Method for quantitatively detecting akkermansia muciniphila and application thereof

Abstract

The application discloses a kind of mucinophilic akkermansia quantitative detection method and application thereof.Mucinophilic akkermansia sample is gradient diluted using gradient protective solution, and gradient diluted bacteria solution is obtained, the gradient diluted bacteria solution is mixed with detection medium, and is poured into container, is placed in anaerobic condition and is cultivated, colony counting is carried out, and the content of mucinophilic akkermansia in mucinophilic akkermansia sample is calculated.The detection method of the application can be widely popularized, the product detection rate is high, parallel detection result has no significant difference, data output is intuitive and reliable, detection process can use automatic equipment, improve detection efficiency, reduce labor cost, and can also be connected to production line production optimization;Compared with flow cytometer equipment detection and other methods, the detection cost is significantly reduced, operation is simple, popularization rate is high, more conducive to the industrialization development of second-generation probiotics, and conducive to the implementation and popularization of quality standard.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a quantitative detection method for Akkermansia myxophilus and its application. Background Technology

[0002] In 2004, researchers at Wageningen University in the Netherlands identified a novel mucus-degrading bacterium from human feces and named it Akkermansia muciniphila (AKK) after a renowned PhD in microbial ecology. Subsequent studies showed that AKK bacteria are closely related to host health, metabolism, and immunity. Using animal models and human trials, researchers found that changes in AkK abundance are significantly associated with numerous diseases, including neurological disorders, hypertension, diabetes, and obesity. Experiments demonstrated that oral administration of Akkermansia muciniphila can induce metabolic disturbances, achieving a preventative effect. It also showed protective effects against cognitive impairment and amyloidosis in Alzheimer's mice, and its combination with Parabacterium difficile provided anti-epileptic protection. This suggests a promising new approach for treating diseases or slowing disease progression.

[0003] Currently, the quality of probiotic products on the market varies greatly, making it difficult for people to choose suitable products. Therefore, testing is particularly important to protect the interests of the public, promote the standardized development of the probiotic market, and drive the healthy development of the probiotic industry.

[0004] Current research on Akkermansia mainly focuses on the correlation and function of its physiological characteristics, which has significant market value for the development of detection methods. Existing probiotic detection methods are mainly divided into two types: plate culture methods and molecular biological detection methods. The culture method allows us to intuitively determine the viability of bacteria based on colony morphology and quantity, while the molecular biological detection method can detect the proportion and relative abundance of different strains in a product. For example, CN110904250A discloses multiplex fluorescent quantitative PCR primers, kits, and detection methods for detecting multiple bacteria, including 16S rDNA primer pairs and probes designed for Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium animalis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Akkermansia. However, this method is highly dependent on molecular biological methods, has high equipment investment costs, low adoption rate, and requires relevant technical background for implementation, which is not conducive to the promotion of quality standards. Conventional plate culture detection methods suffer from detection instability, are easily interfered with, and affect detection accuracy. These problems pose significant difficulties for in-depth research on Akkermansia detection and the formulation of industry standards.

[0005] In conclusion, developing a stable, accurate, and low-cost method for detecting Akkermansia is of great significance for the application of Akkermansia. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a quantitative detection method for Akkermansia myxophilus and its application, achieving stable, accurate, and low-cost detection of Akkermansia myxophilus and promoting its application.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for the quantitative detection of Akkermansia myxophilus, the method comprising:

[0009] A gradient-diluted Akkermansia sample was prepared by serially diluting a gradient-protected solution to obtain a gradient-diluted bacterial solution. The gradient-diluted bacterial solution was mixed with a test culture medium and poured into a container. The container was then incubated under anaerobic conditions, and colony counting was performed to calculate the Akkermansia content in the sample. The gradient-protected solution contained sodium chloride. The test culture medium contained peptone, bovine brain extract, bovine heart extract, sodium chloride, glucose, disodium hydrogen phosphate, mucin, and agar powder.

[0010] In this invention, a novel quantitative method is designed for the second-generation probiotic Akkermansia myxophilus. By controlling a specific culture medium and conducting anaerobic culture, a stable, accurate, and low-cost method for detecting Akkermansia can be achieved, which is significantly superior to existing conventional probiotic plate culture methods and flow cytometry methods.

[0011] Preferably, the concentration of sodium chloride in the gradient protective solution is 8-9 g / L, including but not limited to 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8 or 8.9 g / L, etc.

[0012] Preferably, the gradient protection solution also contains casein peptone.

