Method and kit for detecting cyclamate in baijiu

Abstract

The application discloses a detection method and kit for cyclamate in liquor, and belongs to the technical field of food safety detection. A hapten is obtained, and a cyclamate monoclonal antibody is prepared according to the hapten; further, an enzyme-linked immunosorbent kit is prepared through specific binding of the cyclamate monoclonal antibody and the cyclamate, so that the cyclamate in the liquor can be qualitatively and quantitatively detected. The detection method is rapid, efficient and high in sensitivity; in various types of liquor, the minimum detection limit of the cyclamate is as low as 2.75-2.82 mu g / L, the detection variation coefficient RSD is less than 5%, the detection result is accurate and sensitive, and the problems of complex sample pretreatment steps and high requirements for instrument equipment conditions in the prior art detection method are effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to a method and reagent kit for detecting cyclamate in baijiu (Chinese liquor). Background Technology

[0002] my country's baijiu (white liquor) culture has a long history, and its brewing process is one of my country's precious intangible cultural heritages. Baijiu is made primarily from grains through a process of steaming, saccharification, fermentation, distillation, aging, and blending using a sugar fermentation agent. High-quality baijiu is characterized by its sweet, mellow, and smooth aftertaste, which is related to the flavor compounds such as higher alcohols and polyols naturally produced during the brewing process. Because adding cyclamate to baijiu can make it fuller-bodied and sweeter, some breweries or merchants unauthorizedly add sweeteners to their baijiu, passing it off as high-quality. Furthermore, some merchants use edible alcohol or inferior raw liquor to blend the finished product, adding cyclamate to mask bitterness and other unpleasant tastes. This not only harms the interests of baijiu consumers but also negatively impacts the image of Chinese baijiu culture. Therefore, it is essential and of practical significance to conduct cyclamate detection work and develop an accurate and rapid method for determining cyclamate in baijiu.

[0003] Cyclamate, chemically known as sodium cyclohexylsulfamate, has the following structural formula: Cyclamate is a commonly used food additive, prized for its good taste, low price, and sweetness—30-40 times that of sucrose, yet costing only one-third the price. Since its introduction, the potential harm of cyclamate to human health has been a subject of long-standing debate, with some countries such as the United States, the United Kingdom, Japan, and Southeast Asia banning its use as an additive. In my country, while the use of cyclamate is still permitted, strict limits are imposed. my country's National Food Safety Standard GB2760-2014, "Standard for the Use of Food Additives," specifies the limits for cyclamate in different foods, ranging from 0.65-1.6 g / kg (calculated as cyclohexylsulfamic acid). These limits do not include baijiu (Chinese liquor), where cyclamate is strictly prohibited.

[0004] Currently, the main methods for detecting cyclamate in food both domestically and internationally include high-performance liquid chromatography (HPLC), gas chromatography (GC), spectrophotometry, thin-layer chromatography (TLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and ion chromatography. Each of these methods has its own applicable scope and advantages and disadvantages, suitable for different samples and detection needs. The inventors have discovered that the current national standard methods for detecting cyclamate in baijiu (Chinese liquor) suffer from problems such as a high likelihood of false positives and detection limits that do not meet the requirements. This affects public health and also impacts domestic product export trade. The inventors believe that current equipment and methods for detecting cyclamate are unsuitable for baijiu, as they misidentify cyclamate-like substances produced during baijiu fermentation and storage, leading to false positive results.

[0005] Furthermore, the inventors discovered that existing methods for detecting cyclamate in alcoholic beverages such as baijiu suffer from complex sample pretreatment steps and high requirements for instrumentation. For example, patent document CN 116908317 A discloses a method for detecting food additives in alcoholic beverages using high performance liquid chromatography-triple quadrupole mass spectrometry. The sample pretreatment method involves weighing 2.5g of the alcohol sample, placing it in a 50mL beaker, incubating it in a 60℃ water bath for 30min, washing the residue with the initial mobile phase, transferring the entire sample to a 25mL volumetric flask, making up to volume and shaking well, and filtering through a 0.22μm aqueous microporous membrane. Another example is patent document CN 113295794 A, which discloses a sample pretreatment method where the volume of the sample to be tested is reduced to 10-30% of its original volume using nitrogen blowing. All of these sample pretreatment methods suffer from numerous steps and long processing times, which the inventors believe is related to the low sensitivity of the corresponding detection methods. Regarding detection instruments, patent document CN106324150 A discloses the use of high-performance liquid chromatography with low-temperature evaporation light scattering to determine sweeteners in baijiu; CN 108613969 A discloses the use of Raman spectroscopy to detect cyclamate in baijiu; and CN108508128 A discloses the use of liquid chromatography-tandem mass spectrometry to detect cyclamate in baijiu. In summary, the methods disclosed in the prior art require that the detection work be carried out only in laboratories equipped with the relevant instruments, involving multiple steps, long detection times, and high costs. Currently, there are no reported immunoaffinity detection methods based on cyclamate artificial antigens. Therefore, researching rapid, efficient, and highly sensitive detection methods is crucial for the detection of cyclamate in baijiu. Summary of the Invention

