Nano medaka roe membrane microvillus and application thereof in fresh keeping of fish meat

Abstract

The invention discloses nano medaka roe membrane micro villus and application thereof in fresh keeping of fish meat, and belongs to the technical field of fresh keeping of aquatic products. According to the method, the micro villi on the outer membranes of the medaka roes are separated and extracted from the medaka roes by adopting high-pressure micro-jet homogenization treatment, and meanwhile, the medaka roe membrane micro villi with the nano particle size are obtained by adopting a nozzle with the nano diameter; the obtained nano medaka roe membrane microvilli can provide a protective barrier for fresh fish meat, and the problems that due to the fact that the instant temperature difference is large and heat exchange is violent, the expansion pressure is too large, cells in the fish meat are broken, the tissue structure of the fish meat is damaged, and finally the quality of the fish meat is deteriorated are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic product preservation, and in particular relates to nano-medaka egg membrane microvilli and application thereof in fish meat preservation. Background Art

[0002] Low-temperature preservation technology for aquatic products is the earliest developed, most deeply researched and most widely used preservation technology, with the characteristics of low cost, simple operation and significant effect. According to the different preservation temperatures, low-temperature preservation technology can be divided into refrigeration, ice storage, ice temperature, micro-freezing and frozen storage.

[0003] Frozen preservation technology is to reduce the temperature of aquatic products to below -18℃, freeze most of the water inside, inhibit the growth and reproduction of microorganisms and the speed of enzymatic reactions, thereby slowing down the oxidation of lipids and proteins. Therefore, frozen aquatic products can maintain their original quality for a long time. However, during the frozen preservation process, the freezing method and freezing temperature will cause changes in the color, texture, nutrition and flavor of aquatic products. For example, air freezing and flat contact freezing have slower freezing speeds, usually forming large ice crystals outside the cells of food, destroying the microstructure of the food, and having a great impact on the texture, color and water retention of the food. Frozen storage temperature is also an important factor affecting the quality changes of aquatic products during storage. Generally speaking, during the frozen storage of aquatic products, maintaining a lower frozen storage temperature is conducive to maintaining its original quality. Traditional frozen preservation methods will inevitably cause ice crystals to form in the meat of aquatic products, while protein denaturation and fat autooxidation will occur, thus affecting the cortex of aquatic products. Therefore, the research and application of new frozen preservation technology is a current research hotspot.

[0004] In the freezing processing of aquatic products, liquid nitrogen spray freezing has been successfully applied to the production practice of quick-frozen prepared crayfish or fish fillets. However, the freezing process of liquid nitrogen spray is often difficult to control due to its fast freezing speed. When the liquid nitrogen spray freezing speed is too fast, the instantaneous temperature difference between the surface and the center of the food is large, and the heat exchange is intense, resulting in excessive expansion pressure and cell rupture, which will also destroy the food tissue structure and cause quality deterioration. In addition, the cost of liquid nitrogen spray freezing is relatively high. Studies have shown that the amount of liquid nitrogen required to freeze 1kg of food is about 0.8-2kg, and during the freezing process, liquid nitrogen is used as a one-time freezing medium and cannot be recycled after freezing. Therefore, although liquid nitrogen spray freezing has significant advantages in freezing efficiency, the high cost also limits its application in most foods.

[0005] The medaka belongs to the order Cyprinodontiformes, family Adrianichthyidae, genus Oryzias, and there are 33 species described so far. The utilization and development of medaka resources are limited to the research on the biological development, growth morphology and ecological behavior of newly hatched fry. There are no related reports on the use of medaka for food preservation, especially the preservation of aquatic products.

[0006] Therefore, how to combine the resource utilization of medaka with liquid nitrogen spray freezing technology to reduce the cost of aquatic product preservation while maintaining the quality of aquatic products has become a technical problem that urgently needs to be solved in this field. Summary of the invention

[0007] In order to solve the above technical problems, the present invention proposes nano-medaka egg membrane microvilli and the application thereof in fish meat preservation.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing nano-medaka egg membrane microvilli, comprising the following steps: mixing medaka eggs and water, subjecting them to high-pressure microfluidic homogenization, and then subjecting them to centrifugation, concentration and re-centrifugation to obtain the nano-medaka egg membrane microvilli;

[0010] The high-pressure microfluidization homogenization treatment includes: first treating at 50 bar for 0.1 h, then treating at 80 bar for 0.1 h, and then treating at 100 bar for 0.1 h;

[0011] The diameter of the nozzle used in the high-pressure microfluidic homogenization process is 20 nm.

[0012] Technical principle: The present invention separates and extracts the microvilli on the outer membrane of medaka eggs from medaka eggs by using high-pressure microfluidic homogenization treatment, and at the same time, by using a nozzle with a nanometer diameter, obtains medaka egg membrane microvilli with nanometer particle size; the obtained nano medaka egg membrane microvilli can provide a protective barrier for fresh fish meat, and solves the problem of excessive expansion pressure, cell rupture in fish meat, destruction of fish meat tissue structure, and ultimate deterioration of fish meat quality due to large instantaneous temperature difference and intense heat exchange in liquid nitrogen spray freezing of fish meat.

[0013] Furthermore, the material-liquid ratio of the medaka fish eggs to water is 1:16 g / mL.

[0014] Furthermore, the concentration temperature is 50° C.; and the volume of the concentrated solution obtained by the concentration is 1 / 3 of the volume of the supernatant obtained by centrifugation.

[0015] Furthermore, the rotation speed of the centrifugation and re-centrifugation is 5000 rpm, and the time is 10 min.

[0016] The present invention provides nano-medaka egg membrane microvilli prepared by the preparation method described in the above technical scheme.

