Method for increasing viable count of clostridium butyricum through fermentation-separation coupling

Abstract

The invention discloses a method for increasing the viable count of clostridium butyricum through fermentation-separation coupling, which adopts anion exchange resin to perform in-situ separation on organic acid generated in the fermentation process so as to increase the viable count of clostridium butyricum. The clostridium butyricum is cultured in a constant-pH (7.0) fed-batch fermentation mode, and when the glucose content in fermentation liquor is lower than 5 g / L, a certain amount of fresh culture medium is added to enable the glucose concentration to reach 10-15 g / L; when the pH value of the fermentation environment is lower than 6.5, a proper amount of anion exchange resin is added into the culture medium, and the pH value of the fermentation liquid is adjusted to 7.0. Ion exchange resin is used for establishing a fermentation-separation coupling process, growth inhibition of organic acid on clostridium butyricum in the fermentation process is relieved, the viable count is increased, and a good foundation is laid for the preparation process of the clostridium butyricum microbial inoculum. And the ion exchange resin can be recycled after being eluted, so that the purpose of reducing the cost is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biological fermentation, and in particular relates to a method for increasing the number of live Clostridium butyricum by fermentation-separation coupling. Background Art

[0002] Clostridium butyricum ( Clostridium butyricum ) is also known as Clostridium butyricum. It is a strictly anaerobic spore-forming bacterium that exists in the intestines of healthy humans and animals. It can produce a variety of beneficial biomass such as organic acids, amino acids, vitamins, and antimicrobial peptides. As a potential alternative to antibiotics, Clostridium butyricum has many functions such as regulating the balance of intestinal flora, improving the health of animal intestines, and promoting animal growth. It has broad application prospects in clinical medicine, agriculture, animal husbandry and other industries. European and American countries mostly use Clostridium butyricum as an industrial production strain of 1,3-propanediol and hydrogen. The use of crude raw materials or waste for production is also another research hotspot for this bacterium. At present, my country's research on Clostridium butyricum is still in its early stages. Commercially available bacterial agents and other products are facing the dilemma of low live bacterial counts. The reason is that the biomass of the bacteria cannot reach a high level during the fermentation process. Clostridium butyricum is an acid-producing microorganism. During metabolism, it will produce a variety of organic acids such as acetic acid and butyric acid, which will reduce the pH of the fermentation environment and is not conducive to the growth of bacteria. When the environmental pH is reduced to a certain level, the dissociation of organic acids is inhibited. Molecular organic acids enter the cell by passive transport and dissociate into H + and acid ions, inhibiting the life activities of cells. In addition, pyruvate-ferredoxin oxidoreductase, a key enzyme in the metabolic pathway of Clostridium butyricum, is very sensitive to O2 and loses 50% of its activity after 1 hour in pure oxygen, which greatly increases the difficulty of fermentation. Increasing the biomass of Clostridium butyricum is conducive to the preparation of bacterial agents with high viable counts, and its research and application are of great significance. In the current existing technology, in order to improve the survival rate of bacteria, some sublethal treatments can usually be performed on the bacteria before drying to improve the adaptability of the bacteria in extreme environments. Commonly used sublethal treatments include acid stress, oxygen stress, heat stress and salt stress. Reducing the content of organic acids in the fermentation broth and eliminating its "acid stress effect" on Clostridium butyricum is also an effective method.

[0003] Bioreaction-separation coupling technology, also known as in-situ product separation technology, is to select a suitable separation method during the microbial fermentation process to transfer the metabolites that inhibit microbial growth, ensure the growth of the bacteria and improve productivity. Common separation methods include evaporation, extraction, membrane permeation, adsorption, ion exchange, etc. The selected method must not only be able to effectively separate, but also not have a negative impact on the bacteria. The separation resin used in industry refers to a type of polymer that relies on physical adsorption capacity or electrostatic force to achieve the purpose of separation, which is mainly divided into two categories: adsorption resin and ion exchange resin. Among them, adsorption resins are adsorbed by van der Waals forces or hydrogen bonds, and can be divided into three categories: non-polar, neutral and polar. The advantages are good selectivity, stable physical and chemical properties, mild conditions of use, and easy regeneration and long service life, which is conducive to reducing costs and saving resources. Ion exchange resins are a type of polymer compounds with functional groups in the cross-linked polymer structure. According to the properties of the functional groups carried by the resin, they are divided into anion exchange resins and cation exchange resins. Ion exchange resins are characterized by ion exchange and adsorption, and have advantages in stability, adsorption rate, mechanical strength, etc. They are mainly used in water treatment and environmental protection. Its adsorption force is electrostatic force, which is a reversible reaction. Fermentation-separation coupling is a relatively mature prior art. For example, CN104212858A discloses the addition of resin adsorption during the logarithmic phase of fermentation in a high-yield nisin adsorption-separation coupling fermentation process. The fermentation-separation coupling technology is applied to the Clostridium butyricum fermentation of the present invention, and the main product types and yields in the fermentation process are measured, and the effects on bacterial growth are studied, and the inhibition mechanism is analyzed; at the same time, the adsorption of organic acids by ion exchange resins is investigated, and ion exchange resins are used to remove acid substances in the fermentation broth, remove the inhibitory factors, and achieve high-density fermentation of Clostridium butyricum. Summary of the invention

