Clostridium butyricum culture medium and application thereof

Abstract

The invention belongs to the field of microbial culture media, and particularly relates to a clostridium butyricum culture medium and application thereof. The culture medium comprises glucose, yeast extract powder, fish peptone and sodium citrate, wherein the glucose concentration is greater than or equal to 13g / L. According to the culture medium, the use of beef powder is avoided, the cost is greatly reduced compared with the existing beef powder-containing enhanced clostridium culture medium (RCM), the culture medium has excellent capability of promoting the proliferation of clostridium butyricum, and the OD600 value and the viable count of a thallus are respectively increased by about 1.5 times and 1.3 times.

Description

Technical Field

[0001] The invention belongs to the field of microbial culture media, and in particular relates to a Clostridium butyricum culture medium and application thereof. Background Art

[0002] Clostridium butyricum belongs to the genus Clostridium of the family Bacillaceae. It is also called Clostridium butyricum. It is a strictly anaerobic Gram-positive bacillus and has a certain protective effect on the human body. [1] Short-chain fatty acids such as butyrate, acetate and propionate are its main metabolites, which metabolize energy through β-oxidation and are the main energy source for repairing and regenerating intestinal epithelial cells in farmed animals. It works with other probiotics to improve the balance of intestinal microecology and regulate the body's lipid metabolism and immune function.

[0003] The metabolites of Clostridium butyricum include organic acids, amino acids and nucleosides, among which some active metabolites have the potential to be used as postbiotics. Probiotics are non-pathogenic living microorganisms, while postbiotics are preparations of inanimate microorganisms and their components that promote host health. They mainly include components of the microorganisms themselves, such as cell walls and cell wall peptides, as well as products produced by microbial metabolism. Postbiotics do not rely on the activity of probiotics and have better stability and safety.

[0004] Therefore, it is particularly important to develop a culture medium suitable for the cultivation of Clostridium butyricum. Summary of the invention

[0005] The present invention utilizes design-expert software to explore the response surface optimization conditions, and then obtains the optimal culture medium of Clostridium butyricum through a regression equation.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On one hand, the present invention provides a Clostridium butyricum culture medium, which comprises glucose, yeast extract powder, fish peptone and sodium citrate; wherein the glucose concentration is ≥13g / L.

[0008] Preferably, the above-mentioned glucose concentration may be 13.87 g / L.

[0009] Preferably, the above Clostridium butyricum culture medium may satisfy one or more combinations of the following conditions:

[0010] (a) sodium citrate concentration is 4g / L-10g / L;

[0011] (b) the concentration of yeast extract powder is 5 g / L-20 g / L;

[0012] (c) The concentration of fish peptone is ≥10 g / L.

[0013] Preferably, the above Clostridium butyricum culture medium may satisfy one or more combinations of the following conditions:

[0014] (a) sodium citrate concentration is 10 g / L;

[0015] (b) The concentration of yeast extract powder is 8.76 g / L;

[0016] (c) The concentration of fish peptone is 10 g / L.

[0017] Preferably, the above Clostridium butyricum culture medium further comprises ammonium ions, cysteine ​​hydrochloride and manganese ions.

[0018] Preferably, the above Clostridium butyricum culture medium may also satisfy one or more combinations of the following conditions:

[0019] (a) The concentration of ammonium ions is 2g / L-6g / L;

[0020] (b) the concentration of cysteine ​​hydrochloride is 1 g / L to 2 g / L;

[0021] (e) The concentration of manganese ions is 0.25 g / L-1 g / L.

[0022] Preferably, the above Clostridium butyricum culture medium may also satisfy one or more combinations of the following conditions:

[0023] (c) The concentration of ammonium ions is 2 g / L;

[0024] (d) the concentration of cysteine ​​hydrochloride is 1 g / L;

[0025] (e) The concentration of manganese ions is 0.25 g / L.

[0026] Preferably, the pH of the Clostridium butyricum culture medium is 6-7.

[0027] Another aspect of the present invention provides a use of the Clostridium butyricum culture medium of the present invention in increasing the biomass of Clostridium butyricum.

[0028] In another aspect, the present invention provides a use of the Clostridium butyricum culture medium of the present invention in preparing Clostridium butyricum postbiotics.

[0029] The beneficial effects of the present invention include at least:

[0030] (1) The Clostridium butyricum culture medium provided by the present invention avoids the use of beef powder, and significantly reduces the cost compared with the existing reinforced Clostridium culture medium (RCM) containing beef powder.

