CN113481120A - Culture medium, preparation method thereof and method for culturing bacteroides fragilis by using culture medium - Google Patents
Culture medium, preparation method thereof and method for culturing bacteroides fragilis by using culture medium Download PDFInfo
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- CN113481120A CN113481120A CN202110728771.2A CN202110728771A CN113481120A CN 113481120 A CN113481120 A CN 113481120A CN 202110728771 A CN202110728771 A CN 202110728771A CN 113481120 A CN113481120 A CN 113481120A
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- culture medium
- peptone
- culture
- bacteroides fragilis
- sodium hydroxide
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- 241000606124 Bacteroides fragilis Species 0.000 title claims abstract description 69
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- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Organic Chemistry (AREA)
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- Biotechnology (AREA)
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Abstract
The invention relates to a culture medium, a preparation method thereof and a method for culturing bacteroides fragilis by using the culture medium, wherein the culture medium comprises plant-derived peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, a porphyrin source, glucose and sodium hydroxide. Compared with the prior art, the invention has the following beneficial effects: the culture medium provided by the invention can obtain a microbial culture effect equivalent to that of a culture medium adopting the animal-derived peptone by integrally adjusting the components of the formula under the conditions of improving safety and reducing mixed bacteria by adopting the plant-derived peptone instead of the traditional animal-derived peptone.
Description
Technical Field
The invention relates to the field of microbial culture, in particular to a culture medium, a preparation method thereof and a method for culturing bacteroides fragilis by using the culture medium.
Background
Bacteroides fragilis (Bacteroides fragilis) is one of symbiotic bacteria planted in human intestinal tracts, belongs to strict anaerobes and accounts for about 1% -2% of total colonies of intestinal feces. It is gram-negative bacilli, with size of (0.8-1.3) μm x (1.6-8) μm, uneven staining, round and dark staining at both ends, no staining or light staining in the middle, and vacuole-like. In the past, studies on bacteroides fragilis have generally focused on anaerobic infection and cancer caused by bacteroides fragilis, but with the development of probiotic species, the probiotic properties of bacteroides fragilis have also been reported. In order to support the research of probiotic bacteroides fragilis from laboratory to large-scale culture, a culture medium with reasonable formula and good effect needs to be developed.
Currently, common media that can be used to produce bacteroides fragilis are: modified GAM medium comprising peptone,
Peptone, soybean peptone, yeast extract powder, beef powder, digestive serum powder, beef liver extract powder, glucose, KH2PO4NaCl, soluble starch, L-cysteine, L-arginine, L-tryptophan, sodium thioglycolate and vitamin K1And hemin; the improved PYG culture medium comprises peptone, glucose, yeast extract powder, NaCl, cysteine hydrochloride and CaCl2、MgSO4、K2HPO4、KH2PO4、NaHCO3Vitamin K1, hemin; a medium comprising yeast extract, tryptone, sodium thioglycolate, NaCl, L-cysteine, methylene blue, heme, menadione, and glucose; a medium comprising casein peptone, tryptone, yeast extract, glucose, L-arginine, heme, vitamin K, and sodium pyruvate; the general culture medium can be used for enrichment culture of bacteroides fragilis under special gas conditions, such as TSB and the like.
