Novel dipping type refrigerating fluid as well as preparation method and application thereof

Abstract

The invention provides novel dipping type refrigerating fluid as well as a preparation method and application thereof, and belongs to the technical field of frozen storage and preservation of aquatic products. The novel dipping type refrigerating fluid is prepared from the following raw materials in percentage by weight: 23 to 27 percent of ethanol, 8 to 12 percent of betaine, 9 to 11 percent of propylene glycol and 3 to 5 percent of sodium chloride. The ratio of the absolute value of the freezing point to the viscosity of the obtained novel dipping type freezing fluid is at a high level, which shows that the freezing fluid is very stable in freezing effect, has a good freezing effect, is short in freezing time, keeps various physical and chemical indexes well, and can well inhibit protein oxidation and cathepsin activity of the Chinese penis shrimps through low-temperature cryopreservation; the obtained novel dipping type refrigerating fluid is free of residues, small in volatility and free of obvious corrosion effect on equipment.

Description

Technical Field

[0001] The invention belongs to the technical field of frozen storage and preservation of aquatic products, and in particular relates to a novel immersion freezing liquid and a preparation method and application thereof. Background Art

[0002] Chinese tube whip shrimp is a very delicious food rich in high protein and minerals. It has the function of regulating the heart and protecting the cardiovascular system. It can not only accelerate the regeneration of brain cells, promote brain development, and improve memory, but also prevent Alzheimer's disease and protect eyesight.

[0003] Chinese whip shrimp is the most abundant economic shrimp in the East China Sea and the Yellow Sea. It is the main fishery resource in Zhejiang Province and the East China Sea. Its meat is tender and delicious and rich in nutrients, which is loved by many consumers. Freshness is an important factor affecting the quality of aquatic products. Due to the combined effects of factors such as microorganisms and endogenous proteases, aquatic products are extremely perishable. Therefore, the storage method has a great impact on their quality during transportation and storage. Therefore, it is necessary to study more economical and effective preservation technologies. The most commonly used storage method for Chinese whip shrimp is frozen storage, which can maximize the freshness of Chinese whip shrimp. However, the current methods for quick freezing of aquatic products are very limited. The main methods are freezer freezing and plate freezing, which have problems such as slow freezing rate, large juice loss rate, and poor freezing effect. Since Chinese whip shrimp is rich in protein, there are still many problems during long-term low-temperature storage, such as dry loss, blackening of shrimp heads, softening of texture, and protein denaturation. However, there is still little research on the selection of quick freezing methods for Chinese whip shrimp and the internal causes and mechanisms of corruption and deterioration during storage.

[0004] At present, multiple refrigerants have emerged to improve the quality of seafood, but in actual production and actual use, their development is hindered by some of their own shortcomings. First, due to the repeated contact between food and refrigerant, residual substances will be produced, thereby destroying its physical properties and affecting its safety; second, when food is in long-term contact with refrigerant, it will absorb refrigerant solutes, and excessive absorption will have an adverse effect on the quality and safety of food; third, ethanol and sodium chloride are both common and efficient components of refrigerants, but ethanol is very volatile, and sodium chloride is a common salt, which will cause serious corrosion to production equipment; fourth, in industry, there are high requirements for the quality of refrigerants. If the freezing temperature is too high or the viscosity is too large, it cannot be used to make refrigerants.

[0005] Therefore, it is urgent to develop a coolant with low temperature, low viscosity and good stability to solve the problems of deterioration of storage temperature and storage quality of Chinese tube shrimp. This has very important practical significance for ensuring the value of shrimp products and meeting market needs. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a novel immersion freezing liquid and a preparation method and application thereof. The ratio of the absolute value of the freezing point to the viscosity of the novel immersion freezing liquid obtained by the present invention is at a relatively high level, indicating that the freezing effect of the freezing liquid is very stable, and it has a good freezing effect, a short freezing time, and various physical and chemical indicators are well maintained. Low-temperature freezing storage can better inhibit protein oxidation and tissue protease activity of Chinese tube whip shrimp; and the novel immersion freezing liquid obtained by the present invention has no residue, low volatility, and no obvious corrosion to equipment.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a novel immersion freezing liquid, which is composed of the following raw materials in percentage by weight: 23-27% ethanol, 8-12% betaine, 9-11% propylene glycol, and 4% sodium chloride.

[0009] Preferably, the weight percentages of the raw materials are 25% ethanol, 10% betaine, 10% propylene glycol and 4% sodium chloride.

[0010] Further preferably, the balance of the raw material may be water.

[0011] In a second aspect, the present invention provides a method for preparing the novel immersion freezing liquid, comprising the following steps:

[0012] Weigh the raw materials according to the weight percentage;

[0013] Mix all the ingredients evenly and keep for later use.

[0014] In a third aspect, the present invention provides the use of the novel immersion freezing liquid in quick-freezing Chinese shrimp.

[0015] Preferably, the temperature of the novel immersion freezing liquid for quick-freezing Chinese whip shrimp is -18 to -30°C; and the time required for quick freezing to the target temperature is 15 to 30 minutes.

[0016] Preferably, the storage time of the novel immersion-type freezing liquid quick-frozen Chinese whip shrimp is 120 days.

[0017] In a fourth aspect, the present invention provides a cooling liquid quick-freezing and fresh-keeping method for Chinese prawns, comprising the following steps:

[0018] The fresh Chinese whip shrimp is immersed in the novel immersion freezing liquid described in claim 1, and then the Chinese whip shrimp is quickly frozen to a target temperature using a low-temperature cooling liquid circulation pump.

[0019] Preferably, the ratio of the fresh Chinese whip shrimp to the novel immersion freezing liquid is 5 kg / L.

[0020] Preferably, the target temperature is -18 to -30°C.

[0021] Contains at least the following beneficial technical effects:

[0022] (1) The ratio of the absolute value of the freezing point to the viscosity of the novel immersion freezing liquid obtained by the present invention is at a relatively high level, indicating that the freezing effect of the freezing liquid is very stable and has a good freezing effect; it has a suitable viscosity and can achieve the effect of rapid cooling and convenient reflux.

[0023] (2) The novel immersion freezing liquid obtained by the present invention is suitable for the quick freezing process of Chinese whip shrimp. It takes 5 minutes to quickly freeze through the maximum ice crystal formation zone (0°C to -5°C) at -30°C; it takes 15 minutes to quickly freeze Chinese whip shrimp to -18°C using the freezing liquid, and 30 minutes to -30°C.

[0024] (3) The novel immersion freezing liquid obtained by the present invention has no residue, low volatility, and no obvious corrosive effect on equipment.

[0025] (4) After the novel immersion freezing liquid obtained by the present invention was used for quick freezing of Chinese whip shrimp, it was tested that the physical and chemical indicators in the early stage were well maintained. Low-temperature freezing can effectively inhibit the protein oxidation and cathepsin activity of Chinese whip shrimp, so that its protein structure is well maintained. In the later stage of storage, the inhibitory effect on enzyme activity is reduced, resulting in the hydrolysis of myofibrillar protein. The activity of cathepsin L is higher than that of cathepsin B, which indicates that cathepsin L plays a more important role in the changes of muscle tissue during storage; however, compared with direct storage in a refrigerator, the change range of storage after freezing with freezing liquid is much smaller. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the freezing curve of the refrigerant.

[0027] Figure 2 This is a BSA standard curve.

[0028] Figure 3 A graph showing the effect of freezing methods on the center temperature of Chinese tube shrimp.

[0029] Figure 4 Salt-soluble protein content of Chinese shrimp under different freezing methods.

[0030] Figure 5 Changes in surface hydrophobicity of Chinese tube shrimp under different freezing methods.

[0031] Figure 6 Ca of Chinese troodon sphenanthera under different freezing methods 2+ -ATPase activity change diagram.

[0032] Figure 7 Changes in carbonyl content of Chinese tube shrimp under different freezing methods.

