KR101316450B1 - Aquaculture of low water temperature using fermented milk of mushroom extracts and prophyrins - Google Patents

Abstract

PURPOSE: A cultivation method for eels is provided to reduce fat ingredient of the eels and to enhance growth rates. CONSTITUTION: A cultivation method for eels includes the following steps: adding skimmed milk powder to an Mushroom extract, homogenizing, and sterilizing for 1-2 hours; inoculating a lactic acid bacteria starter which is a mixture strain for Lactobacillus acidophilus (KACC No.12419), Streptococcus thermophillus (KCTC No. 3658), and Bifidobacterium longum (KACC No. 16649) and fermenting for a certain period of time; mixing 1-3 wt% of mushroom extract lactobacillus fermentation milk and 0.5-1 wt% of porphyrin with water; and supplying the mixed feed to water tub once a day.

Description

Eel farming method using bioactive substance as feed additive {Aquaculture of low water temperature using fermented milk of mushroom extracts and prophyrins}

The present invention relates to an eel farming method using mushroom extract lactic acid bacteria fermented milk and porphyrins as feed additives for the development of eel farming technology resistant to low water temperature and development of strong eel farming technology.

As the Korean people's living standards improve and the interest in well-being foods increases, consumers are considering not only taste and nutrition but also functional ingredients in foods. In addition, the preference for aquatic products is reflected in this. Consumers' preferences for aquatic products were found to be unique in taste (28.6%), rich in nutrition (26.5%), and adult disease prevention (21.1%). In this respect, there is a need for continuous research on aquatic foods that are nutritionally superior and contain meat quality, stability, and functional substances for revival, well-being food, and safe food production.

For eel fish, the average cost per unit of production is 33.8% for fry, 20.2% for feed, and 17.2% for labor, and the fuel costs for boilers are 6.5% of the production price of eel (e.g. Standard Guide for Eel Farming, 2010). Since various environmental problems such as carbon dioxide reduction are on the rise, in order to minimize the cost of aquaculture production and realize eco-friendly farming, it is necessary to seek alternative methods of low water temperature breeding through alternative energy development and eel vitality.

It is difficult to accurately identify factors that improve growth, feed availability, and fish quality in aquaculture fish species, but the ingredients and feed systems in the feed are important factors that directly affect feed efficiency and growth. If the proper feeding system is not established, many problems such as economic loss and water pollution caused by loss of feed, or low feed efficiency are caused. Therefore, research to improve the quality of feed is required. It's going on.

The industrialization and internationalization of our society has brought great changes not only to food and nutrient intake of each individual but also to the general food and culture of the people. At the same time, interest in health has reached a level that demands safe foods, and has recently reached its peak by imported fish that have been injected with imported malachide green (prohibited drugs). This phenomenon has begun to strengthen the safety of food in order to lead a healthy life not only in Korea but also in the world.

Therefore, the present invention is to investigate the changes in the body composition and blood components of the cultured eel, such as changes in the weight and weight of the eel cultured by administering a feed additive for the development of high-quality eel culture technology and development of strong eel culture technology, The eel farming method used as feed additive was established.

Korean Patent Publication No. 10-0908928 discloses a feed additive for aquaculture fish, a feed composition, and a method of rearing aquaculture fish using the same, and specifically, Bacillus-subtilus KS-3, Bacillus amylolique Bacillus-amyloliquefaciens KS-4, Bacillus-agaradhaerens KS-5, Paenibacillus-lentimorbus KS-6, Bacillus Laebolactus laevolacticus KS-7, Leuconostoc paramesenteroides KS-9, Kurthia-sibirica KS-13 and Sphingobacterium spiritiborum-GC subgroup B (flavobacte) (Sphingobacterium-spiritivorum-GC subgroup B (Flavobacterium) A composition for providing a feed additive or feed composition for farmed fish comprising at least one microorganism selected from the group consisting of KS-18. Korean Patent Publication No. 10-05336770 relates to a feed additive for fish farming containing Lactobacillus plantarum as an active ingredient, acid resistance at pH 2.5, isolated from kimchi, and bile resistance and antibiotic Bassitra at 500 μg / mL bile acid. A composition for aquaculture feed additives containing Lactobacillus plantarum, which is resistant to leucine, tetracycline, colistin, gentamicin, cephalosporin, and has fish growth promoting activity, is disclosed. However, the prior art is merely to use lactic acid bacteria as feed additives, while producing a functional physiologically active substance by fermenting the lactic acid bacteria as used in the present invention, while using the feed additives, while maintaining the breeding water temperature at low water temperature in general It has a different configuration from the culture method that can have a growth rate as in the culture water temperature of 28 ℃.

The present invention using a fermentation technique using edible lactic acid bacteria, such as Ganoderma lucidum mushroom, shiitake mushroom, ink mushroom, etc. in food and medicinal mushrooms at low temperature (23 ℃) using a functional bioactive material at a culture water temperature of 280,000 The purpose of the present invention is to provide a farming method using porphyrins, which are physiologically active substances, as feed additives to lower the fat content of eel and increase the growth rate.

In the present invention to achieve the above object

Adding 3 to 8% by weight of skim milk powder to the mushroom extract extracted under certain conditions, homogenizing for 3-7 minutes with a blender, and then sterilizing at 90 ° C. for 1-2 hours; After the sterilization in the step to cool to about 37 ~ 40 ℃, inoculating 1% (V / V) of the lactic acid bacteria stater fermentation at 37 ℃ for a certain time; Mixing the fermented mushroom extract lactic acid bacteria fermented milk with the blended feed to prepare a dough feed; It provides an eel low water culture method using the mushroom extract lactic acid bacteria fermented milk as a feed additive comprising the step of feeding the dough feed to the feed of the eel breeding in the tank while maintaining the water temperature of the breeding tank culture tank.

The specific conditions for extracting mushrooms are hot water extraction of mushrooms for 2 hours at 40-60 ° C, 2 hours at 70-90 ° C, and 2 hours at 110-130 ° C. The supernatant is filtered through a filter to remove impurities and then 8,000 rpm By centrifugation for 20-40 minutes to remove the precipitate by mixing the respective extracts, characterized in that sterilized under pressure for 20-40 minutes at 10-130 ℃, but is not limited thereto.

In addition, the mushroom used in the present invention is one or more selected from Ganoderma lucidum mushroom, shiitake mushroom, ink mushroom, lactic acid bacteria stater is Lactobacillus acidophilus (KACC No. 12419) , Streptococcus thermophillus (KCTC No. 3658)) and Bifidobacterium longum (KACC No. 16649) .

Mushroom extract Lactobacillus fermented milk and the active ingredient, which is an active material prepared as an additive material, may be mixed with 1-3.0% by weight based on 100% by weight of compounded feed to supply 1-2% by weight of the fish once a day.

In addition, as an additive material, Porphyrins (porphyrins) 0.5 ~ 1% by weight compared to 100% by weight of the feed mixed with feed once a day and the mixed feed can be supplied 1-2% by weight of the fish body.

By developing high-performance feed additives for eel using products that can make the most of the functions of fermented lactic acid bacteria fermented products using mushroom extracts, economic profits for mass farmers and raw material suppliers (mushroom farmers) are expanded, and the entire fish farm is expanded and exported. It is expected to contribute to the improvement of national competitiveness. In addition, by developing high-performance feed additives for cultured eel using natural-derived physiologically active substances with no side effects, it can be saved at low temperatures (23 ° C) to improve energy efficiency and reduce carbon emissions. In addition, it is expected to produce economic profits of producers of natural products as well as economic profits of producers by improving fish disease resistance by enhancing natural immunity and improving productivity by promoting growth.

1 shows a photograph of a black mushroom which is a material of the present invention.
Figure 2 shows the results of the TLC method (Thin-layer chromatography) for identifying the polysaccharide of the ink mushroom extract.
Figure 3 is a result shown by using the TLC method to confirm the polysaccharide of shiitake extract.
Figure 4 is a result using the TLC method to confirm the polysaccharide of Ganoderma lucidum extract.
Figure 5 is a result of comparing the protein pattern of the yogurt and mushroom extract lactic acid bacteria fermented milk.
Figure 6 is the result of measuring the total equivalent weight of mushroom extract lactic acid bacteria fermented milk by phenol-sulfuric acid method.
Figure 7 is the result of the antioxidant activity of mushroom extract lactic acid bacteria fermented milk.
8 is a result of measuring the weight change of the mouse.
9 is a result of measuring the weight change of the mouse
10 shows the histological pathology of the liver.
11 shows the weight gain and feed efficiency at each temperature.
Figure 12 shows the feed of the eel (23 ℃).
13 shows the appearance and body length (23 ° C.) of the experimental group eel.
14 shows the structural formula of porphyrin.

In order to achieve the object of the present invention, the eel low water temperature culture method using the mushroom extract lactic acid bacteria fermented milk as a feed additive is added to the skim milk powder mushroom extract Lactobacillus asidofyrus ( Lactobacillus acidophilus ( KACC No. 12419)) , Streptococcus thermophillus ( KCTC No. 3658)) and Bifidobacterium longgum ( Bifidobacterium) longum ( KACC No. 16649)) inoculating the mixed strain lactic acid bacteria and fermented with the mushroom lactic acid bacteria fermented milk; Mixing the prepared mushroom extract lactic acid bacteria fermented milk with a blended feed to prepare a dough feed; It comprises a configuration for feeding the dough feed to the feed of the eel breeding in the tank while maintaining the breeding water temperature of the nursing tank culture tank.

As an additive substance, bioactive substance Porphyrins (porphyrins) 0.5 ~ 1% by weight compared to 100% by weight of the feed mixed with feed once a day and the mixed feed can be supplied 1-2% by weight of the fish body.

