Method for detecting heterocyclic amine in fried food
The fried food was pretreated by enzymatic lysis method and the mixed enzymatic lysis of neutral protease and bromelain was solved, and the detection problem of acid hydrolysis was solved when detecting protein-bound heterocyclic amines in fried foods was achieved, efficient and accurate heterocyclic amine detection was achieved, and the reliability of food safety assessment was improved.
Patent Information
- Application Number
- CN202510576053.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-06
- Publication Date
- 2025-07-04
AI Technical Summary
In the prior art, acid hydrolysis method easily destroys the target substance when detecting protein-bound heterocyclic amines in fried foods and has many impurities in the sample, resulting in a decrease in detection sensitivity and accuracy, making it difficult to meet the high requirements of food safety.
The sample is pretreated by enzymatic lysis method, and the mixture of neutral protease and bromelain is used to achieve accurate analysis of heterocyclic amines in fried foods through ultrasound, homogenization, centrifugation, drying, enzymatic lysis, extraction and washing.
It effectively avoids the damage of the target substance, improves the accuracy and sensitivity of detection, and can detect free and protein-bound heterocyclic amines at the same time, comprehensively evaluate the content of heterocyclic amines in food, and provide technical support for food safety risk assessment.
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Figure CN120254126A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for detecting heterocyclic amines in fried foods, belonging to the technical field of heterocyclic amine detection. Background Art
[0002] Fried foods are favored by consumers worldwide due to their unique flavor and texture. However, during the high-temperature cooking process, animal food raw materials are prone to produce heterocyclic amines (HAAs) with potential carcinogenic risks.
[0003] Existing detection technologies mainly focus on the analysis of free HAAs. In recent years, research has found that protein-bound HAAs have gradually become a new focus of food toxicology risk assessment due to their biological significance and metabolic transformation characteristics in food matrices.
[0004] Currently, the pretreatment method for protein-bound HAAs usually uses acid hydrolysis. However, the acid hydrolysis reaction is violent and easily destroys the target substances. In addition, there are many impurities in the sample after acid hydrolysis treatment, which brings difficulties to subsequent detection and reduces the sensitivity and accuracy of the detection method. At the same time, with the increasing attention of people to food safety issues and the continuous increase in the consumption of heat-processed foods, there is an urgent need to develop more sensitive and reliable sample preparation and detection technologies to better ensure food safety.
[0005] Therefore, there is an urgent need to develop a mild and efficient sample preparation method to accurately analyze protein-bound HAAs. Summary of the Invention
[0006] To solve the above problems, the present invention selects enzymatic hydrolysis to pretreat fried samples, and through an efficient and mild sample preparation method, to achieve the accurate analysis and detection of protein-bound heterocyclic amines in fried foods.
[0007] The first object of the present invention is to provide a method for detecting bound peptide heterocyclic amines in fried samples, including the steps of:
[0008] (1) The fried sample is ultrasonically treated, homogenized, centrifuged, and dried with a hydrochloric acid-methanol solution to obtain a dry precipitate;
[0009] (2) The dry precipitate is dissolved in water and homogenized; 4-6 wt% (based on the sample mass) of a mixed enzyme is added and enzymatically hydrolyzed to obtain an enzymatic hydrolysate; wherein, the mass ratio of neutral protease to bromelain in the mixed enzyme is 5-15:5-15;
[0010] (3) The enzymatic hydrolysate is extracted, washed, and eluted to obtain an eluate; the content of bound peptide heterocyclic amines in the eluate is detected by liquid chromatography-mass spectrometry.
[0011] In one embodiment, the mass ratio of neutral protease to bromelain is 5:15 or 15:5 or 10:10.
[0012] In one embodiment, the enzyme activity of neutral protease is 100,000 U / g, and the enzyme activity of bromelain is 100,000 U / g.
[0013] In one embodiment, in step (1), the dosage ratio of the fried sample to the hydrochloric acid-methanol solution is 1-3 g: 10-30 mL.
[0014] In one embodiment, in step (1), the ultrasonic homogenization is carried out at 8000-10000 r / min and 400-600 W for 30-60 min.
