CN106501550A - Evaluation method based on AFM amino acids Fish protein Assembling Behavior - Google Patents

Abstract

The invention discloses an evaluation method for the effect of amino acids on the aggregation behavior of fish protein based on an atomic force microscope, and relates to the field of quality control of fish meat products affected by amino acid additives. The aggregation behavior of aquatic animal protein during processing is not only a necessary process for the formation of high-quality products, but also an intermediate path for quality deterioration. It is the focus of research in the field of gel-based aquatic product production and processing. Free amino acids can change the aggregation behavior of myosin, thereby affecting the quality of gel-like aquatic products. Amino acid additives can realize the production of low-salt soft gel products, and have practical application value. Using atomic force microscope to observe the protein aggregation behavior and the change of aggregate equivalent particle size, by observing the change rule of protein aggregation behavior and counting the change of aggregate particle size, objectively evaluate the effect of different free amino acids on the aggregation behavior of fish myosin , this method can provide a method for quality evaluation and regulation of gel aquatic products.

Description

Evaluation Method of Amino Acid Effect on Aggregation Behavior of Fish Protein Based on Atomic Force Microscopy

technical field

The present invention relates to the field of amino acid additives affecting the aggregation behavior of fish myosin and the quality control of gel-type aquatic products, in particular to the field of aquatic animal protein in different amino acids (concentration: 5 mmol: glutamic acid/cysteine/lysine) /histidine), different heat-induced conditions (no heating as control, heating at 90°C for 30 minutes, heating at 40°C for 60 minutes, heating at 40°C for 60 minutes and then heating at 90°C for 30 minutes) on the quality of aquatic products.

Background technique

my country's fishery resources are very rich. In recent years, the aquatic product processing industry has achieved considerable development, but the processing and comprehensive utilization rate of aquatic products is relatively low, and the product quality is uneven. The quality of the gel, the process of surimi gel formation is the process of deformation and aggregation of fish protein. Currently, turbidity, DLS and other methods are commonly used to measure the change of fish protein aggregation behavior. The test results are not accurate, especially the aggregation behavior after adding additives. It is difficult to accurately reflect the changes in the aggregation behavior of fish protein, but the application of atomic force microscopy to scan the surface morphology and the statistics of aggregate particle size can accurately reflect the influence of additives on the aggregation behavior of fish protein. less. It has been reported in the literature that amino acid additives can improve the solubility of fish protein under low-salt conditions and alleviate the aggregation of fish protein, which is conducive to the formation of low-salt soft gel products. The addition of amino acids will inevitably affect the aggregation behavior of fish protein. The quality of gel products. The present invention will use the advanced technology of atomic force microscope to cut in from the perspective of protein microscopic molecular aggregation behavior changes, study the influence of free amino acids on fish protein aggregation behavior, and objectively evaluate the effect of free amino acids on fish protein aggregation behavior through the size of the equivalent grain size of protein aggregates It provides a theoretical reference for the quality control of gel-type aquatic products to meet people's quality requirements for such products.

Contents of the invention

According to the change of protein aggregation behavior under the condition of different amino acids, the present invention uses the advanced technology of atomic force microscope to study the influence of free amino acids on the aggregation behavior of fish protein from the perspective of the change of protein microscopic molecular aggregation behavior. The size objectively evaluates the effect of free amino acids on the aggregation behavior of fish protein, and provides a theoretical reference for quality control of gel-based aquatic products to meet people's quality requirements for such products.

The present invention includes preparation of fish meat protein samples, preparation of scanning samples, atomic force microscope scanning, and image processing, and the specific operation steps are as follows:

The method for evaluating the effect of amino acids on the aggregation behavior of fish protein based on atomic force microscopy is carried out according to the following steps:

(1) Preparation of fish protein samples: Fresh fish were prepared into fish protein solution according to conventional treatment methods, and corresponding amino acid solution was added to the fish protein solution to obtain protein-amino acid mixture (P-AA).

(2) Preparation of scanning samples: After treating the P-AA solution according to certain heat-induced conditions, drop it on the surface of the newly peeled mica sheet to let it dry naturally, rinse the adsorption layer (1-5) times with pure water, and absorb Excess liquid, then let it dry naturally, to be tested;

(3) Atomic force microscope scanning: the probe model of the atomic force microscope is TAP 150 (MPP-12100-10), the operation mode is molecular force mode, and the scanning range is 5 μm×5 μm;

(4) Image processing: It is carried out on the NanoScope Analysis offline software, and the image is flattened to eliminate the influence of tilt, twist, jumper, etc., and compensate each scanning line to make the obtained data more real, and obtain 2D, 3D pictures, "particle analysis" analysis of two-dimensional images, with its depth diameter as the equivalent particle size;

(5) Finally, the changes in the surface morphology of the aggregation behavior of the protein under different amino acid storage and different heating methods can be observed from the 2D images. From the 3D images, the changes in the three-dimensional morphology such as the height and shape of the aggregates can be observed, and the aggregation can be obtained through particle analysis. The change range and mean value of body equivalent particle size were used as a basis to evaluate the effect of free amino acids on the aggregation behavior of fish myosin, and no amino acids were added as a control.

