Cordyceps sobolifera polysaccharide with effect of reducing blood pressure and microbial fermentation preparation method of cordyceps sobolifera polysaccharide

Abstract

The invention discloses cordyceps sobolifera polysaccharide with an effect of reducing blood pressure and a microbial fermentation preparation method of the cordyceps sobolifera polysaccharide, and belongs to the field of preparation and application of fungal polysaccharide extracts. The cordyceps sobolifera polysaccharide is prepared by adopting a microbial fermentation method, cordyceps sobolifera coremium is used as a fermentation substrate, plant lactobacillus is used as a fermentation strain, liquid fermentation extraction is performed to obtain the cordyceps sobolifera polysaccharide CCP-Lp, the monosaccharide composition and the molar ratio of mannose to glucose to galactose are 1: 0.36: 0.62, and the relative molecular mass is 1.32 * 10 < 5 > Da. Efficacy experiments show that CCP-Lp can significantly inhibit abnormal increase of systolic pressure and diastolic pressure of spontaneously hypertensive rats and regulate the key factor level of a renin-angiotensin-aldosterone system, and the antihypertensive effect of CCP-Lp can be comparable with that of a positive drug captopril. The cordyceps sobolifera polysaccharide CCP-Lp is clear in composition and remarkable in effect, and has wide development and application prospects in the aspect of blood pressure regulation and control.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of preparation and application of fungal polysaccharide extract, and particularly relates to a Cordyceps cicadae polysaccharide with blood pressure lowering effect and a microbial fermentation preparation method thereof. BACKGROUND

[0002] Cordyceps cicadae (Berk.) is a kind of precious fungus for both medicine and food, belonging to Ascomycota and Cordycipitaceae, and also known as golden cicada flower. Isaria cicadae Miq Modern medical research shows that Cordyceps cicadae contains polysaccharides, nucleosides, sterols and other bioactive components, among which polysaccharides as the main active ingredient have immunomodulatory, antioxidant and kidney protection effects. The applicant found that Cordyceps cicadae polysaccharide has a certain blood pressure lowering effect, showing good development potential. However, the current traditional hot water extraction method has problems such as difficulty in efficient extraction of polysaccharides and insufficient biological activity of the obtained polysaccharides, which restricts the development and utilization of high value-added products of Cordyceps cicadae.

[0003] In recent years, microbial fermentation technology has provided a new technical path for the efficient utilization of natural products. Based on the rich enzyme system produced during microbial growth and metabolism, the cell wall can be efficiently degraded, not only significantly improving the yield of polysaccharides, but also through biological transformation to convert the original polysaccharides into new fermentation polysaccharides with higher activity. In addition, the extracellular polysaccharides synthesized by some microorganisms during fermentation may have a synergistic effect with the substrate polysaccharides, thereby comprehensively improving the overall biological efficiency of the fermentation polysaccharides. There is no report on the use of microbial fermentation technology for the preparation of Cordyceps cicadae polysaccharides.

[0004] Therefore, the applicant selected microorganisms (Lactiplantibacillus plantarum, Lactobacillus rhamnosus, Saccharomyces cerevisiae and Red Ganoderma lucidum) with high enzyme production capacity and food safety as fermentation strains, respectively treated Cordyceps cicadae sporocyst bundle powder through liquid fermentation, and evaluated the activity of the obtained Cordyceps cicadae polysaccharides. It was found that the Cordyceps cicadae polysaccharide prepared by Lactiplantibacillus plantarum fermentation showed outstanding performance in reducing the blood pressure of spontaneously hypertensive rats, and its effect was comparable to that of the positive drug captopril. The current patent (CN113396980A) discloses that Cordyceps cicadae fruiting body ultrafine powder is added to skimmed milk, which is claimed to have a blood pressure lowering effect due to the presence of N6-(2-hydroxyethyl) adenosine, and there is no in vivo and in vitro efficacy experiment to verify its blood pressure lowering effect. The blood pressure lowering effect of the active polysaccharide CCP-Lp prepared by the fermentation of Cordyceps cicadae with Lactiplantibacillus plantarum in the present application is fundamentally different from the technical method and active ingredient. SUMMARY

[0005] The present application provides a Cordyceps cicadae polysaccharide with blood pressure lowering effect and a microbial fermentation preparation method and application thereof. The polysaccharide is prepared by fermenting Cordyceps cicadae sporocyst bundle with Lactiplantibacillus plantarum, and can significantly inhibit the abnormal increase of systolic and diastolic blood pressure of spontaneously hypertensive rats.

