CN116114796B - Stress protective agent and preparation method and application thereof - Google Patents

Stress protective agent and preparation method and application thereof Download PDF

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Publication number
CN116114796B
CN116114796B CN202211342555.5A CN202211342555A CN116114796B CN 116114796 B CN116114796 B CN 116114796B CN 202211342555 A CN202211342555 A CN 202211342555A CN 116114796 B CN116114796 B CN 116114796B
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stress
fish
protectant
stress protectant
mass
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CN116114796A (en
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李培玉
宋志东
黄炳山
孙永智
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Shandong Marine Resource and Environment Research Institute
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Shandong Marine Resource and Environment Research Institute
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    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K61/00—Culture of aquatic animals
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    • A01K61/13—Prevention or treatment of fish diseases
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    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
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Abstract

The invention provides a stress protective agent, which belongs to the technical field of aquaculture, and is prepared by compounding resveratrol, beta-nicotinamide mononucleotide and other components, and has stronger targeting property, shorter action time and oxidation resistance and can strengthen the repair capability of damaged mitochondria. The stress protectant can improve the stress resistance and disease resistance of fishes, and has the effects of strengthening the physique of the fishes and reducing the death rate. The invention also provides a preparation method and application of the stress protectant, and the application method can overcome the defect of single application method of the traditional stress protectant, and has the advantages of no toxicity, no harm and environmental protection.

Description

Stress protective agent and preparation method and application thereof
Technical Field
The invention belongs to the field of animal protection, and particularly relates to a stress protective agent and a preparation method and application thereof.
Background
The stress response of fish is a non-specific physiological response generated by ultra-long stimulation (stressor) of fish body to various environmental factors. Too strong or too long stress reaction can cause harm to organisms, so that the growth and development are slow, the reproductive capacity is reduced, the immune function is reduced, the morbidity and mortality are improved, and the like. The stress response of fish is related to various factors, such as farms, nutrition, transportation, diseases, crowded cultivation, manual operation and the like, so that the fish is usually dead in batches, most of injured fish is difficult to recover health, the resulting body wound is difficult to heal, the price of the fish in a pond is reduced, and huge economic loss is brought to the cultivation industry.
Fish stress generally occurs at three levels, namely, at the neuroendocrine system level, at the tissue and organ level, and at the individual and population level. Primary stress is a response at the level of the nervous and endocrine systems of animals such as fish, and is mainly manifested by a significant increase in plasma epinephrine or norepinephrine levels and a significant increase in plasma cortisol levels. Secondary stress is a change in various tissues and organs of the respiratory system, cardiovascular system, immune system, etc. caused by the primary reaction. Tertiary stress refers to changes in individual or population levels, such as changes in growth rate, disease resistance, reproductive capacity, and behavior, that result from secondary stress. Thus, anti-stress agents should begin with the three aspects above to eliminate stress-induced neuroendocrine system disorders and tissue and organ damage, reducing or eliminating individual and population level changes.
Currently, commercial anti-stress agents mainly comprise vitamins and electrolytes (vitamin B2, C, E, K and the like), and most of the formulations of the anti-stress agents come from animal studies, and have poor or no effects. Therefore, it is necessary to develop a stress protectant with obvious anti-stress effect and capable of obviously improving the survival rate of fish.
Disclosure of Invention
Therefore, the invention aims to provide a stress protective agent, a preparation method and application thereof, and aims to achieve the purposes of improving the physical fitness of fishes and reducing the death rate by adopting a high-efficiency antioxidant to target and regulate mitochondrial energy metabolism so as to reduce or eliminate a series of stress signals caused by free radicals and assisting with substances for promoting energy generation, reconstructing the energy metabolism of fishes, improving the stress resistance and disease resistance of the fishes.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the stress protective agent comprises the following raw materials in percentage by mass: resveratrol 0.04-0.08%, beta-nicotinamide mononucleotide 0.08-0.2%, alpha-lipoic acid 0.05-0.15%, coenzyme Q0.1-0.4%, taurine 0.6-2.0%, gamma-aminobutyric acid 0.3-1.0%, glutathione 0.5-1.0%, soybean lecithin 0.5-1.2%, sucrose ester 0.5-2%, adenosine triphosphate 0.2-0.8%, fish oil 10-20% and the balance of water.
