Method for promoting cordyceps militaris to produce bioactive exopolysaccharides by adding traditional Chinese medicine alcohol extracts
The use of traditional Chinese medicine alcohol extracts in the fermentation medium for Cordyceps militaris effectively addresses the mixing issue and enhances EPS production and antibacterial activity, optimizing the fermentation process.
Patent Information
- Application Number
- CN202410428440.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-10
- Publication Date
- 2024-12-13
AI Technical Summary
Existing methods for producing Cordyceps militaris polysaccharides are inefficient and fail to address the issue of polysaccharides mixing with traditional Chinese medicine polysaccharides, affecting mass transfer and EPS production yield.
Using a traditional Chinese medicine alcohol extract as a stimulator/inducer in the fermentation medium to promote EPS production and enhance antibacterial activity, avoiding the influence of water-soluble polysaccharides and optimizing the fermentation process.
The method significantly increases EPS yield and enhances antibacterial activity of Cordyceps militaris polysaccharides, achieving optimal production conditions and improved biological activity.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of industrial production, in particular to a method for promoting Cordyceps militaris to produce bioactive extracellular polysaccharides by adding traditional Chinese medicine alcohol extract. Background Art
[0002] The medicinal and edible fungus Cordyceps militaris, also known as northern cordyceps, is the model species of the genus Cordyceps, along with Cordyceps sinensis, and has been cultivated on a large scale. As one of China's most important traditional Chinese medicines, Cordyceps militaris is widely used due to its rich and diverse active ingredients. Cordyceps polysaccharides are one of the most popular active ingredients in Cordyceps militaris research, primarily composed of mannose, galactose, glucose, arabinose, and fucose. Cordyceps militaris polysaccharides can be divided into three categories based on their source: fruiting body polysaccharides, mycelial intracellular polysaccharides, and extracellular polysaccharides (EPS). EPS, a metabolite secreted by the mycelium during liquid culture, not only has a composition and structure similar to natural Cordyceps polysaccharides but also possesses excellent physiological activities such as antioxidant, immunomodulatory, hypoglycemic, hypolipidemic, and uric acid-lowering properties. Furthermore, EPS obtained through liquid fermentation has the advantages of a short production cycle, convenient extraction and processing, and low cost. At present, domestic and foreign research is mostly focused on the extraction and purification process, structural analysis, physical and chemical properties and functional activity research of Cordyceps militaris polysaccharides. While wild resources are sharply declining, the demand for Cordyceps militaris raw materials for processed foods, health products and pharmaceutical products developed around Cordyceps militaris is increasing, making it an important research direction to further improve the quality of artificially cultivated Cordyceps militaris and increase the output of its key active products.
[0003] Liquid fermentation is a method for quickly, efficiently, and economically obtaining EPS from medicinal fungi. Studies have shown that the composition of the culture medium is crucial to the production of EPS. In order to obtain the maximum amount of EPS, certain exogenous stimulants are added to the liquid fermentation system of medicinal fungi to promote both the growth of bacterial cells and the biosynthesis of secondary metabolites. Many studies have shown that the use of exogenous Chinese medicine as an additive can have a positive regulatory effect on improving the biosynthesis of active metabolites of fermentation strains. Adding appropriate amounts of Chinese herbal ingredients to the culture medium can significantly promote fungal growth or increase the production of active metabolites. The reason may be to induce the gene expression of specific enzymes in the metabolic pathway, or to regulate the activity of key enzymes, or to change cell permeability, thereby maximizing the accumulation of the target product in the fermentation broth and effectively regulating the production of metabolites.
[0004] Existing technical solutions:
[0005] Zhang et al. added an appropriate amount of coix seed powder as an inducer to the fermentation medium, which promoted the production of EPS in Cordyceps militaris, but had little effect on the structure and antioxidant activity of EPS. (Chao Zhang, Lei Jiang, Zi Wang. Effect of coix seed on exopolysaccharide production of Cordyceps militaris in liquid culture[J]. Arabian Journal of Chemistry, 2021, 14:102999.)
[0006] Hou Ruolin et al. added an appropriate amount of Vernonia amygdalina leaf powder to the fermentation medium of Cordyceps militaris. Under optimal conditions, the EPS yield increased by 205% compared with the blank control, with little effect on the EPS structure and antioxidant activity. (Hou Ruolin, Li Lin, Xiang Kaikai, et al. Fermentation conditions for Cordyceps militaris exopolysaccharides using Vernonia amygdalina leaves and their antioxidant activity [J]. Chinese Journal of Biotechnology, 2019, 35(4): 667-676.)
