CN116041560B - Method for preparing shigella-like specific polysaccharide on large scale - Google Patents
Method for preparing shigella-like specific polysaccharide on large scale Download PDFInfo
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 58
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 57
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000000855 fermentation Methods 0.000 claims abstract description 23
- 230000004151 fermentation Effects 0.000 claims abstract description 23
- 230000007062 hydrolysis Effects 0.000 claims abstract description 10
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 10
- 238000000746 purification Methods 0.000 claims abstract description 6
- 239000002994 raw material Substances 0.000 claims abstract description 6
- 230000001580 bacterial effect Effects 0.000 claims description 14
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
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- 238000010438 heat treatment Methods 0.000 claims description 6
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- 239000008215 water for injection Substances 0.000 claims description 6
- 238000004255 ion exchange chromatography Methods 0.000 claims description 5
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 claims description 3
- 239000004380 Cholic acid Substances 0.000 claims description 3
- 229920001425 Diethylaminoethyl cellulose Polymers 0.000 claims description 3
- 230000005526 G1 to G0 transition Effects 0.000 claims description 3
- 241000607768 Shigella Species 0.000 claims description 3
- 230000003698 anagen phase Effects 0.000 claims description 3
- 229940099352 cholate Drugs 0.000 claims description 3
- 235000019416 cholic acid Nutrition 0.000 claims description 3
- 229960002471 cholic acid Drugs 0.000 claims description 3
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 claims description 3
- 230000012010 growth Effects 0.000 claims description 3
- 230000002779 inactivation Effects 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
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- 238000005070 sampling Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 2
- 125000003716 cholic acid group Chemical group 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 238000007710 freezing Methods 0.000 claims description 2
- 230000008014 freezing Effects 0.000 claims description 2
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 2
- 239000001103 potassium chloride Substances 0.000 claims description 2
- 235000011164 potassium chloride Nutrition 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 239000001632 sodium acetate Substances 0.000 claims description 2
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- 239000002904 solvent Substances 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- JVIPLYCGEZUBIO-UHFFFAOYSA-N 2-(4-fluorophenyl)-1,3-dioxoisoindole-5-carboxylic acid Chemical compound O=C1C2=CC(C(=O)O)=CC=C2C(=O)N1C1=CC=C(F)C=C1 JVIPLYCGEZUBIO-UHFFFAOYSA-N 0.000 claims 2
- 239000001768 carboxy methyl cellulose Substances 0.000 claims 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims 1
- 241000607764 Shigella dysenteriae Species 0.000 abstract description 16
- 229940007046 shigella dysenteriae Drugs 0.000 abstract description 16
- 201000010099 disease Diseases 0.000 abstract description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 7
- 239000000427 antigen Substances 0.000 abstract description 5
- 108010060123 Conjugate Vaccines Proteins 0.000 abstract description 4
- 108091007433 antigens Proteins 0.000 abstract description 4
- 102000036639 antigens Human genes 0.000 abstract description 4
- 229940031670 conjugate vaccine Drugs 0.000 abstract description 3
- 239000012467 final product Substances 0.000 abstract description 3
- 241000193830 Bacillus <bacterium> Species 0.000 abstract description 2
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- 239000013558 reference substance Substances 0.000 abstract description 2
- 206010012735 Diarrhoea Diseases 0.000 abstract 2
- 206010016952 Food poisoning Diseases 0.000 abstract 1
- 208000019331 Foodborne disease Diseases 0.000 abstract 1
- 201000009840 acute diarrhea Diseases 0.000 abstract 1
- 229960003964 deoxycholic acid Drugs 0.000 description 13
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 13
- 239000002158 endotoxin Substances 0.000 description 9
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- 108090000623 proteins and genes Proteins 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 229920006008 lipopolysaccharide Polymers 0.000 description 6
- 108010009736 Protein Hydrolysates Proteins 0.000 description 4
- 241000607760 Shigella sonnei Species 0.000 description 4
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- 229940115939 shigella sonnei Drugs 0.000 description 4
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 3
- 241000607762 Shigella flexneri Species 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 208000004429 Bacillary Dysentery Diseases 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
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- 208000035473 Communicable disease Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 241000607000 Plesiomonas Species 0.000 description 1
- 241000446766 Shigella dysenteriae 1 Species 0.000 description 1
- 206010048038 Wound infection Diseases 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
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- 239000000706 filtrate Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
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- 230000008004 immune attack Effects 0.000 description 1
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- 239000011241 protective layer Substances 0.000 description 1
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- 239000013049 sediment Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention aims to provide a method for preparing shigella-like specific polysaccharide on a large scale, wherein the shigella-like specific polysaccharide is gram-negative bacillus and can cause acute diarrhea and food poisoning, and O antigen and various diarrhea serotypes have cross reaction. The specific polysaccharide (also called O-specific polysaccharide) is a component of O antigen, the invention takes shigella dysenteriae as raw material, prepares the specific polysaccharide through fermentation culture, hydrolysis, purification and other steps, and the prepared specific polysaccharide can be used as raw material or final product for disease diagnosis, prevention, treatment and the like, can also be used for preparing standard or reference substance of the specific polysaccharide, and can also be used for preparing conjugate vaccine.
