A method for determining the content of photoinitiators in cell-cultured fish meat

Abstract

The present invention provides a method for determining the content of photoinitiators in cell-cultured fish meat, belonging to the technical field of photoinitiator removal. In the present invention, the cell-cultured fish meat to be tested is mixed with a lysis solution for incubation treatment to obtain a test solution; the test solution is subjected to high-performance liquid chromatography analysis, and according to the photoinitiator standard curve and the chromatogram of the obtained test solution, the content of photoinitiators in the cell-cultured fish meat to be tested is obtained. The present invention uses a lysis solution to treat the cell-cultured fish meat to be tested, which can effectively liquefy the cell-cultured fish meat to be tested and decompose the cell structure, thereby facilitating the accurate determination of photoinitiators through high-performance liquid chromatography analysis. Further, the present invention can use a cyclodextrin metal-organic framework material as an adsorbent material to adsorb the incubation solution obtained after the incubation treatment, and then after desorption treatment, the obtained test solution is subjected to high-performance liquid chromatography analysis, which can further purify the test solution and improve the sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of photoinitiator removal, and particularly relates to a method for determining the content of photoinitiators in cell-cultured fish meat. Background Art

[0002] Cell-cultured fish meat is a new technology for producing edible fish products by culturing animal tissue stem cells, aiming to provide flavors, nutrients, and textures similar to traditional fish meat. Photoinitiators such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) are usually used in the processing of cell-cultured fish meat. LAP releases lithium ions. In medicine, lithium ions can be used to treat bipolar disorder, and the content range of lithium ions in serum is 0.4 - 0.75 mmol / L when used to treat bipolar disorder. However, too high a concentration of lithium ions will affect human health. For example, the acute toxicity of lithium ions starts at 1.4 mmol / L, and a concentration higher than 3.5 mmol / L is considered toxic to patients, which may cause neurological diseases, tubulointerstitial nephropathy, and nephrogenic diabetes insipidus, etc. Therefore, there are certain health risks in consuming cell-cultured fish meat containing residues of photoinitiators such as LAP, and accurately determining the content of photoinitiators in cell-cultured fish meat is a technical problem that needs to be solved currently. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for determining the content of photoinitiators in cell-cultured fish meat, and the method of the present invention can accurately determine the content of photoinitiators in cell-cultured fish meat.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a method for determining the content of photoinitiators in cell-cultured fish meat, including the following steps:

[0006] Mix the cell-cultured fish meat to be tested with a lysis solution for incubation treatment to obtain a test solution; the lysis solution includes methacrylated gelatin, DMEM medium, and water;

[0007] Perform high-performance liquid chromatography analysis on the test solution to obtain a chromatogram of the test solution;

[0008] According to the photoinitiator standard curve and the chromatogram of the test solution, obtain the content of photoinitiators in the cell-cultured fish meat to be tested.

[0009] Preferably, after the incubation treatment, it further includes: mixing the incubation solution obtained after the incubation treatment with a cyclodextrin metal-organic framework material for adsorption treatment to obtain a solid complex; mixing the solid complex with water for desorption treatment to obtain the test solution.

[0010] Preferably, the volume ratio of methacrylated gelatin, DMEM medium and water in the lysis solution is 5-7:1000:250-350; the dosage ratio of the fish meat cultured with the cells to be tested and the lysis solution is 50 mg:150-250 μL.

[0011] Preferably, the temperature of the incubation treatment is 20-30 °C, and the time is 1-2 h; the incubation treatment is carried out under light-shielded conditions.

[0012] Preferably, the cyclodextrin metal-organic framework material is prepared from raw materials including γ-cyclodextrin, and the dosage ratio of the cyclodextrin metal-organic framework material and the incubation solution is 50 mg:150-250 μL; the temperature of the adsorption treatment is 20-30 °C, and the time is 1-840 min; the adsorption treatment is carried out under oscillating conditions, and the rotation speed of the oscillation ≤ 800 rpm.

[0013] Preferably, when performing the desorption treatment, the mass ratio of the solid complex to water is 1-5:5-10; the temperature of the desorption treatment is 20-30 °C, and the time is 30-300 min.

[0014] Preferably, the photoinitiator includes one or more of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, and benzoin diethyl ether.

[0015] Preferably, when the photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, the conditions for the high performance liquid chromatography analysis include:

[0016] The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and mobile phase B is acetonitrile; the volume ratio of mobile phase A to mobile phase B is 60:40; a PDA detector is used, and the detection wavelength is set at 220 nm.

[0017] Preferably, when the photoinitiator is 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, the conditions for the high performance liquid chromatography analysis include:

[0018] The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and mobile phase B is acetonitrile; the volume ratio of mobile phase A to mobile phase B is 60:40; a PDA detector is used, and the detection wavelength is set at 275 nm.

[0019] Preferably, when the photoinitiator is benzoin diethyl ether, the conditions for the high performance liquid chromatography analysis include:

[0020] The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and mobile phase B is acetonitrile. The volume ratio of mobile phase A to mobile phase B is 20:80. A PDA detector is used, and the detection wavelength is set at 252 nm.

[0021] The present invention provides a method for determining the content of photoinitiator in cell-cultured fish meat, which includes the following steps: mixing the cell-cultured fish meat to be tested with a lysis solution for incubation treatment to obtain a test solution; the lysis solution includes methacrylated gelatin, DMEM medium and water; performing high-performance liquid chromatography analysis on the test solution to obtain a chromatogram of the test solution; and obtaining the content of the photoinitiator in the cell-cultured fish meat to be tested according to the photoinitiator standard curve and the chromatogram of the test solution. The present invention uses a lysis solution to treat the cell-cultured fish meat to be tested, which can effectively liquefy the cell-cultured fish meat to be tested and decompose the cell structure, thereby facilitating the accurate determination of the photoinitiator through high-performance liquid chromatography analysis.

