A method for culturing and preserving a clostridium prasrj

Abstract

The application relates to a culture method and a preservation method of a Clostridium prasrj, and belongs to the technical field of microorganisms. The culture method of the Clostridium prasrj comprises the following steps: inoculating glycerol bacteria of the Clostridium prasrj into YCFA liquid culture medium, culturing under anaerobic conditions at 35-37 DEG C for 16-19h, and obtaining a first-stage seed liquid; inoculating the obtained first-stage seed liquid into YCFA liquid culture medium, and statically culturing under anaerobic conditions at 35-37 DEG C for 7-9h to obtain a second-stage seed liquid; inoculating the obtained second-stage seed liquid into YCFA liquid culture medium, and statically culturing under anaerobic conditions at 35-37 DEG C for 16-19h to obtain a Clostridium prasrj fermentation liquid. Through resuscitation, activation and fermentation of the Clostridium prasrj in the YCFA liquid culture medium, the viable bacterial count in the Clostridium prasrj fermentation liquid can be effectively improved, and the highest viable bacterial count can reach 10 9 CFU / mL.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganism, in particular to a culture method and a preservation method of Faecalibacterium prausnitzii. BACKGROUND

[0002] Faecalibacterium prausnitzii (F. prausnitzii) is also known as Faecalibacterium plautii, gram-negative, and is the dominant bacteria of Clostridium leptum group, belonging to Clostridiaceae and Firmicutes. Faecalibacterium prausnitzii F. prausnitzii F. prausnitzii is one of the most important bacteria in human intestinal flora, accounting for 5-15% of the total number of bacteria detected in healthy human fecal samples. However, the abundance of F. prausnitzii in the feces of patients with chronic constipation, irritable bowel syndrome, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), colorectal cancer, type 2 diabetes and metabolic fatty liver disease is significantly reduced. Studies have shown that F. prausnitzii has broad application prospects in anti-inflammatory, anti-tumor and lipid metabolism improvement. However, F. prausnitzii is extremely sensitive to oxygen, and it cannot synthesize essential amino acids such as alanine, cysteine, methionine, serine and tryptophan. The requirements for culture conditions are very harsh, and the conditions for improving the titer of F. prausnitzii in shake flask fermentation are also very harsh. Therefore, it is urgent to develop a culture method that can improve the titer of F. prausnitzii while keeping the number of viable bacteria in the fermentation broth of F. prausnitzii in a relatively stable range. At the same time, there is no suitable preservation method for F. prausnitzii that can maintain a high survival rate. SUMMARY

[0003] The present application aims to overcome the shortcomings of the prior art and provide a culture method that can greatly improve the number of viable F. prausnitzii and a preservation method suitable for F. prausnitzii.

[0004] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:

[0005] In a first aspect, the present application provides a culture method of F. prausnitzii, comprising the following steps:

[0006] (1) inoculating glycerol bacteria of F. prausnitzii into YCFA liquid medium, and culturing at 35-37℃ under anaerobic conditions for 16-19h to obtain a first-stage seed liquid;

[0007] (2) inoculating the first-stage seed liquid obtained in step (1) into YCFA liquid medium, and statically culturing at 35-37℃ under anaerobic conditions for 7-9h to obtain a second-stage seed liquid;

[0008] (3) inoculating the second-stage seed liquid obtained in step (2) into YCFA liquid medium, and statically culturing at 35-37℃ under anaerobic conditions for 16-19h to obtain a F. prausnitzii fermentation broth.

[0009] ​The application can effectively increase the viable cell count in the fermentation liquor of the Prausnitzia by resuscitating, activating and fermenting the Prausnitzia in the YCFA liquid culture medium, and the highest viable cell count can reach 10 9 CFU / mL.

[0010] As a preferred embodiment of the culture method, at least one of the following (I)-(III) is included:

[0011] (I) In step (1), the glycerol inoculation amount of the Prausnitzia is 9-12%;

[0012] (II) In step (2), the inoculation amount of the primary seed liquor is 4-6%;

[0013] (III) In step (3), the inoculation amount of the secondary seed liquor is 0.5-1%.

[0014] As a preferred embodiment of the culture method, the YCFA liquid culture medium includes the following components: 10 g / L tryptone, 2.5 g / L yeast extract, 4.0 g / L sodium bicarbonate, 2.0 g / L glucose, 2.0 g / L maltose, 2.0 g / L cellobiose, 1.0 g / L cysteine hydrochloride, 150 mL / L mineral solution I, 150 mL / L mineral solution II, 1 mg / L resazurin, 6.2 mL / L organic acid mixture, 10.0 mL / L hemin solution, 1.0 mL / L vitamin solution I, and 1.0 mL / L vitamin solution II.

