Cordyceps cicadae polysaccharide with blood pressure lowering efficacy and microbial fermentation preparation method thereof

Abstract

The application discloses a cordyceps cicadae and cordyceps sinensis polysaccharide with blood pressure lowering efficacy and a microbial fermentation preparation method thereof, and belongs to the field of fungal polysaccharide extract preparation and application. The cordyceps cicadae and cordyceps sinensis polysaccharide is prepared by a microbial fermentation method, a cordyceps cicadae and cordyceps sinensis spore bundle is used as a fermentation matrix, and bacillus lactis is used as a fermentation strain; after liquid fermentation extraction, the cordyceps cicadae and cordyceps sinensis polysaccharide CCP-Lp is obtained, the monosaccharide composition and molar ratio of which are as follows: mannose: glucose: galactose = 1: 0.36: 0.62, and the relative molecular mass is 1.32*10 5 Da. Efficacy experiments show that the CCP-Lp can significantly inhibit abnormal increase of systolic pressure and diastolic pressure of spontaneous hypertension rats, and regulate levels of key factors of a renin-angiotensin-aldosterone system; the antihypertensive effect of the CCP-Lp is comparable to that of a positive drug, captopril. The cordyceps cicadae and cordyceps sinensis polysaccharide CCP-Lp has clear composition and significant efficacy, and has a broad development and application prospect in regulating blood pressure.

Description

Technical Field

[0001] This invention belongs to the field of preparation and application of fungal polysaccharide extracts, specifically relating to a Cordyceps militaris polysaccharide with antihypertensive effects and its microbial fermentation preparation method. Background Technology

[0002] Cordyceps sinensis (Cicada Flower Cordyceps) Isaria cicadae Miq Cordyceps militaris, belonging to the Ascomycota phylum and Cordyceps family, is also known as Cordyceps cicadae and is a valuable medicinal and edible fungus. Modern medical research shows that Cordyceps militaris contains various bioactive components such as polysaccharides, nucleosides, and sterols. Among them, polysaccharides, as the main active ingredient, have various pharmacological effects, including immunomodulation, antioxidation, and kidney protection. The applicant's experiments have found that Cordyceps militaris polysaccharides have a certain blood pressure-lowering effect, showing good development potential. However, the current traditional hot water extraction method has problems such as difficulty in efficiently extracting polysaccharides and insufficient bioactivity of the obtained polysaccharides, which restricts the development and utilization of high-value-added products of Cordyceps militaris.

[0003] In recent years, microbial fermentation technology has provided a new technological pathway for the efficient utilization of natural products. This technology, based on the abundant enzyme systems produced during microbial growth and metabolism, can efficiently degrade cell walls, significantly increasing polysaccharide yield. Furthermore, it can transform existing polysaccharides into novel fermented polysaccharides with higher activity through biotransformation. In addition, some microorganisms synthesize extracellular polysaccharides during fermentation, which may have a synergistic effect with matrix polysaccharides, thereby comprehensively enhancing the overall bioavailability of fermented polysaccharides. Currently, there are no reports on the application of microbial fermentation technology for the preparation of Cordyceps militaris polysaccharides.

[0004] Based on this, the applicant selected microorganisms (Lactobacillus plantarum, Lactobacillus rhamnosus, Saccharomyces cerevisiae, and Ganoderma lucidum) with both high enzyme production capacity and food safety as fermentation strains. The applicant treated Cordyceps militaris spore bundle powder through liquid fermentation and evaluated the activity of the resulting Cordyceps militaris polysaccharides. The results showed that the Cordyceps militaris polysaccharides prepared by fermentation with Lactobacillus plantarum showed outstanding performance in lowering blood pressure in spontaneously hypertensive rats, with effects comparable to the positive control drug captopril. Currently, patent (CN113396980A) discloses a method of adding Cordyceps militaris fruiting body ultrafine powder to skim milk, claiming that it has a blood pressure-lowering effect due to the presence of N6-(2-hydroxyethyl) adenosine. However, no in vitro or in vivo efficacy experiments have verified its blood pressure-lowering effect. This is fundamentally different from the present invention's method of preparing Cordyceps militaris active polysaccharide CCP-Lp for lowering blood pressure using Lactobacillus plantarum fermentation. Summary of the Invention

[0005] This invention provides a polysaccharide from Cordyceps militaris with antihypertensive effects, its preparation method by microbial fermentation, and its application. The polysaccharide is obtained by fermenting Cordyceps militaris sporangiophores with Lactobacillus plantarum and can significantly inhibit the abnormal increase in systolic and diastolic blood pressure in spontaneously hypertensive rats.

