CN111820338A

CN111820338A - Natural composite fish-hydrophilic aeromonas resisting agent, natural feed and preparation method thereof

Info

Publication number: CN111820338A
Application number: CN202010708680.8A
Authority: CN
Inventors: 李华涛; 苟丹; 吴敏; 袁佳; 贺扬; 敬晓琴; 刘思妙; 袁萍; 龙娇; 董婷婷; 冯敏; 梁琪
Original assignee: Neijiang Normal University
Current assignee: Neijiang Normal University
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2020-10-27

Abstract

The invention discloses a natural composite fish-hydrophilic monad resisting agent, a natural fish-hydrophilic monad infection resisting feed and a preparation method thereof, wherein the composite fish-hydrophilic monad resisting agent is composed of more than 3 natural medicinal plant extracts. The invention can improve the capability of resisting aeromonas hydrophila infection of the fish in culture and improve the survival rate of the fish in culture, and has no residue, toxic or side effect and no environment pollution.

Description

Natural composite fish-hydrophilic aeromonas resisting agent, natural feed and preparation method thereof

Technical Field

The invention relates to a natural composite fish aeromonas hydrophila resistant agent, a natural product fish aeromonas hydrophila infection resistant feed and a preparation method of the natural product fish aeromonas hydrophila infection resistant feed.

Background

Aeromonas hydrophila belongs to the genus Aeromonas in the family Vibrionaceae and is a gram-negative Brevibacterium. Aeromonas hydrophila is widely distributed in nature and is commonly found in fresh water, sewage, sludge and soil. Cold blood animals such as fish, frog and the like are natural hosts of the bacteria, have pathogenicity on various fishes, amphibians and reptiles, and are main pathogenic bacteria causing diseases of freshwater aquaculture animals. Aeromonas hydrophila can cause red fin disease of eel, erect scale disease of carp and goldfish, stigmatosis of chain and bighead carp, fulminant hemorrhagic disease of silver carp, bacterial enteritis of herring and grass carp, furunculosis of herring, red mouth disease of sweetfish, red neck disease and septicemia of turtle, red leg disease of frog, septicemia and stomatitis of snake, hemorrhagic disease of finless eel and septicemia of fishes such as salmon and trout.

"high density, intensification" is an important feature of current aquaculture. However, the relative living space of the fishes in the water body is reduced in the high-density culture mode, and competition and interference among different individuals are aggravated; the high-density cultivation improves the feed feeding amount, increases the cultivation wastes in the water body, improves the breeding amount of pathogenic microorganisms and deteriorates the water quality. The above conditions improve the stress response and reduce the immunity of the cultured fish, and improve the susceptibility of the fish to aeromonas hydrophila. Aeromonas hydrophila is highly sensitive to antibiotics such as gentamicin, kanamycin, norfloxacin, tobramycin and the like; is moderately sensitive to antibiotics and chemical drugs such as furazolidone, furinas, chloramphenicol, and compound sulfamethoxazole. However, these drugs may increase the drug residue in fish meat, which presents a food safety hazard. For this country, relevant laws and regulations have been issued to prohibit the use of more than one compound drugs such as antibiotics and antibiotics in feed and drinking water for animals, and strictly prohibit the direct sprinkling of antibiotics to aquaculture water. Residual limits in fish products have been specified for most of the antibacterial fisheries drugs currently available. The restrictive laws and regulations reduce the drug residue of fish meat to a certain extent, protect the safety of aquatic products, maintain the health of human bodies, but simultaneously weaken the strength of preventing and treating aquaculture diseases, reduce the aquaculture efficiency and make the market have 'vacancy'. The aquaculture industry has a critical need for alternatives that have the ability to disable fishery drugs without their drawbacks.

High-density intensive culture requires the use of complete formula feed. The addition of antioxidants, mildewcides, growth promoters, phagostimulants and the like to compound feeds is a common phenomenon. The major antioxidants currently used are mainly synthetic antioxidants, including Ethoxyquinoline (EQ), Butylhydroxyanisole (BHA), dibutylhydroxytoluene (BHT), and the like. The fish meal and the grease are important feed raw materials, and the adding amount of the synthetic antioxidant in the fish meal processing process can reach 0.1 percent; synthetic antioxidants are also added to feed-grade oils. However, synthetic antioxidants have been reported to be carcinogenic and genotoxic to animals and to cause residues and enrichment in fish products, creating a food safety hazard and endangering human health. In 2 months in 2017, the European Union issues the G/SPS/EU/190 announcement in 2017: the addition of EQ to the animal feed was stopped. Also, BHT and BHA added to animal feed have been reported to have higher carcinogenic risk than EQ. Chemically synthesized additives such as mildewcides, growth promoters, phagostimulants and the like have also been reported to be potentially toxic to animals and may cause residues in fish meat. The disadvantages of compound feeds will therefore lead to an increased market demand for natural product compound feeds.

