Method and kit for detecting colony count of inactivated bacteria of bifidobacterium YLGB-1496 before inactivation

Abstract

The invention discloses a method and a kit for detecting the colony count of inactivated bacteria of bifidobacterium YLGB-1496 before inactivation, and relates to the technical field of instrument analysis. According to the present invention, at least one of L-methionine, Asp-Phe peptide, prolyl alanine, GPRPK peptide, GP (Hyp) GAG peptide and Asp-Leu peptide is adopted as the target substance, the colony count of the inactivated bacteria before inactivation is calculated by testing the content of the target substance, the detection accuracy is high, the theoretical basis is provided for the quantitative detection of the metagen, and the establishment of the industrial standard for the quantitative detection of the metagen product is easily achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of instrumental analysis, and in particular, to a method and a kit for detecting the number of colonies before inactivation of inactivated cells of Bifidobacterium YLGB-1496. 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 the 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 to be a potential functional food and health management means, and have received special 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 number of colonies in the products, and there is currently no detection standard to follow.

[0004] The strain patent deposit number of Bifidobacterium longum subsp. infantis YLGB-1496 is: CCTCC No: M2011122. The postbiotic product produced by using this exclusive strain has an efficient antioxidant effect, and its antioxidant effect is directly related to the number of its colonies.

[0005] Therefore, there is an urgent need to develop a method for quantitatively analyzing postbiotic products (such as inactivated cells) of YLGB-1496 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 method and a kit for detecting the number of colonies before inactivation of inactivated cells of Bifidobacterium YLGB-1496, aiming to accurately detect the number of colonies before inactivation of inactivated cells.

[0008] The present invention is implemented as follows:

[0009] The present invention provides a method for detecting the number of colonies of inactivated cells of Bifidobacterium YLGB-1496 before inactivation, including: using at least one of L-methionine, Asp-Phe peptide, prolylalanine, GPRPK peptide, GP(Hyp)GAG peptide, and Asp-Leu peptide as a target substance to detect the number of colonies of inactivated cells of Bifidobacterium YLGB-1496 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. The regression equation is y = ax + b, where x represents the concentration of the target substance in mg / 100g, and y represents the number of colonies in 10 9 CFU / mL;

[0011] Preferably, viable Bifidobacterium YLGB-1496 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 cells before inactivation using L-methionine as the target substance, the value of a in the regression equation is 0.010 - 0.015, and the value of b is -0.190 to -0.195. More preferably, when calculating the number of colonies of inactivated cells before inactivation using L-methionine as the target substance, the regression equation used is y = 0.012x - 0.192.

[0013] Preferably, when calculating the number of colonies of inactivated cells before inactivation using Asp-Phe peptide as the target substance, the value of a in the regression equation is 0.110 - 0.115, and the value of b is -0.255 to -0.265. More preferably, when calculating the number of colonies of inactivated cells before inactivation using Asp-Phe peptide as the target substance, the regression equation used is y = 0.113x - 0.259.

[0014] Preferably, when calculating the number of colonies of inactivated cells before inactivation using prolylalanine as the target substance, the value of a in the regression equation is 0.002 - 0.004, and the value of b is -0.270 to -0.280. More preferably, when calculating the number of colonies of inactivated cells before inactivation using prolylalanine as the target substance, the regression equation used is y = 0.003x - 0.274.

[0015] Preferably, when calculating the number of colonies of inactivated cells before inactivation using GPRPK peptide as the target substance, the value of a in the regression equation is 0.040 - 0.050, and the value of b is -0.250 to -0.350. More preferably, when calculating the number of colonies of inactivated cells before inactivation using GPRPK peptide as the target substance, the regression equation used is y = 0.045x - 0.290.

[0016] Preferably, 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 0.230 - 0.240, and the value of b is -0.195 to -0.210; more 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 = 0.234x - 0.202;

[0017] Preferably, when calculating the number of colonies before inactivation of inactivated bacteria using the Asp-Leu peptide as the target, the value of a in the regression equation is 0.105 - 0.110, and the value of b is -0.280 to -0.285; more preferably, when calculating the number of colonies before inactivation of inactivated bacteria using the Asp-Leu peptide as the target, the regression equation used is y = 0.108x - 0.283;

[0018] Preferably, at least one of L-methionine and Asp-Phe peptide is used as the target to detect the number of colonies before inactivation of inactivated Bifidobacterium YLGB-1496.