[0004] The object of the present invention is to provide a method for increasing the number of viable Clostridium butyricum by using a fermentation-separation coupling technology, coupling ion exchange with Clostridium butyricum fermentation, relieving the inhibitory effect of metabolites butyric acid and acetic acid on bacterial growth, and increasing the number of viable bacteria.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for increasing the number of viable Clostridium butyricum by fermentation-separation coupling technology, wherein Clostridium butyricum is cultured by constant pH fed-batch fermentation, when the glucose content in the fermentation broth is lower than 5 g / L, a certain amount of fresh culture medium is added to make the glucose concentration reach 10-15 g / L; when the pH of the fermentation environment is lower than 6.5, an appropriate amount of anion exchange resin is added to the culture medium to adjust the pH of the fermentation broth to 7.0.

[0006] The Clostridium butyricum was activated and inoculated into the fermentation medium.

[0007] The components of the fermentation medium are: glucose 10~30 g / L, tryptone 10~20 g / L, ammonium sulfate 5~10 g / L, CaCl2 0~2 g / L, NaHCO3 0~5 g / L, MgSO4 0~3 g / L, MnSO4 0~1 g / L, and FeSO4 0~5 g / L.

[0008] The specific steps include: Seed culture: Inoculate the activated Clostridium butyricum into the seed culture medium and culture it in an anaerobic workstation at 30~40℃ for 12~48 h.

[0009] The seed liquid obtained in step (1) is inoculated into the fermentation medium for cultivation, the inoculation amount is 1-5% (v / v), the culture temperature is 30-40°C, the rotation speed is 50-200 rpm, and N2 is continuously introduced during the cultivation process.

[0010] When the glucose content in the fermentation broth is lower than 5 g / L, add a certain amount of fresh culture medium to make the glucose concentration reach 10~15 g / L; when the pH of the fermentation environment is lower than 6.5, add an appropriate amount of anion exchange resin to the culture medium to adjust the pH of the fermentation broth to 7.0.

[0011] The fermentation medium described in step (2) comprises 10-30 g / L glucose, 10-20 g / L tryptone, 5-10 g / L ammonium sulfate, 0-2 g / L CaCl2, 0-5 g / L NaHCO3, 0-3 g / L MgSO4, 0-1 g / L MnSO4 and 0-5 g / L FeSO4.

[0012] The feed medium was 40 g of glucose solution.

[0013] In the present invention, the anion exchange resin is at least one of D315, D314, D301, D301G, D311 and 201×7 gel-type adsorption resins, and the adsorption amount of butyric acid and acetic acid is detected. D311 and D301G weakly basic anion exchange resins are further preferred.

[0014] The pretreatment and transformation steps of the anion exchange resin are: (1) Backwash the anion exchange resin with distilled water to remove impurities and fine resin particles until the backwash water is clear.

[0015] (2) Pass 4 to 6 times the volume of 3 to 5% NaOH solution, soak in a shaker at 200 rpm at 20 to 30 °C for 10 to 20 h, drain the alkali solution, and rinse with distilled water until the effluent is neutral.

[0016] (3) Pass 4 to 6 times the volume of 3 to 5% HCl solution, soak at 200 rpm at 20 to 30 °C for 10 to 20 h, drain the acid, and rinse with distilled water until the effluent is neutral.

[0017] (4) Repeat step (2). The pretreated and transformed anion exchange resin is stored in a sterile environment for future use.