[0017] The present invention also provides the use of the nano-medaka egg membrane microvilli described in the above technical solution in the preservation of fish meat, wherein the fish meat is Xing Kangji eel meat.

[0018] Furthermore, the application of the nano-medaka egg membrane microvilli in fish preservation comprises the following steps:

[0019] Fresh live fish are slaughtered and precooled to obtain pretreated fish meat; the nano-medaka egg membrane microvilli described in the above technical solution are smeared on the surface of the pretreated fish meat, and then liquid nitrogen is sprayed and frozen to complete the preservation of the fish meat.

[0020] Furthermore, the application amount is 1.25 mL / g.

[0021] Furthermore, the freezing temperature of the liquid nitrogen spray is -20°C.

[0022] Compared with the prior art, the present invention has the following advantages and technical effects:

[0023] The present invention uniformly covers the surface of fish meat with nano-medaka egg membrane microvilli and then combines the liquid nitrogen spray freezing technology to perform fresh-keeping treatment on Xing Kangji eel meat. Experiments have shown that as the degree of freezing deepens, the thawing loss rate and the cooking loss rate of Xing Kangji eel meat gradually increase, and the centrifugal water holding rate gradually decreases; and in the late freezing period, the water retention of Xing Kangji eel meat in the test group is significantly better than that in the control group; the test group can inhibit the conversion of non-flowing water into free water, and has an inhibitory effect on the water migration of frozen fish meat compared with the control group, and can also inhibit the fluctuation of the brightness value and the change of color of Xing Kangji eel meat during the freezing process, reduce the decline in hardness and elasticity of frozen fish meat, and better maintain the quality of the fish meat.

[0024] The preservation method based on the microvilli structure on the medaka egg membrane combined with the liquid nitrogen spray freezing technology provided by the present invention has a good effect on maintaining the quality of Xingkangji eel meat, and can be used as an efficient and high-quality freezing method in the freezing and preservation of such aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0026] Figure 1 This is a mechanism diagram of the application of nano-medaka egg membrane microvilli in the fish meat preservation of the present invention;

[0027] Figure 2 This is an optical microscope image of medaka fish eggs;

[0028] Figure 3 This is a schematic diagram of the temperature control probe inserted into the back of the fish body;

[0029] Figure 4 : is the thawing loss rate of Example 2 and Comparative Example 1, wherein -20°C LNF is Example 2, and -20°C RF is Comparative Example 1; in the figure, different lowercase letters indicate that the data of different treatment groups frozen at the same temperature are significantly different (P<0.05), and different uppercase letters indicate that the data of the same preservation method frozen at different temperatures are significantly different (P<0.05), the same below;

[0030] Figure 5 is the cooking loss rate of Example 2 and Comparative Example 1, wherein -20°C LNF is Example 2, and -20°C RF is Comparative Example 1;

[0031] Figure 6 is the centrifugal water retention of Example 2 and Comparative Example 1, wherein -20°C LNF is Example 2, and -20°C RF is Comparative Example 1;

[0032] Figure 7 Graphs showing changes in T2 relaxation time of Example 2 and Comparative Example 1, wherein A is Comparative Example 1 and B is Example 2;

[0033] Figure 8 for Figure 7 Schematic diagram of the percentage of the peak area of ​​the moisture signal in the embodiment, wherein A is comparative example 1 and B is embodiment 2;

[0034] Fig. 9 The bar graphs are the texture property results of Example 2 and Comparative Example 1, where A represents hardness and B represents elasticity;

[0035] Fig.10 Schematic diagram of the results of volatile flavor substances in fresh Xing Kang Ji eel meat and Xing Kang Ji eel meat treated by the preservation method of Example 2, wherein A is the GC-IMS spectrum and B is the volatile substance fingerprint. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0038] The embodiment of the present invention provides a method for preparing nano-medaka egg membrane microvilli, comprising the following steps: mixing medaka fish eggs and water and subjecting them to high-pressure microfluidization homogenization, and then subjecting them to centrifugation, concentration and re-centrifugation to obtain the nano-medaka egg membrane microvilli;

[0039] The high-pressure microfluidization homogenization treatment includes: first treating at 50 bar for 0.1 h, then treating at 80 bar for 0.1 h, and then treating at 100 bar for 0.1 h;

[0040] The diameter of the nozzle used in the high-pressure microfluidic homogenization process is 20 nm.

[0041] The invention adopts high-pressure micro-jet homogenization technology to peel off the egg membrane microvilli from the surface of medaka fish eggs, and then removes impurities and unbroken cell fragments through centrifugation, concentration and re-centrifugation, thereby obtaining nano medaka fish egg membrane microvilli; after being treated under different micro-jet pressures, the average particle size of the nano medaka fish egg membrane microvilli changes, and the molecular weight distribution becomes more uniform.

[0042] Studies have shown that the outer surface of some fish eggs (such as silverfish) has an egg membrane silk structure, which covers the entire egg membrane and has the function of classification and reproductive isolation. This egg membrane silk structure, by extending into the egg pore of the fish egg, obviously forms a mechanical barrier, which can protect the fish eggs. The inventor found that the medaka egg membrane also has a microchorionic membrane protrusion structure similar to the silverfish egg membrane silk, and found 9-15 egg pore structures, so it is speculated that this protrusion structure can also provide a mechanical barrier for the medaka eggs and play a protective role, and it is speculated that this mechanical barrier may affect the permeability of the cell membrane at the microscopic level, so that the materials such as water inside and outside the cell membrane cannot be free, thereby affecting the physical properties such as water holding capacity and water distribution. In view of this, the inventor combines the microchorionic membrane protrusion structure on the medaka egg membrane with liquid nitrogen spray freezing technology, thereby achieving better food preservation effect.

