Detection method of hypoglycemic active component trpca of paracasei lactis lc19

Abstract

The application provides a detection method of a blood glucose-lowering active ingredient TrpCA of paracasei Lc19, which comprises the following steps: S1, pretreating a to-be-detected sample by using a methanol aqueous solution to obtain a detection solution; and S2, analyzing the detection solution by using a high performance liquid chromatography-fluorescence detector to determine the content of the TrpCA, wherein an impurity trapping column is arranged between a gradient mixer and a sample injector in the high performance liquid chromatography-fluorescence detector. The application utilizes the structural characteristics (endogenous fluorescence chromophore-indole ring) of the TrpCA, realizes accurate detection of the TrpCA with a sodium molar per liter level by means of the characteristic fluorescence of the indole ring and by optimizing the detection steps and chromatographic conditions. The method provided by the application has high sensitivity, high accuracy and high selectivity, and is simple to operate, economical in cost and widely applicable.

Description

Technical Field

[0001] This application belongs to the field of biological detection technology, specifically relating to a method for detecting tryptophan-binding bile acids, and more specifically to a method for detecting TrpCA, the hypoglycemic active ingredient of Lactobacillus paracasei Lc19. Background Technology

[0002] Tryptophan-cholic acid (TrpCA or TRP-CA) is a newly discovered amino acid-bound bile acid produced by bacteria in recent years. Studies have shown that TrpCA can activate the orphan receptor MRGPRE, promote the secretion of intestinal GLP-1 and insulin, improve glucose tolerance, and play a role in weight loss and blood sugar reduction. Developing a simple, sensitive, economical, practical, and easily scalable method for detecting TrpCA can play an important role in screening specific strains capable of synthesizing this beneficial metabolite, optimizing probiotic production processes, evaluating the TrpCA conversion rate of probiotics, monitoring fermentation broth quality, studying disease mechanisms and discovering biomarkers, and assessing intestinal health and gut microbiota function.

[0003] Therefore, it is essential to develop a simple and easy-to-operate TrpCA detection method. Summary of the Invention

[0004] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a method for detecting tryptophan-binding bile acids, which has advantages such as high sensitivity, high accuracy, and good repeatability.

[0005] This application is based on the following discoveries of the inventors:

[0006] Currently, all existing methods for detecting TrpCA are liquid chromatography-tandem mass spectrometry (LC-MS). However, due to the high cost of the instruments, the complexity of the operation process, and the fact that they usually rely on internal standards for accurate quantification, the application of this method is greatly limited and the detection cost is high.

[0007] Based on the TrpCA endogenous fluorescent chromophore—indole ring ( Figure 1Based on the characteristics of TrpCA, the inventors of this application have developed a high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method for detecting TrpCA. This method utilizes the intrinsic fluorescent chromophore in the molecular structure of TrpCA, which can be directly and sensitively captured by a fluorescence detector, achieving high-sensitivity and high-selectivity direct detection of TrpCA. This method fills the gap in the field of HPLC-FLD for TrpCA detection. It has been validated in terms of specificity, limit of detection, limit of quantitation, accuracy, and repeatability, demonstrating excellent methodological performance. It enables the simple, highly sensitive, highly selective, highly accurate, and cost-effective detection of TrpCA, providing key technical support for the accurate quantification of TrpCA in various matrices such as fermentation broth and biological samples, and for related biological research.

[0008] In one aspect of this application, a method for detecting tryptophan-binding bile acids is provided. According to an embodiment of this application, the method includes:

[0009] S1. The sample to be tested is pretreated with methanol-water solution to obtain the test solution;

[0010] S2. Analyze the test solution using high performance liquid chromatography-fluorescence detection to determine the content of tryptophan-binding cholic acid;

[0011] Among them, an impurity trapping column is added between the gradient mixer and the injector in the high performance liquid chromatography-fluorescence detector;

[0012] The chromatographic conditions of the high-performance liquid chromatography-fluorescence detector include:

[0013] Mobile phase: Mobile phase A is selected from methanol, and mobile phase B is selected from ammonium acetate solution;

[0014] Gradient elution conditions:

[0015] The initial ratio was 40% mobile phase A and 60% mobile phase B, maintained for 4.0 min; from 4.0 to 24.0 min, mobile phase A was increased to 80% and mobile phase B was decreased to 20%, maintained for 26.0 min; at 26.1 min, mobile phase A was increased to 90% and mobile phase B was decreased to 10%, maintained for 28.0 min; from 28.0 to 30.0 min, mobile phase A was decreased to 40% and mobile phase B was increased to 60%, maintained for 38.0 min.

[0016] The fluorescence detection conditions of the high-performance liquid chromatography-fluorescence detector are: excitation wavelength of 280 nm and emission wavelength of 320~380 nm.

