Method and kit for detecting colony count of inactivated bifidobacterium BL-99 cells before inactivation

Abstract

The invention discloses a method and a kit for detecting the colony count of inactivated bifidobacterium BL-99 cells before inactivation, and relates to the technical field of instrument analysis. According to the method, at least one of prolyl alanine, citric acid, Asp-Phe peptide, GP (Hyp) GAG peptide, L-methionine, GPRPK peptide and Asp-Leu peptide is taken as a target substance, the colony count of inactivated thalli before inactivation is calculated by testing the content of the target substance, and the detection accuracy is relatively high. A theoretical basis is provided for quantitative detection of the bifidobacterium BL-99 metagen, and industrial standards are also favorably established for quantitative detection of bifidobacterium BL-99 metagen products.

Description

Technical Field

[0001] The present invention relates to the technical field of instrumental analysis, and in particular, to a detection method and kit for inactivated cells of postbiotics of Bifidobacterium BL-99. Background Art

[0002] Postbiotics refer to preparations of inanimate microorganisms and / or their components that are beneficial to the health of the host. As derivatives of prebiotics and probiotics, postbiotics have multiple potential benefits. They can regulate the intestinal flora, enhance the intestinal barrier function, regulate the intestinal inflammatory response, etc., and then have a positive impact on human intestinal health. In addition, the biological activities of postbiotics are not limited to the intestine, and it also has biological activities such as inhibiting oral pathogenic bacteria and regulating pulmonary inflammatory response. In current postbiotic-related disease research, functional activities such as anti-inflammatory and antioxidant are considered to be the general functions of postbiotics. With the continuous deepening of people's understanding of intestinal health and the microbiome, postbiotics are considered a potential functional food and health management means, and have received particular attention from the industrial community.

[0003] Current postbiotic products mainly include inactivated cells and fermentation broth. Inactivated cells are prepared by processes such as fermentation and inactivation of strains; fermentation broth is obtained by processes such as fermentation and extraction of strains. The components of postbiotic products are relatively complex, and the types of components can reach thousands, making it difficult to quantitatively analyze the colony count in the products, and there is currently no detection standard to follow.

[0004] Bifidobacterium BL-99, deposit number: CGMCC No. 15650, is deposited in the China General Microbiological Culture Collection Center (CGMCC). The postbiotic products produced using this exclusive strain have an efficient anti-inflammatory effect, and its anti-inflammatory effect is directly related to its colony count.

[0005] Therefore, there is an urgent need to develop a method for quantitatively analyzing postbiotic products (such as inactivated cells) of Bifidobacterium BL-99 strain.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The purpose of the present invention is to provide a detection method and kit for inactivated cells of postbiotics of Bifidobacterium BL-99, aiming to accurately detect the colony count before inactivation of inactivated cells of Bifidobacterium BL-99.

[0008] The present invention is implemented as follows:

[0009] The present invention provides a method for detecting the number of colonies of inactivated Bifidobacterium BL-99 before inactivation, including: using at least one of prolylalanine, citric acid, Asp-Phe peptide, GP(Hyp)GAG peptide, L-methionine, GPRPK peptide, and Asp-Leu peptide as a target substance to detect the number of colonies of inactivated Bifidobacterium BL-99 before inactivation.

[0010] In an alternative embodiment, the detection method includes the step of establishing a linear equation between the number of colonies and the content of the target substance to obtain a regression equation, and the regression equation is y = ax + b, where x represents the concentration of the target substance in mg / 100g; y represents the number of colonies in 10 9 CFU / mL;

[0011] Preferably, viable Bifidobacterium BL-99 is provided according to a gradient of the number of colonies, inactivated, and then the target substance is extracted and its content is analyzed. A fitting linear equation is calculated based on the gradient of the number of colonies and the content of the target substance to obtain a regression equation;

[0012] Preferably, when calculating the number of colonies of inactivated bacteria before inactivation using prolylalanine as the target substance, the value of a in the regression equation is 0.065 - 0.070, and the value of b is 0.200 - 0.210; more preferably, when calculating the number of colonies of inactivated bacteria before inactivation using prolylalanine as the target substance, the regression equation used is y = 0.068x + 0.205;

[0013] Preferably, when calculating the number of colonies of inactivated bacteria before inactivation using citric acid as the target substance, the value of a in the regression equation is 0.002 - 0.004, and the value of b is -0.090 to -0.095; more preferably, when calculating the number of colonies of inactivated bacteria before inactivation using citric acid as the target substance, the regression equation used is y = 0.003x - 0.092;

[0014] Preferably, when calculating the number of colonies of inactivated bacteria before inactivation using Asp-Phe peptide as the target substance, the value of a in the regression equation is 1.805 - 1.810, and the value of b is 0.220 - 0.225; more preferably, when calculating the number of colonies of inactivated bacteria before inactivation using Asp-Phe peptide as the target substance, the regression equation used is y = 1.807x + 0.222;

[0015] Preferably, when calculating the number of colonies of inactivated bacteria before inactivation using GP(Hyp)GAG peptide as the target substance, the value of a in the regression equation is 0.085 - 0.095, and the value of b is 0.770 - 0.775; more preferably, when calculating the number of colonies of inactivated bacteria before inactivation using GP(Hyp)GAG peptide as the target substance, the regression equation used is y = 0.090x + 0.773;

[0016] Preferably, when calculating the colony count of inactivated bacteria before inactivation using L-methionine as the target, the value of a in the regression equation is 0.065 - 0.075, and the value of b is 0.525 - 0.535; more preferably, when calculating the colony count of inactivated bacteria before inactivation using L-methionine as the target, the regression equation used is y = 0.071x + 0.529;

[0017] Preferably, when calculating the colony count of inactivated bacteria before inactivation using GPRPK peptide as the target, the value of a in the regression equation is 0.110 - 0.120, and the value of b is -0.235 to -0.245; more preferably, when calculating the colony count of inactivated bacteria before inactivation using GPRPK peptide as the target, the regression equation used is y = 0.115x - 0.241;

[0018] Preferably, when calculating the colony count of inactivated bacteria before inactivation using Asp-Leu peptide as the target, the value of a in the regression equation is 0.235 - 0.247, and the value of b is -0.620 to -0.630; more preferably, when calculating the colony count of inactivated bacteria before inactivation using Asp-Leu peptide as the target, the regression equation used is y = 0.242x - 0.626;

[0019] More preferably, at least one of prolylalanine, citric acid, Asp-Phe peptide, GP(Hyp)GAG peptide, and L-methionine is used as the target to detect the colony count of inactivated Bifidobacterium BL-99 before inactivation;

[0020] Further preferably, at least one of prolylalanine, citric acid, and Asp-Phe peptide is used as the target to detect the colony count of inactivated Bifidobacterium BL-99 before inactivation.