Preferably, the stress protectant comprises the following raw materials in percentage by mass: resveratrol 0.05-0.07%, beta-nicotinamide mononucleotide 0.09-0.15%, alpha-lipoic acid 0.07-0.13%, coenzyme Q0.2-0.3%, taurine 1.0-1.5%, gamma-aminobutyric acid 0.4-0.8%, glutathione 0.6-0.8%, soybean lecithin 0.7-1.1%, sucrose ester 0.8-1.6%, adenosine triphosphate 0.4-0.6%, fish oil 12-16% and the balance of water.
The invention also provides a preparation method of the stress protectant, which comprises the following steps:
(1) Mixing resveratrol, fish oil, soybean lecithin and coenzyme Q, and performing ultrasonic treatment to obtain oil phase emulsion;
(2) Mixing beta-nicotinamide mononucleotide, alpha-lipoic acid, taurine, gamma-aminobutyric acid, adenosine triphosphate, glycal dipeptide and sucrose ester with water to prepare an aqueous phase solution;
(3) And mixing the oil phase emulsion and the aqueous phase solution to obtain the stress protectant.
Preferably, stirring is carried out in the mixing process in the step (1), the rotating speed of stirring is 200-600r/min, and the stirring time is 20-60min;
further, the stirring is magnetic stirring;
preferably, the temperature of the ultrasonic treatment in the step (1) is 35-42 ℃, the power of the ultrasonic treatment is 300-500w, and the time of the ultrasonic treatment is 20-60min;
preferably, the stress protectant prepared in the step (3) is subjected to cold storage, and the temperature of the cold storage is 4-8 ℃.
The invention also provides application of the stress protectant in preventing fish stress reaction.
Preferably, the application method of the stress protectant in application comprises the following steps: mixing and feeding the feed and the stress protectant according to the mass ratio of 25-100:1; during feeding, 1000-1500 fish are fed per 50-100g of stress protectant; the mass of the cultured fish is 150-250 g/tail;
furthermore, the mixture can be fed after being mixed without obvious solution accumulation on the surface of the mixture.
Preferably, the application method of the stress protectant in application comprises the following steps: mixing the stress protectant with water according to the mass ratio of 1:150-200, soaking fish body for 15-25min, and simultaneously carrying out oxygenation treatment; soaking 400-600 tail fishes according to 50-150g of stress protectant during soaking; the fish has a mass of 150-250 g/tail, and the soaking times are 2-3 times.
Compared with the prior art, the invention has the following beneficial effects:
(1) The reagent adopted by the invention is not an anti-stress reagent taking antioxidants such as vitamin C, E as main components, but takes components which can act on mitochondria in a targeting way as main components, such as resveratrol, beta-nicotinamide mononucleotide and the like.
(2) Compared with the traditional anti-stress concept, the invention provides raw materials and energy for the normal functions of cells by adding various mitochondrial metabolism intermediates such as adenosine triphosphate, glutathione and the like, improves the antioxidant enzyme activity of the cells, and ensures that the cells participate in the repair process of mitochondria, thereby achieving the effect of relieving stress response in multiple aspects.
(3) The invention provides two different using methods of the stress protectant in application, namely a feed adding method and an acute soaking method, and the using method overcomes the defect of single using method of the stress protectant and has the advantages of no toxicity, no harm and environmental protection.
(4) The invention adopts a simple and efficient processing method, and utilizes a proper carrier to organically combine the main components together, thereby improving the absorption rate of the organism to the effective components; and by establishing modes of feed addition, acute soaking and the like, the use means of the stress protectant is enlarged, the maximum protection of the fish body under various extreme environments or operation conditions is realized, and the death and injury of the fish are reduced.
