CN110698568A - Extraction method of soybean rhizobium extracellular polysaccharide - Google Patents
Extraction method of soybean rhizobium extracellular polysaccharide Download PDFInfo
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Abstract
The invention discloses a method for extracting rhizobium japonicum extracellular polysaccharide, and belongs to the technical field of rhizobium japonicum extracellular polysaccharide extraction. The invention aims to solve the technical problem that the effect of removing protein in rhizobium extracellular polysaccharide is not ideal. According to the method, the serine protease and the Sevage method are adopted for deproteinizing treatment to treat the crude extracellular polysaccharide of the rhizobium HH103, and the protein removal rate is obviously enhanced along with the increase of the deproteinizing times, so that the linear trend is achieved.
Description
Technical Field
The invention belongs to the technical field of rhizobium extracellular polysaccharide extraction; in particular to a method for extracting rhizobium japonicum extracellular polysaccharide.
Background
Polysaccharides are macromolecular polymers with complex structures formed by dehydrating a plurality of same or different monosaccharides to form glycosidic bonds and connecting the glycosidic bonds in a linear or branched manner, and are widely used as components of cell membranes and cell wall structures in plants, animals, microorganisms (including bacteria and fungi), algae and other organisms. Compared with cell wall polysaccharides extracted from fungi or plants, extracellular polysaccharides secreted into the surrounding environment by bacteria are mainly homogeneous polysaccharides composed of regular repeating units, generally have large molecular mass, and the number of the repeating units is usually more than 1000. Because of the unique physical and chemical properties and biological properties of microbial polysaccharides, more and more microbial polysaccharides are widely applied in various industries.
The rhizobium extracellular polysaccharide can enhance the immunity of organisms and has activities of oxidation resistance, tumor resistance and the like.
The existing method has the problem of non-ideal protein removing effect in rhizobium extracellular polysaccharide.
Disclosure of Invention
The invention aims to solve the technical problem that the effect of removing protein in rhizobium extracellular polysaccharide is not ideal; and provides a method for extracting the soybean rhizobium extracellular polysaccharide.
In order to solve the technical problems, the method for extracting the soybean rhizobium extracellular polysaccharide is carried out according to the following steps:
step one, centrifuging the rhizobium japonicum fermentation liquor for 5min at the speed of 8000rpm, taking supernate, adding trichloroacetic acid with the mass concentration of 80% to the final concentration of 4%, standing for 16h at the temperature of 4 ℃, centrifuging for 5min at the speed of 8000rpm, taking supernate, adding ethanol solution with the volume of 3 times, standing for 24h at the temperature of 4 ℃, centrifuging, dissolving precipitate with deionized water, and dialyzing;
step two, adding serine protease, and oscillating for 80min at 37 ℃ and 140 rpm;
step three, adding a chloroform-n-butanol mixed solution, fully oscillating, centrifuging at 8000rpm for 5min, oscillating again and filtering;
step four, repeating the operation from the step two to the step three until the protein layer is clear, and taking supernatant fluid;
step five, precipitating the supernatant obtained in the step four by using ethanol with the volume concentration of 95%, and reserving the precipitate after centrifugation;
and sixthly, carrying out vacuum drying on the centrifugate obtained in the step five after carrying out reduced pressure concentration and alcohol precipitation treatment to obtain rhizobium crude polysaccharide.
Further defined, the rhizobium fermentation broth in the first step is prepared by the following steps: inoculating Rhizobium japonicum HH103 stored at the temperature of minus 80 ℃ to a TY plate, putting the TY plate into an incubator at the temperature of 28 ℃ for inverted culture for 2-3d, selecting a ring of plate seeds, inoculating the plate seeds into a test tube of 10mL TY liquid culture medium, carrying out shake culture at 140rpm and 28 ℃ for 2d, inoculating 5mL of seed liquid into a triangular flask with the inoculation amount of 1%, wherein the liquid loading amount is 100mL, and the shaking culture at 140rpm and 28 ℃ is 2 d.
Further defined, the volume concentration of the ethanol solution in the first precipitation step is 95% by volume with deionized water.
Further limit, the dialysis time in the first step is 24h, and the water is changed 3 times during the dialysis process.
To further limit, 5mL of deionized water was added to 1g of the precipitate in step one.