[0015] In one embodiment, in step (2), the enzymatic hydrolysis is carried out at pH 4-6 and 45-55 °C for 18-32 h.
[0016] In one embodiment, in step (3), the extraction is carried out using ethyl acetate.
[0017] In one embodiment, in step (3), the washing is carried out by washing successively with an aqueous hydrochloric acid solution and a methanol solution.
[0018] In one embodiment, in step (3), the elution is carried out using an ammonia-methanol solution.
[0019] In one embodiment, the bound peptide heterocyclic amines include one or more of IQ (2-amino-3-methyl-3H-imidazo[4,5-f]quinoline), IQx (2-amino-3-methyl-3H-imidazo[4,5-g]quinoxaline), Glu-P-2 (2-amino-3,8-dimethylimidazo[4,5-f]pyridine), Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole), 4,8-DiMeIQx (2-amino-3,4-dimethyl-3H-imidazo[4,5-g]quinoxaline), 7,8-DiMeIQx (2-amino-3,7,8-trimethyl-3H-imidazo[4,5-g]quinoxaline), PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine), Harman (1-methyl-9H-pyrido[3,4-b]indole) and Norharman (9H-pyrido[3,4-b]indole).
[0020] In one embodiment, the bound peptide heterocyclic amines include Glu-P-2, Trp-P-1, PhIP, Harman and Norharman.
[0021] The second object of the present invention is to provide a method for improving the accuracy of detecting heterocyclic amines in fried samples. The fried samples are pretreated by an enzymatic hydrolysis method to obtain an enzymatic hydrolysate, and then detected by liquid chromatography-mass spectrometry; the pretreatment of the fried samples by the enzymatic hydrolysis method includes the steps:
[0022] (1) The fried sample is ultrasonically treated, homogenized, centrifuged, and dried with a hydrochloric acid - methanol solution to obtain a dry precipitate;
[0023] (2) The dry precipitate is dissolved in water and homogenized; 4 - 6 wt% of a mixed enzyme is added and enzymatic hydrolysis is carried out to obtain an enzymatic hydrolysate; among them, the mass ratio of the neutral protease to bromelain in the mixed enzyme is 70 - 80:20 - 30; the enzymatic hydrolysis is carried out at pH 4 - 6 and 45 - 55 °C for 18 - 32 h.
[0024] The third object of the present invention is to provide a method for simultaneously detecting five heterocyclic amines Glu - P - 2, Trp - P - 1, PhIP, Harman, and Norharman in a sample, including the steps:
[0025] (1) The fried sample is ultrasonically treated, homogenized, centrifuged, and dried with a hydrochloric acid - methanol solution to obtain a dry precipitate;
[0026] (2) The dry precipitate is dissolved in water and homogenized; 4 - 6 wt% of a mixed enzyme is added and enzymatic hydrolysis is carried out to obtain an enzymatic hydrolysate; among them, the mass ratio of the neutral protease to bromelain in the mixed enzyme is 5 - 15:5 - 15;
[0027] (3) The enzymatic hydrolysate is subjected to extraction, washing, and elution to obtain an eluate; the content of the bound peptide heterocyclic amine in the eluate is detected by liquid chromatography - mass spectrometry.
[0028] In one embodiment, the mass ratio of the neutral protease to bromelain is 5:15 or 15:5 or 10:10.
[0029] In one embodiment, the enzyme activity of the neutral protease is 100000 U / g, and the enzyme activity of bromelain is 100000 U / g.
[0030] In one embodiment, in step (1), the dosage ratio of the fried sample to the hydrochloric acid - methanol solution is 1 - 3 g:10 - 30 mL.
[0031] In one embodiment, in step (1), the ultrasonic treatment and homogenization are carried out at 8000 - 10000 r / min and 400 - 600 W for 30 - 60 min.
[0032] In one embodiment, in step (2), the enzymatic hydrolysis is carried out at pH 4 - 6 and 45 - 55 °C for 18 - 32 h.
[0033] In one embodiment, in step (3), the extraction is carried out using ethyl acetate for extraction.