(5) Aiming at the stress reaction of aquatic animals caused by the operations such as transportation, pond moving and the like or the reasons such as too low feed fish meal, extreme climate and the like, the method starts from mitochondrial tissues which are mainly oxidized and powered, and the damaged mitochondria are protected or repaired in a targeted manner by reasonably matching with several reagents such as resveratrol, so that the normal functional state of the damaged mitochondria is promoted, the stress resistance of the aquatic animals is improved, the death rate is reduced, and the survival rate in the pond moving, transportation, fishing and high-vegetable-protein feed feeding process is improved.
Detailed Description
The invention provides a stress protective agent which comprises the following raw materials in percentage by mass: resveratrol 0.04-0.08%, beta-nicotinamide mononucleotide 0.08-0.2%, alpha-lipoic acid 0.05-0.15%, coenzyme Q0.1-0.4%, taurine 0.6-2.0%, gamma-aminobutyric acid 0.3-1.0%, glutathione 0.5-1.0%, soybean lecithin 0.5-1.2%, sucrose ester 0.5-2%, adenosine triphosphate 0.2-0.8%, fish oil 10-20% and the balance of water.
In the invention, the stress protectant comprises resveratrol 0.04-0.08%, preferably 0.05-0.07%, and more preferably 0.06%; the resveratrol is preferably polygonum cuspidatum extract, and the purity is more than or equal to 98%.
In the present invention, the stress protectant comprises 0.08-0.2%, preferably 0.09-0.15%, and more preferably 0.10% of beta-nicotinamide mononucleotide; the beta-nicotinamide mononucleotide is a commercial product, and the purity is more than or equal to 99 percent.
In the present invention, the stress protectant comprises 0.05-0.15%, preferably 0.07-0.13%, and more preferably 0.10% of alpha-lipoic acid; the alpha-lipoic acid is a commercial product, and the purity is more than or equal to 99 percent.
In the present invention, the stress protectant comprises coenzyme Q0.1-0.4%, preferably 0.2-0.3%, and more preferably 0.25%; the coenzyme Q is a commercial product, and the purity is more than or equal to 98 percent.
In the present invention, the stress protectant comprises taurine 0.6-2.0%, preferably 1.0-1.5%, and more preferably 1.3%; the taurine is a commercial product, and the purity is more than or equal to 98 percent.
In the present invention, the stress protectant comprises 0.3-1.0%, preferably 0.4-0.8%, and more preferably 0.5% of gamma-aminobutyric acid; the gamma-aminobutyric acid is a commercial product, and the purity is more than or equal to 98%.
In the present invention, the stress protectant comprises 0.5-1.0%, preferably 0.6-0.8%, and more preferably 0.7% of a glycylglutidine; the glycylglutamine is a commercial product, and the purity is more than or equal to 99 percent.
In the present invention, the stress protectant comprises soybean lecithin 0.5-1.2%, preferably 0.7-1.1%, and more preferably 1.0%; the soybean lecithin is a commercial product, and the purity is more than or equal to 99%.
In the present invention, the stress protectant comprises sucrose esters 0.5-2%, preferably 0.8-1.6%, and more preferably 1.2%; the sucrose ester is a commercial product, and the purity is more than or equal to 99 percent.
In the present invention, the stress protectant comprises adenosine triphosphate 0.2-0.8%, preferably 0.4-0.6%, and more preferably 0.5%; the adenosine triphosphate is a commercial product, and the purity is more than or equal to 99%.
In the invention, the stress protectant comprises 10-20%, preferably 12-16%, and more preferably 14% of fish oil; the fish oil is a commercial product, and the purity is more than or equal to 99 percent.
The invention also provides a preparation method of the stress protectant, which comprises the following steps:
(1) Mixing resveratrol, fish oil, soybean lecithin and coenzyme Q, and performing ultrasonic treatment to obtain oil phase emulsion;
(2) Mixing beta-nicotinamide mononucleotide, alpha-lipoic acid, taurine, gamma-aminobutyric acid, adenosine triphosphate, glycal dipeptide and sucrose ester with water to prepare an aqueous phase solution;
(3) And mixing the oil phase emulsion and the aqueous phase solution to obtain the stress protectant.
