CN118345133A - Preparation method of bacteroides fragilis capsular polysaccharide A - Google Patents
Preparation method of bacteroides fragilis capsular polysaccharide A Download PDFInfo
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 105
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 104
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 104
- 241000606124 Bacteroides fragilis Species 0.000 title claims abstract description 100
- 238000002360 preparation method Methods 0.000 title claims abstract description 50
- 230000001580 bacterial effect Effects 0.000 claims abstract description 47
- 238000000605 extraction Methods 0.000 claims abstract description 37
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 abstract description 28
- 231100000053 low toxicity Toxicity 0.000 abstract description 3
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- 239000010802 sludge Substances 0.000 description 18
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
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- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
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- 241000423333 Bacteroides fragilis NCTC 9343 Species 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
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- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
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- AFYNADDZULBEJA-UHFFFAOYSA-N bicinchoninic acid Chemical compound C1=CC=CC2=NC(C=3C=C(C4=CC=CC=C4N=3)C(=O)O)=CC(C(O)=O)=C21 AFYNADDZULBEJA-UHFFFAOYSA-N 0.000 description 1
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- 238000002329 infrared spectrum Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
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- 238000011068 loading method Methods 0.000 description 1
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- JZMJDSHXVKJFKW-UHFFFAOYSA-N methyl sulfate Chemical compound COS(O)(=O)=O JZMJDSHXVKJFKW-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
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- 229920001542 oligosaccharide Polymers 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- KSSNXJHPEFVKHY-UHFFFAOYSA-N phenol;hydrate Chemical compound O.OC1=CC=CC=C1 KSSNXJHPEFVKHY-UHFFFAOYSA-N 0.000 description 1
- 229940031937 polysaccharide vaccine Drugs 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- -1 small-molecule oligosaccharide Chemical class 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- GRONZTPUWOOUFQ-UHFFFAOYSA-M sodium;methanol;hydroxide Chemical compound [OH-].[Na+].OC GRONZTPUWOOUFQ-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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- 239000012137 tryptone Substances 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
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- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
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Classifications
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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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Sustainable Development (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
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- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The application relates to a preparation method of bacteroides fragilis capsular polysaccharide A, which comprises the following steps: preparing bacterial suspension of bacteroides fragilis with pH less than or equal to 5, heating and extracting, and collecting an extracting solution to obtain a crude sugar solution of capsular polysaccharide A. Compared with the prior art, the application has the following beneficial effects: the inventors have found that, surprisingly, the extraction of the bacterial suspension of Bacteroides fragilis with pH less than or equal to 5 by heating can effectively promote the falling-off of capsular polysaccharide A from the bacteroides fragilis bacterial bodies so as to extract capsular polysaccharide A. Compared with the traditional phenol/water method, the application can obtain the capsular polysaccharide A with low toxicity under the condition of avoiding using phenol. In addition, by adopting the preparation method, the yield of the bacteroides fragilis capsular polysaccharide A is obviously improved.
Description
The application relates to a divisional application, the original application number is 202110660150.5, the application name is a preparation method of bacteroides fragilis capsular polysaccharide A, and the application date is 2021, 6, 15.
Technical Field
The invention relates to a preparation and application technology of bacteroides fragilis capsular polysaccharide, in particular to a preparation method of bacteroides fragilis capsular polysaccharide A.
Background
Bacteroides fragilis (bacteroides fragilis) is a member of the genus Bacteroides among gram-negative anaerobic bacteria belonging to the phylum Bacteroides, bifidobacteria, lactic acid bacteria, and the like, which are completely different from the phylum thick-walled bacteria. The bacteroides has 25 species, 10 species from humans only, 10 species from animals only, and 5 species from humans and animals. Bacteroides fragilis is an obligate anaerobic bacterium, which can be classified into enterotoxigenic bacteroides fragilis (Enterotoxigenic Bacteroides fragilis, ETBF)) and non-enterotoxigenic bacteroides fragilis (Nontoxigenic Bacteroides fragilis, NTBF) depending on whether enterotoxigenic (BFT) can be synthesized or secreted. Bacteroides fragilis is used as part of normal flora in the intestinal tract of humans and animals, and is mainly present in the colon. In addition, the mucous membranes of the respiratory tract, gastrointestinal tract and genitourinary tract can also grow by colonization.
Bacteroides fragilis can express 8 different capsular polysaccharides, designated as a-H, respectively, wherein the effect of capsular polysaccharide a (polysaccharideA, PSA) is determined by its molecular weight, the molecular weight of natural PSA being about 110kDa.