[0018] The most preferred production method is: after the strain is activated, it is cultured at 37°C for 24 h, the fermentation liquid volume is 4 / 7.5L, the inoculation amount is 3% (v / v), the rotation speed is 100 rpm, and N2 is continuously introduced during the culture process to ensure that the fermentation is carried out in an anaerobic environment.

[0019] Among them, the fermentation medium is preferably 20.0 g / L glucose, 15.9 g / L tryptone, 7.5 g / L ammonium sulfate, 1.0 g / L CaCl2, 2.0 g / L NaHCO3, 1.2 g / L MgSO4, 0.45 g / L MnSO4, and 2.97 g / L FeSO4.

[0020] The present invention does not rely on a specific strain of Clostridium butyricum, and may be any strain of Clostridium butyricum that produces organic acids during fermentation, including but not limited to Clostridium butyricum Clostridium butyricum ATCC19398, Clostridium butyricum CGMCC No.1.5205, Clostridium butyricum BNCC 337239.

[0021] The present invention introduces anion exchange resin in the fermentation process of Clostridium butyricum to replace butyric acid and acetic acid produced in the production process of Clostridium butyricum, eliminates the inhibitory effect of organic acid on the bacterial growth process, and increases the number of live fermentation bacteria of Clostridium butyricum.

[0022] Preferably, Clostridium butyricum is Clostridium butyricum Clostridium butyricum ATCC19398 or Clostridium butyricum ( Clostridium butyricum BNCC 337239), when the pH of the fermentation environment is lower than 6.5, add an appropriate amount of D311 resin to the culture medium to increase the number of viable bacteria.

[0023] Preferably, Clostridium butyricum is Clostridium butyricum ( Clostridium butyricum When CGMCC No.1.5205, add D301G resin.

[0024] Compared with the prior art, the present invention has the following advantages: Anion exchange resin was introduced during the fermentation process to achieve in-situ separation of the fermentation products butyric acid and acetic acid, eliminate the inhibitory effect of organic acids on bacterial growth, and increase the number of live bacteria in the fermentation of Clostridium butyricum.

[0025] The anion exchange resin can be reused after regeneration, and the adsorbed butyric acid and acetic acid can be utilized as by-products after desorption, which significantly reduces production costs and saves production resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Adsorption of butyric acid and acetic acid by different resins; Figure 2 The adsorption amount of glucose in fermentation medium by different resins in the presence of butyric acid; Figure 3 The adsorption amount of glucose in fermentation broth by different resins in the presence of acetic acid; Figure 4 OD of Clostridium butyricum during ion exchange high-density culture 600 , pH and glucose concentration changes; Figure 5 Comparison of the fermentation results when pH was adjusted using NaOH and anion exchange resin D311 (total acid ion concentration and osmotic pressure). DETAILED DESCRIPTION

[0027] The present invention can be better understood through the following examples. The specific process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0028] The D315, D314, D301, D301G, D311 and 201×7 gel-type adsorption resins described in the following examples were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0029] The present invention does not rely on a specific strain of Clostridium butyricum, and may be any strain of Clostridium butyricum that produces organic acids in a fermentation process. The Clostridium butyricum strain used in the following examples is Clostridium butyricum Clostridium butyricum ATCC19398, Clostridium butyricum CGMCC No.1.5205, Clostridium butyricum BNCC 337239, and the strains are all existing public strains.

[0030] Example 1 Selection of anion exchange resin Anion exchange resin is alkaline in water and can exchange anions in the solution with OH-. It is often used for water treatment, extraction of rare elements, separation of substances such as amino acids and citric acid. The present invention selects five macroporous adsorption resins D315, D314, D301, D301G and D311 and six anion exchange resins 201×7 gel-type adsorption resins to detect the adsorption of butyric acid and acetic acid. As can be seen from the figure, among the six resins, the adsorption of butyric acid by the three resins D314, D301G and D311 is significantly higher than that of the other three resins, and among these three resins, the adsorption of acetic acid by D301G and D311 is higher. Therefore, D301G and D311 macroporous adsorption resins are selected for subsequent experiments.

