Bifidobacterium culture medium and high-density culture method

Abstract

The application discloses a kind of bifidobacterium culture medium and high-density culture method, belong to microbial fermentation engineering field.The application has obtained the fermentation medium with yeast extract and fish peptone as nitrogen source, glucose as carbon source, compound multiple mineral and growth factor based on the optimization screening of bifidobacterium culture medium component.The application further designs the high-density culture method of bifidobacterium including passing mixed gas, control pH and flow feeding medium based on the fermentation medium.The experimental results show that using the culture method provided by the application, can efficiently amplify bifidobacterium, anaerobic fermentation 24h, and the viable bacterial content of bifidobacterium in fermentation product is as high as 2.11x10 10 CFU / g.The application provides a new strategy for breaking through the upper limit of cell density and shortening the fermentation period by optimizing the culture process of bifidobacterium, which has important significance for the industrial production and promotion of bifidobacterium preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial fermentation engineering, in particular to a Bifidobacterium culture medium and a high-density culture method. BACKGROUND

[0002] Bifidobacterium is (0.5-1.3) μm x (1.5-8.0) μm in size, has a high G+C content (about 55%-67%), and is of a non-typical lactic acid fermentation mode, and its metabolic end products mainly include acetic acid and lactic acid. On different species levels, the cell configurations of Bifidobacterium differ, and even the morphology of the same species is affected by the culture conditions and growth stages. Common morphologies include short rod, curved, stick rod, Y-shaped bifurcation, V-shaped bifurcation, etc. On solid culture medium, Bifidobacterium is of a smooth surface, has protrusions, a neat edge, and a milky white or white colony, and has a soft and easy-to-pick texture. As a strict anaerobe, the growth of Bifidobacterium is affected by the oxygen regulation mechanism, but different species of Bifidobacterium differ in oxygen tolerance. In a high-oxygen environment, the cell will have metabolic disorder to produce excessive hydrogen peroxide, thereby inhibiting the catalytic activity of fructose-6-phosphate-phosphoketolase and hindering proliferation. In terms of physiological adaptability, the optimal growth pH of Bifidobacterium is 6.0-7.0, and the growth is limited when the pH is lower than 4.5 or higher than 8.5. The optimal growth temperature of Bifidobacterium is 37℃-41℃, and the growth cannot be achieved when the temperature is lower than 20℃ or higher than 46℃.

[0003] As a key member of the human intestinal microbial community, Bifidobacterium exhibits its probiotic properties in multiple dimensions such as anti-aging intervention, tumor inhibition, and lipid metabolism regulation through mechanisms such as community homeostasis regulation, metabolic product synthesis, and immune barrier enhancement, and the number level and strain diversity of Bifidobacterium are positively correlated with the health status of the host. At present, Bifidobacterium live bacterial preparations are widely used in many industries such as medicine, food, and feed, but due to the limitations of the anaerobic culture characteristics of the strain itself and the complexity of the production process, the content of Bifidobacterium in some domestic products is low, which makes it difficult for the product to achieve the expected health improvement effect, and to some extent, restricts the development of related industries.

[0004] Therefore, it is urgent to provide a Bifidobacterium high-density culture process and culture medium to solve the problems of limited cell density improvement and long fermentation period of Bifidobacterium. SUMMARY

[0005] The purpose of the present application is to provide a Bifidobacterium culture medium and a high-density culture method to solve the problems existing in the prior art, and the present application provides a new strategy for breaking through the upper limit of cell density improvement and shortening the fermentation period by optimizing the Bifidobacterium culture process. The present application has important significance for the industrial production and popularization of Bifidobacterium preparations.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The present invention provides a culture medium for the fermentation culture of Bifidobacterium, the culture medium comprising a fermentation medium and a fed culture medium;

[0008] The fermentation medium comprises the following components: yeast extract, fish peptone, glucose, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, Tween 80, vitamin B1, glycine, vitamin C, and manganese sulfate monohydrate.

[0009] The fed culture medium comprises the following components: glucose, yeast extract, fish peptone, vitamin B1, glycine, and vitamin C.

[0010] Further, the fermentation medium comprises the following components: yeast extract 33.75 g / L, fish peptone 11.25 g / L, glucose 10 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, triammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, Tween 80 1 g / L, vitamin B1 0.2 g / L, glycine 0.1 g / L, vitamin C 0.06 g / L, and manganese sulfate monohydrate 0.02 g / L.

[0011] Furthermore, the fed culture medium comprises the following components: glucose 150 g / L, yeast extract 66.88 g / L, fish peptone 22.5 g / L, vitamin B1 3 g / L, glycine 1.5 g / L and vitamin C 0.9 g / L.

