CN110711203A - Application of cordycepin polysaccharide in preparation of medicament for resisting gram-negative plant pathogenic bacteria - Google Patents
Application of cordycepin polysaccharide in preparation of medicament for resisting gram-negative plant pathogenic bacteria Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a method for preparing refined polysaccharide with bacteriostatic activity by using cordyceps culture and application thereof. The invention takes cordyceps culture as raw material, extracts crude polysaccharide, and adopts a means of combining a macroporous resin adsorption method and a Sevag method to remove protein in the crude polysaccharide to obtain refined polysaccharide. And further carrying out step-by-step alcohol precipitation to obtain 95% ethanol-precipitated cordycepin polysaccharide CP95 with inhibitory activity on three gram-negative plant pathogenic bacteria. The preparation method has the characteristics of simple operation, stable performance and low cost, and has important economic significance for better developing and utilizing the cordyceps culture and reducing the cleaning cost of the waste cordyceps culture medium.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for preparing refined polysaccharide with bacteriostatic activity by using cordyceps culture and application thereof.
Background
Cordyceps (Cordyceps) is a complex entomomycete complex, including Cordyceps sobolifera, Cordyceps sinensis, Cordyceps militaris and the like, contains various bioactive components, and has important health-care and medicinal values. Cordyceps cicadae (Ophiocerdyceps Sobolifer a, Isaria cicadae, asexual generation) is an entomomycete complex formed by infecting fungus of Clavipitaceae with nymphaea cicadae, and asexual fruiting body (named as peduncle) formed by head hypha is similar to flower bud, so that the Cordyceps cicadae is named as Cordyceps cicadae and is a high-quality Cordyceps sinensis with a long medicinal history in traditional Chinese medicine. Researches show that the Cordyceps sobolifera contains various bioactive substances, the main components of the Cordyceps sobolifera comprise Cordyceps Polysaccharide (CP), cordycepin, cordycepic acid, sterol, uracil, vitamins, alkaloids, inorganic salt mineral elements and the like, and the Cordyceps sobolifera has multiple effects of regulating immunity, resisting fatigue, protecting kidney, improving sleep, resisting tumor, protecting liver, resisting radiation, improving eyesight and the like. Cordyceps (Ophiocerdyceps sinensis) is a complex of fungal stroma developed after the Cordyceps fungi parasitize on the larva body of the hepialus armoricanus and dead larva filled with hyphae, contains cordycepin, Cordyceps polysaccharide, sterol, amino acids, etc., and has effects of immunoregulation, anticancer, endocrine regulation, antibacterial, hematopoiesis promotion, antivirus, liver protection, sedation and anticonvulsion. Cordyceps militaris (Cordyceps militaris L Link) also called Cordyceps militaris and Cordyceps militaris has similar active ingredients and pharmacological action to Cordyceps militaris.
Natural cordyceps is very rare, and in recent years, the development of artificial culture realizes large-scale industrial cultivation of cordyceps. The cordyceps culture medium is prepared by taking grain crops such as rice, wheat and the like as main raw materials, inoculating cordyceps strains, and performing solid fermentation culture to obtain a cordyceps culture, wherein the culture comprises two parts of asexual sporocarp at the upper part and culture residue at the bottom part, and the culture residue consists of transformed culture material, mycelium and conidiospore. In practical application, the asexual fruiting bodies of cordyceps sinensis are generally picked and developed as top quality, so that a large number of culture residues are generated. Detection shows that the culture residual gene after the fruiting body is harvested contains a large amount of mycelia and spores, is rich in adenosine, crude fat, crude protein, crude fiber, polysaccharide, amino acid, calcium, magnesium, potassium, linoleic acid and other substances, and also has a certain application value.
Disclosure of Invention
The invention discloses a method for preparing refined polysaccharide with bacteriostatic activity by using cordyceps culture and application thereof. The invention takes cordyceps culture as raw material, extracts crude polysaccharide, and adopts a means of combining a macroporous resin adsorption method and a Sevag method to remove protein in the crude polysaccharide to obtain refined polysaccharide. Further adopts fractional alcohol precipitation purification, and discloses the application of the 95 percent ethanol precipitation cordycepin polysaccharide extracted from the cordyceps culture in the aspect of inhibiting gram-negative plant pathogenic bacteria.
One of the purposes of the invention is to provide a method for preparing refined polysaccharide with bacteriostatic activity by using cordyceps culture.
Preferably, the method comprises the following steps:
(1) extracting crude polysaccharide: drying a proper amount of cordyceps culture at 70 ℃, adding 10 times of 50% ethanol according to the mass ratio of the culture to the volume of the extracting solution, extracting twice at 80 ℃, stirring for 0.5h each time, preserving heat for 2h, and filtering to obtain 50% ethanol extracting solution; adding 10 times of water solution into the residue after twice extraction with 50% ethanol, extracting at 100 deg.C, stirring for 0.5 hr, keeping the temperature for 1 hr, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide, respectively. Mixing 50% ethanol extraction precipitated polysaccharide and water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps crude polysaccharide;
(2) deproteinizing the crude polysaccharide: dissolving the crude polysaccharide with deionized water, loading the prepared crude polysaccharide solution on macroporous resin, eluting with 3 times volume of deionized water, and collecting the eluate; uniformly mixing the collected eluent and Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at 4000rpm, placing the mixture in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, and repeating the operation for 2 times; concentrating the water phase layer to obtain refined polysaccharide CP;
(3) ethanol precipitation by steps: uniformly stirring the concentrated solution of the refined polysaccharide with ethanol with the final concentration of 50% in volume ratio, standing at 4 ℃ overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and freeze-drying the precipitate at low temperature to obtain CP 50; regulating the volume of ethanol in the supernatant to 75%, standing overnight at 4 deg.C, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 75; concentrating and spin-drying the supernatant, dissolving with 95% ethanol, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 95.
