CN109837217B - High-temperature-resistant Pleurotus ostreatus and screening method and application thereof - Google Patents
High-temperature-resistant Pleurotus ostreatus and screening method and application thereof Download PDFInfo
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Abstract
The invention discloses a high-temperature-resistant Pleurotus ostreatus, a screening method and application thereof, wherein the preservation number of the high-temperature-resistant Pleurotus ostreatus is CGMCC: 16378, has good high temperature resistance, and can grow well when cultured at 30-35 deg.C. The fermentation liquid of high temperature resistant Pleurotus ostreatus contains abundant polysaccharides, and the polysaccharides are in pairs of OH free radical and O2 ‑Free radicals have strong scavenging ability, and high temperature resistant Pleurotus ostreatus can also be used as edible strain.
Description
Technical Field
The invention belongs to the technical field of Pleurotus ostreatus, and particularly relates to a high-temperature-resistant Pleurotus ostreatus and a screening method and application thereof.
Background
Pleurotus ostreatus (Pleurotus ostreatus) belongs to Basidiomycotina, Pleurotus ostreatus, and is widely distributed in Hebei, Shandong, Shanxi, Hunan, Hubei, Heilongjiang, Liaoning, Sichuan and Guizhou in China. Pleurotus ostreatus sporocarp is fat and tender in meat quality, delicious in taste and rich in nutrition, and is a high-protein low-fat food. Pleurotus ostreatus has high nutritive value and certain dietotherapy value, and its sporophore can reduce blood fat and cholesterol, and its polysaccharide has antitumor activity, can scavenge free radical, and has antioxidant effect. At present, in some areas (such as Hunan, Sichuan and the like), due to the fact that summer temperature is too high, excellent high-temperature pleurotus ostreatus strains are lacked, the number of pleurotus ostreatus strains on the market in summer is short, and the quality is not good, and therefore screening of the high-temperature pleurotus ostreatus strains has certain research value.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide high-temperature-resistant Pleurotus ostreatus.
Another purpose of the invention is to provide a screening method of high-temperature-resistant Pleurotus ostreatus.
The invention also aims to provide a fruiting method of the high-temperature-resistant Pleurotus ostreatus.
Another purpose of the invention is to provide a fermentation method of high-temperature-resistant Pleurotus ostreatus.
Another purpose of the invention is to provide a method for obtaining extracellular crude polysaccharide by using high-temperature-resistant Pleurotus ostreatus.
Another objective of the invention is to provide a method for removing O from crude extracellular polysaccharide obtained from Pleurotus ostreatus with high temperature resistance2 -Use in free radicals.
The invention also aims to provide application of the extracellular crude polysaccharide obtained by the high-temperature-resistant pleurotus ostreatus in removing OH free radicals.
The purpose of the invention is realized by the following technical scheme.
The high-temperature-resistant Pleurotus ostreatus is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 10 months and 18 days, and the preservation number is CGMCC: 16378, classified and named Pleurotus ostreatus.
In the technical scheme, 1 mycelium of the high-temperature-resistant Pleurotus ostreatus is placed on a PDA culture medium and grows for 10-15 days at 30-35 ℃ in the dark, the diameter of the formed colony is 78-82 mm, the mycelium is white, and the back of the aerial mycelium is light brown.
The screening method of the high-temperature-resistant Pleurotus ostreatus comprises the following steps:
before the step 1, cutting oyster mushrooms into small pieces of tissues to obtain a plurality of mushroom tissues;
the volume of the mushroom tissue is 0.3-0.6cm3。
Before cutting oyster mushroom into small pieces of tissues, performing surface disinfection on the oyster mushroom, wherein the surface disinfection method comprises the following steps: soaking in 70-75 vol% ethanol water solution for 90-120s, and washing with sterile water for 4-5 times.
in the step 1, the screening medium is a PDA medium.
In the step 1, when mushroom tissue is placed on a screening medium, a cut surface of the mushroom tissue faces the screening medium.
