A method for detecting probiotic activity

Abstract

The present invention relates to a probiotic activity detection method. The probiotic activity detection method comprises: culturing a probiotic sample in a container, extracting gas in the container, detecting the gas using gas chromatography, calculating the CO2 concentration in the gas according to a standard curve, and calculating the probiotic activity in the sample using the CO2 concentration according to formula (1). The present invention designs a new probiotic activity detection method, deeply analyzes the respiration of probiotics, combines gas chromatography detection, constructs the relationship between the CO2 concentration in the culture gas and the bacterial activity, and realizes that the probiotic bacterial activity can be accurately evaluated by detecting the CO2 concentration in the culture gas, which is simple to operate and low in cost.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics and relates to a probiotic activity detection method. Background Art

[0002] Probiotics are a type of active microorganisms that are beneficial to the host, and can improve the balance of the host's gastrointestinal microecology and play a beneficial role. They play multiple important roles in the human intestine, such as maintaining normal intestinal function, promoting digestion and absorption of nutrients, and enhancing immunity. The state of live bacteria is the core feature of probiotics. The number of subcultures of the strain during fermentation, the conditions of fermentation culture, the freeze-drying process of the powder, and the storage method and storage time of the powder will affect the bacterial activity of the finished powder, and the activity of the bacteria has an important influence on the efficacy of probiotics. Therefore, it is crucial to detect and control the bacterial activity of probiotic products.

[0003] Commonly used methods for detecting the activity of probiotics include: (1) plate counting method: by appropriately diluting the sample and then applying it to a plate for culture, the colony forming units (CFU) are counted to estimate the number of live bacteria. This method is low-cost and easy to operate, but time-consuming and difficult to handle slow-growing or uncultivable microorganisms; (2) Raman spectroscopy: by using a microscopic Raman spectrometer to automatically collect all single-cell Raman spectra in the sample, after deducting background signal interference, the total bacterial count, live bacterial count, live bacterial rate and other indicators can be obtained. This method is independent of culture and has rapid detection, but is difficult to operate and expensive; (3) gene expression activity detection: by using PCR and other technologies to detect the nucleic acid of microorganisms, the number of live bacteria is evaluated. This method has high sensitivity, strong specificity and is rapid, but has high requirements for the experimental environment and operators. For example, CN118600054A discloses a triple digital PCR quantitative detection method for active probiotics, in which specific primers and probes are designed to detect Lactobacillus acidophilus, Lactobacillus rhamnosus, and Bifidobacterium animalis.

[0004] In summary, there is still a lack of detection methods for detecting the activity of bacteria in probiotic products that meet multiple requirements. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a probiotic activity detection method, so as to achieve rapid, accurate and low-cost detection of the bacterial activity of probiotic products.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for detecting the activity of probiotics, the method comprising:

[0008] Cultivating a probiotic sample in a container, extracting gas in the container, detecting the gas using a gas chromatograph, calculating the CO2 concentration in the gas according to a standard curve, and calculating the probiotic activity in the sample using the CO2 concentration according to formula (1);

[0009] Formula (1);

[0010] Where P is the probiotic activity (μg·CO2·g·sample -1 ·d -1 ), dc is the CO2 concentration in the container (ppm), dt is the incubation time (days, d), Vh is the gas volume in the container (L), Ws is the dry weight of the sample (g), MV is the volume of 1 mol of gas under standard conditions (L), MW is the relative molecular mass of CO2, Tst is the standard temperature (K), and T is the incubation temperature (℃).

