KR20180092526A - Feed additives for replacement of antibiotics and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a feed additive, which promotes disease prevention and growth of livestock and increases the efficiency of a feed, and more specifically, to a feed additive which can replace antibiotics added to the feed for the livestock by presenting a feed additive comprising a fermented product obtained by fermenting oregano, Syzygium aromaticum and rice bran using a useful microorganism (EM) as a main composition, and to a manufacturing method thereof.

Description

[0001] The present invention relates to a feed additive for antibiotic substitution,

The present invention relates to a feed additive which promotes disease prevention and growth of livestock and increases the efficiency of feed. More specifically, the present invention relates to a feed additive comprising a fermented product prepared by fermenting oregano, cloves and rice bran using a useful microorganism (EM) The present invention relates to a feed additive capable of replacing antibiotics added to feed of livestock, and a method for producing the feed additive.

In general, the feed used for raising livestock, etc., is mainly used as a natural formulated feed mixed with various cereals.

Recently, there have been a lot of studies on feed additive having the function of promoting the prevention and growth of diseases of livestock and enhancing feed efficiency. Antibiotics substitutes are widely used as feed additives, organic acids, enzymes, antioxidants, etc. .

The following is a representative prior art for feed additives to be added to livestock feed.

Korean Patent Publication No. 10-0937552 relates to a feed additive using mugwort and a method for preparing the same, wherein the feed additive comprises 25 to 75% of chopped mugwort, 15 to 50% of rice bran or wheat bran and 10 to 25% Was added to the mixture at a weight ratio of 0.0001 to 5 vol% and stirred. The resultant wormwood mixture was sterilized at 65 ° C to 75 ° C for 40 to 70 minutes, cooled to 30 ° C to 40 ° C, 2 to 7% by weight of microorganisms are added, mixed, sealed and fermented at 25 to 32 ° C for 2 to 5 days. Then, the fermented aged mugwort mixture is added to a conventional dryer to obtain a water content of 20 Dried, pulverized with a conventional pulverizer, packed, aged for 10 to 20 days, and a feed additive prepared using the mugwort was prepared.

In addition, the above-mentioned prior arts have achieved the effect of increasing the physiological activity and feed efficiency of the livestock by feeding the livestock with the commercially available common compound feed at an appropriate ratio. However, the antibiotic can be replaced with antibiotics, antioxidants, And thus it is required to continuously research and solve the problems.

Korean Registered Patent No. 10-0937552 (2010.01.11.) Korean Registered Patent No. 10-1559515 (October 5, 2015) Korean Registered Patent No. 10-1062555 (October 5, 2015)

The present invention relates to a feed additive for feeding antibiotics, which is a method for improving the problems of the conventional feed additive. Conventional antibiotic feed additives include a beneficial microbial agent, a herbal medicine containing a physiologically active substance, an organic acid using citric acid, lactic acid, There was only a problem of presenting a feed additive having only the characteristics of each of them;

Particularly, in animal husbandry farms, since feed additives are used at a preventive level, antibiotic substitute feed additives having antimicrobial, antioxidant and immunity enhancing effects are preferred at the same time. In contrast, It is a main object of the present invention to provide a solution point to the problem that only a feed additive having merely an effect is presented.

The present invention has been made to solve the above-

A feed additive for replacing antibiotics comprising a fermented product fermented with a useful microorganism (EM) as an essential component of oregano, cloves and rice bran;

An oregano fermentation process for fermenting the oregano with the useful microorganism to produce an oregano fermented product; An oriental fermentation process for fermenting the clove with the useful microorganism to produce an oriental fermented product; Wherein the fermented oregano fermented product, the edible fermented product, and the fermented product of the rice bran are further subjected to a stirring and mixing process to be mixed to form a fermented product. A method for preparing a feed additive for substituting an antibiotic is provided.

The feed additive for substituting antibiotics according to the present invention and the method for producing the same according to the present invention can be used not only as a feed source for live bacteria, but also as a bioactive substance such as an enzyme, an antibacterial substance and an organic acid during fermentation An effect that can be generated can be obtained;

As a result, an antibiotic replacement feed additive having antimicrobial, antioxidant, and immunity enhancing properties that can replace antibiotics can be provided.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the storage stability (pH change) of rice bran fermented in a feed additive for antibiotic substitution according to a preferred embodiment of the present invention. FIG.
FIG. 2 is a graph showing the DPPH radical scavenging activity of oregano fermented product contained in the feed additive for antibiotic replacement according to a preferred embodiment of the present invention. FIG.
FIG. 3 is a graph showing DPPH freeradical scavenging activity of an edible fermented product included in a feed additive for antibiotic replacement according to a preferred embodiment of the present invention. FIG.
FIG. 4 is a graph showing DPPH freeradical scavenging activity of rice bran fermented in a feed additive for antibiotic substitution according to a preferred embodiment of the present invention. FIG.
5 is a photograph showing antibacterial activity against intestinal bacteria and food poisoning bacteria of a feed additive for substituting an antibiotic according to a preferred embodiment of the present invention.
FIG. 6 is a graph showing tolerance to bile acid by Lactobacillus casei of a feed additive for replacing antibiotics according to a preferred embodiment of the present invention. FIG.
Fig. 7 is a graph showing the resistance of the yeast (Scahromayces cerevise) of the feed additive for antibiotic replacement according to a preferred embodiment of the present invention to bile acids
8 is an HPLC chromatogram of an organic acid of a feed additive for antibiotic substitution according to a preferred embodiment of the present invention.
9 is a graph showing an organic acid calibration curve of a feed additive for antibiotic substitution according to a preferred embodiment of the present invention.
10 is a graph showing the total polyphenol content of a feed additive for antibiotic substitution according to a preferred embodiment of the present invention.
11 is a graph showing the total flavonoid content of feed additives for antibiotic substitution according to a preferred embodiment of the present invention.
12 is a graph showing DPPH free radical scavenging activity of a feed additive for antibiotic substitution according to a preferred embodiment of the present invention.
13 is a graph showing the superoxide dismutase (SOD) -like activity of a feed additive for substituting an antibiotic according to a preferred embodiment of the present invention.
FIG. 14 is a graph showing the nitrite scavenging ability of a feed additive for substituting antibiotics according to a preferred embodiment of the present invention. FIG.
15 is a graph showing the number of deaths per broiler growth period according to the feeding of antibiotic replacement feed additive according to a preferred embodiment of the present invention.
FIG. 16 is a graph showing ammonia nitrogen of broiler flock according to the feeding of an antibiotic substitute feed additive according to a preferred embodiment of the present invention. FIG.
FIG. 17 is a graph showing the number of eggs scattered according to feeding of an antibiotic substitute feed additive according to a preferred embodiment of the present invention. FIG.
18 is a graph showing the fatty acid analysis of eggs fed with feed additives for antibiotic substitution according to a preferred embodiment of the present invention.
19 is a cholesterol analysis GC chromatogram of a feed additive feed egg replacing antibiotics according to a preferred embodiment of the present invention.

The present invention relates to a feed additive which promotes disease prevention and growth of livestock and increases the efficiency of feed.

Hereinafter, the present invention will be described in detail as a feed additive for replacing antibiotics and a feed additive for substituting antibiotics.

[Feed additives for antibiotic substitution]

The present invention relates to a feed additive for replacing antibiotics comprising a fermented product obtained by fermenting oregano, cloves and rice bran with a useful microorganism (EM) as a main composition.

Specifically, the feed additives of the above-mentioned composition include oregano, cloves and rice bran as main raw materials that can replace the function of antibiotics.

That is, Oregano is .....  Characteristics (Effect)

Further, Cloves .....  Characteristics (Effect)

Furthermore, The rice bran .....  Characteristics (Effect)

In addition, the useful microorganisms (EM) for fermenting oregano, cloves and rice bran with the above-described constitution can be prepared by including live bacteria such as lactic acid bacteria, yeast and the like in the fermented product of the above-mentioned oregano, Such as enzymes, antimicrobials, organic acids, antioxidant components, etc., can be further produced during the fermentation of the oregano, clove and rice gangue.

That is, the useful microorganisms are composed of lactic acid bacteria and yeast as major microorganisms, and it is preferable that the fermented product is further configured to contain an enzyme, an antimicrobial substance, an organic acid, an antioxidant component, . Further, a specific description of the physiologically active substance in the fermentation process by the useful microorganism of the composition will be described below again.

The fermented product may be prepared by first mixing the oregano, cloves and rice bran and then fermenting the fermented product using the useful microorganism. Preferably, the fermented product is fermented as the useful microorganism, and the oregano fermented product, Clove fermented product and rice bran fermented product, and then mixed at a constant composition ratio to form a fermented product.