[0033] Figure 8 Changes in thiol content in Chinese shrimp under different freezing methods.

[0034] Fig. 9 Changes in the activity of tissue plasminogen activator B in Chinese tube shrimp under different freezing methods.

[0035] Fig.10 Changes in tissue proteinase H activity in Chinese tube shrimp under different freezing methods.

[0036] Fig.11 Changes in the activity of tissue proteinase L in Chinese tube shrimp under different freezing methods.

[0037] Fig.12 Changes in NAGase activity of Chinese tube shrimp under different freezing methods.

[0038] Fig.13 Changes in AG enzyme activity of Chinese tube shrimp under different freezing methods.

[0039] Fig.14 SDS-PAGE analysis of myofibrillar proteins in Chinese tube shrimp under different freezing methods.

[0040] Fig.15 Changes in the cross-section of myofibrils of Chinese tube whip shrimp under different freezing methods.

[0041] Fig.16 Changes in the longitudinal section of myofibrils of Chinese tube whip shrimp under different freezing methods. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] The "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C unless otherwise specified.

[0047] The raw materials and instruments used in the following examples of the present invention are commercially available unless otherwise specified.

[0048] Example 1

[0049] Preparation of a new type of immersion freezing liquid

[0050] 1.2 Reagents and instruments

[0051] 1.2.1 Main reagents

[0052] Sodium chloride, Shanghai Sinopharm Group; ethanol, Merck, Germany; propylene glycol, betaine, Henan Chengyi Biotechnology Co., Ltd.

[0053] 1.2.2 Main instruments

[0054] Centrifuge (AvantiJXN-30), Beckman Coulter; analytical balance (ME204E), Mettler Toledo; viscometer (NDJ-8s), Fangrui; ultra-low temperature refrigerator (902GP), Thermo Fisher Scientific; temperature recorder (L93-2L), Luger Technology.

[0055] 1.3 Experimental methods

[0056] 1.3.1 Determination of freezing point of refrigerant

[0057] Put the prepared freezing solution in a beaker, immerse the thermometer probe in the solution and fix it, connect the other end of the probe to the temperature recorder interface, put the beaker containing freezing solution in a -60℃ refrigerator, turn on the recorder to record the temperature change, and the temperature recorder can continuously record the temperature change. As can be seen in the figure, there are two obvious parts in the curve, the steeper part is the cooling curve, and the flatter part is the crystallization curve. Take the average temperature of the flat curve and regard this average temperature as the approximate freezing point.

[0058] 1.3.2 Determination of Viscosity of Refrigerant

[0059] During the freezing period, the viscosity of the freezing liquid will fluctuate greatly. When the temperature drops, its viscosity will gradually increase. In order to accurately measure the viscosity at a specific temperature, the rotor and speed should be changed frequently. The beaker containing the freezing liquid should be placed in a large beaker containing anhydrous ethanol and placed in a refrigerator at -60°C. After each 5°C drop in temperature, take it out immediately, read the viscosity value, and then select the appropriate rotor for measurement.

[0060] 1.3.3 Single Factor Experiment

[0061] Prepare ethanol with concentrations of 0, 10%, 15%, 20%, 25%, and 30%; propylene glycol with concentrations of 0, 2%, 6%, 10%, 14%, and 18%; betaine with concentrations of 0, 5%, 10%, 15%, and 20%; and sodium chloride with concentrations of 0, 2%, 4%, 6%, 8%, and 10% for single-factor experiments. The freezing point, -30°C viscosity, and the ratio of the absolute value of the freezing point to the viscosity at -30°C were used as evaluation indicators. The higher the ratio, the better the quality of the freezing liquid, and the concentration range of the four substances was further determined.

[0062] 1.3.4 Orthogonal test

[0063] Based on the single factor experiment, the concentrations of ethanol, betaine, propylene glycol and sodium chloride were used to make L9(3 4 ) to optimize the best ratio of the four substances. As shown in Table 1:

[0064] Table 1 Orthogonal test factors and levels

[0065]

[0066]

[0067] 1.3.5 Data processing

[0068] Excel was used for statistical data, Origin8.5 was used for chart analysis, and SPSS23.0 was used for variance analysis. The results were expressed as mean ± standard deviation.

[0069] 1.4 Results and Discussion

[0070] 1.4.1 Single factor test and results

[0071] 1.4.1.1 Ethanol addition amount

[0072] According to the data, compared with other current refrigerants, using ethanol as a refrigerant has considerable advantages in terms of relative safety, fast freezing rate and low cost. Moreover, ethanol is a non-toxic solution that can be mixed with water in a certain proportion. It is the main raw material of the refrigerant, but it is easy to volatilize. Prepare ethanol solutions of different concentrations for freezing, and record their freezing point, -30℃ viscosity, and the ratio of the absolute value of the freezing point of the refrigerant to the viscosity, as shown in Table 2.

[0073] Table 2 Effect of ethanol addition on the freezing solution of Chinese whip shrimp

[0074]

[0075] Note: Data in the same column with different letters indicate significant differences (P<0.05).

[0076] As shown in Table 2, the freezing point decreases with the increase of ethanol concentration, and the ratio of the absolute value of the freezing point to the viscosity gradually increases, indicating that the freezing liquid is stable and the freezing effect is good. When the ethanol content is 25%, the freezing point is -45.43℃; when the content is 25% and 30%, the ratio of the absolute value of the freezing point to the viscosity is 0.42 and 0.51, and the ratio is not much different. Reducing the mass fraction of ethanol can reduce the ethanol volatilization, which is easy to produce and apply. Therefore, 25% is selected for the next stage of the experiment.

[0077] 1.4.1.2 Betaine addition amount

[0078] Betaine has high solubility in aqueous solution, up to 160g / 100g. It has a very strong effect on absorbing and retaining water, and can be used as the main component of freezing liquid. Betaine solutions of different concentrations were prepared for freezing, and the freezing point, viscosity at -30℃, and the ratio of the absolute value of the freezing point of the freezing liquid to the viscosity were recorded. The results are shown in Table 4-3. The results show that the freezing point temperature decreases with the increase of betaine content and can reach -50℃. The ratio of the absolute value of the freezing point to the viscosity is from high to low. When the concentration of betaine is 10%, the ratio of the absolute value of the freezing point to the viscosity is the largest, the freezing liquid tends to be stable, and the freezing effect is better. Considering the viscosity and compounding problems of the freezing liquid, 10% is selected for the next stage of the experiment.

[0079] Table 3 Effect of betaine addition on frozen solution of Chinese whip shrimp

[0080]

[0081] Note: Data in the same column with different letters indicate significant differences (P<0.05).

[0082] 1.4.1.3 Propylene glycol addition amount

[0083] Table 4 Effect of propylene glycol addition on the frozen solution of Chinese whip shrimp

[0084]

[0085] Note: Data in the same column with different letters indicate significant differences (P<0.05).

[0086] According to the data, propylene glycol has the advantages of low permeability to food, little effect on food flavor, low freezing point, and low volatility. Therefore, propylene glycol is also an important component in the freezing liquid. Propylene glycol of different concentrations was configured for freezing, and the freezing point, viscosity at -30°C, and the ratio of the absolute value of the freezing point of the freezing liquid to the viscosity were recorded. The results are shown in Table 4-4. As the propylene glycol content increases, the freezing point gradually decreases, and the ratio of the absolute value of the freezing point to the viscosity shows a trend from high to low. Due to the high viscosity of propylene glycol, a high mass fraction will affect the viscosity of the freezing liquid. When the propylene glycol is 6%, the freezing point value reaches -32.42°C, and the ratio of the absolute value of the freezing point to the viscosity is the largest. Therefore, 6% is selected for the next stage of the experiment.

