KR20100101742A - Functional feed additives of marine fisheries and feeds for marine fisheries using thereof - Google Patents

Abstract

PURPOSE: A liquid culture feed additive for marine fishes is provided to reduce the mortality rate of the marine fishes by preventing the acidification of marine fish culture feed, to reduce stress to the marine fishes at a low temperature, and to enhance the immunity of the marine fishes. CONSTITUTION: A liquid culture feed additive for marine fishes 0.05-10.00 weight% of Ganoderma lucidu, 0.05-10.00 weight% of shiitake, 0.05-10.00 weight% of oyster mushroom, purified water, and 1.00-20.00 weight% of powdered skim milk based on the total weight of the additive The additive is manufactured by adding pulverized materials of dried shiitake and the oyster mushroom, hydrothermal extract of the dried ganoderma lucidu and polysaccharide of seaweed to a lactic acid fermented liquid based on the powdered skim milk and sterilizing the mixture at 80-100°C. Lactic bacterium strain is inoculated and cultivated in the mixture.

Description

Cultured feed additives for marine fish and marine fisheries feed comprising the same

The present invention relates to aquaculture feed additives for marine fish and marine fish feed comprising the same, and more specifically, to enhance the natural immunity of marine fish by containing natural physiologically active substances such as mushroom lactic acid fermented milk, algae polysaccharides, elvan Prevents anemia, promotes carbohydrate digestion and promotes absorption of intestines, enhances feed efficiency, improves meat quality by increasing natural minerals and vitamins, and improves the water quality of aquaculture tanks. The present invention relates to aquaculture feed additives for marine fish having an effect of reducing mortality by the marine fish, and to a marine fish feed produced by including the same in general aquaculture feed.

Farmed fish, unlike natural fish, are kept at high density and are always exposed to overcrowded diseases. These diseases are mainly caused by pathogenic bacteria, viruses, and parasites, and there are many difficulties in systematic research on fish diseases due to the diversity of aquaculture environment according to various fish and species. In addition, there are many problems such as deterioration of aquaculture environment due to continuous aquaculture and lower weight gain rate due to inadequate farming. Recently, new fishery products have been developed at home and abroad and applied to aquaculture fish. However, most of them rely on antibiotics.

In particular, indiscriminate use of antibiotics and excessive feed administration are causing other problems such as human health threats due to the accumulation of antibiotics in fish, proliferation of antibiotic resistant strains, export disruptions due to the detection of antibiotics in live fish, and water pollution. Given the global trend in the suppression of antibiotics, the development of new alternatives for natural immunity and disease prevention using natural raw materials is essential at this point. In addition, according to domestic statistics, the production of marine fish is much higher at 29,297 tons in sea level compared to 11,678 tons inland production, which raises the necessity of developing high-performance marine fish feed additives that can be applied to marine fish. R & D is needed.

However, most of the domestic researches mainly focus on the origin and cause analysis of fish disease and do not have a clear solution to the fish virus that is causing fatal damage to marine fish farming. It is, but it is incomplete. In particular, the development of feed additives for improving immunity, increasing feed efficiency, promoting growth, and reducing environmental pollution using natural products are still insignificant. According to the National Fisheries Research and Development Institute announced in January 2003, sea fish production increased from 100,000 tons in 1990 to 100,000 tons in 2001 and consumed 370,000 tons of feed annually. have. Of these, about 300,000 tons (80%) are raw feed, and only 20% (70,000 tons) of mixed feed is very low in terms of feed efficiency. 60,000 tons of raw feed is imported from China and covered. As the fish sinks under the sea without fish intake, it causes pollution of coastal fisheries and causes diseases such as pathogen transmission, and it is urgent to switch to blended feeds. The plan was set to reduce the share of feed value, which accounts for 40% of farming costs, to 50% in 2007 and 100% in 2007. In addition, various diseases that occurred only in summer, when water temperature is high, have been occurring all year round, and as the resistance to antibiotics increases, treatment becomes more difficult and the scale of damage increases. In order to solve these problems, the government is trying to establish a standard of raw materials for formulated feeds and to devote a lot of effort to developing feed containing functional substances. Overseas, many developed countries are focusing on the prevention and treatment of fish diseases using natural products in accordance with the regulations on the use of antibiotics. However, most natural products focus on enhancing immune function, and the scope of application is focused on livestock rather than fish.

Considering that Gyeongsangnam-do and Jeollanam-do occupy about 70% of fish farming in Korea, unlike Jeju-do, sea water temperature drops sharply in winter, which slows down the growth of fish and slows down the growth of fish. It is relatively large. Therefore, there is an urgent need to develop suitable feed and feed additives to reduce mortality and promote activity and growth of fish at low temperatures. This is because in the face of serious changes in the global environment, it leads to the survival of aquaculture, which is directly linked to the increase of fisherman's income. In particular, marine fish feeds contain a lot of protein and fat due to the high percentage of raw feeds. Especially in the hot and humid summer season, high degeneration of the feed itself can lead to fish growth and disease, resulting in a sharp decrease in production, which can increase survival rates. Innovative feed conservation measures should be devised.

Accordingly, the present inventors have found that the use of seaweed polysaccharides, natural bioactive substances, ganban stone, mushroom-containing lactic fermented milk, etc., which are completely harmless to the human body, can solve the problems of conventional marine fish farming feed additives. The present invention has been completed.

That is, the purpose of the present invention is to increase natural immunity, decrease mortality due to hemoglobin levels, increase growth and feed efficiency by promoting digestion and intestinal absorption, decrease mortality by improving the water environment of aquaculture tanks, and thereby using antibiotics. It is to provide a variety of types of aquaculture feed additives for marine fish having an effect such as saving.

Another object of the present invention is to provide a marine fish farming feed comprising the marine fish farming feed additive alone by replacing products such as expensive antibiotics, soy sauces, and nutrients that are currently used as additives.