[0019] More preferably, L-methionine is used as the target to detect the number of colonies before inactivation of inactivated Bifidobacterium YLGB-1496.

[0020] 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, drying it to obtain a powder sample, and re-dissolving and diluting the powder sample.

[0021] 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, dried to obtain a powder sample; the powder sample is re-dissolved and diluted with water;

[0022] 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;

[0023] 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;

[0024] Preferably, the methanol aqueous solution is pre-cooled to 2°C - 6°C before mixing with the inactivated bacteria sample.

[0025] 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. The supernatant is taken and blown with nitrogen until a powder sample is completely obtained;

[0026] Preferably, the powder sample is redissolved in water, the supernatant is taken after centrifugation, and then diluted with 9 to 11 volumes of water.

[0027] In an alternative embodiment, high performance liquid chromatography is used for detection. The standard curve method is adopted to calculate the concentration of the target substance in the inactivated bacteria, and then the number of colonies before inactivation of the inactivated bacteria is calculated from the regression equation;

[0028] Preferably, high performance liquid chromatography is used to test the concentration of the target substance. The testing process includes:

[0029] Preparing a standard working solution: Using L-methionine standard, different dilution multiples of standard working solutions are prepared;

[0030] Preparing a test sample solution: The inactivated bacteria are pretreated to obtain a sample solution;

[0031] Detection: The sample solution and the standard working solution are tested by high performance liquid chromatography. Using the standard working solutions with different concentrations and the corresponding peak areas, a standard curve is plotted; The concentration of the target substance in the inactivated bacteria is calculated using the standard curve.

[0032] In an alternative embodiment, when using high performance liquid chromatography for testing, 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;

[0033] Preferably, the chromatographic column is Poroshell 120Aq-C18;

[0034] Preferably, the injection volume is controlled to be 2 μL to 10 μL.

[0035] In an alternative embodiment, when using high performance liquid chromatography for testing, 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%;

[0036] Among them, the acidic substance is selected from any one of trifluoroacetic acid and phosphoric acid;

[0037] Preferably, the gradient elution program is:

[0038] 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%;

[0039] 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%;

[0040] 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%;

[0041] 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%;

[0042] Preferably, the flow rate is controlled to be 0.5 mL / min - 1.0 mL / min.

[0043] In an alternative embodiment, the process of preparing the standard working solution includes: diluting the L-methionine standard 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 - 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 - 200 μg / mL.

[0044] 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 YLGB-1496, and the kit includes: using at least one of L-methionine, Asp-Phe peptide, prolylalanine, GPRPK peptide, GP(Hyp)GAG peptide, and Asp-Leu peptide as a target substance.

[0045] The present invention has the following beneficial effects: using at least one of L-methionine, Asp-Phe peptide, prolylalanine, GPRPK peptide, GP(Hyp)GAG peptide, and Asp-Leu peptide as a target substance, calculating the number of viable colonies before inactivating the inactivated cells by testing the content of the target substance, and the detection accuracy is relatively high, providing a theoretical basis for the quantitative detection of postbiotics, and also facilitating the establishment of industry standards for the quantitative detection of postbiotic products. Description of the Drawings

[0046] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0047] Figure 1 Is the liquid chromatography diagram of the mixed standard working solution;

[0048] Figure 2 Is the liquid chromatography diagram of the inactivated cells of Bifidobacterium YLGB - 1496. Specific embodiments

[0049] 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. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0050] The embodiments of the present invention provide a method for detecting the number of colonies before inactivation of inactivated cells of Bifidobacterium YLGB - 1496. The inventor creatively uses at least one of L - methionine, Asp - Phe peptide, prolylalanine, GPRPK peptide, GP(Hyp)GAG peptide, and Asp - Leu peptide as a target substance. By detecting the content of the target substance in the inactivated cells of Bifidobacterium YLGB - 1496 and calculating the number of colonies before inactivation of the inactivated cells through an optimized regression equation, the purpose of accurately detecting the number of colonies before inactivation of the inactivated cells is achieved. The specific steps are as follows:

[0051] S1. Use high - performance liquid chromatography to test the concentration of the target substance

[0052] Test the concentration of the target substance in the inactivated cells by high - performance liquid chromatography. The testing process mainly includes the following three steps:

[0053] (1) Prepare the standard working solution

[0054] Use the L - methionine standard product to prepare standard working solutions with different dilution multiples for standby. The concentration range of the standard working solution should cover the concentration of the target substance in the sample to be tested. Use the detection results of different standard working solutions to draw a standard curve, and the content of the target substance in the inactivated cells can be calculated using this standard curve.