[0013] Preferably, the concentration of casein peptone in the gradient protection solution is 0.5 to 1.5 g / L, including but not limited to 0.6, 0.7, 0.8, 0.9, 1, 1.2 or 1.4 g / L, etc.

[0014] In this invention, casein peptone is further added to the gradient protection solution to synergistically improve the detection accuracy.

[0015] Preferably, the concentration of peptone in the detection culture medium is 8-12 g / L, including but not limited to 8.5, 9, 9.5, 10, 10.5, 11 or 11.5 g / L.

[0016] Preferably, the concentration of bovine brain extract powder in the detection culture medium is 11.5–13.5 g / L, including but not limited to 12, 12.5, or 13 g / L.

[0017] Preferably, the concentration of ox heart extract powder in the detection culture medium is 4-6 g / L, including but not limited to 4.5, 5 or 5.5 g / L.

[0018] Preferably, the concentration of sodium chloride in the detection culture medium is 4 to 6 g / L, including but not limited to 4.5, 5 or 5.5 g / L.

[0019] Preferably, the concentration of glucose in the detection culture medium is 1 to 3 g / L, including but not limited to 1.5, 2 or 2.5 g / L.

[0020] Preferably, the concentration of disodium hydrogen phosphate in the detection culture medium is 1.5 to 3.5 g / L, including but not limited to 2, 2.5 or 3 g / L.

[0021] Preferably, the concentration of mucin in the detection culture medium is 1-3 g / L, including but not limited to 1.5, 2 or 2.5 g / L.

[0022] Preferably, the concentration of agar powder in the detection culture medium is 5-20 g / L, including but not limited to 5.5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18 or 19 g / L.

[0023] Preferably, the gradient protection solution contains 8-9 g / L sodium chloride and 0.5-1.5 g / L casein peptone.

[0024] Preferably, the detection culture medium contains 8–12 g / L peptone, 11.5–13.5 g / L bovine brain extract, 4–6 g / L bovine heart extract, 4–6 g / L sodium chloride, 1–3 g / L glucose, 1.5–3.5 g / L disodium hydrogen phosphate, 1–3 g / L mucin, and 5–20 g / L agar powder.

[0025] Preferably, the anaerobic conditions are 80-100% N2, 0-10% H2 and 0-10% CO2.

[0026] In this invention, strictly controlling anaerobic conditions during the cultivation and detection process can further improve the accuracy and stability of the detection.

[0027] Preferably, the culture is carried out in an anaerobic workstation.

[0028] In this invention, an anaerobic workstation is used to ensure that the detection process is conducted in an anaerobic environment, thereby further improving the accuracy and stability of strain detection.

[0029] Preferably, the culture temperature is 35-39°C, including but not limited to 36°C, 37°C or 38°C, etc., preferably 36-38°C; the time is 80-90h, including but not limited to 81, 82, 83, 84, 85, 86, 87, 88 or 89h, etc., preferably 82-86h.

[0030] In this invention, controlling the culture temperature further facilitates the control of detection accuracy and stability.

[0031] As a preferred technical solution, the quantitative detection method for Akkermansia myxophilus includes the following steps:

[0032] A sample of Akkermansia muciniphila was serially diluted using a gradient protection solution to obtain a serially diluted bacterial solution. The gradient protection solution contained 8–9 g / L sodium chloride and 0.5–1.5 g / L casein peptone.

[0033] The graded diluted bacterial solution was mixed with the test culture medium and poured into a container. The container was then placed in an anaerobic workstation for incubation. The gas conditions were controlled at 80–100% N2, 0–10% H2, and 0–10% CO2. The container was incubated at 35–39°C for 80–90 hours. Colony counts were performed, and the content of Akkermansia myxophilus in the sample was calculated.

[0034] The detection culture medium contains 8–12 g / L peptone, 11.5–13.5 g / L bovine brain extract, 4–6 g / L bovine heart extract, 4–6 g / L sodium chloride, 1–3 g / L glucose, 1.5–g / L disodium hydrogen phosphate, 1–3 g / L mucin, and 5–15 g / L agar powder.

[0035] Secondly, the present invention provides the application of the quantitative detection method for Akkermansia myxophilus described in the first aspect in the development of second-generation probiotic products and / or the development of second-generation probiotic production processes.