In the invention, resveratrol, fish oil, soybean lecithin and coenzyme Q are mixed; stirring is carried out in the mixing process, the rotating speed of stirring is 200-600r/min, and the stirring time is 20-60min; further preferably, the stirring is magnetic stirring, the rotating speed of the stirring is 300-500r/min, and the stirring time is 30-50min; still more preferably, the stirring speed is 400r/min, and the stirring time is 40min; the invention carries out ultrasonic treatment after stirring, wherein the temperature of the ultrasonic treatment is 35-42 ℃, the power of the ultrasonic treatment is 300-500w, and the time of the ultrasonic treatment is 20-60min; further preferably, the temperature of the ultrasonic treatment is 37-40 ℃, the power of the ultrasonic treatment is 350-450w, and the time of the ultrasonic treatment is 30-50min; still more preferably, the temperature of the ultrasonic treatment is 38 ℃, the power of the ultrasonic treatment is 400w, and the time of the ultrasonic treatment is 40min;
in the invention, the oil phase emulsion and the aqueous phase solution are mixed to prepare the stress protectant; the stirring treatment is preferably carried out during the mixing, the rotating speed of stirring is 200-600r/min, and the stirring time is 20-60min; further preferably, the stirring speed is 300-500r/min, and the stirring time is 30-50min; still more preferably, the stirring speed is 400r/min and the stirring time is 40min. In the invention, the stress protectant is preferably refrigerated and preserved, and the temperature of the refrigerated and preserved is 4-8 ℃; further preferably, the temperature of the refrigerated storage is 5-7 ℃; still more preferably, the temperature of the refrigerated storage is 6 ℃.
The invention also provides application of the stress protectant in preventing fish stress reaction.
In the invention, the using method of the stress protectant in application comprises a feed adding method: mixing and feeding the feed and the stress protectant according to the mass ratio of 25-100:1; during feeding, 1000-1500 fish are fed per 50-100g of stress protectant; the mass of the cultured fish is 150-250 g/tail. Furthermore, the mixture can be fed after being mixed without obvious solution accumulation on the surface of the mixture. In the present invention, the use time of the stress protectant is preferably 15-30 days before the stress reaction may occur, or long-term feeding.
In the invention, the application method of the stress protectant in application further comprises an acute soaking method: mixing the stress protectant with water according to the mass ratio of 1:150-200, soaking fish body for 15-25min, and simultaneously carrying out oxygenation treatment; soaking 400-600 tail fishes according to 50-150g of stress protectant during soaking; the fish has a mass of 150-250 g/tail, and the soaking times are 2-3 times.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The stress protective agent comprises the following raw material components in percentage by mass: resveratrol 0.06%, beta-nicotinamide mononucleotide 0.10%, alpha-lipoic acid 0.10%, coenzyme Q0.25%, taurine 1.3%, gamma-aminobutyric acid 0.5%, glutathione 0.7%, soybean lecithin 1.0%, sucrose ester 1.2%, adenosine triphosphate 0.5%, fish oil 14% and purified water 80.29%.
A method for preparing a stress protectant, comprising the following steps:
(1) Mixing resveratrol, fish oil, soybean lecithin and coenzyme Q according to mass content, stirring uniformly by a magnetic stirrer, regulating the rotating speed to 350r/min, stirring for 40min, adding into an ultrasonic stirrer, setting the power to 300w, setting the temperature to 40 ℃, and carrying out ultrasonic treatment for 30min to obtain the oil phase emulsion.
(2) Adding beta-nicotinamide mononucleotide, alpha-lipoic acid, taurine, gamma-aminobutyric acid, adenosine triphosphate, glycylglutidine and sucrose ester into purified water which is weighed according to mass fractions, and uniformly stirring to form a water phase solution.