[0031] In addition to the adsorption capacity of organic acids, the selective adsorption of glucose is also an important indicator for selecting resins. Excessive adsorption of glucose by the resin will cause a waste of nutrients, reduce the utilization rate of glucose by the bacteria, and affect the growth of the bacteria. Figure 1 It can be seen that the amount of glucose adsorbed by the resin is proportional to the amount of resin added, and the amount of glucose adsorbed by D301G is higher than that by D311. However, the bacteria will also produce a large amount of butyric acid and acetic acid during the growth process, and the adsorption capacity of the resin for glucose under different conditions may vary greatly. Therefore, in the presence of butyric acid and acetic acid, it is necessary to explore the adsorption capacity and adsorption order of the resin for glucose. Figure 2 and Figure 3 It can be seen that when butyric acid and acetic acid are present in the fermentation medium, the adsorption of glucose increases with the increase in the amount of D311 and D301G resin added, but it is significantly reduced compared to when no organic acid is added. During the experiment, the content of butyric acid and acetic acid in the fermentation medium was tested, and it was found that the presence of both was not detected, indicating that the resin preferentially adsorbs butyric acid and acetic acid.

[0032] The adsorption of glucose by D301G was higher than that by D311 in the fermentation medium with or without organic acid added. However, for the fermentation of different strains, we conducted subsequent experiments on both D311 and D301G.

[0033] Example 2 Clostridium butyricum ( Clostridium butyricum ATCC19398) Fermentation-Separation Coupled Process Seed culture medium: tryptone 10.0 g / L, beef extract 10.0 g / L, glucose 5.0 g / L, sodium chloride 5.0 g / L, yeast powder 3.0 g / L, anhydrous sodium acetate 3.0 g / L, soluble starch 1.0 g / L, L-cysteine ​​hydrochloride 0.5 g / L. Adjust the pH to 7.0±0.2 with NaOH solution.

[0034] Plate culture medium: Add 20.0 g / L agar powder to the seed culture medium.

[0035] Fermentation medium: glucose 20.0 g / L, trypsin 15.9 g / L, ammonium sulfate 7.5 g / L, CaCl2 1.0 g / L, NaHCO3 2.0 g / L, MgSO4 1.2 g / L, MnSO4 0.45 g / L, FeSO4 2.97 g / L.

[0036] The activated Clostridium butyricum ( Clostridium butyricum ATCC19398) was inoculated into the seed culture medium and cultured in an anaerobic workstation at 37℃ for 24 h, then transferred to the fermentation medium for culture. The inoculation amount was 3% (v / v), the culture temperature was 37℃, the culture period was 24 h, the fermentation liquid volume was 4 / 7.5 L, the rotation speed was 100 rpm, and N2 was continuously introduced during the culture process to ensure that the fermentation was carried out in an anaerobic environment. When the pH of the fermentation environment was lower than 6.5, appropriate amounts of anion exchange resins D311, D301G, and 201×7 resins were added to the culture medium separately to adjust the pH of the fermentation liquid to 7.0. When the glucose content in the fermentation liquid was lower than 5g / L, a certain amount of feed medium (40g glucose solution, added twice) was added to the fermentation liquid to make the glucose concentration reach 10 g / L to avoid nutrient deficiency and inhibit bacterial growth. In the control group, 10mol / L sterile NaOH solution was added dropwise using the automatic control module of the fermentation system to adjust the pH of the fermentation liquid to 7.0. Anion exchange resin can separate the acid ions produced during the fermentation process, reduce the osmotic pressure of the fermentation liquid, and eliminate the inhibitory effect on bacterial growth. Clostridium butyricum ATCC19398 entered the logarithmic growth phase after 6 h of fermentation, and the growth slowed down after 12 h. When the OD was 0.04 at 24 h of fermentation, 600 It was 8.82. We tested the viable bacterial count, spore rate, osmotic pressure and acid radical ion concentration of the group using anion exchange resin to adjust the pH and the group using NaOH to adjust the pH after 24 hours of fermentation. Compared with the group using NaOH solution to adjust the pH, the group using ion exchange resin had a higher number of viable bacteria, and lower total acid radical ion concentration and osmotic pressure, indicating that the ion exchange resin separated the acid radical ions in the fermentation broth, reduced the acid radical ion concentration and osmotic pressure of the fermentation broth, and increased the biomass. After 24 hours of fermentation Clostridium butyricum The viable count of ATCC19398 reached 1.18×10 8 cfu / mL, compared with 6.31×10 7 cfu / mL, an increase of 87.0%. Example 3 Clostridium butyricum ( Clostridium butyricum CGMCC No.1.5205) Fermentation-Separation Coupling Process Seed culture medium: tryptone 10.0 g / L, beef extract 10.0 g / L, glucose 5.0 g / L, sodium chloride 5.0 g / L, yeast powder 3.0 g / L, anhydrous sodium acetate 3.0 g / L, soluble starch 1.0 g / L, L-cysteine ​​hydrochloride 0.5 g / L. Adjust the pH to 7.0±0.2 with NaOH solution.

