A method for extracting polysaccharides from Vanilla mulberry and its spore powder and its application

Abstract

The invention discloses an extraction method and application of Vanis lindera and its spore powder polysaccharide. The Vanis lindera spore powder polysaccharide is used for selective separation of polysaccharide in Vanis lindera spore powder sample liquid treated by cellulose enzymolysis, ultrasound, and enzymolysis-assisted ultrasound pretreatment through a separation system composed of tert-butyl alcohol and inorganic salt ammonium sulfate. This is the first time to separate active polysaccharide from lindera spore powder. The Vanis lindera spore powder polysaccharide of the invention has high-efficiency antioxidant, hypoglycemic, immunostimulatory and anti-tumor capabilities, and can be widely used in functional foods, cosmetics, medicines, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungi, and specifically relates to a method for extracting polysaccharides of Vanilla ferns and spore powder thereof and application thereof. Background Art

[0002] Sanghuangporus vaninii, also known as poplar mulberry, belongs to Basidiomycota, Agaricomycetes, Hymenochaetales, Hymenochaetaceae, and Sanghuangporus in taxonomy. It is a rare medicinal fungus growing on poplars in Northeast my country. Modern pharmacological studies have confirmed that polysaccharides are the main active ingredients in the fruiting bodies and mycelium of Sanghuangporus vaninii, which have biological activities such as anti-tumor, hypoglycemic, and immunomodulatory. It is internationally recognized as one of the best medicinal fungi with anti-tumor effects, and is also considered an important raw material for functional foods, medicines, and cosmetics. At present, the extraction of active polysaccharides from Sanghuangporus vaninii spore powder has not yet been carried out. At present, the conventional extraction methods of Sanghuangporus polysaccharides are hot water method and ethanol precipitation method. However, these two extraction methods have the disadvantages of long extraction time, low extraction rate, and high operating cost. In addition, the subsequent polysaccharide purification steps are cumbersome, and a variety of organic reagents are often used, which can easily cause environmental pollution and loss of polysaccharide activity. Therefore, it is particularly important to explore a green, efficient, and rapid extraction strategy for separating polysaccharides from Vanisan chrysospore powder.

[0003] With the continuous innovation and optimization of extraction technology, innovative emerging extraction strategies have been developed. Exploring the separation and extraction of polysaccharides from Vanisan Yellow spore powder and identifying the biological activity of polysaccharides are important scientific issues that need to be solved urgently in order to achieve efficient utilization of Vanisan Yellow medicinal resources. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0005] As one aspect of the present invention, the Vani Sanghuangporus species described in the present invention is Sanghuangporus spp. SV-HN-S1, with a deposit number of CCTCC NO: M 20231010; deposit date: June 12, 2023; deposited at: China Center for Type Culture Collection, address: Wuhan University, Wuhan, China. The species can eject and release spore powder after conventional factory cultivation.

[0006] As one aspect of the present invention, the SV-HN-S1 test tube seed is inoculated in a culture bag and cultured in the dark for 40 days (temperature 26-28°C) to obtain the Sanghuangporus spp.SV-H N-S1 original seed. Subsequently, the original seed is inoculated on the cultivated culture material and cultured in the dark for 40 days under the conditions of 26-28°C to obtain the SV-HN-S1 cultivated seed. After that, the cultivated seed is inoculated in a mulberry basswood bag (temperature 28°C, light-proof) and cultured until the mycelium is full of the fungus bag, and then the sterile blade is used to cut the fungus skin, and buried in a pit, and covered with sand (humidity 70wt%). The fungus bag is stimulated to turn yellow by the temperature difference between day and night (28°C during the day and 20°C at night), and the yellowing place is cut, and the culture is continued (humidity 94wt%, light 850lu×) until the fruiting body of Phellinus igniarius grows. Finally, the fruiting bodies of Sanghuangporus spp. will eject spores in the third quarter of the second year (September-October), and the spore powder of Sanghuangporus spp. will be collected by bagging. According to the spore powder yield per gram of Sanghuangporus spp. fruiting body, the average spore powder yield of Sanghuangporus spp.SV-HN-S1 strain is 0.075±0.006g / g.

