WO2023273019A1 - 培养基及其制备方法、用其培养脆弱拟杆菌的方法 - Google Patents

培养基及其制备方法、用其培养脆弱拟杆菌的方法 Download PDF

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WO2023273019A1
WO2023273019A1 PCT/CN2021/124823 CN2021124823W WO2023273019A1 WO 2023273019 A1 WO2023273019 A1 WO 2023273019A1 CN 2021124823 W CN2021124823 W CN 2021124823W WO 2023273019 A1 WO2023273019 A1 WO 2023273019A1
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peptone
culture
medium
bacteroides fragilis
culture medium
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PCT/CN2021/124823
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French (fr)
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王从峰
丁靖志
李平
郑丽君
吴嘉棋
刘洋洋
潘国平
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广州知易生物科技有限公司
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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
    • 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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  • the invention relates to the field of microorganism culture, in particular to a culture medium, a preparation method thereof, and a method for cultivating Bacteroides fragilis with it.
  • Bacteroides fragilis (Bacteroides fragilis) is one of the commensal bacteria that colonize the human intestinal tract. It is a strict anaerobic bacteria and accounts for about 1%-2% of the total number of colonies in intestinal feces. It is a Gram-negative bacillus with a size of (0.8-1.3) ⁇ m ⁇ (1.6-8) ⁇ m, uneven staining, round and dark staining at both ends, no coloring or light staining in the middle, and seems to be vacuoles.
  • Past research on B. fragilis usually focused on its anaerobic infection and cancer, but with the development of probiotic species, the probiotics of B. fragilis have also been reported. In order to support the probiotic Bacteroides fragilis from laboratory research to large-scale cultivation, it is necessary to develop a well-formulated medium with good effect.
  • modified GAM medium which contains peptone, Peptone, Soytone, Yeast Extract Powder, Beef Powder, Digestive Serum Powder, Beef Liver Extract Powder, Glucose, KH 2 PO 4 , NaCl, Soluble Starch, L-Cysteine, L-Arginine, L-Tryptophan acid, sodium thioglycolate, vitamin K 1 and hemin; modified PYG medium containing peptone, glucose, yeast extract powder, NaCl, cysteine hydrochloride, CaCl 2 , MgSO 4 , K 2 HPO 4.
  • KH 2 PO 4 NaHCO 3 , vitamin K1, hemin
  • anaerobic liquid media including the following raw materials: peptone 10g/L-20g/L, yeast powder 1g/L-10g/ L, glucose 1g/L-10g/L, soybean peptone 1g/L-10g/L, beef powder 1g/L-10g/L, sodium chloride 1g/L-10g/L, soluble starch 1g/L-5g/L L, cysteine 0.1g/L-1.0g/L, potassium dihydrogen phosphate 1g/L-5g/L, vitamin K1 0.001g/L-0.01g/L, hemin 0.001g/L-0.01 g/L, the balance is water.
  • a kind of Bacteroides cellulosilyticus specific screening culture medium comprises enrichment medium, separation medium and growth medium;
  • the formula of described enrichment medium comprises following components: unique carbon source 4g/L-6g/ L, tryptone 15g/L-25g/L, yeast extract 4g/L-6g/L, sodium chloride 4g/L-6g/L, dipotassium hydrogen phosphate 0.04g/L-0.06g/L, diphosphate Potassium Hydrogen 0.04g/L-0.06g/L, Cysteine Hydrochloride 0.5g/L-1g/L, Hemin 0.005g/L-0.01g/L, Vitamin K1 0.001g/L-0.002 g/L, penicillin solution 20ml/L-40ml/L, kanamycin sulfate solution 4ml/L-5ml/L, vancomycin hydrochloride solution 2.0ml/L-2.5ml/L, the balance is none Bacterial water, sodium hydroxide to adjust the pH value to
  • a Bacteroides xylanisolvens specific screening medium said medium includes separation medium and growth medium, said separation medium contains substances such as vancomycin, kanamycin, unique carbon source and acid production indicator .
  • the separation medium includes the following components: the only carbon source arabinose 4g/L-6g/L, tryptone 15g/L-25g/L, yeast extract 4g/L-6g/L, sodium chloride 4g/L -6g/L, dipotassium hydrogen phosphate 0.04g/L-0.06g/L, potassium dihydrogen phosphate 0.04g/L-0.06g/L, cysteine hydrochloride 0.5g/L-1g/L, chlorine Heme 0.005g/L-0.01g/L, vitamin K1 0.001g/L-0.002g/L, acid production indicator bromocresol violet 0.010-0.014g/L, kanamycin sulfate solution 4-5ml /L, vancomycin hydrochloride solution 2.0-2.5ml/L,
  • animal-derived peptone and plant-derived peptone are commonly used organic nitrogen sources, and each has advantages and disadvantages in the application process.
  • Animal-derived peptone is rich in nutrients and is an important supplementary factor for microbial culture. The target microorganisms proliferate rapidly.
  • animal-derived peptone has obvious disadvantages, such as potential virus contamination, unclear composition, and unfavorable product purification.
  • Plant-derived peptone medium has the advantages of no animal-derived pollution, relatively clear ingredients, and high safety, but its nutrition is not as good as animal-derived peptone.
  • Using plant-derived peptone instead of animal-derived peptone to prepare medium for microbial culture can effectively reduce the risk of virus contamination, but the proliferation of microorganisms is relatively slow and the number of viable bacteria is small.
  • one of the objectives of the present invention is to provide a medium formula added with plant-derived peptone, in the process of cultivating Bacteroides fragilis with this medium, Bacteroides fragilis proliferates quickly, and the viable bacteria contained in the culture The number is equivalent to the medium formula supplemented with animal-derived peptone.
  • the present invention provides a culture medium, which comprises plant-derived peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, porphyrin source, glucose and sodium hydroxide.
  • the culture medium comprises water, and in terms of concentration in the culture medium, the culture medium comprises 15g/L-20g/L of the plant-derived peptone, 1g/L-5g/L The yeast powder, 1g/L-10g/L sodium chloride, 1g/L-5g/L dipotassium hydrogen phosphate, 0.001g/L-1g/L porphyrin source, 1g/L- 5g/L of glucose and 0.1g/L-1g/L of sodium hydroxide.