[0031] (2) The Clostridium butyricum culture medium provided by the present invention has an excellent ability to promote the proliferation of Clostridium butyricum. Compared with the RCM culture medium, the OD 600 The value and number of viable bacteria increased by about 1.5 times and 1.3 times, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the effect of glucose concentration on the growth of Clostridium butyricum; NC is the abbreviation of Normal control, representing the blank control group; the values ​​in the figure are all mean ± SD (n = 3), a, b, c, d indicate that there are significant differences between the two groups of data with different letter superscripts (p < 0.05), and when the letters are capitalized, it means that there are extremely significant differences between the two groups (p < 0.01);

[0033] Figure 2 The effect of yeast extract powder concentration on the growth of Clostridium butyricum;

[0034] Figure 3 The effect of fish peptone concentration on the growth of Clostridium butyricum;

[0035] Figure 4 is the effect of ammonium ion concentration on the growth of Clostridium butyricum;

[0036] Figure 5 is the effect of cysteine ​​hydrochloride concentration on the growth of Clostridium butyricum;

[0037] Figure 6 is the effect of manganese ion concentration on the growth of Clostridium butyricum;

[0038] Figure 7 is the effect of sodium citrate concentration on the growth of Clostridium butyricum;

[0039] Figure 8 The effect of different pH values ​​on the growth of Clostridium butyricum;

[0040] Fig. 9 The effect of glucose concentration and yeast extract powder concentration on the growth of Clostridium butyricum;

[0041] Fig.10 The effect of glucose concentration and fish peptone concentration on the growth of Clostridium butyricum;

[0042] Fig.11 is the effect of glucose concentration and sodium citrate concentration on the growth of Clostridium butyricum;

[0043] Fig.12 The effect of yeast extract powder concentration and fish peptone concentration on the growth of Clostridium butyricum;

[0044] Fig.13 The effect of yeast extract powder concentration and sodium citrate concentration on the growth of Clostridium butyricum;

[0045] Fig.14 The effect of fish peptone concentration and sodium citrate concentration on the growth of Clostridium butyricum;

[0046] Fig.15 To compare the effects of CBPM and RCM culture media on the biomass (A) and viable count (B) of Clostridium butyricum; *p<0.05, ***p<0.001. DETAILED DESCRIPTION

[0047] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0048] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0049] The embodiment of the present invention provides a Clostridium butyricum culture medium, which includes glucose, yeast extract powder, fish peptone and sodium citrate; wherein the glucose concentration is ≥13g / L.

[0050] It should be noted that the present invention uses design-expert software to explore the response surface optimization conditions, and then obtains the optimal fermentation conditions of Clostridium butyricum through regression equation. 600 The value is the optimized parameter, and the fermented Clostridium butyricum is suitable for preparing a postbiotic preparation. In addition, the culture medium of the present invention can be named Clostridium butyricum proliferation medium (CBPM).

[0051] It should also be noted that the culture medium of the present invention avoids the use of beef powder, greatly reduces the cost compared with the existing RCM culture medium containing beef powder, and has excellent ability to promote the proliferation of Clostridium butyricum. Compared with RCM culture medium, the OD 600 The value and number of viable bacteria increased by about 1.5 times and 1.3 times, respectively.

[0052] In some specific examples, the glucose concentration may be 13.87 g / L.

[0053] In some specific examples, the Clostridium butyricum culture medium may satisfy one or more combinations of the following conditions:

[0054] (a) the concentration of sodium citrate is 4 g / L-10 g / L, such as 5 g / L, 6 g / L, 7 g / L or 9 g / L;

[0055] (b) the concentration of yeast extract powder is 5 g / L-20 g / L, such as 7 g / L, 10 g / L, 13 g / L, 17 g / L or 19 g / L;

[0056] (c) The concentration of fish peptone is ≥10 g / L, such as 10 g / L, 20 g / L, 30 g / L, 40 g / L, 35 g / L or 40 g / L.

[0057] In some specific examples, the Clostridium butyricum culture medium may satisfy one or more combinations of the following conditions:

[0058] (a) sodium citrate concentration is 10 g / L;

[0059] (b) The concentration of yeast extract powder is 8.76 g / L;

[0060] (c) The concentration of fish peptone is 10 g / L.

[0061] It should be noted that the Clostridium butyricum culture medium in the present invention preferably contains 13.87 g / L glucose, 8.76 g / L yeast extract powder, 10 g / L fish peptone, and 10 g / L sodium citrate. Under this condition, the turbidity of the bacterial solution is 3.57 (OD 600 nm value).

