Freshwater fish partial freezing water-free keep-alive transportation method based on temperature cooperative control

Abstract

The invention discloses a freshwater fish partial freezing water-free keep-alive transportation method based on temperature cooperative control, which comprises the following specific steps: partial freezing treatment: carrying out vacuum-pumping operation on fresh and alive freshwater fish, completely immersing a vacuum bag filled with live fish into a food-grade secondary refrigerant, and setting the temperature of the food-grade secondary refrigerant to be-10 to-20 DEG C; the vacuum packaging bag is removed, the freshwater fish is put into the packaging bag for oxygenation and sealing treatment, and the oxygen concentration is larger than 80%; carrying out water-free keep-alive transportation: placing the fishes on a transport vehicle; rehydration: after the fish arrives at the destination, putting the fish into a water pool, and pre-cooling the water temperature to 10-20 DEG C in advance; the temperature difference value between the rehydration water temperature and the partial freezing treatment water is 20-35 DEG C. Through cooperative control of the partial freezing temperature and the rehydration temperature and assistance of oxygenation and low-temperature transportation, the original longest transportation time can be greatly prolonged to 4 hours, and the rehydration survival rate of long-time transportation is greatly improved. Great market application prospects are realized. In addition, energy consumption is lower, and certain temperature limitation is relieved for research, development and expansion of the secondary refrigerant.

Description

Technical Field

[0001] The present invention relates to the technical field of live fish transportation, and specifically to a method for micro-freezing and waterless preservation of live freshwater fish based on temperature collaborative control. Background Art

[0002] The preservation and transportation of live fish refer to providing the living conditions and environment for fish, or reducing their respiration, metabolic activities and stress responses during transportation through a series of physical or chemical measures, so as to extend their survival time, reduce the mortality rate, ensure their nutritional value and economic benefits, and achieve the purpose of preservation and freshness. Compared with the traditional freezing method, the liquid immersion freezing technology has a faster freezing speed, can significantly shorten the freezing time, and has less energy consumption and lower cost. The characteristic of its fast freezing speed enables the surface of freshwater fish to be frozen in a short time, avoiding the damage caused by self-struggling when it leaves the water; at the same time, the rapid cooling treatment can reduce the continuous pressure accumulation brought by traditional low-temperature dormancy and reduce the degree of lipid peroxidation.

[0003] CN 115067240 B and CN112616726B disclose a method for micro-freezing and fresh preservation transportation of live freshwater fish, and the specific steps are as follows: 1) Pretreatment before micro-freezing: Using a vacuum packaging bag, after putting the live freshwater fish into the bag, quickly perform a vacuum operation with a vacuum packaging machine; 2) Micro-freezing treatment: Put the bag containing the fish into the middle and lower layers of a food-grade cryogen, make the fish body fully contact with the liquid, set the temperature of the food-grade cryogen to -30 to -35°C, and the micro-freezing time is within 2 minutes; 3) Unsealing: Immediately remove the vacuum packaging bag after micro-freezing is completed; 4) Fresh preservation transportation: Place the fish in an anhydrous state evenly on the partition of the transport vehicle; 5) Rehydration: After arriving at the transportation destination, put the fish in the transport vehicle into a large pool. After the micro-freezing and fresh preservation treatment of the present invention, the survival rate of the fish after rehydration is higher, and the longest transportation time can reach 4 hours, with a longer transportation time, greatly reducing the cost of live fish fresh preservation transportation.

[0004] However, the fresh preservation transportation of live fish under a longer transportation time is still a technical problem to be solved urgently. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for micro-freezing and waterless preservation of live freshwater fish based on temperature collaborative control in view of the deficiencies of the prior art, and the specific steps are as follows:

[0006] Micro-freezing treatment: Perform a vacuum operation on the live freshwater fish, and completely immerse the vacuum bag containing the live fish in a food-grade cryogen, and set the temperature of the food-grade cryogen to -10 to -20°C;

[0007] Oxygen filling and packaging: Remove the vacuum packaging bag, put the freshwater fish into a packaging bag for oxygen filling and sealing treatment, and the oxygen concentration > 80%;

[0008] Live transportation without water: Place the fish in a transport vehicle;

[0009] Rehydration: After arriving at the destination, place the fish in a pool, and pre-cool the water temperature to 10 - 20 °C in advance; the temperature difference between the rehydration water temperature and the mild freezing treatment water temperature is 20 - 35 °C.

