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CN111304133B - Culture medium for culturing akkermansia muciniphila and use method and application thereof - Google Patents

Culture medium for culturing akkermansia muciniphila and use method and application thereof Download PDF

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CN111304133B
CN111304133B CN202010200526.XA CN202010200526A CN111304133B CN 111304133 B CN111304133 B CN 111304133B CN 202010200526 A CN202010200526 A CN 202010200526A CN 111304133 B CN111304133 B CN 111304133B
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akkermansia muciniphila
threonine
heart infusion
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CN111304133A (en
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朱永亮
宋伟群
朱浩
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Suzhou Preyson Biotechnology Co ltd
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Abstract

The invention provides a culture medium for culturing akkermansia muciniphila, and a use method and application thereof. The culture medium comprises brain heart infusion, porcine gastric mucin, L-cysteine hydrochloride, threonine, N-acetyl-D-glucosamine and vitamins. The culture medium is added with L-cysteine hydrochloride, threonine, N-acetyl-D-glucosamine and vitamins, so that the akkermansia muciniphila can still obtain enough nutrient substances in the culture medium containing the brain heart infusion solution with lower concentration and the pig stomach mucin, and the normal growth and the propagation of the bacterial strain are ensured. After the culture medium provided by the invention is used for culturing the akkermansia muciniphila, the OD value of the obtained bacterial liquid is 0.8211-0.8741.

Description

Culture medium for culturing akkermansia muciniphila and use method and application thereof
Technical Field
The invention belongs to the technical field of microbial culture media, and particularly relates to a culture medium for culturing akkermansia muciniphila, and a use method and application thereof.
Background
Akkermansia muciniphila (Akkermansia muciniphila) is called Akk bacteria for short, and is a gram-negative strict anaerobe. In 2004, professor delauna and her team isolated Akk bacteria in human intestines, which were ubiquitous in human digestive tracts, accounting for about 3-5%, degraded mucin in human intestines, negatively associated with obesity, diabetes, inflammation and metabolic disorders, and had the effects of delaying aging, inhibiting neurodegenerative diseases, reducing blood lipid and losing weight.
Low levels of Akk bacteria in the gut may cause thinning of the mucosal layer, which results in a reduced barrier function of the gut, making the toxins in the gut more accessible to the human body. Thus, akk bacteria are star bacteria in the last few years of intestinal microbiology research. Meanwhile, akk bacteria have the functions of improving various diseases of the body, a plurality of scholars regard the Akk bacteria as probiotics of the next generation, and the preparation taking the Akk bacteria as the main component must have wide development prospect and market.
Optimization of the Akk bacteria synthetic culture medium is necessary and important content for improving the yield of the Akk bacteria. At present, the culture of Akk at home and abroad mostly depends on a culture medium prepared from bovine brain heart infusion agar dry powder and porcine gastric mucin, and although the culture effect is still good, the cost is higher.
CN109810931A discloses a culture medium for culturing Ackermanella muciniphila and a using method thereof, belonging to the technical field of microbial culture media. The components of the culture medium comprise soybean peptone, threonine, glucose, N-acetylglucosamine, sodium chloride and disodium hydrogen phosphate. The culture medium can be used for culturing the bacterial quantity required by the functional research of the akkermansia muciniphila, the component sources of the culture medium are safe, and potential biological hazards do not exist. However, the culture medium can still culture and culture Akkermansia muciniphila without adding bovine brain heart infusion agar dry powder and porcine gastric mucin, and OD of Akk bacteria is obtained 600 The value is still low.
Therefore, it is reported whether the addition of other reagents is more beneficial to the culture of Akk bacteria and the reduction of the usage amount of bovine brain heart infusion dry powder and porcine gastric mucin and whether a better culture effect can be achieved. Therefore, the development of a culture medium which is beneficial to the growth of Akk bacteria and has low cost is a problem to be solved in the field.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a culture medium for culturing akkermansia muciniphila, a using method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a medium for culturing akkermansia muciniphila, the medium comprising brain heart infusion, porcine gastric mucin, L-cysteine hydrochloride, threonine, N-acetyl-D-glucosamine, and vitamins.
