Counting medium for akkermansia muciniphila and use thereof

Abstract

The application belongs to the field of microorganism counting, and particularly relates to a counting culture medium of Akkermansia muciniphila and application thereof. Components of the counting culture medium comprise: tryptone, tryptone soy broth, brain heart infusion powder, sodium chloride, glucose, sodium bicarbonate (12H2O), special proteose peptone, yeast extract, cysteine hydrochloride, hemin chloride, agar powder, Tris Buffer and sterile defibrinated sheep blood. The counting culture medium provided by the application solves the technical defects of low counting result or incapability of counting caused by unsuitable nutritional components of the existing counting culture medium. In addition, the counting culture medium of the application also significantly shortens the counting period. Therefore, the counting culture medium has high application value in probiotic product inspection and / or quality control.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganism counting, and particularly relates to a counting culture medium of Akkermansia muciniphila and application thereof. BACKGROUND

[0002] As the main component of microecological preparation, probiotics can regulate intestinal flora balance and improve the body's immunity, and has been widely used in ordinary food, health food, medicine, agriculture and other fields. The viable count level of probiotics is directly related to the quality of these products and affects the probiotic effect of probiotics. Therefore, the viable count of probiotics in food is the core content of the inspection of such food. At present, the product standard of our country stipulates that the viable count of lactic acid bacteria in fermented dairy products and live bacteria type lactic acid bacteria beverages shall not be less than 1x10 6 CFU / g (mL), and the detection method standard is generally GB 4789.35-2016.

[0003] At present, the culture medium for counting probiotics in the field is mostly MRS culture medium, modified MRS culture medium, YPD culture medium, etc. Chinese patent application 202010817025.6 discloses a bacillus coagulans counting culture medium, which is YPD solid culture medium with the following formula by mass percentage: 2% glucose, 2% peptone, 1% yeast powder, pH adjusted to 7.0 with sodium hydroxide, 1.5%-2.0% agar powder, sterilized at 121℃ for 20min. Chinese patent application 201610571863.3 discloses a selective counting culture medium for Lactobacillus bulgaricus, which consists of the following components: by weight, glucose 8.8-11.2 parts, soybean peptone 8.5-11.5 parts, yeast powder 4.6-5.4 parts, K2HPO4 1.8-2.2 parts, agar 12.0-18.0 parts, anhydrous sodium acetate 23.0-27.0 parts.

[0004] At present, there is no special viable counting culture medium for Akkermansia muciniphila in the field. SUMMARY

[0005] Akkermansia muciniphila is a kind of intestinal symbiotic bacteria belonging to Verrucomicrobiae, which has multiple genetic branches and phenotypic diversity. With the development of science and technology, more and more studies have shown that Akkermansia muciniphila has unique efficacy in metabolic diseases, cancer and immunotherapy, and is expected to fight diseases at the microbial level and become the next generation of probiotics.

[0006] Since Akkermansia muciniphila has extremely high requirements for nutritional conditions, when tested by using the existing probiotic counting medium, the counting result is low or cannot be counted.

[0007] In order to overcome the defects of the prior art, the present application hereby proposes the following technical solutions:

[0008] In one aspect, the present application provides a counting medium for Akkermansia muciniphila, the components of which include: tryptone, tryptone soy broth, brain heart infusion powder, sodium chloride, glucose, sodium bicarbonate (12H2O), special proteose peptone, yeast extract, cysteine hydrochloride, hemin chloride, agar powder, Tris Buffer and sterile defibrillated sheep blood.

[0009] In some embodiments, the counting medium comprises at least 1.5 g / L special proteose peptone.

[0010] In some embodiments, the counting medium comprises 1.5-3.5 g / L special proteose peptone.

[0011] In some preferred embodiments, the counting medium comprises 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 g / L special proteose peptone.

[0012] In some preferred embodiments, the counting medium comprises 2.5 g / L special proteose peptone.

[0013] In some embodiments, the counting medium comprises at least 4.0 g / L tryptone soy broth.

[0014] In some embodiments, the counting medium comprises 4.0-6.0 g / L tryptone soy broth.

[0015] In some preferred embodiments, the counting medium comprises 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 g / L tryptone soy broth.

[0016] In some embodiments, the counting medium comprises at least 0.3 g / L Tris Buffer.

[0017] In some embodiments, the counting medium comprises 0.3-0.5 g / L Tris Buffer.