[0043] The invention separates and extracts microvilli on the outer membrane of medaka fish eggs from medaka fish eggs by adopting high-pressure micro-jet homogenization treatment, and simultaneously obtains medaka fish egg membrane microvilli with nanometer particle size by adopting a nozzle with nanometer diameter; then, the microvilli are evenly covered on the surface of medaka fish meat, and combined with liquid nitrogen spray freezing technology, the practical application of medaka fish egg membrane microvilli with nanometer particle size in the preservation of Xingkangji eel meat is realized.

[0044] In a preferred embodiment, the solid-liquid ratio of the medaka fish eggs to water is 1:16 g / mL.

[0045] In a preferred embodiment, the equipment for high-pressure microfluidic homogenization is a high-pressure microfluidic homogenizer.

[0046] In a preferred embodiment, the concentration temperature is 50° C.; the volume of the concentrated solution obtained by the concentration is 1 / 3 of the volume of the supernatant obtained by centrifugation; and the concentration is vacuum rotary evaporation concentration.

[0047] In a preferred embodiment, the rotation speed of the centrifugation and re-centrifugation is 5000 rpm, the time is 10 min, and the temperature of the centrifugation and re-centrifugation is 4°C.

[0048] In a preferred embodiment, after the re-centrifugation, the lower layer solution is collected, which is the nano-medaka egg membrane microvilli.

[0049] The present invention provides nano-medaka egg membrane microvilli prepared by the preparation method described in the above technical scheme.

[0050] The present invention also provides the use of the nano-medaka egg membrane microvilli described in the above technical solution in the preservation of fish meat, wherein the fish meat is Xing Kangji eel meat.

[0051] In a preferred embodiment, the application of the nano-medaka egg membrane microvilli in fish meat preservation comprises the following steps:

[0052] Fresh live fish are slaughtered and precooled to obtain pretreated fish meat; the nano-medaka egg membrane microvilli described in the above technical solution are smeared on the surface of the pretreated fish meat, and then liquid nitrogen is sprayed and frozen to complete the preservation of the fish meat.

[0053] The invention realizes the preservation of Xing Kangji eel meat by uniformly covering the surface of fish meat with nano-medaka egg membrane microvilli and combining with liquid nitrogen spray freezing technology.

[0054] The mechanism diagram of the application of nano-medaka egg membrane microvilli in fish meat preservation in the present invention is shown in Figure 1 First, the microvilli structure on the fish eggs is separated and extracted through high-pressure microjet separation, and then evenly covered on the surface of the fish meat. Combined with liquid nitrogen spray freezing technology, ice crystal structure is formed on the surface of the microvilli structure of the fish eggs after liquid nitrogen spray freezing, so that the microvilli are connected to each other and attached to the holes on the surface of the fish tissue, regulating the flow exchange rate of water and other substances inside and outside the fish tissue to achieve the preservation effect.

[0055] In a preferred embodiment, the application amount is 1.25 mL / g.

[0056] In a preferred embodiment, the temperature of the liquid nitrogen spray freezing is -20°C; by adjusting the liquid nitrogen spray rate of the liquid nitrogen spray quick freezing machine, the temperature in the freezing chamber is ensured to be fixed at -20°C during the freezing process.

[0057] Liquid nitrogen quick freezing accelerates the heat transfer process and achieves rapid freezing through the instantaneous vaporization of liquid nitrogen. Compared with traditional freezing technology, liquid nitrogen freezing has the advantages of high heat transfer coefficient, fast freezing speed, low dry loss of frozen products, and small ice crystal formation. Liquid nitrogen spray freezing refers to a method of uniformly dispersing liquid nitrogen into a spray state through a specific device, contacting and heat exchanging with the surface of food, and freezing it quickly. Liquid nitrogen spray freezing can adjust the temperature in the freezing chamber by controlling the rate of liquid nitrogen spraying, so that the food can be quickly frozen without causing low-temperature fracture of the food due to too low temperature. In addition, the liquid nitrogen spray freezing process is easy to control, and the production conditions can be adjusted in time to keep the sample in the best condition. The present invention treats fresh fish meat with nano-microvilli extracted from medaka eggs before liquid nitrogen spray freezing, provides a protective barrier for fresh fish meat, and solves the problem that liquid nitrogen freezing of fish meat due to large instantaneous temperature difference and intense heat exchange leads to excessive expansion pressure, cell rupture in fish meat, destruction of fish meat tissue structure, and ultimately deterioration of fish meat quality.

[0058] The room temperature in the embodiments of the present invention refers to "25±2°C".

[0059] Unless otherwise specified, the raw materials in the examples of the present invention were purchased from commercial sources.

[0060] The Xingkangji eels used in the examples and comparative examples of the present invention were purchased from Guoxin Aquatic Products Direct Store (Qingdao, China), with an average weight of 793.51 g ± 42.07 g. After the seawater was oxygenated, they were transported to the laboratory in a fresh state within 3 hours for experiments.

[0061] The experimental instruments and equipment used in the present invention are shown in Table 1.

[0062] Table 1 Experimental instruments and equipment

[0063]

[0064]

[0065] Example 1

[0066] A method for preparing nano-medaka egg membrane microvilli, the specific steps are as follows:

[0067] Weigh 20 g of medaka roe, add distilled water at a solid-liquid ratio of 1:16 (g / mL), mix well, place the resulting mixture in a high-pressure microfluidizer, first treat it at 50 bar for 0.1 h, then treat it at 80 bar for 0.1 h, and then treat it at 100 bar for 0.1 h to obtain an extract; wherein the nozzle diameter of the high-pressure microfluidizer is 20 nm; the resulting extract is centrifuged at 5000 rpm for 10 min, and the supernatant is collected; the resulting supernatant is concentrated to 1 / 3 of the volume of the supernatant by 50°C vacuum rotary evaporation, and then centrifuged at 4°C and 5000 rpm for 10 min, and the lower layer solution is collected to obtain nano medaka egg membrane microvilli.