[0021] In an alternative embodiment, the inactivated bacteria are pretreated before detection. The pretreatment process includes: extracting the inactivated bacteria sample to obtain an extract, taking the supernatant of the extract and drying it to obtain a powder sample, and re-dissolving and diluting the powder sample.

[0022] In an alternative embodiment, the inactivated bacteria sample is mixed with an organic alcohol aqueous solution for low-temperature extraction to obtain an extract; after centrifuging and separating the extract, the supernatant is taken, and the supernatant is dried to obtain a powder sample; the powder sample is re-dissolved and diluted with water;

[0023] Preferably, the preparation process of the extract includes: mixing the inactivated bacteria sample with an organic alcohol aqueous solution with a volume fraction of 70% - 90%, and performing low-temperature ultrasonic extraction for 20 min - 40 min under the conditions of a frequency of 35 KHz - 45 KHz and a temperature of 2°C - 6°C;

[0024] More preferably, the aqueous organic alcohol solution is an aqueous methanol solution, and the dosage of the aqueous methanol solution corresponding to each 0.1 g of the inactivated cell sample is 1 mL to 3 mL;

[0025] Preferably, the aqueous methanol solution is pre-cooled to 2°C to 6°C before being mixed with the inactivated cell sample.

[0026] In an alternative embodiment, the extract is allowed to stand at a temperature of -15°C to -25°C for 20 min to 40 min, and then centrifuged to obtain the supernatant, which is blown with nitrogen until a powder sample is completely obtained;

[0027] Preferably, the powder sample is re-dissolved in water, the supernatant is taken after centrifugation, and then diluted with 9 to 11 times the volume of water.

[0028] In an alternative embodiment, high performance liquid chromatography is used for detection. The concentration of the target substance in the inactivated cells is calculated by the standard curve method, and then the number of colonies before inactivation of the inactivated cells is calculated from the regression equation;

[0029] Preferably, the concentration of the target substance is measured by high performance liquid chromatography. The measurement process includes:

[0030] Preparing a standard working solution: Using the target substance standard, standard working solutions with different dilution factors are prepared;

[0031] Preparing a test sample solution: The inactivated cells are pretreated to obtain a sample solution;

[0032] Detection: The sample solution and the standard working solution are measured by high performance liquid chromatography. Using the standard working solutions with different concentrations and their corresponding peak areas, a standard curve is plotted; the concentration of the target substance in the inactivated cells is calculated using the standard curve.

[0033] In an alternative embodiment, when using high performance liquid chromatography for measurement, the chromatographic conditions include: the chromatographic column is C18, the inner diameter of the chromatographic column is 4.0 mm to 5.0 mm, the length of the chromatographic column is 140 mm to 160 mm, and the particle size of the chromatographic column packing is 2.5 μm to 3.0 μm; the column temperature is 25°C to 35°C, and the detection wavelength is 200 nm to 220 nm;

[0034] Preferably, the chromatographic column uses Poroshell 120Aq-C18;

[0035] Preferably, the injection volume is controlled to be 2 μL to 10 μL.

[0036] In an alternative embodiment, when tested by high performance liquid chromatography, the mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an acidic substance solution with a volume fraction of 0.05% to 0.20%; mobile phase B is a mixed solution of an acidic substance and acetonitrile, and the volume fraction of the acidic substance is 0.05% to 0.20%.

[0037] Among them, the acidic substance is selected from any one of trifluoroacetic acid and phosphoric acid;

[0038] Preferably, the gradient elution program is as follows:

[0039] When the time is 0.00 min, the volume fraction of mobile phase A in the mobile phase is 98% to 100%, and the volume fraction of mobile phase B is 0% to 2%;

[0040] When the time is 10.00 min, the volume fraction of mobile phase A in the mobile phase is 98% to 100%, and the volume fraction of mobile phase B is 0% to 2%;

[0041] When the time is 33.00 min, the volume fraction of mobile phase A in the mobile phase is 93% to 97%, and the volume fraction of mobile phase B is 3% to 7%;

[0042] When the time is 45.00 min, the volume fraction of mobile phase A in the mobile phase is 93% to 97%, and the volume fraction of mobile phase B is 3% to 7%;

[0043] Preferably, the flow rate is controlled to be 0.5 mL / min to 1.0 mL / min.

[0044] In an alternative embodiment, the process of preparing the standard working solution includes: diluting the target standard product with water to prepare a standard stock solution with a concentration greater than or equal to 5 mg / mL; taking the standard stock solution and diluting it with water to obtain a mixed standard intermediate solution with a concentration of 400 μg / mL to 600 μg / mL; taking the mixed standard intermediate solution and diluting it with water into standard working solutions with different concentrations, and the concentration range of the standard working solutions is 1 μg / mL to 200 μg / mL.

[0045] In a second aspect, the present invention also provides a kit for detecting the number of viable colonies before inactivating the inactivated cells of Bifidobacterium BL-99, and the kit includes: using at least one of prolylalanine, citric acid, Asp-Phe peptide, GP(Hyp)GAG peptide, L-methionine, GPRPK peptide, and Asp-Leu peptide as the target substance.