[0011] In the present invention, a new probiotic activity detection method is designed, the respiration of probiotics is deeply analyzed, and combined with gas chromatography detection, the relationship between the CO2 concentration in the culture gas and the bacterial activity is established, so that the probiotic bacterial activity can be accurately evaluated by detecting the CO2 concentration in the culture gas, which is simple to operate and low in cost. The unit commonly used to measure the activity of probiotic bacteria is the unit viable bacteria count (CFU / mL or CFU / g), but the CFU number can only reflect the number of live probiotics in the product per unit volume or unit mass, but cannot reflect the activity of the live probiotic bacteria. In the present invention, the unit for measuring the bacterial activity of the product per unit volume or unit mass is the amount of carbon dioxide produced by the live probiotic bacteria per unit time (μg·CO2·g·sample -1 ·d -1 ). CO2 is the product of microbial metabolism of carbon-containing nutrients. Both strictly anaerobic microorganisms and facultative anaerobic microorganisms will have microbial respiration processes. Therefore, the amount of CO2 produced is an important indicator for measuring the physiological metabolic activity of microorganisms. The stronger the metabolic activity of live bacteria, the more carbon dioxide is produced in a certain unit time. Therefore, the unit used to measure the activity of the bacteria in the method of the present invention can well reflect the activity of the probiotic bacteria, and gas chromatographs have become conventional instruments and equipment in many enterprises and scientific research institutions; therefore, the method of the present invention can become a conventional method for detecting the activity of probiotic bacteria.

[0012] It can be understood that any microorganism culture container in the art that can be sealed and does not directly replace the culture gas with the outside is applicable to the present invention without special limitation.

[0013] Preferably, the method for constructing the standard curve comprises:

[0014] Use gas chromatography to detect CO2 standard gas of known concentration, use the CO2 peak area signal value detected by gas chromatography as the horizontal axis, and the known CO2 standard gas concentration as the vertical axis to fit and construct a standard curve.

[0015] Preferably, the probiotic-containing sample comprises any one of probiotic powder, probiotic granules or probiotic tablets.

[0016] Preferably, the culturing method includes culturing under strict anaerobic conditions or culturing under facultative anaerobic conditions.

[0017] Preferably, the culturing under strict anaerobic conditions comprises the following steps:

[0018] S1-1: Mix the sample and culture medium and place them in a culture container;

[0019] S1-2: Use nitrogen to purge the culture container;

[0020] S1-3: Culture in the dark and flush with nitrogen again;

[0021] S1-4: Cultivate in the dark.

[0022] Specifically, the culturing under strict anaerobic conditions may include the following steps:

[0023] S1-1: Weigh 5-10 g dry weight of sample and 25-50 mL of trypticase soy broth or thioglycollate broth or MRS broth into a culture bottle and shake to mix;

[0024] S1-2: Use nitrogen to purge the culture bottle for 2-3 minutes;

[0025] S1-3: After culturing in dark for 1-2 days, purge with nitrogen again for 2-3 min;

[0026] S1-4: Place the culture flask in a constant temperature incubator and culture in the dark for 2 to 3 days.

[0027] Preferably, the culturing under facultative anaerobic conditions comprises the following steps:

[0028] S1-5: Mix the sample and culture medium and place them in a culture container;

[0029] S1-6: Use high-purity air to purge the culture container;

[0030] S1-7: Culture in dark environment and purge with high-purity air again;

[0031] S1-8: Cultivate in the dark.

[0032] Specifically, the culturing under facultative anaerobic conditions may include the following steps:

[0033] S1-5: Weigh 5-10 g of dry weight of sample and 10-20 mL of trypticase soy broth or thioglycollate broth or MRS broth into a culture bottle and shake to mix;

[0034] S1-6: The culture bottle is purged with high-purity air for 2 to 3 minutes;

[0035] S1-7: After culturing in dark for 1-2 days, use high-purity air to purge again for 2-3 minutes;

[0036] S1-8: Culture in dark place for 1 to 2 days.

[0037] Preferably, the culture medium in the culture under strict anaerobic conditions or facultative anaerobic conditions can be independently selected from any one of trypticase soy liquid medium, thioglycollate fluid medium or MRS liquid medium.

[0038] Preferably, the tryptic soy liquid culture medium contains 2-3 g / L glucose, 16-18 g / L tryptic soy liquid culture medium, 4-6 g / L sodium chloride, 2-4 g / L soybean papain hydrolysate and 2-3 g / L dipotassium hydrogen phosphate.