[0006] The Cordyceps militaris polysaccharide provided by this invention, abbreviated as CCP-Lp, has a relative molecular mass of 1.32 × 10⁻⁶. 5 Da, its monosaccharide composition and molar ratio are mannose: glucose: galactose = 1: 0.36: 0.62.

[0007] The Cordyceps militaris polysaccharide of this invention is prepared by microbial fermentation, including the following steps:

[0008] Step 1: Crush the Cordyceps militaris spore bundles, pass them through an 80-mesh sieve, add pure water at a mass-to-volume ratio of 1:30-40 (g / mL), stir well, sterilize at 121 ℃ for 30 min, cool to room temperature, and obtain the fermentation culture medium;

[0009] Step 2: Inoculate Lactobacillus plantarum (Lp) into liquid culture medium at an inoculation rate of 1% (v / v) and incubate at 36 ℃ for 48 h to prepare a highly active seed culture.

[0010] The liquid culture medium consisted of: 20 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 10 g / L beef extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diamine citrate, 5 g / L sodium acetate, 0.58 g / L magnesium sulfate heptahydrate, 0.17 g / L manganese sulfate monohydrate, and 1 mL Tween-80.

[0011] The plant lactobacillus ( Lactiplantibacillus plantarum The accession number of the sample is ACCC11095, and it was purchased from the China Agricultural Microbial Culture Collection Center.

[0012] Step 3: Inoculate the seed culture obtained in Step 2 into the fermentation medium obtained in Step 1 at 5% of the total volume of the fermentation medium, place it in an anaerobic incubator, and incubate it at 36 ℃ for 48-72 h to obtain the fermentation broth;

[0013] Step 4: Centrifuge the fermentation broth obtained in Step 3 at 3000 rpm, collect the supernatant, and concentrate it to 1 / 200-300 of the original volume by rotary evaporation at 60-70 ℃. Add 4 times the volume of anhydrous ethanol, stir and mix well, and let it stand at 4 ℃ for 12 h. Centrifuge and collect the precipitate.

[0014] Step 5: Dissolve the precipitate obtained in Step 4 in water to prepare a polysaccharide solution, which is then concentrated and freeze-dried to obtain Cordyceps militaris polysaccharide.

[0015] This invention relates to the application of Cordyceps militaris polysaccharide in the preparation of food, health products, or pharmaceutical formulations with blood pressure-lowering functions.

[0016] Efficacy experiments showed that CCP-Lp could significantly inhibit the abnormal increase in systolic and diastolic blood pressure in spontaneously hypertensive rats, regulate the levels of key factors in the renin-angiotensin-aldosterone system, and its antihypertensive effect was comparable to that of the positive control drug captopril. The Cordyceps militaris polysaccharide CCP-Lp of this invention has a well-defined composition and significant efficacy, and has broad prospects for development and application in regulating blood pressure. Attached Figure Description

[0017] Figure 1 The inhibitory effect of Cordyceps militaris polysaccharides prepared from four strains on in vitro angiotensin-converting enzyme (ACE) activity.

[0018] Figure 2 The effects of each experimental group in Example 3 on systolic blood pressure (SBP) (Figure A) and diastolic blood pressure (DBP) (Figure B) in spontaneously hypertensive rats.

[0019] Figure 3 This describes the effects of each experimental group in Example 3 on the levels of key active factors in the renin-angiotensin-aldosterone (RAAS) system of spontaneously hypertensive rats. Figure 3 A, 3B, 3C, and 3D represent the levels of ACE, Renin, Ang II, and ALD, respectively.

[0020] Figure 4 The molecular weight distribution (Figures A and B) and monosaccharide composition (Figures C and D) of the polysaccharides CCP-WE and CCP-Lp from Cordyceps militaris are shown. Detailed Implementation

[0021] The present invention will be described in detail below through specific implementation examples. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Any non-substantial substitutions or modifications made by those skilled in the art based on the technical solutions of the present invention should be included within the scope of protection of the present invention.

[0022] The Cordyceps sinensis used in this invention is artificially cultivated Isaria cicadae Miq. The process involves inoculating the fungal strain into a specific culture medium, culturing it, harvesting the sporangiophores, and then processing them through drying and other methods to produce Cordyceps sinensis sporangiophore raw materials. The Cordyceps sinensis sporangiophores used in this embodiment were provided by Anhui Cordyceps Source Biotechnology Co., Ltd.

[0023] Example 1: Preparation of Cordyceps militaris polysaccharide CCP-Lp

[0024] Step 1: Crush the spores and stalks of Cordyceps militaris, pass them through an 80-mesh sieve, add pure water at a mass-to-volume ratio of 1:30 (g / mL), stir well, sterilize at 121 ℃ for 30 min, and cool to room temperature to obtain the fermentation culture medium.