[0046] The present invention has the following beneficial effects: Using at least one of prolylalanine, citric acid, Asp-Phe peptide, GP(Hyp)GAG peptide, L-methionine, GPRPK peptide, and Asp-Leu peptide as a target, the colony count before inactivation of inactivated bacteria is calculated by testing the content of the target, and the detection accuracy is relatively high. It provides a theoretical basis for the quantitative detection of postbiotics of Bifidobacterium BL-99, and is also conducive to establishing an industry standard for the quantitative detection of Bifidobacterium BL-99 postbiotic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0048] Figure 1 is the liquid chromatography diagram of the mixed standard working solution;

[0049] Figure 2 is the liquid chromatography diagram of inactivated bacteria of Bifidobacterium BL-99. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0051] The present invention provides a method for detecting the colony count before inactivation of inactivated bacteria of Bifidobacterium BL-99. The inventor creatively uses at least one of prolylalanine, citric acid, Asp-Phe peptide, GP(Hyp)GAG peptide, L-methionine, GPRPK peptide, and Asp-Leu peptide as a target. By detecting the content of the target in the inactivated bacteria of Bifidobacterium BL-99 and calculating the colony count before inactivation of the inactivated bacteria through an optimized regression equation, the purpose of accurately detecting the colony count before inactivation of the inactivated bacteria of Bifidobacterium BL-99 is achieved. The specific steps are as follows:

[0052] S1. Use high-performance liquid chromatography to test the concentration of the target

[0053] The concentration of the target in the inactivated bacteria is tested by high-performance liquid chromatography. The testing process mainly includes the following three steps:

[0054] (1) Prepare the standard working solution

[0055] Using the target standard, prepare standard working solutions with different dilution multiples for standby. The concentration range of the standard working solutions should cover the concentration of the target in the sample to be tested. Use the detection results of different standard working solutions to plot a standard curve, and the content of the target in the inactivated bacteria can be calculated using this standard curve.

[0056] In the actual operation process, the process of preparing the standard working solutions includes: diluting the target standard with water to prepare a standard stock solution with a concentration greater than or equal to 5 mg / mL, and storing it at -20 °C; taking the standard stock solution and diluting it with water to obtain a mixed standard intermediate solution with a concentration of 400 μg / mL - 600 μg / mL, and storing it refrigerated at 4 °C; then taking the mixed standard intermediate solution and diluting it with water into standard working solutions with different concentrations. The concentration range of the standard working solutions is 1 μg / mL - 200 μg / mL, such as 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL. The standard working solutions are prepared before being loaded onto the instrument and are used immediately after preparation.

[0057] (2) Prepare the test sample solution

[0058] Perform pre-treatment on the inactivated bacteria before loading onto the instrument to obtain a sample solution. This pre-treatment process mainly includes processes such as extraction, drying, reconstitution, and dilution.

[0059] In some embodiments, the pre-treatment process before loading onto the instrument includes: extracting the inactivated bacteria sample to obtain an extract, taking the supernatant of the extract and drying it to obtain a powder sample, reconstituting and diluting the powder sample to obtain the sample before loading onto the instrument. If precipitation occurs before loading onto the instrument, it is necessary to process it again by means of vortexing, ultrasonic treatment, centrifugation, etc.

[0060] In the actual operation process, the pre-treatment operation before loading onto the instrument can be carried out according to the following steps: mixing the inactivated bacteria sample with an organic alcohol aqueous solution and performing extraction under low-temperature conditions to obtain an extract; centrifuging the extract to separate and taking the supernatant, drying the supernatant to obtain a powder sample; reconstituting and diluting the powder sample with water to obtain a test sample that meets the requirements for loading onto the instrument.

[0061] In some embodiments, the process of preparing the extract includes: mixing the inactivated bacteria sample with an organic alcohol aqueous solution with a volume fraction of 70% - 90%, and performing low-temperature ultrasonic extraction under the conditions of a frequency of 35 KHz - 45 KHz, a power of 90 W - 120 W, and a temperature of 2 °C - 6 °C for an extraction time of 20 min - 40 min. The inactivated bacteria can be fully extracted through low-temperature ultrasonic extraction. The organic alcohol aqueous solution used in the extraction process can be a methanol aqueous solution, but is not limited thereto; the dosage of the methanol aqueous solution corresponding to every 0.1 g of the inactivated bacteria sample is 1 mL - 3 mL to fully extract the components in the inactivated bacteria.

[0062] Specifically, the volume fraction of the aqueous organic alcohol solution can be 70%, 75%, 80%, 85%, 90%, etc., the frequency can be 35 KHz, 38 KHz, 40 KHz, 43 KHz, 45 KHz, etc., the power can be 90 W, 100 W, 110 W, 120 W, etc., the extraction temperature can be 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, etc., and the extraction time can be 20 min, 30 min, 40 min, etc. The dosage of the methanol aqueous solution corresponding to every 0.1 g of the inactivated bacterial sample can be 1 mL, 2 mL, 3 mL, etc.

[0063] In some embodiments, before being mixed with the inactivated bacterial sample, the methanol aqueous solution can be pre-cooled to 2 °C - 6 °C, and after pre-cooling the methanol aqueous solution to the extraction temperature, it is then mixed with the inactivated bacterial sample.

[0064] In some embodiments, the extract is allowed to stand at a temperature of -15 °C to -25 °C for 20 min to 40 min, and then centrifuged to separate the supernatant. The supernatant is blown with liquid nitrogen until it is completely dried to obtain a powder sample for standby. The whole process is carried out under low-temperature operation to avoid damaging the morphology of the inactivated bacteria at high temperatures. Specifically, when allowing the extract to stand, the undissolved solids can be deposited. The controlled standing temperature can be -15 °C, -20 °C, -25 °C, etc., and the standing time can be 20 min, 30 min, 40 min, etc.; when centrifuging after standing, the operating conditions can be controlled at 9500 rpm / min - 10500 rpm / min, 2 °C - 6 °C.

[0065] In some embodiments, the obtained powder sample is re-dissolved in water, and after centrifugation, the supernatant is taken and diluted with 9 - 11 times (such as 10 times) the volume of water to obtain a sample meeting the requirements for loading onto the machine. When re-dissolving, it can be mixed evenly by vortex mixing, and the vortex mixing time can be 1 min - 3 min. When centrifuging after re-dissolving, the operating conditions can be controlled at 9500 rpm / min - 10500 rpm / min, 2 °C - 6 °C. The supernatant is used as the sample solution for standby and diluted 10 times (i.e., diluted with 10 times the volume of water) before loading onto the machine.