In the step 1, the pure culture method is to repeat the following steps 4-5 times: collecting hypha from the edge of colony, diluting with liquid PDA culture medium at a ratio of 2, 4, 8 and 16 times to obtain second dilution, spreading 0.5-1mL of the second dilution on screening culture medium, and culturing at constant temperature of 25-28 deg.C until colony with diameter of 0.5-1cm is formed.
Step 2, primary screening of high-temperature-resistant strains
The following operations were performed for each colony of the purified strain: taking bacterium blocks from the bacterial colonies of the purified bacterial strains and inoculating the bacterium blocks to a culture medium, culturing at the constant temperature of 25-28 ℃ for 10-15 days to form first bacterial colonies, taking a plurality of bacterium blocks from the first bacterial colonies, respectively inoculating the bacterium blocks taken from the first bacterial colonies to 1 screening culture medium, respectively culturing at the constant temperature of more than 32 ℃ and less than 38 ℃, and selecting bacterial strains with the bacterial colony diameter of more than 4cm after culturing for 14 days as primary screening bacterial strains;
in the step 2, the method for taking a plurality of bacterium blocks on the first colony comprises the following steps: taking 4-8 bacterium blocks on a circumference with the radius of 2cm by taking the inoculation point of the first bacterium colony as a center;
step 3, re-screening the high-temperature resistant strains
The following operations were performed for each primary screened strain: inoculating a bacterial block on the primary screened strain to a culture medium, culturing at the constant temperature of 25-28 ℃ for 10-15 days to form a second bacterial colony, and taking the bacterial block on the second bacterial colony; inoculating the bacterial block obtained from the second colony to a culture medium and culturing at 40 ℃ for 6 hours; and culturing at 25-28 deg.C for 7-10 days, measuring colony diameter, and culturing at high temperature resistant Pleurotus ostreatus.
In the above technical scheme, the screening medium is a PDA medium, and the medium is a PDA medium.
In the technical scheme, the preparation method of the PDA culture medium comprises the following steps: mixing 220 parts by mass of 180-grade potato blocks with 980 parts by volume of 950-grade water, heating to boil and maintaining boiling for 20-30min, filtering while hot, removing filter residues to obtain filtrate, adding 15-25 parts by mass of glucose and 20-25 parts by mass of agar into the filtrate, heating to melt, and fixing the volume to 1000 parts by volume, wherein the pH value is natural.
In the above technical scheme, when the unit of volume parts is mL, the unit of mass parts is g.
The fruiting method of the high-temperature-resistant Pleurotus ostreatus comprises the following steps: inoculating high temperature-resistant Pleurotus ostreatus to fruiting medium, and culturing at 30-35 deg.C for 20-28 days.
In the technical scheme, the preparation method of the mushroom culture medium comprises the following steps: mixing corn cob, cottonseed hull, wood dust, wheat bran, bean cake powder and water, uniformly stirring, fermenting for 10-15 days to obtain the mushroom culture medium, and sterilizing for use, wherein each 100g of the mushroom culture medium is prepared from 30-60g of corn cob, 15-25g of cottonseed hull, 15-25g of wood dust, 5-15g of wheat bran, 2-8g of bean cake powder and 65-70g of water.
The fermentation method of the high-temperature-resistant Pleurotus ostreatus comprises the following steps:
a) under aseptic conditions, inoculating mycelium of high temperature resistant Pleurotus ostreatus to seed culture medium, and culturing in shaking table at constant temperature of 30-35 deg.C and natural humidity for 96-120 hr to obtain seed solution;
b) under aseptic conditions, inoculating the liquid obtained in the step a) to a fermentation medium according to the inoculation amount of 2-7%, and culturing in a shaking table with constant temperature of 28-32 ℃ and natural humidity for 10-15 days to obtain fermentation liquid.
In the above technical scheme, the rotation speed of the shaking table is 150-190rpm/min, and the humidity is naturally 30-60% of the relative humidity.