[0025] Step 2: Inoculate *Lactobacillus plantarum* at a 1% (v / v) inoculation rate into liquid culture medium and incubate at 36 °C for 48 h to prepare a highly active seed culture.

[0026] The liquid culture medium consisted of: 20 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 10 g / L beef extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diamine citrate, 5 g / L sodium acetate, 0.58 g / L magnesium sulfate heptahydrate, 0.17 g / L manganese sulfate monohydrate, and 1 mL Tween-80.

[0027] Step 3: Inoculate the seed culture obtained in Step 2 into the fermentation medium obtained in Step 1 at 5% of the total volume of the fermentation medium, place it in an anaerobic incubator, and incubate it at 36 ℃ for 60 h to obtain the fermentation broth.

[0028] Step 4: Centrifuge the fermentation broth obtained in Step 3 at 3000 rpm, collect the supernatant, concentrate it to 1 / 250 of the original volume, add 4 times the volume of anhydrous ethanol, stir and mix well, let it stand at 4 ℃ for 12 h, and centrifuge to collect the precipitate.

[0029] Step 5: Dissolve the precipitate obtained in Step 4 in water to prepare a polysaccharide solution, which is then concentrated and freeze-dried to obtain Cordyceps militaris polysaccharide CCP-Lp.

[0030] Comparative Example 1: Preparation of Cordyceps militaris polysaccharide CCP-WE

[0031] The preparation process of this embodiment is the same as that of Example 1. The difference between Example 1 and Example 2 is that no bacterial strains are added to the fermentation medium, and the remaining steps are the same as those of Example 1.

[0032] Comparative Example 2: Preparation of Cordyceps militaris polysaccharide CCP-Lr

[0033] The preparation process of this embodiment is the same as that of Example 1, except that Lactobacillus rhamnosus (Lr, commercially available) was inoculated into the fermentation medium, and the rest of the steps were the same as those of Example 1.

[0034] Comparative Example 3: Preparation of Cordyceps militaris polysaccharide CCP-Sc

[0035] The preparation process of this embodiment is the same as that of Example 1. The difference between Example 1 and Example 2 is that the fermentation medium is inoculated with Saccharomyces cerevisiae (Sc, commercially available). All other steps are the same as those of Example 1.

[0036] Comparative Example 4: Preparation of Cordyceps militaris polysaccharide CCP-Gl

[0037] The preparation process of this example is the same as that of Example 1, except that Ganoderma lucidum (Gl, commercially available) is inoculated into the fermentation medium, and the remaining steps are the same as those of Example 1.

[0038] Example 2: Determination of in vitro ACE inhibitory activity of polysaccharides in Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4

[0039] Mix 30 μL of the polysaccharide sample with 100 μL of 50 mU / mL ACE solution in 30 uL of borate buffer. After pre-incubating this mixed system at 37 °C for 10 min, add 200 μL of a substrate solution of HHL ( hippurylhistidylleucine ) with a concentration of 5 mM to initiate the enzymatic reaction, and continue to incubate at 37 °C for 30 min. After the reaction ends, add 250 uL of hydrochloric acid solution (1 mol / L) to terminate the reaction and extract the generated hippuric acid, and then centrifuge at 12000 rpm for 10 min. Transfer the upper organic phase, evaporate and concentrate to dryness by rotary evaporation, dissolve the obtained residue in distilled water, and measure its absorbance at a wavelength of 228 nm.

[0040] Example 3: Hypotensive effects of Cordyceps cicadae polysaccharides CCP-WE and CCP-Lp on spontaneously hypertensive rats

[0041] Spontaneously hypertensive rats (SHR, male, 8 - 10 weeks old) self-bred in the laboratory and WKY blank control rats (male, 8 weeks old) purchased from Hefei Qingyuan Biotechnology Co., Ltd. are selected. The production license number of the experimental animals of WKY rats is: SCXK(Zhe)2025 - 0004. After all animals are adaptively fed in a SPF-level environment for one week, they are randomly divided into 6 groups according to the experimental requirements. They are the control group, the model group, the positive control group Captopril, the CCP-WE group, the CCP-Lp group, and the Lactobacillus plantarum group (Lp). The CCP-WE group is given 300 mg / kg / d of Cordyceps cicadae polysaccharide CCP-WE; the CCP-Lp group is given 300 mg / kg / d of Cordyceps cicadae polysaccharide CCP-Lp, and the Lp group is given 3×10 9 CFU / kg / d of Lactobacillus plantarum, the Captopril group is given 25 mg / kg / d of Captopril, and the control group and the model group are given an equal volume of pure water. They are intragastrically administered once a day for 28 consecutive days. Among them, the control group uses WKY blank control rats, and the remaining groups use spontaneously hypertensive rats. A non-invasive blood pressure monitor for rats is used to monitor the SBP and DBP of rats in each group before and after administration. The rats are fasted for 12 hours (drinking water freely), anesthetized by intraperitoneal injection of a 10% (g / mL) chloral hydrate solution (3.5 mL / kg), and then blood is collected from the eyeballs for subsequent index detection.

