KR20120020823A - Functional feed using natural materials and manufacturing the functional feed - Google Patents

Abstract

PURPOSE: Functional feed using natural ingredients, and a producing method thereof are provided to offer excellent astringency to the feed by using tannin contained in agricultural waste. CONSTITUTION: A producing method of functional feed using natural ingredients comprises the following steps: preparing a tannin by-product extracted from agricultural products; mixing the tannin by-product with germ meal in a weight ratio of 1:1-9; injecting tannin-utilizing bacteria into the mixture, and fermenting the mixture; and drying the fermented feed. The tannin by-product extracted from the dried and crushed agricultural products using water, ethanol, or acetone.

Description

FUNCTIONAL FEED USING NATURAL MATERIALS AND MANUFACTURING THE FUNCTIONAL FEED}

The present invention relates to a functional feed using a natural material and a method of manufacturing the same, and more particularly, by using tannin by-products extracted from agricultural waste and tannin-soluble strains isolated therefrom, without antibiotics and excellent antibacterial and antioxidant activity. A method of making a functional feed and a functional feed produced by the method.

Livestock farmers have tried to improve productivity by adding antibiotics to livestock feeds, since antibiotics can be easily added to livestock feed to prevent disease and increase livestock gains. However, two or more kinds of antibiotics as feed additives are generally added and mixed, and the abuse of these antibiotics causes the problem of antibiotics remaining in livestock and the occurrence of antibiotic resistant bacteria.

Due to these problems, the minimization of antibiotics in the feed is legalized in developed countries, and it is strictly regulated to use antibiotics by self-diagnosis on the farm without prescription. In Korea, regulations on antibiotic use have been tightened, and the use of antibiotics, which was added to feed mixes, has been banned in feed companies.

Various studies have been conducted to replace antibiotics in Korea, but until now, no excellent products have been developed to replace antibiotics, and randomly mixed organic acids, lactic acid bacteria, and immunostimulants, which are considered to be effective in the market, are used. It is true.

To be an antibiotic substitute, it must first have an antimicrobial activity similar to that of antibiotics, and must be a safe substance that is non-toxic and does not accumulate in the body. In addition, in terms of price, it should be more economical than antibiotics in order to have a useful and business product. Therefore, the material that is a raw material of the natural antimicrobial agent should be easily available and low in price.

Tannin is a substance widely distributed in the plant system. It is condensed to produce red or brown precipitates, called probaphene, by polymerization with a hydrolysis type, which is hydrolyzed when heated with dilute acid to produce gallic acid or ellagic acid. Can be classified as a type. In particular, hydrolyzed tannins are used as astringents, hemostatic agents and formal medicines, have an effect of preventing diarrhea, and have been reported to have antiallergic, antiviral and antibacterial effects. Tannins are present in many unripe fruits and seeds, which are a cause of astringent taste and browning in processing, and are largely contained in chestnut peels, green tea waste, dried persimmon and acorn processing waste.

Many studies have been conducted on the recovery and use of tannins from agricultural wastes, but most of them are used as flocculants in wastewater treatment.

Research on various effects of plant extracts has been reported, and the number of related domestic patent applications, which were only 100 cases until the 1990s, has more than doubled since 2000. This is because various studies have been conducted to replace antibiotics and develop new natural products by paying attention to the antioxidant activity, bioactive substance content, anticancer effect, antibacterial and antiviral effects that the plant extract contains. Many domestic native plants and herbs have been reported to have antimicrobial effects in addition to globular tree, red ginseng, baekbaekpi, hwangbaek, eoseongcho, licorice, green tea, sambaekcho, and mugwort.

Chestnut husks containing about 25% of tannins are discarded 35,000 tons annually in Korea, and acorns containing about 6.6% of tannins consume about 14,000 tons annually. Approximately 20 times of wastewater occurs and most tannins are contained in this wastewater. In addition, persimmon husks and tea by-products contain tannins, and about 10,000 tons of tannins are contained in agricultural wastes a year.

The present invention has been made in view of the above problems and the above, the technical problem to be achieved by the present invention is to collect and utilize the tannin that is contained in the agricultural waste is discarded high convergence, excellent antibacterial and antioxidant effect The present invention provides a natural feed material and a method of manufacturing the same.

In order to achieve the above object, the present invention provides a functional feed composition, a functional feed and a method of producing a functional feed.

The present invention provides a functional feed composition comprising tannin by-products, embryos, and tannin-soluble strains extracted from agricultural products.

In addition, according to the present invention, preparing a tannin by-product extracted from agricultural products; Mixing the tannin by-product and the embryo foil in a weight ratio of 1 to 9; Inoculating the mixture with a tannin soluble strain; Fermenting the mixture inoculated with the strain; And it provides a method of producing a functional feed comprising the step of drying the fermented feed and the functional feed produced by the method.

According to the production method of the present invention, it is possible to efficiently recover tannin by-products having high astringent and antibacterial activity from agricultural wastes containing tannins, and fermented them under optimum conditions to produce functional feeds having excellent antibacterial and antioxidant effects. have. According to the present invention, by utilizing the agricultural waste can not only reduce the farm waste, but also through the microbial fermentation to enhance the immunity of livestock without the addition of antibiotics, inexpensively producing a natural feed material with excellent antibacterial and antioxidant activity can do.

