Method and kit for detecting colony count of inactivated bacteria of lactobacillus paracasei K56 before inactivation

Abstract

The invention discloses a method and a kit for detecting the colony count of inactivated bacteria of lactobacillus paracasei K56 before inactivation, and relates to the technical field of instrument analysis. According to the method, at least one of citric acid, GPRPK peptide, GP (Hyp) GAG peptide, L-methionine, Asp-Leu peptide and Asp-Phe peptide is taken as a target substance, the colony count of inactivated bacteria of the lactobacillus paracasei K56 before inactivation is calculated by testing the content of the target substance, the detection accuracy is relatively high, a theoretical basis is provided for quantitative detection of postbiotics, and the method has a good application prospect. The establishment of industrial standards for the quantitative detection of the metastatic products is also facilitated.

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 of inactivated Lactobacillus paracasei K56 before inactivation. 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 lung 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 special attention from the industrial community.

[0003] Current postbiotic products mainly include inactivated bacteria and fermentation broth. Inactivated bacteria 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] Lactobacillus paracasei K56 (deposit number CGMCC No. 15139 or DSM27447) has the effects of significantly reducing the adhesion ability of pathogenic bacteria to intestinal epithelial cells, improving the intestinal barrier function, and activating the intestinal immune ability. The postbiotic products produced by using this exclusive strain have the effects of preventing obesity and reducing intestinal inflammation, and their efficacy is directly related to the number of their colonies.

[0005] Therefore, there is an urgent need to develop a method for quantitatively analyzing postbiotic products (such as inactivated bacteria) of Lactobacillus paracasei K56 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 of inactivated Lactobacillus paracasei K56 before inactivation, aiming to accurately detect the number of colonies of inactivated Lactobacillus paracasei K56 postbiotics before inactivation.

[0008] The present invention is implemented as follows:

[0009] In a first aspect, the present invention provides a method for detecting the number of viable colonies of inactivated Lactobacillus paracasei K56 before inactivation, including: using at least one of citric acid, GPRPK peptide, GP(Hyp)GAG peptide, L-methionine, Asp-Leu peptide, and Asp-Phe peptide as a target substance to detect the number of viable colonies of inactivated Lactobacillus paracasei K56 before inactivation.

[0010] In an alternative embodiment, the detection method includes the step of establishing a linear equation between the number of viable 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 viable colonies, with the unit of 10 9 CFU / mL;

[0011] Preferably, viable Lactobacillus paracasei K56 is provided according to a viable colony number gradient, inactivated, and then the target substance is extracted and its content is analyzed. A fitting linear equation is calculated based on the viable colony number gradient and the target substance content to obtain a regression equation;

[0012] Preferably, when calculating the number of viable colonies of inactivated cells before inactivation using citric acid as the target substance, the value of a in the regression equation is 0.015 - 0.020, and the value of b is 0.018 - 0.023; more preferably, when detecting the number of viable colonies of inactivated cells before inactivation using citric acid as the target substance, the regression equation used is y = 0.017x + 0.021;

[0013] Preferably, when calculating the number of viable colonies of inactivated cells before inactivation using GPRPK peptide as the target substance, the value of a in the regression equation is 0.140 - 0.150, and the value of b is -0.475 to -0.485; more preferably, when detecting the number of viable colonies of inactivated cells before inactivation using GPRPK peptide as the target substance, the regression equation used is y = 0.144x - 0.479;

[0014] Preferably, when calculating the number of viable colonies of inactivated cells before inactivation using GP(Hyp)GAG peptide as the target substance, the value of a in the regression equation is 1.625 - 1.630, and the value of b is -0.975 to -0.985; more preferably, when calculating the number of viable colonies of inactivated cells before inactivation using GP(Hyp)GAG peptide as the target substance, the regression equation used is y = 1.627x - 0.981;