[0018] In some preferred embodiments, the counting medium comprises 0.375 g / L Tris Buffer.

[0019] In some preferred embodiments, the components of the counting medium comprise: Tryptone 5.0 g / L, Tryptone Soy Broth 5.0 g / L, Brain Heart Infusion 8.75 g / L, Sodium Chloride 2.5 g / L, Glucose 3.5 g / L, Sodium Bicarbonate (12H20) 1.25 g / L, Special Peptone 2.5 g / L, Yeast Extract 2.5 g / L, Cysteine Hydrochloride 0.2 g / L, Hematinic Chloride 0.005 g / L, Agar Powder 20 g / L, Tris Buffer 0.375 g / L, 5% (v / v) sterile defibrinated sheep blood.

[0020] In some embodiments, the counting medium further comprises trace element salts.

[0021] In some embodiments, the trace element salts comprise, but are not limited to, K2HPO4·7H2O, MgSO4·7H2O or MnSO4·4H2O.

[0022] In some embodiments, the counting medium has a pH of 7.3-7.7.

[0023] In another aspect, the present application provides the use of any of the foregoing counting media for the enumeration of Akkermansia muciniphila.

[0024] In yet another aspect, the present application provides the use of any of the foregoing counting media for the testing and / or quality control of probiotic products comprising Akkermansia muciniphila.

[0025] In yet another aspect, the present application provides a reagent for the enumeration of Akkermansia muciniphila, the reagent comprising any of the foregoing counting media.

[0026] In yet another aspect, the present application provides a kit for the enumeration of Akkermansia muciniphila, the kit comprising any of the foregoing counting media or any of the foregoing reagents.

[0027] In some embodiments, the kit further comprises a container containing the counting medium or reagent.

[0028] In some embodiments, the kit further comprises a diluent, preferably 0.9% (v / v) physiological saline.

[0029] In some embodiments, the kit further comprises a magnifying glass or a colony counter.

[0030] Compared with the existing counting medium, the counting medium provided by the present application is suitable for counting the viable bacteria of Akkermansia muciniphila, and solves the technical defects of the existing counting medium that the nutritional components are not suitable or cannot culture Akkermansia muciniphila, resulting in low counting results, inaccurate counting or inability to count.

[0031] The counting medium of the present application significantly improves the viability of Akkermansia muciniphila during the counting process, and the viable bacteria of Akkermansia muciniphila are significantly improved by about 4 orders of magnitude compared with the traditional BHI counting medium; through the research of the inventors, the counting medium provided by the national standard GB 4789.35-2016 cannot culture Akkermansia muciniphila. In addition, the counting medium provided by the present application also has the advantage of short counting period, which can shorten the counting period to 3 days. Therefore, the counting medium of the present application has high application value in probiotic product testing and / or quality control.

[0032] Deposit information:

[0033] Biological material: AKK PROBIO

[0034] Classification and naming: Akkermansia muciniphila

[0035] Deposit number: CGMCC No. 20955

[0036] Preservation time: October 26, 2020

[0037] Preservation unit: China General Microbiological Culture Collection Center (CGMCC)

[0038] Preservation address: No. 3, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0039] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. For the purpose of interpreting the present specification, the following definitions will apply, and the terms used in the singular will also include the plural, and vice versa, as appropriate.

[0040] The expressions "a" and "an" as used herein include plural references unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0041] Numerical ranges as used herein are intended to include all numbers and combinations of numbers within the range. For example, a range of 1 to 20 is intended to include any number from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0042] As used herein, the terms "comprises" or "comprising" mean "including, but not limited to." This term is intended to be open-ended to designate any stated feature, element, integer, step, or component, but does not preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of." In one embodiment, the term "comprising" as used throughout the application, and particularly in the claims, is to be interpreted as "consisting of."

[0043] As used herein, the terms "optional," "any of," "any," or "any one of" mean that the subsequently described event or circumstance can or can not occur, and the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally comprising one antibody heavy chain variable region" means that an antibody heavy chain variable region of a particular sequence can or can not be present. As used herein, "a" and "an" are used in the present application to refer to one or more than one grammatical object.

[0044] As used herein, the term "and / or" is to be taken as specific embodiments of the inclusive- or. For example, in the phrase "A and / or B" the events A and B are alternatives that are encompassed by the expression.

