 0.9g/L of potassium dihydrogen phosphate, the cordyceps militaris-ganoderma lucidum inoculation ratio is 1:2, the addition amount of the gastrodia elata is 30.0g/L, the culture time is 15d, the culture temperature is 25 ℃, the shaking table rotates at 200rpm/min, under the conditions, the biomass of a fermentation product is 217.24g/L, the cordycepin yield is 1114.14mg/L, the ganoderma triterpene yield is 10.77mg/L, the predicted value is basically consistent, and the biomass, cordycepin and ganoderma triterpene yields are respectively improved by 76.14%, 319.20% and 13.49% before optimization.
4. Discussion of the related Art
The invention carries out liquid fermentation on cordyceps militaris and lucid ganoderma in a co-culture mode, which is different from the traditional single-fungus two-way fermentation, two medicinal fungi form a stable interaction relation in the co-culture process, and the content of various substances in a fermentation product is also improved through co-culture because a richer enzyme system exists in a co-culture system and an intermediate metabolite is utilized by a biological induction effect, so that the biomass is rapidly increased. The co-culture of fungus and its interaction can regulate bioactive metabolite, induce the production of extracellular secondary metabolite, especially phenolic and quinone compounds, and induce the production of different enzymes. The culture conditions also influence the metabolic characteristics of the microorganisms, the co-culture fermentation in the liquid culture medium can help to find new secondary metabolites and activate the productivity of the microorganisms, and in addition to the co-culture effect, the addition of the drug substrate gastrodia elata also influences the yield of the secondary metabolites in the fermentation products. The invention utilizes the traditional Chinese medicine fermentation technology to ferment the cordyceps militaris, the lucid ganoderma and the tall gastrodia tuber, obtains more cordycepin and lucid ganoderma triterpene, and improves the output of mycelium.
Secondly, the influence of the fermentation product on learning and memory disorder rats caused by lipopolysaccharide
1. Experimental Material
1.1 Experimental animals: SPF grade male eight week old SD rats, 80, initial body weight 180-. Provided by Changsha Duty laboratory animal center, laboratory animal license number: SCXK (Xiang) 2019-. Keeping the temperature in the breeding room at 20-25 deg.C, alternating day and night for 12h/12h, feeding solid granules every day on time to maintain feed and sterile water (autoclaving), and keeping the breeding environment quiet.
1.2 Experimental drugs:
preparing alcohol extract components of gastrodia elata: pulverizing 1500g rhizoma Gastrodiae into powder, adding 10 times volume of 95% ethanol, soaking at 25 deg.C for 24 hr, filtering with Buchner funnel, leaching residue once again, mixing extractive solutions, and concentrating into extract (178.44g), with extraction rate of 11.89%.
Preparing an ethanol extract component of a ganoderma and cordyceps militaris fermentation product: centrifuging the obtained fermentation product (10000rpm/10min) to obtain solid, drying the solid at 60 deg.C (2180g), pulverizing after oven drying (250.57g), adding 10 times volume of 95% ethanol, soaking at 25 deg.C for 24 hr, filtering with Buchner funnel, extracting the residue once, mixing extractive solutions, and concentrating into extract (28.43g), with extraction rate of 11.3%.
Preparing an ethanol extraction component of a gastrodia elata product by co-culture fermentation of ganoderma and cordyceps militaris: centrifuging the obtained fermentation product (10000rpm/10min) to obtain solid, drying the solid at 60 deg.C (6889.11g), pulverizing into powder (791.85g), adding 10 times volume of 95% ethanol, soaking at 25 deg.C for 24 hr, filtering with Buchner funnel, leaching the residue once, mixing extractive solutions, and concentrating into extract (76.77g) with extraction rate of 9.7%. Naofukang tablet (Hangzhou Minsheng pharmaceutical industry Co., Ltd 717H016) is ground into powder for later use.