Further limiting, in the second step, 2.0g of serine protease is added according to the mass ratio of the precipitate in the first step to the serine protease of 10.0: 1.0.
Further limiting, the volume ratio of chloroform to n-butanol in the mixed solution in the third step is 5: 1.
Further limiting, in the step six, the centrifugal treatment is to take 300mL of fermentation liquor, centrifuge the thalli for 5min at 8000r/min and take supernatant; the concentration under reduced pressure is carried out by concentrating to 1/10 with original volume by rotary evaporator, adjusting pH to 7.0, and shaking at 40 deg.C and 150rpm for 100 min. The pH value can be adjusted by using hydrochloric acid with the mass concentration of 10% or NaOH solution with the mass concentration of 10%.
Further, the alcohol precipitation treatment in the sixth step is ethanol precipitation with a volume concentration of 95%.
Further defining the technological parameters of vacuum drying in the step six: the heating temperature was 60 ℃ and the pressure was 50 Pa.
According to the method, the crude extracellular polysaccharide of rhizobia is treated by deproteinizing treatment by adopting serine protease and a Sevage method, and the protein removal rate is obviously enhanced along with the increase of the times of deproteinization, so that the linear trend is formed. The protein removing efficiency of the exopolysaccharide is improved.
Drawings
FIG. 1 is a graph showing the effect of example 1 on protein removal rate;
FIG. 2 is a standard curve of glucose concentration for example 1.
Detailed Description
Example 1: the method for extracting the soybean rhizobium extracellular polysaccharide in the embodiment is carried out according to the following steps:
step one, inoculating rhizobium japonicum HH103 stored at the temperature of-80 ℃ to a TY plate, placing the TY plate on an incubator at the temperature of 28 ℃ for inverted culture for 3d, selecting a ring of plate seeds, inoculating the plate seeds to a test tube of 10mL TY liquid culture medium, carrying out shake culture at 140rpm and 28 ℃ for 2d, inoculating 5mL of seed solution into a 500mL triangular flask by using 1% of inoculation amount, wherein the liquid loading amount is 100mL, and carrying out shake culture at 140rpm and 28 ℃ for 2d to obtain rhizobium japonicum fermentation liquid;
centrifuging the rhizobium japonicum fermentation liquor at 8000rpm for 5min, taking supernatant, adding trichloroacetic acid with the mass concentration of 80% to the final concentration of 4%, standing at 4 ℃ for 16h, centrifuging at 8000rpm for 5min, taking supernatant, adding 3 times of volume of ethanol solution, standing at 4 ℃ for 24h, centrifuging, dissolving precipitate with deionized water, dialyzing for 24h (changing 3 times of water), and taking 20 g;
step two, then 2.0g of serine protease is added, and oscillation is carried out for 80min at 37 ℃ and 140 rpm;
step three, adding a chloroform-n-butanol mixed solution (the volume ratio of chloroform to n-butanol is 5:1), fully shaking, centrifuging at 8000rpm for 5min, shaking at 12000rpm for 2min, and filtering;
step four, repeating the operation from the step two to the step three until the protein layer is clear, and taking supernatant fluid;
step five, precipitating the supernatant obtained in the step four by using ethanol with the volume concentration of 95%, and reserving the precipitate after centrifugation;
step six, taking 300mL of the centrifugate obtained in the step five, centrifuging for 5min at 8000r/min, and taking the supernatant; concentrating to 1/10 of original volume with rotary evaporator, adjusting pH to 7.0, and oscillating at 40 deg.C and 150rpm for 100 min; precipitating with 95 vol% ethanol; vacuum drying at 60 deg.C and 50Pa to obtain crude polysaccharide of Rhizobium.
In the embodiment, the crude extracellular polysaccharide of rhizobium HH103 is treated by deproteinizing treatment by adopting serine protease and Sevage method, the protein removal rate is obviously enhanced along with the increase of the deproteinizing times and has a linear trend, as shown in figure 1, after 6 times of deproteinizing treatment, the protein removal rate reaches 95.2%, and the polysaccharide loss rate also rises to 52.1%.