[0045] Reagents and materials used in the present application:

[0046] Special Peptone, purchased from Thermo Fisher, Cat. No. LP0072B.

[0047] Tryptic Soy Broth, purchased from Thermo Fisher, Cat. No. CM0129R.

[0048] ATCC Standard Strain BAA-835 was purchased from American Type Culture Collection (ATCC).

[0049] MRS Broth was purchased from Qingdao Haibo Bioengineering, Cat. No. HB0384-1.

[0050] Counting medium: Tryptone 4.0-6.0 g / L, Tryptone soya broth 4.0-6.0 g / L, brain heart infusion powder 8-10 g / L, sodium chloride 2.5-3.5 g / L, glucose 2.5-4.5 g / L, sodium bicarbonate (12H2O) 1.0-2.0 g / L, special tryptone 1.5-3.5 g / L, yeast extract 2.0-4.0 g / L, cysteine hydrochloride 0.1-0.3 g / L, hemin chloride 0.004-0.006 g / L, agar powder 10-30 g / L, Tris Buffer 0.3-0.5 g / L, 4-6% (v / v) sterile defibrillated sheep blood.

[0051] The colony counting method of the present embodiment:

[0052] • The dilution fold and the corresponding number of colonies can be observed with the naked eye, if necessary, with a magnifying glass or a colony counter. The colony count is expressed in colony-forming units (CFU).

[0053] • The total number of colonies on the plate is counted, with the number of colonies between 30-300 CFU and without the growth of spreading colonies. The number of colonies of each dilution should be the average of two plates.

[0054] • If one of the plates has a large patch of colony growth, it is not suitable to be used, and the plate without patchy colony growth should be used as the number of colonies of this dilution. If the patchy colony is less than half of the plate, and the remaining half has a very uniform distribution of colonies, the number of colonies of half a plate can be calculated and multiplied by 2 to represent the number of colonies of one plate.

[0055] • When there is chain growth without obvious boundaries between colonies on the plate, each single chain is counted as a colony.

[0056] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. All reagents or instruments not mentioned by the manufacturers are the conventional products that can be purchased in the market. In order to better illustrate the present application, numerous specific details are given in the following specific embodiments. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, the description of the well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present application. Such structures and technologies are also described in many publications, such as Molecular Cloning: A Laboratory Manual (Fourth Edition) (Cold Spring Harbor Laboratory Press), Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience.

[0057] Example 1 Live count of Akkermansia muciniphila

[0058] The cryotubes of Akkermansia muciniphila AKK-PROBIO (the strain is preserved in CGMCC with the preservation number of CGMCC No. 20955) and AKK-835 (ATCC standard strain BAA-835) were selected as the samples to be tested. 1.0 mL of the sample to be tested was taken with a micropipette sterile suction head with a specification of 1.0 mL, and was vertically injected into a sterile test tube containing 9 mL of physiological saline with a concentration of 0.9%, and was uniformly mixed by a vortex oscillator (the rotation of the liquid in the test tube to the bottom was counted as the starting time, and the oscillation was 10 s-15 s), to prepare a 10-fold diluted sample homogenate.

[0059] According to the above operation sequence, the gradient increasing dilution of 10 times was sequentially carried out.

[0060] 100 μL of the sample homogenate from the test tube with an appropriate dilution (10 -5 , 10 -6 , 10 -7 CFU / mL) was respectively taken and was coated on the counting medium of the present application, two parallel samples were prepared for each dilution, and 100 μL of the blank diluent was respectively taken and was coated on the counting medium of the present application as a blank control, and was inverted and cultured at 37°C in anaerobic condition for 3-5 days.

[0061] The components of the counting medium are tryptone 5.0 g / L, tryptone soya broth 5.0 g / L, brain heart infusion powder 8.75 g / L, sodium chloride 2.5 g / L, glucose 3.5 g / L, sodium bicarbonate (12H2O) 1.25 g / L, special proteose peptone 2.5 g / L, yeast extract 2.5 g / L, cysteine hydrochloride 0.2 g / L, hemin chloride 0.005 g / L, agar powder 20 g / L, Tris Buffer 0.375 g / L, and 5% (v / v) sterile defibrinated sheep blood. The pH of the counting medium is adjusted to 7.5±0.2. The medium is sterilized at 121°C for 15 min, and the sterile defibrinated sheep blood is added after the medium is cooled to below 60°C.