[0022] Further, the present invention can use a cyclodextrin metal-organic framework material as an adsorption material to adsorb the incubation solution obtained after the incubation treatment, and then perform desorption treatment, and then perform high-performance liquid chromatography analysis on the obtained test solution, which can further purify the test solution and improve the sensitivity. Description of the Drawings

[0023] Figure 1 It is a graph showing the change of LAP content in cell-cultured fish meat over time;

[0024] Figure 2 It is a standard curve graph of LAP in aqueous solution;

[0025] Figure 3 It is a flow chart of spiked analysis;

[0026] Figure 4 It is a scanning electron microscope image of CD-MOF;

[0027] Figure 5 It is a scanning electron microscope image of LAP-CDMOF;

[0028] Figure 6 It is a scanning electron microscope image of Irgacure 2959-CDMOF;

[0029] Figure 7 It is a scanning electron microscope image of Irgacure 651-CDMOF;

[0030] Figure 8 It is an X-ray diffraction pattern of CD-MOF before and after adsorbing LAP;

[0031] Figure 9X-ray diffraction patterns of CD-MOF before and after adsorbing Irgacure 2959;

[0032] Figure 10 X-ray diffraction patterns of CD-MOF before and after adsorbing Irgacure 651;

[0033] Figure 11 Infrared spectrum diffraction patterns of CD-MOF before and after adsorbing LAP;

[0034] Figure 12 Infrared spectrum diffraction patterns of CD-MOF before and after adsorbing Irgacure 2959;

[0035] Figure 13 Infrared spectrum diffraction patterns of CD-MOF before and after adsorbing Irgacure 651;

[0036] Figure 14 Thermogravimetric analysis diagrams of CD-MOF before and after adsorbing LAP;

[0037] Figure 15 Thermogravimetric analysis diagrams of CD-MOF before and after adsorbing Irgacure 2959;

[0038] Figure 16 Thermogravimetric analysis diagrams of CD-MOF before and after adsorbing Irgacure 651;

[0039] Figure 17 Nitrogen adsorption / desorption isotherm curves of CD-MOF before and after adsorbing LAP;

[0040] Figure 18 Nitrogen adsorption / desorption isotherm curves of CD-MOF before and after adsorbing Irgacure 2959;

[0041] Figure 19 Nitrogen adsorption / desorption isotherm curves of CD-MOF before and after adsorbing Irgacure 651;

[0042] Figure 20 Results diagram of condition optimization for CD-MOF adsorbing LAP;

[0043] Figure 21 Results diagram of condition optimization for CD-MOF adsorbing Irgacure 2959;

[0044] Figure 22 Results diagram of condition optimization for CD-MOF adsorbing Irgacure 651;

[0045] Figure 23 Adsorption performance comparison diagram of γ-CD and CD-MOF prepared with equimolar amount of γ-CD for three photoinitiators;

[0046] Figure 24 The figure shows the results of fitting the adsorption process of LAP by CD-MOF with the Freundlich model;

[0047] Figure 25 The figure shows the results of fitting the adsorption process of Irgacure 2959 by CD-MOF with the Freundlich model;

[0048] Figure 26 The figure shows the results of fitting the adsorption process of Irgacure 651 by CD-MOF with the Freundlich model;

[0049] Figure 27 The figure shows the results of fitting the adsorption process of LAP by CD-MOF with the Langmuir model;

[0050] Figure 28 The figure shows the results of fitting the adsorption process of Irgacure 2959 by CD-MOF with the Langmuir model;

[0051] Figure 29 The figure shows the results of fitting the adsorption process of Irgacure 651 by CD-MOF with the Langmuir model. Detailed implementation method

[0052] The present invention provides a method for determining the content of photoinitiator in cell-cultured fish meat, comprising the following steps:

[0053] Mix the cell-cultured fish meat to be tested with a lysis solution for incubation treatment to obtain a test solution; the lysis solution includes methacrylated gelatin, DMEM medium and water;

[0054] Perform high performance liquid chromatography analysis on the test solution to obtain a chromatogram of the test solution;

[0055] According to the photoinitiator standard curve and the chromatogram of the test solution, obtain the content of the photoinitiator in the cell-cultured fish meat to be tested.

[0056] In the present invention, unless otherwise specified, the raw materials used are commercially available products well-known to those skilled in the art or are prepared by methods well-known to those skilled in the art.

[0057] In the present invention, the fish meat cultured from the cells to be tested is mixed with a lysis solution and incubated to obtain a test solution. As an embodiment of the present invention, the fish meat cultured from the cells to be tested is obtained by referring to the method in the literature (Tissue-like cultured fish fillets through a synthetic food pipeline; Xu, Enbo; Niu, Ruihao; Lao, Jihui; Zhang, Shengliang; Li, Jie; Zhu, Yiyuan; Shi, Huimin; Zhu, Qingqing; Chen, Yijian; Jiang, Yuyan; Wang, Wenjun; Yin, Jun; Chen, Qihe; Huang, Xiao; Chen, Jun; Liu, Donghong; NPJ Science of Food, 2023, Vol 7, Issue 1, p1).

[0058] The lysis solution of the present invention comprises methacrylated gelatin, DMEM medium and water. The volume ratio of methacrylated gelatin (GelMA), DMEM medium and water in the lysis solution is 5-7:1000:250-350, further can be 5.5-6.5:1000:280-320, and specifically can be 6:1000:300; the DMEM medium is a commercially available product well-known to those skilled in the art. As an embodiment of the present invention, the dosage ratio of the fish meat cultured from the cells to be tested to the lysis solution can be 50 mg: 150-250 μL, further can be 50 mg: 180-220 μL, and specifically can be 50 mg: 200 μL.

