Quantitative detection method of Ackermania muciniphila and application of quantitative detection method

Abstract

The invention discloses a quantitative detection method of Ackermania muciniphila and application of the quantitative detection method. The method comprises the following steps: carrying out gradient dilution on an Ackermania muciniphila sample by using a gradient protection solution to obtain a gradient diluted bacterium solution, mixing the gradient diluted bacterium solution with a detection culture medium, pouring the mixture into a container, culturing under an anaerobic condition, and counting bacterial colonies, and calculating the content of the Ackermania muciniphila in the Ackermania muciniphila sample. The detection method can be widely popularized, the product detection rate is high, parallel detection results have no significant difference, data output is visual and reliable, automatic equipment can be adopted in the detection process, the detection efficiency is improved, the labor cost is reduced, and a production line can be connected for production optimization; compared with a flow cytometry equipment detection method and the like, the method has the advantages that the detection cost is obviously reduced, the operation is simple, the popularization rate is high, the industrialization development of the second-generation probiotics is more facilitated, and the promotion and popularization of the quality standard are facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a quantitative detection method of Akkermansia muciniphila and application thereof. Background Art

[0002] In 2004, researchers at Wageningen University in the Netherlands identified a new mucus-degrading bacterium from human feces and named it Akkermansia muciniphila (AKK) after a famous doctor in microbial ecology. Subsequent studies have shown that AKK bacteria are closely related to the health, metabolism and immunity of the host. With the help of animal models and human experiments, researchers have found that changes in the abundance of Akk bacteria are significantly associated with many diseases such as neurological diseases, hypertension, diabetes and obesity. Experiments have shown that oral administration of Akkermansia muciniphila can cause metabolic disorders and achieve a preventive effect. It has a protective effect on the cognitive impairment and pathological phenotype of amyloid protein in Alzheimer's mice, and can achieve anti-epileptic protection when combined with Parabacteroides distichum. It is expected to become a new way to treat diseases or alleviate disease progression.

[0003] At present, the quality of products on the probiotics market varies greatly, and it is difficult for people to choose suitable products. Therefore, testing is particularly important, which is conducive to protecting the interests of the group, promoting the standardized development of the probiotics market, and promoting the healthy development of the probiotics industry.

[0004] At present, the research on Akkermansia mainly focuses on the correlation and function of its physiological characteristics, and has great market value for the development of its detection method. There are two main methods for detecting probiotics: plate culture method and molecular biology detection method. The culture method can help us intuitively determine its vitality according to the morphology and quantity of the colony, and the molecular biology detection method can detect the addition ratio and relative abundance of different strains in the product. For example, CN110904250A discloses multiple fluorescent quantitative PCR primers, kits and detection methods for detecting multiple bacteria, which include 16SrDNA design primer pairs and probes for Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium animalis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri and Akkermansia. However, it is highly dependent on molecular biology methods, the equipment investment cost is high, the popularity rate is low, and the implementation of the detection method requires relevant technical background, which is not conducive to the implementation of quality standards; conventional plate culture detection method has detection instability, is easily interfered, and affects the detection accuracy. The above problems have caused great difficulties for the in-depth research and industry standard formulation of Akkermansia detection.

[0005] In summary, the development of a method that can stably, accurately and cost-effectively detect Akkermansia is of great significance for the application of Akkermansia. Summary of the invention

[0006] In view of the deficiencies of the prior art and actual needs, the present invention provides a quantitative detection method for Akkermansia muciniphila and application thereof, so as to achieve stable, accurate and low-cost detection of Akkermansia and promote the application of Akkermansia.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for quantitatively detecting Akkermansia muciniphila, the method comprising:

[0009] The Akkermansia muciniphila sample is gradiently diluted by using a gradient protection liquid to obtain a gradient diluted bacterial solution, the gradient diluted bacterial solution is mixed with a detection culture medium, poured into a container, cultured under anaerobic conditions, colony counted, and the content of Akkermansia muciniphila in the Akkermansia muciniphila sample is calculated; the gradient protection liquid contains sodium chloride; the detection culture medium contains peptone, bovine brain extract powder, bovine heart extract powder, sodium chloride, glucose, disodium hydrogen phosphate, mucin and agar powder.

[0010] In the present invention, a new quantitative method is designed for the second-generation probiotic Akkermansia muciniphila. By controlling the specific culture medium and performing anaerobic culture, stable, accurate and low-cost detection of Akkermansia can be achieved, which is significantly superior to the existing conventional plate culture method and flow cytometry method for probiotics.