Figure 1 is a photograph showing the results of the antimicrobial activity of the ethanol extract against VRSA strain (Vancomycin-Resistant Staphylococcus Aureus) strain.
Figure 2 is a photograph showing the results of the antimicrobial activity test against MRSA strain (Methicillin-Resistant Staphylococcus Aureus) strains of ethanol extract.
Figure 3 is a photograph showing the antimicrobial activity test results for B. coagulance strains of ethanol extract.
Figure 4 is a photograph showing the antimicrobial activity test results for L. monocytogene strain of ethanol extract.
Figure 5 is a photograph showing the antimicrobial activity test results for the E. coli strain of ethanol extract.
Figure 6 is a photograph showing the antimicrobial activity test results for S. flexneri strain of ethanol extract.
Figure 7 is a photograph showing the results of the antimicrobial activity against the strain VRSA (Vancomycin-Resistant Staphylococcus Aureus) strains.
Figure 8 is a photograph showing the results of the antimicrobial activity of the strain MRSA (Methicillin-Resistant Staphylococcus Aureus) strains of water extract.
Figure 9 is a photograph showing the results of the antimicrobial activity test against B. coagulance strains of water extract.
10 is a photograph showing the results of the antimicrobial activity test against L. monocytogene strain of water extract.
Figure 11 is a photograph showing the results of the antimicrobial activity test against E. coli strains of water extract.
12 is a photograph showing the results of the antimicrobial activity test against S. flexneri strain of water extract.
Figure 13 is a photograph showing the results of the antibacterial activity of the acetone extract against strain VRV (Vancomycin-Resistant Staphylococcus Aureus).
Figure 14 is a photograph showing the results of the antimicrobial activity of the acetone extract MRSA strain (Methicillin-Resistant Staphylococcus Aureus) strain.
Figure 15 is a photograph showing the antimicrobial activity test results for B. coagulance strains of acetone extract.
Figure 16 is a photograph showing the antimicrobial activity test results for L. monocytogene strain of acetone extract.
Figure 17 is a photograph showing the results of the antimicrobial activity test against E. coli strain of acetone extract.
Figure 18 is a photograph showing the antimicrobial activity test results for S. flexneri strain of acetone extract.
19 is a graph showing the results of measuring DPPH radical scavenging ability of ethanol extract for each sample.
20 is a graph showing the results of measuring the DPPH radical scavenging ability of the water extract for each sample.
21 is a graph showing the measurement results of DPPH radical scavenging ability of acetone extract for each sample.
22 is a result of observing the growth of the strain for each medium when the tannin hydrothermal extract is added.
Figure 23 is a graph showing the change in viable cell number of the strain when tannin hydrothermal extract is added according to the embodiment of the present invention.
24 is a graph showing the change in viable cell number according to Bacillus, Lactobacillus and yeast fermentation by raw material of tannin by-products.
Figure 25 is a graph measuring the growth of each strain according to the change in culture temperature for each raw material by-product tannins.
Figure 26 is a graph measuring the growth of each strain according to the change in culture pH for each raw material by-product tannins.
FIG. 27 is a graph illustrating growth growth of strains of tannin by-products from which tannins are removed according to an embodiment of the present invention. FIG.
Figure 28 is a graph measuring the growth of each strain in the feed composition in which the tannin extract removes the tannin component and the excipient according to an embodiment of the present invention in a ratio.
Figure 29 is a graph measuring the growth of each strain in the feed composition of the acorn extract and excipients with the tannin component removed according to an embodiment of the present invention by ratio.
30 is a graph showing a change pattern of microbial species distribution in raw materials according to the fermentation time of tannin by-products.
Figure 31 is a photograph showing the results of the SDS-PAGE experiments with changes in the protein composition according to the presence or absence of strain inoculation of tannin by-products and incubation time. At this time, (a) is a protein marker (marker); (b) is strain free; (c) 1 week after strain addition; (d) is the result for feed two weeks after strain addition.
32 is a photograph of a tannin feed prepared by mixing tannin by-products, excipients and strains. At this time, (a) is the feed added 20% bampi extract; (b) is a feed containing 30% of the chestnut extract; (c) is a feed supplemented with 30% acorn extract.
Figure 33 is the result of measuring the (a) measurement results of the change in moisture content according to the change in the hot air drying temperature and (b) the time to reach the reference moisture content (moisture content 5% by weight or less) during the manufacture of the functional feed according to an embodiment of the present invention to be.
34 is a graph comparing and measuring the initial viable cell number of each strain and the viable cell number after drying according to the change of the hot air drying temperature when preparing the functional feed according to the embodiment of the present invention. At this time, (a) is 40 ℃; (b) is 60 ° C .; (c) is 80 ° C .; (d) was dried at 100 degreeC.
Figure 35 is a graph showing the results of the storage stability confirmation test for each temperature in the functional feed added 20% of the chestnut extract according to an embodiment of the present invention.
Figure 36 is a graph showing the results of the storage stability verification experiments for each temperature in the functional feed added 30% of the chestnut extract according to an embodiment of the present invention.
Figure 37 is a graph showing the results of the storage stability verification experiments for each temperature in the functional feed added 30% acorn extract according to an embodiment of the present invention.
38 is a graph showing the results of the storage stability test according to whether the packaging of the aeration in the functional feed added 30% of the chestnut extract according to an embodiment of the present invention. (a) rent packaging; (b) vacuum packing.
Figure 39 is a photograph showing the results of the antimicrobial activity test for the VRSA strain (Vancomycin-Resistant Staphylococcus Aureus) strain of functional feed prepared by adding tannin water extract (green tea, acorn, chestnut) to the feed according to an embodiment of the present invention.
Figure 40 is a photograph showing the results of the antimicrobial activity test for MRSA strain (Methicillin-Resistant Staphylococcus Aureus) of the functional feed prepared by the addition of tannin water extract (green tea, acorn, chestnut) to the feed according to one embodiment of the present invention.
Figure 41 is a photograph showing the results of the antimicrobial activity test for L. monocytogene strains of functional feed prepared by adding tannin water extract (green tea, acorn, chestnut) to the feed according to an embodiment of the present invention.
42 is a photograph showing the results of the antimicrobial activity test against B. coagulance strains of the functional feed prepared by adding tannin water extract (green tea, acorn, chestnut) to the feed according to one embodiment of the present invention.
Figure 43 is a photograph showing the results of the antimicrobial activity test for E. coli strains of functional feed prepared by adding tannin water extract (green tea, acorns, chestnuts) to the feed according to an embodiment of the present invention.
Figure 44 is a photograph showing the results of the antimicrobial activity test for S. flexneri strain of the functional feed prepared by adding tannin water extract (green tea, acorn, chestnut) to the feed according to one embodiment of the present invention.
Figure 45 is a photograph showing the results of the antimicrobial activity test for the VRSA strain (Vancomycin-Resistant Staphylococcus Aureus) strain of the functional feed prepared by adding tannin ethanol extract (green tea, acorn, chestnut) to the feed according to an embodiment of the present invention.
Figure 46 is a photograph showing the results of the antimicrobial activity of the MRSA strain (Methicillin-Resistant Staphylococcus Aureus) strains of the functional feed prepared by adding tannin ethanol extract (tea, acorn, chestnut) to the feed according to an embodiment of the present invention.
47 is a photograph showing the antimicrobial activity test results for the strain L. monocytogene of the functional feed prepared by adding tannin ethanol extract (green tea, acorn, chestnut) to the feed according to an embodiment of the present invention.
48 is a photograph showing the antimicrobial activity test results for B. coagulance strains of functional feed prepared by adding tannin ethanol extract (green tea, acorn, chestnut) to the feed according to one embodiment of the present invention.
Figure 49 is a photograph showing the results of the antimicrobial activity test for E. coli strains of functional feed prepared by adding tannin ethanol extract (green tea, acorn, chestnut) to the feed according to one embodiment of the present invention.
Figure 50 is a photograph showing the results of the antimicrobial activity test for S. flexneri strain of functional feed prepared by adding tannin ethanol extract (green tea, acorn, chestnut) to the feed according to one embodiment of the present invention.
FIG. 51 is a photograph showing the results of peroxyl radical scavenging activity of a functional feed prepared by adding green tea water extract to a feed according to one embodiment of the present invention. FIG.
52 is a photograph showing the results of peroxyl radical scavenging activity of the functional feed prepared by adding the chestnut water extract to the feed according to one embodiment of the present invention.
FIG. 53 is a photograph showing the results of peroxyl radical scavenging activity of a functional feed prepared by adding acorn water extract to a feed according to one embodiment of the present invention. FIG.