[0015] As a preferred embodiment of the culture method, at least one of the following (IV)-(V) is included:

[0016] (IV) The mineral solution I in the YCFA liquid culture medium is 3.0 g / L potassium phosphate dibasic aqueous solution;

[0017] (V) The mineral solution II in the YCFA liquid culture medium includes the following components: 3.0 g / L potassium dihydrogen phosphate, 6.0 g / L ammonium sulfate, 6.0 g / L sodium chloride, 0.6 g / L magnesium sulfate heptahydrate, and 0.6 g / L calcium chloride dihydrate.

[0018] As a preferred embodiment of the culture method, at least one of the following (IV)-(V) is included:

[0019] (VI) The hemin solution in the YCFA liquid culture medium includes the following components: 2.8 g / L potassium hydroxide, 250 mL / L anhydrous ethanol, and 1.0 g / L chloroferriheme;

[0020] (VII) the organic acid mixture in the YCFA liquid culture medium comprises the following components: 17.0 mL of acetic acid, 6.0 mL of propionic acid, 1.0 mL of n-valeric acid, 1.0 mL of iso-valeric acid, and 1.0 mL of iso-butryic acid.

[0021] As a preferred embodiment of the culture method of the present application, it comprises at least one of the following (VIII)~(IX):

[0022] (VIII) the vitamin solution I in the YCFA liquid culture medium comprises the following components: 0.01 g / L of biotin, 0.01 g / L of vitamin B12, 0.03 g / L of p-aminobenzoic acid, 0.05 g / L of folic acid, and 0.15 g / L of vitamin B6 hydrochloride;

[0023] (IX) the vitamin solution II in the YCFA liquid culture medium comprises the following components: 0.05 g / L of thiamine hydrochloride and 0.05 g / L of riboflavin.

[0024] In a second aspect, the present application provides a preservation method of the Prakramium, which comprises the following steps:

[0025] S1, centrifuging the Prakramium fermentation broth obtained above at 4-8℃ to obtain Prakramium bacteria;

[0026] S2, resuspending the Prakramium bacteria obtained in step S1 by using a phosphate buffer to obtain Prakramium bacteria paste;

[0027] S3, mixing the Prakramium bacteria paste obtained in step S2 with a cryoprotective agent and placing it at -80℃ for cryopreservation; the volume ratio of the Prakramium bacteria paste to the cryoprotective agent is 1:1.

[0028] As a preferred embodiment of the preservation method of the present application, in step S1, the centrifugation conditions are 7000-8000g of centrifugal speed and 6-8min of centrifugation time.

[0029] As a preferred embodiment of the culture method of the present application, in steps S1 and S2, the volume ratio of the Prakramium bacteria paste obtained in step S2 to the Prakramium fermentation broth is Prakramium bacteria paste: Prakramium fermentation broth = 1:(35-45).

[0030] As a preferred embodiment of the preservation method of the present application, in step S3, the cryoprotective agent comprises the following components in mass percentage: 10-15wt% of malt dextrin, 5-10wt% of trehalose, 1-5wt% of arginine, 1-5wt% of histidine, and the balance of water.

[0031] The present application screens the permeability, semi-permeability and non-permeability adjuvants, designs the PB experiment according to the influence of each adjuvant on the number of viable Prasillum, and obtains 12 kinds of cryoprotective agent formulations. The experiment proves that when the cryoprotective agent is 10-15wt% malt dextrin, 5-10wt% trehalose, 1-5wt% arginine and 1-5wt% histidine, the survival rate of Prasillum after low temperature preservation is still as high as 79.32%, which shows that the cryoprotective agent screened by the present application is more suitable for Prasillum.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] (1) The present application can effectively improve the number of viable bacteria in the fermentation broth of Prasillum by resuscitating, activating and fermenting Prasillum in YCFA liquid medium, and the number of viable bacteria can be as high as 10 9 CFU / mL.

[0034] (2) The present application screens the permeability, semi-permeability and non-permeability adjuvants, designs the PB experiment according to the influence of each adjuvant on the number of viable Prasillum, and obtains 12 kinds of cryoprotective agent formulations. The experiment proves that when the cryoprotective agent is 10-15wt% malt dextrin, 5-10wt% trehalose, 1-5wt% arginine and 1-5wt% histidine, the survival rate of Prasillum after low temperature preservation is still as high as 79.32%, which shows that the cryoprotective agent screened by the present application is more suitable for Prasillum. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is the normal graph of the standardization effect of each adjuvant analyzed in the PB experiment design process in Example 2.