[0068] The medaka fish eggs were placed under an optical microscope for observation. Figure 2 .from Figure 2 It can be seen that the medaka egg membrane has a microvilli protrusion structure.

[0069] Example 2

[0070] Example 1 Application of the prepared nano-medaka egg membrane microvilli in the preservation of Xingkangji eel meat:

[0071] (1) fresh conger eels were killed by hitting their heads, the internal organs were removed, the blood stains on the surface of the fish body and the abdominal cavity were washed with tap water, the water on the surface and the abdomen was wiped dry, and the fish were put into a fresh-keeping bag, which was then pre-cooled in a refrigerator at 4° C. to obtain pre-treated fish meat;

[0072] (2) The nano-medaka egg membrane microvilli prepared in Example 1 were evenly applied to the surface of the pretreated fish meat obtained in step (1) at a coating amount of 1.25 mL / g, and then frozen by liquid nitrogen spray at -20°C. That is, by adjusting the liquid nitrogen spray rate of the liquid nitrogen spray quick freezer to ensure that the temperature in the freezing chamber is fixed at -20°C during the freezing process, the fresh-keeping treatment of the fresh conger eel meat was completed, which was recorded as -20°C LNF.

[0073] Comparative Example 1

[0074] (1) fresh conger eels were killed by hitting their heads, the internal organs were removed, the blood stains on the surface of the fish body and the abdominal cavity were washed with tap water, the water on the surface and the abdomen was wiped dry, and the fish were put into a fresh-keeping bag, which was then pre-cooled in a refrigerator at 4° C. to obtain pre-treated fish meat;

[0075] (2) The pretreated fish meat obtained in step (1) is sprayed and frozen with liquid nitrogen at -20°C, that is, the liquid nitrogen spray rate of the liquid nitrogen spray quick freezer is adjusted to ensure that the temperature in the freezing chamber is fixed at -20°C during the freezing process, thereby completing the preservation treatment of the fresh conger eel meat, which is recorded as -20°C RF.

[0076] Insert the thermocouple of the multi-channel temperature measuring instrument into the back muscle behind the head of Xingkangji eel, and the temperature control probe is located at the thickest part of the fish back center. Figure 3 As shown (the temperature control probe is located at the position indicated by the arrow). The temperature was recorded every 10 seconds during the liquid nitrogen spray freezing process, and the temperature was recorded every 30 seconds during the refrigerator thawing process. During the liquid nitrogen spray freezing process, the samples were taken out when the temperature at the center of the fish reached 4°C, 0°C, -4°C, -10°C and -20°C in sequence, which was the Xing Kangji eel meat before thawing; then the samples were placed in a 4°C refrigerator for thawing, and the thawed Xing Kangji eel meat was obtained after the center temperature reached 4°C.

[0077] Testing process and results

[0078] (1) Thawing loss rate

[0079] The weight of the eel meat before thawing was weighed, recorded as W1, and then the eel meat was placed in a 4°C refrigerator and thawed until the center temperature was 4°C. The surface moisture was immediately wiped with filter paper and weighed, recorded as W2. Each group of fish meat was measured in parallel 3 times, and the average value was taken. The thawing loss rate results of Example 2 and Comparative Example 1 are shown in Figure 4 The calculation formula of thawing loss rate is: thawing loss rate (%) = (W1-W2) / W1×100%.

[0080] Figure 4 is the thawing loss rate of Example 2 and Comparative Example 1, wherein -20°C LNF is Example 2 and -20°C RF is Comparative Example 1. The thawing loss rate is closely related to the water holding capacity of fish protein. Thawing loss will cause the loss of water-soluble nutrients in fish, such as water-soluble minerals and proteins. Figure 4 It can be seen that with the deepening of the degree of freezing, the thawing loss rate of the Xing Kangji eel meat in Example 2 and Comparative Example 1 both showed an upward trend, wherein the thawing loss rate changed significantly with the deepening of the degree of freezing during the entire freezing process (P < 0.05). When the temperature at the center of the fish body dropped to -20°C, the thawing loss rate of the Xing Kangji eel meat in Example 2 was 2.89±0.08%, which was significantly lower than the thawing loss rate of 4.11±0.09% in Comparative Example 1 (P < 0.05).

[0081] (2) Cooking loss rate

[0082] The back muscle of the thawed Xingkangji eel meat was weighed and recorded as W0, then placed in a 50mL plastic centrifuge tube, heated in a constant temperature water bath at 95°C for 30 minutes, taken out, and the surface moisture of the sample was dried with filter paper. After cooling to room temperature, it was weighed and recorded as W3. Each group of fish meat was measured in parallel 3 times, and the average value was taken. The cooking loss rate results of Example 2 and Comparative Example 1 are shown in Figure 5 The calculation formula of the cooking loss rate is: cooking loss rate (%) = (W0-W3) / W0×100%.

[0083] Figure 5 The cooking loss rate is the cooking loss rate of Example 2 and Comparative Example 1, wherein -20°C LNF is Example 2 and -20°C RF is Comparative Example 1. The cooking loss rate is also an important indicator reflecting the water holding properties of fish meat. Figure 5 It can be seen that after -4°C, as the degree of freezing deepens, the cooking loss rate begins to show significant differences (P < 0.05). It is possible that the newly generated small ice crystals at this stage gradually grow to form larger ice crystals, causing greater damage to the fish tissue, accelerating the diffusion of water in the cells, and leading to an increase in the cooking loss rate of the fish. When the temperature at the center of the fish body drops to -20°C, there is a significant difference in the cooking loss rate of the two preservation methods. The cooking loss rate of Xing Kangji eel meat treated with the preservation method of Example 2 is 23.62%, which is significantly lower than the cooking loss rate of Xing Kangji eel meat treated with the preservation method of Comparative Example 1, which is 26.36% (P < 0.05).

