KR20030044791A - A preparation method of feed and feed additive comprising mushroom mycelium - Google Patents

Abstract

The present invention relates to a functional feed and a feed additive manufacturing method of culturing mushroom mycelium on various grains, and more particularly, to the culture step of culturing the mycelium produced by spawn culture and fertilization using a mushroom seedling in a composition medium. The culture material is vacuum frozen or hot air dried to form powder and finally feed and feed additives for livestock and fish.

Functional feed and additives using the mushroom mycelium of the present invention can increase the immunity in the breeding and aquaculture of livestock, saltwater fish, freshwater fish and crayfish can prevent disease and prevent bacterial and viral infection.

Description

A preparation method of feed and feed additive comprising mushroom mycelium}

The present invention relates to a method of manufacturing a functional feed and feed additives, and more particularly, to cultivate and modify mushroom spawn, inoculate and incubate mycelia on the composition medium, and to freeze and hot-air dried to enhance the immunity of the functional feed and feed additives It relates to a manufacturing method for manufacturing.

As livestock breeding and fish farming are gradually enlarged and densified, infectious diseases are easily developed due to increased stress due to worsening environmental conditions and lowered immunity.

To this end, there is a need for an effective feed for enhancing autoimmunity and reducing stress caused by the external environment, improving disease defense, increasing resistance to bacteria, and preventing disease and for healthy breeding and farming.

In general, mushrooms are composed of cells called mycelia. A mushroom is a fungus called fungus that forms fruiting bodies (mushrooms). When compared to plants, it is a reproductive organ corresponding to flowers. It is only a short period of time that mushrooms grow and live on the ground. Mushrooms live in a mycelial state and grow in organic matter such as soil, humus and old trees. These mycelia are germinated by spores. When spores germinate, the first mycelium grows, and the first mycelium germinates from another spore, meets, and becomes a secondary mycelium. The secondary mycelium becomes the mycelium of the third, fourth, etc., forming fruiting bodies in a proper environment is called mushroom. Edible mushrooms among these mushrooms are 70 to 95% of moisture and 5 to 30% of organic and inorganic components. Dried mushrooms contain 15-30% protein, 2-10% fat and 50% soluble minerals, and 5-10% crude fiber, calcium, phosphoric acid and ash. In general, tasty edible mushrooms are rich in amino acids, manites, trehalose, and many other vitamins and enzymes such as ergosterine, the precursors of vitamin B ₂ and vitamin D.

The roe deer mushroom ( Hericium erinaceum ) used in the present invention is a mushroom of the Democratic fungi bearded mushroom family and is rich in β-D-glucan component, which is excellent in anticancer effect, anti-inflammatory, antibacterial, promotes digestion, and enhances gastric mucosal protective function. It is also known to be effective in promoting ulcer healing and strengthening immunity.

Shiitake ( Lentinus edodes ) is a fungus of the plepidoptera Oleaceae family, the most widely edible, and contains 27 free amino acids, 9 trace metals, sterols, fatty acids, vitamins, monosaccharides and sugar alcohols.

Schizophyllum commune belongs to the folds of the pleated mushrooms Schizophyllum commune . It is an edible mushroom with good fragrance and taste. It is rich in β-D-glucan and has excellent anti-tumor effect and immune activity effect. .

Auricularia auricula-judae belongs to the tree of the tree of the neck of the neck, and is an edible mushroom that has antitumor, anti-inflammatory and cholesterol-lowering effects.

Ganoderma lucidum ( Ganoderma lucidum ) is a mushroom of the Democratic ulcer, which is widely used for medicinal purposes, and has various pharmacological effects such as anti-tumor, anti-inflammatory, blood pressure control, plasma and hepatic cholesterol suppression, hypoglycemic effect, immune enhancement, and anti-aging.

Pleurotus ostreatus ( Pleurotus ostreatus ) is a fungus of Pleurotus ostreatus , widely edible and has antitumor and nematicidal activity.

Phellinus linteus ( Phellinus linteus ) is a fungus that is also known as woody mud mushroom. Antitumor, immunopotentiation, serum, complement activity, health, detoxification, intestinal, insulin-independent diabetes treatment effect.

