Method for detecting MeIQx in fried aquatic product

Abstract

The invention provides a method for detecting MeIQx in fried aquatic products, and belongs to the technical field of fried aquatic product detection, and the method comprises the following steps: step 1, preparing materials, including preparing experimental materials; step 2, sample preparation: frying the aquatic product at different oil temperatures to obtain a fried sample; and 3, detecting MeIQx, namely detecting the content of MeIQx in the fried sample at different oil temperatures by using UHPLC-MS (Ultra High Performance Liquid Chromatography-Mass Spectrometer). According to the method, the MeIQx in the fried aquatic product is detected, the generation amount of the MeIQx in the aquatic product is obviously influenced by the frying temperature, and the generation amount of the MeIQx is gradually increased along with the increase of the frying temperature and reaches the maximum value 2.12 ng / g at 220 DEG C, which indicates that the temperature is an important factor influencing the formation of the MeIQx.

Description

Technical Field

[0001] The present invention relates to the technical field of fried aquatic product detection, and in particular to a method for detecting MeIQx in fried aquatic products. Background Art

[0002] Aquatic products are rich in protein, unsaturated fatty acids, vitamins and minerals, and have high nutritional value. Among them, fish are rich in protein, unsaturated fatty acids and vitamin D, which help lower cholesterol, prevent cardiovascular and cerebrovascular diseases and promote brain development; shellfish are rich in trace elements such as zinc and selenium, which help improve immunity; shrimps are rich in protein, unsaturated fatty acids and vitamin A, which help protect vision, skin and immune system.

[0003] There are many ways to cook aquatic products, such as steaming, boiling, stir-frying, deep-frying, and baking. Different cooking methods can preserve their nutrients. During the frying process, the oxidation of fats in aquatic products produces acrolein, glyoxal, oxidized triglyceride monomers, conjugated linoleic acid and other lipid oxidation products, which are key factors promoting the formation of MeIQx.

[0004] MeIQx is a class 2B mutagenic and carcinogenic compound. MeIQx is mainly produced during the high-temperature processing of protein-rich aquatic products. After glucose and amino acids (glycine) undergo Strecker degradation reactions, the Strecker degradation products undergo a series of complex chemical reactions such as Amadori rearrangement to form pyrazine and pyridine compounds, which are then reacted with creatinine through the Aldol condensation mechanism. MeIQx is commonly found in fried aquatic products and poses a high risk of harm. Accurate reduction and control of MeIQx is an important problem that plagues the development of the aquatic product processing industry. Therefore, studying the formation factors of MeIQx in fried aquatic products is of great significance to the development of fried aquatic products. Summary of the invention

[0005] The object of the present invention is to provide a method for detecting MeIQx in fried aquatic products, and to reduce the formation of MeIQx in fried aquatic products by studying the factors causing the formation of MeIQx.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for detecting MeIQx in fried aquatic products comprises the following steps:

[0008] Step 1: Prepare materials, including experimental materials;

[0009] Step 2: Sample preparation, frying the aquatic products at different oil temperatures to obtain fried samples;

[0010] Step 3: Detect MeIQx. Use UHPLC-MS to detect the content of MeIQx in fried samples at different oil temperatures.

[0011] Furthermore, the preparation of experimental materials in step 1 includes: fresh squid, soybean oil, hydrochloric acid, methanol, ethanol and distilled water.

[0012] Furthermore, the specific process of preparing the fried sample in step 2 is:

[0013] Step 2.1: The viscera, inner shell and outer skin of the fresh squid were removed and cleaned, and then the squid was cut into small pieces of 3 cm×3 cm, and the muscle tissue was crushed with a meat grinder. The crushed squid was soaked in 25% anhydrous ethanol for 1 hour, and the mixture of ethanol and oil was discarded. The above steps were repeated three times until the desalination was complete and the ethanol in the squid minced meat was completely volatilized. Then 1 kg of squid minced meat and 25 g of salt were put into a minced meat mixer and stirred evenly to obtain a sample;

[0014] Step 2.2: Place the sample in a culture dish with a radius of 35 mm, and place food-grade paper at the bottom of the culture dish to make 1 cm thick patties, and wrap them with sealing film and set them at 4°C for 1 hour. Put the shaped round cakes into an oil bath at an oil temperature of 100-220°C for frying. After the fried squid patties are cooled, vacuum-pack them, and store them in a freezer at a temperature of -18°C.

[0015] Further, the oil temperature of the oil bath frying is 100°C, 120°C, 140°C, 160°C, 180°C, 200°C and 220°C.