[0036] Figure 2 It is the main effect graph of each adjuvant analyzed in the PB experiment design process in Example 2. DETAILED DESCRIPTION

[0037] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific examples.

[0038] Unless otherwise specified, other materials, reagents, etc. used in the examples can be obtained from commercial channels.

[0039] The inoculum is the ratio of the volume of seed liquid transferred to the volume of culture after inoculation, so the corresponding inoculum percentage is the volume percentage.

[0040] The following anaerobic environment is carried out in an anaerobic clean bench, an anaerobic incubator and an anaerobic station.

[0041] Example 1

[0042] The embodiment provides a culture method of the Clostridium prasrj, which comprises the following steps:

[0043] (1) inoculating glycerol bacteria of Clostridium prasrj into YCFA liquid medium at an inoculation amount of 10%, anaerobically and statically culturing for 16-19h to obtain a first-stage seed liquid;

[0044] (2) inoculating the first-stage seed liquid obtained in step (1) into YCFA liquid medium at an inoculation amount of 4%, anaerobically and statically culturing for 7-9h to obtain a second-stage seed liquid;

[0045] (3) inoculating the second-stage seed liquid obtained in step (2) into YCFA liquid medium at an inoculation amount of 0.5%, anaerobically and statically culturing for 16-19h to obtain a Clostridium prasrj fermentation liquid.

[0046] The formula of the YCFA liquid medium is shown in Table 1, and the preparation method is as follows: after mixing tryptone, yeast extract, sodium bicarbonate, glucose, maltose, cellobiose, cysteine hydrochloride, mineral solution I, mineral solution II, resazurin, organic acid mixed solution, hemin solution and vitamin solution I, water is added, the pH value is adjusted to 7.45, sterilization is performed, 1mL of filtered and sterilized vitamin solution II is added, and the medium is subjected to oxygen removal treatment.

[0047] Table 1 Formula of YCFA liquid medium

[0048]

[0049] Comparative Example 1

[0050] The comparative example provides a culture method of Clostridium prasrj, the steps of the culture method are similar to those of Example 1, except that the YCFA liquid medium in steps (1)-(3) is replaced by YCFA+ liquid medium, and the rest of the steps and parameter conditions remain unchanged; the formula of the YCFA+ liquid medium is similar to that of the YCFA liquid medium, except that the concentration of glucose is 0.5g / L, the concentration of maltose is 0, and the concentration of cellobiose is 0, and the rest of the components and their amounts remain unchanged.

[0051] Comparative Example 2

[0052] The comparative example provides a culture method of Clostridium prasrj, the steps of the culture method are similar to those of Example 1, except that the YCFA liquid medium in steps (1)-(3) is replaced by BHI liquid medium, and the rest of the steps and parameter conditions remain unchanged; the BHI liquid medium is prepared by weighing BHI dry powder and mixing with 1L of water, high-pressure sterilization and oxygen removal treatment, and the rest of the components and their amounts remain unchanged.

[0053] Comparative Example 3

[0054] This comparative example provides a culture method of Prasrptococcus, the steps of which are similar to Example 1, except that the YCFA liquid medium in steps (1)-(3) is replaced by mRCM liquid medium, and the remaining steps and parameter conditions remain unchanged; the formula of the mRCM liquid medium is 3 g / L yeast powder, 10 g / L beef powder, 10 g / L tryptone, 5 g / L glucose, 1 g / L soluble starch, 0.5 g / L sodium chloride, 3 g / L sodium acetate, 2 g / L DL-sodium lactate, 0.5 g / L cysteine hydrochloride, 0.2 mL / L 0.5 wt% methylene blue, water to a total volume of 1 L, after autoclaving, add 20 mL of sterile anhydrous ethanol, deoxygenate and reserve.

[0055] Effect Example 1

[0056] In order to screen the culture medium capable of improving the growth of Prasrptococcus, the inventors measured the OD 600 values of the primary seed liquids obtained in Example 1, Comparative Examples 1-3, and measured the OD 600 values of the primary seed liquids after 24 h and 40 h of continuous culture, and the results are shown in Table 2.

[0057] Table 2 Effect of different culture media on the growth of Prasrptococcus

[0058]

[0059] As shown in Table 2, the OD 600 value of Prasrptococcus cultured with YCFA medium is the highest, indicating that the growth rate of Prasrptococcus in YCFA medium is the highest, and therefore YCFA medium is selected as the main culture medium in the culture method of Prasrptococcus.