Other media that have been reported are for example: a method for detecting heat-resistant anaerobic bacteria in live poultry relates to an anaerobic liquid culture medium which comprises the following raw materials: 10g/L-20g/L peptone, 1g/L-10g/L yeast powder, 1g/L-10g/L glucose, 1g/L-10g/L soytone, 1g/L-10g/L beef powder, 1g/L-10g/L sodium chloride, 1g/L-5g/L soluble starch, 0.1g/L-1.0g/L cysteine, 1g/L-5g/L potassium dihydrogen phosphate, 10.001g/L-0.01 g/L vitamin, 0.001g/L-0.01g/L hemin and the balance of water. A bacteriodes cellulolysiticus specificity screening culture medium, which comprises an enrichment culture medium, a separation culture medium and a growth culture medium; the formula of the enrichment medium comprises the following components: 4g/L-6g/L of unique carbon source, 15g/L-25g/L of tryptone, 4g/L-6g/L of yeast extract, 4g/L-6g/L of sodium chloride, 0.04g/L-0.06g/L of dipotassium phosphate, 0.04g/L-0.06g/L of monopotassium phosphate, 0.5g/L-1g/L of cysteine hydrochloride, 0.005g/L-0.01g/L of hemin, K10.001g/L-0.002 g/L of vitamin, 20ml/L-40ml/L of penicillin solution, 4ml/L-5ml/L of kanamycin sulfate solution, 2.0ml/L-2.5ml/L of vancomycin hydrochloride solution and the balance of sterile water, adjusting the pH value to 6.8-7.0 by sodium hydroxide; the sole carbon source is arabinose. A Bacteroides xylanisolvens specificity screening culture medium comprises an isolation culture medium and a growth culture medium, wherein the isolation culture medium contains vancomycin, kanamycin, a unique carbon source, an acid production indicator and the like. The separation culture medium comprises the following components: 4g/L-6g/L of unique carbon source arabinose, 15g/L-25g/L of tryptone, 4g/L-6g/L of yeast extract, 4g/L-6g/L of sodium chloride, 0.04g/L-0.06g/L of dipotassium hydrogen phosphate, 0.04g/L-0.06g/L of potassium dihydrogen phosphate, 0.5g/L-1g/L of cysteine hydrochloride, 0.005g/L-0.01g/L of hemin, K10.001g/L-0.002 g/L of vitamin, 0.010-0.014g/L of acid production indicator bromocresol purple, 4-5ml/L of kanamycin sulfate solution, 2.0-2.5ml/L of vancomycin hydrochloride solution, 15-20g/L of agar, the balance of sterile water and sodium hydroxide to adjust the pH value to 6.8-7.0.
As exemplified above, in the conventional culture media available for the culture of Bacteroides fragilis, peptone of animal origin alone is mostly used, and a combination of peptone of animal origin and peptone of plant origin is used in a small amount. Animal-derived peptone and plant-derived peptone are used as common organic nitrogen sources, and have respective advantages and disadvantages in the application process. Animal-derived peptone is rich in nutrition, is an important supplementary factor for microbial culture, and has obvious defects such as potential virus pollution, undefined components, unfavorable product purification and the like, and target microorganisms are rapidly proliferated. Due to the above-mentioned drawbacks, there is a constant desire among the international pharmaceutical administration and production units to find peptone substitute products of animal origin, ensuring the safety of the biological products. The plant source peptone culture medium has the advantages of no pollution of animal source, clear components, high safety and the like, but has less nutrition than animal source peptone. In the process of preparing the culture medium for culturing the microorganisms by adopting the plant source peptone to replace the animal source peptone, although the risks such as virus pollution and the like can be effectively reduced, the microorganism proliferation is relatively slow and the number of viable bacteria is small.
Based on this, how to combine the proliferation effect of bacteroides fragilis in the case of replacing peptone of animal source with peptone of plant source is a problem that needs to be solved urgently.
Disclosure of Invention
In view of the above-mentioned drawbacks of the background art, it is an object of the present invention to provide a culture medium formulation supplemented with a peptone of plant origin, which allows bacteroides fragilis to proliferate rapidly and allows the number of viable bacteria contained in the culture to be equivalent to that of the culture medium formulation supplemented with a peptone of animal origin during the culture of bacteroides fragilis in the medium.
The purpose of the invention can be realized by the following technical scheme:
in a first aspect, the present invention provides a culture medium comprising a plant-derived peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, a porphyrin source, glucose and sodium hydroxide.