[0059] As an embodiment of the present invention, the temperature of the incubation treatment can be 20-30 °C, specifically can be 25 °C; the time can be 1-2 h, specifically can be 1.5 h; the incubation treatment is preferably carried out under light-shielded conditions. In the examples of the present invention, the lysis solution with the above composition is used for incubation treatment under the above conditions, which can effectively lyse the tissue structure of the fish meat cultured from the cells, and is beneficial to the accurate determination of the residual amount of photoinitiator in the subsequent fish meat tissue. As an embodiment of the present invention, after the incubation treatment, it may further include: performing solid-liquid separation on the product system obtained after the incubation treatment, and collecting the liquid material (i.e., the incubation solution) as the test solution; the method of solid-liquid separation can be centrifugation, the rotation speed of the centrifugation can be 8000-1200 rpm, specifically can be 1000 rpm; the time of the centrifugation can be 5-15 min, specifically can be 10 min.

[0060] Metal-organic framework materials (MOF) are porous coordination materials formed by the coordination or covalent bonds of organic ligands and metal ion centers. They have the advantages of high porosity, large specific surface area, and good stability. They are a rapidly developing new type of porous material with broad application prospects. Cyclodextrin metal-organic framework materials (CD-MOF) are a new type of metal-organic framework material formed by combining cyclodextrin and alkali metal ions through organic coordination. Compared with traditional metal-organic framework materials, CD-MOF not only has the characteristics of high porosity and large specific surface area. For example, taking the CD-MOF prepared with γ-cyclodextrin (γ-CD) as an example, it contains a large number of spherical cavities with a diameter of about 1.7 nm at the center of the cube, and its specific surface area is about 900 m 2 / g. At the same time, CD-MOF also has the characteristics of good biocompatibility and non-toxicity. Its superior cavity structure enables it to be used as an adsorption material. As an implementation mode of the present invention, in order to further purify the test solution and improve the sensitivity, the present invention can use cyclodextrin metal-organic framework materials as an adsorption material to perform adsorption treatment on the incubation solution obtained after incubation treatment, and then obtain the test solution through desorption treatment. Specifically, after the incubation treatment, it may further include: mixing the incubation solution obtained after incubation treatment with cyclodextrin metal-organic framework materials for adsorption treatment to obtain a solid complex; mixing the solid complex with water for desorption treatment to obtain the test solution. The following is a detailed description.

[0061] In the present invention, the incubation solution obtained after incubation treatment is mixed with a cyclodextrin metal-organic framework material for adsorption treatment to obtain a solid complex. As an embodiment of the present invention, the incubation solution is specifically the liquid material obtained by subjecting the product system obtained after incubation treatment to solid-liquid separation with reference to the above scheme. As an embodiment of the present invention, the cyclodextrin metal-organic framework material can be prepared from raw materials including γ-cyclodextrin (γ-CD) (its preparation method will be described in detail later); in the present invention, unless otherwise specified, the cyclodextrin metal-organic framework materials used are all cyclodextrin metal-organic framework materials prepared from raw materials including γ-cyclodextrin; the dosage ratio of the cyclodextrin metal-organic framework material to the incubation solution can be 50 mg: 150 - 250 μL, further can be 50 mg: 180 - 220 μL, and specifically can be 50 mg: 200 μL. As an embodiment of the present invention, the temperature of the adsorption treatment can be 20 - 30 °C, specifically can be 25 °C; the time can be 1 - 840 min, specifically can be 1 min, 10 min, 30 min, 60 min, 180 min, 360 min, 720 min or 840 min. As an embodiment of the present invention, the adsorption treatment can be carried out under oscillating conditions. When the adsorption treatment is carried out under oscillating conditions, the rotation speed of the oscillation ≤ 800 rpm, specifically can be 100 rpm, 200 rpm, 400 rpm, 600 rpm or 800 rpm; the adsorption treatment of the present invention can also be carried out without oscillating conditions (i.e., the rotation speed of the oscillation is 0 rpm). As an embodiment of the present invention, solid-liquid separation is further included after the adsorption treatment, and the solid material collected is the solid complex; the method of the solid-liquid separation can be centrifugation; the rotation speed of the centrifugation can be 8000 - 1200 rpm, specifically can be 1000 rpm; the time of the centrifugation can be 5 - 15 min, specifically can be 10 min. The solid complex of the present invention is a cyclodextrin metal-organic framework material adsorbed with a photoinitiator, denoted as photoinitiator-CD-MOF (specifically can be LAP-CDMOF, Irgacure 2959-CDMOF or Irgacure 651-CDMOF according to the type of the photoinitiator).

[0062] After obtaining the solid complex, the present invention mixes the solid complex with water for desorption treatment to obtain a test solution. As an embodiment of the present invention, when performing the desorption treatment, the mass ratio of the solid complex to water can be 1-5:5-10, further can be 1:5-8, specifically can be 1:5, 1:6, 1:7 or 1:8; the temperature of the desorption treatment can be 20-30°C, specifically can be 25°C; the time of the desorption treatment can be 30-300 min, further can be 45-100 min, specifically can be 50 min, 60 min, 70 min or 80 min. As an embodiment of the present invention, after the desorption treatment, it may further include: performing solid-liquid separation on the product system obtained after the desorption treatment, and collecting the liquid material as the test solution; the method of solid-liquid separation can be centrifugation, the rotation speed of the centrifugation can be 8000-1200 rpm, specifically can be 1000 rpm; the time of the centrifugation can be 5-15 min, specifically can be 10 min.