2. Experimental methods
2.1 animal grouping and dosing: after adaptive feeding for 3 days, the Morris water maze test (MWM) eliminates rats which are over sensitive (find the platform within 10 s) or over slow (cannot find the platform within 2 min), randomly divides 64 selected rats into 8 groups, and each group comprises 8 rats: blank group (physiological saline), model group (physiological saline), positive control group (brain recovery tablet) (0.4g kg) -1 ·d -1 ) Gastrodia elata medium dose group (0.14 g.kg) -1 ·d -1 ) The dosage of the fermentation product of the glossy ganoderma and the cordyceps militaris is 0.13 g.kg -1 ·d -1 ) High-dose group (0.23 g.kg) of ganoderma lucidum cordyceps militaris fermented gastrodia elata product -1 ·d -1 ) The dosage of the product of rhizoma Gastrodiae fermented by Ganoderma and Cordyceps militaris is 0.12 g/kg -1 ·d -1 ) Ganoderma and Cordyceps militaris fermentation rhizoma Gastrodiae product low dose group (0.06 g.kg) -1 ·d -1 )。
After adaptive feeding, rats in each group were given the corresponding drug for 28 consecutive days, starting on day 22, and were subjected to behavioral testing for 7 consecutive days. Half an hour before the behavioral test, lipopolysaccharide is used as a model making medicine, a model group, a positive control group, a gastrodia elata medium dosage group, a ganoderma cordyceps militaris fermentation product medium dosage group, a ganoderma cordyceps militaris fermentation gastrodia elata product high dosage group, a ganoderma cordyceps militaris fermentation gastrodia elata product medium dosage group and a ganoderma cordyceps militaris fermentation gastrodia elata product low dosage group are subjected to intraperitoneal injection of lipopolysaccharide (250 microgram/kg), and a blank group is subjected to intraperitoneal injection of a corresponding dosage of physiological saline. Oral dosing (gavage) was performed in each group 30 minutes before the lipopolysaccharide intraperitoneal injection.
After the intraperitoneal injection is finished on the 22 th day, the learning and memory ability of the rats is measured by adopting an ethological test (Morris water maze and dark avoidance experiment), and the morphological changes of mental state, weight change, hair luster, diet, drinking water and the like of the rats in each group of the drug treatment are observed every day. The material was taken 2 hours after the end of the last dose and animal behavioral testing.
2.2Morris Water maze experiment: before irrigation, the water maze is cleaned, the irrigation depth is about 2cm just submerging the platform, and the water temperature is 23-25 ℃. The placing position is kept fixed in the experiment process, the indoor environment is quiet, light is soft, the swimming track of a mouse can be captured by the camera is taken as a standard, the whole water maze is surrounded by the black curtain, and the black curtain is isolated from the outside to eliminate interference factors.
2.3 dark avoidance experiment: the rats in each group are adapted for 5min in the dark avoiding device, then the face of each rat, facing away from the opening, is placed in a bright room, a timer is started, a dark room power supply is connected, the rats can enter the dark room due to the habit of addiction to darkness, but can return to the bright room after the dark room is shocked, and then the memory of shocking stimulation in the dark room is obtained. The dark latency (i.e., the time from the start of the experiment to the first shock) and the number of dark errors within 5min (i.e., the number of shocks experienced within 5 min) were recorded for each group of rats.
2.4 obtaining animal materials: on the 28 th day of the experiment, after the Morris water maze experiment and the dark avoidance experiment are carried out for 2 hours, after 10 percent chloral hydrate is injected into the abdominal cavity of the rat for anesthesia, blood is taken from the abdominal aorta of the rat, after the blood taking is finished, the rat is kept still for 10min, then serum is separated (3000rpm, 15min and 4 ℃) and is frozen and stored at the temperature of minus 80 ℃ for standby. After blood is taken out by anesthesia, craniotomy is carried out to take out brain, hippocampus is separated from brain tissue, rat hippocampus tissue is put into liquid nitrogen for preservation, and after the material taking is finished, the hippocampus tissue is put into a freezer with the temperature of 80 ℃ below zero for freezing preservation for standby.