Wherein the amino acid added in the step (1) is glutamic acid, cysteine, lysine, histidine, and the concentration of amino acid is 5 millimolar.

The certain thermal induction conditions in the step (2) are heating at 90°C for 30 minutes; heating at 40°C for 60 minutes; heating at 40°C for 60 minutes and then heating at 90°C for 30 minutes.

The step (2) described therein is specifically carried out according to the following steps: cut the mica sheet into a size of 5 mm × 5 mm in advance, and stick it on an iron sheet with a ferromagnetic substance such as a diameter of about 1 cm × 1 cm with double-sided adhesive, and the thickness of the iron sheet is No more than 1mm; dilute the protein solution to 20 μg/mL, peel off the outer layer of the mica sheet with double-sided tape, absorb (1-6 μL) the protein solution and drop it on the surface of the newly peeled mica sheet, and dry it naturally in an ultra-clean bench at room temperature , to form a protein adsorption layer; in order to eliminate the interference of salt, the adsorption layer was washed (1-5) times with pure water (15-30°C, 35 μL), and dried naturally in the ultra-clean bench. All operations were carried out in the ultra-clean bench. Before scanning, the samples were stored in a petri dish with a lid to avoid dust in the air falling on the surface of the samples and interfering with scanning.

The evaluation criteria of step (5) described therein are: the equivalent particle diameter range of pure protein aggregates is 110.193-413.482nm, and the equivalent particle diameter of cysteine aggregates is 110.193-371.402nm (without heating); The equivalent particle size range of protein aggregates is 110.1 93-1266.024nm, the equivalent particle size range of cysteine-added aggregates is 110.193-855.543nm (90°C-30min); the equivalent particle size range of pure protein aggregates is 110.1 93 -862.047nm, the equivalent particle size of cysteine aggregates is 110.193-781.984nm (40°C-60min); the equivalent particle size range of pure protein aggregates is 110.1 93-740.184nm, and cysteine aggregates are added The equivalent particle size is 110.193-490.329nm (40°C-60min and then 90°C-30min). There are significant differences in the equivalent particle size range of aggregates.

It can be seen from the results of this method that the topography of the cysteine solution has no obvious granular impurities, no pollution, and is very smooth, which is obviously different from the surface topography of the protein solution. The dust particles had little effect on the sample, and the presence of salt and amino acids had no effect on the determination of protein (Fig. 1); P-AA solution was treated as follows without heating: it existed in the form of small aggregates, protein clusters The degree of cross-linking among them is relatively low, and the formed small aggregates are dispersed and adsorbed on the surface of the mica sheet; heating at 90°C for 30 minutes: large aggregates appear, and due to the high heating temperature, protein aggregation is very violent, and lateral fusion gradually occurs. As a result, irregular cluster aggregates are formed, which are very unevenly dispersed on the surface of the mica sheet; heating at 40°C for 60 minutes: the protein aggregates, but the aggregation is slow, and the horizontal fusion between aggregates is not obvious, and is unevenly dispersed on the mica sheet surface; heating at 40°C for 60 minutes and then heating at 90°C for 30 minutes: larger aggregates appear, but there are still a large number of small aggregates. At this time, the lateral fusion between aggregates is not obvious, and the large aggregates are in the mica The surface distribution is uneven, while small aggregates are evenly spread across the field of view.

The variation ranges of aggregate equivalent particle size are:

Description of drawings

Figure 1 The 2D and 3D diagrams of the surface morphology of cysteine solution. Figures 2, 4, 6, and 8 show myosin solution without heating, heating at 90°C for 30 minutes, heating at 40°C for 60 minutes, heating at 40°C for 60 minutes, and then heating at 90°C 2D and 3D images of AFM imaging with heating for 30min. Figures 3, 5, 7, and 9 are the 2D and AFM images of the myosin solution without heating in the presence of cysteine, heating at 90°C for 30 minutes, heating at 40°C for 60 minutes, heating at 40°C for 60 minutes, and then heating at 90°C for 30 minutes. 3D graph.

detailed description

Example 1:

The present invention includes preparation of fish meat protein samples, preparation of scanning samples, atomic force microscope scanning, and image processing, and the specific operation steps are as follows:

The method for evaluating the effect of amino acids on the aggregation behavior of fish protein based on atomic force microscopy is characterized in that it is carried out according to the following steps:

(1) Preparation of fish protein samples: fresh fish was prepared into a fish protein solution according to a conventional treatment method, and the corresponding amino acid solution (concentration 5 mmol, cysteine) was added to the fish protein solution to obtain a protein-amino acid mixture (P-AA).

