BACKGROUND OF ART The livestock farming and the fish raising system have been growing in size and crowded in density to the extent that its environment has been getting worse. Increase of stress and decrease of immunity cause the breakout of infectious diseases and therefore, the mass death is inevitable. Up to now, various chemicals such antibiotics and drugs have been used to animals for prevention and treatment of the diseases.

But instead of conventional chemicals, new, natural and effective feed has been required for preventing diseases and robust farming/raising, which by enhancing self- immunity, decreasing the stress by environment and increasing the tolerance for bacteria and virus.

Edible mushroom contains 5-30% of organic compound and minerals and 70- 95% of water. Dried mushroom contains 15-30 % of protein, 2-10% of fat, approximately 50% of soluble minerals, 5-10% of fiber, calcium, phosphoric acid, ash content and so on. Generally good-taste mushroom contains mainly amino acid, manit, trehalose and some enzyme, various vitamin such as vitamin B2 and ergosterin, vitamin D precursor.

Recently, a great many of studies on mushroom are on-going and many pharmaceutical compositions and health care foods comprising mushroom such as Lentinus edodes, Ganoderma lucidum, Agaricus blazei and so on are manufactured and

commercially available. But the feed comprising mushroom is not yet.

WO 02080698 discloses that the method for producing specialty plant bio-feed from the animal MBMs using edible mushroom strains, and the specialty bio-feed produced thereby.

DISCLOSURE OF THE INVENTION Accordingly, it is an object of the present invention to provide a feed and feed additive preparation method characterized by comprising the steps of: (step 1) cultivating and pollinating of mushroom spawn; (step 2) manufacturing medium comprising various grains and other substances; (step 3) inoculating mushroom spawn on the above medium; (step 4) cultivating mushroom mycelium; and (step 5) manufacturing and processing the inventive feed and feed additive by means of lyophilization or hot wind dry of mycelium grown-medium.

It is another object of the present invention to provide a feed and a feed additive for the livestock, the freshwater fish, the seawater fish, the lobster and the like comprising mushroom mycelium as an active ingredient in which, when it is given to the raised fish, the diseases thereof can be suppressed and the growth rate and the raising rate can be increased.

It is another object of the present invention to provide a use for feed and a feed additive comprising mushroom mycelium as an active ingredient to enhance immunity and to prevent from disease.

In accordance with one aspect of the present invention, there are provided a feed and feed additive preparation method, wherein above step 1 is mushroom spawn cultivation and pollination step, which can be executed according to conventional mushroom culture method.

In accordance with another aspect of the present invention, there are provided a

feed and feed additive preparation method, wherein said mushroom can be at least one selected from the group consisting of Lentinus edodes, Hericium erinaceum, Agaricus blazei, Schizophyllum commune, Auricularia auricula judae, Phellinus linteus, Ganoderma lucidum, Inonotus Obliquus, Flammulina vclutipes, Pleurotus ostreatus or Cordyceps sinensis, preferably Hericium erinaceum, and can be selected to inoculate onto medium according to its typical efficacy.

Hericium erinaceum employed in the present invention is belonging to Hericiaceae. P-D-glucan is abundant therein, which is known to have anti-cancer effect, anti-inflammatory/anti-microbial activity, digestion promoting activity, enhancing activity of stomach mucosa protection, ulcer recovery, immunity enhancing activity and so on.

Lentinus edodes belonging to Pleurotaceae is commonly edible mushroom.

Schizophyllum commune belonging to Schizophyllaceae has a good flavor and taste. P-D-glucan is abundant therein, which is known to have anti-cancer effect, activation of immunity activating activity and be good for stamina.

Auricularia auriculajudae belonging to auriculariaceae is commonly edible mushroom having an anti-cancer/anti-inflammatory effect and cholesterol-decreasing effect.

Ganoderma lucidum belonging to Ganodermatoaceae has been widely used as medicine. It is well-known to have anti-cancer effect, anti-inflammatory activity, blood pressure controlling activity, blood glucose level decreasing effect, immunity enhancing effect, aging inhibiting activity and so on.