There has been reported (Dennis L.Kasper,Andrew B.Onderdonk,Joseph Crabb,and John G.BartlettProtective Efficacy of Immunization with Capsular Antigen against Experimental Infection with Bacteroides fragilis.THE JOURNAL OF INFECTIOUS DISEASES.VOL.140,NO.5.NOVEMBER 1979) that Bacteroides fragilis capsular polysaccharide plays a positive role in preventing infection with bacteria of the same genus, which is thought to be the case because Bacteroides capsular polysaccharide has several similar antigenic determinants. This study found that although animals immunized with Bacteroides fragilis capsular polysaccharide were unable to resist infection by other bacteria (e.g., E.coli), the degree of infection was significantly reduced due to immunization against Bacteroides. Meanwhile, the study compares the capsular polysaccharide A and capsular polysaccharide B of bacteroides fragilis, and both capsular polysaccharides show a preventive effect, but the effective dose of capsular polysaccharide A is far smaller than that of capsular polysaccharide B.
At present, there are many cases about the effect of Bacteroides fragilis capsular polysaccharide A in preventing/treating diseases, so how to well realize the preparation of Bacteroides fragilis capsular polysaccharide A is of great significance.
A great deal of research reports on the preparation method of the bacteroides fragilis capsular polysaccharide A (PSA) exist, and the methods disclosed in all the documents are phenol/water methods, and the process is also the process foundation for preparing the bacterial polysaccharide vaccine. The traditional phenol/water-based process for preparing bacteroides fragilis capsular polysaccharide A is as follows:
In the U.S. patent application publication No. US20140243285A1, bacteroides fragilis NCTC 9343 strain is taken as a raw material, phenol/water solution is added for uniform mixing, and the preparation method comprises the steps of continuous stirring extraction at 68 ℃, centrifugation and collection of water phase, diethyl ether extraction for removing phenol, protease/nuclease for removing nucleic acid, ethanol precipitation of polysaccharide, 2% acetic acid high-temperature hydrolysis and molecular sieve or ion exchange chromatography purification process, so as to obtain Bacteroides fragilis capsular polysaccharide A containing trace amount of conjugated lipid, wherein the molecular weight is about 110KD.
The Chinese patent application with publication number CN110025636A, CN109954005A relates to a preparation method of a bacteroides fragilis extract, which comprises the following steps: (1) Centrifuging and precipitating the fermented and cultured bacteroides fragilis bacterial liquid, collecting a first precipitate, adding 65-72 ℃ water into the first precipitate, dissolving, adding a phenol solution, keeping the temperature of 65-72 ℃ and stirring for 25-35 min, centrifuging, and collecting a first supernatant; (2) Extracting the first supernatant collected in the step (1) with diethyl ether to remove phenol, removing residual diethyl ether, and collecting an aqueous phase solution; (3) Adding absolute ethyl alcohol into the water phase solution collected in the step (2) until the final concentration of the ethyl alcohol is 75-85 v/v%, precipitating with alcohol, centrifuging, and collecting a second precipitate; (4) And adding water into the second precipitate to prepare a suspension, regulating the pH to 6.5-7.5, centrifuging, collecting second supernatant, dialyzing for desalting, and freeze-drying to obtain the bacteroides fragilis extract. The technical proposal improves the process of extracting the PSA by a phenol/water method, reduces the steps of enzyme protein removal and nucleic acid removal, and simplifies the process.
However, in essence, the above conventional methods rely on phenol, which is toxic and harmful, and its use is disadvantageous for production safety and environmental protection. Based on the above, it is needed to provide a preparation process of the bacteroides fragilis capsular polysaccharide A with low toxicity.
Disclosure of Invention
In view of the background art, the main purpose of the invention is to provide a preparation method of the capsular polysaccharide A of the bacteroides fragilis, and the capsular polysaccharide A is extracted from the bacteroides fragilis by adopting the preparation method, so that the use of phenol can be avoided, and the process is ensured to be safe and environment-friendly.
The aim of the invention can be achieved by the following technical scheme:
a preparation method of bacteroides fragilis capsular polysaccharide A comprises the following steps:
preparing bacterial suspension of bacteroides fragilis with pH less than or equal to 5, heating and extracting, and collecting an extracting solution to obtain a crude sugar solution of capsular polysaccharide A.
In one embodiment, the pH of the bacterial suspension is between 2.0 and 4.5.
In one embodiment, the pH of the bacterial suspension is between 2.5 and 4.0.
In one embodiment, the temperature used for the heated extraction is 50℃to 120 ℃.
In one embodiment, the temperature used for the heated extraction is 70℃to 120 ℃.
In one embodiment, the duration of the heat extraction is between 0.5h and 3.0h.
In one embodiment, the pH of the bacterial suspension is 2.5-4.0, the temperature adopted for heating extraction is 90-110 ℃, and the heating extraction time is 1.0-2.5 h.
In one embodiment, the method of making further comprises the step of ultrafiltration of the crude sugar solution and collecting the retentate.
In one embodiment, the method further comprises the step of subjecting the retentate to ion exchange chromatography and ultrafiltration of the collected eluate.
