Method for rapidly identifying harmful components in pickled peppers

Abstract

The invention discloses a method for rapidly identifying harmful components in pickled peppers, and belongs to the technical field of food detection. The method comprises the following steps: preparing a reference substance solution, namely preparing a standard substance stock solution and a biogenic amine mixed solution from putrescine, histamine, tyramine and cadaverine standard substances; preparing a test sample solution: preparing the test sample solution by adopting a test sample; the chromatographic conditions are as follows: C18 is used as a filling agent; carrying out gradient elution by taking isopropanol as a mobile phase A and 0.5-2wt% ammonia water as a mobile phase B; the detection wavelength is 254 nm; the column temperature is 25-35 DEG C; and the flow velocity is 0.2-1.0 ml / min. The method has the advantages that (1) the detection speed is high, and the gradient elution speed is high and is only 12 minutes; (2) the number of obvious miscellaneous peaks is small, and only one miscellaneous peak exists; and (3) the method has relatively high precision, relatively low detection limit and good reproducibility.

Description

Technical Field

[0001] The invention relates to the technical field of food detection, and in particular to a method for rapidly identifying harmful components in pickled peppers. Background Art

[0002] Paola is very popular among consumers for its unique flavor, fresh taste, and the effect of increasing appetite and aiding digestion. The lactic acid and lactic acid bacteria contained in it are very beneficial to human health. They can inhibit the growth of putrefactive bacteria in the intestines and weaken the toxic production of putrefactive bacteria in the intestines, lower cholesterol and regulate human physiological functions.

[0003] There are three main principles for making pickled peppers: first, use the high osmotic pressure and strong dehydrating effect of salt to achieve the effects of preservation, dehydration and crispness; second, use the fermentation of microorganisms, mainly lactic acid fermentation, alcohol fermentation and acetic acid fermentation, to convert the sugar substances in fresh chili peppers into lactic acid and a small amount of alcohol and acetic acid; third, use the changes in amino acids produced by the decomposition of protein to increase the color, aroma and taste of the chili peppers, and use spices and seasonings to flavor it.

[0004] The traditional pickled pepper pickling process is to select, cut and clean fresh peppers, put them into a container, add seasoning solution and vegetables such as ginger and garlic (optional), and pickle them. The seasoning solution contains salt, bacteria and additives (including calcium chloride, acetic acid, sodium isovitamin C, sodium metabisulfite), etc.

[0005] The fermentation of pickled peppers is a complex biochemical reaction, which involves changes in the peppers themselves, changes in other vegetables themselves, changes in the flora themselves, interactions between peppers and other vegetables, interactions between flora and peppers, interactions between peppers and additives, interactions between other vegetables and flora, interactions between other vegetables and additives, interactions between additives and flora, etc. Pickled peppers contain complex chemical components, such as nitrates, which are well known to the public. In recent years, due to the emphasis on food safety, it is necessary to analyze the ingredients of pickled peppers.

[0006] Biogenic amine (BA) is a general term for a class of low-molecular-weight organic compounds containing nitrogen and having biological activity. It can be regarded as a substance generated by replacing 1-3 hydrogen atoms in the ammonia molecule with alkyl or aryl groups. It is a low-molecular-weight organic base of aliphatic, ester ring or heterocyclic groups, and is often found in animals, plants and food. Most foods contain biogenic amines, which are mainly generated by the decarboxylation of amino acids by microbial amino acid decarboxylase. Appropriate amounts of biogenic amines can promote normal physiological activities of the human body, while excessive intake will produce adverse reactions.

[0007] According to their structures, biogenic amines can be divided into three parts: aliphatic amines such as putrescine, cadaverin, spermine, and spermidine; aromatic amines such as tyramine and phenylethylamine; and heterocyclic amines such as histamine and tryptamine. A moderate amount of biogenic amines is beneficial to human health, but an excessive amount of biogenic amines can cause human poisoning, leading to serious consequences, such as headaches, changes in blood pressure, respiratory disorders, palpitations, and vomiting. Histamine is the most toxic of the biogenic amines. Excessive histamine can cause headaches, digestive disorders, abnormal blood pressure, and even neurotoxicity. Tyramine is second in toxicity, and excessive amounts can also cause headaches and high blood pressure. Cadaverine and putrescine have low toxicity themselves, but they can inhibit the activity of histamine and tyramine-related metabolic enzymes, increase the amount of histamine and tyramine, and thus enhance the discomfort symptoms of the human body. In addition, putrescine, cadaverine, etc. can react with nitrite to produce carcinogenic nitrosamines.