Cordyceps sinensis is a small mushroom of the genus Cordyceps erectinaceae, most of which parasitic on insects to give fruit to the host body of insects. It is effective as nutrition tonic, immune function enhancement and fatigue recovery, natural healing power, anticancer agent, and inflammation inhibitor.

Agaricus Blazei ( Agaricus Blazei ) belongs to the fungi of the fungi of the fungi, also known as the spirit mushroom. The fruiting bodies of the mushrooms are freshly shaped like mushrooms but have a very unusual pattern and shape and have a unique smell and taste. Agaricus mushrooms are rich in beta-glucans, protein-glucans, lipids, lectins, etc., which are anti-cancer high-molecular polysaccharides.

Currently, research on mushrooms is being carried out all over the world. Among them, various medicines and health supplements using shiitake mushrooms, agaricus mushrooms and ganoderma lucidum mushrooms are being manufactured and marketed. The patent is incomplete. Korean Patent Laid-Open No. 2002-79321 discloses a method for producing vegetable biofeedstock of animal meat and bone meat using edible mushrooms, and a vegetable biofeedstock according to the same.

The present inventors have completed the present invention by producing a functional natural feed excellent in the immune enhancing effect using the mushroom.

Accordingly, an object of the present invention is to enhance the immunity to livestock and saltwater fish, freshwater fish, and soybeans by producing and supplying functional natural feeds in which mushroom mycelium is cultivated to livestock, seawater fish, freshwater fish, and crayfish, which are the main protein sources of humans, thereby enhancing various immune diseases and To prevent bacterial infections and ulcerative diseases, and to improve meat and nutrients.

1 is a flow chart showing a functional feed and additive manufacturing step using the Roe mushroom mycelium.

2 is a diagram showing the length of the flounder with time by the roe deer mushroom feed of the present invention,

Figure 3 is a diagram showing the weight change of the flounder with time by the roe deer mushroom feed of the present invention,

Figure 4 is a diagram showing the survival rate of the flounder fed the functional feed after fish disease bacterial infection,

5 is a diagram showing the number of red blood cells of the flounder ingested the functional feed,

Figure 6 is a diagram showing the hematocrit level of the flounder ingested functional feed of the present invention,

7 is a diagram showing the hemoglobin concentration of the flounder ingested functional feed of the present invention,

8 is a view showing the average red blood cell volume of the olive flounder fed the functional feed of the present invention,

9 is a diagram showing the average amount of hemoglobin hemoglobin in the flounder fed the functional feed,

10 is a view showing the average red blood cell hemoglobin concentration of the flounder fed the functional feed,

Figure 11 is a diagram showing the liver weight index of the flounder ingested the functional feed,

12 is a diagram showing a change in GPT in the flounder fed the functional feed,

13 is a view showing the change of GOT in the flounder fed the functional feed,

14 is a view showing the SOD activity in the flounder fed the functional feed,

Figure 15 is a diagram showing the white blood cell phagocytic ability of the olive flounder fed the functional feed.

Explanation of symbols on important parts of drawing

S100: spawn culture S200: medium composition

S300: inoculation S400: mycelium culture

S500: Machining

In order to achieve the above object, the present invention is (i) spawn culture step of culturing and modifying the spawn of the mushroom and (ii) a medium composition step of formulating a medium by mixing a variety of grains and (iii) mushrooms in the prepared medium Inoculation step of inoculating the spawn of (i) a culture step of culturing the mycelium in the inoculated medium and (iii) a processing step of producing a feed and feed additives by freezing and hot-air drying using a medium cultured mycelium It provides a method of producing a functional feed and feed additives characterized in that.

Functional feed and feed additives of the present invention can be used as livestock and seawater fish, freshwater fish, crayfish feed and additives.

Hereinafter, the present invention will be described in detail.

Functional feed and feed additive using the mushroom mycelium according to the present invention,

For example, the first step of culturing and fertilizing mushroom spawn;

A second step of mixing and preparing the medium composition according to the composition ratio;

A third step of inoculating the cultured spawn in the composition medium,

A fourth step of culturing the mycelium in the inoculated medium and

Powdered medium in which the mycelium is cultured is prepared by a manufacturing method comprising a fifth step of finally obtaining a functional feed and feed additive using the mushroom mycelium.