(2) Preparation of scanning samples: After treating the P-AA solution according to certain heat-induced conditions (no heating as control, heating at 90°C for 30 minutes), drop it on the surface of the newly peeled mica sheet to let it dry naturally, and rinse it with pure water Adsorb the layer 3 times, suck away the excess liquid, and then let it dry naturally. To be tested, cut the mica sheet into a size of 5mm×5mm in advance, and stick it on a ferromagnetic material such as an iron sheet with a diameter of about 1cm×1cm with double-sided adhesive , the thickness of the iron sheet does not exceed 1mm; dilute the protein solution to 20μg/mL, peel off the outer layer of the mica sheet with double-sided tape, absorb (1-6μL) protein solution and drop it on the surface of the newly peeled mica sheet, and put it in an ultra-clean place at room temperature Naturally dry in the bench to form a protein adsorption layer; in order to eliminate the interference of salt, wash the adsorption layer (1-5) times with pure water (15-30°C, 35 μL), and dry naturally in the ultra-clean bench. In-stage. Before scanning, the samples were stored in a petri dish with a lid to avoid dust in the air falling on the surface of the samples and interfering with scanning.

(3) Atomic force microscope scanning: the probe model of the atomic force microscope is TAP 150 (MPP-12100-10), the operation mode is molecular force mode, and the scanning range is 5 μm×5 μm;

(4) Image processing: It is carried out on the NanoScope Analysis offline software, and the image is flattened to eliminate the influence of tilt, twist, jumper, etc., and compensate each scanning line to make the obtained data more real, and obtain 2D, 3D pictures, "particle analysis" analysis of two-dimensional images, with its depth diameter as the equivalent particle size;

(5) Finally, the changes in the surface morphology of the aggregation behavior of the protein under different amino acid storage and different heating methods can be observed from the 2D images. From the 3D images, the changes in the three-dimensional morphology such as the height and shape of the aggregates can be observed, and the aggregation can be obtained through particle analysis. The change range and mean value of body equivalent particle size were used as a basis to evaluate the effect of free amino acids on the aggregation behavior of fish myosin, and no amino acids were added as a control. Figure 5 Add cysteine and heat at 90°C for 30 minutes: large aggregates appear, and myosin aggregation is very violent due to the high heating temperature.

The distribution range of the equivalent particle size of the aggregate is: 110.193-855.543nm, the average value is: 260.396nm, and the distribution range of the equivalent particle size of the pure protein aggregate is: 110.193-1266.024nm, the average value is: 258.595nm, at this time cysteine Acid has obvious effect on the aggregation behavior of fish protein.

Example 2:

The present invention includes preparation of fish meat protein samples, preparation of scanning samples, atomic force microscope scanning, and image processing, and the specific operation steps are as follows:

The method for evaluating the effect of amino acids on the aggregation behavior of fish protein based on atomic force microscopy is characterized in that it is carried out according to the following steps:

(1) Preparation of fish protein samples: Fresh fish were prepared into fish protein solution according to conventional treatment methods, and corresponding amino acid solution (concentration 5 mmol, cysteine) was added to the fish protein solution to obtain protein-amino acid mixture. Liquid (P-AA).

(2) Preparation of scanning samples: After treating the P-AA solution according to certain heat-induced conditions (no heating as control, heating at 40°C for 60 min), drop it on the surface of the newly peeled mica sheet to let it dry naturally, and rinse it with pure water Adsorb the layer 3 times, suck away the excess liquid, and then let it dry naturally. To be tested, cut the mica sheet into a size of 5mm×5mm in advance, and stick it on a ferromagnetic material such as an iron sheet with a diameter of about 1cm×1cm with double-sided adhesive , the thickness of the iron sheet does not exceed 1mm; dilute the protein solution to 20μg/mL, peel off the outer layer of the mica sheet with double-sided tape, absorb (1-6μL) protein solution and drop it on the surface of the newly peeled mica sheet, and put it in an ultra-clean place at room temperature Naturally dry in the bench to form a protein adsorption layer; in order to eliminate the interference of salt, wash the adsorption layer (1-5) times with pure water (15-30°C, 35 μL), and dry naturally in the ultra-clean bench. In-stage. Before scanning, the samples were stored in a petri dish with a lid to avoid dust in the air falling on the surface of the samples and interfering with scanning.

(3) Atomic force microscope scanning: the probe model of the atomic force microscope is TAP 150 (MPP-12100-10), the operation mode is molecular force mode, and the scanning range is 5 μm×5 μm;

(4) Image processing: It is carried out on the NanoScope Analysis offline software, and the image is flattened to eliminate the influence of tilt, twist, jumper, etc., and compensate each scanning line to make the obtained data more real, and obtain 2D, 3D pictures, "particle analysis" analysis of two-dimensional images, with its depth diameter as the equivalent particle size;

(5) Finally, the changes in the surface morphology of the aggregation behavior of the protein under different amino acid storage and different heating methods can be observed from the 2D images. From the 3D images, the changes in the three-dimensional morphology such as the height and shape of the aggregates can be observed, and the aggregation can be obtained through particle analysis. The change range and mean value of body equivalent particle size were used as a basis to evaluate the effect of free amino acids on the aggregation behavior of fish myosin, and no amino acids were added as a control. Figure 7 Adding cysteine and heating at 40°C for 60 minutes: Myosin aggregates, but the aggregation is slow, and the lateral fusion between aggregates is not obvious.

