WO2022262158A1 - Preparation method for capsular polysaccharide a of bacteroides fragilis - Google Patents

Abstract

A preparation method for capsular polysaccharide A of bacteroides fragilis. The preparation method comprises the following steps: preparing a bacterial suspension of bacteroides fragilis having a pH value of less than or equal to 5; performing heating and extraction; and collecting an extract to obtain a crude sugar solution of capsular polysaccharide A.

Description

Preparation method of Bacteroides fragilis capsular polysaccharide A technical field

The invention relates to the preparation and application technology of Bacteroides fragilis capsular polysaccharide, in particular to a preparation method of Bacteroides fragilis capsular polysaccharide A.

Background technique

Bacteroides fragilis (bacteroides fragilis) is a member of the genus Bacteroides among Gram-negative anaerobic bacteria, belonging to the phylum Bacteroidetes, which is completely different from Bifidobacteria and lactic acid bacteria of the phylum Firmicutes. There are 25 species of Bacteroides, 10 species from humans only, 10 species from animals only, and 5 species from both humans and animals. Bacteroides fragilis is an obligate anaerobic bacterium, which can be divided into enterotoxigenic Bacteroides fragilis (ETBF) and non-enterotoxigenic Bacteroides fragilis (ETBF) according to whether it can synthesize and secrete Bacteroides fragilis enterotoxin (BFT) Toxin-type Bacteroides fragilis (Nontoxigenic Bacteroides fragilis, NTBF). Bacteroides fragilis, as part of the normal intestinal flora of humans and animals, mainly exists in the colon. In addition, the mucous membranes of the respiratory tract, gastrointestinal tract, and genitourinary tract can also colonize and grow.

Bacteroides fragilis can express 8 different capsular polysaccharides, which are respectively denoted as A to H. The role of capsular polysaccharide A (polysaccharide A, PSA) is determined by its molecular weight. The molecular weight of natural PSA is about 110kDa.

There have been research reports (Dennis L. Kasper, Andrew B. Onderdonk, Joseph Crabb, and John G. Bartlett Protective Efficacy of Immunization with Capsular Antigen against Experimental Infection with Bacteroides fragilis.THE JOURNAL OF INFECTIOUS.5.1SEAS40.V NOVEMBER 1979) showed that the capsular polysaccharide of Bacteroides fragilis plays an active role in preventing infections caused by bacteria of the same genus, and the study suggested that this is because the capsular polysaccharide of Bacteroides genus has several similar epitopes. The study found that although animals immunized with B. fragilis capsular polysaccharide were unable to fight off infections caused by other bacteria, such as E. coli, there was a significant reduction in infection levels due to immunity to Bacteroides spp. Meanwhile, this study compared B. fragilis capsular polysaccharide A with capsular polysaccharide B. Both capsular polysaccharides showed preventive effects, but capsular polysaccharide A had a much smaller onset dose than capsular polysaccharide B.

At present, there are still many cases about the prevention/treatment of B. fragilis capsular polysaccharide A on diseases, so how to well realize the preparation of B. fragilis capsular polysaccharide A is of great significance.

There have been a large number of research reports on the preparation methods of Bacteroides fragilis capsular polysaccharide A (PSA). The methods disclosed in each document are the phenol/water method, which is also the technological basis for the preparation of bacterial polysaccharide vaccines. The traditional process of preparing Bacteroides fragilis capsular polysaccharide A based on the phenol/water method is as follows:

In the U.S. patent application with the publication number US20140243285A1, the Bacteroides fragilis NCTC 9343 strain is used as raw material, mixed with phenol/water solution, extracted by continuous stirring at 68°C → centrifuged to collect the aqueous phase → ether extraction to remove phenol → protease/nuclease Remove protein and nucleic acid → ethanol precipitation polysaccharide → add 2% acetic acid high temperature hydrolysis → molecular sieve or ion exchange chromatography purification process to obtain Bacteroides fragilis capsular polysaccharide A containing a small amount of bound lipid, with a molecular weight of about 110KD.