In order to achieve the above object, aquaculture feed additives for marine fish according to the present invention may be provided in two types of liquid type and powder type, of which the liquid type is an additive as a lactic acid fermentation product containing mushrooms and algae polysaccharides It includes 0.05 to 10% by weight of Ganoderma lucidum mushroom, 0.05 to 10% by weight of shiitake mushroom, 0.05 to 10% by weight of oyster mushroom, 0.05 to 10% by weight of seaweed extract polysaccharide (liquid), 1.0 to 20% by weight of skim milk powder, Powder type comprises 10 to 90% by weight of elvan, 0.1 to 10% of vitamin C, and 0.1 to 10% by weight of high glutathione-containing yeast.

 Aquaculture feed additives of the present invention is mainly liquid and can be used to mix powder products as needed. Liquid products have the effect of promoting metabolism, such as increased absorption of nutrients due to intestinal environmental improvement effect, improved meat absorption by increasing essential amino acids, increased absorption of digestion by lactic acid bacteria, improved blood circulation, hepatoprotective effect, and immunity. The product has the functions of increasing liver function, protecting liver from harmful substances, increasing minerals such as iron and calcium in the meat, increasing dissolved oxygen and preventing feed waste. When used in combination, synergistic effects of these effects can be expected.

In addition, the marine fish is not limited to this, but refers to a variety of fish and shellfish, such as flounder, stone bream, rockfish, red sea bream, trout, mullet, shrimp, abalone, can be used in land or cage farms.

Specifically, looking at the ingredients added to the aquaculture feed additive for marine fish of the present invention, as a vegetable component, medicinal herbs, edible mushrooms, seaweeds, as a microbial component lactic acid bacteria, yeast and fermented milk using the same, ganban stone is used as a natural mineral component Characterized in that.

Specifically, 0.05-10.00 wt% of Ganoderma lucidum mushroom, 0.05-10.00 wt% of shiitake mushroom, 0.05-10.00 wt% of shiitake mushroom, 0.05-10.00 wt% of seaweed extract polysaccharide, 1.00-20.00 wt% of skim milk powder and the remaining amount Includes purified water.

Mushroom lactic acid bacteria fermented milk as one component of the aquaculture feed additive for marine fish of the present invention is Korean Patent No. 10-0449886, which the inventors have applied for a patent application (name of the invention: by the production method and the production method of mushroom lactic acid fermentation liquid) Mushroom lactic acid fermented milk), Patent No. 10-0534066 (name of the invention: mushroom lactic acid fermentation liquid containing a component effective for lowering blood sugar and a method for producing the fermentation liquid), as described above, by improving the intestinal environment of lactic acid bacteria It inhibits the growth of harmful bacteria, promotes growth by promoting absorption of minerals in the intestine, prevents anemia due to the strong hematopoietic function of the new functional natural physiologically active substance of mushrooms, and inhibits and treats disease occurrence by hyperimmunity.

At this time, the mushroom can be used for all food and medicinal mushrooms, but in the present invention, using the extract of shiitake mushroom or oyster mushroom and extract of ganoderma lucidum mushroom, mushroom lactic fermented milk is dissolved in fermented milk itself or lyophilized dry product form. It is characterized in that it is added to aquaculture feed additives for fish.

Specifically, looking at the method of producing the mushroom-containing lactic fermented milk, first, the dried shiitake mushrooms or oyster mushrooms are called and then pulverized completely with a blender (crusher) to obtain a mushroom pulverized, and then added water to the dried ganoderma lucidum Hot water extraction gives an extract. Seaweed polysaccharide is pulverized after adding the water to water and obtained an extract by pressing for 1 to 12 hours at 121 ℃.

Then, 0.05 to 10% by weight of the prepared shiitake or oyster mushroom pulverized product, 0.05 to 10.0% by weight of Ganoderma lucidum extract, 0.05 to 10.0% by weight of seaweed extract, crude oil or skim milk powder or the total solids content alone 1-20 weight% is added and mixed.

Then Lactobacillus ash FIG filler's (Lactobacillus acidophilus) the culture solution subjected to the sterilization process to 80~100 ℃, Lactobacillus Thermo filler's (Lactobacillus thermophilus), the mixed state of the lactic acid bacteria Bifidobacterium rogeom (Bifido bacterium loggum) 0.001~ Inoculated at 0.01% by weight, and incubated at 37 ° C. for about 3 to 18 hours to finally prepare mushroom lactic acid bacteria fermented milk.

In addition, according to the present invention, it is possible to provide a powder type feed additive, which is specifically 10.00-90.00 wt% of elvan rock, 0.10-10.00 wt% of vitamin C, and high concentration with a particle size of 100-800 mesh relative to the total weight of the additive. It comprises 0.10 to 10.00% by weight of glutathione-containing yeast.

Meanwhile, quartz prophyry is a silicate mineral belonging to quartz rock, mainly composed of alkali feldspar and quartz, and chemical composition is mainly composed of SiO ₂ and Al ₂ O ₃ , and these minerals are recently used as purifiers of livestock manure. Its value is recognized and it is used as a low pollution formula additive for reducing livestock environmental pollution. In addition, they contain abundant natural minerals of their own, promoting metabolism, and are known as a feed additive because they are known to inhibit the growth of common pathogen bacteria by strong antibacterial action. Therefore, in the present invention, it is characterized in that the elvan rock powder having a particle size of 100 to 800 mesh is added at a level of 10 to 90% by weight. Elvan is a rich mineral source that promotes metabolism, increases palatability of meat, and is added to prevent liver damage by selenium.

Therefore, by using inexpensive carbohydrates instead of expensive high proteins, it is possible to lower the price of aquaculture feed and increase production by promoting growth. In addition, mushroom lactic fermented milk increases the secretion of insulin related to carbohydrate metabolism and increases the cellular utilization rate by promoting intestinal absorption.

Vitamin C (ascorbic acid) added as a component of the aquaculture feed additive for marine fish of the present invention is to increase the immune system, increase appetite, improve stress suppression and adaptation by changing the environment (selection, migration, water change, etc.), and It is added within the range of 0.1 to 10% by weight based on the total weight of the additive to inhibit cytotoxicity and prevent disease by the antioxidant action.