The distribution range of the equivalent particle size of the aggregate is: 110.193-781.984nm, the average value is: 202.295nm, and the distribution range of the equivalent particle size of the pure protein aggregate is: 110.193-862.047nm, the average value is: 207.973nm, at this time cysteine Has a slight effect on the aggregation behavior of fish proteins.

Example 3:

The present invention includes preparation of fish meat protein samples, preparation of scanning samples, atomic force microscope scanning, and image processing, and the specific operation steps are as follows:

The method for evaluating the effect of amino acids on the aggregation behavior of fish protein based on atomic force microscopy is characterized in that it is carried out according to the following steps:

(1) Preparation of fish protein samples: Fresh fish were prepared into fish protein solution according to conventional treatment methods, and corresponding amino acid solution (concentration 5 mmol, cysteine) was added to the fish protein solution to obtain protein-amino acid mixture. Liquid (P-AA).

(2) Preparation of scanning samples: After treating the P-AA solution according to certain heat-induced conditions (no heating as control, heating at 40°C for 60 minutes, and then heating at 90°C for 30 minutes), drop it on the surface of the newly peeled mica sheet to make it naturally Dry, wash the adsorption layer with pure water for 3 times, absorb the excess liquid, and then let it dry naturally. To be tested, cut the mica sheet into a size of 5mm×5mm in advance, and stick it on a ferromagnetic material with a diameter of about On a 1cm×1cm iron sheet, the thickness of the iron sheet should not exceed 1mm; dilute the protein solution to 20μg/mL, peel off the outer layer of the mica sheet with double-sided tape, absorb (1-6μL) protein solution and drop it on the surface of the newly peeled mica sheet , dry naturally in the ultra-clean bench at room temperature to form a protein adsorption layer; in order to eliminate the interference of salt, wash the adsorption layer (1-5) times with pure water (15-30°C, 35 μL), and dry naturally in the ultra-clean bench. All operations are performed in a clean bench. Before scanning, the samples were stored in a petri dish with a lid to avoid dust in the air falling on the surface of the samples and interfering with scanning.

(3) Atomic force microscope scanning: the probe model of the atomic force microscope is TAP 150 (MPP-12100-10), the operation mode is molecular force mode, and the scanning range is 5 μm×5 μm;

(4) Image processing: It is carried out on the NanoScope Analysis offline software, and the image is flattened to eliminate the influence of tilt, twist, jumper, etc., and compensate each scanning line to make the obtained data more real, and obtain 2D, 3D pictures, "particle analysis" analysis of two-dimensional images, with its depth diameter as the equivalent particle size;

(5) Finally, the changes in the surface morphology of the aggregation behavior of the protein under different amino acid storage and different heating methods can be observed from the 2D images. From the 3D images, the changes in the three-dimensional morphology such as the height and shape of the aggregates can be observed, and the aggregation can be obtained through particle analysis. The change range and mean value of body equivalent particle size were used as a basis to evaluate the effect of free amino acids on the aggregation behavior of fish myosin, and no amino acids were added as a control. Figure 9 Add cysteine and heat at 40°C for 60 minutes, then heat at 90°C for 30 minutes: larger aggregates appear, but there are still a large number of small aggregates, and the lateral fusion between aggregates is not obvious at this time.

The distribution range of the equivalent particle size of the aggregate is: 110.193-490.329nm, the average value is: 186.933nm, the distribution range of the equivalent particle size of the pure protein aggregate is: 110.193-740.184nm, the average value is: 226.070nm, at this time cysteine Has a significant effect on the aggregation behavior of fish proteins.

Example 4:

The present invention includes preparation of fish meat protein samples, preparation of scanning samples, atomic force microscope scanning, and image processing, and the specific operation steps are as follows:

The method for evaluating the effect of amino acids on the aggregation behavior of fish protein based on atomic force microscopy is characterized in that it is carried out according to the following steps:

(1) Preparation of fish protein samples: Fresh fish were prepared into fish protein solution according to conventional treatment methods, and corresponding amino acid solution (concentration 5 mmol, cysteine) was added to the fish protein solution to obtain protein-amino acid mixture. Liquid (P-AA).