In one embodiment, the medium comprises water, and the medium comprises 15g/L to 20g/L of the plant-derived peptone, 1g/L to 5g/L of the yeast powder, 1g/L to 10g/L of the sodium chloride, 1g/L to 5g/L of the dipotassium hydrogen phosphate, 0.001g/L to 1g/L of the porphyrin source, 1g/L to 5g/L of the glucose, and 0.1g/L to 1g/L of the sodium hydroxide, in terms of concentration in the medium.
In one embodiment, the plant-derived peptone comprises at least one of soybean peptone, rice peptone, pea peptone, wheat peptone and cotton seed peptone, in terms of concentration in the medium.
In one embodiment, the medium comprises 16g/L to 19g/L of the pea peptone, 2g/L to 4g/L of the yeast powder, 3g/L to 7g/L of the sodium chloride, 2g/L to 4g/L of the dipotassium hydrogen phosphate, 0.002g/L to 0.9g/L of the porphyrin source, 2g/L to 4g/L of the glucose, and 0.2g/L to 0.8g/L of the sodium hydroxide, measured in concentrations in the medium.
In a second aspect, the present invention provides a method for preparing a culture medium as described above, comprising the steps of:
mixing the plant source peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, porphyrin source, glucose, sodium hydroxide and water.
In one embodiment, the preparation method comprises the following steps:
mixing the sodium hydroxide with a portion of the water to produce a sodium hydroxide solution;
mixing the sodium hydroxide solution and the porphyrin source and mixing the resulting mixed solution with the plant-derived peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, glucose and the remainder of the water.
In one embodiment, the preparation method further comprises the step of sterilizing the product obtained by mixing.
In a third aspect, the present invention provides a method for culturing bacteroides fragilis, comprising the step of inoculating bacteroides fragilis to the culture medium as described above for culturing.
In one embodiment, the bacteroides fragilis is a bacteroides fragilis strain with the preservation number of CGMCC No. 10685.
In one embodiment, the culture is performed by anaerobic static culture.
In one embodiment, the culture mode adopts anaerobic shaking culture.
In one embodiment, the rotation speed for shaking culture is 50-500 rpm.
In one embodiment, the temperature used for the incubation is 36.5 ℃ to 37.5 ℃.
Compared with the prior art, the invention has the following beneficial effects:
the culture medium provided by the invention can obtain the bacteroides fragilis culture effect which is superior to the traditional animal source peptone culture medium in safety, growth state and bacterial activity under the condition of similar viable bacteria amount by integrally adjusting the components of the formula and adopting plant source peptone to replace animal source peptone.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the microscopic examination result of Bacteroides fragilis isolated and purified according to formula II in example 2;
FIG. 2 shows the microscopic examination result of Bacteroides fragilis isolated and purified in example 2 by using the formulation IV of example 2;
FIG. 3 shows the results of microscopic examination of isolated and purified Bacteroides fragilis from example 2 using the comparative formulation of example 2.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Term(s) for
Unless otherwise stated or contradicted, terms or phrases used herein have the following meanings:
as used herein, the term "and/or", "and/or" includes any one of two or more of the associated listed items, as well as any and all combinations of the associated listed items, including any two of the associated listed items, any more of the associated listed items, or all combinations of the associated listed items.
As used herein, "one or more" means any one, any two, or any two or more of the listed items. Wherein, the 'several' means any two or more than any two.
As used herein, "a combination thereof," "any combination thereof," and the like, includes all suitable combinations of any two or more of the listed items.
In the present specification, the term "suitable" in "a suitable combination, a suitable manner," any suitable manner "and the like shall be construed to mean that the technical solution of the present invention can be implemented, the technical problem of the present invention can be solved, and the technical effect of the present invention can be achieved.
Herein, "preferred" merely describes a more effective embodiment or example, and it should be understood that the scope of the present invention is not limited thereto.
The terms "first aspect," "second aspect," "third aspect," and the like, in the description herein are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor is it to be construed as implicitly indicating the importance or quantity of the technical feature indicated.