[0007] Hu Yongle et al. used Magnolia officinalis as the medicinal matrix to perform bidirectional liquid fermentation of Cordyceps militaris. 5g of Magnolia officinalis was added to the fermentation medium to make the EPS content in the fermentation product reach 3.11g / L. (Hu Yongle, Zhang Chuanhai, Lin Chongzhan, et al. Optimization of bidirectional liquid fermentation process of Cordyceps militaris and Magnolia officinalis by response surface methodology [J]. Journal of Mycology, 2020, 39(5): 944-954.).
[0008] The main disadvantages of the above technology are: 1. Directly adding traditional Chinese medicine or its aqueous extract may cause the fungal polysaccharides obtained by fermentation to be mixed with traditional Chinese medicine polysaccharides; 2. Directly adding too much traditional Chinese medicine powder will affect the mass transfer effect of fermentation, or the powder will produce a shearing effect on the mycelium during shake flask fermentation; 3. Directly adding EPS obtained by traditional Chinese medicine fermentation will increase the yield, but the enhancement effect of its biological activity is not obvious.
[0009] The main reasons for the above shortcomings include: 1. The polysaccharides in traditional Chinese medicine itself or its water extract are soluble in water. If there are residual traditional Chinese medicine polysaccharides at the end of fermentation, it will affect the determination of fungal polysaccharides obtained by fermentation; 2. The effective substances that can promote EPS production in a certain amount of traditional Chinese medicine are limited. Only by adding a large amount of traditional Chinese medicine powder can the effect of increasing EPS production be achieved. Too much traditional Chinese medicine powder will affect the mass transfer effect of nutrients, oxygen, etc., and even cause shear damage to the mycelium; 3. Directly adding EPS obtained by traditional Chinese medicine fermentation will result in less structural changes and cause less changes in biological activity.
[0010] Therefore, how to quickly and efficiently obtain Cordyceps militaris EPS has always been a research focus in this field. Summary of the Invention
[0011] The purpose of the present invention is to efficiently obtain EPS from Cordyceps militaris. The present invention adds an appropriate amount of a traditional Chinese medicine alcohol extract as a stimulator / inducer to a fermentation medium, thereby eliminating the influence of the traditional Chinese medicine itself or polysaccharides in its aqueous extract on the EPS obtained by fermentation of Cordyceps militaris. The present invention aims to regulate the secondary metabolic pathway, promote the synthesis and secretion of EPS, increase EPS production and enhance the antibacterial activity of EPS.
[0012] In order to solve the above technical problems, the present invention adopts the following technical solutions:
[0013] The invention discloses an application of a traditional Chinese medicine alcohol extract for promoting the production of extracellular polysaccharides by Cordyceps militaris.
[0014] Among them, the alcohol extract of traditional Chinese medicine is a substance or a class of substances with clear chemical composition obtained by extracting, separating, and purifying traditional Chinese medicine using alcohol organic solvents. This or such extracts usually have high purity and clear chemical composition, which makes them more operable in terms of dosage and efficacy. In contrast, the ingredients of traditional Chinese medicine are relatively complex, the required dosage is large, and the efficacy is often difficult to accurately control. Moreover, polysaccharides are soluble in water and insoluble in alcohol. Using alcohol to extract the effective substances of traditional Chinese medicine to explore the production of extracellular polysaccharides in Cordyceps militaris greatly avoids the impact of traditional Chinese medicine polysaccharides on the study of Cordyceps militaris fungal polysaccharides. The study unexpectedly found that the active ingredients in traditional Chinese medicine that are soluble in alcohol can effectively promote the production of extracellular polysaccharides in Cordyceps militaris, improve the quality of the obtained extracellular polysaccharides, and improve the antibacterial activity.
[0015] Preferably, the Chinese medicine ethanol extract includes one or more of coix seed ethanol extract, magnolia bark ethanol extract, red wolfberry ethanol extract, black wolfberry ethanol extract, Acanthopanax senticosus ethanol extract, and Plantago asiatica ethanol extract.
[0016] Preferably, the Chinese medicine ethanol extract includes red wolfberry ethanol extract.
[0017] The EPS-L fermented by adding the red wolfberry alcohol extract to the Cordyceps militaris liquid fermentation of the present invention has a stronger antibacterial effect on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis than the blank group EPS-C, indicating that the addition of the red wolfberry alcohol extract has a significant enhancing effect on the antibacterial activity of the fermented EPS.
[0018] Preferably, the concentration of the Chinese medicine ethanol extract is 0.5-10 g / L.
[0019] Preferably, the concentration of the Chinese medicine ethanol extract is 2 g / L.