[0042] Changes in SBP and DBP in rats of different groups were detected using a non-invasive tail artery pressure measurement method. The BP-2010 system (rat non-invasive blood pressure analysis system, BP-2010, Beijing Ruanlong Biotechnology Co., Ltd.) was used. Measurements were performed in a quiet environment. Rats underwent 3-5 acclimatization training sessions before the formal measurement. During measurement, the rats were placed in a special restraint device, and the tail-neck sensor was placed at the point of obvious tail artery pulsation. Blood pressure values ​​were measured after the rats calmed down, and the measurements were repeated 8-10 times. Outliers were removed, and the average value was taken as the final result. The activity of the RAAS system in SHR rats was assessed by detecting the levels of ACE, Renin, Ang II, and ALD in serum. After centrifugation at 3000 rpm for 10 min, the supernatant serum was aliquoted, and the concentrations of the above indicators in serum were determined strictly according to the instructions of the kit (rat ACE, Renin, Ang II, ALD detection kit, Shanghai Qifa Experimental Reagent Co., Ltd.).

[0043] Example 4: Molecular weight determination and monosaccharide composition analysis of Cordyceps militaris polysaccharides CCP-WE and CCP-Lp

[0044] (1) Molecular weight determination

[0045] Cordyceps militaris polysaccharides CCP-WE and CCP-Lp, as well as dextran standards of different molecular weights (T10, T50, T500, T1000, T2000), were prepared into 5 mg / mL solutions with double-distilled water and analyzed using an Agilent high-performance liquid chromatography-evaporative light scattering detector. Chromatographic separation was performed using a TSK-Gel G6000 PWXL column with ultrapure water as the mobile phase, N2 as the carrier gas, a gas flow rate of 2.5 L / min, and an injection volume of 10 μL. A standard curve was established using the logarithm of the molecular weight (Lg Mw) and retention time (Rt) of the dextran standards to determine the molecular weight range of Cordyceps militaris polysaccharides CCP-WE and CCP-Lp.

[0046] (2) Monosaccharide composition analysis

[0047] Monosaccharide composition analysis of CCP-WE and CCP-Lp samples after acid hydrolysis and pre-column PMP (1-phenyl-3-methyl-5-pyrazolone) derivatization was performed using an Agilent high-performance liquid chromatography-diode array detector. Eight mg of CCP-WE and CCP-Lp samples were accurately weighed and dissolved in 5 mL of 2 mol / L trifluoroacetic acid. The solutions were sealed in nitrogen-filled, stoppered, and hydrolyzed in an oil bath at 110 °C for 8 h. After the reaction, methanol was added repeatedly, and residual acid was removed by rotary evaporation until the pH of the system returned to neutral. The solutions were then reconstituted with 1 mL of ultrapure water to obtain the hydrolysate for later use. Take 1 mL each of 5 mg / mL monosaccharide standard solution and 1 mL each of CCP-WE and CCP-Lp hydrolysate, and add 1 mL of sodium hydroxide solution (0.3 mol / L) and 1 mL of PMP solution (0.5 mol / L) sequentially. After shaking and mixing, react in a water bath at 70 °C for 1 h to perform PMP derivatization. Adjust the pH to neutral using hydrochloric acid solution (1 mL, 0.3 mol / L), and then extract three times with chloroform to remove excess PMP reagent. Collect the aqueous phase, filter it through a 0.22 μm filter membrane, and perform HPLC-DAD analysis.

[0048] Figure 1 The inhibitory effect of Cordyceps militaris polysaccharides prepared from four different bacterial strains on in vitro ACE activity was investigated. The results showed that, compared with the CCP-WE group, each fermentation group exhibited varying degrees of ACE inhibition, with the CCP-Lp group showing the most significant inhibitory effect, achieving an ACE inhibition rate 1.5 times that of the CCP-WE group, demonstrating the best antihypertensive potential.