[0015] Preferably, when calculating the number of viable colonies of inactivated cells before inactivation using L-methionine as the target substance, the value of a in the regression equation is 0.125 - 0.135, and the value of b is -0.935 to -0.945; more preferably, when calculating the number of viable colonies of inactivated cells before inactivation using L-methionine as the target substance, the regression equation used is y = 0.129x - 0.939;

[0016] 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 ranges from 0.35 to 0.45, and the value of b ranges from -0.980 to -0.985; 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 = 0.409x - 0.983;

[0017] Preferably, 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 ranges from 0.425 to 0.430, and the value of b ranges from 2.450 to 2.550; more 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 = 0.428x + 2.500;

[0018] More preferably, at least one of citric acid and GPRPK peptide is used as the target to detect the number of colonies before inactivation of inactivated Lactobacillus paracasei K56.

[0019] 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 redissolving and diluting the powder sample.

[0020] 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, taking the supernatant, drying the supernatant to obtain a powder sample; redissolving and diluting the powder sample with water;

[0021] 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% to 90%, and performing low-temperature ultrasonic extraction for 20 min to 40 min under the conditions of a frequency of 35 KHz to 45 KHz and a temperature of 2°C to 6°C;

[0022] 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 to 3 mL;

[0023] Preferably, the methanol aqueous solution is pre-cooled to 2°C to 6°C before mixing with the inactivated bacteria sample.

[0024] In an alternative embodiment, the extract is left standing at a temperature of -15°C to -25°C for 20 min to 40 min, then centrifuged and separated, the supernatant is taken, and nitrogen blown until completely dry to obtain a powder sample;

[0025] Preferably, the powder sample is redissolved with water, the supernatant is taken after centrifuging and separating, and then diluted with 9 to 11 times the volume of water.

[0026] In an alternative embodiment, detection is performed using high performance liquid chromatography (HPLC). The concentration of the target substance in the inactivated bacteria is calculated using the standard curve method, and then the number of colonies in the inactivated bacteria before inactivation is calculated from the regression equation.

[0027] Preferably, the concentration of the target substance is measured using HPLC, and the measurement process includes:

[0028] Preparing a standard working solution: Using the target substance standard, prepare standard working solutions with different dilution factors.

[0029] Preparing a test sample solution: Pretreat the inactivated bacteria to obtain a sample solution.

[0030] Detection: Using HPLC, measure the sample solution and the standard working solution. Using the standard working solutions with different concentrations and their corresponding peak areas, plot a standard curve; calculate the concentration of the target substance in the inactivated bacteria using the standard curve.

[0031] In an alternative embodiment, when using HPLC for measurement, the chromatographic conditions include: The chromatographic column is Poroshell 120 Aq-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.

[0032] Preferably, the injection volume is controlled to be 2 μL to 10 μL.

[0033] In an alternative embodiment, when using HPLC for measurement, 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%.

[0034] Wherein, the acidic substance is selected from any one of trifluoroacetic acid and phosphoric acid;

[0035] Preferably, the gradient elution program is:

[0036] 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%;

[0037] 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%;

[0038] 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%.

[0039] 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%.

[0040] Preferably, the flow rate is controlled to be 0.5 mL / min - 1.0 mL / min.

[0041] In an alternative embodiment, the process of preparing the standard working solution includes: diluting the target 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 to form standard working solutions with different concentrations, and the concentration range of the standard working solutions is 1 μg / mL - 200 μg / mL.

[0042] In a second aspect, the present invention also provides a kit for detecting the number of colony-forming units of viable cells of Lactobacillus paracasei K56 before inactivation. The kit includes: using at least one of citric acid, GPRPK peptide, GP(Hyp)GAG peptide, L-methionine, Asp-Leu peptide, and Asp-Phe peptide as the target substance.

[0043] The present invention has the following beneficial effects: using at least one of citric acid, GPRPK peptide, GP(Hyp)GAG peptide, L-methionine, Asp-Leu peptide, and Asp-Phe peptide as the target substance, calculating the number of colony-forming units of viable cells of Lactobacillus paracasei K56 before inactivation 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore 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.

