Animal-origin-free culture medium and method for culturing Cristinella parvum by using animal-origin-free culture medium

Abstract

According to the invention, a proper amount of yeast extract powder, soy peptone, fructose, anhydrous magnesium sulfate, L-cysteine hydrochloride monohydrate, sodium carbonate and monopotassium phosphate are used to form the non-animal-origin liquid culture medium, and in the process of culturing Cristinia parvata by using the non-animal-origin liquid culture medium, the Cristinia parvata proliferates quickly. And the kristinia parvata cultured by adopting the non-animal-origin culture medium is good in growth state, good in safety, easy to operate and control and long in culture time, and meets the industrial requirements of medicinal values. In addition, when the non-animal-origin culture medium is used for culturing the kristinia parvata, the viable count is high, the strain activity is high, the passage stability is good, and the kristinia parvata can be subjected to large-scale industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a culture medium without animal origin and a method for culturing Christensenella parvum using the culture medium. Background Art

[0002] Christensenella minua is a Gram-negative bacterium belonging to the Firmicutes, Fusobacteria, and Fusobacteriales. It is one of the most abundant symbiotic bacteria in the intestine. Christensenella minua has glycolytic properties and can produce a large amount of acetate and a small amount of butyrate. Studies have shown that Christensenella minua is associated with a variety of metabolic diseases, such as obesity, diabetes, and hyperlipidemia. It is also associated with immune diseases such as inflammatory bowel disease. In addition, it is also associated with melanoma and Parkinson's disease. Christensenella minua is one of the iconic microorganisms for measuring the balance of intestinal microecology. Due to a series of important discoveries on Christensenella minua in recent years, especially its therapeutic effect in metabolic-related diseases, it has become an important candidate strain of "second-generation probiotics (NGP)" with great application and development value-living therapeutic drugs (LBP). As a new candidate probiotic, the cultivation of Christensenella minua is particularly important.

[0003] Traditional culture media for culturing Christensenella contain animal-derived ingredients and are relatively nutritious, such as PYG, modified GAM and other culture media, in which fructose, glucose and the like are good carbon sources, and beef extract is a good nitrogen source. Although the culture results are acceptable, the traditional culture media for culturing Christensenella have many components, and the preparation process is cumbersome, which is not conducive to large-scale production. In addition, although beef extract is a good nitrogen source, it comes from animal tissue or digested matter, and the ingredients from animals may contain contaminants from viruses, prions or bacteria, or contain antiallergens, antigenic peptides or other undesirable ingredients, and may also carry unknown viruses and other dangerous ingredients; animal-derived culture media mainly use beef extract, and its composite components and their proportions are not clear or fixed, which is more likely to produce unstable performance, resulting in inconsistent results between batches, unstable and non-standard quality of the culture media, and directly affecting the accuracy of microbial inspection and research results. As a result, the test results are not comparable. Therefore, the safety and stability of culture media with animal-derived ingredients cannot be fully guaranteed. Summary of the invention

[0004] Based on the above technical problems, one of the objectives of the present invention is to provide an animal-origin-free culture medium, in the process of culturing Christensenella parvum with the animal-origin-free culture medium, the Christensenella parvum reproduces quickly.

[0005] First aspect

[0006] The present invention provides a culture medium which does not contain animal-derived components.

[0007] In some embodiments, the animal-derived component is a bovine-derived component, such as beef extract, beef meal, beef liver extract, and the like.

[0008] In some embodiments, the culture medium is used to grow Christensenella parvum.

[0009] In some embodiments, the culture medium comprises the following components:

[0010] 25g / L~50g / L yeast extract powder, 15g / L~45g / L plant peptone, 5g / L~8g / L sugars, 0.01g / L~0.08g / L anhydrous magnesium sulfate, 0.5g / L~3g / L sodium carbonate, 1g / L~3g / L dipotassium hydrogen phosphate.

[0011] In some embodiments, the culture medium comprises the following components:

[0012] 25g / L~50g / L yeast extract powder, 15g / L~45g / L plant peptone, 5g / L~8g / L sugars, 0.01g / L~0.08g / L anhydrous magnesium sulfate, 0.5g / L~3g / L sodium carbonate, 1g / L~3g / L dipotassium hydrogen phosphate, 1.5g / L~4g / L L-cysteine ​​hydrochloride monohydrate.

[0013] In some embodiments, the plant peptone is soy peptone.

[0014] In some embodiments, the sugar is selected from soluble starch, sucrose, glucose, fructose, preferably fructose.

[0015] In some embodiments, the culture medium comprises the following components:

[0016] 25g / L~50g / L yeast extract powder, 15g / L~45g / L plant peptone, 5g / L~8g / L sugars, 0.01g / L~0.08g / L anhydrous magnesium sulfate, 0.5g / L~3g / L sodium carbonate, 1g / L~3g / L dipotassium hydrogen phosphate, 1.5g / L~4g / L L-cysteine ​​hydrochloride monohydrate, water.

[0017] In some embodiments, the culture medium comprises the following components:

[0018] 25g / L~50g / L yeast extract powder, 15g / L~45g / L soy peptone, 5g / L~8g / L fructose, 0.01g / L~0.08g / L anhydrous magnesium sulfate, 0.5g / L~3g / L sodium carbonate, 1g / L~3g / L dipotassium hydrogen phosphate, 1.5g / L~4g / L L-cysteine ​​hydrochloride monohydrate, water.

[0019] In some embodiments, water is added to 1 L.