[0039] Preferably, the thioglycollate fluid culture medium contains trypticase, sodium chloride, yeast extract powder, glucose, L-cystine, sodium thioglycollate, 0.1% resazurin solution and agar.

[0040] Preferably, the MRS liquid culture medium contains peptone, beef extract powder, yeast extract powder, glucose, Tween 80, K2HPO4·7H2O, sodium acetate·3H2O, triammonium citrate, MgSO4·7H2O and MnSO4·4H2O.

[0041] Preferably, the pH of the trypticase soy liquid culture medium is 7.1-7.5.

[0042] Preferably, the trypticase soytone liquid culture medium needs to be sterilized in a high pressure sterilization container at 121° C. for 15 min.

[0043] Preferably, the thioglycollate fluid culture medium contains 15 g / L trypticase, 2.5 g / L sodium chloride, 5 g / L yeast extract powder, 5.5 g / L glucose, 0.5 g / L L-cystine, 0.5 g / L sodium thioglycollate, 1 mL / L freshly prepared 0.1% resazurin solution and 0.75 g / L agar.

[0044] Preferably, the pH of the thioglycollate fluid culture medium is 6.9-7.3, for example, it can be 7.0, 7.1 or 7.2.

[0045] Preferably, the thioglycollate fluid culture medium needs to be sterilized using an autoclave container at 121° C. for 15 min.

[0046] Preferably, the MRS liquid culture medium contains 10 g / L peptone, 10 g / L beef extract powder, 5 g / L yeast extract powder, 20 g / L glucose, 1 mL / L Tween 80, 2 g / L K2HPO4·7H2O, 5 g / L sodium acetate·3H2O, 2 g / L triammonium citrate, 0.1 g / L MgSO4·7H2O and 0.05 g / L MnSO4·4H2O.

[0047] Preferably, the pH of the MRS liquid culture medium is 6.0-6.4.

[0048] Preferably, the MRS liquid culture medium needs to be sterilized in a high pressure sterilization container at 121° C. for 15 min.

[0049] Preferably, the high-purity air contains O2 and N2, but does not contain CO2. The specific volume ratio of O2 and N2 can be adjusted according to actual needs, for example, the high-purity air contains 21% O2 and 79% N2.

[0050] Preferably, the temperature of the culturing under strict anaerobic conditions or the culturing under facultative anaerobic conditions in the dark is independently selected from 28°C to 37°C, for example, it can be 29, 30, 31, 32, 33, 34, 35 or 36°C.

[0051] Preferably, when extracting the gas in the container, the ambient pressure is maintained at 101.3 kPa±1 kPa and the ambient temperature is maintained at 25°C±3°C.

[0052] Preferably, the conditions for gas chromatography detection include: the carrier gas is nitrogen, the nitrogen flow rate is 1-2 mL / min, the temperature of the hydrogen flame ionization detector is 200°C-250°C, the starting temperature of the chromatographic column is 40°C-50°C, the temperature of the chromatographic column is gradually increased to 200°C-250°C, and the chromatographic column is a capillary column.

[0053] As a preferred technical solution, the probiotic activity detection method comprises the following steps:

[0054] S1: Cultivate the probiotic sample in a container under strict anaerobic conditions or facultative anaerobic conditions;

[0055] S2: Use gas chromatography to detect CO2 standard gas of known concentration, use the CO2 peak area signal value detected by gas chromatography as the horizontal coordinate, and use the known CO2 standard gas concentration as the vertical coordinate to fit and construct a standard curve;

[0056] S3: extracting gas from the container, detecting the gas using a gas chromatograph, calculating the CO2 concentration in the gas according to a standard curve, and calculating the probiotic activity in the sample using the CO2 concentration according to formula (1);

[0057] Formula (1);