[0045] Figure 1 It is the liquid chromatogram of the mixed standard working solution;

[0046] Figure 2 It is the liquid chromatogram of inactivated cells of Lactobacillus paracasei K56. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] 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 described clearly and completely below. For those not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchases.

[0048] The embodiments of the present invention provide a method for detecting the number of colonies before inactivation of inactivated cells of Lactobacillus paracasei K56. The inventor creatively uses at least one of citric acid, GPRPK peptide, GP(Hyp)GAG peptide, L-methionine, Asp-Leu peptide, and Asp-Phe peptide as a target substance, and by testing the content of the target substance in the inactivated cells of Lactobacillus paracasei K56 and optimizing the regression equation to calculate the number of colonies before inactivation of the inactivated cells, the purpose of accurately detecting the number of colonies before inactivation of the inactivated cells is achieved. The specific steps are as follows:

[0049] S1. Use high-performance liquid chromatography to test the concentration of the target substance

[0050] The concentration of the target substance in the inactivated cells is tested by high-performance liquid chromatography. The testing process mainly includes the following three steps:

[0051] (1) Prepare the standard working solution

[0052] Using the target substance standard, 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. The detection results of different standard working solutions are used to draw a standard curve, and the content of the target substance in the inactivated cells can be calculated using this standard curve.

[0053] In the actual operation process, the process of preparing the standard working solution includes: diluting the target substance 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 solution 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 solution is prepared before being loaded onto the machine and used immediately after preparation.

[0054] (2) Prepare the test solution

[0055] The inactivated cells are pre-treated before being loaded onto the machine to obtain a sample solution. This pre-treatment process mainly includes processes such as extraction, drying, reconstitution, and dilution.

[0056] In some embodiments, the pre-processing process includes: extracting the inactivated bacterial sample to obtain an extract, drying the supernatant of the extract to obtain a powder sample, and re-dissolving and diluting the powder sample to obtain a sample before the machine. If precipitation occurs before the machine, it needs to be processed again by vortexing, ultrasound, centrifugation, etc.

[0057] In actual operation, the pre-processing operations can be carried out according to the following steps: the inactivated bacterial sample is mixed with an organic alcohol aqueous solution and extracted under low temperature conditions to obtain an extract; the extract is centrifuged to separate the supernatant, and the supernatant is dried to obtain a powder sample; the powder sample is re-dissolved and diluted with water to obtain a sample to be tested that meets the requirements of the machine.

[0058] In some embodiments, the preparation process of the extract includes: mixing the inactivated bacterial sample with an organic alcohol aqueous solution with a volume fraction of 70% to 90%, performing low-temperature ultrasonic extraction at a frequency of 35KHz to 45KHz, a power of 90W-120W, and a temperature of 2°C to 6°C, and the extraction time is 20min to 40min. 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 thereto; the amount of methanol aqueous solution corresponding to each 0.1g of inactivated bacterial sample is 1mL to 3mL, so as to fully extract the components in the inactivated bacteria.

[0059] 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., the extraction time can be 20min, 30min, 40min, etc. The amount of methanol aqueous solution corresponding to each 0.1g inactivated bacterial sample can be 1mL, 2mL, 3mL, etc.

[0060] In some embodiments, the methanol aqueous solution can be precooled to 2°C to 6°C before being mixed with the inactivated bacteria sample. The methanol aqueous solution is precooled to the extraction temperature before being mixed with the inactivated bacteria sample.

[0061] In some embodiments, the extract is left standing at a temperature of -15°C to -25°C for 20 min to 40 min, and then centrifuged. The supernatant is taken, and the supernatant is blown with liquid nitrogen until completely dried to obtain a powder sample for standby. The whole process is carried out under low-temperature operation to avoid destroying the morphology of inactivated bacteria at high temperature. Specifically, when the extract is left standing, undissolved solids can be deposited. The temperature for standing 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 to 6°C.