[0006] Based on the above background, this invention provides a method for detecting cyclamate in baijiu (Chinese liquor). This method achieves qualitative and quantitative detection of cyclamate through the specific binding of a cyclamate monoclonal antibody. The method is rapid, efficient, and highly sensitive, with a detection limit as low as 0.75-0.82 μg / L for cyclamate in various types of baijiu. Furthermore, it exhibits good accuracy and precision. Because it utilizes the specific binding of antibody and antigen, it effectively avoids false positives caused by cyclamate-like substances produced during baijiu fermentation and storage, resulting in high detection accuracy. In a second aspect, this invention provides a rapid detection kit for cyclamate in baijiu. In a third aspect, this invention provides a hapten for preparing cyclamate monoclonal antibodies and a method for preparing the same. In a fourth aspect, this invention provides a cyclamate monoclonal antibody prepared based on the hapten.

[0007] This invention includes the following technical solutions:

[0008] In a first aspect, the present invention provides a method for detecting cyclamate in baijiu (Chinese liquor), characterized in that the method comprises: using an indirect competitive ELISA method, using a cyclamate-coated enzyme-labeled plate, adding the sample solution to be tested, and simultaneously adding cyclamate monoclonal antibody (primary antibody), incubating, washing and patting dry, adding enzyme-labeled goat anti-mouse antibody (secondary antibody), developing color with substrate, terminating the reaction, and measuring the absorbance value with an enzyme-labeled microplate reader.

[0009] Preferably, the detection method further includes preparing an ELISA standard curve, and substituting the percentage absorbance value of the sample to be tested into the ELISA standard curve to obtain the cyclamate concentration of the sample to be tested.

[0010] The method for preparing the ELISA standard curve includes: using a cyclamate-coated microplate, adding cyclamate standard solutions of varying concentrations, simultaneously adding cyclamate monoclonal antibody, incubating, washing and drying, adding enzyme-labeled goat anti-mouse antibody, performing substrate color development, terminating the reaction, and measuring the absorbance using a microplate reader; plotting the ELISA standard curve with standard concentration as the x-axis and percentage absorbance as the y-axis.

[0011] The formula for calculating the percentage absorbance value described in this invention is as follows:

[0012] Percent absorbance value = B / B0 × 100%

[0013] Wherein: B - average absorbance value of standard (or sample); B0 - average absorbance value of standard with a concentration of 0 ppb.

[0014] The cyclamate monoclonal antibody of the present invention highly specifically binds to cyclamate contained in a sample, and the cyclamate monoclonal antibody is obtained by the following preparation method:

[0015] (a1) An immunogen is prepared by conjugating the hapten shown in Formula I with a carrier protein;

[0016] (I)

[0017] (a2) Prepare cyclamate monoclonal antibody according to the immunogen described in step (a1);

[0018] The carrier protein is selected from any one or a combination of two or more of the following: thyroglobulin (BTG), bovine serum albumin (BSA), keyhole hemocyanin (KLH), ovalbumin (OVA), diphtheria toxoid, non-toxic mutants of diphtheria toxoid, tetanus toxoid, and proteins expressed by bacteria.

[0019] In a specific embodiment of the present invention, the carrier protein used to prepare the immunogen is bovine serum albumin (BSA).

[0020] The preparation of cyclamate monoclonal antibody based on the immunogen described in step (a2) is a routine operation in the art, and the present invention does not limit the specific preparation method of the monoclonal antibody. Those skilled in the art should understand that any substitution or modification of the carrier protein and / or preparation method or other technical features in the preparation of cyclamate monoclonal antibody from the hapten provided by the present invention is within the scope of the present invention.