  • the plant-derived peptone includes at least one of soy peptone, rice peptone, pea peptone, wheat peptone and cottonseed peptone.
  • the culture medium comprises 16g/L-19g/L of the pea peptone, 2g/L-4g/L of the yeast powder, 3g/L -7g/L said sodium chloride, 2g/L-4g/L said dipotassium phosphate, 0.002g/L-0.9g/L said porphyrin source, 2g/L-4g/L said glucose and The sodium hydroxide of 0.2g/L-0.8g/L.
  • the present invention provides a method for preparing the culture medium as described above, the preparation method comprising the following steps:
  • the said plant source peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, porphyrin source, glucose, sodium hydroxide and water are mixed.
  • the preparation method comprises the following steps:
  • the preparation method further includes the step of sterilizing the mixed product.
  • the present invention provides a method for cultivating Bacteroides fragilis, the culturing method comprising the step of inoculating Bacteroides fragilis to the above-mentioned medium for culturing.
  • the Bacteroides fragilis is a strain of Bacteroides fragilis with the deposit number CGMCC No.10685.
  • the cultivation method adopts anaerobic static cultivation.
  • the culture method adopts anaerobic shaking culture.
  • the rotational speed adopted for shaking culture is 50 rpm-500 rpm.
  • the temperature used for culturing is 36.5°C-37.5°C.
  • the present invention has the following beneficial effects:
  • the culture medium provided by the present invention through the overall adjustment of formula components, in the case of using plant-derived peptone instead of animal-derived peptone, can obtain safety, growth status and bacterial vigor better than traditional animal under the condition of a similar amount of viable bacteria.
  • the effect of Bacteroides fragilis culture on peptone-derived medium can obtain safety, growth status and bacterial vigor better than traditional animal under the condition of a similar amount of viable bacteria.
  • Fig. 1 is the Bacteroidetes fragilis microscopic examination result that adopts embodiment 2 formula two separation and purification in embodiment 2;
  • Fig. 2 is the microscopic examination result of Bacteroides fragilis isolated and purified by formula four of Example 2 in Example 2;
  • Fig. 3 is the microscopic examination result of Bacteroides fragilis isolated and purified using the comparative formula of Example 2 in Example 2.
  • the optional range of the terms “and/or”, “or/and”, “and/or” includes any of two or more of the associated listed items, and also includes any of the associated listed items. Any and all combinations of any and all of the relevant listed items include any combination of any two of the relevant listed items, any more of the relevant listed items, or all of the relevant listed items.
  • the first aspect is used for descriptive purposes only, and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the indicated The importance or number of technical characteristics.
  • the technical features described in open form include closed technical solutions consisting of the enumerated features, as well as open technical solutions including the enumerated features.
  • the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid phase mixing, and refers to volume percentage for liquid-liquid phase mixing.
  • the percentage concentration involved in the present invention refers to the final concentration unless otherwise specified.
  • the final concentration refers to the proportion of the added component in the system after the component is added.
  • the temperature parameters in the present invention allow either constant temperature treatment or treatment within a certain temperature range.
  • the isothermal treatment allows the temperature to fluctuate within the precision of the instrument control.
  • the present invention provides a culture medium, which comprises plant-derived peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, porphyrin source, glucose and sodium hydroxide.
  • the present invention has the following beneficial effects: the medium provided by the present invention, through the overall adjustment of the formula components, can obtain the equivalent of the animal-source peptone medium when plant-source peptone is used instead of animal-source peptone. Microbial (especially Bacteroides fragilis) culture effect.
  • the main sources of the plant-derived peptone in the present invention are corn gluten, pea protein, soybean protein (non-genetically modified), wheat gluten, rice gluten, cottonseed protein or a mixture of various vegetable proteins. Plant-derived peptone medium has the advantages of no animal-derived pollution, relatively clear components, and high safety.
  • plant-derived peptone instead of animal-derived peptone in the medium can effectively reduce various risks caused by the introduction of animal-derived peptone during the development of live bacterial drugs.
  • the types of plant-derived peptones in the present invention include, but are not limited to, soybean peptones, rice peptones, pea peptones, wheat peptones, and cottonseed peptones.
  • the porphyrin source in the present invention refers to the substance that provides porphyrin, including but not limited to hemin, serum, protoporphyrin and the like.
  • the present invention takes hemin as an example to explain the technical solution of the present invention, but this is not a limitation to the technical solution of the present invention.
  • the culture medium comprises water, and in terms of concentration in the culture medium, the culture medium comprises 15g/L-20g/L of the plant-derived peptone, 1g/L-5g/L The yeast powder, 1g/L-10g/L sodium chloride, 1g/L-5g/L dipotassium hydrogen phosphate, 0.001g/L-1g/L porphyrin source, 1g/L- 5g/L of glucose and 0.1g/L-1g/L of sodium hydroxide.
  • the culture medium comprises 16g/L-19g/L of the pea peptone, 2g/L-4g/L of the yeast powder, 3g/L-7g/L
  • the sodium chloride described in L the dipotassium hydrogen phosphate described in 2g/L-4g/L, the porphyrin source described in 0.002g/L-0.9g/L, the glucose described in 2g/L-4g/L and 0.2g/L
  • dipotassium hydrogen phosphate in the present invention can be added in the form of dipotassium hydrogen phosphate trihydrate.
  • the culture medium comprises 17g/L-18.5g/L of the pea peptone, 2.5g/L-3.5g/L of the yeast powder, 4g/L-6g/L said sodium chloride, 2.5g/L-3g/L said dipotassium hydrogen phosphate, 0.003g/L-0.85g/L said porphyrin source, 2.5g/L-3g/L L said glucose and 0.3g/L-0.6g/L said sodium hydroxide.
  • the culture medium does not contain animal peptone.
  • the porphyrin source is hemin.
  • the present invention provides a method for preparing the culture medium as described above, the preparation method comprising the following steps:
  • the said plant source peptone, yeast powder, sodium chloride, dipotassium hydrogen phosphate, porphyrin source, glucose, sodium hydroxide and water are mixed.