[0012] The present invention also provides an application of the above-mentioned culture medium in the fermentation culture of Bifidobacteria.

[0013] The present invention also provides a method for culturing Bifidobacteria, comprising the step of anaerobic fermentation culture of Bifidobacteria using the above-described culture medium.

[0014] Furthermore, it includes the following steps:

[0015] Bifidobacterium was inoculated into MRS medium and cultured for 15 h to prepare a seed culture.

[0016] The seed culture is inoculated into the fermentation medium for anaerobic fermentation; the feed medium is added during the anaerobic fermentation process.

[0017] Furthermore, anaerobic conditions are maintained by introducing a mixed gas; the mixed gas consists of 80% nitrogen, 10% hydrogen and 10% carbon dioxide by volume fraction.

[0018] Furthermore, the mixed gas consists of 80% nitrogen, 10% hydrogen and 10% carbon dioxide by volume fraction.

[0019] Furthermore, the feed rate of the culture medium is 12.5 mL / h.

[0020] Furthermore, the initial pH value of the anaerobic fermentation culture system is 6-7; the anaerobic fermentation culture also includes the step of adding a 20% by mass potassium hydroxide solution to make the pH value of the anaerobic fermentation culture system not lower than 6.

[0021] Furthermore, the anaerobic fermentation culture was conducted at a temperature of 37°C and a rotation speed of 90 r / min.

[0022] The present invention discloses the following technical effects:

[0023] Through optimized screening of Bifidobacterium culture medium components, this invention yielded a Bifidobacterium fermentation medium using yeast extract and fish peptone as nitrogen sources, glucose as a carbon source, and a combination of various minerals and growth factors. Based on this fermentation medium, this invention further designed a high-density Bifidobacterium culture method including the introduction of a mixed gas, pH control, and fed-batch culture. Experimental results show that using the culture method provided by this invention, Bifidobacterium can be efficiently amplified; after 24 hours of anaerobic fermentation, the viable Bifidobacterium content in the fermentation product reaches as high as 2.11 × 10⁻⁶. 10 CFU / g. This invention provides a new strategy for overcoming the upper limit of cell density and shortening the fermentation cycle by systematically optimizing the Bifidobacterium culture process; this invention is of great significance for the industrial production and promotion of Bifidobacterium preparations. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 A statistical graph showing the effect of different carbon source concentrations on the viable count of Bifidobacteria;

[0026] Figure 2 A statistical graph showing the effect of different carbon source conditions on the viable count of Bifidobacteria;

[0027] Figure 3 A statistical graph showing the effect of different nitrogen source conditions on the viable count of Bifidobacteria;

[0028] Figure 4A statistical chart showing the effects of different minerals and growth factors on the viable count of Bifidobacteria;

[0029] Figure 5 A statistical graph showing the effect of different culture conditions on the viable count of Bifidobacterium. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] The yeast extract powder used in this invention is yeast extract powder FM828, purchased from Angel Yeast Co., Ltd. All reagents used in this invention are publicly available products that can be purchased by the public.

[0036] Example 1

[0037] 1. Isolation of Bifidobacteria

[0038] 1.1 Preparation of basal culture medium

[0039] Basic culture medium: tryptone 10 g / L, beef extract 5 g / L, yeast extract 5 g / L, glucose 20 g / L, CH3COONa 5 g / L, K2HPO4 2 g / L, C6H5O7(NH4)3 2 g / L, MgSO4 0.2 g / L, MnSO4·H2O 0.02 g / L, V B1 0.5 g / L, Tween 80 1 g / L. Adjust pH to 6.5, sterilize at 110℃ for 15 min.

[0040] 1.2 Isolation and Screening of Strains

[0041] Collect sufficient fecal samples from healthy infants (without gastrointestinal symptoms and who have not taken any medications affecting the gut microbiota prior to sampling). Quickly transfer the collected fecal samples to sterile containers, add sterile saline for dilution and mixing to reduce impurities and microbial concentration variations. Perform serial dilutions of the pretreated fecal suspension to obtain different concentrations of bacterial suspension. Select an appropriate dilution of 10-1. -4 -10 -5 The bacterial culture was evenly spread onto MRS medium. Multiple parallel plates were prepared for each dilution to increase the reliability of the screening. The spread plates were placed in a constant temperature biochemical incubator and anaerobically cultured at 37°C for 48 h.

[0042] 1.3 Isolation and purification of strains

[0043] After incubation, observe the colony morphology on the plates and select colonies that resemble Bifidobacterium. Bifidobacterium colonies are characterized by a smooth, raised surface, neat edges, and are milky white or white in color; they are also soft and easily picked. The selected colonies are then inoculated onto fresh MRS agar plates using the four-zone streak method for further purification. This step is repeated until a pure culture is obtained.