Specifically, the oregano fermented product is contained in a feed additive and acts as a main substance containing an antimicrobial agent, an antioxidant substance, and an organic acid. The edible fermented product acts as a main substance containing an antimicrobial agent and an antioxidant substance, The fermented product acts as a main material containing probiotics and organic acids.

At this time, the oregano fermented product, the edible fermented product and the rice bran fermented product may be stirred and mixed in a proper composition ratio according to the judgment of the skilled artisan. Preferably, 7 to 13% by weight of the oregano fermented product, 2 to 8% And the fermented rice bran can be mixed at a composition ratio of 80 to 90% by weight to form a fermented product.

At this time, if the composition ratio of the oregano fermented product is less than 7% by weight, the composition ratio of the fermented oregano fermented product is insufficient and the effect of the fermented product of the fermented oregano fermented product becomes insufficient and 13% , It is preferable that the composition ratio of the above-mentioned oriental fermented product or rice bran fermented product is insufficient, so that the oregano fermented product preferably maintains the composition ratio within the above range.

If the composition of the above-mentioned edible fermented product is less than 2% by weight, the composition of the edible fermented product is insufficient, and the effect of the edible fermented product on the antimicrobial activity and antioxidative effect of the edible fermented product is insufficient. It is preferable that the composition of the fermented oregano fermented product or the fermented product thereof is insufficient, so that the edible fermented product maintains the composition ratio within the above range.

If the composition ratio of the rice bran fermented product is less than 80% by weight, the composition of the rice bran fermented product may be insufficient and the effect of the feed additive due to the rice bran fermented product may be insufficient. It is preferable that the composition ratio of the fermented oregano fermented product or the edible fermented product is relatively insufficient so that the fermented product of the fermented product maintains the composition ratio within the above range.

Test examples for confirming the respective functionalities of the fermented oregano fermented products, edible fermented products and fermented rice bran contained in the fermented product and test examples for testing the prepared feed additives include the following feed additives for antibiotic substitution I will reiterate in the concrete explanation about.

[Preparation method of feed additive for antibiotic substitution]

The present invention relates to a fermentation process for fermenting oregano, cloves and rice bran using a useful microorganism (EM) containing lactic acid bacteria and yeast as major microorganisms, and a fermentation process for preparing feed additives for replacing antibiotics.

Specifically, the fermentation step is a step of obtaining a fermented product by fermenting the oregano, clove and rice bran using a useful microorganism. The oregano, clove and rice bran can be firstly mixed and fermented, but preferably the oregano, The cloves and the rice bran are respectively fermented to obtain the oregano fermented product, the oriental fermented product and the rice bran fermented product, and the oregano fermented product, the oriental fermented product and the bivalent fermented product may be stirred and mixed at a constant composition ratio to make a fermented product.

That is, the fermentation process comprises an oregano fermentation process in which the oregano is fermented with the useful microorganism to produce an oregano fermented product; An oriental fermentation process for fermenting the clove with the useful microorganism to produce an oriental fermented product; Wherein the fermented oregano fermented product, the edible fermented product, and the fermented product of the rice bran are further subjected to a stirring and mixing process to be mixed to form a fermented product. Lt; / RTI >

At this time, the oregano fermentation process can be performed by mixing oregano and useful microorganisms at a constant composition ratio and fermenting them at a predetermined temperature for a predetermined period of time. Preferably, the oregano fermentation process is carried out by mixing 100 parts by weight of the oregano And 35 to 45 parts by weight of a microorganism mixed solution to prepare an oregano fermentation raw material; Fermenting the fermented oregano raw material in an incubator maintained at 30 to 38 DEG C for 15 to 25 days to prepare an oregano fermented product; The useful microorganism mixture may be prepared by mixing 2 to 5 parts by weight of useful microorganisms and 2 to 5 parts by weight of molasses in relation to 100 parts by weight of distilled water.

Specifically, the oregano fermentation raw material mixing step is a process for preparing an oregano fermentation raw material by mixing a beneficial microorganism mixture containing beneficial microorganisms in oregano with a predetermined size, wherein the oregano is ground to a size within a range of 100 to 600mesh It is preferable to increase the fermentability by securing the exposed area of the oregano particle to the useful microorganism.

In addition, the useful microorganism mixture may be composed of various compositions according to the judgment of a person skilled in the art, provided that an environment for allowing the useful microorganism to grow smoothly can be provided. Preferably, 5 parts by weight of the molasses and 2 to 5 parts by weight of the molasses.

At this time, if the composition ratio of the molasses is less than 2 parts by weight with respect to the composition ratio of the beneficial microorganisms, there is a problem that the propagation of useful microorganisms is insufficient due to insufficient feeding for proliferation of beneficial microorganisms, and when more than 5 parts by weight, It is preferable that the molasses is contained in the useful microorganism mixture at a composition ratio within the above range because the proliferation rate of useful microorganisms in the case of exceeding 5 parts by weight is not greatly changed as compared with the proliferation rate of useful microorganisms in molasses contained in parts by weight or less Do.

In addition, the beneficial microorganism may be diluted to a concentration of 2 to 5%.

If the composition ratio of the useful microorganism mixture is less than 35 parts by weight, the fermentability of oregano is somewhat insufficient. If the composition contains more than 45 parts by weight of the beneficial microorganism mixture, the oregano fermentation raw material becomes oregano It is preferable to keep the composition ratio between the mixture of the oregano and the useful microorganism within the above-mentioned range.

The oregano fermentation raw material fermentation step is a process for producing an oregano fermented product by enclosing the oregano fermentation raw material in a container and then fermenting the fermented product for 15 to 25 days in an incubator maintained at 30 to 38 ° C to produce an oregano fermented product, It is a step to ferment oregano.

At this time, if the treatment temperature of the oregano fermentation raw material fermentation step is less than 30 ° C or more than 38 ° C, the temperature for proliferation of useful microorganisms contained in the oregano fermentation raw material becomes low or high, It is preferable to have a fermentation period of 15 to 25 days within the above temperature range.

In addition, the clod fermentation process may be performed by mixing the clods and the useful microorganisms at a constant composition ratio and fermenting them at a predetermined temperature for a predetermined period of time. Preferably, the clod fermentation process comprises the steps of: And 35 to 45 parts by weight of a mixed solution to prepare an edible fermentation raw material; Fermenting the raw material of clove fermentation raw material in a container and sealing it and then fermenting it for 15 to 25 days in an incubator maintained at 30 to 38 ° C to produce an oriental fermented product; The useful microorganism mixture may be prepared by mixing 2 to 5 parts by weight of useful microorganisms and 2 to 5 parts by weight of molasses in relation to 100 parts by weight of distilled water.

Specifically, in the step of mixing the oriental fermentation raw material, a process for preparing an oriental fermentation raw material by mixing a useful microorganism mixture containing a useful microorganism in a clot having a predetermined size, the clove is ground to a size within a range of 100 to 600mesh It is desirable to increase the fermentability by securing the exposed area of the clinging particles to the useful microorganism.

In addition, the useful microorganism mixture may be composed of various compositions according to the judgment of a person skilled in the art, provided that an environment for allowing the useful microorganism to grow smoothly can be provided. Preferably, 5 parts by weight of the molasses and 2 to 5 parts by weight of the molasses.

At this time, if the composition ratio of the molasses is less than 2 parts by weight with respect to the composition ratio of the beneficial microorganisms, there is a problem that the propagation of useful microorganisms is insufficient due to insufficient feeding for proliferation of beneficial microorganisms, and when more than 5 parts by weight, It is preferable that the molasses is contained in the useful microorganism mixture at a composition ratio within the above range because the proliferation rate of useful microorganisms in the case of exceeding 5 parts by weight is not greatly changed as compared with the proliferation rate of useful microorganisms in molasses contained in parts by weight or less Do.

In addition, the beneficial microorganism may be diluted to a concentration of 2 to 5%.

If the composition ratio of the useful microorganism mixture is less than 35 parts by weight, the fermentability of the clove becomes somewhat insufficient. If the composition is more than 45 parts by weight, the composition ratio of the useful microorganism mixture is excessive, It is preferable to keep the composition ratio between the clove and the useful microorganism mixture liquid within the above range because there is a possibility of corruption during the fermentation step of the fermentation material.

The fermentation step of the raw material for fermentation of oriental fermented raw materials is carried out in an incubator kept at 30 to 38 ° C after containing the oriental fermentation raw material in the container and then fermented for 15 to 25 days to produce an oriental fermented product, It is a step of fermenting cloves.