[0021] In a specific embodiment of the present invention, the method for preparing cyclamate monoclonal antibodies based on immunogens includes the following steps:

[0022] (1) Immunization of mice: Immunogen was dissolved in physiological saline at 100 μg / mouse and mixed with an equal volume of Freund's adjuvant to immunize BALB / c mice. The first immunization used complete Freund's adjuvant. On days 7, 14 and 28 after the first immunization, the immunogen was mixed with an equal volume of incomplete Freund's adjuvant and immunized again. The last immunization was performed without Freund's adjuvant. The serum titer was detected by indirect ELISA.

[0023] (2) Cell fusion and cloning: The screened mouse spleen cells and mouse myeloma cells were fused and cultured. Positive cell wells were detected by indirect ELISA. Cell wells with strong positive results and high inhibition rate were further screened by indirect competitive ELISA. The positive cell wells with the best inhibition were subcloned multiple times by limiting dilution method. Finally, the hybridoma cell line 24D1 was obtained.

[0024] (3) Preparation of monoclonal antibodies: Hybridoma cells were injected into the peritoneum of BALB / c mice using an in vivo induction method, ascites fluid was collected, purified, and cyclamate monoclonal antibodies were obtained.

[0025] The cyclamate coating agent for coating enzyme-labeled plates described in this invention is prepared by coupling the hapten represented by Formula I with a carrier protein.

[0026] The carrier protein is selected from any one or a combination of two or more of the following: thyroglobulin (BTG), bovine serum albumin (BSA), keyhole hemocyanin (KLH), ovalbumin (OVA), diphtheria toxoid, non-toxic mutants of diphtheria toxoid, tetanus toxoid, and proteins expressed by bacteria.

[0027] In a specific embodiment of the present invention, the carrier protein used to prepare the coating antigen is ovalbumin (OVA).

[0028] The enzyme-labeled goat anti-mouse antibody described in this invention refers to a horseradish peroxidase (HRP)-labeled goat anti-mouse antibody.

[0029] Secondly, the present invention provides a rapid detection kit for cyclamate in baijiu (Chinese liquor), characterized in that the kit includes an antibody working solution, an enzyme-labeled plate, an enzyme-labeled working solution, and a cyclamate standard working solution.

[0030] The antibody working solution is obtained by diluting the cyclamate monoclonal antibody prepared in this invention by 5000-6000 times.

[0031] The enzyme-labeled plate was obtained by coating the original plate with cyclamate.

[0032] The enzyme-labeled working solution was obtained by diluting horseradish peroxidase (HRP)-labeled goat anti-mouse antibody.

[0033] The cyclamate standard working solution is a PBS solution with cyclamate concentrations of 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, and 8.1 ppb.

[0034] Furthermore, the kit also includes sample diluent, antibody diluent, washing solution, substrate chromogenic solution, and stop solution.

[0035] The sample diluent was a PBS buffer containing a light stabilizer, bovine serum albumin, and a surfactant, with a pH of 7.2, wherein the mass concentrations of the light stabilizer, bovine serum albumin, and surfactant were 0.2 g / L, 0.1 g / L, and 0.1 g / L, respectively.

[0036] In the kit provided by this invention, antibodies, including cyclamate monoclonal antibody and enzyme-labeled antibody, are diluted using an antibody diluent. The antibody diluent is a PBS buffer containing Proclin 300 and Triton X-100, wherein the mass concentrations of Proclin 300 and Triton X-100 are 200 μL / L and 500 μL / L, respectively.

[0037] The washing solution is a PBS buffer containing Tween 20 and Proclin 300, wherein the concentrations of Tween 20 and Proclin 300 are 20 mL / L and 300 μL / L, respectively.

[0038] The substrate colorimetric solution is an aqueous solution of 1.0 g / L urea peroxide, 5.0 g / L sodium acetate, 0.5 g / L light stabilizer, 2.5 mL / L phosphoric acid, and 5.0 g / L tetramethylbenzidine.

[0039] The terminating solution is a 0.05 mol / L aqueous solution of sulfuric acid.

[0040] In a specific embodiment of the present invention, the light stabilizer is selected from UV328, namely 2-(2'-hydroxy-3',5'-dipentylphenyl)benzotriazole, and the surfactant is selected from Tween-20.

[0041] The kit provided by this invention for detecting cyclamate operates on the following principle: An indirect competitive ELISA method is used, employing a coating antigen to coat the ELISA plate. A cyclamate monoclonal antibody is used as the first antibody, and the solid-phase antigen competes with the liquid-phase antigen in the sample for binding to the first antibody. A goat anti-mouse antibody labeled with horseradish peroxidase (HRP) is used as the second antibody. After the reaction, substrate solution is added for color development, and the reaction is terminated. The absorbance values ​​of the ELISA plate are read at dual wavelengths of 450 nm and 630 nm to obtain the cyclamate absorbance value of the sample. An ELISA standard curve is plotted, and the absorbance value is substituted into the standard curve to obtain the cyclamate concentration in the sample.