Pleurotus ostreatus belonging to Hymenochaetaceae has been also widely used as medicine. It is known to have anti-cancer effect, immunity enhancing effect, detoxification, treating effect of non-insulin dependent diabetes melitus and so on.

Cordyceps sinensis belonging to Cordyceps of Clavicipitales is parasitic on insect. It is well-known to have anti-cancer effect, anti-inflammatory activity, blood

pressure control, blood glucose level decreasing effect, immunity enhancing effect, inhibition of senile change and so on.

In aforementioned medium, suitable starch-bearing substances are those commonly used as feed components and are generally derived from grains selected from the group consisting of corn, soybean, wheat, sorghum, barley, oat, rye, potato, tapioca, sugarcane molasses and the mixture thereof. Examples of suitable starch-bearing substances include, but are not limited to, corn flour, ground corn, soybean flour, wheat flour, ground oat flour, wheat middling, soybean meal, corn grit, and the mixture thereof.

A variety of protein-bearing substances can be used as a component of present invention as long as the protein-bearing substance can support the animal growth. The protein content of protein-bearing substances can be varied over the range of from about 10-w/w% to about 90%. For economical aspects, a crude protein-bearing substances such as corn gluten meal, a soybean meal, rape seed meal, coconut meal, cotton seed meal, a fish meal, blood meal, pupa of insect and the mixture thereof are preferable.

Other suitable protein-bearing substances include, but are not limited to, soybean protein concentrate, dried whey, soy flour, plasma protein, dried skim milk, whey protein concentrate, canola meal, wheat gluten meal, yeast, sunflower meal, and the mixture thereof.

Any fat-containing substance that can support the animal growing can be used.

Suitable fat-containing substances include, but are not limited to, animal fat such as lard and tallow and the like, soybean oil, lecithin, coconut oil, whey-fat blend, and the mixture thereof.

. And other substances such as wheat bran, rice bran, barley bran, beetpulp, cotton pellet and the mixture thereof can be used to make inventive feed and feed additive.

The water-and fat-soluble vitamins and trace minerals can also be comprised in present invention. Suitable vitamins include vitamin A, vitamin D, vitamin E, vitamin K, riboflavin, pantothenic acid, niacin, vitamin B, 2, folic acid, biotin, vitamin C, and the

mixture thereof. Suitable trace elements include copper, zinc, iodine, selenium, manganese, iron, cobalt, compounds thereof, or the mixture thereof. The term"trace" used herein denotes the quantity of these components used in present composition that is substantially quietly smaller than that of other components. Generally the vitamins- trace minerals are present in the composition in the range of from about 0.0001 to about 5% based on total weight % of the composition.

The feed and feed additive of the present invention can be prepared by any mixing means known to one skilled in the art such as, for example, mechanical blending, extrusion, pelletizing, and spray drying. The order of adding individual components for mixing generally does not alter the physical characteristics or feeding efficiency of the composition. However, it is preferred that the medium be mixed with a carrier such as, for example, rice bran, grain, wheat bran or mixtures thereof to facilitate even mixing and distribution. Any physical form of the composition such as, for example, powder, pellet, cube, semi-solid, and combinations thereof can be used.

In accordance with still another aspect of the present invention, there are provided a feed and feed additive preparation method, wherein medium composition can be, preferably, comprised with at least one component selected from the group consisting of corn, soybean, wheat, rye, oat, potato, barley, tapioca, sugarcane molasses, bakery by-product, brewing by-product, sesame by-product, bean-curd by-product, soy and sesame oil by-product, rapeseed meal, cone gluten meal, soybean meal, fish meal, blood meal, pupa of insect, coconut meal, cotton seed meal, animal fat, wheat bran, rice bran, barley bran, beer draff, beetpulp, cotton pellet and mixtures thereof. These components are commercially available.

The ratio of the components is not so important and it is changeable as the need arises. Preferably, the ratio is generally ranging from about 0 to 30 w/w % in present composition.