At this time, if the processing temperature of the fermentation source of the oriental fermentation raw material is less than 30 ° C or exceeds 38 ° C, the temperature for proliferation of useful microorganisms contained in the oriental fermentation raw material becomes low or high, It is preferable to have a fermentation period of 15 to 25 days within the above temperature range.

That is, the object of the oriental fermentation process having the above-described constitution can be processed in the same conditions as those of the oregano fermentation process, with the object being cloven.

In addition, the rice bran fermentation process can be performed by mixing rice bran and useful microorganisms at a constant composition ratio and fermenting them at a predetermined temperature for a predetermined period of time. Preferably, 100 parts by weight of rice bran is diluted with distilled water to 2 to 5% 18 to 61 parts by weight of a useful microorganism solution and 54 to 183 parts by weight of molasses diluted to 2 to 5% with distilled water to prepare an undiluted fermentation raw material; Fermenting raw material fermenting raw material in which the raw materials for rice germ fermentation are put in a container and sealed and then fermented for 15 to 25 days in an incubator maintained at 30 to 38 ° C to produce a fermented raw material.

Specifically, the raw rice fermentation raw material mixing step is a step of preparing a raw material for rice bran by mixing raw rice bran, useful microbial broth and molasses diluted in distilled water, and the rice bran can be a powder having a generally known particle size.

The useful microorganism solution contained in the raw materials for rice germ fermentation is preferably diluted with distilled water to 2 to 5%, and the molasses is also diluted to 2 to 5% with distilled water.

In addition, it is preferable that the useful microorganism solution contained in the raw material for rice germanium fermentation is 18 to 61 parts by weight with respect to 100 parts by weight of rice bran. If the composition ratio of the useful microbial solution is less than 18 parts by weight, And when the composition ratio of the useful microbial liquid is more than 61 parts by weight, the fermentation effect of rice bran is not greatly increased due to the increase in the number of useful microbes. Therefore, the composition ratio of the useful microbial liquid to the rice bran ranges from It is preferable to have the inside.

It is preferable that the molasses contained in the rice germ fermentation raw material has 54 to 183 parts by weight based on 100 parts by weight of the rice bran. If the composition ratio of the molasses is less than 54 parts by weight, The useful microorganism growth rate is insufficient. When the content exceeds 183 parts by weight, the useful microorganism growth rate when the content exceeds 183 parts by weight is not significantly changed compared with the proliferation rate of the useful microorganism with respect to molasses containing 183 parts by weight or less. It is preferable that the molasses is included in the useful microorganism mixture at a composition ratio within the above range.

In connection with the above, in the present invention, the rice bran is a major factor in the constitution of producing an organic acid of an antibiotic substitute. In order to optimize the conditions for producing the organic acid, Can be used.

Hereinafter, a detailed description according to the water content of rice bran will be given below.

Also, the fermentation step of raw rice fermentation raw material is carried out by closing the raw rice fermented raw material with the above-mentioned constitution and sealing it and then fermenting the raw rice fermented in an incubator maintained at 36 ° C for 21 days, to be.

At this time, if the processing temperature of the rice bran fermentation raw material fermentation step is less than 30 ° C. or more than 38 ° C., the temperature for proliferation of the useful microorganisms contained in the raw material of rice bran fermentation becomes low or high, It is preferable to have a fermentation period of 15 to 25 days within the above temperature range.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Test Examples, which are for the purpose of preparing an antimicrobial substitute feed additive according to the present invention.

[ Example ]

Ⅰ. Preparation and characterization of fermented material

1. Manufacture of oregano fermented products and edible fermented products

end. Useful microorganism

A useful microorganism (EM-1) used for the fermentation of raw materials is a combination of several microorganisms beneficial to humans among many microorganisms present in the natural world. It is a major microorganism in which yeast, lactic acid bacteria, and photosynthetic bacteria constitute EM. Coexistence coexistence The antioxidant power of the fermentation product produced by the public relations is an important effect of EM and it is used in various fields such as agriculture, fishery industry, living environment and pollution purification. Purchase of EM-1 produced by EVERMIRACLE CO., LTD. Respectively.

The total microbial counts of the useful microorganisms (EM-1) used in the test were 1.79 × 10 ^{10 for} Lactobacillus casei and 3.5 × 10 ⁴ for yeast (Schematics cerevise), pH 3.36, EC (ms / cm) 3.35 Respectively.

I. Raw materials and fermentation conditions

Origenum majorana L. was purchased from Taiyuan Foods in Turkey and used as a raw material. 200 ml of a useful microorganism mixture (188 ml of distilled water, useful microorganism (EM-1, 6 ml) at a concentration of 3%, molasses (6 g) at a concentration of 3%) was added to 500 g of the raw materials (each of which was subjected to oregano and clove) The mixture was well mixed and then pressed into the container. The top was covered with filter paper, sealed with a stopper, and fermented for 21 days in an incubator at 36 ° C.

All. Extraction of useful microbial fermentation product

1 liter of 95% ethanol was added to 100 g of each of the fermented oregano fermented product and fermented fermented product as a useful microorganism, followed by shaking for 2 hours, followed by filtration. Then, 1 L of ethanol was added to the residue again, The extract was repeatedly filtered to remove impurities. The filtrate was concentrated under reduced pressure at 50 ° C using a vacuum concentrator to remove ethanol. The concentrate was diluted to 20 ml with distilled water, dried using a freeze dryer, and then diluted to a constant concentration while being frozen. The ethanol extraction yield of the oregano fermented product was 10.6% and the ethanol extraction yield of the oriental fermented product was 18.1%.

la. Determination of antioxidant activity (DPPH free radical scavenging activity)

DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity was obtained by dissolving 0.1 g of each of the freeze-dried oregano fermented product and the oriental fermented product in 10 ml of ethanol to make a stock solution.

That is, 1 ml of each of the samples of the fermented oregano fermented product and the edible fermented product was added to 1 ml of a 2.0 × 10 ^-4 M DPPH solution dissolved in ethanol, and the final reaction solution was added to 2 ml. The mixture was shaken with a vortex mixer for 10 seconds, For 30 minutes, and the absorbance was measured at 517 nm using a spectrophotometer. DPPH radical scavenging activity of each sample was calculated by the following equation.

     SA (%) = (A-B) / A x 100

     A: Absorbance of DPPH EtOH solution

     B: absorbance of DPPH + sample

2. Preparation of rice fermented rice

end. Raw material

  The rice bran used for the fermentation of useful microorganism (EM-1) was purchased from RPC of Sosung-myeon, Jungpup-si and purchased organic rice bran within 3 days. The water content of the organic rice bran used for the test was 11.37%.

  I. Useful Microbial Fermentation and Extraction

The rice fermented product was optimized for the production of organic acid by focusing on the role of organic acid in antibiotic substitute. In order to select optimum conditions for organic acid production, molasses was used as a substrate for the fermentation of useful microorganisms according to the water content of the rice bran, and EM-1 as the useful microorganism was diluted to 3%, and the water content of the rice bran was 25% %, 35%, 40% and 45%, respectively. Finally, it was fermented for 21 days in an incubator at 36 ° C with anaerobic digestion as shown in Table 1 (input conditions according to the water content of organic rice bran).

All. pH measurement

The pH of the fermented rice bran fermented according to the fermentation period of the fermented rice bran according to the fermentation period of the fermented rice bran was taken from 5 g of the fermented rice bran sample, 25 ml of distilled water was added, and the mixture was shaken in a homogenizer for 30 minutes. 2 filter paper, and then measured using a pH meter (Mettler, MP 220) and an EC meter.

la. Total acidity measurement

For total acidity measurement, 5 g of corn steep liquor was sampled, 25 ml of distilled water was added, and the mixture was shaken in a homogenizer for 30 minutes. 2 filter paper, then dilute with 10 ml of distilled water, add 2 to 3 drops of 1% phenolphthalein indicator to 10 ml of sample, add 0.1 N (w / v) NaOH solution through buret until the sample turns red And 5 times repeated measurements. After measuring the appropriate consumption (ml), the total acidity in the sample was converted into lactic acid by the following equation

×100Acidity (% lactic acid) =

× 100

3. Characteristics of Fermented Water

1) Characterization of Fermented Rice Bran by Water Content of Rice Rice

In the antibiotic substitute, acidity is a very important factor. Various methods have been tried to control the acidity. However, in the invention, the most natural and environmentally friendly method is to use organic acid which is produced when fermenting microorganisms. Therefore, since the information of the organic acid generated during fermentation is important, the following test was conducted to select optimum microorganism fermentation conditions for organic matter.

  end. Changes in pH of fermented rice bran during fermentation

  The changes of pH of fermented rice bran were investigated according to the water content of rice bran during fermentation. As shown in Table 2 (pH change by fermentation period according to the water content of rice bran), the pH of the rice microbial fermentation tended to decrease with increasing fermentation period.