[0036] This invention presents an anaerobic culture and quantitative detection method for the second-generation probiotic Akkermansia myxophilus. This detection method can be widely applied, has a high product detection rate, and shows no significant differences in parallel test results. Based on the growth characteristics of the strain, an anaerobic workstation is used to ensure that the detection process is conducted in an anaerobic environment, thereby improving the accuracy of strain detection. The data output is intuitive and reliable. The detection process can be automated, improving detection efficiency and reducing labor costs. It can also be integrated into production lines for optimization. Compared with methods such as flow cytometry, this method significantly reduces detection costs, is simple to operate, and has a high adoption rate, which is more conducive to the industrialization of second-generation probiotics and the implementation and promotion of quality standards.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] This invention presents a novel quantitative method for the second-generation probiotic Akkermansia myxophilus. Based on the characteristics of the strain, it innovatively develops an industry-wide standard. By controlling specific culture media and conducting anaerobic culture, it can achieve stable, accurate, and low-cost detection of Akkermansia, which is significantly superior to existing conventional standard methods and flow cytometry methods for probiotics. In addition, by combining specific gradient protection solutions and culture conditions, the accuracy and stability of the detection can be further improved. Detailed Implementation

[0039] To further illustrate the technical means and effects of this invention, the following embodiments are provided for further explanation. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0040] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0041] In a specific embodiment of the present invention, peptone was purchased from Qingdao Haibo Biotechnology, bovine brain extract powder was purchased from Beijing Punade, bovine heart extract powder was purchased from Qingdao Haibo Biotechnology, mucin was purchased from Pusitang, and trypsin was purchased from Qingdao Haibo Biotechnology.

[0042] Example 1

[0043] This embodiment demonstrates the quantitative detection of Akkermansia myxophilus.

[0044] S1 Preliminary preparations: preparation and sterilization of culture medium, preparation and sterilization of gradient protection solution, and dispensing of test tubes and blue-capped bottles.

[0045] 1) Preparation of culture medium for detecting Akkermansia myxophilus: peptone: 10.0g, bovine brain extract: 12.5g, bovine heart extract: 5.0g, sodium chloride: 5g, glucose: 2.0g, disodium hydrogen phosphate: 2.5g, mucin: 2g; agar powder: 10g; add to 1000mL of distilled water, heat and stir to dissolve, adjust pH to 7.4±0.2, and autoclave at 121℃ for 15min.

[0046] 2) Preparation of Akkermansia muciniphila gradient protection solution: Add 8.5g NaCl and 0.5g casein peptone to 1000mL distilled water, stir to dissolve, dispense into containers, and autoclave at 121℃ for 15min.

[0047] 3) An automated dispensing robot was used to dispense the test tubes and initial dissolution blue-capped bottles used in the experiment. All dispensing was done using a gradient preservative solution after sterilization. The required number of gradient dilution test tubes was calculated based on the viable cell count range of the samples. The automated dispensing robot was set with dispensing parameters. After sterilization, 9.0 mL of gradient preservative solution was dispensed into the test tubes using a peristaltic pump. Then, 198.0 mL of gradient preservative solution was dispensed into the blue-capped bottle using the same peristaltic pump. The test tubes were gently capped with sterile rubber stoppers, and the blue-capped bottles were tightened. This prepared seven 9.0 mL test tubes and one 198.0 mL blue-capped bottle.

[0048] All the following operations are performed inside the anaerobic workstation. Before the operation, put the necessary items into the workstation in advance (ensure that the workstation is clean and disinfected and free of contaminants).

[0049] S2 detection process: initial solution preparation, gradient dilution, sample transfer.

[0050] 1) Initial solution preparation:

[0051] Take 2g of the mixed Akkermansia muciniphila sample into a sterile blue-capped bottle containing 198mL (with an appropriate amount of sterile glass beads pre-placed in the bottle), mix the sample thoroughly, and prepare a 1:100 sample homogenate.

[0052] 2) Gradient dilution

[0053] Slowly pipette 1 mL from the middle of the 1:100 sample homogenate and gently pour it along the wall of a test tube containing 9 mL of gradient protection solution (be careful not to let the pipette tip touch the gradient protection solution). Mix using a vortex mixer, starting the timer when the liquid reaches the bottom of the test tube, and vortex for 3 seconds. Repeat this vortexing process 8 times to prepare a 1:1000 sample homogenate. Using a 1 mL sterile pipette tip, perform 10-fold increments of sample homogenate, changing to a 1 mL sterile pipette tip for each increment. Dilute a total of 7 test tubes. This will prepare a 1:1000 sample homogenate. 9 The sample is homogenized.