Hereinafter, the present invention will be described in detail.

The present invention provides a functional feed composition comprising tannin by-products, embryos, and tannin-soluble strains extracted from agricultural products.

Specifically, in the present invention, the tannin by-products extracted from the agricultural products are dry grinding of agricultural products containing tannins; And extracting the dry pulverized product with water, ethanol or acetone, preferably further comprising inactivating the tannin component after preparing the extract with water, ethanol or acetone. Good to do.

In the present invention, the agricultural product is characterized in that any one of gampi, bampi, acorny or green tea gourd, the tannin by-product and the embryo gourd is mixed in a weight ratio of 1 to 9, the tannin soluble strain is Bacillus subtilis, Lactobacillus plantarum and yeast (yeast) is characterized in that.

In another aspect, the present invention provides a method of producing a functional feed, comprising the steps of: (1) preparing a tannin by-product extracted from agricultural products; (2) mixing the tannin by-product and the embryo foil in a weight ratio of 1 to 9; (3) inoculating the mixture with a tannin soluble strain; (4) fermenting the mixture inoculated with the strain; And (5) provides a method of producing a functional feed comprising the step of drying the fermented feed.

Tannin by-products extracted from the agricultural product by-products are dry grinding of agricultural products containing tannins; Extracting the dry pulverized product with water, ethanol or acetone; And inactivating the tannin component in the extract, wherein the agricultural product is characterized in that any one of gampi, bampi, acorny or green tea foil.

In the present invention, the fermentation step is a fermentation for 2 to 4 days at 20 ~ 40 ℃, pH 3.5 ~ 5.5, the drying step is characterized in that the hot air drying step for 90 to 180 minutes at 35 ~ 65 ℃.

In another aspect, the present invention further comprises a tannin extract prepared by extracting one or more agricultural products selected from the group consisting of gampi, bampi, acorn and green tea foil after the fermentation step with water, ethanol or acetone to increase antibacterial and antioxidant activity. It provides a method for producing a functional feed characterized in that.

At this time, the tannin extract is characterized in that the green tea foil extract 1 to 3% by weight, acorny extract 0.2 to 0.6% by weight or 1 to 8% by weight of the chestnut extract.

The present invention also provides a functional feed produced by the above method.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited to the following examples.

Green tea used in this experiment was purchased from Boseong, Jeollanam-do, Sangju-si, Gyeongsangbuk-do, chestnuts, Salmi, Chungju-si, Chungcheongbuk-do, and acorns were purchased from Asan-si, Chungcheongnam-do, and dried and finely ground to prepare a pulverized sample.

Example 1 Preparation of Sample Extract

(1) Preparation of Water Extract

1000 ml of distilled water (DW) was added to 100 g of the dry pulverized sample for extraction, and the inlet was blocked, followed by bathing in a water bath at 90 ° C. for 1 hour. The mixture of the heated sample and distilled water was filtered, filtered, and concentrated under reduced pressure at 85 to 90 ° C. in a deep freezer. The mixture was frozen at −42 ° C. for 24 hours, lyophilized at −52 ° C. for 48 hours and then ground. An extract in the form of a powder was obtained.

(2) Preparation of Ethanol Extract

1000 ml of ethanol was added to 100 g of the dry pulverized sample for extraction, and the inlet was blocked, followed by bathing in a water bath at 90 ° C. for 1 hour. The mixture was filtered and filtered under reduced pressure at 85 to 90 ° C., filtered through a filter paper, and placed in a deep freezer. The mixture was frozen at −42 ° C. for 24 hours, lyophilized at −52 ° C. for 48 hours and then ground. An extract in the form of a powder was obtained.

(3) Preparation of Acetone Extract

1000 ml of acetone was added to 100 g of the dry pulverized sample for extraction, and the inlet was blocked, followed by bathing in a water bath at 90 ° C. for 1 hour. The sample was agitated for 30 minutes at 1690 × g to obtain a supernatant and a precipitate. 1000 ml of acetone was added to the precipitate, and the above procedure was repeated four times. The supernatant was collected and concentrated and filtered. The concentrated filtered mixture was placed in a deep freezer, and the mixture was frozen for 24 hours at -42 ° C and lyophilized at -52 ° C for 48 hours and then ground to obtain an extract in powder form.

Experimental Example 1. Determination of tannin content of each sample extract

Tannin content of each extract prepared in Example 1 was measured according to the oxidative titration method of Lowenthal.

First, the reagents used in the experiment were prepared by the following method.

1) KMnO ₄ solution is prepared by dissolving 1.33 g of KMnO ₄ in water to 1 l. The expression of KMnO ₄ solution is as follows. That is, take 10 ml of 0.1N oxalic acid solution into an Erlenmeyer flask, add 2-3 drops of concentrated H ₂ SO ₄ , warm it to 80 ° C, and titrate with KMnO ₄ solution until it becomes pink. Find the amount of oxalic acid. The 0.1N oxalic acid solution was prepared by dissolving 6.3 g of pure crystalline oxalic acid in water to make 1 L.

2) The indigo carmine aqueous solution was dissolved in 5 g of indigo carmine in 375 ml of water, 50 ml of concentrated sulfuric acid (H ₂ SO ₄ ) was carefully added, diluted with 575 ml of water and filtered.

3) Acid saturated salt solution was prepared by adding 25 mL of concentrated sulfuric acid ((H ₂ SO ₄ ) to 970 mL of saturated saline solution.

4) The gelatin solution is softened by soaking 25 g of gelatin in the acidic saturated salt solution for 1 hour in advance and then dissolving by heating. After cooling, the gelatin solution is adjusted to 1 L with saturated salt solution.

5) Purified kaolin was dried at 110 ° C. and used as a filter aid.