Description
Technical Field
The invention relates to a method for preparing shigella-like specific polysaccharide (also called O-specific polysaccharide) on a large scale, belonging to the field of biological products.
Technical Field
Shigella is a gram-negative bacillus, and is widely existed in nature, mainly causes gastrointestinal tract diseases and wound infection, and also possibly causes diseases such as meningitis, bacteremia and the like. According to O antigen, the serotype can be divided into more than 100 serotypes, some serotypes have a cross reaction (Linnerborg M et al.,Structure elucidation of O-antigen lipopolysaccharide from two strains of Plesiomona shigelloides that share a type specific antigen with Shigella flexneri 6and the common group 1antigen with shigella flexneri spp and shigella dysenteriae 1[J].Eur J Biochem 1995,231:839-844). of serotypes with shigella sonnei, shigella dysenteriae, shigella pallidus, shigella flexneri and other shigella dysenteriae, for example, 7-63-5 strains of shigella sonnei can be used for replacing shigella sonnei to prepare shigella sonnei dysentery polysaccharide (Du Lin and the like, research on 7-63-5 strains of shigella sonnei and polysaccharide thereof, microbiological immunology progress 2006, 34 (3): 1-4) to prepare shigella sonnei dysentery polysaccharide protein conjugate vaccine.
The specific polysaccharide of gram-negative bacteria is part of the lipopolysaccharide. Lipopolysaccharide is the most major component outside the outer membrane of gram-negative bacteria, consisting of 3 parts, respectively specific polysaccharide, core polysaccharide and lipid a. The specific polysaccharide is a structure positioned at the outermost side of the bacteria, is a protective layer for the bacteria to evade host immune attack, is also a target point for host immune cells to capture the bacteria, determines the antigenic property of the bacteria and reflects the serotype of the bacteria; the core polysaccharide is the part linking the specific polysaccharide and lipid a; lipid a is the pyrogenic moiety of lipopolysaccharide. Since the specific polysaccharide moiety determines the serotype of bacteria, it is structurally type-specific and is the main antigenic component for bacterial diagnosis, pathogen prevention and infectious disease treatment.
The existing preparation method of the specific polysaccharide mainly comprises the steps of preparing the lipopolysaccharide, hydrolyzing the lipopolysaccharide, and purifying through centrifugation, dialysis, precipitation and the like to obtain the specific polysaccharide. In the patent (CN 101693747A), the thalli are used for direct hydrolysis, the thalli are removed by centrifugation, the crude product of the specific polysaccharide is collected by ethanol precipitation, and the specific polysaccharide is obtained by centrifugation, ultrafiltration, chromatography and other steps after re-dissolution.
Disclosure of Invention
The invention aims to provide a method for preparing shigella dysenteriae specific polysaccharide on a large scale.
The method for preparing the shigella dysenteriae specific polysaccharide on a large scale takes shigella dysenteriae as a raw material, and the specific polysaccharide is obtained through the steps of fermentation culture, hydrolysis, purification and the like.