[0011] Preferably, the concentration of sodium chloride in the gradient protection 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 and the like.

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

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

[0014] In the present invention, casein peptone is further added to the gradient protection solution to cooperate with each other and further improve the detection accuracy.

[0015] Preferably, the concentration of peptone in the detection 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, etc.

[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 the beef heart extract powder in the detection 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 medium is 4-6 g / L, including but not limited to 4.5, 5 or 5.5 g / L.

[0019] Preferably, the concentration of glucose in the detection medium is 1-3 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 medium is 1.5-3.5 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 medium is 5 to 20 g / L, including but not limited to 5.5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18 or 19 g / L, etc.

[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 powder, 4-6 g / L bovine heart extract powder, 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 the present invention, strict anaerobic conditions are controlled during the culture and detection process, which can further improve the detection accuracy and stability.

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

[0028] In the present invention, an anaerobic workstation is used to ensure that the detection process is 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 culture 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 the present invention, controlling the culture temperature is further beneficial to controlling the detection accuracy and stability.

[0031] As a preferred technical solution, the quantitative detection method of Akkermansia muciniphila comprises the following steps:

[0032] Using a gradient protection solution to gradient dilute the Akkermansia muciniphila sample to obtain a gradient diluted bacterial solution, wherein the gradient protection solution contains 8-9 g / L sodium chloride and 0.5-1.5 g / L casein peptone;

[0033] The gradient diluted bacterial solution is mixed with the detection culture medium, poured into a container, placed in an anaerobic workstation for culture, the gas conditions are controlled to be 80-100% N2, 0-10% H2 and 0-10% CO2, cultured at 35-39° C. for 80-90 hours, colony counting is performed, and the content of Akkermansia muciniphila in the Akkermansia muciniphila sample is calculated;

[0034] The detection culture medium contains 8-12 g / L peptone, 11.5-13.5 g / L ox brain extract powder, 4-6 g / L ox heart extract powder, 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] In a second aspect, the present invention provides application of the quantitative detection method of Akkermansia muciniphila described in the first aspect in the development of second-generation probiotic products and / or the development of second-generation probiotic production processes.

[0036] In the present invention, an anaerobic culture and quantitative detection method for the second-generation probiotic Akkermansia muciniphila is designed. The detection method can be widely promoted, has a high product detection rate, and has no significant difference in parallel detection results. According to the growth characteristics of the strain, an anaerobic workstation is used to ensure that the detection process is in an anaerobic environment, thereby improving the accuracy of strain detection, and the data output is intuitive and reliable. The detection process can use automated equipment to improve detection efficiency and reduce labor costs, and can also be connected to production line production optimization; compared with methods such as flow cytometer equipment detection, the detection cost is significantly reduced, the operation is simple, and the popularization rate is high, which is more conducive to the industrialization development of the second-generation probiotics and the promotion and promotion of quality standards.

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

[0038] In the present invention, a new quantitative method is designed for the second-generation probiotic Akkermansia muciniphila, and innovative development of industry-wide standards is carried out based on the characteristics of the strain. By controlling a specific culture medium and conducting anaerobic culture, stable, accurate and low-cost detection of Akkermansia can be achieved, which is significantly superior to existing conventional standard methods and flow cytometry methods for probiotics. In addition, combined with a specific gradient protective solution and culture conditions, the accuracy and stability of the detection can be further improved. DETAILED DESCRIPTION

[0039] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0040] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

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

[0042] Example 1

[0043] This example performs quantitative detection of Akkermansia muciniphila.

[0044] S1 Preliminary preparation: preparation and sterilization of culture medium, preparation and sterilization of gradient protective solution, and packaging of test tubes and blue-cap bottles.

[0045] 1) Akkermansia muciniphila detection medium configuration: peptone: 10.0 g, ox brain extract powder: 12.5 g, ox heart extract powder: 5.0 g, sodium chloride: 5 g, glucose: 2.0 g, disodium hydrogen phosphate: 2.5 g, mucin: 2 g; agar powder: 10 g; add into 1000 mL of distilled water, heat and stir to dissolve, adjust pH to 7.4 ± 0.2, and sterilize under high pressure at 121 °C for 15 min.