[0060] Effect Example 2

[0061] The OD 600 value and viable cell count of the Prasrptococcus fermentation broth obtained in Example 1 were detected, and the culture method of Example 1 was repeated 3 times, the OD 600 value and viable cell count of the Prasrptococcus fermentation broth were detected, and the results are shown in Table 3.

[0062] Table 3 OD 600 and viable cell count of Prasrptococcus fermentation broth at different fermentation times

[0063]

[0064] As shown in Table 3, the viable cell count of the Prasrptococcus fermentation broth prepared by the culture method of Prasrptococcus of the present application can reach 10 8 ~10 9 CFU / mL.

[0065] Example 2

[0066] In order to screen the optimal cryoprotectant composition to improve the freeze protection effect of Prasrptococcus, in this embodiment, the permeability, semi-permeability and non-permeability adjuvants are screened, and the parameter requirement is that the viable bacteria number of the freeze-dried powder is ≥10 10 CFU / mL, and the survival rate is ≥10%, and the specific operation of PB experimental design by using Minitab is as follows: open the Minitab software, execute: statistics > DOE > screening > create screening factors, and the design type is selected as Plackett-Burman, and the factor number is selected as the 8 factors listed in table 4. The normal chart and the main effect chart obtained by PB experimental design are shown in Figures 1-2 , 12 formulations are obtained, and water is set as a control group, and the above cryoprotectant formulations are shown in table 4.

[0067] Table 4 Formulations of different cryoprotectants

[0068]

[0069] The above cryoprotectant is mixed with the Prasrptococcus fermentation broth to prepare a freeze-dried powder, and the specific steps are as follows:

[0070] S1, centrifuge the Prasrptococcus fermentation broth obtained in example 1 at 4℃, 8000 rpm for 7 min to obtain Prasrptococcus bacteria;

[0071] S2, resuspend the Prasrptococcus bacteria obtained in step S1 by using phosphate buffer to obtain Prasrptococcus bacteria paste, and the volume ratio of the Prasrptococcus bacteria paste to the Prasrptococcus fermentation broth in step S1 is 1:40;

[0072] S3, mix the Prasrptococcus bacteria paste obtained in step S2 with the above cryoprotectant respectively, and freeze at -80℃, calculate the viable bacteria number after freezing, and the results are shown in table 5; the volume ratio of the Prasrptococcus bacteria paste to the cryoprotectant is 1:1.

[0073] Table 5 Effect of different cryoprotectants on the viable bacteria number of Prasrptococcus after freezing

[0074]

[0075] As shown in table 5, the survival rate of Prasrptococcus of test No. 1 is the highest, so the formulation of 10wt% malt dextrin, 5wt% trehalose, 1wt% arginine and 1wt% histidine is used as the cryoprotectant in the Prasrptococcus preservation method of the application.

[0076] Example 3

[0077] The application provides a Prasrptococcus preservation method, which comprises the following steps:

[0078] S1, centrifuging the Clostridium prasrifermentans fermentation broth obtained in Example 1 at 4°C to obtain Clostridium prasrifermentans bacteria;

[0079] S2, resuspending the Clostridium prasrifermentans bacteria obtained in step S1 with phosphate buffer to obtain Clostridium prasrifermentans bacteria slurry, wherein the volume ratio of the Clostridium prasrifermentans bacteria slurry to the Clostridium prasrifermentans fermentation broth is Clostridium prasrifermentans bacteria slurry: Clostridium prasrifermentans fermentation broth = 1:40;

[0080] S3, mixing the Clostridium prasrifermentans bacteria slurry obtained in step S2 with cryoprotective agent and storing at -80°C; wherein the volume ratio of the Clostridium prasrifermentans bacteria slurry to the cryoprotective agent is 1:1, and the components of the cryoprotective agent are shown in Table 6.

[0081] Table 6 Components and amounts of cryoprotective agent

[0082]

[0083] Example 4

[0084] This example provides a Clostridium prasrifermentans preservation method, which is similar to Example 3, except that the amounts of the components of the cryoprotective agent in step S3 are different, as shown in Table 6, and the other steps and parameter conditions remain unchanged. According to the method of Example 2, the survival rate of the Clostridium prasrifermentans of this example is 67.9%, and the number of viable bacteria after freezing is 3.6x10 10 CFU / mL.

[0085] Comparative Example 4

[0086] This comparative example provides a Clostridium prasrifermentans preservation method, which is similar to Example 3, except that the amounts of the components of the cryoprotective agent in step S3 are different, as shown in Table 6, and the other steps and parameter conditions remain unchanged. According to the method of Example 2, the survival rate of the Clostridium prasrifermentans of this example is 6.05%, and the number of viable bacteria after freezing is 3.21x10 9 CFU / mL.