The culture medium comprises L-cysteine hydrochloride, threonine, N-acetyl-D-glucosamine and vitamins in addition to brain heart infusion and porcine gastric mucin. Cysteine is a reducing agent, can consume oxygen in a culture medium, and is beneficial to anaerobic growth of akkermansia muciniphila. Threonine, N-acetyl-D-glucosamine and vitamins can also improve the content of carbon and nitrogen in the culture medium, the N-acetyl-D-glucosamine is a basic composition unit of a plurality of important polysaccharides in organisms and can provide a carbon source, and the vitamins are used as growth factors and can promote the growth of bacteria and are all nutrient substances required for maintaining the normal growth and reproduction of the strains.
As a preferable technical scheme of the invention, the culture medium comprises 25-40g/L brain heart infusion, 1.5-6g/L pig gastric mucin, 0.1-5g/L L-cysteine hydrochloride, 1-10g/L threonine, 1-10g/L N-acetyl-D-glucosamine and vitamin with the mass fraction of 0.01-0.05%, wherein the mass fraction represents the ratio of the vitamin to the total mass of the culture medium.
When the concentration of each component in the culture medium is in the range, the culture medium has a good effect of culturing Akk bacteria, if the concentration is lower than the minimum concentration, the nutrient of the culture medium is possibly insufficient, and if the concentration is higher than the maximum concentration, the growth and the reproduction of the Akk bacteria are not coordinated, so that the growth condition is not good. In order to ensure that the strain can normally grow and develop, the culture medium must have enough carbon and nitrogen nutrients, and the proportion of carbon and nitrogen should be paid attention to, so that the requirements of the vegetative growth stage on the nutrients can be ensured, and the reproductive growth can be smoothly carried out.
In the present invention, the concentration by mass of the brain-heart infusion in the medium is 25 to 40g/L, and may be, for example, 25g/L, 26g/L, 28g/L, 30g/L, 32g/L, 35g/L, 36g/L, 38g/L or 40 g/L.
The mass concentration of the porcine gastric mucin in the culture medium is 0.5-2g/L, and can be, for example, 0.5g/L, 0.6g/L, 0.8g/L, 1g/L, 1.2g/L, 1.4g/L, 1.5g/L, 1.6g/L, 1.8g/L or 2 g/L.
The mass concentration of L-cysteine hydrochloride in the medium is 0.1-5g/L, and may be, for example, 0.1g/L, 0.5g/L, 1g/L, 1.2g/L, 1.5g/L, 1.8g/L, 2g/L, 2.4g/L, 3g/L, 3.5g/L, 4g/L, 5.5g/L, or 5 g/L.
The mass concentration of threonine in the culture medium is 1-10g/L, and can be 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L or 10g/L, etc.
The mass concentration of N-acetyl-D-glucosamine in the culture medium is 1-10g/L, and may be, for example, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, or 10 g/L.
The mass fraction of the vitamin in the medium may be 0.01 to 0.05%, for example, 0.01%, 0.012%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, or the like.
Preferably, the vitamin comprises vitamin K 1 。
Preferably, the medium is water as a solvent.
As a preferable technical scheme of the invention, the culture medium also comprises trace elements.
Preferably, the trace elements include any one or a combination of two or more of zinc, boron, copper, iron, molybdenum, nickel, manganese or cobalt.
Preferably, the content of the trace element in the medium is 0.05-0.15g/L, and may be, for example, 0.06g/L, 0.07g/L, 0.08g/L, 0.09g/L, 0.1g/L, 0.12g/L, 0.14g/L, or 0.15 g/L.
By adding the trace elements, the survival activity of the Akk bacteria can be further improved, and the Akk bacteria can grow more advantageously during culture.