Wherein, the fermentation culture comprises the following steps:
(1) Resuscitating strain, inoculating the strain to solid culture medium, standing and culturing at 35-37 deg.C,
(2) Subculturing, inoculating resuscitating strain into liquid culture medium, culturing at 35-37 deg.c and 100-200 rpm for 4-12 hr,
(3) Culturing in a fermentation tank, inoculating the subculture bacterial liquid into the fermentation tank, and fermenting under the condition: stirring at 35-37 deg.C and pH 6-8 and 100-300 rpm, introducing compressed air for culture,
(4) Sampling, detecting and drawing a growth curve, selecting inactivation from the late phase of the logarithmic growth phase to the early phase of the stationary phase,
(5) Centrifuging or filtering the inactivated fermentation liquor, collecting precipitate to obtain Shigella-like thallus,
Wherein, the fermentation tank can be used for culturing, and 10-100L single tank fermentation or multi-stage tank continuous fermentation can be used.
Wherein, the hydrolysis comprises the following steps:
(1) Adding thalli into a solvent to prepare bacterial suspension with the concentration of 5-10%,
(2) The pH value of the bacterial suspension is regulated to 2-4,
(3) Heating to 90-100 ℃, continuously stirring and reacting for 60-200 minutes,
(4) The reaction solution is placed in an ice-water bath, a precipitator is added,
(5) Standing for 1-3 hours, centrifuging or filtering, collecting hydrolysate,
Wherein, pure water and water for injection can be used for preparing the bacterial suspension, and salt solutions such as sodium chloride solution, potassium chloride solution, sodium acetate solution and the like can also be used. Preferably pure water or water for injection.
Wherein the precipitant is cholic acid or cholate and its derivative, and the addition concentration is 0.1% -2%, preferably 0.5% -1%.
Wherein, the purification comprises the following steps:
(1) The pH value of the hydrolysis supernatant is regulated to be neutral,
(2) Ultrafiltering and concentrating the supernatant of the hydrolysate by using an ultrafiltration membrane bag, collecting the trapped component,
(3) Dialyzing and washing the trapped component by using 0.01mol/L phosphate buffer solution, collecting dialysate,
(4) Separating dialysate by ion exchange chromatography medium, collecting flow-through component at 206nm,
(5) Ultrafiltering and concentrating the flow-through components by using an ultrafiltration membrane bag, replacing salt in a sample by water for injection, collecting polysaccharide solution,
(6) And (3) freezing the polysaccharide solution at the temperature below minus 30 ℃ for 2 to 6 hours, and then freeze-drying to obtain the specific polysaccharide.
Wherein, the ultrafiltration adopts a combination form of any group of ultrafiltration membrane bags of 100Kd-5Kd, 50Kd-5Kd, 30Kd-5Kd, 100Kd-3Kd, 50Kd-3Kd and 30Kd-3 Kd. Preferably 50Kd-3Kd, 30Kd-3Kd ultrafiltration membrane packets.
Wherein, the ion exchange chromatography medium adopts CM (carboxymethyl) cellulose, DEAE (diethyl aminoethyl) cellulose, hydroxyapatite and other mediums, which can be singly used or matched with each other. DEAE is preferred.
The shigella-like specific polysaccharide can be used as a raw material or a final product for diagnosing, preventing, treating diseases caused by shigella-like, and the like, can also be used for preparing a standard or reference substance of the specific polysaccharide, and can be used for preparing conjugate vaccines. Meanwhile, the method can also be used for replacing specific polysaccharide of shigella dysenteriae with serum cross reaction with shigella dysenteriae, and can be used as a raw material or a final product for diagnosing, preventing, treating diseases caused by shigella dysenteriae, and the like, and preparing a standard or reference product of the specific polysaccharide for preparing conjugate vaccines.
Compared with the existing method, the preparation method has the advantages that:
1) Can be produced in large scale, can harvest more than 2kg of thalli per 100L of fermentation liquor, can harvest more than 1.5g of specific polysaccharide per 1kg of thalli,
2) The operation process is simple, the operators in the field can easily grasp,
3) Avoiding the use of harmful organic solvents such as phenol, alcohols, ketones and the like, reducing the harm to operators and the pollution to the environment,
4) The prepared specific polysaccharide can be used for diagnosing, preventing and treating shigella dysenteriae related diseases, and also can be used for diagnosing, preventing and treating specific serotype shigella dysenteriae related diseases.
Drawings
FIG. 1 is a nuclear magnetic pattern of specific polysaccharides.