[0008] Therefore, it is very necessary to detect the content of putrescine, histamine, tyramine and cadaverine in pickled peppers. In the prior art, all biogenic amines are usually detected. For example, the patent with application number CN201610447198.7 discloses a method for rapid detection of 7 biogenic amines in liquor by ultra-high performance convergence chromatography in series with QDa. For example, the patent with application number CN201410120955.0 discloses a method for determining the content of biogenic amines in food by ultra-high performance convergence chromatography. For example, the patent with application number CN 201010111733.4 discloses a method for detecting biogenic amines in bean paste. For example, the patent with application number CN201811372977.0 discloses a method for detecting biogenic amines in Pueraria root. For example, the patent with application number CN202211688102.8 discloses a method for detecting biogenic amines in fermented soy products. Summary of the invention

[0009] The present invention provides a method for quickly identifying harmful components in pickled peppers, which can quickly identify harmful bioamines in pickled peppers with few interfering peaks. The scheme is as follows: The embodiment of the present invention provides a method for quickly identifying harmful components in pickled peppers, the method comprising: Preparation of reference solution: Putrescine, histamine, tyramine and cadaverine standards were prepared into standard stock solutions and biogenic amine mixtures.

[0010] The preparation process of the reference solution is as follows: add the reference substance to 0.1 mol / L hydrochloric acid to prepare single standard and mixed standard solutions of different concentrations, add 10 μL benzoyl chloride and 1 mL 2 mol / L sodium hydroxide solution to 2 mL of the above solution, mix well, and react at 30 ° C in the dark for 40 minutes. After the reaction is completed, add 2 mL of 5 wt% sodium chloride solution and 3 mL of anhydrous ether for extraction and centrifugation. Separate the ether layer, extract the residue again according to the above process, combine the two ether layers, purge with nitrogen, dry at 35 ° C, and finally dissolve in isopropanol.

[0011] Preparation of test sample solution: Prepare the test sample solution using the test sample.

[0012] The preparation process of the test solution is as follows: mince the test sample, take 10 g, add 20 mL of 0.1 mol / L hydrochloric acid, homogenize for 10-20 minutes, let stand for 5-10 minutes, centrifuge at high speed, and take the supernatant; add 20 mL of 0.1 mol / L hydrochloric acid again, repeat the above process once; combine the two supernatants; the supernatant is diluted to 50 mL with 0.1 mol / L hydrochloric acid, take 2 mL of the above solution, add 10 μL of benzoyl chloride and 1 mL of 2 mol / L sodium hydroxide solution, mix well, and react at 30°C in the dark for 40 minutes; after the reaction is completed, add 2 mL of 5 wt% sodium chloride solution and 3 mL of anhydrous ether for extraction, and centrifuge; separate the ether layer, extract the residue again according to the above process, combine the two ether layers, purge with nitrogen, dry at 35°C, and finally dissolve with isopropanol.

[0013] Chromatographic conditions: C18 as filler; isopropanol as mobile phase A, 0.5-2wt% ammonia water as mobile phase B, gradient elution; the gradient elution procedure is: 0-3min, the volume ratio of mobile phase A:mobile phase B is 50-60%:40-50%; 3-5min, the volume ratio of mobile phase A:mobile phase B is 15-20%:80-85%; 5-7min, the volume ratio of mobile phase A: mobile phase B is 5-10%:90-95%; 7-8min, the volume ratio of mobile phase A:mobile phase B is 40-48%:52-60%; 8-12min, the volume ratio of mobile phase A: mobile phase B is 25-30%: 70-75%; The detection wavelength is 254nm; the column temperature is 25-35℃; the flow rate is 0.2-1.0ml / min.

[0014] Preferably, the procedure of gradient elution is: using isopropanol as mobile phase A and 1 wt % ammonia water as mobile phase B for gradient elution; the procedure of gradient elution is: 0-3min, the volume ratio of mobile phase A:mobile phase B was 55%:45%; 3-5min, the volume ratio of mobile phase A:mobile phase B is 20%:80%; 5-7min, the volume ratio of mobile phase A:mobile phase B is 8%:92%; 7-8min, the volume ratio of mobile phase A:mobile phase B is 45%:55%; 8-12min, the volume ratio of mobile phase A:mobile phase B is 25%:75%; The detection wavelength was 254 nm; the column temperature was 30°C; the flow rate was 0.5 ml / min.