As a preferred technical scheme of the invention, the solution containing the trace elements comprises zinc chloride (ZnCl) 2 ) Boric acid (H) 3 BO 3 ) Copper chloride dihydrate (CuCl) 2 ·2H 2 O), ferrous chloride tetrahydrate (FeCl) 2 ·4H 2 O), sodium molybdate dihydrate (Na) 2 MoO 4 ·2H 2 O), nickel chloride hexahydrate (NiCl) 2 ·6H 2 O), manganese chloride tetrahydrate (MnCl) 2 ·4H 2 O) or cobalt chloride hexahydrate (CoCl) 2 ·6H 2 O) or two of themCombinations of the above.
Preferably, the first and second electrodes are formed of a metal, the formulation of the solution containing the trace elements is 0.05-0.1g/L (e.g., 0.05g/L, 0.06g/L, 0.07g/L, 0.08g/L, 0.09g/L, or 0.1 g/L) zinc chloride, 0.05-0.1g/L (e.g., 0.05g/L, 0.06g/L, 0.07g/L, 0.08g/L, 0.09g/L, or 0.1 g/L) boric acid, 0.01-0.02g/L (e.g., 0.01g/L, 0.012g/L, 0.014g/L, 0.016g/L, 0.018g/L, or 0.02 g/L) copper chloride dihydrate, 1-2g/L (e.g., 1g/L, 1.2g/L, 1.4g/L, 1.6g/L, or 1.02 g/L) copper chloride 1.8g/L, 2g/L, etc.) ferrous chloride tetrahydrate, 0.02 to 0.03g/L (for example, 0.02g/L, 0.022g/L, 0.024g/L, 0.026g/L, 0.028g/L, 0.03g/L, etc.) sodium molybdate dihydrate, 0.02 to 0.03g/L (for example, 0.02g/L, 0.022g/L, 0.024g/L, 0.026g/L, 0.028g/L, 0.03g/L, etc.) nickel chloride hexahydrate, 0.05 to 0.15g/L (for example, 0.05g/L, 0.07g/L, 0.08g/L, 0.1g/L, 0.12g/L, 0.15g/L, etc.) manganese chloride tetrahydrate, and 0.1 to 0.15g/L cobalt chloride hexahydrate.
Preferably, the formulation of the solution containing the trace elements is: 0.07g/L of zinc chloride, 0.06g/L of boric acid, 0.015g/L of copper chloride dihydrate, 1.5g/L of ferrous chloride tetrahydrate, 0.025g/L of sodium molybdate dihydrate, 0.025g/L of nickel chloride hexahydrate, 0.1g/L of manganese chloride tetrahydrate and 0.12g/L of cobalt chloride hexahydrate.
In a preferred embodiment of the present invention, the culture medium further comprises a surfactant.
Preferably, the surfactant comprises tween-80. Tween-80 is a good emulsifier and dispersant, and can make nutrient components in the culture medium more uniform.
Preferably, the mass fraction of the surfactant (ratio of surfactant to the total weight of the medium) is 0.01 to 0.02%, and may be, for example, 0.01%, 0.012%, 0.014%, 0.015%, 0.016%, 0.017%, 0.018%, 0.019%, 0.02%, or the like.
As a preferable technical scheme of the invention, the content of each component in the culture medium is as follows:
25-40g/L brain heart infusion, 1.5-5g/L pig gastric mucin, 0.5-2g/L L-cysteine hydrochloride, 3-10g/L threonine, 3-10g/L N-acetyl-D-glucosamine, and the mass fraction is 0.01-0.02%Vitamin K 1 0.05-0.15g/L of trace elements and 0.01-0.02% of Tween-80 by mass fraction.
Preferably, the content of each component in the culture medium is as follows: 27-30g/L brain and heart infusion, 2-2.5g/L pig gastric mucin, 1.4-1.6g/L L-cysteine hydrochloride, 4-6g/L threonine, and vitamin K with mass fraction of 0.014-0.016% 1 4-6g/L of N-acetyl-D-glucosamine, 0.08-0.12g/L of trace elements and 0.014-0.016% of Tween-80.