  • the preparation method includes the following steps:
  • the preparation method includes the following steps:
  • step (2) Dissolving the porphyrin source with the sodium hydroxide solution described in step (1), preparing a porphyrin/sodium hydroxide solution, keeping it away from light, and standing;
  • step (3) Mixing the porphyrin/sodium hydroxide solution described in step (2) with other materials to prepare the culture medium.
  • 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 is 2%.
  • step (2) the standing time is 10min-20min. Further, the standing time is 10 minutes.
  • water described in the present invention may be double distilled water or the like.
  • the preparation method further includes the step of sterilizing the mixed product.
  • the method of sterilization is not particularly limited in the present invention, for example, high-pressure steam sterilization is used, and the sterilization condition is high-pressure steam sterilization at 121° C. for 30 minutes.
  • the present invention provides a method for cultivating Bacteroides fragilis, the culturing method comprising the step of inoculating Bacteroides fragilis to the above-mentioned medium for culturing.
  • the present invention does not specifically limit the type of Bacteroides fragilis to be cultured, for example, it may be a strain of Bacteroides fragilis with a preservation number of CGMCC No.10685.
  • the present invention does not specifically limit the specific steps and conditions of cultivation, and any suitable conditions for anaerobic bacteria (such as Bacteroides fragilis) can be used for cultivation.
  • the mode of cultivation can be anaerobic static cultivation, or anaerobic shaking cultivation, etc. If it is anaerobic shaking cultivation, the rotating speed used in shaking cultivation is 50rpm-500rpm, such as 50rpm, 100rpm, 200rpm, 250rpm, 300rpm, 350rpm, 400rpm, 450rpm, 500rpm, etc.
  • the temperature used for culturing can be 36.5°C, 37°C, 37.5°C or any range between these values.
  • the scale of culture can be adjusted according to needs, which can be on the scale of shake tubes/flasks, or 30L, 300L, etc. It is understood that according to the adjustment of the culture scale, the culture conditions can also be adjusted accordingly. Make adaptive adjustments.
  • test methods described in the following examples are conventional methods; the reagents and biological materials, unless otherwise specified, can be obtained from commercial sources.
  • Embodiment 1 the impact of different kinds of peptones on the culture effect of Bacteroides fragilis
  • the culture medium formula is shown in Table 1 below.
  • the concentration of hemin contained in the culture medium is 0.006g/L.
  • Bacteroides fragilis ZY-312 with the preservation number of Bacteroides fragilis No. 10685 was used.
  • the cultivation effect of the medium using pea peptone is equivalent to that of the medium using animal-source protein, and higher than the medium using other plant peptone.
  • the overall culture effect is better than the effect of adopting the same proportioning animal-derived medium formula (formula 4); (2) in the formula 1.
  • formula 2 and the control formula there is no significant difference between the control formula and formula 2, but there is a significant difference between the control formula and formula 1 and formula 3, which shows that there is a preferred solution for the culture medium formula of the present invention
  • Pea peptone is preferably selected as the plant peptone.
  • Embodiment 2 Morphological comparison and viability determination of Bacteroides fragilis obtained from medium containing plant-derived peptone and medium containing animal-derived peptone
  • Test object the culture obtained by using the medium formula 2 of Example 1, the culture obtained by the medium formula 4 of Example 1, and the culture obtained by the control formula of Example 1.
  • the prepared skimmed milk powder solution requires: specific gravity 1.033-1.034; acidity ⁇ 20°T; temperature 20°C.
  • Inoculation Preheat the viability tube to 37°C before inoculation, and add 3% (v/v) of the above culture into the sterilized viability tube.
  • the inoculation operation is required to be completed in an ultra-clean bench.
  • the operation process is an aseptic operation, and the inoculation straw must be sterilized.
  • Fermentation was carried out at a constant temperature of 37° C. for 3.5 hours.
  • Acid measurement After 3.5 hours of fermentation and cultivation, the vitality tube was immediately taken out for acidity measurement. Use a pipette to transfer 10 mL of the sample into a 100 mL Erlenmeyer flask, rinse the pipette with 20 mL of pure water, pour it into a 100 mL Erlenmeyer flask, add 3 drops of 0.5% phenolphthalein, and start the titration. Titrate with 0.1mol/mL NaOH standard solution until reddish, and the color will not fade within 30s. The milliliter of 0.1mol/mL NaOH standard solution consumed is multiplied by 10, which is the acidity.
  • the microscopic examination results of culture medium formula 2 are shown in Figure 1; the microscopic examination results of culture medium formula 4 are shown in Figure 2; the microscopic examination results of culture medium formula 4 are shown in Figure 3.
  • Figure 1, Figure 2, and Figure 3 it can be seen that the shape of Bacteroides fragilis contained in the culture medium formula 2 obtained in Example 1 is relatively short and round, which is close to the normal shape of Bacteroides fragilis; while the culture obtained from formula 4 and The shape of Bacteroides fragilis contained in the culture obtained from the comparison formula is relatively elongated.
  • the shape of Bacteroides fragilis is short and round, and elongated indicates that the growth state is not good. It can be seen that the plant-derived peptone medium is more conducive to the growth of Bacteroides fragilis.
  • the Bacteroides fragilis was cultivated with the second formula of the present invention, compared with the medium containing animal peptone, the bacterial viability was significantly improved, specifically, P ⁇ 0.05 compared with the control formula, and P ⁇ 0.01 compared with the fourth formula.
  • this example also tested the viability of the cultures obtained from formula 1 and formula 3 in Example 1, and the results obtained showed that the mean bacterial viability corresponding to formula 1 was 0.82, and the mean bacterial viability corresponding to formula 3 was 0.85. It can be seen that the bacterial activity obtained by culturing with plant-derived peptone medium is higher.
  • Embodiment 3 the safety comparison of different culture medium formulations
  • Strain expansion culture method 3.50 ⁇ 10 8 CFU/mL Bacteroides fragilis seeds were inoculated into a 1L-scale medium at a ratio of 10%, and binary gas (7% (v/v) CO 2 , 93 % (v/v) N 2 ) as anaerobic protective gas, at 37°C for static or shaking tube culture for 48 hours. The culture broth was used for 30L fermentation inoculation.
  • the number of miscellaneous bacteria in the expanded culture medium obtained by using 30 L of plant-derived peptone medium is far less than that of the traditional animal-derived peptone medium, and its safety is better than that of the traditional animal-derived peptone medium.