[0010] Furthermore, before live transportation of fresh-water fish after mild freezing treatment, fast and temporarily raise them for 12 - 48 h. The temperature change range during the fasting and temporary raising stage is < 3 °C compared with the daily living water temperature, and the daily living water temperature < 23 °C.

[0011] Furthermore, the fresh-water fish is crucian carp.

[0012] Furthermore, the temperature of the transport vehicle is lower than 4 °C.

[0013] Furthermore, the mild freezing time is 60 - 120 s.

[0014] Furthermore, change the water body 10 - 20 min after rehydration.

[0015] Furthermore, during rehydration, pre-cool the water temperature to 11 - 14 °C in advance.

[0016] Furthermore, the temperature difference between the rehydration water temperature and the mild freezing treatment water temperature is 22 - 30 °C.

[0017] Beneficial effects: By synergistically controlling the mild freezing temperature and the rehydration temperature, supplemented by oxygenation and low-temperature transportation, the original maximum transportation time of up to 4 hours is greatly extended, and the rehydration survival rate during long-term transportation is extremely improved. It has great market application prospects. In addition, compared with the coolant temperature of -30 - -35 °C in the prior art, the coolant temperature of this application does not need to be too low, has lower energy consumption, and also removes certain temperature restrictions for further research and development of the types of coolants. Description of the Drawings

[0018] Figure 1 Shows the change in the respiratory frequency of crucian carp after rehydration at different immersion freezing temperatures.

[0019] Figure 2 Shows the change in the pH value of the water quality after rehydration at different immersion freezing temperatures.

[0020] Figure 3 Shows the change in the ammonia nitrogen content of the water quality after rehydration at different immersion freezing temperatures.

[0021] Figure 4 Shows the survival state of the fish body after rehydration for different live preservation times.

[0022] Figure 5 Shows the effect of different rehydration water temperatures on tissue damage of liquid immersion frozen live crucian carp.

[0023] Figure 6Effect of different rehydration water temperatures on the antioxidant stress system of liquid immersion frozen crucian carp preserved alive without water

[0024] Figure 7 Schematic diagram of the operation process of the method for preserving and transporting fresh water fish in micro-freezing and anhydrous state based on temperature co-control Specific implementation mode

[0025] For the convenience of understanding by those skilled in the art, the present invention will be further described below in conjunction with embodiments. The content mentioned in the implementation mode does not limit the present invention

[0026] Example 1

[0027] Fresh crucian carp was purchased from Lao Cai Freshwater Fish Shop at Dongbozi Market, Laishan District, Yantai City, Shandong Province. The average weight was (380±50) g, the average body length was (25.9±0.8) cm, and the average body width was (8.8±0.6) cm. It was transported to the laboratory (short-distance transportation time was 5 - 6 min) in a double-layer plastic bag with water and placed in a temporary culture pond (56 cm×45.4 cm×35 cm) for temporary culture. During the experiment, the temporary culture conditions were as follows: tap water that had been exposed to the sun for 1 - 2 days was used, and the air pump was turned on for 24 hours. The dissolved oxygen level was (7.56±1.65) mg / L, and the fish-water ratio was 1:5. The quality of the temporary culture water: the ammonia nitrogen content was (0.02±0.02) mg / L, the nitrite nitrogen content was (0.001±0.001) mg / L, the sulfide content was (0.000±0.000) mg / L, and the pH value was 7.96±0.04. After 24 hours of temporary culture, the experiment was carried out, and healthy, disease-free, active, and fish with intact scales and no external injuries were selected