[0062] In some embodiments, the obtained powder sample is re-dissolved in water. After centrifugation, the supernatant is taken, and then diluted with 9 to 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 to 3 min. When centrifuging after re-dissolving, the operating conditions can be controlled at 9500 rpm / min - 10500 rpm / min, 2°C to 6°C. The supernatant is used as a sample solution for standby and diluted 10 times (i.e., diluted with 10 times the volume of water) before loading onto the machine.

[0063] 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 Lactobacillus paracasei K56 and then performing enlarged cultivation, followed by inactivation treatment and drying to obtain an inactivated bacteria sample. In some embodiments, the enlarged cultivation can be to cultivate Lactobacillus paracasei K56 until the late logarithmic phase, and the quantity level can reach 10 9 above, and then centrifuging to wash the bacteria, inactivation treatment, and freeze-drying are carried out in sequence. Centrifuging the bacteria can be to wash multiple times with sterile water and then resuspend with sterile water. During the inactivation treatment, the inactivation temperature can be controlled at 95°C to 105°C, and the inactivation time can be 10 min to 20 min to better ensure the anti-inflammatory effect of Lactobacillus paracasei K56.

[0064] Specifically, during the inactivation treatment, the inactivation temperature can be controlled at 95°C, 100°C, 105°C, etc.; the inactivation time can be 10 min, 15 min, 20 min, etc.

[0065] (3) Detection and analysis

[0066] 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. Using 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 in combination with the standard curve, the concentration of the target substance in the inactivated bacteria is calculated.

[0067] It should be noted that the prepared test sample solution is injected into a high-performance liquid chromatograph, and the retention time and peak area of the chromatographic peaks are recorded for quantitative analysis by the external standard method. The response value of the target 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 diluted by an appropriate multiple according to the measured concentration before analysis. The calculation results are expressed as the arithmetic mean of three independent determinations obtained under repeatability conditions, and the results are retained to three significant figures.

[0068] The content of each target in the sample is calculated according to formula (1):

[0069]

[0070] In the formula:

[0071] 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);

[0072] 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);

[0073] V—the volume of the sample extraction solution made up to a constant volume, in milliliters (mL);

[0074] m—the mass or volume of the sample taken, in grams or milliliters (g or mL);

[0075] 10—the unit conversion factor;

[0076] f—the dilution factor.

[0077] In some embodiments, when using high-performance liquid chromatography for testing, 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 at 2 μL to 10 μL, and the flow rate is controlled at 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.

[0078] 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.

[0079] Further, 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 in mobile phase B is 0.05% - 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.

[0080] 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.

[0081] 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.

[0082] S2. Calculate the number of colonies of inactivated bacteria before inactivation

[0083] According to the content of the target substance detected, calculate the number of colonies of inactivated cells of Lactobacillus paracasei K56 before inactivation. The regression equations corresponding to different target substances are different:

[0084] Preferably, when calculating the colony count of inactivated bacteria before inactivation with citric acid as the target substance, the value of a in the regression equation is 0.015 - 0.020 (such as 0.016, 0.017, 0.018, 0.019, 0.020, etc.), and the value of b is 0.018 - 0.023 (such as 0.018, 0.019, 0.020, 0.021, 0.022, 0.023, etc.); more preferably, when detecting the colony count of inactivated bacteria before inactivation with citric acid as the target substance, the regression equation used is y = 0.017x + 0.021.

[0085] Preferably, when calculating the colony count of inactivated bacteria before inactivation with GPRPK peptide as the target substance, the value of a in the regression equation is 0.140 - 0.150 (such as 0.140, 0.142, 0.144, 0.146, 0.148, 0.150), and the value of b is -0.475 to -0.485 (such as -0.475, -0.477, -0.479, -0.480, -0.482, -0.485, etc.); more preferably, when detecting the colony count of inactivated bacteria before inactivation with GPRPK peptide as the target substance, the regression equation used is y = 0.144x - 0.479.