  • Embodiment 4 the influence of the culture medium of different proportioning on the culture of Bacteroides fragilis
  • Embodiment 5 and embodiment 6 the influence of different culture medium formulations on the culture result of Bacteroides fragilis
  • Embodiments 5 and 6 respectively provide a culture medium.
  • the formulas are shown in Table 7, which are respectively recorded as formula 8 and formula 9.
  • the formula 8 and formula 9 are respectively used to cultivate Bacteroides fragilis ZY-312, and the cultivation steps refer to Example 1:
  • Embodiment 1 formula two Embodiment 5 formula eight Embodiment 6 formula nine Plant source peptone pea peptone 18 Pea peptone 25 pea peptone 10 yeast 3 3 3 Sodium chloride 5 5 5 Dipotassium 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 Make up 1L Make up 1L Make up 1L
  • Viable bacteria unit CFU/mL
  • Comparative example 1 Comparative example 1, comparative example 2 and comparative example 3 provide a kind of culture medium respectively, formula is shown in Table 9, is recorded as formula ten, formula eleven and formula twelve respectively, adopts formula ten, formula eleven and formula twelve pairs respectively Bacteroides fragilis ZY-312 is cultivated, and the cultivation steps refer to Example 1:
  • Bacteroides fragilis ZY-312 was cultured using formula 10, formula 11 and formula 12 respectively, and the results are shown in the following table.

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Abstract

提供一种培养基及其制备方法、用其培养脆弱拟杆菌的方法。该培养基包含植物源蛋白胨、酵母粉、氯化钠、磷酸氢二钾、卟啉源、葡萄糖和氢氧化钠。该培养基，通过配方成分的整体调整，采用植物源蛋白胨代替传统动物源蛋白胨，，能够获得与采用动物源蛋白胨培养基相当的微生物培养效果。

Description

培养基及其制备方法、用其培养脆弱拟杆菌的方法 技术领域
本发明涉及微生物培养领域，具体而言，涉及一种培养基及其制备方法、用其培养脆弱拟杆菌的方法。
背景技术
脆弱拟杆菌(Bacteroides fragilis)是人类肠道中定植的共生菌之一，属严格厌氧菌，大约占肠道粪便总菌落数的1％-2％。其为革兰氏阴性杆菌，大小为(0.8-1.3)μm×(1.6-8)μm，染色不均，两端圆而深染，中间不着色或染色较浅，似为空泡。过去对脆弱拟杆菌的研究通常集中在其导致的厌氧菌感染和癌症等方面，但随着益生菌种类的开发，脆弱拟杆菌的益生性也被报道。为了支持益生性脆弱拟杆菌从实验室研究到大规模培养，有必要开发一种配方合理，效果良好的培养基。
目前，可以用于生产脆弱拟杆菌的常见培养基有：改良GAM培养基，该培养基包含蛋白胨、
Figure PCTCN2021124823-appb-000001
胨、大豆胨、酵母浸粉、牛肉粉、消化血清粉、牛肝浸粉、葡萄糖、KH 2PO 4、NaCl、可溶性淀粉、L-半胱氨酸、L-精氨酸、L-色氨酸、硫乙醇酸钠、维生素K 1和氯化血红素；改良PYG培养基，该培养基包含蛋白胨、葡萄糖、酵母浸粉、NaCl、盐酸半胱氨酸、CaCl 2、MgSO 4、K 2HPO 4、KH 2PO 4、NaHCO 3、维生素K1、氯化血红素；包含酵母提取物、胰蛋白胨、硫代乙醇酸钠、NaCl、L-半胱氨酸、亚甲蓝、血红素、甲萘醌和葡萄糖的培养基；包含酪蛋白胨、胰蛋白胨、酵母提取物、葡萄糖、L-精氨酸、血红素、维生素K和丙酮酸钠的培养基；通用培养基在特殊气体条件下可用于脆弱拟杆菌增菌培养，如TSB等。