[0046] 2) Preparation of gradient protective solution for Akkermansia muciniphila: Add 8.5 g NaCl and 0.5 g casein peptone into 1000 mL distilled water, stir to dissolve, and sterilize under high pressure at 121° C. for 15 min after aliquoting.

[0047] 3) Use an automatic dispensing robot to complete the dispensing of the test tubes used in the experiment and the blue-capped bottles for initial dissolution. All of them adopt the method of dispensing after the gradient protection liquid is sterilized. The number of gradient dilution test tubes required is calculated according to the range of viable bacteria counts of the sample. The automatic dispensing robot sets the dispensing parameters. After sterilization, the liquid is dispensed into 9.0 mL of gradient protection liquid in the test tube through a peristaltic pump. After sterilization, the liquid is dispensed into 198.0 mL of gradient protection liquid in the blue-capped bottle through a peristaltic pump. The test tube is gently buckled with a sterile rubber test tube stopper, and the blue-capped bottle is tightened with the bottle cap. Prepare 7 9.0 mL test tubes and 1 198.0 mL blue-capped bottle.

[0048] The following operations are all completed in the anaerobic workstation. Before the operation, the required items should be placed in the workstation in advance (make sure the workstation is clean and disinfected and free of contaminants).

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

[0050] 1) Preparation of initial solution:

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

[0052] 2) Gradient dilution

[0053] Slowly draw 1 mL from the middle of the 1:100 sample solution, and slowly inject it into the test tube containing 9 mL of gradient protection solution along the test tube wall (be careful not to let the tip of the gun touch the gradient protection solution), mix it with a vortex oscillator, start timing from the time the liquid in the test tube rotates to the bottom, oscillate for 3 seconds, and repeat the oscillation 8 times to make a 1:1000 sample solution. Take another 1 mL sterile pipette tip and make 10-fold incremental sample solutions according to the above operation sequence. Each time the incremental dilution is made, the 1 mL sterile pipette tip is used once, and a total of 7 test tubes are diluted. At this time, a 1:1000 sample solution has been made. 9 Homogenize the sample.

[0054] 3) Sample transfer

[0055] Option 10 8 and 10 9 For two test tubes, pipette 1 mL of sample solution for each dilution into sterile plates, and make three plates for each dilution.

[0056] S3 medium pouring and culture

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

[0058] 2) After the culture medium solidifies, the plate is inverted and placed in an anaerobic workstation (80N2, 10% H2 and 10% CO2), cultured at 37°C for 84 hours, and the colonies are counted.

[0059] Example 2

[0060] This example performs quantitative detection of Akkermansia muciniphila.

[0061] S1 Preliminary preparation: preparation and sterilization of culture medium, preparation and sterilization of gradient protective solution, and packaging of test tubes and blue-cap bottles.

[0062] 1) Akkermansia muciniphila detection medium configuration: peptone: 8 g, ox brain extract powder: 11.5 g, ox heart extract powder: 4, sodium chloride: 4 g, glucose: 1 g, disodium hydrogen phosphate: 1.5 g, mucin: 1 g; agar powder: 5 g; add into 1000 mL of distilled water, heat and stir to dissolve, adjust pH to 7.4 ± 0.2, and sterilize under high pressure at 121 °C for 15 min.

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

[0064] 3) Use an automatic dispensing robot to complete the dispensing of the test tubes used in the experiment and the blue-capped bottles for initial dissolution. All of them adopt the method of dispensing after the gradient protection liquid is sterilized. The number of gradient dilution test tubes required is calculated according to the range of viable bacteria counts of the sample. The automatic dispensing robot sets the dispensing parameters. After sterilization, the liquid is dispensed into 9.0 mL of gradient protection liquid in the test tube through a peristaltic pump. After sterilization, the liquid is dispensed into 198.0 mL of gradient protection liquid in the blue-capped bottle through a peristaltic pump. The test tube is gently buckled with a sterile rubber test tube stopper, and the blue-capped bottle is tightened with the bottle cap. Prepare 7 9.0 mL test tubes and 1 198.0 mL blue-capped bottle.

[0065] The following operations are all completed in the anaerobic workstation. Before the operation, the required items should be placed in the workstation in advance (make sure the workstation is clean and disinfected and free of contaminants).

