Bifidobacterium culture medium and high-density culture method

Abstract

The invention discloses a bifidobacterium culture medium and a high-density culture method, and belongs to the field of microbial fermentation engineering. By optimizing and screening components of the bifidobacterium culture medium, the fermentation culture medium which takes yeast extract powder and fish peptone as nitrogen sources and glucose as a carbon source and is compounded with various mineral substances and growth factors is obtained. Based on the fermentation culture medium, the invention further designs a bifidobacterium high-density culture method which comprises the steps of introducing mixed gas, controlling pH and feeding a fed-batch culture medium. Experimental results show that by using the culture method provided by the invention, the bifidobacteria can be efficiently amplified, anaerobic fermentation is performed for 24 hours, and the viable content of the bifidobacteria in a fermentation product is up to 2.11 * 10 < 10 > CFU / g. According to the method, a bifidobacterium culture process is systematically optimized, a new strategy is provided for breaking through the upper limit of thallus density increase and shortening the fermentation period, and the method is of great significance to industrial production and popularization of bifidobacterium preparations.

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 has a non-typical lactic acid fermentation mode, and its metabolic end products mainly include acetic acid and lactic acid. On the species level, the cell configuration of Bifidobacterium differs, and even within the same species, the morphology of the bacterial cells is affected by the culture conditions and the growth stage. Common morphologies include short rod-shaped, curved, stick-shaped, Y-shaped bifurcated, V-shaped bifurcated, etc. On solid culture medium, Bifidobacterium has a smooth surface, protrusions, and a neat edge, and forms a milky white or white colony with soft texture and easy to pick up. As an obligate 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 bacterial cells undergo 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 bacterial cells cannot grow 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 strengthening, 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 strains and the complexity of the production process, the content of Bifidobacterium in some domestic products is low, which makes it difficult for the products to achieve the expected health improvement effect, and to some extent, restricts the development of related industries.

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

[0005] The present application aims to provide a Bifidobacterium culture medium and a high-density culture method to solve the problems existing in the prior art, and to provide a new strategy for breaking through the upper limit of bacterial 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 object, the present application provides the following scheme:

[0007] The present application provides a culture medium for fermentation culture of Bifidobacterium, which comprises a fermentation culture medium and a feeding culture medium.

[0008] The fermentation culture medium comprises the following components: yeast extract powder, 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 feeding culture medium comprises the following components: glucose, yeast extract powder, fish peptone, vitamin B1, glycine and vitamin C.

[0010] Further, the fermentation culture medium comprises the following components: yeast extract powder 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] Further, the feeding culture medium comprises the following components: glucose 150 g / L, yeast extract powder 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 application also provides the use of the above-mentioned culture medium in fermentation culture of Bifidobacterium.

[0013] The present application also provides a method for culturing Bifidobacterium, which comprises the step of anaerobic fermentation culture of Bifidobacterium using the above-mentioned culture medium.

[0014] Further, the method comprises the following steps:

[0015] Inoculating Bifidobacterium into MRS culture medium for culture for 15 h to prepare seed liquid;

[0016] Inoculating the seed liquid into the fermentation culture medium for anaerobic fermentation culture; and feeding the feeding culture medium during the anaerobic fermentation culture.

[0017] Further, the anaerobic condition is maintained by passing mixed gas; the mixed gas comprises 80% nitrogen, 10% hydrogen and 10% carbon dioxide by volume fraction.

[0018] Further, the mixed gas comprises 80% nitrogen, 10% hydrogen and 10% carbon dioxide by volume fraction.

[0019] Further, the flow rate of the feed medium is 12.5 mL / h.

[0020] Further, the initial pH value of the anaerobic fermentation culture system is 6-7; the anaerobic fermentation culture further comprises the step of adding potassium hydroxide solution with a mass fraction of 20% to keep the pH value of the anaerobic fermentation culture system not lower than 6.

[0021] Further, the temperature of the anaerobic fermentation culture is 37℃, and the rotation speed is 90 r / min.