[0028] Test Example 1: After the crucian carp used in the test was vacuum-packed, it was immersed and frozen for 1 min 30 s at a cryogen temperature of -10°C, and then rehydrated. After 15 minutes of rehydration, the respiratory rate was recorded, and the middle-layer water sample was taken to detect the ammonia nitrogen content and pH value of the water quality with a water quality analyzer. There were 6 fish in each group, the rehydration temperature was 11.0 - 14.0°C, and the fish-water ratio was 1:5. The immersion freezing temperature was determined according to the freezing degree of the crucian carp at different temperatures and the recovery situation after rehydration. The control group for the respiratory rate was the respiratory rate of the live crucian carp without treatment in a calm state; the control group for the water quality was the initial water quality, that is, a blank water sample without crucian carp

[0029] Example 2

[0030] Same as Example 1, the difference is that the cryogen temperature is -15°C

[0031] Example 3

[0032] Same as Example 1, the difference is that the cryogen temperature is -20°C

[0033] Comparative Example 1

[0034] It is the same as Example 1, except that the temperature of the secondary refrigerant is -25°C.

[0035] Comparative Example 2

[0036] It is the same as Example 1, except that the temperature of the secondary refrigerant is -30°C.

[0037] Comparative Example 3

[0038] It is the same as Example 1, except that the temperature of the secondary refrigerant is -5°C.

[0039] From Figure 1-3 it can be seen the change of the respiration frequency of crucian carp after immersion freezing and rehydration at different temperatures. It can be seen that compared with the control group, there is no significant difference in immersion freezing at -5°C, but as the immersion temperature further decreases, the respiration frequency of crucian carp increases significantly. There is no significant difference among the three groups of immersion freezing at -10°C, -15°C and -20°C. After that, the respiration frequency of crucian carp increases significantly again, and the respiration frequency is the fastest under the condition of immersion freezing at -30°C. Crucian carp will swim rapidly in water after being stimulated by low temperature and rehydrated. The lower the temperature, the stronger the stimulating effect, and the higher the respiration frequency.

[0040] The influence of different immersion freezing temperatures on the metabolism of crucian carp after rehydration. It can be seen from the figure that the pH value of the water quality decreases significantly after treatment at different immersion freezing temperatures, corresponding to the change of the respiration frequency, indicating that the respiration frequency increases after immersion freezing and rehydration, and the discharged CO2 increases, resulting in a rapid decrease in the pH value of the water quality; while the ammonia nitrogen content increases significantly compared with the control group, showing a trend of first increasing and then decreasing with the decrease of the immersion temperature, and the content is the highest at -15°C. The increase of ammonia nitrogen content is related to metabolism. Low temperature stimulation leads to an increase in the metabolism of crucian carp after rehydration, and the ammonia nitrogen content in the water also increases accordingly; while the decrease in content is mainly affected by the degree of freezing. From the freezing situation, it can be seen that the immersion temperature below -20°C can completely freeze the crucian carp, and it may take some time for the crucian carp to recover normal metabolism after freezing and rehydration. Therefore, the ammonia nitrogen content in the latter three groups is significantly lower than that at -15°C, which also reflects from the side that the immersion freezing temperature below -20°C causes greater damage to crucian carp, and there is a large temperature difference with the rehydration temperature, and the time required for the crucian carp to thaw and the body to recover after rehydration is longer. Therefore, the ammonia nitrogen content of metabolites in the water is less. From the results, except for the highest ammonia nitrogen content in the -15°C group, there is basically no significant difference among other groups of different temperature immersion freezing groups. At the same time, after immersion freezing and rehydration, the metabolism of crucian carp is vigorous, the water quality deteriorates seriously, the turbidity of the water body increases, and a large amount of metabolic wastes appear. The turbidity situation is basically consistent with the change of the ammonia nitrogen content in the water quality.