已被报道的其他培养基例如：一种生禽中耐热厌氧菌的检测方法，涉及厌氧液体培养基包括以下原料：蛋白胨10g/L-20g/L、酵母粉1g/L-10g/L、葡萄糖1g/L-10g/L、大豆胨1g/L-10g/L、牛肉粉1g/L-10g/L、氯化钠1g/L-10g/L、可溶性淀粉1g/L-5g/L、半胱氨酸0.1g/L-1.0g/L、磷酸二氢钾1g/L-5g/L、维生素K1 0.001g/L-0.01g/L、氯化血红素0.001g/L-0.01g/L，余量为水。一种Bacteroides cellulosilyticus特异性筛选培养基，所述培养基包括富集培养基、分离培养基和生长培养基；所述富集培养基的配方包括如下组分：唯一碳源4g/L-6g/L，胰蛋白胨15g/L-25g/L，酵母提取物4g/L-6g/L，氯化钠4g/L-6g/L，磷酸氢二钾0.04g/L-0.06g/L，磷酸二氢钾0.04g/L-0.06g/L，半胱氨酸盐酸盐0.5g/L -1g/L，氯化血红素0.005g/L-0.01g/L，维生素K1 0.001g/L-0.002g/L，青霉素溶液20ml/L-40ml/L，卡那霉素硫酸盐溶液4ml/L-5ml/L，万古霉素盐酸盐溶液2.0ml/L-2.5ml/L，余量为无菌水，氢氧化钠调节pH值为6.8-7.0；所述唯一碳源为阿拉伯糖。一种Bacteroides xylanisolvens特异性筛选培养基，，所述培养基包括分离培养基和生长培养基，所述分离培养基中含有万古霉素、卡那霉素、唯一碳源及产酸指示剂等物质。所述分离培养基包括如下组分：唯一碳源阿拉伯糖4g/L-6g/L，胰蛋白胨15g/L-25g/L，酵母提取物4g/L-6g/L，氯化钠4g/L-6g/L，磷酸氢二钾0.04g/L-0.06g/L，磷酸二氢钾0.04g/L-0.06g/L，半胱氨酸盐酸盐0.5g/L-1g/L，氯化血红素0.005g/L-0.01g/L，维生素K1 0.001g/L-0.002g/L，产酸指示剂溴甲酚紫0.010-0.014g/L，卡那霉素硫酸盐溶液4-5ml/L，万古霉素盐酸盐溶液2.0-2.5ml/L，琼脂15-20g/L，余量为无菌水，氢氧化钠调节pH值为6.8-7.0。
如上例举的传统的可用于脆弱拟杆菌培养的培养基中，绝大多数采用单纯动物源蛋白胨，少部分采用动物源蛋白胨和植物源蛋白胨的组合。动物源蛋白胨和植物源蛋白胨作为常用有机氮源，在应用过程中各有利弊。动物源蛋白胨营养丰富，是微生物培养的重要补充因子，目标微生物增殖快，但是动物源蛋白胨存在明显的缺点，如可能存在潜在的病毒污染、成分不明确、不利于产物纯化等。由于上述缺陷，国际上药品管理机构和生产单位一直希望能够找到动物源蛋白胨替代产品，确保生物制品的安全。植物源蛋白胨培养基具有无动物源存在的污染、成分较为明确、高安全性等优点，但营养不及动物源蛋白胨。采用植物源蛋白胨代替动物源蛋白胨制备培养基用于微生物培养的过程中，虽然能够有效降低病毒污染等风险，但微生物增殖相对较慢、活菌数少。
基于此，如何在植物源蛋白胨代替动物源蛋白胨的情况下兼顾脆弱拟杆菌增殖效果是迫切需要解决的问题。
发明内容
鉴于以上背景技术的不足，本发明的目的之一是提供一种添加植物源蛋白胨的培养基配方，用该培养基培养脆弱拟杆菌的过程中，脆弱拟杆菌增殖快，培养物所含活菌数与添加动物源蛋白胨的培养基配方相当。
本发明的目的可以通过以下技术方案实现：
第一方面，本发明提供一种培养基，所述培养基包含植物源蛋白胨、酵母粉、氯化钠、磷酸氢二钾、卟啉源、葡萄糖和氢氧化钠。
在其中一个实施例中，所述培养基包含水，以在所述培养基中的浓度计，所述培养 基包含15g/L-20g/L所述植物源蛋白胨、1g/L-5g/L所述酵母粉、1g/L-10g/L所述氯化钠、1g/L-5g/L所述磷酸氢二钾、0.001g/L-1g/L所述卟啉源、1g/L-5g/L所述葡萄糖和0.1g/L-1g/L所述氢氧化钠。
在其中一个实施例中，以在所述培养基中的浓度计，所述植物源蛋白胨包含大豆蛋白胨、大米蛋白胨、豌豆蛋白胨、小麦蛋白胨和棉籽蛋白胨中的至少一种。
在其中一个实施例中，以在所述培养基中的浓度计，所述培养基包含16g/L-19g/L所述豌豆蛋白胨、2g/L-4g/L所述酵母粉、3g/L-7g/L所述氯化钠、2g/L-4g/L所述磷酸氢二钾、0.002g/L-0.9g/L所述卟啉源、2g/L-4g/L所述葡萄糖和0.2g/L-0.8g/L所述氢氧化钠。
第二方面，本发明提供一种如上所述的培养基的制备方法，所述制备方法包含如下步骤：
将所述的植物源蛋白胨、酵母粉、氯化钠、磷酸氢二钾、卟啉源、葡萄糖、氢氧化钠与水混合。
在其中一个实施例中，所述制备方法包含如下步骤：
混合所述氢氧化钠与部分所述水，制备氢氧化钠溶液；
混合所述氢氧化钠溶液和所述卟啉源并将所得混合溶液与所述的植物源蛋白胨、酵母粉、氯化钠、磷酸氢二钾、葡萄糖和剩余的所述水混合。
在其中一个实施例中，所述制备方法还包含对混合所得产物进行灭菌的步骤。
第三方面，本发明提供一种脆弱拟杆菌的培养方法，所述培养方法包含将脆弱拟杆菌接种至如上所述的培养基进行培养的步骤。
在其中一个实施例中，所述脆弱拟杆菌为保藏号为CGMCC No.10685的脆弱拟杆菌菌株。
在其中一个实施例中，培养的方式采用厌氧静态培养。
在其中一个实施例中，培养的方式采用厌氧振荡培养。
在其中一个实施例中，震荡培养所采用的转速为50rpm-500rpm。
在其中一个实施例中，培养所采用的温度为36.5℃-37.5℃。
与现有技术相比，本发明具备如下有益效果：
本发明提供的培养基，通过配方成分的整体调整，在采用植物源蛋白胨代替动物源蛋白胨的情况下，能够在活菌量相似的情况下，获得安全性、生长状态和菌活力优于传 统动物源蛋白胨培养基的脆弱拟杆菌培养效果。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为实施例2中采用实施例2配方二分离纯化的脆弱拟杆菌镜检结果；
图2为实施例2中采用实施例2配方四分离纯化的脆弱拟杆菌镜检结果；
图3为实施例2中采用实施例2对比配方分离纯化的脆弱拟杆菌镜检结果。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
术语