The glutathione-containing yeast added to the marine fish feed additive of the present invention refers to a yeast that is about 2.27 times more glutathione-containing than the general yeast, and the glutathione-containing yeast refers to a paper published by the present inventors in 2004. (Optimal Fermentation Conditions for Enhanced Glutathione Production by Saccharomyces cerevisiae FF-8, The Journal of Microbiology, March 2004, p. 51-55 Vol. 42, No. 1). The glutathione-containing yeast is added within the range of 0.1 to 10% by weight relative to the total weight of the powder additive in order to suppress the accumulation of fish in the liver and reduce mortality caused by liver disease.

Looking at the main effects of the above ingredients in the marine fish culture feed additive of the present invention, the drug, food mushrooms containing the immune enhancing substance, by reducing the mortality by strengthening the anti- potentiation by the natural immunity enhancing action through the use of seaweed and natural by mushrooms and elvan Antibacterial effect can prevent fish disease.

The present invention also relates to aquaculture feed comprising the aquaculture feed additive for marine fish, general marine fish aquaculture feed (for example, blended wet feed (MP; moist pellet) commercially available) ), Blended dry matter (EP; extruded pellet feed, etc.) is added in the range of 0.1 to 5.0% by weight based on the total weight of the feed.

In particular, the aquaculture feed additive mixture for marine fish can be used by coating on aquaculture fish feed, the coating conditions are coated with water and liquid type additives or powder type additives in a weight ratio of 3: 1 and then coated for 3 minutes 1 It is sufficient to repeat the process of stopping for 3 times three times.

As described above, many deaths in fish farming have occurred due to the change of feed which is easy to occur in high-fat, high-protein feeds (including live feeds), but as exemplified in the following examples, the fat is oxidized as a result of the present invention. As a result of measuring the amount of peroxide deteriorating, it was confirmed that the anti-oxidative effect of 5% when stored for 6 hours at room temperature and 16.2% when stored frozen at -20 degrees Celsius for 5 days. In addition, to further prove this, the mortality rate of the marine fish was reduced in winter, and the low-temperature farming experiments were conducted at Whayama Fisheries and Fisheries Research Center in Wakayama Prefecture, Japan. It was possible to increase the economics of fish farmers.

Furthermore, in order to examine the pollutant emission level of phosphorus (P), which is the main cause of environmental pollution in the land and cage farms, the present invention was added to the control compared with the control group. In one sphere, 13% higher body phosphorus (P) was found to be used as energy. Therefore, the feed additive of the present invention and the cultured feed containing the same have been found to reduce marine pollution caused by eutrophication of the feed in the farm.

Aquaculture feed additives for marine fish according to the present invention can be used alone by replacing products such as antibiotics, soy sauces, and nutrients that are expensive, and reduce mortality by inhibiting rancidity of marine fish farming feed (including feed). Not only can it reduce the mortality caused by stress at low water temperature, it can prevent disease by strengthening immunity, promote digestion by intestinal lactic acid bacterium and increase hemoglobin level in blood, increase the rate of feeding and promote growth. As a result, feed efficiency is increased, natural immunity can be enhanced, and hemoglobin is increased to prevent anemia and promote growth.

Preparation of the ingredients

1) Manufacture of liquid mushroom lactic acid bacteria fermented milk (hereinafter referred to as 'BH1000' )

As experimental materials, dried products of Lentinus edodes and Ganoderma lucidum , Pleurotus ostreatus and green seaweed were purchased from the general market.

The dried shiitake mushrooms were added with twice as much water and soaked, and then completely ground using a blender. In addition, a small amount of water was added to the dried ganoderma lucidum, and then heat-treated at 80 to 120 ° C. for about 30 to 240 minutes to obtain an extract. In addition, the oyster mushroom was pulverized completely by the addition of a small amount of water.

In addition, after the addition of three times the weight of the water to the green pulverized with a blender and it was pressurized at 121 ℃ for 3 to 12 hours to obtain an extract was used as a liquid.

Then, 0.05 to 10% by weight of the prepared shiitake or oyster mushroom pulverized, 0.05-10.0% by weight of Ganoderma lucidum extract, 0.05-10.0% by weight of seaweed extract, crude or de-fatted oil, or the total solids alone, respectively. The amount was added 1 to 20% by weight and sterilized at 80 ° C to 100 ° C.

Inject 0.001 ~ 0.01 % by weight of Lactobacillus acidophilus , Lactobacillus thermophilus , and Bifidobacterium loggum , which are widely used as commercial lactic acid bacteria, to sterilized culture medium. Incubated at 37 ℃ for about 3 to 18 hours to prepare a mushroom lactic acid bacteria fermented milk (BH1000).

Thus prepared mushroom lactic acid bacteria fermented milk itself or lyophilized was used as aquaculture feed additive for the marine fish of the liquid type.

2) Preparation of Powder Type Additives

Vitamin C, which is sold for general food use [Product type; Food additive, Manufacturer: China, Company: Shijazhuang pharma. Weisheng pharmaceutical (Shijazhuang) Co. Ltd. Where to buy: Elka Trading Co., Ltd., pulverized ganban stone (product name: Medilite C-325, manufacturer: Lifelong (Korea)), prepared as ganban stone powder having a particle size of 100 to 800 mesh, and containing glutathione This high yeast ( Saccharomyces cerevisiae ) (see Paper Optimal Fermentation Conditions for Enhanced Glutathione Production by Saccharomyces cerevisiae FF-8, The Journal of Microbiology, March 2004, p. 51-55 Vol. 42, No. 1) % glucose, 3% yeast extract, 0.06% potassium dihydrogenphosphate, 0.06% L-cystein) were incubated at 30 ° C. for 72 hours. The yeasts thus prepared were dried, and then the ingredients were uniformly mixed.

Example 1 Preparation of Aquaculture Feed Additives for Marine Fish

The amount of each component added when preparing 1 kg of aquaculture feed mixed with a typical liquid type and powder type seafood fish feed additive according to the present invention is as follows. For reference, powder products are based on elvan, vitamin C and yeast:

Mushroom lactic acid fermented milk liquid (BH 1000) ------------- 5-100 g

Powder product --------------------------- 1 ~ 50 g

Elvan rock ------------------------ 0.5-50 g

Vitamin C (Ascorbic acid) ---------- 0.01-10 g

Yeast ------------------------- 0.01 ~ 10 g

As described above, the powder product was used by uniformly mixing each component.