The Chinese invention patent application whose publication numbers are CN110025636A and CN109954005A relates to the preparation method of the Bacteroides fragilis extract comprising the following steps: (1) centrifuging the Bacteroides fragilis bacterial liquid after fermentation and cultivation, collecting the first precipitate, and taking the Add water at 65°C to 72°C for the first precipitate, add phenol solution after dissolving, keep stirring at 65°C to 72°C for 25min to 35min, centrifuge, and collect the first supernatant; (2) In step (1), The first supernatant of collection removes phenol with ether extraction, then removes residual ether, collects aqueous phase solution; (3) in the aqueous phase solution collected in step (2), add dehydrated alcohol to the final concentration of ethanol is 75-85v/v%, alcohol precipitation, centrifugation, and collect the second precipitate; (4) take the second precipitate, add water to prepare a suspension, then adjust the pH to 6.5-7.5, centrifuge, and collect the second precipitate The supernatant liquid was dialyzed to remove salt and freeze-dried to obtain the Bacteroides fragilis extract. These technical proposals improve the process of extracting PSA by the phenol/water method, reduce the steps of enzymatic protein removal and nucleic acid removal, and simplify the process.

But in essence, the above traditional methods rely on phenol, which is toxic and harmful, and its use is not conducive to production safety and environmental protection. Based on this, it is urgent to provide a preparation process of Bacteroides fragilis capsular polysaccharide A with low toxicity.

Contents of the invention

In view of the above background technology, the main purpose of the present invention is to provide a preparation method of capsular polysaccharide A from Bacteroides fragilis, which can avoid the use of phenol and ensure a safe and environmentally friendly process.

The purpose of the present invention can be achieved through the following technical solutions:

A preparation method of Bacteroides fragilis capsular polysaccharide A, said preparation method comprising the following steps:

Prepare a bacterial suspension of Bacteroides fragilis with pH ≤ 5, extract by heating, and collect the extract to obtain a crude sugar solution of capsular polysaccharide A.

In one of the embodiments, the pH of the bacterial suspension is 2.0-4.5.

In one of the embodiments, the pH of the bacterial suspension is 2.5-4.0.

In one embodiment, the temperature used for heating and extraction is 50°C-120°C.

In one embodiment, the temperature used for heating and extraction is 70°C-120°C.

In one of the embodiments, the duration of heating extraction is 0.5h-3.0h.

In one embodiment, the pH of the bacterial suspension is 2.5-4.0, the temperature used for heating extraction is 90°C-110°C, and the duration of heating extraction is 1.0h-2.5h.

In one of the embodiments, the preparation method further includes the step of ultrafiltering the crude sugar solution and collecting the retentate.

In one embodiment, the preparation method further includes the steps of performing ion exchange chromatography on the retentate and ultrafiltration on the collected eluate.

In one embodiment, the heating extraction method is water bath heating, air bath heating or/and steam heating.

In one of the embodiments, the extraction solution is collected by centrifugation.

In one of the embodiments, the Bacteroides fragilis is a strain of Bacteroides fragilis with the deposit number CGMCC No.10685.

Compared with the prior art, the present invention has the following beneficial effects:

The inventors unexpectedly found that heating and extracting the bacterial suspension of Bacteroides fragilis with pH ≤ 5 can effectively promote the shedding of capsular polysaccharide A from the Bacteroides fragilis cells, thereby extracting capsular polysaccharide A. Compared with the traditional phenol/water method, the present invention avoids the use of phenol and has low toxicity. Moreover, by adopting the preparation method of the present invention, the yield of Bacteroides fragilis capsular polysaccharide A is obviously improved.