[0007] As one aspect of the present invention, the Vanisan yellow spore powder of the present invention is provided by the Edible Fungi Research Institute of Shanghai Academy of Agricultural Sciences, and the spore powder is bright yellow in color (see Figure 1 ).

[0008] As one aspect of the present invention, the present invention provides a method for preparing the Vanisan chrysanthemum spore powder polysaccharide, which comprises the following steps:

[0009] (1) Preparation of Vanisan chrysanthemum spore powder: Grind the collected Vanisan chrysanthemum spore powder in liquid nitrogen for 10-30 minutes;

[0010] (2) Pretreatment of Vanisan chrysospore powder samples: including enzymatic hydrolysis (time 1-3 h, temperature 20-40 °C), ultrasound (frequency 10-30 kHz, power 40-60 W / L), and enzymatic hydrolysis combined with ultrasound;

[0011] (3) Construction of three-phase separation system: 10-30% (w / v) ammonium sulfate is added to the Vanisan yellow spore powder sample solution; then tert-butanol is slowly added according to the ratio of spore powder sample solution to tert-butanol of 1:0.5-1:2 (v / v); the mixture is gently stirred on a magnetic stirrer at 20°C-40°C for a certain time of 10-40 minutes, and after being fully mixed, the mixture is centrifuged at 4500rpm for 10 minutes to form a clear three-phase. Finally, the lower phase solution is taken out and dialyzed in a dialysis bag with running water, concentrated under reduced pressure, and freeze-dried to obtain Vanisan yellow spore powder polysaccharide.

[0012] As one aspect of the present invention, the present invention provides the use of the Vanisan chrysospore powder polysaccharide in the preparation of antioxidant, blood sugar-lowering, immunomodulatory, and anti-cancer foods or drugs.

[0013] Beneficial effects of the present invention:

[0014] 1. The enzymatic hydrolysis / ultrasound-assisted three-phase separation system of the present invention can significantly improve the yield of polysaccharides from the yellow spore powder of Vanisan mulberry, that is, enzymatic hydrolysis assisted by ultrasound-assisted three-phase separation> ultrasound-assisted three-phase separation> enzymatic hydrolysis assisted by three-phase separation> three-phase separation;

[0015] 2. The polysaccharide of the powder of Vanisan spores of the present invention has high-efficiency antioxidant, hypoglycemic, immunostimulatory and anti-tumor activities in in vitro experiments, and can be developed and applied as daily anti-aging, diabetes prevention and treatment, and anti-cancer food, health care products and medicines.

[0016] 3. The Vani Sanguisorba officinalis strain of the present invention can produce high spore powder and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0018] Figure 1 This is a real picture of Vanisan yellow spore powder;

[0019] Figure 2 The immunomodulatory results of Vanisan chrysanthemum spore powder polysaccharide;

[0020] Figure 3 The inhibitory effect of Vanisan spore powder polysaccharide on the proliferation activity of tumor cells.

[0021] Figure 4 This is the technical route of the present invention. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0023] The Sanghuangporus spp.SV-HN-S1 strain and its spore powder described in the present invention are provided by the Edible Fungi Research Institute of Shanghai Academy of Agricultural Sciences. The spore powder is bright yellow in color (see Figure 1 ), the factory-based cultivation of this Vanilla fungus species is capable of ejecting spores.

[0024] The Sanghuangporus spp. SV-HN-S1 of the present invention was confirmed by |TS identification, and its sequencing results showed that it had the highest homology with the |TS sequence of Sanghuangporus vaninii. The upstream primer sequence was 5'-TGCGCATGTGCACGGCCTT-3'; the downstream primer sequence was 5'-AGGAGGTAACAACAACTCCTT-3'.