For example, the content of each component in the culture medium can be 29.6g/L brain heart infusion, 1.152g/L pig gastric mucin, 1.5g/L L-cysteine hydrochloride, 5g/L threonine, and 0.015% by mass of vitamin K 1 5g/L of N-acetyl-D-glucosamine, 0.1g/L of trace elements and 0.015 percent of Tween-80 by mass fraction.
In a second aspect, the present invention provides a method of using a culture medium as in the first aspect, the method comprising the steps of:
the bacterial liquid of akkermansia muciniphila is inoculated into the culture medium according to the first aspect for anaerobic culture.
In the invention, the adhesive protein akkermansia anserina feces is extracted and separated, and the specific steps are as follows:
(1) Sample treatment: putting 150mL of physiological saline solution into a 250mL conical flask, sealing, putting the conical flask and the culture medium into an autoclave together, sterilizing at 121 ℃ for 20 minutes, taking out, and cooling. Adding 25g of feces of normal healthy people, shaking, filtering to remove insoluble substances, performing gradient dilution, and selecting 10 -3 To 10 -9 And (3) performing seven gradients, centrifuging at 3000r/min, cooling the culture medium to 55 ℃, pouring 15-20mL of the culture medium into the flat plate, then respectively injecting 0.2mL of the culture medium into the flat plate containing the culture medium by using a pipette, uniformly coating, solidifying, and pouring into a closed container containing an anaerobic gas generating bag at 37 ℃ for culturing.
(2) And (3) separation culture: after culturing for 2 weeks at 37 ℃, observing colony morphology, performing gram staining microscopy, selecting a milky colony without impurities around the colony from a solid medium plate, taking 0.2mL of bacterial liquid, coating the bacterial liquid on the plate, and screening until only one bacterium is on the plate, wherein the obtained strain is akkermansia muciniphila.
As a preferable technical scheme of the invention, the concentration of the bacterial liquid is (0.8-1.2) multiplied by 10 8 cfu/mL, for example, may be 0.8X 10 8 cfu/mL、0.85×10 8 cfu/mL、0.9×10 8 cfu/mL、0.95×10 8 cfu/mL、1×10 8 cfu/mL、1.05×10 8 cfu/mL、1.1×10 8 cfu/mL or 1.2X 10 8 cfu/mL, etc.
Preferably, the inoculum size of the bacterial suspension is 100-200. Mu.L, such as 110. Mu.L, 120. Mu.L, 130. Mu.L, 140. Mu.L, 150. Mu.L, 160. Mu.L, 170. Mu.L, 180. Mu.L, 190. Mu.L or 200. Mu.L.
Preferably, the temperature of the anaerobic culture is 35-40 ℃, for example, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃ or 40 ℃.
Preferably, the anaerobic culture time is 72-96h, such as 72h, 75h, 78h, 80h, 82h, 85h, 88h, 90h, 92h, 94h or 96h.
As a preferable technical scheme of the invention, the using method comprises the following steps: inoculating a bacterial liquid of Ackermanella muciniphila into the culture medium of the first aspect, wherein the concentration of the bacterial liquid is (0.8-1.2). Times.10 8 cfu/mL, the inoculum size is 100-200. Mu.L, then anaerobic culture is carried out for 72-96h at 35-40 ℃.
In a third aspect, the present invention also provides a use of akkermansia muciniphila cultured by the culture medium according to the first aspect in the preparation of a medicament for treating metabolic diseases.
The recitation of numerical ranges herein includes not only the above-recited numerical values, but also any numerical values between any of the above-recited numerical ranges not recited, and for the sake of brevity and clarity, the present invention is not intended to be exhaustive of the specific numerical values encompassed within the range.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) The culture medium provided by the invention comprises brain-heart infusion and porcine gastric mucinL-cysteine hydrochloride, vitamin K 1 Threonine and N-acetyl-D-glucosamine, wherein the culture medium can provide required nutrient elements for the growth and the propagation of Akk bacteria, and meanwhile, the culture medium can obtain more viable count than that obtained by culturing in a mucin culture medium by further adding trace elements and a Tween-80 reagent, which shows that the culture medium provided by the invention can completely realize the in-vitro amplification culture of Akk.