[0020] In some embodiments, the culture medium

[0021] Yeast extract powder is 35g / L to 50g / L, preferably 35g / L to 40g / L;

[0022] and / or, soy peptone is 25 g / L to 40 g / L, preferably 25 g / L to 35 g / L;

[0023] and / or, fructose is 5 g / L to 8 g / L, preferably 5 g / L to 6 g / L;

[0024] and / or, anhydrous magnesium sulfate is 0.03 g / L to 0.08 g / L, preferably 0.03 g / L to 0.05 g / L;

[0025] and / or, L-cysteine ​​hydrochloride monohydrate is 0 g / L to 4.0 g / L, preferably 2.0 g / L to 2.5 g / L;

[0026] and / or, sodium carbonate is 0.8 g / L to 3.0 g / L, preferably 0.8 g / L to 1.2 g / L;

[0027] And / or, dipotassium hydrogen phosphate is 1.5 g / L to 3.0 g / L, preferably 1.5 g / L to 2.0 g / L.

[0028] In some embodiments, the culture medium comprises the following components:

[0029] 35g / L~50g / L yeast extract powder, 25g / L~40g / L soy peptone, 5g / L~8g / L fructose, 0.03g / L~0.08g / L anhydrous magnesium sulfate, 0.8g / L~3.0g / L sodium carbonate, 1.5g / L~3.0g / L dipotassium hydrogen phosphate, 0g / L~4.0g / L L-cysteine ​​hydrochloride monohydrate, water;

[0030] Make up to 1 L with water.

[0031] In some embodiments, the culture medium comprises the following components:

[0032] 35g / L~40g / L yeast extract powder, 25g / L~35g / L soy peptone, 5g / L~6g / L fructose, 0.03g / L~0.05g / L anhydrous magnesium sulfate, 0.8g / L~1.2g / L sodium carbonate, 1.5g / L~2.0g / L dipotassium hydrogen phosphate, 2.0g / L~2.5g / L L-cysteine ​​hydrochloride monohydrate, water;

[0033] Make up to 1 L with water.

[0034] In some embodiments, the culture medium further comprises Tween 80, preferably 1 g / L to 3 g / L Tween 80, such as 1 g / L Tween 80.

[0035] In some embodiments, the culture medium comprises the following components:

[0036] 35g / L~50g / L yeast extract powder, 25g / L~40g / L soy peptone, 5g / L~8g / L fructose, 0.03g / L~0.08g / L anhydrous magnesium sulfate, 0.8g / L~3.0g / L sodium carbonate, 1.5g / L~3.0g / L dipotassium hydrogen phosphate, 0g / L~4.0g / L L-cysteine ​​hydrochloride·monohydrate, 1g / L~3g / L Tween 80, water;

[0037] Make up to 1 L with water.

[0038] In some embodiments, the culture medium comprises the following components:

[0039] 35g / L~40g / L yeast extract powder, 25g / L~35g / L soy peptone, 5g / L~6g / L fructose, 0.03g / L~0.05g / L anhydrous magnesium sulfate, 0.8g / L~1.2g / L sodium carbonate, 1.5g / L~2.0g / L dipotassium hydrogen phosphate, 2.0g / L~2.5g / L L-cysteine ​​hydrochloride·monohydrate, 1g / L Tween 80, water;

[0040] Make up to 1 L with water.

[0041] In the animal-derived-free culture medium of the present invention, the concentration of each component is the concentration during use.

[0042] Second aspect

[0043] The present invention also provides a method for preparing the above culture medium, the method comprising:

[0044] The components in the above culture medium are mixed, dissolved and sterilized at high temperature.

[0045] The third aspect

[0046] The present invention also provides application of the culture medium in culturing Christensenella microphylla.

[0047] The fourth aspect

[0048] The present invention also provides a method for culturing Christensenella parvum, the method comprising:

[0049] Christensenella parvum was inoculated into the above culture medium for cultivation.

[0050] In some embodiments, the culturing method comprises:

[0051] inoculating Christensenella parvum into a seed culture medium for activation culture to prepare a seed solution, and inoculating the seed solution into a fermentation culture medium for fermentation culture;

[0052] The seed culture medium and / or the fermentation culture medium is the above-mentioned culture medium.

[0053] In some embodiments, the fermentation culture conditions are: anaerobic culture, the fermentation culture temperature is 36°C to 38°C, the fermentation culture rotation speed is 100rpm to 350rpm, the fermentation culture time is 30h to 50h, and the fermentation culture inoculation concentration is 2.0E+08CFU / mL to 8.0E+08CFU / mL.

[0054] In some embodiments, the fermentation temperature is, for example, 36°C, 36.5°C, 37°C, 37.5°C, or 38°C; the fermentation speed is, for example, 100 rpm, 150 rpm, 200 rpm, 250 rpm, 300 rpm, or 350 rpm; and the fermentation duration is, for example, 30 h, 32 h, 34 h, 36 h, 38 h, 40 h, 42 h, 44 h, 46 h, 48 h, or 50 h.

[0055] In some embodiments, the conditions for activation culture are: anaerobic culture, the temperature of activation culture is 36°C to 38°C, the activation culture is static culture, the number of activation culture is 2 to 3 times, and the duration of each activation culture is 28h to 70h.

[0056] In some embodiments, the temperature of the activation culture is, for example, 36°C, 36.5°C, 37°C, 37.5°C, or 38°C.