The current regulations in China clearly stipulate that natural plants with homology of medicine and food and extracts thereof can be used as feed raw materials. After the medicinal plants (Chinese herbal medicines) are subjected to the extraction process, most of ineffective components are removed, so that the activity is enhanced. The medicinal plants are extracted to decompose the comprehensive functions and concentrate specific functions, thereby improving the effect of a certain function. The plant extract has reduced volume compared with Chinese herbal medicine, and is convenient for storage, transportation and transaction. The planting of Chinese herbal medicine has a long history in China, rich resources and wide sources. Some medicinal plants have become regional weeds, agricultural waste or municipal waste. The utilization of the leftovers of the non-genuine medicinal materials and the Chinese herbal medicines after processing is very limited. The active ingredients contained in the medicinal plants are obtained by an extraction process and are used for developing natural functional feeds, increasing the new application of the medicinal plants, improving the traditional utilization structure of the medicinal plants, exerting the comprehensive utilization potential of the medicinal plants, changing waste into valuable and improving the economic and social benefits of the medicinal plants, so that the method has important significance.

Disclosure of Invention

Based on the above problems, in one aspect, the present invention provides a natural composite fish-resistant aeromonas hydrophila agent, which can improve the ability of fish to resist infection by aeromonas hydrophila in culture, improve the survival rate of fish in culture, and has no residue, no toxic or side effect, and no environmental pollution.

The technical scheme is as follows: a natural composite agent for resisting fish hydrophilic monad is prepared from more than 3 kinds of natural plant extracts.

Further, in the present invention, when the complex anti-fish aeromonas hydrophila agent contains an ethanol extract of pomegranate rind, an ethanol extract of turmeric, an ethanol extract of myrobalan, and an ethanol extract of scutellaria, a good anti-fish aeromonas hydrophila infection effect can be obtained.

Furthermore, when the weight ratio of the pomegranate peel ethanol extract to the turmeric ethanol extract to the myrobalan ethanol extract to the scutellaria ethanol extract is 1-2: 1-2, the survival rate of the fish in the culture can be effectively improved by 8.74-41.06%.

Furthermore, in the invention, when the compound anti-fish-aeromonas hydrophila stress agent contains the water extract of the myrobalan, the water extract of the dark plum and the water extract of the baical skullcap root, better anti-fish-aeromonas hydrophila infection effect can be obtained.

Furthermore, according to the invention, when the weight ratio of the myrobalan water extract to the smoked plum water extract to the scutellaria baicalensis water extract is 1-2: 1-2, the survival rate of the fish in the culture can be effectively increased by 11.44-45.76%.

Furthermore, in the invention, when the compound anti-fish-aeromonas hydrophila stress agent contains the pomegranate rind ethanol extract, the turmeric ethanol extract, the myrobalan ethanol extract, the scutellaria ethanol extract, the myrobalan water extract, the dark plum water extract and the scutellaria water extract, on one hand, a better anti-fish-aeromonas hydrophila infection effect can be obtained, and on the other hand, a little dosage can be used, so that the cost can be saved.

Furthermore, the survival rate of the fish in the culture can be effectively improved by 48.43% when the weight ratio of the pomegranate peel ethanol extract, the turmeric ethanol extract, the myrobalan ethanol extract, the scutellaria ethanol extract, the myrobalan water extract, the smoked plum water extract and the scutellaria water extract is 1-2: 2-4.

In one aspect, the invention also provides a natural product feed for resisting the infection of the aeromonas hydrophila of the fish.

The technical scheme is as follows: a natural product feed for resisting fish aeromonas hydrophila infection comprises a basic feed and the natural compound agent for resisting fish aeromonas hydrophila.

Furthermore, the natural composite fish-hydrophilic aeromonas sobria agent is 0.4-1.2 wt% of the basic feed.

Further, when the compound anti-fish-hydrophilic single cell bacterium agent contains pomegranate peel ethanol extract, turmeric ethanol extract, myrobalan ethanol extract and scutellaria ethanol extract, the natural compound anti-fish-hydrophilic single cell bacterium agent accounts for 0.4-0.6 wt% of the basic feed.

Further, when the compound fish-resistant aeromonas hydrophila stress agent contains a myrobalan water extract, a dark plum water extract and a scutellaria baicalensis water extract, the natural compound fish-resistant aeromonas hydrophila stress agent accounts for 0.6-1.2 wt% of the basic feed.