In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
In the present invention, the numerical range is defined to include both end points of the numerical range unless otherwise specified.
The percentage contents referred to in the present invention mean, unless otherwise specified, mass percentages for solid-liquid mixing and solid-solid phase mixing, and volume percentages for liquid-liquid phase mixing.
The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system to which the component is added.
The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a certain temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.
In a first aspect, the present invention provides a culture medium comprising a plant-derived peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, a porphyrin source, glucose and sodium hydroxide.
Compared with the prior art, the invention has the following beneficial effects: the culture medium provided by the invention can obtain the culture effect of microorganisms (particularly bacteroides fragilis) equivalent to that of a culture medium of peptone of animal source under the condition of adopting peptone of plant source to replace peptone of animal source through the overall adjustment of the formula components. The main sources of the plant source peptone are corn gluten, pea protein, soybean protein (without transgene), wheat gluten, rice gluten, cotton seed protein or the mixture of various plant proteins. The plant source peptone culture medium has the advantages of no pollution of animal source, clear components, high safety and the like. The culture medium adopting the plant source peptone to replace the animal source peptone can effectively reduce various risks caused by introducing the animal source peptone in the process of developing the viable bacteria medicament. The kinds of the plant-derived peptone include, but are not limited to, soybean peptone, rice peptone, pea peptone, wheat peptone, cotton seed peptone and the like.
The porphyrin source in the invention refers to a substance providing porphyrin, including but not limited to hemin, serum, protoporphyrin and the like. The present invention is explained by taking hemin as an example, but this is not a limitation to the present invention.
In one embodiment, the medium comprises water, and the medium comprises 15g/L to 20g/L of the plant-derived peptone, 1g/L to 5g/L of the yeast powder, 1g/L to 10g/L of the sodium chloride, 1g/L to 5g/L of the dipotassium hydrogen phosphate, 0.001g/L to 1g/L of the porphyrin source, 1g/L to 5g/L of the glucose, and 0.1g/L to 1g/L of the sodium hydroxide, in terms of concentration in the medium.
Further preferably, the culture medium comprises 16g/L to 19g/L of the pea peptone, 2g/L to 4g/L of the yeast powder, 3g/L to 7g/L of the sodium chloride, 2g/L to 4g/L of the dipotassium hydrogen phosphate, 0.002g/L to 0.9g/L of the porphyrin source, 2g/L to 4g/L of the glucose and 0.2g/L to 0.8g/L of the sodium hydroxide, in terms of concentration in the culture medium.
It is to be understood that the dipotassium phosphate according to the present invention may be added in the form of dipotassium phosphate trihydrate.
In one embodiment, the medium comprises 17g/L to 18.5g/L of the pea peptone, 2.5g/L to 3.5g/L of the yeast powder, 4g/L to 6g/L of the sodium chloride, 2.5g/L to 3g/L of the dipotassium hydrogen phosphate, 0.003g/L to 0.85g/L of the porphyrin source, 2.5g/L to 3g/L of the glucose, and 0.3g/L to 0.6g/L of the sodium hydroxide, in terms of concentration in the medium.
In one embodiment, the medium is free of animal-derived peptones.
In one embodiment, the porphyrin source is hemin.
In a second aspect, the present invention provides a method for preparing a culture medium as described above, comprising the steps of:
mixing the plant source peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, porphyrin source, glucose, sodium hydroxide and water.
In one embodiment, the preparation method comprises the following steps:
mixing the sodium hydroxide and a portion of the water to produce a sodium hydroxide solution;
mixing the sodium hydroxide solution and the porphyrin source, and mixing the resulting mixed solution with the plant-derived peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, glucose, and the remainder of the water.
In one embodiment, the preparation method comprises the following steps:
(1) taking sodium hydroxide and a proper amount of water to prepare a sodium hydroxide solution;
(2) dissolving a porphyrin source by using the sodium hydroxide solution obtained in the step (1) to prepare a porphyrin/sodium hydroxide solution, keeping out of the sun, and standing;
(3) and (3) mixing the porphyrin/sodium hydroxide solution obtained in the step (2) with other materials to prepare the culture medium.