In the above technical scheme, the preparation method of the seed culture medium comprises the following steps: mixing 5-15 parts by mass of bran and 10-30 parts by mass of bean cake powder, fixing the volume to 1000 parts by volume with a constant volume solution, and sterilizing for use, wherein the constant volume solution is a mixed solution of water, monopotassium phosphate and magnesium sulfate heptahydrate, the concentration of monopotassium phosphate in the constant volume solution is 1.5-4.5g/L, the concentration of magnesium sulfate heptahydrate is 0.5-2.5g/L, and when the unit of the volume parts is mL, the unit of the mass parts is g.
In the above technical scheme, the preparation method of the fermentation medium comprises the following steps: uniformly mixing 25-60 parts by mass of maltodextrin, 10-25 parts by mass of glycerol, 40-60 parts by mass of yeast peptone, 1.5-4.5 parts by mass of monopotassium phosphate, 0.5-2.5 parts by mass of magnesium sulfate heptahydrate, 1-3 parts by mass of ammonium dihydrogen phosphate, 0.3-1.5 parts by mass of cysteine, 1.5-3.5 parts by mass of methionine and 0.00005-0.001 parts by mass of vitamin B1, fixing the volume to 1000 parts by volume by using deionized water, and sterilizing for use.
In the technical scheme, the content of extracellular crude polysaccharide in the fermentation liquor is 3g/L, and the total sugar content is 2.4 g/L.
The method for obtaining the extracellular crude polysaccharide by using the high-temperature-resistant Pleurotus ostreatus comprises the following steps: centrifuging the fermentation liquid for 10-20min, removing precipitate, adding equal volume of anhydrous ethanol into supernatant, performing alcohol precipitation at 22-27 deg.C for 16-24 hr, filtering, and collecting precipitate as extracellular crude polysaccharide.
In the technical scheme, the centrifugal speed is 3000-6000 rmp/min.
Removal of O from crude extracellular polysaccharide obtained from the high temperature resistant Pleurotus ostreatus2 -Use in free radicals.
In the technical scheme, the crude extracellular polysaccharide is mixed with water to prepare a polysaccharide aqueous solution, and when the mass concentration of the crude extracellular polysaccharide in the polysaccharide aqueous solution reaches 0.45mg/mL, O is contained2 -The clearance was (31.53 ± 1.89)%.
Use of extracellular crude polysaccharide obtained from said thermophilic Pleurotus ostreatus for scavenging OH free radicals.
In the above technical scheme, the crude extracellular polysaccharide is mixed with water to prepare a polysaccharide aqueous solution, and when the mass concentration of the crude extracellular polysaccharide in the polysaccharide aqueous solution reaches 0.45mg/mL, the OH clearance is (22.81 ± 1.27)%.
The invention has the following beneficial effects: the Pleurotus ostreatus has good high temperature resistance, can grow well when cultured at 32 deg.C, and provides a good variety for summer market. The Pleurotus ostreatus fermentation liquor contains abundant extracellular crude polysaccharides, and the extracellular crude polysaccharides have strong antioxidant capacity, and the fermentation liquor has good prospect in antioxidant application.
Drawings
FIG. 1 shows the colony morphology characteristics of the RSL-HM2 strain;
FIG. 2 is a microscopic image of the mycelium of the RSL-HM2 strain;
FIG. 3 is a photograph after fruiting of RSL-HM2 strain;
FIG. 4 is a glucose standard curve;
FIG. 5 shows the ability of polysaccharide to scavenge free radicals in the fermentation broth of the RSL-HM2 strain.
Detailed Description
The experimental procedures in the following examples are, unless otherwise specified, conventional procedures, and can be referred to from the procedures described in biochemistry and related books.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified, and the reagents are of a chemically pure grade and are not designated by the manufacturer.
In the phosphate buffer solution, the first solution is disodium hydrogen phosphate, and the second solution is sodium dihydrogen phosphate.