In accordance with another aspect of the present invention, there are provided

the inventive feed preparation method, wherein preferred medium can be made by mixing 10-30 w/w % of corn, 10-30 w/w % of soybean meal, 5-20 w/w % of potato, 5- 20 w/w % of Sorghum, 5-30 w/w % of cotton pellet, 5-20 w/w % of fish meal, 5-30 w/w % of barley and 1-10 w/w % of additives such as vitamins.

All weight % disclosed herein based on the total weight % of the composition equaling 100%.

In accordance with another aspect of the present invention, there are provided inventive preparation method, wherein step 2 is medium preparation step, in which medium is put into 500-2000cc container for mushroom cultivation, sterilized at the temperature ranging from 50 to 121°C for the period ranging from 30 min to 5 hours and let cool down; step 3 is spawn inoculation step, in which mushroom spawn is inoculated in amount of about 5 to 30g/container onto autoclaved medium in clean room; step 4 is mushroom cultivation step, in which above inoculated medium is incubated in 50-80% of humidity room at the temperature ranging from about 20 to 25°C for the period ranging from 5 to 90 days; step 5 is pulverization, mixing and formation of feed and feed additive, in which medium and mycelium are separated, vacuum lyophilization at-70°C to-120°C or hot wind dry above 60°C followed by pulverization, mixing and formation.

Dried mushroom mycelium is comprised in the inventive feed ranging about 1 to 50 w/w% based on total weight %.

Inventive feed comprising mushroom mycelium according to the inventive preparation method contains many kinds of protein, carbohydrates, mineral, vitamin and polysaccharides of mushroom and also contains P-glucan, which has immunity enhancing-effect, to prevent disease and improve flesh and nutrients thereby.

When inventive feed was treated to vibrio infected-flatfish, it caused to the increase of growth rate and immunity against vibrio, therefore, showed 100% viability.

It is very effective to prevent fishes from being affected by diseases and to raise the

robust fishes.

In accordance with another aspect of the present invention, there are provided a feed manufactured by above procedure for the livestock, the freshwater fish, the seawater fish, the lobster and the like comprising mushroom mycelium as a active ingredient in which, when it is given to the raised fish, the diseases thereof are suppressed and the growth rate and the raising rate can be increased.

In accordance with another aspect of the present invention, there are provided a feed additive manufactured by aforementioned procedure for the livestock, the freshwater fish, the seawater fish, the lobster and the like comprising mushroom mycelium as a active ingredient.

Inventive feed additive can be added with the range from 0.1 to 20 w/w% of feed to enhance the immunity of the livestock, the freshwater fish, the seawater fish, the lobster and the like.

In accordance with another aspect of the present invention, there are provided a use for feed and a feed additive comprising the composition prepared by the aforementioned process to enhance immunity and to prevent from bacterial or viral disease.

In accordance with another aspect of the present invention, there are provided a use for animal feed additive by adding 0.1 to 20 w/w% of the composition prepared by the aforementioned process based on total weight to enhance immunity and to prevent from bacterial or viral disease.

The following specific Figures and Examples are provided to assist the understanding of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, in which: Fig. 1 presents the flow chart of the feed and feed additive preparation with Hericium erinaceum mycelium; Fig. 2 represents body length growth of flatfish treated with the inventive feed comprising Hericium erinaceum according to feeding period ; Fig. 3 depicts body weight change of flatfish treated with the inventive feed comprising Hericium erinaceum according to feeding period; Fig. 4 shows the survival rate (%) of flatfish treated with the inventive feed after vibrio sp. Infection; Fig. 5 presents the red blood cell (RBC) level of flatfish treated with the inventive feed; Fig. 6 shows the hematocrit level (%) of flatfish treated with the inventive feed; Fig. 7 exhibits the hemoglobin concentration of flatfish treated with the inventive feed; Fig. 8 represents the average RBC volume of flatfish treated with the inventive feed; Fig. 9 presents the average RBC hemoglobin level of flatfish treated with the inventive feed; Fig. 10 shows the average RBC hemoglobin concentration of flatfish treated with the inventive feed; Fig. 11 depicts the liver mass index (%) of flatfish treated with the inventive feed; Fig. 12 shows the GPT change of flatfish treated with the inventive feed;

Fig. 13 exhibits the GOT change of flatfish treated with the inventive feed; Fig. 14 presents the SOD (Superoxide anion radical) activity of flatfish treated with the inventive feed; Fig. 15 depicts the white blood cell (WBC) phagocytosis activity of flatfish treated with the inventive feed.