[0058] Among them, P is the probiotic activity, dc is the CO2 gas concentration in the container, dt is the incubation time, Vh is the gas volume in the container, Ws is the dry weight of the sample, MV is the volume of 1 mol of gas under standard conditions, MW is the relative molecular mass of CO2, Tst is the standard temperature, and T is the incubation temperature.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] The present invention provides a novel probiotic activity detection method, which can detect the activity of probiotics cultured under strictly anaerobic conditions and the activity of probiotics cultured under facultative anaerobic conditions. The CO2 concentration is measured by gas chromatograph to judge the respiration of microorganisms, thereby measuring the activity of the probiotics, thereby achieving effective detection of the activity of the probiotics with high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a diagram of the culture bottle used in the probiotic culture experiment of the present invention. DETAILED DESCRIPTION

[0062] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0063] 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.

[0064] In the present invention, in view of the current lack of a good detection method for measuring the activity of bacteria in probiotic products, based on the fact that microorganisms produce carbon dioxide during their metabolic nutrition and reproduction stages, an in-depth analysis of the relationship between microbial activity and the production of carbon dioxide is conducted, and a method for detecting microbial activity by detecting carbon dioxide is designed.

[0065] Example 1

[0066] This embodiment provides a method for detecting probiotic activity, comprising the following steps:

[0067] S1: Probiotic powder is cultured in glass bottles;

[0068] S2: Use gas chromatograph to make a standard curve using CO2 standard gas; the correlation coefficient of the standard curve ranges from 0.99 to 1.00, and the confidence level of the standard curve is ≥95%;

[0069] S3: Use a medical syringe to extract 5 mL of culture gas in the bottle, inject the extracted gas into the gas chromatograph, use the gas chromatograph to detect, and calculate the concentration of CO2 in the bottle based on the prepared standard curve;

[0070] S4: Use the following formula to calculate the activity of probiotic powder:

[0071]

[0072] In the formula, dc is the CO2 gas concentration in the culture bottle (ppm), dt is the culture time (days, d), Vh is the gas volume in the culture bottle (L), Ws is the sample mass (g), MV is the volume of 1 mol of culture gas under standard conditions (L), MW is the relative molecular mass of CO2, Tst is the standard temperature (K), T is the culture temperature (℃), and P is the activity of the probiotic powder (μg·CO2·g·sample -1 ·d -1 ).

[0073] Under strict anaerobic conditions, the cultivation of probiotic powder includes the following steps:

[0074] S1-1: Weigh a sample equivalent to 10 g of dry weight, take 50 mL of tryptic soytone liquid culture medium, put it into a culture bottle, and shake to mix; the preparation method of the tryptic soytone liquid comprises the following steps: 1) take 17.0 g of tryptic soytone, 5.0 g of sodium chloride, 3.0 g of soybean papain hydrolyzate, 2.5 g of potassium dihydrogen phosphate, and 1000 mL of purified water, and place them in a container for lukewarm dissolution; 2) adjust the pH of the mixed liquid to 7.1-7.5 at 25°C; 3) add 2.5 g of glucose to the mixed liquid and stir evenly; 4) sterilize the container at 121°C for 15 min using an autoclave;

[0075] S1-2: Use high-purity nitrogen to purge the culture bottle for 2 minutes;

[0076] S1-3: After culturing in dark for 1 day, high-purity nitrogen was used for purging again for 2 min;

[0077] S1-4: Place the culture flask in a 37°C constant temperature incubator and culture in the dark for 3 days;

[0078] The glass bottles used for culture can be found in Figure 1The specification of the culture bottle is 0.15 L, the bottle body is made of glass, and the top of the bottle mouth is sealed with a butyl rubber stopper. Before use, the glass bottle and the butyl rubber stopper are sterilized at 121°C in an autoclave for 15 min. When in use, the needle of the medical syringe is connected to the plastic three-way valve and then inserted into the butyl rubber stopper.

[0079] Then, when extracting gas in step S3, the ambient pressure needs to be maintained at 101.3 kPa±1 kPa and the ambient temperature needs to be maintained at 25°C±3°C. Secondly, the gas chromatography conditions in step S3 are as follows: the carrier gas is nitrogen (flow rate: 1.5 mL / min), the temperature of the hydrogen flame ionization detector is 240°C, the starting temperature of the chromatographic column is 45°C, the temperature of the chromatographic column is gradually increased to 240°C, and the chromatographic column is a capillary column.