[0087] 1.4.1.4 Amount of sodium chloride added

[0088] At present, sodium chloride is the most commonly used freezing liquid. Sodium chloride solution has long been used for freezing and preservation of seafood such as fish and shrimp on fishing boats. It is the most common component of the freezing liquid. Although it has a low freezing point, it is easy to corrode instruments and often penetrates into the frozen items, thus affecting their taste and quality. Sodium chloride solutions of different concentrations were prepared for freezing, and the freezing point, viscosity at -30°C, and the ratio of the absolute value of the freezing point of the freezing liquid to the viscosity were recorded. The results are shown in Table 4-5. The freezing point gradually decreases with the increase of sodium chloride content. When the sodium chloride concentration is 4%, the freezing point drops to -36.1°C, and the ratio of the absolute value of the freezing point to the viscosity is the largest. Due to the problems that sodium chloride is easy to corrode instruments and penetrate into items, 4% was selected for the next stage of the experiment.

[0089] Table 5 Effect of sodium chloride addition on frozen solution of Chinese whip shrimp

[0090]

[0091] Note: Data in the same column with different letters indicate significant differences (P<0.05).

[0092] 1.4.2 Orthogonal test results

[0093] Based on the results of single factor experiments, the ratio of the absolute value of the freezing point of the freezing liquid to its viscosity was selected as an indicator, and the orthogonal experiment was used to optimize the freezing liquid formula suitable for the rapid freezing of Chinese whip shrimp. The results of the orthogonal experiment are shown in Table 6.

[0094] It can be seen from the table that the best additive combination of the Chinese whip shrimp freezing liquid formula is A3B2C3D3, that is, the additive amounts are 25% ethanol, 10% betaine, 10% propylene glycol, and 4% sodium chloride respectively.

[0095] Table 6 Orthogonal test design and results of frozen solution of Chinese whip shrimp

[0096]

[0097]

[0098] Note: Data in the same column with different letters indicate significant differences (P<0.05).

[0099] A: ethanol concentration; B: betaine concentration; C: propylene glycol concentration; D: sodium chloride concentration.

[0100] Table 7 | Freezing point | / Viscosity variance analysis results

[0101]

[0102] Note: A: ethanol concentration; B: betaine concentration; C: propylene glycol concentration; D: sodium chloride concentration.

[0103] The results in Table 7 show that the concentrations of ethanol, betaine, propylene glycol and sodium chloride solution have a great influence on the ratio of the absolute value of the freezing point and viscosity of the freezing liquid (P<0.05). The range and variance are used to measure the influence of the four groups of factors on the experimental results. The comprehensive analysis results show that the order of influence of A, B, C, and D on the ratio of the absolute value of the freezing point and viscosity is: A>C>D>B. Among them, the amount of ethanol added is the most important factor affecting the optimization of the freezing liquid formula. Therefore, the optimal addition level of ethanol solution is 25%; the amount of propylene glycol added is also the main factor affecting the optimization of the freezing liquid formula. Propylene glycol has the advantage of low volatility, and the addition of propylene glycol is considered to be 10%; since sodium chloride solution is easy to corrode equipment, the addition of sodium chloride solution is selected to be 4%; betaine has a strong hygroscopic and water-retaining property. Considering that the addition of betaine is 10%, the optimal addition amount combination A3B2C3D3 of the freezing formula of Chinese whip shrimp freezing liquid is selected, and a verification test is required.

[0104] Table 8 Verification test results

[0105]

[0106] As shown in Table 8, when the temperature of the freezing liquid is frozen to -10℃, -20℃, -30℃, -40℃ and -50℃, the viscosity continues to increase. At -40℃, the viscosity rises sharply, which is 63.92% higher than that at -30℃. At -30℃, the ratio of the absolute value of the freezing point to the viscosity reaches the maximum, indicating that the freezing effect is best at this temperature. At each temperature, the viscosity value is lower than the test value of Yang Xianqing et al. Therefore, the formula of the freezing liquid is better.

[0107] 1.5 Summary of this chapter

[0108] This chapter aims to screen and optimize the formula of a new type of immersion freezing liquid. It studies the selection of ethanol, betaine, propylene glycol and sodium chloride to form a multi-component coolant and applies it to the rapid freezing of Chinese whip shrimp.

[0109] The experiment shows that the addition range of the four substances is determined by a single factor test. On the basis of the single factor test, the orthogonal test is used to analyze the effect of the addition amount of the four substances on the ratio of the absolute value of the freezing point of the freezing liquid to the viscosity, and the optimal addition ratio of the four substances is determined: a multi-component coolant composed of 25% ethanol, 10% betaine, 10% propylene glycol, and 4% sodium chloride, based on the total mass fraction of the freezing liquid. Under this condition, a confirmation test was carried out. At -30℃, the ratio of the absolute value of the freezing point to the viscosity reached the maximum, indicating that the freezing liquid at this temperature is stable and has a good freezing effect. This provides scientific theoretical data for the subsequent comparative analysis of the quality changes of Chinese whip shrimp frozen liquid quick freezing and refrigerator direct freezing of shrimp meat.

[0110] Example 2

[0111] Changes of protein oxidation-related indicators in Chinese prawns under different frozen storage conditions

[0112] 2.1 Introduction

[0113] The protein content of Chinese whip shrimp is rich, reaching about 18%. The change of protein has an important influence on the changes of muscle tissue of Chinese whip shrimp, among which protein oxidation is an important change. Studies have shown that protein oxidation will affect carbonyl, sulfhydryl, ATPase activity and surface hydrophobicity. Since refrigeration conditions are not suitable for long-term storage of Chinese whip shrimp, based on the previous chapter, the freezing liquid with the optimal temperature and the best ratio is selected to explore the changes of protein oxidation during storage of Chinese whip shrimp by quick freezing of freezing liquid and refrigerator freezing. Frozen storage can inhibit the activity of microorganisms for a long time and avoid deterioration caused by microorganisms to the greatest extent, thereby ensuring the flavor and nutritional value of food. Malik et al. studied the physical and chemical properties of different species of fish under frozen storage conditions. The results showed that during frozen storage, the protein of fish meat denatured, which may be related to the formation and growth of ice crystals during freezing. Therefore, the research on new quick freezing methods and processes is particularly important. Compared with refrigerator freezing, quick freezing with freezing liquid can better maintain the quality of raw materials. In a very short time, tiny ice crystals will be produced. These ice crystals are numerous and evenly distributed, and do not cause much damage to the food. The food can also maintain its original texture and taste after thawing.

[0114] The present invention selects four different freezing conditions (-18°C direct freezing, -18°C quick freezing, -30°C direct freezing, -30°C quick freezing), and measures various indexes of protein oxidation of myofibrillar protein of Chinese tube whip shrimp as the storage time is prolonged, explores and compares the effects of various freezing methods on the oxidation of myofibrillar protein of Chinese tube whip shrimp, and provides a theoretical basis for the storage period of Chinese tube whip shrimp in future production processes.

[0115] 2.2 Materials and instruments

[0116] 2.2.1 Experimental Materials

[0117] Chinese whip shrimp were caught in Zhoushan waters of the East China Sea, with an average body length of (12.5±2.2) cm and an average weight of (15.5±2.3) g. After being caught, they were placed in ice packs and placed in an incubator, and then quickly sent back to the laboratory after landing.

[0118] 2.2.2 Main reagents

[0119] Copper sulfate pentahydrate, potassium sodium tartrate, disodium hydrogen phosphate, bromophenol blue, Shanghai Sinopharm Group; bovine serum albumin, Beijing Solebow Company; Tris-maleic acid, Maclean Company; ATPase kit, carbonyl kit, thiol kit, Nanjing Jiancheng Institute.

[0120] 2.2.3 Main instruments

[0121] UV-visible spectrophotometer (Cary 50), Varian; centrifuge (AvantiJXN-30), Beckman Coulter; analytical balance (ME204E), Mettler Toledo; vortex mixer (LPD2500), Leppert; electric constant temperature water bath (DF-101S), Bonsi Instruments; low temperature coolant circulation pump (DLSB-30 / 40), Gengyu Instruments.