[0016] Furthermore, the specific process of using UHPLC-MS to detect the content of MeIQx in the fried sample in step 3 is as follows:

[0017] Step 3.1: Dry and grind the fried squid patties into powder, weigh 4 g and add them into 25 mL of 0.1 mol / L hydrochloric acid for homogenization for 1 min, 15 s / time, for 4 times in total, and then ultrasonically extract for 15 min;

[0018] Step 3.2: Then, the mixture was centrifuged at 10,000 r / min for 20 min at 4°C, and the supernatant was collected. 25 mL of hydrochloric acid was added to extract the precipitate again. The supernatant was rotary evaporated and concentrated. The volume was fixed to 25 mL with distilled water. The protein was removed by ethanol-freezing method, and then filtered with a 0.22 μm filter membrane. UHPLC-MS detection was performed using a Waters AtlanticsdC18 column (250 mm×4.6 mm×3.0 μm).

[0019] Furthermore, the UHPLC chromatographic conditions are as follows:

[0020] Mobile phase: 0.1% formic acid aqueous solution (A)-methanol (B); gradient elution: 0-0.1min, 5% B; 0.1-10min, 5%-100% B; 10-10.5min, 100%-5% B; 10.5-15.5min, 5% B; flow rate was 0.5mL / min, injection volume was 1μL, and column temperature was 35°C.

[0021] Furthermore, the mass spectrometry detection conditions were: electrospray ionization source ionization, positive ion mode, source temperature 130°C, MRM mode, capillary voltage 3.5V; in addition, nitrogen was used as the desolvation gas and cone gas, with flow rates of 800L / h and 50L / h respectively, the desolvation temperature was 350°C, and argon was used as the collision gas, with a flow rate of 0.13mL / min.

[0022] The present invention has the following beneficial effects due to the adoption of the above technical solution:

[0023] In the present invention, MeIQx in fried aquatic products was detected and it was found that the frying temperature significantly affected the amount of MeIQx generated in aquatic products. The amount of MeIQx generated gradually increased with the increase of frying temperature and reached a maximum value of 2.12 ng / g at 220°C, indicating that temperature is an important factor affecting the formation of MeIQx. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic diagram of the amount of MeIQx generated in fried squid at different temperatures of the present invention;

[0025] Figure 2 It is a schematic diagram of the microstructure of fried squid at different temperatures according to the present invention;

[0026] Figure 3 It is a schematic diagram of infrared spectra of fried squid at different temperatures according to the present invention;

[0027] Figure 4 It is a schematic diagram of the interaction force of the present invention on fried squid at different temperatures;

[0028] Figure 5 It is a schematic diagram of the Zeta potential of fried squid at different temperatures according to the present invention;

[0029] Figure 6 It is a schematic diagram of the contact angle of squid processed at different temperatures according to the present invention (the numbers in A represent the temperature). DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.

[0031] Example 1

[0032] A method for detecting MeIQx in fried aquatic products comprises the following steps:

[0033] Step 1: Prepare materials, fresh squid, soybean oil, hydrochloric acid, methanol, ethanol and distilled water;

[0034] Step 2: Sample preparation, the specific process is:

[0035] Step 2.1: The viscera, inner shell and outer skin of the fresh squid were removed and cleaned, and then the squid was cut into small pieces of 3 cm×3 cm, and the muscle tissue was crushed with a meat grinder. The crushed squid was soaked in 25% anhydrous ethanol for 1 hour, and the mixture of ethanol and oil was discarded. The above steps were repeated three times until the desalination was complete and the ethanol in the squid minced meat was completely volatilized. Then 1 kg of squid minced meat and 25 g of salt were put into a minced meat mixer and stirred evenly to obtain a sample;

[0036] Step 2.2: Place the sample in a culture dish with a radius of 35 mm, and place food-grade paper at the bottom of the culture dish to make 1 cm thick patties, and wrap them with sealing film and set at 4°C for 1 hour. Put the shaped round cakes into an oil bath at an oil temperature of 100°C for frying. After the fried squid patties are cooled, vacuum-pack them, and store them in a freezer at a temperature of -18°C.

[0037] Step 3: Detect MeIQx, the specific process is as follows: Step 3.1: Dry and crush the fried squid patties into powder, weigh 4 g and add them into 25 mL of 0.1 mol / L hydrochloric acid for homogenization for 1 min, 15 s / time, 4 times in total, and then ultrasonically extract for 15 min;

[0038] Step 3.2: Then, centrifuge at 10,000 r / min for 20 min at 4°C and collect the supernatant, add 25 mL of hydrochloric acid to extract the precipitate again, rotary evaporate the supernatant, combine and concentrate, fix the volume to 25 mL with distilled water, remove the protein by ethanol-freezing method, and then filter with 0.22 μm filter membrane and perform UHPLC-MS detection using Waters AtlanticsdC18 column.

[0039] UHPLC chromatographic conditions are as follows:

[0040] Mobile phase: 0.1% formic acid aqueous solution (A)-methanol (B); gradient elution: 0-0.1min, 5% B; 0.1-10min, 5%-100% B; 10-10.5min, 100%-5% B; 10.5-15.5min, 5% B; flow rate was 0.5mL / min, injection volume was 1μL, and column temperature was 35°C.