Further, when the compound anti-fish-hydrophilic aeromonas hydrophila agent contains pomegranate rind ethanol extract, turmeric ethanol extract, myrobalan ethanol extract, scutellaria ethanol extract, myrobalan water extract, dark plum water extract and scutellaria water extract, the natural compound anti-fish-hydrophilic aeromonas hydrophila agent is 0.4-0.6% of the basal feed.

On one hand, the invention also provides a preparation method of the natural product feed for resisting the infection of the aeromonas hydrophila of the fish.

The technical scheme is as follows: the preparation method of the natural product feed for resisting fish aeromonas hydrophila infection comprises the steps of firstly calculating the mass of oil and/or water required by unit mass of basic feed according to a feed formula, adding the natural compound agent for resisting fish aeromonas hydrophila into the oil and/or water, and then uniformly mixing the oil and/or water into the basic feed to prepare the pellet feed.

The basic feed adopts natural raw materials, and other substances are not artificially added; the additive component meets the requirements of national standard NY/T471-once 2018, and has no residue, no toxic or side effect and no environmental pollution.

The principle and the beneficial effects of the invention are as follows:

the inventors of the present invention found in long-term studies that: the death rate of the fish after the toxic challenge of the aeromonas hydrophila can be reduced by feeding the composite plant extract. The pomegranate peel ethanol extract, the myrobalan ethanol extract, the scutellaria ethanol extract and the curcuma ethanol extract play a role in promoting the anti-infection effect of other components of the compound extract; the ethanol extracts of eucommia ulmoides and dark plum have the inhibiting effect. The water extracts of the myrobalan, the scutellaria baicalensis and the dark plum have a synergistic effect on the anti-infection effect of other components of the compound extract; the turmeric water extract, pomegranate rind water extract and eucommia water extract have inhibiting effect.

The natural functional feed can improve the ability of the fish to resist the aeromonas hydrophila during cultivation and can effectively improve the survival rate of the fish during cultivation.

The inventor of the invention also finds that the natural composite fish aeromonas hydrophila resisting agent has the effects of resisting feed oxidation and preventing mildew, and when the natural composite fish aeromonas hydrophila resisting agent is added into natural basic feed, non-natural substances such as antioxidant, mildew preventive and the like are not added.

Drawings

FIG. 1 shows the effect of the feed of the compound alcohol extract on the death rate of crucian after toxin attack;

FIG. 2 shows the effect of the feed of the compound water extract on the death rate of crucian after toxicity attack;

fig. 3 shows the effect of the feed of the compound anti-infective agent on the death rate of crucian after toxicity attack.

Detailed Description

The invention will be further explained with reference to the drawings.

In the following examples, the basic feed formulation is as follows:

in the above table:

the fish meal, the bean pulp, the peanut pulp, the cottonseed meal, the wheat flour and the rapeseed oil contain nutrients and are not added with other substances artificially, wherein:

the fish meal contains 89.88% of dry matter, 51.70% of crude protein, 15.01% of crude fat and 18.63% of crude ash, and is prepared from silver carp, crucian carp and grass carp by air-blast drying at 80 deg.C.

The soybean meal contains 89.13% of dry matter, 46.92% of crude protein, 1.53% of crude fat and 5.46% of crude ash, and is a byproduct of soybean after heating, crushing, oil extraction and solvent evaporation.

The peanut meal contains 88.21% of dry matter, 47.54% of crude protein, 1.42% of crude fat and 5.33% of crude ash, and is a byproduct of peanut kernels after heating, crushing, oil extraction and solvent evaporation.

The cottonseed meal contains 89.46% of dry matter, 44.37% of crude protein, 0.54% of crude fat and 5.82% of crude ash, and is a byproduct of cottonseed kernels after heating, crushing, oil extraction and solvent evaporation.

DL-methionine meets the requirement of NY/T471-2018.

Wheat flour is obtained by pulverizing wheat, and contains dry matter 87.60%, crude protein 13.32%, crude fat 2.03%, and crude ash 1.84%.

Canola oil, containing 99.03% dry matter, 0.00% crude protein, 98.00% crude fat and 0.53% crude ash, was prepared from canola oil by pressing.

The vitamin supplement comprises per kilogram: vitamin A acetate 500,000IU, vitamin D₃240,000IU, 20.00g DL-alpha-tocopherol, vitamin K₃0.10g, vitamin B₁0.10g, 0.50g of riboflavin, 0.92g of pyridoxine hydrochloride, 0.001g of cyanocobalamin, 2.46g of D-calcium pantothenate, 2.80g of nicotinic acid, 0.10g of D-biotin, 51.81g of inositol and 0.50g of folic acid, and the carrier is defatted rice bran. The components meet the requirements of NY/T471-2018.