[0034] In one embodiment, in step (3), the washing is carried out by washing successively with an aqueous hydrochloric acid solution and a methanol solution.
[0035] In one embodiment, in step (3), the elution is carried out using an ammonia-methanol solution.
[0036] The fourth object of the present invention is to provide the application of any of the described methods in the detection of heterocyclic amines.
[0037] In one embodiment, the heterocyclic amines include IQ, IQx, Glu-P-2, Trp-P-1, 4,8-DiMeIQx, 7,8-DiMeIQx, PhIP, Harman, and Norharman.
[0038] In one embodiment, the peptide-bound heterocyclic amines include Glu-P-2, Trp-P-1, PhIP, Harman, and Norharman.
[0039] Advantages of the present invention
[0040] The present invention uses a mild protease hydrolysis method to replace the traditional acid hydrolysis, avoiding the destruction of the target substances. The total amount of heterocyclic amines detected is significantly higher than that of the acid hydrolysis method and is closer to the actual value. At the same time, the method of the present invention can detect more types of heterocyclic amines.
[0041] The present invention effectively realizes the simultaneous detection of free and protein-bound heterocyclic amines, comprehensively evaluates the content of heterocyclic amines in food, and provides technical support for food safety risk assessment. The method of the present invention is applicable to the analysis and detection of bound heterocyclic amines in various fried foods and has broad practical application prospects. Description of the drawings
[0042] Figure 1 It is the liquid-phase detection results of Example 1, Comparative Example 1, and heterocyclic amine standards. Detailed implementation manners
[0043] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.
[0044] Raw materials used in the examples:
[0045] Method for preparing crispy fried pork:
[0046] From 1 kg of packaged Meihao brand original flavor crispy fried pork stored at -4°C, take out long strip-shaped crispy fried pork with uniform thickness and width. Fry it in Jinlongyu sunflower seed oil at 200°C for 210 s. After cooling to room temperature, put it in a -80°C refrigerator and freeze for 1 hour. Then put it into a freeze dryer and freeze-dry for 24 h. After taking it out, use a blender to grind it into a uniform powder and store it at -20°C for standby.
[0047] Neutral protease is purchased from Yuanye Biotechnology Co., Ltd., and its enzyme activity is 100000 U / g;
[0048] Bromelain was purchased from Shanghai Macklin Biochemical Co., Ltd., and its enzyme activity was 100000 U / g;
[0049] The remaining reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.
[0050] Example 1
[0051] 1. Sample pretreatment
[0052] Weigh 2 g of the crushed deep-fried pork sample, add 20 mL of hydrochloric acid-methanol mixture (5 mL of 5 mol / L HCl + 95 mL of methanol), homogenize at 10000 r / min, perform ultrasonic treatment at 500 W for 30 min, centrifuge at 5000 r / min for 20 min, and discard the supernatant; repeat the extraction process once to obtain a precipitate; vacuum-dry the precipitate at 60 °C to obtain a dry precipitate;
[0053] Add 20 mL of deionized water to the dry precipitate, homogenize for 30 s; add a mixed enzyme (mass ratio of neutral protease to bromelain is 10:10), the total enzyme concentration is 5% (w / w) based on the sample mass, adjust the pH to 7, and enzymatically hydrolyze at 50 °C for 24 h; after the reaction, add 2 mL of 1 mol / L NaOH to terminate the enzyme activity to obtain an enzymatic hydrolysate.
[0054] 2. Extraction and purification
[0055] Add 20 mL of ethyl acetate to the enzymatic hydrolysate, perform ultrasonic extraction for 30 min, centrifuge at 5000 r / min for 10 min, and take the supernatant;
[0056] Activate and equilibrate the SelectPrep MCX column (60 mg / 3 mL) with 6 mL of methanol and 6 mL of 0.1 mol / L HCl in sequence, load 10 mL of the extract, wash with 3 mL of 0.1 mol / L HCl and 3 mL of methanol in sequence, and elute the bound heterocyclic amines with 6 mL of ammonia-methanol (90:10, ammonia concentration is 29%) to obtain an eluate.