The mushroom of the first step is selected from at least one of shiitake, roe deer mushroom, agaricus mushroom, skirt mushroom, sour mushroom, situation mushroom, ganoderma lucidum mushroom, chaga, enoki mushroom, oyster mushroom or cordyceps mushroom, preferably roe deer The fungus is used and can be inoculated into the medium according to the unique efficacy of each mushroom.

In addition, the medium composition of the present invention of the second step is corn, bran, corn gluten wheat, sugar cane molasses, soybean meal, rapeseed meal, coconut foil, palm wheat, animal fats, wheat, tapioca, baking by-products, brewing, rye, oats It is selected from potato, barley, sorghum, wild fish, cottonseed foil, rice bran, barley bran, beer foil, beet pulp, cotton pellet, long milk gourd, tofu meal, fish meal, pupa, and blood meal, and the composition ratio is changed as needed. It is possible.

In a preferred embodiment of the present invention, 20% corn, 20% soybean meal, 10% potato, 10% sorghum, 10% cotton pellets, 10% fishmeal, 10% barley are mixed, the medium composition to 70% moisture content Manufacture.

In the production method of the present invention, the second step is a step of preparing the medium by placing the composition in a 500-2000 cc size mushroom culture vessel, sterilized mushroom culture medium at 50 to 121 ℃ for 30 minutes to 5 hours In the third step, inoculate the mushroom spawn, and in this case, the sterilized medium is preferably inoculated 5-30 g of the mushroom spawn per cultivation container in a clean room (third step). In the fourth step, the medium inoculated with the spawn is incubated for about 5 days to 90 days in a culture chamber of 20-25 ° C. and 50-80% humidity. Provided is a functional feed manufacturing method for powdering, mixing and molding after vacuum freeze drying at 120 ℃ or hot air drying at 60 ℃ or more.

The weight ratio of the mushroom mycelium in the prepared final feed of the present invention is characterized in that 1 to 50% by weight.

Functional feed using mushroom mycelium according to the method of the present invention contains various proteins, carbohydrates, minerals, vitamins and polysaccharides contained in the mushrooms, and also contains β-glucan which is excellent in immunostimulating effect in polysaccharides to prevent various diseases. And improves meat and nutrients.

When the functional feed of the present invention is administered as a feed to the flounder infected with Vibrio bacteria, the growth rate is increased, the immunity against Vibrio bacteria is increased, and the survival rate is excellent, which is effective for preventing fish diseases and farming healthy fish.

In addition, the present invention provides a functional feed and feed additives for increasing the immunity and disease prevention of livestock and seawater fish, freshwater fish, mandarin prepared by the manufacturing method.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Culture and Modification of Roebuck Mushroom Spawn

The seed of the Roe deer fungi (Korean Mushroom Farming Association) was distributed and incubated at 20-23 ° C. and 70% humidity in a sterile culture room according to a conventional method.

Example 2. Preparation of Media Compositions and Sterile Media

A medium composition was prepared by mixing 20% corn, 20% soybean meal, 10% potato, 10% sorghum, 10% cotton pellets, 10% fish meal, 10% barley. The medium composition was put into a 1000cc mushroom cultivation container, the stopper was closed, and sterilized at 65 ° C. for 1 hour to prepare a sterile medium.

Example 3. Spawn Inoculation and Cultivation

10 g per cultivation container was inoculated into the sterile medium of Example 1 in the aseptic medium of Example 2. The culture was transferred to a sterile culture room, and the spawn was incubated until the mycelia grew widely throughout the medium. The culture was incubated for 30 days in the temperature 20-23 ℃, humidity 70%, no light.

Example 4 Medium Fodder Cultured with Mycelia

The medium from which the roe deer mushroom and mushrooms were cultured was separated, powdered by hot air drying with a dryer, mixed to contain 3% by weight and 5% by weight of roe deer mushroom, and compression molded to be fed.

Example 5 Experimental Word Preparation

It was used to confirm that pathogens were not detected by receiving halibut with a body weight of 67.7g and a body length of 20.2cm received from Sinyang Fisheries (Sinyang-ri, Seongsan-eup). 12 flounders were placed in a 150 L water tank equipped with a circulating filtration system and aerated. The water temperature during the experiment ranged from 27 to 28 ° C, and the feed was administered twice in 1% of body weight per day. The breeder was fully reclaimed once a week.