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

[0067] 1) Preparation of initial solution:

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

[0069] 2) Gradient dilution

[0070] Slowly draw 1 mL from the middle of the 1:100 sample solution, and slowly inject it into the test tube containing 9 mL of gradient protection solution along the test tube wall (be careful not to let the tip of the gun touch the gradient protection solution), mix it with a vortex oscillator, start timing from the time the liquid in the test tube rotates to the bottom, oscillate for 3 seconds, and repeat the oscillation 8 times to make a 1:1000 sample solution. Take another 1 mL sterile pipette tip and make 10-fold incremental sample solutions according to the above operation sequence. Each time the incremental dilution is made, the 1 mL sterile pipette tip is used once, and a total of 7 test tubes are diluted. At this time, a 1:1000 sample solution has been made. 9 Homogenize the sample.

[0071] 3) Sample transfer

[0072] Option 10 8 and 10 9 For two test tubes, pipette 1 mL of sample solution for each dilution into sterile plates, and make three plates for each dilution.

[0073] S3 medium pouring and culture

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

[0075] 2) After the culture medium solidifies, the plate is inverted and placed in an anaerobic workstation. Incubate at 36°C for 96 hours and count the colonies.

[0076] Example 3

[0077] This example performs quantitative detection of Akkermansia muciniphila.

[0078] S1 Preliminary preparation: preparation and sterilization of culture medium, preparation and sterilization of gradient protective solution, and packaging of test tubes and blue-cap bottles.

[0079] 1) Akkermansia muciniphila detection medium configuration: peptone: 12 g, bovine brain extract powder: 13.5 g, bovine heart extract powder: 6 g, sodium chloride: 6 g, glucose: 3 g, disodium hydrogen phosphate: 3.5 g, mucin: 3 g; agar powder: 20 g; add into 1000 mL of distilled water, heat and stir to dissolve, adjust pH to 7.4 ± 0.2, and sterilize under high pressure at 121 °C for 15 min.

[0080] 2) Preparation of gradient protective solution for Akkermansia muciniphila: Add 9 g NaCl and 0.5 g casein peptone into 1000 mL distilled water, stir to dissolve, and sterilize under high pressure at 121° C. for 15 min after aliquoting.

[0081] 3) Use an automatic dispensing robot to complete the dispensing of the test tubes used in the experiment and the blue-capped bottles for initial dissolution. All of them adopt the method of dispensing after the gradient protection liquid is sterilized. The number of gradient dilution test tubes required is calculated according to the range of viable bacteria counts of the sample. The automatic dispensing robot sets the dispensing parameters. After sterilization, the liquid is dispensed into 9.0 mL of gradient protection liquid in the test tube through a peristaltic pump. After sterilization, the liquid is dispensed into 198.0 mL of gradient protection liquid in the blue-capped bottle through a peristaltic pump. The test tube is gently buckled with a sterile rubber test tube stopper, and the blue-capped bottle is tightened with the bottle cap. Prepare 7 9.0 mL test tubes and 1 198.0 mL blue-capped bottle.

[0082] The following operations are all completed in the anaerobic workstation. Before the operation, the required items should be placed in the workstation in advance (make sure the workstation is clean and disinfected and free of contaminants).

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

[0084] 1) Preparation of initial solution:

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

[0086] 2) Gradient dilution

[0087] Slowly draw 1 mL from the middle of the 1:100 sample solution, and slowly inject it into the test tube containing 9 mL of gradient protection solution along the test tube wall (be careful not to let the tip of the gun touch the gradient protection solution), mix it with a vortex oscillator, start timing from the time the liquid in the test tube rotates to the bottom, oscillate for 3 seconds, and repeat the oscillation 8 times to make a 1:1000 sample solution. Take another 1 mL sterile pipette tip and make 10-fold incremental sample solutions according to the above operation sequence. Each time the incremental dilution is made, the 1 mL sterile pipette tip is used once, and a total of 7 test tubes are diluted. At this time, a 1:1000 sample solution has been made. 9 Homogenize the sample.

[0088] 3) Sample transfer

[0089] Option 10 8 and 10 9 For two test tubes, pipette 1 mL of sample solution for each dilution into sterile plates, and make three plates for each dilution.

[0090] S3 medium pouring and culture

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

[0092] 2) After the culture medium solidifies, the plate is inverted and placed in an anaerobic workstation. Incubate at 38°C for 80 hours and count the colonies.

[0093] Example 4

[0094] This example performs quantitative detection on Akkermansia muciniphila at different concentrations.

[0095] A1: Weigh 10 g of Akkermansia muciniphila sample and mix thoroughly, which is recorded as A1.