2.5 ELISA assay for detecting SOD, MDA, IL-6 and IL-10 content in rat serum: and (3) taking the kit out of a refrigerator at 4 ℃, standing at room temperature for 10min, and designing standard sample holes, blank holes and sample holes on an ELISA plate. Adding the prepared sample and standard substance, covering the reaction hole with a sealing plate glue, placing in a 37 ℃ incubator, and reacting for 1 hour. Washing the plate for 5 times, drying, adding an enzyme-labeled reagent, covering the reaction hole with a sealing plate rubber, placing in a 37 ℃ thermostat, and reacting for 30 minutes. Washing the plate for 5 times, adding color developing solution, covering the reaction hole with sealing plate glue, placing in a 37 ℃ incubator, and reacting for 15 minutes. Adding stop solution, placing in an enzyme-linked immunosorbent assay device for 15 minutes, measuring the absorbance value, and recording.
2.6 real-time fluorescent quantitative PCR method for detecting the expression of IL-10, TNF-alpha and BDNF in the hippocampus of rats
Extraction of RNA: the operation steps are carried out according to the instruction of the kit, a part of the obtained RNA is used, and the rest RNA is stored at-80 ℃ for later use.
The reverse transcription reaction
[ solution ] the following mixture was prepared in a 200. mu. LEP tube of RNase-free (Table 2-3)
TABLE 2-3 reverse transcription reaction System
② centrifuging and mixing the liquid in the EP tube evenly, incubating for 15min at 42 ℃, incubating for 2min at 85 ℃ to inactivate All in one reverse transcriptase, cooling to room temperature on ice, and storing at-20 ℃ for later use.
Three-step fluorescent quantitative (qRT-PCR) detection
The primer tubes are instantaneously centrifuged, the triple distilled water with the volume above the tubes is added, the mixture is fully mixed, 10 mu L of the mixture is sucked out, the mixture is added into an EP tube filled with 90 mu L of the triple distilled water, and the mixture is stored at the temperature of minus 20 ℃.
② construction of q-PCR System (tables 2-4)
Tables 2-4 reaction dosage table
The specific design of primers required for the reaction is shown in tables 2-5. Primers were designed by Primer software and synthesized by Shanghai Biotechnology Ltd.
TABLE 2-5 mRNA primer sequences to be tested
And (3) amplification procedure: stage 1: pre-denaturation, at 95 deg.C for 5min, and circulating for 1 time; and (2) stage: thermal dissolution: 95 ℃ for 15 sec; 60 ℃ for 30 sec; 72 ℃ for 30 sec; circulating for 40 times; and (3) stage: dissolution curve: 95 ℃ for 15 sec; 60 ℃ for 1 min; 95 ℃ for 15 sec; the cycle was 1 time.
2.7 data processing: statistical analysis is carried out on the data by adopting a sps 17.0, and mean values are represented by +/-standard deviation (X +/-S); comparing data which are in accordance with normal distribution among groups by adopting a one-factor analysis of variance test, and if the data are in non-normal distribution, comparing whether the difference between the two groups has statistical significance by adopting a rank sum test; the difference was considered statistically significant with P <0.05 and with P <0.01, as the test level α ═ 0.05.
3. Results of the experiment
3.1 general status of rats in each group: during the experiment, the rats in the administration group have good mental state, normal water intake and food intake, free movement, normal behavior and glossy hair. In the later stage of injecting lipopolysaccharide, the model group rats are listened, intake and intake of water are reduced, behaviors are tired, hair is not lusterless, and weight is slowly increased. The general state of the rats in the other groups is good, and the rats in each group do not die in the experiment.
3.2 Effect of fermentation products on rat body weight: the mean body weight changes of the experimental rats after two phases of administration and molding are shown in tables 2-6. The data show that the body weight of rats in each group increased after administration and after molding. After molding is finished, the weight of the rats in the Gastrodia elata medium-dose group, the Gastrodia elata fermentation product high-dose group, the Gastrodia elata fermentation product medium-dose group and the Gastrodia elata fermentation product low-dose group have no significant difference with the weight of the rats in the blank group and the positive control group; the weight of the rat in the model group slowly increases, and is lower than that of the blank group and the positive control group; the weight of rats in the middle-dose group of the gastrodia elata, the high-dose group of the gastrodia elata product fermented by the lucid ganoderma and cordyceps militaris and the middle-dose group of the gastrodia elata product fermented by the lucid ganoderma and cordyceps militaris is higher than that of the rats in the model group.