Preferably, the method comprises the following steps:
(1) extracting crude polysaccharide: drying a proper amount of cordyceps culture at 70 ℃, adding 10 times of 50% ethanol according to the mass ratio of the culture to the volume of the extracting solution, extracting twice at 80 ℃, stirring for 0.5h each time, preserving heat for 2h, and filtering to obtain 50% ethanol extracting solution; adding 10 times of water solution into the residue after twice extraction with 50% ethanol, extracting at 100 deg.C, stirring for 0.5 hr, keeping the temperature for 1 hr, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide, respectively. Mixing 50% ethanol extraction precipitated polysaccharide and water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps crude polysaccharide;
(2) deproteinizing the crude polysaccharide: dissolving the crude polysaccharide powder by using deionized water, uniformly mixing the prepared crude polysaccharide solution and a Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at 4000rpm, placing in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, and repeating the operation for 3 times; loading the aqueous phase layer treated by the Sevag method to macroporous resin, eluting by using deionized water with the volume of 3 times, and concentrating the aqueous phase to obtain refined polysaccharide CP;
(3) ethanol precipitation by steps: uniformly stirring the concentrated solution of the refined polysaccharide with ethanol with the final concentration of 50% in volume ratio, standing at 4 ℃ overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and freeze-drying the precipitate at low temperature to obtain CP 50; regulating the volume of ethanol in the supernatant to 75%, standing overnight at 4 deg.C, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 75; concentrating and spin-drying the supernatant, dissolving with 95% ethanol, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 95.
Preferably, the CP95 has use in the preparation of a medicament for combating gram-negative phytopathogenic bacteria.
Preferably, the gram-negative phytopathogen is a rice bacterial brown streak RS-1 strain, a tomato bacterial wilt MYA-1 strain or a sweet potato stem rot Dd-1 strain.
Preferably, the cordyceps culture comprises cordyceps cicadae miq, cordyceps sinensis or cordyceps militaris and the like.
Preferably, the cordyceps culture comprises a culture residue, a fruit body, a liquid fermentation mycelium or a liquid fermentation mycelium filtrate.
The invention also aims to provide application of the 95% ethanol precipitated cordycepin polysaccharide CP95 in preparing a medicament for resisting gram-negative plant pathogenic bacteria.
Preferably, the CP95 is prepared from Cordyceps culture.
Preferably, the preparation method of the CP95 comprises the following steps:
(1) extracting crude polysaccharide: drying a proper amount of cordyceps culture at 70 ℃, adding 10 times of 50% ethanol according to the mass ratio of the culture to the volume of the extracting solution, extracting twice at 80 ℃, stirring for 0.5h each time, preserving heat for 2h, and filtering to obtain 50% ethanol extracting solution; adding 10 times of water solution into the residue after twice extraction with 50% ethanol, extracting at 100 deg.C, stirring for 0.5 hr, keeping the temperature for 1 hr, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide, respectively. Mixing 50% ethanol extraction precipitated polysaccharide and water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps crude polysaccharide;
(2) deproteinizing the crude polysaccharide: dissolving the crude polysaccharide with deionized water, loading the prepared crude polysaccharide solution on macroporous resin, eluting with 3 times volume of deionized water, and collecting the eluate; uniformly mixing the collected eluent and Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at 4000rpm, placing the mixture in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, and repeating the operation for 2 times; concentrating the water phase layer to obtain refined polysaccharide CP;
(3) ethanol precipitation by steps: uniformly stirring the concentrated solution of the refined polysaccharide with ethanol with the final concentration of 50% in volume ratio, standing at 4 ℃ overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and freeze-drying the precipitate at low temperature to obtain CP 50; regulating the volume of ethanol in the supernatant to 75%, standing overnight at 4 deg.C, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 75; concentrating and spin-drying the supernatant, dissolving with 95% ethanol, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 95.
Preferably, the preparation method of the CP95 comprises the following steps:
(1) extracting crude polysaccharide: drying a proper amount of cordyceps culture at 70 ℃, adding 10 times of 50% ethanol according to the mass ratio of the culture to the volume of the extracting solution, extracting twice at 80 ℃, stirring for 0.5h each time, preserving heat for 2h, and filtering to obtain 50% ethanol extracting solution; adding 10 times of water solution into the residue after twice extraction with 50% ethanol, extracting at 100 deg.C, stirring for 0.5 hr, keeping the temperature for 1 hr, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide, respectively. Mixing 50% ethanol extraction precipitated polysaccharide and water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps crude polysaccharide;
(2) deproteinizing the crude polysaccharide: dissolving the crude polysaccharide powder by using deionized water, uniformly mixing the prepared crude polysaccharide solution and a Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at 4000rpm, placing in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, and repeating the operation for 3 times; loading the aqueous phase layer treated by the Sevag method to macroporous resin, eluting by using deionized water with the volume of 3 times, and concentrating the aqueous phase to obtain refined polysaccharide CP;
(3) ethanol precipitation by steps: uniformly stirring the concentrated solution of the refined polysaccharide with ethanol with the final concentration of 50% in volume ratio, standing at 4 ℃ overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and freeze-drying the precipitate at low temperature to obtain CP 50; regulating the volume of ethanol in the supernatant to 75%, standing overnight at 4 deg.C, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 75; concentrating and spin-drying the supernatant, dissolving with 95% ethanol, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 95.