[0041] The mass spectrometry detection conditions were as follows: electrospray ionization source ionization, positive ion mode, source temperature 130°C, MRM mode, capillary voltage 3.5V; in addition, nitrogen was used as the desolvation gas and cone gas, with flow rates of 800L / h and 50L / h respectively, the desolvation temperature was 350°C, and argon was used as the collision gas, with a flow rate of 0.13mL / min.

[0042] Example 2

[0043] A method for detecting MeIQx in fried aquatic products comprises the following steps:

[0044] Step 1: Prepare materials, fresh squid, soybean oil, hydrochloric acid, methanol, ethanol and distilled water;

[0045] Step 2: Sample preparation, the specific process is:

[0046] Step 2.1: The viscera, inner shell and outer skin of the fresh squid were removed and cleaned, and then the squid was cut into small pieces of 3 cm×3 cm, and the muscle tissue was crushed with a meat grinder. The crushed squid was soaked in 25% anhydrous ethanol for 1 hour, and the mixture of ethanol and oil was discarded. The above steps were repeated three times until the desalination was complete and the ethanol in the squid minced meat was completely volatilized. Then 1 kg of squid minced meat and 25 g of salt were put into a minced meat mixer and stirred evenly to obtain a sample;

[0047] Step 2.2: Place the sample in a culture dish with a radius of 35 mm, and place food-grade paper at the bottom of the culture dish to make 1 cm thick patties, and wrap them with sealing film and set them at 4°C for 1 hour. Put the shaped round cakes into an oil bath at an oil temperature of 120°C for frying. After the fried squid patties are cooled, vacuum-pack them, and store them in a freezer at a temperature of -18°C.

[0048] Step 3: Detect MeIQx, the specific process is as follows: Step 3.1: Dry and crush the fried squid patties into powder, weigh 4 g and add them into 25 mL of 0.1 mol / L hydrochloric acid for homogenization for 1 min, 15 s / time, 4 times in total, and then ultrasonically extract for 15 min;

[0049] Step 3.2: Then, centrifuge at 10,000 r / min for 20 min at 4°C and collect the supernatant, add 25 mL of hydrochloric acid to extract the precipitate again, rotary evaporate the supernatant, combine and concentrate, fix the volume to 25 mL with distilled water, remove the protein by ethanol-freezing method, and then filter with 0.22 μm filter membrane and perform UHPLC-MS detection using Waters AtlanticsdC18 column.

[0050] UHPLC chromatographic conditions are as follows:

[0051] Mobile phase: 0.1% formic acid aqueous solution (A)-methanol (B); gradient elution: 0-0.1min, 5% B; 0.1-10min, 5%-100% B; 10-10.5min, 100%-5% B; 10.5-15.5min, 5% B; flow rate was 0.5mL / min, injection volume was 1μL, and column temperature was 35°C.

[0052] The mass spectrometry detection conditions were as follows: electrospray ionization source ionization, positive ion mode, source temperature 130°C, MRM mode, capillary voltage 3.5V; in addition, nitrogen was used as the desolvation gas and cone gas, with flow rates of 800L / h and 50L / h respectively, the desolvation temperature was 350°C, and argon was used as the collision gas, with a flow rate of 0.13mL / min.

[0053] Example 3

[0054] A method for detecting MeIQx in fried aquatic products comprises the following steps:

[0055] Step 1: Prepare materials, fresh squid, soybean oil, hydrochloric acid, methanol, ethanol and distilled water;

[0056] Step 2: Sample preparation, the specific process is:

[0057] Step 2.1: The viscera, inner shell and outer skin of the fresh squid were removed and cleaned, and then the squid was cut into small pieces of 3 cm×3 cm, and the muscle tissue was crushed with a meat grinder. The crushed squid was soaked in 25% anhydrous ethanol for 1 hour, and the mixture of ethanol and oil was discarded. The above steps were repeated three times until the desalination was complete and the ethanol in the squid minced meat was completely volatilized. Then 1 kg of squid minced meat and 25 g of salt were put into a minced meat mixer and stirred evenly to obtain a sample;

[0058] Step 2.2: Place the sample in a culture dish with a radius of 35 mm, and place food-grade paper at the bottom of the culture dish to make 1 cm thick patties, and wrap them with sealing film and set them at 4°C for 1 hour. Put the shaped round cakes into an oil bath at an oil temperature of 140°C for frying. After the fried squid patties are cooled, vacuum-pack them, and store them in a freezer at a temperature of -18°C.