[0020] The present invention adds an appropriate amount of a Chinese herbal medicine alcohol extract as a stimulator / inducer to the fermentation medium, eliminating the influence of the Chinese herbal medicine itself or its aqueous extract polysaccharides on the EPS obtained from Cordyceps militaris fermentation, which may be mixed with Chinese herbal medicine polysaccharides. This regulates secondary metabolic pathways, promotes the synthesis and secretion of EPS, and increases EPS yield. The most significant EPS production-promoting effect was achieved by adding 2g / L of red wolfberry alcohol extract to Cordyceps militaris during 5 days of liquid fermentation at the beginning of fermentation.
[0021] Preferably, the preparation of the Chinese medicine ethanol extract comprises the following steps:
[0022] S1. Wash, dry and crush the Chinese medicine to obtain Chinese medicine powder;
[0023] S2, soaking the Chinese medicine powder in an organic solvent for extraction, and then separating the solid and liquid to obtain a first separated liquid and a separated residue;
[0024] S3, soaking the separation residue in an organic solvent for extraction, and then performing solid-liquid separation to obtain a second separation liquid;
[0025] S4. Combining the first separated liquid and the second separated liquid, removing the organic solvent, and re-dissolving with water to obtain the traditional Chinese medicine alcohol extract.
[0026] Preferably, the organic solvent includes anhydrous ethanol; in step S1, the Chinese medicine is washed and dried at 45°C; in step S2, the Chinese medicine powder and the organic solvent are soaked in a mass volume ratio of 1g:10mL, and the extraction time includes 24h; the solid-liquid separation method includes filtration; in step S3, the amount of organic solvent is the same as that in step S2; the solid-liquid separation method includes filtration; in step S4, the organic solvent is removed by rotary evaporation.
[0027] Preferably, the method comprises the following steps:
[0028] A. Activation of the strain: Inoculate the Cordyceps militaris strain into the activation medium on the activation plate for activation culture, and store at low temperature after each passage;
[0029] B. Preparing seed solution: placing the Cordyceps militaris strains on the activated plate in sterile water, breaking them up and diluting them for microscopic examination to obtain a spore suspension; mixing the spore suspension with a seed culture medium and performing seed culture to obtain a seed solution;
[0030] C. Preparing a fermentation system: sterilizing the Chinese medicine alcohol extract and then adding it to a fermentation medium to obtain a fermentation system;
[0031] D. Fermentation: mixing the seed liquid with the fermentation system, performing fermentation culture, and obtaining a fermentation filtrate.
[0032] Preferably, in step A, the Cordyceps militaris strains are stored in an environment of -80°C before use; the activation plate includes a PDA plate; the activation culture medium includes potatoes, glucose, and agar; the activation culture conditions include: after inoculating the Cordyceps militaris strains, culturing them at 25°C in the dark for 7 days, which is recorded as the first generation strains; after re-inoculating the first generation strains, culturing them at 25°C in the dark for 7 days, which is recorded as the second generation strains; after re-inoculating the second generation strains, culturing them at 25°C in the dark for 14 days, which is recorded as the third generation strains, which are used for the subsequent preparation of the seed liquid; in step B, the Cordyceps militaris strains on the activation plate are dispersed with glass beads; the number of spores in the spore suspension includes 1×10 6 / mL; the volume ratio of the spore suspension to the seed culture medium includes 1:9; the seed culture medium includes glucose, yeast extract, KH2PO4, and MgSO4·7H2O; the seed culture conditions include shaking culture at 25°C and 140 rpm in a shake flask for 2 days; in step C, the sterilization includes sterilization by filtration using a 0.22 μm filter membrane; the fermentation medium includes glucose, yeast extract, KH2PO4, and MgSO4·7H2O; in step D, the fermentation culture conditions include shaking culture at 25°C and 140 rpm in a shake flask for 5 days.
[0033] Preferably, the activation culture medium comprises 200 g / L potato, 20 g / L glucose, and 20 g / L agar; the seed culture medium comprises 20 g / L glucose, 1 g / L yeast extract, 2 g / L KH2PO4, and 1 g / LMgSO4·7H2O; the fermentation culture medium comprises 40 g / L glucose, 2 g / L yeast extract, 2 g / L KH2PO4, and 1 g / L MgSO4·7H2O; and the activation culture medium, the seed culture medium, and the fermentation culture medium are all sterilized at 121°C for 20 minutes before use.
[0034] Compared with the prior art, the present invention has the following beneficial effects:
[0035] 1. The present invention adds an appropriate amount of Chinese herbal medicine alcohol extract as a stimulator / inducer to the fermentation medium to eliminate the influence of the polysaccharides in the Chinese herbal medicine itself or its aqueous extract on the possible mixing of Chinese herbal medicine polysaccharides in the fermented fungal polysaccharides, aiming to regulate the secondary metabolic pathway, promote the synthesis and secretion of EPS, increase EPS production, and efficiently obtain Cordyceps militaris EPS.