[0057] The activation described in the present invention is a step-by-step activation. A first-level seed liquid is prepared by activating once, and the first-level seed liquid is activated for a second time to prepare a second-level seed liquid, and so on.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] The present invention forms an animal-free liquid culture medium by adding appropriate amounts of yeast extract powder, soy peptone, fructose, anhydrous magnesium sulfate, L-cysteine ​​hydrochloride monohydrate, sodium carbonate and potassium dihydrogen phosphate. In the process of culturing Christensenella minor by using the animal-free culture medium, the Christensenella minor proliferates quickly. In addition, the Christensenella minor cultured by using the animal-free culture medium has a good growth state, good safety, easy operation and control, and a short culture time, thus meeting the industrial requirements of medicinal value. In addition, the animal-free culture medium of the present invention is used to culture Christensenella minor, and the number of viable bacteria is high, the strain activity is high, the passage stability is good, and the scale-up can be carried out for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the microscopic examination result of Christensenella microti cultured in the medium formula of Example 1;

[0061] Figure 2 This is the microscopic examination result of Christensenella microti cultured in the medium formula of Example 2;

[0062] Figure 3 This is the microscopic examination result of Christensenella microti cultured in the medium formula of Example 3;

[0063] Figure 4 This is the microscopic examination result of Christensenella microti cultured in the medium formula of Example 4;

[0064] Figure 5 This is the microscopic examination result of Christensenella microti cultured in the medium formula of Example 5;

[0065] Figure 6 This is the microscopic examination result of Christensenella microti cultured in the medium formula of Example 6;

[0066] Figure 7 This is the microscopic examination result of Christensenella microti cultured with the medium formula of Comparative Example 1;

[0067] Figure 8 This is the microscopic examination result of Christensenella microti cultured with the medium formula of Comparative Example 2;

[0068] Fig. 9 This is the microscopic examination result of Christensenella microti cultured with the medium formula of Comparative Example 3;

[0069] Fig.10 This is the microscopic examination result of Christensenella microti cultured with the medium formula of Comparative Example 4;

[0070] Fig.11 This is the microscopic examination result of Christensenella microti cultured in the medium formula of Comparative Example 5;

[0071] Fig.12This is the microscopic examination result of Christensenella microti cultured with the medium formula of Comparative Example 6;

[0072] Fig.13 This is the microscopic examination result of Christensenella microti cultured with the medium formula of Comparative Example 7;

[0073] Fig.14 This is the microscopic examination result of Christensenella microti cultured with the medium formula of Comparative Example 8;

[0074] Fig.15 This is the microscopic examination result of Christensenella microti cultured in the medium formula of Comparative Example 9;

[0075] Fig.16 These are the microscopic examination results of Christensenella microti cultured in the culture medium formula of Comparative Example 10. DETAILED DESCRIPTION

[0076] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific implementation or embodiment, and are not intended to limit the present invention. The optional scope of the term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the combinations include any two related listed items, any more related listed items, or all related listed items.

[0078] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0079] The percentage contents involved in the present invention, unless otherwise specified, refer to mass percentage for solid-liquid mixture and solid-solid mixture, and refer to volume percentage for liquid-liquid mixture.

[0080] The percentage concentrations involved in the present invention, unless otherwise specified, refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0081] Example 1

[0082] 1. Culture medium and its preparation

[0083] The animal-derived-free culture medium contained 35 g / L yeast extract, 25 g / L soy peptone, 5 g / L fructose, 0.03 g / L anhydrous magnesium sulfate, 2.0 g / L L-cysteine ​​hydrochloride monohydrate, 0.8 g / L sodium carbonate, and 1.5 g / L dipotassium hydrogen phosphate.

[0084] After the ingredients are mixed with water, dissolution is completed and sterilized at high temperature.

[0085] 2. Cultivation of Christensenella

[0086] (1) Inoculate the bacterial seeds of Christensenella parvula into 10 mL of animal-free culture medium at a ratio of 5% (v / v) with an inoculation concentration of 5.0E+08 CFU / mL, and culture the culture anaerobically at 37±1°C for 48 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas to obtain a primary seed solution;

[0087] (2) The first-level seed solution that passed the microscopic examination was inoculated into 200 mL of animal-free culture medium at a ratio of 5% (v / v), with an inoculation concentration of 2.0E+08 CFU / mL, and cultured anaerobically at 37±1°C for 36 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas. After the culture, the second-level seed solution was obtained;

[0088] (3) Animal-free culture medium was added to the fermenter, the anaerobic protective gas was a binary mixed gas (7% CO2 / 93% N2), and the secondary seed liquid was inoculated at a ratio of 8% (v / v) with an inoculation concentration of 6.0E+08 CFU / mL. The culture was carried out at 37±1°C and anaerobic stirring at 200 rpm for an average of 36 hours.

[0089] Example 2

[0090] 1. Culture medium and its preparation

[0091] The animal-origin-free culture medium contained 35 g / L yeast extract, 25 g / L soy peptone, 5 g / L fructose, 0.03 g / L anhydrous magnesium sulfate, 2.0 g / L L-cysteine ​​hydrochloride monohydrate, 0.8 g / L sodium carbonate, 1.5 g / L dipotassium hydrogen phosphate, and 1.0 g / L Tween 80.

[0092] After the ingredients are mixed with water, dissolution is completed and sterilized at high temperature.

[0093] 2. Cultivation of Christensenella

[0094] (1) Inoculate the bacterial seeds of Christensenella parvum into 10 mL of animal-free culture medium at a ratio of 5% (v / v) with an inoculation concentration of 5.0E+08 CFU / mL, and culture the culture anaerobically at 37±1°C for 54 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas to obtain a primary seed solution;

[0095] (2) The first-level seed solution that passed the microscopic examination was inoculated into 200 mL of animal-free culture medium at a ratio of 5% (v / v), with an inoculation concentration of 2.0E+08 CFU / mL, and cultured anaerobically at 37±1°C for 33 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas. After the culture, the second-level seed solution was obtained;

[0096] (3) Animal-free culture medium was added to the fermenter, the anaerobic protective gas was a binary mixed gas (7% CO2 / 93% N2), and the secondary seed liquid was inoculated at a ratio of 8% (v / v) with an inoculation concentration of 6.0E+08 CFU / mL. The culture was carried out at 37±1°C and anaerobic stirring at 200 rpm for an average of 38 hours.