[0063] After obtaining the test solution, the present invention performs high performance liquid chromatography analysis on the test solution to obtain a chromatogram of the test solution. As an embodiment of the present invention, specifically, appropriate conditions are selected for high performance liquid chromatography analysis according to the specific type of initiator. As an embodiment of the present invention, the photoinitiator can include one or more of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone and benzoin diethyl ether, specifically can be lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP, CAS No. 85073-19-4), 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (Irgacure 2959, CAS No. 106797-53-9) or benzoin diethyl ether (Irgacure 651, CAS No. 24650-42-8).

[0064] As an embodiment of the present invention, when the photoinitiator is LAP, the conditions for high performance liquid chromatography analysis can include: the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and the mobile phase B is acetonitrile; the volume ratio of the mobile phase A to the mobile phase B is 60:40; a PDA detector is used, and the detection wavelength is set to 220 nm. As an embodiment of the present invention, when the photoinitiator is LAP, the high performance liquid chromatography system used for high performance liquid chromatography analysis can be a Shimadzu LC-20 high performance liquid chromatography system, equipped with a Welch XB-C18 chromatographic column (4.6×250 mm, 5 μm); the elution time can be 15 min; the injection volume can be 5 μL; the flow rate of the mobile phase can be 1 mL / min, and the column temperature can be 30°C.

[0065] As an embodiment of the present invention, when the photoinitiator is Irgacure 2959, the conditions for the high performance liquid chromatography analysis include: the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and the mobile phase B is acetonitrile; the volume ratio of the mobile phase A to the mobile phase B is 60:40; a PDA detector is used, and the detection wavelength is set to 275 nm. As an embodiment of the present invention, when the photoinitiator is Irgacure 2959, the high performance liquid chromatography system used for the high performance liquid chromatography analysis can be a Shimadzu LC-20 high performance liquid chromatography system, equipped with a Welch XB-C18 chromatographic column (4.6×250 mm, 5 μm); the elution time can be 10 min; the injection volume can be 10 μL; the flow rate of the mobile phase can be 1 mL / min, and the column temperature can be 30 °C.

[0066] As an embodiment of the present invention, when the photoinitiator is Irgacure 651, the conditions for the high performance liquid chromatography analysis include: the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and the mobile phase B is acetonitrile; the volume ratio of the mobile phase A to the mobile phase B is 20:80; a PDA detector is used, and the detection wavelength is set to 252 nm. As an embodiment of the present invention, when the photoinitiator is Irgacure 651, the high performance liquid chromatography system used for the high performance liquid chromatography analysis can be a Shimadzu LC-20 high performance liquid chromatography system, equipped with a Welch XB-C18 chromatographic column (4.6×250 mm, 5 μm); the elution time can be 10 min; the injection volume can be 20 μL; the flow rate of the mobile phase can be 1 mL / min, and the column temperature can be 30 °C.

[0067] After obtaining the chromatogram of the test solution, the present invention obtains the content of the photoinitiator in the test cell-cultured fish meat according to the photoinitiator standard curve and the chromatogram of the test solution. As an embodiment of the present invention, the photoinitiator standard curve can include a LAP standard curve, an Irgacure 2959 standard curve or an Irgacure 651 standard curve. Taking the LAP standard curve as an example, specifically, the LAP standard product is dissolved in water to obtain a continuous dilution solution with a concentration of 15.625 - 2000 μg / mL as the LAP standard solution, and then the high performance liquid chromatography analysis is carried out with reference to the above technical solution. The LAP standard curve is plotted according to the LAP chromatographic peak area (ordinate) and the mass concentration of the LAP standard solution (abscissa); the Irgacure 2959 standard curve and the Irgacure 651 standard curve are plotted with reference to the LAP standard curve plotting method, which will not be elaborated here.

[0068] Based on the standard curve of the photoinitiator and the chromatogram of the test solution, the content of the photoinitiator in the test solution can be obtained, and on this basis, the content of the photoinitiator in the test cell-cultured fish meat can be obtained.

[0069] Based on the residual problem of photoinitiators in cell-cultured fish meat, the present invention constructs a method for quantitatively detecting photoinitiators (LAP, Irgacure 2959, and Irgacure 651) in cell-cultured fish meat by high performance liquid chromatography (HPLC). When pretreating the test cell-cultured fish meat, after treating the test cell-cultured fish meat with a lysis solution, cyclodextrin metal-organic framework material (CD-MOF) is used as an adsorbent material to adsorb the incubated solution obtained after incubation treatment. After desorption treatment, the obtained test solution is then subjected to high performance liquid chromatography analysis, which can further purify the test solution and improve the sensitivity. Specifically, in the test examples of the present invention, the structure and physical properties of CD-MOF after adsorbing photoinitiators were characterized, and the adsorption law and adsorption capacity of CD-MOF were explored. The adsorption isotherm was used to clarify the adsorption mechanism, quantify the affinity between CD-MOF and photoinitiators, and explore the adsorption kinetics of CD-MOF for photoinitiators based on the pseudo-first-order kinetic model and the pseudo-second-order kinetic model. Taking LAP as the photoinitiator as an example, the experimental results show that the Freundlich model is more suitable than the Langmuir model for describing the adsorption process of CD-MOF for LAP (R 2 = 0.9927), the pseudo-second-order kinetic model has a higher fitting degree for the experimental data (R 2 = 0.9999), and the maximum adsorption capacity of CD-MOF for LAP can reach 88.19 ± 3.65 mg / g. Finally, a standard addition recovery experiment was carried out on the cell-cultured fish meat sample in the present invention, and the results show that the recovery rate of CD-MOF for LAP in the cell-cultured fish meat sample is as high as 97.14 - 98.78%, verifying the accuracy of the method of the present invention.

