KR100573759B1 - An animal feed additive having immune stimulating effects on white spot syndrome virus(wssv) - Google Patents

Abstract

The present invention relates to an animal feed additive having an immune enhancing effect on shrimp infected with White Spot Syndrome Virus (WSSV), and specifically, the licorice, cheongung, peanut, buckwheat, hearing and laver of the present invention. Animal feed additives containing natural substances, such as extracts, increase resistance to white spot syndrome virus against shrimp, thereby reducing the occurrence of White Spot Syndrome Disease (WSSD) disease and increasing immunity. Since it has the effect of improving the survival rate and increasing the feeding rate to promote growth, it is possible to prevent disease of individuals and infection of bacteria and viruses in the breeding and farming of shellfish.

White Spot Syndrome Virus, Immune Enhancement, Feed Additive

Description

An animal feed additive having immune stimulating effects on white spot syndrome virus (WSSV)}             

FIG. 1 is a diagram showing the daily feeding amount of one lobster (Penaeus chinensis) for feed added with 5 ml of SP-04 to 100 g of normal feed.

2 is a diagram showing the change in the daily feed intake of one of the crayfish according to the amount of SP-04 added to the general feed,

Figure 3 is a view showing the change in growth rate of lobster according to the amount of SP-04 added to the general feed,

Figure 4 is a diagram observing the effect of the SP-04-added diet on the survival rate of the crayfish infected with White Spot Syndrome Virus (WSSV),

FIG. 6 is a diagram illustrating the effect of enhancing the resistance to WSSV of white mulberry shrimp ( Penaeus vannamei ) by feed with SP-04. FIG.

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The present invention relates to an animal feed additive having an immune enhancing effect against white spot syndrome virus (WSSV) infected with shrimp.

White Spot Syndrome Virus (WSSV) is a virus that is widely distributed around the world. It has a tail-like attachment at the end of the viron, and has rod-shaped capsids and envelopes. It has an ovary form similar to Bacillus, and has been called baculovirus or bacillus-like formed virus, but recently it is named whispovirus, a genetically new virus group. van Hulten MC, et al., J. Gen. Virol. , 81 (Pt2) , pp307-16, 2000). The virus has a length of about 275 nm, a diameter of about 120 nm, and consists of double-stranded DNA having a size of about 290 kb.

More than a decade ago, WSSV-induced white spot syndrome disease (WSSD) occurred in shrimps for the first time in China, resulting in massive deaths of shrimp farming (Inouye et al., Fish Pathol. 29 , pp149-158, 1994; Nakano et al., Fish Pathol . 29 , pp 135-139, 1994). Since then, WSSV has spread rapidly throughout Asia, including Thailand and India, causing enormous damage to the shrimp farming industry (Huang et al., Marine Fisher. Res. 16 , pp 1-10, 1995; Krishna et al., World Aquacult. , Pp . 14-19, 1997; Lightner, J. Appl. Aquacult. 9 , pp27-52, 1999; Park et al., Dis. Aquat . Org. 34 , pp71-75, 1998).

WSSV mainly infects crustaceans such as shrimp, crayfish, and crabs. In particular, the shrimp's carapace, appendage, and cuticle show white spots, and shrimp's hepatopancreas is red. The disease is deadly within 3 to 10 days and 100 to 80% mortality within 20 to 10 days, and it is a fatal disease for shrimp that dies in 20 days. (Lightner, (editor), World Aquaculture Society , Baton Rouge, LA, USA, p 160, 1996).

In Korea, since the damage caused by WSSV disease has been shown in some shrimp farms in Chungnam and Jeonbuk in 1993, it has increased every year, and in 1996, the yield of lobster has been reduced by 30 ~ 75% depending on the region. The mortality rate is 68%, causing economic damage to shrimp farmers and related industrial workers (Marine Fisheries Agency, 2001). Recently, there have been many studies on the diagnosis of WSSV by genetic engineering methods, but the prevention and treatment of this disease has not been developed yet, so it is irrelevant to the disease caused by WSSV worldwide. To date, to reduce the damage from diseases caused by WSSV, only the disinfection and cleanliness of the breeding facilities and the management of the breeding water are being done.