[0097] Example 3

[0098] This embodiment is a variation of Embodiment 1.

[0099] 1. Culture medium and its preparation

[0100] The animal-derived-free culture medium contained 40 g / L yeast extract, 35 g / L soy peptone, 6 g / L fructose, 0.05 g / L anhydrous magnesium sulfate, 2.5 g / L L-cysteine ​​hydrochloride monohydrate, 1.2 g / L sodium carbonate, and 2 g / L dipotassium hydrogen phosphate.

[0101] After the ingredients are mixed with water, dissolution is completed and sterilized at high temperature.

[0102] 2. Cultivation of Christensenella

[0103] (1) Inoculating the bacterial seeds of Christensenella parvum into 10 mL of animal-free culture medium at a ratio of 5% (v / v) with an inoculation concentration of 5.0E+08 CFU / mL, and culturing the culture medium anaerobically at 37±1°C for 63 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas to obtain a primary seed solution;

[0104] (2) The first-level seed solution that passed the microscopic examination was inoculated into 200 mL of animal-free culture medium at a ratio of 5% (v / v), with an inoculation concentration of 2.0E+08 CFU / mL, and cultured anaerobically at 37±1°C for 40 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas. After the culture, the second-level seed solution was obtained;

[0105] (3) Animal-free culture medium was added to the fermenter, the anaerobic protective gas was a binary mixed gas (7% CO2 / 93% N2), and the secondary seed liquid was inoculated at a ratio of 8% (v / v) with an inoculation concentration of 6.0E+08 CFU / mL. The culture was carried out at 37±1°C and anaerobic stirring at 200 rpm for an average of 42 hours.

[0106] Example 4

[0107] This embodiment is a variation of the second embodiment.

[0108] 1. Culture medium and its preparation

[0109] Animal-origin-free media includes

[0110] 40g / L yeast extract powder, 35g / L soy peptone, 6g / L fructose, 0.05g / L anhydrous magnesium sulfate, 2.5g / L L-cysteine ​​hydrochloride monohydrate, 1.2g / L sodium carbonate, 2g / L dipotassium hydrogen phosphate and 1.0g / L Tween 80.

[0111] After the ingredients are mixed with water, dissolution is completed and sterilized at high temperature.

[0112] 2. Cultivation of Christensenella

[0113] (1) Inoculating the bacterial seeds of Christensenella parvum into 10 mL of animal-free culture medium at a ratio of 5% (v / v) with an inoculation concentration of 5.0E+08 CFU / mL, and culturing the culture medium anaerobically at 37±1°C for 63 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas to obtain a primary seed solution;

[0114] (2) The first-level seed solution that passed the microscopic examination was inoculated into 200 mL of animal-free culture medium at a ratio of 5% (v / v), with an inoculation concentration of 2.0E+08 CFU / mL, and cultured anaerobically at 37±1°C for 40 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas. After the culture, the second-level seed solution was obtained;

[0115] (3) Animal-free culture medium was added to the fermenter, the anaerobic protective gas was a binary mixed gas (7% CO2 / 93% N2), and the secondary seed liquid was inoculated at a ratio of 8% (v / v) with an inoculation concentration of 6.0E+08 CFU / mL. The culture was carried out at 37±1°C and anaerobic stirring at 200 rpm for an average of 42 hours.

[0116] Example 5

[0117] 1. Culture medium and its preparation

[0118] The animal-derived-free culture medium contained 50 g / L yeast extract, 40 g / L soy peptone, 8 g / L fructose, 0.08 g / L anhydrous magnesium sulfate, 4 g / L L-cysteine ​​hydrochloride monohydrate, 3 g / L sodium carbonate, and 3 g / L dipotassium hydrogen phosphate.

[0119] After the ingredients are mixed with water, dissolution is completed and sterilized at high temperature.

[0120] 2. Cultivation of Christensenella

[0121] (1) Inoculate the bacterial seeds of Christensenella parvum into 10 mL of animal-derived-free culture medium at a ratio of 5% (v / v) with an inoculation concentration of 5.0E+08 CFU / mL, and culture the culture anaerobically at 37±1°C for 70 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas to obtain a primary seed solution;

[0122] (2) The first-level seed solution that passed the microscopic examination was inoculated into 200 mL of animal-free culture medium at a ratio of 5% (v / v), with an inoculation concentration of 2.0E+08 CFU / mL, and cultured anaerobically at 37±1°C for 43 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas. After the culture was completed, the second-level seed solution was obtained;

[0123] (3) Animal-free culture medium was added to the fermenter, the anaerobic protective gas was a binary mixed gas (7% CO2 / 93% N2), and the secondary seed liquid was inoculated at a ratio of 8% (v / v) with an inoculation concentration of 6.0E+08 CFU / mL. The culture was carried out at 37±1°C and anaerobic stirring at 200 rpm for an average of 42 hours.

[0124] Example 6

[0125] 1. Culture medium and its preparation

[0126] The animal-derived-free culture medium contains 35 g / L yeast extract, 25 g / L soy peptone, 5 g / L fructose, 0.03 g / L anhydrous magnesium sulfate, 0.8 g / L sodium carbonate, and 1.5 g / L dipotassium hydrogen phosphate.

[0127] After the ingredients are mixed with water, dissolution is completed and sterilized at high temperature.