Example 2: Preparation of aquaculture feed for marine fish

In blending commercially available marine fish feed (EP; dominant feed) 1,000 kg of the liquid type and powder type additive mixtures prepared in Example 1, the compounding content is dissolved by mixing 20 kg of liquid type and 5 kg of powder type. Fish farming feed (hereinafter referred to as 'BHG') was prepared.

Experimental Example 1: Small Scale Marine Fish Basic Culture Experiment

Marine fish basic farming conditions

(1) Marine fish farming facility: A tank for marine fish farming was leased for 3 mx 3 mx 1 m tanks in the Gyeongnam Fisheries Research and Development Institute for rock dome, and 6 mx 6 in the Pacific fishery in Geoje, Gyeongnam for flounder. A water bath of mx 1 m was used.

(2) Experimental fish and aquaculture management: Experimental fish were purchased from 1,000 nurseries of 100 ~ 120g and 2,000 flounders prepared in nursery and pre-cultured with commercially mixed feed (EP; dominant feed) and then experimented with them to adapt to experimental feed. Feed was given for 2 weeks. After the adaptation of the experimental feed was measured the body fasted for about a day, the average weight was equally divided into the control, BH1000 alone, BHG and the experiment was started. Feeding was provided twice a day (9 am, 5 pm) close to full stomach. Airstones were installed in each aquaculture tanks for smooth oxygen supply to the aquaculture tanks, and the water temperature was maintained at 19-24 ℃.

(3) Preparation of sample: Heparinized blood to prevent blood coagulation by weighing and screening in the state of paralyzing on ice in the state of fasting for one day on the morning of the first month of administration of the experimental diet. Heparin-free blood for serum separation was obtained and serum was obtained by allowing the heparin-free blood to stand at room temperature for 30 minutes and then centrifuging at 3,000 rpm for 15 minutes.

Marine fish basic farming result

In order to measure the immune-related biochemical index, the blood was separated and examined for blood and serological clinical biochemical index. Blood was treated with heparin to prevent coagulation, and serum was obtained by allowing heparin-free blood to stand at room temperature for 30 minutes and then centrifuging at 3,000 rpm for 15 minutes. The obtained blood and serum were analyzed in Neodin vetlab, a specialized commissioning institution.

For reference, as can be seen in Figure 1, in the case of stone bream after the BHG diet, vivid streaks appeared clearly, and the liver size was also slightly larger and red and there was no congestion.

Example of rock dome farming

Specifically, the whole amount of EP feed was used as a control for the sea bream during the 30-day diet. In addition, as an experimental example of the present invention, EP feed was used by coating 2 wt% BH1000 and 0.5 wt% powder products, respectively. In the coating, water and each additive were mixed at a weight ratio of 3: 1, and then coated for 3 minutes, followed by a 3-minute process of stopping for 1 minute. Table 1 summarizes the index of growth of sea bream, and blood and serum clinical biochemical index.

Control BHG Body weight (g) 172.44 179.44 Length (cm) 17.89 18.11 Glucose (mg / dl) 35.66 37.16 GOT (U / l) 267.87 225.12 GPT (U / l) 31.87 20.87 amylase (U / l) 18.36 20.63 T. chol (mg / dl) 417 382 Triglyceride (mg / dl) 549 552 cortisol (μg / dl) 2.70 2.40 RBC (x10 ⁶⁾ 3.03 3.12 Hb (g / dl) 12.25 12.33 HCT (%) 45.28 45.36

As shown in Table 1, it was confirmed that the BHG diet stone dome improved body weight 4%, body length 1.2% compared to the normal control. In the blood and serum clinical biochemical index surveys, total Cholesterol was 8.4% lower but neutral lipid was 0.54% higher. GOT and GPT, which are indicators of liver function, were 16% and 34.6% lower than those of the control, respectively, and hemoglobin was 0.65% higher. Cortisol was found to be about 11% lower and erythrocyte count was 3% higher, but hematocrit (HCT), which represents the proportion of red blood cells in a certain volume of blood, was 0.17% higher.

Case of flounder farming

The diet period and conditions of the flounder were the same as the above conditions. The size of the flounder liver was similar in size but the thickness of the BHG diet group was a little thick, compared to the congestion shown in the control group was found to be significantly less in the BHG diet group through the photo of FIG. In addition, Table 2 below summarizes the indexes on the flounder growth.

Control BHG Length (cm) 24.9 25.5 Body weight (g) 178.9 198.8 Soy Weight (g) 2.78 3.26 Relative weight (%) 1.55 1.63 Feed Intake (g / day) 3.85 4.13 Feed efficiency (%) 68.39 79.90 Increase rate (%) 79.0 99.0 Obesity degree (CF) 1.14 1.20

※ Relative liver weight (%) = liver weight / weight x 100, feed efficiency (%) = (weight gain / feed intake) x 100

※ obesity degree = [weight / length (cm) ³ ] x100, N = 20, dietary 35 days, feed additive: 0.5%

As shown in Table 2, the BHG diet group was found to increase overall, such as 2.4% body weight, 1.1% body weight, feed intake 7.2%, feed efficiency 16.8%, weight gain 25.3%, obesity 5.2% compared to the control group.

Table 3 summarizes the results of the blood and serum clinical biochemical index survey of the flounder.

Control BHG Protein (g / dl) 5.54 6.25 Albumin (g / dl) 1.54 1.78 Triglyceride (mg / dl) 569 622 T. Chol. (mg / dl) 453 392 GOT (U / l) 23.8 18.1 GPT (U / l) 13.3 12.1 T. Bilirubin (mg / dl) 0.39 0.58 D. Bilirubin (mg / dl) 0.24 0.34 RBC (x10 ⁶⁾ 3.01 3.72 Hemoglobin (g / dl) 6.91 7.88 WBC (x10 ³⁾ 19.5 16.9 Platelet (x10 ³⁾ 262.7 250.6 Lymphocyte (%) 83.6 91.4 Monocyte (%) 11.0 10.5

As shown in Table 3, the total Cholesterol was 13.5% lower, and the liver function indicators GOT and GPT were 34% and 8.9% lower than the control, respectively. Erythrocyte count and hemoglobin were 23.5% and 14% higher, respectively. However, leukocytes and platelets decreased by 13.4% and 4.7%, respectively. Lymphocytes were up 9.3%, but monocytes were down 4.6%.