[0122] 2.3 Experimental methods

[0123] 2.3.1 Pretreatment of raw materials

[0124] Quick freezing in freezing liquid: Use a low-temperature coolant circulation pump to quickly freeze the Chinese whip shrimp packed in sealed bags to -18℃ and -30℃, and then store them in a refrigerator at -18℃ and -30℃ for 120 days, respectively, and mark them as Group A and Group C, respectively.

[0125] Refrigerator storage: The Chinese whip shrimp were packaged into sealed bags and stored in refrigerators at -18℃ and -30℃ for 120 days. The samples were marked as group B and group D. The indicators of the four groups of Chinese whip shrimp A, B, C, and D were measured every 20 days.

[0126] 2.3.2 Determination of protein content

[0127] The content of myofibrillar protein in Chinese shrimp was determined by biuret method.

[0128] Drawing of BSA standard curve: Take 18 test tubes and divide them into three groups, add 0.0ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml of 10mg / ml bovine serum protein solution, add water to 1ml, add 4ml of biuret reagent, shake well, color at 20℃ to 25℃ for 30min, and measure at 540nm wavelength. The first test tube without protein solution is the blank control, take the average value of the three groups, use protein content as the horizontal axis and light absorption value as the vertical axis to draw the standard curve, see Figure 2 .

[0129] 2.3.3 Determination of salt-soluble protein content

[0130] According to the method of Tadpitchayangkoon et al. Take 2g shrimp meat, add 30mL cold water, homogenize at 10000r / min for 10s, centrifuge at 4℃ and 12000r / min for 5min, collect the precipitate and supernatant respectively. Add 30mL 0.3% NaCl solution to the precipitate, repeat the above operation, and collect the precipitate again. Continue to add 30mL ice-cold 0.6M NaCl-20mM Tris-maleic acid buffer (pH=7.0) to the precipitate, homogenize at 10000r / min for 5s, and extract protein at 4℃ for 1h. The homogenate at this time is salt-soluble protein, and the salt-soluble protein content is calculated using the double contraction pulse method.

[0131] 2.3.4 Extraction of myofibrillar proteins

[0132] According to the method of Jiang et al., 10 ml of salt-soluble protein was taken, 30 mL of cold water was added, the supernatant was diluted to precipitate myofibrillar protein, centrifuged at 12000 r / min for 5 min at 4°C, and the supernatant was discarded. The above steps were repeated twice. The precipitate was dissolved with 0.6 M NaCl and stored in a refrigerator at 4°C for subsequent experiments.

[0133] 2.3.5 Determination of surface hydrophobicity

[0134] According to the method of Zhou Jingli et al., the myofibrillar protein extracted in Section 5.3.4 was diluted to 2 mg / mL with 10 mmol / L phosphate buffer, and 2 mL was taken from it and 40 μL of 1 mg / mL bromophenol blue solution was added. The control group was added with 2 mL of phosphate buffer and then 40 μL of bromophenol blue solution. After mixing, it was allowed to stand for 15 minutes, centrifuged at 5000 r / min for 10 minutes, and the absorbance was measured at 595 nm. Since bromophenol blue can bind to hydrophobic groups, it can bind to exposed amino acid residues in proteins. When the number of them bound to bromophenol blue increases, the surface hydrophobicity will gradually increase. The formula is:

[0135]

[0136] Where: B is the binding amount of bromophenol blue / μg.

[0137] 2.3.6Ca 2+ -ATPase activity assay

[0138] Refer to the instructions of the ATPase kit for determination. The calculation formula is:

[0139]

[0140] 2.3.7 Determination of carbonyl content

[0141] Refer to the instructions of the carbonyl kit for determination. The calculation formula is:

[0142]

[0143] 2.3.8 Determination of thiol content

[0144] Refer to the instructions of the thiol kit for determination. The calculation formula is:

[0145]

[0146] 2.3.9 Data processing

[0147] Excel was used for statistical data, Origin8.5 was used for chart analysis, and SPSS23.0 was used for variance analysis. The results were expressed as mean ± standard deviation.

[0148] 2.4 Results and Discussion

[0149] 2.4.1 Effects of different freezing conditions on the central temperature of Chinese shrimp

[0150] Freezing rate is an important factor affecting the quality of frozen aquatic products. It is closely related to the water transfer rate and ice crystal formation during the freezing process. Figure 3 As shown in the figure, the changes in the center temperature of Chinese whip shrimp under -30℃ direct freezing and -30℃ quick freezing.

[0151] Depend on Figure 3 It can be seen that it takes 5 minutes to pass the maximum ice crystal formation zone (0℃ to -5℃) by -30℃ quick freezing, 15 minutes to quick freeze the Chinese whip shrimp with freezing liquid to -18℃, and 30 minutes to -30℃, and its center temperature drops linearly; the effect of -30℃ direct freezing is lower than quick freezing, and it takes 15 minutes to pass the maximum ice crystal formation zone, and the temperature drops slowly, requiring 35 minutes to freeze to -18℃ and 50 minutes to freeze to -30℃. Comparative analysis of the freezing rate under the two methods shows that quick freezing with freezing liquid can complete the freezing of Chinese whip shrimp faster than direct freezing in the refrigerator.

[0152] 2.4.2 Salt-soluble protein content

[0153] In the muscles of Chinese whip shrimp, salt-soluble protein is the main protein, accounting for about 65% to 75%. Figure 4The salt-soluble protein of fresh shrimp was 120.4 mg / g, and its content showed a trend of gradual decrease with the increase of storage time. However, the salt-soluble protein content of Chinese whip shrimp after quick freezing in freezing liquid was always higher than that under refrigerator freezing during the frozen storage period, which means that the faster the freezing rate, the less the denaturation of salt-soluble protein. At 120 days, the salt-soluble protein content under -30℃ refrigerator storage was 77.9 mg / g, which was 35.3% lower than the initial value, and the difference was significant (P<0.05). On the 60th day of storage, the salt-soluble protein content of shrimp meat directly stored in -18℃ refrigerator was significantly reduced to 78.2 mg / g (P<0.05). Until 120 days, the protein content showed a slow downward trend, but there was no significant difference (P>0.05). Although the protein content under -30℃ quick freezing and -18℃ quick freezing also decreased, there was no significant difference during the entire storage period (P>0.05). At the same time, we can see that at the 20th, 60th and 120th day of storage, the protein content of group C was significantly different from that of group B (P<0.05). This shows that the solubility of salt-soluble protein in shrimp meat directly stored in a -18℃ refrigerator decreases faster than that in the other three storage methods. Therefore, it shows that low storage temperature has a greater impact on the solubility of salt-soluble protein.

[0154] Liu et al. analyzed the biochemical and physical properties of Procambarus clarkii under frozen storage conditions and found that after 24 weeks of storage, the content of salt-soluble protein extracted from Procambarus clarkii also decreased significantly, which is similar to the experimental results of this paper. Studies have found that there are many reasons for the decrease in protein content caused by low-temperature freezing. Lin Hong et al. showed that ice crystal precipitation occurs in some of the bound water in the muscles of aquatic products, causing non-covalent bonds between actomyosin molecules, forming insoluble aggregates of large molecules, resulting in reduced actomyosin dissolution. Zeng Mingyong et al. found that actomyosin degrades and denatures to generate alkaline proteins. This protein cannot be dissolved at high ion concentrations, but can be dissolved in alkaline solutions, thereby reducing its solubility during frozen storage. The heavy chain of myosin is polymerized, and the content of salt-soluble protein decreases. From the results of the determination of the salt-soluble protein content in this experiment, it can be seen that the freezing speed has a certain influence on the content of salt-soluble protein during frozen storage. The reason is probably that quick freezing generates tiny ice crystals in cells, which in turn changes their microenvironment.