[0128] 2. Cultivation of Christensenella

[0129] (1) Inoculate the bacterial seeds of Christensenella parvum into 10 mL of animal-free culture medium at a ratio of 5% (v / v) with an inoculation concentration of 5.0E+08 CFU / mL, and culture the culture anaerobically at 37±1°C for 69 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas to obtain a primary seed solution;

[0130] (2) The first-level seed solution that passed the microscopic examination was inoculated into 200 mL of animal-free culture medium at a ratio of 5% (v / v), with an inoculation concentration of 2.0E+08 CFU / mL, and cultured anaerobically at 37±1°C for 48 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas. After the culture, the second-level seed solution was obtained;

[0131] (3) Animal-free culture medium was added to the fermenter, the anaerobic protective gas was a binary mixed gas (7% CO2 / 93% N2), and the secondary seed liquid was inoculated at a ratio of 8% (v / v) with an inoculation concentration of 6.0E+08 CFU / mL. The culture was carried out at 37±1°C and anaerobic stirring at 200 rpm for an average of 42 hours.

[0132] Table 1 Summary of animal-free liquid culture medium formulations in Example

[0133]

[0134] Comparative Example 1

[0135] 1. Culture medium and its preparation

[0136] The medium contained water and the following components at the following concentrations: 5 g / L fructose, 60 g / L beef extract, 2.0 / L L-cysteine ​​hydrochloride·monohydrate, 0.03 g / L anhydrous magnesium sulfate, 1.5 g / L dipotassium hydrogen phosphate, and 0.8 g / L sodium carbonate.

[0137] After the culture medium components and water are mixed and dissolved, they are sterilized at high temperature.

[0138] 2. Comparative Example 1 The specific steps of culturing Christensen bacteria are as follows:

[0139] (1) Inoculate the bacterial seeds of Christensenella parvum into 10 mL of culture medium at a ratio of 5% (v / v) with an inoculation concentration of 2.0E+08 CFU / mL, and culture the culture anaerobically at 37±1°C for 58 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas to obtain a primary seed solution;

[0140] (2) The first-level seed solution that passed the microscopic examination was inoculated into 200 mL of culture medium at a ratio of 5% (v / v), with an inoculation concentration of 3.0E+08 CFU / mL, and cultured anaerobically at 37±1°C for 28 h under ternary gas (5% CO2 / 10% H2 / 85% N2) as anaerobic protective gas. After the culture was completed, the second-level seed solution was obtained;

[0141] (3) Add culture medium to the fermenter, use binary mixed gas (7% CO2 / 93% N2) as anaerobic protective gas, inoculate secondary seed solution at a ratio of 8% (v / v) with an inoculation concentration of 5.0E+08 CFU / mL, and culture at 37°C with anaerobic stirring at 200 rpm for 38.8 h.

[0142] Comparative Example 2

[0143] Comparative Example 2 is a comparative example of Example 1, and the difference relative to Example 1 mainly includes a different formula of animal-derived culture medium. Specifically, the animal-derived culture medium of Comparative Example 2 contains water and 60 g / L yeast extract powder, 5 g / L fructose, 0.03 g / L anhydrous magnesium sulfate, 2.0 g / L L-cysteine ​​hydrochloride monohydrate, 0.8 g / L sodium carbonate and 1.5 g / L dipotassium hydrogen phosphate.

[0144] Comparative Example 3

[0145] Comparative Example 3 is a comparative example of Example 1. The difference relative to Example 1 mainly includes a different formula of animal-free culture medium. Specifically, the animal-free culture medium of Comparative Example 3 contains water and 60 g / L soy peptone, 5 g / L fructose, 0.03 g / L anhydrous magnesium sulfate, 2.0 g / L L-cysteine ​​hydrochloride monohydrate, 0.8 g / L sodium carbonate, and 1.5 g / L dipotassium hydrogen phosphate.

[0146] Comparative Example 4

[0147] Comparative Example 4 is a comparative example of Example 1, and the difference relative to Example 1 mainly includes a different formula of animal-derived culture medium. Specifically, the animal-derived culture medium of Comparative Example 4 contains water and 35 g / L yeast extract powder, 25 g / L soy peptone, 5 g / L fructose, 0.03 g / L anhydrous magnesium sulfate, 2.0 g / L L-cysteine ​​hydrochloride monohydrate, 0.8 g / L sodium carbonate, 1.5 g / L dipotassium hydrogen phosphate and 0.2 g / L potassium dihydrogen phosphate.

[0148] Comparative Example 5

[0149] Comparative Example 5 is a comparative example of Example 1, and the difference relative to Example 1 mainly includes a different formula of animal-derived culture medium. Specifically, the animal-derived culture medium of Comparative Example 5 contains water and 35 g / L yeast extract powder, 25 g / L soy peptone, 5 g / L fructose, 0.03 g / L anhydrous magnesium sulfate, 2.0 g / L L-cysteine ​​hydrochloride monohydrate, 0.8 g / L sodium carbonate, 1.5 g / L dipotassium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate and 1 g / L sodium chloride.

[0150] Comparative Example 6

[0151] Comparative Example 6 is a comparative example of Example 1. The difference relative to Example 1 mainly includes a different formula of animal-derived culture medium. Specifically, the animal-derived culture medium of Comparative Example 6 contains water and 60 g / L yeast extract powder, 20 g / L soy peptone, 5 g / L fructose, 0.03 g / L anhydrous magnesium sulfate, 2.0 g / L L-cysteine ​​hydrochloride monohydrate, 0.8 g / L sodium carbonate, and 1.5 g / L dipotassium hydrogen phosphate.

[0152] Comparative Example 7

[0153] Comparative Example 7 is a comparative example of Example 1. The difference compared with Example 1 mainly includes a different formula of animal-free culture medium. Specifically, the animal-free culture medium of Comparative Example 7 includes water and 20 g / L yeast extract, 60 g / L soy peptone, 5 g / L fructose, 0.03 g / L anhydrous magnesium sulfate, 2.0 g / L L-cysteine ​​hydrochloride monohydrate, 0.8 g / L sodium carbonate and 1.5 g / L dipotassium hydrogen phosphate.