[0006] The invention provides a cicada manjita cordyceps polysaccharide, which is abbreviated as CCP-Lp, and has a relative molecular mass of 1.32*10 5 Da, and its monosaccharide composition and molar ratio are composed of mannose: glucose: galactose = 1: 0.36: 0.62.

[0007] The cicada manjita cordyceps polysaccharide is prepared by a microbial fermentation method, and comprises the following steps:

[0008] Step 1: The cicada manjita cordyceps spore bundle is crushed, and is passed through an 80-mesh sieve. Pure water is added in a mass-volume ratio of 1:30-40 (g / mL), and is stirred uniformly. Sterilization is performed at 121 DEG C for 30 min, and cooling is performed to room temperature. A fermentation medium is prepared.

[0009] Step 2: The plant lactobacillus (Lp) is inoculated into the liquid medium at an inoculation amount of 1% (v / v), and is cultured at 36 DEG C for 48 h. A high-activity seed solution is prepared.

[0010] The liquid medium comprises the following components: glucose 20 g / L, yeast powder 5 g / L, protein peptone 10 g / L, beef extract 10 g / L, potassium phosphate dibasic 2 g / L, citric acid diamine 2 g / L, sodium acetate 5 g / L, magnesium sulfate heptahydrate 0.58 g / L, manganese sulfate monohydrate 0.17 g / L, and Tween-80 1 mL.

[0011] The plant lactobacillus (Lp) has a preservation number of ACCC11095, and is purchased from the China Agricultural Microbial Culture Collection Center. Lactiplantibacillus plantarum

[0012] Step 3: The seed solution obtained in step 2 is inoculated into the fermentation medium obtained in step 1 at 5% of the total volume of the fermentation medium, and is placed in an anaerobic incubator. Culturing is performed at 36 DEG C for 48-72 h. A fermentation liquid is obtained.

[0013] Step 4: The fermentation liquid obtained in step 3 is centrifuged at 3000 rpm. The supernatant is collected, and is concentrated to 1 / 200-300 of the original volume by rotary evaporation at 60-70 DEG C. Four times the volume of anhydrous ethanol is added. After stirring and mixing, standing is performed at 4 DEG C for 12 h. The precipitate is collected by centrifugation.

[0014] Step 5: The precipitate obtained in step 4 is dissolved in water to prepare a polysaccharide solution. Concentration, freeze-drying, and the like are performed. The cicada manjita cordyceps polysaccharide is prepared.

[0015] The application of the cicada manjita cordyceps polysaccharide in the preparation of food, health products or pharmaceutical preparations with a blood pressure-lowering function.

[0016] ​The efficacy experiment shows that the CCP-Lp can significantly inhibit the abnormal increase of systolic pressure and diastolic pressure of the spontaneously hypertensive rats, regulates the key factor level of the renin-angiotensin-aldosterone system, and the antihypertensive effect can be comparable to that of the positive drug captopril. The chanshu chongcao polysaccharide CCP-Lp has clear composition and significant efficacy, and has a broad development and application prospect in regulating blood pressure. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The inhibitory effect of the chanshu chongcao polysaccharide prepared by fermentation of the four strains on the activity of angiotensin converting enzyme (ACE) in vitro.

[0018] Figure 2 The influence of each experimental group in example 3 on the systolic pressure (SBP) (A figure) and diastolic pressure (DBP) (B figure) of the spontaneously hypertensive rats.