[0155] 2.4.3 Changes in surface hydrophobicity

[0156] The degree of exposure of groups in proteins can indirectly reflect changes in surface hydrophobicity. Figure 5In the first 60 days, the surface hydrophobicity of the four groups A, B, C, and D showed an increasing trend, from 310.23 when fresh to 395.62, 485.21, 386.74, and 435.64, with the maximum value appearing on the 60th day. From 0 to 60 days, the surface hydrophobicity of each group changed greatly, and from 60 to 120 days, the change tended to be stable. The increase in surface hydrophobicity is because the protein is oxidized in the frozen state, resulting in structural changes, thereby exposing the hydrophobic amino acids hidden in the protein. During storage for 60-120 days, although the surface hydrophobicity decreased slightly, it was still higher than the initial value. The results showed that when the Chinese whip shrimp was stored for 60, 80, and 120 days, although the surface hydrophobicity change trend of myofibrils under the four groups of frozen storage methods was basically the same, there were still significant differences between them. Groups B and D were significantly higher than groups A and C, indicating that the protein structure of shrimp meat under refrigerator storage has undergone significant changes. When stored at -30°C, the surface hydrophobicity is the lowest, thus ensuring that the spatial conformation of the protein does not change.

[0157] Jarenback found that during low-temperature storage, the surface hydrophobicity of cod first showed an upward trend, and then began to show a downward trend at the 12th week. This may be because further oxidation occurred, the hydrophobic groups became hydrophobic, and aggregation occurred, which reduced its value. Adriana found that after 120 days of freezing at -20℃, the muscle fibers of Penaeus tenuissima stored with tocopherol remained intact, and the surface hydrophobicity increased slowly. Chen et al. found that freezing technology is very important for storing animal products such as surimi. However, mechanical damage caused by ice crystals can lead to quality degradation. Therefore, he studied the protective effect of antifreeze peptides on the quality of surimi during frozen storage and its possible mechanism. It was found that antifreeze peptides showed a strong inhibitory effect on ice crystal recrystallization, which can maintain the surface hydrophobicity of myofibrillar protein, thereby protecting surimi from losing bound water during frozen storage, which is similar to the results of this article.

[0158] 2.4.4Ca 2+ -ATPase activity

[0159] Ca 2+ -ATPase activity can also be used to evaluate the integrity of myosin and myofibrillar protein molecules. Figure 6 During storage, Ca 2+ -ATPase activity was significantly reduced, indicating that the oxidation process continued under the frozen state, but there were great differences at different temperatures. During the period, the activities of groups A and C were higher than those of groups B and D, and there was a significant difference between the two. 2+The decreasing trend of -ATPase activity is faster than that in the later stage of storage, which may be because the ice crystals produced by Chinese whip shrimp are smaller after rapid freezing, which can better protect the integrity of myosin in muscle. 2+ -ATPase activity was 0.98U / mgprot. After 120 days, the Ca of groups A, B, C, and D 2+ -ATPase activity was 0.21, 0.13, 0.32, and 0.18 U / mgprot, respectively, down by 78.5%, 86.7%, 67.3%, and 81.6%. It can be seen that the Ca content in the muscle of Chinese whip shrimp was increased after quick freezing with freezing solution and then stored. 2+ -ATPase activity remained the highest and decreased the least.

[0160] Shui et al. found that after 120 days of frozen storage of shrimp slips mixed with carrageenan, the Ca 2+ -ATPase activity decreases slowly. Niu et al. discussed the changes in sensory and biochemical properties of fresh scallop meat during storage. Microscopic observation of myofibrils extracted from scallop meat showed that the myofibril proteins constituting the myofibrils changed to a certain extent due to freezing. Although the myofibrillar proteins in the head region remained stable, the Ca 2+ -ATPase activity indicated that the interaction between myofibrillar proteins was weakened by freezing. 2+ There are many factors that affect the activity of -ATPase. Chen et al. believe that during the frozen storage, the formation and growth of ice crystals lead to the destruction of the spatial structure of the protein, thereby reducing the Ca 2+ -ATPase activity. This experiment shows that quick freezing in cryogenic liquid can inhibit the growth of ice crystals during storage to a certain extent. Therefore, it can better inhibit protein oxidation and cause less damage to protein structure.

[0161] 2.4.5 Carbonyl content

[0162] After protein oxidation, amino acids can be converted into carbonyl derivatives, among which carbonyl can be used as an indicator of the degree of protein oxidation. During the entire storage period, the carbonyl content of myofibrils of Chinese whip shrimp increased with time under the four storage methods. Figure 7. The carbonyl content increased from 1.25 nmol / mgprot in fresh shrimp samples to 4.02 nmol / mgprot, indicating that under frozen storage conditions, the protein of Chinese whip shrimp underwent oxidative changes. As can be seen from the figure, the carbonyl content of the muscle protein of Chinese whip shrimp in groups A and C increased sharply on the 60th day, and reached a maximum value of 4.02 and 3.85 nmol / mgprot on the 120th day. The change trends of groups B and D were basically the same. From 0 to 60 days, there was no significant difference in the carbonyl content of the two groups, indicating that storage after freezing in the freezing liquid can significantly inhibit protein oxidation, prevent denaturation, and reduce carbonyl synthesis. Experiments show that quick freezing in freezing liquid is better than direct freezing in the refrigerator.

[0163] Sam et al. found that after storing fish meat at -30°C for six months, the protein carbonyl content increased by 23.9%. Li et al. found that protein oxidation is considered to be an important factor affecting the quality of shrimp paste. They oxidized the myofibrillar protein of white shrimp with different concentrations of hydroxyl free radicals. After oxidation, it was found that the carbonyl content and surface hydrophobicity increased. At the same time, it was found that microstructure, carbonyl content and enzyme activity play a key role in the performance of myofibrillar protein, which provides a certain theoretical basis for the production of shrimp products in the future.

[0164] 2.4.6 Thiol content

[0165] Thiol is an important substance in myofibrillar protein, which can maintain the structural stability of muscle protein. During the frozen storage process, the oxidation level of protein can be evaluated by the change of thiol content in muscle. As shown in the figure, under different storage methods, the thiol content gradually decreased, indicating that the protein was gradually oxidized and denatured, and also showed that even after quick freezing in the freezing liquid, the muscle protein of Chinese whip shrimp could not be completely protected from oxidation. From 0 to 60 days, the thiol content decreased rapidly. Zeng Mingyong et al. found that this change was because some thiol groups in myofibrils existed outside the molecules and would be oxidized at the beginning of storage, but no obvious changes in protein structure would occur. From the 60th day to the 100th day, the thiol content increased slightly, which was due to the increase of thiol groups in the protein. Liu et al. measured the thiol content of silver carp fillets stored at -20°C for nine months and found that with the extension of time, the thiol first increased and then decreased significantly, which was consistent with this experimental study. Compared with groups A and C, the thiol content of groups B and D decreased sharply in the first 40 days and the last 20 days. In the later stage of storage, there was a significant difference between groups B and D (P<0.05). The thiol content after quick freezing in frozen liquid was significantly higher than that after direct freezing. On the 60th day, the thiol content of groups A, B, C, and D decreased by 0.71, 1.29, 0.59, and 1.12 mmol / gprot. On the 120th day, compared with fresh shrimp samples, the thiol content of the four groups was significantly reduced (P<0.05). In short, although the thiol content decreased during the entire storage period, the protein would also undergo oxidative decomposition, but the faster the freezing rate and the lower the freezing temperature, the lower the protein denaturation, and the less likely the thiol group would be oxidized.