[0042] Thirdly, the present invention provides a hapten for preparing cyclamate whole antigen, characterized in that the hapten is a compound represented by Formula I:

[0043] (I).

[0044] Fourthly, the present invention provides a method for preparing a hapten represented by Formula I, characterized in that the preparation method comprises the following steps:

[0045] (S1) Dissolve 4-aminocyclohexanecarboxylic acid in acetonitrile solution, add chlorosulfonic acid dropwise at a molar mass ratio of 1:1 under ice bath, react for two hours, after the reaction is completed, carefully quench with ice water dropwise, and then extract with dichloromethane several times to obtain 4-(sulfonamide)cyclohexane-1-carboxylic acid.

[0046] (S2) 4-(sulfonamide)cyclohexane-1-carboxylic acid was dissolved in DMF solution, activated with NHS and EDC, and methyl 4-aminobutyrate was added at a molar ratio of 1:1. The reaction was allowed to proceed overnight to obtain the compound shown in Formula II, with the structure shown below. (II);

[0047] (S3) Dissolve the compound shown in Formula II in MeOH solution, add sodium hydroxide according to Formula II and sodium hydroxide aqueous solution at a molar mass ratio of 1:5, react overnight, and then adjust the pH of the solution to 3 with dilute HCl to obtain the compound shown in Formula I.

[0048] Fifthly, the present invention provides a cyclamate monoclonal antibody, characterized in that the cyclamate monoclonal antibody is prepared by a monoclonal antibody preparation technique from an immunogen obtained by conjugating a hapten of Formula I with a carrier protein.

[0049] (I)

[0050] The carrier protein is selected from any one or a combination of two or more of the following: thyroglobulin (BTG), bovine serum albumin (BSA), keyhole hemocyanin (KLH), ovalbumin (OVA), diphtheria toxoid, non-toxic mutants of diphtheria toxoid, tetanus toxoid, and proteins expressed by bacteria.

[0051] Fifthly, the present invention provides a rapid test kit as described in the second aspect of the present invention, a hapten as described in the third aspect of the present invention, and the cyclamate monoclonal antibody as described in the fifth aspect of the present invention, in at least one of the following applications:

[0052] (b1) Application in the detection of cyclamate in baijiu;

[0053] (b2) Application in the preparation of products for detecting cyclamate in baijiu.

[0054] The products include, but are not limited to, test strips, membrane strips, reagent kits, chips, models, or detection platforms.

[0055] The method and kit for detecting cyclamate in baijiu provided by this invention have the following beneficial technical effects:

[0056] In GB5009.97-2023, the standard for the determination of cyclohexylsulfamic acid in food, the detection limit for gas chromatography is 0.01 g / kg, the detection limit for high-performance liquid chromatography (HPLC) is 0.01 g / kg, and the detection limit for HPLC is 0.03 mg / kg. Patent CN 106324150 A discloses a detection limit of 0.6 mg / kg for cyclamate in baijiu using HPLC low-temperature evaporation light scattering, while patent CN 108613969 A discloses a detection limit of 0.5 mg / L for cyclamate in baijiu using Raman spectroscopy. Patent CN117147512A discloses a rapid detection method for cyclamate using a photoprobe method, achieving a detection limit of 0.95 μmol / L. However, these methods do not meet the detection limit standards. The literature (Research on the method for simultaneous determination of six trace sweeteners in baijiu by ultra-high pressure liquid chromatography-mass spectrometry, *Journal of Food and Biotechnology*, Wu Shijia, Vol. 29, No. 5, p. 670) uses liquid chromatography-mass spectrometry (LC-MS) and achieves a detection limit of 0.01 mg / L for cyclamate. The literature (Research on the method for simultaneous determination of four sweeteners in baijiu by liquid chromatography-tandem mass spectrometry, Xia Yulin, *China Brewing*, 2011, No. 3, p. 156) describes a detection limit of 2 μg / L for cyclamate in baijiu. The method provided by this invention for the detection of cyclamate in baijiu has a detection limit of 2.75-2.82 μg / L, a quantitation limit of 5.05-5.29 μg / L, and method recoveries between 98-105%, with coefficients of variation (RSD) of less than 5% for each added concentration.