[0036] 2. The present invention optimizes the conditions for adding the alcohol extract of traditional Chinese medicine (optimally, red wolfberry) to the liquid fermentation of Cordyceps militaris. Adding 2g / L red wolfberry alcohol extract at the beginning of fermentation has the most significant effect on promoting EPS production in Cordyceps militaris for 5 days of liquid fermentation.
[0037] 3. The EPS-L fermented by adding red wolfberry ethanol extract to the liquid fermentation of Cordyceps militaris in the present invention has a stronger antibacterial effect on Escherichia coli, Staphylococcus aureus and Bacillus subtilis than the blank group EPS-C. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 This is a diagram showing the effect of the concentration of coix seed ethanol extract on the biomass and EPS yield of Cordyceps militaris.
[0039] Figure 2 This is a diagram showing the effect of the concentration of Magnolia officinalis ethanol extract on the biomass and EPS yield of Cordyceps militaris.
[0040] Figure 3 This is a diagram showing the effect of the concentration of red wolfberry alcohol extract on the biomass and EPS yield of Cordyceps militaris.
[0041] Figure 4 This is a diagram showing the effect of the concentration of black wolfberry alcohol extract on the biomass and EPS yield of Cordyceps militaris.
[0042] Figure 5 This is a diagram showing the effect of the concentration of Acanthopanax senticosus ethanol extract on the biomass and EPS yield of Cordyceps militaris.
[0043] Figure 6 This is a diagram showing the effect of the concentration of Plantain ethanol extract on the biomass and EPS yield of Cordyceps militaris.
[0044] Figure 7 This is a diagram showing the effects of adding red wolfberry, black wolfberry and their combined alcohol extracts on the biomass and EPS yield of Cordyceps militaris.
[0045] Figure 8 This is a diagram showing the effect of the addition time of red wolfberry alcohol extract on the biomass and EPS yield of Cordyceps militaris.
[0046] Figure 9 This is a diagram showing the effect of adding red wolfberry alcohol extract on the biomass and EPS production during the Cordyceps militaris fermentation process.
[0047] Figure 10 This is a graph showing the inhibitory effect of different concentrations of EPS on Escherichia coli.
[0048] Figure 11 This is a graph showing the inhibitory effect of different concentrations of EPS on Staphylococcus aureus.
[0049] Figure 12 This is a graph showing the inhibitory effect of different concentrations of EPS on Bacillus subtilis. DETAILED DESCRIPTION
[0050] To make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be described in further detail below with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto.
[0051] Example 1:
[0052] 1 Materials and Methods
[0053] 1.1 Materials
[0054] Cordyceps militaris (C. militaris) was collected in our laboratory. Coix seed, Magnolia officinalis, red wolfberry, black wolfberry, Acanthopanax senticosus, and Plantago were purchased from Beijing Tong Ren Tang.
[0055] Culture medium: ①PDA: 200 g / L potato, 20 g / L glucose, 20 g / L agar, natural pH; ②Seed medium: 20 g / L glucose, 1 g / L yeast extract, 2 g / L KH2PO4, 1 g / L MgSO4·7H2O, natural pH; ③Fermentation medium: 40 g / L glucose, 2 g / L yeast extract, 2 g / L KH2PO4, 1 g / LMgSO4·7H2O, natural pH; All the above culture media were sterilized at 121°C for 20 min and set aside.
[0056] 1.2 Methods
[0057] 1.2.1 Preparation of Cordyceps militaris seed solution
[0058] (1) Activation of strains: Inoculate the Cordyceps militaris strain stored at -80°C onto a PDA plate, incubate at 25°C in the dark for 7 days, and record it as the first generation; inoculate the first generation onto a new PDA plate, incubate at 25°C in the dark for 7 days, and record it as the second generation; inoculate the second generation onto a new PDA plate, incubate at 25°C in the dark for 14 days, and record it as the third generation for subsequent seed solution preparation.
[0059] (2) Preparation of seed solution: Cut the Cordyceps militaris spawn on the PDA plate into pieces and place them in 50 mL of sterile water. Use glass beads to break them up and then dilute them for microscopic examination so that the spore count reaches 1 × 10 6 / mL to obtain a spore suspension; 15mL of the spore suspension was placed in 135mL of seed culture medium, and cultured at 25°C and 140rpm in a shake flask for 2 days to obtain a seed solution.
[0060] 1.2.2 Preparation of Chinese herbal medicine ethanol extracts
[0061] Clean Chinese herbal medicine was dried at 45°C and then pulverized. 10 g of the powder was added to 100 mL of anhydrous ethanol and extracted for 24 hours. The first filtrate was collected by filtration. The residue was then added to 100 mL of anhydrous ethanol and extracted for 24 hours. The second filtrate was collected by filtration. The combined filtrates were then rotary evaporated to remove the ethanol. The mixture was reconstituted with deionized water and the volume was adjusted to 100 mL. This yielded 10 mL of ethanol extract (100 g / L ethanol extract) per 1 g of Chinese herbal medicine. After sterilization through a 0.22 μm filter, the ethanol extracts were added in varying amounts as exogenous additives to the Cordyceps militaris liquid fermentation medium described in 1.2.3.