[0062] In some specific examples, the Clostridium butyricum culture medium further includes ammonium ions, cysteine ​​hydrochloride and manganese ions.

[0063] In some specific examples, the above Clostridium butyricum culture medium may also satisfy one or more combinations of the following conditions:

[0064] (a) The concentration of ammonium ions is 2 g / L-6 g / L, such as 3 g / L, 4 g / L or 5 g / L;

[0065] (b) the concentration of cysteine ​​hydrochloride is 1 g / L-2 g / L, such as 1.2 g / L, 1.5 g / L or 1.7 g / L;

[0066] (e) The concentration of manganese ions is 0.25 g / L-1 g / L, such as 0.5 g / L, 0.7 g / L or 0.9 g / L.

[0067] In some specific examples, the above Clostridium butyricum culture medium may also satisfy one or more combinations of the following conditions:

[0068] (c) The concentration of ammonium ions is 2 g / L;

[0069] (d) the concentration of cysteine ​​hydrochloride is 1 g / L;

[0070] (e) The concentration of manganese ions is 0.25 g / L.

[0071] In some specific examples, the pH of the Clostridium butyricum culture medium is 6-7.

[0072] It should be noted that the change of the initial pH value of the culture medium in the range of 6.0-7.0 has little effect on the bacterial concentration, but when the pH value exceeds 7.0, the bacterial concentration decreases significantly.

[0073] The embodiment of the present invention also provides a use of the Clostridium butyricum culture medium of the present invention in increasing the biomass of Clostridium butyricum.

[0074] The embodiment of the present invention also provides a use of the Clostridium butyricum culture medium of the present invention in preparing Clostridium butyricum postbiotics.

[0075] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0076] In the following example, Clostridium butyricum HADIG-CB004CB004 was deposited by Hunan Xiangnong Animal Pharmaceutical Co., Ltd. in the General Microbiology Center of China National Microorganism Culture Collection on July 10, 2023, with a deposit number of CGMCC No. 27853. The deposit information is as follows: The Clostridium butyricum provided by the present invention was deposited in the General Microbiology Center of China National Microorganism Culture Collection on July 10, 2023. The address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 27853, and the taxonomic name is Clostridium butyricum.

[0077] In the following examples, the culture medium information used is as follows:

[0078] RCM medium: peptone 10g / L, beef powder 10g / L, yeast powder 3g / L, glucose 5g / L, soluble starch 1g / L, sodium chloride 5g / L, sodium acetate 3g / L, L-cysteine ​​hydrochloride 0.5g / L, pH 6.8±0.1; autoclave at 121℃ for 15 minutes and set aside;

[0079] Clostridium butyricum proliferation medium (CBPM): glucose 13.87 g / L, yeast extract powder 8.76 g / L, fish peptone 10 g / L, sodium citrate 10 g / L, ammonium sulfate 1 g / L, manganese sulfate monohydrate 0.25 g / L, cysteine ​​hydrochloride 1 g / L, pH 6.8. Autoclave at 121°C for 15 minutes and set aside.

[0080] Clostridium butyricum counting medium (double-layer TSC solid medium): The composition of the tryptone-sulfite-cycloserine agar base (TSC) medium is 15g / L tryptone, 5g / L soytone, 5g / L yeast powder, 1g / L sodium pyrosulfite, 1g / L ammonium ferric citrate, 15g / L agar, pH 7.6±0.2; the lower layer of the TSC double-layer medium is TSC medium + 5g / L agar; the upper layer is 20g / L sodium alginate or gelatin + 30g / L TSC medium (the upper layer needs to be stirred and dissolved on a magnetic stirrer before sterilization). Autoclave at 121℃ for 15 minutes. When cooled to 50±1℃, add 80ml of 0.5% D-cycloserine solution that has been sterilized by filtration to each 1L of the medium, mix well, and set aside.

[0081] In the following examples, TSC culture medium and D-cycloserine were purchased from Qingdao Haibo Biotechnology Co., Ltd. Sodium citrate (analytical grade) was purchased from Tianjin Dingshengxin Chemical Co., Ltd. Other components in the above culture medium were purchased from Shanghai Bioengineering Co., Ltd. Stab S2T small-capacity stackable full-temperature oscillating incubator and Herotherm 180 constant temperature incubator were purchased from Shanghai Rundu Biotechnology Co., Ltd. 2.5L sealed anaerobic culture tank and anaerobic gas production bag were purchased from Mitsubishi Corporation of Japan; LX-B100L vertical automatic electric heating pressure steam sterilizer was purchased from Hefei Huatai Medical Equipment Co., Ltd.; UV-1601 ultraviolet spectrophotometer was purchased from Beijing Beifen Rayleigh Analytical Instrument (Group) Co., Ltd.