(2) Preparation of scanning samples: After treating the P-AA solution according to a certain heat-induced condition (without heating), drop it on the surface of the newly peeled mica sheet to let it dry naturally, rinse the adsorption layer with pure water for 3 times, and suck it away. Let the excess liquid dry naturally. To be tested, cut the mica sheet into a size of 5mm×5mm in advance, and stick it on a ferromagnetic material such as an iron sheet with a diameter of about 1cm×1cm with double-sided adhesive. The thickness of the iron sheet does not exceed 1mm. ; Dilute the protein solution to 20 μg/mL, peel off the outer layer of the mica sheet with double-sided tape, absorb (1-6 μL) the protein solution and drop it on the surface of the newly peeled mica sheet, and dry naturally in the ultra-clean bench at room temperature to form protein Adsorption layer: In order to eliminate the interference of salt, the adsorption layer was washed (1-5) times with pure water (15-30°C, 35 μL), and dried naturally in the ultra-clean bench. All operations were carried out in the ultra-clean bench. Before scanning, the samples were stored in a petri dish with a lid to avoid dust in the air falling on the surface of the samples and interfering with scanning.

(3) Atomic force microscope scanning: the probe model of the atomic force microscope is TAP 150 (MPP-12100-10), the operation mode is molecular force mode, and the scanning range is 5 μm×5 μm;

(4) Image processing: It is carried out on the NanoScope Analysis offline software, and the image is flattened to eliminate the influence of tilt, twist, jumper, etc., and compensate each scanning line to make the obtained data more real, and obtain 2D, 3D pictures, "particle analysis" analysis of two-dimensional images, with its depth diameter as the equivalent particle size;

(5) Finally, the changes in the surface morphology of the aggregation behavior of the protein under different amino acid storage and different heating methods can be observed from the 2D images. From the 3D images, the changes in the three-dimensional morphology such as the height and shape of the aggregates can be observed, and the aggregation can be obtained through particle analysis. The change range and mean value of body equivalent particle size were used as a basis to evaluate the effect of free amino acids on the aggregation behavior of fish myosin, and no amino acids were added as a control. Figure 3 Addition of cysteine without heating: it exists in the form of small aggregates, the degree of cross-linking between myosin clusters is relatively low, and the formed small aggregates are dispersed and adsorbed on the surface of the mica sheet.

The distribution range of the equivalent particle size of the aggregate is: 120.115-371.402nm, the average value is: 165.579nm, the distribution range of the equivalent particle size of the pure protein aggregate is: 110.193-413.482nm, the average value is: 191.334nm, at this time cysteine Has a slight effect on the aggregation behavior of fish proteins.

Example 5:

The present invention includes preparation of fish meat protein samples, preparation of scanning samples, atomic force microscope scanning, and image processing, and the specific operation steps are as follows:

The method for evaluating the effect of amino acids on the aggregation behavior of fish protein based on atomic force microscopy is characterized in that it is carried out according to the following steps:

(1) Preparation of fish protein samples: fresh fish was prepared into fish protein solution according to conventional treatment methods, and corresponding amino acid solution (concentration 5 mmol, glutamic acid) was added to the fish protein solution to obtain protein-amino acid mixture (P-AA).

(2) Preparation of scanning samples: After treating the P-AA solution according to certain heat-induced conditions (no heating as control, heating at 90°C for 30 minutes), drop it on the surface of the newly peeled mica sheet to let it dry naturally, and wash it with pure water Rinse the adsorption layer 3 times, absorb excess liquid, and then let it dry naturally. To be tested, cut the mica sheet into a size of 5mm×5mm in advance, and stick it on a ferromagnetic material such as a ferromagnetic material with a diameter of about 1cm×1cm. On the sheet, the thickness of the iron sheet should not exceed 1 mm; dilute the protein solution to 20 μg/mL, peel off the outer layer of the mica sheet with double-sided tape, absorb (1-6 μL) protein solution and drop it on the surface of the newly peeled mica sheet, and place it in an ultra- Naturally dry in the clean bench to form a protein adsorption layer; in order to eliminate the interference of salt, wash the adsorption layer (1-5) times with pure water (15-30°C, 35 μL), and dry naturally in the ultra-clean bench. In the clean bench. Before scanning, the samples were stored in a petri dish with a lid to avoid dust in the air falling on the surface of the samples and interfering with scanning.

(3) Atomic force microscope scanning: the probe model of the atomic force microscope is TAP 150 (MPP-12100-10), the operation mode is molecular force mode, and the scanning range is 5 μm×5 μm;

(4) Image processing: It is carried out on the NanoScope Analysis offline software, and the image is flattened to eliminate the influence of tilt, twist, jumper, etc., and compensate each scanning line to make the obtained data more real, and obtain 2D, 3D pictures, "particle analysis" analysis of two-dimensional images, with its depth diameter as the equivalent particle size;

(5) Finally, the changes in the surface morphology of the aggregation behavior of the protein under different amino acid storage and different heating methods can be observed from the 2D images. From the 3D images, the changes in the three-dimensional morphology such as the height and shape of the aggregates can be observed, and the aggregation can be obtained through particle analysis. The change range and mean value of body equivalent particle size were used as a basis to evaluate the effect of free amino acids on the aggregation behavior of fish myosin, and no amino acids were added as a control. Add glutamic acid at 90°C-30min: large aggregates appear, and the transverse cross-linking is not obvious.