[0025] The collected Vanisan spore powder was ground in liquid nitrogen for 30 min, then 100 mL of distilled water was added and mixed to obtain the spore powder sample solution, and the polysaccharides were immediately extracted by the following four methods.

[0026] Embodiment 1:

[0027] Three-phase separation and extraction: Take 10mL of spore powder sample solution, add ammonium sulfate to make its mass fraction 20%, and add 10mL of tert-butyl alcohol at the same time, and place it in a 35℃ water bath for stirring and incubation for 30min. Subsequently, the mixed solution is centrifuged at 4500rpm for 10min to form a clear three-phase solution; the lower phase solution is dialyzed in a dialysis bag with a relative molecular mass of 8000-10000 for 3 days to remove impurities with a smaller relative molecular mass, and the dialyzate is concentrated under reduced pressure and freeze-dried. The obtained polysaccharide is named SVSPs-C.

[0028] The yield of SVSPs-C was 12.97%, and the monosaccharide composition was galactose: glucose: mannose = 0.46: 6.97: 1.00.

[0029] Embodiment 2:

[0030] Enzyme-assisted three-phase separation and extraction: Take 15.0g of Vanisan spore powder ground under liquid nitrogen for 30min, add 100mL of acetic acid-phosphate buffer (0.1M, pH 6.0: the optimal pH for cellulase), and add 4% (w / v) cellulase in a water bath at 35℃ (the optimal temperature for cellulase) for 2h, and then place it in a 90℃ water bath to inactivate the enzyme for 10min. Subsequently, the enzymatic hydrolyzed spore powder sample solution was centrifuged at 4500rpm for 10min and the precipitate was discarded to obtain the enzymatic hydrolyzate. Take 10mL of the enzymatic hydrolyzate, add 20% (w / v) ammonium sulfate and tert-butanol (the volume ratio of tert-butanol to the enzymatic hydrolyzate is 1:1), and stir and incubate in a 35℃ water bath for 30min. Then, the mixed solution was centrifuged at 4500rpm for 10min to form three clear phases; the lower phase solution was dialyzed in a dialysis bag with a relative molecular mass of 8000-10000 for 3 days, the dialysate was concentrated under reduced pressure and freeze-dried, and the obtained polysaccharide was named SVSPs-E.

[0031] The yield of SVSPs-E was 15.28%, and the monosaccharide composition was galactose: glucose: mannose = 0.29: 2.25: 1.00.

[0032] Embodiment 3:

[0033] Ultrasonic-assisted three-phase separation and extraction: 100 mL of Vanisan spore powder sample solution was placed in a 35°C water ultrasonic tank, the ultrasonic frequency was set to 20kHz, the ultrasonic power density was 50W / L, and after stirring and ultrasonicating for 60 minutes, the supernatant was taken after centrifugation at 4500rpm for 10 minutes to obtain the ultrasonic pre-treated sample solution. Take 10 mL of the ultrasonic pre-treated sample solution, add 20% ammonium sulfate and tert-butyl alcohol (the volume ratio of tert-butyl alcohol to the ultrasonic pre-treated sample solution is 1:1), stir and incubate in a 35°C water bath for 30 minutes, and then centrifuge at 4500rpm for 10 minutes to form three phases. Take the lower phase solution and dialyze it in a dialysis bag with a relative molecular mass of 8000-10000 for 3 days. After the dialysate is concentrated under reduced pressure, it is freeze-dried, and the obtained polysaccharide is named SVSPs-U.

[0034] The yield of SVSPs-U was 19.09%, and the monosaccharide composition was glucose: mannose = 9.09: 1.00.