[0041] Test Example 2: Determination of the longest survival time of crucian carp without water

[0042] Perform liquid immersion freezing and anhydrous live preservation simulation transportation on crucian carp according to the optimized process, observe the situation within 72 hours after rehydration, and determine the longest anhydrous live preservation time node. According to the pre-experiment situation, start observing from 14 hours of anhydrous live preservation under summer temperature conditions and from 24 hours of anhydrous live preservation under other temperature conditions, and use 2 hours as the time interval to find the live preservation time node when the rehydration state deteriorates.

[0043] Table 1 Evaluation scoring criteria for the rehydration status of crucian carp after anhydrous live preservation

[0044]

[0045] Table 2 Comparison of state scores after rehydration at different live preservation times

[0046]

[0047]

[0048] Note: The values are expressed as mean ± standard deviation. Different superscript letters in the same column represent significant differences in data (p < 0.05).

[0049] From Table 1, Table 2 and Figure 4 It can be seen that after anhydrous live preservation for 24 - 26 hours, they all quickly recovered after rehydration and always remained upright without tipping over, with good activity; in the 28-hour group, only the state of one crucian carp deteriorated after rehydration, and tipping over began to occur but it was sensitive to stimuli and recovered to the normal state within 24 hours after rehydration, with a survival rate of 100%; in the 30-hour group and 32-hour group, the state of crucian carp deteriorated severely after rehydration, showing tipping over without swimming, weak breathing, and reduced sensitivity to stimuli, and some crucian carp died within 24 hours after rehydration. Generally, the state scores in all aspects of crucian carp after rehydration gradually decreased with the extension of the anhydrous live preservation time. The recovery state, appearance, eyes, and whether there was bleeding on the body surface in the 28-hour anhydrous live preservation group decreased significantly. Considering the comprehensive score and survival rate, the longest time for liquid immersion freezing and anhydrous live preservation transportation was determined to be 26 hours, or even 28 hours.

[0050] Experimental Example 3: Effect of rehydration temperature on the physiological recovery of crucian carp

[0051] Fresh crucian carp was purchased from the wholesale market in Laishan District, Yantai City, Shandong Province, with an average weight of (380±50) g, an average body length of (25.9±0.8) cm, and an average body width of (8.8±0.6) cm. It was transported to the laboratory in a double-layer plastic bag with water (short-distance transportation time was 5 - 6 min) and temporarily raised in a temporary raising pond (56 cm×45.4 cm×35 cm). During the experiment, the temporary raising conditions were as follows: tap water that had been sun-dried for 1 - 2 days was used, and the air pump was turned on 24 hours a day. The dissolved oxygen level was (7.56±1.65) mg / L, the water temperature was 10°C, and the fish-to-water ratio was 1:5. The quality of the temporarily raised water: the ammonia nitrogen content was (0.02±0.02) mg / L, the nitrite nitrogen content was (0.001±0.001) mg / L, the sulfide content was (0.000±0.000) mg / L, and the pH value was 7.96±0.04. After 48 hours of temporary raising, the experiment was carried out, and healthy, disease-free, active, and scale-intact crucian carp without external injuries were selected.

[0052] The fresh water fish was subjected to a vacuum operation, and the vacuum bag containing the live fish was completely immersed in a food-grade secondary refrigerant. The temperature of the food-grade secondary refrigerant was set at -15°C, and the micro-freezing time was 90 s. The vacuum packaging bag was removed, and the fresh water fish was put into a packing bag for oxygen filling and sealing. The oxygen concentration was >80%, and it was placed in a 4°C refrigerator to simulate anhydrous live transportation for 20 h. Subsequently, rehydration was carried out at rehydration temperatures of 10°C, 15°C, 20°C, and 25°C respectively.

[0053] It can be seen from Figure 5 , Figure 6 that ACP and AKP are related to tissue damage and the immune system of the body. The increase in the enzyme activities of AKP and ACP indicates that the body is trying to repair tissue damage and is accompanied by the regulation and response of the immune system. The ACP and AKP in the AC and WT groups increased significantly, indicating that the stress of rapid cooling and anhydrous transportation significantly stimulated the immune system of crucian carp. After 72 h of rehydration, the ACP and AKP of crucian carp dropped back to levels close to those of the NC group, indicating the recovery of ACP and AKP after rehydration. Among the four temperature differences, the ACP and AKP in the 15°C group were the lowest, showing that the recovery effect at 15°C was the best.