[0062] 1.2.3 Optimization of the addition conditions of Chinese medicine alcohol extracts
[0063] The following experiment was set up with the fermentation medium as the blank group and the fermentation medium supplemented with Chinese medicine alcohol extract as the supplemented group:
[0064] (1) Add different types and concentrations of Chinese herbal alcohol extracts
[0065] At the beginning of fermentation, alcohol extracts of various Chinese medicinal herbs such as coix seed, magnolia bark, red wolfberry, black wolfberry, Acanthopanax and plantain were added to the fermentation medium with final concentrations of 0, 0.5, 1, 2, 6, and 10 g / L, respectively, to obtain fermentation systems. 15 mL of Cordyceps militaris seed liquid was taken and placed in 90 mL of fermentation medium. After shaking and culturing at 25°C and 140 rpm in a shake flask for 5 days, the bacteria were collected to determine the dry weight (biomass), and the fermentation filtrate was obtained to determine the EPS yield.
[0066] (2) Adding red wolfberry, black wolfberry and their compound alcohol extracts
[0067] At the beginning of fermentation, red wolfberry, black wolfberry and a traditional Chinese medicine alcohol extract of the two were added to the fermentation medium at a final concentration of 2 g / L to obtain fermentation systems. 15 mL of Cordyceps militaris seed liquid was taken and placed in 90 mL of fermentation medium. After shaking and culturing at 25°C and 140 rpm in a shake flask for 5 days, the bacteria were collected to determine the biomass, and the fermentation filtrate was obtained to determine the EPS yield.
[0068] (3) Adding red wolfberry alcohol extract at different addition times
[0069] 15 mL of Cordyceps militaris seed liquid was taken and placed in 90 mL of fermentation medium. Red wolfberry ethanol extract with a final concentration of 2 g / L was added on the 0th, 1st, 2nd, 3rd, and 4th day after the start of fermentation to obtain fermentation systems. After shaking culture at 25°C and 140 rpm in a shake flask for 5 days, the bacteria were collected to determine the biomass, and the fermentation filtrate was obtained to determine the EPS yield.
[0070] (4) Fermentation time of adding red wolfberry alcohol extract
[0071] At the beginning of fermentation, a red wolfberry alcohol extract at a final concentration of 2 g / L was added to the fermentation medium to obtain a fermentation system. 15 mL of Cordyceps militaris seed liquid was aspirated and placed in 90 mL of fermentation medium. The system was cultured at 25°C and 140 rpm in a shake flask. The cells were collected after 1, 2, 3, 4, 5, 6, and 7 days of culture to determine the biomass, and the fermentation filtrate was obtained to determine the EPS yield.
[0072] 1.2.4 Determination of EPS yield in biomass and fermentation filtrate
[0073] (1) Biomass determination
[0074] After the fermentation, the cells were collected, washed with deionized water three times, dried at 45 °C to a constant weight, and the dry weight of the cells was weighed using an analytical balance and recorded as biomass.
[0075] (2) Determination of EPS yield in fermentation filtrate
[0076] After fermentation, the fermentation broth was collected and centrifuged at 6000 rpm for 10 min. The supernatant was added with three times the volume of anhydrous ethanol and precipitated overnight. The precipitate was collected by centrifugation at 6000 rpm for 5 min, dried at 45°C to constant weight, and the dry weight of the bacteria was weighed on an analytical balance and recorded as crude extracellular polysaccharide (EPS).
[0077] 1.2.5 Effect of adding red wolfberry alcohol extract on the antibacterial activity of EPS after fermentation
[0078] The antibacterial activity of EPS of Cordyceps militaris was investigated using Escherichia coli, Staphylococcus aureus and Bacillus subtilis as indicator bacteria.
[0079] Preparation of indicator bacterial suspension: Escherichia coli, Staphylococcus aureus, and Bacillus subtilis stored in glycerol tubes were inoculated into nutrient broth for recovery and cultured at 37°C for 24 hours. The bacterial suspension was subcultured and the bacterial suspension cultured to the logarithmic phase was diluted to 1×10 7 CFU / mL is the indicator bacteria suspension, which is ready for use.
[0080] Preparation of EPS solution: Weigh 100 mg of polysaccharide (the polysaccharide in the blank group is marked as EPS-C, and the polysaccharide in the group with red wolfberry alcohol extract is marked as EPS-L), dissolve it in deionized water and make it up to 10 mL, that is, 10 mg / mL EPS solution. Sterilize it at 121℃ for 20 min and set aside.