[0066] It should be noted that the inactivated bacteria tested in the embodiments of the present invention can be prepared by a conventional preparation process. The main steps include: activating Bifidobacterium BL-99 and then performing enlarged cultivation, followed by inactivation treatment and drying to obtain an inactivated bacterial sample. In some embodiments, the enlarged cultivation can be culturing Bifidobacterium BL-99 until the late logarithmic phase, and the quantity level can reach 10 9Above, centrifugal washing of bacteria, inactivation treatment, and freeze-drying are then carried out in sequence. The centrifuged bacteria can be washed multiple times with sterile water and then resuspended with sterile water. During the inactivation treatment, the inactivation temperature can be controlled at 85°C to 95°C, and the inactivation time can be 10 min to 20 min to better ensure the anti-inflammatory effect of Bifidobacterium BL-99.

[0067] Specifically, during the inactivation treatment, the inactivation temperature can be controlled at 85°C, 90°C, 95°C, etc.; the inactivation time can be 10 min, 15 min, 20 min, etc.

[0068] (3) Detection and analysis

[0069] The sample solution and the standard working solution are tested by high performance liquid chromatography, and the peak areas of the corresponding chromatograms are measured. The concentration of the standard working solution is used as the abscissa, and the peak area of the chromatogram is used as the ordinate to draw a standard curve. According to the detection results of the sample solution and combined with the standard curve, the concentration of the target substance in the inactivated bacteria is calculated.

[0070] It should be noted that the prepared test sample solution is injected into the high performance liquid chromatograph, the retention time and peak area of the chromatographic peak are recorded, and the external standard method is used for quantification. The response value of the target substance in the test sample solution should be within the linear range of quantitative determination by the instrument. When it exceeds the linear range, it should be appropriately diluted according to the measured concentration and then analyzed. The calculation result is expressed as the arithmetic mean of three independent determinations obtained under repeatability conditions, and the result is retained to three significant figures.

[0071] The content of each target substance in the sample is calculated according to formula (1):

[0072]

[0073] In the formula:

[0074] X—the content of a certain target component in the sample, in milligrams per 100 grams or milligrams per 100 milliliters (mg / 100g or mg / 100mL);

[0075] c i —the mass concentration of each target component in the sample solution obtained from the standard working curve, in micrograms per milliliter (μg / mL);

[0076] V—the volume of the sample extraction solution made up to volume, in milliliters (mL);

[0077] m—the mass or volume of the sample taken, in grams or milliliters (g or mL);

[0078] 10—the unit conversion factor;

[0079] f—the dilution factor.

[0080] In some embodiments, when tested by high performance liquid chromatography, the chromatographic conditions using the LC-20A analysis system include: the chromatographic column is C18, the inner diameter of the chromatographic column is 4.0 mm to 5.0 mm, the length of the chromatographic column is 140 mm to 160 mm, and the particle size of the chromatographic column packing is 2.5 μm to 3.0 μm; the column temperature is 25°C to 35°C, and the detection wavelength is 200 nm to 220 nm; the injection volume is controlled to be 2 μL to 10 μL, and the flow rate is controlled to be 0.5 mL / min to 1.0 mL / min. Preferably, the chromatographic column uses Poroshell 120Aq-C18. By optimizing the chromatographic conditions, the accuracy of detection can be further improved.

[0081] Specifically, the inner diameter of the chromatographic column can be 4.0 mm, 4.6 mm, 5.0 mm, etc., the length can be 140 mm, 150 mm, 160 mm, etc., and the particle size of the chromatographic column packing can be 2.5 μm, 2.8 μm, 3.0 μm, etc. For example, the chromatographic column parameters can be 4.6 mm × 150 mm, 2.7 μm. The column temperature can be 25°C, 30°C, 35°C, etc., the detection wavelength can be 200 nm, 210 nm, 220 nm, etc., and the injection volume can be 2 μL, 5 μL, 8 μL, 10 μL, etc. The flow rate can be 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min.

[0082] Further, when tested by high performance liquid chromatography, the mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an acidic substance solution with a volume fraction of 0.05% to 0.20%; mobile phase B is a mixed solution of an acidic substance and acetonitrile, and the volume fraction of the acidic substance in mobile phase B is 0.05% to 0.20%. Among them, the acidic substance is selected from any one of trifluoroacetic acid and phosphoric acid, and can be any one of the above.

[0083] Specifically, mobile phase A can be an aqueous solution of an acidic substance, and the volume fraction of the acidic substance can be 0.05%, 0.10%, 0.15%, 0.20%, etc.; mobile phase B is obtained by mixing an acidic substance and acetonitrile, and the volume fraction of the acidic substance can be 0.05%, 0.10%, 0.15%, 0.20%, etc.

[0084] Further, the gradient elution program is as follows: when the time is 0.00 min, the volume fraction of mobile phase A in the mobile phase is 98% - 100%, and the volume fraction of mobile phase B is 0% - 2%; when the time is 10.00 min, the volume fraction of mobile phase A in the mobile phase is 98% - 100%, and the volume fraction of mobile phase B is 0% - 2%; when the time is 33.00 min, the volume fraction of mobile phase A in the mobile phase is 93% - 97%, and the volume fraction of mobile phase B is 3% - 7%; when the time is 45.00 min, the volume fraction of mobile phase A in the mobile phase is 93% - 97%, and the volume fraction of mobile phase B is 3% - 7%. By controlling the conditions of gradient elution, a better elution effect can be achieved to improve the accuracy of detection.

[0085] S2. Calculate the number of colonies of inactivated bacteria before inactivation

[0086] Using at least one of prolylalanine, citric acid, Asp-Phe peptide, GP(Hyp)GAG peptide, L-methionine, GPRPK peptide, and Asp-Leu peptide as the target substance, detect the number of colonies of the inactivated bacteria prepared from Bifidobacterium BL-99 before inactivation. By studying the relationship between the content of the target substance and the number of colonies, establish a linear equation between the number of colonies and the content of the target substance to obtain the regression equation. The regression equation is y = ax + b, where x represents the concentration of the target substance, with the unit of mg / 100 g; y represents the number of colonies, with the unit of 10 9 CFU / mL.