In one example, in step (1), the concentration of sodium hydroxide in the sodium hydroxide solution is 0.5-2.5%. Further, the concentration of sodium hydroxide in the sodium hydroxide solution was 2%.
In one example, in the step (2), the standing time is 10min to 20 min. Further, the standing time was 10 min.
It is understood that the water according to the present invention may be double distilled water or the like.
In one embodiment, the preparation method further comprises the step of sterilizing the product obtained by mixing. It is understood that the sterilization mode is not limited in the present invention, for example, autoclaving is adopted, and the sterilization condition is autoclaving at 121 ℃ for 30 min.
In a third aspect, the present invention provides a method for culturing bacteroides fragilis, comprising the step of inoculating bacteroides fragilis to the culture medium as described above for culturing.
The species of the Bacteroides fragilis to be cultured in the present invention is not particularly limited, and for example, the Bacteroides fragilis strain with the collection number of CGMCC No.10685 can be used.
The specific steps and conditions for the culture are not particularly limited in the present invention, and any anaerobic bacteria (e.g., bacteroides fragilis) can be used under appropriate conditions for the culture. For example, the culture method may be anaerobic static culture or anaerobic shaking culture, and in the case of anaerobic shaking culture, the rotation speed used for shaking culture is 50rpm to 500rpm, for example, 50rpm, 100rpm, 200rpm, 250rpm, 300rpm, 350rpm, 400rpm, 450rpm, 500rpm, or the like. For example, the temperature used for the culture may be 36.5 ℃, 37 ℃, 37.5 ℃ or any range between these values. For example, the scale of the culture can be adjusted as necessary, and the scale may be a shake tube/flask scale, or a scale of 30L, 300L or the like, and it is understood that the culture conditions can be adjusted adaptively according to the adjustment of the culture scale.
The test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biomaterials, if not specifically mentioned, are commercially available.
Example 1 Effect of different kinds of peptones on the Effect of Bacteroides fragilis cultivation
(1) The media formulations are shown in table 1 below. In Table 1, the medium contained hemin at a concentration of 0.006 g/L.
(2) The culture medium formula I to the culture medium formula IV are prepared by the following method:
preparing sodium hydroxide into a 2% sodium hydroxide solution by using part of water, mixing the sodium hydroxide solution with hemin, keeping the mixture out of the sun, standing the mixture for 10 minutes, and completely dissolving the mixture to obtain a material a;
mixing peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate trihydrate, glucose and residual water to obtain a material b;
mixing the material a and the material b, dissolving, and sterilizing at high temperature.
(3) The Bacteroides fragilis adopts Bacteroides fragilis ZY-312 with preservation number of CGMCC No. 10685.
(4) The specific steps for culturing Bacteroides fragilis in this example are as follows:
mixing 3.50X 108The Bacteroides fragilis seeds were inoculated at a 10% (v/v) ratio into 10mL of medium under binary gas (7% v/vCO)2，93％v/vN2) The culture results were evaluated by conducting static culture at 37 ℃ for 48 hours under anaerobic atmosphere and measuring the viable cell count.
The experiment was repeated 3 times. The culture results are shown in Table 2.
TABLE 1 culture medium formulation of bacteroides fragilis containing different peptone species
TABLE 2 Effect of different peptones on Bacteroides fragilis culture results
According to the data in the table, the culture effect of the culture medium using pea peptone is equivalent to that of the culture medium using animal-derived protein traditionally, and is higher than that of the culture medium using other plant peptone. Specifically, it can be seen through the significance analysis that: (1) by adopting the culture medium formula containing the plant source peptone, the overall culture effect is superior to that of the formula (formula four) adopting the animal source culture medium with the same proportion; (2) in the formula I, the formula II, the formula III and the control formula, the control formula and the formula II have no significant difference, but the control formula, the formula I and the formula III have significant difference, which shows that the culture medium formula of the invention has a preferred scheme, and preferably selects the pea peptone as the plant peptone.