[0070] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

[0071] The preparation method of CD-MOF used in the following experiments of the present invention includes the following steps:

[0072] Add γ-cyclodextrin (2962 mg, 2.28 mmol), potassium hydroxide (1024 mg, 18.24 mmol) and ultrapure water (80 mL) into a beaker, ultrasonically oscillate for 1 min at room temperature and filter through a 0.45 μm aqueous filter membrane to obtain Solution 1; pre-place methanol (48 mL) in a 250 mL conical flask, then add the above Solution 1 to obtain a milky white Solution 2, place the conical flask in a water bath at 90 °C, and react for 4 min at a rotation speed of 400 rpm to obtain a clear and transparent Solution 3; quickly add polyethylene glycol (PEG-8000, 1024 mg) to the above Solution 3, react at 90 °C for 10 min, after the reaction, let the obtained product system stand in a cold water bath (24 °C) for 12 h, after standing, collect the precipitate and wash it 3 times by centrifugation with methanol, put the washed precipitate into a vacuum drying oven, dry it at 60 °C under vacuum conditions for 5 h, and then cool it to room temperature to obtain CD-MOF (particle size 5 - 10 μm).

[0073] In the following experiments of the present invention, CD-MOF is used to adsorb the photoinitiator, and the adsorption capacity Q of CD-MOF for the photoinitiator is calculated according to Equation 1:

[0074] Q=(C o −C e )V / m Equation 1;

[0075] In Equation 1, Q is the adsorption capacity of CD-MOF for the photoinitiator at the adsorption time t, mg / g; C o is the initial concentration of the photoinitiator, mg / mL; C e is the equilibrium concentration of the photoinitiator at the adsorption time t, mg / mL; V is the volume of the reaction system, mL; m is the mass of the used CD-MOF, mg.

[0076] Example 1

[0077] Weigh 50 mg of the cell-cultured fish meat sample (obtained by referring to the method in the literature: Tissue-like cultured fish fillets through a synthetic food pipeline; Xu, Enbo; Niu, Ruihao; Lao, Jihui; Zhang, Shengliang; Li, Jie; Zhu, Yiyuan; Shi, Huimin; Zhu, Qingqing; Chen, Yijian; Jiang, Yuyan; Wang, Wenjun; Yin, Jun; Chen, Qihe; Huang, Xiao; Chen, Jun; Liu, Donghong; NPJ Science of Food, 2023, Vol 7, Issue 1, p1) and transfer it to a centrifuge tube. Add 200 μL of gelatin methacrylate (GelMA) lysis solution, which is prepared by mixing 6 μL of GelMA, 1 mL of DMEM medium, and 300 μL of pure water. Then, incubate the centrifuge tube at room temperature (25 °C) in the dark for 90 min. After the incubation, centrifuge the resulting liquid at 1000 rpm for 10 min, and collect the supernatant as the test solution. Subsequently, perform HPLC analysis on the test solution to determine the photoinitiator content in the cell-cultured fish meat sample;

[0078] The photoinitiator includes LAP, Irgacure 2959, or Irgacure 651. The relevant HPLC analysis conditions are as follows:

[0079] The conditions for quantitative detection of LAP are as follows: Use a Shimadzu LC-20 high-performance liquid chromatography system equipped with a Welch XB-C18 chromatographic column (4.6×250 mm, 5 μm); The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and mobile phase B is acetonitrile; Isocratic elution, the volume ratio of mobile phase A to mobile phase B is 60:40, and the elution time is 15 min; The injection volume is 5 μL, the flow rate is 1 mL / min, and the column temperature is 30 °C; Use a PDA detector for detection, and set the detection wavelength to 220 nm;

[0080] The conditions for quantitative detection of Irgacure 2959 refer to the conditions for quantitative detection of LAP, with the differences being: the elution time is 10 min, the injection volume is 10 μL, and the detection wavelength is set to 275 nm;

[0081] The conditions for quantitative detection of Irgacure 651 were similar to those for quantitative detection of LAP, except that the volume ratio of mobile phase A to mobile phase B was 20:80, the elution time was 10 min, the injection volume was 20 μL, and the detection wavelength was set at 252 nm.

[0082] When quantitatively detecting photoinitiators, taking LAP as an example, the LAP standard is dissolved in pure water and a series of dilutions with concentrations of 15.625~2000μg / mL are set as standard solutions to prepare the LAP standard curve. The Irgacure2959 standard curve and the Irgacure 651 standard curve follow the same conditions.

[0083] Example 2

[0084] The incubation treatment was performed according to the method of Example 1, specifically, 50 mg of the cell culture fish sample was weighed and transferred to a centrifuge tube, and 200 μL of methacrylated gelatin (GelMA) lysate was added, wherein the GelMA lysate was mixed with 6 μL of GelMA, 1 mL of DMEM culture medium and 300 μL of purified water; then the centrifuge tube was incubated at room temperature (25°C) and in the dark for 90 minutes. After the incubation treatment, the obtained feed solution was centrifuged at a speed of 1000 rpm for 10 minutes, and the supernatant was collected; 200 μL of the supernatant was mixed with 50 mg of CD-MOF , adsorption treatment was carried out at a temperature of 25°C and an oscillation speed of 600 rpm for 6 hours, and then centrifuged at 1000 rpm for 10 minutes, and the solid material was collected as the initiator-CD-MOF; the initiator-CD-MOF was mixed with water in a mass ratio of 1:5, and desorption treatment was carried out at a temperature of 25°C for 60 minutes. After the desorption treatment, the obtained feed liquid was centrifuged at a speed of 1000 rpm for 10 minutes, and the supernatant was collected as the test liquid; then the test liquid was subjected to HPLC analysis according to the method of Example 1 to determine the content of the photoinitiator in the cell cultured fish meat sample.