[0087] The regression equations corresponding to different target substances are different:

[0088] When calculating the number of colonies of inactivated bacteria before inactivation using prolylalanine as the target substance, the value of a in the regression equation is 0.065 - 0.070 (such as 0.066, 0.067, 0.068, 0.069, 0.070, etc.), and the value of b is 0.200 - 0.210 (such as 0.200, 0.203, 0.205, 0.208, 0.210, etc.); more preferably, when calculating the number of colonies of inactivated bacteria before inactivation using prolylalanine as the target substance, the regression equation used is y = 0.068x + 0.205.

[0089] When calculating the number of colonies of inactivated bacteria before inactivation using citric acid as the target substance, the value of a in the regression equation is 0.002 - 0.004 (such as 0.002, 0.003, 0.004, etc.), and the value of b is -0.090 - -0.095 (such as -0.090, -0.091, -0.092, -0.0903, -0.094, -0.095, etc.); preferably, when calculating the number of colonies of inactivated bacteria before inactivation using citric acid as the target substance, the regression equation used is y = 0.003x - 0.092.

[0090] When calculating the number of colonies before inactivation of inactivated bacteria using the Asp-Phe peptide as the target, the value of a in the regression equation is 1.805 - 1.810 (such as 1.805, 1.806, 1.807, 1.808, 1.809, 1.810, etc.), and the value of b is 0.220 - 0.225 (such as 0.220, 0.221, 0.222, 0.223, 0.224, 0.225, etc.); preferably, when calculating the number of colonies before inactivation of inactivated bacteria using the Asp-Phe peptide as the target, the regression equation used is y = 1.807x + 0.222.

[0091] When calculating the number of colonies before inactivation of inactivated bacteria using the GP(Hyp)GAG peptide as the target, the value of a in the regression equation is 0.085 - 0.095 (such as 0.085, 0.087, 0.090, 0.092, 0.095, etc.), and the value of b is 0.770 - 0.775 (such as 0.770, 0.771, 0.772, 0.773, 0.774, 0.775, etc.); preferably, when calculating the number of colonies before inactivation of inactivated bacteria using the GP(Hyp)GAG peptide as the target, the regression equation used is y = 0.090x + 0.773.

[0092] When calculating the number of colonies before inactivation of inactivated bacteria using L-methionine as the target, the value of a in the regression equation is 0.065 - 0.075 (such as 0.065, 0.067, 0.070, 0.071, 0.073, 0.075, etc.), and the value of b is 0.525 - 0.535 (such as 0.525, 0.527, 0.529, 0.530, 0.532, 0.535, etc.); preferably, when calculating the number of colonies before inactivation of inactivated bacteria using L-methionine as the target, the regression equation used is y = 0.071x + 0.529.

[0093] When calculating the number of colonies before inactivation of inactivated bacteria using the GPRPK peptide as the target, the value of a in the regression equation is 0.110 - 0.120 (such as 0.110, 0.113, 0.115, 0.118, 0.120, etc.), and the value of b is -0.235 - -0.245 (such as -0.235, -0.237, -0.240, -0.243, -0.245, etc.); preferably, when calculating the number of colonies before inactivation of inactivated bacteria using the GPRPK peptide as the target, the regression equation used is y = 0.115x - 0.241.

[0094] When calculating the colony count of inactivated bacteria before inactivation using the Asp-Leu peptide as the target, the value of a in the regression equation ranges from 0.235 to 0.247 (such as 0.235, 0.237, 0.240, 0.242, 0.245, 0.247, etc.), and the value of b ranges from -0.620 to -0.630 (such as -0.620, -0.623, -0.625, -0.626, -0.628, -0.630, etc.); preferably, when calculating the colony count of inactivated bacteria before inactivation using the Asp-Leu peptide as the target, the regression equation used is y = 0.242x - 0.626.

[0095] In a preferred embodiment, using at least one of prolylalanine, citric acid, Asp-Phe peptide, GP(Hyp)GAG peptide, and L-methionine as the target to detect the colony count of inactivated Bifidobacterium BL-99 before inactivation can further improve the accuracy of detection. More preferably, using at least one of prolylalanine, citric acid, and Asp-Phe peptide as the target to detect the colony count of inactivated Bifidobacterium BL-99 before inactivation, and using the above three targets for detection can further improve the accuracy.

[0096] According to the concentration of the target obtained in S1, substitute it into the regression equation to calculate the colony count of inactivated bacteria before inactivation.

[0097] It should be noted that the process of obtaining the regression equation is as follows: Provide Bifidobacterium BL-99 live bacteria according to the colony count gradient, perform inactivation treatment, then extract and analyze the content of the target. Calculate the fitting linear equation based on the colony count gradient and the target content to obtain the regression equation. When prolylalanine, L-methionine, citric acid, GPRPK, GP(Hyp)GAG, Asp-Leu peptide, and Asp-Phe peptide are used as the targets respectively, the corresponding relationships between the colony count and the content of the target are as follows:

[0098] When the colony count of Bifidobacterium BL-99 is 2×10 ∧ 9 CFU / mL, the contents of prolylalanine, L-methionine, citric acid, GPRPK, GP(Hyp)GAG, Asp-Leu peptide, and Asp-Phe peptide are 22.69 mg / 100 g, 12.30 mg / 100 g, 720.38 mg / 100 g, 44.70 mg / 100 g, 4.56 mg / 100 g, 20.91 mg / 100 g, and 0.83 mg / 100 g in sequence.

[0099] For different colony counts (1 - 300, unit: 10^ 9The heat-inactivated bacterial sample BL-99 (CFU / mL) was detected by the above liquid chromatography method, and the HPLC fingerprint and the analysis results of the contents of 7 target components were established, so as to study the relationship between the contents of the target components and the main index of colony count, in order to identify the quality differences of postbiotics products.

[0100] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0101] It should be noted that the preparation process of the heat-inactivated cells of Bifidobacterium BL-99 tested in the following examples is as follows: Take 1.5 ml of the preserved strain at -80°C, thaw it at room temperature, aseptically take 200 μL of the bacterial liquid and inoculate it into 10 mL of the seed liquid medium, and culture it statically at 37°C for 11-13 h. Then, transfer it to a large flask with an inoculation amount of 2%, culture it for 2 days until the late logarithmic phase, centrifuge (4500 r / min, 10 min), wash it 3 times with sterile water, and resuspend it with 0.1 times the volume of sterile water (10-fold concentration). Then, inactivate it at 90°C for 15 min and freeze it at -80°C overnight until it is completely freeze-dried.