TABLE 2-6 influence of fermented Gastrodia elata product of Ganoderma and Cordyceps militaris on rat body weight (unit: g, X + -S, n ═ 8)
Note: comparison with blank group * P<0.05, ** P<0.01; comparison with model group ▲ P<0.05， ▲▲ P<0.01; comparison with Positive control group # P<0.05， ## P<0.01。
3.3Morris Water maze test of the Effect of fermentation products on rat behaviours
Compared with a model control group, the escape latency of the administration group is shortened, the escape latency of the blank group and the positive control group is obviously shortened on 23 th and 24 th days, and the escape latency of the blank group and the positive control group is obviously shortened on 25 th to 28 th days; in the Gastrodia elata medium dose group, the escape latency is obviously shortened on the 27 th and 28 th days; the escape latency is obviously shortened on days 27 and 28 by the dosage group in the fermentation product of the lucid ganoderma and the cordyceps militaris; the escape latency is obviously shortened on 25 th and 26 th days, and the escape latency is obviously shortened on 27 th and 28 th days when the lucid ganoderma cordyceps militaris fermented gastrodia elata product is prepared in high and medium dosage groups; in the low-dose group of the ganoderma lucidum cordyceps militaris fermentation gastrodia elata product, the escape latency period is shortened from 25 days. Compared with a positive control group, the escape latency of the gastrodia elata medium-dosage group is obviously prolonged on the 28 th day; the dosage of the ganoderma lucidum and cordyceps militaris fermentation product is set, and the dosage is prolonged from 26 days; the ganoderma lucidum cordyceps militaris fermented gastrodia elata product has high and medium dosage groups, and the escape latency period has no obvious difference; the escape latency of the low-dose group of ganoderma lucidum cordyceps militaris fermented gastrodia elata products is prolonged from 26 days.
The results indicate that the high and medium dosage composition of the ganoderma lucidum and cordyceps militaris fermented gastrodia elata product can improve the learning and memory capacity of rats with learning and memory disorders caused by lipopolysaccharide.
3.4 dark avoidance experiments to test the Effect of fermentation products on rat behaviourology
The rats were tested for 7 days in this experiment and the results are shown in tables 2-7.
Table 2-7 dark avoidance experiments to test the effect of fermented Gastrodia elata product of Ganoderma and Cordyceps militaris on rat behavioristics (X + -S, n ═ 8)
Note: comparison with blank group * P<0.05， ** P<0.01; comparison with model group ▲ P<0.05， ▲▲ P<0.01; comparison with Positive control group # P<0.05， ## P<0.01。
Compared with a blank group, the model group has shortened dark avoidance latency and increased dark avoidance error times, which indicates that the model building is successful; the dark-avoiding latency period of the gastrodia elata medium-dose group, the ganoderma cordyceps militaris fermentation product medium-dose group and the ganoderma cordyceps militaris fermentation gastrodia elata product high-dose and low-dose group is shortened, and the number of dark-avoiding errors is increased; the positive control group and the dosage group in the gastrodia elata product fermented by the lucid ganoderma and the cordyceps militaris have slightly prolonged dark-avoiding latency, slightly increased times of dark-avoiding errors and no statistical significance in difference; compared with the model group, the rats in each group have prolonged dark avoidance latency and reduced dark avoidance error times; compared with a positive control group, the gastrodia elata medium-dosage group has the advantages that the dark-avoiding latency is shortened, and the number of times of dark-avoiding errors is increased; the dark-avoiding latency period is shortened and the number of dark-avoiding errors is increased by using the dosage group in the ganoderma lucidum and cordyceps militaris fermentation product; the lucid ganoderma cordyceps militaris fermentation gastrodia elata product high-dose group has the advantages that the dark-avoiding latency is shortened, and the number of times of dark-avoiding errors is slightly increased; in the dosage group of the ganoderma lucidum cordyceps militaris fermented gastrodia elata product, the dark-avoiding latency is slightly shortened, the number of times of dark-avoiding errors is slightly increased, and the difference has no statistical significance; the dark-avoiding incubation period is shortened and the number of dark-avoiding errors is slightly increased in the low-dose group of the ganoderma lucidum cordyceps militaris fermentation gastrodia elata product.