[0085] Test Example 1

[0086] Taking LAP as the photoinitiator as an example, in order to evaluate the changes in the LAP content in the cell-cultured fish meat during the culture process of the cell-cultured fish meat, samples were collected at four time points: day 0 (initial), day 1, day 10 and day 20 of culture, and the samples were treated by the GelMA lysis method in Example 1 and analyzed by HPLC.

[0087] Figure 1Figure showing the change in LAP content in cell-cultured fish meat over time. DMEM medium was used as a control. The results showed that on day 0, the LAP concentration ranged from 211.28 to 339.30 μg / mL. From day 1 to day 20, the LAP concentration in cell-cultured fish meat decreased to 33 - 105 μg / mL, but its content was still much higher than the 1.1 - 1.7 μg / mL in the DMEM medium control. This indicates that LAP residues were present throughout the entire culture process of the cell-cultured fish meat samples.

[0088] Test Example 2

[0089] The LAP standard was dissolved in pure water and a series of dilutions with concentrations ranging from 15.625 to 2000 μg / mL were set as the LAP standard solution. Then, the LAP standard solution was detected using the HPLC method in Example 1, and the LAP standard curve was plotted.

[0090] Figure 2 Figure of the LAP standard curve in aqueous solution. The results showed that R 2 = 0.9999, indicating that the standard curve is reliable.

[0091] Test Example 3

[0092] To verify the accuracy of the detection method of the present invention, according to the Figure 3 flow chart shown, a spiking analysis was carried out in this test example. Specifically, incubation treatment was performed according to the method in Example 1. After the incubation treatment, the resulting liquid was centrifuged at 1000 rpm for 10 min, and the supernatant was collected. 190 μL of the supernatant was mixed with 10 μL of an LAP aqueous solution with a concentration of 2 mg / mL, and then 50 mg of CD-MOF was added. Adsorption treatment was carried out at a temperature of 25°C and an oscillation speed of 600 rpm for 6 h. Then, it was centrifuged at 1000 rpm for 10 min, and the solid material collected was LAP-CD-MOF. The LAP-CD-MOF was mixed with water in a mass ratio of 1:5 and desorption treatment was carried out at a temperature of 25°C for 60 min. After the desorption treatment, the resulting liquid was centrifuged at 1000 rpm for 10 min, and the supernatant collected was the spiked sample to be tested. Then, the spiked sample to be tested was analyzed by HPLC according to the method in Example 1 and compared with the detection results of the sample to be tested in Example 1.

[0093] Table 1 shows the recovery data of LAP added to cell-cultured fish meat samples in the present invention. The test concentrations and recovery data in Table 1 are expressed as mean ± deviation. The results showed that the recovery rate of LAP in cell-cultured fish meat samples by CD-MOF was as high as 97.14 - 98.78%, indicating a good recovery effect, which shows that CD-MOF can adsorb and recover the residual photoinitiator in cell-cultured fish meat well.

[0094] Table 1 Recovery data of adding LAP to cell-cultured fish meat samples

[0095]

[0096] Test Example 4

[0097] When the present invention verifies the adsorption effect of the CD-MOF on different types of photoinitiators, specifically, a certain amount of CD-MOF is mixed with an ethanol solution of the photoinitiator and subjected to adsorption treatment to obtain three complexes of LAP-CDMOF, Irgacure 2959-CDMOF, and Irgacure 651-CDMOF adsorbed with photoinitiators LAP, Irgacure 2959, and Irgacure 651, respectively. A single-factor experiment is used to optimize the molar ratio of CD-MOF (based on the molar amount of γ-CD used) to the photoinitiator, the adsorption treatment time, and the oscillation speed during the adsorption treatment. At the same time, the appearance morphology of the complex is observed by scanning electron microscopy, the crystal properties of the complex are analyzed by X-ray diffractometer, the changes in functional groups of the complex are analyzed by Fourier transform infrared spectrometer, and the thermal stability and specific surface area of the complex are analyzed by thermogravimetric analyzer and nitrogen adsorption / desorption curve. Moreover, the adsorption capacities of equimolar amounts of γ-CD and CD-MOF prepared from γ-CD for LAP, Irgacure 2959, and Irgacure are compared. Finally, the adsorption processes of CD-MOF for three different photoinitiators are fitted by Freundlich and Langmuir models. The following is a specific description.

[0098] 1. Complex characterization

[0099] 50 mg of CD-MOF was added to 5 mL of an LAP ethanol solution with a concentration of 0.4 mg / mL (the molar ratio of γ-CD to LAP was 5:1), and adsorption treatment was carried out at 25 °C under the condition of an oscillation speed of 400 rpm for 12 h. The obtained product system was centrifuged at 5000 rpm for 5 min, and the precipitate was collected and vacuum-dried at 60 °C for 6 h to obtain CD-MOF adsorbed with LAP, denoted as LAP-CDMOF.

[0100] Referring to the above method, the difference is that LAP was replaced with Irgacure 2959 and Irgacure 651, respectively, and finally Irgacure 2959-CDMOF and Irgacure 651-CDMOF were obtained.