[0059] Step 3: Detect MeIQx, the specific process is as follows: Step 3.1: Dry and crush the fried squid patties into powder, weigh 4 g and add them into 25 mL of 0.1 mol / L hydrochloric acid for homogenization for 1 min, 15 s / time, 4 times in total, and then ultrasonically extract for 15 min;

[0060] Step 3.2: Then, centrifuge at 10,000 r / min for 20 min at 4°C and collect the supernatant, add 25 mL of hydrochloric acid to extract the precipitate again, rotary evaporate the supernatant, combine and concentrate, fix the volume to 25 mL with distilled water, remove the protein by ethanol-freezing method, and then filter with 0.22 μm filter membrane and perform UHPLC-MS detection using Waters AtlanticsdC18 column.

[0061] UHPLC chromatographic conditions are as follows:

[0062] Mobile phase: 0.1% formic acid aqueous solution (A)-methanol (B); gradient elution: 0-0.1min, 5% B; 0.1-10min, 5%-100% B; 10-10.5min, 100%-5% B; 10.5-15.5min, 5% B; flow rate was 0.5mL / min, injection volume was 1μL, and column temperature was 35°C.

[0063] The mass spectrometry detection conditions were as follows: electrospray ionization source ionization, positive ion mode, source temperature 130°C, MRM mode, capillary voltage 3.5V; in addition, nitrogen was used as the desolvation gas and cone gas, with flow rates of 800L / h and 50L / h respectively, the desolvation temperature was 350°C, and argon was used as the collision gas, with a flow rate of 0.13mL / min.

[0064] Example 4

[0065] A method for detecting MeIQx in fried aquatic products comprises the following steps:

[0066] Step 1: Prepare materials, fresh squid, soybean oil, hydrochloric acid, methanol, ethanol and distilled water;

[0067] Step 2: Sample preparation, the specific process is:

[0068] Step 2.1: The viscera, inner shell and outer skin of the fresh squid were removed and cleaned, and then the squid was cut into small pieces of 3 cm×3 cm, and the muscle tissue was crushed with a meat grinder. The crushed squid was soaked in 25% anhydrous ethanol for 1 hour, and the mixture of ethanol and oil was discarded. The above steps were repeated three times until the desalination was complete and the ethanol in the squid minced meat was completely volatilized. Then 1 kg of squid minced meat and 25 g of salt were put into a minced meat mixer and stirred evenly to obtain a sample;

[0069] Step 2.2: Place the sample in a culture dish with a radius of 35 mm, and place food-grade paper at the bottom of the culture dish to make 1 cm thick patties, and wrap them with sealing film to set at 4°C for 1 hour. Put the shaped round cakes into an oil bath at an oil temperature of 160°C for frying. After the fried squid patties are cooled, vacuum pack them, and store them in a freezer at a temperature of -18°C.

[0070] Step 3: Detect MeIQx, the specific process is as follows: Step 3.1: Dry and crush the fried squid patties into powder, weigh 4 g and add them into 25 mL of 0.1 mol / L hydrochloric acid for homogenization for 1 min, 15 s / time, 4 times in total, and then ultrasonically extract for 15 min;

[0071] Step 3.2: Then, centrifuge at 10,000 r / min for 20 min at 4°C and collect the supernatant, add 25 mL of hydrochloric acid to extract the precipitate again, rotary evaporate the supernatant, combine and concentrate, fix the volume to 25 mL with distilled water, remove the protein by ethanol-freezing method, and then filter with 0.22 μm filter membrane and perform UHPLC-MS detection using Waters AtlanticsdC18 column.

[0072] UHPLC chromatographic conditions are as follows:

[0073] Mobile phase: 0.1% formic acid aqueous solution (A)-methanol (B); gradient elution: 0-0.1min, 5% B; 0.1-10min, 5%-100% B; 10-10.5min, 100%-5% B; 10.5-15.5min, 5% B; flow rate was 0.5mL / min, injection volume was 1μL, and column temperature was 35°C.

[0074] The mass spectrometry detection conditions were as follows: electrospray ionization source ionization, positive ion mode, source temperature 130°C, MRM mode, capillary voltage 3.5V; in addition, nitrogen was used as the desolvation gas and cone gas, with flow rates of 800L / h and 50L / h respectively, the desolvation temperature was 350°C, and argon was used as the collision gas, with a flow rate of 0.13mL / min.

[0075] Example 5

[0076] A method for detecting MeIQx in fried aquatic products comprises the following steps:

[0077] Step 1: Prepare materials, fresh squid, soybean oil, hydrochloric acid, methanol, ethanol and distilled water;

[0078] Step 2: Sample preparation, the specific process is:

[0079] Step 2.1: The viscera, inner shell and outer skin of the fresh squid were removed and cleaned, and then the squid was cut into small pieces of 3 cm×3 cm, and the muscle tissue was crushed with a meat grinder. The crushed squid was soaked in 25% anhydrous ethanol for 1 hour, and the mixture of ethanol and oil was discarded. The above steps were repeated three times until the desalination was complete and the ethanol in the squid minced meat was completely volatilized. Then 1 kg of squid minced meat and 25 g of salt were put into a minced meat mixer and stirred evenly to obtain a sample;

[0080] Step 2.2: Place the sample in a culture dish with a radius of 35 mm, and place food-grade paper at the bottom of the culture dish to make 1 cm thick patties, and wrap them with sealing film and set them at 4°C for 1 hour. Put the shaped round cakes into an oil bath at an oil temperature of 180°C for frying. After the fried squid patties are cooled, vacuum-pack them, and store them in a freezer at a temperature of -18°C.