The results of the darkness-avoiding experiments show that the intervention of the dose group in the product of fermenting the gastrodia elata by the ganoderma lucidum and cordyceps militaris improves the learning and memory capacity of rats with learning and memory disorders caused by lipopolysaccharide.
3.5 content changes of SOD, MDA, IL-6, IL-10 in rat serum
3.5.1 content change of SOD and MDA in rat serum: see tables 2-8. Compared with the blank group, the SOD content of the rat in the model group is obviously reduced, which indicates that the model building is successful; compared with the model group, the content of SOD in the positive control group, the gastrodia elata medium dosage group, the ganoderma lucidum cordyceps militaris fermented gastrodia elata product is high, and the content of SOD in the medium dosage group is obviously increased; the SOD content of the product of Gastrodia elata fermented by Ganoderma and Cordyceps militaris in low-dose group is increased. Compared with a positive control group, the SOD content of rats in the Gastrodia elata medium-dose group, the Gastrodia elata and Cordyceps militaris fermentation product medium-dose group and the Gastrodia elata and fermentation product high, medium and low-dose groups is obviously reduced.
Compared with the blank group, the model group rat has obviously increased MDA content, which indicates that the model is successfully made; compared with the model group, the positive control group, the gastrodia elata medium dose group, the ganoderma cordyceps militaris fermentation product medium dose group, the ganoderma cordyceps militaris fermentation gastrodia elata product high and the medium dose group rat MDA content is obviously reduced. Compared with a positive control group, the MDA content of rats in a gastrodia elata medium-dose group and a ganoderma and cordyceps militaris fermented gastrodia elata product low-dose group is remarkably increased; the MDA content of rats in dosage group in the ganoderma lucidum and cordyceps militaris fermentation product is increased.
TABLE 2-8 rat serum SOD, MDA content (X + -S, n ═ 8)
Note: comparison with blank group * P<0.05， ** P<0.01; comparison with model group ▲ P<0.05， ▲▲ P<0.01; comparison with Positive control group # P<0.05， ## P<0.01。
3.5.2 content changes of IL-6 and IL-10 in rat serum: see tables 2-9. Compared with the blank group, the IL-6 content of rats in the model group, the gastrodia elata middle dose group, the ganoderma cordyceps militaris fermentation product middle dose group and the ganoderma cordyceps militaris fermentation gastrodia elata product high and low dose group is remarkably increased; the IL-6 content of rats in the dosage group in the ganoderma lucidum cordyceps militaris fermented gastrodia elata product is increased. Compared with the model group, the IL-6 content of rats in the positive control group, the gastrodia elata medium dose group, the ganoderma cordyceps militaris fermentation product medium dose group and the ganoderma cordyceps militaris fermentation gastrodia elata product high, medium and low dose group is obviously reduced. Compared with a positive control group, the content of IL-6 in rats in a gastrodia elata medium-dose group, a ganoderma lucidum and cordyceps militaris fermentation product medium-dose group and a ganoderma lucidum and cordyceps militaris fermentation gastrodia elata product high-dose and low-dose group is remarkably increased.
Compared with the blank group, the IL-10 content of rats in the model group, the positive control group, the gastrodia elata medium dosage group, the ganoderma cordyceps militaris fermentation product medium dosage group and the ganoderma cordyceps militaris fermentation gastrodia elata product high, medium and low dosage group is obviously reduced. Compared with the model group, the positive control group, the gastrodia elata medium-dose group, the ganoderma and cordyceps militaris fermented gastrodia elata product is high, and the IL-10 content of rats in the medium-dose group is obviously increased; the content of IL-10 in the rat in the dosage group in the ganoderma lucidum and cordyceps militaris fermentation product is increased. Compared with the positive control group, the IL-10 content of rats in the Gastrodia elata medium-dose group, the Gastrodia elata fermentation product medium-dose group and the Gastrodia elata fermentation product high-dose and low-dose groups is obviously reduced (the IL-10 content of rats in the Gastrodia elata fermentation product medium-dose group is reduced).