BEST MODE FOR CARRING OUT THE INVENTION The following Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Example 1 : Hericium erinaceum spawn cultivation and pollination Hericium erinaceum spawn was distributed from Korea Mushroom Corporation and cultivated at 20-23°C, 70% of humidity in cleanroom according to the conventional mushroom culture method.

Example 2: Preparation of medium composition and sterile medium The medium was prepared by mixing 20w/w% corn, 20w/w% soy bean meal, 10w/w% potato, 10w/w% sorghum, 10w/w% fish meal and 10w/w% barley and medium composition was put into 1000cc container for mushroom cultivation. After the stopper was put on the bottle, medium was autoclaved at 65°C for 1 hour to make sterile medium and cooled down.

Example 3: Spawn inoculation and cultivation Hericium erinaceum spawn of Example 1 was inoculated by lOg/container onto sterile medium of Example 2. It was moved to and incubated at the temperature ranging from 20 to 23°C with 70% of humidity and dark condition in the cleanroom for 30 days.

Example 4: Preparation of inventive feed Grown Hericium erinaceum mycelium and medium were separated. Those were hot wind-dried with dryer, pulverized, mixed mycelium and medium in ratio of 3: 97 or 5: 95 by weight and formed to make an inventive feed.

Example 5: Preparation of fishes Flatfishes (average body weight 67.7g, average body length 20.2cm) were given from Shinyang-Susan (Sungsan-eub Shinyang-ri, Korea) and confirmed that fished did not have any pathogen.

12 flatfishes were set in a 150 L of water tank for raising with circulating filtration and aeration apparatus and acclimated for 7 days. Water temperature ranged from 27 to 28°C and conventional feed was fed to the fishes twice a day by lw/w% of body weight.

Water was completely changed once a week.

Example 6: Preparation of Pathogen Vibrio sp. was given from Jeju Province Fisheries Resource Research Institute in 2002, stored at-70°C. After culturing in marine broth (DIFCO, France) for 18 hours, it was used for fishes to be infected with

Example 7: Vibrio sp. Infection Vibrio sp. of Example 6 was incubated with shaking at 200rpm, 27°C for 18 hours in marine broth and directly diluted with 0.85% saline or collected by centrifugation (3000rpm, 30 min) followed by dilution.

Vibrio sp. was diluted in the concentration of 1 X 103, 1 X 104 and 1 x 106 cfu/m. .

Flatfishes were moved into another 20L of water tank, infected with diluted vibrio sp and stayed for 12 hours. Finally flatfishes were moved back to the water tank for raising and observed for 1 week.

In case that flatfishes were still alive, fishes have been infected with higher vibrio concentration once again.

Example 8: Confirmation of vibrio infection Dead flatfish's organs were aseptically removed to streak on Marine agar (DIFCO, France). And the color of colony was observed on BTB teeple agar (DIFCO, France), vibrio selection medium. Biochemical character and morphology were refered to Bergeys manual of systematic bacteriology.

Experimental Example 1 : Observation the growth of the vibrio-infected flatfish treated with inventive feed After 50 flatfishes of Example 5 were infected with vibrio sp. of Example 6 according to Example 7, the fishes were observed with feeding 500g of the inventive feed comprising 3w/w% Hericium erinaceum and 5w/w% Hericium erinaceum twice a day for 8 weeks.

The growth of flatfish that was infected with vibrio and then taken inventive feed for 8 weeks was shown in Fig. 2 and Fig. 3.

The result of flatfish body length change was shown in Fig. 2.