[0082] 1. Activation of strains

[0083] The Clostridium butyricum HADIG-CB004 glycerol bacteria were streaked on the RCM solid plate and incubated upside down in a constant temperature anaerobic culture tank at 37°C. After culturing overnight, a single colony was picked and inoculated into the RCM liquid culture medium as seed solution for later use.

[0084] 2. Single factor experiment to optimize culture medium formula

[0085] (I) Effects of gradient glucose concentration on the growth of Clostridium butyricum

[0086] According to the above preparation method, a Clostridium butyricum proliferation medium was prepared, wherein glucose was used as a carbon source for a single factor experiment (medium formulation optimization), and its concentration gradient was set to 5 g / L, 10 g / L, 15 g / L, 20 g / L, and 25 g / L. In addition, the yeast extract concentration in the medium was 5 g / L, the fish peptone concentration was 10 g / L, and the pH was 6.8.

[0087] The prepared Clostridium butyricum proliferation medium was used to culture Clostridium butyricum HADIG-CB004. After static culture at 37°C for 16 hours, 1 ml of the fermentation liquid was taken and centrifuged at 8000 rpm for 5 minutes. The bacteria were resuspended with an equal volume of physiological saline, diluted 5-10 times, and the bacterial concentration (OD 600 value); the result is Figure 1 As shown in the figure, within the range of 5g / L-15g / L glucose concentration, as the glucose concentration increases, the bacterial concentration also increases; when the glucose concentration exceeds 15g / L, the bacterial concentration does not change much, and even shows a slight downward trend. This may be because Clostridium butyricum requires a certain concentration of glucose to grow, but when the glucose concentration is too high, the osmotic pressure of the culture medium is too high, causing the bacteria to dehydrate, thereby inhibiting the growth of the strain.

[0088] (II) Effects of gradient yeast extract concentration on the growth of Clostridium butyricum

[0089] According to the above preparation method, a Clostridium butyricum proliferation medium was prepared, wherein yeast extract powder was used as an organic nitrogen source, and vitamins and amino acids were provided for the growth of Clostridium butyricum, and the concentration gradient was set to 2.5 g / L, 5 g / L, 10 g / L, 15 g / L, and 20 g / L, and a single factor experiment (medium formulation optimization) was performed. In addition, the glucose concentration in the medium was 15 g / L, the fish peptone concentration was 10 g / L, and the pH was 6.8.

[0090] The prepared Clostridium butyricum proliferation medium was used to culture Clostridium butyricum HADIG-CB004. After static culture at 37°C for 16 hours, 1 ml of the fermentation liquid was taken and centrifuged at 8000 rpm for 5 minutes. The bacteria were resuspended with an equal volume of physiological saline, diluted 5-10 times, and the bacterial concentration (OD 600 value); the result is Figure 2 As shown in the figure, when the yeast extract powder concentration is 2.5 to 15g / L, the bacterial concentration increases with the increase of yeast extract powder concentration; when it exceeds 15g / L, the bacterial growth is inhibited; this shows that yeast extract powder, as a nutrient, has a promoting effect on bacteria, and its concentration exceeding a certain level will affect bacterial growth.

[0091] 3. Effect of gradient concentration of fish peptone on the growth of Clostridium butyricum

[0092] According to the above preparation method, a Clostridium butyricum proliferation medium was prepared, wherein fish peptone was used as the second organic nitrogen source and provided growth factors for the growth of Clostridium butyricum, and its concentration gradient was set to 10 g / L, 20 g / L, 30 g / L, 35 g / L, and 40 g / L, and a single factor experiment (medium formula optimization) was performed. In addition, the glucose concentration in the medium was 15 g / L, the yeast extract concentration was 5 g / L, and the pH was 6.8.