2-5 g of the extract prepared in Example 1 was taken, 400 ml of distilled water was added, boiled for 30 minutes, and leached. The leaching solution was filtered through a dry filter paper and 20 ml of an indigo carmine aqueous solution and 750 ml of distilled water were added to 10 µl of the filtrate to prepare a sample solution. The sample solution is titrated with KMnO ₄ solution, but the KMnO ₄ solution is quickly added dropwise until the sample solution turns green, and then slowly added dropwise until the solution turns yellow or the periphery of the liquid surface becomes non-red. . The consumption amount of the KMnO ₄ solution at this time is A (ml).

Next, 100 ml of the sample solution is taken, 50 ml of gelatin solution, 100 ml of acidic saturated saline solution, and 10 g of powdered kaolin are added, shaken for several minutes, and the supernatant is filtered. Take 25 ml of the filtrate, filter this leaching solution with dry filter paper, add 20 ml of indigo carmine solution and 750 ml of distilled water to 10 µl of the filtrate, and titrate with KMnO ₄ solution, until the solution turns green. After the dropwise addition, the solution is slowly added dropwise and titrated until the solution becomes yellow or the periphery of the liquid surface becomes non-red. The consumption amount of the KMnO ₄ solution at this time is B (ml). Tannin content (%) of each extract was calculated by the following equation.

Tannin Content (%) = {(AB) × 0.043} × 50

As a result of analyzing the tannin content of each sample extract, green tea showed the highest content of ethanol extract, and acorn, chestnut and persimmon showed the highest tannin content of acetone extract. The experimental results are shown in Table 1 below.

[Table 1] Tannin content by sample extract

The superscripts A, B, and C in the table indicate statistical significance, meaning that statistically significant differences are recognized between different alphabets.

Experimental Example 2. Measurement of antimicrobial activity of each sample extract

The antimicrobial activity of each extract prepared in Example 1 was tested using a paper disc method, strains were used as a total of six species, two gram-positive bacteria, two gram-negative bacteria, two gram-resistant bacteria. The growth medium of the bacteria was incubated for 15 to 18 hours in a 37 ℃ incubator using LB agar medium as a solid medium.

The strains used in the antimicrobial experiments of tannin extracts were L. monocytogene, B. coagulance, E. coli, S. flexneri, Methicillin-Resistant Staphylococcus Aureus (MRSA), and Vancomycin-Resistant Staphylococcus Aureus (VRSA).

The solid medium was prepared by mixing 10 g of trypton, 10 g of NaCl, 15 g of agar, and 5 g of yeast in 1 liter of distilled water. Pour 20 ml of solid medium into a petri dish and solidify. Take 20 μl of 6 strains cultured in a liquid medium, spread it uniformly, and sterilize filter paper disc (diameter 8) in an autoclave. Mm). Sample extracts (water, acetone, and ethanol extracts of Gampi, Bampi, Acorn, and Green Tea Fries, respectively) were injected on the filter paper disc at 2, 3, 4, and 5 mg / disc for each concentration. After 48 hours of incubation, the diameter (mm) of the clear zone around the disc was measured.

As a control group, ampicillin and gentamicin were used as antibiotics for each bacterium.

Specific results of the antimicrobial activity of each sample are shown in Tables 2 to 4 and FIGS. 1 to 18. In Tables 2 to 4, when the diameter of the growth inhibitory ring (Inhibitory zone) is 8 mm or more, ++++; In the case of 5 to 7 mm, +++; ++ for 2-4.5 mm; In the case of 0.2 to 1.5 mm; In the case of 0.1 mm or less, it was represented by-.

In the case of ethanol extract, antibacterial activity was highest in green tea and lowest in persimmon. In particular, green tea showed high antimicrobial activity against MRSA (Methicillin Resistant Staphylococcus Aureus), which is resistant to an antibiotic called methicillin. Green tea water extract showed particularly strong antimicrobial activity in VRSA, B. coagulance and S. flexneri , and water extract of persimmon showed no antimicrobial activity against other pathogens except S. flexneri . The antibacterial activity of the acetone extract of each sample showed a similar tendency to that of the ethanol extract, and the antimicrobial activity of the green tea extract showed the greatest results.

[Table 2] Antimicrobial Activity of Ethanol Extracts

Table 3 Antimicrobial Activity of Distilled Water Extracts

Table 4 Antimicrobial Activity of Acetone Extract

Experimental Example 3. Measurement of antioxidant activity of each sample extract

The antioxidant capacity of each extract prepared in Example 1 was tested using a method of measuring DPPH radical scavenging activity (DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity).

30 μl of DPPH solution (30 μM) was added to 30 μl of the sample solution in which 2 mg of each extract was dissolved in 1 ml of distilled water, stirred for 10 seconds, and then the mixed solution was transferred to a quartz capillary tube and measured by ESR after 2 minutes. It was. The spectrum was recorded under the conditions of field: 337.1 ± 5 mT, power: 4 mW, scan time: 2 minutes, amplitude: 1 × 500, time constant: 0.3 seconds. The scavenging activity of DPPH radicals for each sample was expressed as follows. Calculated using.

DPPH radical scavenging activity (%) = 100-(ESR signal intensity for medium containing the samples / ESR signal intensity for the control medium) × 100

Specific results of DPPH radical scavenging activity of each sample extract are shown in FIGS. 19 to 21.

As a result of analyzing the IC ₅₀ value of each sample in the antioxidant activity measurement experiment, ethanol extract showed the highest antioxidant effect in the order of green tea 0.0022 mg / ml, chestnut 0.0076 mg / ml, acorn 0.0106 mg / ml, persimmon 0.576 mg / ml. Appeared. In particular, green tea showed a lower IC ₅₀ value compared to vitamin C (IC ₅₀ = 0.003 mg / ml), which is a representative natural antioxidant, indicating that the antioxidant effect of green tea ethanol extract is very high. In the case of water extract, IC ₅₀ values of each sample were in order of acorn 0.011 mg / ml, green tea 0.020 mg / ml, chestnut 0.065 mg / ml, and persimmon 0.576 mg / ml. . In the case of acetone extract, the IC ₅₀ value of each sample was 0.021 mg / ml of green tea, 0.037 mg / ml of acorn, 0.044 mg / ml of chestnut, and 0.143 mg / ml of persimmon.

From the above results, antioxidant effects were green tea, acorn, chestnut and persimmon, and it was found that the antioxidant effect of ethanol extract was the highest by extract.