[0055] In the actual operation process, the process of preparing the standard working solution includes: diluting the L-methionine standard with water to prepare a standard stock solution with a concentration of 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 to 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, and the concentration range of the standard working solutions is 1 μg / mL to 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 solution is prepared before being loaded onto the machine and used immediately after preparation.

[0056] (2) Preparation of the test sample solution

[0057] The inactivated bacteria are pretreated before being loaded onto the machine to obtain a sample solution, and this pretreatment process mainly includes processes such as extraction, drying, reconstitution, and dilution.

[0058] In some embodiments, the pretreatment process before loading onto the machine 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 a sample before loading onto the machine. If precipitation occurs before loading onto the machine, it is necessary to process it again by means of vortexing, ultrasonic treatment, centrifugation, etc.

[0059] In the actual operation process, the operation of the pretreatment before loading onto the machine can be carried out according to the following steps: mixing the inactivated bacteria sample with an organic alcohol aqueous solution and carrying out 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 machine.

[0060] 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% to 90%, and carrying out low-temperature ultrasonic extraction under the conditions of a frequency of 35 KHz to 45 KHz, a power of 90 W - 120 W, and a temperature of 2 °C to 6 °C, and the extraction time is 20 min to 40 min. The inactivated bacteria can be fully extracted by low-temperature ultrasonic extraction. The organic alcohol aqueous solution used in the extraction process can be a methanol aqueous solution, but is not limited to this; the dosage of the methanol aqueous solution corresponding to every 0.1 g of the inactivated bacteria sample is 1 mL to 3 mL to fully extract the components in the inactivated bacteria.

[0061] Specifically, the volume fraction of the organic alcohol aqueous solution can be 70%, 75%, 80%, 85%, 90%, etc., the frequency can be 35KHz, 38KHz, 40KHz, 43KHz, 45KHz, etc., the power can be 90W, 100W, 110W, 120W, etc., the extraction temperature can be 2°C, 3°C, 4°C, 5°C, 6°C, etc., and the extraction time can be 20min, 30min, 40min, etc. The dosage of the methanol aqueous solution corresponding to every 0.1g of the inactivated bacterial cell sample can be 1mL, 2mL, 3mL, etc.

[0062] In some embodiments, before the methanol aqueous solution is mixed with the inactivated bacterial cell sample, it can be pre-cooled to 2°C - 6°C first, and then mixed with the inactivated bacterial cell sample after being pre-cooled to the extraction temperature.

[0063] In some embodiments, the extract is allowed to stand at a temperature of -15°C to -25°C for 20min to 40min, and then centrifuged to obtain the supernatant, and the supernatant is blown with liquid nitrogen until completely powdered to obtain a powdered sample for standby. The whole process is carried out under low-temperature operation to avoid destroying the morphology of the inactivated bacterial cells at high temperature. Specifically, when the extract is allowed to stand, the undissolved solids can be deposited, and the temperature for standing can be -15°C, -20°C, -25°C, etc., and the standing time can be 20min, 30min, 40min, etc.; when centrifuging after standing, the operating conditions can be controlled at 9500rpm / min - 10500rpm / min, 2°C - 6°C.

[0064] In some embodiments, the obtained powdered sample is re-dissolved in water, the supernatant is taken after centrifugation, and then 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 1min - 3min. When centrifuging after re-dissolving, the operating conditions can be controlled at 9500rpm / min - 10500rpm / min, 2°C - 6°C, and 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.

[0065] It should be noted that the inactivated bacterial cells tested in the embodiments of the present invention can be prepared by a conventional preparation process. The main steps include: activating Bifidobacterium YLGB - 1496 and then performing enlarged cultivation, followed by inactivation treatment and drying to obtain an inactivated bacterial cell sample. In some embodiments, the enlarged cultivation can be to cultivate Bifidobacterium YLGB - 1496 until the late logarithmic phase, and the order of magnitude can reach 10 9After the above steps, centrifugal washing of bacteria, inactivation treatment, and freeze-drying are 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 - 95°C, and the inactivation time can be 10 min - 20 min to better ensure the antioxidant effect of Bifidobacterium YLGB-1496.

[0066] 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.