[0093] The prepared Clostridium butyricum proliferation medium was used to culture Clostridium butyricum HADIG-CB004. After static culture at 37°C for 16 hours, 1 ml of the fermentation liquid was taken and centrifuged at 8000 rpm for 5 minutes. The bacteria were resuspended with an equal volume of physiological saline, diluted 5-10 times, and the bacterial concentration (OD 600 value); the result is Figure 3 As shown in the figure, the results show that when the fish peptone concentration increases within the range of 10 to 30 g / L, the bacterial concentration also increases; when the fish peptone concentration exceeds 30 g / L, its effect on the bacterial concentration is not significant. As a nutrient, fish peptone mainly provides nitrogen source for microorganisms. If its concentration is too high, the osmotic pressure of the culture medium will increase, which will also inhibit the growth of microorganisms.

[0094] 4. Effect of gradient concentration of ammonium ions on the growth of Clostridium butyricum

[0095] According to the above preparation method, a Clostridium butyricum proliferation medium was prepared, wherein ammonium sulfate was used as an inorganic nitrogen source, and its concentration gradient was set to 1 g / L, 2 g / L, 4 g / L, 6 g / L, and 8 g / L, and a single factor experiment (medium formulation optimization) was performed. In addition, the glucose concentration in the medium was 15 g / L, the yeast extract concentration was 5 g / L, the fish peptone concentration was 20 g / L, and the pH was 6.8.

[0096] The prepared Clostridium butyricum proliferation medium was used to culture Clostridium butyricum HADIG-CB004. After static culture at 37°C for 16 hours, 1 ml of the fermentation liquid was taken and centrifuged at 8000 rpm for 5 minutes. The bacteria were resuspended with an equal volume of physiological saline, diluted 5-10 times, and the bacterial concentration (OD 600 value); the result is Figure 4 As shown, the results show that, relative to organic nitrogen sources, with the increase of ammonium ion concentration, the change in bacterial concentration is relatively small; ammonium ions mainly provide inorganic nitrogen sources for the culture medium, which may also have a regulating effect on the pH value and osmotic pressure of the culture medium. The optimal concentration is 2g / L, but excessive addition will cause certain toxicity to microbial growth.

[0097] (V) Effect of gradient concentration of cysteine ​​hydrochloride on the growth of Clostridium butyricum

[0098] According to the above preparation method, a Clostridium butyricum proliferation medium was prepared, wherein cysteine ​​hydrochloride was used as a reducing agent, and its concentration gradient was set to 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, and 4 g / L, and a single factor experiment (culture condition optimization) was performed. In addition, the glucose concentration in the medium was 15 g / L, the yeast extract concentration was 5 g / L, the fish peptone concentration was 20 g / L, the ammonium sulfate concentration was 2 g / L, and the pH was 6.8.

[0099] The prepared Clostridium butyricum proliferation medium was used to culture Clostridium butyricum HADIG-CB004. After static culture at 37°C for 16 hours, 1 ml of the fermentation liquid was taken and centrifuged at 8000 rpm for 5 minutes. The bacteria were resuspended with an equal volume of physiological saline, diluted 5-10 times, and the bacterial concentration (OD 600 value); the result is Figure 5 As shown in the figure, cysteine ​​hydrochloride with a gradient concentration of 0.25g / L-1g / L has a very significant effect on bacterial growth, and the optimal cysteine ​​hydrochloride concentration for preparing Clostridium butyricum culture medium is 1g / L; this is because in order to cultivate anaerobic bacteria, an anaerobic environment must be created because an oxygen environment will inhibit its growth. L-cysteine ​​hydrochloride added to the culture medium can act as a reducing agent, has an antioxidant effect, can remove free oxygen, reduce the redox potential, and help the reproduction of anaerobic bacteria.

[0100] (VI) Effect of gradient concentration of manganese sulfate on the growth of Clostridium butyricum

[0101] According to the above preparation method, a Clostridium butyricum proliferation medium was prepared, wherein manganese sulfate monohydrate was used as a trace element to promote the growth of Clostridium butyricum, and its concentration gradient was set to 0.25 g / L, 0.5 g / L, 1 g / L, and 2 g / L, and a single factor experiment (medium formula optimization) was performed. In addition, the glucose concentration in the medium was 15 g / L, the yeast extract concentration was 5 g / L, the fish peptone concentration was 20 g / L, the ammonium sulfate concentration was 2 g / L, the cysteine ​​hydrochloride concentration was 1 g / L, and the pH was 6.8.