除非另外说明或存在矛盾之处，本文中使用的术语或短语具有以下含义：
本文所使用的术语“和/或”、“或/和”、“及/或”的可选范围包括两个或两个以上相关所列项目中任一个项目，也包括相关所列项目的任意的和所有的组合，所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。
本文中，“一种或几种”指所列项目的任一种、任两种或任两种以上。其中，“几种”指任两种或任两种以上。
本文中所使用的“其组合”、“其任意组合”、“其任意组合方式”等中包括所列项目中任两个或任两个以上项目的所有合适的组合方式。
本文中，“合适的组合方式”、“合适的方式”、“任意合适的方式”等中所述“合适”，以能够实施本发明的技术方案、解决本发明的技术问题、实现本发明预期的技术效果为准。
本文中，“优选”仅为描述效果更好的实施方式或实施例，应当理解，并不构成对本发明保护范围的限制。
本发明中，“第一方面”、“第二方面”、“第三方面”、等仅用于描述目的，不能理解为指示或暗示相对重要性或数量，也不能理解为隐含指明所指示的技术特征的重要性或数量。
本发明中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。
本发明中，涉及到数值区间，如无特别说明，则包括数值区间的两个端点。
本发明中涉及的百分比含量，如无特别说明，对于固液混合和固相-固相混合均指质量百分比，对于液相-液相混合指体积百分比。
本发明中涉及的百分比浓度，如无特别说明，均指终浓度。所述终浓度，指添加成分在添加该成分后的体系中的占比。
本发明中的温度参数，如无特别限定，既允许为恒温处理，也允许在一定温度区间内进行处理。所述的恒温处理允许温度在仪器控制的精度范围内进行波动。
第一方面，本发明提供一种培养基，所述培养基包含植物源蛋白胨、酵母粉、氯化钠、磷酸氢二钾、卟啉源、葡萄糖和氢氧化钠。
与现有技术相比，本发明具备如下有益效果：本发明提供的培养基，通过配方成分的整体调整，在采用植物源蛋白胨代替动物源蛋白胨的情况下，能够获得与动物源蛋白胨培养基相当的微生物(特别是脆弱拟杆菌)培养效果。本发明所述的植物源蛋白胨的主要来源有玉米面筋、豌豆蛋白、大豆蛋白(无转基因)、小麦面筋、大米面筋、棉籽蛋白或各类植物蛋白的混合物。植物源蛋白胨培养基具有无动物源存在的污染、成分较为明确、高安全性等优点。采用植物源蛋白胨代替动物源蛋白胨的培养基能够有效降低活菌药物开发过程中引入动物源蛋白胨造成的各类风险。本发明植物源蛋白胨的种类包括但不限于大豆蛋白胨、大米蛋白胨、豌豆蛋白胨、小麦蛋白胨和棉籽蛋白胨等。
本发明所述的卟啉源是指提供卟啉的物质，包括但不限于氯化血红素、血清、原卟啉等。本发明以氯化血红素为例对本发明技术方案进行解释说明，但这并非是对本发明技术方案的限制。
在其中一个实施例中，所述培养基包含水，以在所述培养基中的浓度计，所述培养基包含15g/L-20g/L所述植物源蛋白胨、1g/L-5g/L所述酵母粉、1g/L-10g/L所述氯化钠、1g/L-5g/L所述磷酸氢二钾、0.001g/L-1g/L所述卟啉源、1g/L-5g/L所述葡萄糖和0.1g/L-1g/L所述氢氧化钠。
进一步优选地，以在所述培养基中的浓度计，所述培养基包含16g/L-19g/L所述豌 豆蛋白胨、2g/L-4g/L所述酵母粉、3g/L-7g/L所述氯化钠、2g/L-4g/L所述磷酸氢二钾、0.002g/L-0.9g/L所述卟啉源、2g/L-4g/L所述葡萄糖和0.2g/L-0.8g/L所述氢氧化钠。
可以理解的是，本发明所述磷酸氢二钾可以以三水合磷酸氢二钾的形式加入。
在其中一个实施例中，以在所述培养基中的浓度计，所述培养基包含17g/L-18.5g/L所述豌豆蛋白胨、2.5g/L-3.5g/L所述酵母粉、4g/L-6g/L所述氯化钠、2.5g/L-3g/L所述磷酸氢二钾、0.003g/L-0.85g/L所述卟啉源、2.5g/L-3g/L所述葡萄糖和0.3g/L-0.6g/L所述氢氧化钠。
在其中一个实施例中，所述培养基不含动物源蛋白胨。
在其中一个实施例中，所述卟啉源为氯化血红素。
第二方面，本发明提供一种如上所述的培养基的制备方法，所述制备方法包含如下步骤：
将所述的植物源蛋白胨、酵母粉、氯化钠、磷酸氢二钾、卟啉源、葡萄糖、氢氧化钠与水混合。
在其中一个实施例中，所述制备方法包括如下步骤：
混合所述氢氧化钠和部分所述水，制备氢氧化钠溶液；
混合所述氢氧化钠溶液和所述卟啉源，并将所得混合溶液与所述的植物源蛋白胨、酵母粉、氯化钠、磷酸氢二钾、葡萄糖和剩余的所述水混合。
在其中一个实施例中，所述制备方法包括如下步骤：
(1)取氢氧化钠和适量水，配置成氢氧化钠溶液；
(2)以步骤(1)所述氢氧化钠溶液溶解卟啉源，制备卟啉/氢氧化钠溶液，避光，静置；
(3)将步骤(2)所述卟啉/氢氧化钠溶液与其他物料混合，制得所述培养基。
在其中一个示例中，步骤(1)中，所述氢氧化钠溶液中氢氧化钠的浓度为0.5-2.5％。进一步地，所述氢氧化钠溶液中氢氧化钠的浓度为2％。
在其中一个示例中，步骤(2)中，静置的时间为10min-20min。进一步地，静置的时间为10min。
可以理解的是，本发明所述的水，可以是双蒸水等。
在其中一个实施例中，所述制备方法还包含对混合所得产物进行灭菌的步骤。可以理解的是，本发明对灭菌的方式不做特别限定，例如采用高压蒸汽灭菌，灭菌条件为在121℃高压蒸汽灭菌30min。
第三方面，本发明提供一种脆弱拟杆菌的培养方法，所述培养方法包含将脆弱拟杆菌接种至如上所述的培养基进行培养的步骤。
本发明对培养对象脆弱拟杆菌的种类不做特别限定，例如可以为保藏号为CGMCC No.10685的脆弱拟杆菌菌株。