[0019] Figure 3 The influence of each experimental group in example 3 on the key active factor level of the renin-angiotensin-aldosterone (RAAS) system of the spontaneously hypertensive rats, Figure 3 A, 3B, 3C, 3D are the levels of ACE, Renin (renin), Ang Ⅱ (angiotensin Ⅱ) and ALD (aldosterone).

[0020] Figure 4 The molecular weight distribution (A, B figure) and monosaccharide composition (C, D figure) of the chanshu chongcao polysaccharide CCP-WE and CCP-Lp. DETAILED DESCRIPTION

[0021] The present application will be described in detail below through specific implementation cases. It should be understood that the following examples are only used to illustrate the present application, and are not used to limit the protection scope of the present application. Any non-essential replacement or modification made by a person skilled in the art on the basis of the technical solutions of the present application should be included in the protection scope of the present application.

[0022] The chanshu chongcao used in the present application is artificially cultivated Isaria cicadae Miq., and the spore bundle is collected after being cultured by inoculating the strain into a specific culture medium, and then dried to prepare chanshu spore bundle raw materials. The chanshu spore bundle used in this embodiment is provided by "Anhui Chongcao Source Biological Technology Co., Ltd.".

[0023] Example 1: Preparation of chanshu chongcao polysaccharide CCP-Lp

[0024] Step 1: The chanshu chongcao spore bundle is crushed, passed through an 80-mesh sieve, added with pure water at a mass-volume ratio of 1:30 (g / mL), stirred uniformly, sterilized at 121 ℃ for 30 min, and cooled to room temperature to prepare a 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 referred to Example 1, except that the fermentation medium is inoculated with red Ganoderma lucidum (Gl, commercially available), and the remaining steps are the same as Example 1.

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

[0039] Mix 30 μL of polysaccharide sample with 100 μL of 50 mU / mL ACE solution in 30 uL of sodium borate buffer. After pre-incubation at 37 ℃ for 10 min, add 200 μL of HHL (hippuryl glycyl leucine) substrate solution with a concentration of 5 mM to start the enzymatic reaction, and continue incubation at 37 ℃ for 30 min. After the reaction is completed, add 250 uL of hydrochloric acid solution (1 mol / L) to terminate the reaction and extract the hippuric acid generated, followed by centrifugation at 12000 rpm for 10 min. Remove the upper organic phase, concentrate and dry by rotary evaporation, and then dissolve the residue with distilled water, and measure the absorbance at a wavelength of 228 nm.

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

[0041] Spontaneously hypertensive rats (SHR, male, 8-10 weeks old) were selected from the laboratory and WKY blank control rats (male, 8 weeks old) were purchased from Hefei Qingyuan Biotechnology Co., Ltd. The experimental animal production license number of WKY rats is: SCXK (Zhejiang) 2025-0004. After all animals were adaptively fed in SPF level environment for one week, they were randomly divided into 6 groups according to the experimental requirements. They were control group, model group, positive control group Captopril, CCP-WE group, CCP-Lp group, and plant lactobacillus group (Lp). The CCP-WE group was given 300 mg / kg / d of cicadas Cordyceps polysaccharide CCP-WE; the CCP-Lp group was given 300 mg / kg / d of cicadas Cordyceps polysaccharide CCP-Lp, the Lp group was given 3×10 9 CFU / kg / d of plant lactobacillus, the Captopril group was given 25 mg / kg / d of Captopril, and the control group and the model group were given equal volume of pure water, once a day for 28 days. The control group used WKY blank control rats, and the other groups used spontaneously hypertensive rats. The SBP and DBP of rats in each group before and after administration were monitored by rat non-invasive blood pressure instrument. The rats were fasted for 12 hours (free water), anesthetized by intraperitoneal injection of 10% (g / mL) hydrated chloral solution (3.5 mL / kg), and then the eyeball blood was collected for subsequent index detection.