[0035] Embodiment 4:

[0036] Enzymatic hydrolysis and ultrasonic assisted three-phase separation and extraction: 15.0 g of Vanisan yellow spore powder ground under liquid nitrogen for 30 minutes was added to 100 mL of acetic acid-phosphate buffer, and 4% cellulase was added to shake and incubate in a 35°C water bath for 2 hours, and then the enzyme was inactivated in a 90°C water bath for 10 minutes to obtain an enzymatic solution. After that, 100 mL of the enzymatic solution was placed in a 35°C water ultrasonic tank (frequency 20kHz, power 50W / L) and stirred while ultrasonicated for 60 minutes, centrifuged at 4500rpm for 10 minutes, and the precipitate was discarded, and the supernatant was taken to obtain an enzymatic hydrolysis and ultrasonic pretreatment sample solution. Take 10 mL of the enzymatic hydrolysis and ultrasonic pretreatment sample solution, add 20% ammonium sulfate and 10 mL tert-butanol by mass fraction, mix well, place in a 35°C water bath, stir and incubate for 30 minutes, and form three phases after centrifugation at 4500rpm for 10 minutes. The lower phase solution was dialyzed in a dialysis bag with a relative molecular mass of 8000-10000 for 3 days. The dialysate was concentrated under reduced pressure and freeze-dried. The obtained polysaccharide was named SVSPs-E / U.

[0037] The yield of SVSPs-E / U was 25.40%, and the monosaccharide composition was galactose: glucose: mannose = 0.19: 1.98: 1.00.

[0038] The following examples were tested using the Vanisan chrysospore powder polysaccharide prepared in Examples 1-4:

[0039] Embodiment 5:

[0040] Antioxidant activity of polysaccharides from Vanilla chrysanthemum spore powder:

[0041] (1) Determination of DPPH free radical scavenging rate: 1 mL of DPPH (0.1 mmol / L)-ethanol solution was mixed with 3 mL of Vanisan chrysospore polysaccharide solution of different concentrations (0.1, 0.5, 1, 1.5, 2 mg / mL), and the mixture was allowed to react for 30 min in the dark. The absorbance was measured at 517 nm. C As a positive control.

[0042] The DPPH scavenging rates of SVSPs-C, SVSPs-E, SVSPs-U and SVSPs-E / U at a concentration of 2 mg / mL were measured to be 42.61%, 53.09%, 64.81% and 74.42%, respectively.

[0043] (2) Determination of Trolox antioxidant capacity (TEAC): Weigh 4 kinds of Vanisan chrysanthemum spore powder polysaccharides and add 4 times the volume of PBS solution to fully dissolve. Take 100 μL of Vanisan chrysanthemum spore powder polysaccharide solution in a test tube, add 3.9 mL of ABTS solution to the test tube, mix well and incubate for 20 minutes, and measure the absorbance value at a wavelength of 734 nm.

[0044] The equivalent antioxidant capacity of SVSPs-C, SVSPs-E, SVSPs-U and SVSPs-E / U was measured to be 280.58, 299.56, 345.86 and 376.39 μmol Trolox / g sample, respectively.

[0045] (3) Determination of ferric iron reducing ability (FRAP): Weigh four kinds of Vanisan chrysanthemum spore powder polysaccharides and add 4 times the volume of PBS to fully dissolve them. Accurately pipette 50 μL of Vanisan chrysanthemum spore powder polysaccharide solution into a test tube, add 5 mL of FRAP working solution, mix thoroughly, incubate at 37°C for 5 min, and measure its absorbance at a wavelength of 593 nm.

[0046] The equivalent ferrous ion capacities of SVSPs-C, SVSPs-E, SVSPs-U, and SVSPs-E / U were 20.61, 26.67, 30.18, and 35.53 μmol Fe, respectively. 2+ / g sample.