[0054] 3) Sample transfer

[0055] Choose 10 8 and 10 9 Two test tubes are used. For each dilution, 1 mL of sample is pipetted into a sterile agar plate and homogenized. Three plates are prepared for each dilution.

[0056] S3 medium pouring and incubation

[0057] 1) Pour the culture medium cooled to 48±2℃ into the culture dish, and rotate the culture dish 10 times clockwise and 10 times counterclockwise to mix the sample evenly.

[0058] 2) After the culture medium solidifies, the plate is inverted and placed in an anaerobic workstation (80% N2, 10% H2 and 10% CO2) and incubated at 37°C for 84 hours, and then colony counting is performed.

[0059] Example 2

[0060] This embodiment demonstrates the quantitative detection of Akkermansia myxophilus.

[0061] S1 Preliminary preparations: preparation and sterilization of culture medium, preparation and sterilization of gradient protection solution, and dispensing of test tubes and blue-capped bottles.

[0062] 1) Preparation of culture medium for detecting Akkermansia myxophilus: peptone: 8g, bovine brain extract: 11.5g, bovine heart extract: 4g, sodium chloride: 4g, glucose: 1g, disodium hydrogen phosphate: 1.5g, mucin: 1g; agar powder: 5g; add to 1000mL of distilled water, heat and stir to dissolve, adjust pH to 7.4±0.2, and autoclave at 121℃ for 15min.

[0063] 2) Preparation of Akkermansia muciniphila gradient protection solution: Add 8g NaCl and 1.5g casein peptone to 1000mL distilled water, stir to dissolve, dispense into containers, and autoclave at 121℃ for 15min.

[0064] 3) An automated dispensing robot was used to dispense the test tubes and initial dissolution blue-capped bottles used in the experiment. All dispensing was done using a gradient preservative solution after sterilization. The required number of gradient dilution test tubes was calculated based on the viable cell count range of the samples. The automated dispensing robot was set with dispensing parameters. After sterilization, 9.0 mL of gradient preservative solution was dispensed into the test tubes using a peristaltic pump. Then, 198.0 mL of gradient preservative solution was dispensed into the blue-capped bottle using the same peristaltic pump. The test tubes were gently capped with sterile rubber stoppers, and the blue-capped bottles were tightened. This prepared seven 9.0 mL test tubes and one 198.0 mL blue-capped bottle.

[0065] All the following operations are performed inside the anaerobic workstation. Before the operation, put the necessary items into the workstation in advance (ensure that the workstation is clean and disinfected and free of contaminants).

[0066] S2 detection process: initial solution preparation, gradient dilution, sample transfer.

[0067] 1) Initial solution preparation:

[0068] Take 2g of the mixed Akkermansia muciniphila sample into a sterile blue-capped bottle containing 198mL (with an appropriate amount of sterile glass beads pre-placed in the bottle), mix the sample thoroughly, and prepare a 1:100 sample homogenate.

[0069] 2) Gradient dilution

[0070] Slowly pipette 1 mL from the middle of the 1:100 sample homogenate and gently pour it along the wall of a test tube containing 9 mL of gradient protection solution (be careful not to let the pipette tip touch the gradient protection solution). Mix using a vortex mixer, starting the timer when the liquid reaches the bottom of the test tube, and vortex for 3 seconds. Repeat this vortexing process 8 times to prepare a 1:1000 sample homogenate. Using a 1 mL sterile pipette tip, perform 10-fold increments of sample homogenate, changing to a 1 mL sterile pipette tip for each increment. Dilute a total of 7 test tubes. This will prepare a 1:1000 sample homogenate. 9 The sample is homogenized.

[0071] 3) Sample transfer

[0072] Choose 10 8 and 10 9 Two test tubes are used. For each dilution, 1 mL of sample is pipetted into a sterile agar plate and homogenized. Three plates are prepared for each dilution.

[0073] S3 medium pouring and incubation

[0074] 1) Pour the culture medium cooled to 48±2℃ into the culture dish, and rotate the culture dish 10 times clockwise and 10 times counterclockwise to mix the sample evenly.

[0075] 2) After the culture medium solidifies, the plate is inverted and placed in the anaerobic workstation. It is then incubated at 36°C for 96 hours, and colony counting is performed.

[0076] Example 3

[0077] This embodiment demonstrates the quantitative detection of Akkermansia myxophilus.

[0078] S1 Preliminary preparations: preparation and sterilization of culture medium, preparation and sterilization of gradient protection solution, and dispensing of test tubes and blue-capped bottles.