[0084] (3) Centrifugal water retention

[0085] Take the back muscle of the thawed Xingkangji eel meat, record it as W4. Cut the filter paper to a height slightly lower than the height of a 50mL plastic centrifuge tube and a width twice the circumference of the centrifuge tube mouth. Wrap the thawed Xingkangji eel meat back muscle with the filter paper of the above specifications and place it in a centrifuge tube. Centrifuge at 5000r / min for 20min at 4°C. Weigh the fish meat mass after centrifugation and record it as W5. Each group of samples was measured in parallel 3 times, and the average value was taken. The centrifugal water retention results of Example 2 and Comparative Example 1 are shown in Figure 6 The calculation formula of centrifugal water retention is: centrifugal water retention (%) = W5 / W4×100%.

[0086] Figure 6 The centrifugal water holding capacity of Example 2 and Comparative Example 1, wherein -20°C LNF is Example 2 and -20°C RF is Comparative Example 1. Water holding capacity refers to the ability of meat to retain original or added water. Figure 6It can be seen that in terms of water holding capacity, the water holding capacity of the Xing Kangji eel meat treated by the two preservation methods in the early stage of freezing is not much different, but as the degree of freezing deepens, when the temperature of the center point of the fish body reaches -4°C, the centrifugal water holding rate of the -20°C RF group (Comparative Example 1) decreases significantly, and decreases by 11.29% compared with the initial value. This may be due to the formation of ice crystals during the freezing process, which destroys the cell structure of the fish meat, further causing myofibril dehydration, aggravating protein denaturation, and thus reducing the water holding performance of the fish meat; while the centrifugal water holding rate of the -20°C LNF group (Example 2) changes less, and only decreases by 4.90% compared with the initial value. This shows that the degree of damage to the fish cells and tissues of Xing Kangji eel meat by ice crystals using the preservation method of Example 2 is small, and as the degree of freezing deepens, the size of ice crystals does not increase significantly, and the water holding capacity of Xing Kangji eel meat in Example 2 is significantly better than that of Comparative Example 1. When the temperature at the center of the fish body dropped to -20°C, the water holding capacity of Example 2 was 85.76%, which was significantly better than the water holding capacity of 80.03% of Comparative Example 1 (P<0.05). The above results show that the preservation method provided by the present invention can significantly reduce the centrifugal loss of Xingkangji eel meat.

[0087] (4) Water distribution

[0088] The back muscle of the thawed Xingkangji eel meat was cut into strips of 2.0 cm × 0.6 cm × 0.6 cm, then wrapped in plastic wrap and placed in a 15 mm NMR tube for NMR. The results are shown in Figure 7 Among them, the test conditions of the low-field nuclear magnetic resonance instrument are set as follows: T2 spectrum inversion parameters: magnet temperature—35°C; main frequency—SF=22MHz; offset frequency O1—971582.1; 90° pulse time—10μs; 180° pulse time—23μs; signal sampling points TD—323006; sampling repetition time TW—5000ms; RG1—20.0db; DRG1—3; repeated scanning times NS—8 times; echo number NECH—5000.

[0089] Figure 7 2 is a graph showing the change in T2 relaxation time of Example 2 and Comparative Example 1, wherein A is Comparative Example 1 and B is Example 2. In the graph, the four peaks T 2b1 、T 2b2 、T 21 and T 22 They represent four different types of water in the back muscle of Xingkangji eel meat, T 2b1 and T 2b2 Represents bound water, collectively referred to as T 2b It is the water that is tightly bound to the biomacromolecules in fish meat, mainly through hydrogen bonds. It is distributed in the matrix of sarcoplasm, sarcolemma and myofibril protein. 2b1 (0~2ms) and T2b2 (2-10ms) represent strongly bound water and weakly bound water, which are most tightly bound or relatively tightly bound to biological macromolecules; T 21 (10-100ms) is immobile water, also known as fixed water, which is mainly embedded or fixed in the tissue structure, such as adsorbed in the tertiary and quaternary structures of proteins, or located in the high-density myofibril reticular space structure; T 22 (100-1000ms) represents free water, which is located outside the myofibrillar protein network and is the most active water in the tissue. It is easy to freeze at 0°C. The peak width represents the transverse relaxation time. The shorter the peak width, the denser the muscle tissue and the stronger the water binding. Figure 7 As can be seen from the figure, as the degree of freezing deepens, the two preservation methods T 21 The increase in transverse relaxation time indicates that the water holding capacity of fish meat is reduced. Figure 7 It can be seen from Part A and Part B in that the preservation method of Example 2 can significantly inhibit the migration of water inside the Xingkangji eel meat during frozen storage, reduce the conversion of non-flowing water into free water, and improve the water binding ability of fish meat cell tissue.