The mineral supplement comprises per kilogram: FeSO₄37.90g, 1.69g of copper methionine, 23.71g of zinc methionine, 7.15g of manganese methionine, 0.06g of selenium methionine and 0.134g of KI, and the diluent is limestone powder. The components meet the requirements of NY/T471-2018.

In the following examples:

the pomegranate peel ethanol extract is obtained by analytical grade ethanol extraction, is yellow red extract, and contains the polyphenols of 137.47-148.75 mg/g.

The Curcuma rhizome ethanol extract is obtained by extracting with analytical grade ethanol, and is yellowish brown extract, wherein the content of polyphenols is 90.56-105.33 mg/g.

The fructus Chebulae ethanol extract is obtained by analytical grade ethanol extraction, and is brown extract, wherein the content of polyphenols is 142.80-161.19 mg/g.

The Scutellariae radix ethanol extract is obtained by analytical grade ethanol extraction, and is yellow extract, wherein the content of flavonoids is 126.32-140.48 mg/g.

The eucommia ulmoides ethanol extract is obtained by analytical grade ethanol extraction, is a brownish red extract, and has the polyphenol content of 80.44-99.18 mg/g.

The fructus mume ethanol extract is obtained by analytical grade ethanol extraction, is dark red extract, and has polyphenol content of 63.54-67.61 g/kg.

The water extract of Eucommiae cortex is obtained by extracting with distilled water, and is brown powder, with crude protein content of 15.53-16.74% and total sugar content of 17.95-20.29%.

The water extract of fructus Chebulae is obtained by extracting with distilled water, and is brown powder with crude protein content of 24.88-27.50% and total sugar content of 25.57-29.96%.

The dark plum water extract is obtained by extracting with distilled water, and is dark purple red powder, crude protein content is 16.58-18.26%, and total sugar content is 15.24-17.08%.

The Scutellariae radix water extract is obtained by extracting with distilled water, and is brown powder, with crude protein content of 16.14-18.78% and total sugar content of 28.64-31.22%.

The water extract of pericarpium Granati is obtained by extracting with distilled water, and is brown powder with crude protein content of 18.75-20.96% and total sugar content of 23.84-25.06%.

The Curcuma rhizome water extract is obtained by extracting Curcuma rhizome water extract with distilled water, and is brown powder, with crude protein content of 18.22-21.19% and total sugar content of 30.66-33.42%.

Example 1: preparation of natural product feed for resisting fish aeromonas hydrophila infection

1) The 6 extracts were added to the oil and fat at a concentration of 1g/kg (one extract per kg of basal feed) respectively according to table 1 below, mixed well and added to the basal feed to make 8 corresponding pellet feeds identified as a0, a1, a2, A3, a4, a5, a6, a7, respectively.

TABLE 1

Additive material

A0

A1

A2

A3

A4

A5

A6

A7

Pomegranate rind ethanol extract

×

Ethanol extract of Curcuma rhizome

×

Ethanol extract of eucommia ulmoides

×

Fructus Chebulae ethanol extract

×

Fructus mume ethanol extract

×

Ethanol extract of Scutellaria baicalensis Georgi

×

In table 1:

a0 refers to basal feed without any added extract. In the examples which follow, A0 was taken in two portions, designated A0(1) and A0(2), respectively.

A1 indicates that 1g of pericarpium Granati ethanol extract, 1g of Curcuma rhizome ethanol extract, 1g of Eucommiae cortex ethanol extract, 1g of fructus Chebulae ethanol extract, 1g of mume fructus ethanol extract and 1g of Scutellariae radix ethanol extract are added into each kg of basal feed.

A2 is prepared by adding ethanol extract of Curcuma rhizome 1g, ethanol extract of Eucommiae cortex 1g, ethanol extract of fructus Chebulae 1g, ethanol extract of mume fructus 1g and ethanol extract of Scutellariae radix 1g into basic feed per kg.

A3 indicates that 1g of pericarpium Granati ethanol extract, 1g of Eucommiae cortex ethanol extract, 1g of fructus Chebulae ethanol extract, 1g of mume fructus ethanol extract and 1g of Scutellariae radix ethanol extract are added into each kg of basal feed.

A4 indicates that 1g of pericarpium Granati ethanol extract, 1g of Curcuma rhizome ethanol extract, 1g of fructus Chebulae ethanol extract, 1g of mume fructus ethanol extract and 1g of Scutellariae radix ethanol extract are added into each kg of basal feed.