In other words, when the water content was 25%, the pH change increased until the 11th day of fermentation, but the decrease width did not increase after that, but the pH value remained constant. Respectively.

When the moisture content was 30%, the pH value decreased gradually until the 11th day of fermentation and showed the lowest pH value of 4.59.

In addition, the pH values of 35%, 40% and 45% of water contents decreased rapidly until the third day of fermentation. After 7 days, the pH values remained constant and stabilized. 4.5% for 35%, 4.39 for 40% and 4.34 for 45%. Therefore, the pH value of each test sphere according to fermentation period showed the lowest pH value between 7th day and 11th day of fermentation.

The pH value of fermented microorganism fermented according to the moisture content of rice bran was lower as the moisture content was higher than that of control fermented at 18th day of fermentation. 1.86, 35% for 1.98, 40% for 2.15 and 45% for 2.2. In the case of fermentation using useful microorganisms (EM), it is known that organic acid is generated during the decomposition of organic substances by the lactic acid bacteria present in the useful microorganisms, and the pH tends to be kept low (Higa, 2001; Xu, 2000). Therefore, it was confirmed that the fermentation of rice bran using the useful microorganism proceeded well.

I. Storage stability of fermented rice bran

 The storage stability of the useful microorganism fermented rice bran was analyzed by comparing the pH value of the 18th day of fermentation with the pH value of 180 days of storage at room temperature, and the result as shown in Fig. 1 was obtained.

That is, the pH value of the fermented corn steels with a moisture content of 25% was decreased by 0.11 compared to the fermentation of the 18th day of fermentation, 0.19 for the moisture content of 30% and 0.15 for the moisture content of 35% 45% showed almost no change in pH value. Therefore, fermented rice fermented with useful microorganisms showed very low pH value even after 6 months of storage.

All. Changes in total acidity of fermented rice bran during fermentation

In the fermentation of rice microbial fermentation, the changes in total acidity of fermentation period of each sample were investigated according to the water content of rice bran, and the results were as shown in Table 3 (change in total acidity by fermentation period according to the water content of rice bran).

In other words, total acidity increased gradually as fermentation progressed in all the test sites. Total acidity was maintained constantly after reaching the peak. The total acidity of the fermented control group showed a maximum value at the 11th day of fermentation. When the water content was 25% and 30%, the total acidity was steadily increased. Respectively.

The total acidity showed the highest value at the 7th day of fermentation when the moisture content was 35% and 40%, and the highest value at the fermentation 3 days when the moisture content was 45%. As the moisture content increased in the microbial fermentation of rice bran The fermentation period, which indicates the highest total acidity value, was shortened.

In addition, the total acidity value of the rice microbial fermentation according to the water content was increased 2.7% in the case of 25% moisture content, 3.6% in the moisture content 30%, 3.6% in the moisture content 35% 40% and 45%, respectively, showing a rise of 3.3% to 3.72%. Therefore, the highest total acidity value was obtained when the moisture content of rice bran fermentation was above 35%.

2) DPPH freeradical scavenging activity of fermented material

DPPH is a stable free radical, reduced by sulfur amino acids such as cysteine and glutathione, ascorbic acid, tocopherol, and so on. Therefore, DPPH is used not only for measuring the antioxidant activity of hydrogen donor or free radical scavenging activity, Simple and reproducible, it is widely used in various fields including food.

end. DPPH freeradical scavenging activity of fermented oregano

In order to examine the antioxidative activity of oregano fermented products, DPPH radical scavenging activities of extracts of oregano fermented products were measured. The results were as shown in Fig. 2 (DPPH radical scavenging activity of oregano fermented water extract).

As a result, it was found that the untreated control (the known oregano extract without fermentation) showed 55.4 ug / ml and the useful fermented microorganism extract had 38.2 ug / ml as compared with the IC ₅₀ showing the scavenging activity of 50% Fermentation was shown to enhance antioxidant activity. The DPPH radical scavenging activity of each concentration was 6.7% at 10 ug / ml, 8.0% at 20 ug / ml, 14.1% at 40 ug / ml and 60 ug / ml 18.7% higher scavenging activity.

I. DPPH freeradical scavenging activity of oriental fermented material

In order to examine the antioxidant activity of the clove fermented product, DPPH radical scavenging activity was measured by the concentration of the extract extracted from the clove fermentation product. The result was as shown in Fig. 3 (DPPH radical scavenging activity of the clove fermented water extract).

In other words, the IC ₅₀ values exhibiting 50% scavenging activity were 9.8 μg / ml in the control (the known clove extract in the unfermented state) and 9.45 μg / ml in the test sample, and the 50% Respectively. The concentration of IC ₅₀ value is about 10 μg / ml, which is very high among the extracts derived from natural products, indicating that the antioxidant ability of the clove is excellent. The DPPH radical scavenging activity of each concentration was slightly higher than that of control, but the difference was not significant.

All. DPPH freeradical scavenging activity of rice bran fermented

In order to examine the antioxidant activity of the fermented rice bran, DPPH radical scavenging activity was measured by the concentration of extract of the fermented rice bran. The results were as shown in Fig. 4 (DPPH radical scavenging activity of fermented rice bran extract).

In other words, when compared with the IC ₅₀ showing the scavenging activity of 50%, the control (non-fermented rice bran extract) of 1.4 mg / ml and the useful fermentation extract of fermented microorganism 0.5 mg / Fermentation was shown to enhance antioxidant activity. The DPPH radical scavenging activity of each concentration was 8.2% at the concentration of 0.1 mg / ml, 25.4% at the concentration of 0.5 mg / ml and 34.8% higher at the concentration of 1.0 mg / ml compared to the control . These results indicate that fermentation of rice microbial fermentation (EM-1) significantly enhances antioxidant activity, which appears to be due to the effect of organic acids produced during the fermentation process.

Ⅱ. Preparation and Characteristics of Feed Additives

1. Preparation of Feed Additives

1) Useful microorganism (EM-1) Characteristics of fermented feed additive raw material

Fermentation was carried out using useful microorganisms in the production of feed additive for antibiotic substitution. The fermentation efficiency of lactic acid bacteria and yeast was increased during the fermentation process as shown in Table 4 (mixing ratio and role of feed additive raw materials) Microbial fermented rice bran contains a large amount of lactic acid and acetic acid produced during the fermentation process, so that the role of organic acid in the antibiotic substitute and the role of antioxidant and antibacterial activity of oregano and clove are added to feed additives for fermented microbial fermentation antibiotic substitute Respectively.

2) Useful microorganism (EM-1) Fermented feed additive Ratio of ingredients

For the preparation of feed additive for the substitution of antibiotics, a fermented product anaerobically fermented with microorganisms such as rice bran, oregano and clove was used. The mixing ratio of the beneficial microorganism (EM-1) fermented feed additive was oregano and the clove fermented product was added at a concentration that antimicrobial and antioxidative effect was recognized. That is, the mixture was placed in a stirrer at a composition ratio of 10% by weight of the fermented oregano, 5% by weight of the oriental fermented product, and 85% by weight of the fermented rice bran product and stirred evenly for 30 minutes.

2. Characteristics of feed additive

1) Test method

end. Extraction of feed additive

100 g of a feed additive, which is a fermented product obtained by mixing oregano fermented product, clove fermented product and rice bran fermented product, was added with 1 L of 95% ethanol and extracted by shaking at room temperature for 2 hours. The extract obtained by repeating this process 3 times was filtered with a filter paper (Whatman No. 2) to remove impurities. The filtrate was concentrated under reduced pressure at 50 ° C using a vacuum concentrator to remove ethanol. The concentrate was diluted to 20 ml with distilled water, dried using a freeze dryer, and then diluted to a constant concentration while being frozen. The ethanol extraction yield of the feed additive was 12.6%.

I. Organic acid analysis

0.5 g of the reagents of D, L-tartaric acid, oxalic acid, formic acid, D, L-malic acid, lactic acid, succinic acid, citric acid and acetic acid were dissolved in 100 ml of distilled water, And diluted to a standard curve.

For the analysis of the sample, 100 ml of acetonitrile was added to 50 g of the feed additive sample, and the mixture was crushed at 5,000 rpm for 3 minutes in a homogenizer. After shaking for 30 minutes in a shaker, the mixture was centrifuged at 3000 rpm for 5 minutes using a centrifuge and the supernatant was used for purification Respectively.

5 ml of methanol and 5 ml of distilled water were sequentially added to the SPE C18 cartridge to activate the sample. 1 ml of the sample was loaded and then eluted with 4 ml of distilled water, followed by filtration with an RC filter (regenerated cellulose, 0.2 μ m) .