[0081] Step 3: Detect MeIQx, the specific process is as follows: Step 3.1: Dry and crush the fried squid patties into powder, weigh 4 g and add them into 25 mL of 0.1 mol / L hydrochloric acid for homogenization for 1 min, 15 s / time, 4 times in total, and then ultrasonically extract for 15 min;

[0082] Step 3.2: Then, centrifuge at 10,000 r / min for 20 min at 4°C and collect the supernatant, add 25 mL of hydrochloric acid to extract the precipitate again, rotary evaporate the supernatant, combine and concentrate, fix the volume to 25 mL with distilled water, remove the protein by ethanol-freezing method, and then filter with 0.22 μm filter membrane and perform UHPLC-MS detection using Waters AtlanticsdC18 column.

[0083] UHPLC chromatographic conditions are as follows:

[0084] Mobile phase: 0.1% formic acid aqueous solution (A)-methanol (B); gradient elution: 0-0.1min, 5% B; 0.1-10min, 5%-100% B; 10-10.5min, 100%-5% B; 10.5-15.5min, 5% B; flow rate was 0.5mL / min, injection volume was 1μL, and column temperature was 35°C.

[0085] The mass spectrometry detection conditions were as follows: electrospray ionization source ionization, positive ion mode, source temperature 130°C, MRM mode, capillary voltage 3.5V; in addition, nitrogen was used as the desolvation gas and cone gas, with flow rates of 800L / h and 50L / h respectively, the desolvation temperature was 350°C, and argon was used as the collision gas, with a flow rate of 0.13mL / min.

[0086] Example 6

[0087] A method for detecting MeIQx in fried aquatic products comprises the following steps:

[0088] Step 1: Prepare materials, fresh squid, soybean oil, hydrochloric acid, methanol, ethanol and distilled water;

[0089] Step 2: Sample preparation, the specific process is:

[0090] Step 2.1: The viscera, inner shell and outer skin of the fresh squid were removed and cleaned, and then the squid was cut into small pieces of 3 cm×3 cm, and the muscle tissue was crushed with a meat grinder. The crushed squid was soaked in 25% anhydrous ethanol for 1 hour, and the mixture of ethanol and oil was discarded. The above steps were repeated three times until the desalination was complete and the ethanol in the squid minced meat was completely volatilized. Then 1 kg of squid minced meat and 25 g of salt were put into a minced meat mixer and stirred evenly to obtain a sample;

[0091] Step 2.2: Place the sample in a culture dish with a radius of 35 mm, and place food-grade paper at the bottom of the culture dish to make 1 cm thick patties, and wrap them with sealing film to set at 4°C for 1 hour. Put the shaped round cakes into an oil bath at an oil temperature of 200°C for frying. After the fried squid patties are cooled, vacuum-pack them, and store them in a freezer at a temperature of -18°C.

[0092] Step 3: Detect MeIQx, the specific process is as follows: Step 3.1: Dry and crush the fried squid patties into powder, weigh 4 g and add them into 25 mL of 0.1 mol / L hydrochloric acid for homogenization for 1 min, 15 s / time, 4 times in total, and then ultrasonically extract for 15 min;

[0093] Step 3.2: Then, centrifuge at 10,000 r / min for 20 min at 4°C and collect the supernatant, add 25 mL of hydrochloric acid to extract the precipitate again, rotary evaporate the supernatant, combine and concentrate, fix the volume to 25 mL with distilled water, remove the protein by ethanol-freezing method, and then filter with 0.22 μm filter membrane and perform UHPLC-MS detection using Waters AtlanticsdC18 column.

[0094] UHPLC chromatographic conditions are as follows:

[0095] Mobile phase: 0.1% formic acid aqueous solution (A)-methanol (B); gradient elution: 0-0.1min, 5% B; 0.1-10min, 5%-100% B; 10-10.5min, 100%-5% B; 10.5-15.5min, 5% B; flow rate was 0.5mL / min, injection volume was 1μL, and column temperature was 35°C.

[0096] The mass spectrometry detection conditions were as follows: electrospray ionization source ionization, positive ion mode, source temperature 130°C, MRM mode, capillary voltage 3.5V; in addition, nitrogen was used as the desolvation gas and cone gas, with flow rates of 800L / h and 50L / h respectively, the desolvation temperature was 350°C, and argon was used as the collision gas, with a flow rate of 0.13mL / min.