Example 2 Microbial Analysis in Tannin By-Product Raw Materials

Before separating the microorganisms that can grow using tannin by-products, the distribution of microorganisms present in the raw material itself was checked for future reference.

Four kinds of tannin by-products (gampi, bampi, acorn and dry green crushed samples) were taken 5 g each, suspended in 45 ml sterile saline, and vortexed for 30 minutes. Each tannin by-product suspension is serially diluted and spread on TSA, MRS-BCP, and PDA media, respectively, and cultured for 24 or 48 hours to measure viable cell count. The composition for each medium is shown in Table 5 below.

[Table 5] Composition Table of Media for Strain Separation from Tannin By-Products

Example 3. Isolation of Tannin Soluble Strains

(1) Tannin hydrothermal extract preparation

Four kinds of tannin by-products (gampi, bampi, acorn and dry green crushed green tea samples) were collected 10 g each, mixed with 250 ml of distilled water, and heated at 50 ° C. for 1 hour. After the heated aqueous solution was collected using a vacuum pump, 250 ml of distilled water was further mixed and heated to 90 to 100 ° C to prepare a tannin hot water extract.

(2) Preparation of strain sample

Four kinds of tannin by-products (gampi, bampi, acorn and dry green crushed samples), which were not subjected to hot water extraction, were collected 10 g each, suspended in 90 ml of sterile saline, and vortexed for 30 minutes. Was prepared.

(3) strain isolation

To prepare a solid medium for each strain type to be separated as shown in Table 6, the high-temperature high-pressure sterilization was carried out at 121 ℃. After sterilization, the tannin hot water extract was added at 20 vol% to a medium cooled to about 60 ° C., mixed, and then dispensed into a plate to prepare a solid medium.

After spreading the sample of the strain in multiple dilutions in each medium and incubated for 24 or 48 hours to determine the number of colonies (colon) and viable growth.

[Table 6] Culture Conditions of Strains of Medium Containing Tannin Hot Water Extract

In order to isolate the available strains of tannins, the results of observing the growth of each strain by adding 20% of tannin hydrothermal extract to the three media are shown in Table 7 and FIG. 22. When the bampi hot water extract was added, it was confirmed that the strains were grown in TSA medium and MRS-BCP medium, and in the case of the acorni hot water extract, it was confirmed that the strains were grown in PDA medium. On the other hand, strains were not detected in the green tea foil and gampi hot water extract with strong antibacterial effect.

Then, biochemical tests (API kit, Biomerieux, France) or microscopic observation were performed to confirm the species of the strains grown in the medium to which the hot water extract was added. As a result of the API test, the strains grown in TSA medium were Bacillus subtilis (94% of match) and the strains grown in MRS-BCP medium were Lactobacillus plantarum (99% of match). Strains grown in PDA medium were observed under an optical microscope, and many of the budding forms were confirmed, which may be presumed to be yeast strains.

Based on the above results, it was confirmed that one strain of Bacillus, one strain of Lactobacillus, and one strain of yeast may be grown in a medium containing tannin hydrothermal extract. May be used for feed fermentation with tannin by-products.

[Table 7] Growth status by strain when tannin hot water extract is added

Example 4 Isolation of Growth Strains by Tannin Hot Water Extract Concentration

Proceed in the same manner as in the strain isolation method of Example 3, the concentration of tannin hydrothermal extract in the solid medium was changed by varying the content of the tannin hydrothermal extract added to each medium as shown in Table 8 below. Each of the strain samples (pretreated tannin by-product raw material) was diluted and spread in multiple solid media prepared, and cultured for 24 or 48 hours to determine the number of colonies and viable cells grown.

[Table 8] Hot Water Extract Amount for Experiments by Tannin Hot Water Extract Concentration

In particular, when 50% of tannin hydrothermal extract was added in TSA medium, the number of viable cells was reduced from 2.6 × 10 ³ to 5.5 × 10 ² , and in case of MRS medium, 4.4 × 10 ³ when tannin hydrothermal extract was added. The number of viable cells was reduced below, and the number of viable cells of 5.0 × 10 ² or less was confirmed in PDA medium regardless of the concentration of tannin hydrothermal extract. The results are shown in FIG.

Example 5 Tannin By-Product Fermentation and Sampling Analysis

Four kinds of tannin by-products (gampi, bampi, acorny and green tea powder dry ground samples) were prepared as shown in Table 9 below, and then placed in a photocursor bottle, and then Bacillus, lactic acid bacteria, and yeast cultures were added. The mixed strain sample is mixed well with a reagent spoon or the like, and then fermented in a 30 ° C. incubator.

The pH and viable cell counts of day 0, 1, 2, 3, and 7 samples of the tannin by-products during fermentation were analyzed. For each fermentation time, 5 g of each tannin by-product sample was taken, suspended in 45 ml saline and vortexed for 30 minutes. After serial dilution with sterile saline, smear the TSA, MRS-BCP, PDA medium and check the viable cell count of Bacillus, lactic acid bacteria and yeast.

[Table 9] Raw Material and Species Composition for Fermentation by Tannin By-Products

The results are shown in FIG. Most Bacillus spp. Cultured in TSA medium showed less than 10 ⁴ viable cell counts after 2 days of fermentation. In the case of Lactobacillus spp. In the medium containing tannin by-products such as gourd, acorn, and gampi, the number of viable cells decreased from 10 ² to 10 ³ after one day of culture. In yeast cultured in PDA media, viable cell count was reduced to 10 ² in tannin by-product fermentation media except for acorns, and the growth rate of the strain was drastically reduced due to the antibacterial effect of tannins contained in each sample. Is considered.

Example 6 Tannin By-Product Fermentation According to Culture Temperature

When fermentation of tannin by-products of Example 5 was confirmed by the growth temperature according to the culture temperature was to determine the optimum culture temperature. However, as shown in Example 5, since the growth of microorganisms and fermentation by microorganisms were suppressed due to the antibacterial effect of a considerable amount of tannins present in the tannin by-products, the tannin components were removed from each tannin by-product samples, and Bacillus, The fermentation was carried out by inoculating lactic acid bacteria and yeast.

After removing the tannin component as shown in Example 8 to prepare a fermentation sample as in Example 5, and then fermented at a temperature of 20, 30, 40, 50, 60 ℃, to prepare for the number of viable cells at the initial inoculation The optimum fermentation temperature was determined by measuring the change in viable cell count after 7 days of fermentation. The results are shown in FIG.