Example 6 Experimental Strains

In early 2002, the Vibrio sp . Was isolated from Jeju Provincial Resources Research Institute and stored at -70 ℃ before incubating for 18 hours in marine broth (Marine broth, DIFCO, France).

Example 7. Infection Experiment

After the above strains were shaken in marine broth at 27 ° C. and 200 rpm for 18 hours, 1 × 10 ³ and 1 × 10 ⁴ CFU / mL of the infection were inoculated by diluting in 0.85% saline solution. × 10 ⁶ CFU / ㎖ was collected by centrifugation (3000rpm, 30 minutes) and then diluted with physiological saline. Soaking was carried in a 20 liter tank of the flounder and inoculated with the bacteria incubated in the tank, and soaked for 12 hours and then transferred to a breeding tank. If the flounder did not die after 1 week of observation, the infection was increased by increasing the bacterial concentration.

Example 8. Infection Identification Test

Aseptically removed organs of dead flounder and spread them to marine agar medium (Marine agar. DIFCO, France), and colony color on BTB teeple agar (BTB teeple Agar, DIFCO, France) Was observed. Biochemistry followed the method of the Berges manual of Sytemetic Bacteriology.

Experimental Example 1 Observation of Growth Change after Vibrio Infection

After the 50 experimental fish of Example 5 were infected with the Vibrio bacteria of Example 6, 3% and 5% of the roe deer mushroom of the present invention was observed for 8 weeks while being administered 500g twice a day.

Results of growth during breeding for 8 weeks after the infection of fish disease bacteria (Vibrio bacteria) by the feedstock of Roeden beetle mushrooms are shown in FIGS. 2 and 3.

As shown in Figure 2, 3% and 5% of the scavengerae mushroom fungus was increased by 4 cm and 6.4 cm than the initial length, respectively, and the control group showed a 3.6 cm increase. These results indicate that the mushroom 3% and 5% addition groups increased 1.3% and 11.3%, respectively, compared to the control group.

In the result of the weight gain during the flounder (FIG. 3), the control group showed an increase of 15.3 g compared to the initial weight, whereas the increase of 91.2 g and 102.9 g increased in the 3% and 5% mushroom added groups, respectively. The increase was 9.6% and 23.7%, respectively. Therefore, it was found that dietary supplementation of scavengerae mushrooms was effective in weight gain in breeding flounder. However, the overall growth rate was very slow, and these results show that the flounder suffered a lot of stress because it was raised after being infected with fish disease bacteria for 8 weeks.

Figure 4 is a count of the survival rate observed during the breeding of flounder infected with fish disease bacteria for 8 weeks, the control group continued to die for 3 weeks from the beginning of infection, and died again from 6 to 8 weeks after about 8 weeks 300,000 survived. On the other hand, in the group where the roe deer mushroom was added, no individuals died during the eight-week breeding period, so the halibut in these mushroom groups was very resistant to fish disease bacteria.

Experimental Example 2 Analysis of Changes in Blood Characteristics of Olive Flounder Fed Functional Feed

Hematocrit, erythrocyte count, hemoglobin concentration, mean erythrocyte volume, mean erythrocyte hemoglobin concentration, and mean erythrocyte hemoglobin concentration were examined and analyzed for the changes in blood characteristics of Vibrio spp.

Blood samples were taken from the blood vessels of the flounder tail using a syringe. Hematocrit (Ht) was obtained as the ratio of erythrocytes to the length of erythrocytes and plasma sections after centrifugation at 4000 to 4500 rpm for 30 minutes in a micro-hematocrit plate. Erythrocyte count was diluted with diluent and counted using hemocytometer. Hemoglobin concentration was measured by absorbance at 540nm after reacting the blood in diluent using the cyan metahemoglobin method.

As a result, in the blood physiological measurement used as an indicator of blood oxygen transport capacity, the erythrocyte count did not show a significant change in all the experimental groups (see FIG. 5), but the hemacrit value was the lowest in the 0.5% addition group. (See FIG. 6). This phenomenon is thought to have resulted in low hematcretic levels, although the overall number of red blood cells did not change due to the generation of new red blood cells. On the other hand, in the results of Figure 8 the average erythrocyte volume was the highest value in the 3% addition group, the control group and the 5% addition group did not show a significant difference. As such, the 3% addition group exhibited the highest value in the number and surface area of red blood cells, which is considered to have the highest activity of red blood cells.