[0166] 2.5 Summary of this chapter

[0167] This chapter focuses on the content of salt-soluble protein, changes in surface hydrophobicity, Ca 2+ -ATPase activity, carbonyl and thiol content were used as indicators to study the changes in protein quality indicators of Chinese shrimp during frozen storage by four freezing methods: -18℃ direct freezing, -18℃ quick freezing, -30℃ direct freezing and -30℃ quick freezing. The following conclusions were drawn:

[0168] 1. After 60 days of frozen storage, the protein oxidation rate decreased significantly, and the carbonyl, thiol, surface hydrophobicity and Ca 2+ -ATPase activity had no significant change, indicating that the oxidation and degradation of myofibrillar protein were not significant. In addition, storage in frozen solution had a good inhibitory effect on the oxidation of muscle protein in Chinese whip shrimp.

[0169] 2. There is a significant correlation between a series of quality indicators of Chinese tube whip shrimp and the degree of oxidation of its protein, which fully demonstrates that oxidation in the frozen state has a great impact on its preservation quality.

[0170] From the experimental results, it can be seen that although the four freezing methods failed to inhibit the decline of various protein indicators of Chinese tube whip shrimp during storage, compared with the direct freezing treatment group at -18℃ and -30℃, the protein oxidation and denaturation rate was slowed down after quick freezing in freezing liquid, and the meat quality, taste and flavor of Chinese tube whip shrimp were significantly improved.

[0171] Example 3

[0172] Changes of enzyme activity and tissue microstructure of Chinese shrimp under different frozen storage conditions

[0173] 3.1 Introduction

[0174] Fresh shrimps have high water content and rich nutrients, making them very prone to spoilage. Studies have shown that shrimp muscle spoilage may be caused by a variety of reasons. In recent years, people have paid more and more attention to the impact of cathepsins on the quality of aquatic products, especially during postmortem storage, which has a great impact on muscle quality. Cathepsins are the main cause of muscle protein degradation. Since cathepsins in shrimps will affect muscle protein to a certain extent, they will destroy cell and myofibrillar proteins.

[0175] Cathepsin has strong thermal stability and can block the internal peptide bonds of proteins, so it plays a big role in the degradation of proteins. Its activity is very high in various aquatic products. According to existing research, 13 different cathepsins have been found, of which, except for cathepsin D, the rest are cysteine ​​proteases. Only after the lysosomes are destroyed, cathepsins will have an effect on muscle tissue, etc. In addition, there is an enzyme in the mitochondria and lysosomes of cells, which will be released once the cells are destroyed, such as AG and NAG enzymes. Studies have shown that [144-146] Because the cell structure of aquatic products is damaged during freezing and thawing, the activity of AG enzyme and NAG enzyme changes. Therefore, freezing speed, storage temperature, etc. will have a certain impact on the enzyme activity of aquatic products, causing it to change during storage.

[0176] According to the research results in the previous chapter, the protein oxidation index of Chinese whip shrimp is indeed gradually deteriorating during freezing. On this basis, we also want to explore other reasons for the deterioration of shrimp meat quality. Soottawat et al. found that during freezing, the formation of ice crystals will destroy cells, release enzymes, decompose proteins, and cause changes in protein and muscle tissue. Therefore, we want to explore the changes in AG, NAG, and tissue proteases in the muscles of Chinese whip shrimp under freezing conditions and the changes in muscle microstructure. The present invention selects four different freezing conditions (-18℃ direct freezing, -18℃ quick freezing, -30℃ direct freezing, -30℃ quick freezing) to determine the AG enzyme, NAG enzyme and tissue protease activity of Chinese whip shrimp, and observes the changes in its muscle tissue microstructure by SDS-PAGE gel electrophoresis and scanning electron microscopy.

[0177] 3.2 Materials and instruments

[0178] 3.2.1 Experimental Materials

[0179] Chinese whip shrimp were caught in Zhoushan waters of the East China Sea, with an average body length of (12.5±2.2) cm and an average weight of (15.5±2.3) g. After being caught, they were placed in ice bags and in an incubator, and were quickly sent back to the laboratory after landing.

[0180] 3.2.2 Main reagents

[0181] Sodium citrate, p-nitrophenyl-α-pyranoglucoside, disodium hydrogen phosphate, ethanol, Shanghai Sinopharm Group; Coomassie Brilliant Blue R-250, glycine, NAGase kit, SDS-PAGE gel preparation kit, Solebao Co., Ltd.; 2.5% glutaraldehyde, Feijing Co., Ltd.; Cathepsin H kit, Cathepsin B kit, Cathepsin L kit, Xingyi Technology Co., Ltd.

[0182] 3.2.3 Main instruments

[0183] UV-visible spectrophotometer (Cary 50), Varian; centrifuge (AvantiJXN-30), Beckman Coulter; analytical balance (ME204E), Mettler Toledo; vortex mixer (LPD-2500), Leppert; electric constant temperature water bath (DF-101S), Bonsi Instrument; low temperature coolant circulation pump (DLSB-30 / 40), Gengyu Instrument; electrophoresis instrument (Mini-PROTEAN), Bio-Rad Co., Ltd.; microplate reader (MK3), Regal Instrument; scanning electron microscope (EVPMA25 / LS25), Zeiss, Germany.

[0184] 3.3 Experimental methods

[0185] 3.3.1 Pretreatment of raw materials

[0186] Quick freezing in freezing liquid: Use a low-temperature coolant circulation pump to quickly freeze the Chinese whip shrimp packed in sealed bags to -18℃ and -30℃, and then store them in a refrigerator at -18℃ and -30℃ for 120 days, respectively, and mark them as Group A and Group C, respectively.

[0187] Refrigerator storage: The Chinese whip shrimp were packaged and placed in sealed bags, and then stored in refrigerators at -18℃ and -30℃ for 120 days, respectively. The samples were marked as group B and group D. The various indicators of the four groups of Chinese whip shrimp A, B, C, and D were measured every 20 days.

[0188] 3.3.2 Determination of cathepsin activity

[0189] The assay was performed according to the instructions of the cathepsin B, cathepsin L and cathepsin H kits.

[0190] 3.3.3 Determination of NAGase and AGase activities

[0191] NAG enzyme activity: refer to the instructions of the NAG enzyme kit for determination. The calculation formula is:

[0192]

[0193] AG enzyme activity: refer to the method of Soottawat et al. Weigh 24g of shrimp meat and homogenize it evenly, centrifuge it at 8000r / min for 30min, and obtain shrimp exudate. Add water to make up to 25mL, take 2mL in a test tube, add 0.6mL 0.1mol / L sodium citrate (pH=4) and 0.4mL 1.0mol / L NaCI to the test tube, shake to mix, preheat at 37℃ for 10min, add 2mL p-nitrobenzene-α-pyranoglucoside (4.2mmol / L), and start the reaction. After mixing, place it in a 37℃ water bath for 1h, and then add 2mL KOH (0.3mol / L) to terminate the reaction. There are 3 parallels in each group. Add KOH as a negative control before the experiment. Replace the sample solution with distilled water to form a blank control. After the reaction, measure the absorbance at 405nm.

[0194] 3.3.4 SDS-PAGE electrophoresis analysis

[0195] Referring to the method of Laemmli et al., SDS-PAGE gel electrophoresis was used to analyze the changes in muscle protein of Chinese whip shrimp. The concentration of separation gel was 12%, and the concentration of concentrated gel was 5%. The concentration of 5.3.4 was adjusted to 3 mg / mL, and 10 μL of sample was taken for loading. The voltage was 90V. After the indicator entered the separation gel, the voltage was adjusted to 120V. The whole electrophoresis process took about 2.5 hours. After the electrophoresis, stain for 2 hours, decolorize for 4-6 hours, until the bands were clear, and observe the protein bands.