A5 indicates that 1g of pericarpium Granati ethanol extract, 1g of Curcuma rhizome ethanol extract, 1g of Eucommiae cortex ethanol extract, 1g of mume fructus ethanol extract and 1g of Scutellariae radix ethanol extract are added into each kg of basal feed.

A6 indicates that 1g of pericarpium Granati ethanol extract, 1g of Curcuma rhizome ethanol extract, 1g of Eucommiae cortex ethanol extract, 1g of fructus Chebulae ethanol extract and 1g of Scutellariae radix ethanol extract are added into each kg of basal feed.

A7 is prepared by adding 1g pericarpium Granati ethanol extract, 1g Curcuma rhizome ethanol extract, 1g Eucommiae cortex ethanol extract, 1g fructus Chebulae ethanol extract and 1g mume fructus ethanol extract into each kg basal feed.

2) The 6 extracts were added to water at a concentration of 2g/kg (one extract per kg of basal feed) according to table 2 below, mixed well and added to the basal feed to make 8 corresponding pellet feeds identified as B0, B1, B2, B3, B4, B5, B6, B7, respectively.

TABLE 2

Additive material

B0

B1

B2

B3

B4

B5

B6

B7

Pomegranate rind water extract

×

Turmeric water extract

×

Aqueous extract of eucommia ulmoides

×

Aqueous extract of Terminalia chebula

×

Aqueous extract of dark plum fruit

×

Aqueous extract of Scutellaria baicalensis Georgi

×

In table 2:

b0 refers to basal feed without any added extract. In the examples that follow, B0 was divided into two portions, designated B0(1) and B0 (2).

B1 is prepared by adding 2g of pericarpium Granati water extract, 2g of Curcuma rhizome water extract, 2g of Eucommiae cortex water extract, 2g of fructus Chebulae water extract, 2g of mume fructus water extract and 2g of Scutellariae radix water extract into each kg of basal feed.

B2 is prepared by adding 2g Curcuma rhizome water extract, 2g Eucommiae cortex water extract, 2g fructus Chebulae water extract, 2g mume fructus water extract and 2g Scutellariae radix water extract into each kg basal feed.

B3 is prepared by adding 2g of pericarpium Granati water extract, 2g of Eucommiae cortex water extract, 2g of fructus Chebulae water extract, 2g of mume fructus water extract and 2g of Scutellariae radix water extract into each kg of basal feed.

B4 is prepared by adding 2g of pericarpium Granati water extract, 2g of Curcuma rhizome water extract, 2g of fructus Chebulae water extract, 2g of mume fructus water extract and 2g of Scutellariae radix water extract into each kg of basal feed.

B5 is prepared by adding 2g of pericarpium Granati water extract, 2g of Curcuma rhizome water extract, 2g of Eucommiae cortex water extract, 2g of mume fructus water extract and 2g of Scutellariae radix water extract into each kg of basal feed.

B6 is prepared by adding 2g of pericarpium Granati water extract, 2g of Curcuma rhizome water extract, 2g of Eucommiae cortex water extract, 2g of fructus Chebulae water extract and 2g of Scutellariae radix water extract into each kg of basal feed.

B7 is prepared by adding 2g of pericarpium Granati water extract, 2g of Curcuma rhizome water extract, 2g of Eucommiae cortex water extract, 2g of fructus Chebulae water extract and 2g of mume fructus water extract into one kg of basal feed.

3) The following table 3 shows that 12 kinds of extracts are added to water at a concentration of 1 or 2g/kg (one extract is added per kg of basal feed) respectively, and the ethanol extract is added to oil and fat, mixed uniformly and then added to the basal feed to prepare corresponding 6 kinds of pellet feeds which are respectively marked as C0, C1, C2, C3, C4 and C5, wherein the adding amount of C5 is 0.5 g/kg.

TABLE 3

Additive material

C0

C1

C2

C3

C4

C5

Pomegranate rind ethanol extract

×

Ethanol extract of Curcuma rhizome

×

Ethanol extract of eucommia ulmoides

×

Fructus Chebulae ethanol extract

×

Fructus mume ethanol extract

×

Ethanol extract of Scutellaria baicalensis Georgi

×

Pomegranate rind water extract

×

Turmeric water extract

×

Aqueous extract of eucommia ulmoides

×

Aqueous extract of Terminalia chebula

×

Aqueous extract of dark plum fruit

×

Aqueous extract of Scutellaria baicalensis Georgi

×

In table 3:

c0 refers to basal feed without any added extract. In the examples that follow, C0 was divided into two portions, designated C0(1) and C0 (2).