Test Example 2: Bulk Form Experiment

Seafood farming conditions

(1) Large-scale aquaculture experiment fish species: flounder is 12 mx 12 mx 0.8 m land tank of Pacific fishery of Geoje, Gyeongnam, and Dome is 10 mx 10 mx 6 m cage of longevity fish in Gyeongnam Seopo. The density was determined and experimented for 3 months to obtain the result.

(2) Experimental fish and aquaculture management: The flounder was about 120g and the sea bream was about 50g and was pre-cultured with commercially mixed feed and then fed for 2 weeks as experimental feed to adapt to the experimental feed. After the adaptation of the experimental feed was divided into a control group, BH1000 + powder product (BHG) addition group, and experimented for 3 months. Feeding was provided twice a day (9 am, 5 pm) close to full stomach. For the smooth supply of oxygen to the aquaculture tanks, the flounder was installed with an air stone in the aquaculture tank, and the water temperature was maintained at 19 ~ 24 ℃. The stone dome was installed with an air pump under the cage and the water temperature was specified as the natural water temperature of June ~ August. Managed.

(3) Sample preparation: Heparinized blood to prevent blood coagulation by weighing and screening in the state of paralyzing on ice in the state of fasting for one day in the morning of the third month after the administration of the experiment. Heparin-free blood for serum separation was obtained and serum was obtained by allowing the heparin-free blood to stand at room temperature for 30 minutes and then centrifuging at 3,000 rpm for 15 minutes.

Large Sea Bream Case

The feeding period of rock bream was 3 months, and total EP diet was used. EP feed was used to coat the BH1000 2% and 0.5% powder product of the present invention. As a coating method, the mixture was mixed with water 3: 1 and coated for 3 minutes, and then prepared for 1 minute rest 3 times. Growth index of rock dome is summarized in Table 4 below.

Control BHG Length (cm) 22.2 24.7 Body weight (g) 198 211 Soy Weight (g) 2.96 3.22 Relative weight (%) 1.49 1.52

※ Relative liver weight (%) = liver weight / weight x100

As shown in Table 4, the BHG diet group of the present invention was confirmed that the body weight is 6.56%, 1.26% improved compared to the control group.

And the blood and serum clinical biochemical index survey results of sea bream are summarized in Table 5 below.

Control BHG Glucose (mg / dl) 335.3 242 GOT (U / l) 67.5 63.3 GPT (U / l) 7.2 7.0 amylase (U / l) 10.5 11.95 T. chol (mg / dl) 234.5 215 Triglyceride (mg / dl) 1229.4 1076.6 cortisol (μg / dl) 13.71 13.64

As shown in Table 5, in the BHG diet group of the present invention, the neutral lipid was 12.5% and the total cholesterol was 8.4% lower than the control group, and the liver function indicators GOT and GPT were 6.3% and 2.8% lower than the control, respectively. It appears that liver function has improved. Glucose was 28% lower in blood and Amylase was 13.8% higher. Cortisol was 0.6% lower.

Case of flounder large-scale farming

The feeding period and conditions of the flounder were the same as those of the flounder above, and the growth index of the flounder observed was summarized in Table 6 below.

Control BHG Length (cm) 29.2 30.2 Body weight (g) 300 331 Soy Weight (g) 4.84 5.26 Relative weight (%) 1.61 1.59 Obesity degree (CF) 1.20 1.19

※ Relative liver weight (%) = liver weight / weight x100, obesity degree = [weight / length (cm) ³ ] x100, N = 20

As shown in Table 6, the BHG diet group flounder of the present invention was confirmed to improve by 10.3% body weight, 3.4% body length compared to the control group. Furthermore, the BHG diet group was thicker than the control group, so obesity was slightly lower than the control group, but the actual meat quality was excellent.

In addition, the blood and serum clinical biochemical index investigation results of the flounder are summarized in Table 7 below.

Control BHG Protein (g / dl) 5.90 6.07 Albumin (g / dl) 2.39 2.56 Triglyceride (mg / dl) 514.1 488.6 T. Chol. (mg / dl) 539 518.6 GOT (U / l) 43.8 36 GPT (U / l) 6.4 5.5 T. Bilirubin (mg / dl) 0.84 0.77 Glucose (mg / dl) 100 68.4 Insuline (μg / ml) 1.58 1.84 Amylase (U / l) 1.00 1.37

As shown in Table 7, the protein content was 2.88%, albumin was 7.1% higher than the control group, neutral lipid was 5%, total cholesterol was 3.8% lower, liver function indicators GOT, GPT was found in the control Compared to 18% and 14% lower than liver function can be seen that. In addition, Glucose in blood was 31.6% lower, Insulin was 16.5% higher, and Amylase 37% higher.

Test Example 3 Inhibitory Effect of Irido Virus

For reference, iridovirus is known as a causative agent of iridovirus, a DNA virus, and the main onset time is around 25 ° C. Iridovirus infection causes the fish to swim and die, and the appearance is black and sometimes ocular hyperemia occurs. Preventive measures include prohibiting the stocking of seedlings in the affected area, increasing the anti-medical history by administering an immune enhancer before the high temperature, and preventing stress during the high temperature.

Therefore, this test example examined the inhibitory effect of the iridovirus, a pathogenic microorganism.

Specifically, about 100 g of rock dome was selected, BHG of the present invention was fed for 1 month, the spleen was extracted, ground using a pastle, and extracted with a supernatant, and the degree of infection of iridovirus was measured by PCR.