[0054] MDA is a product of cell membrane lipid peroxidation, and its level reflects the degree of lipid oxidation damage. The content of MDA in the liver increased after AC and WT treatments. Compared with the WT group, there was no significant difference in MDA between the 10°C rehydration group and the 20°C rehydration group, indicating that these two water temperatures had no obvious recovery effect on lipid peroxidation of crucian carp liver cells; the MDA in the 25°C rehydration group increased significantly, which was related to the heat stress at a higher survival water temperature; the 15°C rehydration group decreased, approaching the NC level, indicating that 15°C was more conducive to the recovery of lipid peroxidation of crucian carp liver cells.

[0055] T-AOC reflects the overall antioxidant capacity of the body. Freezing and anhydrous transportation stress significantly reduced the antioxidant capacity of crucian carp; the T-AOC of the 10°C rehydration group recovered to the NC level, the T-AOC of the 15°C rehydration group did not change significantly compared with the WT group, while the T-AOC under the rehydration conditions of 20°C and 25°C was significantly lower than that of the WT group. T-AOC reflects the overall antioxidant capacity of the body, and the decrease in its value indicates damage to the antioxidant stress system. It can be seen that 10-15°C is more conducive to the recovery of the body's antioxidant system. AC and WT treatments inhibited the activities of SOD and CAT enzymes, and rehydration to 10°C effectively restored the activities of antioxidant enzymes and improved the stress state. Higher water temperatures and larger temperature differences led to a decrease in enzyme activity, indicating that rehydration to higher water temperatures may exacerbate oxidative stress and damage the activity of antioxidant enzymes.

[0056] Those skilled in the art of the present technology can understand that, unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as the general understanding of those of ordinary skill in the art to which the present invention belongs. It should also be understood that terms such as those defined in a general dictionary should be understood to have a meaning consistent with the meaning in the context of the prior art, and will not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0057] It should be understood that the detailed description of the technical solutions of the present invention with the aid of the preferred embodiments above is illustrative rather than restrictive. Those of ordinary skill in the art can modify the technical solutions recorded in each embodiment on the basis of reading the specification of the present invention, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of each embodiment of the present invention.

Claims

1. A method for transporting freshwater fish in a partially frozen state without water and keeping them alive based on coordinated temperature control, characterized in that: The specific steps are as follows: Micro-freezing treatment: vacuumize the fresh water fish and completely immerse the vacuum bag containing the live fish in a food-grade refrigerant. The temperature of the food-grade refrigerant is set to -10~-20℃. Oxygen-filled packaging: remove the vacuum packaging bag, put the freshwater fish into the packaging bag for oxygenation and sealing, and the oxygen concentration should be >80%; Waterless alive transportation: place the fish in the transport vehicle; Rehydration: After arriving at the destination, place the fish in a pool and pre-cool the water to 10-20°C; the difference between the rehydration water temperature and the slightly frozen water temperature is 20-35°C.

2. The method according to claim 1, characterized in that Freshwater fish that have been partially frozen should be fasted for 12 to 48 hours before being transported alive. The water temperature during the fasting period should vary by less than 3°C from the daily water temperature, and the daily water temperature should be less than 23°C.

3. The method according to claim 1, characterized in that The freshwater fish is crucian carp.

4. The method according to claim 1, characterized in that The temperature of the transport vehicle is below 4°C.

5. The method according to claim 1, characterized in that The micro-freezing time is 60~120 s.

6. The method according to claim 1, characterized in that Replace the water after 10-20 min of rehydration.

7. The method according to claim 1, characterized in that When rehydrating, precool the water temperature to 11~14℃.

8. The method according to claim 1, characterized in that The difference between the rehydration water temperature and the slightly frozen water temperature is 22~30℃.

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