[0017] This application's method achieves accurate detection of TrpCA at sodium molar per liter by utilizing the characteristic fluorescence of the indole ring and optimizing the detection steps and chromatographic conditions. This method possesses TrpCA detection capabilities comparable to LC-MS, overcoming the limitations of LC-MS methods such as high instrument cost, complex operation procedures, and high detection costs. It provides an efficient, economical, and reliable solution for relevant analytical needs. Therefore, this method exhibits high sensitivity, high accuracy, and high selectivity, while also being simple to operate, cost-effective, and widely applicable.

[0018] In one optional embodiment of this application, the impurity collecting column has an inner diameter of 4.6 mm, a column length of 50 mm, and a pressure resistance of 40 MPa.

[0019] In one optional embodiment of this application, the impurity trapping column is selected from the Ghost-Buster Column impurity trapping column.

[0020] In an optional embodiment of this application, the gain PMT in the chromatographic conditions is 8 to 10.

[0021] In an optional embodiment of this application, the mobile phase B is selected from an ammonium acetate solution with a concentration of 5-20 mmol / L and a pH value of 3.0-5.0.

[0022] In an optional embodiment of this application, the chromatographic conditions used are a C18 column, a flow rate of 0.3~0.6 mL / min, a column temperature of 30~45℃, and an injection volume of 20~50 μL.

[0023] In one optional embodiment of this application, the C18 chromatographic column has a length of 150 mm, an inner diameter of 3.0 mm, a porous surface packing material, and a packing particle size of 2.7 μm.

[0024] In one optional embodiment of this application, the C18 column is selected from Agilent, Infinity LabPoroshell 120 EC-C18.

[0025] In an optional embodiment of this application, step S1, the preprocessing includes:

[0026] S1-1. The sample to be tested is mixed with a methanol-water solution, wherein the sample to be tested is in liquid state and the volume ratio of the sample to the methanol-water solution is 1:(1~3), or the sample to be tested is in solid state and the volume ratio of the sample to the methanol-water solution is 1:(8~12).

[0027] S1-2. Centrifuge the mixed product and collect the supernatant to obtain the detection solution.

[0028] In one optional embodiment of this application, the methanol aqueous solution is selected from 40-60% methanol aqueous solution.

[0029] In one optional embodiment of this application, the sample to be tested is liquid, and the mixing process is selected from vortex oscillation.

[0030] In one optional embodiment of this application, the sample to be tested is solid, and the mixing process is selected from ultrasonic processing.

[0031] In one optional embodiment of this application, the centrifugation process is carried out at a speed of 10,000 to 15,000 rpm, a temperature of 4°C, and a time of 5 to 15 minutes.

[0032] In one optional embodiment of this application, the vortex oscillation time is 5 to 15 minutes.

[0033] In one optional embodiment of this application, the ultrasonic treatment time is 30-60 minutes.

[0034] In an optional embodiment of this application, step S1, the preprocessing further includes:

[0035] S1-3. The supernatant from step S1-2 is concentrated by nitrogen blowing.

[0036] In one optional embodiment of this application, the temperature of the nitrogen blowing concentration is 30~40°C.

[0037] Beneficial effects:

[0038] 1. This application presents a novel method for detecting TrpCA using high-performance liquid chromatography (HPLC) separation and fluorescence detection, leveraging the characteristic fluorescence of the indole ring in the TrpCA molecular structure. This method directly and efficiently utilizes the physicochemical properties of the target analyte and is unaffected by impurities such as tryptophan (which also has an indole ring in its molecular structure) and non-fluorescent impurities. This method fills a gap in HPLC-FLD technology for TrpCA detection. Furthermore, this method has been validated in terms of specificity, limit of detection, limit of quantitation, accuracy, and repeatability, demonstrating excellent methodological performance. The method achieves simple operation, high sensitivity, high selectivity, high accuracy, and cost-effectiveness for the detection of TrpCA.

[0039] 2. The method of this application is simple to operate, cost-effective, highly practical, and widely applicable, while also possessing high sensitivity. In particular, compared with the previously reported LC-MS method, the method of this application has significant comprehensive advantages, specifically manifested in: 1) more convenient instrument operation and lower requirements for operators; 2) less data generated, reducing the burden of data analysis; 3) the purchase and maintenance cost of HPLC-FLD instruments is much lower than that of LC-MS, and the instrument is more widely used in routine analytical laboratories, greatly improving the practicality and scalability of this method; 4) the limit of quantitation of this method is as low as 2 nmol / L, which can effectively cover the content range of TrpCA synthesized by probiotics (3.9~140.43 nmol / L) in existing technologies (patent numbers CN119215078A, CN119235873A).