The biochemical incubator used in the examples described below was Shanghai saltating SPX-150-II, the shaker was Tianjin Ono HNY-2102, and the spectrophotometer was Pujingyou UV spectrophotometer, Tu-1901.
The preparation method of the PDA culture medium comprises the following steps: taking 200g of peeled potatoes, cutting into small pieces (potato pieces), putting into a pot, adding 980ml of water, heating on a heater until boiling, maintaining the boiling for 30min, pouring into a measuring cup, filtering on the measuring cup with 2 layers of gauze while the materials are hot, and removing filter residues to obtain filtrate. Adding 20g of glucose and 25g of agar into the filtrate, heating to melt, fixing the volume to 1000mL, naturally adjusting the pH value, sterilizing at 121 +/-1 ℃ for 30min under high temperature and high pressure, and pouring the mixture into a flat plate for later use.
In the examples described below, the autoclave pressure is about 0.1 MPa. The humidity is about 30% when natural.
The technical scheme of the invention is further explained by combining specific examples.
The high-temperature-resistant Pleurotus ostreatus is preserved in the China general microbiological culture Collection center (CGMCC) in 2018, 10 months and 18 days, and the preservation number is CGMCC: 16378, classified and named Pleurotusostreatus, with the collection address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.
The screening method of the high-temperature-resistant Pleurotus ostreatus comprises the following steps:
Step 2, primary screening of high-temperature-resistant strains
The following operations were performed for each colony of the purified strain: bacterial colonies of the purified strains from eggplant-type bottles with a sterile spatulaTaking 0.5-1cm3Inoculating the bacterial block on PDA culture medium, culturing at 26 deg.C for 14 days to form the first colony, taking 6 0.5-1cm on the circumference with radius of 2cm and the center of the inoculation point on the first colony3The pellet of (1) is prepared by inoculating 6 pellets taken from a first colony on 1 screening medium respectively, placing the 6 pellets in constant temperature culture at different temperatures, observing the growth conditions of hyphae on the pellets every day at the constant temperature culture temperature of 6 screening media of 28 ℃, 30 ℃, 32 ℃, 34 ℃, 36 ℃ and 38 ℃ (the conditions of 28 ℃ and 30 ℃ are used for comparison), measuring and recording the diameter of the colonies, selecting strains with the diameter of more than 4cm after the strains are cultured for 14 days at the temperature of more than 32 ℃ as primary screening strains, obtaining 5 primary screening strains, and sequentially naming the 5 primary screening strains as RSL-HM1, RSL-HM2, RSL-HM3, RSL-HM4 and RSL-HM5 as shown in Table 1. Wherein, the screening culture medium is a PDA culture medium.
TABLE 1
Note: the colonies are neat and the hyphae are dense; the colonies are neat and dense; "+ +" hypha sparse; "+" has growth; "-" no growth.
Step 3, re-screening the high-temperature resistant strains
The following operations were performed for each primary screened strain: taking 0.5-1cm of the bacterial colony of the primary screening strain by using a sterile shovel3Inoculating the bacterial block on PDA culture medium, culturing at 26 deg.C for 15 days to form a second colony, taking 6 0.5-1cm on the circumference with radius of 2cm and the center of the inoculation point on the second colony3The fungal mass of (a); inoculating 6 bacterium blocks obtained by the second bacterial colony to 1 culture medium respectively and culturing under different culture environments, wherein the culture environments are as follows in sequence: culturing at 38 ℃ for 2 hours, at 38 ℃ for 4 hours, at 38 ℃ for 6 hours, at 40 ℃ for 2 hours, at 40 ℃ for 4 hours, and at 40 ℃ for 2 hours6 hours; culturing at 26 ℃ for 7 days after being placed in different culture environments, and then restoring to culture for 7 days, measuring the diameter of a bacterial colony, selecting a strain which is cultured at 40 ℃ for 6 hours in the culture environment and then placed at 26 ℃ for 7 days for restoring to culture, wherein the bacterial colony still can grow is pleurotus ostreatus, in the embodiment, the RSL-HM2 strain is cultured at 40 ℃ for 6 hours and then placed at 26 ℃ for 7 days for restoring to culture, and the growth state after other conditions are better than that of other strains, which indicates that the bacterial strain has strong heat resistance.