The distribution range of the equivalent particle size of the aggregate is: 110.193-450.042nm, the average value is: 196.479nm, and the distribution range of the equivalent particle size of the pure protein aggregate is: 110.193-1266.024nm, the average value is: 258.595nm, at this time glutamic acid is Fish protein aggregation behavior was slightly affected.

Embodiment 6:

The present invention includes preparation of fish meat protein samples, preparation of scanning samples, atomic force microscope scanning, and image processing, and the specific operation steps are as follows:

The method for evaluating the effect of amino acids on the aggregation behavior of fish protein based on atomic force microscopy is characterized in that it is carried out according to the following steps:

(1) Preparation of fish protein samples: fresh fish was prepared into fish protein solution according to conventional treatment methods, and corresponding amino acid solution (concentration 5 mmol, glutamic acid) was added to the fish protein solution to obtain protein-amino acid mixture (P-AA).

(2) Preparation of scanning samples: After treating the P-AA solution according to certain heat-induced conditions (no heating as control, heating at 40°C for 60 minutes, and then heating at 90°C for 30 minutes), drop it on the surface of the newly peeled mica sheet to make it naturally Dry, wash the adsorption layer with pure water for 3 times, absorb the excess liquid, and then let it dry naturally. To be tested, cut the mica sheet into a size of 5mm×5mm in advance, and stick it on a ferromagnetic material with a diameter of about On a 1cm×1cm iron sheet, the thickness of the iron sheet should not exceed 1mm; dilute the protein solution to 20μg/mL, peel off the outer layer of the mica sheet with double-sided tape, absorb (1-6μL) protein solution and drop it on the surface of the newly peeled mica sheet , dry naturally in the ultra-clean bench at room temperature to form a protein adsorption layer; in order to eliminate the interference of salt, wash the adsorption layer (1-5) times with pure water (15-30°C, 35 μL), and dry naturally in the ultra-clean bench. All operations are performed in a clean bench. Before scanning, the samples were stored in a petri dish with a lid to avoid dust in the air falling on the surface of the samples and interfering with scanning.

(3) Atomic force microscope scanning: the probe model of the atomic force microscope is TAP 150 (MPP-12100-10), the operation mode is molecular force mode, and the scanning range is 5 μm×5 μm;

(4) Image processing: It is carried out on the NanoScope Analysis offline software, and the image is flattened to eliminate the influence of tilt, twist, jumper, etc., and compensate each scanning line to make the obtained data more real, and obtain 2D, 3D pictures, "particle analysis" analysis of two-dimensional images, with its depth diameter as the equivalent particle size;

(5) Finally, the changes in the surface morphology of the aggregation behavior of the protein under different amino acid storage and different heating methods can be observed from the 2D images. From the 3D images, the changes in the three-dimensional morphology such as the height and shape of the aggregates can be observed, and the aggregation can be obtained through particle analysis. The change range and mean value of body equivalent particle size were used as a basis to evaluate the effect of free amino acids on the aggregation behavior of fish myosin, and no amino acids were added as a control. Add glutamic acid for 40°C-60min and then 90°C-30min: the spacing between large aggregates decreases, and there is a tendency to produce transverse crosslinks.

The distribution range of the equivalent particle size of aggregates is: 110.193-439.670nm, the average value is: 201.85nm, the distribution range of the equivalent particle size of pure protein aggregates is: 110.193-740.184nm, the average value is: 226.070nm, at this time glutamic acid is Fish protein aggregation behavior has a significant effect.

Embodiment 7:

The present invention includes preparation of fish meat protein samples, preparation of scanning samples, atomic force microscope scanning, and image processing, and the specific operation steps are as follows:

The method for evaluating the effect of amino acids on the aggregation behavior of fish protein based on atomic force microscopy is characterized in that it is carried out according to the following steps:

(1) Preparation of fish protein samples: fresh fish was prepared into fish protein solution according to conventional treatment methods, and corresponding amino acid solution (concentration 5 mmol, histidine) was added to the fish protein solution to obtain protein-amino acid mixture (P-AA).