[0057] This invention's detection method does not require large-scale chromatographic or mass spectrometric instruments. It uses a low-cost, small-sized, and portable detection kit to achieve the detection standards of liquid chromatography-mass spectrometry (LC-MS). It improves the specificity, sensitivity, and detection speed of cyclamate in baijiu (Chinese liquor), while reducing detection costs and operational difficulty. This makes the detection method for cyclamate in baijiu faster and more accurate, facilitating food safety monitoring and supervision, and effectively combating such illegal additives. Attached Figure Description

[0058] Figure 1 The standard curve for cyclamate ELISA. Detailed Implementation

[0059] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] I. Preparation of cyclamate hapten and whole antigen

[0061] (1) Preparation and structural identification of cyclamate hapten

[0062] The reaction route diagram is as follows:

[0063]

[0064]

[0065] ;

[0066] S1: Dissolve 4-aminocyclohexanecarboxylic acid in acetonitrile solution, add chlorosulfonic acid dropwise at a molar mass ratio of 1:1-2 under ice bath conditions, react for two hours, quench the reaction dropwise with ice water after the reaction is complete, and then extract with dichloromethane several times to obtain compound 1, namely 4-(sulfonamide)cyclohexane-1-carboxylic acid;

[0067] S2: 4-(sulfonamide)cyclohexane-1-carboxylic acid (compound 1) was dissolved in DMF solution, activated with NHS and EDC, and 4-(sulfonamide)cyclohexane-1-carboxylic acid (compound 1) and methyl 4-aminobutyrate (compound 2) were added at a molar mass ratio of 1:1. The reaction was carried out overnight to obtain the compound shown in Formula II.

[0068] S3: Dissolve the compound shown in Formula II in MeOH solution, add sodium hydroxide according to Formula II and sodium hydroxide at a molar ratio of 1:5, react overnight, and then adjust the pH of the solution to 3 with dilute HCl to obtain the compound shown in Formula I.

[0069] The hydrogen nuclear magnetic resonance data of the compound represented by Formula I are as follows: 1 H NMR (500 MHz, Chloroform ) δ4.17 (dt, J = 16.1, 11.8 Hz, 2H), 3.43 (t, J = 15.0 Hz, 2H), 3.29 (t, J = 15.2Hz, 1H), 2.76 (dt, J = 16.1, 11.8 Hz, 2H), 2.55 – 2.37 (m, 1H), 2.31 (t, J =10.8 Hz, 2H), 2.17 – 1.59 (m, 4H), 1.25 (dt, J = 15.2, 11.8 Hz, 2H).

[0070] (2) Preparation of cyclamate whole antigen

[0071] 2.1 Synthesis of Immunogens

[0072] S1: Dissolve 8.25 mg of cyclamate hapten in 1.5 mL of DMF, stir at 200 rpm for 10 min, add 12.7 mg of EDC to dissolve, then add 7.4 mg of NHS, and activate at room temperature for 2-3 h;

[0073] S2: Weigh 50 mg BSA and dissolve it in 3.5 mL of 0.1 M sodium bicarbonate solution. Stir at 200 rpm for 10 min to ensure complete dissolution. Cool the solution in an ice bath at 0-4 ℃. Add the reaction solution from step (1) dropwise (1 mL / min) while stirring at 1000 rpm. Stir at 500 rpm for 24 h.

[0074] S3: Place the reaction product into a dialysis bag rinsed with distilled water, add 1 L of 0.01 M PBS (1×, pH 7.2) and stir at 4 ℃ (100 rpm) for 3 days, changing the medium 3 times a day. Centrifuge the dialysis product at 5000 rpm for 6 min, aliquot, and store at -20 ℃ for later use.

[0075] 2.2 Synthesis of Coated Origin

[0076] S1: Dissolve 8.25 mg of cyclamate hapten in 1.5 mL of DMF, stir at 200 rpm for 10 min, add 12.7 mg of EDC to dissolve, then add 7.4 mg of NHS, and activate at room temperature for 2-3 h;

[0077] S2: Weigh 33.6 mg of OVA and dissolve it in 3.5 mL of 0.1 M sodium bicarbonate solution. Stir at 200 rpm for 10 min to ensure complete dissolution. Cool the solution in an ice bath at 0-4 ℃. Add the reaction solution from step 1 dropwise (1 mL / min) while stirring at 1000 rpm. Stir at 500 rpm for 24 h.