C1 indicates that 1g of pericarpium Granati ethanol extract, 1g of Curcuma rhizome ethanol extract, 1g of fructus Chebulae ethanol extract, 1g of mume fructus ethanol extract and 1g of Scutellariae radix ethanol extract are added into each kg of basal feed.

C2 indicates that 1g of pericarpium Granati ethanol extract, 1g of Curcuma rhizome ethanol extract, 1g of fructus Chebulae ethanol extract and 1g of Scutellariae radix ethanol extract are added into each kg of basal feed.

C3 is obtained by adding 2g Curcuma rhizome water extract, 2g Eucommiae cortex water extract, 2g fructus Chebulae water extract, 2g mume fructus water extract and 2g Scutellariae radix water extract into each kg basal feed.

C4 is prepared by adding fructus Chebulae water extract 2g, fructus mume water extract 2g, and radix Scutellariae water extract 2g into basic feed per kg.

C5 is prepared by adding 0.5g ethanol extract of pericarpium Granati, 0.5g ethanol extract of Curcuma rhizome, 0.5g ethanol extract of fructus Chebulae, 0.5g ethanol extract of Scutellariae radix, 1g water extract of fructus Chebulae, 1g water extract of mume fructus and 1g water extract of Scutellariae radix into each kg basal feed.

Example 2

The purchased crucian fries are cultured in a culture room for a week, 405 crucian fries with the same weight are randomly selected and divided into 9 treatment groups, and each group has 3 repeated fish tanks. The shape, size and color of each fish tank are the same, the size is 30 multiplied by 40cm, and an 800L/h flow aerator is arranged in each fish tank. The water in the fish tank takes drinkable tap water as a water source, and the volume of the water in the fish tank is 30L. The temperature of the culture room is controlled to be 22 +/-1 ℃, and the oxygen is continuously supplied by the oxygen increasing machine. The 9 treatment groups were fed with the feeds labeled "a 0 (1)", "a 0 (2)", "a 1", "a 2", "A3", "a 4", "a 5", "a 6" and "a 7" in example 1, respectively, and fed with satiety 4 times per day. Stopping feeding for 1 day after 7 days of feeding and carrying out toxicity attack experiments.

The toxicity counteracting experiment adopts an intraperitoneal injection method. The number of crucian carps in each fish tank is 15. 0.1mL of distilled water was injected into fish fed with "A0 (1)" feed, and 1.0X 10 was injected into fish in other fish tanks⁸0.1mL of aeromonas hydrophila solution at a concentration of one/mL. After injection and sterilization, the fish are returned to the original aquarium, and the death number of the fish in each aquarium is observed and recorded in the next week.

The test results are shown in table 4 below and fig. 1.

TABLE 4 influence of the feed of the compound alcohol extract on the death number of crucian after challenge

In table 4, data are presented as mean ± standard deviation of 3 replicates; the difference in the upper-case letters in the same column data indicates significant difference (P < 0.05).

As can be seen from table 4 and fig. 1, the mortality sequence: a2 > a5 ═ a7 > A3 > a1 > a6 > a4 at 68.89%, 64.44%, 62.22%, 60.00%, 53.33%, 48.89%, 44.44%, respectively.

Example 3

The purchased crucian fries are cultured in a culture room for a week, 405 crucian fries with the same weight are randomly selected and divided into 9 treatment groups, and each group has 3 repeated fish tanks. The shape, size and color of each fish tank are the same, the size is 30 multiplied by 40cm, and an 800L/h flow aerator is arranged in each fish tank. The water in the fish tank takes drinkable tap water as a water source, and the volume of the water in the fish tank is 30L. The temperature of the culture room is controlled to be 22 +/-1 ℃, and the oxygen is continuously supplied by the oxygen increasing machine. The 9 treatment groups were fed with the feeds labeled "B0 (1)", "B0 (2)", "B1", "B2", "B3", "B4", "B5", "B6" and "B7" in example 1, respectively, and fed with satiety 4 times per day. Stopping feeding for 1 day after 7 days of feeding and carrying out toxicity attack experiments.

The toxicity counteracting experiment adopts an intraperitoneal injection method. The number of crucian carps in each fish tank is 15. 0.1mL of distilled water was injected into the fish fed with the feed "B0 (1)", and 1.0X 10 was injected into the fish in the other fish tanks⁸0.1mL of aeromonas hydrophila solution at a concentration of one/mL. After injection and sterilization, the fish are returned to the original aquarium, and the death number of the fish in each aquarium is observed and recorded in the next week.