[0040] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0041] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0042] Figure 1 This is the molecular structure diagram of TrpCA in this application;

[0043] Figure 2 The image shows the detection results of MRS medium containing TrpCA with 1 mmol / L bile acid and 1 mmol / L tryptophan added in Example 1 of this application, with and without a contaminant trapping column connected between the gradient mixer and the injector.

[0044] Figure 3 The above is a superimposed chromatogram of the TrpCA standard solution (blue line, 100 nmol / L), the MRS medium containing TrpCA with 1 mmol / L bile acid and 1 mmol / L tryptophan (green line), and the BHI medium containing TrpCA with 0.1 mmol / L bile acid and 0.1 mmol / L tryptophan (purple line) in Example 1 of this application.

[0045] Figure 4 This is a superimposed chromatogram of the TrpCA standard solution (green line, 100 nmol / L) and the tryptophan standard solution (blue line, 100 nmol / L) in Example 1 of this application;

[0046] Figure 5 This is a superimposed chromatogram of the TrpCA standard solution in Example 2 of this application at emission wavelengths of 350 nm (blue line) and 380 nm (green line);

[0047] Figure 6 The image shows a superimposed chromatogram of the TrpCA standard solution in Example 2 of this application at emission wavelengths of 350 nm (blue line) and 320 nm (green line);

[0048] Figure 7 The image shows a superimposed chromatogram of the following solutions in Example 2 of this application: TrpCA standard solution (blue line, 50 nmol / L), TrpCA in MRS medium with 1 mmol / L bile acid and 1 mmol / L tryptophan (green line), and TrpCA in BHI medium with 0.1 mmol / L bile acid and 0.1 mmol / L tryptophan (purple line) (flow rate 0.3 mL / min).

[0049] Figure 8 The above is a superimposed chromatogram of the following in Example 2 of this application: TrpCA standard solution (blue line, 50 nmol / L), TrpCA in MRS medium with 1 mmol / L bile acid and 1 mmol / L tryptophan (green line), and TrpCA in BHI medium with 0.1 mmol / L bile acid and 0.1 mmol / L tryptophan (purple line) (column temperature 30°C).

[0050] Figure 9 The above is a superimposed chromatogram of the following in Example 2 of this application: TrpCA standard solution (blue line, 50 nmol / L), TrpCA in MRS medium with 1 mmol / L bile acid and 1 mmol / L tryptophan (green line), and TrpCA in BHI medium with 0.1 mmol / L bile acid and 0.1 mmol / L tryptophan (purple line) (column temperature 45°C).

[0051] Figure 10 This is a superimposed chromatogram of 100 nmol / L TrpCA standard solutions with injection volumes of 10 μL (green line), 20 μL (purple line), 30 μL (orange line), 40 μL (orange-red line) and 50 μL (blue line) in Example 2 of this application;

[0052] Figure 11 The image shows a superimposed chromatogram of the 1 nmol / L TrpCA standard solution in Example 2 of this application at gain PMT 10 (blue line) and gain PMT 8 (green line).

[0053] Figure 12 The image shows a superimposed chromatogram of the 100 nmol / L TrpCA standard solution in Comparative Example 1 of this application at emission wavelengths of 350 nm (blue line) and 310 nm (green line).

[0054] Figure 13The image shows the superimposed chromatograms of the 100 nmol / L TrpCA standard solution in Comparative Example 2 of this application at flow rates of 0.6 mL / min (blue line) and 0.2 mL / min (green line).

[0055] Figure 14 The above are superimposed chromatograms of the following in Comparative Example 3 of this application: 20 nmol / L TrpCA standard solution (blue line), TrpCA in MRS medium with 1 mmol / L bile acid and 1 mmol / L tryptophan (green line), and TrpCA in BHI medium with 0.1 mmol / L bile acid and 0.1 mmol / L tryptophan (purple line) (column temperature 55℃).

[0056] Figure 15 The image shows the superimposed chromatograms of the 1 nmol / L TrpCA standard solution in Comparative Example 5 of this application at gain PMT 10 (blue line) and gain PMT 5 (green line).

[0057] in, Figures 2-15 The horizontal axis represents retention time, and the vertical axis represents response time. Detailed Implementation

[0058] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0059] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0060] Detection method for tryptophan-binding bile acids

[0061] In one aspect of this application, a method for detecting tryptophan-binding bile acids is proposed. According to an embodiment of this application, the method includes: S1, pretreating the sample to be tested with a methanol-water solution to obtain a detection solution; S2, analyzing the detection solution using a high-performance liquid chromatography-fluorescence detector to determine the content of tryptophan-binding cholic acid; wherein, an impurity trapping column is added between the gradient mixer and the injector in the high-performance liquid chromatography-fluorescence detector; the chromatographic conditions of the high-performance liquid chromatography-fluorescence detector include: mobile phase: mobile phase A is selected from methanol, and mobile phase B is selected from ammonium acetate solution; gradient elution conditions: the initial ratio is 40% mobile phase A and 60% mobile phase B, maintained for 4.0 min; from 4.0 to 24.0 min, mobile phase A increases to 80% and mobile phase B decreases to 20%, maintained for 26.0 min; at 26.1 min, mobile phase A increases to 90% and mobile phase B decreases to 10%, maintained for 28.0 min; from 28.0 to 30.0 min, mobile phase A decreases to 40% and mobile phase B increases to 60%, maintained for 38.0 min. min; The fluorescence detection conditions of the high performance liquid chromatography-fluorescence detector are: excitation wavelength of 280 nm and emission wavelength of 320~380 nm.