(2) Preparation of scanning samples: After treating the P-AA solution according to certain heat-induced conditions (no heating as control, heating at 40°C for 60 minutes, and heating at 40°C for 60 minutes), drop it on the surface of the newly peeled mica sheet to let it dry naturally , rinse the adsorption layer with pure water for 3 times, absorb the excess liquid, and then let it dry naturally. To be tested, cut the mica sheet into a size of 5mm×5mm in advance, and stick it on a ferromagnetic material such as a diameter of about 1cm with double-sided adhesive ×1cm iron sheet, the thickness of the iron sheet does not exceed 1mm; dilute the protein solution to 20μg/mL, peel off the outer layer of the mica sheet with double-sided tape, absorb (1-6μL) protein solution and drop it on the surface of the newly peeled mica sheet, Naturally dry in the ultra-clean bench at room temperature to form a protein adsorption layer; in order to eliminate the interference of salt, wash the adsorption layer (1-5) times with pure water (15-30°C, 35 μL), and dry naturally in the ultra-clean bench. All operations were performed in a clean bench. Before scanning, the samples were stored in a petri dish with a lid to avoid dust in the air falling on the surface of the samples and interfering with scanning.

(3) Atomic force microscope scanning: the probe model of the atomic force microscope is TAP 150 (MPP-12100-10), the operation mode is molecular force mode, and the scanning range is 5 μm×5 μm;

(4) Image processing: It is carried out on the NanoScope Analysis offline software, and the image is flattened to eliminate the influence of tilt, twist, jumper, etc., and compensate each scanning line to make the obtained data more real, and obtain 2D, 3D pictures, "particle analysis" analysis of two-dimensional images, with its depth diameter as the equivalent particle size;

(5) Finally, the changes in the surface morphology of the aggregation behavior of the protein under different amino acid storage and different heating methods can be observed from the 2D images. From the 3D images, the changes in the three-dimensional morphology such as the height and shape of the aggregates can be observed, and the aggregation can be obtained through particle analysis. The change range and mean value of body equivalent particle size were used as a basis to evaluate the effect of free amino acids on the aggregation behavior of fish myosin, and no amino acids were added as a control. Add histidine for 40°C-60min, and then 90°C-30min, the small aggregates formed will form larger aggregates, but the transverse cross-linking is not obvious.

The distribution range of the equivalent particle size of the aggregate is: 110.193-348.462nm, the average value is: 144.762nm, the distribution range of the equivalent particle size of the pure protein aggregate is: 110.193-740.184nm, the average value is: 226.070nm, at this time, histidine is Fish protein aggregation behavior has a significant effect.

Embodiment 8:

The present invention includes preparation of fish meat protein samples, preparation of scanning samples, atomic force microscope scanning, and image processing, and the specific operation steps are as follows:

The method for evaluating the effect of amino acids on the aggregation behavior of fish protein based on atomic force microscopy is characterized in that it is carried out according to the following steps:

(1) Preparation of fish protein samples: fresh fish was prepared into fish protein solution according to conventional treatment methods, and corresponding amino acid solution (concentration 5 mmol, histidine) was added to the fish protein solution to obtain protein-amino acid mixture (P-AA).

(2) Preparation of scanning samples: After treating the P-AA solution according to certain heat-induced conditions (no heating as control, heating at 90°C for 30 minutes), drop it on the surface of the newly peeled mica sheet to let it dry naturally, and rinse it with pure water Adsorb the layer 3 times, suck away the excess liquid, and then let it dry naturally. To be tested, cut the mica sheet into a size of 5mm×5mm in advance, and stick it on a ferromagnetic material such as an iron sheet with a diameter of about 1cm×1cm with double-sided adhesive , the thickness of the iron sheet does not exceed 1mm; dilute the protein solution to 20μg/mL, peel off the outer layer of the mica sheet with double-sided tape, absorb (1-6μL) protein solution and drop it on the surface of the newly peeled mica sheet, and put it in an ultra-clean place at room temperature Naturally dry in the bench to form a protein adsorption layer; in order to eliminate the interference of salt, wash the adsorption layer (1-5) times with pure water (15-30°C, 35 μL), and dry naturally in the ultra-clean bench, all operations are performed in ultra-clean In-stage. Before scanning, the samples were stored in a petri dish with a lid to avoid dust in the air falling on the surface of the sample and interfering with scanning.

(3) Atomic force microscope scanning: the probe model of the atomic force microscope is TAP 150 (MPP-12100-10), the operation mode is molecular force mode, and the scanning range is 5 μm×5 μm;

(4) Image processing: It is carried out on the NanoScope Analysis offline software, and the image is flattened to eliminate the influence of tilt, twist, jumper, etc., and compensate each scanning line to make the obtained data more real, and obtain 2D, 3D pictures, "particle analysis" analysis of two-dimensional images, with its depth diameter as the equivalent particle size;

(5) Finally, the changes in the surface morphology of the aggregation behavior of the protein under different amino acid storage and different heating methods can be observed from the 2D images. From the 3D images, the changes in the three-dimensional morphology such as the height and shape of the aggregates can be observed, and the aggregation can be obtained through particle analysis. The change range and mean value of body equivalent particle size were used as a basis to evaluate the effect of free amino acids on the aggregation behavior of fish myosin, and no amino acids were added as a control. Adding histidine at 90°C-30min: small aggregates spread all over the field of view, and no obvious aggregation behavior occurred.