Since 1994, research has been conducted on white spot syndrome, and the gene sequence of the causative bacterium WSSV has been revealed, and many studies such as PCR detection methods, histological lesions, and infectious processes have been conducted (Lo et al., Dis. Aquat . Org. 30 , pp 53-72, 1997; Supamattaya, et al., Dis. Aquat . Org. 32 , pp79-85, 1998; Hossain et al., Aquaculture, 198 , pp 1-11, 2001). Recently, a lot of researches have been made for the development of immune enhancing agents against vibrio and WSSV, and in these studies, glucan and Bifidobacterium thermophilum extracted from yeast cell wall. Immunosuppressive effects of peptidoglycan extracted from algae), lipopolysaccaride extracted from wheat, fucoidan and brown algae extracts have been reported (Chang et al., Dis. Aquat). Org. 36 , pp163-168, 1999, Fish & Shellfish Immun. 10 , pp505-514, 2000; Henning et al., Fish Pathol. 33 , pp389-393, 1998; Itami et al., Aquaculture 164 , pp277- 288, 1998; Song et al., Fish Vaccinol . 90 , pp413-421, 1997; Su et al., J. Taiwan Fisher. Res. 3 , pp125-132, 1995; Sung et al., Fish Pathol. 29 , pp 11-17, 1994). However, the effects of these materials are not significant, and reports of the use of these materials to reduce the mass sensitization by WSSV have not been published.

It is known that there are about 100 kinds of diseases for shrimps, but the damage caused by WSSV is known to be the most serious. In many countries, many studies have been conducted on the virus, studies on the virus's test methods and biological characteristics are being actively conducted, and studies on the immune-enhancing effects of feed addition such as β-glucan are being conducted. The effect is low and is not used in the field. The groundbreaking method for the prevention or treatment of the virus has not been developed yet, and only the cleanliness of the breeding facilities and the management of the breeding water have been made.

Therefore, the present inventors have a feeding effect, a growth effect and resistance to white spot syndrome virus and a resistance to shrimps, using a feed additive mixed with natural substances such as licorice, cheonggung, peanut, buckwheat, auditory and seaweed extract. The present invention was completed by confirming the enhancement effect.

The purpose of the present invention is to increase the resistance to the white spot syndrome virus infected with shrimp to reduce the occurrence of white spot syndrome disease, and to increase the immunity to this to improve the survival rate of the individual, increase the rate of feeding effect to promote growth In the breeding and farming of crustaceans having an animal feed additive that can prevent disease of individuals and infection of bacteria and viruses.

In accordance with the above object, the present invention is an animal for increasing immunity and disease prevention containing licorice, cheongung, peanut, buckwheat, auditory and seaweed extracts with immunopotentiating effect against White Spot Syndrome Virus (WSSV) Provide feed additives.

The animal feed additive can be used as feed and additives for fish and shellfish, preferably shrimp, including livestock, marine fish and freshwater fish, including mammals and poultry.

Hereinafter, the present invention will be described in detail.

Licorice, Cheongung, Peanut, Buckwheat, Hearing and Seaweed extracts of the present invention contain lower alcohols such as methanol, ethanol and butanol in volumes of about 2 to 10 times, preferably about 2 to 5 times the weight of each sample. Or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably ethanol at an extraction temperature of −30 to 30 ° C., preferably at −20 to 10 ° C., hot water extraction, reflux extraction, and reflux cooling. After extraction by continuous extraction once or five times, preferably two to three times by extraction method such as extraction or ultrasonic extraction, it is filtered through filtrate, the filtrate is concentrated under reduced pressure with a rotary vacuum concentrator, and the residue is vacuum freeze-dried. Extraction of each sample available in water, lower alcohol, or a mixed solvent thereof can be obtained through hot air drying or drying by spraying.

In addition, the present invention provides an animal feed additive for increasing immunity and disease prevention containing licorice, cheonggung, peanut, buckwheat, auditory and seaweed extract prepared by the above method.