Description of drawings

Fig. 1 is the colony characteristic figure of Bacteroides fragilis ZY-312 of embodiment one;

Fig. 2 is the microscopic observation figure after Gram staining of Bacteroides fragilis ZY-312 in Example 1;

Fig. 3 is the 1H spectrum analyzed by the capsular polysaccharide A (PSA) nuclear magnetic resonance spectrometer of the second embodiment of the present invention;

Fig. 4 is the 13C spectrum analyzed by the capsular polysaccharide A (PSA) NMR spectrometer of the second embodiment of the present invention;

Fig. 5 is the COZY spectrum analyzed by the capsular polysaccharide A (PSA) nuclear magnetic resonance spectrometer of the second embodiment of the present invention;

Fig. 6 is the HSQC spectrum analyzed by the capsular polysaccharide A (PSA) NMR spectrometer of the second embodiment of the present invention;

Fig. 7 is the HMBC spectrum analyzed by the capsular polysaccharide A (PSA) nuclear magnetic resonance spectrometer of the second embodiment of the present invention;

Fig. 8 is the chemical structural formula of Bacteroides fragilis capsular polysaccharide A prepared in Example 2 of the present invention.

detailed description

In order to facilitate the understanding of the present invention, the present invention will be described in more detail below. It should be understood, however, that the present invention may be embodied in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the disclosure of the present invention more thorough and comprehensive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used herein in the description of the present invention are only for the purpose of describing specific embodiments or examples, and are not intended to limit the present invention. The optional range of the term "and/or" used herein includes any one of two or more of the relevant listed items, and also includes any and all combinations of the relevant listed items, and any and all of the Combinations include combinations of any two of the related listed items, any more of the related listed items, or all of the related listed items.

The invention provides a method for preparing Bacteroides fragilis capsular polysaccharide A, the preparation method comprising the following steps:

Prepare a bacterial suspension of Bacteroides fragilis with pH ≤ 5, extract by heating, and collect the extract to obtain a crude sugar solution of capsular polysaccharide A.

In the preparation method provided by the present invention, the specific preparation method of the bacterial suspension of pH≤5 Bacteroides fragilis is not particularly limited, including but not limited to the following methods: (1) cultivating Bacteroides fragilis and collecting the collected thalli (can be Called bacteria slime, water content can be controlled at 50% ~ 95%) resuspended in a solvent such as water (for example, resuspended in purified water whose quality is 3 to 10 times the weight of the thallus), and then added to adjust the pH The pH of the bacterial suspension was adjusted to ≤5. The pH regulator can be any one or a combination of inorganic acids, organic acids and acidic buffers. The inorganic acid mentioned here is, for example, hydrochloric acid, sulfuric acid, phosphoric acid, etc., and the organic acid mentioned here is, for example, acetic acid, citric acid, etc. (2) Cultivate Bacteroides fragilis and resuspend the collected bacteria in inorganic acid, organic acid or/and acidic buffer solution with pH≤5.

In the preparation method provided by the present invention, the pH of the bacterial suspension of Bacteroides fragilis can be adjusted to be 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 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 3.5-4.0. Using a better pH can further increase the yield of the crude Bacteroides fragilis capsular polysaccharide A.

In the preparation method provided by the present invention, the temperature used for heating and extraction is 50°C to 120°C, for example, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, 120°C or any two temperatures The temperature range between the values. Preferably, the temperature used for the heating extraction is 70°C to 120°C. Further preferably, the temperature used for the heating extraction is 90°C to 110°C. Using a better pH can further increase the yield of the crude Bacteroides fragilis capsular polysaccharide A.

In the preparation method provided by the present invention, the heating extraction time is 0.5h to 3.0h, 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 duration of heating extraction is 1.0h-2.5h.

In the preparation method provided by the invention, the pH of the bacterial suspension is 2.5-4.0, the temperature used for heating extraction is 90°C-110°C, and the duration of heating extraction is 1.0h-2.5h. In the present invention, by resuspending the bacteria in water, adjusting the pH to 2.5-4.0, extracting at 90°C-110°C for 1.0h-2.5h, and then centrifuging to get the supernatant, it can effectively promote the shedding of capsular polysaccharides from the bacteria. It is beneficial to promote the denaturation and precipitation of proteins and nucleic acids, thereby reducing the residues of proteins and nucleic acids in the capsular polysaccharide solution.