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

[0023] Through optimization and screening of the components of the Bifidobacterium culture medium, the present application obtains a Bifidobacterium fermentation culture medium with yeast extract powder and fish peptone as nitrogen sources, glucose as carbon source, and multiple minerals and growth factors combined. Based on the fermentation culture medium, the present application further designs a Bifidobacterium high-density culture method including passing mixed gas, controlling pH and adding feed medium. The experimental results show that using the culture method provided by the present application, Bifidobacterium can be efficiently expanded, and the viable Bifidobacterium content in the fermentation product is as high as 2.11 x 10 10 CFU / g after 24 h of anaerobic fermentation. The present application provides a new strategy for breaking through the upper limit of cell density and shortening the fermentation period by systematically optimizing the Bifidobacterium culture process; and the present application has important significance for the industrial production and popularization of Bifidobacterium preparations. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0025] Figure 1 Statistical diagram of the influence of different carbon source concentrations on the viable Bifidobacterium count;

[0026] Figure 2 Statistical diagram of the influence of different carbon source conditions on the viable Bifidobacterium count;

[0027] Figure 3 Statistical diagram of the influence of different nitrogen source conditions on the viable Bifidobacterium count;

[0028] Figure 4 Statistical diagram of the influence of different minerals and growth factors on the viable Bifidobacterium count;

[0029] Figure 5This is a statistical chart showing the effects of different culture conditions on the number of viable bifidobacteria. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting 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 terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

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

[0035] The yeast extract powder of the present invention is yeast extract powder FM828, which is purchased from Angel Yeast Co., Ltd. The reagents used in the present invention are all publicly available and can be purchased by the public.

[0036] Example 1

[0037] 1. Bifidobacterium Isolation

[0038] 1.1 Preparation of basal culture medium

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

[0040] 1.2 Isolation and screening of strains

[0041] Collect sufficient fecal samples from healthy infants (without gastrointestinal discomfort symptoms and without taking medications that affect intestinal flora before sampling). Transfer the collected fecal samples quickly to a sterile container and add sterile saline to dilute and mix to reduce impurities and microbial concentration differences in the sample. Perform gradient dilutions on the pretreated fecal suspension to obtain bacterial suspensions of different concentrations. Select an appropriate dilution of 10 -4 -10 -5 Evenly spread the bacterial solution onto MRS medium. Set up multiple parallel plates for each dilution to increase screening reliability. Place the coated plates in a constant-temperature biochemical incubator and incubate anaerobically at 37°C for 48 hours.

[0042] 1.3 Isolation and purification of strains

[0043] After the incubation period, observe the colony morphology on the plate and select colonies that resemble Bifidobacterium. Bifidobacterium colonies are characterized by a smooth surface, raised bumps, neatly margined, milky white or white colonies, and a soft texture that is easy to pick. The selected colonies are inoculated onto new MRS medium plates using the four-zone streak method for further purification. Repeat this step until a pure culture is obtained.

[0044] 1.4 Identification and verification of strains

[0045] DNA from the pure culture was extracted and amplified by PCR using primers specific for Bifidobacterium. The amplified products were then analyzed by gel electrophoresis. Simultaneously, 16S rRNA gene sequencing was performed. The results were compared with sequences of known strains, confirming the strain as Bifidobacterium animalis subsp. lactis, designated Bifidobacterium lactis BB-12.

[0046] The Bifidobacterium animalis subsp. BB-12 strain has been deposited in the China Center for Type Culture Collection with the deposit number: CCTCC NO: M 20251070 and the deposit date: May 15, 2025; the deposit address is Wuhan University, Wuhan, China.

[0047] 2. Optimization of culture medium components

[0048] 2.1 Preparation of seed solution

[0049] Bifidobacterium stored in -80°C glycerol tubes was inoculated into Erlenmeyer flasks (MRS medium) and cultured in a bacterial incubator at 37°C for 15 hours for later use. The experiments in this example to optimize the medium composition were all inoculated with the seed solution prepared in this way.

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

[0051] 2.2.1 Optimization of carbon source concentration

[0052] Tryptone 10g / L, beef extract powder 5g / L, yeast extract powder 5g / L, glucose 20g / L, CH3COONa 5g / L, K2HPO4 2g / L, C6H5O7(NH4)3 2g / L, MgSO4 0.2g / L, MnSO4·H2O 0.02g / L, V B1 0.5 g / L of dapoxetine and 1 g / L of Tween 80, adjust the pH to 6.5, and sterilize at 110°C for 15 min. Anaerobic fermentation was carried out at 37°C for 24 h. Culture media were prepared by changing the glucose concentration in the above basal medium 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).

[0053] The results are as follows Figure 1 As shown in the figure, when the glucose concentration was 0.5%-1.0%, the number of viable bifidobacteria was the largest. After comprehensive consideration, the carbon source concentration of 1.0% (10 g / L) was selected as the optimal carbon source concentration for subsequent experiments.