[0080] Example 2

[0081] This embodiment provides a method for detecting probiotic activity, comprising the following steps:

[0082] S1: Probiotic powder is cultured in glass bottles;

[0083] S2: Use gas chromatograph to make a standard curve using standard gas of carbon dioxide; the correlation coefficient of the standard curve ranges from 0.99 to 1.00, and the confidence level of the standard curve is ≥95%;

[0084] S3: Use a medical syringe to extract 5 mL of culture gas in the bottle, inject the extracted gas into the gas chromatograph, use the gas chromatograph to detect, and calculate the concentration of CO2 in the bottle based on the prepared standard curve;

[0085] S4: Use the following formula to calculate the activity of probiotic powder:

[0086]

[0087] In the formula, dc is the CO2 gas concentration in the culture bottle (ppm), dt is the culture time (days, d), Vh is the gas volume in the culture bottle (L), Ws is the sample mass (g), MV is the volume of 1 mol of culture gas under standard conditions (L), MW is the relative molecular mass of carbon dioxide, Tst is the standard temperature (K), T is the culture temperature (℃), and P is the activity of the probiotic powder (μg·CO2·g·sample -1 ·d -1 ).

[0088] Under facultative anaerobic conditions, the cultivation of probiotic powder includes the following steps:

[0089] S1-5: Weigh a sample equivalent to 10 g of dry weight, take 20 mL of tryptic soytone liquid culture medium, put it into a culture bottle, and shake to mix; the preparation method of the tryptic soytone liquid comprises the following steps: 1) take 17.0 g of tryptic soytone, 5.0 g of sodium chloride, 3.0 g of soybean papain hydrolyzate, 2.5 g of potassium dihydrogen phosphate, and 1000 mL of purified water, and place them in a container for slight dissolution; 2) adjust the pH of the mixed liquid to 7.1-7.5 at 25°C; 3) add 2.5 g of glucose to the mixed liquid and stir evenly; 4) sterilize the container at 121°C for 15 min using an autoclave;

[0090] S1-6: The culture bottle is purged with high-purity air for 2 min;

[0091] S1-7: After culturing in dark for 1 day, high-purity air was used to purge again for 2 min;

[0092] The high-purity air composition in steps S1-6 and S1-7 is 21% O2, 79% N2, and 0% CO2;

[0093] S1-8: Culture in the dark for 2 days.

[0094] Then, when extracting gas in step S3, the ambient pressure needs to be maintained at 101.3 kPa±1 kPa and the ambient temperature needs to be maintained at 25°C±3°C. Secondly, the gas chromatography conditions in step S3 are as follows: the carrier gas is nitrogen (flow rate: 1.5 mL / min), the temperature of the hydrogen flame ionization detector is 240°C, the starting temperature of the chromatographic column is 45°C, the temperature of the chromatographic column is gradually increased to 240°C, and the chromatographic column is a capillary column.

[0095] Example 3

[0096] This example uses the probiotic Lactobacillus acidophilus LA85 powder as an example to verify the probiotic activity detection method. The method includes the following steps:

[0097] S1: Prepare tryptic soy casein liquid culture medium. The preparation method includes the following steps: 1) Take 17.0 g tryptic soy casein, 5.0 g sodium chloride, 3.0 g soybean papain hydrolyzate, 2.5 g potassium dihydrogen phosphate, and 1000 mL purified water, place in a container for lukewarm dissolution; 2) Adjust the pH of the mixed liquid to 7.1-7.5 at 25°C; 3) Add 2.5 g glucose to the mixed liquid and stir evenly; 4) Use an autoclave to sterilize the container at 121°C for 15 min;