[0081] Turbidimetric analysis of the antibacterial activity of EPS: 100 μL of indicator bacterial suspension was respectively aspirated into a 96-well plate, and 100 μL of 10 mg / mL polysaccharide solution was added. The final concentrations of the polysaccharide solution were adjusted to 10, 5, 2.5, 1.25, 0.625, and 0.3125 mg / mL using the half-dilution method. The plates were incubated at 37°C for 12 h, and the absorbance was measured at 600 nm to calculate the inhibition rate and half-inhibitory concentration (IC). 50 .
[0082] Wherein: A is the blank control group: 200 μL logarithmic phase indicator bacteria solution; B is the blank background group: 200 μL nutrient broth; C is the experimental group: 100 μL logarithmic phase indicator bacteria solution + 100 μL EPS solution; D is the experimental background group: 100 μL nutrient broth + 100 μL EPS solution.
[0083] 1.2.6 Statistical analysis
[0084] Data were analyzed using Origin 2022. All data are presented as mean ± standard deviation. Significant differences between groups were analyzed using one-way analysis of variance and t-test. Statistically significant effects were defined as * indicating P < 0.05 and ** indicating P < 0.01.
[0085] Effect Example 1
[0086] Add different types and concentrations of Chinese herbal alcohol extracts
[0087] The effects of different types and concentrations of Chinese herbal medicine alcohol extracts on Cordyceps militaris liquid fermentation were investigated. Figures 1 to 6 As shown in the figure, the blank group was a group with the same conditions except that no Chinese herbal alcohol extract was added. The results showed that compared with the blank group (EPS yield of 1.70g / L), the addition of Chinese herbal alcohol extracts such as coix seed, magnolia bark, red wolfberry, black wolfberry, Acanthopanax senticosus and plantain at the beginning of fermentation had an improved effect on EPS yield within a certain concentration range. The optimal addition concentrations were 1, 2, 2, 2, 1, and 2g / L, corresponding to maximum EPS yields of 2.14, 1.73, 2.41, 2.35, 2.13, and 1.99g / L, respectively, and the yield increased by 25.88%, 1.76%, 41.76%, 38.24%, 25.29%, and 17.06%, respectively. Among them, the addition of 2g / L red wolfberry alcohol extract had the most significant effect, which may be because certain components in the red wolfberry alcohol extract are involved in the metabolic pathways of Cordyceps militaris polysaccharide synthesis or secretion, affecting EPS biosynthesis, secretion, and accumulation.
[0088] Effect Example 2
[0089] Add red wolfberry, black wolfberry and their compound alcohol extracts
[0090] The effects of adding red wolfberry, black wolfberry and their combined alcohol extracts on the liquid fermentation of Cordyceps militaris were investigated. Figure 7 As shown, the blank group is a group with the same conditions except that no Chinese medicine alcohol extract is added. The results show that compared to the blank group (EPS output 1.70g / L), adding a final concentration of 2g / L red wolfberry, 2g / L black wolfberry, 2g / L red wolfberry and 2g / L black wolfberry compound Chinese medicine alcohol extract at the beginning of fermentation has an improving effect on EPS output, with corresponding EPS outputs of 2.54, 2.21, and 2.33g / L, respectively, and the yields increased by 49.41%, 30.00%, and 37.06%, respectively. Compared with the blank group and the addition of a single black wolfberry alcohol extract, it was found that the compound alcohol extract can increase EPS output, but the promoting effect is not as good as that of adding a single red wolfberry alcohol extract, that is, the compound has no further promoting effect on EPS output. The subsequent experiment selects a single addition of 2g / L red wolfberry alcohol extract as the experimental parameter for studying the promotion of EPS output.
[0091] Effect Example 3
[0092] Adding red wolfberry ethanol extract at different adding times
[0093] The effect of adding time of red wolfberry alcohol extract on liquid fermentation of Cordyceps militaris was investigated. Figure 8 The blank group was the group with the same conditions except that no red wolfberry ethanol extract was added. The results showed that compared to the blank group (EPS yield of 1.70 g / L), adding red wolfberry ethanol extract at a final concentration of 2 g / L on days 0, 1, 2, 3, and 4 after the start of fermentation increased EPS production, with corresponding EPS yields of 2.42, 1.77, 1.93, 1.98, and 1.96 g / L, respectively, representing yield increases of 42.35%, 4.12%, 13.53%, 16.47%, and 15.29%, respectively. Adding red wolfberry ethanol extract on days 0 to 4 increased EPS production, with the most significant effect observed on day 0 (i.e., the beginning of fermentation). This may be due to the fact that certain components in the red wolfberry ethanol extract significantly promote the activation of key enzymes in the early stages of EPS synthesis.