[0054] 2.2.2 Fermentation medium configuration and culture conditions

[0055] Tryptone 10g / L, beef extract powder 5g / L, yeast extract powder 5g / L, glucose 10g / L, CH3COONa 5g / L, K2HPO4 2g / L, C6H5O7(NH4)3 2g / L, MgSO4 0.2g / L, MnSO4·H2O 0.02g / L, V B1 0.5 g / L of glucose and 1 g / L of Tween 80, adjust the pH to 6.5, and sterilize at 110°C for 15 min. Anaerobic fermentation was carried out at 37°C for 24 h. The carbon source (glucose 10 g / L) in the above basal medium was replaced with the carbon source in the following carbon source conditions 1-7 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 condition 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 bacteria count

[0064] Culture media were prepared according to carbon source conditions 1-7, and bifidobacteria were fermented using different culture media. The fermented bifidobacterium bacterial liquid was diluted with sterile water, and the viable bacteria were counted according to the dilution spread plate method.

[0065] The statistical results of the number 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 have different viable bacterial counts after fermentation. The highest viable bacterial count of the Bifidobacterium strain cultured under carbon source condition 1 is 2.1×10 9 CFU / mL, and this carbon source culture condition was selected for subsequent culture.

[0066] 2.3 Optimization of nitrogen source screening for culture medium

[0067] 2.3.1 Fermentation medium configuration and culture conditions

[0068] Tryptone 10g / L, beef extract powder 5g / L, yeast extract powder 5g / L, glucose 10g / L, CH3COONa 5g / L, K2HPO4 2g / L, C6H5O7(NH4)3 2g / L, MgSO4 0.2g / L, MnSO4·H2O 0.02g / L, V B1 0.5 g / L of ethanol and 1 g / L of Tween 80, adjust the pH to 6.5, and sterilize at 110°C for 15 min. Anaerobic fermentation was carried out at 37°C for 24 h. The nitrogen sources in the above basal medium (10 g / L of tryptone, 5 g / L of beef extract powder, and 5 g / L of yeast extract powder) were replaced with the nitrogen sources in the following nitrogen source conditions 1-7 to prepare the culture medium.

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

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

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

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

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

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

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

[0076] 2.3.2 Viable bacteria count

[0077] Culture media were prepared according to nitrogen source conditions 1-7, and bifidobacteria were fermented using different culture media. The fermented bifidobacterium bacterial liquid was diluted with sterile water, and the viable bacteria were counted according to the dilution spread plate method.

[0078] The statistical results of the number of viable bifidobacteria after fermentation are as follows: Figure 3 As shown by Figure 3 It can be seen that the number of live bifidobacteria after fermentation is different with different nitrogen source components. When the nitrogen source concentration is 33.75 g / L yeast extract powder and 11.25 g / L fish peptone (nitrogen source condition 7), the content of live bifidobacteria in the fermentation product is the highest, which is 3.6×10 9 CFU / mL, and this nitrogen source culture condition was selected for subsequent culture.

[0079] 2.4 Screening of different minerals and growth factors

[0080] 2.4.1 Preparation of fermentation medium

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

[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 0.2g / L;

[0086] Mineral and Growth Factor Condition 5: V C 0.06g / L;

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

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

[0089] 2.4.2 Viable bacteria count

[0090] Culture media were prepared according to mineral and growth factor conditions 1-7, and bifidobacteria were fermented using different culture media. The fermented bifidobacterium culture liquid was diluted with sterile water, and live bacteria were counted according to the dilution spread plate method.

[0091] The statistical results of the number of viable bifidobacteria after fermentation are as follows: Figure 4 As shown by Figure 4 It can be seen that the culture medium prepared with minerals and growth factors in condition 1 has the best type and combination of minerals and growth factors for fermentation of bifidobacteria, and the fermentation effect is significant, with the highest viable bacterial count reaching 5.6×10 9 CFU / mL.

[0092] 2.5 Screening of Bifidobacterium Culture Conditions

[0093] Bifidobacterium was cultured in a fermenter in a fed-batch culture medium with constant pH.

[0094] The optimized culture medium formula is as follows: yeast extract powder 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 0.2g / L, glycine 0.1g / L, V C 0.06g / L and MnSO4·H2O 0.02g / L.