Preferably, the gram-negative phytopathogen is a rice bacterial brown streak RS-1 strain, a tomato bacterial wilt MYA-1 strain or a sweet potato stem rot Dd-1 strain.
Preferably, the cordyceps culture comprises cordyceps cicadae miq, cordyceps sinensis or cordyceps militaris and the like.
Preferably, the cordyceps culture comprises a culture residue, a fruit body, a liquid fermentation mycelium or a liquid fermentation mycelium filtrate.
The invention provides a method for preparing refined polysaccharide with bacteriostatic activity by using cordyceps culture and application thereof. The preparation method has the characteristics of simple operation, stable performance and low cost, and the protein removal method combining the Sevag method and the macroporous resin adsorption method can reduce the protein removal times of the Sevag method by 3-4 times, thereby not only saving the use amount of the Sevag reagent and reducing the treatment cost, but also effectively removing the protein in the polysaccharide, and the protein removal rate reaches more than 96 percent, thereby laying a good foundation for further separation and purification of polysaccharide components and research on physiological functions. The cordyceps sinensis essential polysaccharide CP95 prepared by the invention has stronger inhibiting effect on three important gram-negative plant pathogenic bacteria which are harmful and difficult to prevent and treat in agricultural production, and the antibacterial activity of the cordyceps sinensis essential polysaccharide CP95 is obviously higher than 5% of amino-oligosaccharide and 5% of chitosan, so that the cordyceps sinensis essential polysaccharide has important economic significance for better developing and utilizing cordyceps sinensis cultures and reducing the cleaning cost of cordyceps sinensis waste culture media.
Drawings
FIG. 1 is a schematic diagram of the preparation process of refined polysaccharide with bacteriostatic activity by using Cordyceps culture.
FIG. 2 is a graph showing the effect of deproteinization of crude polysaccharide, in which (A) shows deproteinization by Sevag method, (B) shows deproteinization by macroporous resin adsorption method, (C) shows deproteinization by Sevag method after macroporous resin adsorption, and (D) shows deproteinization by Sevag method before macroporous resin adsorption.
FIG. 3 is the elution curve of macroporous resin in Cordyceps cicadae polysaccharide solution, wherein (A) and (B) show the change of polysaccharide content, and (C) and (D) show the change of protein content.
FIG. 4 is a graph of the bacteriostatic effect of cordyceps cicadae miq polysaccharide on rice bacterial brown streak fungus RS-1, wherein 1 represents 2000mg/mL cordyceps cicadae polysaccharide solution, 2 represents 1000mg/mL cordyceps cicadae polysaccharide solution, 3 represents 500mg/mL cordyceps cicadae polysaccharide solution, 4 represents 100mg/mL cordyceps cicadae polysaccharide solution, and 5 represents negative control sterilized water.
FIG. 5 is a graph showing the inhibitory effect of OSP95 on three gram-negative plant pathogens, wherein (A) is the inhibitory effect of OSP95 on rice bacterial brown streak RS-1 strain, (B) is the inhibitory effect of OSP95 on tomato bacterial wilt MYA-1 strain, (C) is the inhibitory effect of OSP95 on sweet potato stem rot Dd-1 strain, 1 represents 25mg/ml OSP95-A, 2 represents 80mg/ml OSP95-A, 3 represents 20mg/ml OSP95-B, 3' represents 40mg/ml 95-B, 4 represents 40mg/ml 5% amino-oligosaccharin, 5 represents sterilized distilled water, and 6 represents 20mg/ml 5% OSP.
FIG. 6 is a graph showing the effect of different concentrations of 5% amino-oligosaccharin and OSP95 on bacterial inhibition and growth of tomato bacterial wilt MYA-1 strain, wherein (A) is 5% amino-oligosaccharin with different concentrations, (B) is OSP95-A with different concentrations, and (C) is OSP95-B with different concentrations, and 0-6 respectively represent concentrations of 0, 0.625mg/ml, 1.25mg/ml, 2.5mg/ml, 5mg/ml, 10mg/ml and 20 mg/ml.
Detailed Description
The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the present invention, all the equipment, materials and the like are commercially available or commonly used in the industry, if not specified. Unless otherwise indicated, the examples employ methods that are within the ordinary skill in the art.
The cordyceps sobolifera culture medium used in the invention adopts wheat as a main material, and the strain adopted in the artificial cultivation of cordyceps sobolifera is cordyceps sobolifera (O.sobolifera)022007-9 strain, is obtained by separation of plant protection and entomogenous fungi research laboratories of subtropical crop research institute in Zhejiang province, and is stored in China general microbiological culture Collection center (CGMCC No: 8989).