[0062] This application utilizes the characteristic fluorescence of the indole ring in the TrpCA molecular structure. It is the first application of high-performance liquid chromatography-fluorescence detection to detect TrpCA, and it was discovered that connecting an impurity trapping column between the gradient mixer and the injector can effectively separate impurities from TrpCA. The inventors found through chromatographic results that even with a mass spectrometry-grade mobile phase, fluorescent impurities still interfere, severely affecting the identification and separation of TrpCA characteristic peaks. By adding an impurity trapping column between the gradient mixer and the injector, the applicant found that this fluorescent impurity interference could be effectively removed, thus achieving clear elution of TrpCA peaks under conventional liquid chromatography-grade mobile phase conditions. Furthermore, by optimizing the chromatographic gradient elution conditions, baseline separation of tryptophan, which also contains an indole ring in its molecular structure, was achieved. Therefore, the method of this application has high sensitivity, high accuracy, high selectivity, and is simple to operate, cost-effective, and widely applicable.

[0063] According to embodiments of this application, the above method may further include at least one of the following technical features:

[0064] In an optional embodiment of this application, the impurity trapping column has an inner diameter of 4.6 mm, a column length of 50 mm, and a pressure resistance of 40 MPa. This allows for effective separation of impurities from TrpCA in the sample, improving detection accuracy.

[0065] In one optional embodiment of this application, the impurity trapping column is selected from the Ghost-Buster Column impurity trapping column. This allows for effective separation of impurities from TrpCA, improving detection accuracy.

[0066] In an optional embodiment of this application, the gain PMT in the chromatographic conditions is 8-10, for example, 8, 9, or 10. The inventors discovered during experiments that a suitable gain range can achieve an optimal balance between signal amplification and background noise: it can sufficiently amplify the characteristic fluorescence signal of TrpCA to ensure high-sensitivity detection, while effectively suppressing the synchronous amplification of baseline noise. This improves the signal-to-noise ratio, resulting in a more stable chromatographic baseline, clearer target peak separation, and increased detection sensitivity.

[0067] In an optional embodiment of this application, the mobile phase B is selected from an ammonium acetate solution with a concentration of 5-20 mmol / L and a pH value of 3.0-5.0.

[0068] In a preferred embodiment of this application, the mobile phase B is selected from an ammonium acetate solution with a concentration of 10 mmol / L and a pH of 4.0.

[0069] In an optional embodiment of this application, the chromatographic conditions employ a C18 column, a flow rate of 0.3-0.6 mL / min (e.g., 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min), a column temperature of 30-45℃ (e.g., 30℃, 32℃, 35℃, 38℃, 40℃, 42℃, 45℃), and an injection volume of 20-50 μL (e.g., 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL). Therefore, this method can shorten the elution time of TrpCA, reduce interference from impurity peaks, increase peak intensity, further save detection time, and improve detection accuracy and sensitivity.

[0070] In a preferred embodiment of this application, the chromatographic conditions used are a C18 column, a flow rate of 0.4~0.6 mL / min, a column temperature of 35~45℃, and an injection volume of 30~50 μL.

[0071] In one optional embodiment of this application, the C18 chromatographic column has a length of 150 mm, an inner diameter of 3.0 mm, a porous surface packing material, and a packing particle size of 2.7 μm.

[0072] In one optional embodiment of this application, the C18 column is selected from Agilent, Infinity LabPoroshell 120 EC-C18.

[0073] In an optional embodiment of this application, the emission wavelength of the fluorescence detection condition is 320~350 nm. This ensures the detection of fluorescent groups in TrpCA and improves detection sensitivity.

[0074] In an optional embodiment of this application, step S1, the preprocessing includes:

[0075] S1-1. The sample to be tested is mixed with a methanol-water solution, wherein the sample to be tested is in liquid state and the volume ratio of the sample to the methanol-water solution is 1:(1~3), or the sample to be tested is in solid state and the volume ratio of the sample to the methanol-water solution is 1:(8~12).

[0076] S1-2. Centrifuge the mixed product and collect the supernatant to obtain the detection solution.