[0097] Example 7

[0098] A method for detecting MeIQx in fried aquatic products comprises the following steps:

[0099] Step 1: Prepare materials, fresh squid, soybean oil, hydrochloric acid, methanol, ethanol and distilled water;

[0100] Step 2: Sample preparation, the specific process is:

[0101] Step 2.1: The viscera, inner shell and outer skin of the fresh squid were removed and cleaned, and then the squid was cut into small pieces of 3 cm×3 cm, and the muscle tissue was crushed with a meat grinder. The crushed squid was soaked in 25% anhydrous ethanol for 1 hour, and the mixture of ethanol and oil was discarded. The above steps were repeated three times until the desalination was complete and the ethanol in the squid minced meat was completely volatilized. Then 1 kg of squid minced meat and 25 g of salt were put into a minced meat mixer and stirred evenly to obtain a sample;

[0102] Step 2.2: Place the sample in a culture dish with a radius of 35 mm, and place food-grade paper at the bottom of the culture dish to make 1 cm thick patties, and wrap them with sealing film and set them at 4°C for 1 hour. Put the shaped round cakes into an oil bath at an oil temperature of 220°C for frying. After the fried squid patties are cooled, vacuum pack them, and store them in a freezer at a temperature of -18°C.

[0103] Step 3: Detect MeIQx, the specific process is as follows: Step 3.1: Dry and crush the fried squid patties into powder, weigh 4 g and add them into 25 mL of 0.1 mol / L hydrochloric acid for homogenization for 1 min, 15 s / time, 4 times in total, and then ultrasonically extract for 15 min;

[0104] Step 3.2: Then, centrifuge at 10,000 r / min for 20 min at 4°C and collect the supernatant, add 25 mL of hydrochloric acid to extract the precipitate again, rotary evaporate the supernatant, combine and concentrate, fix the volume to 25 mL with distilled water, remove the protein by ethanol-freezing method, and then filter with 0.22 μm filter membrane and perform UHPLC-MS detection using Waters AtlanticsdC18 column.

[0105] UHPLC chromatographic conditions are as follows:

[0106] Mobile phase: 0.1% formic acid aqueous solution (A)-methanol (B); gradient elution: 0-0.1min, 5% B; 0.1-10min, 5%-100% B; 10-10.5min, 100%-5% B; 10.5-15.5min, 5% B; flow rate was 0.5mL / min, injection volume was 1μL, and column temperature was 35°C.

[0107] The mass spectrometry detection conditions were as follows: electrospray ionization source ionization, positive ion mode, source temperature 130°C, MRM mode, capillary voltage 3.5V; in addition, nitrogen was used as the desolvation gas and cone gas, with flow rates of 800L / h and 50L / h respectively, the desolvation temperature was 350°C, and argon was used as the collision gas, with a flow rate of 0.13mL / min.

[0108] The fried squid patties obtained in Example 1-7 were observed by SEM.

[0109] The obtained fried squid patties were cut into small pieces, vacuum freeze-dried and sent to the electron microscope platform of Huazhong Agricultural University for sample preparation. After gold plating, they were observed by a scanning electron microscope (JSM-6390LV), and the appropriate field of view and magnification were selected to record the image.

[0110] Microstructural changes such as Figure 2 As shown in the figure, the squid minced fish has a loose network structure, a rough and uneven surface, many wrinkles, and a large pore size. Its microstructure does not change significantly with increasing temperature.

[0111] The fried squid patties obtained in Examples 1-7 were subjected to infrared spectroscopy analysis.

[0112] The fried squid patties were freeze-dried, and appropriate amounts of dried potassium bromide powder and fried squid patties samples (potassium bromide: minced fish = 100:1) were fully ground in a mortar and tableted. After tableting, the FT-IR spectrum of the samples was measured using a Fourier transform infrared spectrometer (IS50 Suzhou Ops Plasma Technology Co., Ltd.). The attenuated total reflectance mode was used to measure the FT-IR spectrum of the samples at 4000 to 400 cm -1 The measurement is carried out within the wave number range of 4cm -1 Each spectrum was collected with 64 scans at a resolution of 1000 nm and air as the background.

[0113] Near infrared spectroscopy changes such as Figure 3 As shown in the figure, all squid surimi have characteristic absorption peaks of infrared spectrum of protein (Amide A, B, I, II and III), and the change trend of infrared spectrum is basically the same, but the absorption intensity of each absorption peak is different under different frying temperatures; the absorption peak of amide A of all surimi is at 2920cm -1 The amide I band is red-shifted to 1750 cm -1A strong absorption peak appears at , which is manifested as the C=O stretching vibration of the amide I band.

[0114] The fried squid patties obtained in Examples 1-7 were subjected to interaction force measurement.

[0115] Ionic bond determination: Weigh 1 g of the fried squid patties sample obtained in Example 1-6, add 10 mL of A1 extract (0.6 mol / L NaCl), homogenize at 5 000 r / min for 2 min, place at 4°C for 1 h, and centrifuge at 4°C and 18 600 r / min for 25 min. The supernatant (A) is stored at 4°C.