[0098] S2: Randomly select 5 production batches of Lactobacillus acidophilus LA85 powder, take 10 g of Lactobacillus acidophilus LA85 powder from each batch, take 5 portions of 20 mL of tryptic soytone liquid culture medium, and add each batch of powder and each medium into a glass bottle for culture;

[0099] S3: The culture bottle is purged with high-purity air for 2 min;

[0100] S4: After culturing in dark for 1 day, high-purity air is used for purging again for 2 min; the high-purity air composition in steps S3 and S4 is 21% O2, 79% N2, and 0% CO2;

[0101] S5: Place the culture bottle in a 32°C constant temperature incubator and culture in the dark for 2 days;

[0102] S6: Use gas chromatograph to make a standard curve using CO2 standard gas; the correlation coefficient of the standard curve ranges from 0.99 to 1.00, and the confidence level of the standard curve is ≥95%;

[0103] S7: Use a medical syringe to extract 5 mL of culture gas in the bottle, inject the extracted gas into the gas chromatograph, use the gas chromatograph to detect, and calculate the concentration of CO2 in the bottle based on the prepared standard curve;

[0104] When extracting gas in step S7, the ambient pressure needs to be maintained at 101.3 kPa±1 kPa and the ambient temperature needs to be maintained at 25°C±3°C. Secondly, the gas chromatography conditions in step S7 are as follows: the carrier gas is nitrogen (flow rate: 1.5 mL / min), the temperature of the hydrogen flame ionization detector is 240°C, the starting temperature of the chromatographic column is 45°C, the temperature of the chromatographic column is gradually increased to 240°C, and the chromatographic column is a capillary column.

[0105] S8: Use the following formula to calculate the activity of Lactobacillus acidophilus LA85 powder:

[0106]

[0107] In the formula, dc is the CO2 gas concentration in the culture bottle (ppm), dt is the culture time (days, d), Vh is the gas volume in the culture bottle (L), Ws is the sample mass (g), MV is the volume of 1 mol of culture gas under standard conditions (L), MW is the relative molecular mass of carbon dioxide, Tst is the standard temperature (K), T is the culture temperature (℃), and P is the activity of the probiotic powder (μg·CO2·g·sample -1 ·d -1 ).

[0108] S9: Take the same 5 production batches of Lactobacillus acidophilus LA85 powder, and test the total number of viable bacteria of each batch of Lactobacillus acidophilus LA85 powder according to Appendix 2: Determination of viable bacteria count of microecological live bacteria products in Part III of the 2020 edition of the Chinese Pharmacopoeia "General Introduction to Microecological Live Bacteria Products". Then, divide the result of the powder activity by the result of the viable bacteria count to obtain the total number of viable bacteria per 1×10 per gram of sample. 11 The results of the activity and total viable count of Lactobacillus acidophilus LA85 powder are shown in Table 1.

[0109] Table 1

[0110]

[0111] As can be seen from the test results in Table 1, there is no significant difference in the total number of viable bacteria in the 5 batches of Lactobacillus acidophilus LA85 powder, but the activity of the 5 batches of Lactobacillus acidophilus LA85 powder detected according to the method in Example 3 is significantly different. In addition, the activity of the same number of colonies in each gram of powder is also significantly different. It can be seen that if the traditional viable bacteria count detection method is used, only the total number of viable bacteria in the product can be obtained, and the activity difference between the probiotic powders cannot be reflected. However, this method can well make up for the shortcomings of the traditional viable bacteria count detection, can reflect the true activity of the bacteria, and can well reflect the state difference of the activity of the bacteria between different batches of products.