The inventive feed comprising 3% and 5% Hericium erinaceum caused the vibrio infected-flatfish to grow to the size of about 4cm and 6.4cm, respectively, compared with the conventional feed 3.6cm. These results showed that inventive feed led 1.3% and 11.3% of growth increase compared with control group.

And the increasing result of flatfish body weight (Fig. 3) showed that the inventive feed comprising 3% and 5% Hericium erinaceum caused the infected-flatfish to gain 91.2g and 102.9g of weight, however the conventional feed caused the infected flatfish to gain only 15.2g of weight.

These results showed that inventive feed led 9.6% and 23.7% of weight increase comparing with control group.

Therefore, it is confirmed that the inventive feed comprising 3% and 5% Hericium erinaceum is effective to growth and weight increase.

Fig. 4 exhibits the viability of vibrio infected-flatfishes for 8 weeks. The flatfishes of control group died of infection continuously for early 3 weeks and also died from 6th to 8"week. Finally, in 8th week, only 30 flatfishes were survived.

On the contrary, all of the flatfishes taken the inventive feed were survived.

It is meant that the inventive feed can make the fishes strong against the infectious

disease.

Experimental Example 2: Characterization of Flatfish Blood Flatfishes infected with vibrio were fed up the inventive feed comprising Hericium erinaceum. And then the changes of their blood were analyzed on hematocrit level, red blood cell (RBC) level, hemoglobin concentration, average RBC volume, average RBC hemoglobin quantity and average RBC hemoglobin concentration.

Blood sample was collected from the blood vessel in the tail of fish using syringe.

Hematocrit level was analyzed by the ratio of RBC part against RBC and plama part. Blood was separated into two parts by centrifugation (4000-4500rpm, 30mins) in micro-hematocrit.

RBC level was determined with the diluted blood by using Neubaur chamber.

Hemoglobin concentration was measured by the absorbance (540nm) of reaction mixture of blood and diluent according to cyanmetahemoglobin method.

As the results, RBC level was not significantly changed (Fig. 5), wherein RBC level is the index of oxygen delivery capability. But the hematocrit level was the lowest in the group taken inventive feed comprising 5% of Hericium erinaceum (Fig. 6). These results were meant that newly generated RBC led the low hematocrit level, while total RBC number was not changed.

And average RBC volume was the largest in the group taken inventive feed comprising 3% of Hericium erinaceum, however the control group and the group of inventive feed comprising 5% of Hericium erinaceum had not shown any significance in Fig. 8.

The inventive feed comprising 3% of Hericium erinaceum had the highest level of RBC and surface dimension therefore it was confirmed that its RBC was the highest

activity to deliver oxygen.

And then the results of hemoglobin concentration (Fig. 7), average RBC hemoglobin level (Fig. 9) and average RBC hemoglobin concentration (Fig. 10) showed the groups taken inventive feed comprising 3% and 5% of Hericium erinaceum had the greater ability to deliver oxygen in blood comparing with control group.

The greater oxygen delivery capability of blood can lead to resist the stress such as hypooxygen circumstances to fishes, to prevent the death in raising and to lessen the stress during transport.

Experimental Example 3: Analysis of Change in Liver Function Liver function is the index of health. Fundamental liver mass index, GPT (Glutamic pyruvic transaminase) and GOT (Glutamic oxalacetic transaminase) were measured to analyze the change of liver function.

Blood sample was collected from the blood vessel in the tail of fish using syringe.

Liver mass index was calculated the percentage of liver weight against total body weight.

GPT and GOT was measured by using commercial GPT and GOT kit (ASAN Pharmaceuticals Co. , Ltd.; Reitman-Frankel method).

As shown in Fig. 11, the liver mass index was the highest in the group of inventive feed comprising 5% mushroom and that of the group taken 3% mushroom containing-feed showed the significance compared with control group. These results were caused by inventive feed to increase of liver activity.

As shown in Fig. 12, GPT level was not significantly changed while that of the group taken 3% mushroom containing-feed showed a little increase. Also, GOT did not show any significant difference (Fig. 13).