[0116] Hydrogen bond determination: Add 10 mL of A2 extract (a mixture of 1.5 mol / L urea and 0.6 mol / L NaCl) to the precipitate obtained by the above centrifugation, homogenize at 5 000 r / min for 2 min, place at 4°C for 1 h, centrifuge at 4°C and 18 600 r / min for 25 min, and store the supernatant (B) at 4°C.

[0117] Hydrophobic interaction determination: Add 10 mL of A3 extract (a mixture of 8 mol / L urea and 0.6 mol / L NaCl) to the above centrifugal precipitate, homogenize at 5,000 r / min for 2 min, place at 4°C for 1 h, centrifuge at 4°C and 18,600 r / min for 25 min, and store the supernatant at 4°C. Repeat this step, and combine the supernatants (C) obtained from the two centrifugations and store them at 4°C.

[0118] Disulfide bond determination: continue to add 10mL of A4 extract (a mixture of 0.5mol / L β-mercaptoethanol, 0.6mol / L NaCl and 8mol / L urea, pH 7) to the precipitate, homogenize at 5000r / min for 2min, place at 4℃ for 1h, centrifuge at 4℃ and 18,600r / min, and store the supernatant (D) at 4℃.

[0119] After centrifugation in the above steps, the supernatant obtained was added with an equal volume of 20% TCA, centrifuged at 3950r / min for 15min, the supernatant was discarded, 1mL of 1mol / L NaOH solution was added to the precipitate and placed at 4°C, and the protein content was determined by the biuret method. The contribution of ionic bonds, hydrogen bonds, hydrophobic interactions, and disulfide bonds were expressed as the relative mass fractions of proteins dissolved in A, B, C, and D, respectively. The interaction between protein molecules determines the stability of the fish paste network structure.

[0120] Figure 4The intermolecular interactions of squid surimi at different frying temperatures were shown. Among the chemical interactions between surimi protein molecules in fried squid patties, hydrophobic interactions are dominant, followed by disulfide bonds, ionic bonds, and hydrogen bonds. The hydrophobic interactions at 120°C are significantly higher than those at other frying temperatures.

[0121] Zeta potential measurement of the fried squid patties obtained in Example 1-7

[0122] The fried squid patties thawed in the refrigerator were cut into small pieces and crushed in a chopper. A certain amount of pure water was added to prepare a solution with a mass concentration of 10 mg / mL. The solution was stirred at 5000 r / min for 2 min using a high-speed homogenizer, and then the solution was centrifuged (8000 r / min, 2 min) to obtain the sample to be tested. The Zeta potential of each group of solutions was measured using a Malvern nanoparticle size potential analyzer. The solution to be tested was carefully injected into the sample pool to avoid bubbles that would affect the results. The two sections of the electrode were kept flat during the test. The test temperature was set to 25°C, the equilibrium time was 30 s, and each sample was measured 3 times to take the average value.

[0123] like Figure 5 As shown in the figure, the Zeta potential of all samples is negative, indicating that most of the amino acids on the surface of the samples are negatively charged. The higher the absolute value of the potential, the stronger its ability to resist intermolecular aggregation through electrostatic repulsion. As the frying temperature increases, the Zeta potential of the surimi decreases first, then increases, and then decreases. The absolute value of the Zeta potential of the surimi reaches a maximum value of 16.27mV at 120℃. The results show that the frying temperature affects the Zeta potential of squid surimi.

[0124] The fried squid patties obtained in Examples 1-7 were analyzed for hydrophobicity

[0125] After the fried squid patties were freeze-dried, 500 mg of the sample was ground in a mortar and then made into thin slices by an infrared tablet press. The contact angle of ultrapure water on the fish paste thin slices was measured by a contact angle analyzer (Dataphysics OCA15EC). The volume of ultrapure water was 3 μL, and three parallel measurements were made for each sample. The contact angle formed by ultrapure water on the thin slice was calculated by the online professional software SCA20. The contact angle describes the ability of a liquid to spread on a solid surface and reflects the hydrophobicity of the surface of an object.

[0126] Figure 6 The contact angles of squid minced fish under different treatments are shown. All θ values ​​are less than 90°, indicating that all surimi samples are dominated by hydrophilic behavior. At different frying temperatures, the contact angle change trends are basically the same, and the hydrophobicity increases with the increase of frying temperature. The results show that frying temperature affects the hydrophobicity of squid minced fish.

[0127] Therefore, it can be concluded that high temperature treatment weakens the protein structure and the non-covalent forces between and within protein molecules, resulting in the expansion of the pore size of surimi and the loosening of the ultramicrostructure.

[0128] High temperature treatment induces the network structure "skeleton" of fish paste gel to change from tight to loose, and the pore size expands and becomes hollow. Under high temperature, the microscopic network structure of squid paste gel is destroyed and becomes loose, and the pore size increases.