[0077] In one optional embodiment of this application, the volume ratio of the sample to be tested to the methanol aqueous solution is 1:1.

[0078] In one optional embodiment of this application, the sample to be tested is a solid, and the volume ratio of the sample to the methanol aqueous solution is 1:10.

[0079] In an optional embodiment of this application, the methanol aqueous solution is selected from 40-60% methanol aqueous solutions, such as 40%, 45%, 50%, 55%, and 60% methanol aqueous solutions.

[0080] In one optional embodiment of this application, the sample to be tested is liquid, and the mixing process is selected from vortex oscillation.

[0081] In one optional embodiment of this application, the sample to be tested is solid, and the mixing process is selected from ultrasonic processing.

[0082] In one optional embodiment of this application, the centrifugation process is carried out at a speed of 10,000 to 15,000 rpm (e.g., 10,000, 11,000, 12,000, 13,000, 14,000, 15,000), at a temperature of 4°C, and for a time of 5 to 15 min (e.g., 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min).

[0083] In an optional embodiment of this application, the vortex oscillation time is 5 to 15 min (e.g., 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min).

[0084] In one optional embodiment of this application, the ultrasonic treatment time is 30 to 60 minutes (e.g., 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes).

[0085] In an optional embodiment of this application, step S1, the preprocessing further includes:

[0086] S1-3. The supernatant from step S1-2 is concentrated by nitrogen blowing.

[0087] In one optional embodiment of this application, the nitrogen blowing concentration temperature is 30~40°C (e.g., 30°C, 32°C, 35°C, 38°C, 40°C).

[0088] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0089] Example 1: Detection method of TrpCA, the hypoglycemic active ingredient of Lactobacillus paracasei Lc19

[0090] 1. Since TrpCA is usually produced by bacteria under culture conditions in the presence of tryptophan and bile acids, and the molecular structure of tryptophan contains an indole ring, it can be detected by a fluorescence detector, while the molecular structure of bile acids does not contain an endogenous luminescent group and cannot be detected by a fluorescence detector, it is necessary to investigate whether tryptophan and TrpCA can be separated on the chromatogram, and also to investigate whether other small molecule compounds containing indole rings that may be present in the fermentation broth will interfere with the quantification of TrpCA.

[0091] This application employs the following steps for testing:

[0092] (1) Take 0.5 mL of liquid sample (TrpCA added to MRS medium containing 1 mmol / L bile acid and 1 mmol / L tryptophan (disclosed in Example 3 of patent CN119235873A, where the final concentration of TrpCA is 100 nmol / L), add 0.5 mL of methanol, and vortex for 5 min.

[0093] (2) Centrifuge the sample after step (1) at 12000 rpm, 4℃, for 10 min, and collect the supernatant.

[0094] (3) The supernatant obtained in step (2) is concentrated by nitrogen blowing at a temperature of 35°C, and the concentrated supernatant is filtered.

[0095] (4) The sample obtained in step (3) was analyzed by high performance liquid chromatography-fluorescence detection instrument and quantified by external standard method;

[0096] The conditions for high-performance liquid chromatography-fluorescence detection analysis are as follows:

[0097] The analytical column was an Infinitylab Poroshell 120 EC-C18 (3.0 x 150 mm, particle size 2.7 μm).

[0098] Mobile phase: Phase A was methanol, and Phase B was 10 mmol / L ammonium acetate solution at pH 4.0. The initial ratio was 40% Phase A and 60% Phase B, maintained for 4.0 min. From 4.0 to 24.0 min, Phase A increased to 80% and Phase B decreased to 20%, maintained for 26.0 min. At 26.1 min, Phase A increased to 90% and Phase B decreased to 10%, maintained for 28.0 min. From 28.0 to 30.0 min, Phase A decreased to 40% and Phase B increased to 60%, maintained for 38.0 min.

[0099] The chromatographic conditions were: flow rate 0.6 mL / min, column oven temperature 40℃, injection volume 50 μL, and gain PMT 10.

[0100] The fluorescence detection conditions were: excitation wavelength of 280 nm and emission wavelength of 350 nm.

[0101] The results showed that there were spurious peaks near TrpCA, which interfered with the quantification of TrpCA. (See details...) Figure 2 The green line.

[0102] 2. Furthermore, connecting an impurity trapping column (Yuexu Ghost-Buster Column, inner diameter: 4.6 mm, column length: 50 mm) before the injector can eliminate interference from impurity peaks caused by the mobile phase. Figure 4 ).

[0103] (1) Take 0.5 mL of the liquid sample (TrpCA added to MRS medium containing 1 mmol / L bile acid and 1 mmol / L tryptophan (disclosed in Example 3 of patent CN119235873A, where the final concentration of TrpCA is 100 nmol / L), add 0.5 mL of methanol, and vortex for 5 min.