In one embodiment, the heated extraction is performed by water bath heating, gas bath heating, or/and steam heating.
In one embodiment, the extraction fluid is collected by centrifugation.
In one embodiment, the bacteroides fragilis is a bacteroides fragilis strain with a preservation number of CGMCC No. 10685.
Compared with the prior art, the invention has the following beneficial effects:
The inventors have found that, surprisingly, the extraction of the bacterial suspension of Bacteroides fragilis with pH less than or equal to 5 by heating can effectively promote the falling-off of capsular polysaccharide A from the bacteroides fragilis bacterial bodies so as to extract capsular polysaccharide A. Compared with the traditional phenol/water method, the method avoids using phenol and has low toxicity. In addition, by adopting the preparation method, the yield of the bacteroides fragilis capsular polysaccharide A is obviously improved.
Drawings
FIG. 1 is a colony characterization of Bacteroides fragilis ZY-312 of example one;
FIG. 2 is a view of a gram-stained Bacteroides fragilis ZY-312 of example I;
FIG. 3 is a 1H spectrum of capsular polysaccharide A (PSA) analysis by nuclear magnetic resonance spectrometer according to example two of the present invention;
FIG. 4 is a 13C spectrum of capsular polysaccharide A (PSA) analysis by nuclear magnetic resonance spectrometer according to example two of the present invention;
FIG. 5 is a COSY spectrum of capsular polysaccharide A (PSA) analysis by nuclear magnetic resonance spectroscopy according to example two of the present invention;
FIG. 6 is a spectrum of HSQC analyzed by a capsular polysaccharide A (PSA) nuclear magnetic resonance spectrometer according to example two of the present invention;
FIG. 7 is a HMBC spectrum of capsular polysaccharide A (PSA) analyzed by nuclear magnetic resonance spectrometer in accordance with example two of the present invention;
FIG. 8 shows the chemical structural formula of the Bacteroides fragilis capsular polysaccharide A prepared in the second embodiment of the invention.
Detailed Description
The present invention will be described in more detail below in order to facilitate understanding of the present invention. It should be understood, however, that the invention may be embodied in many different forms and should not be limited to the implementations or embodiments described herein. Rather, these embodiments or examples are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments or examples only and is not intended to be limiting of the invention. As used herein, the optional scope of the term "and/or" includes any one of the two or more related listed items, as well as any and all combinations of related listed items, including any two or more of the related listed items, or all combinations of related listed items.
The invention provides a preparation method of bacteroides fragilis capsular polysaccharide A, which comprises the following steps:
Preparing bacterial suspension of bacteroides fragilis with pH less than or equal to 5, heating and extracting, and collecting an extracting solution to obtain a crude sugar solution of capsular polysaccharide A.
In the preparation method provided by the invention, the specific preparation method of the bacterial suspension of the bacteroides fragilis with the pH less than or equal to 5 is not particularly limited, and comprises the following modes: (1) Culturing Bacteroides fragilis and resuspending the collected thallus (which can be called as bacterial mud, the water content can be controlled between 50% and 95%) in a solvent such as water (for example, in purified water with the mass 3-10 times that of the thallus), and then adding a pH regulator to adjust the pH of the bacterial suspension to be less than or equal to 5. The pH regulator may be any one or a combination of several of inorganic acid, organic acid and acid buffer. The inorganic acid is, for example, hydrochloric acid, sulfuric acid, phosphoric acid or the like, and the organic acid is, for example, acetic acid, citric acid or the like. (2) Culturing Bacteroides fragilis and suspending the collected bacteria in inorganic acid, organic acid or/and acid buffer solution with pH less than or equal to 5.
In the preparation method provided by the invention, the pH of the bacterial suspension of the bacteroides fragilis can be adjusted to be 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5 or a pH range between any two pH values. Preferably, the pH of the bacterial suspension is adjusted to 2.0-4.5. Further preferably, the pH of the bacterial suspension is 2.5-4.0. More preferably, the pH of the bacterial suspension is 3.5 to 4.0. The yield of the crude product of the bacteroides fragilis capsular polysaccharide A can be further improved by adopting the better pH.
The preparation method provided by the invention adopts the temperature of 50-120 ℃ for heating and extracting, for example, the temperature can be 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃,100 ℃, 110 ℃, 120 ℃ or a temperature range between any two temperature values. Preferably, the temperature used for the heat extraction is 70-120 ℃. Further preferably, the temperature used for the heat extraction is 90℃to 110 ℃. The yield of the crude product of the bacteroides fragilis capsular polysaccharide A can be further improved by adopting the better pH.
According to the preparation method provided by the invention, the heating and extraction time is 0.5-3.0 h, for example, 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, 3.0h or a time range value between any two time values. Preferably, the heating extraction is performed for a period of time ranging from 1.0h to 2.5h.