The test results are shown in table 5 below and fig. 2.

TABLE 5 influence of feed fed with compound water extract on death number of crucian after toxicity attack

In table 5, data are presented as mean ± standard deviation of 3 replicates; the difference in the upper-case letters in the same column data indicates significant difference (P < 0.05).

As can be seen from table 5 and fig. 2, feeding the complex extract reduced the post-challenge mortality rate of the fish, in order of mortality rate, to a different extent compared to B0 (2): b6 > B5 > B7 > B1 > B3 > B2 ═ B4, 68.89%, 62.22%, 60.00%, 51.11%, 48.89%, 42.22%, 42.22%, respectively.

Example 4

The purchased crucian fries are cultured in a culture room for one week, 315 crucian fries with the same weight are selected and randomly divided into 7 treatment groups, and each group has 3 repeated fish tanks. The shape, size and color of each fish tank are the same, the size is 30 multiplied by 40cm, and an 800L/h flow aerator is arranged in each fish tank. The water in the fish tank takes drinkable tap water as a water source, and the volume of the water in the fish tank is 30L. The temperature of the culture room is controlled to be 22 +/-1 ℃, and the oxygen is continuously supplied by the oxygen increasing machine. The 9 treatment groups were fed with the feeds labeled "C0 (1)", "C0 (2)", "C1", "C2", "C3", "C4" and "C5" in example 1, respectively, and fed for 4 times per day with satiation. Stopping feeding for 1 day after 7 days of feeding and carrying out toxicity attack experiments.

The toxicity counteracting experiment adopts an intraperitoneal injection method. The number of crucian carps in each fish tank is 15. 0.1mL of distilled water was injected into fish fed with "C0 (1)" feed, and 1.0X 10 was injected into fish in other fish tanks⁸0.1mL of aeromonas hydrophila solution at a concentration of one/mL. After injection and sterilization, the fish are returned to the original aquarium, and the death number of the fish in each aquarium is observed and recorded in the next week.

The test results are shown in table 6 below and fig. 3.

TABLE 6 influence of feed with composite anti-infective agent on death number of crucian after challenge

In table 6, data are presented as mean ± standard deviation of 3 replicates; the difference in the upper-case letters in the same column data indicates significant difference (P < 0.05).

As can be seen from table 6 and fig. 3, feeding the complex extract reduced the mortality rate of the fish after challenge to a different extent compared to C0 (2). Mortality sequence: c2 < C1; c4 ═ C3; c5 ═ C2 < C4, 37.78%, 44.44%, 42.22%, 44.44%, 37.78%, 42.22%, respectively.

In the invention, the anti-infection effect of C2 is higher than that of C1; although the anti-infection effect of the C4 is equivalent to that of the C3, the C4 is lower in dosage, so that the cost can be saved. The effect of C5 is better than that of C4, and not better than that of C2, but there is still some advantage in cost saving.

Example 5

The non-dead fish after challenge in example 4 were bled from the tail vein, red blood cells were isolated, and the apoptosis rate of red blood cells was determined using Annexin V-FITC kit and flow cytometer, the results of which are shown in table 7.

TABLE 7 Effect of Compound extract on toxin-attacking-induced apoptosis of Carassius auratus erythrocytes

In table 7, data are presented as mean ± standard deviation of 3 replicates, 4 fish per replicate; the difference in the upper-case letters in the same column data indicates significant difference (P < 0.05).

As can be seen from table 7, feeding of the complex extract inhibited toxicity-challenge-induced apoptosis of the red blood cells of crucian; the apoptosis rate of C5 was lower than that of C4 and C2, i.e., the mixture of the complex alcohol extract and the complex water extract had better anti-apoptotic effect.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A natural composite agent for resisting fish hydrophilic monad is prepared from more than 3 kinds of natural plant extracts.

2. The natural complex fish aeromonas hydrophila-resistant agent according to claim 1, wherein:

the compound fish aeromonas hydrophila resisting agent contains pomegranate peel ethanol extract, turmeric ethanol extract, myrobalan ethanol extract and scutellaria ethanol extract:

the weight ratio of the pomegranate peel ethanol extract to the turmeric ethanol extract to the myrobalan ethanol extract to the scutellaria ethanol extract is 1-2: 1-2.

Preferably, the pomegranate peel ethanol extract is yellow red extract, wherein the content of polyphenol substances is 137.47-148.75 mg/g; the Curcuma rhizome ethanol extract is yellow brown extract, wherein the content of polyphenols is 90.56-105.33 mg/g; the fructus Chebulae ethanol extract is brown extract, wherein the content of polyphenols is 142.80-161.19 mg/g; the Scutellariae radix ethanol extract is yellow extract, wherein the content of flavonoids is 126.32-140.48 mg/g.