The analytical apparatus was analyzed with a Varian 920-LC. The columns were ACE 5 C18 (4.6 × 250 mm, particle size 5 μL) and column oven temperature was 24 ° C. and flow rate was 0.7 mL / min at 214 nm. At this time, 50% of 20 mM KH ₂ PO ₄ (pH 2.4), 49% of DW and 1% of acetonitrile were used as a mobile phase.

All. Antimicrobial activity measurement

Three commercial strains ( Clostridium perfringens (CP), Escherichia coli (EC), and Salmonella typhimurium (ST)) used in the antimicrobial activity test were purchased from the Rural Development Administration of Korea. First, the seed culture of the test strain was activated by subculture in Nutrient Agar (NA) medium three times and used as a test strain.

The antimicrobial activity test was carried out by using a disk diffusion method in which the degree of growth inhibition was measured by comparing the proliferation degree of the test strain by contacting a paper disk treated with the useful microorganism fermented extract at a concentration of 1 mg, 5 mg, .

Namely, 20 mL of each sterilized NA medium was dispensed into a petri dish, and the plates were coagulated to prepare a plate culture medium. Then, the culture broth activated in NA medium was plated thereon, and sterilized 8 mm paper disks 4 After incubation, 50 μL of sample solution was inoculated with a 0.45 μm membrane filter and incubated at 28 ° C for 24 or 48 hours. The clear zone ) Diameter.

 la. Investigation of acid resistance of feed additive

To investigate the tolerance of lactic acid bacteria and yeasts in the feed additives, lactic acid bacteria were cultured in MRS broth and yeast strains were cultured in NB for 18 hours. 1 ml of the culture broth was added to phosphate buffered saline (PBS) adjusted to pH 2.0, 2.5 and 3.0 , PBS: 0.8% NaCl, 0.02% KH2PO4, 0.015% Na2HPO4). Each sample was incubated at 36 ° C for 48 hours and the number of viable cells was measured.

All. Stability of bile acids

To investigate the resistance of the strains present in the feed additives to bile acids, 1 ml of culture medium containing 0.3%, 0.6% and 1.0% (w / v) of oxgall (Sigma Co., USA) 9 ml of MRS broth was inoculated, and the growth curve of the bacteria was measured by spectrophotometer for 48 hours. The total number of bacteria after culturing was measured, and the culture solution containing no bile acid was compared with the control group.

hemp. Total polyphenol content measurement

The total polyphenol content of the feed additive was determined by adding 2 ml of 2% Na ₂ Cl ₃ to 100 μl of the sample, adding 100 μl of 50% Folin-Ciocalteu reagent, mixing with a vortex for 30 seconds, placing in a dark place for 30 minutes, Absorbance was measured at 750 nm and gallic acid was used as a standard.

bar. Total flavonoid content measurement

For the analysis of total flavonoid content, 0.1 mL of 1 N NaOH was mixed with 1.0 mL of the diluted diluted feed additive extract and the same amount of diethylene glycol, and the mixture was reacted at 37 ° C for 1 hour. Absorbance was measured. The total flavonoid content of the feed additives for antibiotic substitution was calculated using a standard calibration curve obtained by analyzing gallic acid as a reference material.

four. Superoxide Dismutase (SOD) -like activity measurement

SOD activity was measured by Marklund and Marklund method using color development by auto - oxidation of pyrogallol in alkaline state.

In other words, 2.8 ml of 50 mM Tris-HCl buffer and 0.1 ml of pyrogallol were mixed and reacted at 5 ° C for 5 minutes. Then, 0.1 ml of the sample was added, reacted at 25 ° C for 10 minutes, and 0.1 ml of 1 M HCl was added thereto. After termination, the absorbance change was measured at 420 nm. tris-HCl buffer and distilled water. L-ascorbic acid was prepared at four concentrations of 125, 250, 500 and 1000 ppm (99.5% ethanol) as a positive control.

     SOD-like activity (%) = (1-A / B) 100

      A: Absorbance after incubation with sample in tris-HCl buffer

      B: Absorbance after incubation of control group

Ah. Nitrite scavenging ability measurement

Nitrite scavenging of the feed additive extract was carried out by mixing 40 μl of the sample extract, 140 μl of 0.1 N HCl (pH 1.2) and 0.2 μM citrate buffer (pH 4.2, 6.0) in 20 μl of 1 mM NaNO ₂ according to Kato's method Was adjusted to 200 ul. The reaction mixture was reacted in a constant temperature water bath at 37 ° C for 1 hour. Then, 1000 μl of 2% acetic acid and 1% sulfanilic acid (1% sulfanilic acid, 30% acetic acid) 80 μl) were mixed and reacted at room temperature for 15 minutes, and the absorbance was measured at 520 nm. As a control, distilled water was added instead of Griess reagent, and the same method as above was used.

2) General ingredients of feed additives

As shown in Table 5 (general composition of feed additive), the feed additive showed 5.86% crude protein, 3.65% crude fiber and 1.45% ash, and all of the heavy metals were below the standard value, All the values are below the reference value. Also, Escherichia coli and Salmonella were not detected.

3) Antimicrobial activity and total number of microorganisms in feed additives

end. Antibacterial activity against intestinal bacteria and food poisoning bacteria

The antimicrobial activities of the feed additive extracts against the intestinal bacteria Clostridium perfringens (CP), Escherichia coli (EC) and food poisoned Salmonella typhimurium (ST) were measured.

That is, the antimicrobial activity against the Clostridium perfringens (CP) of the feed additive extract, as shown in Table 6 and FIG. 5 (antimicrobial activity against intestinal bacteria and food poisoning bacteria in the feed additive) , And 6 mg, respectively. The antimicrobial activity against Escherichia coli (EC) was 32 mm at the concentration of 6 mg and 30 mm at the Salmonella typhimurium (ST) of the food poisoning bacteria. Thus, the JEM probiotics extract was very strong against intestinal harmful bacteria Indicating that it exhibited an antibacterial effect.

I. Microbial total number of feed additive

The total number of microorganisms in the feed additive was measured to be 3.8 × 10 ^{6 for the} lactobacillus casei and 3.9 × 10 ⁴ for the yeast (Scahromayces cerevise) as shown in Table 7 (total number of microorganisms in the feed additive).

4) Evaluation of stability against acid resistance of feed additive

In order to exert a function as a probiotic probiotic agent, it is necessary to reach the final target site through the pancreas and duodenum, which secrete strong gastric juice and bile. Therefore, ability to survive in the intestine. Stability of the strain should be considered first.

In other words, it is known that lactic acid bacteria generally produce a variety of metabolites such as lactic acid and acetic acid, organic acids such as hydrogen peroxide and antibacterial substances such as bacteriocin, and inhibit the growth of intestinal decay bacteria and harmful pathogenic bacteria. Lactobacillus casei present in the useful microorganism (EM-1) was almost unaffected by 4.6 × 10 ⁸ cfu / ml at pH 3.0, as shown in Table 8 (evaluation of acid resistance of feed additive) 10 ⁶ cfu / ml at pH 2.0 and 3.8 × 10 ⁴ cfu / ml at pH 2.0, indicating that the number of cells decreased with decreasing pH. In the case of the yeast (Scahromayces cerevise), 2.09 × 10 ⁵ cfu / ml at pH 3.0, 3.4 × 10 ⁴ cfu / ml at pH 2.5 and 2.2 × 10 ⁴ cfu / ml at pH 2.0, .

 5) Evaluation of stability of the feed additive for bile acid

end. Assessment of tolerance to bile acids using absorbance

The tolerance of the feed additives to bile acids was measured by a spectrophotometer for 24 and 48 hours, and the survival rate of the bacteria after 24 and 48 hours was evaluated to evaluate the stability against bile acids.

That is, as shown in FIG. 6 (tolerance to bile acid of the feed additive Lactobacillus casei), the resistance of the lactic acid bacterium (Lactobacillus casei), the maximum number of bacteria in the feed additives, to bile acid was evaluated. As a result, %, And 0.6%, the absorbance was similar to that of the control without bile acid. However, the absorbance was increased in the culture medium containing 1.0% and the absorbance value was increased as the content of bile acid was increased in the culture for 48 hours The results showed that the lactic acid bacteria present in the feed additives showed high stability against bile acids.

In addition, as shown in FIG. 7 (tolerance to bile acid by the feed additive yeast Scahromayces cerevise), the tolerance of the yeast (Scahromayces cerevise) to the bile acids in the feed additives decreased as the bile acid content increased for 24 hours , And the same tendency was observed in the culture for 48 hours. In the case of yeast, the stability against bile acid was not recognized.