The LB solid culture medium used in the invention consists of 5.0g of yeast powder, 10.0g of peptone, 5.0g of NaCl, 17.0g of agar powder, 1000mL of distilled water, pH 7.0-7.2, high-pressure steam sterilization (121 ℃, 20 min); the NA solid medium consists of 3.0g of beef extract, 1g of yeast extract, 5g of peptone, 10g of glucose (or sucrose), 15g of agar powder and 1000mL of distilled water, and the pH value is 6.8-7.0. The rice bacterial brown streak (Acidovorax avenae sub sp. avenae) RS-1 strain used in the invention is given by professor plum, a institute of biotechnology, Zhejiang university, sweet potato stem rot (Dickeya dadantii) Dd-1 strain is given by microorganisms of the institute of agricultural sciences, Zhejiang and a Cayan researchers of the institute of plant protection, and the tomato bacterial wilt (Ralstoniasolanacearum) MYA-1 strain is collected by a research laboratory of the institute of subtropical crops, Zhejiang and is separated from a tomato bacterial wilt plant disease strain in the Town of Thyama. The Sevag reagent is prepared by mixing chloroform and n-butanol at a volume ratio of 4:1, and macroporous adsorption resin AB-8 is purchased from Cangzhou Baoyin adsorption material science and technology Limited.
Example 1 extraction of crude polysaccharide from Cordyceps cicadae Miq culture residue and measurement of polysaccharide content
Taking a certain amount of culture residue obtained after picking cordyceps sobolifera sporocarp, drying the culture residue in a 70-DEG C oven, feeding the culture residue into a 10-ton extraction tank, adding 50% ethanol in an amount which is 10 times the mass of the culture residue and the volume of an extracting solution, heating the mixture to a micro-boiling state (80-90 ℃), stirring the mixture for 0.5h, preserving the heat for 2h, filtering the mixture to obtain an ethanol extracting solution, continuously adding 50% ethanol in an amount which is 10 times the mass of the filter residue into the filter residue for secondary extraction, and combining the two filtrates to; adding 10 times of water solution into the filter residue after two times of extraction with 50% ethanol, heating to boiling (90-100 deg.C), stirring for 0.5h, keeping the temperature for 1h, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide. Mixing the 50% ethanol extraction precipitated polysaccharide and the water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps cicadae crude polysaccharide powder.
During the culture process of the cordyceps sinensis, the main materials constituting the culture medium are converted and utilized by the cordyceps sinensis, so that the extracted polysaccharide mainly originates from cordyceps sinensis mycelia and conidia.
And detecting the polysaccharide content by adopting a phenol-sulfuric acid method. Accurately weighing 100mg of anhydrous glucose dried to constant weight to prepare 0.1 mg/mL of standard glucose solution-1Respectively sucking 0mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0mL of standard glucose solution, placing the standard glucose solution in a test tube, adding 1.0mL of distilled water in each tube, respectively adding 1mL of 5% phenol reagent, uniformly mixing, then quickly adding 5.0mL of concentrated sulfuric acid, standing for 10min, shaking and mixing, placing the mixture in a water bath at 30 ℃ for reaction for 20min, measuring the absorbance at 490nm by using an ultraviolet spectrophotometer, and preparing a standard curve by using the glucose concentration and the absorbance as horizontal and vertical coordinates to obtain a regression equation. Respectively taking a certain amount of 50% alcohol extraction precipitated polysaccharide and water extraction precipitated polysaccharide to dissolve in water to obtain sample solutions, and measuring the polysaccharide content of 1mL of the sample solutions according to the method. The content of 50% alcohol extraction precipitation polysaccharide is 44.7%, and the content of water extraction concentration precipitation polysaccharide is 45.3%.
The yield of the crude polysaccharide prepared by utilizing cordyceps sobolifera culture residues is calculated to be 14.2% by weighing the obtained crude polysaccharide powder.
Example 2 preparation of Cordyceps cicadae Miquel Cordyceps sinensis extract polysaccharide
The crude polysaccharide is deproteinized by adopting a Sevag method, a macroporous resin adsorption method and a method combining the Sevag method and the macroporous resin adsorption method respectively to obtain the refined polysaccharide.
1. Sevag deproteinization
Dissolving the crude polysaccharide powder by using deionized water according to the mass concentration ratio of 5%, uniformly mixing the prepared crude polysaccharide solution and Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at the rotating speed of 4000rpm, and separating in a separating funnel to remove the middle denatured protein layer and the lower organic solution layer. Repeating the above operation for multiple times until the middle denatured protein layer is not appeared, and determining the protein content each time the sample is left.
Protein content determination using coomassie brilliant blue method: preparing Bovine Serum Albumin (BSA) standard solution of 1 mg/mL -10, 0.02, 0.04, 0.06, 0.08 and 0.1mL of standard BSA were respectively sucked, added to 1.0mL with distilled water, shaken well, added with 5mL of Coomassie brilliant blue G-250 solution, left for 30min, and then the absorbance at 595nm was measured with an ultraviolet spectrophotometer. The regression equation was obtained by plotting A595 against the milligrams of BSA. A sample solution of 1.0mL in an appropriate concentration was taken and measured in the manner described above. Wherein the protein removal rate calculation formula is as follows:
wherein: m0Is the protein mass of the sample before treatment, M1Is the protein content of the treated sample.
As can be seen from FIG. 2A, the protein removal rate of the cordyceps sobolifera crude polysaccharide after deproteinization for 1 time by the Sevag method is about 40%, the protein removal rate for 2 times is about 79%, the protein removal rate for 3 times is about 80%, and the effect is not obvious after 4-6 times, which indicates that the Sevag method cannot completely remove the protein in the cordyceps sobolifera crude polysaccharide.