[0101] Figure 4 is the scanning electron microscope image of CD-MOF, Figure 5Scanning electron microscope image of LAP - CDMOF, Figure 6 Scanning electron microscope image of Irgacure 2959 - CDMOF, Figure 7 Scanning electron microscope image of Irgacure 651 - CDMOF. The results show that after the CD - MOF adsorbs the photoinitiator to form a complex, there is a slight deformation, but it still presents a relatively uniform cubic shape, which is basically the same as the shape of the CD - MOF without adsorbed photoinitiator, indicating that the adsorption of the photoinitiator has little effect on the morphology of CD - MOF.

[0102] Figure 8 X - ray diffraction patterns of CD - MOF before and after adsorbing LAP, Figure 9 X - ray diffraction patterns of CD - MOF before and after adsorbing Irgacure 2959, Figure 10 X - ray diffraction patterns of CD - MOF before and after adsorbing Irgacure 651. The results show that after the CD - MOF adsorbs the photoinitiator, the obtained complex has obvious characteristic peaks at specific angles, which indicates that the CD - MOF has successfully adsorbed the photoinitiator and the formed complexes are all in a crystalline state; however, the characteristic peaks of the complex are weaker than those of CD - MOF and the corresponding photoinitiator before adsorption, which indicates that the adsorption process has changed the crystal structures of CD - MOF and the photoinitiator to a certain extent.

[0103] Figure 11 Infrared spectrum diffraction patterns of CD - MOF before and after adsorbing LAP, Figure 12 Infrared spectrum diffraction patterns of CD - MOF before and after adsorbing Irgacure 2959, Figure 13 Infrared spectrum diffraction patterns of CD - MOF before and after adsorbing Irgacure 651. As Figure 11 shown, LAP - CDMOF has a characteristic peak at 1200 - 1250 cm -1 which may be related to the stretching of the P = O bond, and a weaker characteristic peak at 700 - 800 cm -1 which may be the stretching vibration of the P = C bond. The appearance of these characteristic peaks indicates that LAP has been successfully adsorbed by CD - MOF. Figure 12 And Figure 13 The results also show the characteristic peaks of Irgacure 2959 - CDMOF and Irgacure 651 - CDMOF respectively, which indicates that CD - MOF has also successfully adsorbed two photoinitiators, Irgacure 2959 and Irgacure 651.

[0104] Figure 14 Thermogravimetric analysis diagrams of CD - MOF before and after adsorbing LAP, Figure 15Thermogravimetric analysis diagrams of CD-MOF before and after adsorbing Irgacure 2959 Figure 16 Thermogravimetric analysis diagrams of CD-MOF before and after adsorbing Irgacure 651. The results show that the thermal stability of the complex obtained after CD-MOF adsorbs the photoinitiator is lower than that of CD-MOF, indicating that the thermal stability of CD-MOF is reduced after adsorbing the photoinitiator.

[0105] Figure 17 Nitrogen adsorption / desorption isotherm diagrams of CD-MOF before and after adsorbing LAP Figure 18 Nitrogen adsorption / desorption isotherm diagrams of CD-MOF before and after adsorbing Irgacure 2959 Figure 19 Nitrogen adsorption / desorption isotherm diagrams of CD-MOF before and after adsorbing Irgacure 651. The results show that after CD-MOF adsorbs the photoinitiator, the specific surface area and pore volume of the obtained complex are much lower than those of CD-MOF, indicating that CD-MOF successfully adsorbs the photoinitiator and the adsorbed photoinitiator occupies the internal pore cavities of CD-MOF.

[0106] 2. Condition optimization

[0107] Single-factor experiments were used to optimize the process of CD-MOF adsorbing the photoinitiator to achieve the maximum adsorption capacity of the photoinitiator. Taking the photoinitiator LAP as an example, LAP-CDMOF was prepared according to the method in "1. Complex characterization". Specifically, first, the molar ratio of CD-MOF (based on the molar amount of γ-CD used) to LAP (5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, 1:6, 1:8, 1:10) was optimized; after determining the optimal molar ratio of CD-MOF to adsorb LAP, based on this optimal molar ratio, different adsorption treatment times (1 min, 10 min, 30 min, 60 min, 180 min, 360 min, 720 min, 840 min) were optimized; after determining the optimal molar ratio and adsorption treatment time, based on this, the oscillation speed (0 rpm, 100 rpm, 200 rpm, 400 rpm, 600 rpm, 800 rpm) during the adsorption treatment process was optimized. After each adsorption treatment experiment, the precipitate was collected by centrifugation (5000 rpm, 5 min) and vacuum dried at 60 °C for 6 h to obtain LAP-CDMOF.

[0108] The optimization methods for constructing Irgacure 2959-CDMOF and Irgacure 651-CDMOF are the same as those for LAP-CDMOF, and then the adsorption abilities of CD-MOF for different photoinitiators were compared.

[0109] Figure 20This is the graph of the optimized conditions for the adsorption of LAP by CD-MOF. From left to right, it corresponds to the molar ratio of γ-CD to LAP, the adsorption treatment time, and the oscillation speed in sequence. The results show that when the molar ratio of γ-CD to LAP is 1:5, the adsorption treatment time is 360 min, and the oscillation speed during the adsorption treatment is 600 rpm, the adsorption capacity of CD-MOF for LAP reaches the maximum value of 82.62 ± 3.52 mg / g.

[0110] Figure 21 This is the graph of the optimized conditions for the adsorption of Irgacure 2959 by CD-MOF. From left to right, it corresponds to the molar ratio of γ-CD to LAP, the adsorption treatment time, and the oscillation speed in sequence. The results show that when the molar ratio of γ-CD to Irgacure 2959 is 1:3, the adsorption treatment time is 180 min, and the oscillation speed during the adsorption treatment is 400 rpm, the adsorption capacity of CD-MOF for Irgacure 2959 reaches the maximum value of 60.54 ± 6.39 mg / g.