(2) After the Akk bacteria are cultured by the culture medium through a proper culture method, the OD value of the obtained strain is 0.8211-0.8741, and the strain in the culture medium is identified to be the Akk bacteria through a PCR technology, so that the culture medium provided by the invention is beneficial to the culture of the Akk bacteria, is convenient for preparing the Akk bacteria into preparations at the later stage, and can realize subsequent functional research and industrial development.
Drawings
FIG. 1 is a DNA sequence comparison of Akk bacteria cultured in example 1 and standard Akk bacteria in a database.
Detailed Description
The technical solutions of the present invention are further described by the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.
In the following examples, the strains and experimental reagents used were purchased from conventional manufacturers, and the specific sources are shown in table 1 below:
TABLE 1
Laboratory strains or reagents Company (SA)
Physiological saline Guangzhou Ruishu biological reagent Co., ltd
Brain heart infusion Solarbio
Porcine gastric mucin SIGMA
Threonine Shanghai Bioengineering Co., ltd
N-acetyl-D-glucosamine Shanghai Bioengineering Co., ltd
Ethanol Shanghai national medicine group
Tween-80 Shanghai Bioengineering Co., ltd
Fast pfu DNA polymerase kit Beijing Quan Shijin Biotechnology Co., Ltd.
Primer and method for producing the same Shanghai Bioengineering Co., ltd
ZnCl 2 Tianjin Yuanzhi chemical reagents Co., ltd
H 3 BO 3 Tianjin Chengyuan chemical reagent Co., ltd
CuCl 2 ·2H 2 O Tianjin Chengyuan chemical reagent Co., ltd
FeCl 2 ·4H 2 O Tianjin Chengyuan chemical reagent Co., ltd
Na 2 MoO 4 ·2H 2 O Tianjin Chengyuan chemical reagent Co., ltd
NiCl 2 ·6H 2 O Shanghai national medicine group
MnCl 2 ·4H 2 O Tianjin Yuanzhi chemical reagents Co., ltd
CoCl 2 ·6H 2 O Tianjin Chengyuan chemical reagent Co., ltd
In the following examples, the formulation of the trace elements is shown in table 2 below:
TABLE 2
Chemical composition Content (g/L)
ZnCl 2 0.07
H 3 BO 3 0.06
CuCl 2 ·2H 2 O 0.015
FeCl 2 ·4H 2 O 1.5
Na 2 MoO 4 ·2H 2 O 0.025
NiCl 2 ·6H 2 O 0.025
MnCl 2 ·4H 2 O 0.1
CoCl 2 ·6H 2 O 0.12
In the following examples, the sources of the experimental instruments are shown in table 3 below:
TABLE 3
Laboratory apparatus Company(s)
Magnetic stirrer Hangzhou rice Europe apparatus LimitedCompany(s)
Electronic balance SHANGHAI SUNNY HENGPING SCIENTIFIC INSTRUMENT Co.,Ltd.
High-speed centrifugal machine ThermoFisher
Vertical high-pressure steam sterilizer Shanghai Shen'an medical device factory
Enzyme mark instrument ZHENGZHOU BOSAI BIOENGINEERING Co.,Ltd.
Example 1
This embodiment provides a culture medium comprising:
7.4g of brain-heart infusion, 0.8g of porcine gastric mucin, 0.3g of L-cysteine hydrochloride, 1.0g of threonine and 0.015 percent of vitamin K by mass fraction 1 Adding 1.0g of N-acetyl-D-glucosamine, adding water to a constant volume of 200mL, stirring for 30min, mixing, transferring to a conical flask, and sterilizing.