2. Deproteinization by macroporous resin adsorption method
A weak-polarity macroporous adsorption resin AB-8 is selected and filled into a column (the diameter of the column is 50mm multiplied by 600 mm). Pretreating and regenerating macroporous resin: the pretreatment is carried out by soaking for 12h with 95% ethanol, washing with deionized water until no alcohol smell exists, soaking for 4h with 5% hydrochloric acid solution by mass, and washing with deionized water until the pH value is about 6.0; then soaking the mixture for 4 hours by using a sodium hydroxide solution with the mass fraction of 2 percent, and then washing the mixture by using deionized water until the pH value is about 8.0. Putting the cordyceps sobolifera crude polysaccharide solution into a column, eluting by using deionized water with 3 times of volume, and collecting eluent to measure protein content; the collected eluates were further loaded onto a column and the protein contents thereof were measured, respectively.
As can be seen from FIG. 2B, the protein removal rate of the cordyceps sobolifera culture medium crude polysaccharide after being subjected to the adsorption deproteinization for 1 time by the macroporous resin is about 86%, and no obvious effect is obtained when the eluent is continuously applied to the column for 1 time, which indicates that the macroporous resin adsorption method cannot completely remove the protein in the cordyceps sobolifera culture medium crude polysaccharide.
3. Deproteinizing by macroporous resin adsorption method, and deproteinizing by Sevag method (method one)
Dissolving the crude polysaccharide powder by using deionized water according to the mass concentration ratio of 5%, loading the prepared crude polysaccharide solution on macroporous resin, eluting by using deionized water with the volume of 3 times, and collecting eluent to measure the protein content. Mixing the collected eluent and Sevag reagent uniformly according to a volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at a rotating speed of 4000rpm, placing the mixture in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, repeating the operation for 2 times, and collecting an aqueous phase layer each time to measure the protein content. As can be seen from FIG. 2C, the protein removal rate after 1 time of desorption by macroporous resin adsorption is about 86%, the protein removal rate after 1 time of desorption by Sevag method reaches 96%, and the protein removal rate after 2 times of desorption by Sevag method reaches more than 97%, which indicates that the protein in the crude polysaccharide is almost completely removed. In addition, the color of the polysaccharide adsorbed by the macroporous resin is changed from brown to yellow brown, and the decolorization is obvious.
And concentrating the water phase layer after the protein is effectively removed to obtain the cordyceps soboliferｃA extract polysaccharide OSP, wherein the OSP is named as OSP-A.
4. Deproteinizing by Sevag method, and deproteinizing by macroporous resin adsorption method (method two)
Dissolving the crude polysaccharide powder with deionized water according to the mass concentration ratio of 5%, uniformly mixing the prepared crude polysaccharide solution and Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at the rotating speed of 4000rpm, placing the mixture in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, repeating the operation for 3 times, and collecting an aqueous phase layer for protein content determination; and (3) loading the aqueous phase layer treated by the Sevag method to macroporous resin, eluting by using deionized water with 3 times of volume, and collecting eluent to determine the protein content. As can be seen from FIG. 2D, the protein removal rate after 3 times of Sevag method removal was about 80%, and the protein removal rate reached 98% by once macroporous resin adsorption, indicating that the protein in the crude polysaccharide was almost completely removed. In addition, the color of the polysaccharide adsorbed by the macroporous resin is changed from brown to yellow brown, and the decolorization is obvious.
And concentrating the water phase after the protein is effectively removed to obtain cordyceps sobolifera extract polysaccharide OSP, wherein the OSP is named as OSP-B.
Example 3 two treatment methods combining macroporous resin adsorption method and Sevag method for Cordyceps cicadae Miquel polysaccharide
Influence of (2)
A, B two processing groups are set, wherein, the processing group A is that crude polysaccharide solution without deproteinization is directly loaded on macroporous resin, the processing group B is that polysaccharide solution collected after deproteinization for 3 times by utilizing Sevag method is loaded on macroporous adsorption resin, deionized water with 3 times volume, 20% ethanol and 70% ethanol are used for elution in sequence, finally 95% ethanol is used for cleaning, eluent is collected, 250ml of eluent is collected in each part, and the content of polysaccharide and protein in the collected solution are respectively measured to obtain a polysaccharide elution curve.
From the polysaccharide change curves in fig. 3A and fig. 3B, it can be seen that the polysaccharides of the treatment groups a and B can be separated by macroporous resin adsorption and elution to obtain two main polysaccharide display peaks at the water phase portion and the 20% ethanol portion, and a polysaccharide small peak at the 70% ethanol portion, and the separated polysaccharide peaks have similar types, indicating whether the crude polysaccharide is deproteinized by Sevag method before loading on the macroporous resin, and the influence on the polysaccharide components is not large, and also indicating that the deproteinization by Sevag method has mild effect on the polysaccharides. And it can also be seen that the cordyceps sobolifera polysaccharide component is very soluble in water and is most likely to have a glycoprotein component.
As can be seen from the protein change curve 3C and FIG. 3D corresponding to the A, B treatment group, the protein fraction separated in the A treatment group was more abundant and more heterogeneous than that in the B treatment group, and a small amount of protein was detected in both major polysaccharide fractions separated in each of the two treatment groups, i.e., the aqueous phase fraction and the 20% ethanol fraction, wherein a portion of free protein was also clearly heterogeneous in both polysaccharide fractions separated in the A treatment group. The macroporous resin achieves better protein removing effect by different adsorption degrees of different proteins mixed in the macroporous resin in the separation process of polysaccharide, and the Sevag method can remove most of free proteins in crude polysaccharide. Therefore, the polysaccharide components of the crude polysaccharide which is not deproteinized by the Sevag method and is directly separated by the macroporous resin need to be subjected to the Sevag method to remove part of free protein, so that the ideal deproteinizing effect can be achieved.