[0094] Effect Example 4
[0095] Fermentation time after adding red wolfberry alcohol extract
[0096] After determining the addition amount (2g / L) and addition time (at the beginning of fermentation) of red wolfberry alcohol extract, the effect of alcohol extract on the fermentation time of Cordyceps militaris was investigated. Figure 9As shown, the blank group is a group with the same conditions except that no red wolfberry alcohol extract was added. The results showed that adding red wolfberry alcohol extract with a final concentration of 2g / L at the beginning of fermentation and culturing for 1, 2, 3, 4, 5, 6, and 7 days had an effect on improving EPS production. The corresponding EPS production of the blank group was 0.68, 1.32, 1.36, 1.56, 1.70, 1.68, and 1.66g / L, respectively. The corresponding EPS production of the addition group was 0.67, 1.81, 1.96, 2.44, 2.44, 2.15, and 1.83g / L, respectively. The yield increased by -1.47%, 37.12%, 44.12%, 56.41%, 43.53%, 27.98%, and 10.24%, respectively. As the fermentation progressed, the bacteria grew, EPS gradually accumulated and reached peak values on the 4th and 5th days. The red wolfberry group exceeded the highest level of the blank group on the 5th day on the 2nd day, indicating that the addition of red wolfberry alcohol extract can change the growth and metabolic rhythm of Cordyceps militaris.
[0097] Taking into account the biomass and EPS yield, adding 2 g / L red wolfberry ethanol extract to the fermentation medium at the beginning of fermentation and conducting liquid fermentation for 5 days was selected as the most suitable condition for Cordyceps militaris to produce EPS.
[0098] Effect Example 5
[0099] Turbidimetric analysis of the effect of adding red wolfberry alcohol extract on the antibacterial activity of EPS after fermentation
[0100] Turbidimetric determination of the inhibition rate and half inhibitory concentration IC of EPS-C and EPS-L against Escherichia coli, Staphylococcus aureus and Bacillus subtilis 50 The concentration of the polysaccharide solution was controlled between 0.3125 and 10 mg / mL using the half-dilution method. The polysaccharide concentration was plotted as the horizontal axis and the inhibition rate of the polysaccharide on the indicator bacteria as the vertical axis. The half-inhibitory concentration (IC) was calculated. 50 value.
[0101] Effect Example 6
[0102] Inhibition of EPS on Escherichia coli
[0103] The inhibition rate of EPS at different concentrations on Escherichia coli is as follows Figure 10 As shown in the figure, the inhibitory effect of EPS-L on E. coli is better than that of EPS-C, and the inhibition rate of EPS-L on E. coli can reach more than 85% when the concentration is 10 mg / mL. The IC 50 (0.60 mg / mL) was lower than that of EPS-C (1.02 mg / mL).
[0104] Effect Example 7
[0105] Inhibition of EPS on Staphylococcus aureus
[0106] The inhibition rate of different concentrations of EPS on Staphylococcus aureus is as follows Figure 11 As shown in the figure, the inhibitory effect of EPS-L on Staphylococcus aureus is better than that of EPS-C, and the inhibition rate of EPS-L on Staphylococcus aureus can reach more than 80% at 10 mg / mL. The IC 50 (0.42 mg / mL) was lower than that of EPS-C (0.80 mg / mL).
[0107] Effect Example 8
[0108] Inhibition of EPS on Bacillus subtilis
[0109] The inhibition rate of different concentrations of EPS on Bacillus subtilis is as follows Figure 12 As shown in the figure, the inhibitory effect of EPS-L on Bacillus subtilis is better than that of EPS-C, and the inhibition rate of EPS-L on Bacillus subtilis can reach more than 80% at 10 mg / mL. The IC 50 (0.70 mg / mL) was lower than that of EPS-C (1.55 mg / mL).
[0110] It can be seen that EPS-L fermented with red wolfberry alcohol extract has a stronger antibacterial effect on Escherichia coli, Staphylococcus aureus and Bacillus subtilis than EPS-C in the blank group.
[0111] Effect Example 9
[0112] in conclusion
[0113] The study optimized the addition conditions of an alcohol extract of a traditional Chinese medicine (optimally, red wolfberry) during the liquid fermentation process of Cordyceps militaris. Adding 2g / L of red wolfberry alcohol extract at the beginning of fermentation was most effective in promoting EPS production in Cordyceps militaris over a five-day liquid fermentation. The addition also significantly impacted the antibacterial activity of the Cordyceps militaris EPS against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. Subsequent research will screen and validate effective EPS-promoting substances in the alcohol extract and elucidate their mechanisms of production, aiming to provide new strategies for regulating EPS metabolism. Furthermore, the study will explore the relationship between EPS structure and biological activity, providing a solid theoretical foundation for the further development of Cordyceps militaris EPS products.