I. Survival number and survival rate of bile acid for feed additive

In case of Lactobacillus casei, the number of viable cells increased in the culture for 48 hours compared to that of 24 hours in the case of feed additives such as Table 9 (Survival number of the feed additive to bile acid) and Table 10 (Survival rate of the feed additive to bile acid) (0.3%, 0.6%) was similar to that of control (0%) without bile acid, but increased to 6.67 CFU / mL at 1% concentration and increased by 11% compared with the control . In the case of yeast, the number of viable cells increased at 24 hours after culture but decreased at 48 hours. Survival rate decreased 3.1% at 1.0% concentration after 48 hours of incubation.

In order to perform the function as a probiotic, it is necessary to have tolerance to grow in a medium higher than the concentration of intestinal bile (0.6%). Therefore, the feed additive is resistant to bile and can function as a probiotic .

6) Storage stability of feed additive (pH, lactic acid and total acidity)

The total acidity was 0.987%, lactic acid was 0.3%, and pH was 3.26 for the useful microorganism (EM-1) as shown in Table 11 (pH, lactic acid and total acidity change according to storage of feed additive). The total acidity, lactic acid content, and acetic acid content of EM-1 fermented feed additives were 5.25%, 1.31%, and 4.32, respectively, And pH slightly increased.

In the case of feed additive, there was little change in pH and slightly decreased total acidity and acetic acid content compared with 1 month and 6 months after fermentation, but the overall change was insignificant and the storage stability of feed additive probiotics was stable until 6 months.

  7) Organic acid content of feed additive

  end. HPLC chromatogram of organic acids

Acetic acid> acetic acid> lactic acid> lactic acid> lactic acid> lactic acid> lactic acid> lactic acid> lactic acid> lactic acid> lactic acid was determined as shown in FIG. 8 (HPLC chromatogram of organic acid of feed additive), FIG. 9 (organic acid calibration curve of feed additive) DL-tartaric acid and DL-tartaric acid in the order of lactic acid> acetic acid> DL-tartaric acid> DL-malic acid in the order of feed additive. Lactic acid showed higher content of feed additive than EM-1 but higher content of acetic acid than EM-1.

 8) Total polyphenol content of feed additive

The total polyphenol contents of the feed additives were analyzed by thermogravimetric analysis of the feed additive and the heat treatment at 100 ℃ for 10 min. As shown in Fig. 10 (total polyphenol content of the feed additive), the total polyphenol content of the heat-treated feed additive was 2 μg / ml at 0.01%, 56 μg / ml at 0.1% and 81 μg / And there was no difference in contents between the samples.

9) Total flavonoid content of feed additives

As shown in FIG. 11 (total flavonoid content of the feed additive), the total flavonoid content of the heat-treated feed additive was 8.5 μg / ml at 0.1%, 13.0 μg / ml at 0.1% and 80.5 μg / ml at 1%. The total flavonoid content of the heat - treated samples was the same as that of the untreated samples.

 10) DPPH free radical scavenging activity of feed additive

DPPH free radical scavenging activity of DPPH free radical scavenging activity of feed additive extract was measured as 42% at 0.25 mg / ml and 71% at 0.5 mg / ml after 30 minutes of reaction, as shown in FIG. 12 (DPPH free radical scavenging activity of feed additive) 1.00% showed an elimination activity of 92 mg / ml, and the IC ₅₀ showing a 50% scavenging activity was 0.36 mg / ml.

 11) Superoxide Dismutase (SOD) -like activity of feed additives

Superoxide dismutase (SOD) is O ₂ in vivo ^- an enzyme that serves to convert the (superoxide) by H ₂ O ₂ and O ₂ in a normal state. Since the active oxygen generated in vivo causes an oxidative disorder in the body, development of a material having SOD - like activity is required to suppress this phenomenon.

- CuZn-SOD / Mn-SOD / Extracellular SOD

₂ O ₂ ^- + 2H ^{+ -} & gt ^; H ₂ O ₂ + O ₂

The SOD-like active substance is not an enzyme but is a low-molecular substance having a role similar to that of SOD, mainly belonging to the pytochemical and inhibiting the reactivity of superoxide. Therefore, ingestion of this SOD - like substance can prevent the oxidative damage by removing superoxide in the living body, and antioxidant effect can be expected.

That is, SOD-like activities of the feed additive extracts were measured by the autoxidation reaction of yrogallol, as shown in FIG. 13 (superoxide dismutase (SOD) -like activity of the feed additive) % To 20%, and SOD-like activity from 0.1% to 41%. The SOD - like activity of the non - heat treated samples was similar to that of the heat treated samples. Thus, the stability of the feed additive to heat was superior to the SOD-like activity.

 12) Nitrite scavenging ability of feed additive

Since nitric oxide (NO) produced by NO synthase (NO) is rapidly converted to nitrate (NO ₃ ^- ) and nitrite (NO ₂ ^- ), the total NO ₃ ^- / NO ₂ ^- Can be used as a marker. After reduction of NO ₃ ^- to NO ₂ ^- using nitrate reductase, the amount of NO ₂ ^- can be measured with Griess reagent. It is known that the color reaction using Griess reagent is inhibited by NADPH, which is essential for NO synthase and nitrate reductase reaction.

Namely, as shown in FIG. 14 (nitrite scavenging ability of feed additive), nitrite scavenging ability of the feed additives was 25% at the concentration of 0.1%, 43% at the concentration of 0.5%, 81% Respectively. The difference in nitrite scavenging ability between the heat treated sample and the nitrite scavenging ability was not recognized in the case of the non-heat treated sample, and the stability against heat was confirmed.

Ⅲ. Assessment of Livestock Specification by Feed Additive Feeding

(1) Evaluation of the productivity and characteristics of broiler chickens

1) Disclosure animal and specification management

The productivity of broiler chickens was evaluated at the farms in Ju - po, Bu - gun, Jeonbuk, Korea. The broiler broiler chicks were distributed at Dongwoo, Dongwoo, Korea for testing. The experimental group was tested for 26,554 in 2 dogs and 30,282 in 2 dogs Respectively.

The experimental diets were fed with broiler diets containing no antibiotics. The diets were fed at an initial feed rate of 1 to 7 days, an electric feed rate of 8 to 21 days, and a late feed rate of 22 to 30 days. Feed additives were fed at a ratio of 0.5% based on the basic feed. During the test period, treatments and water were free from vegetarian diet.

2) Test methods and survey items

end. Feed intake and feed conversion rate

The feed intake rate for the entire test period was measured by subtracting the feed amount from the feed amount, and the feed conversion rate was calculated by the feed amount (feed intake / gain amount) used for the growth.

I. Determination of ammonia nitrogen in flour

Ammonia nitrogen (NH ₃ -N) in the feces is oxidized with a basic hypochlorous acid solution to produce monochloramine, followed by phenol, nitroprusside, and excess hypochlorous acid to produce indole phenol At this time, the absorbance of the final color developed indene phenol was measured at 640 nm, which is the maximum absorption wavelength, using a spectrophotometer (Mecasys, OPTIZEN 2120UV).

All. Investigation of microorganisms in cecum and feces

After completion of the specimen test, 3 chicks closely approximating the ending weight were selected for each treatment, and the cecum was collected after selection and slaughtered for analysis. 1 g of the contents of the cecum was continuously diluted to 9 mL of sterilized peptone (Merck) in weight-to-volume to determine the total microflora, Lactobacillus and E. coli . The medium contained Tryptic Soy Agar (Difco), MRS Agar and MacConkey Agar. Continuous dilution was carried out to inoculate 0.1 mL of each of the diluted solutions at a time point containing 10 ⁷ to 10 ⁹ microorganisms on a petri dish (SPL 90 × 15) by a flaked flat plate method. After inoculation, the number of colonies was measured after incubation at 37 ° C for 48 hours. In order to measure the number of E. coli in the feces, 3 replicates were collected at the end of the specimen and stored at -70 ℃ until analysis. A total of 1 g of each sample was collected and the total number of microorganisms was measured as in the method of analysis in the contents of the cecum.

la. Total IgG and total IgM content in serum

Total IgG and IgM levels were measured in order to evaluate the immune response of broiler chickens. After completion of the specimen test, 5 chicks of average body weight per treatment were selected, and after fasting for 24 hours, 4 mL of blood was collected from the vein and the serum was separated. The separated serum was stored at -70 ° C. until analysis. Chicken IgG ELISA Quantitation Kit (Bethyl laboratories, Inc., U.S.A) was used for total IgG analysis and Chicken IgM ELISA Quantitation Kit (Bethyl laboratories, Inc., U.S.A) was used for IgM analysis. Absorbance was measured at 470 nm using an ELISA reader (Biotek, powerwave XS, U.S.A).