Bacillus sp. Strains showed viable cell counts of about 6.6 × 10 ⁴ above 30 ° C and the highest viable cell counts at night. Lactobacillus spp. Also showed the highest viable cell counts at night, but showed a high viable cell number at 20 to 30 ° C in temperature conditions, and reduced viable cell numbers in proportion to temperature above 40 ° C. On the other hand, yeast showed the highest viable cell count in acorns and the optimum temperature range was 20 ~ 30 ℃.

Example 7 Tannin By-Product Fermentation According to Culture pH

When fermentation of tannin by-product of Example 5, it was intended to confirm the growth according to the pH of the initial culture. However, as shown in Example 5, since the growth of microorganisms and fermentation by microorganisms were suppressed due to the antibacterial effect of a considerable amount of tannins present in the tannin by-products, the tannin components were removed from each tannin by-product samples, and Bacillus, The fermentation was carried out by inoculating lactic acid bacteria and yeast.

After removing the tannin component as shown in Example 8 to prepare a fermentation sample as in Example 5, and then adjusted to pH 4, 5, 7, 9 using 18% HCl solution and 6 N NaOH solution, respectively give. The fermentation was carried out for 7 days in an incubator at 30 ° C. and the optimal fermentation pH was determined by measuring the change in viable cell number. The results are shown in FIG.

Bacillus sp. Strains showed the highest viable cell counts at night and maintained a viable cell count of 3.8 × 10 ⁴ at pH 7. The Lactobacillus spp. Also showed the highest viable cell counts at night, and showed a relatively high viable cell count of 7.1 × 10 ⁵ in the pH 4 range, but rapidly decreased to 10 ⁴ or lower at pH 5 and above. On the other hand, in case of yeast, the number of viable cells was the highest in acorn, and the number of viable cells decreased rapidly from pH 5 and above, and no yeast was found in pH 9.

After removal of the tannin component having antimicrobial activity from a tannin-products Given the above results, a relatively when to efficiency but check to be increased compared to when the actual live cell number of 10 ⁷ or more if you feel that the fermentation bacteria for fermented feeds prepared Is considered insufficient. This may be due to the lack of various nutrients for fermentation in the samples of green tea, acorn, chestnut and gampi. Therefore, in the subsequent experimental step, four kinds of tannin by-products and excipients from which the tannin components were removed were mixed by ratio to confirm the mixing conditions that may have optimum growth properties in feed.

Example 8. Confirmation of growth growth according to removal of tannin component

To determine whether the growth of each strain is changed depending on the removal of tannin components presumed to be present in the tannin by-products, inactivation of tannin components was carried out by the following method.

An appropriate amount of each tannin by-product raw material (gampi, bampi, acorny and green tea foil dry ground sample) was taken and mixed with water about three times the amount of raw material. The mixture was adjusted to pH 3 using 10% H ₂ SO ₄ . It was heated for 1 hour and cooled to room temperature, and then the supernatant was removed. Three times of water was mixed with the precipitated raw material, adjusted to pH 7, and heated for about 1 hour. After cooling to room temperature, the supernatant was removed and the precipitated raw material was dried at 60 ° C. in a dry oven. Tannin by-products after drying are used to inoculate the strain and proceed with fermentation.

As a result of culturing each strain in a medium in which 16.2 g of tannin by-products from which tannin was removed were excised, 46.2 g of embryobacterium as an excipient, 1 ml of each of Bacillus, Lactobacillus and yeast, and 31 ml of distilled water, Bacillus, Both lactobacillus and yeast showed high viable cell count of at least 2.4 × 10 ⁶ and maintained high viable cell count until 3 days after fermentation. In particular, in the case of Lactobacillus (lactobacillus), the number of viable cells increased steadily until 1.7 × 10 ¹⁰ until 3 days of fermentation at night and then maintained a high viable count in the range of 10 ⁹ to 10 ¹⁰ until 7 days of fermentation. In addition, overall viable cell counts were higher in bampi and acorns. The result is shown in FIG. 27.

Example 9 Confirmation of Growth Change According to Excipient Addition Ratio

The excipients added during feed preparation were added to tannin by-products in proportion, and the growth rate of each strain was determined according to the addition ratio. As an added excipient, our embryo battery was used, as shown in Table 10 below, by varying the addition ratio of tannin by-products and mixing, fermentation was carried out to measure the number of live bacteria of strains from day 0 to day 7, and changes in appearance and flavor Confirmed.

[Table 10] Composition for growth confirmation by mixing ratio of tannin by-products and excipients

Tannin or acorn extract was added to the excipients by concentration to determine the optimal mixing condition of chestnut, acorn and excipients, which showed high viable cell counts of bacillus, lactobacillus and yeast among tannin by-products from which tannin was removed By culturing the strain to confirm the growth is shown in Figures 28 and 29.

In the Bacillus and lactobacillus genus, the number of viable cells increased rapidly after 1 day of fermentation, showing a high viable count of 10 ⁹ to 10 ¹⁰ after 3 days of fermentation, especially when 20% of chestnut was added. The high viable cell count was maintained. In the case of acorns, the number of viable cells was 10 ⁸ ~ 10 ^{9 after} 3 days of fermentation, and the highest viable cell number was maintained when 30% was added.

Based on the above results, it was decided to proceed with the experiment for the establishment of the commercialization conditions by manufacturing the bampi 20%, bampi 30% products and acorn 30% products.

Experimental Example 4. Variation of Microbial Species Distribution in Raw Materials According to Tannin By-Product Fermentation Time

Based on the results of the fermentation using tannin by-products for future reference in developing mass production and feed process through scale-up, the microbial species distribution pattern was analyzed according to fermentation time. It was.

As shown in FIG. 30, it was confirmed that Bacillus species rapidly increased at the initial stage of culture (day 0 to day 1), and lactic acid bacteria and yeast strains were increased from the second day of culture. Lactobacillus was found to have a high occupancy rate on the 7th day of culture, but considering that Bacillus is a spore-producing bacterium, it is estimated that there will be other changes in the distribution of the species after a long period of more than 7 days. It seems to be verified in the experiment.

Experimental Example 5. Changes in Protein Content in Raw Material by Tannin By-Product Fermentation

When the tannin by-products were fermented, we tried to check the change in composition of the by-products depending on the inoculation of the species. Thus, the samples without added the species and the sample added three species to the water extract of the chestnut were prepared, respectively, and the change of protein according to the incubation time was measured. 1 g of each sample without strain, one week after strain addition, and two weeks after strain addition were collected and suspended in 9 ml of distilled water and bathed at 50 ° C for 20 minutes. After centrifugation at 3,000 rpm for 10 minutes, the supernatant was recovered and transferred to 100 μl in an ephendorf tube, and 100 μl of sample buffer was added and mixed at 1: 1. Heat treated at 100 ° C. for 5 minutes, left at 4 ° C. for 5 minutes, and 20 μl were loaded onto a 12% SDS-PAGE gel. The 4% stacking gel and the separation gel were electrophoresed at 100 V for 1 hour and 20 minutes, and then stained with Coomassie blue to identify the band tendency.