More specifically,

a) Extract one or more mushrooms selected from Ganoderma lucidum mushroom, shiitake mushroom, ink mushroom, hot water extract for 2 hours at 40-60 ℃, 2 hours at 70-90 ℃, and 2 hours at 110-130 ℃ and filter the supernatant with a filter. After removing the impurities, centrifuged again at 8,000 rpm for 20-40 minutes to remove the precipitates, and each extract was mixed and 3-8% by weight of skim milk powder was added to the mushroom extract obtained by autoclaving at 10-130 ° C. for 20-40 minutes. Homogenizing for 3-7 minutes and then sterilizing 1-2 hours at 90 ℃;

b) After sterilization, the mixture is cooled to about 37-40 ° C., and then Lactobacillus acido pyrus ( Lactobacillus) acidophilus (KACC No. 12419) ) , Streptococcus thermophillus (KCTC No. 3658)) and bifidobacteria tumefaciens ronggeom (Bifidobacterium Lactobacillus starter which is a mixed strain of longum (KACC No. 16649) ) was inoculated at 1% (V / V) and fermented at 37 ° C. for a predetermined time;

c) 1-3% by weight of the mushroom extract lactic acid bacteria fermented milk prepared in step b), and 0.5-1% by weight of porphyrin (porphyrins) 100% by weight of the blended feed additionally as a dough feed Manufacturing;

d) Feeding the dough feed to the feed of eel bred in the tank while maintaining the breeding water temperature of 23-28 ° C. of the nursing tank culture tank may be made to feed 1 to 3% by weight of the fish once a day .

In order to achieve the object of the present invention, when described in detail with the embodiment as described above as follows.

I. Aquaculture Method Using Mushroom Extract as Additive to Eel Feed

1. Extraction and fermentation of cold-resistant additives

The following materials and methods were used to develop low temperature resistant eel feed additive using mushroom polysaccharides.

end. mushroom

(1) ink mushrooms

Black mushroom ( Coprinus comatus ) is an edible mushroom belonging to the pleated fungi family, which grows on the grass stumps, compost heaps, or humus on the edges of the road from spring to autumn. This mushroom is widely grown in Korea, and has been known for its pharmacological effects of psoriasis and central nervous system stimulation, and has been used as an agent for promoting digestion and treating hemorrhoids. At present, there is no place to produce ink mushrooms in large quantities, and it is the only one that has been successfully grown and produced in Baekhwa Forest Farm in Jinju, Gyeongnam.

(A) Analysis of General Components of Ink Mushrooms

The general component analysis of ink mushrooms was analyzed by entrusting the inspection to Entec Research Institute of KFDA. As a result, carbohydrates were 7.53 g / 100 g, crude protein 2.47 g / 100 g, crude ash 1.38 g / 100 g, crude fat 0.6 g / 100 g (Table 1).

Results of Analysis of General Ingredients of Ink Mushrooms (g / 100 g) moisture Views min Crude fat Crude protein carbohydrate 88 1.38 0.6 2.47 7.53

Analysis of the main mineral content of the black ink mushrooms showed that the potassium content is quite high. Potassium is an essential microelement in human nutritional aspects and plays an important role in maintaining fluid and electrolyte balance, and is known to be able to significantly improve kidney function and reduce the risk of hypertension. The next components were magnesium, sodium, and calcium, and a small amount of iron, zinc, manganese, and copper (Table 2). Therefore, the ink mushrooms contain most of the essential minerals, so it may be helpful for various physiological activities in the human body.

Analysis of Major Mineral Contents of Black Mushroom (mg / 100 g) K Ca Na Mg Fe Zn Mn Cu 11713.98 122.08 132.32 144.38 6.38 4.04 0.78 1.09

Analysis of the vitamin content of the black ink mushroom revealed that 11.61 mg / 100 g of vitamin C, which prevents scurvy, prevents antioxidant activity and removes free radicals, and 1.78 mg / 100 g of niacin, which has great nutrition for the skin, digestive system, and nervous system. (Table 3).

Vitamin Content Analysis of Black Mushrooms (mg / 100 g) A (μg / 100 g) B1 B2 B6 C Niacin 2.46 0.04 0.27 0 11.61 1.78

Niacin is vitamin B3, has an effect on schizophrenia and has been found to be Pellagra disease. Niacin does a lot of work in our bodies because it acts as a coenzyme of enzymes that participate in over 50 chemical and redox reactions in vivo. It is involved in the energy metabolism of the three major nutrients, producing energy, and also involved in the synthesis of fatty acids and steroids, which contributes to the production of cholesterol, sex hormones and subsignal hormones. It is involved in fat metabolism, lowers blood pressure, treats atherosclerosis, reduces cholesterol levels and treats hyperlipidemia.

(B) Extraction of ink mushrooms

After thoroughly washing the fruiting bodies of the ink mushrooms, completely removing the water, and then fully ripening the fruiting bodies at 37 ° C for one week to induce self-extinguishing, ripening for 10 weeks at 10 ° C, and then standing at 4 ° C for 2 weeks to clean the cell walls of the ink mushrooms. The mushrooms were digested and the remaining mushrooms were thoroughly ground in a blender to obtain mushroom grounds. After 3-12 hours at 50 ° C, 3-12 hours at 80 ° C and 3-12 hours at 121 ° C, hot water was extracted and the supernatant was filtered through 3M filter paper to remove impurities, followed by centrifugation at 8,000 rpm for 30 minutes. The precipitate was removed. Each extract was mixed and autoclaved at 121 ° C. for 3 to 8 hours to be used as an experimental material.

(C) whole equivalence measurement

The total equivalent of the black mushroom extract was measured by phenol-sulfuric acid method (Dubois, M. et . Al . , 1956). In other words, 1 ml of 5% purified phenolic water was added to the test tube, each containing 1 ml of ink extract and standard solution, and mixed well. 5 ml of concentrated sulfuric acid was added to develop color by exothermic reaction. Was measured and analyzed. To quantify the total equivalent glucose, glucose, a standard substance, was prepared for each concentration, and the experiment was performed in the same manner as the sample to prepare a calibration curve and to measure the total equivalent of the sample. The total equivalent weight of the black mushroom extract was found to be 10 mg / ml.

(D) Thin layer chromatography (TLC)

The TLC method was used to identify the polysaccharide of the black mushroom extract. TLC was performed using a silica gel plate (25DC-Alufolien Kieselgel 60, Merck Co., ltd.) And the developing solvent was developed using 1-butanol / ethanol / water (5: 5: 3). After complete development, the plate was completely dried, and then a color developing solution containing 3% sulfuric acid was applied to methanol, and then placed on a hot plate of 100 ° C. or higher and heated for 10 minutes. As a result, many high-molecular-weight sugars and low-molecular-weight sugars such as glucose (G1) molecular weight did not develop on TLC, and many sugars were also mixed between G2 and G7 (FIG. 2). The standard materials used were standard malto-oligosaccharides (G1 to G7), and G1, G2, G3, G4, G5, G6 and G7 were glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose, respectively.

(2) Shiitake mushrooms ( Lentinus edodes )

Shiitake is a fungus belonging to the genus Oysteraceae. It occurs in dead or dead branches of broad-leaved trees such as oaks (auburn tree, oak tree, oyster oak, prunus oak, etc.), oak tree and chestnut tree from spring to autumn. It is excellent in taste and has been treated as one of the three major edible mushrooms in Korea, so that it is called 1 tong, 2 shiitake and 3 bunches. It has been found that shiitake mushrooms have a special ingredient that suppresses the accumulation of cholesterol in the blood, and is widely used in foods that prevent high blood pressure.

Shiitake mushrooms have a component called ritadenine (Eritadenine) that removes blood cholesterol and lowers blood pressure. Eritadenin melts when dried mushrooms are soaked in water, so do not discard the juice and use it for cooking. Shiitake mushrooms contain a relatively large amount of nutrients. Recently, it has been found that it contains substances with various pharmacological effects, such as anticancer substances and inhibitors of blood pressure rise, and thus has received great attention as a health promoting food. In addition, it contains a lot of protein, fat and sugar, and vitamin B1, B2 content is more than twice higher than vegetables, contains niacin, and contains a lot of ergosterine, which has the effect of vitamin D. Promotes absorption. It does not contain vitamins A and C.

Tables 4 and 5 show general ingredient analysis tables of shiitake mushrooms listed in the Food Composition Table (Fifth Edition, 1996) of the Rural Development Institute.

Table of General Components of Shiitake Mushrooms mushroom Energy (Kcal)  moisture (%) Protein (g) Lipid (g)  carbohydrate Ash (g) Carbohydrate (g) Fiber (g) Raw altitude 27 90.8 2.0 0.3 5.4 0.7 0.8 Dried Elevation 272 10.6 18.1 3.1 57.0 6.7 4.5

Table of Mineral Composition of Shiitake Mushrooms mushroom   Inorganic matter (mg)   vitamin calcium  sign  iron salt  potassium A (I.U.) B1 (mg) B2 (mg) Niacin (mg) C (mg) Raw altitude 6 28 0.6 5 180 0 0.08 0.23 4.0 0 Dried Elevation 19 268 3.3 25 2140 0 0.48 1.57 19.0 0

Shiitake mushrooms used in this experiment were purchased from the Buong Pyo High Agricultural Cooperative Corporation (Mungyeong, Gyeongbuk), and the mushroom polysaccharides were induced by hot water extraction. Hot water was extracted for 2 hours at 50 ° C., 2 hours at 80 ° C., and 2 hours at 121 ° C., and the supernatant was filtered through 3M filter paper to remove impurities, followed by centrifugation at 8,000 rpm for 30 minutes to remove precipitates. Each extract was mixed and autoclaved at 121 ° C. for 30 minutes to be used as an experimental material. Figure 3 is a result shown by using the TLC method to confirm the polysaccharide of shiitake extract. As a result, many high-molecular-weight sugars and low-molecular-weight sugars such as maltose (G2) molecular weight were not developed on TLC. In addition, many sugars were mixed between G3 and G7. .