Capsular polysaccharides are macromolecular substances, and their separation and preparation are more complicated than monosaccharides and small molecule oligosaccharides. During the separation and purification of capsular polysaccharides, the main impurities are proteins, nucleic acids, endotoxins and other substances; in addition, Bacteroides fragilis can express multiple types of capsular polysaccharides at the same time, and some types of capsular polysaccharides do not present the required Bioactivity, which also brings great challenges to the preparation of high-purity active capsular polysaccharides. For safety considerations, during the preparation of capsular polysaccharides, impurities and non-target capsular polysaccharide residues should be reduced as much as possible. In addition, the prepared capsular polysaccharide also needs to maintain its inherent natural structure to ensure its high immunogenicity or other biological activities. The biological activity of capsular polysaccharide has obvious structure-activity relationship with its structure, charge properties, functional groups, molecular weight, etc. are the basis of affecting the biological activity of capsular polysaccharide. For this reason, the present invention further purifies the crude sugar solution as described above.

The above-mentioned preparation method provided by the present invention also includes the step of ultrafiltering the crude sugar solution and collecting the retentate. The molecular weight cut-off of the ultrafiltration membrane used in the ultrafiltration here can be 100KD, 50KD, 30KD, 10KD, 5KD, 3KD or a range between any two molecular weight values.

Further, the preparation method provided by the present invention also includes the steps of performing ion exchange chromatography on the retentate and ultrafiltration on the collected eluate. Preferably: the ion-exchange chromatography is anion-exchange chromatography, such as DEAE Sepharose Fast Flow ion-exchange column chromatography (16mm×200mm); the elution method adopts gradient elution, and the pH of the purified eluent is 5.0-9.0 ; The eluent flow rate is 15mL/min-25mL/min (for example, 15, 20, 25mL/min).

For the convenience of storage, the preparation method of the present invention can also dry (for example freeze-dry) the product obtained through the purification steps such as ultrafiltration as described above to obtain dry powder of capsular polysaccharide A.

In the preparation method provided by the present invention, the heating method of heating extraction is not particularly limited, and water bath heating, air bath heating, steam heating, etc. can be used, and of course two or more heating methods can also be used in combination. It can be understood that during the heating extraction process, some auxiliary means can also be added to improve the extraction effect, and these auxiliary means include but are not limited to ultrasound and pressure.

In the preparation method of the present invention, the collection method used for collecting the extract is not particularly limited, including but not limited to collecting the extract by centrifugation, and the centrifugation conditions can be 20°C-30°C, 11000g-13000g Centrifuge for 8 minutes to 12 minutes. For example, centrifuge at 13000g for 8min at 20°C, centrifuge at 12000g for 10min at 25°C, and centrifuge at 11000g for 12min at 30°C.

The preparation method provided by the present invention can be used to extract capsular polysaccharide A from any Bacteroides fragilis, and there is no special limitation on the specific Bacteroides fragilis strain, for example, it can be applied to the Bacteroides fragilis strain with the deposit number of CGMCC No.10685.

The test methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and biological materials, unless otherwise specified, can be obtained from commercial sources.

In the following examples, the percentages are mass percentages unless otherwise specified.

Embodiment 1, the preparation of Bacteroides fragilis bacterium slime

Streak inoculation of Bacteroides fragilis ZY-312 strain on blood plate, anaerobic culture for 48h. Observe the colony morphological characteristics, staining characteristics, size, club shape and distribution, etc.

Colony characteristics: After Bacteroides fragilis ZY-312 was cultured on a blood plate at 37°C for 48 hours, it was slightly convex, translucent, white, smooth, non-hemolytic, and the diameter of the colony was between 1mm and 3mm, see Figure 1.

Morphology under the microscope: Bacteroides fragilis ZY-312 was examined by Gram staining. It is a Gram-negative bacterium with a typical rod shape, blunt rounded ends and dense staining. The uncolored part in the middle of the bacteria is like a vacuole. figure 2.