Example 2 comparison of Bacteroides fragilis morphology and determination of viability obtained from Medium containing peptone of plant origin and Medium containing peptone of animal origin
(1) The test object is: the cultures obtained using the culture of medium formulation two of example 1, the culture of medium formulation four of example 1 and the control formulation of example 1 were used.
(2) The experimental method comprises the following steps:
1) and (3) morphology comparison: and separating and purifying the culture, and performing microscopic examination.
2) And (3) activity determination: the above culture was isolated and purified, and examined as follows.
Preparing a live tube: weighing 11g of skimmed milk powder, adding 89mL of distilled water, stirring for 10-15min to dissolve completely, standing for 1h, filtering with double-layer gauze, and removing insoluble substances. The solution was then dispensed into 10mL tubes and sealed with stoppers. Intermittent three-time sterilization is adopted: sterilizing in water bath at 90-95 deg.C for 20min, taking out, naturally cooling to room temperature, and storing in refrigerator at 4-6 deg.C. And performing secondary sterilization on the second day by the same method as the primary sterilization, taking out, naturally cooling to room temperature, and storing in a refrigerator at 4-6 ℃. And performing third sterilization on the third day by the same method as the first sterilization, taking out, naturally cooling to room temperature, and storing in a refrigerator at 4-6 ℃ for later use.
The prepared skim milk powder solution requires: the specific gravity is 1.033-1.034; the acidity is less than or equal to 20 degrees T; the temperature was 20 ℃.
② determination of vitality
Inoculation: the viability tubes were preheated to 37 ℃ before inoculation and 3% (v/v) of the above culture was added to the sterilized viability tubes. The inoculation operation is required to be finished in a super clean bench, the operation process is aseptic operation, and the inoculation sucker is required to be sterilized.
Fermentation: fermenting and culturing at constant temperature of 37 deg.C for 3.5 h.
Acid measurement: after fermentation culture for 3.5h, immediately taking out the activity tube for acidity determination. Transferring 10mL of sample into a 100mL triangular flask by using a pipette, rinsing the pipette by using 20mL of purified water, pouring the pipette into the 100mL triangular flask together, adding 3 drops of 0.5% phenolphthalein, and starting titration. Titrate to reddish with 0.1mo1/mL NaOH standard solution and do not fade within 30 s. The acidity is determined by multiplying the number of milliliters of 0.1mol/mL NaOH standard solution consumed by 10.
And (3) calculating: the strain activity is 0.087X and the sodium hydroxide is consumed in milliliters.
(3) The experimental results are as follows:
1) and (3) morphology comparison:
the microscopic examination result of the culture obtained by the culture medium formula II is shown in figure 1; the microscopic examination result of the culture obtained by the medium formula IV is shown in figure 2; the microscopic examination results of the cultures obtained from the comparative formulation are shown in FIG. 3. As can be seen from fig. 1, 2, and 3: example 1 culture medium formula two the culture obtained contained a relatively short circle of bacteroides fragilis morphology, which was close to the normal form of bacteroides fragilis; the culture obtained by the formula IV and the culture obtained by the comparative formula have slender shapes of bacteroides fragilis. The shape of Bacteroides fragilis is preferably short circular, and slender, indicating poor growth state. As can be seen, the plant-derived peptone medium is more favorable for the growth of Bacteroides fragilis.
2) And (3) activity determination: the results are given in the table below.