[0067] (3) Detection and analysis

[0068] 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. With the concentration of the standard working solution as the abscissa and the peak area of the chromatogram as the ordinate, a standard curve is plotted. 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.

[0069] It should be noted that the prepared test sample solution is injected into the high-performance liquid chromatograph, and the retention time and peak area of the chromatographic peak are recorded for external standard 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 repetitive conditions, and the result is retained to three significant figures.

[0070] The content of each target substance in the sample is calculated according to formula (1):

[0071]

[0072] In the formula:

[0073] 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);

[0074] 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);

[0075] V—the volume of the sample extraction solution made up to volume, in milliliters (mL);

[0076] m—the mass or volume of the sample taken, in grams or milliliters (g or mL);

[0077] 10—the unit conversion factor;

[0078] f—the dilution factor.

[0079] In some embodiments, when tested by high performance liquid chromatography, the chromatographic conditions of 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. By optimizing the chromatographic conditions, the accuracy of detection can be further improved. Preferably, the chromatographic column uses Poroshell 120Aq-C18 to further improve the detection precision.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] S2. Calculate the number of colonies of the inactivated bacteria before inactivation

[0085] At least one of L-methionine, Asp-Phe peptide, prolylalanine, GPRPK peptide, GP(Hyp)GAG peptide, and Asp-Leu peptide is used as a target substance. By detecting the content of the target substance in the inactivated bacteria of Bifidobacterium YLGB-1496, the number of colonies of the inactivated bacteria before inactivation is calculated through an optimized regression equation. By studying the relationship between the content of the target substance and the number of colonies, a linear equation between the number of colonies and the content of the target substance is established 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.

[0086] The regression equations corresponding to different target substances are different:

[0087] When calculating the number of colonies of the inactivated bacteria before inactivation with L-methionine as the target substance, the value of a in the regression equation is 0.010 - 0.015 (such as 0.010, 0.011, 0.012, 0.013, 0.014, 0.015, etc.), and the value of b is -0.190 to -0.195 (such as -0.190, -0.191, -0.192, -0.193, -0.194, -0.195, etc.); preferably, when calculating the number of colonies of the inactivated bacteria before inactivation with L-methionine as the target substance, the regression equation used is y = 0.012x - 0.192.

[0088] When calculating the number of colonies before inactivation of inactivated bacteria using Asp-Phe peptide as the target, the value of a in the regression equation is 0.110 - 0.115 (such as 0.110, 0.111, 0.112, 0.113, 0.114, 0.115, etc.), and the value of b is -0.255 to -0.265 (such as -0.255, -0.257, -0.259, -0.260, -0.262, -0.265, etc.); preferably, when calculating the number of colonies before inactivation of inactivated bacteria using Asp-Phe peptide as the target, the regression equation used is y = 0.113x - 0.259.

[0089] When calculating the number of colonies before inactivation of inactivated bacteria using prolylalanine as the target, 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 -0.270 to -0.280 (such as -0.272, -0.274, -0.276, -0.278, -0.280, etc.); preferably, when calculating the number of colonies before inactivation of inactivated bacteria using prolylalanine as the target, the regression equation used is y = 0.003x - 0.274.

[0090] When calculating the number of colonies before inactivation of inactivated bacteria using GPRPK peptide as the target, the value of a in the regression equation is 0.040 - 0.050 (such as 0.040, 0.043, 0.045, 0.047, 0.050, etc.), and the value of b is -0.250 to -0.350 (such as -0.250, -0.260, -0.270, -0.280, -0.290, -0.300, -0.310, -0.320, -0.330, -0.340, -0.350, etc.); preferably, when calculating the number of colonies before inactivation of inactivated bacteria using GPRPK peptide as the target, the regression equation used is y = 0.045x - 0.290.

[0091] When calculating the number of colonies before inactivation of inactivated bacteria using GP(Hyp)GAG peptide as the target, the value of a in the regression equation is 0.230 - 0.240 (such as 0.230, 0.232, 0.234, 0.236, 0.238, 0.240, etc.), and the value of b is -0.195 to -0.210 (such as -0.195, -0.197, -0.200, -0.202, -0.205, -0.207, -0.210, etc.); preferably, when calculating the number of colonies before inactivation of inactivated bacteria using GP(Hyp)GAG peptide as the target, the regression equation used is y = 0.234x - 0.202.

[0092] 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 0.105 - 0.110 (such as 0.105, 0.106, 0.107, 0.108, 0.109, 0.110, etc.), and the value of b is -0.280 to -0.285 (such as -0.280, -0.281, -0.282, -0.283, -0.284, -0.285, etc.); 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 = 0.108x - 0.283.