[0104] (2) Centrifuge the sample after step (1) at 12000 rpm, 4℃, for 10 min, and collect the supernatant.

[0105] (3) The supernatant obtained in step (2) is concentrated by nitrogen blowing at a temperature of 35°C, and the concentrated supernatant is filtered.

[0106] (4) The sample obtained in step (3) was analyzed by high performance liquid chromatography-fluorescence detection instrument and quantified by external standard method;

[0107] The conditions for high-performance liquid chromatography-fluorescence detection analysis are as follows:

[0108] A Ghost-Buster Column (4.6 x 50) impurity trapping column is added between the gradient mixer and the injector. mm) ;

[0109] The analytical column was an Infinitylab Poroshell 120 EC-C18 (3.0 x 150 mm, particle size 2.7 μm).

[0110] Mobile phase: Phase A was methanol, and Phase B was 10 mmol / L ammonium acetate solution at pH 4.0. The initial ratio was 40% Phase A and 60% Phase B, maintained for 4.0 min. From 4.0 to 24.0 min, Phase A increased to 80% and Phase B decreased to 20%, maintained for 26.0 min. At 26.1 min, Phase A increased to 90% and Phase B decreased to 10%, maintained for 28.0 min. From 28.0 to 30.0 min, Phase A decreased to 40% and Phase B increased to 60%, maintained for 38.0 min.

[0111] The chromatographic conditions were: flow rate 0.6 mL / min, column oven temperature 40℃, injection volume 50 μL, and gain PMT 10.

[0112] The fluorescence detection conditions were: excitation wavelength of 280 nm and emission wavelength of 350 nm.

[0113] The results showed that connecting an impurity trapping column (Yuexu, Ghost-Buster Column, inner diameter: 4.6 mm, column length: 50 mm) before the injector could eliminate interference from impurity peaks introduced by the mobile phase. See details... Figure 2 The blue line.

[0114] 3. Further, this application uses the above two methods to verify the effects of tryptophan standard solution and two typical fermentation broth substrates (both containing tryptophan). One fermentation broth substrate is disclosed in Example 3 of patent CN119235873A (i.e., MRS medium with 1 mmol / L bile acid and 1 mmol / L tryptophan added), and the other is reported in the literature (10.1016 / j.cell.2025.05.010) (i.e., BHI medium with 0.1 mmol / L bile acid and 0.1 mmol / L tryptophan added). The results are as follows: Figure 3 As shown.

[0115] Figure 3 It can be seen that the fermentation broth matrix has no impurities interfering with the TrpCA elution time; such as Figure 4 As shown, under this mobile phase gradient condition, the chromatographic peak of TrpCA was baseline separated from the tryptophan chromatographic peak. This indicates that tryptophan and other small molecule compounds containing indole rings in the fermentation broth matrix do not interfere with the quantification of TrpCA. Therefore, this further demonstrates the high selectivity of this method.

[0116] Example 2: Screening of analytical conditions for high performance liquid chromatography-fluorescence detection

[0117] 1. Compared with the second scheme in Example 1, the difference is that the emission wavelength in the fluorescence detection conditions described in step (4) is changed from 350 nm to 380 nm. The detection results are as follows: Figure 5 As shown, the response of the TrpCA chromatographic peak at this wavelength decreases by 34%.

[0118] 2. Compared with the second scheme in Example 1, the difference is that the emission wavelength in the fluorescence detection conditions described in step (4) is changed from 350 nm to 320 nm. The detection results are as follows: Figure 6 As shown, the response of the TrpCA chromatographic peak at this wavelength will decrease by 70%.

[0119] 3. Compared with the second scheme in Example 1, the difference is that the flow rate in the high-performance liquid chromatography conditions described in step (4) is changed from 0.6 mL / min to 0.3 mL / min. The detection results are as follows: Figure 7 As shown, this indicates that at this flow rate, the elution time of the TrpCA chromatographic peak is delayed from 25.5 min to 31.5 min, and the specificity is good, with no impurities interfering near the TrpCA elution time.

[0120] 4. Compared with the second scheme in Example 1, the difference is that the column temperature in the high-performance liquid chromatography conditions described in step (4) is changed from 40℃ to 30℃. The detection results are as follows: Figure 8As shown, this indicates that the elution time of the TrpCA chromatographic peak at this column temperature is delayed from 25.5 min to 26.5 min, and the specificity is good, with no impurities interfering near the TrpCA elution time.

[0121] 5. Compared with the second scheme in Example 1, the difference is that the column temperature in the high-performance liquid chromatography conditions described in step (4) is changed from 40℃ to 45℃. The detection results are as follows: Figure 9 As shown, this indicates that the elution time of the TrpCA chromatographic peak at this column temperature will be advanced from 25.5 min to 24.8 min, with good specificity and no impurities interfering with the TrpCA elution time.