TABLE 2-9 IL-6 and IL-10 content in rat serum (X. + -. S, n. gtoreq.8)
Note: comparison with blank group * P<0.05， ** P<0.01; comparison with model group ▲ P<0.05， ▲▲ P<0.01; comparison with Positive control group # P<0.05， ## P<0.01。
3.6 TNF- α, BDNFmRNA expression levels in rat hippocampus: see tables 2-10. Compared with the blank group, the expression levels of TNF-alpha mRNA in the model group, the middle dosage group of the ganoderma lucidum cordyceps militaris fermentation product and the high and low dosage groups of the ganoderma lucidum cordyceps militaris fermentation gastrodia elata product are obviously increased; the expression level of TNF-alpha mRNA in the dose group in the gastrodia elata is increased. Compared with the model group, the positive control group, the gastrodia elata medium dose group, the ganoderma lucidum cordyceps militaris fermentation product medium dose group and the ganoderma lucidum cordyceps militaris fermentation gastrodia elata product high, medium and low dose groups have obviously reduced rat TNF-alpha mRNA expression levels. Compared with a positive control group, the expression level of rat TNF-alpha mRNA in the dosage group of the gastrodia elata and the dosage group of the ganoderma lucidum and cordyceps militaris fermented gastrodia elata product is slightly increased, and the difference has no statistical significance; the expression level of rat TNF-alpha mRNA is obviously increased in the middle dose group of the lucid ganoderma cordyceps militaris fermentation product and the high and low dose groups of the lucid ganoderma cordyceps militaris fermentation gastrodia elata product.
Compared with the blank group, the BDNFmRNA expression levels of the model group, the positive control group, the gastrodia elata middle dose group, the ganoderma cordyceps militaris fermentation product middle dose group and the ganoderma cordyceps militaris fermentation gastrodia elata high, middle and low dose groups are remarkably reduced. Compared with the model group, the BDNFmRNA expression levels of the positive control group, the gastrodia elata medium dose group, the ganoderma cordyceps militaris fermentation product medium dose group and the ganoderma cordyceps militaris fermentation gastrodia elata product high, medium and low dose groups are obviously increased. Compared with a positive control group, the BDNFmRNA expression levels of the gastrodia elata medium-dose group, the ganoderma cordyceps militaris fermentation product medium-dose group and the ganoderma cordyceps militaris fermentation gastrodia elata high-dose and low-dose groups are remarkably reduced; the BDNFmRNA expression level of a dosage group in the product of Gastrodia elata fermented by lucid ganoderma and cordyceps militaris is slightly reduced, and the difference has no statistical significance.
Table 2-10 rat hippocampus expression levels of TNF- α, BDNFmRNA (X ± S, n ═ 8)
Note: comparison with blank group * P<0.05， ** P<0.01; comparison with model group ▲ P<0.05， ▲▲ P<0.01; comparison with Positive control group # P<0.05， ## P<0.01。
4. Conclusion
The medicine dry prognosis can improve the symptoms of listlessness, inappetence and weight loss of model rats.
In the Morris water maze experiment, the high and medium dose groups of the ganoderma and cordyceps militaris fermented gastrodia elata product can obviously shorten the escape latency of experimental animals (P is less than 0.01). In the darkness-avoiding experiment, the darkness-avoiding incubation period of the administration group is increased, the number of times of memory errors is reduced, and the effect of the dosage group in the ganoderma lucidum cordyceps militaris fermentation gastrodia elata product is most obvious.
The high and medium dose groups of the ganoderma lucidum and cordyceps militaris fermented gastrodia elata product can reduce the content of MDA (P is less than 0.01) in serum of experimental rats, and the medium dose effect is most obvious. The dosage of the product of Gastrodia elata fermented by Ganoderma and Cordyceps militaris can reduce IL-6 content in model rat (P is less than 0.01). The content of IL-10 in the serum of the experimental rat is improved (P is less than 0.01), and the effect of the medium-dose group is most obvious.