[0015] Among them, for the test solution, taking tyramine as the reference peak, the relative retention times of the four characteristic peaks (usually 2-5 detection peaks are detected, hiding the peak of the mobile phase) are as follows: Tyramine: relative retention time is 1; Putrescine: relative retention time is 2.968-2.984; Cadaverine: relative retention time is 3.386-3.393; Histamine: relative retention time is 4.180-4.187.

[0016] The applicant found that tyramine was always present in all samples (the applicant tested 68 samples from 7 provinces). The first characteristic peak was used as the reference peak, and the number of reference peaks was at most 5 and at least 2. There was only one interfering peak (other biogenic amines), which was very intuitive.

[0017] Figure 1 Among them, peak 1 is tyramine, peak 2 is putrescine, peak 3 is cadaverine, peak 4 is a miscellaneous peak, and peak 5 is histamine.

[0018] Figure 2 Among them, peak 1 is tyramine, peak 2 is putrescine, peak 3 is a miscellaneous peak, and peak 4 is histamine.

[0019] Advantages of this patent: (1) Fast detection speed and gradient elution speed, only 12 minutes.

[0020] (2) There are few obvious impurity peaks, only one.

[0021] (3) It has high precision, low detection limit and good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1is the liquid chromatogram of sample 6; Figure 2 This is the liquid chromatogram of sample 4. DETAILED DESCRIPTION

[0023] The protection scope of the present invention is not limited to the following embodiments. Those skilled in the art may adjust one or several steps. These changes that do not deviate from the essential content of the present invention are all within the protection scope of this application.

[0024] Embodiment 1: Take 7 samples and conduct three parallel experiments on each sample. The samples are as follows: Table 1

[0025] The chromatographic column used was: C18 (2.1 mm × 100 mm, 1.7 μm), with isopropanol as mobile phase A and 1 wt% ammonia water as mobile phase B, for gradient elution; the gradient elution procedure was: 0-3min, the volume ratio of mobile phase A:mobile phase B was 55%:45%; 3-5min, the volume ratio of mobile phase A:mobile phase B is 20%:80%; 5-7min, the volume ratio of mobile phase A:mobile phase B is 8%:92%; 7-8min, the volume ratio of mobile phase A:mobile phase B is 45%:55%; 8-11min, the volume ratio of mobile phase A:mobile phase B is 25%:75%.

[0026] The preparation process of the reference solution is as follows: The reference substances were putrescine, histamine, tyramine and cadaverine standards.

[0027] The reference substance was added to 0.1 mol / L hydrochloric acid to prepare single standards of 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 5.0 μg / L, 10.0 μg / L and 20.0 μg / L. 2 mL of the above solution was added with 10 μL of benzoyl chloride and 1 mL of 2 mol / L sodium hydroxide solution. After mixing, the mixture was reacted at 30°C in the dark for 40 minutes. After the reaction was completed, 2 mL of 5 wt% sodium chloride solution and 3 mL of anhydrous ether were added for extraction, and the mixture was centrifuged at 1500 r / min for 12 minutes. The ether layer was separated, and the residue was extracted once more according to the above process. The two ether layers were combined, purged with nitrogen, dried at 35°C, and finally dissolved with isopropanol and filtered through a 0.22 μm membrane.

[0028] The single standard solutions were mixed and made up to volume to obtain a mixed standard solution.

[0029] The preparation process of the test solution is as follows: The test samples are shown in Table 1.

[0030] The sample was minced, 10 g was taken, 20 mL of 0.1 mol / L hydrochloric acid was added, homogenized for 15 minutes, allowed to stand for 7 minutes, and then centrifuged at 80000 r / min for 20 minutes to take the supernatant. 20 mL of 0.1 mol / L hydrochloric acid was added to the precipitate again, and the above process was repeated once; the two supernatants were combined; the supernatant was made up to 50 mL with 0.1 mol / L hydrochloric acid, 2 mL of the above solution was taken, 10 μL of benzoyl chloride and 1 mL of 2 mol / L sodium hydroxide solution were added, mixed, and reacted at 30 ° C in the dark for 40 minutes; after the reaction was completed, 2 mL of 5 wt% sodium chloride solution and 3 mL of anhydrous ether were added for extraction, and centrifuged at 1500 r / min for 12 minutes; the ether layer was separated, and the residue was extracted again according to the above process, and the two ether layers were combined, purged with nitrogen, dried at 35 ° C, and finally dissolved with isopropanol and filtered with a 0.22 μm membrane.