(3) Ganoderma lucidum mushroom lucidum )

Ganoderma lucidum is a mushroom belonging to the Democratic Mushrooms Cloudy Mushroom and Bulchocho (Gamnoaceae, Ganodema). In Korea, it is commonly called Ganoderma or Bulgocho. It is recorded. Most of them are parasitic in hardwoods, but G. neo - japonicum , G. tsugae and G. atkinonii occur in conifers. Cultivation species is Ganoderma lucidum , G. tsugae to be.

General composition of Ganoderma lucidum is shown in Table 6. Crude fiber per building was the highest at 43.5%, followed by carbohydrates at 25.3%, crude protein at 13.6%, crude fat at 3.6% and crude ash at 1.1%.

General properties of Ganoderma lucidum (%) moisture Views min Crude fat Crude protein Crude fiber carbohydrate 12.9 1.1 3.6 13.6 43.5 25.3

Inorganic components include K, Na, Ca, Mg, P, Fe, Cu and the like and their contents are shown in Table 7. The highest K content was 3,598 ppm, followed by phosphorus (P) 3,030 ppm, magnesium (Mg) 772 ppm, calcium (Ca) 163 ppm and sodium (Na) 148 ppm.

Mineral content of Ganoderma lucidum (ppm) K Na Ca Mg P Fe Cu Zn Mn Ni Co Cr 3,598 148 163 772 3,030 36 22 20 16 4 8 One

Ganoderma lucidum used in this experiment was purchased from Baekhwarim Farm (Gyeongnam Jinju) and the mushroom polysaccharides were induced by hot water extraction method combined with low temperature extraction. Hot water was extracted for 2 hours at 50 ° C, 2 hours at 80 ° C, and 2 hours at 121 ° C. The supernatant was filtered through Whatman filter paper (England, Cat No 1002 185) to remove impurities, and then centrifuged at 8,000 rpm for 30 minutes. The precipitate was removed. Each extract was mixed and autoclaved at 121 ° C. for 30 minutes to be used as an experimental material. Figure 4 is a result using the TLC method to confirm the polysaccharide of Ganoderma lucidum extract. As a result, many high molecular weight sugars and low molecular weight sugars such as glucose (G1) and maltose (G2) molecular weights were not developed on TLC. It was confirmed that it was done.

I. Fermentation Conditions of Lactic Acid Bacteria Using Mushroom Polysaccharides

In general, yogurt is a product that is fermented by inoculating lactic acid bacteria with milk or skim milk powder as raw materials, and it is an excellent food and nutritional food because it contains active ingredients such as lactic acid, peptone, peptides, and oligosaccharides produced by lactic acid bacteria during fermentation. At the same time, it is recognized as a representative health drink. In addition, lactic acid bacteria are known to have a strong effect on the digestion and absorption by inhibiting the growth of harmful bacteria, enhance immunity, and help the peristalsis of the intestines by strongly settling in the stomach or intestine.

In the present invention, the mushroom extract Lactobacillus fermented milk was prepared using the mushroom extract exhibiting a natural immune enhancing effect. The prepared mushroom extract lactic acid bacteria fermented milk was used as a feed additive for eel low temperature culture.

(1) Strain

The strain is Lactobacillus acidophilus ( KACC No. 12419)) , Streptococcus thermophillus ( KCTC No. 3658)) and Bifidobacterium longgum ( Bifidobacterium) longum ( KACC No. 16649)) was used. The strain can be searched and distributed in the Agricultural Genetic Resource Information Center (http://www.genebank.go.kr) and the Korea Life Resource Center (http://kctc.kribb.re.kr). Skim milk was used as TTC test negative product produced by Seoul Milk.

(2) Mushroom Extract Lactic Acid Bacteria Fermented Milk

3-8% by weight of skim milk powder was added to the mushroom extract of Ganoderma lucidum and shiitake mushrooms under certain conditions, homogenized with a blender for 5 minutes, and sterilized at 90 ° C. for 1 hour. After sterilization, the mixture was cooled to about 37-40 ° C., and then inoculated with 1% of the lactic acid bacterium pre-cultured with mixed lactic acid bacteria, followed by fermentation at 37 ° C., and the pH, titratable acidity, and viable cell number were observed from 0 to 6 hours.

(3) Change of pH during fermentation of mushroom extract lactic acid bacteria fermented milk

In order to investigate the acid production capacity of lactic acid bacteria, 100 g of the fermentation sample solution was collected by time and measured directly with a pH meter (Thermo Orion, Model 520, USA). The initial pH of the fermentation broth was 6.53 and 6.38, which was slightly lower than that of the mushroom. As the number of viable cells increased, the pH decreased by the same tendency in all experiments, and after 6 hours, the pH of fermented Lactobacillus Lactobacillus extract was slightly lower than that of pH 4.45. Table 8).

PH Change during Fermentation of Mushroom Extract Lactic Acid Bacteria Fermented Milk Fermented Time (h) Control Mushroom Extract Lactobacillus Fermented Milk 0 6.50 6.38 One 6.35 6.16 2 6.00 5.82 3 5.42 4.88 4 4.93 4.52 5 4.87 4.42 6 4.45 4.37

(4) Change of titratable acidity during fermentation of mushroom extract lactic acid bacteria fermented milk

The tendency of titratable acidity increase with the addition of mushroom extract increased steadily in each experiment until 6 hours after fermentation, similar to the pH change (Table 9). The titratable acidity of fermented broth was slightly higher (0.528%) compared to 0.485% of control. These results suggest that the growth of lactic acid bacteria is promoted by various minerals and amino acids contained in mushrooms, resulting in increased acid production.

The titratable acidity was dissolved well after adding 50 ml of distilled water to 9 g of fermentation sample solution, dropping 4-5 drops of 1% phenolphtalein solution, titrating with 0.1 N NaOH, and measuring the consumption.

Titratable acidity = (0.1 N NaOH consumption-0.1 N NaOH factor * 0.09) / sample weight (g) * 100

Changes in Titration of Fermented Milk Extracted from Mushrooms during Fermentation at 37 ℃ for 6 Hours Fermented time (h) Control Mushroom Extract Lactobacillus Fermented Milk 0 0.118 0.115 One 0.1202 0.128 2 0.154 0.163 3 0.272 0.313 4 0.387 0.405 5 0.450 0.447 6 0.473 0.528

(5) Change in the Number of Viable Cells during Fermentation of Mushroom Fermented Milk

The viable cell count of Lactobacillus Lactobacillus fermented milk was measured at 37 ℃ after aseptically taking an amount of fermented sample solution at each time and diluting the diluted sample to the appropriate concentration in BCP (Bromocresol purple) agar plate (Eiken Chemicha Co., Japan) medium. Yellow colony after 72 hours of incubation was measured.

As a result of measuring the change in the number of lactic acid bacteria during the fermentation process of the mushroom extract addition, it was found that it continues to increase with time. At 6 hours of fermentation, the number of lactic acid bacteria was 8.89∼8.94 log cfu / mL. The number of lactobacillus of fresh liquid and staple fermented milk is more than 10 ⁷ and 10 ⁸ cfu / ml, respectively. In this experiment, the amount of lactic acid bacteria of fresh liquid and staple fermented milk is higher than the standard value in the control and added mushroom extract lactic acid bacteria fermented milk. Suited.

Measurement result of the number of lactic acid bacteria (log cfu / mL) during the fermentation process with mushroom extract Fermented time (h) Control Mushroom Extract Lactobacillus Fermented Milk 0 6.49 6.48 One 7.22 7.25 2 7.98 8.12 3 8.47 8.79 4 8.69 8.89 5 8.87 8.91 6 8.89 8.94

(6) Analysis of General Components of Mushroom Extract Lactic Acid Bacteria Fermented Milk

Nutritional analysis of mushroom extract Lactobacillus fermented milk was analyzed by requesting the Korean Sweet Feed Association. As a result, water content was 92.3%, carbohydrate 5.3%, crude protein 2.53%, and very small amount of 12.01 ppm of cholesterol was detected (Table 11).

Analysis of General Components of Mushroom Extract Lactic Acid Bacteria Fermented Milk (%) moisture Views min Crude fat Crude protein carbohydrate cholesterol 92.3 0.65 0.25 2.53 5.3 12.01 ppm

(7) Determination of Mineral Content in Mushroom Extract Lactic Acid Bacteria Fermented Milk

As a result of analyzing the major mineral contents of Lactobacillus Lactobacillus extract and fermented yogurt, the contents of potassium, sodium and magnesium were detected in Lactobacillus Lactobacillus fermented milk containing mushroom extract more than regular yoghurt. Contained. These results are believed to be extracted from the mineral content of the mushrooms added to the yogurt culture (Table 12).

Analysis of Mineral Components of Lactic Acid Bacteria Fermented Milk (mg / 100 g) Minerals K Ca Na Mg Fe Zn Mn Cu Yogurt 78.9 22.1 10.8 6.6 0.9 - - - Mushroom Extract Lactobacillus Fermented Milk 149.26 6.614 35.681 9.749 0.283 0.343 - -

※-; not detected

(8) Determination of Vitamin Content in Lactic Acid Bacteria Fermented Milk

The vitamin content of mushroom extract Lactobacillus fermented milk was found to be 55,298.95 IU / kg. Vitamin B2 and C were detected at 1.14 mg / kg and 0.52 mg / kg, respectively (Table 13).

Vitamin Content Measurement of Fermented Milk Lactobacillus Extract A (IU / kg) B1 B2 (mg / kg) B6 C (mg / kg) Niacin 55298.95 - 1.14 - 0.52 -

※-; not detected

(9) amino acid content

The constituent amino acids of the mushroom extract lactic acid bacteria fermented milk were measured. Among the 18 amino acids, Glutamic acid was detected the most and Cystine and Tryptophan were less detected, but all 18 amino acids were found to be evenly distributed (Table 14).