[0078] S3: Place the reaction product into a dialysis bag rinsed with distilled water, add 1 L of 0.01 M PBS (1×, pH 7.2) and stir at 4 ℃ (100 rpm) for 3 days, changing the medium 3 times a day. Centrifuge the dialysis product at 5000 rpm for 6 min, aliquot, and store at -20 ℃ for later use.

[0079] II. Preparation of Monoclonal Antibodies by Immunizing Animals with Cyclamate Immunogen

[0080] The specific steps for preparing cyclamate monoclonal antibodies using conventional methods are as follows:

[0081] 1. Animal immunization

[0082] Prepare an immunogen at 100 μg / mouse, dissolve the immunogen in physiological saline and mix it with an equal volume of Freund's complete adjuvant, and subcutaneously inject it into the neck and back of 6-8 week old Balb / c female mice. On days 7, 14 and 28 after the initial immunization, administer an additional immunization with an equal volume of immunogen and Freund's incomplete adjuvant. Three days before fusion, administer an additional immunization with 100 μg / mouse of immune complex without Freund's adjuvant.

[0083] 2. Cell fusion and cloning

[0084] Following standard procedures, spleen cells from immunized mice were mixed with myeloma cells (SP2 / 0) in the logarithmic growth phase. Preheated fusion agent (PEG4000) was then slowly added over 30 seconds for fusion. The cells were then suspended in HAT medium and homogenized. An appropriate amount of feeder cells was added, and the mixture was cultured in 96-well plates at 37°C in a 5% CO2 incubator. After 7 days, the medium was partially replaced with HT medium, and after 9 days, the medium was completely replaced.

[0085] After cell fusion, hybridoma cells were screened using a stepwise selection method after 7-10 days of cell growth. Initial selection was performed using indirect ELISA, screening 834 positive wells. These positive wells were then further screened using indirect competitive ELISA, with wells exhibiting strong positivity and high inhibition rates selected for subclonal selection. After multiple subclonal selections, the positive rate reached 100%, ultimately yielding multiple hybridoma cell lines capable of secreting cyclamate monoclonal antibodies. Among these, cell line 24D1, with the highest antibody titer and best inhibition, was identified. 50 It was 0.56 ng / mL.

[0086] Cyclamate monoclonal antibodies were prepared using an in vivo ascites in mouse method. 8-10 week old Balb / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin per mouse, followed by intraperitoneal injection of 1-2 × 10⁻⁶ hybridoma cells 7-10 days later. 5 Ascites fluid was extracted from each mouse 7-10 days later. The fluid was centrifuged at 5000 rpm for 10 min at 4°C to remove the upper oil and lower precipitate. The acesulfame potassium monoclonal antibody was then purified using the caprylic acid-saturated ammonium sulfate method and stored at -20°C.

[0087] III. Preparation of an enzyme-linked immunosorbent assay kit for cyclamate in baijiu (Chinese liquor)

[0088] The enzyme-linked immunosorbent assay (ELISA) kit for cyclamate in baijiu includes: antibody working solution, ELISA plate, enzyme-labeled working solution, working solution containing cyclamate standards of different concentrations, sample diluent, antibody diluent, washing solution, substrate chromogenic solution, and stop solution.

[0089] (1) Preparation of ELISA plates

[0090] Cyclamate microplate: Dilute the obtained cyclamate coating agent to 10.0 μg / mL, add 100 μL of coating agent to each well, incubate at 37℃ for 2 h, discard the liquid, wash twice with washing buffer diluted 20 times, 30 seconds each time, pat dry, then add 150 μL of blocking buffer to each well, incubate at 37℃ for 1 h, discard the liquid in the well, dry to obtain cyclamate microplate, and store in an aluminum foil vacuum seal.

[0091] (2) Preparation of antibody working solution

[0092] The cyclamate monoclonal antibody was diluted 6000 times to obtain the cyclamate antibody working solution.

[0093] (3) Enzyme-labeled working solution

[0094] Horseradish peroxidase (HRP)-labeled goat anti-mouse antibody.

[0095] (4) Preparation of standard working solution

[0096] The concentrations of the working solutions of cyclamate standard were 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, and 8.1 ppb.

[0097] (5) Preparation of other reagents

[0098] The sample diluent was a PBS buffer containing the light stabilizer UV328, bovine serum albumin, and surfactant Tween 20, with a pH of 7.2. The mass concentrations of the light stabilizer, bovine serum albumin, and surfactant were 0.2 g / L, 0.1 g / L, and 0.1 g / L, respectively.