[0090] Figure 8 for Figure 7 Schematic diagram of the percentage of the peak area of ​​the moisture signal in the example 1, wherein A is comparative example 1 and B is example 2. Figure 8 It can be seen that when the temperature at the center of the fish body reaches -4°C (Fresh 4°C), the percentage of free water content in the conger eel meat of Comparative Example 1 significantly increases (P < 0.05), and then continues to increase, while the free water content in Example 2 does not change much during the entire freezing process. When the temperature at the center of the fish body reaches -20°C, the free water content of the conger eel meat in Comparative Example 1 reaches 6.17%, which is 7.81 times higher than 0.70% when the temperature at the center of the fish body reaches -4°C (Fresh 4°C), while the free water content in the conger eel meat sample of Example 2 is only 2.56%, which is significantly lower than that of Comparative Example 1 (P < 0.05). In summary, the preservation method of Example 2 can maintain the internal moisture state of the fish meat, reduce water loss, and has little effect on the quality of the conger eel meat after freezing.

[0091] (5) Color difference value

[0092] The surface moisture of the thawed Xingkangji eel meat was wiped clean with filter paper, and then the color of the back muscle of Xingkangji eel meat was measured using a colorimeter, and the L*, a* and b* values ​​were recorded. Each group of samples was measured in parallel 5 times and the average value was taken. Color is the most important attribute characteristic of meat products and is directly related to consumer acceptance. The changes in the color of the Xingkangji eel meat during the freezing process in Example 2 and Comparative Example 1 are shown in Table 2.

[0093] Table 2 Changes in color difference of Xingkangji eel meat

[0094]

[0095] Note: Different lowercase letters in the table indicate that the data of different treatment groups frozen at the same temperature are significantly different (P < 0.05), and different uppercase letters indicate that the data of the same preservation method frozen at different temperatures are significantly different (P < 0.05), the same below.

[0096] As shown in Table 2, during the freezing process, the L* value of the -20℃RF group was higher than that of the -20℃LNF group (P<0.05). This may be because the traditional freezing method has a slow freezing speed, produces larger ice crystals during the freezing process, and causes more severe mechanical damage to the tissue cell structure, resulting in more contents flowing out of the cells. Combined with the results of the aforementioned fish meat moisture distribution state, that is, after freezing in the -20℃RF group, more bound water in the Xingkangji eel meat is converted into free water and migrates to the surface of the fish meat, which leads to a higher L* value of the sample. The a* values ​​of the two groups of samples did not change much before and after freezing. In general, the preservation method of Example 2 helps to maintain the stability of the brightness and red-green value of the Xingkangji eel meat after freezing.

[0097] (6) Texture Profile Analysis (TPA)

[0098] Take the back muscle of the thawed Xingkangji eel meat and cut it into small cubic pieces of 15mm×15mm×15mm in size. Select cylindrical and flat-bottomed probes with a diameter of 40mm for measurement. The cylindrical probe is used to measure hardness, and the flat-bottomed probe is used to measure elasticity. The parameters are set as follows: probe descent speed before measurement: 60mm / min, measurement speed: 1mm / s; probe return speed after measurement: 1mm / s; compression deformation amount is 50%; trigger force: 0.15N. Each group of samples was repeated 5 times and the average value was taken. Texture properties are closely related to the taste of food and can be used to characterize the freshness of food. The present invention selects two TPA-related parameters, hardness and elasticity, to evaluate the quality of Xingkangji eel meat. The texture property results of Example 2 and Comparative Example 1 are shown in Tables 3 and Fig. 9 .

[0099] Table 3 Changes in fish meat texture characteristics

[0100]

[0101] Fig. 9 The bar graph of the texture properties of Example 2 and Comparative Example 1 shows that A is hardness and B is elasticity. Hardness is the ability of fish meat to resist deformation caused by external forces. Fig. 9As can be seen from Part A in Figure 3 and Table 3, the hardness of fish meat treated by the two preservation methods showed a trend of first increasing and then decreasing with the freezing temperature. The hardness was higher when the temperature at the center of the fish body reached 4°C. At this time, the muscle fibers of the fish meat were not damaged by ice crystals and the structure was more compact. The hardness of the fish meat increased slightly at 0°C, which may be due to the glycolysis reaction after the fish died to produce lactic acid, and the large-scale decomposition of ATP promoted the formation of actomyosin complex, resulting in a sharp contraction of the muscle, entering a state of rigidity, and increasing the hardness. After that, the hardness of the Xingkangji eel meat decreased with the deepening of the freezing degree, and the hardness of the fish meat showed a continuous downward trend. When frozen to -10°C, the hardness of the Xingkangji eel meat treated by the two preservation methods was significantly different (P < 0.05). The hardness of the fish meat in the -20°C LNF group after thawing was 33.66 ± 1.72N, which was 12.39% lower than that of the fresh sample. The hardness of the fish meat in the -20°C RF group after thawing was 28.04 ± 1.49N, which was 27.01% lower than that of the fresh sample. During the entire freezing process, the decrease in fish meat hardness in the -20℃LNF group was lower than that in the -20℃RF group.

[0102] Elasticity is the ability of fish meat to deform under external force and return to its original shape after the external force is removed. Fig. 9 It can be seen from Part B and Table 3 that when the center temperature of the fish body just reaches 4°C, the elasticity of the fish meat is the highest. The elasticity of the fish meat treated by the two preservation methods decreases with the freezing temperature and basically shows a positive correlation with the hardness change. When the center temperature of the fish body is frozen to -20°C, the elasticity of the Xingkangji eel meat in the -20°C LNF group decreases to 2.62±0.03N, which is 32.17% lower than the fresh sample, and there is no significant difference with the -20°C RF group (P>0.05).

[0103] Water plays an important role in maintaining the cell and tissue structure of Xing Kangji eel meat. Therefore, the changes in the texture characteristics of Xing Kangji eel meat are related to the size of ice crystals formed inside the fish meat during the freezing process and the integrity of the tissue. In addition, protein denaturation caused by ice crystal formation will also affect the texture of the fish meat. In general, freezing can reduce the hardness and elasticity of fish meat to a certain extent, and the preservation method provided by the present invention produces smaller ice crystals during the freezing process, which causes less damage to the tissue and can effectively maintain the original texture characteristics of the fish meat.