The distribution range of the equivalent particle size of the aggregate is: 110.193-296.5nm, the average value is: 144.762nm, and the distribution range of the equivalent particle size of the pure protein aggregate is: 110.193-1266.024nm, the average value is: 258.595nm, at this time glutamic acid is Fish protein aggregation behavior has a significant effect.

Claims (7)

1. the evaluation method based on AFM amino acids Fish protein Assembling Behavior, it is characterised in that according to following steps Suddenly carry out：

（1）The preparation of fish protein sample：Fresh fish is made fish protein solution according to conventional treatment method, in flesh of fish egg Corresponding Freamine Ⅲ is added in white solution, albumen-amino acid mixed liquor is obtained（P-AA）；

（2）The preparation of scanning sample：After P-AA solution is processed, the mica sheet table of new stripping is dropped in by certain thermal induction condition Face spontaneously dries which, secondary with pure water flushing adsorption layer (1 ~ 5), siphons away surplus liquid, then spontaneously dries which, to be measured；

（3）Afm scan：Atomic force microscope probe model TAP 150（MPP-12100-10）, operator scheme For molecule force mode, sweep limits is 5 μm of 5 μ m；

（4）Picture processing：Carry out on the offline softwares of NanoScope Analysis, picture carries out Flatten planarizing process, Eliminate the impact of inclination, distortion, wire jumper etc., every scan line compensated, so as to get data truer, obtain 2D, 3D pictures, carry out " particle analysis " analysis, using its depth diameter as equivalent grain size to two dimensional image；

（5）Finally albumen can be observed from 2D pictures deposit Assembling Behavior surface topography under different heating mode in different aminoacids Change, the change of the stereoscopic pattern such as the height form that can observe aggregation from 3D pictures can be gathered by grading analysis The excursion and average of collective's equivalent grain size, and affect fish myosin Assembling Behavior to enter free amino acid on this basis Row is evaluated, and is not added with amino acid as control.

2. the evaluation method based on AFM amino acids Fish protein Assembling Behavior according to claim 1, It is characterized in that wherein described the step of（2）Specifically carry out as steps described below：Mica sheet is cut into 5 mm × 5 mm in advance big Little, with two-sided gluing on the iron plate for being about 1 cm × 1 cm with ferromagnetic material such as diameter, iron plate thickness is less than 1 mm； Protein solution is diluted to 20 μ g/mL, with double faced adhesive tape by mica sheet extexine peel off, draw (1 ~ 6 μ L) protein solution drip in The new mica sheet surface that peels off, spontaneously dries in super-clean bench under room temperature, forms protein adsorption layer；For eliminating the interference of salinity, Use pure water（15 ~ 30 DEG C, 35 μ L）Rinse adsorption layer (1 ~ 5) secondary, spontaneously dry in super-clean bench, all operations are in super-clean bench Inside carry out；Before scanning, sample is stored in culture dish with cover, it is to avoid the dust in air falls in sample surfaces interference scanning.

3. the evaluation method based on AFM amino acids Fish protein Assembling Behavior according to claim 1, It is characterized in that wherein described the step of（5）Evaluation criterion is：Pure protein aggregation equivalent grain size scope is 110.1 93- 413.482 nm, it is 110.193-371.402 nm to add cysteine aggregation equivalent grain size（Do not heat）；Pure protein is assembled Body equivalent grain size scope is 110.1 93-1266.024 nm, and it is 110.193- to add cysteine aggregation equivalent grain size 855.543 nm（90 ℃-30 min）；Pure protein aggregation equivalent grain size scope is 110.1 93-862.047nm, adds half Cystine aggregation equivalent grain size is 110.193-781.984 nm（40 ℃-60 min）；Pure protein aggregation equivalent grain size model Enclose for 110.1 93-740.184 nm, it is 110.193-490.329 nm to add cysteine aggregation equivalent grain size（40 ℃- 60 min and then 90 DEG C of -30 min）, there is notable difference in aggregation equivalent grain size scope.

4. the evaluation method based on AFM amino acids Fish protein Assembling Behavior according to claim 1, It is characterized in that wherein described the step of（1）The amino acid of middle interpolation be glutamic acid, cysteine, lysine, histidine, amino The concentration of acid is 5 mMs.

5. the evaluation method based on AFM amino acids Fish protein Assembling Behavior according to claim 1, It is characterized in that wherein described the step of（2）Described certain thermal induction condition is 90 DEG C of 30 min of heating；40 DEG C of heating 60 min；40 DEG C heating 60 min after again 90 DEG C heating 30 min.

6. the evaluation method based on AFM amino acids Fish protein Assembling Behavior according to claim 2, It is characterized in that wherein described the step of（2）Draw 1 ~ 6 μ L protein solutions.

7. the evaluation method based on AFM amino acids Fish protein Assembling Behavior according to claim 2, It is characterized in that wherein described the step of（2）Draw the pure water that 35 μ L are 15 ~ 30 DEG C.