In this case, 5'-GTGACTGCACACCAATGGAC-3 '(forward) and 5'-GGCTTTCTCAATCAGCTTGC-3' (reverse) were used as PCR primers. The size of the PCR product of iridovirus is 698 bp. PCR amplification was performed in 0.2 ml PCR tubes with 20 pmol of each primer, 2.5 μl of 10X PCR buffer, 2.5 μl of 25 mM MgCl _2, 2.5 μl of dNTP, 1 μl forward primer, 1 μl reverse primer, 0.5 μl Taq polymerase (5 U / μl, Takara, Tokyo, Japan) and DNA or tissue template were mixed and finally sterile water was added to make 25 μl.

The PCR reaction conditions were first denatured at 95 ° C. for 1 minute, followed by 30 cycles of 94 ° C. 30 sec, 58 ° C. 45 sec, and 72 ° C. 45 sec. Final elongation was performed at 72 degreeC for 6 minutes. The amplified PCR product was confirmed using 1.5% agarose gel. As can be seen in Figures 3 and 4, in the initial stage of the sea bream all 10 were infected with iridovirus, but BHG upset 50% disappeared after one month.

In addition, as can be seen in Figures 5 and 6, in the case of the flounder all 10 were confirmed to be normal. In general, olive flounder is known to have low mortality and low infection rate. The results also showed that the infection rate against iridovirus was quite low.

Test Example 4: Prevention of rancidity of commercial feed (raw feed)

For reference, the commercially available marine fish feed contains a lot of protein and fat due to the high rate of raw feed. Especially, in the hot and humid summer season, high degeneration of feed itself leads to fish growth disorders and disease, resulting in a sharp decrease in production. As a result, groundbreaking feed preservation methods should be devised to increase survival rates.

 In addition, long-term administration of rancidity, such as imported megali (Netherlands) or poorly measured feed due to a lack of domestic feed, may cause damage to hematopoietic organs (kidney, liver, spleen tissue) of cultured fish, Reducing the resistance is causing various diseases. When fat is oxidized and decayed, the amount of fat decay can be determined by measuring the amount of peroxide value, which is expressed in mg per 1 kg of feed by adding potassium iodine to the fat contained in the feed.

In this test example, BHG according to the present invention was administered to raw feeds used in general aquaculture fish and their storage time (1, 3, 6 hours, 28 ℃), by date (1, 3, 5 days, -20) Peroxide values were measured.

   Peroxide value was measured by Entec Analysis Institute, a registered agency of KFDA. As a specific experimental method, put an appropriate amount of the sample in a 1000 ml Erlenmeyer flask and pour enough to immerse the ether. The oil was extracted by standing for 2 hours. Filter with NO.4 filter paper. The filtrate was transferred to a separatory funnel and washed three times with water. Only the ether layer was taken and dehydrated with anhydrous sodium sulfate. Concentrate on water bath at 40 ℃, measure fat weight and precisely weigh 1 ~ 5 g of sample (lipid) and place in a stoppered flask. It was dissolved in 25 ml of acetic acid: chloroform (3: 2), 1 ml of saturated potassium iodide solution was mixed and left in the dark for 10 minutes. 30 ml of distilled water was added and shaken vigorously, and 1 ml of starch indicator was added and titrated with 0.01 N sodium thiosulfate. This developed product was mixed with feed and examined the effect of rancidity at room temperature after feed molding.

As a result, as shown in Figure 7, up to 1 hour feed molding showed a significant rancidence inhibitory effect of 48%, over time showed a rancidence inhibitory effect of 5 ~ 10%.

In addition, the peroxide value was measured by freezing storage at -20 ℃ immediately after the feed molding, and as a result, the peroxide price did not increase as stored at room temperature. ). The equation used is:

과산화물가 = x

Peroxide value = x

Where a is the 0.01 N sodium thiosulfate consumption (ml), b is the 0.01 N sodium thiosulfate consumption (ml) in the blank test, f is the 0.01 N sodium thiosulfate titer, and s is the sample amount (g) .)

Experimental Example 5. Effect of reducing fish mortality in winter

For reference, marine cultured fish have seasonal characteristics in which the sea water temperature drops sharply in winter, which slows growth and causes more than 70% of death due to a decline in fish activity. Therefore, it is urgently needed to reduce the mortality rate of fish at low temperature, and to develop appropriate feed and feed additives that can promote activity and growth, which directly leads to the survival of aquaculture in the face of serious changes in the global environment. Because it becomes. Therefore, we used BHG, a developed feed additive, to measure the average fish weight, feed, growth factor, blood and serological clinical biochemistry index.

(1) Average fish stocks, feed, and weighting factors

The fish used in this experiment were 183 sea bream with an average body weight of about 205 g in a 3 x 3 x 3 m cage. The breeding period was 3 months and the temperature was 12 ~ 20.3 ℃. Sup was given only three days a week because of the low water temperature. Feed was mixed with water for general red snapper feed [MP raw material (salt raw fish), feed oil] and then used in pellet form in granulator. The experimental group was added with BH1000 2% and powder product 0.5% (BHG). The test site was conducted at the Fisheries Research and Development Center, Agricultural Research and Fisheries General Technology Center, Sangsan, Japan. The results of observing the growth pattern of the sea bream are summarized in Table 8 below.

attempt Average fish weight (g) Feed amount (g) Growth Factor start end start end Control 183 175 205.3 261.0 38,798 3.25 BHG 183 179 206.5 268.7 40,899 2.73

※ growth factor; Amount of food needed to increase fish weight by 1 kg

As shown in Table 8, the survival rate of the control group was 95.6%, the experimental group was 97.8%, and the average fish body weight was increased by 3% (130.12%) in the experimental group compared to the control group (127.13%). The feed rate was 2,101 g in the experimental group and the increase in coefficient was 2.73 in the experimental group.

The results of wintering experiments were observed from Tongyeong, Gyeongnam until mid-December, and the results of blood and serum clinical biochemical indexes of sea bream are summarized in Table 9 below.