[0049] Figure 2 The effects of each experimental group in Example 3 on SBP and DBP in SHR rats were shown. Compared with the control group, the SBP and DBP of the model group rats were significantly increased. Compared with the model group, the CCP-Lp group significantly reduced the levels of SBP and DBP, and its blood pressure value was close to that of the positive drug group, and significantly better than that of the CCP-WE and Lp groups, indicating that CCP-Lp has a significant antihypertensive effect.

[0050] Figure 3 This section describes the effects of each experimental group in Example 3 on the levels of key active factors in the RAAS system of SHR rats. Compared with the control group, the serum levels of ACE, Renin, Ang II, and ALD in the model group rats were significantly increased. Compared with the model group, the CCP-Lp group significantly reduced the serum levels of ACE, Renin, Ang II, and ALD, and all levels were superior to those in the CCP-WE and Lp groups, consistent with the in vitro ACE activity results. This indicates that CCP-Lp may exert a significant regulatory effect on spontaneous hypertension by effectively inhibiting the expression of key active factors such as ACE, Renin, Ang II, and ALD in the RAAS system.

[0051] Figure 4 The molecular weight distribution (Figures A and B) and monosaccharide composition (Figures C and D) of the Cordyceps militaris polysaccharides CCP-WE and CCP-Lp are shown in the figures. As shown, CCP-WE exhibits two chromatographic peaks with retention times of 14.892 min and 17.581 min, and its monosaccharide composition and molar ratio are mannose:glucose:galactose = 1:3.44:0.69. CCP-Lp shows a single absorption peak with a retention time of 15.187 min and a relative molecular mass of 1.32 × 10⁻⁶. 5 The monosaccharide composition and molar ratio of Da were mannose:glucose:galactose = 1:0.36:0.62. The results showed that although *Lactobacillus plantarum* fermentation did not change the types of monosaccharides in *Cordyceps militaris* polysaccharides, it significantly altered their molar ratios, with a relatively increased ratio of mannose to galactose and a significantly decreased ratio of glucose. This specific change in monosaccharide composition is the key structural basis for the significantly superior antihypertensive activity of CCP-Lp compared to CCP-WE.

Claims

1. A method for preparing Cordyceps militaris polysaccharide by microbial fermentation, characterized in that... Includes the following steps: Step 1: Crush the Cordyceps militaris spore bundles, pass them through an 80-mesh sieve, add pure water and stir evenly. After sterilization, cool to room temperature to obtain the fermentation culture medium. Step 2: Inoculate *Lactobacillus plantarum* into liquid culture medium and culture to prepare a highly active seed culture; Step 3: Inoculate the seed culture obtained in Step 2 into the fermentation medium obtained in Step 1, place it in an anaerobic incubator, and let it stand for culture to obtain the fermentation broth; Step 4: Centrifuge the fermentation broth obtained in Step 3, collect the supernatant, concentrate it to 1 / 200-300 of the original volume, add 4 times the volume of anhydrous ethanol, stir and mix well, let it stand at 4 ℃ for 12 h, and centrifuge to collect the precipitate. Step 5: Dissolve the precipitate obtained in Step 4 in water to prepare a polysaccharide solution, which is then concentrated and freeze-dried to obtain Cordyceps militaris polysaccharide. The plant lactobacillus ( Lactiplantibacillus plantarum The accession number of the sample is ACCC11095, and it was purchased from the China Agricultural Microbial Culture Collection Center.

2. The preparation method according to claim 1, characterized in that: In step 1, add pure water at a mass-to-volume ratio of 1g:30-40mL.

3. The preparation method according to claim 1, characterized in that: In step 2, *Lactobacillus plantarum* is inoculated into the liquid culture medium at an inoculation rate of 1%.

4. The preparation method according to claim 3, characterized in that: The incubation temperature was 36℃, and the incubation time was 48h.

5. The preparation method according to claim 1, characterized in that: In step 3, the seed culture obtained in step 2 is inoculated into the fermentation medium obtained in step 1 at 5% of the total volume of the fermentation medium.

6. The preparation method according to claim 5, characterized in that: The incubation temperature was 36℃, and the incubation time was 48-72 h.

7. A polysaccharide from Cordyceps militaris, abbreviated as CCP-Lp, prepared according to any one of the preparation methods in claims 1-6, characterized in that: The relative molecular mass of the Cordyceps militaris polysaccharide is 1.32 × 10⁻⁶. 5 Da, its monosaccharide composition and molar ratio are mannose: glucose: galactose = 1: 0.36: 0.

62.

8. The use of the Cordyceps militaris polysaccharide according to claim 7 in the preparation of food or health products with auxiliary blood pressure lowering function.

9. The use of the Cordyceps militaris polysaccharide according to claim 7 in the preparation of a pharmaceutical preparation with antihypertensive function.

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