[0111] Figure 22 This is the graph of the optimized conditions for the adsorption of Irgacure 651 by CD-MOF. From left to right, it corresponds to the molar ratio of γ-CD to LAP, the adsorption treatment time, and the oscillation speed in sequence. The results show that when the molar ratio of γ-CD to Irgacure 651 is 1:3, the adsorption treatment time is 360 min, and the oscillation speed during the reaction is 400 rpm, the adsorption capacity of CD-MOF for Irgacure 651 reaches the maximum value of 45.88 ± 4.27 mg / g.

[0112] 3. Comparison of the adsorption performance of γ-CD and CD-MOF for photoinitiators

[0113] Three photoinitiators were adsorbed using γ-CD and CD-MOF prepared with an equimolar amount of γ-CD. The conditions used for the adsorption treatment of the three photoinitiators were respectively based on Figures 20 - 22 the optimal conditions obtained.

[0114] Figure 23 This is the graph comparing the adsorption performance of γ-CD and CD-MOF prepared with an equimolar amount of γ-CD for three photoinitiators. From left to right, it corresponds to LAP, Irgacure 2959, and Irgacure 651 in sequence. The results show that the adsorption capacity of CD-MOF prepared with an equimolar amount of γ-CD for photoinitiators is much greater than that of γ-CD.

[0115] 4. Adsorption mechanism study

[0116] To quantify the affinity between CD-MOF and photoinitiators and determine the maximum adsorption capacity of CD-MOF for photoinitiators, adsorption isotherms were used to elucidate the adsorption mechanism, and the Freundlich and Langmuir models were used to fit the adsorption processes of CD-MOF for three different photoinitiators. The specific results are as follows:

[0117] Figure 24 Figure showing the result of fitting the adsorption process of CD-MOF for LAP using the Freundlich model Figure 25 Figure showing the result of fitting the adsorption process of CD-MOF for Irgacure 2959 using the Freundlich model Figure 26 Figure showing the result of fitting the adsorption process of CD-MOF for Irgacure 651 using the Freundlich model Figure 27 Figure showing the result of fitting the adsorption process of CD-MOF for LAP using the Langmuir model Figure 28 Figure showing the result of fitting the adsorption process of CD-MOF for Irgacure 2959 using the Langmuir model Figure 29 Figure showing the result of fitting the adsorption process of CD-MOF for Irgacure 651 using the Langmuir model. The results show that, compared with the Langmuir model, the Freundlich model is more suitable for fitting the adsorption process of CD-MOF for photoinitiators. In addition, the maximum adsorption amounts of CD-MOF for photoinitiators were quantified using the Langmuir model. The results indicate that the maximum adsorption amount of CD-MOF for LAP is 106.79 mg / g, for Irgacure 2959 is 52.63 mg / g, and for Irgacure 651 is 21.05 mg / g.

[0118] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for determining the content of photoinitiator in cell-cultured fish meat, comprising the following steps: Mix the cell-cultured fish meat to be tested with a lysis solution for incubation treatment, mix the incubation solution obtained after the incubation treatment with a cyclodextrin metal-organic framework material for adsorption treatment to obtain a solid complex; mix the solid complex with water for desorption treatment to obtain a test solution; the lysis solution includes methacrylated gelatin, DMEM medium and water; when performing the desorption treatment, the mass ratio of the solid complex to water is 1-5:5; the temperature of the desorption treatment is 20-30 °C, and the time is 30-60 min; Perform high-performance liquid chromatography analysis on the test solution to obtain a chromatogram of the test solution; According to the photoinitiator standard curve and the chromatogram of the test solution, obtain the content of the photoinitiator in the cell-cultured fish meat to be tested, and the photoinitiator is one or more of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone and benzoin diethyl ether.

2. The method according to claim 1, wherein The volume ratio of methacrylated gelatin, DMEM medium and water in the lysis solution is 5-7:1000:250-350; the dosage ratio of the cell-cultured fish meat to be tested to the lysis solution is 50 mg:150-250 μL.

3. The method according to claim 1, characterized in that, The temperature of the incubation treatment is 20-30 °C, and the time is 1-2 h; the incubation treatment is carried out under light-proof conditions.

4. The method according to claim 1, characterized in that, The cyclodextrin metal-organic framework material is prepared from raw materials including γ-cyclodextrin, and the dosage ratio of the cyclodextrin metal-organic framework material to the incubation solution is 50 mg:150-250 μL; the temperature of the adsorption treatment is 20-30 °C, and the time is 1-840 min; the adsorption treatment is carried out under oscillation conditions, and the rotation speed of the oscillation ≤ 800 rpm.

5. The method according to claim 1, wherein When the photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, the conditions for the high-performance liquid chromatography analysis include: The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and mobile phase B is acetonitrile; the volume ratio of mobile phase A to mobile phase B is 60:40; a PDA detector is used, and the detection wavelength is set to 220 nm.

6. The method according to claim 1, wherein When the photoinitiator is 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, the conditions for the high-performance liquid chromatography analysis include: The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and mobile phase B is acetonitrile; the volume ratio of mobile phase A to mobile phase B is 60:40; a PDA detector is used, and the detection wavelength is set to 275 nm.

7. The method according to claim 1, wherein When the photoinitiator is benzoin diethyl ether, the conditions for the high-performance liquid chromatography analysis include: The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and mobile phase B is acetonitrile. The volume ratio of mobile phase A to mobile phase B is 20:

80. A PDA detector is used, and the detection wavelength is set at 252 nm.

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