Control BHG HCT (%) 27.31 29.06 Hb (mg / dl) 4.27 4.51 RBC (10 ⁴ / mm ³ ) 270.8 276.7 TP (g / dl) 2.62 3.3 GLU (mg / dl) 52.6 66.3 TCHO (mg / dl) 299.8 269.4 TG (mg / dl) 222.42 211.71 AMYL (U / l) 50.28 51.14 ALP (mg / dl) 115 110.75 BUN (mg / dl) 6.86 5.67 GOT (U / l) 27.83 26.5 GPT (U / l) 5.0 4.83 Ca (mg / dl) 12.92 13.02 Mg (mg / dl) 2.18 2.13 P (mg / dl) 14.87 15.0

As shown in Table 9, hematocrit (HCT) was 6.4% higher, Hemoglobin was 5.62% higher. Erythrocyte count was 2.1% and total protein mass was 26%. Glucose content increased 26% and Amylase was 1.7% higher, indicating that carbohydrate utilization was significantly increased, similar to the flounder results. Neutral lipid was 4.8% and total cholesterol was 10.2% lower, and liver function indexes GOT and GPT were 4.8% and 3.4% lower than control, respectively. Inorganic salts increased by 0.8% for calcium and 0.87% for phosphorus, but 0.3% for magnesium.

Test Example 6: Reduction of eutrophication by aquaculture feed

The degree of reduction of eutrophication by commercially produced aquaculture feed by the feed additive of the present invention was measured in fish bodies and feeds and summarized in Table 10 below.

*. Breeding conditions: 183 sea bream fish with an average fish weight of 205g are placed in a marine cage of 3 × 3 × 3m and the test is started.

*. Breeding period and place: December 3, 2004 ~ March 22, 2005

   Fisheries Research and Development Center, Wakayama Prefecture Agricultural Technology Center

*. Water temperature during breeding period: 12 ~ 20.3 ℃

*. Days of feeding: 48 days (3 days a week due to low water temperature)

Body at start (%) % At end of body feed(%) Control BHG moisture 73.7 74.9 73.2 30.3 Crude protein 16.5 15.3 15.7 36.1 Crude fat 5.1 4.9 5.7 18.9 Ash 4.6 4.8 5.3 8.9 sign 0.87 0.86 0.96 1.3

As shown in Table 10, in order to examine the pollutant emissions of phosphorus (P), a major source of environmental pollution in land and cage farms, a comparison of the content of phosphorus (P) in the feed used and released in fish bodies Compared with the present invention, it was confirmed that 13% higher phosphorus (P) was accumulated and used as energy in the sphere containing the present invention. Therefore, the feed additive of the present invention and the cultured feed containing the same have been found to reduce marine pollution caused by eutrophication of the feed in the farm.

Test Example 7: Antibiotic replacement effect and mortality reduction effect

This test example is a result of observing the mortality of the olive flounder in order to examine the antibiotic replacement effect of the additive according to the present invention, Table 11 is the result of the BHG addition sphere according to the present invention, Table 12 summarizes the results of the control group will be.

*. Breeding condition: 130,000 and 160,000 halibuts of the flounder around 230 ~ 250g of average fish weight are put into the land farm and the test is started.

*. Breeding period and place: June 2007 ~ August 2007 Ilosam Fisheries (present, Hanseong Fisheries), Jangheung-gun, Jeonnam

*. Water temperature during breeding period: 18 ~ 28 ℃

*. Days of Feeding: 93 days

BHG
Addition Total feed
Feed amount (Kg) Dead water Mortality
(%) Average weight
(g) A heavy dose
(Kg) Total breeding quantity
(Kg) Total attempt
(beauty) Antibiotic
Usage (times) May 07 1,500 1.2 250 4,940 30,875 123,500 One June 07 35,000 1,800 1.5 300 6,100 36,600 122,000 0 Jul 07 40,000 2,400 2.0 360 7,176 43,200 119,870 0 Aug 07 45,000 2,500 2.1 450 10,539 52,700 119,500 0

Control Total feed
Feed amount (Kg) Dead water Mortality
(%) Average weight
(g) A heavy dose
(Kg) Total breeding quantity
(Kg Total attempt
(beauty) Antibiotic
Usage (times) May 07 2,400 1.4 230 4,100 39,543 162,920 One June 07 23,000 5,008 3.0 250 4,400 41,730 157,800 One Jul 07 26,000 6,420 4.0 280 5,000 44,940 151,300 One Aug 07 30,000 10,500 7.0 320 6,000 48,000 140,200 One

As shown in Tables 11 and 12, in the case of Table 11, mortality was reduced by about 10% or more compared to Table 12, and antibiotics were found to be sufficient even if they were not used monthly.

Test Example 8: Low water temperature culture experiment in flounder culture

This test example summarizes the results of the low water temperature experiment of the additive according to the present invention, Table 13 below is the result of the BHG addition sphere according to the present invention, Table 14 below summarizes the results of the control group.

*. Breeding condition: Start the test by putting 120,000 pieces of flounder around 30g of average fish weight into aquaculture farm

*. Breeding period and place: November 2007 ~ February 2008 Hawon Fisheries, Wando-gun, Jeonnam

*. Water temperature during breeding period: 7 ~ 12 ℃

*. Days of Feeding: 115 days

BHG
Addition Total feed
Feed amount (Kg) Our company
attempt Mortality
(%) Temperature Average weight
(g) A heavy dose
(Kg) Total breeding quantity
(Kg) Total attempt
(beauty) Antibiotic
Usage (times) November 07 2,500 930 0.775 12 30 3,600 120,000 0 Dec 07 2,300 560 0.470 10 52 2,125 5,725 119,070 0 Jan 08 1,600 820 0.692 8 65 1,909 7,634 118,510 0 Feb 08 1,600 940 0.799 7 76 1,280 8,914 117,690 0

Control Total feed
Feed amount (Kg) Our company
attempt Mortality
(%) Temperature Average weight
(g) A heavy dose
(Kg) Total breeding quantity
(Kg) Total attempt
(beauty) Antibiotic
Usage (times) November 07 2,400 1,280 1.067 12 30 3,600 120,000 0 Dec 07 1,600 1,820 1.517 10 45 1,742 5,342 118,720 One Jan 08 1,300 2,500 2.083 8 58 1,278 6,620 114,400 0 Feb 08 1,200 2,880 2.400 7 66 1,040 7,660 111,520 0

As shown in Table 13 and 14, in the case of Table 13 as a result of the present invention, mortality was reduced by about 5% or more compared to Table 14 and also in the BHG addition group (Table 13) it can be confirmed that no antibiotics were treated separately because there is no disease there was. Not only that, but the seawater temperature up to 10 ° C is more than 2 ° C (for example, the same feed rate as 10 ° C to 12 ° C is possible). As a result, the overall farming period was increased, which shortened the shipment time, thereby confirming economic aspects.