[0047] Embodiment 6:

[0048] Hypoglycemic activity of polysaccharides from Vanisan chrysospore powder:

[0049] (1) Determination of α-amylase inhibitory activity: 20.00 μL of Vanisan chrysospore polysaccharide solution of different concentrations (0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg / mL) and 20.00 μL of α-amylase solution (5.00 U / mL) were vortexed in a 1.50 mL centrifuge tube and incubated in a water bath at 37°C for 10 min; after completion, 40.00 μL of 1.00% starch solution was added and the reaction was continued at 37°C for 10 min; then 80.00 μL of DNS reagent was added to terminate the reaction, and the mixture was heated in a boiling water bath for 5 min, taken out, quickly cooled in an ice water bath, and finally diluted with 1.00 mL of water. Acarbose was used as a positive control, and the absorbance was measured at a wavelength of 540 nm.

[0050] The inhibitory rates of SVSPs-C, SVSPs-E, SVSPs-U and SVSPs-E / U on α-amylase were measured. 50 The values ​​were 5.20, 4.68, 2.60 and 1.93 mg / mL respectively.

[0051] (2) Determination of α-glucosidase inhibition rate: 0.70 mL of phosphate buffer (0.10 mol / L, pH = 6.80), 0.10 mL of Vanisan chrysospore powder polysaccharide solution of different concentrations (0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg / mL) and 0.10 mL of α-glucosidase solution (0.24 U / mL) were vortexed and mixed in a 1.50 mL centrifuge tube, and incubated in a water bath at 37°C for 10 min; then 0.10 mL of pNPG solution (2.50 mmol / L) was added, and the reaction was continued at 37°C for 20 min; finally, 0.40 mL of Na2CO3 solution (0.20 mol / L) was added to terminate the reaction. Acarbose was used as a positive control, and the absorbance was measured at a wavelength of 400 nm.

[0052] The inhibitory rates of SVSPs-C, SVSPs-E, SVSPs-U and SVSPs-E / U on α-glucosidase were measured. 50 The values ​​were 6.78, 5.26, 2.88 and 2.01 mg / mL respectively.

[0053] Embodiment 7:

[0054] Immunomodulatory activity of polysaccharides from Vanisan chrysanthemum spore powder:

[0055] (1) Toxicity test of Vanisan spore powder polysaccharide on RAW264.7 cells: The cell concentration was adjusted to 1×10 5After adding 100 μL / well of cell suspension of 100 μg / mL to a 96-well plate, the plate was placed in a cell culture incubator (37°C, 5% CO2) for 24 hours. The culture solution was then aspirated and 100 μL of 25, 50, 100, 200, and 500 μg / mL Vanisan spore powder polysaccharide DMEM solutions were added. The blank group replaced the spore powder polysaccharide solution with DMEM, with 6 parallels in each group. After 24 hours of culture, 10 μL of CCK-8 solution was added to each well 1 hour before the end, and the absorbance was measured at 450 nm.

[0056] The results showed that the four polysaccharides from the spore powder of Vanilla mulberry had a promoting effect on the proliferation activity of RAW264.7 cells (see Figure 2 ).

[0057] (2) Determination of nitric oxide (NO) release in RAW264.7 cells: Cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 1×10 5 / mL, add the cell suspension (100μL / well) to a 24-well plate and culture for 24h. Then aspirate the culture medium and replace it with serum-free DMEM solution, starve and culture for 2h to keep the cells at the same level. Set up a blank group (DMEM culture medium), LPS group (1μg / mL LPS stimulation), and Vanisan spore powder polysaccharide intervention group (concentrations of 25, 50, 100 and 20μg / mL). Add 1mL of each to the 24-well plate and incubate in an incubator for 48h, and then measure the NO release amount according to the NO kit.

[0058] The results showed that the four polysaccharides from the yellow spore powder of Vanilla mulberry significantly promoted the release of NO from RAW264.7 cells in a dose-dependent manner (see Figure 2 ).