Detailed Description
The invention is further illustrated by the following examples. The present embodiment is merely an example, which is one of the solutions of the present invention, and the protection scope of the present invention is not limited to the example.
Example 1 Shigella-like O.sp.specific polysaccharide
(1) Fermentation culture
Taking 1 strain of Shigella dysenteriae (strain number CMCC 10709), inoculating on a solid culture medium, standing and culturing at 35-37 ℃ for about 14 hours, transferring into 1L liquid culture medium, shaking and culturing at 35-37 ℃ and 120rpm for about 6 hours, inoculating into a 10L fermentation tank, culturing for about 4 hours, inoculating into a 60L fermentation tank, culturing for about 4 hours, inoculating into a 300L fermentation tank, and culturing the same conditions in the fermentation tank: stirring at 35-37 deg.c and 120rpm to maintain pH value of 6.6-7.4, introducing compressed air and adding glucose as supplementary material. Sampling, detecting and drawing a growth curve in a 300L fermentation tank, selecting late logarithmic growth phase or early stationary phase for inactivation, and centrifugally collecting about 8kg of bacterial sediment.
(2) Hydrolysis
Taking 800g of shigella dysenteriae, adding pure water to prepare bacterial suspension with the concentration of 5%, adjusting the pH value to 3.2, heating to 100 ℃, stirring at 120rpm for reaction for 120 minutes, placing in an ice water bath for cooling for 60 minutes, adding sodium deoxycholate to the final concentration of 1%, and centrifugally collecting the supernatant of the hydrolysate.
(3) Purification
Ultrafiltering the hydrolysate with 30Kd ultrafilter membrane, collecting the filtered components, ultrafiltering the filtrate with 3Kd ultrafilter membrane to concentrate to 1/10 of the original volume, ultrafiltering and dialyzing with 10 times of 0.01mol/L phosphate buffer solution, collecting the dialysate, separating with chromatography column with DEAE as chromatography medium, and collecting the flow-through components at 206 nm. The collected flow-through components are ultrafiltered and concentrated, the replacement salt is washed by water for injection, frozen below minus 30 ℃ for 2 to 6 hours and then freeze-dried, and the specific polysaccharide is obtained.
Example 2 specific polysaccharide detection method and pass criteria
All detection items of the harvested specific polysaccharide, protein, nucleic acid, endotoxin, molecular size and the like are carried out according to related items in the pharmacopoeia of the people's republic of China. The protein content is measured according to Fu Lin Fenfa, also called Lowry method; the nucleic acid content is measured according to an ultraviolet-visible spectrophotometry; bacterial endotoxin test was performed according to bacterial endotoxin test method; the polysaccharide molecular size is determined by a group A meningococcal polysaccharide molecular size determination method, and the recovery rate of the polysaccharide with the K D value not higher than 0.85 is not less than 70%.
Example 3 specific polysaccharide yield and detection results
EXAMPLE 4 Effect of different sodium deoxycholate concentrations
Taking 800g of shigella dysenteriae, adding pure water to prepare bacterial suspension with the concentration of 5%, adjusting the pH value to 3.2, heating to 100 ℃, stirring at 120rpm for reaction for 120 minutes, placing in ice water bath for cooling for 60 minutes, adding sodium deoxycholate to the final concentration of 0.1%, centrifuging, collecting the supernatant of the hydrolysate, and determining the ratio of protein to polysaccharide.
EXAMPLE 5 Effect of different sodium deoxycholate concentrations
Taking 800g of shigella dysenteriae, adding pure water to prepare bacterial suspension with the concentration of 5%, adjusting the pH value to 3.2, heating to 100 ℃, stirring at 120rpm for reaction for 120 minutes, placing in ice water bath for cooling for 60 minutes, adding sodium deoxycholate to the final concentration of 0.5%, centrifuging, collecting the supernatant of the hydrolysate, and determining the ratio of protein to polysaccharide.
EXAMPLE 6 Effect of different sodium deoxycholate concentrations
Taking 800g of shigella dysenteriae, adding pure water to prepare bacterial suspension with the concentration of 5%, adjusting the pH value to 3.2, heating to 100 ℃, stirring at 120rpm for reaction for 120 minutes, placing in ice water bath for cooling for 60 minutes, adding sodium deoxycholate to the final concentration of 1.5%, centrifuging, collecting the supernatant of the hydrolysate, and determining the ratio of protein to polysaccharide.