Determination of Amino Acid Contents of Mushroom Extract Lactic Acid Bacteria Fermented Milk Amino acid type content (%) Amino acid type content (%) Aspartic acid 0.19 Leucine 0.22 Threonine 0.11 Tyrosine 0.12 Serine 0.14 Phenylalanine 0.12 Glutamic acid 0.51 Histidine 0.07 Proline 0.23 Lysine 0.19 Glycine 0.05 Arginine 0.08 Alanine 0.09 Cystine 0.02 Valine 0.13 Methionine 0.06 Isoleucine 0.10 Tryptophan 0.02

※ written in bold; Essential amino acid

(10) protein pattern comparison

In order to compare protein patterns of normal yogurt and Lactobacillus extract fermented milk, each sample was electrophoresed on SDS-polyacryamide gel to compare protein patterns. Protein electrophoresis was performed with 15% polyacryamide gel. After electrophoresis, the gel was stained with 0.1% coomassie brilliant blue 250R and decolorized with 10% acetic acid and 30% methanol. Molecular weights were expressed as the ratio of the moving distance of the sample according to the relative moving distance of the standard protein (Precision Plus Protein Standards, Bio-Rad Co. Ltd) after development (Laemmli, UK 1970). The protein of Lactobacillus Lactobacillus fermented milk was 50 kDa or more less than the general yogurt but 38 kDa or more was detected more than the general yogurt (FIG. 5).

(11) Whole equivalent of mushroom extract lactic acid bacteria fermented milk

The total equivalent of Lactobacillus extract fermented milk was measured by phenol-sulfuric acid method (Dubois, M. et. Al . , 1956). In other words, 1 ml of 5% purified phenolic water was added to the test tube, each containing 1 ml of ink extract and standard solution, and mixed well. 5 ml of concentrated sulfuric acid was added to develop color by exothermic reaction. Was measured and analyzed. To quantify the total equivalent glucose, glucose, a standard substance, was prepared for each concentration, and the experiment was performed in the same manner as the sample to prepare a calibration curve and to measure the total equivalent of the sample. The total equivalent of Lactobacillus mushroom fermented milk was found to be 13.75 mg / ml.

TLC method was used to confirm the polysaccharide of the mushroom extract lactic acid bacteria fermented milk. As a result, sugars were found near G2 and G3 in general yogurt, and more sugar was found in G2 than G3. On the other hand, mushroom yogurt (Mushroom Extract Lactic Acid Bacteria Fermented Milk) also showed high molecular weight sugars that did not develop on TLC, and showed more sugar in G3 than G2 (Fig. 6).

(12) Determination of Polyphenol Contents of Mushroom Extract Lactic Acid Bacteria Fermented Milk

Polyphenols, which are present in nature a lot, have two or more hydroxyl groups (-OH) attached to the benzene ring. These compounds include green tea, oolong tea, persimmon leaf tea, coffee, apples, immature fruits, It is found in vegetable foods such as strawberries, eggplants, grapes, black beans, red beans, vegetables and fruits, cacao, and red wine. The compound is known to have antioxidant action, lowering blood cholesterol levels because it prevents antioxidant function and digestive tract from absorbing cholesterol.

The content of the polyphenolic compound was measured by slightly modifying the Folin-Denis method using a phenomenon in which the phenolic substance reacted with phosphomolybdate to appear blue (Swain, T. et . Al., 1959). That is, 0.2 ml of Folin-ciocalteau's phenol reagent was added to 2 ml of mushroom extract lactic acid bacteria fermented milk and mixed well, and the mixture was left to stand at room temperature for 3 minutes. After exactly 3 minutes of reaction, 0.4 ml of saturated Na ₂ CO ₃ solution was added, mixed, distilled water was added to make 4 ml, and the mixture was left at room temperature for 1 hour to measure absorbance at 725 nm. In order to quantify the polyphenol content, tannic acid, a standard substance, was prepared at a constant concentration, and was tested in the same manner as the sample to prepare a calibration curve to measure the polyphenol content of the sample. The total polyphenol content of the black mushroom extract used in this experiment was found to be 372.8 μg / ml.

(13) Antioxidant Activity of Mushroom Extract Lactic Acid Bacteria Fermented Milk

The DPPH method is a method of measuring the antioxidant effect by hydrogen donating ability according to the degree of dark purple decolorization as it is reduced by antioxidants such as tocopherol, ascorbate, flavonoid compound, sulfur-containing amino acids such as glutathion, maillard type browning substance, and peptide. Known as (Blois, MS 1958). Free radical activity was measured by slightly modifying the method of Ozcelik (2003). The sample was dissolved in 1 ml of 100% methanol and filtered through a 0.2 μm filter. DPPH (α, α-diphenyl-β-picrylhydrazyl) was dissolved in 0.5% of 100% methanol. 3.75 ml of 0.5 mM DPPH was mixed with 0.25 ml of a sample dissolved in methanol and left for 30 minutes, and then the absorbance decrease was measured at 528 nm. The control group used BHT (butylate hydroxytoluene) which is an antioxidant and used to prevent oxidation of fat. Antioxidant activity of the mushroom extract Lactobacillus fermented milk was significantly higher than the general yogurt, and showed higher antioxidant activity than the representative antioxidant BHT (Fig. 7).

All. Safety review through animal experiment

Repeated dose toxicity tests for the safety evaluation of mushroom-derived Lactobacillus fermented milk were conducted in accordance with the Toxicological Test Standards of the Korea Food and Drug Administration (Notice No. 2009-60, Revised October 21, 2005). Black mushroom fruit body is registered as a vegetable raw material in the food industry.

The rodent mouse was used to purchase 6 weeks of male ICR mouse from Hyochang Science of Daegu. Purified mice were fed for 1 week with normal diet, dispensed per group, placed in cages, temperature 22 ± 2 ℃, humidity 50 ± 5%, contrast ± cycle 12 hours (specification: 07: 00 ~ 19: 00, Memorization: 19:00 ~ 07:00) was raised in the animal control room automatically controlled. ICR mouse (n = 8) was mixed with feed of lactic acid bacteria fermented milk to 10% and fed daily for 5 weeks. Observed general behaviors such as rejection of animal samples every hour up to 12 hours on the day of diet, response to various stimuli at least twice daily (morning and afternoon) for 5 weeks from the day following diet, exercise, breathing, convulsions, General conditions such as hair, skin, and excreta were carefully observed. As a result of observing the general condition of the experimental animals, no apparent abnormalities were observed after the diet of mushroom extract lactic acid bacteria.

As a result of measuring the weight fluctuation of the mouse three times a week, the average increase over the 5 weeks was 6.31 g of control male (CM) and 5.68 g of fermented mushroom Lactobacillus fermented milk. There was no significant difference (Table 15, Figures 8, 9). In addition, no animals died during the experiment, and there was no difference in activity. After the experiment, all mice were necropsied and blood and organs were extracted. There was no difference between the groups in the weighing results of the organs, and it was considered safe from the animal experiments (Table 16).

Weight fluctuation measurement result of mouse Day CM TM One 27.75 ± 0.80 27.88 ± 0.92 3 27.81 ± 0.88 27.56 ± 0.98 5 28.06 ± 0.82 28.00 ± 1.25 8 28.44 ± 1.27 28.69 ± 1.36 10 28.75 ± 1.07 29.00 ± 1.41 12 29.56 ± 1.21 29.00 ± 1.31 15 30.38 ± 1.03 29.25 ± 1.22 17 30.88 ± 0.69 29.75 ± 0.80 19 30.94 ± 0.68 29.74 ± 0.73 22 31.88 ± 0.74 29.75 ± 0.96 24 32.06 ± 0.32 30.63 ± 0.83 26 32.69 ± 0.71 31.69 ± 0.88 29 32.69 ± 1.00 32.25 ± 0.76 31 33.06 ± 1.05 32.81 ± 0.70 33 33.56 ± 1.08 33.25 ± 0.96 36 34.06 ± 0.86 33.56 ± 0.73

* CM; control male, TM; Mushroom Extract Lactobacillus Fermented Milk fed male. n = 8.

Weight measurement result of isolated organ Liver Heart Kidney Spleen Testis Stomach CM 1.82 ± 0.04 0.17 ± 0.01 0.53 ± 0.02 0.15 + - 0.01 0.21 + 0.04 0.84 ± 0.07 TM 1.82 ± 0.04 0.15 + 0.02 0.52 + 0.02 0.14 + 0.02 0.21 ± 0.02 0.82 ± 0.05

* Weight: g, CM; control male, TM; Mushroom Extract Lactobacillus Fermented Milk fed male. n = 8.

Serum is separated from blood from each group, which accounts for approximately 60% of liver damage, GOT, GPT, and serum proteins, and measures albumin, a protein nutritional source for albumin, cholesterol, and triglycerides. The change in chemical value was measured through. As a result, there was no significant difference between the control group and the mushroom extract lactic acid bacteria fermented milk diet group (Table 17). Therefore, it was confirmed that the diet of mushroom extract Lactobacillus fermented milk did not cause serological abnormalities in mice.

Results of chemical measurements as indicators of liver damage GOT
(U / L) GPT
(U / L) TCHO
(Mg / dL) TG
(Mg / dL) CRE
(Mg / dL) BUN
(Mg / dL) ALB
(g / dL) ALP
(U / L) LDH
(U / L) CM 51.67 ± 9.6 48.0 ± 16.5 95.0 ± 6.4 208.5 ± 5.6 0.4 ± 0.0 27.9 ± 0.8 2.9 ± 0.1 77.2 ± 8.7 1067.2 ± 289.9 TM 45.5 ± 13.4 34.17 ± 12.6 92.3 ± 6.4 181.7 ± 10.0 0.4 ± 0.0 25.7 ± 2.1 2.9 ± 0.1 74.2 ± 7.5 886.3 ± 426.0

* CM; control male, TM; Mushroom Extract Lactobacillus Fermented Milk fed male. n = 8.

When the appearance and dissected organs of the experimental animals were visually observed, no abnormal signs were observed. Among the organs extracted for histopathological examination, livers were fixed in formalin to make 5 μm sections, and then HE As a result of staining (hematoxylin and eosin), liver damage was not observed in all the male and female animals (FIG. 10). In the above results, oral administration of the black mushroom extract to rodents (ICR mouse) confirmed the safety of the animal through general safety test methods such as general condition, serological analysis, and histopathological analysis, and it was very safe. No effect was found.