[0079] 1) Preparation of culture medium for detecting Akkermansia myxophilus: peptone: 12g, bovine brain extract: 13.5g, bovine heart extract: 6g, sodium chloride: 6g, glucose: 3g, disodium hydrogen phosphate: 3.5g, mucin: 3g; agar powder: 20g; add to 1000mL of distilled water, heat and stir to dissolve, adjust pH to 7.4±0.2, and autoclave at 121℃ for 15min.

[0080] 2) Preparation of Akkermansia muciniphila gradient protection solution: Add 9g NaCl and 0.5g casein peptone to 1000mL distilled water, stir to dissolve, dispense into containers, and autoclave at 121℃ for 15min.

[0081] 3) An automated dispensing robot was used to dispense the test tubes and initial dissolution blue-capped bottles used in the experiment. All dispensing was done using a gradient preservative solution after sterilization. The required number of gradient dilution test tubes was calculated based on the viable cell count range of the samples. The automated dispensing robot was set with dispensing parameters. After sterilization, 9.0 mL of gradient preservative solution was dispensed into the test tubes using a peristaltic pump. Then, 198.0 mL of gradient preservative solution was dispensed into the blue-capped bottle using the same peristaltic pump. The test tubes were gently capped with sterile rubber stoppers, and the blue-capped bottles were tightened. This prepared seven 9.0 mL test tubes and one 198.0 mL blue-capped bottle.

[0082] All the following operations are performed inside the anaerobic workstation. Before the operation, put the necessary items into the workstation in advance (ensure that the workstation is clean and disinfected and free of contaminants).

[0083] S2 detection process: initial solution preparation, gradient dilution, sample transfer.

[0084] 1) Initial solution preparation:

[0085] Take 2g of the mixed Akkermansia muciniphila sample into a sterile blue-capped bottle containing 198mL (with an appropriate amount of sterile glass beads pre-placed in the bottle), mix the sample thoroughly, and prepare a 1:100 sample homogenate.

[0086] 2) Gradient dilution

[0087] Slowly pipette 1 mL from the middle of the 1:100 sample homogenate and gently pour it along the wall of a test tube containing 9 mL of gradient protection solution (be careful not to let the pipette tip touch the gradient protection solution). Mix using a vortex mixer, starting the timer when the liquid reaches the bottom of the test tube, and vortex for 3 seconds. Repeat this vortexing process 8 times to prepare a 1:1000 sample homogenate. Using a 1 mL sterile pipette tip, perform 10-fold increments of sample homogenate, changing to a 1 mL sterile pipette tip for each increment. Dilute a total of 7 test tubes. This will prepare a 1:1000 sample homogenate. 9 The sample is homogenized.

[0088] 3) Sample transfer

[0089] Choose 10 8 and 10 9 Two test tubes are used. For each dilution, 1 mL of sample is pipetted into a sterile agar plate and homogenized. Three plates are prepared for each dilution.

[0090] S3 medium pouring and incubation

[0091] 1) Pour the culture medium cooled to 48±2℃ into the culture dish, and rotate the culture dish 10 times clockwise and 10 times counterclockwise to mix the sample evenly.

[0092] 2) After the culture medium solidifies, the plate is inverted and placed in the anaerobic workstation. It is then incubated at 38°C for 80 hours, and colony counting is performed.

[0093] Example 4

[0094] This embodiment demonstrates the quantitative detection of Akkermansia myxophilus at different concentrations.

[0095] A1: Weigh 10g of Akkermansia myxophilus sample, mix thoroughly, and record as A1.

[0096] A2: Weigh 1g of Akkermansia muciniphila sample and 9g of maltodextrin into pre-labeled aluminum foil bags. Perform a 10-fold serial dilution of the bacterial powder and mix thoroughly. This is recorded as A2.

[0097] A3: Weigh 0.1g of Akkermansia muciniphila sample and 9.9g of maltodextrin into pre-labeled aluminum foil bags, perform a 100-fold serial dilution of the bacterial powder, mix thoroughly, and record as A3.

[0098] S1: Preliminary preparation: preparation and sterilization of culture medium, preparation and sterilization of gradient protection solution, and dispensing of test tubes and blue-capped bottles.