3. The natural complex fish aeromonas hydrophila agent according to claim 2, wherein: the weight ratio of the pomegranate peel ethanol extract to the turmeric ethanol extract to the myrobalan ethanol extract to the scutellaria ethanol extract is 1:1:1: 1.

4. The natural complex fish aeromonas hydrophila agent according to claim 1, wherein:

the compound fish-hydrophilic aeromonas sobria agent contains water extract of myrobalan, water extract of dark plum and water extract of scutellaria;

the weight ratio of the myrobalan water extract to the dark plum water extract to the scutellaria baicalensis water extract is 1-2: 1-2.

Preferably, the water extract of fructus Chebulae is brown powder with crude protein content of 24.88-27.50% and total sugar content of 25.57-29.96%; the mume fructus water extract is dark purple red powder, crude protein content is 16.58-18.26%, and total sugar content is 15.24-17.08%; the Scutellariae radix water extract is brown powder, and has crude protein content of 16.14-18.78% and total sugar content of 28.64-31.22%.

5. The natural complex fish aeromonas hydrophila agent according to claim 4, wherein: the weight ratio of the myrobalan water extract to the smoked plum water extract to the scutellaria baicalensis water extract is 1:1: 1.

6. The natural complex fish aeromonas hydrophila agent according to claim 1, wherein:

the compound fish aeromonas hydrophila resisting agent contains pomegranate rind ethanol extract, turmeric ethanol extract, myrobalan ethanol extract, scutellaria ethanol extract, myrobalan water extract, dark plum water extract and scutellaria water extract;

the weight ratio of the pericarpium granati ethanol extract to the curcuma longa ethanol extract to the myrobalan ethanol extract to the scutellaria baicalensis ethanol extract to the myrobalan water extract to the fructus mume water extract to the scutellaria baicalensis water extract is 1-2: 2-4;

preferably, the pomegranate peel ethanol extract is yellow red extract, wherein the content of polyphenol substances is 137.47-148.75 mg/g; the Curcuma rhizome ethanol extract is yellow brown extract, wherein the content of polyphenols is 90.56-105.33 mg/g; the fructus Chebulae ethanol extract is brown extract, wherein the content of polyphenols is 142.80-161.19 mg/g; the Scutellariae radix ethanol extract is yellow extract, wherein the content of flavonoids is 126.32-140.48 mg/g; the fructus Chebulae water extract is brown powder with crude protein content of 24.88-27.50% and total sugar content of 25.57-29.96%; the mume fructus water extract is dark purple red powder, crude protein content is 16.58-18.26%, and total sugar content is 15.24-17.08%; the Scutellariae radix water extract is brown powder, and has crude protein content of 16.14-18.78% and total sugar content of 28.64-31.22%.

7. The natural complex fish aeromonas hydrophila agent according to claim 1, wherein:

the weight ratio of the pericarpium granati ethanol extract to the curcuma longa ethanol extract to the myrobalan ethanol extract to the scutellaria baicalensis ethanol extract to the myrobalan water extract to the fructus mume water extract to the scutellaria baicalensis water extract is 1:1:1:2:2: 2.

8. A natural product feed for resisting fish aeromonas hydrophila infection, which comprises a basal feed and the natural compound aeromonas hydrophila resisting agent as claimed in any one of claims 1 to 7, wherein the natural compound aeromonas hydrophila resisting agent is 0.4 to 1.2 wt%, preferably 0.4 to 0.6 wt% or 0.6 to 1.2 wt% of the basal feed.

9. The natural feed for combating aeromonas hydrophila infection in fish according to claim 8, wherein: the basic feed comprises the following components in parts by weight: 15-19 parts of fish meal, 20-26 parts of soybean meal, 13-19 parts of peanut meal, 3-7 parts of cottonseed meal, 0.3-0.7 part of DL-methionine, 33-37 parts of wheat flour, 1.0-2.0 parts of rapeseed oil, 0.5-2.0 parts of vitamin additive and 0.5-2.0 parts of mineral additive.

10. A process for preparing a feed as claimed in any one of claims 8 to 9 for naturally fighting against infection by Aeromonas hydrophila in fish, which comprises calculating the amount of oil and/or water required per unit mass of basal feed based on the feed formulation, adding the natural complex Aeromonas hydrophila agent as claimed in any one of claims 1 to 7 to the oil and/or water, mixing the mixture uniformly with the basal feed, and granulating the mixture.