[0031] The upper column conditions are: The detection wavelength was 254 nm; the column temperature was 30°C; the flow rate was 0.5 ml / min; and the injection volume was 10 μL. The test results are shown in Table 2: Table 2

[0032] Table 3

[0033] As can be seen from Table 3, the R 2 All are above 0.995, with stable linearity and small error.

[0034] As can be seen from Table 3, the detection limit of this method is 20-62ug / kg, and the quantification limit is 65-205ug / kg, which has high sensitivity.

[0035] It can be seen from Table 3 that the results have good reproducibility and repeatability.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for quickly identifying harmful components in pickled peppers, characterized in that: The method comprises: Preparation of reference solution: Putrescine, histamine, tyramine and cadaverine standards were prepared into standard stock solutions and biogenic amine mixed solutions; Preparation of test solution: Prepare the test solution using the test sample; Chromatographic conditions: C18 as filler; isopropanol as mobile phase A, 0.5-2.0wt% ammonia water as mobile phase B, gradient elution; the gradient elution procedure is: 0-3min, the volume ratio of mobile phase A:mobile phase B is 50-60%:40-50%; 3-5min, the volume ratio of mobile phase A:mobile phase B is 15-20%:80-85%; 5-7min, the volume ratio of mobile phase A: mobile phase B is 5-10%:90-95%; 7-8min, the volume ratio of mobile phase A:mobile phase B is 40-48%:52-60%; 8-12min, the volume ratio of mobile phase A: mobile phase B is 25-30%: 70-75%; The detection wavelength is 254nm; the column temperature is 25-35℃; the flow rate is 0.2-1.0ml / min.

2. The method according to claim 1, characterized in that The reference substance was added to 0.1 mol / L hydrochloric acid to prepare single standard substances of different concentrations. 2 mL of the above solution was added with 10 μL of benzoyl chloride and 1 mL of 2 mol / L sodium hydroxide solution, mixed well, and reacted at 30°C in the dark for 40 minutes. After the reaction was completed, 2 mL of 5 wt% sodium chloride solution and 3 mL of anhydrous ether were added for extraction, and centrifuged. The ether layer was separated, and the residue was extracted once more according to the above process. The two ether layers were combined, purged with nitrogen, dried at 35°C, and finally dissolved with isopropanol.

3. The method according to claim 2, characterized in that Chop the sample, take 10g, add 20mL of 0.1mol / L hydrochloric acid, homogenize for 10-20 minutes, let stand for 5-10 minutes, centrifuge at high speed, and take the supernatant; add 20mL of 0.1mol / L hydrochloric acid again, repeat the above process once; combine the two supernatants; use 0.1mol / L hydrochloric acid to make the supernatant volume 50mL, take 2mL of the above solution, add 10μL of benzoyl chloride and 1mL2mol / L sodium hydroxide solution, mix well, and react at 30℃ in the dark for 40 minutes; after the reaction is completed, add 2mL of 5wt% sodium chloride solution and 3mL of anhydrous ether for extraction, and centrifuge; The ether layer was separated, and the residue was extracted once more according to the above process. The two ether layers were combined, purged with nitrogen, dried at 35°C, and finally dissolved with isopropanol.

4. The method according to claim 1, characterized in that: The procedure of gradient elution is: using isopropanol as mobile phase A and 1wt% ammonia water as mobile phase B for gradient elution; the procedure of gradient elution is: 0-3min, the volume ratio of mobile phase A:mobile phase B was 55%:45%; 3-5min, the volume ratio of mobile phase A:mobile phase B is 20%:80%; 5-7min, the volume ratio of mobile phase A:mobile phase B is 8%:92%; 7-8min, the volume ratio of mobile phase A:mobile phase B is 45%:55%; 8-12min, the volume ratio of mobile phase A:mobile phase B is 25%:75%; The detection wavelength was 254 nm; the column temperature was 30°C; the flow rate was 0.5 ml / min.

5. The method according to claim 1, characterized in that For the test solution, taking tyramine as the reference peak, the relative retention times of the four characteristic peaks are as follows: Tyramine: relative retention time is 1; Putrescine: relative retention time is 2.968-2.984; Cadaverine: relative retention time is 3.386-3.393; Histamine: relative retention time is 4.180-4.187.

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