On the other hand, in the hemoglobin concentration (see Fig. 7) and the mean erythrocyte hemoglobin concentration (see Fig. 9) and the mean erythrocyte hemoglobin concentration (see Fig. 10) used as a direct indicator for the transport of oxygen, the experimental group added the feeder containing the worm fungus Compared to the high level, the oxygen transport capacity of blood was excellent. When the blood is excellent in oxygen transport ability, the fish is expected to increase the resistance from stress such as the external low oxygen environment to prevent the death caused during the breeding and reduce the stress during transportation.

Experimental Example 3 Analysis of Changes in Liver Function of Olive Flounder Fed Functional Feed

Liver function is a frequently used item in the human body as an indicator of health. In this study, we analyzed the differences in liver function by measuring GPT and GOT, which are basic liver weight index and indicators of liver function. Blood samples were taken from the blood vessels of the flounder tail using a syringe. Liver weight index expressed liver weight as a percentage of total weight. GPT (Glutamic pyruvic transminase) and GOT (Glutamic oxalacetic transminase) were measured using an Asan commercial kit (Reitman-Frankel method).

Referring to the results of FIG. 11, the liver weight index according to the addition of the roe deer mushroom was the highest in the 5% added group, and the 3% added group showed a significant increase compared to the control group. This result is believed to be due to the increase in liver activity with the addition of roe deer mushroom containing EP feed.

GPT showed a tendency to increase slightly in the 3% addition group, but did not show a significant difference from the control group (see FIG. 12). In addition, the GOT also showed a tendency to increase in the 3% and 5% addition group, but did not show a significant difference (see Fig. 13). These trends are thought to be a series of processes to restore the hematological imbalance caused by Vibrio bacteria by increasing liver weight index and increasing activity.

Experimental Example 4. Analysis of changes in free radicals of olive flounder fed functional feed

Active oxygen refers to chemicals that contain oxygen and are highly reactive. In the narrow sense, a super oxide anion radical (superoxide anion radical) and the like, hydrogen peroxide (H ₂ O _2), hydroxyl radicals (hydroxyl radical) (and OH) (and O ₂ ^{- -} or O _2). These free radicals are produced by stress and are known to have various physiological adverse effects such as genetic variation. To prevent the action of free radicals, the liver produces and secretes enzymes such as superoxid dismutase (SOD) in the liver. Therefore, in this experiment, we measured the activity of SOD that plays a physiological defense. Blood samples were taken from the blood vessels of the flounder tail using a syringe. Superoxide dismutase (SOD) is a Marklund method for measuring the amount of inhibition of the autooxidation rate of pyrogallol, a polyhydric phenol (Marklund S and Marklund G; Eur J Biochem. , 16; 47 (3) , pp469-474 ) . , 1974), a liver homogenate was added to 8.7 ml of 50 mM phosphate buffer (pH 8.24), and 0.3 ml of 3 mM pyrogallol solution was added at 325 nm.

Referring to the results of FIG. 14, a large amount of SOD activity was shown in the control group to remove superoxide anion radicals generated by the stress action generated by the infection of Vibrio. However, the activity of SOD was drastically decreased by the addition of Roebuck mushroom. These results are thought to be due to the decrease in stress in the fish body by the addition of the scavenger mushroom and increased resistance to Vibrio bacteria.

Experimental Example 5. Analysis of Phagocytic Changes in White Blood Cells of Olive Flounder Fed Functional Feed

The phagocytic activity of leukocytes is generally used as an indicator of immunological activity. In this study, leukocytes were isolated from blood and examined for phagocytosis of E. coli.

5-1. Separation of blood cells

After the flounder was anesthetized, blood was collected from caudate blood vessels, mixed with 1 ml of blood and 3-4 ml of HBSS (Hank's Balanced Salt Solution), placed in a silicone coated test tube, and centrifuged at 5 ° C. at 300 to 500 rpm. Thereafter, only the supernatant was used as phagocytic suspension.