[0154] Comparative Example 8

[0155] Comparative Example 8 is a comparative example of Example 1, and the difference relative to Example 1 mainly includes a different formula of animal-derived culture medium. Specifically, the animal-derived culture medium of Comparative Example 8 contains water and 35 g / L yeast extract powder, 25 g / L soy peptone, 5 g / L fructose, 0.03 g / L anhydrous magnesium sulfate, 2.0 g / L L-cysteine ​​hydrochloride monohydrate, 0.5 g / L sodium carbonate and 1.0 g / L dipotassium hydrogen phosphate.

[0156] Comparative Example 9

[0157] Comparative Example 9 is a comparative example of Example 1. The difference compared with the example mainly includes a different formula of animal-free culture medium. Specifically, the animal-free culture medium of Comparative Example 9 includes water and 35 g / L yeast extract powder, 25 g / L soy peptone, 3 g / L fructose, 0.03 g / L anhydrous magnesium sulfate, 2.0 g / L L-cysteine ​​hydrochloride monohydrate, 0.8 g / L sodium carbonate, and 1.5 g / L dipotassium hydrogen phosphate.

[0158] Comparative Example 10

[0159] Comparative Example 10 is a comparative example of Example 1. The difference compared with Example 1 mainly includes a different formula of animal-derived culture medium. Specifically, the animal-derived culture medium of Comparative Example 10 includes water and 35 g / L yeast extract powder, 25 g / L soy peptone, 10 g / L fructose, 0.03 g / L anhydrous magnesium sulfate, 2.0 g / L L-cysteine ​​hydrochloride monohydrate, 0.8 g / L sodium carbonate and 1.5 g / L potassium dihydrogen phosphate.

[0160]

[0161] Test Example 1 Christensenella microflora culture fluid detection

[0162] 1. Test object: Cultures obtained from the culture medium formulations of Examples 1 to 6 and Comparative Examples 1 to 10.

[0163] 2. Experimental methods:

[0164] 1) Morphological comparison: The above cultures were separated, purified, and examined under a microscope.

[0165] 2)OD 600 Value detection: Dilute the above culture with purified water to an appropriate concentration, use purified water as a control, and measure the optical density value at 600nm (1cm optical path) with a UV spectrophotometer.

[0166] 3) Flow cytometry detection of viable bacteria count:

[0167] ① Preparation of mixed dye: Take the dye stored at -20℃ and place it on ice to melt. Then take 500μL of SYBR Green I solution and add 30μL of PI stock solution, mix well and set aside.

[0168] ② Preparation of test solution: Take 1 mL of the test bacterial solution in a 15 mL centrifuge tube, dilute it 10 times, and then dilute it to an appropriate multiple with ultrapure water in a 250 mL volumetric flask as the test solution.

[0169] ③ Sample staining: Take 0.5 mL of the test solution, add 50 μL of mixed dye, stain for 30 minutes in the dark, and analyze the samples. Perform 2 parallel tests in total, and take the average of the 2 parallel test results as the final result.

[0170] ④Flow cytometry detection conditions: gain FSC 300, SSC 300, FITC 300, PE 300; threshold PE-A 800, FITC-A 500. Flow rate: low speed (10 μL / min), recording time 180 s.

[0171] ⑤ Number of viable bacteria (cells / mL) = flow cytometry response value (cells / μL) × 10 3 ×1.1×dilution factor.

[0172] 3. Experimental results

[0173] 1) Morphological comparison:

[0174] The microscopic examination results of the culture obtained from the animal-free liquid culture medium described in Examples 1-6 are as follows: Figure 1-Figure 6 As shown, the microscopic examination results of the culture obtained from the culture medium of Comparative Examples 1-10 are as follows Figure 7-16 The analysis showed that the morphology of Christensen bacteria contained in the cultures obtained in Comparative Examples 1, 4-10 and Examples 1-6 was G - , slender straight rods or short straight rods with cone ends, arranged individually or in chains, this morphology is close to the normal morphology of Kristensen; combined with Figure 8 , Fig. 9 It can be seen that the morphology of Christensenella minor contained in the cultures obtained in Comparative Examples 2 and 3 is G + , rod-shaped, arranged singly or in clusters, which does not conform to the morphology of Christensenella parvum.

[0175] It can be seen that the selection of yeast extract powder as the sole nitrogen source or the selection of soy peptone as the sole nitrogen source resulted in the morphology of Christensenella minor contained in the culture obtained being G + , which does not conform to the morphology of Christensenella microphylla.

[0176] 2)OD 600 The values, viable counts and bacterial activity were tested, and the results are shown in Table 3.

[0177] Table 3 Test results of cultures obtained by culturing Christensenella in culture medium of Examples 1-6 and Comparative Examples 1-10

[0178]

[0179] From the results in Table 3, it can be seen that the culture medium described in Examples 1 and 2 of the present invention is used to culture Christensenella microti, and the culture time is shorter, less than 40 hours. Compared with the culture medium described in Comparative Example 1, the OD 600 The values ​​were increased by 22.48% and 19.06% on average, and the number of viable bacteria was increased by 23.11% and 12.60% respectively; the culture medium described in Examples 3-6 cultured Christensenella microphylla, and the culture time was slightly longer than that of the culture medium described in Comparative Example 1, about 42h, and its OD 600 The values ​​increased by an average of 10.57%, 17.67%, 9.63% and 9.53% respectively, and the number of viable bacteria increased by 3.78%, 7.14%, 0.84% ​​and 2.52% respectively.