[0093] In a preferred embodiment, using at least one of L-methionine and Asp-Phe peptide as the target to detect the number of colonies before inactivation of inactivated Bifidobacterium YLGB-1496 can provide the accuracy of detection. In a more preferred embodiment, using L-methionine as the target to detect the number of colonies before inactivation of inactivated Bifidobacterium YLGB-1496 can further improve the accuracy of detection.

[0094] According to the concentration of the target calculated in S1, substitute it into the regression equation to calculate the number of colonies before inactivation of the inactivated bacteria.

[0095] It should be noted that the process of obtaining the regression equation is as follows: Provide live Bifidobacterium YLGB-1496 with a gradient of colony numbers, perform inactivation treatment, then extract and analyze the content of the target. Calculate the fitting linear equation based on the colony number 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 target respectively, the corresponding relationship between the colony number and the content of the target is as follows:

[0096] When the colony number of Bifidobacterium YLGB-1496 is 1×10 ∧ 9 CFU / mL, the contents of prolylalanine, L-methionine, GPRPK, GP(Hyp)GAG, Asp-Leu peptide, and Asp-Phe peptide are 673.72 mg / 100 g, 128.81 mg / 100 g, 44.55 mg / 100 g, 9.79 mg / 100 g, 20.10 mg / 100 g, and 15.84 mg / 100 g in sequence.

[0097] For different colony numbers (1 - 300, unit: 10^ 9The inactivated bacteria sample YLGB-1496 (CFU / mL) was detected by the above liquid chromatography method to establish the HPLC fingerprint and the analysis results of the contents of 7 target components, 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.

[0098] The embodiment of the present invention also provides a kit for detecting the colony count of Bifidobacterium YLGB-1496 inactivated bacteria before inactivation. The kit includes at least one of L-methionine, Asp-Phe peptide, prolylalanine, GPRPK peptide, GP(Hyp)GAG peptide and Asp-Leu peptide as a target substance. The kit may also include other conventional reagents for detection, such as reagents for pretreatment of inactivated bacteria.

[0099] The features and properties of the present invention will be further described in detail below in conjunction with embodiments.

[0100] It should be noted that the preparation process of the inactivated Bifidobacterium YLGB-1496 bacteria 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, 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 completely freeze-dried.

[0101] 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 11NO2S, 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 purified water (Wahaha Group Co., Ltd., Hangzhou).

[0102] 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 blower; Vortex mixer; High - speed centrifuge; Ultrasonic cleaner; Analytical balance.

[0103] Example 1

[0104] This example provides a method for detecting the colony count of inactivated Bifidobacterium YLGB - 1496 before inactivation. The specific steps are as follows:

[0105] (1) Preparation of standard working solution

[0106] Standard stock solution: Weigh appropriate amounts of standard products separately and accurately (accurate to 0.1 mg), dissolve them in water and prepare standard stock solutions with a concentration of 5 mg / mL respectively, and store them at - 20 °C.

[0107] Mixed standard intermediate solution: Accurately pipette appropriate volumes of 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.

[0108] 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 freshly before use.

[0109] (2) Preparation of elution solution

[0110] 0.1% Aqueous Phosphoric Acid Solution: Take 1 mL of phosphoric acid, dilute it with water and make up the volume to 1000 mL, mix well, and use it immediately after preparation.

[0111] 0.1% Acetonitrile-Phosphoric Acid Solution: Take 1 mL of phosphoric acid, dilute it with acetonitrile and make up the volume to 1000 mL, mix well, and use it immediately after preparation.

[0112] (3) Preparation of Test Solution

[0113] 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:

[0114] Accurately 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, gently blow it dry with nitrogen at 40 °C, make up the volume to 0.2 mL with pure water, vortex and mix well for 1 min, ultrasonicate for 2 min, centrifuge at 10000 rpm / min for 10 min, aspirate the supernatant and dilute it 10 times for standby.

[0115] (4) Detection and Analysis

[0116] Use high performance liquid chromatography to test the sample solution and the standard working solution, measure the peak areas of the corresponding chromatograms. Take the concentration of the standard working solution as the abscissa and the peak area of the chromatogram as the ordinate to plot the standard curve. According to the detection results of the sample solution in step (3), calculate the concentration of the target substance in the inactivated bacteria in combination with the standard curve.