[0037] Plate culture medium: Add 20.0 g / L agar powder to the seed culture medium.

[0038] Fermentation medium: glucose 20.0 g / L, trypsin 15.9 g / L, ammonium sulfate 7.5 g / L, CaCl2 1.0 g / L, NaHCO3 2.0 g / L, MgSO4 1.2 g / L, MnSO4 0.45 g / L, FeSO4 2.97 g / L.

[0039] The activated Clostridium butyricum CGMCC No.1.5205 was inoculated into the seed culture medium and cultured in an anaerobic workstation at 37℃ for 24 h, then transferred to the fermentation medium for culture. The inoculation amount was 3% (v / v), the culture temperature was 37℃, the culture period was 24 h, the fermentation liquid volume was 4 / 7.5 L, the rotation speed was 100 rpm, and N2 was continuously introduced during the culture process to ensure that the fermentation was carried out in an anaerobic environment. When the pH of the fermentation environment was lower than 6.5, an appropriate amount of anion exchange resin was added to the culture medium to adjust the pH of the fermentation liquid to 7.0. When the glucose content in the fermentation liquid was lower than 5 g / L, a certain amount of feed medium (glucose solution) was added to the fermentation liquid to make the glucose concentration reach 10 g / L to avoid nutrient deficiency and inhibit bacterial growth. In the control group, 10 mol / L sterile NaOH solution was added dropwise using the automatic control module of the fermentation system to adjust the pH of the fermentation liquid to 7.0. Anion exchange resin can separate the acid ions produced during the fermentation process, reduce the osmotic pressure of the fermentation liquid, and eliminate the inhibitory effect on bacterial growth. Clostridium butyricum The highest number of viable bacteria in CGMCC No.1.5205 reached 1.35×10 8 cfu / mL, compared with 7.22×10 7 cfu / mL, an increase of 87.0%. Example 4 Clostridium butyricum ( Clostridium butyricum BNCC337239) Fermentation-separation coupling process Seed culture medium: tryptone 10.0 g / L, beef extract 10.0 g / L, glucose 5.0 g / L, sodium chloride 5.0 g / L, yeast powder 3.0 g / L, anhydrous sodium acetate 3.0 g / L, soluble starch 1.0 g / L, L-cysteine ​​hydrochloride 0.5 g / L. Adjust the pH to 7.0±0.2 with NaOH solution.

[0040] Plate culture medium: Add 20.0 g / L agar powder to the seed culture medium.

[0041] Fermentation medium: glucose 20.0 g / L, trypsin 15.9 g / L, ammonium sulfate 7.5 g / L, CaCl2 1.0 g / L, NaHCO3 2.0 g / L, MgSO4 1.2 g / L, MnSO4 0.45 g / L, FeSO4 2.97 g / L.

[0042] The activated Clostridium butyricum ( Clostridium butyricum BNCC 337239) was inoculated into the seed culture medium and cultured in an anaerobic workstation at 37℃ for 24 h, then transferred to the fermentation medium for culture. The inoculation amount was 3% (v / v), the culture temperature was 37℃, the culture period was 24 h, the fermentation liquid volume was 4 / 7.5 L, the rotation speed was 100 rpm, and N2 was continuously introduced during the culture process to ensure that the fermentation was carried out in an anaerobic environment. When the pH of the fermentation environment was lower than 6.5, an appropriate amount of anion exchange resin (D311, D301G, 201×7) was added to the culture medium to adjust the pH of the fermentation liquid to 7.0. When the glucose content in the fermentation liquid was lower than 5 g / L, a certain amount of feed medium (40g glucose solution, added twice) was added to the fermentation liquid to make the glucose concentration reach 10 g / L to avoid nutrient deficiency and inhibit bacterial growth. In the control group, 10 mol / L sterile NaOH solution was added dropwise using the automatic control module of the fermentation system to adjust the pH of the fermentation liquid to 7.0. Anion exchange resin can separate the acid ions produced during the fermentation process, reduce the osmotic pressure of the fermentation liquid, and eliminate the inhibitory effect on bacterial growth. Clostridium butyricum The highest viable count of BNCC 337239 reached 6.09×10 8 cfu / mL, compared with 3.18×10 8 cfu / mL, an increase of 91.5%. The detection method of organic acid and acid radical ion involved in the present invention is as follows: (1) Sample treatment: After the fermentation broth was centrifuged, the supernatant was mixed with methanol in a ratio of 1:1, the pH was adjusted to neutral with NaOH, and then filtered through a 0.22 μm micromembrane.