2. Test words  And breeding management

One) Test words

The test eel used 3,200 animals ( Anguilla . Japonica ), which are being cultivated in eel farms (average 10 ~ 30g / mar), and purchased commercial eel food (more than 48% protein, more than 5% fat) for food. Was used. As an additive, the mushroom extract Lactobacillus fermented milk, which is an active substance, was mixed with 2% of the blended feed and supplied once a day (09:00 grade), and the mixed feed was supplied with 1 ~ 2% of the fish body. The 28 ℃ water tank supplied only nutrients (vitamins, digestives, and soy sauces) to general feed, and the mixed feed amount was 1 ~ 2% of the fish weight.

2) Circulation Breeding  Water quality and basic water quality investigation

The circulating filtration system reused breeding water through drum filter, sedimentation tank, fixed filtration material and fluid filtration material, and the breeding water for each tank was rotated 6 times a day, and oxygen was supplied by using a ring blower. Mean DO was maintained at 8 (± 1). Breeding water temperature was maintained in three sections of water temperature 20 ℃, 22 ℃ and 28 ℃ by heating using boiler as a test breeding in circulation system. During the test period, a small amount of return was conducted at regular intervals (1-10% of the total volume once a week) to remove the bottom sludge in the settling tank and the filtration tank. The basic water quality of breeding water was measured using YSI550A and YSI60 products, and dissolved oxygen, pH, etc., and ammonia, nitrous acid, etc., were used for measuring water quality (CHEMetrics).

The water quality measurement result of each section during the study period was 20 ℃, 22 ℃, 28 ℃ using boiler and heater, and dissolved oxygen was 7.5 ~ 8.5 through liquefied oxygen and blower. mg / L, pH 5.3-5.6, ammonia 2-11 mg / L and nitrite 0.1-2.3 mg / L. When a certain amount of ammonia and nitrite rose, the farming method was chosen to reduce the concentration of ammonia and nitrite in the water as much as possible. No chemicals or sodium chloride were used. However, after the water exchange, a small amount of EM bacteria powder was used to make water needed for eel farming. Table 18 shows the results of measuring the water quality such as water temperature, pH, DO, ammonia, nitrite amount for eel growth during the test period. During the breeding period, there was no significant difference, but ammonia and nitrite were slightly higher at 28 ℃.

Water quality results during the test month Water temperature (℃) pH DO (mg / L) Ammonia (mg / L) Nitrous acid (mg / L) 20 22 28 20 22 28 20 22 28 20 22 28 20 22 28 10 20 22 28 5.6 5.6 5.6 7.5 7.5 7.5 2.0 4.0 5.0 0.1 0.2 0.3 11 20 22 28 5.5 5.5 5.5 8.5 8.5 8.5 3.0 5.0 6.0 0.1 0.2 0.3 12 20 22 28 5.6 5.6 5.6 7.9 7.9 7.9 4.0 5.0 6.0 0.2 0.4 0.8 One 20 22 28 5.3 5.3 5.3 8.0 8.0 8.0 2.0 6.0 6.0 0.1 1.2 2.3 2 20 22 28 5.5 5.5 5.5 8.5 8.5 8.5 8.0 8.0 8.0 1.2 2.0 2.0 3 20 22 28 5.5 5.5 5.5 8.5 8.5 8.5 4.0 6.0 8.0 0.8 1.5 2.1 4 20 22 28 5.6 5.6 5.6 8.0 8.0 8.0 6.0 4.0 6.0 0.8 1.6 0.8 5 20 22 28 5.3 5.3 5.3 8.2 8.2 8.2 2.0 4.0 4.0 0.6 0.8 1.7 6 20 22 28 5.3 5.3 5.3 8.4 8.4 8.4 2.0 4.0 4.0 0.2 0.6 1.6 Average 20 22 28 5.5 5.5 5.5 8.2 8.2 8.2 3.7 5.1 6.1 0.46 0.94 1.3 maximum - - - 5.6 5.6 5.6 8.5 8.5 8.5 8.0 8.0 8.0 1.2 2.0 2.3 at least - - - 5.3 5.3 5.3 7.5 7.5 7.5 2.0 2.0 4.0 0.1 0.2 0.3

3) Experimental facilities and Test words  arrangement

The test facility was a circulation system breeding tank, and the breeding water was passed through the settling tank, filtered, and reused. The water temperature was maintained in three sections at 20 ° C, 22 ° C and 28 ° C for each test section by using a boiler and a heater, and dissolved oxygen (DO) using a blower and liquefied oxygen was maintained at 8 or more. The breeding tank used a 2m x 2m x 0.6m tank.

The test fish were 3,200 animals ( Anguilla . Japonica ), which are being cultivated in the eel farm, and 10,30g / million on average.

4) feed

The feed used for the test was purchased commercial eel powder feed (more than 48% protein, more than 5% fat) and supplied 1 ~ 2% of fish body once a day (09:00). Was fed by mixing 2% of dry feed by temperature setting section, and vitamin E, C, digestive, and other nutrients, which are generally added at the time of feeding, were added at a certain period.

3. Eel Ingredient Analysis

The body composition analysis, fat and amino acid analysis of the eel bred in the test were commissioned by Chonnam National University Equipment Management Center, and blood component analysis was commissioned by Neodin Betlab.

 One) Body composition  analysis

As for the body composition of the eels tested, the crude protein content was slightly higher than that of the other parts of the Lactobacillus fermented milk fermented milk at the breeding water temperature of 22 ℃. The content value was relatively low. The high protein content and low fat content mean that the eel can be tasted light. In addition, the water content was the highest in the breeding water temperature 28 ℃. An important result here is that the diet of mushroom extract Lactobacillus fermented milk as an additive was tested at low temperature and proved that there was no significant difference from the normal breeding at 28 ℃, and the energy saving effect (heating fuel) could be clearly confirmed. As a general component of the analysis method, the moisture content was 105 ℃ drying method, crude fat was Soxhlet method, ash content was analyzed by ash method at 550 ℃ ash content. Carbohydrates were limited to the values of water, crude protein, crude fat, and ash at 100.

Body composition analysis (per 100 g) division moisture Ash Crude fat Crude protein carbohydrate 20 ℃ 63.96 1.03 15.60 19.35 0.06 22 ℃ 63.51 1.02 15.85 19.56 0.05 28 ℃ 64.02 1.03 15.78 19.12 0.05

Free amino acid analysis result (Unit: mg / 100g) sample
ingredient 20 ℃ 22 ℃ 28 ℃ Phosphoserine 0.816 0.809 0.819 Taurine 51.843 52.182 45.509 Phosphoethanolamine 3.571 3.459 2.771 Urea 15.119 12.224 12.296 Threonine 3.658 3.046 3.729 Serine 2.765 2.793 2.415 Glutamic acid 4.798 7.066 6.950 Glycine 15.407 17.131 17.671 Alanine 10.805 12.186 13.060 Valine 2.901 3.075 2.687 Methionine 2.098 1.780 1.695 Cysthathionine 0.393 0.388 0.445 Isoleucine 1.671 1.648 1.675 Leucine 3.055 2.926 2.871 Tyrosine 2.030 1.932 1.709 피 phenylalanine 2.487 2.212 1.954 β-alanine 1.102 1.216 1.193 γ-amino-n-butyric acid 0.371 0.288 0.313 Histidine 17.988 19.925 21.744 1-methylhistidine 0.205 0.142 0.239 Carnosine 357.243 366.382 373.722 Anserine 7.486 4.913 9.594 Ornitine 0.337 0.416 0.982 Lysine 6.198 7.841 15.897 Ammonia 18.649 17.655 18.524 Ethanolamine 4.522 4.161 3.789 Arginine 2.742 2.786 8.086 Total 540.258 550.583 572.338

 2) amino acid analysis

The amino acids of eel treated with mushroom extract Lactobacillus fermented milk at different temperature were analyzed for free amino acid and constituent amino acid. The free amino acids and constituent amino acids of the eel at different temperatures were analyzed. As a result of the amino acid content analysis, it was found to be generally evenly distributed including various amino acids, and it was considered that the food value was very high as a source of various nutrients beneficial to the human body (Tables 20 and 21).

Free amino acid analysis conditions      Instrument S433-H (SYKAM)      Column Cation separation column (LCA K07 / Li)      Column size 4.6 ㅧ 150mm      열 온도 37 ~ 74 ℃      Flow rate Buffer 0.45ml / min, reagent 0.25ml / min      Buffer pH range 2.90-7.95      Wavelength 440nm and 570nm

In the pretreatment method for the analysis of constituent amino acids, 0.5 g of the sample was weighed in an 18 ml test tube, 3 ml of 6 N HCl was added thereto, and the resultant was sealed under reduced pressure, and then hydrolyzed in a heating block set at 120 ° C. for at least 24 hours. The hydrolyzed sample was analyzed by removing the acid at 50 ° C., 10 ml of sodium loading buffer, and then filtering 1 ml of the solution using a 0.2 μm membran filter and quantitating with an amino acid autoanalyzer (S433-H) (Tables 22 and 23). ).