TABLE 2
Culture and morphological characterization of RSL-HM2
1. Culture of high temperature resistant RSL-HM2
Passage in a test tube: under aseptic conditions, 1 piece of mycelium of RSL-HM2 strain is taken out from PDA culture medium and placed on slant culture medium of 1 test tube, and cultured in 32 deg.C incubator for 15 days, and after the mycelium grows over the whole slant culture medium surface, it is stored in refrigerator at 4 deg.C for use.
Passage in a eggplant-shaped bottle: under aseptic condition, taking 1 piece of mycelium from the slant culture medium of the test tube, placing on the slant culture medium of 1 eggplant-shaped bottle, placing in an incubator at 32 ℃, culturing for 15 days until the mycelium grows over the surface of the whole slant culture medium, and storing in a refrigerator at 4 ℃ for later use.
The preparation method of the slant culture medium comprises the following steps: cutting 200g of peeled potatoes into small pieces, putting the small pieces into a pot, adding 980mL of water, heating on a heater until the potatoes are boiled, maintaining the temperature for 20min, filtering the small pieces on a measuring cup while the potatoes are hot by using 2 layers of gauze, removing filter residues, adding 20g of glucose and 25g of agar into filtrate, heating and melting the filtrate, fixing the volume to 1000mL, filling the filtrate into corresponding 7 mL/test tube or 70 mL/eggplant bottle, plugging a rubber plug, sterilizing the filtrate at the high temperature of 121 +/-1 ℃ for 30 minutes, and paving an inclined plane for later use.
The RSL-HM2 strain grows faster on PDA culture medium (potato dextrose agar culture medium), 1 piece of mycelium grows for 10 days at 32 ℃ in the dark, the diameter of the colony is about 80mm, the mycelium is white, the aerial mycelium is flourishing, the back is light brown, and the colony morphology is shown in figure 1. The hyphae are observed to be in a central compact and cluster shape under a microscope, the branch hyphae are short and strong, and the photograph of the demethylated blue staining is shown in figure 2.
2. Fruiting test of high temperature resistant RSL-HM2
Fruiting and culturing: under aseptic conditions, the RSL-HM2 strain was inoculated onto mushroom-growing medium in a blue-capped flask, and cultured in an incubator at 32 ℃ for 25 days. The RSL-HM2 strain grows hypha in a blue-cap bottle and then grows mushroom, and the mushroom growth form of the strain is shown in figure 3.
And (3) mushroom culture medium growth: every 100g of mushroom culture medium is prepared by 50g of corncobs, 20g of cottonseed hulls, 20g of sawdust, 10g of wheat bran, 6g of bean cake powder and 70g of water, and the preparation method comprises the following steps: according to the proportion, corncobs, cottonseed hulls, sawdust, wheat bran, bean cake powder and water are mixed uniformly and fermented for 10 days (24 hours per day) to obtain a mushroom culture medium, 60g of the mushroom culture medium is taken and filled into 100mL volumetric flasks, and the flasks are sterilized at the high temperature of 121 +/-1 ℃ for 60 minutes under high pressure and cooled for later use.
Extraction and antioxidation of RSL-HM2 polysaccharide
1. A fermentation method of high temperature resistant Pleurotus ostreatus (RSL-HM2 strain) comprises the following steps:
a) seed bottle culture: aseptically, 3 mycelia (0.5-1 cm) of RSL-HM2 strain were scooped up using aseptic inoculation2) Inoculating to a seed culture medium, and culturing in a shaking table with constant temperature of 32 ℃ and natural humidity (humidity of 30%) for 96h to obtain a seed solution, wherein the rotation speed of the shaking table is 150 rpm/min.