[0099] The antibody diluent is a PBS buffer containing Proclin 300 and Triton X-100, wherein the mass concentrations of Proclin 300 and Triton X-100 are 200 μL / L and 500 μL / L, respectively.

[0100] The washing solution is a PBS buffer containing Tween 20 and Proclin 300, wherein the concentrations of Tween 20 and Proclin 300 are 20 mL / L and 300 μL / L, respectively.

[0101] The substrate colorimetric solution is an aqueous solution of 1.0 g / L urea peroxide, 5.0 g / L sodium acetate, 0.5 g / L light stabilizer UV328, 2.5 mL / L phosphoric acid, and 5.0 g / L tetramethylbenzidine.

[0102] The terminating solution is a 0.05 mol / L aqueous solution of sulfuric acid.

[0103] IV. Instructions for using the cyclamate enzyme-linked immunosorbent assay kit

[0104] (I) Detection methods using reagent kits

[0105] S1: Insert the strips into the microplate holder and record the positions of each standard and sample. Perform parallel double-well printing. Seal unused strips in a self-sealing bag and store them immediately at 4°C.

[0106] S2: Add 50 μL of each standard working solution / sample solution to the corresponding standard / sample well;

[0107] S3: Add 50 μL of antibody working solution to each well;

[0108] S4: Gently shake the microplate for 10 seconds to mix thoroughly, and react at room temperature (25±2℃) in the dark for 30 minutes.

[0109] S5: Discard the liquid in the wells, add 260 μL of washing working solution to each well, wash thoroughly 4 times, soaking for 15-30 seconds each time; discard the liquid in the wells and pat the microplate dry.

[0110] S6: Add 50 μL of enzyme-labeled working solution to each well, and repeat steps S4-S5;

[0111] S7: Add 100 μL of substrate solution to each well;

[0112] S8: Gently shake the microplate for 10 seconds to mix thoroughly, and react at room temperature (25±2℃) in the dark for 15 minutes;

[0113] S9: Add 50 μL of stop solution to each well, gently shake the microplate for 10 s to mix thoroughly;

[0114] S10: Within 5 minutes after termination, read the absorbance value of the ELISA plate using a microplate reader at dual wavelengths of 450 nm and 630 nm.

[0115] (II) Analysis of test results

[0116] 2.1 Calculate the percentage absorbance value

[0117] The percentage of absorbance of each standard (or sample to be tested) can be obtained by dividing the average absorbance value of each standard (or sample to be tested) by the absorbance value of the zero standard (standard with a concentration of 0 μg / L) and multiplying by 100%.

[0118] Absorbance percentage = B / B0 × 100%

[0119] Wherein: B - average absorbance value of standard (or sample); B0 - average absorbance value of standard with a concentration of 0 ppb.

[0120] 2.2 Creating a standard curve

[0121] A standard curve was plotted with the percentage absorbance of each standard on the ordinate and the concentration of cyclamate (μg / L) in the working solution of each standard on the x-axis. Figure 1 As shown.

[0122] 2.3 Calculate the cyclamate content in the sample.

[0123] By substituting the percentage absorbance value of the sample into the standard curve, the residual concentration of the sample can be obtained. Multiplying this by the dilution factor of the sample gives the actual content of cyclamate in the original sample.

[0124] (III) Specificity, accuracy, and precision testing of the cyclamate enzyme-linked immunosorbent assay kit

[0125] 1. Specificity test of the kit:

[0126] The specificity of the cyclamate enzyme-linked immunosorbent assay kit is determined by cross-reactivity tests with the corresponding substance.

[0127] Casserole was serially diluted with sodium saccharin and acesulfame potassium, and the detection methods of each reagent kit were followed to construct standard curves. The 50% inhibitory concentration (IC50) of each reagent was then determined from the curves.50 The specific method is as follows: Obtain the concentration of cyclamate (μg / L) corresponding to a vertical axis value of 50%, i.e., IC50. 50 Value. Calculate the cross-reactivity of the kit with cyclamate, sodium saccharin, and acesulfame potassium using the following formula:

[0128] Cross-reactivity rate (%) = (concentration of cyclamate causing 50% inhibition / concentration of cross-reactant causing 50% inhibition) × 100%. The results are shown in Table 1.

[0129] Table 1. Specificity of the kit

[0130] .

[0131] Experiments show that the kit of the present invention has good specificity for cyclamate and can be used for the detection of cyclamate.