[0086] Preferably, when calculating the colony count of inactivated bacteria before inactivation with GP(Hyp)GAG peptide as the target substance, the value of a in the regression equation is 1.625 - 1.630 (such as 1.625, 1.626, 1.627, 1.628, 1.629, 1.630, etc.), and the value of b is -0.975 to -0.985 (such as -0.975, 0.977, 0.980, 0.981, 0.983, 0.985, etc.); more preferably, when calculating the colony count of inactivated bacteria before inactivation with GP(Hyp)GAG peptide as the target substance, the regression equation used is y = 1.627x - 0.981.

[0087] Preferably, when calculating the colony count of inactivated bacteria before inactivation with L-methionine as the target substance, the value of a in the regression equation is 0.125 - 0.135 (such as 0.125, 0.127, 0.129, 0.130, 0.132, 0.135, etc.), and the value of b is -0.935 to -0.945 (such as -0.935, -0.937, -0.939, -0.940, -0.942, -0.945, etc.); more preferably, when calculating the colony count of inactivated bacteria before inactivation with L-methionine as the target substance, the regression equation used is y = 0.129x - 0.939.

[0088] 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 0.35 - 0.45 (such as 0.350, 0.370, 0.409, 0.420, 0.450, etc.), and the value of b is -0.980 to -0.985 (-0.980, -0.981, -0.982, -0.983, -0.984, -0.985, etc.); 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 = 0.409x - 0.983.

[0089] Preferably, when calculating the colony count of inactivated bacteria before inactivation using Asp-Phe peptide as the target, the value of a in the regression equation is 0.425 - 0.430 (such as 0.425, 0.426, 0.427, 0.428, 0.429, 0.430, etc.), and the value of b is 2.450 to 2.550 (such as 2.450, 2.470, 2.500, 2.520, 2.550, etc.); more preferably, when calculating the colony count of inactivated bacteria before inactivation using Asp-Phe peptide as the target, the regression equation used is y = 0.428x + 2.500.

[0090] In a preferred embodiment, using at least one of citric acid and GPRPK peptide as the target, the colony count of inactivated Lactobacillus paracasei K56 before inactivation is detected. By optimizing the selection of the target, the accuracy of the detection can be further improved.

[0091] Among them, x represents the concentration of the target, in mg / 100g; y represents the colony count, in 10 9 CFU / mL. According to the concentration of the target calculated in S1, substitute it into the regression equation to calculate the colony count of inactivated bacteria before inactivation.

[0092] It should be noted that the process of obtaining the regression equation is as follows: Provide live Lactobacillus paracasei K56 with a gradient of colony counts, 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 target respectively, the corresponding relationship between the colony count and the content of the target is as follows:

[0093] When the colony count of Lactobacillus paracasei K56 is 5×10 ∧When the number of colonies is 9 CFU / mL, the contents of prolylalanine, L-methionine, citric acid, GPRPK, GP(Hyp)GAG, and Asp-Leu peptide are 43.59 mg / 100 g, 62.60 mg / 100 g, 293.51 mg / 100 g, 43.79 mg / 100 g, 5.75 mg / 100 g, and 20.56 mg / 100 g in sequence.

[0094] For the inactivated bacteria sample K56 with different colony numbers (1 - 300, unit: 10^ 9 CFU / mL), the above liquid chromatography method was used for detection 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 number, in order to achieve the purpose of identifying the quality differences of postbiotics products.

[0095] The embodiment of the present invention also provides a kit for detecting the colony number before inactivation of inactivated cells of Lactobacillus paracasei K56. The kit includes: at least one of citric acid, GPRPK peptide, GP(Hyp)GAG peptide, L-methionine, Asp-Leu peptide, and Asp-Phe peptide as the target substance. Other conventional reagents for detection can also be included in the kit, such as reagents for pretreatment of inactivated cells, etc.