According to the preparation method provided by the invention, the pH of the bacterial suspension is 2.5-4.0, the temperature adopted by heating extraction is 90-110 ℃, and the heating extraction time is 1.0-2.5 h. According to the invention, the bacterial body is suspended in water, the pH is regulated to 2.5-4.0, the bacterial body is extracted for 1.0-2.5 hours at the temperature of 90-110 ℃, and the supernatant is obtained by centrifugation, so that the capsular polysaccharide can be effectively promoted to fall off from the bacterial body, and the denaturation and precipitation of protein and nucleic acid are facilitated, thereby reducing the protein and nucleic acid residues of capsular polysaccharide solution.
Capsular polysaccharide is a macromolecular substance, and separation and preparation are more complex than those of monosaccharide and small-molecule oligosaccharide. In the separation and purification process of capsular polysaccharide, main impurities are protein, nucleic acid, endotoxin and other substances; in addition, bacteroides fragilis can express multiple types of capsular polysaccharides simultaneously and some types of capsular polysaccharides do not exhibit the desired biological activity, which also presents a great challenge for the preparation of active capsular polysaccharides of high purity. For safety reasons, impurities, non-target capsular polysaccharide residues should be reduced as much as possible during the capsular polysaccharide preparation process. In addition, the capsular polysaccharide prepared also needs to retain its natural structure inherent to ensure its high immunogenicity or other biological activity. The biological activity of the capsular polysaccharide has obvious structure-activity relation with the structure thereof, and charge property, functional group, molecular weight and the like are all the basis for influencing the biological activity of the capsular polysaccharide. For this purpose, the invention further provides for the purification of the crude sugar solution as described above.
The preparation method provided by the invention further comprises the steps of ultrafiltering the crude sugar solution and collecting trapped liquid. The molecular weight cut-off of the ultrafiltration membrane used for ultrafiltration can be 100KD, 50KD, 30KD, 10KD, 5KD, 3KD or a range between any two molecular weight values.
Further, the preparation method provided by the invention further comprises the steps of carrying out ion exchange chromatography on the trapped liquid and carrying out ultrafiltration on the collected eluent. Preferably: the ion exchange chromatography is anion exchange chromatography, for example DEAE Sepharose Fast Flow ion exchange column chromatography (16 mm. Times.200 mm); gradient elution is adopted in the elution mode, and the pH value of the purified eluent is 5.0-9.0; the eluent flow rate is 15 mL/min-25 mL/min (e.g. 15, 20, 25 mL/min).
For convenience of preservation, the preparation method of the present invention may further dry (e.g. freeze-dry) the product obtained by the purification steps such as ultrafiltration as described above to obtain the capsular polysaccharide A dry powder.
The heating method for heating and extracting provided by the invention is not particularly limited, and water bath heating, gas bath heating, steam heating and the like can be adopted, and two or more heating methods can be combined for use. It will be appreciated that during the heating extraction process, some auxiliary means may be added to enhance the extraction effect, including but not limited to ultrasound, pressurization.
In the preparation method of the invention, the collection mode adopted for collecting the extracting solution is not particularly limited, and comprises but is not limited to adopting a centrifugal mode to collect the extracting solution, wherein the centrifugal condition can be 20-30 ℃ and 11000-13000 g for 8-12 min. For example 13000g at 20℃for 8min, 12000g at 25℃for 10min, 11000g at 30℃for 12min.
The preparation method provided by the invention can be used for extracting capsular polysaccharide A from any bacteroides fragilis, is not particularly limited to specific bacteroides fragilis strains, and can be suitable for bacteroides fragilis strains with the preservation number of CGMCCNo.10685.
The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials are commercially available unless otherwise indicated.
In the following examples, the percentages are mass percentages unless otherwise indicated.
Example one, preparation of Bacteroides fragilis mud
The bacteroides fragilis ZY-312 strain is streaked and inoculated on a blood plate for anaerobic culture for 48 hours. Colony morphology, staining characteristics, size, sphere shape, distribution, etc. were observed.
Colony characteristics: after the bacteroides fragilis ZY-312 is cultured on a blood plate for 48 hours at 37 ℃, the bacteroides fragilis ZY-312 is slightly convex, semitransparent, white, smooth in surface and free from hemolysis, and the colony diameter is between 1mm and 3mm, as shown in figure 1.
Morphology under microscope: the bacteroides fragilis ZY-312 was subjected to gram-stain microscopic examination to show a typical rod shape for gram-negative bacteria, and was rounded at both ends to be densely stained, and the non-colored part in the middle of the thallus was formed as a cavitation, see FIG. 2.
And (3) inoculating a single colony into tryptone broth for fermentation culture for 8 hours (the temperature is 37 ℃), centrifuging and precipitating the obtained bacterial liquid at the rotating speed of 3000r/min for 15min, removing the supernatant, and collecting the precipitate to obtain the bacteroides fragilis ZY-312 bacterial sludge.