[0062] The number of colonies of Akkermansia muciniphila is selected to be between 30-300 CFU, and the plate without the growth of spreading colonies is selected. The total number of colonies of Akkermansia muciniphila is calculated, and the number of colonies of each dilution is the average of two plates.

[0063] If only the number of colonies on the plate of one dilution is within the appropriate counting range, the average of the number of colonies on two plates is calculated first, and then the average is multiplied by the corresponding dilution factor to obtain the total number of colonies per milliliter of the sample to be tested.

[0064] If the number of colonies on the plates of two consecutive dilutions is within the appropriate counting range, the following formula is used for calculation:

[0065]

[0066] In the formula:

[0067] N is the number of viable Akkermansia muciniphila (CFU) in the sample to be tested;

[0068] ∑C is the sum of the number of colonies on the plates (including the plates with the number of colonies within the appropriate range);

[0069] n1 is the number of plates of the first dilution (low dilution factor);

[0070] n2 is the number of plates of the second dilution (high dilution factor);

[0071] d is the dilution factor (first dilution).

[0072] Comparative Example 1

[0073] The difference between this comparative example and Example 1 is that the special proteose peptone is replaced by proteose peptone, and the other processes remain unchanged.

[0074] Comparative Example 2

[0075] The difference between the present comparative example and Example 1 is that the special proteose peptone is replaced by soybean proteose peptone, and the other processes are unchanged.

[0076] Comparative Example 3

[0077] The difference between the present comparative example and Example 1 is that the special proteose peptone is replaced by casein peptone, and the other processes are unchanged.

[0078] Comparative Example 4

[0079] The difference between the present comparative example and Example 1 is that the special proteose peptone is not added, and the other processes are unchanged.

[0080] Comparative Example 5

[0081] The difference between the present comparative example and Example 1 is that the Tris Buffer is replaced by PBS Buffer, and the other processes are unchanged.

[0082] Comparative Example 6

[0083] The difference between the present comparative example and Example 1 is that the Tris Buffer is not added, and the other processes are unchanged.

[0084] Comparative Example 7

[0085] The difference between the present comparative example and Example 1 is that the Tryptic Soy Broth is not added, and the other processes are unchanged.

[0086] Comparative Example 8

[0087] The difference between the present comparative example and Example 1 is that the Tryptic Soy Broth is replaced by MRS Broth.

[0088] Comparative Example 9

[0089] The BHI counting medium disclosed in Chinese Patent Application 202011071502.5 is used for counting, and the components thereof are as follows:

[0090] Tryptic Soy Broth 10.0 g / L, Brain Heart Infusion 17.5 g / L, Sodium Chloride 5.0 g / L, Yeast Extract 5.0 g / L, Glucose 2.0 g / L, Disodium Phosphate Dibasic Dodecahydrate 2.5 g / L, L-Cysteine Hydrochloride Monohydrate 0.4 g / L, 0.5% Vitamin K1, Hematin Chloride 1 mL / L, 5% Sterile Defibrinated Sheep Blood (v / v), pH 7.2-7.4.

[0091] Comparative Example 10

[0092] The counting medium provided in the national standard GB 4789.35-2016 is used for counting, and the components thereof are as follows:

[0093] Tryptone 10.0 g / L, beef powder 5.0 g / L, yeast powder 4.0 g / L, glucose 20.0 g / L, Tween 80 1.0 mL, K2HPO4·7H2O 2.0 g / L, sodium acetate·3H2O 5.0 g / L, triammonium citrate 2.0 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·4H2O 0.05 g / L, agar powder 15.0 g / L, mupirocin lithium salt 0.05 g / L, cysteine hydrochloride 0.5 g / L.

[0094] The viable cell numbers in Example 1 and Comparative Examples 1-10 were counted to evaluate the influence of different counting culture media on the viable cell number of Akkermansia muciniphila. The results are shown in Table 1.

[0095] Table 1 Influence of different counting culture media on the viable cell number of Akkermansia muciniphila

[0096]

[0097] The counting results show that the counting culture medium of the present application significantly improves the viable cell number of Akkermansia muciniphila compared with BHI counting culture medium, which is increased by about 4 orders of magnitude; compared with the counting culture medium provided in the national standard GB 4789.35-2016, the viable cell number of Akkermansia muciniphila is counted.