Analysis of constituent amino acids (Unit: mg / 100g) sample
ingredient 20 ℃ 22 ℃ 28 ℃ Aspatic acid 1,537.488 1,559.316 1,601.183 Threonine 704.802 711.037 734.890 Serine 656.044 661.766 686.476 Glutamic acid 2,408.582 2,437.474 2,523.878 Proline 601.567 648.857 652.526 Glycine 811.283 841.132 864.642 Alanine 936.346 953.780 975.185 Cystine 80.611 79.314 107.338 Valine 783.730 776.403 790.500 Methionine 464.872 470.624 481.713 Isoleucine 712.166 716.516 726.779 Leucine 1,240.652 1,261.050 1,293.400 Tyrosine 520.799 536.994 551.549 Phenylalanine 635.931 638.096 656.889 Histidine 743.512 749.035 782.045 Lysine 1,432.444 1,446.303 1,499.544 Ammonia 603.294 595.647 608.783 Arginine 1,038.561 1,065.315 1,081.013 Total 15912.684 16148.661 16618.333

Compositional amino acid analysis conditions      Instrument S433 (SYKAM)      Column Cation separation column (LCA K06 / Na)      Column size 4.6 ㅧ 150mm      열 온도 37 ~ 74 ℃      Flow rate Buffer 0.45ml / min, reagent 0.25ml / min      Buffer pH range 3.45-10.85      Wavelength 440nm and 570nm

3) fatty acid analysis

After breeding test in which mushroom extract Lactobacillus fermented milk was administered in each section, the fat content was slightly lower in 20 ℃ section than in 28 ℃, which is the general nurture water temperature (Table 24), and the saturated fatty acid content was 20 ℃ section ( 47.78 mg / 100 g), 28 ° C section (49.94 mg / 100 g), 22 ° C section (52.87 mg / 100 g), etc.Alkenes with only one double bond (hydrocarbon compounds belonging to ethylenic hydrocarbons) ) Monoenes were found at 28 ° C (40.97 mg / 100 g), 22 ° C (44.57 mg / 100 g), and 20 ° C (45.02 mg / 100 g). Polyenes, polyunsaturated organic compounds, were found at 28 ° C (5.49 mg / 100 g), 22 ° C (6.16 mg / 100 g), and 20 ° C (7.2 mg / 100 g). In conclusion, 47.78% of saturated fatty acids and 52.22% of unsaturated fatty acids. Detailed fatty acid content of each section is shown in Table 24.

Fatty Acid Analysis (Unit: mg / 100g total fatty acids) sample
ingredient 20 ℃ 22 ℃ 28 ℃ Caprylic Acid (C8: 0) 0.21 0.14 0.18 Capric acid (C10: 0) 0.16 0.10 0.08 Undecanoic acid (C11: 0) 0.06 0.03 0.02 Lauric acid (C12: 0) 0.21 0.15 0.13 Tridecanoic acid (C13: 0) 0.06 0.06 0.05 Myristic acid (C14: 0) 6.63 7.01 6.29 Pentadecanoic acid (C15: 0) 0.82 0.75 0.62 Palmitic acid (C16: 0) 24.12 24.45 24.90 Heotadecanoic acid (C17: 0) 1.11 1.06 0.66 Stearic acid (C18: 0) 7.20 8.54 7.88 Arachidic acid (C20: 0) 2.18 1.30 0.33 Heneicosanoic acid (C21: 0) 4.46 8.79 8.33 Behenic acid (C22: 0) 0.14 0.11 0.12 Tricosanoic acid (C23: 0) 0.24 0.23 0.24 Lignoceric acid (C24: 0) 0.18 0.16 0.11 Saturates 47.78 52.87 49.94 Myristoleic acid (C14: 1) 0.27 0.30 0.38 cis-10-pentadecenoic acid (C15: 1) 0.02 0.02 0.01 Palmitoleic acid (C16: 1) 7.78 6.63 8.03 cis-10-Heptadecenoic acid (C17: 1) 0.54 0.70 0.22 Elaidic acid (C18: 1n9t) 7.40 5.90 5.02 Oleic acid (C18: 1n9c) 26.57 25.48 27.82 cis-11-Eicosenoic acid (C20: 1) 1.80 1.24 2.29 Erucic acid (C22: 1n9) 0.25 0.25 0.24 Nervonic acid (C24: 1) 0.38 0.45 0.57 Monoenes 45.02 40.97 44.57 Linolelaidic acid (C18: 2n6t) 3.54 2.47 2.10 Linoleic acid (C18: 2n6c) 1.11 1.34 0.96 cis-11,14-Eicosadienoic acid (C20: 2) 0.48 0.23 0.26 cis-13,16-Docosadienoic acid (C22: 2) 0.03 0.07 0.05 γ-Linolenic acid (C18: 3n6) 0.26 0.42 0.58 Linolenic acid (C18: 3n3) 0.53 0.66 0.70 cis-8,11,14-Eicosatrienoic acid (C20: 3n6) 0.51 0.24 0.23 cis-11,14,17-Eicosatienoic acid (C20: 3n3) 0.27 0.35 0.34 cis-5,8,11,14,17-Eicosapentaenoic acid (C20: 5n3) 0.23 0.15 0.15 cis-4,7,10,13,16,19-Docosahexaenoic acid (C22: 6n3) 0.24 0.22 0.13 Polyenes 7.20 6.16 5.49

4) Serum biochemical test

Serum biochemical tests of eel at different temperatures of the circulatory system were performed. Serum is separated from blood from each group, which accounts for approximately 60% of liver damage, GOT, GPT, and serum proteins, and measures albumin, a protein nutritional source for albumin, cholesterol, and triglycerides. The change in chemical value was measured through. As a result, GOT and GPT levels, which are indicators of liver damage, were significantly lower in the Lactobacillus fermented milk diet group. The total cholesterol level (TCHO) was lower than 28 ℃ but the triglyceride content was slightly higher. The detailed serum biochemical values for each section are shown in Table 25. As a result of analysis of blood composition of eel treated with mushroom extract Lactobacillus fermented milk by circulation temperature, lipid was highest in 20 ℃ and glucose (blood glucose level) was lowest in 28 ℃ breeding temperature. It was smooth and activated to supply energy to fish, and the feeding rate was high, so it was good to grow.

Blood component analysis division LDH  Amylase Creatinine T.chol TG GLU ALBUMIN AST ALT 20 ℃ 782.7 560.7 0.50 353.7 820 110.0 1.6 456.3 20.7 22 ℃ 251.0 378.8 0.50 382.0 660 125.0 1.37 74.3 3.3 28 ℃ 508.0 213.8 0.13 473.3 542 89.7 1.6 183.0 25.9 unit U / L mg / dL U / L mg / dL mg / dL U / L U / L mg / dL mg / dL

Test equipment: Forcyte (CDC tech., USA), BS-400 (MINDRAY, CHINA) LDH: Glycolytic enzyme, Amylase: Enzyme, Creatinine: Protein waste, T.chol: Total cholesterol, TG: Neutral lipid, GLU: Glucose (blood sugar level), RBC: total red blood cell count, Hb: hemoglobin

 5) Weight gain

The test fish were 3,200 animals ( Anguilla . Japonica ), which are being cultivated in the eel farm. The feed was fed without feeding, and the remaining amount was discarded after collection. For food, commercial eel sacred feed (over 48% protein, over 5% fat) was purchased and used. As an additive, the mushroom extract Lactobacillus fermented milk, which is an active substance, was supplied once a day (09:00 grade) by mixing 2.0% of the mushroom extract Lactobacillus fermented milk with the feed added. Suddenly.

The growth rate of eel administered at different temperatures was slightly lower than that of the control (28 ℃) and the feed efficiency was slightly lower than that of the control (28 ℃). However, the reduction of carbon emissions due to the energy (heating fuel) saving effect, which is a high cost in eel farming and causes environmental problems, was considered to be more effective than the control (28 ℃). In addition, there was no significant difference in mortality during the breeding period.

Weight gain and feed efficiency at each temperature Purchase test words
(October 28, 2010) Primary measurement
(2011. 01. 31) Secondary measurement
(2011. 04. 28) Final measurement
(2011.07.15) Temperature weight weight Feed efficiency weight Feed efficiency weight Feed efficiency 20 ℃ 16 kg 52.1 kg 54% 120.5 kg 61% 176.8 kg 64% 22 ℃ 16 kg 52.8 kg 55% 118.9 kg 59% 176.1 kg 65% 28 ℃ 16 kg 57.5 kg 62% 130.5 kg 66% 189.5 kg 67%

4 . Example - Exponential Equation Eel Breeding at the Pond

Eel low-temperature mass culture experiments were carried out in the exponential type yangjang, and was carried out in a water bath of about 10 x 10 m at 23 ℃. In the eel culture, the water level of the cultured tank was adjusted to about 1 m, and a liquefied oxygen system and a circulation pump were installed to supply oxygen. The temperature of each tank was adjusted to about 23 ° C and 28 ° C, and about 60 to 90 g in each tank. Eel was added to the density and cultured. Feed feeding method was used after attaching the food container to the wall of the culture tank and making mushroom extract lactic acid bacteria fermented milk into the form of dough mixed with feed. The dose was supplied in a range of 1 to 3% of the body weight to eat all within 1 hour.

Three months after the administration of the experimental diet, the eel weighed from 200 to 300g was fasted a day before, and then taken in a culture tank, weighed in the state of paralyzing on ice, and selected only eels with a long body length and heparin treatment to prevent blood coagulation. Heparin-free blood was collected for blood and serum separation, and muscle was taken from the end of the intestine and used as an analytical material. Serum for lipid measurement was heparin untreated blood was allowed to stand at room temperature for 30 minutes and then centrifuged for 15 minutes at 3,000 rpm. Blood clinical biochemical index was analyzed by Neodine BetLab Co., Ltd. The measured results are as follows.

(1) weight and length measurement

In the cultivation of eel in aquaculture experiment, mushroom extract lactobacillus fermented milk, which was developed at 23 ° C, was added 2.0% to the diet, and the mushroom extract lactobacillus fermented milk diet group showed blacker color and larger size. Mushroom Lactobacillus fermented milk diet group showed 45 g (about 24%) in length and 4.84 cm (about 10%) in growth rate compared to the control (Table 27).

Eel weight and body length (23 ℃) Control Test Weight (g) Length (cm) Weight (g) Length (cm) One 186.6 48 228.1 54 2 193.5 49 236.8 55 3 192.2 49 223.5 52 4 172.3 48 201.2 50 5 188.8 49 234.6 53 6 164.6 47 247.3 55 Average 183.00 48.33 228.58 53.17

In FIG. 11, the difference in meat color is that the control is gray in the back and the pear is white, and the fin of the treatment is black, which is a color of natural eel, and the pear is slightly golden. This result was attributed to the fact that the blended feed supplemented with Lactobacillus Lactobacillus fermented milk had an effect on the synergistic effect of the contents of inorganic substances (Fe, Zn) and the synergistic effects of RBC and Hb on the production and transport of blood. In addition, some malformations (curved back) appeared in the control, but the BHT-Vem diet group showed little such phenomenon (FIG. 12).