[0129] The mesh structure "skeleton" of the squid sample fried at high temperature changes from tight to loose, and the pore size expands. The formation of MeIQx is through the weakening of non-covalent forces by high temperature, which destroys the microstructure of the squid paste gel, degrades the MeIQx precursor, and transforms into MeIQx through the MeIQx generation cascade chemical reaction.

[0130] High temperature induces the hydrophobic groups of surimi molecules to condense into aggregates. The generated MeIQx groups may be wrapped in the aggregates, and the interaction between the aggregates is destroyed by high temperature, resulting in the loosening of the ultrafine network structure and forming a single aggregate.

[0131] The chemical forces of surimi, such as hydrophobic interaction, ionic bond, hydrogen bond, disulfide bond, etc., are the key to the stability of surimi network structure. Ionic bond affects the interaction between protein molecules, and hydrogen bond mainly maintains the secondary structure of protein. High temperature leads to an increase in the content of hydrogen bond and disulfide bond in surimi, a decrease in hydrophobic interaction, and a loose network structure. As a result, the amount of MeIQx generated gradually increases with the increase of frying temperature.

[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting MeIQx in fried aquatic products, characterized in that: The following steps are involved: Step 1: Prepare materials, including experimental materials; Step 2: Sample preparation, frying the aquatic products at different oil temperatures to obtain fried samples; Step 3: Detect MeIQx. Use UHPLC-MS to detect the content of MeIQx in fried samples at different oil temperatures.

2. The method for detecting MeIQx in fried aquatic products according to claim 1, characterized in that: The preparation of experimental materials in step 1 includes: fresh squid, soybean oil, hydrochloric acid, methanol, ethanol and distilled water.

3. The method for detecting MeIQx in fried aquatic products according to claim 1, characterized in that: The specific process of preparing the fried sample in step 2 is: Step 2.1: The viscera, inner shell and outer skin of the fresh squid were removed and cleaned, and then the squid was cut into small pieces of 3 cm×3 cm, and the muscle tissue was crushed with a meat grinder. The crushed squid was soaked in 25% anhydrous ethanol for 1 hour, and the mixture of ethanol and oil was discarded. The above steps were repeated three times until the desalination was complete and the ethanol in the squid minced meat was completely volatilized. Then 1 kg of squid minced meat and 25 g of salt were put into a minced meat mixer and stirred evenly to obtain a sample; Step 2.2: Place the sample in a culture dish with a radius of 35 mm, and place food-grade paper at the bottom of the culture dish to make 1 cm thick patties, and wrap them with sealing film and set them at 4°C for 1 hour. Put the shaped round cakes into an oil bath at an oil temperature of 100-220°C for frying. After the fried squid patties are cooled, vacuum-pack them, and store them in a freezer at a temperature of -18°C.

4. The method for detecting MeIQx in fried aquatic products according to claim 1, characterized in that: The oil temperature for oil bath frying is 100℃, 120℃, 140℃, 160℃, 180℃, 200℃ and 220℃.

5. The method for detecting MeIQx in fried aquatic products according to claim 1, characterized in that: The specific process of using UHPLC-MS to detect the content of MeIQx in fried samples in step 3 is as follows: Step 3.1: Dry and grind the fried squid patties into powder, weigh 4 g and add them into 25 mL of 0.1 mol / L hydrochloric acid for homogenization for 1 min, 15 s / time, for 4 times in total, and then ultrasonically extract for 15 min; Step 3.2: Then, centrifuge at 10,000 r / min for 20 min at 4°C and collect the supernatant, add 25 mL of hydrochloric acid to extract the precipitate again, rotary evaporate the supernatant, combine and concentrate, fix the volume to 25 mL with distilled water, remove the protein by ethanol-freezing method, and then filter with 0.22 μm filter membrane and perform UHPLC-MS detection using Waters Atlantics dC18 column.

6. The method for detecting MeIQx in fried aquatic products according to claim 5, characterized in that: UHPLC chromatographic conditions are as follows: Mobile phase: 0.1% formic acid aqueous solution (A)-methanol (B); gradient elution: 0-0.1min, 5% B; 0.1-10min, 5%-100% B; 10-10.5min, 100%-5% B; 10.5-15.5min, 5% B; flow rate was 0.5mL / min, injection volume was 1μL, and column temperature was 35°C.

7. The method for detecting MeIQx in fried aquatic products according to claim 5, characterized in that: The mass spectrometry detection conditions were as follows: electrospray ionization source ionization, positive ion mode, source temperature 130°C, MRM mode, capillary voltage 3.5V; in addition, nitrogen was used as the desolvation gas and cone gas, with flow rates of 800L / h and 50L / h respectively, the desolvation temperature was 350°C, and argon was used as the collision gas, with a flow rate of 0.13mL / min.

Images