TABLE 3 viability of the culture media of different formulations
Through significance analysis: the bacteroides fragilis is cultured by adopting the formula II, the bacterial activity is obviously improved compared with that of a culture medium containing animal source peptone, and specifically, the bacterial activity is less than 0.05 compared with a control formula P and less than 0.01 compared with a formula IV. In addition, in this example, the culture obtained from the first and third formulations of example 1 was subjected to viability tests, and the results obtained were that the mean value of the viability of the bacteria corresponding to the first formulation was 0.82, and the mean value of the viability of the bacteria corresponding to the third formulation was 0.85. Therefore, the bacteria cultured by adopting the plant source peptone culture medium have higher activity.
Example 3 safety comparison of different media formulations
(1) The strain expanding culture method comprises the following steps: mixing 3.50X 108The C.fragilis seeds were inoculated at a rate of 10% into 1L scale medium in a binary gas (7% (v/v) CO)2，93％(v/v)N2) The culture was carried out by standing or shaking at 37 ℃ for 48 hours under anaerobic atmosphere. The culture broth was used for 30L fermentation inoculation.
(2)30L of fermentation, the method is as follows: adding culture medium and heme into a 50L fermentation tank, culturing for 30L after digestion, inoculating 2L culture solution obtained in the step (1) after 24h in the same way with anaerobic protective gas, culturing for 8h at 37 ℃ under anaerobic stirring at 500rpm, supplementing a carbon source according to a proper time in fermentation liquor during the period (2.0L of 1.0M glucose solution, beginning to supplement when the glucose concentration is less than 5mM, completing supplement within 1 h), and taking the culture solution obtained in 8h for carrying out mixed bacteria detection.
By adopting the method, 30L-grade fermentation culture solution of the bacteroides fragilis strain is prepared by using a second culture medium formula and a reference formula in table 1, and the culture solution is subjected to mixed bacteria detection, wherein the detection results are as follows:
TABLE 4 results of detection of infectious microbes in the expanded culture broth obtained with different kinds of peptone medium
Note: as compared to the control formulation,.: p < 0.01.
As can be seen from the data in the table, the number of the mixed bacteria in the enlarged culture solution obtained by using 30L of the culture medium containing the plant source peptone is far less than that of the traditional animal source peptone culture medium, and the safety is better than that of the traditional animal source peptone culture medium.
Example 4 Effect of different ratios of Medium on Bacteroides fragilis culture
(1) The formulation of the medium of this example is shown in Table 5 below, and the medium was prepared in the same manner as in example 1.
(2) The Bacteroides fragilis adopts Bacteroides fragilis ZY-312 with preservation number of CGMCC No. 10685.
(3) This example describes the procedure of culturing Bacteroides fragilis in the same manner as in example 1.
The results of Bacteroides fragilis culture are shown in Table 6 below.
TABLE 5 culture medium formula of different proportions
TABLE 6 influence of different formulations on Bacteroides fragilis culture results (viable count units: CFU/mL)
According to the data in the table, the following data are obtained: compared with the formula five, the formula six and the formula seven, the culture effect of the culture medium adopting the formula two is relatively good, which shows that the culture medium formula of the invention has a preferred scheme, namely the formula two.
Examples 5 and 6, Effect of different media formulations on Bacteroides fragilis culture results
Examples 5 and 6 provide a medium having the formula shown in Table 7, which is expressed as formula eight and formula nine, respectively, and Bacteroides fragilis ZY-312 was cultured using formula eight and formula nine, respectively, according to the culturing procedure described in example 1:
TABLE 7 influence of different peptone to sodium hydroxide ratios on Bacteroides fragilis culture results
| Example 1 formulation two | Example 5 formulation eight | Example 6 formulation nine | |
| Plant source peptone | Pea peptone 18 | Pea peptone 25 | Pea peptone 10 |
| Yeast powder | 3 | 3 | 3 |
| Sodium chloride | 5 | 5 | 5 |
| Dipotassium hydrogen phosphate | 2.5 | 2.5 | 2.5 |
| Glucose | 2.5 | 2.5 | 2.5 |
| Hemin | 0.006 | 0.006 | 0.006 |
| Sodium hydroxide | 0.4 | 2 | 0.05 |
| Water (W) | Complement 1L | Complement 1L | Complement 1L |
With reference to example 1, Bacteroides fragilis ZY-312 was cultured using formulation eight and formulation nine, respectively, and the results are shown in the following table.