[0043] (2) Detection was performed using high performance liquid chromatography (HPLC).

[94] The method described detects the content of organic acid and acid radical ion in the fermentation broth.

[0044] (3) Standard sample preparation: Weigh 1 g of glacial acetic acid and n-butyric acid respectively and dissolve them in 100 mL of methanol to prepare 10 g / L glacial acetic acid and n-butyric acid standards. Dilute them to prepare 8 g / L, 4 g / L, 2 g / L and 1 g / L standards, and filter them through a 0.22 μm micromembrane.

Claims

1. A method for increasing the number of viable Clostridium butyricum by fermentation-separation coupling, characterized in that: Clostridium butyricum was cultured by constant pH fed-batch fermentation. When the glucose content in the fermentation broth was lower than 5 g / L, a certain amount of fresh culture medium was added to make the glucose concentration reach 10-15 g / L. When the pH of the fermentation environment was lower than 6.5, an appropriate amount of anion exchange resin was added to the culture medium to adjust the pH of the fermentation broth to 7.

0.

2. The method for increasing the number of viable Clostridium butyricum by fermentation-separation coupling according to claim 1, characterized in that: The anion exchange resin used is at least one of D315, D314, D301, D301G, D311 and 201×7.

3. The method for increasing the number of viable Clostridium butyricum by fermentation-separation coupling according to claim 1, characterized in that: The components of the culture medium used are as follows: glucose 10~30 g / L, tryptone 10~20 g / L, ammonium sulfate 5~10 g / L, CaCl2 0~2 g / L, NaHCO3 0~5 g / L, MgSO4 0~3 g / L, MnSO4 0~1 g / L, FeSO4 0~5 g / L.

4. The method for increasing the number of viable Clostridium butyricum by fermentation-separation coupling according to claim 1, characterized in that: The butyric Clostridium strains include Clostridium butyricum ATCC19398, Clostridium butyricum CGMCC No.1.5205, Clostridium butyricum At least one of BNCC 337239.

5. The method for increasing the number of viable Clostridium butyricum by fermentation-separation coupling according to claim 1, characterized in that: After activating Clostridium butyricum, it is added into the fermentation medium at a ratio of 1-5% (v / v). The culture temperature is 30-40°C, the rotation speed is 50-200 rpm, and N2 is continuously introduced during the culture process.

6. The method for increasing the number of viable Clostridium butyricum by fermentation-separation coupling according to claim 1, characterized in that: The fermentation medium is: 20.0 g / L glucose, 15.9 g / L tryptone, 7.5 g / L ammonium sulfate, 1.0 g / L CaCl2, 2.0 g / L NaHCO3, 1.2 g / L MgSO4, 0.45 g / L MnSO4, and 2.97 g / L FeSO4.

7. The method for increasing the number of viable Clostridium butyricum by fermentation-separation coupling technology according to claim 2, characterized in that: The anion exchange resin is D311 or D301G weakly basic anion exchange resin.

8. The method for increasing the number of viable Clostridium butyricum by fermentation-separation coupling technology according to claim 4, characterized in that: The butyric clostridium is butyric clostridium ( Clostridium butyricum When CGMCC No.1.5205 is used, D301G resin is added to increase the number of viable bacteria.

9. The method for increasing the number of viable Clostridium butyricum by fermentation-separation coupling technology according to claim 4, characterized in that: The butyric clostridium is butyric clostridium Clostridium butyricum ATCC19398 or Clostridium butyricum ( Clostridium butyricum BNCC 337239), add an appropriate amount of D311 resin to the culture medium to increase the number of viable bacteria.

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