(2) feeding and mortality measurements

Feeding rate and pesa rate were measured at 23 ° C and 28 ° C in aquaculture eels. Based on the feed rate at the time of the final inspection, the feeding rate of mushroom extract lactic acid bacteria fermented milk was added 2.0% to the feed, and the control group did not add the mortality rate. The total number of dead deaths was added together and measured. Although there were some differences depending on the water quality of each farm, the feeding rate increased 31% compared to the control at 28 ℃, and 43% higher than the control at 23 ℃. It was 7% lower than the control and 13% lower than the control at 28 ° C (Table 28). As shown in the gene expression studies, growth and feed-related gene changes were caused by the development of high growth factor receptor protein and glucokinase protein. In addition, the decrease in mortality rate is thought to be the result of increased immunity due to high expression level of antigen (antibody-forming protein).

Feed rate and mortality for each temperature
Feed rate Mortality rate Temperature size
(4 ~ 5) Control Test Control Test 23 ℃ 2.6 ton
(14,200) 0.9% 1.29% 13%
(1,846) 6%
(852) 28 ℃ 2.7 ton
(14,500) 1.31% 1.72% 22%
(3,190) 9%
(1,305)

(3) Serum biochemical test

Serum is separated from blood from each group, which accounts for approximately 60% of liver damage, GOT, GPT, and serum proteins, and measures albumin, a protein nutritional source for albumin, cholesterol, and triglycerides. The change in chemical value was measured through. As a result, GOT and GPT levels, which are indicators of liver damage, were significantly lower in the Lactobacillus fermented milk diet group. However, total cholesterol (TCHO) and triglyceride (TG) contents were slightly higher. This result is considered to be a phenomenon that the mushroom extract fermentation lactic acid bacteria group grows faster than the control group, resulting in the increase of the overall weight and body length. Total erythrocyte counts (Hb) and hemoglobin (Hb), which are directly related to the growth of eel, were about two to three times higher (Table 29).

Measurement result of change of chemical value of eel blood (23 ℃) Glucose
(Mg / dL) GOT
(U / L) GPT
(U / L) TCHO
(Mg / dL) TG
(Mg / dL) CRE
(Mg / dL) ALB
(g / dL) Amylase
(U / L) LDH
(U / L) RBC
(M / uL) Hb
(g / dL) CM 109.25 226.3 14.5 308.7 620 0.26 0.83 247.5 643.4 0.725 3.71 TM 122.4 142.3 3.03 467.7 727.3 0.17 1.1 201.0 555.2 1.443 9.28

* CM; control eel, TM; BHT-Vem fed eel. n = 6.

(4) Analysis of Eel Meat Composition

(A) Analysis of general components of eel

Analysis of the general constituents of the eel in the mushroom-derived Lactobacillus fermented milk was commissioned by Entec Research Institute. As a result, carbohydrate and crude ash increased in small amount, crude fat decreased by about 5 g and crude protein increased by about 3 g (Table 30).

Common Components of Mushroom Extract Lactic Acid Bacteria Fermented Eel (g / 100 g, 23 ℃) moisture Views min Crude fat Crude protein carbohydrate Control 69.29 1.21 18.34 14.07 0.09 BHT-Vem 68.06 1.23 13.38 17.17 0.3

Analysis of the major mineral contents of eel dietary extract Lactobacillus fermented milk showed that the calcium content (83.78 mg / kg increase) and magnesium content (78.61 mg / kg increase) were significantly higher than the control. Iron and zinc were increased in small amounts and potassium, sodium, manganese and copper were decreased (Table 31).

Mineral Content of Mushroom Extract Lactic Acid Bacteria Fermented Eel (mg / kg, 23 ℃) K Ca Na Mg Fe Zn Mn Cu Control 2271.26 316.13 1091.55 259.45 2.93 17.42 0.25 0.23 BHT-Vem 2005.23 399.91 956.33 338.06 3.25 18.48 0.22 0.16

The vitamin content of eel dietary extracts from Lactobacillus Lactobacillus fermented milk was 0.24 mg / 100 g of scurvy prevention, antioxidant effects, and free radicals, and niacin with greater nutrition to the skin, digestive system, and nervous system. This increased by 0.87 mg / 100 g (Table 32).

Vitamin Content of Mushroom Extract Lactic Acid Bacteria Fermented Eel (mg / 100g, 23 ℃) A (μg / 100 g) B1 B2 B6 C Niacin Control 1095.23 0.42 0 0 0.10 2.51 Mushroom Extract Lactobacillus Fermented Milk 962.96 0.34 0.16 0 0.34 3.38

The constituent amino acid of the eel in which the mushroom extract lactic acid bacteria fermented milk was measured was measured. Glutamic acid was the most detected among the amino acids in the experimental and control groups, while Cystine and Lysine were the least detected. The experimental group detected about 20-30% more amino acids than the control group.

Amino Acid Measurement Results of Mushroom Extract Lactic Acid Bacteria Fermented Eel (23 ℃) Amino acid type Content (mg / kg) Amino acid type Content (mg / kg) Control BHT-Vem Control BHT-Vem Aspartic acid 946.67 1235.84 Leucine 833.8 1075.63 Threonine 436.45 560.31 Tyrosine 373.29 477.1 Serine 342.48 452.48 Phenylalanine 429.14 544.93 Glutamic acid 1352.79 1796.32 Histidine 903.91 1156.95 Proline 466.38 622.17 Lysine 102.63 135.33 Glycine 660.31 919.37 Arginine 675.89 897.34 Alanine 652.38 865.91 Cystine 146.73 171.0 Valine 505.65 628.99 Methionine 323.03 411.52 Isoleucine 489.0 608.74

※ written in bold; Essential amino acid

(B) fatty acid composition analysis

Mushroom extract Lactobacillus fermented milk diet eel and fatty acids of the control were compared and analyzed. The result is a 50% decrease in saturated fatty acid capric acid (C10: 0), 18% increase in myristic acid (C14: 0), and 0.7 for palmitic acid (C16: 0). % Increase, stearic acid (18: 0) increased 12%, and arachidic acid (C20: 0) increased 62.5%. In polyunsaturated fatty acids, alpha-linolenic acid (18: 3) increased by 17.2%, docosahexaenoic acid (DHA, C22: 6) increased by about 4.8%, and ω- The 6 fatty acid arachidonic acid (C20: 4) increased by 40%. However, the ω-9 fatty acid oleic acid (C18: 1) decreased 6.3% and eicosanoic acid (C20: 1) decreased 14.7% (Table 34).

Comparison of Fatty Acids in Mushroom Extract Lactic Acid Bacteria Fermented Eel (23 ℃) Composition of fatty acids (%) C10: 0 C12: 0 C14: 0 C15: 0 C16: 0 C16: 1 C17: 0 C17: 1 C18: 0 C18: 1 C18: 2 C18: 3 C20: 0 C20: 1 C20: 4 C22: 6 Control 0.02 0.10 5.14 0.22 30.96 10.07 0.38 0.23 6.35 31.88 1.48 0.29 0.16 3.39 0.47 8.19 S.D. 0.01 0.01 0.12 0.03 0.02 0.23 0.20 0.20 0.30 0.30 0.06 0.01 0.01 0.21 0.02 0.31 Test 0.01 0.10 6.07 0.22 31.18 10.64 0.37 0.37 7.11 29.87 1.40 0.34 0.26 2.89 0.66 8.58 S.D. 0.00 0.02 0.24 0.02 0.65 1.05 0.02 0.02 0.62 1.52 0.25 0.01 0.02 0.20 0.03 0.23

(C) meat elasticity test

Six samples of eel (12 samples) were used for the analysis of the texture of the eel. The eel sample for tissue elasticity test was used by cutting 5 cm in the tail direction from the dorsal fin and removing the skin of the cut tissue. The eel elasticity was measured by shear force and data analysis using a texture analyzer, and the texture conditions are shown in Table 35.

Eel texture test conditions (Texture tester operating conditions) Operating conditions Option Puncture test Force unit g Control probe 2 mm needle (stainless steel) Pre-test speed 1.0 mm / sec Test speed 1.0 mm / sec Post-test speed 1.0 mm / sec Trigger type Auto Trigger force 5 g

The elasticity analysis results according to the conditions of Table 35. are as follows (Table 36.). As a result of analyzing the elasticity, the test group (added mushroom extract Lactobacillus fermented milk) showed 17% higher elasticity than the control group. This result is considered to be one of the key key points when branding the eel that feeds this product.

Eel Elasticity Analysis Results (23 ℃) No. Shear force (g force) Average Force (g) Control-1 167.1 210.5 Control-2 235.0 Control-3 209.6 Control-4 217.3 Control-5 208.1 Control-6 226.3 Test -1 276.0 247.6 Test -2 234.3 Test -3 258.8 Test -4 225.0 Test -5 226.0 Test -6 265.4

(D) Sensory tests

The results of sensory evaluation through the panel (university professor, broadcasting director, doctor, lawyer, grilled eel, etc.) showed that the treatment group responded well to all items of fragrance, taste, juiciness, texture, and general symbol. In particular, the odor, meat quality, taste, etc., which are the most problematic problems in eel meat, were confirmed.

II . Aquaculture method using bioactive substance as eel feed additive

1. Test words and additives

The test fish were 7,360 animals ( Anguilla . Japonica ), which are being cultivated at our test site, and the food was purchased from commercial eel sacred feed (more than 48% protein, more than 5% fat). It was.

As the additives, biophysical porphyrins were mixed with 0.1% by weight, 0.5% by weight, and 1% by weight of the feed to the feed once a day (09:00 grade). Feeding amounted to 2% of the fish.