[0098] The counting culture medium provided by the present application contains special tryptone, which contains a variety of tryptone and is rich in nutrients, effectively promotes the survival of Akkermansia muciniphila, can quickly recover Akkermansia muciniphila, and increases the number of cultivable Akkermansia muciniphila. The special tryptone of the present application significantly improves the viable cell number of Akkermansia muciniphila. For example, AKK-PROBIO, compared with tryptone, soybean tryptone and casein tryptone, is increased by about 5.6 times, 7.7 times and 2.4 times, respectively.

[0099] The counting culture medium provided by the present application contains tryptone soy broth, and the tryptone soy broth of the present application significantly improves the viable cell number of Akkermansia muciniphila. On the contrary, the counting result of MRS broth is not accurate, which is 2 orders of magnitude different from the counting culture medium of the present application.

[0100] In addition, the counting medium provided by the present application also significantly shortens the counting time, reduces the error, and ensures the accuracy of the counting result. Taking the BHI medium of Comparative Example 9 as an example, it usually needs to grow for about 5 days before counting, while the counting medium provided by the present application can be used for viable cell counting after 3 days of culture, which shortens the culture time by at least 2 days, significantly improves the counting efficiency, and is more suitable for the industrialized production activities and quality control of probiotics, and has obvious advantages.

[0101] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A Akkermansia muciniphila The counting medium is characterized in that The counting medium comprises the following components: tryptone, tryptone soy broth, brain heart extract powder, sodium chloride, glucose, sodium bicarbonate·12H2O, special peptone, yeast extract, cysteine ​​hydrochloride, hemin, agar powder, Tris Buffer and sterile defibrinated sheep blood; The special peptone was purchased from Thermo Fisher with the product number LP0072B.

2. The counting medium according to claim 1, wherein The counting medium contains at least 1.5 g / L special peptone.

3. The counting medium according to claim 2, wherein The counting medium contains 1.5-3.5 g / L special peptone.

4. The counting medium according to claim 2, wherein The counting medium contains at least 4 g / L Tryptone Soy Broth.

5. The counting medium according to claim 4, wherein The counting medium contained 4-6 g / L Tryptone Soy Broth.

6. The counting medium according to any one of claims 1 to 5, characterized in that The counting culture medium contains at least 0.3 g / L Tris Buffer.

7. The counting medium according to claim 6, wherein The counting medium contains 0.3-0.5 g / L Tris Buffer.

8. The counting medium according to claim 6, wherein The counting medium comprises the following components: 5.0 g / L tryptone, 5.0 g / L tryptone soy broth, 8.75 g / L brain heart extract powder, 2.5 g / L sodium chloride, 3.5 g / L glucose, 1.25 g / L sodium bicarbonate·12H2O, 2.5 g / L special peptone, 2.5 g / L yeast extract, 0.2 g / L cysteine ​​hydrochloride, 0.005 g / L hemin, 20 g / L agar powder, 0.375 g / L Tris buffer, and 5% (v / v) sterile defibrinated sheep blood.

9. The counting culture medium according to claim 6, characterized in that The counting medium further comprises trace element salts; and / or The pH of the counting medium is 7.3-7.

7.

10. The counting culture medium according to claim 9, characterized in that The trace element salt includes K2HPO4·7H2O, MgSO4·7H2O or MnSO4·4H2O.

11. according to any one of claims 1-10 counting culture medium in Akkermansia muciniphila Application in counting.

12. A method for Akkermansia muciniphila A counting reagent, characterized in that The reagent comprises the counting culture medium according to any one of claims 1 to 10.

13. A method for Akkermansia muciniphila A counting kit, characterized in that The kit comprises the counting medium according to any one of claims 1 to 10.

14. The kit according to claim 13, characterized in that The kit further comprises at least one of a container containing the counting medium, a diluent, a magnifying glass, and a colony counter.

15. The kit according to claim 14, characterized in that The diluent is 0.9% (v / v) physiological saline.

16. A method for Akkermansia muciniphila A counting kit, characterized in that The kit comprises the reagent according to claim 12.

17. The kit according to claim 16, characterized in that The kit further comprises at least one of a container for containing the reagent, a diluent, a magnifying glass, and a colony counter.

18. The kit according to claim 17, characterized in that The diluent is 0.9% (v / v) physiological saline.

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