[0104] (7) Volatile flavor substances

[0105] 5.00 g of fresh Xing Kangji eel meat (Fresh) and Xing Kangji eel meat treated by the preservation method of Example 2 (-20°C LNF) were weighed, minced and placed in a headspace injection bottle for volatile flavor substance testing, and each sample was measured 3 times in parallel. The determination of volatile substances in Xing Kangji eel meat was analyzed by GC-IMS, wherein the analysis conditions of the gas chromatography (GC) system, ion mobility spectrometry (IMS) system and headspace automatic injection system were as follows:

[0106] Gas chromatography system: The chromatographic column selected was MXTWAX (15m×0.53mm ID, GAS Dortmund, Germany); the chromatographic separation conditions were as follows: the injection port temperature T3 was 50°C; the chromatographic column transmission temperature T4 was 80°C; the GC column temperature T2 was 60°C; the chromatographic column transmission tube temperature T5 was 50°C. The carrier gas flow rate EPC2 was set as follows: the initial flow rate was 2mL / min, and it was maintained at 2mL / min for the first two minutes. The flow rate increased to 20mL / min, 50mL / min, 100mL / min, and 150mL / min at 10min, 20min, 25min, and 30min, respectively.

[0107] Ion mobility spectrometry system: The temperature T1 in the IMS drift tube was 45 °C, the EPC1 flow rate was fixed at 150 mL / min, the radioactive source in the tube was a deuterium source, and the ionization mode was set to positive ion mode.

[0108] Headspace automatic sampling system: incubation temperature 60°C, oscillation incubation time 20 min, automatic injection needle syringe temperature 65°C, injection speed 60 mL / min, sample injection volume 0.8 mL.

[0109] The retention index (RI) of volatile substances in Xingkangji eel meat was calibrated by using the curve obtained under the same analytical conditions with orthoketones (C4-C9) as external standards. The measured volatile substances were qualitatively identified by comparing the retention time (Rt) and migration time (Dt) of the compounds that have been identified in the GC-IMS database. The signal intensity of volatile flavor substances in each sample was calculated by the instrument's own software VOCal 0.4.03.

[0110] Fig.10 Schematic diagram of the results of volatile flavor substances in fresh conger eel meat and conger eel meat treated by the preservation method of Example 2, wherein A is a GC-IMS spectrum, B is a volatile substance fingerprint, Fresh represents fresh conger eel meat, and -20°C LNF represents conger eel meat treated by the preservation method of Example 2. Fig.10In part A of the graph, the ordinate is retention time (RT), the abscissa is ion migration time (DT), the background of the spectrum is blue, and the red vertical line at 8.0 on the abscissa represents the RIP peak (reaction ion peak). Each bright spot on the right side of the RIP peak represents a volatile substance, and the redder the color, the stronger the signal intensity of the substance. There may be multiple signals at the same retention time and different migration times, representing the monomer, dimer and trimer forms of a compound, which is related to the content of the substance itself and the affinity with hydrogen protons. The volatile substances in the -20℃ LNF group showed significant differences compared with the Fresh group. The volatile substances in the fish meat of the Fresh group were mainly between the retention time of 100-400s and the migration time of 8.0-15.0ms, while the number of volatile substances in the fish meat of the -20℃LNF group increased significantly within the retention time of 100-400s, the peak signal was significantly enhanced, and the retention time of volatile flavor substances was correspondingly prolonged. The retention time of volatile flavor substances was extended from mainly concentrated in 100-400s to 100-700s. The content of volatile flavor substances in Xingkangji eel meat showed an upward trend. The reason may be that the ice crystals formed during the freezing process destroyed the original structure of fish meat cells and tissues, resulting in the outflow of contents. The lower the liquid nitrogen spray temperature, the faster the freezing. Rapid freezing formed small ice crystals with uniform distribution and regular shape, which was beneficial to maintaining the original cell and tissue structure of fish meat.

[0111] right Fig.10 The qualitative analysis was performed after integrating all signal peaks within the retention time and migration time in the GC-IMS spectrum, and the fingerprint of volatile flavor substances of Xingkangji eel meat before and after freezing was constructed, such as Fig.10 As shown in part B. Fig.10 In Part B, each column represents fish samples with different treatments, and each group of samples was tested three times in parallel. Each row represents a substance and is labeled with the corresponding name. The change in the content of volatile substances before and after treatment can be seen by the depth of color. Fig.10 As can be seen from part B in the figure, the number of volatile substances in the fish meat of the Fresh group is relatively small, and the signal intensity is relatively low. After being treated by the preservation method in Example 2, the volatile substances in the Xing Kangji eel meat changed significantly, and the types and signal intensity of volatile substances in the Xing Kangji eel meat of the -20℃ LNF group increased.

[0112] The signal intensity of volatile substances in different samples was calculated using the LAV software provided by the instrument. The results are shown in Table 4.