Example 7 results of detection of supernatants of hydrolysates of different sodium deoxycholate concentrations
The results showed that the mean protein/polysaccharide value after centrifugation of the hydrolysate was 12.68% using 0.1% concentration of sodium deoxycholate; the average value of protein/polysaccharide after the hydrolysate is centrifuged is 8.88% by using 0.5% concentration of sodium deoxycholate; the average value of protein/polysaccharide after the hydrolysate is centrifugated is 8.30% by using 1.0% concentration of sodium deoxycholate; the average value of protein/polysaccharide after the hydrolysate is centrifuged is 8.73% by using 1.5% concentration of sodium deoxycholate; in conclusion, the preferable concentration of the sodium deoxycholate is 0.5% -1%.
Claims (8)
1. A method for preparing shigella-like specific polysaccharide on a large scale is characterized in that shigella-like specific polysaccharide is obtained by taking shigella as a raw material through fermentation culture, hydrolysis and purification steps, wherein the hydrolysis comprises the following steps:
(1) Adding thalli into a solvent to prepare bacterial suspension with the concentration of 5-10%,
(2) The pH value of the bacterial suspension is regulated to 2-4,
(3) Heating to 90-100 ℃, continuously stirring and reacting for 60-200 minutes,
(4) Placing the reaction solution in ice-water bath, adding precipitant which is cholic acid or cholate and its derivative, the adding concentration is 0.1% -2%,
(5) Standing for 1-3 hours, centrifuging or filtering, and collecting hydrolysate;
Wherein the purification comprises the steps of:
(1) The pH value of the hydrolysis supernatant is regulated to be neutral,
(2) Ultrafiltering and concentrating the supernatant of the hydrolysate by using an ultrafiltration membrane bag, collecting the trapped component,
(3) Dialyzing and washing the trapped component by using 0.01mol/L phosphate buffer solution, collecting dialysate,
(4) Separating dialysate by ion exchange chromatography medium, collecting flow-through component at 206nm, (5) ultrafiltering and concentrating the flow-through component by ultrafiltration membrane bag, replacing salt in sample with water for injection, collecting polysaccharide solution,
(6) And (3) freezing the polysaccharide solution at the temperature below minus 30 ℃ for 2 to 6 hours, and then freeze-drying to obtain the specific polysaccharide.
2. The method according to claim 1, characterized in that: wherein, in the hydrolysis step, the precipitant is cholic acid or cholate and its derivative, and the adding concentration is 0.5% -1%.
3. The method according to claim 1, characterized in that: wherein, the fermentation culture comprises the following steps:
(1) Resuscitating strain, inoculating the strain to solid culture medium, standing and culturing at 35-37 deg.C,
(2) Subculturing, inoculating resuscitating strain into liquid culture medium, culturing at 35-37 deg.c and 100-200 rpm for 4-12 hr,
(3) Culturing in a fermentation tank, inoculating the subculture bacterial liquid into the fermentation tank, and fermenting under the condition: stirring at 35-37 deg.C and pH 6-8 and 100-300 rpm, introducing compressed air for culture,
(4) Sampling, detecting and drawing a growth curve, selecting inactivation from the late phase of the logarithmic growth phase to the early phase of the stationary phase,
(5) Centrifuging or filtering the inactivated fermentation liquor, and collecting the precipitate to obtain the shigella-like thallus.
4. A method according to claim 3, wherein the fermenter culture is performed using 10 to 100L of single-tank fermentation or using multistage tank continuous fermentation.
5. The method of claim 1, wherein the bacterial suspension is prepared by pure water, water for injection, or sodium chloride solution, potassium chloride solution, sodium acetate solution.
6. The method according to claim 1, wherein the ultrafiltration is performed by using any one of the group consisting of 100Kd-5Kd, 50Kd-5Kd, 30Kd-5Kd, 100Kd-3Kd, 50Kd-3Kd, and 30Kd-3 Kd.
7. The method of claim 1, wherein the ion exchange chromatography medium is carboxymethyl cellulose, diethyl aminoethyl cellulose, or hydroxyapatite, singly or in combination.
8. The method of claim 7, wherein the ion exchange chromatography medium is diethyl aminoethyl cellulose.
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