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

[0096] Anaerobic fermentation was carried out at a constant temperature of 37°C for 24 hours. The culture conditions were set to fermentation conditions 1 to 6, respectively, and the ratio of each gas in the mixed gas in fermentation conditions 1 to 6 was a volume ratio.

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

[0098] Fermentation condition 2: mixed gas (nitrogen: hydrogen: carbon dioxide = 8:1:1), supplemented with 20% NaOH to maintain a constant pH of 6.0, rotation speed 30 r / min, temperature 38°C;

[0099] Fermentation condition 3: mixed gas (nitrogen: hydrogen: carbon dioxide = 8:1:1), supplemented with 20% NaOH to maintain a constant pH of 6.0, rotation speed 30 r / min, temperature 37°C;

[0100] Fermentation condition 4: mixed gas (nitrogen: hydrogen: carbon dioxide = 8:1:1), supplemented with 20% NaOH to maintain a constant pH of 6.0, rotation speed 90 rpm, temperature 37°C;

[0101] Fermentation condition 5: mixed gas (nitrogen: hydrogen: carbon dioxide = 8:1:1), supplemented with 20% KOH to maintain a constant pH of 6.0, rotation speed 90 rpm, temperature 37°C;

[0102] Fermentation Condition 6: Mixed gas (nitrogen:hydrogen:carbon dioxide = 8:1:1) was passed through the fermentation chamber, pH was maintained at 6.0 with 20% KOH, the fermentation speed was 90 r / min, the temperature was 37°C, 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 method in fermentation conditions 1 to 6. The obtained bifidobacterium bacterial solution was diluted with sterile water and then the viable bacteria were counted according to the dilution plate method. The statistical results of the viable bacteria count after fermentation are as follows: Figure 4 As shown by Figure 4 It can be seen that the number of viable bifidobacteria obtained by fermentation is different under different culture conditions. Among them, the content of viable bifidobacteria in the fermentation product of the bifidobacterium screened by the present invention is the highest when cultured under condition 6, which is 2.11×10 10CFU / g.

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

Claims

1. A culture medium for bifidobacterium fermentation culture, characterized in that The culture medium includes a fermentation medium and a feed medium; The fermentation medium comprises the following components: yeast extract powder, fish peptone, glucose, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, Tween 80, vitamin B1, glycine, vitamin C and manganese sulfate monohydrate; The feed medium comprises the following components: glucose, yeast extract powder, fish peptone, vitamin B1, glycine and vitamin C.

2. The culture medium according to claim 1, wherein The fermentation medium includes the following components: 33.75 g / L yeast extract, 11.25 g / L fish peptone, 10 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.5 g / L magnesium sulfate, 1 g / L Tween 80, 0.2 g / L vitamin B1, 0.1 g / L glycine, 0.06 g / L vitamin C, and 0.02 g / L manganese sulfate monohydrate.

3. The culture medium according to claim 1, wherein The feed medium includes the following components: 150 g / L glucose, 66.88 g / L yeast extract powder, 22.5 g / L fish peptone, 3 g / L vitamin B1, 1.5 g / L glycine and 0.9 g / L vitamin C.

4. Use of the culture medium according to any one of claims 1 to 3 in bifidobacterium fermentation culture.

5. A method for culturing bifidobacteria, characterized in that: The method comprises the step of anaerobic fermentation culturing bifidobacteria using the culture medium according to any one of claims 1 to 3.

6. The method according to claim 5, wherein The following steps are involved: Bifidobacterium was inoculated into MRS medium and cultured for 15 h to prepare seed solution; The seed liquid is inoculated into the fermentation medium to carry out anaerobic fermentation culture; and the feed medium is added during the anaerobic fermentation culture.

7. The method according to claim 6, wherein Anaerobic conditions were maintained by introducing a mixed gas, which consisted of 80% nitrogen, 10% hydrogen and 10% carbon dioxide by volume.

8. The method according to claim 6, wherein The feed medium was added at a rate of 12.5 mL / h.

9. The method according to claim 6, wherein The initial pH value of the anaerobic fermentation culture system is 6-7; the anaerobic fermentation culture further comprises the step of adding a potassium hydroxide solution with a mass fraction of 20% to make the pH value of the anaerobic fermentation culture system not lower than 6.

10. The method according to claim 6, wherein The temperature of the anaerobic fermentation culture is 37° C. and the rotation speed is 90 r / min.

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