[0099] 1) Preparation of culture medium for detecting Akkermansia myxophilus: peptone: 10.0g, dehydrated calf brain extract powder: 12.5g, dehydrated calf heart extract powder: 5.0g, sodium chloride: 5g, glucose: 2.0g, disodium hydrogen phosphate: 2.5g, mucin: 2g; agar powder: 10g; add to 1000mL of distilled water, heat and stir to dissolve, adjust pH to 7.4±0.2, and autoclave at 121℃ for 15min.

[0100] 2) Preparation of Akkermansia muciniphila gradient protection solution: Add 8.5g NaCl and 1.0g casein peptone to 1000mL distilled water, stir to dissolve, dispense into containers, and autoclave at 121℃ for 15min.

[0101] 3) An automated dispensing robot was used to dispense the test tubes and initial dissolution blue-capped bottles used in the experiment. All dispensing was done using a gradient preservative solution after sterilization. The required number of gradient dilution test tubes was calculated based on the viable cell count range of the samples. The automated dispensing robot was set with dispensing parameters. After sterilization, 9.0 mL of gradient preservative solution was dispensed into the test tubes using a peristaltic pump. Then, 198.0 mL of gradient preservative solution was dispensed into the blue-capped bottles using the same peristaltic pump. The test tubes were gently capped with sterile rubber stoppers, and the blue-capped bottles were tightened. For sample A1, 7 9.0 mL test tubes and 1 198.0 mL blue-capped bottle were prepared; for sample A2, 6 9.0 mL test tubes and 1 198.0 mL blue-capped bottle were prepared; and for sample A3, 5 9.0 mL test tubes and 1 198.0 mL blue-capped bottle were prepared.

[0102] All the following operations are performed inside the anaerobic workstation. Before the operation, put the necessary items into the workstation in advance (ensure that the workstation is clean and disinfected and free of contaminants).

[0103] S2 detection process: initial solution preparation, gradient dilution, sample transfer.

[0104] 1) Initial solution preparation:

[0105] Take 2g of each of the three mixed samples and put them into a 198.0mL sterile blue-capped bottle (with an appropriate amount of sterile glass beads pre-placed in the bottle) with a pre-labeled serial number. Mix the samples thoroughly to make a 1:100 sample homogenate.

[0106] 2) Gradient dilution

[0107] For each sample, slowly pipette 1 mL from the middle of its 1:100 homogenate and gently inject it along the wall of the test tube into a tube containing 9 mL of gradient protection solution (ensure the pipette tip does not touch the gradient protection solution). Mix using a vortex mixer, starting the timer when the liquid reaches the bottom of the tube, and vortex for 3 seconds. Repeat this vortexing process 8 times to prepare a 1:1000 homogenate. Using a 1 mL sterile pipette tip, perform 10-fold increments of homogenate, changing to a 1 mL sterile pipette tip for each increment. A total of 7 test tubes were diluted using A1, resulting in a 1:1000 homogenate. 9 The sample was homogenized, and A2 was used to dilute 6 test tubes in total, thus preparing a 1:10 solution. 8 The sample was homogenized, and A3 was used to dilute 5 test tubes in total, thus preparing a 1:10 solution. 7 The sample is homogenized.

[0108] 3) Sample transfer

[0109] A1 Choose 10 8 and 10 9 Two test tubes are used. For each dilution, 1 mL of sample is pipetted into a sterile agar plate and homogenized. Three plates are prepared for each dilution.

[0110] A2 Choose 10 7 and 10 8 Two test tubes are used. For each dilution, 1 mL of sample is pipetted into a sterile agar plate and homogenized. Three plates are prepared for each dilution.

[0111] A3 Choose 10 6 and 10 7 Two test tubes are used. For each dilution, 1 mL of sample is pipetted into a sterile agar plate and homogenized. Three plates are prepared for each dilution.

[0112] S3 medium pouring and incubation

[0113] 1) Pour the culture medium cooled to 48±2℃ into the culture dish, and rotate the culture dish 10 times clockwise and 10 times counterclockwise to mix the sample evenly.

[0114] 2) After the culture medium solidifies, the plate is inverted and placed in the anaerobic workstation. It is then incubated at 37°C for 96 hours, and colony counting is performed.

[0115] Example 5

[0116] This embodiment demonstrates the quantitative detection of Akkermansia myxophilus.

[0117] Compared with Example 1, the only difference is that in the S3 medium pouring and culturing, after the medium solidifies, the plate is inverted and placed in an anaerobic box (with an internal anaerobic bag - Thermo Fisher AN0035A-Oxoid AnaeroGen 3.5L) and incubated at 37°C for 84 hours.