本发明对培养的具体步骤和条件不做特别限定，可以采用任意厌氧菌(例如脆弱拟杆菌)适宜的条件进行培养。例如培养的方式可以为厌氧静态培养，也可以为厌氧振荡培养等，如果是厌氧振荡培养，振荡培养所采用的转速为50rpm-500rpm，例如为50rpm、100rpm、200rpm、250rpm、300rpm、350rpm、400rpm、450rpm、500rpm等。例如培养所采用的温度可以为36.5℃、37℃、37.5℃或者这些值之间的任意范围值。例如可以根据需要，对培养的规模进行调整，可以是摇管/摇瓶规模的，也可以30L、300L等规模的，可以理解的是，根据培养规模的调整，也可以相应地对培养的条件进行适应性调整。
下述实施例中所述试验方法，如无特别说明，均为常规方法；所述试剂和生物材料，如无特别说明，均可从商业途径获得。
实施例1、不同种类的蛋白胨对脆弱拟杆菌培养效果的影响
(1)培养基配方如下表1。表1中，培养基所含氯化血红素的浓度为0.006g/L。
(2)培养基配方一至培养基配方四是通过以下方法制备的：
先将氢氧化钠用部分水配置成2％的氢氧化钠溶液，并将其与氯化血红素混合，避光，静置10分钟，完全溶解后，制得物料a；
蛋白胨、酵母粉、氯化钠、三水磷酸氢二钾、葡萄糖和剩余水混合，制得物料b；
混合物料a、物料b，溶解后，高温灭菌。
(3)脆弱拟杆菌采用保藏号为CGMCC No.10685的脆弱拟杆菌ZY-312。
(4)本实施例培养脆弱拟杆菌的具体步骤如下：
将3.50×10 8CFU/mL脆弱拟杆菌种子按照10％(v/v)比例接种至10mL培养基中，在以二元气体(7％v/vCO 2，93％v/vN 2)作为厌氧保护气下，37℃进行静置培养48小时，检测活菌数，以评估培养结果。
实验重复3次。培养结果见表2。
表1、含不同蛋白胨种类的脆弱拟杆菌培养基配方
Figure PCTCN2021124823-appb-000002
表2、不同蛋白胨对脆弱拟杆菌培养结果的影响
Figure PCTCN2021124823-appb-000003
根据表中数据可知，采用豌豆蛋白胨的培养基培养效果与传统应用动物源蛋白的培养基相当，高于采用其他植物蛋白胨的培养基。具体地，通过显著性分析可知：(1)采用本发明含植物源蛋白胨的培养基配方，整体培养效果优于采用相同配比动物源培养基配方(配方四)的效果；(2)在配方一、配方二、配方三和对照配方中，对照配方与配方二间不具有显著性差异，但对照配方与配方一、配方三间具有显著性差异，这说明本发明培养基配方存在优选方案，较优地选择豌豆蛋白胨作为植物蛋白胨。
实施例2、含植物源蛋白胨培养基与含动物源蛋白胨培养基所得脆弱拟杆菌形态对 比和活力测定
(1)检验对象：对采用实施例1培养基配方二所得培养物、实施例1培养基配方四所得培养物及实施例1对照配方所得培养物。
(2)实验方法：
1)形态对比：对上述培养物分离纯化，镜检。
2)活力测定：对上述培养物进行分离纯化，按下述步骤进行检验。
①制备活力管：称取脱脂奶粉11g加入89mL蒸馏水，搅拌10-15min使之充分溶解，溶解后静置1h后用双层纱布过滤，除去不溶物。然后将溶液分装试管10mL，盖上塞子密封。采用间歇三次灭菌：第一次灭菌90℃-95℃水浴灭菌20min，取出自然冷却至室温然后放置于4℃-6℃冰箱保存。第二天进行第二次灭菌，方法同第一次灭菌，取出自然冷却至室温然后放置于4℃-6℃冰箱保存。第三天进行第三次灭菌，方法同第一次灭菌，取出自然冷却至室温然后放置于4℃-6℃冰箱保存备用。
制得的脱脂奶粉溶液要求：比重1.033-1.034；酸度≤20°T；温度为20℃。
②活力测定
接种：接种前将活力管预热至37℃，在灭菌的活力管中加入3％(v/v)的上述培养物。接种操作要求在超净台完成，操作过程为无菌操作，使用接种吸管必须经过灭菌。
发酵：在恒温37℃进行发酵培养3.5h。
测酸：发酵培养3.5h后，立即取出活力管进行酸度测定。用移液管移取10mL样品至100mL三角瓶中，用20mL纯净水将移液管润洗干净后，一并倒入100mL三角瓶中，加入3滴0.5％酚酞，开始滴定。用0.1mo1/mL NaOH标准溶液滴定至微红色，并在30s内不退色。消耗的0.1mol/mL NaOH标准溶液毫升数乘以10，即为酸度。
计算：菌种活力＝0.087×消耗氢氧化钠毫升数。
(3)实验结果：
1)形态对比：
培养基配方二所得培养物的镜检结果如图1所示；培养基配方四所得培养物的镜检结果如图2所示；对比配方所得培养物的镜检结果如图3所示。结合图1、图2、图3可知：实施例1培养基配方二所得培养物所含脆弱拟杆菌的形态较为短圆，这种形态接近脆弱拟杆菌的正常形态；而配方四所得培养物和对比配方所得培养物所含脆弱拟杆菌的形态较为细长。脆弱拟杆菌的形态呈短圆较佳，呈细长说明生长状态不佳。可见，植物源蛋白胨培养基更利于脆弱拟杆菌的生长。
2)活力测定：结果如下表。
表3、不同配方培养基所得菌活力
Figure PCTCN2021124823-appb-000004
经显著性分析：采用本发明配方二配方培养脆弱拟杆菌，相对于采用含动物源蛋白胨培养基，菌活力显著提升，具体地，相对于对照配方P＜0.05，相对于配方四P＜0.01。另外，本实施例也对实施例1的配方一、配方三所得培养物进行了活力测试，所得结果为配方一对应的菌活力均值为0.82，配方三对应的菌活力均值为0.85。可见，采用植物源蛋白胨培养基培养得到的菌活力更高。
实施例3、不同培养基配方的安全性比较
(1)菌种扩大培养方法：将3.50×10 8CFU/mL脆弱拟杆菌种子按照10％比例接种至1L规模培养基中，在以二元气体(7％(v/v)CO 2，93％(v/v)N 2)作为厌氧保护气下，37℃进行静置或摇管培养48小时。培养液用于30L发酵接种。
(2)30L发酵，方法如下：50L发酵罐，加入培养基及血红素，消后共30L，厌氧保护气同上，接种2L步骤(1)培养24h时所得培养液，37℃，厌氧搅拌500rpm培养8h，期间根据发酵液中控在合适的时机补充碳源(1.0M葡萄糖溶液2.0L，葡萄糖浓度小于5mM时开始补加，1h内补完)，取8h时所得培养液进行杂菌检测。