[0044] 1.4 Identification and Verification of Strains

[0045] DNA was extracted from the pure culture and amplified by PCR using primers specific to Bifidobacterium. The amplified products were then detected by gel electrophoresis. Simultaneously, 16S rRNA gene sequencing was performed, and the sequencing results were compared with those of known strains to identify it as *Bifidobacterium lactis* subspecies, named *Bifidobacterium lactis* subspecies BB-12.

[0046] The strain of Bifidobacterium animalis subsp. BB-12 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251070, deposited on May 15, 2025, at Wuhan University, Wuhan, China.

[0047] 2. Optimization of culture medium components

[0048] 2.1 Preparation of Seed Liquid

[0049] Bifidobacteria preserved in glycerol tubes at -80℃ were inoculated into Erlenmeyer flasks (MRS medium) and cultured at 37℃ in a bacterial incubator for 15 h for later use. In this example, the experiments to optimize the composition of the culture medium were all conducted using seed culture prepared by this method for inoculation.

[0050] 2.2 Optimization of carbon source screening for culture media

[0051] 2.2.1 Optimization of carbon source concentration

[0052] Tryptone 10 g / L, beef extract 5 g / L, yeast extract 5 g / L, glucose 20 g / L, CH3COONa 5 g / L, K2HPO4 2 g / L, C6H5O7(NH4)3 2 g / L, MgSO4 0.2 g / L, MnSO4·H2O 0.02 g / L, V B1 0.5 g / L glucose and 1 g / L Tween 80 were added, and the pH was adjusted to 6.5. The mixture was then sterilized at 110℃ for 15 min. The mixture was then incubated at 37℃ for anaerobic fermentation for 24 h. The glucose in the basal medium was then changed to different concentrations: 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, and 7.0% (w / v) to prepare additional culture media.

[0053] The results are as follows Figure 1 As shown, the number of viable Bifidobacteria was highest when the glucose concentration was 0.5%-1.0%. Taking all factors into consideration, a carbon source concentration of 1.0% (10 g / L) was selected as the optimal carbon source concentration for subsequent experiments.

[0054] 2.2.2 Preparation of fermentation medium and culture conditions

[0055] Tryptone 10 g / L, beef extract 5 g / L, yeast extract 5 g / L, glucose 10 g / L, CH3COONa 5 g / L, K2HPO4 2 g / L, C6H5O7(NH4)3 2 g / L, MgSO4 0.2 g / L, MnSO4·H2O 0.02 g / L, V B1 Sterilize at 110℃ for 15 min with 0.5 g / L glucose and 1 g / L Tween 80, adjust pH to 6.5, and incubate at 37℃ for anaerobic fermentation for 24 h. Replace the carbon source (10 g / L glucose) in the above basal medium with the carbon source in conditions 1-7 below to prepare the culture medium.

[0056] Carbon source condition 1: Glucose 10 g / L;

[0057] Carbon source condition 2: Stachyose 10 g / L;

[0058] Carbon source condition 3: maltose 10 g / L;

[0059] Carbon source condition 4: lactose 10 g / L;

[0060] Carbon source conditions 5: glucose 3.3 g / L, maltose 3.3 g / L, lactose 3.3 g / L;

[0061] Carbon source condition 6: glucose 5 g / L, maltose 5 g / L;

[0062] Carbon source condition 7: glucose 5 g / L, lactose 5 g / L.

[0063] 2.2.3 Viable cell count

[0064] Culture media were prepared according to carbon source conditions 1-7, and Bifidobacteria were fermented using different culture media. The fermented Bifidobacteria broth was diluted with sterile water, and viable bacteria were counted according to the dilution plating method.

[0065] The results of the count of viable Bifidobacteria after fermentation are as follows: Figure 2 As shown, by Figure 2 It can be seen that different carbon source culture conditions result in different viable cell counts after fermentation. The Bifidobacterium strain cultured under carbon source condition 1 had the highest viable cell count of 2.1 × 10⁻⁶. 9 CFU / mL, and this carbon source was selected for subsequent culture conditions.