3) Evaluation of productivity of broiler chickens

end. Feed intake

The feed intake of broiler chickens during the test period was measured as in Table 13 (feed intake of broiler chickens during feeding of feed additive). As a result, 2,256 g of chickens fed the control diet and 2,221 g of marine fermented feed additive And intake was 89g less than the control. Therefore, feeding of feed additive to 30,282 broilers in the control can save 2,695 kg of feed.

I. Feed rate

As shown in Table 14 (feed conversion ratio of feed additives), the feed conversion ratio was calculated by dividing the feed intake by the amount of feed required to breed 1 kg of broiler meat. The feeding rate of the feed additive was 1.390, The feed requirement ratio was 0.062 less than 1.452. This result is similar to the report that lactic acid bacterium is effective in improving the weight gain and feed efficiency when added to broiler chickens.

All. Mortality rate of broiler chickens

As shown in FIG. 15 (number of deaths per broiler growth period according to feeding of feed additive), the number of deaths of broiler chickens during the growing period was high until the second day in the control group, but thereafter decreased. But after 25 days of age, it gradually increased again. In the case of feed additive, the mortality rate after 2 days was drastically decreased, especially after 16 days.

As shown in Table 15, the mortality rate of broiler chickens in the control group was 7.18% in the initial feed group and 2.65% in the feed group, as shown in Table 15 (feed mortality of feed additives) %, But decreased slightly in late-stage feeds and decreased by 56.9% compared to the initial feeds. In the experimental group, the mortality was 2.94% in the initial feeding group, 1.46% in the feeding group, 50.4% in the feeding group, and 87.6% in the late feeding group.

Compared with the control, mortality was lower in the initial, feed and late feed than in the control. The mortality rate of the experimental group was 2.44 times higher than that of the control group, 1.82 times higher than that of the control group, and 4.69 times lower than that of the control group. In addition, the mortality rate in the test period was 4.68% in the test group and 12.28% in the control group, which was 7.6% lower than the control group. The results showed that the feed additive significantly reduced the mortality rate, and the higher the feeding effect, the lower the mortality rate.

4) The ammonia nitrogen

As shown in FIG. 16 (the result of measuring the content of ammonia nitrogen in broiler flocks by feeding of feed additive), the content of ammonia nitrogen in broiler flour 16 days and 30 days was measured. As a result, The content of nitrogen was 1.5 mg / L in the control and 0.8 mg / L in the test. The nitrogen content in the test group was lower than that in the control group, and the control group was 1.0 mg / L in the control group and 0.8 mg / The content was 20% lower. Therefore, feeding of feed additive decreased the amount of ammonia nitrogen and decreased odor generation.

5) Total number of microorganisms in cecum and feces

end. Total number of microorganisms in the cecum

The total number of microorganisms in the cecum of broiler chickens fed with feed additives, such as the total number of cecal microorganisms in broiler chickens at the time of feeding the feed additive, was analyzed. As a result, the total number of microorganisms in the broilers was similar to the total number of microorganisms In case of Lactobacillus , however, Coli form, which is a harmful organism, showed lower total number of test group than control group. As a result of this study, it was found that the feeding effect of feed additive suppresses harmful bacteria in cecum and increases beneficial bacteria in broiler chickens as a result of Kiberwetter-Swida and Binek (2009). These results suggest that the organic acids in the feed additives increase the resistance of the broiler to intestinal infectious agents and regulate the mucosal immune system, which promotes the dominance of the beneficial bacteria and acts as an immunity enhancer to reduce pathogenic microorganisms compared to the control.

I. Total number of microorganisms in feces

As shown in Table 17 (total number of microorganisms in the feces of the broiler when feeding the feed additive), the fecal microorganisms were counted on the 30th day of the experiment in order to measure the number of microorganisms in the broiler feces fed the feed additive Respectively. As for the total number of microorganisms in the feces, the number of lactic acid bacteria, Lactobacillus, which is a beneficial bacteria in the feeding additive of the feed additives, is increased and coli form number, which is a harmful microorganism, is decreased. As the number of coli forms decreased and Lactobacillus increased significantly, the addition of feed additives in the feed increased the number of beneficial bacteria, decreased the number of coli forms, It is believed that maintaining the bacterial gun in the intestine results in improved productivity and reduced mortality.

6) Total IgG and total IgM content in serum

Total IgG and IgM levels in serum were measured to evaluate the immune response of broiler chickens, such as Table 18 (total IgG and total IgM content in broiler chickens during feed additives). Serum immunoglobulin IgG and IgM levels were significantly increased in the experimental group fed the feed additive compared to the control group. Serum IgG was increased by 18.4% and serum IgM by 21.1% compared with the control. These results suggest that the administration of probiotics affects the enhancement of the immunity of broiler chicks. As a result, it is considered that the feeding of feed additives gives a positive nutritional function to immune function.

(2) Evaluation of laying productivity and egg characteristics

1) Disclosure animal and specification management

Productivity and characterization of laying hens were carried out at a poultry farm in the munjung-myeon, Iksan-si, Jeonbuk, Korea, and a 35-week-old Hy-Line Brown laying hens were used as test animals. In the breeding farm, 120 specimens (115 hens and 5 cocks) were housed in a 4 m wide, 7.5 m high and 2 m high, and feeds were fed in the control diet, Feed additives were added at 0.5%, and about 120 g of feed was added per day.

2) Test methods and survey items

end. Egg weight, egg weight, egg yolk, egg white and eggshell weight

Egg production was measured using eggs produced from 20 days to 50 days after feeding of feed additive. The egg production rate was measured at 11:00 am and 4:00 pm during the test period. The egg weight and the number of egg laying were examined by each test, and the egg production rate was calculated by dividing the egg weight by the number of eggs. Egg yolks, egg whites, and egg shells were recovered in batches of 10 eggs. Egg yolks, egg whites and egg shells were separated and counted with electronic scales.

I. Feed rate

After 24 hours, the amount of feed was collected and the remaining amount was checked. The feed conversion ratio was calculated by dividing the daily feed intake (g) by the daily egg weight (g) per day, by comparing the scattering effect with the feed intake of the laying hens, and by verifying the nutritional value of the feed . The amount of egg laying was calculated by egg production rate (%) × average egg weight (g).

Feed demand ratio =

       Feed intake (g) / egg production (g) × 100

All. Inorganic content

The content of inorganic components was measured by incubating egg yolk at 660 ° C for 4 hours, dissolving in HCl: H ₂ O (1: 1, v / v) solution, leaving overnight, filtering and analyzing using ICP (Varian 720-ES) .

la. Determination of antioxidant activity (DPPH free radical scavenging activity) of eggs

DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity was determined by using lyophilized chloroform: methanol (2: 1, v / v) And homogenized for 3 minutes with a homogenizer adjusted to 2,500 rpm. 1 filter paper and used as an analytical sample. 1 ml of each sample was added to 1 ml of 2.0 × 10 ^-4 M DPPH solution dissolved in ethanol, added to 2 ml of final reaction solution, shaken for 10 seconds with vortex mixer, allowed to react at room temperature for 30 minutes, and then spectrophotometer And the absorbance at 517 nm was measured. DPPH radical scavenging activity of each sample was calculated by the following equation.

   SA (%) = (A-B) / A x 100

    A: Absorbance of DPPH EtOH solution

    B: absorbance of DPPH + sample

hemp. Composition and content of fatty acids

Analysis of fatty acids in egg yolk was carried out by hydrolysis of egg yolk and methyl esterification to make derivative, followed by GC analysis. To 10 g of the lyophilized sample, 50 mL of chloroform: methanol (2: 1, v / v) was added and homogenized for 3 minutes with a homogenizer adjusted to 2,500 rpm. 1 filter paper, and then concentrated using a rotary vacuum evaporator at 50 ° C. Approximately 10 mg of the egg yolk extract was measured, and 1 mL of a methanol solution containing 0.5 N NaOH was added thereto. The resulting solution was sieved in a water bath at 60 ° C for 15 minutes and transferred to a separating funnel to remove impurities. After 2 ml of BF3-methanol was added to the filtrate from which the impurities were removed, it was sealed and then methylated by heating in a 60 ° C water bath for 15 minutes. After cooling at room temperature, 1 mL of hexane and 8 mL of NaCl saturated solution were added and shaken. After the layer was divided, the upper layer was transferred to a 10 mL tube, dried with Na ₂ SO ₄ , and used as a GC sample. The analysis conditions of gas chromatography (BRUKER 450-GC) are as follows. Detector: FID, Column: VF-5 (0.32 x 30 m), Tem: 80 캜 (30 min hold), 20 캜 / min, (280 캜, 10 min hold) Inj.temp: 250 캜, Det.temp: 300 캜

bar. Total cholesterol analysis

Cholesterol content was determined by lyophilization and 0.3 g of egg yolk was dissolved in 10 mL of a hydrolysis solvent (95% ethanol: 33% KOH = 94: 6, v / v) and then 5α-cholestane (2.5 mg / mL) 0.5 mL was added thereto, and the mixture was subjected to sifting under a reflux condenser at 60 DEG C for 1 hour. After the saponification, 10 mL of water and 5 mL of hexane were added and centrifuged at 1,000 rpm to obtain supernatant. Hexane layers were collected and concentrated completely with nitrogen gas and re-dissolved in 1 mL of hexane and analyzed by gas chromatography (BRUKER 450-GC). The analysis conditions are as follows. Detector: FID, Column: VF-5 (0.32 x 30 m), Tem: 80 캜 (30 min hold), 20 캜 / min, (280 캜, 10 min hold) Inj.temp: 250 캜, Det.temp: 300 캜