Select a single colony and inoculate it in tryptone broth for fermentation and culture for 8 hours (at a temperature of 37°C). The obtained bacterial solution is centrifuged and precipitated at a speed of 3000r/min, centrifuged for 15 minutes, the supernatant is removed, and the precipitate is collected to obtain Bacteroides fragilis ZY -312 Slime.

Example 2, comparison of preparation methods of Bacteroides fragilis capsular polysaccharide A

The bacteria slime prepared in Example 1 was used to carry out the experiment.

(1) Method 1: Prepare Bacteroides fragilis capsular polysaccharide A using the method provided by the invention

①Take 50g of bacteria slime (obtained by Example 1), add 300g of purified water to resuspend the bacteria, adjust its pH to 3.5 with 1mol/L hydrochloric acid solution, extract at 100°C for 1.5h, cool to room temperature, and centrifuge at 12000g for 10min at room temperature. Take the supernatant to obtain a crude sugar solution.

②The crude sugar solution is concentrated by 10KD ultrafiltration membrane ultrafiltration to remove small molecular impurities until the conductivity is stable, and the reflux liquid is collected;

③ Add an equal volume of 40mmol/L Tris-HCl (pH8.5) to the reflux liquid to convert salt; DEAE Sepharose Fast Flow ion exchange column chromatography (16mm×200mm), flow rate 20mL/min, use 0.2mol/L sodium chloride 20mmol/L Tris-HCl (pH8.5) linear gradient elution of 25 column volumes (within 25 column volumes, the concentration of sodium chloride in the mobile phase rises from 0mol/L to 0.2mol/L), segmented collection, 100mL/bottle (component), tracked and monitored by SEC-HPLC, combined 206nm absorption peak into a single, symmetrical peak component, 10KD ultrafiltration membrane ultrafiltration, added purified water and repeated ultrafiltration until the conductivity was stable, collected the reflux liquid, Freeze-dried;

Two experiments were performed in parallel, and the capsular polysaccharide A of Bacteroides fragilis was obtained and named "S1" and "S2", respectively.

(2) Method 2: Preparation of Bacteroides fragilis capsular polysaccharide A by phenol/water method

① Take 50g of bacteria sludge (obtained by Example 1), add 200mL of 0.15mol/L sodium chloride solution, and after washing, centrifuge at 12000g for 20min at room temperature to collect the bacteria sludge;

②Add 750mL of purified water to resuspend the bacteria, then add an equal volume of 75% phenol aqueous solution (m/m), stir and extract at 68°C for 30min, centrifuge at 16000g for 30min at room temperature, and remove the precipitate;

③The supernatant was extracted three times with an equal volume of ether, and the aqueous phase was collected; the aqueous phase was concentrated by a rotary evaporator, dialyzed and then freeze-dried.

④ Dissolve the above sample with 0.1mol/L sodium acetate solution (containing 10mmol/L CaCl ₂ and 10mmol/L MgCl ₂ ), add 2mg deoxyribonuclease and 10mg ribonuclease, stir at 37°C for 2h, then add 2mg deoxyribonucleic acid again Enzyme and 10mg ribonuclease, stir overnight at 37°C; adjust pH to 7.0, add 20mg pronase, stir at 37°C for 2h, add 20mg pronase again, stir overnight at 37°C; add absolute ethanol to a final concentration of 80% , overnight at 4°C, centrifuged at 12,000 g for 10 min, and the precipitate was collected.

⑤ Add 5% acetic acid solution to the precipitate to redissolve, hydrolyze at 100°C for 1 hour, and desalinize by dialysis, then conduct chromatography on DEAE Sepharose Fast Flow ion exchange column, collect in sections, track and monitor with SEC-HPLC, and merge the 206nm absorption peak into a single, symmetrical peak The components were desalted by ultrafiltration and freeze-dried; 2 experiments were performed in parallel, and the capsular polysaccharide A of Bacteroides fragilis was obtained and named "S3" and "S4", respectively.