[0112] Example 4

[0113] This example uses the probiotics Pediococcus acidilactici PA53 particles as an example to verify the probiotic activity detection method. The method includes the following steps:

[0114] S1: Prepare tryptic soy casein liquid culture medium. The preparation method includes the following steps: 1) Take 17.0 g tryptic soy casein, 5.0 g sodium chloride, 3.0 g soybean papain hydrolyzate, 2.5 g potassium dihydrogen phosphate, and 1000 mL purified water, place in a container for lukewarm dissolution; 2) Adjust the pH of the mixed liquid to 7.1-7.5 at 25°C; 3) Add 2.5 g glucose to the mixed liquid and stir evenly; 4) Use an autoclave to sterilize the container at 121°C for 15 min;

[0115] S2: Randomly select 5 production batches of Pediococcus acidilactici PA53 granules, take 10 g of Pediococcus acidilactici PA53 granules from each batch, take 5 portions of 50 mL of tryptic soytone liquid culture medium, add each batch of bacterial powder and each medium into a glass bottle for culture;

[0116] S3: The culture bottle is purged with high-purity nitrogen for 2 min;

[0117] S4: After culturing in dark for 1 day, high-purity nitrogen was used to purge again for 2 min;

[0118] S5: Place the culture flask in a 37°C constant temperature incubator and culture in the dark for 3 days;

[0119] S6: Use gas chromatograph to make a standard curve using CO2 standard gas; the correlation coefficient of the standard curve ranges from 0.99 to 1.00, and the confidence level of the standard curve is ≥95%;

[0120] S7: Use a medical syringe to extract 5 mL of culture gas in the bottle, inject the extracted gas into the gas chromatograph, use the gas chromatograph to detect, and calculate the concentration of CO2 in the bottle based on the prepared standard curve;

[0121] When extracting gas in step S7, the ambient pressure needs to be maintained at 101.3 kPa±1 kPa and the ambient temperature needs to be maintained at 25°C±3°C. Secondly, the gas chromatography conditions in step S7 are as follows: the carrier gas is nitrogen (flow rate: 1.5 mL / min), the temperature of the hydrogen flame ionization detector is 240°C, the starting temperature of the chromatographic column is 45°C, the temperature of the chromatographic column is gradually increased to 240°C, and the chromatographic column is a capillary column.

[0122] S8: Use the following formula to calculate the activity of Lactobacillus acidophilus LA85 powder:

[0123]

[0124] In the formula, dc is the CO2 gas concentration in the culture bottle (ppm), dt is the culture time (days, d), Vh is the gas volume in the culture bottle (L), Ws is the sample mass (g), MV is the volume of 1 mol of culture gas under standard conditions (L), MW is the relative molecular mass of carbon dioxide, Ts is the standard temperature (K), T is the culture temperature (℃), and P is the activity of the probiotic powder (μg·CO2·g·sample -1 ·d -1 ).

[0125] S9: Take the same 5 production batches of Pediococcus acidilactici PA53 granules and each batch of Lactobacillus acidophilus LA85 powder according to Appendix 2: Determination of viable count of microecological live bacteria products in Part III of the 2020 edition of the Chinese Pharmacopoeia "General Introduction to Microecological Live Bacteria Products" to test the total number of viable bacteria of the 5 batches of Pediococcus acidilactici PA53 granules. The results are shown in Table 2. Then, the result of the particle activity was divided by the result of the viable count to obtain the total number of viable bacteria per 1×10 per gram of sample. 11 The activity of CFU colony count, the activity of Pediococcus acidilactici PA53 particles and the total number of viable bacteria are shown in Table 2.

[0126] Table 2

[0127]

[0128] In summary, the present invention designs a new probiotic activity detection method, deeply analyzes the respiration of probiotics, combines gas chromatography detection, constructs the relationship between the CO2 concentration in the culture gas and the bacterial activity, and realizes the evaluation of the probiotic activity by detecting the CO2 concentration in the culture gas. The operation is simple and the cost is low.