[0096] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0097] It should be noted that the preparation process of the inactivated cells of Lactobacillus paracasei K56 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 solution 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.

[0098] 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 purities 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.) standard products 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).

[0099] 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.

[0100] Example 1

[0101] This example provides a method for detecting the colony count of inactivated cells of Lactobacillus paracasei K56 before inactivation, using citric acid as the target to detect the colony count of inactivated cells before inactivation. The specific steps are as follows:

[0102] (1) Preparation of standard working solution

[0103] Standard stock solution: Weigh an appropriate amount of standard product (i.e., the target standard product, 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.

[0104] Mixed standard intermediate solution: Accurately pipette an appropriate volume of standard stock solution 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.

[0105] 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 for immediate use.

[0106] (2) Preparation of elution solution

[0107] 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.

[0108] 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.

[0109] (3) Preparation of Test Solution

[0110] Perform pre-treatment on the inactivated bacteria before loading onto the instrument to obtain a sample solution. The pre-treatment process is as follows:

[0111] Accurately weigh 0.3 g of the inactivated bacteria sample into a centrifuge tube, add 6 mL of methanol-aqueous solution pre-cooled to 4 °C with a volume fraction of 80%, 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, take the supernatant and dilute it 10 times for standby.

[0112] (4) Detection and Analysis

[0113] 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.

[0114] 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.

[0115] Table 1 Gradient Elution Program Table

[0116] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 33 95 5 45 95 5

[0117] (5) Calculate the Number of Colony-Forming Units of the Inactivated Bacteria before Inactivation

[0118] When calculating the number of colony-forming units 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, in mg / 100 g; y represents the number of colony-forming units, in 109 CFU / mL. Based on 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.

[0119] Example 2

[0120] The difference from Example 1 is only that: the target substance is replaced with GPRPK peptide, and the regression equation used is y = 0.144x - 0.479.

[0121] Examples 3 - 7

[0122] The differences between Examples 3 - 7 and Example 1 are only that: the target substances are replaced, and in Examples 3 - 7, the target substances are replaced with prolylalanine, L - methionine, GP(Hyp)GAG, Asp - Leu peptide, and Asp - Phe peptide respectively, and the regression equations are adjusted accordingly.

[0123] Test Example 1

[0124] 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. Take 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.

[0125] The limit of detection (LOD) and limit of quantitation (LOQ) are used to evaluate the sensitivity of the method. The limit of detection is the lowest concentration at which the analyte can be detected, and the limit of quantitation 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.

[0126] The test results are shown in Table 2:

[0127] Table 2 Establishment of HPLC standard curves for different target substances

[0128]

[0129] The elution peaks of seven standard products in 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 In 1 - 7 in the figure are: 1 - prolylalanine; 2 - L - methionine; 3 - citric acid; 4 - GPRPK; 5 - GP(Hyp)GAG; 6 - Asp - Leu; 7 - Asp - Phe.

[0130] The liquid chromatogram of inactivated cells of Lactobacillus paracasei K56 is as Figure 2as shown Figure 2 Among them, 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.

[0131] Test Example 2

[0132] The accuracy was evaluated by the recovery test, and the precision was evaluated by the coefficient of variation (RSD). For the test, the recovery rates were investigated by adding standard solutions with 3 different concentrations to the test samples, that is, the ratio of the added amount of the standard shown in the test to the true added amount of the standard, expressed as a percentage. Three parallels were made for each concentration level, and the recovery rates and coefficients of variation were calculated. According to the requirements of "GB / T 27417 Conformity assessment - Guidelines for the validation and verification of chemical analysis methods", the added recovery rates should be within 80% - 110%, and the precision should be less than 20%. The test results are shown in Table 3:

[0133] Table 3 Results of the spike test

[0134]

[0135] 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 added recovery rate is 80% - 110%, and the relative standard deviation (RSDRSD) is within 10%.