[0102] The materials used in the following examples are as follows: Prolylalanine (C8H 14 N2O3, CAS: 6422-36-2): purity ≥ 96.5%; L-Methionine (Methionine, C5H 11 NO2S, CAS: 63-68-3): purity ≥ 98.5%; Citric acid (C6H8O7, CAS: 77-92-9): purity ≥ 99.5%; Asp-Leu (Aspartic acid-Leucine, purchased from Beijing Bio-Tech Pack Technology Co., Ltd.); Asp-Phe (Aspartic acid-Phenylalanine, purchased from Beijing Bio-Tech Pack Technology Co., Ltd.); The purity of the standard products of GPRPK peptide (Glycine-Proline-Arginine-Proline-Lysine, purchased from Beijing Bio-Tech Pack Technology Co., Ltd.) and GP(Hyp)GAG peptide (Glycine-Proline (Hydroxyproline)-Glycine-Alanine-Glycine, purchased from Beijing Bio-Tech Pack Technology Co., Ltd.) are all ≥ 98.0%; Acetonitrile, methanol and phosphoric acid (chromatographic grade, Thermo fisher brand); Formic acid and acetonitrile (mass spectrometry grade, Thermo fisher brand); The experimental water is Wahaha pure water (Wahaha Group Co., Ltd., Hangzhou).

[0103] The instruments and equipment used in the following examples are as follows: High performance liquid chromatograph, with diode array detector: Shimadzu LC-20A, SPD-M20A; High resolution quadrupole time-of-flight liquid chromatography-mass spectrometry: Agilent 6546LC / Q-TOF; Water bath nitrogen blowing instrument; Vortex mixer; High speed centrifuge; Ultrasonic cleaner; Analytical balance.

[0104] Example 1

[0105] This example provides a method for detecting the colony count of inactivated Bifidobacterium BL-99 before inactivation, using prolylalanine as the target substance. The specific steps are as follows:

[0106] (1) Preparation of standard working solution

[0107] Standard stock solution: Weigh an appropriate amount of the standard substance (i.e., the target substance standard, accurate to 0.1 mg) separately and precisely, dissolve it in water, and prepare standard stock solutions with a concentration of 5 mg / mL respectively, and store them at -20 °C.

[0108] Mixed standard intermediate solution: Accurately pipette appropriate volumes of the standard stock solutions respectively, make up the volume with water, and prepare a mixed standard intermediate solution with a concentration of 500 μg / mL, and store it refrigerated at 4 °C.

[0109] Mixed standard working solution: Gradually dilute the mixed standard intermediate solution with water as needed to prepare mixed standard working solutions with concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL respectively, and prepare them immediately before use.

[0110] (2) Preparation of elution solution

[0111] 0.1% phosphoric acid aqueous solution: Take 1 mL of phosphoric acid, dilute it with water and make up the volume to 1000 mL, mix well, and prepare it immediately before use.

[0112] 0.1% phosphoric acid acetonitrile solution: Take 1 mL of phosphoric acid, dilute it with acetonitrile and make up the volume to 1000 mL, mix well, and prepare it immediately before use.

[0113] (3) Preparation of test sample solution

[0114] Perform pre-treatment on the inactivated bacteria before loading onto the machine to obtain a sample solution. The pre-treatment process before loading onto the machine is as follows:

[0115] Precisely weigh 0.3 g of the inactivated bacteria sample into a centrifuge tube, add 6 mL of methanol aqueous solution with a volume fraction of 80% pre-cooled to 4 °C, shake well, extract ultrasonically at low temperature for 30 min (100 W, 40 KHz), let the sample stand at -20 °C for 30 min, centrifuge at 4 °C and 10000 rpm / min for 10 min, collect the supernatant, dry it slowly with nitrogen at 40 °C, make up the volume with 0.2 mL of pure water, vortex and mix well for 1 min, ultrasonicate for 2 min, centrifuge at 10000 rpm / min for 10 min, and pipette the supernatant and dilute it 10 times for standby.

[0116] (4) Detection and analysis

[0117] The sample solution and the standard working solution were tested by high performance liquid chromatography, and the peak areas of the corresponding chromatograms were determined. The concentration of the standard working solution was used as the abscissa, and the peak area of the chromatogram was used as the ordinate to plot the standard curve. According to the test results of the sample solution in step (3), the concentration of the target substance in the inactivated bacteria was calculated by combining with the standard curve.

[0118] Chromatographic analysis conditions: Use the LC-20A analysis system, the chromatographic column is Poroshell 120Aq-C18 column (4.6mm×150mm, 2.7μm); mobile phase A is 0.1% phosphoric acid aqueous solution; mobile phase B is 0.1% phosphoric acid acetonitrile solution, and the gradient elution program is shown in Table 1; the flow rate is 0.7mL / min; the detection wavelength is 210nm; the column temperature is 30°C; the injection volume is 5μL.

[0119] Table 1 Gradient elution program table

[0120] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 33 95 5 45 95 5

[0121] (5) Calculate the number of colonies before inactivation of the inactivated bacteria

[0122] When calculating the number of colonies using the concentration of the target substance in the inactivated bacteria, the regression equations corresponding to the three target substances are different: when calculating the number of colonies before inactivation of the inactivated bacteria using prolylalanine as the target substance, the regression equation used is y = 0.068x + 0.205. Among them, x represents the concentration of the target substance, in mg / 100g; y represents the number of colonies, in 10 9 CFU / mL. According to the calculated concentration of the target substance, substitute it into the regression equation to calculate the number of colonies before inactivation of the inactivated bacteria.

[0123] Example 2

[0124] The difference from Example 1 is only that: the target substance is replaced with citric acid. When calculating the number of colonies before inactivation of the inactivated bacteria using citric acid as the target substance, the regression equation used is y = 0.003x - 0.092.

[0125] Example 3

[0126] The difference from Example 1 is only that: the target substance is replaced with Asp-Phe peptide. When calculating the number of colonies before inactivation of the inactivated bacteria using Asp-Phe peptide as the target substance, the regression equation used is y = 1.807x + 0.222.