The animal feed additive of the present invention is an essential component of licorice extract 0-8% by weight, cheongung extract 0-50% by weight, peanut extract 0-90% by weight, buckwheat extract 0-90% by weight, auditory extract 1-10% by weight And seaweed extract is characterized in that consisting of 0-12% by weight.

When the animal feed additive of the present invention is administered to feed shrimp infected with white spot syndrome virus, the feed rate and growth rate are improved, and the survival rate is increased due to the increase of immunity against the white spot syndrome virus. It is effective for farming healthy fish.

Licorice, cheongung, peanut, buckwheat, auditory and laver extracts for animal feed additives may be 20 to 90% high concentrate or powder or granule form.

The licorice, cheongung, peanut, buckwheat, auditory and seaweed extracts for animal feed additives are organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymeric phosphate), etc. Any one or more of natural antioxidants such as phosphates, polyphenols, catechins, alpha-tocopherols, rose mary extracts, vitamin C, green tea extracts, licorice extracts, chitosan, tannic acid and phytic acid It may further include.

Animal feed additives containing licorice, cheonggung, peanut, buckwheat, auditory and seaweed extracts of the present invention and feed containing them are amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal, enzymes, Various adjuvants, such as live microbial preparations, may be used for the production of grains, for example milled or crushed wheat, oats, barley, corn and rice; Vegetable protein feed, such as rape, soybeans and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Sugars and dairy products, for example, a dry ingredient consisting of various powdered milk and whey powder, and a drying additive, all mixed together with a liquid component, and a component which becomes a liquid after heating, that is, a lipid, for example, an animal liquefied by heating. In addition to the main components such as fats and vegetable fats can be used with substances such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like.

Licorice, Cheongung, Peanut, Buckwheat, Hearing and Seaweed extract for animal feed additives can be administered to animals alone in combination with other feed additives in an edible carrier.

In addition, the licorice, cheongung, peanut, buckwheat, auditory and laver extracts for animal feed additives can be mixed as a top dressing or directly in animal feed or separately from the feed, in a separate oral formulation, by injection or transdermal or with other ingredients. It can be easily administered in combination. Typically, single or divided daily dosages can be used as is well known in the art.

When the licorice, cheongung, peanut, buckwheat, auditory and laver extracts for animal feed additives are administered separately from animal feed, the dosage form of the extracts, as is well known in the art, combines them with a non-toxic pharmaceutically acceptable edible carrier. It can be prepared in an immediate release or sustained release formulation. Such edible carriers may be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the dosage form of the extract may be tablets, capsules, powders, torokies or sugar-containing tablets or top dressings in microdisperse form. If a liquid carrier is used, it may be in the form of soft gelatin capsules or syrups or liquid suspensions, emulsions or solutions. In addition, the dosage form may contain auxiliaries such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters and the like. They may also contain substances useful for improving and preventing other allergic and nonallergic inflammatory diseases.

In addition, the animal feed, which includes licorice, cheonggung, peanut, buckwheat, auditory, and seaweed extracts as animal feed additives, may be any protein-containing organic cereal meal commonly used to meet the dietary needs of animals. Such protein-containing flours typically consist mainly of corn, soy flour, or corn / bean flour mixtures.

The animal feed additive may be used by adding to the animal feed by dipping, spraying or mixing.

The invention is applicable to a number of animal diets, including mammals, poultry, fish and crustaceans. More specifically, the diet is commercially important mammals such as pigs, cows, sheep, goats, laboratory rodents (rats, mice, hamsters and gerbils), furry animals (eg, mink and fox), and Zoo animals (eg monkeys and tailless monkeys), as well as livestock (eg cats and dogs). Commercially important poultry typically include chickens, turkeys, ducks, geese, pheasants and quails. Commercially raised fish and crustaceans such as trout or crayfish may also be included.

In the animal feed blending method including the extract according to the present invention, licorice, cheongung, peanut, buckwheat, auditory and seaweed extracts are incorporated into the animal feed in an amount of about 5 g to 100 g per kg of feed on a dry weight basis.