[0096] A2: Weigh 1 g of Akkermansia muciniphila sample and 9 g of maltodextrin into a pre-numbered aluminum foil bag, make a 10-fold serial dilution of the bacterial powder, mix thoroughly, and record it as A2.

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

[0098] S1: Preliminary preparation: preparation and sterilization of culture medium, preparation and sterilization of gradient protective solution, and packaging of test tubes and blue-cap bottles.

[0099] 1) Akkermansia muciniphila detection medium configuration: peptone: 10.0 g, dehydrated calf brain extract powder: 12.5 g, dehydrated ox heart extract powder: 5.0 g, sodium chloride: 5 g, glucose: 2.0 g, disodium hydrogen phosphate: 2.5 g, mucin: 2 g; agar powder: 10 g; add into 1000 mL of distilled water, heat and stir to dissolve, adjust pH to 7.4 ± 0.2, and sterilize under high pressure at 121 °C for 15 min.

[0100] 2) Preparation of gradient protective solution for Akkermansia muciniphila: Add 8.5 g NaCl and 1.0 g casein peptone into 1000 mL distilled water, stir to dissolve, and sterilize under high pressure at 121° C. for 15 min after aliquoting.

[0101] 3) Use an automatic dispensing robot to complete the dispensing of the test tubes used in the experiment and the blue-capped bottles for initial dissolution. All of them adopt the method of dispensing after the gradient protection liquid is sterilized, and the number of gradient dilution test tubes required is calculated according to the range of viable bacteria count of the sample. The automatic dispensing robot sets the dispensing parameters, and the liquid after sterilization is dispensed into 9.0mL gradient protection liquid in the test tube through a peristaltic pump, and the liquid after sterilization is dispensed into 198.0mL gradient protection liquid in the blue-capped bottle through a peristaltic pump. The test tube is gently buckled with a sterile rubber test tube stopper, and the blue-capped bottle is tightened with the bottle cap. For the A1 sample, 7 9.0mL test tubes and 1 198.0mL blue-capped bottle are prepared, for the A2 sample, 6 9.0mL test tubes and 1 198.0mL blue-capped bottle are prepared, and for the A3 sample, 5 9.0mL test tubes and 1 198.0mL blue-capped bottle are prepared.

[0102] The following operations are all completed in the anaerobic workstation. Before the operation, the required items should be placed in the workstation in advance (make sure the workstation is clean and disinfected and free of contaminants).

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

[0104] 1) Preparation of initial solution:

[0105] Take 2 g of each of the three mixed samples and put them into a pre-numbered 198.0 mL sterile blue-cap bottle (with an appropriate amount of sterile glass beads pre-placed in the bottle), mix the samples thoroughly to make a 1:100 sample solution.

[0106] 2) Gradient dilution

[0107] For each sample, slowly draw 1 mL from the middle of the 1:100 sample solution, and slowly inject it into the test tube containing 9 mL of gradient protection solution along the test tube wall (be careful not to let the tip of the gun touch the gradient protection solution), mix it with a vortex oscillator, start timing from when the liquid in the test tube rotates to the bottom, oscillate for 3 seconds, repeat the oscillation 8 times, and make a 1:1000 sample solution. Take another 1 mL sterile pipette tip, and make 10-fold incremental sample solutions according to the above operation sequence. Each time the incremental dilution is made, the 1 mL sterile pipette tip is used once. A1 has diluted 7 test tubes in total, and now a 1:1000 sample solution has been made. 9 Sample homogenate, A2 diluted 6 test tubes, now has a 1:10 8 Sample homogenate, A3 diluted 5 test tubes, now has a 1:10 7 Homogenize the sample.

[0108] 3) Sample transfer

[0109] A1 Select 10 8 and 10 9 For two test tubes, pipette 1 mL of sample solution for each dilution into sterile plates, and make three plates for each dilution.

[0110] A2 Select 10 7 and 10 8 For two test tubes, pipette 1 mL of sample solution for each dilution into sterile plates, and make three plates for each dilution.

[0111] A3 Select 10 6 and 10 7 For two test tubes, pipette 1 mL of sample solution for each dilution into sterile plates, and make three plates for each dilution.

[0112] S3 medium pouring and culture

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

[0114] 2) After the culture medium solidifies, the plate is inverted and placed in an anaerobic workstation. Incubate at 37°C for 96 hours and count the colonies.

[0115] Example 5

[0116] This example performs quantitative detection of Akkermansia muciniphila.