[0180] The formulas described in Comparative Examples 2 to Comparative Examples 10 were used to culture Christensenella microphylla, and the average culture time was 48h, 50h, 40h, 40h, 50h, 48h, 50h, 40h and 40h, respectively, which were extended by 33.34%, 38.89%, 11.11%, 11.11%, 38.89%, 33.34%, 38.89%, 11.11% and 11.11% respectively compared with Example 1 (36h); OD 600 The values ​​were 1.533, 1.601, 1.746, 1.684, 1.588, 1.594, 1.635, 1.724 and 1.676, respectively, which were higher than those in Example 1 (2.468). 600 The values ​​decreased by 37.88%, 35.13%, 29.25%, 33.23%, 35.65%, 35.41%, 33.75%, 30.15% and 32.09% respectively; the viable counts were 8.71E+08CFU / mL, 9.14E+08CFU / mL, 1.81E+09CFU / mL, 1.69E+09CFU / mL, 8.92E+08CFU / mL, 9 .03E+08CFU / mL, 1.61E+09CFU / mL, 1.76E+09CFU / mL and 1.60E+09CFU / mL, the viable counts were reduced by 70.27%, 68.81%, 38.23%, 42.32%, 69.56%, 69.18%, 45.05%, 39.93% and 45.39% respectively compared with Example 1 (2.93E+09CFU / mL).

[0181] It can be seen that the animal-free culture medium formulas described in Examples 1 and 2 can culture Christensenella microti for a relatively short culture time and the obtained culture OD 600 The value and viable count were higher than those of Comparative Examples 1-10; the animal-free culture medium formula of Example 3-6 was used to culture Christensenella microti, and the obtained culture OD 600 The values ​​and viable counts are higher than those in Comparative Examples 1-10.

[0182] By comparing Example 1 with Comparative Example 1, it can be seen that even when the average culture time is longer, using yeast extract powder and soy peptone as the nitrogen source is more conducive to the proliferation of Christensenella microti, and the number of viable bacteria is improved to a certain extent compared to using beef extract as the nitrogen source.

[0183] By comparing Example 1 with Comparative Examples 2 and 3, it can be seen that even when the average culture time is longer, the combination of yeast extract powder and soy peptone as the nitrogen source is more conducive to the proliferation of Christensenella microsporum than the use of a single yeast extract powder or soy peptone as the nitrogen source, and the number of viable bacteria is significantly increased.

[0184] Comparison of Example 1 with Comparative Examples 6 and 7 shows that even when the average culture time is longer, the ratio of yeast extract powder to soy peptone as nitrogen sources will affect the proliferation and viable count of Christensenella parvum.

[0185] Comparison of Comparative Examples 4-5 and Example 1 shows that the addition of additional inorganic salts, such as potassium dihydrogen phosphate and sodium chloride, will lead to a significant decrease in the number of viable bacteria even when the average culture time is longer.

[0186] Comparison of Example 1 and Comparative Examples 1-10 shows that the average viable counts of Comparative Examples 1-10 are significantly lower than that of Example 1 even when the average culture time is longer.

[0187] Comparison of Example 1 and Comparative Examples 9-10 shows that the amount of fructose added exceeds the range, resulting in a longer culture time and a decrease in the number of viable bacteria.

[0188] It can be seen that when the culture medium with the formula of the present invention is used to culture Christensenella minor, the proliferation of Christensenella minor is fast, the culture time is short, and the number of viable bacteria is high.

[0189] Test Example 2 Safety Comparison of Culture Medium Formulas

[0190] 1) Test object: cultures obtained from the culture medium formulations of Examples 1-6 and Comparative Examples 1-10.

[0191] 2) Sampling: Take the culture fluid obtained after 20h and 36h for bacteria detection.

[0192] The above method was adopted to prepare Christensenella microphylla fermentation culture broth using the culture medium formulas of Comparative Examples 1 to Comparative Example 10 and Example 1 to Example 6, and the culture broth was tested for foreign bacteria. The test results are shown in Table 4 below.

[0193] Table 4 Results of detection of foreign bacteria in fermentation broth of Christensenella

[0194]

[0195]

[0196] According to the data results in Table 4, the detection results of miscellaneous bacteria in the culture fluid obtained by the animal-free culture medium formula described in Examples 1-6 and Comparative Examples 2-10 meet the requirements; the detection results of non-pathogenic miscellaneous bacteria in the culture fluid obtained by culturing with the animal-derived culture medium described in Comparative Example 1 exceeded the standard and did not meet the requirements.

[0197] The difference between Comparative Example 1 and Example 1 lies in the selection of nitrogen source, where beef extract is used in Comparative Example 1 while yeast extract powder and soy peptone are used in Example 1. It can be seen that beef extract as a nitrogen source component of the culture medium will cause non-pathogenic bacteria to appear in the fermentation culture broth of Christensenella parvum, while yeast extract powder and soy peptone will not appear non-pathogenic bacteria and are safer.

[0198] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A culture medium, wherein the culture medium does not contain animal-derived components; Preferably, the animal-derived component is a bovine-derived component, such as beef extract, beef powder, beef liver extract, etc.; Preferably, the culture medium is used for culturing Christensenella parvum.

2. The culture medium according to claim 1, characterized in that The culture medium comprises the following components: 25g / L~50g / L yeast extract powder, 15g / L~45g / L plant peptone, 5g / L~8g / L sugars, 0.01g / L~0.08g / L anhydrous magnesium sulfate, 0.5g / L~3g / L sodium carbonate, 1g / L~3g / L dipotassium hydrogen phosphate; Preferably, the culture medium comprises the following components: 25g / L~50g / L yeast extract powder, 15g / L~45g / L plant peptone, 5g / L~8g / L sugars, 0.01g / L~0.08g / L anhydrous magnesium sulfate, 0.5g / L~3g / L sodium carbonate, 1g / L~3g / L dipotassium hydrogen phosphate, 1.5g / L~4g / L L-cysteine ​​hydrochloride monohydrate; Preferably, the plant peptone is soy peptone; Preferably, the sugar is selected from soluble starch, sucrose, glucose, fructose, preferably fructose.