In addition, the feed mixture is thoroughly mixed and then a viscous granulated or granular material is obtained depending on the degree of grinding of the components. It is fed as a mash or formed into the desired discrete shape for further processing and packaging. At this time, to prevent separation during storage, it is preferable to add water to the animal feed and then go through the usual pelletization, expansion, or extrusion process. Excess water may be removed to dryness.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1. Preparation of Sample (SP-04)

In order to search for natural substances that have an immune-boosting effect on shrimp-infected white spot syndrome virus (WSSV), 7 kinds of cereals (Mungung Agricultural Products Market, Busan) and 5 kinds of vegetables (Mungung Agricultural Products Market, Busan) ), Five herbal medicines (Wongwangdang, Busanjin-gu, Busan), and 10 kinds of seaweeds (collected from Cheongsapo Coast, Busan, and Kim Eun-mung purchased at the Ummung Agricultural Products Market) from these plants and extracted by organic solvents such as alcohol. Six kinds of licorice, Cheongung, Peanut, Buckwheat, Hearing, and Laver, which showed the effect of feeding on the larvae and the resistance to the virus, were selected by adding to the general feed after extracting by the same method.

After drying and slicing the selected six kinds of samples (Liquorice, Cheongung, Peanut, Buckwheat, Hearing, Seaweed), 100g of 100g each was added to 0.15ℓ of ethanol and left at room temperature for 24 hours, followed by 2 hours at 60 ~ 70 ℃ temperature. After refluxing three times, the filtrate was concentrated in a vacuum condenser and freeze-dried to obtain 2.0g, 1.9g, 2.3g, 2.1g, 2.5g, 1.8g of each extract.

Each of the extracts extracted in the same volume (volume) to each of the volume (volume) of 200 ml these were prepared as a standard drug mixture in the mixing ratio as shown in Table 1 to make a feed additive Shrimpower-04 (hereinafter, SP-04). This mixed feed additive (SP-04) was used as a sample of the following experimental example.

Pharmaceutical Range of mixing ratio Licorice 0-8 Celestial palace 0-60 peanut 0-95 Buckwheat 1-95 Hearing 1-10 Kim 0-12 Sum 100 ml

Experimental Example 1. Feeding effect of SP-04 on lobster

In order to grasp the possibility of using shrimp as an additive for general fish feed for SP-04, the mixed feed additive of the present invention, to investigate palatability for SP-04, 5 ml of SP-04 in 100 g of general feed In addition to the addition was administered to the crayfish for 15 days to investigate the daily feeding amount per crayfish. In addition, the addition amount of SP-04 was added in 5 ml, 10 ml, 20 ml and 40 ml, and then administered for 15 days to investigate the feeding amount for each. In addition, only the normal feed was compared to compare as a control.

As a result of the experiment, the feeding amount of the SP-04 added feed was 0.24 ± 0.03 g, which was significantly higher than the feeding amount of 0.13 ± 0.02g of the control group fed only the general feed (p < 0.01) (see FIG. 1).

In addition, when the addition amount of SP-04 was 5ml, 10ml, 20ml and 40ml, as shown in FIG. 2, the feed dosage of 5ml of SP-04 was 0.24 ± 0.02g and 10ml Feed added with 0.26 ± 0.02g showed a significant difference compared to the control without the addition of SP-04. However, in the case of the increase in the amount of SP-04 added to 20ml and 40ml, the feed intake of the crayfish was 0.20 ± 0.02g and 0.15 ± 0.02g, respectively. It could be seen (see Fig. 2).

The experimental group added 40 ml of SP-04 was not significantly different from the control group, but the experimental group added 20 ml showed a significant difference from the control group. As a result, it was found that the proper range of the amount of SP-04 added was 5 ml to 20 ml per 100 g of feed.

Experimental Example 2. Growth effect of SP-04 on the lobster

In order to investigate the growth effect according to the addition amount of feed additive SP-04, the amount of SP-04 was added in 10ml, 20ml and 40ml, respectively, for 20 days at the natural water temperature. After feeding with feed, the change in body length after 20 days was measured. In addition, only the normal feed for the same period as a control group was compared and compared.