Example two preparation method of Bacteroides fragilis capsular polysaccharide A comparison
Experiments were performed using the bacterial sludge prepared in example one.
(1) Method 1: the method provided by the invention is used for preparing the bacteroides fragilis capsular polysaccharide A
① 50G of bacterial sludge (obtained by preparation of example 1) was taken, 300g of purified water was added to suspend the bacterial mass, the pH was adjusted to 3.5 with 1mol/L hydrochloric acid solution, extraction was carried out at 100℃for 1.5 hours, cooling to room temperature, and centrifugation was carried out at 12000g for 10 minutes at room temperature, and a crude sugar solution was obtained from the supernatant.
② Ultrafiltering and concentrating the crude sugar solution with 10KD ultrafilter membrane to remove small molecular impurities until the conductivity is stable, and collecting the reflux;
③ Adding an equal volume of 40mmol/L Tris-HCl (pH 8.5) salt to the reflux liquid; DEAE Sepharose Fast Flow ion exchange column chromatography (16 mm. Times.200 mm), flow rate 20mL/min, linear gradient eluting with 20mmol/L Tris-HCl (pH8.5) containing 0.2mol/L sodium chloride for 25 column volumes (in 25 column volumes, sodium chloride concentration in mobile phase increases from 0mol/L to 0.2 mol/L), sectional collecting, 100 mL/bottle (component), SEC-HPLC tracking monitoring, combining 206nm absorption peak as single and symmetrical peak component, 10KD ultrafiltration membrane ultrafiltration, adding purified water for repeated ultrafiltration until conductivity is stable, collecting reflux liquid, and lyophilizing;
In parallel 2 experiments, the obtained bacteroides fragilis capsular polysaccharide A is named as 'S1' and 'S2', respectively.
(2) Method 2: preparation of bacteroides fragilis capsular polysaccharide A by phenol/water method
① Taking 50g of bacterial sludge (obtained by the preparation of the example 1), adding 200mL of 0.15mol/L sodium chloride solution, washing, centrifuging at normal temperature for 20min by 12000g, and collecting bacterial sludge;
② Adding 750mL of purified water to allow the bacteria to be resuspended, adding an equal volume of 75% phenol water solution (m/m), stirring at 68 ℃ to extract for 30min, centrifuging at 16000g at room temperature for 30min, and removing precipitate;
③ Extracting the supernatant with an equal volume of diethyl ether for 3 times, and collecting a water phase; the aqueous phase was concentrated by rotary evaporator, dialyzed and lyophilized.
④ The sample was dissolved in 0.1mol/L sodium acetate solution (containing 10mmol/L CaCl 2 and 10mmol/L LMgCl 2), 2mg of DNase and 10mg of RNase were added, stirred at 37℃for 2 hours, 2mg of DNase and 10mg of RNase were again added, and stirred at 37℃overnight; adjusting pH to 7.0, adding 20mg pronase, stirring at 37deg.C for 2 hr, adding 20mg pronase again, and stirring at 37deg.C overnight; absolute ethanol was added to a final concentration of 80%, and the mixture was centrifuged at 12000g for 10min at 4℃overnight to obtain a precipitate.
⑤ Adding 5% acetic acid solution into the precipitate for redissolution, hydrolyzing at 100deg.C for 1 hr, dialyzing for desalting, separating with DEAE Sepharose Fast Flow ion exchange column chromatography, collecting in sections, tracking and monitoring by SEC-HPLC, mixing components with 206nm absorption peak as single and symmetrical peak, ultrafiltering for desalting, and lyophilizing; in parallel 2 experiments, the obtained bacteroides fragilis capsular polysaccharide A is named as 'S3' and 'S4', respectively.
(3) Experimental results
The comparison results of the capsular polysaccharide A of Bacteroides fragilis obtained by the two preparation methods are shown in Table 1. Wherein:
Protein content testing: the fourth part <0731> protein content assay of the chinese pharmacopoeia (2020 edition) -the fourth method 2,2 '-biquinoline-4, 4' -dicarboxylic acid method (BCA method);
nucleic acid content testing: ultraviolet-visible spectrophotometry of the fourth part <0401> of the Chinese pharmacopoeia (2020 edition);
combined lipid content test: ① Weighing capsular polysaccharide A10mg, adding 2% acetic acid 20ml, refluxing at 90deg.C, and adding free lipid for 2 hr; ② Adding 50ml of n-hexane, sufficiently shaking, sucking an organic phase, drying by nitrogen, and extracting free lipid; ③ 10ml of 4% sodium hydroxide-methanol solution is added and the mixture is saponified for 1h at 80 ℃; ④ Adding 10ml of n-hexane, sufficiently shaking, sucking an organic phase, drying by nitrogen, and extracting fatty acid; ⑤ Adding 10ml of 1% methanol sulfate, and reacting at 80 ℃ for 30min to esterify fatty acid methyl; ⑥ 20ml of n-hexane was added, and fatty acid methyl ester was extracted for GC-MS analysis.