[0102] The prepared Clostridium butyricum proliferation medium was used to culture Clostridium butyricum HADIG-CB004. After static culture at 37°C for 16 hours, 1 ml of the fermentation liquid was taken and centrifuged at 8000 rpm for 5 minutes. The bacteria were resuspended with an equal volume of physiological saline, diluted 5-10 times, and the bacterial concentration (OD 600 value); the result is Figure 6As shown, 0.25 g / L manganese sulfate (manganese sulfate is an inorganic trace element, among which manganese is one of the nutrients necessary for microbial growth) was the most appropriate to be added to the culture medium. Compared with the NC group without addition, it had a significant promoting effect on the growth of Clostridium butyricum (p<0.05).

[0103] 7. Effect of gradient buffer concentration on the growth of Clostridium butyricum

[0104] According to the above preparation method, a Clostridium butyricum proliferation medium was prepared, wherein sodium citrate was used as a promoter for the growth of Clostridium butyricum, and its gradient concentration was set to 2 g / L, 4 g / L, 8 g / L, 10 g / L, and 12 g / L, and a single factor experiment (medium formula optimization) was performed. In addition, the concentration of glucose in the medium was 15 g / L, the concentration of yeast extract powder was 5 g / L, the concentration of fish peptone was 20 g / L, the concentration of ammonium sulfate was 2 g / L, the concentration of cysteine ​​hydrochloride was 1 g / L, the concentration of manganese sulfate monohydrate was 0.25 g / L, and the pH was 6.8;

[0105] The prepared Clostridium butyricum proliferation medium was used to culture Clostridium butyricum HADIG-CB004. After static culture at 37°C for 16 hours, 1 ml of the fermentation liquid was taken and centrifuged at 8000 rpm for 5 minutes. The bacteria were resuspended with an equal volume of physiological saline, diluted 5-10 times, and the bacterial concentration (OD 600 value); the result is Figure 7 As shown in the figure, when sodium citrate increases in the range of 2g / L-10g / L, the bacterial concentration also increases. When too much sodium citrate is added, the concentration of Clostridium butyricum instead shows a downward trend. Sodium citrate is currently the most important citrate, with good pH regulating effect and buffering properties. In addition, whether Clostridium butyricum can use sodium citrate to enter the citric acid cycle (tricarboxylic acid cycle) to provide energy for bacterial growth is also worthy of further study.

[0106] 8. Effect of initial pH value of culture medium on the growth of Clostridium butyricum

[0107] A Clostridium butyricum proliferation medium is prepared according to the above preparation method, wherein the components of the Clostridium butyricum proliferation medium include: a glucose concentration of 15 g / L, a yeast extract concentration of 5 g / L, a fish peptone concentration of 20 g / L, an ammonium sulfate concentration of 2 g / L, a cysteine ​​hydrochloride concentration of 1 g / L, a manganese sulfate monohydrate concentration of 0.25 g / L, a sodium citrate concentration of 10 g / L, and the pH values ​​are set to 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5, respectively;

[0108] The prepared Clostridium butyricum proliferation medium was used to culture Clostridium butyricum HADIG-CB004. After static culture at 37°C for 16 hours, 1 ml of the fermentation liquid was taken and centrifuged at 8000 rpm for 5 minutes. The bacteria were resuspended with an equal volume of physiological saline, diluted 5-10 times, and the bacterial concentration (OD 600 value); the result is Figure 8 As shown, the change of the initial pH value of the culture medium in the range of 6.0-7.0 has little effect on the bacterial concentration, but when the pH value exceeds 7.0, the bacterial concentration drops significantly; generally speaking, too high or too low pH values ​​are not conducive to the growth of Clostridium butyricum; when the pH is close to the neutral value of 7.0, Clostridium butyricum can achieve the best growth state.

[0109] 3. Optimization of culture medium by response surface analysis

[0110] Based on the results of single factor experiments, glucose (carbon source), yeast extract powder (nitrogen source and vitamins), fish peptone (nitrogen source and growth factor) and sodium citrate (buffer salt and growth promoter) were selected as the factors that significantly affected the growth of Clostridium butyricum HADIG-CB004. The response surface analysis software Design Expert (V8.0.6.1) was used to analyze the effect of fermentation culture broth cell concentration (OD 600 value) as the response value, and optimize the conditions to obtain the optimal fermentation conditions and verify them.

[0111] Glucose concentration, yeast extract powder concentration, fish peptone concentration and sodium citrate concentration were selected as four factors and three levels in the response surface experiment. The factors and levels of the response surface experiment are shown in Tables 1 and 2.