The preparation method of the seed culture medium comprises the following steps: respectively weighing 10g of bran and 20g of bean cake powder into 1000mL triangular bottles, metering the volume to 1000mL by using a constant volume solution, accurately metering 300mL by using a metering cylinder, respectively filling the metering cylinder into a 1L shake flask, covering 4 layers of white cloth, sterilizing at the high temperature of 121 +/-1 ℃ for 30 minutes, and cooling for later use, wherein the constant volume solution is a mixed solution of water, potassium dihydrogen phosphate and magnesium sulfate heptahydrate, the concentration of the potassium dihydrogen phosphate in the constant volume solution is 3.5g/L, and the concentration of the magnesium sulfate heptahydrate is 2 g/L.
b) Fermentation culture: under the aseptic condition, inoculating the seed solution onto a fermentation medium in a fermentation bottle by using a pipette according to the inoculation amount of 5%, and culturing in a natural shaking table at the constant temperature of 30 ℃ and the humidity for 14d to obtain a fermentation solution, wherein the rotation speed of the shaking table is 150 rpm/min.
The preparation method of the fermentation medium comprises the following steps: uniformly mixing 45g of maltodextrin, 20g of glycerol, 50g of yeast peptone, 4g of potassium dihydrogen phosphate, 2g of magnesium sulfate heptahydrate, 1g of ammonium dihydrogen phosphate, 1g of cysteine, 1g of methionine and 1mg of vitamin B1, metering to 1000mL by using deionized water, accurately metering 150mL by using a measuring cylinder, subpackaging in 500mL triangular bottles, covering with four layers of white cloth, sterilizing at the high temperature of 121 +/-1 ℃ for 30 minutes, and cooling for later use.
2. Obtaining crude polysaccharide of fermentation liquor
Centrifuging the fermentation liquor at 4000rmp/min for 10min, removing the precipitate, adding equal volume of anhydrous ethanol into the supernatant, performing alcohol precipitation at 25 ℃ for 18h, filtering, collecting the precipitate as crude extracellular polysaccharide, vacuum drying the crude extracellular polysaccharide at-10 ℃, and weighing to obtain the dry weight of the crude extracellular polysaccharide. The crude extracellular polysaccharide yield was calculated as follows:
crude extracellular polysaccharide yield (g/L) ═ dry weight of crude extracellular polysaccharide (g)/volume of fermentation broth (L) × 1000
The content of crude extracellular polysaccharide in the fermentation broth of RSL-HM2 strain was calculated to be 3 g/L.
3, determination of total sugar content in RSL-HM2 fermentation liquor
(1) Drawing of standard curve
Respectively sucking 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL of glucose standard solution (solvent is water, glucose concentration is 1g/L) into a 25mL colorimetric tube with a plug, adding distilled water to supplement the distilled water to 1.0mL, then adding 1.0mL of phenol aqueous solution (phenol volume concentration is 5%), then quickly adding 5.0mL of concentrated sulfuric acid (adding the concentrated sulfuric acid perpendicular to the liquid level to avoid adherence so as to fully react with the liquid in the colorimetric tube with the plug), and standing for 10 min. Mixing, reacting in 30 deg.C water bath for 20min, measuring absorbance at 490nm, determining glucose standard curve with glucose mass concentration as abscissa and absorbance as ordinate, and obtaining linear regression equation from glucose standard curve as shown in FIG. 4y＝0.0111x-0.0098，R2＝0.9971。
(2) Sample assay
Preparing a fermentation liquid as a sample, diluting the sample with water to different times (between 20 and 200 times) to obtain a sample solution, sucking 1.0mL of the sample solution into a 25mL test tube with a plug, adding 1.0mL of a phenol aqueous solution (the volume concentration of phenol is 5%), rapidly adding 5.0mL of concentrated sulfuric acid, and standing for 10 min. Mixing, reacting in water bath at 30 deg.C for 20min, and measuring absorbance at 490 nm.