[0127] Examples 4-7

[0128] The difference between Examples 4-7 and Example 1 is only that: the target substance is replaced, and in Examples 4-7, the target substances are replaced with L-methionine, GPRPK, GP(Hyp)GAG, and Asp-Leu peptide respectively, and the regression equations are adjusted accordingly.

[0129] Test Example 1

[0130] Inject the series of standard working solutions prepared in step (1) into the high performance liquid chromatograph respectively, inject samples in parallel three times for each concentration, and measure the peak areas of the corresponding chromatograms. Use the concentration of the standard working solution as the abscissa and the peak area of the chromatogram as the ordinate to draw the standard curve.

[0131] The limit of detection (LOD) and limit of quantitation (LOQ) are used to evaluate the sensitivity of the method. The LOD is the lowest concentration at which the analyte can be detected, and the LOQ is the lowest concentration at which the analyte can be quantitatively detected. By continuously injecting the mixed standard product with gradually decreasing concentration, the LOD is the concentration of the target component corresponding to S / N (the ratio of the signal value to the noise value) ≥ 3, and the LOQ is the concentration of the target component corresponding to S / N ≥ 10.

[0132] The test results are shown in Table 2:

[0133] Table 2 Establishment of HPLC standard curves for different target substances

[0134]

[0135] The elution peaks of seven standard products by HPLC under the optimized chromatographic conditions are as Figure 1 shown, and it can be seen that the 7 target substances can be effectively baseline separated. Figure 1 1 - 7 in the figure are in turn: 1 - prolylalanine; 2 - L - methionine; 3 - citric acid; 4 - GPRPK; 5 - GP(Hyp)GAG; 6 - Asp - Leu; 7 - Asp - Phe.

[0136] The liquid chromatogram of inactivated cells of Bifidobacterium BL - 99 is as Figure 2 shown, Figure 2 1 - 7 in the figure are in turn: 1 - prolylalanine; 2 - L - methionine; 3 - citric acid; 4 - GPRPK peptide; 5 - GP(Hyp)GAG peptide; 6 - Asp - Leu peptide; 7 - Asp - Phe peptide.

[0137] Test Example 2

[0138] The recovery test was used to evaluate the accuracy, and the coefficient of variation (RSD) was used to evaluate the precision. In the test, the recovery rate was investigated by adding standard solutions with three different concentrations to the test samples, that is, the ratio of the added amount of the standard substance shown by the detection to the true added amount of the standard substance, expressed as a percentage. Three parallels were made for each concentration level, and the recovery rate and the coefficient of variation were calculated. According to the requirements of "GB / T 27417 Guidelines for the Verification and Validation of Chemical Analysis Methods in Conformity Assessment", the added recovery rate should be within 80% - 110%, and the precision should be less than 20%. The test results are shown in Table 3 as follows:

[0139] Table 3 Results of the spike test

[0140]

[0141] It can be seen that the accuracy of the detection method provided by the embodiment of the present invention meets the requirements of "GB / T 27417". The average recovery rate of the spike recovery is 80% - 110%, and the relative standard deviation (RSDRSD) is within 10%.

[0142] Test Example 3

[0143] Test the detection accuracy of the method provided by the test example, and use the correlation coefficient R 2 Evaluate the effects of different test methods, and the results are shown in Table 4

[0144] Correlation coefficient R 2 Is a calculation method using a conventional linear regression model fitting metric

[0145] Table 4 Comparison of the effects of different target substances in the embodiment

[0146]

[0147]

[0148] It can be seen that the detection accuracy using at least one of prolylalanine, citric acid, Asp-Phe peptide, GP(Hyp)GAG peptide, and L-methionine as the target substance is relatively high, and the correlation coefficient R 2 Can reach above 0.90. Using at least one of prolylalanine, citric acid, and Asp-Phe peptide as the target substance has higher detection accuracy, and the correlation coefficient R 2 Can reach above 0.99, and the test accuracy using citric acid as the target substance is the highest

[0149] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention

Claims

1. A method for detecting the number of colonies of live cells of Bifidobacterium BL-99 before inactivation, characterized in that, Including: Using at least one of prolylalanine, citric acid, Asp-Phe peptide, GP(Hyp)GAG peptide, L-methionine, GPRPK peptide and Asp-Leu peptide as a target substance to detect the colony count of inactivated cells of Bifidobacterium BL-99 before inactivation.

2. The detection method according to claim 1, characterized in that The detection method includes the step of establishing a linear equation between the number of colonies and the content of the target substance to obtain a regression equation, where the regression equation is y = ax + b. Here, x represents the concentration of the target substance, with the unit of mg / 100g; y represents the number of colonies, with the unit of 10 9 CFU / mL; Preferably, when calculating the colony count of inactivated cells before inactivation using prolylalanine as the target substance, the value of a in the regression equation is 0.065 - 0.070, and the value of b is 0.200 - 0.210; more preferably, when calculating the colony count of inactivated cells before inactivation using prolylalanine as the target substance, the regression equation used is y = 0.068x + 0.205; Preferably, when calculating the colony count of inactivated cells before inactivation using citric acid as the target substance, the value of a in the regression equation is 0.002 - 0.004, and the value of b is -0.090 - -0.095; more preferably, when calculating the colony count of inactivated cells before inactivation using citric acid as the target substance, the regression equation used is y = 0.003x - 0.092; Preferably, when calculating the colony count of inactivated cells before inactivation using Asp-Phe peptide as the target substance, the value of a in the regression equation is 1.805 - 1.810, and the value of b is 0.220 - 0.225; more preferably, when calculating the colony count of inactivated cells before inactivation using Asp-Phe peptide as the target substance, the regression equation used is y = 1.807x + 0.222; Preferably, when calculating the colony count of inactivated cells before inactivation using GP(Hyp)GAG peptide as the target substance, the value of a in the regression equation is 0.085 - 0.095, and the value of b is 0.770 - 0.775; more preferably, when calculating the colony count of inactivated cells before inactivation using GP(Hyp)GAG peptide as the target substance, the regression equation used is y = 0.090x + 0.773; Preferably, when calculating the colony count of inactivated cells before inactivation using L-methionine as the target substance, the value of a in the regression equation is 0.065 - 0.075, and the value of b is 0.525 - 0.535; more preferably, when calculating the colony count of inactivated cells before inactivation using L-methionine as the target substance, the regression equation used is y = 0.071x + 0.529; Preferably, when calculating the colony count of inactivated cells before inactivation using GPRPK peptide as the target substance, the value of a in the regression equation is 0.110 - 0.120, and the value of b is -0.235 - -0.245; more preferably, when calculating the colony count of inactivated cells before inactivation using GPRPK peptide as the target substance, the regression equation used is y = 0.115x - 0.241; Preferably, when calculating the number of colonies before inactivation of inactivated bacteria using Asp-Leu peptide as the target, the value of a in the regression equation is 0.235 - 0.247, and the value of b is -0.620 to -0.630; more preferably, when calculating the number of colonies before inactivation of inactivated bacteria using Asp-Leu peptide as the target, the regression equation used is y = 0.242x - 0.626; More preferably, at least one of prolylalanine, citric acid, Asp-Phe peptide, GP(Hyp)GAG peptide, and L-methionine is used as the target to detect the number of colonies before inactivation of inactivated Bifidobacterium BL-99; Further preferably, at least one of prolylalanine, citric acid, and Asp-Phe peptide is used as the target to detect the number of colonies before inactivation of inactivated Bifidobacterium BL-99.