[0113] Table 4 Volatile substance information

[0114]

[0115]

[0116] from Fig.10 As shown in Table 4, 16 volatile flavor substances were identified in fresh Xing Kangji eel meat (Fresh), and 32 were detected after the preservation method in Example 2, including 5 alcohols, 6 aldehydes, 1 acid, 3 ketones, 6 esters, 2 ethers, 5 other substances and 4 substances that could not be identified (marked as 1, 2, 3, 4 in the fingerprint). The 5 alcohols are 1-phenylethanol, 2-heptanol, benzyl alcohol, ethanol, and n-butanol. The signal intensity of benzyl alcohol and ethanol before and after the preservation treatment of Xing Kangji eel meat is high. Ethanol is an important alcohol substance in aquatic products. Researchers have detected the presence of ethanol in a variety of aquatic products or frozen fish paste products such as rainbow trout, tilapia, and bighead carp. The content of 1-phenylethanol, 2-heptanol and n-butanol in the Fresh group is very low or not detected, and the content increases significantly after the preservation treatment. This may be due to the ice crystals produced by freezing causing certain mechanical damage to cells or tissue structures, thereby leading to the outflow of volatile substances. Aldehydes are the main volatile flavor substances in aquatic products. Due to their low odor threshold, even a small amount of aldehyde substances have an important impact on the flavor of fish. The six aldehyde substances in Xingkangji eel meat are 2-hexenal, benzaldehyde, trans-2-pentenal, heptaldehyde, nonanal, and octanal, and these aldehyde substances all show an upward trend after being treated with the fresh-keeping method in Example 2. 2-hexenal, as a typical straight-chain unsaturated aldehyde, belongs to unsaturated aldehydes (often showing grassy or fishy smell). After being treated with the fresh-keeping method in Example 2, its content increases, and it is one of the substances that cause fish to have a fishy smell. The heptaldehyde content increases significantly after being treated with the fresh-keeping method in Example 2. As an important volatile flavor substance in fish, it may be one of the main reasons for the increase in the fishy smell of fish after freeze-thaw. Ketones are mainly derived from the thermal oxidation degradation of unsaturated fatty acids and have unique aromas and fruity flavors. Although their thresholds are high, they can enhance the overall flavor of fish meat to a certain extent. Three ketone compounds were detected in each group, among which the signal intensities corresponding to 2,3-butanedione and acetone did not change much before and after treatment. 3-Hydroxy-2-butanone increased significantly after treatment, which mainly originated from the oxidation of palmitic acid, stearic acid and oleic acid. In summary, the types and signal intensities of volatile substances in Xingkangji eel meat increased after -20℃ LNF treatment. 1-phenylethanol, 2-heptanol, n-butanol, 2-hexenal, heptanal, dimethyl disulfide, and dimethyl trisulfide were extremely low or undetectable in fresh fish meat (Fresh), and increased significantly after -20℃ LNF treatment, which may be an important reason for the change in fish flavor after freeze-thaw.

[0117] In summary, in terms of water holding capacity, the water holding capacity of Xing Kangji eel meat treated by the two preservation methods in the early stage of freezing was not much different, but with the deepening of freezing, the water holding capacity of Xing Kangji eel meat in the -20℃LNF group was significantly better than that in the -20℃RF group. When the temperature of the center point of the fish body dropped to -20℃, the thawing loss rate and cooking loss rate of the -20℃LNF group were reduced by 10.39% and 29.68% respectively compared with the -20℃RF group, and the centrifugal water holding rate increased by 7.16%. LF-NMR results showed that the content of immobile water in Xing Kangji eel meat gradually decreased during freezing, and the free water content increased. The -20℃LNF group could inhibit the migration of water in the fish meat and the conversion of immobile water to free water. When the temperature of the center point of the fish body dropped to -20℃, the free water content was reduced by 66.23% compared with the -20℃RF group. In addition, the -20℃ LNF group was able to delay the deterioration of the texture properties of the Xing Kangji eel meat, such as hardness and elasticity, and inhibit the fluctuation of the brightness value and the change of color of the Xing Kangji eel meat during the freezing process. After the preservation treatment, the types and signal intensity of volatile substances in the Xing Kangji eel meat increased, which better maintained the quality of the fish meat.

[0118] The above are only preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing nano-medaka egg membrane microvilli, characterized in that: The following steps are involved: The medaka fish eggs and water are mixed and subjected to high-pressure microfluidization homogenization treatment, and then the mixture is subjected to centrifugation, concentration and re-centrifugation to obtain the nano medaka fish egg membrane microvilli; The high-pressure microfluidization homogenization treatment includes: first treating at 50 bar for 0.1 h, then treating at 80 bar for 0.1 h, and then treating at 100 bar for 0.1 h; The diameter of the nozzle used in the high-pressure microfluidic homogenization process is 20 nm.

2. The method for preparing nano medaka egg membrane microvilli according to claim 1, characterized in that: The solid-liquid ratio of the medaka fish eggs to water is 1:16 g / mL.

3. The method for preparing nano medaka egg membrane microvilli according to claim 1, characterized in that: The concentration temperature is 50° C.; the volume of the concentrated solution obtained by the concentration is 1 / 3 of the volume of the supernatant obtained by centrifugation.

4. The method for preparing nano medaka egg membrane microvilli according to claim 1, characterized in that: The rotation speed of the centrifugation and re-centrifugation was 5000 rpm, and the time was 10 min.

5. Nano-medaka egg membrane microvilli prepared according to the preparation method according to any one of claims 1 to 4.

6. An application of the nano-medaka egg membrane microvilli according to claim 5 in fish meat preservation, characterized in that: The fish meat is Xing Kangji eel meat.

7. The use of the nano-medaka egg membrane microvilli in fish preservation according to claim 6, characterized in that: The following steps are involved: Slaughtering and precooling fresh live fish to obtain pretreated fish meat; The nano-medaka egg membrane microvilli described in claim 5 are applied to the surface of the pre-treated fish meat, and then the fish meat is sprayed and frozen with liquid nitrogen to preserve the fish meat.

8. The use of the nano-medaka egg membrane microvilli in fish preservation according to claim 7, characterized in that: The application amount of the application is 1.25 mL / g.

9. The use of nano medaka egg membrane microvilli in fish preservation according to claim 7, characterized in that: The freezing temperature of the liquid nitrogen spray is -20°C.

Images