[0118] Example 6

[0119] This embodiment demonstrates the quantitative detection of Akkermansia myxophilus.

[0120] Compared with Example 1, the only difference is that the gradient protection solution is replaced with an equal amount of 0.85% sodium chloride solution, and the rest is the same as Example 1.

[0121] Example 7

[0122] This embodiment demonstrates the quantitative detection of Akkermansia myxophilus.

[0123] Compared with Example 1, the only difference is that the gradient protection solution is replaced with an equal amount of solution containing 0.85% sodium chloride and 0.1% tryptone, otherwise it is the same as Example 1.

[0124] Comparative Example 1

[0125] This comparative example demonstrates the quantitative detection of Akkermansia myxophilus.

[0126] Compared with Example 1, the only difference is that the bovine brain extract in the Akkermansia myxophilus detection medium is replaced with an equal amount of bovine heart extract; otherwise, it is the same as Example 1.

[0127] Comparative Example 2

[0128] This comparative example demonstrates the quantitative detection of Akkermansia myxophilus.

[0129] Compared with Example 1, the only difference is that the peptone in the Akkermansia myxophilus detection medium is replaced with an equal amount of tryptone, and the rest is the same as Example 1.

[0130] Comparative Example 3

[0131] This comparative study used flow cytometry to quantitatively detect Akkermansia myxophilus.

[0132] S1 Preliminary preparations: preparation and sterilization of gradient protection solution, dispensing of test tubes and blue-capped bottles.

[0133] 1) Preparation of Akkermansia myxophilus gradient protection solution: Add 8.5g NaCl and 1.0g casein peptone to 1000mL distilled water, stir to dissolve, dispense into containers, and autoclave at 121℃ for 20min.

[0134] 2) An automated dispensing robot was used to dispense the test tubes and initial dissolution blue-capped bottles used in the experiment. All dispensing was done using a gradient preservative solution after sterilization. The required number of gradient dilution test tubes was calculated based on the viable cell count range of the samples. The automated dispensing robot was set with dispensing parameters. After sterilization, 9.0 mL of gradient preservative solution was dispensed into the test tubes using a peristaltic pump. Then, 198.0 mL of gradient preservative solution was dispensed into the blue-capped bottle using the same peristaltic pump. The test tubes were gently capped with sterile rubber stoppers, and the blue-capped bottles were tightened. This prepared three 9.0 mL test tubes and one 198.0 mL blue-capped bottle.

[0135] S2 detection process: initial solution preparation, gradient dilution, sample transfer, staining, and instrument loading.

[0136] 1) Initial solution preparation:

[0137] Take 2g of the mixed sample into a sterile blue-capped bottle containing 198mL (with an appropriate amount of sterile glass beads pre-placed in the bottle), mix the sample thoroughly, and prepare a 1:100 sample homogenate.

[0138] 2-gradient dilution

[0139] Slowly pipette 1 mL from the middle of the 1:100 sample homogenate and gently pour it along the wall of a test tube containing 9 mL of gradient protection solution (be careful not to let the pipette tip touch the gradient protection solution). Mix using a vortex mixer, starting the timer when the liquid reaches the bottom of the test tube, and vortex for 3 seconds. Repeat this vortexing process 8 times to prepare a 1:1000 sample homogenate. Using a 1 mL sterile pipette tip, perform 10-fold increments of sample homogenate, changing to a 1 mL sterile pipette tip for each increment. Dilute a total of 3 test tubes. This will prepare a 1:1000 sample homogenate. 5 The sample is homogenized.

[0140] 3) Sample transfer

[0141] Choose 10 4 and 10 5 Two test tubes were used. After vortexing each test tube (3 seconds, 8 times), 1 mL of the homogenate was drawn from each dilution and injected into a 2 mL sterile centrifuge tube pre-labeled with the dilution. Three sterile centrifuge tubes were prepared for each dilution.

[0142] 4) Staining

[0143] Mix SYTO9 nucleic acid stain and propidium iodide 1:1 thoroughly (vortex and centrifuge), then add 3 μL of the dye mixture to a 2 ml sterile centrifuge tube containing the sample homogenate, and vortex to mix. Place the prepared sample solution in a constant temperature incubator at 37°C and in the dark for 15 min (the flow cytometer can be turned on at this time).

[0144] 5) Computer Lab

[0145] When using a flow cytometer for detection, the instrument detection of stained samples must be completed within 45 minutes.