[0066] 2.3 Optimization of Nitrogen Source Screening in Culture Media

[0067] 2.3.1 Preparation of fermentation medium and culture conditions

[0068] Tryptone 10 g / L, beef extract 5 g / L, yeast extract 5 g / L, glucose 10 g / L, CH3COONa 5 g / L, K2HPO4 2 g / L, C6H5O7(NH4)3 2 g / L, MgSO4 0.2 g / L, MnSO4·H2O 0.02 g / L, V B1 0.5 g / L, Tween 80 1 g / L, adjust pH to 6.5, sterilize at 110℃ for 15 min. Incubate at 37℃ for anaerobic fermentation for 24 h; then replace the nitrogen sources (tryptone 10 g / L, beef extract 5 g / L, yeast extract 5 g / L) in the above basal medium with the nitrogen sources in conditions 1-7 below to prepare the culture medium.

[0069] Nitrogen source condition 1: 20 g / L yeast extract;

[0070] Nitrogen source condition 2: Fish peptone 20 g / L;

[0071] Nitrogen source condition 3: 20 g / L beef meal;

[0072] Nitrogen source condition 4: peptone 20 g / L;

[0073] Nitrogen source condition 5: yeast extract 10 g / L, fish peptone 10 g / L;

[0074] Nitrogen source condition 6: yeast extract 15 g / L, fish peptone 5 g / L;

[0075] Nitrogen source condition 7: yeast extract 33.75 g / L, fish peptone 11.25 g / L.

[0076] 2.3.2 Viable cell count

[0077] Culture media were prepared according to nitrogen source conditions 1-7, and Bifidobacteria were fermented using different culture media. The fermented Bifidobacteria broth was diluted with sterile water, and viable bacteria were counted according to the dilution plating method.

[0078] The results of the count of viable Bifidobacteria after fermentation are as follows: Figure 3 As shown, by Figure 3 It can be seen that different nitrogen source compositions result in different numbers of viable Bifidobacteria after fermentation. When the nitrogen source concentration is 33.75 g / L yeast extract and 11.25 g / L fish peptone (nitrogen source condition 7), the content of viable Bifidobacteria in the fermentation product is the highest, at 3.6 × 10⁻⁶. 9 CFU / mL, and this nitrogen source was selected for subsequent culture conditions.

[0079] 2.4 Screening of different minerals and growth factors

[0080] 2.4.1 Preparation of fermentation medium

[0081] Yeast extract 33.75 g / L, fish peptone 11.25 g / L, glucose 10 g / L, CH3COONa 5 g / L, K2HPO4 2 g / L, C6H5O7(NH4)3 2 g / L, MgSO4 0.2 g / L, MnSO4·H2O 0.02 g / L, V B1 0.5 g / L, Tween 80 1 g / L, adjust pH to 6.5, sterilize at 110℃ for 15 min. Maintain constant temperature at 37℃ for anaerobic fermentation for 24 h; remove minerals and growth factors (MgSO4 0.2 g / L, MnSO4·H2O 0.02 g / L, V...) from the fermentation medium. B1Culture media were prepared according to the following mineral and growth factor conditions 1-7, with 0.5 g / L and Tween 80 1 g / L respectively.

[0082] Mineral and growth factor conditions 1: MgSO4 0.5 g / L;

[0083] Mineral and growth factor condition 2: MnSO4·H2O 0.02 g / L;

[0084] Mineral and growth factor condition 3: Glycine 0.1 g / L;

[0085] Mineral and growth factor condition 4: V B1 0.2 g / L;

[0086] Mineral and growth factor conditions 5: V C 0.06 g / L;

[0087] Mineral and growth factor condition 6: Tween 80 1 g / L;

[0088] Mineral and growth factor conditions 7: MgSO4 0.5 g / L, MnSO4·H2O 0.02 g / L, glycine 0.1 g / L, V B1 0.2 g / L, V C 0.06 g / L, Tween 80 1 g / L.

[0089] 2.4.2 Viable cell count

[0090] Culture media were prepared according to mineral and growth factor conditions 1-7, respectively. Bifidobacteria were fermented using different culture media. The fermented Bifidobacteria broth was diluted with sterile water and viable bacteria were counted according to the dilution plating method.

[0091] The results of the count of viable Bifidobacteria after fermentation are as follows: Figure 4 As shown, by Figure 4 It can be seen that the culture medium prepared using mineral and growth factor condition 7 for fermentation of Bifidobacteria showed the best combination of minerals and growth factors, resulting in significant fermentation effect and a maximum viable count of 5.6 × 10⁻⁶. 9 CFU / mL.

[0092] 2.5 Screening of Bifidobacterium culture conditions

[0093] Bifidobacteria were cultured in a fermenter at constant pH using an optimized culture medium;

[0094] The optimized culture medium formula is as follows: yeast extract 33.75 g / L, fish peptone 11.25 g / L, glucose 10 g / L, K2HPO4 2 g / L, CH3COONa 5 g / L, C6H5O7(NH4)3 2 g / L, MgSO4 0.5 g / L, Tween 80 1 g / L, V B1 0.2 g / L, glycine 0.1 g / L, V C 0.06 g / L and MnSO4·H2O 0.02 g / L.