[0196] 3.3.5 Microstructure observation

[0197] Cut the shrimp into 1mm pieces 3 The small pieces were immersed in 2.5% glutaraldehyde and placed in a 4°C refrigerator overnight. They were then dehydrated with different concentrations of ethanol (50%, 70%, 80%, 90%) for 15 minutes and dehydrated with anhydrous ethanol three times for 15 minutes each. They were placed in a freeze dryer and vacuum dried. After being taken out, they were sprayed with 10nm thick gold. Finally, the cross-section and longitudinal section of the body of the Chinese whip shrimp were observed by scanning electron microscopy at 500 times.

[0198] 3.3.6 Data processing

[0199] Excel was used for statistical data, Origin8.5 was used for chart analysis, and SPSS23.0 was used for variance analysis. The results were expressed as mean ± standard deviation.

[0200] 3.4 Results and Discussion

[0201] 3.4.1 Changes in the activities of cathepsins B, L, and H

[0202] The activities of cathepsin B and H in Chinese whip shrimp stored for 120 days Fig. 9 , 10 As shown. Fig.10 It can be seen that the activity of cathepsin H is significantly lower than that of the other two groups of enzymes, and the change is not large. Therefore, we believe that cathepsin H has little effect on protein degradation during freezing, so this article will not discuss it. Fig. 9 The activity of cathepsin B changed significantly during the storage period. On the 20th and 40th day, the enzyme activity of groups A and C was significantly lower than that of groups B and D, indicating that storage after freezing in the freezing solution inhibited the activity of cathepsin B. In the later stage of storage, starting from the 60th day, the enzyme activity of each group showed an upward trend, but there was no significant difference between the groups (P>0.05). The results showed that short-term low-temperature freezing could significantly inhibit the activity of cathepsin in Chinese whip shrimp, but with the increase of freezing time, the activity of cathepsin B was no longer effectively inhibited, and the increase in activity further enhanced the degradation of its muscle protein. On the 120th day, the activity of cathepsin B in groups A, B, C and D increased from 0.145U / g of fresh shrimp samples to 0.161, 0.167, 0.158 and 0.164U / g, respectively.

[0203] Nasin measured the changes of cathepsin B and B+L in goat meat stored at -15°C for 120 days and found that the activity decreased on the 6th day compared with the 0th day. When stored for 80 days, the activity was significantly lower than that of fresh samples, which is similar to the results of the present invention. Ge et al. found that endogenous cathepsins and microorganisms would cause the texture of grass carp fillets to soften during storage, so they immersed the fillets in iodoacetic acid solution to eliminate the activity of cathepsins. During the entire storage period, treatment with iodoacetic acid solution reduced the activity of cathepsin B and B+L, but had no significant effect on microbial growth. It shows that protein hydrolysis induced by endogenous cathepsins rather than microorganisms plays an important role in the softening of the texture of frozen grass carp fillets.

[0204] Depend on Fig.11 It can be seen that the activity of cathepsin L changes significantly. In the first 40 days, its activity shows a downward trend, which may be because cryopreservation reduces the activity of the enzyme, or it may be that the cathepsin in the lysosome is released into other subcellular

[31] At 40 days, the enzyme activities of groups A, B, C, and D were lower than those of fresh shrimp. At the end of storage, the enzyme activities of the four groups continued to rise. At 120 days, the activities of cathepsin L in groups A, B, C, and D increased from 0.213 U / g of fresh shrimp to 0.258, 0.271, 0.252, and 0.262 U / g, respectively, with significant differences, but the differences among the four groups were not significant (P>0.05). At the end of storage, the activity of cathepsin increased, the permeability of cell membranes increased, and the organizational structure of cells changed, thus leading to the degradation of myofibrillar proteins, which is consistent with the changes in protein oxidation described in the previous chapter. By comparing the activities of cathepsin B and L, we found that the activity of cathepsin L was higher than that of cathepsin B, which indicates that cathepsin L plays a more important role in the changes of muscle tissue during storage, which is consistent with the results of Takahiko's research. As can be seen from the chart, at 20th, 40th and 100th days, the enzyme activities of groups A and C were lower than those of groups B and D, and that of group C was even lower than that of group A (P<0.05). The results showed that freezing in cryogenic fluid had a good inhibitory effect on tissue proteases, and as the temperature decreased, the degradation rate would also decrease.

[0205] Zhao measured the changes of cathepsin B, L and H in sturgeon meat at -3℃ and found that the activity began to decline at the 6th day. When stored for 18 days, the activity was significantly lower than that of fresh samples, which is similar to the results of the present invention. In order to study the effects of refrigeration and freezing on the stiffness of bighead carp meat, Lu et al. measured the activity of cathepsin L, protein oxidation and microstructural changes. It was found that for refrigerated and frozen bighead carp, the activity of cathepsin L showed a continuous upward trend in the first 12 hours, while the enzyme activity began to gradually decline from 12 to 24 hours. There was no significant difference in the activity of lysosomal cathepsins between bighead carp meat under refrigeration and freezing (P>0.05). From 48 to 72 hours, the activity of cathepsins in fish meat under refrigeration was significantly higher than that under freezing. Among them, the enzyme activity in sarcoplasm was higher than that in myofibril. The reason may be that the cell structure of bighead carp is more complete under freezing, indicating that low temperature is more conducive to maintaining the integrity of muscle structure.

[0206] 3.4.2 Changes in NAGase and AGase activities

[0207] like Fig.12 As shown in the results, under the four freezing methods, the NAGase activity of Chinese whip shrimp increased with the extension of storage time. In the early stage of storage, the NAGase activity increased slowly, and there was no significant difference among the groups (P>0.05). However, after 120 days of storage, the activity of NAGase continued to increase, which means that the cell tissue structure is still changing. The NAGase activity increased from 2.30U / gprot of fresh shrimp to 4.15, 4.80, 4.00, and 4.35U / gprot respectively. The results showed that during the frozen storage, the formation and growth of ice crystals had a certain effect on the muscle tissue of Chinese whip shrimp, but the enzyme activity of shrimp was significantly lower than that of shrimp directly stored in the refrigerator after freezing in freezing liquid. Xie found that betaine had a certain effect on the NAGase activity in shrimp meat. The results showed that appropriate concentrations of betaine can lead to inhibition of NAGase activity. Inhibition kinetics showed that betaine is a mixed inhibitor, and the time course of NAGase catalyzed hydrolysis by betaine at different concentrations showed that the rate decreased with time until it approached a straight line at different concentrations of betaine, indicating that the inhibitory effect of betaine on NAGase was a slow reaction. This showed that freezing with freezing fluid could relatively reduce the amount of ice crystals generated without damaging the cell tissue structure.

[0208] like Fig.13As shown in the figure, both AG enzyme and NAG enzyme activity can be used to judge the integrity of cells. The higher the activity, the greater the damage to the cells. From 80 to 120 days, the activity of AG enzyme increased slowly and showed a stable trend. There was no obvious change during this period. It may be due to the long-term storage that the AG enzyme began to decompose. At 120 days, the activity of AG enzyme increased from 0.24U / gprot of fresh samples to 1.78, 1.82, 1.75, and 1.80U / gprot.

[0209] Soottawat et al. investigated the changes in AG and NAG enzymes in Penaeus monodon and Penaeus vannamei after they were frozen for 20 weeks. The results showed that compared with Penaeus monodon, the changes in AG and NAG enzyme activities in Penaeus vannamei were more significant, but after 20 weeks of storage, the enzyme activities of both shrimps increased significantly, which was consistent with the change trend of this experiment. Therefore, with the extension of freezing time, the increase of ice crystals, changes in muscle tissue, the release of enzymes in lysosomes and mitochondria into other subcells, the cells will lose integrity, and the muscle tissue of Chinese whip shrimp will deteriorate. In short, from the changes in AG and NAG enzyme activities, it can be seen that storage after freezing in frozen liquid is still better than direct storage in the refrigerator in terms of structural integrity.