Porphyrin, used as an additive, is a compound containing various side chains in porphine, and constitutes pigment components such as hemoglobin, cytochrome, and chlorophyll, which play an important role in the redox reaction in vivo. The structure has been studied since the beginning of the 20th century, and H. Fischer succeeded in the synthesis. 14 shows the general structural formula of porphyrin.

It is a compound which comprises pigment | dye components, such as hemoglobin, cytochrome, and chlorophyll which play an important role in the redox reaction in a living body. Naturally found porphyrins and simple derivatives thereof are classified into thioporphyrin, mesoporphyrin, protoporphyrin, deuteroporphyrin, hematoporphyrin, uroporphyrin, etc., depending on the type of side chain, and depending on the location of the side chain. It is called Type I, II, III, etc. Porphyrin is available from http://www.sigmaaldrich.com/.

2. Breeding management

1) Breeding management and basic water quality survey

The circulating filtration system reused the breeding water through drum filter, sedimentation tank, fixed filtration material, and fluid filtration material. Breeding water temperature was maintained at 27 (1) by heating using a blower heater and a boiler as a test breeding in the circulation system.

During the test period, a small amount of return was conducted at regular intervals (1 ~ 3% of the total volume once a week) to remove the bottom sludge of the sedimentation tank. The basic water quality of breeding water was measured using YSI550A and YSI60 products, and dissolved oxygen, pH, etc., and ammonia, nitrous acid, etc., were used for measuring water quality (CHEMetrics).

During the study period, the water quality was maintained at 26 to 29 by hot air and boiler operation as shown in Table 37. The dissolved oxygen was 6.5 ~ 7.2 / L, the pH was 4.7 5.3, the ammonia was 5 11 / L and the nitrite was 0.1 2.3 / L.

Water quality results during the test date Water temperature () pH DO (/ L) Ammonia (/ L) Nitrous acid (/ L) March 3 ~ 8 28 (1) 5.0 (0.3) 7.0 (0.2) 6.0 (1) 0.2 (0.1) March 9-15 27 (1) 5.2 (0.2) 6.9 (0.2) 8.0 (2) 0.2 (0.1) March 16-22 27 (1) 4.9 (0.2) 6.7 (0.2) 10.0 (2) 1.5 (0.1) March 23-29 27 (1) 5.1 (0.2) 7.0 (0.2) 9.0 (1) 1.8 (0.1) March 30-April 5
April 6-12
April 13-19
April 20-24 27 (1)
28 (1)
28 (1)
27 (1) 5.1 (0.2)
5.0 (0.2)
5.1 (0.2)
5.1 (0.2) 7.0 (0.2)
6.8 (0.2)
6.9 (0.2)
6.7 (0.2) 10.0 (1)
9.0 (1)
10.0 (1)
10.0 (1) 2.1 (0.1)
1.9 (0.1)
2.0 (0.1)
2.2 (0.1) Average 27.4 5.06 6.88 9.00 1.46 maximum 29.0 5.3 7.2 11.0 0.1 at least 26.0 4.7 6.5 5.0 2.3

2) Experimental breeding facility and Test words  arrangement

The test facility was a circulation system breeding tank, and the breeding water was passed through the settling tank, filtered, and reused. The water temperature was maintained at 26 ~ 28, and dissolved oxygen (DO) using the blower was maintained at 6.5 or higher. The breeding tank used 4.54.51m tank.

The test fish were placed in four groups of 160kg of eel ( A. japonica ), which is about 87g from 2008, being grown in Korea at the test site, and the test period was 53 days from March 3 to April 24 by the concentration of functional substances. Additive dosing tests were performed.

3) feed

The feed used for the test was purchased commercial eel powder feed (more than 48% protein, more than 5% fat) and supplied 2% of the fish's weight once a day (09:00). 0.1%, 0.5%, and 1% were mixed and fed, and vitamin E, C, digestive and other nutrients that are commonly added at feed level were not added, and no antibiotics or other drugs were used. .

3. Eel ingredient analysis and A heavy dose

The body composition analysis of the eel bred for the test was commissioned by the equipment management department of Chonnam National University, and the blood component analysis was commissioned by Neodin Betlab Co., Ltd.

 One) Body composition  analysis

As shown in Table 2, the cholesterol content of the test eel was lower than that of the control and other functional substances in the 0.1% of the sample, and the fat content of the 0.5% of the sample was relatively low.

Body composition analysis (per 100 g) division carbohydrate Fat protein Ash cholesterol moisture Control 0.070 18.512 16.204 0.918 435 64.296 0.1% 0.248 18.600 17.366 0.807 355 62.979 0.5% 0.842 16.582 15.188 0.855 387 66.533 One% 0.343 16.827 15.846 0.886 511 66.099

Chonnam National University Equipment Management System, Inspection Equipment: Forcyte (CDC tech., USA), BS-400 (MINDRAY, CHINA), Electric Furnace, Elemental Analyzer

2) Blood component analysis

As a result of analysis of blood composition of eel administered by the concentration of functional substance, lipid was highest in the control as shown in Table 3, and glucose (blood glucose level) was the lowest in the 0.5% addition dose section, so blood supply was smooth and supply of energy to fish was reduced. It was activated, and the feeding rate was high, and the growth was good.

Blood component analysis division LDH  Amylase Creatinine T.chol TG GLU RBC Hb Control 2,183 439.7 2.90 435 1209.7 145.0 1.73 7.7 0.1% 2,505 286.4 2.47 355 915.3 158.7 1.96 12.8 0.5% 1,717 300.0 1.33 387 759.3 188.7 1.78 12.5 One% 2,661 288.6 1.67 511 1078.7 108.7 2.60 17.0

Neodin Bevel Lab, Inspection Equipment: Forcyte (CDC tech., USA), BS-400 (MINDRAY, CHINA)

LDH: Glycolytic enzyme, Amylase: enzyme, Creatinine: protein waste, T.chol: total cholesterol, TG: neutral lipid, GLU: glucose (blood sugar), RBC: total red blood cell count, Hb: hemoglobin

3) Weight gain

The growth rate after the administration of functional substance concentration was increased by 53kg of 160kg eel in each test group, which was slightly higher than the control group. The efficiency was 73.5%, the highest growth rate. In addition, mortality during breeding period was not significantly different in each section, but overall was lower than control.

As a result of the test with the administration of bioactive substance in the eel, the functional material used in the eel was tested by the addition concentration using the bioactive substance to increase the activation and feeding rate of the energy supply in the fish of the eel. It was found that the added dose section had low fat content and good growth rate.

In addition, the eel ingested with the bioactive substance was lower in glucose than the control group, so the blood supply was smooth, and the energy supply in the eel was activated, thus the feeding rate was high, and no fish disease occurred during the test period. It is thought to be effective for aquaculture product safety and branding of low fat eel.

The method of eel feeding using physiologically active substances derived from natural products with no side effects using mushroom extracts can improve energy efficiency and reduce carbon emissions by saving energy for warming the farm by raising them at low temperature (23 ℃). In addition, it is expected to produce economic profits of producers of natural products as well as economic profits of producers by improving fish disease resistance by enhancing natural immunity and improving productivity by promoting growth. In addition, it will be able to create economic profits for farmers and raw materials suppliers (mushroom farmers) and expand fishery farming as a whole, and seek to boost domestic demand, replace imports, and export, ultimately contributing to national competitiveness.

Claims (3)

a) Extract one or more mushrooms selected from Ganoderma lucidum mushroom, shiitake mushroom, ink mushroom, hot water extract for 2 hours at 40-60 ℃, 2 hours at 70-90 ℃, and 2 hours at 110-130 ℃ and filter the supernatant with a filter. After removing the impurities, centrifuged again at 8,000 rpm for 20-40 minutes to remove the precipitates, and each extract was mixed and 3-8% by weight of skim milk powder was added to the mushroom extract obtained by autoclaving at 10-130 ° C. for 20-40 minutes. Homogenizing for 3-7 minutes and then sterilizing 1-2 hours at 90 ℃;
b) Lactobacillus acidophilus (KACC No. 12419 ) , Streptococcus thermophillus (KCTC No. 3658) ) and Bifidobacterium longgum after cooling to 37-40 ° C after sterilization Inoculating 1% (V / V) of lactic acid bacteria starter, which is a mixed strain of Bifidobacterium longum (KACC No. 16649) , and fermenting at 37 ° C. for a predetermined time;
c) 1-3% by weight of the mushroom extract lactic acid bacterium fermented milk prepared in step b) and 0.5-1% by weight of porphyrin 100% by weight of the blended feed is additionally mixed with dough water to form a dough feed. Manufacturing;
d) Feeding the dough feed to the feed of the eel is kept in the tank while maintaining the breeding water temperature of 23-28 ° C. of the cultivation tank, characterized in that the step of feeding 1 to 3% by weight of the fish once a day Eel farming method using bioactive substance
0.5-1% by weight of porphyrin in addition to 100% by weight of the blended feed is prepared by kneading feed with the dough water, while maintaining the breeding water temperature of the culture tank in the nursing tank at 23-28 ℃ Eel farming method using physiologically active substances, characterized in that feed 1 to 3% by weight of the fish once a day to feed the eel
The method of claim 1 or 2 wherein the number of dough is at least one mushroom selected from Ganoderma lucidum mushroom, shiitake mushroom, ink mushrooms for 2 hours at 40-60 ℃, 2 hours at 70-90 ℃, 2 hours at 110-130 ℃ Mushroom extract obtained by hot water extraction, filtering the supernatant with a filter to remove impurities, centrifugation at 8,000 rpm for 20-40 minutes to remove precipitates, mixing each extract and autoclaving at 10-130 ℃ for 20-40 minutes Eel culture method using a bioactive material, characterized in that obtained by adding 3 to 8% by weight of skim milk powder to the homogenized for 3-7 minutes and then sterilized 1-2 hours at 90 ℃

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