[0146] The colony count results of each embodiment and comparative example are statistically analyzed and are shown in Table 1. Each gradient shows the results of three parallel experiments.

[0147] Table 1

[0148]

[0149] As shown in Table 1, the quantitative detection method for Akkermansia myxophilus designed in this invention can accurately and stably detect Akkermansia myxophilus, with results comparable to those obtained by flow cytometry, but at a significantly lower cost.

[0150] Furthermore, comparing the results of Example 1 and Example 5, it can be seen that strictly controlling the anaerobic environment during the culture and detection process in this invention can significantly improve the stability of the detection; comparing the results of Example 1 and Examples 6 and 7, it can be seen that utilizing a specific component gradient protective solution in this invention can further improve the detection accuracy; comparing the results of Example 1 and Comparative Examples 1 and 2, it can be seen that utilizing a specific component culture medium in this invention can further improve the detection accuracy.

[0151] The results of Example 4 are shown in Table 2.

[0152] Table 2

[0153]

[0154] As shown in Table 2, the quantitative detection method for Akkermansia myxophilus designed in this invention has wide applicability and can be adapted to samples of different concentrations.

[0155] In summary, this invention presents an anaerobic culture and quantitative detection method for the second-generation probiotic Akkermansia myxophilus. This detection method can be widely applied, has a high product detection rate, shows no significant differences in parallel test results, provides intuitive and reliable data output, and allows for automated testing equipment to improve detection efficiency and reduce labor costs. It can also be integrated into production lines for optimization. Compared to methods such as flow cytometry, this method significantly reduces detection costs, is simple to operate, and has a high adoption rate, making it more conducive to the industrialization of second-generation probiotics and to the implementation and promotion of quality standards.

[0156] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A quantitative detection method for Akkermansia myxophilus for non-disease diagnosis purposes, characterized in that, The method includes: The Akkermansia myxophilus sample was serially diluted using a gradient protection solution to obtain a serially diluted bacterial solution. The serially diluted bacterial solution was mixed with the test culture medium and poured into a container. The container was then incubated under anaerobic conditions, and colony counting was performed to calculate the Akkermansia myxophilus content in the sample. The gradient protection solution contains 8-9 g / L sodium chloride and 0.5-1.5 g / L casein peptone; The detection culture medium contains 8-12 g / L peptone, 11.5-13.5 g / L bovine brain extract, 4-6 g / L bovine heart extract, 4-6 g / L sodium chloride, 1-3 g / L glucose, 1.5-3.5 g / L disodium hydrogen phosphate, 1-3 g / L mucin, and 5-20 g / L agar powder. The anaerobic conditions are 80-100% N2, 0-10% H2 and 0-10% CO2.

2. The quantitative detection method for Akkermansia myxophilus for non-disease diagnosis purposes according to claim 1, characterized in that, The culture was carried out in an anaerobic workstation.

3. The quantitative detection method for Akkermansia myxophilus for non-disease diagnosis purposes according to claim 1, characterized in that, The culture temperature is 35~39℃ and the time is 80~90 h.

4. The quantitative detection method for Akkermansia myxophilus for non-disease diagnosis purposes according to claim 3, characterized in that, The culture temperature was 36~38℃ and the time was 82~86 h.

5. The quantitative detection method for Akkermansia myxophilus for non-disease diagnosis purposes according to any one of claims 1-4, characterized in that, The method includes the following steps: A sample of Akkermansia muciniphila was serially diluted using a gradient protection solution to obtain a serially diluted bacterial solution. The gradient protection solution contained 8-9 g / L sodium chloride and 0.5-1.5 g / L casein peptone. The graded diluted bacterial solution was mixed with the test culture medium and poured into a container. The container was then placed in an anaerobic workstation for incubation. The gas conditions were controlled at 80-100% N2, 0-10% H2 and 0-10% CO2. The container was incubated at 35-39℃ for 80-90 h. Colony counts were performed, and the content of Akkermansia myxophilus in the sample was calculated. The detection culture medium contains 8-12 g / L peptone, 11.5-13.5 g / L bovine brain extract, 4-6 g / L bovine heart extract, 4-6 g / L sodium chloride, 1-3 g / L glucose, 1.5-3.5 g / L disodium hydrogen phosphate, 1-3 g / L mucin, and 5-20 g / L agar powder.

6. The quantitative detection method for Akkermansia myxophilus as described in any one of claims 1-5 for non-disease diagnosis purposes is applied in the development of second-generation probiotic products and / or the development of second-generation probiotic production processes.

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