[0122] 6. Compared with the second scheme in Example 1, the difference is that the injection volume in the high-performance liquid chromatography conditions described in step (4) is changed from 50 μL to 20 μL. The detection results are as follows: Figure 10 As shown, the response of the TrpCA chromatographic peak decreases by 60% at this injection volume, but the specificity is good, and there is no impurity interference near the TrpCA elution time.

[0123] 7. Compared with the second scheme in Example 1, the difference is that the gain PMT in the high-performance liquid chromatography conditions described in step (4) is changed from 10 to 8. For example... Figure 11 As shown, the response of the TrpCA chromatographic peak decreases by 81% under this gain PMT, but 1 nmol / L TrpCA can still be accurately quantified.

[0124] Comparative Example 1:

[0125] Compared to the second scheme in Example 1, the difference lies in that the emission wavelength in the fluorescence detection conditions described in step (4) is changed from 350 nm to 310 nm. Figure 12 As shown, the response of the TrpCA chromatographic peak at this wavelength will decrease by 91%, and under this condition, it is impossible to determine the content of TrpCA synthesized by probiotics (3.9~140.43 nmol / L) disclosed in the prior art (patent numbers CN119215078A, CN119235873A).

[0126] Comparative Example 2:

[0127] Compared to the second scheme in Example 1, the difference lies in that the flow rate in the high-performance liquid chromatography conditions described in step (4) is changed from 0.6 mL / min to 0.2 mL / min. For example... Figure 13 As shown, at this flow rate, the elution time of the TrpCA chromatographic peak is delayed from 25.5 min to 36.5 min.

[0128] Comparative Example 3:

[0129] Compared to the second scheme in Example 1, the difference lies in that the column temperature in the high-performance liquid chromatography conditions described in step (4) is changed from 40°C to 55°C. Figure 14 As shown, impurities interfere with the elution time of TrpCA at this column temperature, affecting accurate quantification.

[0130] Comparative Example 4:

[0131] Compared to the second scheme in Example 1, the difference is that the injection volume in the high-performance liquid chromatography conditions described in step (4) is changed from 50 μL to 10 μL. Figure 10 As shown, the response of the TrpCA chromatographic peak will decrease by 80% under this injection volume. Under this condition, it is impossible to determine the content of TrpCA synthesized by probiotics (3.9~140.43 nmol / L) disclosed in the prior art (patent numbers CN119215078A, CN119235873A).

[0132] Comparative Example 5:

[0133] The difference between this and the second scheme in Example 1 is that the gain PMT in the high-performance liquid chromatography conditions described in step (4) is changed from 10 to 5. Figure 15 As shown, less than 1 nmol / L of TrpCA can be detected under this gain PMT. Under these conditions, the content of TrpCA synthesized by probiotics (3.9~140.43 nmol / L) disclosed in the prior art (patent numbers CN119215078A, CN119235873A) cannot be determined.

[0134] Test example:

[0135] 1. Sensitivity:

[0136] Sensitivity testing was performed based on the method of the second scheme in Example 1 of this application. The limit of detection (LOD) and limit of quantitation (LOQ) were determined by adding TrpCA to a blank matrix, and the concentrations corresponding to signal-to-noise ratios of 3 and 10 were used as the estimated LOD and LOQ, respectively. The LOD was confirmed by measuring the detection probability of the target analyte at that concentration level, and the LOQ was verified by adding the target analyte at a concentration corresponding to 10 times the signal-to-noise ratio to the blank matrix, performing 6 independent measurements within 3 days, and evaluating its recovery rate.

[0137] For the limit of detection (LOD), 25 parallel samples were prepared from a blank sample matrix. The target analyte at an estimated LOD concentration (0.6 nmol / L) was added to each sample. The detection probability of TrpCA (greater than 3 times the signal-to-noise ratio) was 100%, meeting the requirements of GB 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods of National Food Safety Standard" (not less than 95%), and was therefore designated as the LOD. For the limit of quantitation (LOQ), the target analyte at a LOQ concentration (2 nmol / L) was added to the blank matrix. The results of six independent determinations within 3 days are shown in Table 1. The recoveries of the LOQ spiked samples were 104.5%–107.5%, with a relative standard deviation of 1.1%, meeting the requirements of GB 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods of National Food Safety Standard" (mass fraction 1 μg / kg < p ≤ 10 μg / kg, recovery rate 60%–120%, relative standard deviation ≤ 20%).

[0138] Table 1: Validation results of the limit of quantitation

[0139]

[0140] The method of this application has a detection limit of 0.6 nmol / L and a quantitation limit of 2 nmol / L in the sample. The contents of TrpCA synthesized by various probiotics disclosed in the examples of patents CN119215078A and CN119235873A range from 3.9 to 140.43 nmol / L. Therefore, it can be further demonstrated that the sensitivity of the method of this application is superior to that of the prior art.