[0095] The feed culture medium formula is: glucose 150 g / L, yeast extract 66.88 g / L, fish peptone 22.5 g / L, vitamin C... B1 3 g / L, glycine 1.5 g / L, V C 0.9 g / L. Adjust pH to 6.5 and sterilize at 110℃ for 15 min.

[0096] The mixture was anaerobic fermented at a constant temperature of 37℃ for 24 h. The fermentation conditions were set as fermentation condition 1 to fermentation condition 6, and the proportions of each gas in the mixed gas mixture in fermentation condition 1 to fermentation condition 6 were by volume.

[0097] Fermentation conditions 1: mixed gas (nitrogen:hydrogen:carbon dioxide = 8:1:1), rotation speed 30 r / min, temperature 38℃;

[0098] Fermentation conditions 2: A mixed gas (nitrogen:hydrogen:carbon dioxide = 8:1:1) was introduced, 20% NaOH was added to maintain a constant pH of 6.0, the rotation speed was 30 r / min, and the temperature was 38℃;

[0099] Fermentation conditions 3: mixed gas (nitrogen:hydrogen:carbon dioxide = 8:1:1), 20% NaOH added to maintain pH at 6.0, rotation speed 30 r / min, temperature 37℃;

[0100] Fermentation conditions 4: mixed gas (nitrogen:hydrogen:carbon dioxide = 8:1:1), 20% NaOH added to maintain pH at 6.0, rotation speed 90 r / min, temperature 37℃;

[0101] Fermentation conditions 5: mixed gas (nitrogen:hydrogen:carbon dioxide = 8:1:1), 20% KOH added to maintain pH at 6.0, rotation speed 90 r / min, temperature 37℃;

[0102] Fermentation conditions 6: A mixed gas (nitrogen:hydrogen:carbon dioxide = 8:1:1) was introduced, 20% KOH was added to maintain the pH at 6.0, the rotation speed was 90 r / min, the temperature was 37℃, and 300 mL of feed medium was added over 24 h, i.e., the feed rate was 12.5 mL / h.

[0103] Fermentation was carried out according to the methods described in fermentation conditions 1-6. The resulting Bifidobacterium bacterial suspension was diluted with sterile water, and viable cell counts were performed using the dilution plating method. The viable cell count results after fermentation are as follows: Figure 5 As shown, by Figure 5 It can be seen that the number of viable Bifidobacteria obtained from fermentation varies under different culture conditions. Among them, the Bifidobacteria screened in this invention, cultured under condition 6, had the highest content of viable bacteria in the fermentation product, which was 2.11 × 10⁻⁶. 10 CFU / g.

[0104] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A culture medium for the fermentation culture of Bifidobacteria, characterized in that, The culture medium includes a fermentation medium and a fed culture medium; The fermentation medium comprises the following components: yeast extract 33.75 g / L, fish peptone 11.25 g / L, glucose 10 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, triammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, Tween 80 1 g / L, vitamin B1 0.2 g / L, glycine 0.1 g / L, vitamin C 0.06 g / L, and manganese sulfate monohydrate 0.02 g / L; The supplemental culture medium comprises the following components: Glucose 150 g / L, yeast extract 66.88 g / L, fish peptone 22.5 g / L, vitamin B1 3 g / L, glycine 1.5 g / L and vitamin C 0.9 g / L.

2. The application of the culture medium as described in claim 1 in the fermentation culture of Bifidobacteria.

3. A method for culturing Bifidobacteria, characterized in that, Includes the step of anaerobic fermentation culture of Bifidobacteria using the culture medium described in claim 1; Specifically, the following steps are included: Bifidobacterium was inoculated into MRS medium and cultured for 15 h to prepare a seed culture. The seed culture is inoculated into the fermentation medium for anaerobic fermentation; the feed medium is added during the anaerobic fermentation process. Anaerobic conditions are maintained by introducing a mixed gas; the mixed gas consists of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide by volume fraction. The feed rate of the culture medium is 12.5 mL / h.

4. The method as described in claim 3, characterized in that, The initial pH value of the anaerobic fermentation culture system is 6-7; the anaerobic fermentation culture also includes the step of adding a 20% potassium hydroxide solution to make the pH value of the anaerobic fermentation culture system not lower than 6.

5. The method as described in claim 3, characterized in that, The anaerobic fermentation culture was conducted at a temperature of 37°C and a rotation speed of 90 r / min.

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