采用上述方法，用表1培养基配方二和对照配方制备脆弱拟杆菌菌种30L级发酵培养液，对上述培养液进行杂菌检测，检测结果如下表：
表4、含不同种类的蛋白胨培养基所得扩大培养液中杂菌检测结果
Figure PCTCN2021124823-appb-000005
Figure PCTCN2021124823-appb-000006
注：与对照配方比较，**：P<0.01。
根据表中数据可知，用含植物源蛋白胨培养基30L所得扩大培养液中的杂菌数远少于传统动物源蛋白胨培养基，其安全性优于传统动物源蛋白胨培养基。
实施例4、不同配比的培养基对脆弱拟杆菌培养的影响
(1)本实施例培养基配方见下表5，培养基制备方法同实施例1。
(2)脆弱拟杆菌采用保藏号为CGMCC No.10685的脆弱拟杆菌ZY-312。
(3)本实施例培养脆弱拟杆菌的具体步骤同实施例1。
脆弱拟杆菌培养结果如下表6。
表5、不同配比的培养基配方
Figure PCTCN2021124823-appb-000007
表6、不同配方对脆弱拟杆菌培养结果的影响(活菌数单位：CFU/mL)
Figure PCTCN2021124823-appb-000008
根据表中数据可知：相对于配方五、配方六、配方七，采用配方二的培养基培养效 果相对较好，这说明本发明培养基配方存在优选方案即配方二。
实施例5和实施例6、不同培养基配方对脆弱拟杆菌培养结果的影响
实施例5和6分别提供一种培养基，配方见表7，分别记作配方八和配方九，分别采用配方八和配方九对脆弱拟杆菌ZY-312进行培养，培养步骤参照实施例1：
表7、不同蛋白胨与氢氧化钠比例对脆弱拟杆菌培养结果的影响
  实施例1配方二 实施例5配方八 实施例6配方九
植物源蛋白胨 豌豆蛋白胨18 豌豆蛋白胨25 豌豆蛋白胨10
酵母粉 3 3 3
氯化钠 5 5 5
磷酸氢二钾 2.5 2.5 2.5
葡萄糖 2.5 2.5 2.5
氯化血红素 0.006 0.006 0.006
氢氧化钠 0.4 2 0.05
水 补足1L 补足1L 补足1L
参照实施例1，分别采用配方八、配方九培养脆弱拟杆菌ZY-312，结果如下表。
表8、不同蛋白胨与氢氧化钠比例对脆弱拟杆菌培养结果的影响
Figure PCTCN2021124823-appb-000009
注：与配方二比较，**：P<0.01。活菌数单位：CFU/mL
可见，培养基所含植物源蛋白胨和氢氧化钠的浓度对脆弱拟杆菌的培养结果具有明显影响。
对比例1、对比例2和对比例3、不同培养基配方对脆弱拟杆菌培养结果的影响
对比例1、对比例2和对比例3分别提供一种培养基，配方见表9，分别记作配方 十、配方十一和配方十二，分别采用配方十、配方十一和配方十二对脆弱拟杆菌ZY-312进行培养，培养步骤参照实施例1：
表9、不同培养基配方对脆弱拟杆菌培养结果的影响
Figure PCTCN2021124823-appb-000010
参照实施例1，分别采用配方十、配方十一和配方十二培养脆弱拟杆菌ZY-312，结果如下表。
表10、不同培养基配方对脆弱拟杆菌培养结果的影响
Figure PCTCN2021124823-appb-000011
注：与配方二比较，**：P<0.01；活菌数单位：10 9CFU/mL
可见，氢氧化钠在脆弱拟杆菌的培养中是必要的。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种培养基，其特征在于，所述培养基包含植物源蛋白胨、酵母粉、氯化钠、磷酸氢二钾、卟啉源、葡萄糖和氢氧化钠。
  2. 根据权利要求1所述的培养基，其特征在于，所述培养基包含水，以在所述培养基中的浓度计，所述培养基包含15g/L-20g/L所述植物源蛋白胨、1g/L-5g/L所述酵母粉、1g/L-10g/L所述氯化钠、1g/L-5g/L所述磷酸氢二钾、0.001g/L-1g/L所述卟啉源、1g/L-5g/L所述葡萄糖和0.1g/L-1g/L所述氢氧化钠。
  3. 根据权利要求1或者2所述的培养基，其特征在于，所述植物源蛋白胨包含大豆蛋白胨、大米蛋白胨、豌豆蛋白胨、小麦蛋白胨和棉籽蛋白胨中的至少一种。
  4. 根据权利要求3所述的培养基，其特征在于，以在所述培养基中的浓度计，所述培养基包含16g/L-19g/L所述豌豆蛋白胨、2g/L-4g/L所述酵母粉、3g/L-7g/L所述氯化钠、2g/L-4g/L所述磷酸氢二钾、0.002g/L-0.9g/L所述卟啉源、2g/L-4g/L所述葡萄糖和0.2g/L-0.8g/L所述氢氧化钠。
  5. 权利要求1至4任一项所述的培养基的制备方法，其特征在于，所述制备方法包括如下步骤：将所述的植物源蛋白胨、酵母粉、氯化钠、磷酸氢二钾、卟啉源、葡萄糖、氢氧化钠与水混合。
  6. 根据权利要求5所述的培养基的制备方法，其特征在于，所述制备方法包括如下步骤：
    混合所述氢氧化钠与部分所述水，制备氢氧化钠溶液；
    混合所述氢氧化钠溶液和所述卟啉源并将所得混合溶液与所述的植物源蛋白胨、酵母粉、氯化钠、磷酸氢二钾、葡萄糖和剩余的所述水混合。
  7. 根据权利要求5所述的培养基的制备方法，其特征在于，所述制备方法还包含对混合所得产物进行灭菌的步骤。
  8. 一种脆弱拟杆菌的培养方法，其特征在于，所述培养方法包含将脆弱拟杆菌接种至权利要求1至4任一项所述的培养基进行培养的步骤。
  9. 根据权利要求7所述的脆弱拟杆菌的培养方法，其特征在于，所述脆弱拟杆菌为保藏号为CGMCC No.10685的脆弱拟杆菌菌株。
  10. 根据权利要求8或者9所述的脆弱拟杆菌的培养方法，其特征在于，培养的方式采用厌氧静态培养或/和厌氧振荡培养；或/和，培养所采用的温度为36.5℃-37.5℃。
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