(3) Experimental results

Table 1 shows the comparison results of B. fragilis capsular polysaccharide A obtained by the two preparation methods. in:

Protein content test: Chinese Pharmacopoeia (2020 Edition) Part IV <0731> Determination of protein content - the fourth method 2,2'-biquinoline-4,4'-dicarboxylic acid method (BCA method);

Nucleic acid content test: Chinese Pharmacopoeia (2020 Edition) Part IV <0401> UV-Vis Spectrophotometry;

Bound lipid content test: ①Weigh 10mg of capsular polysaccharide A, add 20ml of 2% acetic acid, add 2h under reflux at 90°C, and free lipid; Lipid; ③Add 10ml of 4% sodium hydroxide-methanol solution, saponify at 80°C for 1h; ④Add 10ml of n-hexane, shake fully, absorb the organic phase, blow dry with nitrogen, and extract fatty acid; ⑤Add 1% methanol sulfuric acid 10ml, 80 React at ℃ for 30 minutes to esterify the fatty acid methyl ester; ⑥Add 20ml of n-hexane to extract the fatty acid methyl ester and analyze it by GC-MS.

Yield of crude capsular polysaccharide A: Yield = weight of crude product after freeze-drying/mass of bacteria slime × 100%.

Table 1. Comparison of quality attributes of Bacteroides fragilis capsular polysaccharide A obtained by two preparation methods

In this example, for the capsular polysaccharide A (PSA) prepared by method 1, the 1H spectrum analyzed by the NMR spectrometer is shown in Figure 3, the 13C spectrum is shown in Figure 4, the COZY spectrum is shown in Figure 5, the HSQC spectrum is shown in Figure 6, and the HMBC spectrum is shown in Figure 6. See Figure 7, and see Figure 8 for the chemical structure.

Example 3, the influence of the pH of the bacteria sludge suspension on the yield of capsular polysaccharide A

Bacteroides fragilis ZY-312 was fermented, and after centrifugation, the precipitate was collected as a sludge (obtained by the preparation in Example 1), and stored at -20°C.

Take 8 parts of bacteria slime, 10g each, add 50g of purified water to resuspend the bacteria, adjust the pH to 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 6.5 respectively;

Heat extraction at 105°C for 60 minutes, take it out immediately after the end, cool to room temperature, centrifuge at 12000g at room temperature for 10 minutes, and take the supernatant;

The 10KD ultrafiltration centrifuge tube was concentrated to remove salt, and freeze-dried to obtain the crude Bacteroides fragilis capsular polysaccharide A.

See Table 2 for the yields of Bacteroides fragilis capsular polysaccharide A crude sugar obtained under different pH conditions.

Table 3 shows the effect of different pH values of extracts on the quality attributes of pure Bacteroides fragilis capsular polysaccharide A.

According to Table 2, it can be seen that in the range of pH 2.5 to 4.0, the yield of Bacteroides fragilis capsular polysaccharide A crude sugar is higher than 0.8%, the yield is 0.48% at pH 2.0, and the yield drops significantly at pH 5.0 and above. Therefore, during the preparation of Bacteroides fragilis capsular polysaccharide, the pH of the suspension should be controlled at 2.5-4.0.

According to Table 3, it can be seen that in the range of pH 2.5-4.0, the protein content of Bacteroides fragilis capsular polysaccharide A is 0.45-0.85%, the nucleic acid content is less than 0.1%, and the binding lipid content is 0.05-0.25%. The content is 0.45%, the nucleic acid content is 0.07%, and the binding lipid content is 0.01%. When the pH is 5.0 and above, the content of protein, nucleic acid and bound lipid increases obviously, especially the bound lipid rises to 1.89%. Therefore, during the preparation of Bacteroides fragilis capsular polysaccharide, the pH of the suspension should be controlled at 2.5-4.0.