[0114] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.
Claims
1. An application of a Chinese medicine alcohol extract, characterized in that: Used to promote the production of extracellular polysaccharides by Cordyceps militaris.
2. The use according to claim 1, characterized in that: The Chinese medicine alcohol extract includes one or more of coix seed alcohol extract, magnolia bark alcohol extract, red wolfberry alcohol extract, black wolfberry alcohol extract, Acanthopanax senticosus alcohol extract, and Plantago asiatica alcohol extract.
3. The use according to claim 1, characterized in that: The traditional Chinese medicine alcohol extract comprises red wolfberry alcohol extract.
4. The use according to claim 1, characterized in that: The concentration of the Chinese medicine alcohol extract is 0.5-10 g / L.
5. The use according to claim 1, characterized in that: The concentration of the Chinese medicine alcohol extract includes 2g / L.
6. The use according to claim 1, characterized in that: The preparation of the Chinese medicine alcohol extract comprises the following steps: S1, washing, drying and crushing the Chinese medicine to obtain Chinese medicine powder; S2, soaking the Chinese medicine powder in an organic solvent for extraction, and then separating the solid and liquid to obtain a first separation liquid and separation residue; S3, soaking the separation residue in an organic solvent for extraction, and then separating the solid and liquid to obtain a second separation liquid; S4. Combining the first separated liquid and the second separated liquid, removing the organic solvent, and re-dissolving with water to obtain the traditional Chinese medicine alcohol extract.
7. The use according to claim 6, characterized in that The organic solvent includes anhydrous ethanol; in step S1, the Chinese medicine is washed and then dried at 45°C; in step S2, the Chinese medicine powder and the organic solvent are soaked in a mass volume ratio of 1g:10mL, and the extraction time includes 24h; the solid-liquid separation method includes filtration; in step S3, the amount of the organic solvent is the same as that in step S2; the solid-liquid separation method includes filtration; in step S4, the organic solvent is removed by rotary evaporation.
8. The use according to claim 1, characterized in that: The steps include: A. Activated strains: Inoculate the Cordyceps militaris strains into the activation medium on the activation plate for activation culture, and store at low temperature after each passage; B. preparing seed solution: placing the Cordyceps militaris strains on the activation plate in sterile water, breaking them up and diluting them for microscopic examination to obtain a spore suspension; mixing the spore suspension with a seed culture medium, performing seed culture to obtain a seed solution; C. Preparing a fermentation system: sterilizing the Chinese medicine alcohol extract, and then adding it to a fermentation medium to obtain a fermentation system; D. Fermentation: mixing the seed liquid with the fermentation system, performing fermentation culture, and obtaining a fermentation filtrate.
9. The use according to claim 8, characterized in that In step A, the Cordyceps militaris strains are stored in an environment of -80°C before use; the activation plate includes a PDA plate; the activation culture medium includes potatoes, glucose, and agar; the conditions of the activation culture include: the Cordyceps militaris strains are inoculated and cultured at 25°C in the dark for 7 days, which is recorded as the first generation strains; the first generation strains are re-inoculated and cultured at 25°C in the dark for 7 days, which is recorded as the second generation strains; the second generation strains are re-inoculated and cultured at 25°C in the dark for 14 days, which is recorded as the third generation strains, which are used for the subsequent preparation of the seed liquid; each passage is stored at 4°C; in step B, the Cordyceps militaris strains on the activation plate are dispersed with glass beads; the number of spores in the spore suspension includes 1×10 6 / mL; the volume ratio of the spore suspension to the seed culture medium includes 1:9; the seed culture medium includes glucose, yeast extract, KH2PO4, and MgSO4·7H2O; the seed culture conditions include 25°C and shaking 140rpm for 2 days; in step C, the sterilization includes filtering and sterilizing with a 0.22μm filter membrane; the fermentation medium includes glucose, yeast extract, KH2PO4, and MgSO4·7H2O; in step D, the fermentation culture conditions include 25°C and shaking 140rpm for 5 days.
10. The use according to claim 8, characterized in that The activation culture medium comprises 200 g / L potato, 20 g / L glucose and 20 g / L agar; the seed culture medium comprises 20 g / L glucose, 1 g / L yeast extract, 2 g / L KH2PO4 and 1 g / L MgSO4·7H2O; the fermentation culture medium comprises 40 g / L glucose, 2 g / L yeast extract, 2 g / L KH2PO4 and 1 g / L MgSO4·7H2O; the activation culture medium, the seed culture medium and the fermentation culture medium are all sterilized at 121°C for 20 minutes before use.