[0112] Table 1 Response surface design factors and levels

[0113]

[0114]

[0115] Table 2 Response surface test results

[0116] Group No. Glucose concentration Yeast extract powder concentration Fish peptone concentration Sodium citrate concentration <![CDATA[OD 600 ]]> 1 0 0 -1 -1 2.34 2 -1 0 -1 0 1.66 3 0 1 0 1 2.88 4 1 1 0 0 2.69 5 -1 0 1 0 1.48 6 0 0 0 0 3.07 7 1 0 0 1 3.63 8 0 0 0 0 3.07 9 0 -1 0 -1 2.22 10 0 0 0 0 3.07 11 0 0 1 -1 2.50 12 -1 0 0 1 1.62 13 0 -1 0 1 2.63 14 1 0 0 -1 2.26 15 0 -1 1 0 2.89 16 1 0 -1 0 2.87 17 -1 1 0 0 1.54 18 0 0 -1 1 3.13 19 -1 -1 0 0 1.38 20 0 1 -1 0 2.56 21 1 -1 0 0 2.92 22 -1 0 0 -1 1.62 23 0 1 1 0 2.83 24 0 0 1 1 2.33 25 0 1 0 -1 2.00 26 1 0 1 0 3.02 27 0 -1 -1 0 3.08

[0117] Design Expert software was used to analyze the experimental data in Table 2 and establish a multivariate regression model. The cell concentration (OD 600 The quadratic multinomial regression equation between the concentration of glucose (g / L), yeast extract powder (g / L), fish peptone (g / L) and sodium citrate (g / L) is as follows:

[0118] Bacterial concentration (OD 600Value)=3.07+0.67*A-0.052*B-0.049*C+0.27*D–0.097*A*B+0.082*A*C+0.34*A*D+0.11*B*C+0.12*B*D–0.24*C*D–0.62*A 2 -0.25*B 2 -0.12*C 2 –0.31*D 2 ;

[0119] As shown in Table 3, the F of the model is 17.89, p<0.0001, indicating that the difference of the model is significant. As shown in Table 3, the first-order term A (glucose concentration) of the independent variable is significant (p<0.01), and D (sodium citrate concentration) is significant (p<0.01). The model established by Clostridium butyricum has significant variation among different treatment groups (p<0.05), indicating that the linear relationship between its dependent variable and all independent variables is significant, that is, the experimental method is reliable and the equation is reasonable. 2 , B 2 With D 2 The quadratic terms of glucose concentration and sodium citrate concentration were significantly different (p<0.05); the quadratic terms of fish peptone concentration and sodium citrate concentration were significantly different (p<0.01), and the quadratic terms of fish peptone concentration and sodium citrate concentration were significantly different (p<0.05). The F value also shows that the order of influence of each factor on the change of bacterial liquid turbidity in the experiment is: glucose concentration> sodium citrate concentration> yeast extract powder concentration> fish peptone concentration.

[0120] Table 3 Analysis of variance of response surface quadratic model

[0121] source sum of squares Degrees of Freedom Mean square sum F-number P-value Significance Model 9.49 14 0.68 17.89 <0.0001 *** A-Glucose concentration 5.45 1 5.45 143.99 <0.0001 *** B- Yeast extract powder concentration 0.032 1 0.032 0.85 0.3759 C-fish peptone concentration 0.029 1 0.029 0.77 0.3987 D-Sodium Citrate Concentration 0.90 1 0.90 23.67 0.0004 *** AB 0.038 1 0.038 1.00 0.3362 AC 0.027 1 0.027 0.72 0.4131 AD 0.47 1 0.47 12.39 0.0042 ** BC 0.053 1 0.053 1.40 0.2602 BD 0.055 1 0.055 1.46 0.2505 CD 0.23 1 0.23 6.08 0.0297 * <![CDATA[A 2 ]]> 2.04 1 2.04 53.91 <0.0001 *** <![CDATA[B 2 ]]> 0.34 1 0.34 8.98 0.0111 * <![CDATA[C 2 ]]> 0.075 1 0.075 1.99 0.1842 <![CDATA[D 2 ]]> 0.51 1 0.51 13.53 0.0032 ** Residual 0.45 12 0.038 Lack of Fit 0.45 10 0.045 Pure Error 0.000 2 0.000 Total error 9.94 26

[0122] Note: ** means p<0.01, extremely significant; * means p<0.05, significant.