Blank control: 1.0mL of pure water was taken in a 25mL stoppered test tube, and 1.0mL of an aqueous phenol solution (5% by volume of phenol) was added to the pure water, followed by rapid addition of 5.0mL of concentrated sulfuric acid and standing for 10 min. Mixing, reacting in water bath at 30 deg.C for 20min, and measuring absorbance at 490 nm.
And substituting the absorbance of the sample solution into a linear regression equation of a glucose standard curve, and calculating to obtain the glucose amount in the sample solution.
The total sugar content is calculated as glucose according to the following formula:
In the formula: c, total sugar content in the sample solution, mg/L;
m is the amount of glucose, μ g, measured for the sample solution;
v-volume of sample solution, mL
And (5) calculating the total sugar content of the sample solution diluted by different times, and calculating the average value.
The total sugar content in the RSL-HM2 strain fermentation liquor is 2.4g/L, and the total sugar content in the extracellular crude polysaccharide is about 80%.
Antioxidant experiment of RSL-HM2 strain polysaccharide
(1) O scavenging by RSL-HM2 polysaccharide2 -Determination of free radicals
Preparing samples to be tested with different extracellular crude polysaccharide concentrations: the crude extracellular polysaccharide of RSL-HM2 strain was formulated into polysaccharide aqueous solutions of different mass concentrations of 0.15mg/mL, 0.20mg/mL, 0.25mg/mL, 0.30mg/mL, 0.35mg/mL, 0.40mg/mL and 0.45mg/mL, respectively. The following operations were performed for each concentration of aqueous polysaccharide solution: 2.4mL of 62.5mmol/L sodium phosphate buffer (pH 7.8) (solvent is water) was aspirated, and 0.20mL of 0.06mmol/L aqueous riboflavin solution, 0.20mL of 30mmol/L aqueous methionine solution, 0.10mL of 0.003mmol/L aqueous ethylenediaminetetraacetic acid (EDTA) solution, 0.050mL of aqueous polysaccharide solution, and 0.20mL of 1.125mmol/L aqueous NBT (nitrotetrazolium chloride) solution (ready for use) were added.
Blank sample: and replacing the polysaccharide water solution in the sample to be detected with sodium phosphate buffer solution as a blank sample for comparison.
And (3) respectively reacting the sample to be detected and the blank sample under 4000Lx sunlight for 25min, measuring the light absorption value under 560nm, repeating for 3 times, and calculating the average value. Expressed as percent (%) clearance:
O2 -clearance ═ a (blank) -a (sample to be tested)]A (blank) x 100%
A (blank sample) is the average value of the absorbance of the blank sample, and A (sample to be measured) is the average value of the absorbance of the sample to be measured.
0.15-0.45mg/mL of isolated crude extracellular polysaccharide couple O of RSL-HM2 strain2 -Has good effect of removing the crude polysaccharide from the extracellular portion of the high temperature resistant Pleurotus ostreatus which can increase O2 -Radical scavenging rate) and the scavenging rate gradually increases with the increase of the extracellular crude polysaccharide mass concentration in the aqueous polysaccharide solution, and the results are shown in fig. 5, when the extracellular crude polysaccharide concentration in the aqueous polysaccharide solution reaches 0.45mg/mL, O2 -The clearance was (31.53 ± 1.89)%.