The expression detection results of TNF-alpha and BDNFmRNA in an experimental rat hippocampus by a real-time fluorescent quantitative PCR technology show that the dosage group in the ganoderma lucidum cordyceps militaris fermentation gastrodia elata product can improve the learning and memory disorder rat caused by lipopolysaccharide by reducing the expression level of TNF-alpha mRNA of the learning and memory disorder model rat and increasing the expression level of BDNFmRNA of the model rat.
Claims (2)
1. A co-fermentation product ethanol extract with memory improving and improving effects is prepared by taking Ganoderma and Cordyceps militaris as fermentation strains, and rhizoma Gastrodiae as medicinal substrate, and performing bidirectional liquid co-culture fermentation; the cordyceps militaris strain is B1528, and the strain preservation number is as follows: CCTCC NO: m2019789, deposited at the China center for type culture Collection on 2019, 10/9; the ganoderma lucidum strain is B1.4, and the preservation number of the strain is as follows: CCTCC NO: m2019790, deposited at 9.10.2019 in the center of China center for type culture Collection; the preparation method of the extract comprises the following steps:
(1) preparing a culture medium:
preparing a PDA culture medium, namely cleaning and peeling 200g of potatoes, cutting the potatoes into small pieces, putting the small pieces into a pot, adding 1000mL of water, boiling for 20-30 minutes, filtering, adding 20g of agar into filtrate, heating and stirring, adding 20g of glucose after the agar is dissolved, stirring uniformly, cooling, keeping the pH natural, and sterilizing at 121 ℃ for 30min for later use;
preparing a seed liquid culture medium: taking 20g of potatoes, cleaning, peeling, cutting into small pieces, putting into a pot, adding 100mL of water, boiling for 10-15 minutes, filtering, adding 1.82g of cane sugar, 3.00g of peptone, 0.09g of monopotassium phosphate and 3.00g of rhizoma gastrodiae powder into filtrate, heating and stirring uniformly, cooling, keeping the pH natural, and sterilizing at 121 ℃ for 30min for later use;
preparation of initial liquid culture medium: taking 20g of potatoes, cleaning, peeling, cutting into small pieces, putting into a pot, adding 100mL of water, boiling for 10-15 minutes, filtering, adding 2g of glucose and 3g of rhizoma gastrodiae into filtrate, heating, stirring uniformly, cooling, naturally adjusting pH, and sterilizing at 121 ℃ for 30min for later use;
(2) activating strains: respectively transferring Ganoderma strain B1.4 and Cordyceps militaris strain B1528 to PDA plate, and culturing at 25 deg.C in dark for 7 days;
(3) preparing a seed solution: under the aseptic condition, punching an activated ganoderma lucidum strain B1.4 and a cordyceps militaris strain B1528 with the diameter of 5mm from the flat plate in the step (2) by using a puncher, inoculating the activated ganoderma lucidum strain B1.4 and the cordyceps militaris strain B1528 into a 500mL conical flask filled with 100mL of seed liquid culture medium, and performing shake culture for 5 days in a shaking incubator at the temperature of 25 ℃ at 200r/min in the dark condition for later use;
(4) liquid fermentation: inoculating activated ganoderma lucidum strain B1.4 and cordyceps militaris strain B1528 into an initial liquid culture medium, wherein the cordyceps militaris-ganoderma lucidum inoculation ratio is 1:2, the inoculation amount is 3mL/100mL, after shaking up gently, putting into a constant-temperature culture shaking table, and carrying out dark culture at 25 ℃ for 15d at 200rpm/min to obtain a gastrodia elata product obtained by co-culturing and fermenting ganoderma lucidum and cordyceps militaris;
(5) preparing an ethanol extract of a fermentation product: centrifuging the obtained fermentation product at 10000rpm/10min to obtain solid, drying the solid at 60 ℃, pulverizing into powder, adding 10 times of 95% ethanol, soaking at 25 ℃ for 24h, filtering with Buchner funnel, leaching the residue once again, mixing the extractive solutions, and concentrating into extract with extraction rate of 9.7%.
2. Use of ethanol extract of co-fermentation product with memory improving and improving effects as claimed in claim 1 in preparation of health products for improving and improving memory and medicines for treating diseases or symptoms of dysmnesia.
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