3) Evaluation of productivity of laying hens

 end. Number of laying eggs and egg production by feed additives

As a result of evaluating the number of eggs and the egg production rate for 30 days from 20 days to 50 days after the feeding of the feed additive, as shown in FIG. 17 (number of eggs to be fed with feed additive) and Table 19 Of the control group increased by 10.97 compared to the control, and that of the control group increased by 9.54% compared to that of the control group.

 I. Feeding amount of feed additive

As shown in Table 20 (amount of egg production by feed additives), daily egg production was shown using egg production and egg weight. The amount of spawning increased by 12.2% in the experimental group fed the feed additives compared to the control. These results showed that the mean egg weight of the experimental group did not show any significant difference from the control, but the egg production rate appeared to be different.

All. Feed intake and feed conversion rate

As shown in Table 21 (egg production rate by feed additives), the feed intake was increased by 2.6 g per day in the test group fed with the feed additive compared to the control group. However, the daily feed intake and the feed intake were used to calculate the feed intake One feed conversion ratio was lower in the test group than in the control group. Therefore, feeding of feed additives seems to increase the efficiency of the feed.

4) Evaluation of egg quality and quality

end. Egg yolk, egg white, egg shell weight

As shown in Table 22 (weight of egg yolk, egg white, eggshell of feed additive feed egg), egg yolk content of egg yolk was higher in the control group than in the control group, But the egg shell was higher in the test group.

 I. Inorganic and harmful components of egg yolk

The effects of feed additives on the mineral content of egg yolk, such as Table 23 (yolk inorganic component of feed additive feed egg) and Table 24 (egg yolk heavy metal of feed additive feed egg), were examined, mg / kg, and the experimental group was 382.57 mg / kg, which was 2.3 times higher than the control group.

The contents of major inorganic components of egg yolk were Ca, followed by K, Al, Na, Mg and Fe. The content of each major inorganic component was 2.08 times for Ca, 2.43 times for K, 2.36 times for Al, 2.51 times for Na, 2.43 times higher than that of Fe and 3.06 times higher than that of Fe. Therefore, feeding of feed additives significantly increased the inorganic content of egg yolk. In addition, the analysis of harmful components of egg yolk showed the value lower than the standard value of food revolution and safety of feed additive was recognized.

All. DPPH free radical scavenging activity of eggs

To evaluate the antioxidative activity of egg yolk and egg white, as in Table 25 (DPPH free radical scavenging activity of feed additive-fed egg yolk) and Table 26 (DPPH free radical scavenging activity of feed additive-supplemented egg white) 1-diphenyl-2-picrylhydrazyl) free radical scavenging activity. The free radical scavenging activity of egg yolk was increased by 5.6% at 10% concentration compared to the control, but no significant difference was observed between egg yolk and control.

la. Composition and content of fatty acids in eggs

As shown in Table 27 (fatty acid composition of feed additive feed egg) and FIG. 18 (fatty acid analysis GC chromatogram of feed additive feed egg), the fatty acid content of egg yolk was the same as that of oleic acid, stearic acid, palmitoleic acid acid, palmitic acid, linoleic acid and myristic acid. There was no significant difference in oleic acid among unsaturated fatty acids, but palmitoleic acid and linoleic acid were slightly increased in the feed additive group compared to the control group. The ratio of saturated fatty acid: unsaturated fatty acid (SFA: USFA) in egg yolks was 43.3: 56.7 in the control group but 42.1: 57.9 in the control group. These results showed that feeding of feed additives increased the content of unsaturated fatty acids.

hemp. The total cholesterol content of eggs

As shown in Table 28 (total cholesterol content of feed additive feed egg) and FIG. 19 (cholesterol analysis GC chromatogram of feed additive feed egg), cholesterol plays an important role in maintaining life. Higher cholesterol levels lead to arteriosclerosis, hypertension And it is necessary to take an appropriate amount of intake.

Total cholesterol level of egg yolk was 26.65 mg / g, which was 12.37% less than that of the control. These results suggest that fatty acid content of feed additive is high in fatty acid analysis. Therefore, feeding of feed additives lowers the cholesterol content of egg yolk and low cholesterol can be produced.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, It is possible to carry out various changes in the present invention.

Claims (10)

A feed additive for substituting antibiotics, characterized by comprising a fermented product fermented with useful microorganisms (EM) of oregano, cloves and rice bran as a main composition.
The method according to claim 1,
The useful microorganism may be,
Wherein the lactic acid bacteria and yeast are composed of major microorganisms.
The method according to claim 1,
The oregano, cloves and rice bran,
Wherein the microorganism is fermented as a useful microorganism and then mixed to form a fermented product.
The method of claim 3,
The fermented product may contain,
7 to 13% by weight of oregano fermented product obtained by fermenting oregano as a useful microorganism, 2 to 8% by weight of an oriental fermented product obtained by fermenting cloves with a useful microorganism, and 80 to 90% by weight of fermented rice bran fermented with a useful microorganism, Wherein the feed additive is adapted to replace the antibiotics.
A fermentation process for producing fermented products by fermenting oregano, cloves and rice bran using a useful microorganism (EM) having lactic acid bacteria and yeast as main microorganisms.
6. The method of claim 5,
The fermentation process comprises:
An oregano fermentation process for fermenting the oregano with the useful microorganism to produce an oregano fermented product;
An oriental fermentation process for fermenting the clove with the useful microorganism to produce an oriental fermented product;
And a microbial fermentation step of fermenting the rice bran with the useful microorganism to produce a rice bran fermented product,
Wherein the oregano fermented product, the edible fermented product and the unfermented fermented product are further mixed and stirred to form a fermented product.
The method according to claim 6,
In the oregano fermentation process,
With respect to 100 parts by weight of the ground oregano,
And 35 to 45 parts by weight of a useful microorganism mixture to prepare an oregano fermentation raw material;
Fermenting the oregano fermentation raw material in an incubator maintained at 30 to 38 ° C for 15 to 25 days to produce an oregano fermented product,
The useful microorganism mixture may contain,
Wherein the microorganism is composed of 2 to 5 parts by weight of useful microorganisms and 2 to 5 parts by weight of molasses in 100 parts by weight of distilled water.
The method according to claim 6,
The clove fermentation process comprises:
With respect to 100 parts by weight of the crushed clove,
Fermentation raw material mixing step of mixing 35 to 45 parts by weight of a useful microorganism mixture to produce an oriental fermentation raw material;
Fermenting the raw material of clove fermentation material by enclosing the clove material in a container and sealing it and then fermenting it for 15 to 25 days in an incubator maintained at 30 to 38 ° C to produce an oriental fermentation product,
The useful microorganism mixture may contain,
Wherein the microorganism is composed of 2 to 5 parts by weight of useful microorganisms and 2 to 5 parts by weight of molasses in 100 parts by weight of distilled water.
The method according to claim 6,
In the above rice bran fermentation process,
With respect to 100 parts by weight of rice bran,
18 to 61 parts by weight of a useful microorganism solution diluted to 3% with distilled water, and 54 to 183 parts by weight of molasses diluted with 3% to distilled water to prepare a raw material for rice bran fermentation;
Fermenting raw material fermenting raw material, which comprises fermenting raw rice fermented raw material in a container and sealing the raw rice fermented raw material and fermenting the fermented raw material for 15 to 25 days in an incubator maintained at 30 to 38 ° C to produce a raw fermented raw material. ≪ / RTI >
10. The method of claim 9,
The above-
To 25% to 45%. ≪ RTI ID = 0.0 > 21. < / RTI >

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