(3) Mixing the oil phase emulsion and the aqueous phase solution, stirring for 40min at a rotation speed of 350r/min by a magnetic stirrer to obtain the stress protectant, and refrigerating at 6deg.C for use.
The application method of the stress protectant in the application of preventing the fish stress reaction comprises the following steps:
200g of stress protectant is taken and added into 30kg of water to soak fish bodies, and oxygenation treatment is carried out at the same time.
Example 2
The stress protective agent comprises the following raw material components in percentage by mass: resveratrol 0.04%, beta-nicotinamide mononucleotide 0.08%, alpha-lipoic acid 0.06%, coenzyme Q0.1%, taurine 0.6%, gamma-aminobutyric acid 0.4%, glutathione 0.5%, soybean lecithin 0.6%, sucrose ester 2.0%, adenosine triphosphate 0.2%, fish oil 20% and purified water 75.42%.
A method for preparing a stress protectant, comprising the following steps:
(1) Mixing resveratrol, fish oil, soybean lecithin and coenzyme Q according to mass content, stirring uniformly by a magnetic stirrer, regulating the rotating speed to 200r/min, stirring for 20min, adding into an ultrasonic stirrer, setting the power to 500w, and carrying out ultrasonic treatment for 20min at 35 ℃ to obtain the oil phase emulsion.
(2) Adding beta-nicotinamide mononucleotide, alpha-lipoic acid, taurine, gamma-aminobutyric acid, adenosine triphosphate, glycylglutidine and sucrose ester into purified water which is weighed according to mass fractions, and uniformly stirring to form a water phase solution.
(3) Mixing the oil phase emulsion and the aqueous phase solution, stirring at a rotation speed of 200r/min by a magnetic stirrer for 60min to obtain the stress protectant, and refrigerating at 4deg.C for use.
The application method of the stress protectant in the application of preventing the fish stress reaction comprises the following steps:
100g of stress protectant is taken and added into 5kg of feed, and the mixture is stirred and fed when no obvious solution is accumulated on the surface of the mixture.
Example 3
The stress protective agent comprises the following raw material components in percentage by mass: resveratrol 0.08%, beta-nicotinamide mononucleotide 0.2%, alpha-lipoic acid 0.15%, coenzyme Q0.4%, taurine 2.0%, gamma-aminobutyric acid 1.0%, glutathione 1.0%, soybean lecithin 1.2%, sucrose ester 0.5%, adenosine triphosphate 0.8%, fish oil 10% and purified water 82.67%.
A method for preparing a stress protectant, comprising the following steps:
(1) Mixing resveratrol, fish oil, soybean lecithin and coenzyme Q according to mass content, uniformly stirring by a magnetic stirrer, adjusting the rotating speed to 600r/min, stirring for 60min, putting into an ultrasonic stirrer, setting the power to 400w, setting the temperature to 42 ℃, and carrying out ultrasonic treatment for 60min to obtain an oil phase emulsion;
(2) Adding beta-nicotinamide mononucleotide, alpha-lipoic acid, taurine, gamma-aminobutyric acid, adenosine triphosphate, glycylglutidine and sucrose ester into purified water which is weighed according to mass fractions, and uniformly stirring to form a water phase solution.
(3) Mixing the oil phase emulsion and the aqueous phase solution, stirring for 20min at 600r/min of a magnetic stirrer to obtain the stress protectant, and refrigerating at 8deg.C.
Protection test one
The stress protectant prepared in the examples 1-3 of the invention is mixed with black to form pellet feed for feeding 10 months 2021.
The experiment is provided with a control group which is fed with pellet feed without adding a protective agent. And feeding the black in front of the pond moving of a certain culturing farm in the east camping for 11 months and 20 days, and fishing out the pond moving according to normal operation after feeding for one month, wherein the pond moving distance is about 10 km. The protection results are shown in table 1:
table 1 protection results
As a result, it is found that the survival rate of black of the fish stress protection emulsion can reach more than 95.96%, and the fish stress protection emulsion can quickly adapt to the new pond environment after the fish is transplanted, and no sequelae exists. The survival rate of the fish in the control group is only about 81.19-89.23%, and some fish cannot adapt to the new pond, and then the fish in the control group die about 4% in two or three days.