Table 2. Effect of pH of the extract on the yield of crude Bacteroides fragilis capsular polysaccharide A

Table 3. The effect of the pH of the extract on the quality attributes of the pure Bacteroides fragilis capsular polysaccharide A

pH	protein content%	Nucleic acid content%	Bound lipid content %
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

Embodiment 4, the influence of extraction temperature on the yield of capsular polysaccharide A

Get 8 parts of bacteria slime (obtained by the preparation of Example 1), each part of 10g, add 50g of purified water to resuspend the bacteria, and adjust its pH to about 3.5;

Continue to stir and heat extraction at 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, 120°C for 60 minutes, take it out immediately after the end, cool to room temperature, centrifuge at 12000g for 10 minutes at room temperature, and take the supernatant;

The 10KD ultrafiltration centrifuge tube was used for ultrafiltration to remove small molecular impurities, and then freeze-dried to obtain the crude Bacteroides fragilis capsular polysaccharide A.

Table 4 shows the yields of B. fragilis capsular polysaccharide A obtained under different extraction temperature conditions.

According to Table 4, it can be seen that in the temperature range of 90°C to 120°C, the yield of rough sugar of Bacteroides fragilis capsular polysaccharide A is higher than 0.80%. During the preparation of membrane polysaccharide A, the extraction temperature should be controlled within the range of 90°C to 120°C, preferably 90°C to 110°C.

Table 4. Effect of extraction temperature on yield of crude Bacteroides fragilis capsular polysaccharide A

Embodiment 5, the influence of extraction time on the yield of capsular polysaccharide A

Get 6 parts of bacteria slime (obtained by the preparation of Example 1), each part of 10g, add 50g of purified water to resuspend the bacteria, adjust its pH to about 4.0;

Put it in a vertical autoclave, extract at 110°C for 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, and 3.0h, take it out immediately after the end, cool to room temperature, and centrifuge to get the supernatant;

The 10KD ultrafiltration centrifuge tube was used for ultrafiltration to remove small molecular impurities, and then freeze-dried to obtain the crude Bacteroides fragilis capsular polysaccharide A.

Table 5 shows the yields of Bacteroides fragilis capsular polysaccharide A crude sugar obtained under different extraction time conditions.

According to Table 5, it can be seen that when the extraction time is 0.5h, the yield of Bacteroides fragilis capsular polysaccharide is 0.74%, and in the range of 1.0h to 2.5h extraction time, the yield is higher than 0.80%. When the extraction time is 3.0h , the yield decreased, which may be due to the degradation of capsular polysaccharide. Therefore, the extraction time during the preparation of the capsular polysaccharide of Bacteroides fragilis is preferably 1.0 h to 2.5 h.

Table 5. The effect of extraction time on the yield of Bacteroides fragilis capsular polysaccharide A crude sugar

Example 6. Preparation of Bacteroides fragilis capsular polysaccharide A with different molecular weight distributions

(1) Experimental method

Using the method provided by the invention to prepare Bacteroides fragilis capsular polysaccharide A with different molecular weight distributions, the specific steps are as follows:

① Take 100g of fermented ZY-312 Bacteroides fragilis sludge (obtained by Example 1), add 500mL of purified water, stir evenly, and adjust the pH to 3.0-3.1 with 1M hydrochloric acid solution;

②Continue stirring and heating at 100°C for 2 hours, cool to room temperature, centrifuge at 12000g at room temperature for 10 minutes, and obtain 500mL supernatant;

③ Add 5L of purified water, concentrate to about 100mL with 10KD ultrafiltration membrane; add 5L of purified water again, and ultrafilter to 100mL; repeat 3 times, the final volume is about 100mL, and the conductivity is 25μs/cm;

④ Add 100mL 40mmol/L Tris-HCl (pH8.5), mix well, and pass through a 0.45μm filter membrane;

⑤ Load the sample to the ion-exchange chromatography column (50mm×150mm, UniGel 80Q filler, Suzhou Nawei) at a flow rate of 20mL/min, and rinse with 20mmol/L Tris-HCl for 2 column volumes; then within 25 column volumes, Gradient elution of Tris-HCl (pH8.5) solution containing 0.2M sodium chloride, that is, the concentration of sodium chloride increased from 0 to 0.2M.