Example 2
This embodiment provides a culture medium, the culture medium comprising:
7.4g of brain-heart infusion, 0.8g of porcine gastric mucin, 0.3g of L-cysteine hydrochloride, 1.0g of threonine and 0.015 percent of vitamin K 1 Adding water to a constant volume of 200mL, stirring for 30min, transferring into a conical flask, and sterilizing.
Example 3
This embodiment provides a culture medium comprising:
7.4g brain heart infusion, 0.8g pig stomach mucin, 0.3g L-cysteine hydrochloride, 1.0g threonine0.015 percent of vitamin K by mass fraction 1 1.0g of N-acetyl-D-glucosamine, 0.1mL of trace elements and 0.015% of Tween-80 by mass fraction, adding water to a constant volume of 200mL, stirring for 30min, uniformly mixing, transferring into a conical flask, and sterilizing.
Example 4
This example provides a medium which differs from example 3 only in that the amount of porcine gastric mucin is 0.48g.
Example 5
This example provides a medium which differs from example 3 only in that the amount of porcine gastric mucin is 0.4g.
Example 6
This embodiment provides a culture medium comprising:
7.4g of brain-heart infusion, 0.4g of porcine gastric mucin, 0.45g of L-cysteine hydrochloride, 1.5g of threonine and 0.02 mass percent of vitamin K 1 1.5g of N-acetyl-D-glucosamine, 0.15mL of trace elements and 0.02% of Tween-80 by mass fraction, adding water to a constant volume of 200mL, stirring for 30min, uniformly mixing, transferring into a conical flask, and sterilizing.
Example 7
This example provides a medium which differs from example 3 in that: the weight of the brain-heart infusion is 5.92g, and the weight of the pig stomach mucin is 0.48g.
Example 8
This example provides a medium which differs from example 3 in that: the weight of the brain-heart infusion is 5.5g, and the weight of the pig stomach mucin is 0.48g.
Example 9
The difference from example 3 is that the mass of L-cysteine hydrochloride in the medium is 1g.
Example 10
The difference from example 3 is that the mass of L-cysteine hydrochloride in the medium is 0.1g.
Example 11
The difference from example 3 is that the mass of threonine in the medium is 2g.
Example 12
The difference from example 3 is that the mass of threonine in the medium is 0.5g.
Example 13
The difference from example 3 is that the medium vitamin K 1 Is 0.05% by mass.
Example 14
The difference from example 3 is that the mass of N-acetyl-D-glucosamine in the medium is 2g.
Comparative example 1
This comparative example provides a culture medium comprising:
adding water into 7.4g brain-heart infusion and 0.8g pig stomach mucin, diluting to constant volume of 200mL, stirring for 30min, transferring into conical flask, and sterilizing.
Comparative example 2
This comparative example provides a culture medium comprising:
7.4g of brain-heart infusion, 0.8g of porcine gastric mucin and 0.3g of L-cysteine hydrochloride, adding water to a constant volume of 200mL, stirring for 30min, uniformly mixing, transferring into a conical flask, and sterilizing.
Comparative example 3
This comparative example provides a culture medium comprising:
adding water to a volume of 200mL for 7.4g of brain-heart infusion, 0.8g of pig gastric mucin, 0.3g of L-cysteine hydrochloride and 1.0g of threonine, stirring for 30min, transferring into a conical flask, and sterilizing.
Comparative example 4
The present comparative example provides a culture medium comprising:
7.4g brain heart infusion, 0.8g pig stomach mucin, 0.3g L-cysteine hydrochloride, 1.0g threonine, 0.2mL vitamin K 1 Adding water to a constant volume of 200mL, stirring for 30min, mixing, transferring to a conical flask, and sterilizing.
Comparative example 5
The difference from example 3 is that L-cysteine hydrochloride is replaced by lysine hydrochloride in the medium.
Comparative example 6
The difference from example 3 is that N-acetyl-D-glucosamine is replaced by glucose in the medium.