As described above, according to the present invention, carbohydrates instead of the high fat high protein components of the feed additives to improve the fact that a lot of deaths in fish farming caused by the change of the feed that is likely to occur in the existing high fat high protein feed (including raw feed) In addition, it was possible to improve the economic efficiency of farmed fish prices by improving the anti- rancidity effect, and improving the intestinal environment of fish and increasing the absorption rate such as reducing the mortality rate of marine fish in winter.

In addition, as a result of comparing the pollutant emission content of phosphorus (P) which is the main cause of environmental pollution in land and cage farms, in the present invention, by accumulating phosphorus (P) in the body by about 13% higher, It has proved to be an efficient product that can reduce marine pollution caused by nutrition.

The high-performance marine fish farming feed additive according to the present invention is developed in the form of fermented milk using natural materials as raw materials, so it has excellent natural immunity, improves liver function, and improves digestive absorption rate, but there is no worry about side effects and the rancidity of feed. It has the function to reduce not only the disease caused by denatured feed but also the mortality caused by stress at low water temperature, which is effective in improving the water environment and promoting growth of aquaculture tanks, thus reducing the economic loss of producers due to mass mortality. In addition, it is possible to contribute to the production of economic profits of the producers by improving the resistance to fish disease by enhancing the natural immunity and improving the productivity by promoting growth.

1 is a photograph for contrasting the appearance and liver form when A: feed + BHG, B: feed respectively for the sea bream.

Figure 2 is a photo of the flounder for A: feed + BHG, B: feed when each diet.

Figure 3 is a PCR electrophoresis picture of sea bream spleen tissue, M is the marker; C1 represents tissues infected with iridovirus; C2 is iridovirus DNA and 1-10 is a sample number of randomly selected rockfish spleen tissue.

Figure 4 is a PCR electrophoresis picture of rockfish spleen tissue fed a BHG diet for 1 month, M is the marker; C1 represents tissues infected with iridovirus; C2 is iridovirus DNA and 1-10 is a sample number of randomly selected rockfish spleen tissue.

Figure 5 is a PCR electrophoresis picture of the olive flounder spleen tissue fed a BHG diet for 1 month, M is the marker; C1 represents tissues infected with iridovirus; C2 is iridovirus DNA and 1-10 is a sample number of randomly selected flounder spleen tissue.

Figure 6 is a PCR electrophoresis picture of the olive flounder spleen tissue fed a BHG diet for 1 month, M is the marker; C1 represents tissues infected with iridovirus; C2 is iridovirus DNA and 1-10 is a sample number of randomly selected flounder spleen tissue.

7 is a graph over time of the peroxide value of the control and BHG control group the raw feed; BHG means feed + BH1000 + Gluta-MC.

8 is a graph of the control group and the peroxide value of BHG over time as the control group the raw feed; BHG means feed + BH1000 + Gluta-MC.

Claims (11)

It comprises 0.05 to 10.00% by weight of Ganoderma lucidum mushroom, 0.05 to 10.00% by weight of shiitake mushroom, 0.05 to 10.00% by weight of oyster mushroom, 0.05 to 10.00% by weight of seaweed extract polysaccharide and the remaining amount of purified water, The skimmed milk powder is cultured feed additives for marine fish of the liquid type, characterized in that used within the range of 1.00 to 20.00% by weight relative to the total weight of the additive. The method of claim 1, wherein the additive is added to the skimmed milk powder-based lactic acid bacteria fermentation broth, dried shiitake mushrooms and oyster mushrooms, hot water extracts of dried Ganoderma lucidum mushrooms, and seaweed extract polysaccharides, and then sterilized at 80 to 100 ° C. Cultured feed additives for marine fish, characterized in that the inoculated and cultured lactic acid bacteria. The aquatic feed additive for marine fish according to claim 2, wherein the seaweed extract polysaccharide is a polysaccharide extracted from an extract obtained by crushing after adding water to the shell and pressing for 1 to 12 hours at 121 ° C. According to claim 2, wherein the lactic acid bacteria Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus thermophilus ( Lactobacillus thermophilus ), Bifidobacterium loggum for a marine fish characterized in that the mixed strain of Aquaculture feed additives. The cultured feed additive for marine fish according to claim 2, wherein the lactic acid bacteria fermentation broth is in the form of fermented milk itself or lyophilized dried product. The aquaculture feed additive for marine fish according to claim 2, wherein the inoculation and culture conditions are incubated for 3 to 8 hours at 37 ° C. An additive for powdered marine fish comprising 10.00 to 90.00% by weight of ganbanite having a particle size of 100 to 800 mesh, 0.10 to 10.00% by weight of vitamin C, and 0.10 to 10.00% by weight of high concentration glutathione-containing yeast. 8. The aquaculture feed additive for marine fish according to claim 1 or 7, wherein the marine fish is selected from flounder, rock bream, mullet, shrimp, abalone and the like. The marine fish cultured feed having a synergistic effect on the improvement of liver function and immunity by mixing the aquaculture feed additive for marine fish of the liquid type of claim 1 with the cultured feed additive for marine fish of the powder type of claim 7. 10. The method according to claim 9, wherein the liquid feed additives for marine fish cultured and the powdered feed additives for marine fish of the powder type are each mixed in the range of 0.1 to 10% by weight relative to the total weight of the cultured feed. Marine fish feed. The method of claim 10, wherein the fish feed additive mixture for marine fish is coated on the marine fish feed, At this time, the coating is mixed with water and liquid-type additives or powder-type additives at a weight ratio of 3: 1, and the marine fish farming feed, characterized in that it is carried out three times the process of three minutes of coating and resting for 1 minute.

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