[0210] 3.4.3 SDS-PAGE electrophoresis analysis of myofibrillar proteins

[0211] Depend on Fig.14 It can be seen that the myofibrillar protein of Chinese whip shrimp has a large number of protein bands between 10kDa and 50kDa and 90kDa and 220kDa. Due to the increase in storage time, myosin heavy chain, actin, and tropomyosin have all changed, and some small molecular protein bands have been formed. At 40 days, the bands of myosin heavy chain and actin in the refrigerator storage group became narrower, indicating that the protein had been degraded, while there was no change in groups A and C. At 60 days, the protein bands of groups A and C began to narrow slightly, indicating that the protein had changed, but not as obviously as groups B and D. Delbarre's study found that the degradation of myosin heavy chain, actin, and tropomyosin are all related to cathepsins B and L. Compared with 6.4.1, the experimental results show that from the 60th day, the activity of cathepsins B and L increased, and the activity of the refrigerator direct freezing group was significantly higher than that of the freezing liquid quick freezing group. In summary, the structure of the protein changed after storage for 60 days. The degradation rate of the protein of the Chinese whip shrimp after freezing in the cryosol was much slower than that of direct refrigerator storage. Moreover, the degradation of the protein would be aggravated by the increase of temperature. The above results are consistent with the changes in the activities of AG enzyme, NAG enzyme and cathepsin, indicating that the degradation of muscle protein of the Chinese whip shrimp is affected by cathepsin.

[0212] Wang et al. studied the changes in molecular weight of grass carp myofibrillar protein during storage and found that its myofibrillar protein changed significantly after 20 weeks of storage, especially the high molecular weight protein, which was almost completely degraded in the later stage of storage, while the low molecular weight protein was relatively stable. Zhang et al. found that temperature fluctuations are a common problem in frozen storage of shrimp products, so they investigated the effects of oligosaccharides and oligoxylose on ice crystal growth and recrystallization in frozen peeled shrimp exposed to temperature fluctuations. The data showed that compared with the control samples, shrimp treated with oligosaccharides and oligoxylose had significant improvements in thawing, cooking loss, protein content and enzyme activity when exposed to temperature fluctuations. SDS-PAGE analysis confirmed that treatment with oligosaccharides had a significant effect on the stability of muscle protein and inhibited the degradation of muscle protein affected by temperature fluctuations. This may be because the incorporated oligosaccharides and oligoxylose bind to muscle protein through hydrogen bonds and capture water molecules in myofibrils, thereby effectively inhibiting the growth and recrystallization of ice crystals, reducing the degeneration of myofibrils and the destruction of tissue structure. Therefore, we found that the development of more economical and efficient preservation technologies, such as the use of a combination of refrigerants and refrigerator storage, can better maintain the quality of frozen foods and extend their shelf life.

[0213] 3.4.4 Analysis of microstructure results

[0214] like Fig.15 , Fig.16As shown in the figure, after 60 days of storage, there was no obvious change in the cross section of the Chinese whip shrimp in groups A and C. The longitudinal section of the muscle was still very neat, with only some small intercellular gaps, which may be caused by the loss of shrimp juice. After freezing the shrimp, the water in the shrimp meat will also be frozen, and the free water in the shrimp body will be frozen into ice, which will increase the volume and cause the myofibrils to be squeezed. After thawing, the ice in the muscle melted into water again, so the gaps between the myofibrils became larger. Compared with the fresh samples, a large number of gaps appeared in the cross section of the refrigerator storage group, and the longitudinal section of the myofibrils was chaotically distributed. The myofibrils of the shrimp meat at -18℃ even broke. After 120 days of storage, the tissue structure of the four groups of Chinese whip shrimp changed significantly. There were many large gaps in the cross section of groups B and D, and the gaps between groups A and C gradually increased. Observation of the longitudinal section revealed that the muscle fibers changed due to the action of tissue proteases, which separated them from the endomysium. In addition, connective tissues such as endomysium and perimysium will also degrade. As the shrimp meat thaws, the myofibril ruptures and a large amount of water flows out of the cells. Groups B and D have a large range of muscle fiber ruptures and blurred myometrial striations. Even if they are stored after freezing in the cryosol, the interfascicular gaps are increased compared to the early stage of storage. The above results show that storage after freezing in the cryosol is conducive to maintaining the muscle tissue structure, because the freezing speed is fast, the ice crystals formed are small and evenly distributed, and will not cause mechanical damage to the body of the Chinese whip shrimp. It can effectively inhibit the denaturation and degradation of muscle protein in the Chinese whip shrimp, and low temperature is conducive to maintaining the integrity of the myofibrils of the Chinese whip shrimp. The results show that in the middle and late stages of frozen storage, with the gradual oxidation of protein and the action of tissue proteases, the muscle tissue structure of the shrimp changes, the functional properties of the protein change, and the increase in the activity of AG and NAG enzymes leads to the destruction of cell integrity. Therefore, the texture, quality and tissue structure integrity of the Chinese whip shrimp are all affected to a certain extent.

[0215] 3.5 Summary of this chapter

[0216] This chapter studies the changes of enzyme activity and tissue microstructure of Chinese shrimp during frozen storage by four freezing methods: -18℃ direct freezing, -18℃ quick freezing, -30℃ direct freezing and -30℃ quick freezing. The results are as follows:

[0217] 1. Storage after freezing in cryosol has a significant inhibitory effect on enzyme activity, but the enzyme activity still slowly increases in the later stage of storage, which indicates that freezing in cryosol has a reduced inhibitory effect on cathepsin. Since the change in cathepsin activity is not significant, it is speculated that it plays a certain auxiliary role in protein degradation. Compared with storage in a refrigerator, storage after freezing in cryosol has a significant inhibitory effect.

[0218] 2. In the late storage period, the cell tissue of Chinese whip shrimp was damaged, and the activities of AG, NAG enzymes and cathepsin increased, which led to the degradation of myofibrillar protein and the change of muscle tissue microstructure. Therefore, cathepsin can promote protein degradation and destroy the structure of muscle fibers.

[0219] 3. Although ice crystals will form after freezing, the particles are smaller and more evenly distributed, so the mechanical damage to the muscle cells of Chinese whip shrimp is much less than that of refrigerator storage. The experimental results show that the effect of freezing with freezing liquid is significantly better than direct storage in the refrigerator.

[0220] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A novel immersion freezing liquid, characterized in that: The invention is composed of the following raw materials in percentage by weight: 23-27% of ethanol, 8-12% of betaine, 9-11% of propylene glycol and 3-5% of sodium chloride.

2. The novel immersion freezing liquid according to claim 1 is characterized in that: The weight percentages of the raw materials are 25% ethanol, 10% betaine, 10% propylene glycol and 4% sodium chloride.

3. The method for preparing the novel immersion freezing liquid according to claim 1 or 2, characterized in that: The following steps are involved: Weigh the raw materials according to the weight percentage; Mix all the ingredients evenly and keep for later use.

4. Use of the novel immersion freezing liquid according to claim 1 or 2 in quick-freezing Chinese shrimp.

5. The use according to claim 4, characterized in that: The temperature of the novel immersion freezing liquid for quick freezing of Chinese whip shrimp is -18 to -30°C; and the time required for quick freezing to the target temperature is 15 to 30 minutes.

6. The use according to claim 5, characterized in that: The storage time of the novel immersion-type freezing liquid quick-frozen Chinese whip shrimp is 120 days.

7. A cooling liquid quick freezing and preservation method for Chinese whip shrimp, characterized in that: The following steps are involved: The fresh Chinese whip shrimp is immersed in the novel immersion freezing liquid described in claim 1, and then the Chinese whip shrimp is quickly frozen to a target temperature using a low-temperature cooling liquid circulation pump.

8. The cooling liquid quick freezing and preservation method according to claim 7, characterized in that: The ratio of the fresh Chinese whip shrimp to the novel immersion freezing liquid is 5kg / L.

9. The cooling liquid quick freezing and preservation method according to claim 7 or 8, characterized in that: The target temperature is -18 to -30°C.

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