3. The culture medium according to claim 1 or 2, characterized in that The culture medium comprises the following components: 25g / L~50g / L yeast extract powder, 15g / L~45g / L plant peptone, 5g / L~8g / L sugars, 0.01g / L~0.08g / L anhydrous magnesium sulfate, 0.5g / L~3g / L sodium carbonate, 1g / L~3g / L dipotassium hydrogen phosphate, 1.5g / L~4g / L L-cysteine ​​hydrochloride·monohydrate, water; Preferably, the culture medium comprises the following components: 25g / L~50g / L yeast extract powder, 15g / L~45g / L soy peptone, 5g / L~8g / L fructose, 0.01g / L~0.08g / L anhydrous magnesium sulfate, 0.5g / L~3g / L sodium carbonate, 1g / L~3g / L dipotassium hydrogen phosphate, 1.5g / L~4g / L L-cysteine ​​hydrochloride monohydrate, water; Preferably, fill up to 1 L with water.

4. The culture medium according to any one of claims 1 to 3, characterized in that In the culture medium, Yeast extract powder is 35g / L to 50g / L, preferably 35g / L to 40g / L; and / or, soy peptone is 25 g / L to 40 g / L, preferably 25 g / L to 35 g / L; and / or, fructose is 5 g / L to 8 g / L, preferably 5 g / L to 6 g / L; and / or, anhydrous magnesium sulfate is 0.03 g / L to 0.08 g / L, preferably 0.03 g / L to 0.05 g / L; and / or, L-cysteine ​​hydrochloride monohydrate is 0 g / L to 4.0 g / L, preferably 2.0 g / L to 2.5 g / L; and / or, sodium carbonate is 0.8 g / L to 3.0 g / L, preferably 0.8 g / L to 1.2 g / L; And / or, dipotassium hydrogen phosphate is 1.5 g / L to 3.0 g / L, preferably 1.5 g / L to 2.0 g / L.

5. The culture medium according to any one of claims 1 to 4, characterized in that The culture medium comprises the following components: 35g / L~50g / L yeast extract powder, 25g / L~40g / L soy peptone, 5g / L~8g / L fructose, 0.03g / L~0.08g / L anhydrous magnesium sulfate, 0.8g / L~3.0g / L sodium carbonate, 1.5g / L~3.0g / L dipotassium hydrogen phosphate, 0g / L~4.0g / L L-cysteine ​​hydrochloride·monohydrate, water; add water to 1L; Preferably, the culture medium comprises the following components: 35g / L~40g / L yeast extract powder, 25g / L~35g / L soy peptone, 5g / L~6g / L fructose, 0.03g / L~0.05g / L anhydrous magnesium sulfate, 0.8g / L~1.2g / L sodium carbonate, 1.5g / L~2.0g / L dipotassium hydrogen phosphate, 2.0g / L~2.5g / L L-cysteine ​​hydrochloride monohydrate, water; add water to 1L.

6. The culture medium according to any one of claims 1 to 5, characterized in that The culture medium further comprises Tween 80, preferably 1 g / L to 3 g / L Tween 80, for example 1 g / L Tween 80; Preferably, the culture medium comprises the following components: 35g / L~50g / L yeast extract powder, 25g / L~40g / L soy peptone, 5g / L~8g / L fructose, 0.03g / L~0.08g / L anhydrous magnesium sulfate, 0.8g / L~3.0g / L sodium carbonate, 1.5g / L~3.0g / L dipotassium hydrogen phosphate, 0g / L~4.0g / L L-cysteine ​​hydrochloride·monohydrate, 1g / L~3g / L Tween 80, water; add water to 1L; Preferably, the culture medium comprises the following components: 35g / L~40g / L yeast extract powder, 25g / L~35g / L soy peptone, 5g / L~6g / L fructose, 0.03g / L~0.05g / L anhydrous magnesium sulfate, 0.8g / L~1.2g / L sodium carbonate, 1.5g / L~2.0g / L dipotassium hydrogen phosphate, 2.0g / L~2.5g / L L-cysteine ​​hydrochloride·monohydrate, 1g / L Tween 80, water; Make up to 1 L with water.

7. A method for preparing the culture medium according to any one of claims 1 to 6, comprising: The components in the culture medium are mixed, dissolved and sterilized at high temperature.

8. Use of the culture medium according to any one of claims 1 to 6 in culturing Christensenella parvum.

9. A method for culturing Christensenella parvum, the method comprising: Inoculating Christensenella parvum into the culture medium according to any one of claims 1 to 6 for culturing; Preferably, the culturing method comprises: inoculating Christensenella parvum into a seed culture medium for activation culture to prepare a seed solution, and inoculating the seed solution into a fermentation culture medium for fermentation culture; The seed culture medium and / or the fermentation culture medium is the culture medium according to any one of claims 1 to 6.

10. The culture method according to claim 9, characterized in that: The fermentation culture conditions are: anaerobic culture, the fermentation culture temperature is 36°C to 38°C, the fermentation culture speed is 100rpm to 350rpm, the fermentation culture time is 30h to 50h, and the fermentation culture inoculation concentration is 2.0E+08CFU / mL to 8.0E+08CFU / mL; And / or, the conditions of activation culture are: anaerobic culture, the temperature of activation culture is 36°C to 38°C, the activation culture is static culture, the number of activation culture is 2 to 3 times, and the duration of each activation culture is 28h to 70h.

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