[0136] Test Example 3

[0137] 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.

[0138] Correlation coefficient R 2 It is a calculation method using the conventional linear regression model fitting metric.

[0139] Table 4 Comparison of the effects of different target substances in the embodiment

[0140]

[0141] It can be seen that when citric acid or GPRPK peptide is used as the target substance to detect the colony count of inactivated cells of Lactobacillus paracasei K56 before inactivation, the accuracy is significantly higher, and the accuracy can be significantly improved compared with other target substances in other embodiments. The accuracy of using citric acid as the target substance for detection is the highest.

[0142] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, various modifications and variations can be made to the present invention. 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 Lactobacillus paracasei K56 inactivated cells before inactivation, characterized in that, Including: Using at least one of citric acid, GPRPK peptide, GP(Hyp)GAG peptide, L-methionine, Asp-Leu peptide and Asp-Phe peptide as a target substance to detect the colony count of inactivated cells of Lactobacillus paracasei K56 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, 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 citric acid as the target substance, the value of a in the regression equation is 0.015 - 0.020, and the value of b is 0.018 - 0.023; more preferably, when detecting the colony count of the inactivated cells before inactivation using citric acid as the target substance, the regression equation used is y = 0.017x + 0.021; 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 0.140 - 0.150, and the value of b is -0.475 - -0.485; more preferably, when detecting the colony count of the inactivated cells before inactivation using GPRPK peptide as the target substance, the regression equation used is y = 0.144x - 0.479; 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 1.625 - 1.630, and the value of b is -0.975 - -0.985; 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 = 1.627x - 0.981; 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 0.125 - 0.135, and the value of b is -0.935 - -0.945; 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 = 0.129x - 0.939; 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 0.35 - 0.45, and the value of b is -0.980 - -0.985; 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 = 0.409x - 0.983; 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 0.425 - 0.430, and the value of b is 2.450 - 2.550; 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 = 0.428x + 2.500; More preferably, using at least one of citric acid and GPRPK peptide as a target substance to detect the colony count of inactivated cells of Lactobacillus paracasei K56 before inactivation.

3. The detection method according to claim 1, characterized in that, Before detection, pretreatment is performed on the inactivated bacterial cells. The process of the pretreatment includes: extracting the inactivated bacterial cell 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, characterized in that, Mix the inactivated bacterial cell sample with an organic alcohol aqueous solution and perform low-temperature extraction to obtain an extract; after centrifugally separating the extract, 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 bacterial 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 bacterial cell sample is 1 mL - 3 mL; Preferably, the methanol aqueous solution is pre-cooled to 2°C - 6°C before being mixed with the inactivated bacterial cell 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 perform centrifugal separation, take the supernatant, and blow it with nitrogen until it is completely dry to obtain the powder sample; Preferably, redissolve the powder sample with water, after centrifugal separation, 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 bacterial cells by the standard curve method, and then calculate the number of colonies before inactivation of the inactivated bacterial cells 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 a target substance standard product to prepare standard working solutions with different dilution multiples; Prepare a test sample solution: perform pretreatment on the inactivated bacterial cells to obtain a sample solution; Detection: Use high-performance liquid chromatography to test the sample solution and the standard working solution, use the standard working solutions with different concentrations and the corresponding peak areas to draw a standard curve; calculate the concentration of the target substance in the inactivated bacterial cells using the standard curve.

7. The detection method according to claim 6, wherein When using high-performance liquid chromatography for testing, the chromatographic conditions include: the chromatographic column uses Poroshell 120Aq-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, 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: 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, wherein 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 - 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 colony count of Lactobacillus paracasei K56 inactivated cells before inactivation, characterized in that, The kit includes: using at least one of citric acid, GPRPK peptide, GP(Hyp)GAG peptide, L-methionine, Asp-Leu peptide, and Asp-Phe peptide as the target.

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