[0059] Embodiment 8:

[0060] Antitumor activity of polysaccharide from Vanisan chrysanthemum spore powder:

[0061] Huh-7 (human liver cancer cells), MCF-7 (human breast cancer cells), and 95-D (human lung cancer cells) cells in the logarithmic growth phase were collected, counted, and the cell density was adjusted to about 5×10 3 / mL, add 100μL to each well in a 96-well plate. Vanisan spore powder polysaccharide treatment group (500μg / mL), positive control group (doxorubicin hydrochloride, 2μmol / L), and blank group are culture medium without cells. Set up 5 replicate wells, culture for 24h, add 10μL CCK-8 solution to each well and culture for 4h in the dark, use an enzyme reader to measure the optical density (D) value at a wavelength of 450nm, and calculate the cell inhibition rate. Cell survival rate = [1-(experimental group D value-blank group D value) / (control group D value-blank group D value)]×100%.

[0062] It was found that SVSPs-C, SVSPs-E, SVSPs-U and SVSPs-E / U had a significant inhibitory effect on the proliferation of tumor cells at a concentration of 500 μg / mL (see Figure 3 ).

[0063] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A kind of Vanilla yellow ( Sanghuangporus vaninii ) strain, characterized in that: The Vani Sang Yan strain is Vani Sang Yan Sanghuangporus spp. SV-HN-S1, its deposit number is CCTCC NO: M 20231010, and it was deposited in the China Center for Type Culture Collection on June 12, 2023.

2. A method for preparing spore powder polysaccharide using the Vaniella fusiformis species of claim 1, characterized in that: Made by the following steps, Step 1, grinding the spore powder of the Vani Sanguisorba species described in claim 1 under liquid nitrogen conditions; Step 2, adding distilled water to the spore powder sample obtained in step 1 and mixing thoroughly to obtain a Vanisan spore powder sample solution; Step 3, adding acetate-phosphate buffer to the spore powder obtained in step 1, then adding cellulase for incubation, centrifuging, and taking the supernatant enzymatic solution; Step 4, placing the spore powder sample solution obtained in step 2 in water for ultrasonic pretreatment, centrifuging, and taking the supernatant; Step 5, adding acetic acid-phosphate buffer to the spore powder obtained in step 1, then adding cellulase for incubation, centrifuging, taking the supernatant enzymatic solution, placing it in water for ultrasonic pretreatment, centrifuging, and taking the supernatant; Step six, adding ammonium sulfate and tert-butanol to the solution obtained in step two, step three, step four or step five, stirring in a water bath, centrifuging, taking the lower phase solution for dialysis, concentrating under reduced pressure, and freeze-drying to obtain spore powder polysaccharide.

3. The method according to claim 2, characterized in that: The third step is to add acetate-phosphate buffer to the spore powder obtained in the first step, then add 4% cellulase and incubate for 2 hours, centrifuge the enzymatically hydrolyzed spore powder sample solution at 4500 rpm for 10 minutes, and take the supernatant enzymatic hydrolyzate.

4. The method according to claim 2 or 3, characterized in that: The fourth step is to place the spore powder sample solution obtained in the second step in 35° C. water for ultrasonic pretreatment for 1 hour, centrifuge at 4500 rpm for 10 minutes, and take the supernatant.

5. The method according to claim 2 or 3, characterized in that: In the step six, the mass fraction of ammonium sulfate is 20%, and the volume of the tert-butanol is equal to the volume of the solution.

6. Use of spore powder polysaccharide prepared by the Vani Sanguisorba officinalis species described in claim 1 in the preparation of antioxidant drugs.

7. Use of spore powder polysaccharide prepared by the Vani Sanguisorba officinalis species described in claim 1 in the preparation of hypoglycemic drugs.

8. Use of spore powder polysaccharide prepared from the Vaniella fusiformis species of claim 1 in the preparation of immunomodulatory drugs.

9. Use of spore powder polysaccharide prepared by the Vaniella fusiformis species described in claim 1 in the preparation of anti-tumor drugs.

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