Comparative example 7
The difference from example 3 is that the medium has trace elements replaced by a single iron element.
Performance test
Ackermanella muciniphila was cultured using the media provided in examples 1 to 16 and comparative examples 1 to 7.
OD value detection
Sterilizing the culture medium at high temperature, cooling, inoculating Ackermansia muciniphila (1 × 10) 8 cfu/mL, 200. Mu.L), inoculating, placing in a closed container, placing two anaerobic gas generating bags at the same time, culturing at 37 ℃, taking out after 4 days, centrifuging at 3000rpm for 10min, then resuspending the precipitate with 2mL sterile PBS, adding into a 96-well plate, 200. Mu.L per well, and using PBS as blank control.
OD value of the bacterial liquid was measured at 630nm using a microplate reader, and the measurement was repeated three times, and the average value was obtained, and the results are shown in Table 4.
TABLE 4
Figure BDA0002419205750000131
Figure BDA0002419205750000141
As shown in the table, the Akk bacteria culture medium provided by the invention can ensure that the bacterial strains can normally grow, and the OD values of the Akk bacteria culture medium are all larger than 0.82; and as can be seen from examples 3, 4 and 5, when the content of the porcine gastric mucin was changed alone, the culture effect was best when the mass of the porcine gastric mucin was 0.48 g; from examples 4, 7 and 8, it is understood that the culture effect is best when the brain-heart infusion content is changed alone, and the brain-heart infusion content is 5.92.
As can be seen from the above table, the optimal scheme of the culture medium prepared in the invention is as follows: 5.92g brain heart infusion, 0.48g pig stomach mucin, 0.3g L-cystatinAmino acid hydrochloride, 1.0g threonine, 0.2mL vitamin K 1 1.0g of N-acetyl-D-glucosamine, 0.1mL of trace elements and 0.1mL of Tween-80, and adding water to a constant volume of 200mL.
2. Purification of mucins
The mucin has more impurities, the impure culture medium is turbid and opaque, the impurities can be removed after purification, and the culture medium is transparent and not turbid.
Dissolving mucin in water, and stirring at 30-35 deg.C for 5 hr to obtain mixed solution; centrifuging the mixed solution at 3000rpm for 10min, collecting supernatant, adding ethanol, standing for 4h, removing supernatant to obtain precipitate, dissolving with water, repeatedly washing with ethanol for 2 times, and dissolving the precipitate with water to obtain purified mucin.
3. Identification of the type of Strain in the Medium
Extracting the cultured AKK bacteria DNA, detecting the DNA concentration by using nanodrop 2000, taking 5 mu L of DNA, and amplifying the AKK bacteria DNA sequence by using the following primers:
upstream primer (SEQ ID NO. 1): CAAGTCGAACGAGAGAATTGCTAGC,
downstream primer (SEQ ID NO. 2): CATCCCAGTTACAGTCCACCTT.
The PCR reaction system is shown in Table 5 below:
TABLE 5
Reagent Content (μ L)
Fast pfu DNA polymerase 1
5X Fast pfu buffer 10
dNTP(2.5mM) 2
AKK upstream primer (10. Mu.M) 2
AKK downstream primer (10. Mu.M) 2
AKK bacteria DNA (34 ng/. Mu.L) 5
3dH 2 O 28
Total volume 50
The PCR reaction conditions are shown in Table 6 below:
TABLE 6
Figure BDA0002419205750000151
Figure BDA0002419205750000161
Adding 5 mu L of total PCR products into 1 mu L of DNA loading buffer, performing electrophoresis by using 1% agarose gel, collecting pictures by using a Chemi Doc XRS + imager, sending the rest PCR products to Shanghai biological engineering Limited company for sequencing by using an upstream primer, wherein the obtained sequence is SEQ ID NO.3, comparing the obtained result by using a BLAST program of NCBI, and the result of comparing the sequencing result with the data of Akk bacteria in the NCBI is shown in figure 1, wherein the data similarity is 100%, and the cultured strains are all Akk bacteria.