(2) Determination of scavenging of OH free radicals by RSL-HM2 polysaccharide
The crude extracellular polysaccharide of RSL-HM2 strain was formulated into polysaccharide aqueous solutions of different mass concentrations of 0.15mg/mL, 0.20mg/mL, 0.25mg/mL, 0.30mg/mL, 0.35mg/mL, 0.40mg/mL and 0.45mg/mL, respectively. 0.20mL of FeSO was taken4EDTA Mixed aqueous solution (FeSO)4FeSO and EDTA in a molar ratio of 1:14To a concentration of 10mmol/L), 0.50mL of α -deoxyribose aqueous solution (10mmol/L) was added, followed by 0.05mL of polysaccharide aqueous solution, and phosphate buffer (pH)7.4, 0.1mol/L) to 1.8mL, and finally adding 0.20mL of H2O2Uniformly mixing an aqueous solution (10mmol/L), keeping the temperature in a water bath at 37 ℃ for 1h, then adding 1.00mL of a 2.8% (w/w) trichloroacetic acid (TCA) aqueous solution and 1.00mL of a 1.0% (w/w) thiobarbituric acid (TBA) aqueous solution, uniformly mixing, heating in a boiling water bath for 15min, cooling to room temperature of 20-25 ℃, measuring an absorbance value As of the solution at a wavelength of 532nm by using a spectrophotometer, measuring an absorbance value Ac when no polysaccharide aqueous solution is added, replacing the polysaccharide aqueous solution with a phosphate buffer solution (pH7.4, 0.1mol/L) for a blank sample, and expressing the measured absorbance value As Ao, wherein the free radical Scavenging capacity (Scavengiactivity SA) of the polysaccharide aqueous solution can be expressed As:
SA(％)＝[1-(As-Ao)/(Ac-Ao)]×100
the separated crude extracellular polysaccharide of RSL-HM2 strain has better effect of removing OH (the removal rate of OH free radicals can be improved by the crude extracellular polysaccharide of high temperature resistant Pleurotus ostreatus), and the removal rate is gradually increased from 0.15-0.45mg/mL along with the increase of the mass concentration of the crude extracellular polysaccharide in the polysaccharide aqueous solution, and the result is shown in FIG. 5, when the mass concentration of the crude extracellular polysaccharide in the polysaccharide aqueous solution reaches 0.45mg/mL, the removal rate of OH is (22.81 +/-1.27)%.
In conclusion, the crude extracellular polysaccharide obtained from the RSL-HM2 strain of the present invention has a high ability to scavenge free radicals.
The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.
Claims (8)
1. The high-temperature-resistant Pleurotus ostreatus is characterized in that the preservation number is CGMCC: 16378.
2. the method for producing Pleurotus ostreatus according to claim 1, comprising the steps of: inoculating high temperature-resistant Pleurotus ostreatus to fruiting medium, and culturing at 30-35 deg.C for 20-28 days.
3. The method for fermenting pleuromus ostreatus according to claim 1, comprising the steps of:
a) under aseptic conditions, inoculating mycelium of high temperature resistant Pleurotus ostreatus to seed culture medium, and culturing in shaking table at constant temperature of 30-35 deg.C and natural humidity for 96-120 hr to obtain seed solution;
b) under aseptic conditions, inoculating the liquid obtained in the step a) to a fermentation medium according to the inoculation amount of 2-7%, and culturing in a shaking table with constant temperature of 28-32 ℃ and natural humidity for 10-15 days to obtain fermentation liquid.
4. The method for obtaining crude extracellular polysaccharide of pleuromus ferrugineus according to claim 1, comprising the steps of: centrifuging the fermentation liquid of the high-temperature-resistant Pleurotus ostreatus for 10-20min, removing the precipitate, adding the supernatant into equal volume of anhydrous ethanol, performing alcohol precipitation at 22-27 deg.C for 16-24h, filtering, and collecting the precipitate as extracellular crude polysaccharide.
5. Removal of O from crude extracellular polysaccharide obtained from Pleurotus ostreatus of claim 12 -Use in free radicals.
6. The use of claim 5, wherein the crude extracellular polysaccharide is mixed with water to prepare an aqueous polysaccharide solution, and when the crude extracellular polysaccharide concentration in the aqueous polysaccharide solution reaches 0.45mg/mL, O is present2 -The clearance was 31.53 ± 1.89%.
7. Use of crude extracellular polysaccharide obtained from Pleurotus ostreatus of claim 1 for scavenging OH radicals.
8. The use according to claim 7, wherein the crude extracellular polysaccharide is mixed with water to prepare an aqueous polysaccharide solution, and when the crude extracellular polysaccharide concentration in the aqueous polysaccharide solution reaches 0.45mg/mL, the. OH clearance is 22.81 ± 1.27%.
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