[0057] 3. Detection and analysis:
[0058] The eluate was concentrated to nearly dry by nitrogen blowing, redissolved in 300 μL of methanol, filtered through a 0.22 μm nylon needle, and then loaded;
[0059] Using a UPLC-MS / MS device, detection was performed on a Waters BEH C18 chromatographic column (2.1×150 mm, 1.7 μm), and 9 typical heterocyclic amines (IQ, IQx, Glu-P-2, Trp-P-1, 4,8-DiMeIQx, 7,8-DiMeIQx, PhIP, Harman, Norharman) were successfully detected.
[0060] Example 2
[0061] (1) On the basis of Example 1, change the mixed enzyme in Step 1 to a single neutral enzyme (i.e., neutral protease: bromelain = 20:0), and the remaining steps are the same as those in Example 1, and detect the content of heterocyclic amines.
[0062] (2) On the basis of Example 1, change the mixed enzyme in Step 1 to a single bromelain enzyme (i.e., neutral protease: bromelain = 0:20), and the remaining steps are the same as those in Example 1, and detect the content of heterocyclic amines.
[0063] Example 3
[0064] (1) On the basis of Example 1, change the mass ratio of neutral protease to bromelain in Step 1 to 15:5, and the total enzyme dosage is 5%, and the remaining steps are the same as those in Example 1, and detect the content of heterocyclic amines.
[0065] (2) On the basis of Example 1, change the mass ratio of neutral protease to bromelain in Step 1 to 5:15, and the total enzyme dosage is 5%, and the remaining steps are the same as those in Example 1, and detect the content of heterocyclic amines.
[0066] Comparative Example 1
[0067] Use acid hydrolysis method for pretreatment, and the steps are as follows:
[0068] Weigh 2 g of the crushed deep-fried pork sample, add 20 mL of hydrochloric acid-methanol mixture (5 mL of 5 mol / L HCl + 95 mL of methanol), homogenize and ultrasonically treat for 30 min, centrifuge at 5000 r / min for 20 min, and discard the supernatant; repeat the extraction process once to finally obtain a precipitate; vacuum dry the precipitate at 60 °C to obtain a dry precipitate.
[0069] Transfer the dry precipitate to a pressure-resistant bottle, add 20 mL of HCl (6 mol / L), and blow nitrogen into the mixture for 1 min; hydrolyze the mixture at 110 °C for 24 hours, then centrifuge the solution at 10000 r / min for 10 minutes at 4 °C; collect the supernatant obtained after centrifugation and dilute it 10 times before solid-phase extraction.
[0070] The steps of extraction, purification and detection analysis are the same as those in Example 1.
[0071] Comparative Example 2
[0072] On the basis of Example 1, replace the mixed enzyme with an equal amount of proteinase K, and the remaining steps are the same as those in Example 1.
[0073] Example 4
[0074] 1. Optimization of enzyme inoculum amount
[0075] On the basis of Example 1, the enzyme inoculum amounts were changed to 1%, 2%, 3%, 4%, 5%, and 6% respectively, and the remaining steps were the same as those in Example 1.
[0076] 2. Optimization of enzyme reaction pH
[0077] On the basis of Example 1, the pH values were changed to 5, 6, 7, 8, and 9 respectively, and the remaining steps were the same as those in Example 1.
[0078] 3. Optimization of enzymolysis time
[0079] On the basis of Example 1, the enzymolysis times were changed to 6 h, 12 h, 24 h, 36 h, and 48 h respectively, and the remaining steps were the same as those in Example 1.
[0080] Example 5
[0081] The detection results of Examples 1 - 3 and Comparative Examples 1 - 2 are shown in Table 1.