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

[0118] Example 6

[0119] This example performs quantitative detection of Akkermansia muciniphila.

[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 example performs quantitative detection of Akkermansia muciniphila.

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

[0124] Comparative Example 1

[0125] This comparative example was used to quantitatively detect Akkermansia muciniphila.

[0126] Compared with Example 1, the only difference is that the bovine brain extract powder in the Akkermansia muciniphila detection medium is replaced with an equal amount of bovine heart extract powder, and the rest is the same as Example 1.

[0127] Comparative Example 2

[0128] This comparative example was used to quantitatively detect Akkermansia muciniphila.

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

[0130] Comparative Example 3

[0131] In this comparative example, flow cytometry was used to perform quantitative detection of Akkermansia muciniphila.

[0132] S1 Preliminary preparation: preparation and sterilization of gradient protective solution, and packaging of test tubes and blue-cap bottles.

[0133] 1) Preparation of Akkermansia muciniphila gradient protection solution: 8.5 g NaCl and 1.0 g casein peptone were added to 1000 mL distilled water, stirred to dissolve, and sterilized at 121° C. for 20 min after aliquoting.

[0134] 2) Use an automatic dispensing robot to complete the dispensing of the test tubes used in the experiment and the blue-capped bottles for initial dissolution. The gradient protection liquid is sterilized and then dispensed. The number of gradient dilution test tubes required is calculated according to the range of viable bacteria in the sample. The automatic dispensing robot sets the dispensing parameters. After sterilization, the liquid is dispensed into 9.0 mL of gradient protection liquid in the test tube through a peristaltic pump. After sterilization, the liquid is dispensed into 198.0 mL of gradient protection liquid in the blue-capped bottle through a peristaltic pump. The test tube is gently covered with a sterile rubber test tube stopper, and the blue-capped bottle is tightened with a bottle cap. Three 9.0 mL test tubes and one 198.0 mL blue-capped bottle are prepared.

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

[0136] 1) Preparation of initial solution:

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

[0138] 2 Serial dilutions

[0139] Slowly draw 1 mL from the middle of the 1:100 sample solution, and slowly inject it into the test tube containing 9 mL of gradient protection solution along the test tube wall (be careful not to let the tip of the gun touch the gradient protection solution), mix it with a vortex oscillator, start timing from the time the liquid in the test tube rotates to the bottom, oscillate for 3 seconds, repeat the oscillation 8 times, and make a 1:1000 sample solution. Take another 1 mL sterile pipette tip, and make 10-fold incremental sample solution according to the above operation sequence. Each time the incremental dilution is made, the 1 mL sterile pipette tip is used once, and a total of 3 test tubes are diluted. At this time, a 1:1000 sample solution has been made. 5 Homogenize the sample.

[0140] 3) Sample transfer

[0141] Option 10 4 and 10 5 For each of the two test tubes, vortex mix them evenly (3s, 8 times), then pipette 1mL of the sample solution for each dilution into a 2mL sterile centrifuge tube pre-marked with the dilution. Make three sterile centrifuge tubes for each dilution.

[0142] 4) Dyeing

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

[0144] 5) On the machine

[0145] When using flow cytometry for testing, instrument testing of stained samples must be completed within 45 minutes.

[0146] The test results of each embodiment and comparative example were statistically analyzed, and the colony count results are shown in Table 1. Each gradient shows 3 parallel test results.

[0147] Table 1

[0148]

[0149] It can be seen from the results in Table 1 that the quantitative detection method for Akkermansia muciniphila designed in the present invention can accurately and stably detect Akkermansia muciniphila quantitatively, which is comparable to the detection result of flow cytometry, but the cost is significantly reduced.

[0150] In addition, by comparing the results of Example 1 and Example 5, it can be seen that in the present invention, the anaerobic environment is strictly controlled during the culture and detection process, which can significantly improve the stability of the detection; by comparing the results of Example 1 with Examples 6 and 7, it can be seen that the present invention uses a specific component gradient protective solution to further improve the detection accuracy; by comparing the results of Example 1 with Comparative Examples 1 and 2, it can be seen that the present invention uses a specific component culture medium to further improve the detection accuracy.

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

[0152] Table 2

[0153]

[0154] It can be seen from the results in Table 2 that the quantitative detection method for Akkermansia muciniphila designed in the present invention has wide applicability and can be adapted to samples of different concentrations and specifications.