Example 4 stepwise alcohol precipitation of Cordyceps sobolifera extract polysaccharide
And (2) carrying out ethanol precipitation on the cordyceps soboliferｃA extract polysaccharide OSP-A and the cordyceps soboliferｃA extract polysaccharide OSP-B obtained in the embodiment 2 sequentially by using 50%, 75% and 95% ethanol in volume ratio step by step, namely uniformly stirring the extract polysaccharide concentrated solution by using the ethanol with the final volume concentration of 50%, standing overnight at 4 ℃, carrying out centrifugal separation (8000r/min, 15min), collecting precipitates, freeze-drying at low temperature, adjusting the volume of the ethanol in the supernatant to 75%, standing overnight at 4 ℃ for centrifugation (8000r/min, 15min), collecting the precipitates, freeze-drying the precipitates at low temperature, concentrating and spin-drying the supernatant, dissolving the precipitates by using 95% ethanol, standing overnight at 4 ℃ for centrifugation (8000r/min, 15min), collecting the precipitates, and freeze-drying at low temperature. The three-stage alcohol precipitation collected precipitates are cordyceps sobolifera extract polysaccharides with three molecular weight segments, which are respectively marked as 50% ethanol precipitation cordyceps sobolifera extract polysaccharides OSP50 (named as OSP50-A and OSP50-B), 75% ethanol precipitation cordyceps sobolifera extract polysaccharides OSP75 (named as OSP75-A and OSP75-B) and 95% ethanol precipitation cordyceps sobolifera extract polysaccharides OSP95 (named as OSP95-A and OSP 95-B).
Example 5 bacteriostatic effect of Cordyceps cicadae Miquel polysaccharide on Rice bacterial brown streak disease RS-1
Melting the solid LB medium, cooling to about 45 deg.C, adding 30 μ L of test bacteriogenic bacteria solution (OD600 ═ 0.3) respectively, mixing, embedding 5 Oxford cups in each plate before the bacterial plates are solidified, and taking out the culture medium in the Oxford cups and the round holes by using tweezers after the bacterial plates are solidified. And respectively adding 100 mu L of cordyceps sobolifera crude polysaccharide solutions of 100mg/mL, 500mg/mL, 1000mg/mL and 2000mg/mL into the round holes, taking sterile water treatment as a control, repeating for 3 times, placing the sample in an incubator at 30 ℃ for culturing for 48h after the sample is completely diffused at room temperature, taking out the sample, observing and measuring the diameter of the inhibition zone.
As can be seen from FIG. 4, the cordyceps sobolifera crude polysaccharide (the concentration is more than or equal to 100mg/mL) has a certain degree of inhibition effect on rice bacterial brown streak germ RS-1, and the bacteriostatic effect and the crude polysaccharide concentration are in positive correlation; during high concentration (2000mg/mL), can reach better antibacterial effect, smooth in the antibacterial zone, basically there is not the bacterial colony to appear, but along with the reduction of polysaccharide concentration, antibacterial effect weakens, shows that antibacterial zone diminishes and marginal definition variation, and along with the extension of culture time, bacterial colony quantity also constantly increases in the antibacterial zone, and negative control sterile water treatment then does not appear antibacterial zone.
Example 6 ethanol precipitation of Cordyceps sobolifera extract polysaccharide, crude polysaccharide, amino-oligosaccharin and Cordyceps sobolifera extract polysaccharide with three molecular weight ranges
Bacteriostatic effect of chitosan
Melting solid LB and NA culture media, cooling to about 45 deg.C, adding bacterial growth-testing liquid (OD)6000.3)30 μ L (wherein, the rice bacterial brown streak RS-1 strain is inoculated in LB culture medium, the sweet potato stem rot Dd-1 strain and the tomato bacterial wilt MYA-1 strain are inoculated in NA culture medium), mixing, before bacterial plate solidification, each plate is embedded with 5 Oxford cups, after solidification, the culture medium in the Oxford cups and the round holes is taken out by tweezers. And respectively adding 100 mu L of cordyceps sobolifera polysaccharide solution into the round holes, repeating the experiment for 3 times, placing the sample in an incubator at 30 ℃ for culturing for 48 hours after the sample is completely diffused at room temperature, taking out the sample, observing and measuring the diameter of the bacteriostatic zone. And sterilized water was used as a negative control, and a 5% amino-oligosaccharin aqueous solution and a chitosan aqueous solution were used as positive controls, and the experimental results are shown in the following table:
TABLE 1 inhibitory effect of OSP50, OSP75, OSP95, crude polysaccharide, amino-oligosaccharin and chitosan on three strains
Note: "ND" means no zone of inhibition.
From the above results, it can be seen that the fine polysaccharides OSP95-A and OSP95-B obtained by ethanol precipitation with 95% ethanol have strong inhibitory effects on rice bacterial brown streak RS-1 strain, tomato bacterial wilt MYA-1 strain and sweet potato stem rot Dd-1 strain, while 50% ethanol precipitated fine polysaccharides (OSP50-A and OSP50-B) and 75% ethanol precipitated fine polysaccharides (OSP75-A and OSP75-B) with the same concentration have no bacteriostatic effects on tomato bacterial wilt MYA-1 strain and sweet potato stem rot Dd-1 strain.