Protection test II
The stress protectant prepared in the invention example 1-2 is applied to the process of transporting weever from a smoke counter to east camping on 4 days of 2022, the water tanks in the transport vehicle are divided into two rows, the protectant is directly added into the water tank on one side for dipping, and the protectant is not added into the water tank on the other side. Pure oxygen is filled in the transportation process, and the transportation distance is 300 km or more. The protection results are shown in table 2:
table 2 protection results
After the transportation is finished, the survival rate of the weever on one side added with the protective agent is more than 95.84%, the state is good, and the new environment adaptability is strong. The survival rate of the weever on the side without the protective agent is only 79.28-84.08%, the fish body is blackened, and the action is slow. By contrast, the fish stress protective agent has remarkable protective effect on fish stress.
In conclusion, the stress protectant in the application can obviously relieve the stress response of fish and improve the survival rate of the fish when the stress response occurs.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (9)

1. The fish stress protective agent is characterized by comprising the following raw materials in percentage by mass: resveratrol 0.04-0.08%, beta-nicotinamide mononucleotide 0.08-0.2%, alpha-lipoic acid 0.05-0.15%, coenzyme Q0.1-0.4%, taurine 0.6-2.0%, gamma-aminobutyric acid 0.3-1.0%, glutathione 0.5-1.0%, soybean lecithin 0.5-1.2%, sucrose ester 0.5-2%, adenosine triphosphate 0.2-0.8%, fish oil 10-20% and the balance of water.
2. The stress protectant of claim 1, comprising the following raw materials in mass percent: resveratrol 0.05-0.07%, beta-nicotinamide mononucleotide 0.09-0.15%, alpha-lipoic acid 0.07-0.13%, coenzyme Q0.2-0.3%, taurine 1.0-1.5%, gamma-aminobutyric acid 0.4-0.8%, glutathione 0.6-0.8%, soybean lecithin 0.7-1.1%, sucrose ester 0.8-1.6%, adenosine triphosphate 0.4-0.6%, fish oil 12-16% and the balance of water.
3. A method of preparing the stress protectant of claim 1 or 2, comprising the steps of:
(1) Mixing resveratrol, fish oil, soybean lecithin and coenzyme Q, and performing ultrasonic treatment to obtain oil phase emulsion;
(2) Mixing beta-nicotinamide mononucleotide, alpha-lipoic acid, taurine, gamma-aminobutyric acid, adenosine triphosphate, glycal dipeptide and sucrose ester with water to prepare an aqueous phase solution;
(3) And mixing the oil phase emulsion and the aqueous phase solution to obtain the stress protectant.
4. The method according to claim 3, wherein the stirring is carried out during the mixing in the step (1), the stirring speed is 200-600r/min, and the stirring time is 20-60min.
5. A method according to claim 3, wherein the temperature of the ultrasonic treatment in step (1) is 35-42 ℃, the power of the ultrasonic treatment is 300-500w, and the time of the ultrasonic treatment is 20-60min.
6. The method according to claim 3, wherein the stress protectant obtained in step (3) is followed by cold storage at a temperature of 4-8deg.C.
7. Use of the stress protectant of claim 1 or 2 for preventing stress in fish.
8. The use according to claim 7, characterized in that the feed is mixed with the stress protectant in a mass ratio of 25-100:1, fed; during feeding, 1000-1500 fish are fed per 50-100g of stress protectant; the mass of the cultured fish is 150-250 g/tail.
9. The use according to claim 7, wherein the stress protectant is mixed with water in a mass ratio of 1:150-200, the fish body is soaked for 15-25min, and oxygenation is performed simultaneously; soaking 400-600 tail fishes according to 50-150g of stress protectant during soaking; the fish has a mass of 150-250 g/tail, and the soaking times are 2-3 times.
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