[0042] The changes of SBP and DBP of rats in each group were detected by noninvasive tail artery tonometry. The BP-2010 system (rat noninvasive blood pressure analysis system, BP-2010, Beijing Ruolong Biotechnology Co., Ltd.) was used to measure the blood pressure of rats in a quiet environment. Before formal measurement, the rats were given 3-5 times of adaptive training. During measurement, the rats were placed in a special fixator, and the tail cuff sensor was placed at the site of obvious tail artery pulsation. After the rats were calm, the blood pressure value was measured, and the average value after 8-10 times of repeated measurement and removal of abnormal values was taken as the final result. The levels of ACE, Renin, Ang Ⅱ and ALD in serum were detected to evaluate the activity state of the RAAS system of SHR rats. After centrifugation at 3000 rpm for 10 min, the upper serum was obtained and strictly according to the instructions of the kit (rat ACE, Renin, Ang Ⅱ, ALD detection kit, Shanghai Qifa Experimental Reagent Co., Ltd.), the concentrations of each index in serum were determined.

[0043] Example 4: Determination of molecular weight and monosaccharide composition analysis of Cordyceps cicadae Cordyceps polysaccharides CCP-WE and CCP-Lp

[0044] (1) Determination of molecular weight

[0045] Cordyceps cicadae Cordyceps polysaccharides CCP-WE and CCP-Lp and dextran standard (T10, T50, T500, T1000, T2000) of different molecular weights were respectively prepared into 5 mg / mL solution with double distilled water, and Agilent high performance liquid-evaporative light scattering detector was used for determination. TSK-Gel G6000 PWXL column was used for chromatographic separation, ultrapure water was used as mobile phase, N2 was used as carrier gas, gas flow rate was set to 2.5 L / min, and injection volume was 10 μL. The standard curve was established with the logarithm of molecular weight (Lg Mw) and retention time (Rt) of dextran standard, and the molecular weight range of Cordyceps cicadae Cordyceps polysaccharides CCP-WE and CCP-Lp was measured.

[0046] (2) Monosaccharide composition analysis

[0047] The sample of CCP-WE and CCP-Lp acid hydrolysis-pre-column PMP (1-phenyl-3-methyl-5-pyrazolone) derivatization was analyzed by Agilent high performance liquid chromatography-diode array detector. 8 mg of CCP-WE and CCP-Lp samples were accurately weighed and dissolved in 5 mL of 2 mol / L trifluoroacetic acid, and then sealed with nitrogen in a test tube with a stopper, and hydrolyzed at 110 ℃ in an oil bath for 8 h. After the reaction was completed, methanol was added several times and the residual acid was removed by rotary evaporation until the pH value of the system returned to neutral, 1 mL of ultrapure water was added for redissolution, and the hydrolyzate was obtained for use. 1 mL of 5 mg / mL monosaccharide standard solution and CCP-WE and CCP-Lp hydrolyzate was removed, respectively, sodium hydroxide solution (1 mL, 0.3 mol / L) and PMP solution (1 mL, 0.5 mol / L) were added in turn, shaken and mixed, and then PMP derivatization was carried out at 70 ℃ water bath for 1 h. The pH was adjusted to neutral with hydrochloric acid solution (1 ml, 0.3 mol / L), and then excess PMP reagent was removed by extraction with chloroform three times. The aqueous phase was collected, filtered through a 0.22 μm filter membrane, and then subjected to HPLC-DAD analysis.

[0048] Figure 1 The ACE activity in vitro was inhibited by the polysaccharides prepared by fermentation of the four strains. The results showed that compared with the CCP-WE group, each fermentation group showed different degrees of ACE inhibition effect, and the CCP-Lp group showed the most significant inhibition effect, with an ACE inhibition rate of 1.5 times that of the CCP-WE group, showing the best potential for lowering blood pressure.

[0049] Figure 2 The effects of each experimental group in Example 3 on the SBP and DBP of SHR rats. 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 the blood pressure value was close to that of the positive drug group, and was significantly better than that of the CCP-WE and Lp groups, indicating that CCP-Lp had a significant effect on lowering blood pressure.