[0117] Chromatographic analysis conditions: Use the LC-20A analysis system, the chromatographic column is Poroshell 120Aq-C18 column (4.6 mm × 150 mm, 2.7 μm); mobile phase A is 0.1% aqueous phosphoric acid solution; mobile phase B is 0.1% acetonitrile-phosphoric acid solution, and the gradient elution program is shown in Table 1; the flow rate is 0.7 mL / min; the detection wavelength is 210 nm; the column temperature is 30 °C; the injection volume is 5 μL.

[0118] Table 1 Gradient Elution Program Table

[0119] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 33 95 5 45 95 5

[0120] (5) Calculate the Colony Count of the Inactivated Bacteria before Inactivation

[0121] When calculating the colony count using the concentration of the target substance in the inactivated bacteria, the regression equation used is y = 0.012x - 0.192; where x represents the concentration of the target substance, with the unit of mg / 100 g; y represents the colony count, with the unit of 10 9CFU / mL. According to the calculated concentration of the target substance, substitute it into the regression equation to calculate the number of colonies before the inactivated bacteria were inactivated.

[0122] Examples 2 - 7

[0123] The differences between Examples 2 - 7 and Example 1 are only as follows: Replace the target substance. In Examples 2 - 7, the target substances are respectively replaced with prolylalanine, citric acid, GPRPK, GP(Hyp)GAG, Asp - Leu peptide, and Asp - Phe peptide, and the regression equation is adjusted accordingly.

[0124] Test Example 1

[0125] Inject the series of standard working solutions prepared in step (1) into the high - performance liquid chromatograph respectively. Inject each concentration in parallel three times 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.

[0126] 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.

[0127] The test results are shown in Table 2:

[0128] Table 2 Establishment of HPLC standard curves for different target substances

[0129]

[0130] The elution peaks of seven standard products by HPLC under the optimized chromatographic conditions are as Figure 1 shown. It can be seen that the 7 target substances can be effectively baseline - separated. Figure 1 In it, 1 - 7 are in sequence: 1 - prolylalanine; 2 - L - methionine; 3 - citric acid; 4 - GPRPK; 5 - GP(Hyp)GAG; 6 - Asp - Leu; 7 - Asp - Phe.

[0131] The liquid chromatogram of the inactivated bacteria of Bifidobacterium YLGB - 1496 is as Figure 2 shown, Figure 2 In it, 1 - 7 are in sequence: 1 - prolylalanine; 2 - L - methionine; 3 - citric acid; 4 - GPRPK peptide; 5 - GP(Hyp)GAG peptide; 6 - Asp - Leu peptide; 7 - Asp - Phe peptide.

[0132] Test Example 2

[0133] The accuracy was evaluated by a recovery test, and the precision was evaluated by the coefficient of variation (RSD). In the test, the recovery rate was investigated by adding standard solution with three different concentrations to the test samples, that is, the ratio of the added amount of the standard substance shown in 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:

[0134] Table 3 Results of the spike test

[0135]

[0136] 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%.

[0137] Test Example 3

[0138] The detection accuracy of the method provided by the test example was tested, and the correlation coefficient R 2 was used to evaluate the effects of different test methods, and the results are shown in Table 4.

[0139] Correlation coefficient R 2 is a calculation method for fitting and measuring using a conventional linear regression model.

[0140] Table 4 Comparison of the effects of different target substances in the embodiment

[0141]

[0142] It can be seen that using L-methionine or Asp-Phe peptide as the target substance can provide the detection accuracy, and the correlation coefficient R 2 can reach more than 0.9. Using L-methionine as the target substance to detect the colony count of inactivated cells of Bifidobacterium YLGB-1496 before inactivation, the accuracy is further improved, and the correlation coefficient R 2 can reach more than 0.95, and the accuracy can be significantly improved compared with other target substances.

[0143] 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 in the protection scope of the present invention.

Claims

1. A method for detecting the number of colonies of inactivated Bifidobacterium YLGB-1496 before inactivation, characterized in that, Including: Using at least one of L-methionine, Asp-Phe peptide, prolylalanine, GPRPK peptide, GP(Hyp)GAG peptide and Asp-Leu peptide as a target substance to detect the colony count of inactivated cells of Bifidobacterium YLGB-1496 before inactivation.