[0155] In summary, the present invention designs an anaerobic culture and quantitative detection method for the second-generation probiotic Akkermansia muciniphila. The detection method can be widely promoted, the product detection rate is high, there is no significant difference in parallel detection results, the data output is intuitive and reliable, and the detection process can use automated equipment to improve detection efficiency and reduce labor costs. It can also be connected to the production line for production optimization; compared with methods such as flow cytometer equipment detection, the detection cost is significantly reduced, the operation is simple, and the popularity rate is high, which is more conducive to the industrialization development of the second-generation probiotics and the promotion and promotion of quality standards.

[0156] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various 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 muciniphila, characterized in that: The method comprises: Using a gradient protection solution to gradient dilute the Akkermansia muciniphila sample to obtain a gradient diluted bacterial solution, mixing the gradient diluted bacterial solution with a detection culture medium, pouring the solution into a container, culturing under anaerobic conditions, counting colonies, and calculating the content of Akkermansia muciniphila in the Akkermansia muciniphila sample; The gradient protection solution contains sodium chloride; The detection culture medium contains peptone, ox brain extract powder, ox heart extract powder, sodium chloride, glucose, disodium hydrogen phosphate, mucin and agar powder.

2. The quantitative detection method of Akkermansia muciniphila according to claim 1, characterized in that: The concentration of sodium chloride in the gradient protection solution is 8-9 g / L.

3. The quantitative detection method of Akkermansia muciniphila according to claim 1 or 2, characterized in that: The gradient protection solution also contains casein peptone; Preferably, the concentration of casein peptone in the gradient protection solution is 0.5-1.5 g / L.

4. The quantitative detection method of Akkermansia muciniphila according to any one of claims 1 to 3, characterized in that: The concentration of peptone in the detection medium is 8-12 g / L; Preferably, the concentration of bovine brain extract powder in the detection medium is 11.5-13.5 g / L; Preferably, the concentration of the beef heart extract powder in the detection medium is 4 to 6 g / L; Preferably, the concentration of sodium chloride in the detection medium is 4 to 6 g / L; Preferably, the concentration of glucose in the detection medium is 1 to 3 g / L; Preferably, the concentration of disodium hydrogen phosphate in the detection medium is 1.5 to 3.5 g / L; Preferably, the concentration of mucin in the detection medium is 1 to 3 g / L; Preferably, the concentration of agar powder in the detection medium is 5-20 g / L.

5. The quantitative detection method of Akkermansia muciniphila according to any one of claims 1 to 4, characterized in that: The gradient protection solution contains 8-9 g / L sodium chloride and 0.5-1.5 g / L casein peptone; Preferably, the detection culture medium contains 8-12 g / L peptone, 11.5-13.5 g / L bovine brain extract powder, 4-6 g / L bovine heart extract powder, 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 of Akkermansia muciniphila according to any one of claims 1 to 5, characterized in that: The anaerobic conditions are 80-100% N2, 0-10% H2 and 0-10% CO2; Preferably, the culturing is carried out in an anaerobic workstation.

7. The quantitative detection method of Akkermansia muciniphila according to any one of claims 1 to 6, characterized in that: The culture temperature is 35-39° C. and the culture time is 80-90 hours.

8. The quantitative detection method of Akkermansia muciniphila according to claim 7, characterized in that: The culture temperature is 36-38° C. and the culture time is 82-86 hours.

9. The quantitative detection method of Akkermansia muciniphila according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: Using a gradient protection solution to gradient dilute the Akkermansia muciniphila sample to obtain a gradient diluted bacterial solution, wherein the gradient protection solution contains 8-9 g / L sodium chloride and 0.5-1.5 g / L casein peptone; The gradient diluted bacterial solution is mixed with the detection culture medium, poured into a container, placed in an anaerobic workstation for culture, the gas conditions are controlled to be 80-100% N2, 0-10% H2 and 0-10% CO2, cultured at 35-39° C. for 80-90 hours, colony counting is performed, and the content of Akkermansia muciniphila in the Akkermansia muciniphila sample is calculated; The detection culture medium contains 8-12 g / L peptone, 11.5-13.5 g / L ox brain extract powder, 4-6 g / L ox heart extract powder, 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.

10. Use of the quantitative detection method of Akkermansia muciniphila according to any one of claims 1 to 9 in the development of second-generation probiotic products and / or the development of second-generation probiotic production processes.

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