⑥The eluate was collected in sections. A total of 3 components were collected and identified by infrared spectroscopy, all of which were determined to be capsular polysaccharide A of Bacteroides fragilis.

⑦ Each component was desalted by ultrafiltration with a 10KD filter membrane, and the conductivity was stable when the volume reached 100mL, and then freeze-dried to obtain Bacteroides fragilis capsular polysaccharide A with different weight-average molecular weights, which were 168mg, 213mg, and 197mg respectively; the samples were named P1 , P2, P3.

(2) Experimental results

Table 6. Capsular polysaccharide A of Bacteroides fragilis with different weight-average molecular weights

In summary, the capsular polysaccharide A prepared by the preparation method of the present invention: its molecular weight distribution in the part of 70KD～100KD accounts for 70%～80% of the total; molecular weight is 80KD～90KD; its weight average molecular weight/number average molecular weight (Mw /Mn) ratio is 1.0-1.3; the fatty acid content is less than 0.02% or does not contain fatty acid. The above process of the present application simplifies the preparation process of the Bacteroides fragilis capsular polysaccharide, does not involve the use of organic reagents such as phenol, ether, ethanol, etc., is green and environmentally friendly, and can be industrialized.

The above-mentioned embodiments only express several implementation modes of the present invention, which are convenient for a specific and detailed understanding of the technical solution of the present invention, but should not be construed as limiting the protection scope of the invention patent. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. It should be understood that technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are within the protection scope of the appended claims of the present invention. Therefore, the scope of protection of the patent for the present invention shall be based on the content of the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims (10)

1. A preparation method of Bacteroides fragilis capsular polysaccharide A, characterized in that the preparation method comprises the following steps:

   Prepare a bacterial suspension of Bacteroides fragilis with pH ≤ 5, extract by heating, and collect the extract to obtain a crude sugar solution of capsular polysaccharide A.
2. The method for preparing Bacteroides fragilis capsular polysaccharide A according to claim 1, characterized in that the pH of the bacterial suspension is 2.0-4.5.
3. The preparation method of Bacteroides fragilis capsular polysaccharide A according to claim 2, characterized in that the pH of the bacterial suspension is 2.5-4.0.
4. The method for preparing Bacteroides fragilis capsular polysaccharide A according to claim 1, characterized in that the temperature used for heating and extracting is 50°C-120°C.
5. The method for preparing Bacteroides fragilis capsular polysaccharide A according to claim 4, characterized in that the temperature used for heating and extracting is 70°C-120°C.
6. The preparation method of Bacteroides fragilis capsular polysaccharide A according to claim 1, characterized in that the heating extraction time is 0.5h-3.0h.
7. According to the preparation method of Bacteroides fragilis capsular polysaccharide A according to any one of claims 1 to 6, it is characterized in that the pH of the bacterial suspension is 2.5-4.0, and the temperature used for heating and extraction is 90°C-110°C , the heating extraction time is 1.0h ~ 2.5h.
8. The preparation method of Bacteroides fragilis capsular polysaccharide A according to any one of claims 1 to 6, characterized in that the preparation method further comprises the step of ultrafiltering the crude sugar solution and collecting the retentate.
9. The preparation method of Bacteroides fragilis capsular polysaccharide A according to claim 8, is characterized in that, described preparation method also comprises carrying out ion exchange chromatography to described retentate and carries out ultrafiltration to the eluent collected. step.
10. According to the preparation method of Bacteroides fragilis capsular polysaccharide A according to any one of claims 1 to 6 and 9, it is characterized in that the heating extraction method is water bath heating, air bath heating or/and steam heating;

    Or/and, the method of collecting the extract is centrifugation;

    Or/and, said Bacteroides fragilis is a strain of Bacteroides fragilis with the preservation number CGMCC No.10685.

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