TABLE 8 influence of different peptone to sodium hydroxide ratios on Bacteroides fragilis culture results
Note: compared to formulation two, a: p < 0.01. Viable count unit: CFU/mL
It can be seen that the concentrations of the plant-derived peptone and sodium hydroxide contained in the medium have a significant effect on the culture results of Bacteroides fragilis.
Comparative example 1, comparative example 2 and comparative example 3, Effect of different Medium formulations on Bacteroides fragilis culture results
Comparative example 1, comparative example 2 and comparative example 3 each provide a culture medium, the formulation is shown in table 9, and is respectively denoted as formulation ten, formulation eleven and formulation twelve, and the bacteroides fragilis ZY-312 is cultured by using formulation ten, formulation eleven and formulation twelve, the culture steps refer to example 1:
TABLE 9 influence of different media formulations on Bacteroides fragilis culture results
With reference to example 1, Bacteroides fragilis ZY-312 was cultured using formulation ten, formulation eleven and formulation twelve, respectively, and the results are shown in the following table.
TABLE 10 influence of different media formulations on Bacteroides fragilis culture results
Note: compared to formulation two, a: p<0.01; viable count unit: 109CFU/mL
It can be seen that sodium hydroxide is essential in the culture of bacteroides fragilis.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A culture medium, comprising a plant-derived peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, a porphyrin source, glucose, and sodium hydroxide.
2. The culture medium according to claim 1, wherein the culture medium comprises water, and the culture medium comprises 15g/L to 20g/L of the plant-derived peptone, 1g/L to 5g/L of the yeast powder, 1g/L to 10g/L of the sodium chloride, 1g/L to 5g/L of the dipotassium hydrogenphosphate, 0.001g/L to 1g/L of the porphyrin source, 1g/L to 5g/L of the glucose, and 0.1g/L to 1g/L of the sodium hydroxide, in terms of concentration in the culture medium.
3. The culture medium according to claim 1 or 2, wherein the peptone of plant origin comprises at least one of soybean peptone, rice peptone, pea peptone, wheat peptone and cotton seed peptone.
4. The culture medium according to claim 3, wherein the culture medium comprises 16g/L to 19g/L of the pea peptone, 2g/L to 4g/L of the yeast powder, 3g/L to 7g/L of the sodium chloride, 2g/L to 4g/L of the dipotassium hydrogen phosphate, 0.002g/L to 0.9g/L of the porphyrin source, 2g/L to 4g/L of the glucose and 0.2g/L to 0.8g/L of the sodium hydroxide, in terms of concentration in the culture medium.
5. The method for preparing a culture medium according to any one of claims 1 to 4, comprising the steps of: mixing the plant source peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, porphyrin source, glucose, sodium hydroxide and water.
6. The method for preparing a culture medium according to claim 5, comprising the steps of:
mixing the sodium hydroxide with a portion of the water to produce a sodium hydroxide solution;
mixing the sodium hydroxide solution and the porphyrin source and mixing the resulting mixed solution with the plant-derived peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, glucose and the remainder of the water.
7. The method for preparing a culture medium according to claim 5, further comprising a step of sterilizing the product obtained by mixing.
8. A method for culturing Bacteroides fragilis, comprising the step of inoculating Bacteroides fragilis into the medium according to any one of claims 1 to 4 and culturing.
9. The Bacteroides fragilis culture method of claim 7, wherein the Bacteroides fragilis is a Bacteroides fragilis strain with a collection number of CGMCC No. 10685.
10. The method for culturing Bacteroides fragilis according to claim 8 or 9, wherein the culturing is performed by anaerobic static culture or/and anaerobic shaking culture; or/and the temperature adopted by the culture is 36.5-37.5 ℃.
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