Crude capsular polysaccharide A yield: yield = crude weight/mass of bacterial sludge after lyophilization x 100%.
Table 1 comparison of quality Properties of Bacteroides fragilis capsular polysaccharide A obtained by two preparation methods
In this example, the capsular polysaccharide A (PSA) prepared by the method 1 has a 1H spectrum shown in FIG. 3, a 13C spectrum shown in FIG. 4, a COSY spectrum shown in FIG. 5, an HSQC spectrum shown in FIG. 6, an HMBC spectrum shown in FIG. 7, and a chemical structural formula shown in FIG. 8.
Example III influence of pH of bacterial sludge suspension on capsular polysaccharide A yield
Bacteroides fragilis ZY-312 is fermented, centrifuged, and the precipitate is collected to obtain bacterial sludge (obtained by the preparation of example 1), which is preserved at-20 ℃.
Taking 8 parts of bacterial sludge, adding 50g of purified water into 10g of each part to suspend the bacterial sludge again, and respectively adjusting the pH to 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 and 6.5;
Heating at 105deg.C for 60min, immediately taking out, cooling to room temperature, centrifuging at 12000g for 10min, and collecting supernatant;
concentrating and desalting by a 10KD ultrafiltration centrifuge tube, and freeze-drying to obtain a crude product of the bacteroides fragilis capsular polysaccharide A.
The yields of crude polysaccharide A from Bacteroides fragilis at different pH conditions are shown in Table 2.
The effect of different extract pH on the quality attributes of Bacteroides fragilis capsular polysaccharide A pure quality is shown in Table 3.
As can be seen from table 2: in the pH range of 2.5-4.0, the yield of the crude polysaccharide A of the bacteroides fragilis capsular polysaccharide is higher than 0.8%, the yield is 0.48% when the pH is 2.0, and the yield is reduced obviously when the pH is 5.0 or above, so that the pH of the suspension liquid in the preparation process of the bacteroides fragilis capsular polysaccharide should be controlled to be 2.5-4.0.
As can be seen from table 3: the content of the protein of the bacteroides fragilis capsular polysaccharide A is 0.45-0.85% within the pH range of 2.5-4.0; the nucleic acid content is less than 0.1%, the conjugated lipid content is 0.05-0.25%, the protein content is 0.45% at pH2.0, the nucleic acid content is 0.07%, and the conjugated lipid content is 0.01%. pH5.0 and above, the protein, nucleic acid and bound lipid content increased significantly, especially to 1.89%. Therefore, the pH of the suspension is controlled to be 2.5-4.0 in the preparation process of the bacteroides fragilis capsular polysaccharide.
TABLE 2 influence of pH of the extract on crude yield of Bacteroides fragilis capsular polysaccharide A
TABLE 3 influence of pH of the extract on quality attributes of pure Bacteroides fragilis capsular polysaccharide A
| pH | Protein content% | Nucleic acid content% | Content of bound lipid% |
| 2.0 | 0.45 | 0.07 | 0.01 |
| 2.5 | 0.51 | 0.06 | 0.01 |
| 3.0 | 0.48 | 0.06 | 0.03 |
| 3.5 | 0.61 | 0.05 | 0.09 |
| 4.0 | 0.85 | 0.09 | 0.25 |
| 4.5 | 0.89 | 0.12 | 1.01 |
| 5.0 | 1.56 | 0.18 | 1.89 |
| 6.0 | 2.98 | 0.29 | 2.32 |
| 6.5 | 4.51 | 0.42 | 2.34 |
Example IV influence of extraction temperature on capsular polysaccharide A yield
Taking 8 parts of bacterial sludge (obtained by preparing in example 1), adding 50g of purified water to each 10g of bacterial sludge to re-suspend the bacterial sludge, and regulating the pH of each bacterial sludge to be about 3.5;
Extracting at 50deg.C, 60deg.C, 70deg.C, 80deg.C, 90deg.C, 100deg.C, 110deg.C, 12000g, centrifuging at room temperature for 10min, collecting supernatant;
And ultra-filtering small molecular impurities by a 10KD ultra-filtration centrifuge tube, and freeze-drying to obtain a crude product of the bacteroides fragilis capsular polysaccharide A.
The yields of the obtained bacteroides fragilis capsular polysaccharide A under different extraction temperature conditions are shown in Table 4.
From table 4, it can be seen that: the yield of crude sugar of the bacteroides fragilis capsular polysaccharide A is higher than 0.80 percent in the temperature range of 90-120 ℃, and the yield of the crude sugar is obviously reduced at 80 ℃ or below, so that the extraction temperature should be controlled in the range of 90-120 ℃, preferably 90-110 ℃ in the preparation process of the bacteroides fragilis capsular polysaccharide A.