The crude polysaccharide without deproteinization also shows a certain inhibition effect on three strains, but the effect is obviously lower than that of the fine polysaccharide OSP95, the effect is possibly influenced by mixing impurities such as protein and the like, and the effect is possibly influenced by mixing polysaccharide without bacteriostatic activity, and the specific reason is to be further analyzed. And the antibacterial effect of the OSP95 is obviously higher than that of chitosan and higher than that of 5% amino-oligosaccharin.
Generally, the lower the concentration of ethanol, the higher the molecular weight of the polysaccharide precipitated, and the higher the concentration of ethanol, the smaller molecular weight polysaccharide can be precipitated. The inventor finds that the cordyceps sobolifera extract polysaccharide OSP95 subjected to ethanol precipitation with 95% of ethanol in stepwise ethanol precipitation has strong antibacterial activity on three gram-negative plant pathogenic bacteria, and in addition, the polysaccharide component is very easy to dissolve in water, so that the polysaccharide which plays a role in inhibiting the gram-negative plant pathogenic bacteria in the cordyceps sobolifera polysaccharide is probably water-soluble micromolecule polysaccharide.
Example 7 inhibitory Effect of different concentrations of OSP95-A and OSP95-B on three gram-negative plant bacteria
Three gram-negative plant virus bacteria were treated with different concentrations of OSP95-a and OSP95-B, respectively, in the same manner as described in example 6, three replicates were set, sterilized water was used as a negative control, the conventional control agent was 5% amino-oligosaccharin aqueous solution, the experimental results are shown in fig. 5, and the specific inhibition circle diameter data are shown in the following table:
TABLE 2 inhibitory Effect of different concentrations of OSP95 on three strains
Note: "ND" means no zone of inhibition; "-" indicates not tested; the capital letters show significant difference (P < 0.05), and the lowercase letters
The letters indicate that the difference is significant.
From the results, the 95% ethanol-precipitated cordyceps sobolifera extract polysaccharides OSP95-A and OSP95-B obtained by stepwise ethanol precipitation of cordyceps sobolifera extract polysaccharides have strong inhibition effects on the rice bacterial brown streak RS-1 strain, the tomato bacterial wilt MYA-1 strain and the sweet potato stem rot Dd-1 strain, and the antibacterial effect is enhanced along with the increase of the polysaccharide concentration (figure 5).
For the rice bacterial brown streak RS-1 strain, the result of significant anova shows that the diameter of an inhibition zone of OSP95-A (25mg/ml) is about 10.83mm, and the difference between the diameter of the inhibition zone and the diameter of a conventional control 5% amino-oligosaccharin (40mg/ml) which is 10.67mm is not significant; the diameters of inhibition zones of OSP95-A (80mg/ml) and OSP95-B (40mg/ml) reach 14.00mm and 13.17mm respectively, the difference between the two is not obvious, but the two are extremely obvious level higher than that of the low-concentration treatment group and the conventional control treatment group.
Similarly, for tomato bacterial wilt MYA-1 strain and sweet potato stem rot Dd-1 strain, the significant variance analysis result shows that the bacteriostasis effects of OSP95-A (25mg/ml) and control 5% amino-oligosaccharin (40mg/ml) are equivalent, and no significant difference exists between the bacteriostasis effects, but the significant level of the two is superior to that of OSP95-B (20mg/ml), and the significant level is lower than that of OSP95-A (80 mg/ml).
EXAMPLE 8 varying concentrations of OSP95-A, OSP95-B and 5% amino-oligosaccharin on tomato bacterial wilt MYA-1 Strain
Inhibiting effect
The tomato bacterial wilt MYA-1 strain is treated by respectively adopting OSP95-A, OSP95-B and 5% amino-oligosaccharin with the concentrations of 0, 0.625, 1.25, 2.5, 5, 10 and 20mg/ml, and the results show that for the tomato bacterial wilt MYA-1 strain, OSP95-A and OSP95-B only show very weak inhibition zones under the concentration of 5mg/ml, and the inhibition zones of OSP95-A and OSP95-B higher than 10mg/ml are clear; while 5% amino-oligosaccharin had no inhibitory effect at 5mg/ml concentration, the boundary line of the zone at 10mg/ml was somewhat blurred, and the inhibitory effect was lower than that of OSP95 (see Table 3 and FIG. 6). The results show that OSP95-A and OSP95-B have strong inhibitory activity on tomato bacterial wilt MYA-1 strains, and obvious bacteriostasis can be realized when the concentration is more than 10mg/ml, so that the compounds can be used for preparing medicaments for resisting tomato bacterial wilt.
TABLE 3 inhibition of MYA-1 strain by different concentrations of OSP95-A, OSP95-B and 5% amino-oligosaccharin
Through the above embodiments, the inventor finds that the 95% ethanol-precipitated cordyceps sobolifera extract polysaccharide OSP95 obtained by fractional alcohol precipitation of the extract polysaccharide prepared from cordyceps sobolifera culture residues has a strong inhibiting effect on rice bacterial brown streak RS-1 strains, tomato bacterial wilt MYA-1 strains and sweet potato stem rot Dd-1 strains, and can be used for preparing a medicament for resisting gram-negative plant pathogenic bacteria. The yield of OSP95, a polysaccharide having antibacterial activity, was isolated from the culture residue by weighing the resulting precipitate in a yield of about 4.52% to 4.63%.