5-2. Leukocyte phagocytosis test

The leukocyte bactericidal activity was measured by phagocytosis of leukocytes and then by examining whether bacteria survive in the leukocytes. Bacteria to be used in the bactericidal test were cultured E. coli and adjusted to a concentration of 10 ⁷ / ml after centrifugal washing. Fresh serum was collected for opsonin and then stored at 80 ° C. 0.3 ml of HBBS, 0.1 ml of bacteria and 0.1 ml of serum were added to a silicone coated test tube, followed by shaking culture for 10 minutes at 25 °. Then, 0.5 ml (2.5 × 10 ⁶ ) of phagocyte suspension was added and cultured continuously. Immediately after the addition of the phagocytes, 0.1 ml was collected and transferred to a test tube containing 9.9 ml of sterile distilled water stored at 4 ° C, and used as a sample for 0 minutes. After 30 minutes, 90 minutes and 120 minutes of incubation, 0.1 ml of the sample was collected in the same manner. Three centrifugal washings (500 rpm, 10 minutes) with cold HBSS, and 10 ml of sterile distilled water were added thereto. The number of colonies was counted after inoculation and incubation at 25 ° C.

After 0.5 hour, the phagocytosis of the control group was 100, and the immunological activity of the locust mushroom was evaluated by indicating the ratio for the phagocytosis for 2 hours. The results are shown in FIG. The 5% group showed 155% at 0.5 hours, indicating the highest phagocytic activity of leukocytes. This trend was maintained for 2 hours after the experiment. Even in the 3% added group, the phagocytic activity was higher from 120% to 146% compared to the control group. As a result, the immunological activity in the blood tends to be increased by the addition of the roe deer mushroom, and it is estimated that the constituent of the roe deer mushroom acts in the prevention of diseases caused by pathogenic microorganisms.

The above experiments were carried out to investigate whether the locust mushroom was effective in the prevention of fish diseases and the cultivation of healthy flounder in the flounder. The roe deer mushroom was infected with fish disease bacteria for 8 weeks to keep the flounder cultivated immunologically healthy to prevent any death. These results were found in the results of the white blood cell phagocytic ability to kill bacteria invading the body, the activity of SOD, a biological defense enzyme, the oxygen transport ability of blood. In addition, roe deer mushrooms seemed to play a positive role in improving the growth rate of flounder infected with fish disease bacteria.

As a result of the above, it was confirmed that the functional feed containing the roe deer mushroom of the present invention is effective for growth, stress reduction and increased immunity. Functional feed and additives using the mushroom mycelium of the present invention exhibits the effect of increasing immunity in the breeding and aquaculture of livestock, sea fish, freshwater fish and crayfish, thereby preventing disease and preventing bacteria and virus infection.

Claims (7)

(I) a seed culture step of culturing and modifying the seed spawn of the mushroom, (ii) a medium composition step of forming a medium by combining various grains, and (iii) an inoculation step of inoculating the seed spawn of the mushroom on the prepared medium; A functional feed comprising a culture step of culturing the mycelium in the inoculated medium for 5 to 90 days and a processing step of preparing a feed by vacuum freeze drying or hot air drying using the medium in which the mycelium is cultured. Manufacturing method. The method of claim 1, wherein the mushroom is selected from the group consisting of shiitake, roe beet mushrooms, agaricus mushrooms, skirt mushrooms, woody mushrooms, situation mushrooms, ganoderma lucidum mushrooms, chaga mushrooms, enoki mushrooms, oyster mushrooms and cordyceps Manufacturing method. The method according to claim 2, wherein the mushroom is a roe deer mushroom. The medium of claim 1, wherein the medium is corn, bran, corn gluten, sugar cane molasses, soybean meal, rapeseed meal, coconut gourd, palm wheat, animal fat, wheat, tapioca, baking by-products, spirits, rye, oats, potatoes, barley , Sorghum, wild jelly, cottonseed gourd, rice bran, barley bran, beer gourd, beet pulp, cotton pellets, long milk gourd, tofu gourd, fish meal, pupa and blood meal. The method of claim 1, wherein the weight ratio of the mushroom mycelium in the feed is characterized in that 1 to 50% by weight. A functional feed having an effect of increasing immunity and preventing disease prepared by the method of any one of claims 1 to 5. The feed of claim 6 for livestock and saltwater fish, freshwater fish and crayfish.