TABLE 4 influence of extraction temperature on crude yield of Bacteroides fragilis capsular polysaccharide A
Example five, influence of extraction time on capsular polysaccharide A yield
Taking 6 parts of bacterial sludge (obtained by the preparation of example 1), adding 50g of purified water to each 10g of bacterial sludge to re-suspend the bacterial sludge, and adjusting the pH of the bacterial sludge to about 4.0;
Placing into a vertical autoclave, extracting at 110deg.C for 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, and 3.0h respectively, immediately taking out after finishing, cooling to room temperature, centrifuging, and collecting supernatant;
And ultra-filtering small molecular impurities by a 10KD ultra-filtration centrifuge tube, and freeze-drying to obtain a crude product of the bacteroides fragilis capsular polysaccharide A.
The yields of crude polysaccharide A from Bacteroides fragilis at different extraction times are shown in Table 5.
From table 5, it can be seen that: when the extraction time is 0.5h, the yield of the crude polysaccharide of the bacteroides fragilis capsular polysaccharide is 0.74%, the yield is higher than 0.80% in the extraction time range of 1.0-2.5 h, and when the extraction time is 3.0h, the yield is reduced, which is probably that the capsular polysaccharide is degraded. Therefore, the extraction time in the preparation process of the bacteroides fragilis capsular polysaccharide is preferably 1.0-2.5 h.
TABLE 5 influence of extraction time on crude saccharide yield of Bacteroides fragilis capsular polysaccharide A
EXAMPLE six preparation of Bacteroides fragilis capsular polysaccharide A with different molecular weight distribution
(1) Experimental method
The method provided by the invention is used for preparing the bacteroides fragilis capsular polysaccharide A with different molecular weight distribution, and comprises the following specific steps:
① Taking 100g of fermented ZY-312 bacteroides fragilis mud (prepared by the method of example 1), adding 500mL of purified water, uniformly stirring, and adjusting the pH to 3.0-3.1 by using 1M hydrochloric acid solution;
② Continuously stirring and heating for 2h at 100 ℃, cooling to room temperature, and centrifuging at 12000g for 10min at normal temperature to obtain 500mL of supernatant;
③ Adding 5L of purified water, and concentrating to about 100mL by a10 KD ultrafiltration membrane; adding 5L of purified water again, and ultrafiltering to 100mL; repeating 3 times, and the final volume is about 100mL, and the conductivity is 25 mu s/cm;
④ 100mL of 40mmol/L Tris-HCl (pH 8.5) was added, mixed well and filtered through a 0.45 μm filter;
⑤ Loading onto ion exchange chromatography column (50 mm. Times.150 mm, unigel 80Q packing, suzhou Nami) at 20mL/min flow rate, rinsing 2 column volumes with 20mmol/L Tris-HCl; then, tris-HCl (pH 8.5) solution containing 0.2M sodium chloride was eluted in a gradient, i.e., the concentration of sodium chloride was increased from 0 to 0.2M, over 25 column volumes.
⑥ And collecting the eluate in a segmented way, and collecting 3 components in total, and identifying by infrared spectrum to determine that the components are all bacteroides fragilis capsular polysaccharide A.
⑦ The 10KD filter membrane of each component is subjected to ultrafiltration desalination, the conductivity is stable when the volume reaches 100mL, and the polysaccharide A is freeze-dried to obtain the bacteroides fragilis capsular polysaccharides A with different weight average molecular weights, wherein the capsular polysaccharides A are 168mg, 213mg and 197mg respectively; the samples were designated P1, P2, P3, respectively.
(2) Experimental results
TABLE 6 Bacteroides fragilis capsular polysaccharide A with different weight average molecular weights
In conclusion, the capsular polysaccharide A prepared by the preparation method of the application: the molecular weight of the catalyst is distributed in 70 KD-100 KD, and the part accounts for 70% -80% of the total amount; the molecular weight is 80 KD-90 KD; the ratio of the weight average molecular weight to the number average molecular weight (Mw/Mn) is 1.0 to 1.3; fatty acid content is less than 0.02% or no fatty acid is present. The process of the application simplifies the preparation process of the bacteroides fragilis capsular polysaccharide, does not involve the use of organic reagents such as phenol, diethyl ether, ethanol and the like, is green and environment-friendly, and can be industrialized.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. It should be understood that, based on the technical solutions provided by the present invention, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.
Claims (10)
1. The preparation method of the bacteroides fragilis capsular polysaccharide A is characterized by comprising the following steps of:
(1) Preparing bacterial suspension of bacteroides fragilis with pH less than or equal to 5, heating and extracting, and collecting an extracting solution to obtain a crude sugar solution of capsular polysaccharide A;
(2) A step of ultrafiltration of the crude sugar solution and collecting a retentate; and
(3) And (3) performing ion exchange chromatography on the trapped liquid and performing ultrafiltration on the collected eluent.