Consequently, above results is a phenomenon for recovery of hematological imbalance generated by vibrio, wherein said phenomenon is the increase of liver mass index and activity.

Experimental Example 4: Analysis of Active Oxygen in Flatfish Blood Active oxygen is a kind of chemical which is highly active and containing oxygen such as superoxide anion radical ('02-or °2-), hyperoxide (H202), hydroxyl radical (*OH) and so on. Active oxygen is generated by stress and has the physiologically bad effect, e. g. genetic mutation. To inhibit the toxic effect of active oxygen, liver organ generates and secrets such enzyme as SOD (superoxide dismutase) in the body.

On the ground of that, SOD activity was measured to detect physiological defense mechanism in the flatfish fed up with the inventive feed.

SOD was measured according to the Marklund's method (Marklund S and Marklund G; Eur. R Biochem., 16, 47 (3), pp469-474,1974) determining the inhibition rate of autooxidant rate of pyrogallol, i. e. , one of the polyphenol.

8. 7mQ of 50mM phosphate buffer (pH 8.24) was added to flatfish liver homogenate and then 0. 3mQ of 3mM pyrogallol solution was added. Absorbance was measured at 325nm.

As shown in Fig. 14, control group showed high SOD activity in order to remove superoxide anion radical owing to stress by vibrio infection, however, the group treated with the inventive feed comprising Hericium erinaceum showed low SOD activity.

It was confirm that the inventive feed increased the tolerance against vibrio and decreased the stress, so SOD activity was diminished.

Experimental Example 5 : Phagocytosis activity of Flatfish white blood cell Phagocytosis activity of white blood cell (WBC) is generally used as the immunological activity index and then that of flatfish WBC against E. coli was measured.

5-1. Hemocvte isolation Blood was collected from the vessel of tail of anesthetized flatfish.

Im of blood and 3-4moi of HBSS (Hank's balanced salt solution) were mixed, moved into silicone-coated tube and centrifuged with the speed of 300-500rpm at 5°C.

And the supernatant was used as macrophage-containing solution.

5-2. Assav of phaaocvtic activity of WBC WBC bactericidal activity was measured by the detection of live bacteria in WBC after phagocytosis of bacteria into WBC.

The preparation of bacteria was performed by culturing and diluting to the concentration of 107/moi.

Fresh serum was prepared and stored at-80°C for opsonization.

0. 3mQ of HBSS, 0. 1mQ of bacteria and 0. 1mQ of serum were put into silicone- coated tube and incubated with shaking at 25°C for 10 mm. And 0. 5mQ of above macrophage-containing solution (2.5 X 106) was added thereto. Incubation was performed continuously. Shortly after the addition of macrophage-containing solution, 0. 1 iflg of culture solution was collected and mixed with 9. 9m of distilled water in tube to use as a sample of time zero.

At the interval of each 30,90, 120 mins after culture, 0. 1mQ sample was collected, mixed with HBSS and centrifuged (500rpm, 10 mins) three times. And then distilled water was added thereto. The mixture was vortexed to disrupt WBC, inoculated on agar medium, incubated at 25°C and calculated the number of colonies.

Immunological activity of inventive feed was evaluated by the ratio of determining phagocytosis, assuming the phagocytic activity of control group at 30 min was 100% (Fig.

15).

In the group treated with 5% Hericium erinaceum-containing feed, the ratio was the highest level of phagocytic activity at 30 mins and it was maintained for 2 hours.

In the group treated with 3% Hericium erinaceum-containing feed, the phagocytic activity ranged from 120 to 146%, which was higher than that of control.

As above results, immunological activity of the blood was increased by the addition of Hericium erinaceum and inventive feed comprising Hericium erinaceum was effective to the growth, the stress decrease, the enhancement of immunity and the prevention of diseases by pathogen.

INDUSTRIAL APPLICABILITY Inventive feed and feed additive can be used to farm and raise the livestock, the freshwater fish, the seawater fish, the lobster and the like for enhancing immunity and therefore, the infection and disease by bacteria or virus can be prevented.