[0141] 2. Accuracy:

[0142] Accuracy tests were conducted based on the method of the second scheme in Example 1 of this application. Correctness and repeatability were evaluated by spiking, and the recovery rate and relative standard deviation of six parallel independent experiments within 3 days were calculated. As shown in Table 2, the recovery rate was 99.1%–107.5%, and the relative standard deviation was 0.8%–1.8%, both meeting the requirements of GB 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods in National Food Safety Standards".

[0143] Table 2: Validation results of accuracy and repeatability

[0144]

[0145] 3. Selectivity:

[0146] This method utilizes the characteristics of the indole ring, an endogenous fluorescent chromophore in the TrpCA molecule. This indole ring possesses characteristic excitation and emission wavelengths, enabling it to generate a specific fluorescent signal that can be directly captured by a fluorescence detector. As demonstrated by the detection results in Example 1, the method of this application effectively reduces sample matrix interference, and tryptophan, which also possesses an indole ring, can be completely separated from TrpCA in this method. Therefore, the method of this application exhibits high selectivity.

[0147] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0148] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for detecting tryptophan-conjugated bile acids, characterized by, The method comprises the following steps: S1, using methanol aqueous solution to pretreat the sample to be detected to obtain a detection solution; S2, analyzing the detection solution by high performance liquid chromatography-fluorescence detector to determine the content of tryptophan combined with cholic acid; Wherein, an impurity trapping column is added between the gradient mixer and the sample injector of the high performance liquid chromatography-fluorescence detector; The chromatographic conditions of the high performance liquid chromatography-fluorescence detector include: Mobile phase: the mobile phase A is selected from methanol, and the mobile phase B is selected from ammonium acetate solution; Gradient elution conditions: The initial proportion is that the mobile phase A is 40% and the mobile phase B is 60%, which is maintained for 4.0 min; within 4.0-24.0 min, the mobile phase A rises to 80% and the mobile phase B drops to 20%, which is maintained for 26.0 min; at 26.1 min, the mobile phase A rises to 90% and the mobile phase B drops to 10%, which is maintained for 28.0 min; within 28.0-30.0 min, the mobile phase A drops to 40% and the mobile phase B rises to 60%, which is maintained for 38.0 min; The fluorescence detection conditions of the high performance liquid chromatography-fluorescence detector are that the excitation wavelength is 280 nm and the emission wavelength is 320-380 nm.

2. The detection method according to claim 1, characterized in that, The inner diameter of the impurity trapping column is 4.6 mm, the column length is 50 mm, and the pressure resistance is 40 Mpa; The impurity trapping column is selected from the Ghost-Buster Column impurity trapping column.

3. The method of claim 1, wherein The gain PMT in the chromatographic conditions is 8-10.

4. The detection method according to claim 1, characterized in that, The mobile phase B is selected from the ammonium acetate solution with a concentration of 5-20 mmol / L and a pH value of 3.0-5.

0.

5. The detection method according to claim 1, characterized in that, The chromatographic column used in the chromatographic conditions is a C18 chromatographic column, the flow rate is 0.3-0.6 mL / min, the column temperature is 30-45℃, and the injection volume is 20-50 μL; The C18 chromatographic column has a column length of 150 mm, an inner diameter of 3.0 mm, a surface porous filler, and a filler particle size of 2.7 μm; The C18 chromatographic column is selected from Agilent, InfinityLab Poroshell 120 EC-C18.

6. The method of claim 1, wherein, In step S1, the pretreatment comprises: S1-1, mixing the sample to be measured with the methanol aqueous solution, wherein the sample to be measured is in liquid state, and the volume ratio of the sample to be measured to the methanol aqueous solution is 1: (1-3), or the sample to be measured is in solid state, and the volume ratio of the sample to be measured to the methanol aqueous solution is 1: (8-12); S1-2, centrifuging the mixed product to obtain the detection solution.

7. The detection method according to claim 6, characterized in that, The methanol aqueous solution is selected from a 40-60% methanol aqueous solution.

8. The detection method according to claim 6, characterized in that, The centrifuging is performed at a speed of 10,000-15,000 rpm and a temperature of 4℃ for 5-15 min.

9. The detection method according to claim 6, characterized in that, The mixing treatment is selected from vortex oscillation, and the vortex oscillation is performed for 5-15 min; or The mixing treatment is selected from ultrasonic treatment, and the ultrasonic treatment is performed for 30-60 min.

10. The method of claim 6, wherein, In step S1, the pretreatment further comprises: S1-3, the supernatant in step S1-2 is subjected to nitrogen blowing concentration treatment, and the temperature of the nitrogen blowing concentration is 30-40℃.

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