2. The method of claim 1, wherein the pH of the bacterial suspension is selected from the group consisting of 2.0 to 4.5, 2.5 to 4.0, and 3.5 to 4.5.
3. The method of claim 2, wherein the pH of the bacterial suspension is 2.0, 2.5, 3.0, 3.5, 4.0 or 4.5.
4. The process according to claim 1, wherein the temperature used for the heat extraction is selected from the group consisting of 90 ℃ to 120 ℃ and 90 ℃ to 110 ℃.
5. The process according to claim 4, wherein the heating extraction is carried out at a temperature of 90℃at 100℃at 110℃or 120 ℃.
6. The preparation method according to claim 1, wherein the duration of the heat extraction is 0.5h to 3.0h, preferably 0.5h, 1.0h, 1.5h, 2.0h, 2.5h or 3.0h.
7. The preparation method according to claim 1, wherein the pH of the bacterial suspension is 2.5-4.0, the temperature used for heating extraction is 90-110 ℃, and the duration of heating extraction is 1.0-2.5 h.
8. The process according to any one of claims 1 to 7, wherein the ultrafiltration of the crude sugar solution is carried out using an ultrafiltration membrane having a molecular weight cut-off of 10KD.
9. The method according to any one of claims 1 to 6, wherein the ion exchange chromatography is anion exchange chromatography;
Preferably, anion exchange chromatography employs DEAE Sepharose Fast Flow ion exchange column chromatography columns, 16mm by 200mm; further preferably, the elution mode is gradient elution;
More preferably, the pH of the eluent is 5.0-9.0, and the flow rate of the eluent is 15-25 mL/min.
10. The method according to any one of claims 1 to 6, wherein the heating extraction is performed by water bath heating, gas bath heating or/and steam heating;
or/and, collecting the extracting solution by centrifugation;
Or/and the bacteroides fragilis is a bacteroides fragilis strain with a preservation number of CGMCC No. 10685;
preferably, the extract is cooled prior to the centrifugation;
More preferably, the conditions of centrifugation include: the temperature is 20-30 ℃, and the centrifugation is carried out for 8-12 min at 11000-13000 g.
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| CN114699423B (en) * | 2022-02-16 | 2023-06-23 | 广州知易生物科技有限公司 | Application of capsular polysaccharide extract of bacteroides fragilis in preparation of medicines for preventing and treating schizophrenia |
| CN114699422B (en) * | 2022-02-16 | 2023-07-18 | 广州知易生物科技有限公司 | Application of capsular polysaccharide extract of bacteroides fragilis in preparation of medicines for preventing and treating Alzheimer disease |
| CN116327809A (en) * | 2023-02-27 | 2023-06-27 | 广州知易生物科技有限公司 | Application of bacteroides fragilis and extract capsular polysaccharide A thereof in preparation of product for preventing and treating autoimmune arthritis |
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| GB0502096D0 (en) * | 2005-02-01 | 2005-03-09 | Chiron Srl | Purification of streptococcal capsular polysaccharide |
| JP5286089B2 (en) * | 2006-01-13 | 2013-09-11 | バクスター・インターナショナル・インコーポレイテッド | Method for purifying polysaccharides |
| AU2010310919B2 (en) * | 2009-10-30 | 2015-05-07 | Glaxosmithkline Biologicals S.A. | Purification of Staphylococcus aureus type 5 and type 8 capsular saccharides |
| CN102660603B (en) * | 2012-04-17 | 2015-03-25 | 江苏康泰生物医学技术有限公司 | Method for rapid purification of bacterial capsular polysaccharide |
| EP3034516A1 (en) * | 2014-12-19 | 2016-06-22 | Novartis AG | Purification of streptococcal capsular polysaccharide |
| CN105434476B (en) * | 2015-10-29 | 2019-02-15 | 广州知易生物科技有限公司 | A kind of application of bacteroides fragilis in prevention and or treatment inflammation enteropathy |
| CN107082819A (en) * | 2017-01-22 | 2017-08-22 | 华兰生物工程股份有限公司 | A kind of purification process of streptococcus pneumoniae capsular polysaccharide |
| CN107043431B (en) * | 2017-02-23 | 2020-06-30 | 上海瑞宙生物科技有限公司 | Purification method of bacterial capsular polysaccharide |
| CN109897117B (en) * | 2017-12-08 | 2021-08-06 | 广州知易生物科技有限公司 | Phosphorylated TP2 polysaccharide, and preparation method and application thereof |
| CN118345133A (en) * | 2021-06-15 | 2024-07-16 | 广州知易生物科技有限公司 | Preparation method of bacteroides fragilis capsular polysaccharide A |
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