[0082] Table 1 Detection results
[0083]
[0084] Note: N.D. indicates not detected
[0085] Comparing Example 1 and Example 2, it can be seen that the double - enzyme synergistic treatment has better heterocyclic amine release ability, especially outstanding in the signal response of PhIP and Norharman;
[0086] Comparing Example 3 and Comparative Example 1, it can be seen that compared with acid hydrolysis, the enzymatic hydrolysis method of double - enzyme synergistic treatment has better release ability for bound heterocyclic amines, and the obtained heterocyclic amines are superior to acid hydrolysis in terms of the detected heterocyclic amine concentration;
[0087] Comparing Example 3(1) and Example 3(2), it can be seen that when double - enzyme synergistic treatment is carried out, when the ratio of neutral protease to bromelain is 15:5, the heterocyclic amine release ability is the best.
[0088] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.
Claims
1. A method for detecting bound peptide heterocyclic amines in fried samples, characterized in that, Including the steps: (1) The fried sample is ultrasonically treated, homogenized, centrifuged, and dried with a hydrochloric acid - methanol solution to obtain a dried precipitate; (2) The dried precipitate is dissolved in water and homogenized; a 4 - 6 wt% mixed enzyme is added and enzymatically hydrolyzed to obtain an enzymolysis solution; wherein, the mass ratio of neutral protease to bromelain in the mixed enzyme is 5 - 15:5 - 15; (3) The enzymolysis solution is subjected to extraction, washing, and elution to obtain an eluate; the content of bound peptide heterocyclic amines in the eluate is detected by liquid chromatography - mass spectrometry.
2. The method according to claim 1, characterized in that In step (1), the dosage ratio of the fried sample to the hydrochloric acid - methanol solution is 1 - 3 g:10 - 30 mL.
3. The method according to claim 1, wherein In step (1), the homogenization is at 8000 - 10000 r / min and 400 - 600 W, and ultrasonic treatment is carried out for 30 - 60 min.
4. The method according to claim 1, wherein In step (2), the enzymatic hydrolysis is carried out at pH 4 - 6 and 45 - 55 °C for 18 - 32 h.
5. The method according to claim 1, characterized in that, In step (3), the extraction is carried out using ethyl acetate.
6. The method according to claim 1, characterized in that In step (3), the washing is carried out successively with an aqueous hydrochloric acid solution and a methanol solution, and the elution is carried out using an ammonia - methanol solution.
7. The method according to claim 1, wherein The bound peptide heterocyclic amines include one or more of IQ, IQx, Glu - P - 2, Trp - P - 1, 4,8 - DiMeIQx, 7,8 - DiMeIQx, PhIP, Harman, and Norharman.
8. A method for simultaneously detecting five heterocyclic amines, namely Glu-P-2, Trp-P-1, PhIP, Harman, and Norharman, in a sample, characterized in that, Including the steps: (1) The fried sample is ultrasonically treated, homogenized, centrifuged, and dried with a hydrochloric acid - methanol solution to obtain a dried precipitate; (2) The dried precipitate is dissolved in water and homogenized; a 4 - 6 wt% mixed enzyme is added and enzymatically hydrolyzed to obtain an enzymolysis solution; wherein, the mass ratio of neutral protease to bromelain in the mixed enzyme is 5 - 15:5 - 15; (3) The enzymolysis solution is subjected to extraction, washing, and elution to obtain an eluate; the content of bound peptide heterocyclic amines in the eluate is detected by liquid chromatography - mass spectrometry.
9. A method for improving the accuracy of detecting heterocyclic amines in fried samples, characterized in that, The fried sample is pretreated by an enzymatic method to obtain an enzymolysis solution, and then detected by liquid chromatography - mass spectrometry; The pretreatment of the fried sample by the enzymatic method includes the steps: (1) The fried sample is ultrasonically treated, homogenized, centrifuged, and dried with a hydrochloric acid - methanol solution to obtain a dried precipitate; (2) The dried precipitate is dissolved in water and homogenized; a 4 - 6 wt% mixed enzyme is added and enzymatically hydrolyzed to obtain an enzymolysis solution; wherein, the mass ratio of neutral protease to bromelain in the mixed enzyme is 5 - 15:5 - 15; the enzymatic hydrolysis is carried out at pH 4 - 6 and 45 - 55 °C for 18 - 32 h.
10. Use of the method according to any one of claims 1 - 9 in the detection of heterocyclic amines.