TABLE 4 yield of bacteriostatic polysaccharide (OSP95) with bacteriostatic activity
The inventor adopts cordyceps sobolifera sporocarp, cordyceps militaris culture residue, cordyceps militaris sporocarp and cordyceps liquid fermentation mycelium liquid to carry out experiments respectively, similar experimental effects are obtained, and the experiments are not described one by one.
While the present invention has been described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (7)
1. An application of 95% ethanol-precipitated Cordyceps sinensis extract polysaccharide CP95 in preparing medicine for resisting gram-negative plant pathogenic bacteria is provided.
2. The use according to claim 1, wherein the CP95 is prepared from a culture of Cordyceps sinensis.
3. The use according to claim 2, wherein the CP95 is prepared by a method comprising the steps of:
(1) extracting crude polysaccharide: drying a proper amount of cordyceps culture at 70 ℃, adding 10 times of 50% ethanol according to the mass ratio of the culture to the volume of the extracting solution, extracting twice at 80 ℃, stirring for 0.5h each time, preserving heat for 2h, and filtering to obtain 50% ethanol extracting solution; adding 10 times of water solution into the residue after twice extraction with 50% ethanol, extracting at 100 deg.C, stirring for 0.5 hr, keeping the temperature for 1 hr, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide, respectively. Mixing 50% ethanol extraction precipitated polysaccharide and water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps crude polysaccharide;
(2) deproteinizing the crude polysaccharide: dissolving the crude polysaccharide with deionized water, loading the prepared crude polysaccharide solution on macroporous resin, eluting with 3 times volume of deionized water, and collecting the eluate; uniformly mixing the collected eluent and Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at 4000rpm, placing the mixture in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, and repeating the operation for 2 times; concentrating the water phase layer to obtain refined polysaccharide CP;
(3) ethanol precipitation by steps: uniformly stirring the concentrated solution of the refined polysaccharide with ethanol with the final concentration of 50% in volume ratio, standing at 4 ℃ overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and freeze-drying the precipitate at low temperature to obtain CP 50; regulating the volume of ethanol in the supernatant to 75%, standing overnight at 4 deg.C, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 75; concentrating and spin-drying the supernatant, dissolving with 95% ethanol, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 95.
4. The use according to claim 2, wherein the CP95 is prepared by a method comprising the steps of:
(1) extracting crude polysaccharide: drying a proper amount of cordyceps culture at 70 ℃, adding 10 times of 50% ethanol according to the mass ratio of the culture to the volume of the extracting solution, extracting twice at 80 ℃, stirring for 0.5h each time, preserving heat for 2h, and filtering to obtain 50% ethanol extracting solution; adding 10 times of water solution into the residue after twice extraction with 50% ethanol, extracting at 100 deg.C, stirring for 0.5 hr, keeping the temperature for 1 hr, and filtering to obtain water extract. And respectively concentrating the 50% ethanol extract and the water extract to obtain 50% ethanol concentrated solution and water concentrated solution. Adding 3 times of 95% ethanol into the two concentrated solutions, standing overnight, and precipitating polysaccharide to obtain 50% ethanol-extracted precipitated polysaccharide and water-extracted precipitated polysaccharide, respectively. Mixing 50% ethanol extraction precipitated polysaccharide and water extraction precipitated polysaccharide, and spray drying to obtain Cordyceps crude polysaccharide;
(2) deproteinizing the crude polysaccharide: dissolving the crude polysaccharide powder by using deionized water, uniformly mixing the prepared crude polysaccharide solution and a Sevag reagent according to the volume ratio of 5:1, violently oscillating for 30min, centrifuging for 15min at 4000rpm, placing in a separating funnel for separating to remove an intermediate denatured protein layer and a lower organic solution layer, and repeating the operation for 3 times; loading the aqueous phase layer treated by the Sevag method to macroporous resin, eluting by using deionized water with the volume of 3 times, and concentrating the aqueous phase to obtain refined polysaccharide CP;
(3) ethanol precipitation by steps: uniformly stirring the concentrated solution of the refined polysaccharide with ethanol with the final concentration of 50% in volume ratio, standing at 4 ℃ overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and freeze-drying the precipitate at low temperature to obtain CP 50; regulating the volume of ethanol in the supernatant to 75%, standing overnight at 4 deg.C, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 75; concentrating and spin-drying the supernatant, dissolving with 95% ethanol, standing at 4 deg.C overnight, centrifuging at 8000r/min for 15min, collecting precipitate, and lyophilizing the precipitate at low temperature to obtain CP 95.
5. Use according to claim 1, wherein the gram-negative phytopathogenic bacterium is the RS-1 strain of rice bacterial brown streak, the MYA-1 strain of tomato bacterial wilt or the Dd-1 strain of sweet potato stem rot.
6. The method of claim 3 or 4, wherein the cordyceps culture comprises cordyceps culture such as cordyceps cicadae, cordyceps sinensis or cordyceps militaris.
7. The method of claim 3 or 4, wherein the cordyceps culture comprises a culture residue, a fruit body, a liquid fermentation mycelium, or a liquid fermentation mycelium filtrate.
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| CN113893385A (en) * | 2021-10-09 | 2022-01-07 | 南京师范大学 | Composition for preparing hydrogel, hydrogel and preparation method and application thereof |
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