In conclusion, the culture medium provided by the invention can provide required nutrient elements for the growth and propagation of Akk bacteria, can obtain more viable bacteria than the number of the viable bacteria cultured in a mucin culture medium, completely realizes the in-vitro expansion culture of the Akk bacteria, and has the advantages of good culture effect and low culture cost.
The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must be implemented depending on the above detailed structural features. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> Suzhou Prisisen Gene science and technology, inc
<120> culture medium for culturing akkermansia muciniphila, and use method and application thereof
<130> 20200313
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> Artificial Synthesis
<400> 1
caagtcgaac gagagaattg ctagc 25
<210> 2
<211> 24
<212> DNA
<213> Artificial Synthesis
<400> 2
catcccagtt accagtctca cctt 24
<210> 3
<211> 480
<212> DNA
<213> Akkermansia muciniphila
<400> 3
gggtgagtaa cacgtgagta acctgccccc cagagcggga tagccctggg aaactgggat 60
taataccgca tagtatcgaa agattaaagc agcaatgcgc ttggggatgg gctcgcggcc 120
tattagttag ttggtgaggt aacggctcac caaggcgatg acgggtagcc ggtctgagag 180
gatgtccggc cacactggaa ctgagacacg gtccagacac ctacgggtgg cagcagtcga 240
gaatcattca caatggggga aaccctgatg gtgcgacgcc gcgtggggga atgaaggtct 300
tcggattgta aacccctgtc atgtgggagc aaattaaaaa gatagtacca caagaggaac 360
agacggctaa ctctgtgcca gcagccgcgg taatacagag gtctcaagcg ttgttcggaa 420
tcactgggcg taaagcgtgc gtaggctgtt tcgtaagtcg tgtgtgaaag gcgcgggctc 480

Claims (8)

1. A culture medium for culturing Ackermanella muciniphila, which is characterized by comprising the following components:
28-30g/L brain-heart infusion, 2-2.5g/L pig gastric mucin, 1.4-1.6g/L L-cysteine hydrochloride, 4-6g/L threonine, 4-6g/L N-acetyl-D-glucosamine, 0.014-0.016% of vitamin K1, 0.08-0.12g/L of trace elements and 0.014-0.016% of tween-80;
wherein, the formula of the solution containing the trace elements is as follows: 0.05-0.1g/L of zinc chloride, 0.05-0.1g/L of boric acid, 0.01-0.02g/L of copper chloride dihydrate, 1-2g/L of ferrous chloride tetrahydrate, 0.02-0.03g/L of sodium molybdate dihydrate, 0.02-0.03g/L of nickel chloride hexahydrate, 0.05-0.15g/L of manganese chloride tetrahydrate and 0.1-0.15g/L of cobalt chloride hexahydrate.
2. The culture medium according to claim 1, wherein the culture medium is water as a solvent.
3. A method of using the medium according to claim 1 or 2, comprising the steps of:
the culture medium according to claim 1 or 2, wherein a bacterial solution of akkermansia muciniphila is inoculated, and anaerobic culture is performed.
4. The use method according to claim 3, wherein the concentration of the bacterial liquid is (0.8-1.2) × 10 8 cfu/mL。
5. The use method according to claim 3, wherein the inoculation amount of the bacterial liquid is 100 to 200. Mu.L.
6. Use according to claim 3, wherein the temperature of the anaerobic cultivation is 35-40 ℃.
7. The use according to claim 3, wherein the anaerobic cultivation is carried out for 72-96h.
8. Use according to claim 3, characterized in that it comprises the following steps:
inoculating the culture medium according to claim 1 or 2 with a bacterial solution of Ackermanella muciniphila, the concentration of which is (0.8-1.2). Times.10 8 cfu/mL, the inoculation amount is 100-200 mu L, and then anaerobic culture is carried out for 72-96h at 35-40 ℃.
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