[0132] 2. Determination of the lowest detection limit

[0133] Blank samples of soy sauce aroma, strong aroma, and light aroma baijiu were taken and tested according to the reagent kit method. The measured values ​​were obtained based on the standard curve, and the average value was calculated. The limit of detection (LOD) was then calculated by adding three times the standard deviation. The results are shown in Table 2.

[0134] Table 2. Statistical table of detection limit and quantitation limit results (μg / L)

[0135] .

[0136] The results showed that the limit of detection (LOD) for cyclamate in Maotai-flavor baijiu was 2.86 μg / L and the limit of quantification (LOQ) was 5.29 μg / L; the LOD for cyclamate in strong-flavor baijiu was 2.82 μg / L and the LOQ was 5.12 μg / L; and the LOD for cyclamate in light-flavor baijiu was 2.75 μg / L and the LOQ was 5.05 μg / L.

[0137] 3. Accuracy and precision tests of the reagent kit

[0138] Blank samples of soy sauce aroma type, strong aroma type, and light aroma type baijiu were pretreated and then cyclamate was added to obtain test sample solutions. The concentrations of cyclamate in the samples were low concentration group (5 μg / L), medium concentration group (30.0 μg / L), and high concentration group (100.0 μg / L). Each batch of samples was tested in parallel 5 times, and the average value was taken.

[0139] Table 3 Accuracy and Precision

[0140] .

[0141] The results showed that the recoveries of all samples at various spiking concentrations were between 98% and 105%. The coefficients of variation (RSD) for each spiking concentration were all below 5%. This indicates that the method of the present invention has good accuracy and precision, and can effectively and accurately detect trace amounts of cyclamate in baijiu (Chinese liquor).

Claims

1. A method for detecting cyclamate in baijiu (Chinese liquor), characterized in that, The method includes: using an indirect competitive ELISA method, a cyclamate-coated enzyme-linked immunosorbent assay (ELISA) plate is coated with a cyclamate-coated plate, the sample solution to be tested is added, and cyclamate monoclonal antibody is added simultaneously. The plate is incubated, washed, and dried. Enzyme-labeled goat anti-mouse antibody is added, followed by substrate color development and termination. The absorbance is measured using an ELISA reader. The cyclamate-coated plate is prepared by conjugating the hapten shown in Formula I with ovalbumin. The cyclamate monoclonal antibody is obtained by the following preparation method: (a1) An immunogen was prepared by conjugating the hapten shown in Formula I with bovine serum albumin; (a2) Prepare cyclamate monoclonal antibody according to the immunogen described in step (a1); （I）； The detection method has a detection limit of 2.75-2.82 μg / L for cyclamate in baijiu.

2. The detection method according to claim 1, characterized in that, The detection method also includes preparing an ELISA standard curve, and substituting the percentage absorbance value of the sample to be tested into the ELISA standard curve to obtain the cyclamate concentration of the sample to be tested; The method for preparing the ELISA standard curve includes: using a cyclamate-coated microplate, adding cyclamate standard solutions of varying concentrations, simultaneously adding cyclamate monoclonal antibody, incubating, washing and drying, adding enzyme-labeled goat anti-mouse antibody, performing substrate color development, terminating the reaction, and measuring the absorbance using a microplate reader; plotting the ELISA standard curve with standard concentration as the x-axis and percentage absorbance as the y-axis.

3. A rapid detection kit for cyclamate in baijiu (Chinese liquor), characterized in that, The kit includes an antibody working solution, an enzyme-labeled plate, an enzyme-labeled working solution, and a cyclamate standard working solution; the antibody working solution is obtained by diluting the cyclamate monoclonal antibody prepared according to claim 1 by 5000-6000 times.

4. The reagent kit according to claim 3, characterized in that, The ELISA plate is obtained by coating the cyclamate-coated substrate prepared according to claim 1; the enzyme-labeled working solution is obtained by diluting horseradish peroxidase-labeled goat anti-mouse antibody; the cyclamate standard working solution is a PBS solution with cyclamate concentrations of 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, and 8.1 ppb.

5. The reagent kit according to claim 3, characterized in that, The kit also includes sample diluent, antibody diluent, washing solution, substrate chromogenic solution, and stop solution.

6. The use of the kit according to any one of claims 3-5 in at least one of the following: (b1) Application in the detection of cyclamate in baijiu; (b2) Application in the preparation of products for detecting cyclamate in baijiu; The products include test strips, membrane strips, reagent kits, chips, models, or detection platforms.

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