[0050] Figure 3 The effects of each experimental group in Example 3 on the key active factor levels of the RAAS system of SHR rats. Compared with the control group, the levels of ACE, Renin, Ang Ⅱ and ALD in the serum of the model group rats were significantly increased. Compared with the model group, the CCP-Lp group could significantly reduce the levels of ACE, Renin, Ang Ⅱ and ALD active factors in the serum, and was better than the CCP-WE and Lp groups, which was consistent with the in vitro ACE activity results. It was indicated that CCP-Lp could effectively inhibit the expression of key active factors such as ACE, Renin, Ang Ⅱ and ALD in the RAAS system, and had a significant regulatory effect on spontaneous hypertension.

[0051] Figure 4 The molecular weight distribution (A, B) and monosaccharide composition (C, D) of CCP-WE and CCP-Lp. As shown in the figure, CCP-WE showed two chromatographic peaks with retention times of 14.892 min and 17.581 min, and the monosaccharide composition and molar ratio were composed of mannose: glucose: galactose = 1: 3.44: 0.69; CCP-Lp was a single absorption peak with a retention time of 15.187 min, and the relative molecular mass was 1.32 x 10 5 Da, the monosaccharide composition and molar ratio were composed of mannose: glucose: galactose = 1: 0.36: 0.62. The results showed that the fermentation of plant lactobacillus did not change the type of monosaccharide composition of cicada worm grass polysaccharide, but significantly changed the molar ratio. The proportion of mannose and galactose was relatively increased, while the proportion of glucose was significantly reduced. This specific change of monosaccharide composition is the key structural basis for the significantly better blood pressure lowering activity of CCP-Lp than CCP-WE.

Claims

1. A microbial fermentation method for preparing Cordyceps cicadae polysaccharide, characterized in that It comprises the following steps: Step 1: crushing Cordyceps cicadae spore bundle through 80 mesh screen, adding pure water and stirring uniformly, sterilizing and cooling to room temperature to prepare fermentation medium; Step 2: inoculating Bacillus velezensis in liquid medium to prepare high-activity seed liquid; Step 3: inoculating the seed liquid obtained in step 2 into the fermentation medium obtained in step 1 and placing in an anaerobic incubator to obtain fermentation liquid; Step 4: centrifuging the fermentation liquid obtained in step 3, collecting supernatant, concentrating to 1 / 200-300 of the original volume, adding 4 times volume of anhydrous ethanol, stirring uniformly, standing at 4℃ for 12 hours, and centrifuging to collect precipitate; Step 5: dissolving the precipitate obtained in step 4 in water to prepare polysaccharide solution, concentrating and freeze-drying to prepare Cordyceps cicadae polysaccharide.

2. The preparation method according to claim 1, characterized in that: In step 1, 1g of the Cordyceps cicadae spore bundle is added to 30-40 mL of pure water.

3. The preparation method according to claim 1, characterized in that: In step 2, the Bacillus velezensis is inoculated into the liquid medium at an inoculation amount of 1%.

4. The preparation method according to claim 3, characterized in that: The culture temperature is 36℃, and the culture time is 48 hours.

5. The preparation method according to claim 3, characterized in that: The preservation number of the Bacillus velezensis is ACCC11095.

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

7. The preparation method according to claim 6, characterized in that: The culture temperature is 36℃, and the culture time is 48-72 hours.

8. A Cordyceps cicadae polysaccharide, abbreviated as CCP-Lp, prepared according to the preparation method of any one of claims 1-7, characterized in that: The relative molecular mass of the Cordyceps cicadae polysaccharide is 1.32 x 10 5 Da, the monosaccharide composition and molar ratio thereof are mannose: glucose: galactose = 1 : 0.36 : 0.

62.

9. The use of the Cordyceps cicadae polysaccharide of claim 8 in the preparation of food, health products or pharmaceutical preparations with blood pressure lowering function.

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