Fermentation culture of strain and extraction result of extracellular polysaccharide
Drawing a standard curve by taking the standard glucose concentration as an abscissa and the absorbance value as an ordinate, and establishing a linear regression equation y of 4.71x +0.068, R20.9925, as shown in fig. 2. Under the initial fermentation conditions, HH103 reached stationary phase 64h after growth and the rhizobium exopolysaccharide yield was 3.32 g/L.
Claims (10)
1. The extraction method of the soybean rhizobium extracellular polysaccharide is characterized by comprising the following steps:
step one, centrifuging the rhizobium japonicum fermentation liquor for 5min at the speed of 8000rpm, taking supernate, adding trichloroacetic acid with the mass concentration of 80% to the final concentration of 4%, standing for 16h at the temperature of 4 ℃, centrifuging for 5min at the speed of 8000rpm, taking supernate, adding ethanol solution with the volume of 3 times, standing for 24h at the temperature of 4 ℃, centrifuging, dissolving precipitate with deionized water, and dialyzing;
step two, adding serine protease, and oscillating for 80min at 37 ℃ and 140 rpm;
step three, adding a chloroform-n-butanol mixed solution, fully oscillating, centrifuging at 8000rpm for 5min, oscillating again and filtering;
step four, repeating the operation from the step two to the step three until the protein layer is clear, and taking supernatant fluid;
step five, precipitating the supernatant obtained in the step four by using ethanol with the volume concentration of 95%, and reserving the precipitate after centrifugation;
and sixthly, carrying out vacuum drying on the centrifugate obtained in the step five after carrying out reduced pressure concentration and alcohol precipitation treatment to obtain rhizobium crude polysaccharide.
2. The extraction process according to claim 1, wherein the fermentation broth of rhizobia in step one is obtained by the steps of: inoculating Rhizobium japonicum HH103 stored at the temperature of minus 80 ℃ to a TY plate, putting the TY plate into an incubator at the temperature of 28 ℃ for inverted culture for 2-3d, selecting a ring of plate seeds, inoculating the plate seeds into a test tube of 10mL TY liquid culture medium, carrying out shake culture at 140rpm and 28 ℃ for 2d, inoculating 5mL of seed liquid into a triangular flask with the inoculation amount of 1%, wherein the liquid loading amount is 100mL, and the shaking culture at 140rpm and 28 ℃ is 2 d.
3. The extraction process according to claim 1, wherein the ethanol solution is dissolved in deionized water at a concentration of 95% by volume in the precipitation step.
4. The extraction method according to claim 1, wherein the dialysis time in step one is 24h, and the water is changed 3 times during dialysis.
5. The extraction process according to claim 1, wherein 5mL of deionized water is added to 1g of the precipitate in step one.
6. The extraction method according to claim 1, wherein in the second step, 2.0g of serine protease is added according to the mass ratio of the precipitate in the first step to the serine protease of 10.0: 1.0.
7. The extraction method according to claim 1, wherein the volume ratio of chloroform to n-butanol in the mixed solution of step three is 5: 1.
8. The extraction method according to claim 1, wherein the centrifugation in the sixth step is to take 300mL of fermentation broth, centrifuge the thallus for 5min at 8000r/min, take the supernatant; vacuum concentrating by rotary evaporator to 1/10, adjusting pH to 7.0, and shaking at 40 deg.C and 150rpm for 100 min.
9. The method according to claim 1, wherein the ethanol precipitation in the sixth step is performed by using 95 vol.% ethanol.
10. The extraction method according to claim 1, wherein the vacuum freeze-drying process parameters in step six are as follows: the heating temperature was 60 ℃ and the pressure was 50 Pa.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112940973A (en) * | 2021-02-23 | 2021-06-11 | 黑龙江省科学院微生物研究所 | High-density culture method for slow-growing soybean rhizobium |
| CN115044522A (en) * | 2022-05-18 | 2022-09-13 | 东北农业大学 | Rhizobium HH103 for expressing fluorescent gene and construction method and application thereof |
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Cited By (3)
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| CN112940973A (en) * | 2021-02-23 | 2021-06-11 | 黑龙江省科学院微生物研究所 | High-density culture method for slow-growing soybean rhizobium |
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| CN115044522A (en) * | 2022-05-18 | 2022-09-13 | 东北农业大学 | Rhizobium HH103 for expressing fluorescent gene and construction method and application thereof |
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