[0129] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting probiotic activity, characterized in that: The probiotic activity detection method comprises: Cultivating a probiotic sample in a container, extracting gas in the container, detecting the gas using a gas chromatograph, calculating the CO2 concentration in the gas according to a standard curve, and calculating the probiotic activity in the sample using the CO2 concentration according to formula (1); Formula (1); Wherein, P is the probiotic activity, dc is the CO2 gas concentration in the container, dt is the incubation time, Vh is the gas volume in the container, Ws is the dry weight of the sample, MV is the volume of 1 mol of gas under standard conditions, MW is the relative molecular mass of CO2, Tst is the standard temperature, and T is the incubation temperature; The method for constructing the standard curve comprises: Use gas chromatography to detect CO2 standard gas of known concentration, use the CO2 peak area signal value detected by gas chromatography as the horizontal coordinate, and use the known CO2 standard gas concentration as the vertical coordinate to fit and construct a standard curve; The probiotic sample includes any one of probiotic powder, probiotic granules or probiotic tablets; The culturing method includes culturing under strict anaerobic conditions or culturing under facultative anaerobic conditions; The culturing under the strict anaerobic conditions comprises the following steps: S1-1: Mix the sample and culture medium and place them in a culture container; S1-2: Use nitrogen to purge the culture container; S1-3: Culture in the dark and flush with nitrogen again; S1-4: dark culture; The culturing under the facultative anaerobic conditions comprises the following steps: S1-5: Mix the sample and culture medium and place them in a culture container; S1-6: Use high-purity air to purge the culture container; S1-7: Culture in dark environment and purge with high-purity air again; S1-8: Cultivate in the dark.

2. The probiotic activity detection method according to claim 1, characterized in that: The culture medium in the culture under strict anaerobic conditions or facultative anaerobic conditions can be independently selected from any one of trypticase soytone liquid culture medium, thioglycollate fluid culture medium or MRS liquid culture medium; The tryptic soy casein liquid culture medium contains glucose, tryptic soy casein, sodium chloride, soybean papain hydrolyzate and dipotassium hydrogen phosphate; The thioglycollate fluid medium contains trypticase, sodium chloride, yeast extract powder, glucose, L-cystine, sodium thioglycollate, 0.1% resazurin solution and agar; The MRS liquid culture medium contains peptone, beef extract powder, yeast extract powder, glucose, Tween 80, K2HPO4·7H2O, sodium acetate·3H2O, triammonium citrate, MgSO4·7H2O and MnSO4·4H2O.

3. The probiotic activity detection method according to claim 1, characterized in that: The high-purity air contains O2 and N2 but does not contain CO2.

4. The probiotic activity detection method according to claim 1, characterized in that: The temperature of the culturing under strict anaerobic conditions or the culturing under facultative anaerobic conditions in the dark is independently selected from 28° C. to 37° C.

5. The probiotic activity detection method according to claim 1, characterized in that: When extracting the gas in the container, the ambient pressure is maintained at 101.3 kPa±1 kPa and the ambient temperature is maintained at 25°C±3°C.

6. The probiotic activity detection method according to claim 1, characterized in that: The gas chromatography detection conditions include: the carrier gas is nitrogen, the nitrogen flow rate is 1-2 mL / min, the temperature of the hydrogen flame ionization detector is 200°C-250°C, the starting temperature of the chromatographic column is 40°C-50°C, the temperature of the chromatographic column is gradually increased to 200°C-250°C, and the chromatographic column is a capillary column.

7. The method for detecting probiotic activity according to any one of claims 1 to 6, characterized in that: The probiotic activity detection method comprises the following steps: S1: Cultivate the probiotic sample in a container under strict anaerobic conditions or facultative anaerobic conditions; S2: Use gas chromatography to detect CO2 standard gas of known concentration, use the CO2 peak area signal value detected by gas chromatography as the horizontal coordinate, and use the known CO2 standard gas concentration as the vertical coordinate to fit and construct a standard curve; S3: extracting gas from the container, detecting the gas using a gas chromatograph, calculating the CO2 concentration in the gas according to a standard curve, and calculating the probiotic activity in the sample using the CO2 concentration according to formula (1); Formula (1); Among them, P is the probiotic activity, dc is the CO2 gas concentration in the container, dt is the incubation time, Vh is the gas volume in the container, Ws is the dry weight of the sample, MV is the volume of 1 mol of gas under standard conditions, MW is the relative molecular mass of CO2, Tst is the standard temperature, and T is the incubation temperature.

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