2. The detection method according to claim 1, wherein 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, 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 the inactivated cells before inactivation using L-methionine as the target substance, the value of a in the regression equation is 0.010 - 0.015, and the value of b is -0.190 to -0.195; more preferably, when calculating the colony count of the inactivated cells before inactivation using L-methionine as the target substance, the regression equation used is y = 0.012x - 0.192; Preferably, when calculating the colony count of the inactivated cells before inactivation using Asp-Phe peptide as the target substance, the value of a in the regression equation is 0.110 - 0.115, and the value of b is -0.255 to -0.265; more preferably, when calculating the colony count of the inactivated cells before inactivation using Asp-Phe peptide as the target substance, the regression equation used is y = 0.113x - 0.259; Preferably, when calculating the colony count of the inactivated cells before inactivation using prolylalanine as the target substance, the value of a in the regression equation is 0.002 - 0.004, and the value of b is -0.270 to -0.280; more preferably, when calculating the colony count of the inactivated cells before inactivation using prolylalanine as the target substance, the regression equation used is y = 0.003x - 0.274; Preferably, when calculating the colony count of the inactivated cells before inactivation using GPRPK peptide as the target substance, the value of a in the regression equation is 0.040 - 0.050, and the value of b is -0.250 to -0.350; more preferably, when calculating the colony count of the inactivated cells before inactivation using GPRPK peptide as the target substance, the regression equation used is y = 0.045x - 0.290; Preferably, when calculating the colony count of the inactivated cells before inactivation using GP(Hyp)GAG peptide as the target substance, the value of a in the regression equation is 0.230 - 0.240, and the value of b is -0.195 to -0.210; more preferably, when calculating the colony count of the inactivated cells before inactivation using GP(Hyp)GAG peptide as the target substance, the regression equation used is y = 0.234x - 0.202; Preferably, when calculating the colony count of the inactivated cells before inactivation using Asp-Leu peptide as the target substance, the value of a in the regression equation is 0.105 - 0.110, and the value of b is -0.280 to -0.285; more preferably, when calculating the colony count of the inactivated cells before inactivation using Asp-Leu peptide as the target substance, the regression equation used is y = 0.108x - 0.283; Preferably, using at least one of L-methionine and Asp-Phe peptide as a target substance to detect the colony count of inactivated cells of Bifidobacterium YLGB-1496 before inactivation; More preferably, using L-methionine as the target substance, the colony count of the inactivated cells of Bifidobacterium YLGB-1496 before inactivation is detected.

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

4. The detection method according to claim 3, wherein Mix the inactivated cell sample with an organic alcohol aqueous solution for low-temperature extraction to obtain an extract; after centrifuging and separating the extract, take the supernatant, dry the supernatant to obtain the powder sample; re-dissolve and dilute the powder sample with water. Preferably, the preparation process of the extract includes: mixing the inactivated cell 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 cell sample is 1 mL - 3 mL. Preferably, before mixing with the inactivated cell sample, the methanol aqueous solution is pre-cooled to 2°C - 6°C.

5. The detection method according to claim 4, characterized in that Let the extract stand at a temperature of -15°C - -25°C for 20 min - 40 min, then centrifuge and separate, take the supernatant, and blow it with nitrogen until it is completely dry to obtain the powder sample. Preferably, re-dissolve the powder sample with water, centrifuge and separate, take the supernatant, and then dilute it 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 substance in the inactivated cells by the standard curve method, and then calculate the colony count of the inactivated cells before inactivation from the regression equation. Preferably, use high-performance liquid chromatography to test the concentration of the target substance. The testing process includes: Prepare a standard working solution: Use L-methionine standard to prepare standard working solutions with different dilution multiples. Prepare a test sample solution: Pretreat the inactivated cells 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 their corresponding peak areas; calculate the concentration of the target substance in the inactivated cells using the standard curve.

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

8. The detection method according to claim 7, characterized in that When using high-performance liquid chromatography for testing, 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% - 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% - 0.20%. Among them, the acidic substance is selected from any one of trifluoroacetic acid and phosphoric acid; Preferably, 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%; Preferably, the flow rate is controlled to be 0.5 mL / min - 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 L-methionine 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 - 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 - 200 μg / mL.

10. A kit for detecting the number of colonies before inactivation of inactivated cells of Bifidobacterium YLGB-1496, characterized in that, The kit includes: at least one of L-methionine, Asp-Phe peptide, prolylalanine, GPRPK peptide, GP(Hyp)GAG peptide, and Asp-Leu peptide as the target substance.

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