3. The detection method according to claim 1, characterized in that, Before detection, the inactivated bacteria are pretreated, and the process of the pretreatment includes: extracting the inactivated bacteria sample to obtain an extract, taking the supernatant of the extract, drying it to obtain a powder sample, and redissolving and diluting the powder sample.

4. The detection method according to claim 3, wherein Mix the inactivated bacteria sample with an organic alcohol aqueous solution for low-temperature extraction to obtain an extract; centrifuge the extract and take the supernatant, dry the supernatant to obtain the powder sample; redissolve and dilute the powder sample with water; Preferably, the preparation process of the extract includes: mixing the inactivated bacteria sample with an organic alcohol aqueous solution with a volume fraction of 70% - 90%, and performing low-temperature ultrasonic extraction for 20 min - 40 min under the conditions of a frequency of 35 KHz - 45 KHz and a temperature of 2°C - 6°C; More preferably, the organic alcohol aqueous solution is a methanol aqueous solution, and the dosage of the methanol aqueous solution corresponding to every 0.1 g of the inactivated bacteria sample is 1 mL - 3 mL; Preferably, the methanol aqueous solution is precooled to 2°C - 6°C before mixing with the inactivated bacteria sample.

5. The detection method according to claim 4, wherein Let the extract stand at a temperature of -15°C - -25°C for 20 min - 40 min, then centrifuge and take the supernatant, and blow it with nitrogen until it is completely dry to obtain the powder sample; Preferably, the powder sample is redissolved with water, the supernatant is taken after centrifugation, and then diluted with 9 - 11 times the volume of water.

6. The detection method according to any one of claims 2-5, characterized in that, Use high-performance liquid chromatography for detection, calculate the concentration of the target in the inactivated bacteria by the standard curve method, and then calculate the number of colonies before inactivation of the inactivated bacteria from the regression equation; Preferably, use high-performance liquid chromatography to test the concentration of the target, and the testing process includes: Prepare a standard working solution: use the target standard product to prepare standard working solutions with different dilution multiples; Prepare a test sample solution: pretreat the inactivated bacteria to obtain a sample solution; Detection: Use high-performance liquid chromatography to test the sample solution and the standard working solution, draw a standard curve using the standard working solutions with different concentrations and the corresponding peak areas; calculate the concentration of the target in the inactivated bacteria using the standard curve.

7. The detection method according to claim 6, wherein When tested by high performance liquid chromatography, the chromatographic conditions include: the chromatographic column is C18, the inner diameter of the chromatographic column is 4.0 mm to 5.0 mm, the length of the chromatographic column is 140 mm to 160 mm, and the particle size of the chromatographic column packing is 2.5 μm to 3.0 μm; the column temperature is 25°C to 35°C, and the detection wavelength is 200 nm to 220 nm; Preferably, the chromatographic column is Poroshell 120Aq-C18; Preferably, the injection volume is controlled to be 2 μL to 10 μL.

8. The detection method according to claim 7, characterized in that, When tested by high performance liquid chromatography, the mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an acidic substance solution with a volume fraction of 0.05% to 0.20%; mobile phase B is a mixed solution of an acidic substance and acetonitrile, and the volume fraction of the acidic substance is 0.05% to 0.20%; Among them, the acidic substance is selected from any one of trifluoroacetic acid and phosphoric acid; Preferably, the gradient elution program is: When the time is 0.00 min, the volume fraction of mobile phase A in the mobile phase is 98% to 100%, and the volume fraction of mobile phase B is 0% to 2%; When the time is 10.00 min, the volume fraction of mobile phase A in the mobile phase is 98% to 100%, and the volume fraction of mobile phase B is 0% to 2%; When the time is 33.00 min, the volume fraction of mobile phase A in the mobile phase is 93% to 97%, and the volume fraction of mobile phase B is 3% to 7%; When the time is 45.00 min, the volume fraction of mobile phase A in the mobile phase is 93% to 97%, and the volume fraction of mobile phase B is 3% to 7%; Preferably, the flow rate is controlled to be 0.5 mL / min to 1.0 mL / min.

9. The detection method according to claim 6, characterized in that The process of preparing the standard working solution includes: diluting the target standard product with water to prepare a standard stock solution with a concentration greater than or equal to 5 mg / mL; taking the standard stock solution and diluting it with water to obtain a mixed standard intermediate solution with a concentration of 400 μg / mL to 600 μg / mL; taking the mixed standard intermediate solution and diluting it with water into standard working solutions with different concentrations, and the concentration range of the standard working solutions is 1 μg / mL to 200 μg / mL.

10. A kit for detecting the number of colonies before inactivating the inactivated cells of Bifidobacterium BL-99, characterized in that, The kit includes: using at least one of prolylalanine, citric acid, Asp-Phe peptide, GP(Hyp)GAG peptide, L-methionine, GPRPK peptide, and Asp-Leu peptide as the target substance.

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