[0123] The response surface graph shows the interaction between two response factors and their impact on the response value. The steeper the graph, the more obvious the interaction, and the smoother the graph, the weaker the interaction. Figures 9 to 14 It can be seen that the interaction between glucose concentration and sodium citrate concentration is the strongest, and the interaction between yeast extract powder concentration and fish peptone concentration is the weakest. The above results are consistent with the prediction model. The prediction model shows that the optimal culture conditions are: glucose 13.87g / L, yeast extract powder 8.76g / L, fish peptone 10g / L, sodium citrate 10g / L. Under this condition, the turbidity of the bacterial solution is 3.57.

[0124] 4. TSC double-layer agar plate counting Clostridium butyricum vegetative cells

[0125] The seed liquid of Clostridium butyricum HADIG-CB004 was inoculated into fresh sterile RCM and CBPM medium at an inoculation volume of 5%. After 18 hours of static culture in a 37°C constant temperature incubator, 1 ml of the fermentation liquid was taken and diluted to 10% with sterile saline. -6 and 10 -7 There are 2 gradient concentrations in total. Take 1 ml of fermentation dilution liquid and add it to the bottom of the dish. Pour the TSC lower layer medium by pouring. After the lower layer medium solidifies, pour the upper layer TSC medium containing sodium alginate (20g / L) to the lid of the dish and cover the dish. After the medium solidifies, place it in a 37℃ constant temperature incubator overnight and count the number of black colonies.

[0126] The results are as follows Fig.15 The results show that the predicted values ​​of CBPM medium are basically consistent with the actual values ​​after verification test. In three repeated experiments, the average OD 600 The value is 3.98, and the number of viable bacteria is 5×10 8 CFU / ml. In contrast, the average OD of Clostridium butyricum HADIG-CB004 cultured in RCM medium 600 The value was 2.37, and the number of viable bacteria was 3.9×10 8 CFU / ml. It can be seen that the use of CBPM medium can increase the OD of Clostridium butyricum HADIG-CB004 600 The value increased by about 1.5 times, and the number of viable bacteria increased by about 1.3 times. In addition, compared with RCM medium, CBPM medium is cheaper because it only contains 7 medium components and does not contain expensive beef powder.

[0127] In summary, CBPM medium showed superior performance in the cultivation of Clostridium butyricum and can be used as an alternative to RCM medium for the proliferation of Clostridium butyricum vegetative cells and the preparation of postbiotics.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A Clostridium butyricum culture medium, characterized in that Comprising glucose, yeast extract powder, fish peptone and sodium citrate; wherein the glucose concentration is ≥13g / L.

2. The Clostridium butyricum culture medium according to claim 1, characterized in that The glucose concentration was 13.87 g / L.

3. The Clostridium butyricum culture medium according to claim 1 or 2, characterized in that The Clostridium butyricum culture medium meets one or more combinations of the following conditions: (a) sodium citrate concentration is 4g / L-10g / L; (b) the concentration of yeast extract powder is 5 g / L-20 g / L; (c) The concentration of fish peptone is ≥10 g / L.

4. The Clostridium butyricum culture medium according to claim 3, characterized in that The Clostridium butyricum culture medium meets one or more combinations of the following conditions: (a) sodium citrate concentration is 10 g / L; (b) The concentration of yeast extract powder is 8.76 g / L; (c) The concentration of fish peptone is 10 g / L.

5. The Clostridium butyricum culture medium according to claim 1, 2 or 4, characterized in that The Clostridium butyricum culture medium also includes ammonium ions, cysteine ​​hydrochloride and manganese ions.

6. The Clostridium butyricum culture medium according to claim 5, characterized in that The Clostridium butyricum culture medium meets one or more combinations of the following conditions: (a) The concentration of ammonium ions is 2g / L-6g / L; (b) the concentration of cysteine ​​hydrochloride is 1 g / L to 2 g / L; (e) The concentration of manganese ions is 0.25 g / L-1 g / L.

7. The Clostridium butyricum culture medium according to claim 5, characterized in that The Clostridium butyricum culture medium meets one or more combinations of the following conditions: (c) The concentration of ammonium ions is 2 g / L; (d) the concentration of cysteine ​​hydrochloride is 1 g / L; (e) The concentration of manganese ions is 0.25 g / L.

8. The Clostridium butyricum culture medium according to claim 1, 2, 4, 6 or 7, characterized in that Its pH is 6-7.

9. Use of the Clostridium butyricum culture medium according to claims 1 to 8 in increasing the biomass of Clostridium butyricum.

10. Use of the Clostridium butyricum culture medium according to claims 1 to 8 in the preparation of Clostridium butyricum postbiotics.

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