The change of protein composition with the passage of strain and culture time at the time of tannin by-product fermentation is shown in FIG. 31. The band patterns of (b) without adding any strains, (c) after one week of addition of strains, and (d) after two weeks of addition of strains were confirmed, and 14.5 kDa (kilo Dalton) and 21.5. It was confirmed that there is a difference in the band existing between kDa.

Based on these results, it was confirmed that there was a change in the unknown protein content having a size between 14.5 kDa and 21.5 kDa depending on the presence or absence of the inoculation and the incubation time of the strain. ) Is estimated to be used by microorganisms.

Example 10 Preparation of Functional Feed Using Natural Tannin By-Products

Based on the optimum growth conditions of the strains identified in the previous experiment, tannin by-products, embryos and strains of tannin by removing the tannin component from the pulpy and acorn extract dry grinding samples were prepared as shown in Table 11 below. After inoculating the strain into the raw material was subjected to fermentation for 3 days at 30 ℃ to increase the number of viable cells, and the final tannin feed was prepared by hot air drying at a temperature of 60 ℃. The prepared feed is shown in FIG.

Table 11 Preparation of Tannin Feed

Experimental Example  6. Tannins  Confirmation of stability of hot air drying by feed temperature

The stability of the tannin feed prepared by culturing the strain in tannin by-products during drying was determined. When the tannin feed prepared in Example 10 was dried at 40, 60, 80, and 100 ° C., respectively, the time taken to reduce the moisture content to 5% or less was measured. For this, 100 g of each tannin feed was placed in an aluminum plate. 5 g was taken every 30 minutes and the moisture in the feed was measured with a moisture meter. In addition, by measuring the number of live bacteria after the water content is reduced to 5% or less to confirm the stability according to hot air drying.

The change in moisture content and the time to reach the reference moisture content (5%) according to the change of hot air drying temperature are shown in FIG. 33, and the feed containing 20% of bampi by-products (hereinafter referred to as 'Bampi 20') and the feed containing 30% of bampi by-products (hereinafter, 'Bampi 30') and feed containing 30% of acorn by-products (hereinafter, 'acorn 30') were found to be insignificant.

The optimum hot air drying temperature was determined by comparing with the stability results performed after measuring the hot air drying time, and the stability results according to the hot air drying temperature are shown in FIG. 34. As shown in the results, the stability at 40 ° C was the best, and the stability of Lactobacillus and yeast decreased rapidly at 80 ° C and 100 ° C. Although the yeast stability was slightly decreased at 60 ℃, the stability of Bacillus and Lactobacillus was relatively good and considering the economical efficiency and mass production according to the drying time, it would be advantageous to dry at 60 ℃ for 2 hours.

Experimental Example 7. Confirmation of storage stability of tannin feed

The storage stability of the tannin feed prepared in Example 10 was measured by measuring the change in viable cell count according to the storage method and period.

First, take 100 g of tannin feed prepared in order to check the stability according to the storage temperature, put it in a vacuum zipper bag, and store it in an incubator at 20, 25, 30, and 40 ° C., respectively. The number of viable cells was measured.

The results are shown in FIGS. 35 to 37. Both Pampi 20, Bampi 30 and Acorn 30 feeds had excellent stability at 20 ° C and decreased with increasing temperature. In the case of acorn 30 feed, it was confirmed that lactic acid bacteria and yeast stability were lower at the same temperature than the feed mixed with chestnut. Stability at 20 ° C was similar for Bampi 20 and Bampi 30, but at higher temperatures, Lactobacillus and yeast stability were significantly better in Bampi 30 than at Bampi 20. Bacillus strains were not significantly different from each other by feed and temperature, and the number of viable bacteria was generally maintained at about 10 ⁹ . Considering the result of maintaining the viable cell number by strain and the economics according to the temperature maintenance, storage at 25 ° C may be most advantageous.

Next, in order to confirm the storage stability according to the aeration or not, 100 grams were produced by collecting 30 g of Pampi, which was proven to be stable in the previous experiment, and then packed in a zone (10 cm × 8 cm) and a vacuum bag (vacuum zipper bag). Divided into and stored in a 25 ℃ incubator. The changes in viable cell counts at the beginning, 4th, 8th and 12th weeks were measured to compare the storage stability according to aeration.

The results are shown in FIG. 38, and as a result of measuring the number of viable cells up to 12 weeks, it was confirmed that the Bacillus strains were not significantly different, but the vacuum packaging was somewhat disadvantageous in the stability of lactic acid bacteria and yeast. Therefore, it is expected that the field packaging will be excellent for mass production in the future.

Example 11.Evaluation of Antibacterial and Antioxidant Activity According to the Blending Ratio of Tannin Fermented Feed and Tannin By-Product

The tannin feed prepared in Example 10 was prepared after inactivating tannin having an antimicrobial effect for the first time for fermentation by microorganisms, and the feed containing 30% of bampi by-products showed the best results in viable cell count and storage stability. . In order to enhance the antimicrobial and antioxidant effects in feeds prepared by adding 30% of bampi by-products, green tea flakes and acorns were found to have high antibacterial and antioxidant effects in tannin by-products (gampi, bampi, acorny and dry green tea samples). Or we evaluated the antimicrobial and antioxidant activity of feed supplemented with chestnut by-products to determine the optimal tannin concentration for the final product.

The blending ratio of tannin by-products to the fermented feed was determined by varying the amount of tannins based on the amount of tannins contained in the tannin by-products.

The antimicrobial activity was measured using the tannin fermented feed and the tannin by-product blending ratio using the same paper disc method as in Experimental Example 2, and the antioxidant activity was measured using an electronic spin resonance device (ESR). Evaluation was made by measuring scavenging activity. 200 μl PBS solution (pH 7.4) was added to 100 μl of the sample solution in which 4 mg of each extract was dissolved in 1 ml of distilled water, and stirred in a constant temperature water bath at 37 ° C. for 30 minutes, and then 100 μl of the mixed solution was transferred to a quartz capillary tube. Two minutes after the transfer was measured by ESR. Spectra were recorded under conditions of central field: 3475 G, modulation frequency: 110 kHz, modulation amplitude: 2 G, microwave power: 1 mW, gain: 6.3 × 10 ⁵ , and temperature: 298 K.