As a result, the average body length of the control group fed the non-SP-04 diet was increased to 73.2 ± 2.8 mm after 20 days, and the feed containing 10-mL, 20-mL and 40-mL of SP-04, respectively. In the experimental groups, the average body length of the larvae grew to 82.4 ± 1.8mm, 79.3 ± 3.2mm and 75.1 ± 2.9mm, respectively. Feed grade added with 10ml and 20ml SP-04 The experimental group showed more significant growth effect than the control body length of the control group, but did not show significant growth effect in the experimental group fed 40ml (see Fig. 3). ).

Experimental Example 3 Resistance of White Spot Syndrome Virus (WSSV) to SP-04

In order to investigate the effect of SP-04 on resistance to white spot syndrome virus (WSSV) in the crayfish, a diet containing SP-04 was bred for 10 days. The survival rate was investigated. The experimental group was artificially infected with WSSV and then fed the feed containing 5 ml of SP-04 (SP experimental group) and the feeding group with the addition of 5 ml of SP-04 from 2 weeks before WSSV infection. SP-2 experimental group) and the experimental group (negative control, negative control), which did not artificially infect WSSV and added SP-04, and the general diet without artificially infecting WSSV and added SP-04. Survival was observed by comparing with the experimental group (positive control, positive control).

As a result, in the SP experimental group, the movements of the larvae dropped markedly as in the positive control group on the 2nd day after the WSSV infection, and deaths began to develop. After 3 days, the survival rate was 80%. After 6 days, the survival rate was 40%. Showed. After 7 days, the survival rate was 30%, but after 8 days, all the subjects became active and the movements returned to normal. Thereafter, no individuals died until the end of the breeding experiment. In addition, the SP-2 experimental group showed a much better survival rate than the SP experimental group. Four days after the first infection, the feeding activity was slow, but after 6 days, the feeding activity was normal, and the survival rate was not decreased further. After 10 days, the survival rate was 70%. On the other hand, the negative control group showed a survival rate of 100%, but in the positive control group, the death of the subjects started from the day after infection and showed a mortality rate of 100% after 4 days (see FIG. 4).

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Experimental Example 5. White Real Shrimp with Feed Added with SP-04 Penaeus vannamei Of resistance to WSSV

Four experimental groups were set up by receiving 200 P. vannamei from each of two ton FRP tanks. In the two experimental groups, 5 ml and 10 ml of SP-04 were added to 100 g of feed, respectively, and the feed was fed two weeks before the experiment began, and general feed without SP-04 was not artificially infected with WSSV. The experimental group (negative control group) and the WSSV were artificially infected with the feed group without SP-04 and compared with the experimental group (positive control group). All experimental groups except the negative control group were artificially infected with WSSV, and then 30-day breeding experiment was observed for the change in survival rate.

As a result, the positive control group showed a mortality rate of 100% in 7 days, and the feeding group with 10 ml of SP-04 showed 82% survival rate, similar to the 79% survival rate of the negative control group. In addition, the feeding test group added 5 ml of SP-04 showed a high survival rate of 75% (see Fig. 6).

As described above, animal feed additives containing natural substances, such as licorice, cheonggung, peanut, buckwheat, auditory and seaweed extracts according to the present invention, white spots by increasing the immunity and resistance to white spot syndrome virus against shrimp White Spot Syndrome Disease (WSSD) By reducing the incidence of disease or various diseases, improving the survival rate of individuals and promoting growth, production of cultured shrimp can be increased.

Claims (5)

Licorice ethanol extract 1-8%, Cheongung ethanol extract 10-50%, peanut ethanol extract 17-90%, buckwheat ethanol extract 18-90%, auditory ethanol extract 1-10% and seaweed ethanol extract 1- Animal feed additive containing 12% by weight. delete delete delete The animal feed additive of claim 1 wherein the animal is shrimp or crustaceans.

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