[0087] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and do not limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A method for culturing a strain of Faecalibacterium prausnitzii, characterized in that, The method comprises the following steps: (1) inoculating glycerol bacteria of the Prasrptococcus sp. into YCFA liquid medium, and statically culturing at 35-37 DEG C under anaerobic conditions for 16-19 hours to obtain a first-stage seed liquid; (2) inoculating the first-stage seed liquid obtained in step (1) into YCFA liquid medium, and statically culturing at 35-37 DEG C under anaerobic conditions for 7-9 hours to obtain a second-stage seed liquid; (3) inoculating the second-stage seed liquid obtained in step (2) into YCFA liquid medium, and statically culturing at 35-37 DEG C under anaerobic conditions for 16-19 hours to obtain a Prasrptococcus sp. fermentation liquid; The YCFA liquid medium comprises the following components: 10 g / L of tryptone, 2.5 g / L of yeast extract, 4.0 g / L of sodium bicarbonate, 2.0 g / L of glucose, 2.0 g / L of maltose, 2.0 g / L of cellobiose, 1.0 g / L of cysteine hydrochloride, 150 mL / L of mineral solution I, 150 mL / L of mineral solution II, 1 mg / L of resazurin, 6.2 mL / L of organic acid mixture, 10.0 mL / L of hemin solution, 1.0 mL / L of vitamin solution I, and 1.0 mL / L of vitamin solution II; The mineral solution I in the YCFA liquid medium is a 3.0 g / L potassium phosphate dibasic aqueous solution; The mineral solution II in the YCFA liquid medium comprises the following components: 3.0 g / L of potassium dihydrogen phosphate, 6.0 g / L of ammonium sulfate, 6.0 g / L of sodium chloride, 0.6 g / L of magnesium sulfate heptahydrate, and 0.6 g / L of calcium chloride dihydrate; The hemin solution in the YCFA liquid medium comprises the following components: 2.8 g / L of potassium hydroxide, 250 mL / L of anhydrous ethanol, and 1.0 g / L of chloroferrihematin; The organic acid mixture in the YCFA liquid medium comprises the following components: 17.0 mL of acetic acid, 6.0 mL of propionic acid, 1.0 mL of n-valeric acid, 1.0 mL of iso-valeric acid, and 1.0 mL of iso-butyric acid; The vitamin solution I in the YCFA liquid medium comprises the following components: 0.01 g / L of biotin, 0.01 g / L of vitamin B12, 0.03 g / L of p-aminobenzoic acid, 0.05 g / L of folic acid, and 0.15 g / L of vitamin B6 hydrochloride; The vitamin solution II in the YCFA liquid medium comprises the following components: 0.05 g / L of thiamine hydrochloride and 0.05 g / L of riboflavin.

2. The culture method according to claim 1, wherein At least one of the following (I)-(III) is comprised: (I) in step (1), the inoculation amount of the glycerol bacteria of the Prasrptococcus sp. is 9-12%; (II) in step (2), the inoculation amount of the first-stage seed liquid is 4-6%; (III) in step (3), the inoculation amount of the second-stage seed liquid is 0.5-1%; The inoculation amount refers to the ratio of the volume of the seed liquid transferred and the volume of the culture liquid after inoculation, and thus the corresponding inoculation amount percentage is a volume percentage.

3. A method for preserving a Clostridium prasrificum, characterized by, The method comprises the following steps: S1, centrifuging the Prasrptococcus sp. fermentation liquid obtained in claim 1 at 4-8 DEG C to obtain Prasrptococcus sp. bacteria; S2, resuspending the Prasrptococcus sp. bacteria obtained in step S1 by using a phosphate buffer to obtain Prasrptococcus sp. bacteria paste; S3, mixing the obtained p. pullorum slurry with cryoprotectant, and storing at -80℃; the volume ratio of the p. pullorum slurry to the cryoprotectant is 1:1; In step S3, the cryoprotectant comprises the following components by mass percentage: 10-15wt% maltodextrin, 5-10wt% trehalose, 1-5wt% arginine, 1-5wt% histidine, and the balance is water.

4. The preservation method according to claim 3, wherein In step S1, the centrifugal condition is 7000-8000g of centrifugal speed and 6-8min of centrifugal time.

5. The method of claim 3, wherein, In steps S1 and S2, the volume ratio of the p. pullorum slurry obtained in step S2 to the p. pullorum fermentation liquid is p. pullorum slurry:p. pullorum fermentation liquid=1:(35-45).

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