The results of measuring the antimicrobial activity by adding the tannin by-products to the tannin feed by concentration by the above method are shown in Table 12, Table 13, and FIGS. 39 to 50. If the diameter of the growth inhibitory zone (inhibitory zone) is more than 8 mm ++++; From 5 to 7.5 mm +++; ++ from 2.5 to 4.5 mm; If it is 2-1 mm; If it is 1 mm or less, it is represented by-.

When the green tea water extract was added to the tannin feed, it did not show any difference according to the added concentration, but overall, it showed that the green tea by-products showed antibacterial activity against all pathogens except E. coli . Acorn water extract showed strong antibacterial activity against VRSA and S. flexneri at 0.6% concentration. The addition of Bampi water extract showed antimicrobial activity against the pathogens except B. coagulance , but did not show any difference according to the concentration.

On the other hand, when the ethanol extract of green tea was added to the fermented feed, it was shown to have antimicrobial activity against all pathogens except E. coli . Acorns showed antimicrobial activity against L. monocytogene and B. coagulance at 0.2% of tannin, whereas they showed antimicrobial activity against all pathogens at 0.3% of tannin. Bampi showed slightly weaker antimicrobial activity than green tea and acorn, but it showed antimicrobial activity against most pathogens including VRSA at a concentration of more than 5% of tannin.

In view of the above results, the optimum concentration for improving the antibacterial activity by adding tannin by-products to general fermented feed is considered to be 1 ~ 3% green tea, 0.3 ~ 0.6% acorn, 1 ~ 5% chestnut.

Table 12 Antimicrobial Activities of Fermented Feeds Added with Tannin By-Product (Distilled Water Extract)

[Table 13] Antimicrobial Activity of Fermented Feeds Added with Tannin By-Product (Ethanol Extract)

The results of evaluating the peroxyl radical scavenging ability while varying the concentration of tannin by-products (green tea, acorn and chestnut) in the fermented feed are shown in FIGS. 51 to 53. The IC ₅₀ value of each fermented feed was analyzed through antioxidant experiments. The results showed that the antioxidant effect increased by 0.052 mg / ml for 1% green tea and 0.031 mg / ml for 2% green tea. Seemed. The IC ₅₀ values of fermented feed added with chestnut were 1% (0.569 mg / mL), 4% (0.488 mg / mL), and 6% (0.042 mg / mL), respectively. As the increase of antioxidant activity increased significantly. For chestnut acorns, the IC ₅₀ value at 0.4% was 0.211 mg / ml.

Claims (24)

Functional feed composition comprising tannin by-products, embryos and tannin soluble strains extracted from agricultural products. The method of claim 1,
Tannin by-products extracted from the agricultural product is a functional feed composition, characterized in that the dry ground samples of agricultural products containing tannins. The method of claim 1,
Tannin by-products extracted from the agricultural products are dry grinding of agricultural products containing tannins; And extracting the dry pulverized product into water, ethanol or acetone. The method of claim 3,
Tannin by-products extracted from the produce is a functional feed composition, characterized in that further comprising the step of inactivating the tannin component after preparing the extract with water, ethanol or acetone. The method of claim 1,
Tannin by-products extracted from the agricultural products are dry grinding of agricultural products containing tannins; And inactivating the tannin component in the dry ground product. The method according to any one of claims 1 to 5,
The agricultural product is a functional feed composition, characterized in that any one of gampi, bampi, acorny or green tea foil. The method of claim 1,
Functional feed composition, characterized in that the tannin by-products and the embryos are mixed in a weight ratio of 1: 1 to 9. The method of claim 1,
The tannin soluble strains are Bacillus subtilis, Lactobacillus plantarum and yeast (yeast) functional feed composition, characterized in that. (1) preparing tannin by-products extracted from agricultural products;
(2) mixing the tannin by-product and the embryo foil in a weight ratio of 1 to 9;
(3) inoculating the mixture with a tannin soluble strain;
(4) fermenting the mixture inoculated with the strain; And
(5) drying the fermented feed;
Method of producing a functional feed comprising a. The method of claim 9,
Tannin by-products extracted from the agricultural product is a functional feed composition, characterized in that the dry ground samples of agricultural products containing tannins. The method of claim 9,
Tannin by-products extracted from the agricultural products are dry grinding of agricultural products containing tannins; And extracting the dry pulverized product into water, ethanol or acetone. 12. The method of claim 11,
Tannin by-products extracted from the produce is a functional feed composition, characterized in that further comprising the step of inactivating the tannin component after preparing the extract with water, ethanol or acetone. The method of claim 9,
Tannin by-products extracted from the agricultural products are dry grinding of agricultural products containing tannins; And inactivating the tannin component in the dry ground product. The method according to any one of claims 9 to 13,
The agricultural product is a method of producing a functional feed, characterized in that any one of gampi, bampi, acorns or green tea foil. The method of claim 9,
The tannin soluble strain is Bacillus subtilis, Lactobacillus plantarum and yeast (yeast) method of producing a functional feed characterized in that. The method of claim 9,
The fermentation step is a method of producing a functional feed, characterized in that the fermentation for 2 to 4 days at 20 ~ 40 ℃, pH 3.5 ~ 5.5. The method of claim 9,
The drying step is a method of producing a functional feed, characterized in that for drying the hot air for 90 to 180 minutes at 35 ~ 65 ℃. A functional feed produced by the method of any one of claims 9-14. The method of claim 9,
After the fermentation step further comprises a tannin extract to produce a functional feed characterized in that to increase the antimicrobial and antioxidant activity. The method of claim 19,
The tannin extract is a method of producing a functional feed, characterized in that the extraction of one or more agricultural products selected from the group consisting of gampi, bampi, acorn and green tea foil with water, ethanol or acetone. The method of claim 19,
The agricultural product extract is a method of producing a functional feed, characterized in that containing 1 to 3% by weight of green tea leaf water extract, green tea leaf ethanol extract or green tea leaf acetone extract. The method of claim 19,
The agricultural product extract is a method for producing a functional feed, characterized in that it comprises 0.2 to 0.6% by weight of acorn extract, acorn extract ethanol extract or acorn extract acetone. The method of claim 19,
The agricultural product extract is a method of producing a functional feed, characterized in that it comprises 1 ~ 8% by weight of the chestnut water extract, chestnut ethanol extract or chestnut acetone extract. Functional food produced by increasing the antibacterial and antioxidant activity prepared by the method of any one of claims 19 to 23.

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