KR20080005037A - Functional fermented green tea microorganisms using green tea and useful microorganisms and manufacturing process of the same and feeding method of pig and pork acquired of the same - Google Patents

Abstract

A functional fermented green tea microorganism agent prepared from green tea and useful microorganisms is provided to inhibit the intestinal harmful bacteria, increase the cell-mediated immunity and humoral immunity and be used for raising a pig, thereby capable of increasing the productivity of the pig by significantly improving the weight gain of the pig and the feed demanding rate and providing the pig with high protein content, low fat content, good flesh color, low cholesterol and good tenderness of a meat. A method for preparing a functional fermented green tea microorganism agent comprises the steps of: (a) mixing powdered dried green tea leaves, non-fat rice bran and wheat bran in the weight ratio of 3:5:2; (b) after evenly spraying and inoculating each Lactobacillus acidophilus KCTC 3111 and Lactobacillus plantarum KCTC 3104 in a level of 1x10^7/ml, respectively into the mixture to make the water content percentage of the total mixture become about 40% water content, subjecting the total mixture to an anaerobic and an aerobic lactobacillus fermentation process at a temperature of 37 deg.C for 3 days to prepare a solid fermented product; and (c) after evenly spraying and inoculating each Saccharomyces cerevisiae KCTC 7915, Bacillus subtilis KCTC 3239, and Bacillus coagulans KCTC 1015, in a level of 1x10^12/ml, respectively into the solid fermented product, drying the product at a temperature of 35-40 deg.C for 2 days. A pig is characterized in that it is limitlessly fed by a feed containing 0.1-1 wt.% of the functional fermented green tea microorganism agent and has the protein content of 19.7-22.5 %/g, the fat content of 1.7-2.7 %/g, and the cholesterol content of 78-81.8 mug/g.

Description

FUNCTIONAL FERMENTED GREEN TEA MICROORGANISMS USING GREEN TEA AND USEFUL MICROORGANISMS AND MANUFACTURING PROCESS OF THE SAME AND FEEDING METHOD OF PIG AND ACQUIRED OF THE SAME}

1 is a manufacturing process of the fermented green tea microbial agent using the green tea and the useful microorganism of the present invention.

The present invention relates to a fermented green tea microbial agent using green tea and useful microorganisms, a method for preparing the same, and a method of raising pigs using the same, and more specifically, to dried pork powder, dried green tea powder, and then degreasing rice as a grain by-product. Inoculate two kinds of lactic acid bacteria into the mixture mixed with the wheat vein at a predetermined ratio, make a solid fermentation product of lactic acid bacteria through anaerobic and aerobic effect tablets, and then inoculate two kinds of yeast bacteria and two kinds of Bacillus subtilis at 35-40 The daily drying process is carried out to produce fermented green tea microorganisms, a feed additive for livestock.

In addition, the present invention by using the fermented green tea microbial agent in the breeding period to feed pigs added in 0.1% to 1% by weight based on the total amount of feed based on the total feed amount to breeding pigs, the weight gain and feed during the same breeding period Significantly improved demand to increase productivity, high protein content, low fat content, good meat color, low cholesterol, tender and tender pork can be obtained.

In recent years, the pig industry has increased in size due to the large scale of breeding, resulting in increased diarrhea and pneumonia, and increased productivity due to the use of antibiotics in pig feed and the use of chemical treatments. Recently, however, regulations on the use of antibiotics have been tightened around the world, and as an alternative, microbial agents on non-antibiotic biological methods are used as antibiotic substitutes.

In particular, since May 2006, the addition of antibiotics in formulated feeds has been greatly reduced, and antibiotics have been tightened to be used for therapeutic purposes, not for the purpose of promoting livestock growth. Therefore, there is an urgent need for alternatives to antibiotics. It is true. Furthermore, in recent years, the importance of microbial agents and fermented feeds using natural functional substances and useful microorganisms has emerged again in terms of changing national consciousness, demand for safe livestock, demand for high quality livestock or branding, and improving the livestock environment. .

The microbial agent is a substitute for antibiotics and is a live microbial feed additive that can have a beneficial effect on the animal by balancing the intestinal microflora of the animal and inducing beneficial gut benefits of the microorganisms to promote animal health and promote animal growth. It is known that this can be done (Fuller, 1989). Feeding microorganisms has been shown to prevent diarrhea and improve piglet productivity (Pollman et al., 1980; Collington et al., 1988), and to improve growth capacity not only in piglets, but also in growing and finishing pigs (Baird, 1977). In addition, the addition of microbial agents was able to improve the utilization of nutrients in feed by activating intestinal enzymes (Collinston et al., 1988; Scheuermann, 1993), and have been reported to enhance animal health by improving immunity (Kato et al. , 1983; Fuller, 1989).

Recently, the development of functional foods has been very active due to the increasing interest and demand of consumers for health-oriented foods. Recently, a lot of researches have been made to utilize vegetable food as a material of health food (Park et al., 2002), and much attention has been focused on the practical use attempts and researches in the food-related industry and medical field on extracts of plants. (Yoon et al., 1977).

The main physiologically active substance of green tea is catechin, a kind of polyphenol, which accounts for more than 70% of green tea polyphenol content (Khokhar and Magnusdottir, 2002). (Graham, 1992). Useful ingredients of green tea have antioxidant activity (Kim et al., 2004; Robak and Gryglewski, 1998), antimicrobial activity (Shi, 2002), cholesterol lowering effect (Yang and Koo, 1997; Jin et al., 2004). It has been shown to be prophylactic (Yang and Laudau, 2002) and has also been reported to reduce LDL and total cholesterol levels (Davies et al., 2003).

In general, pork is a popular food for consumers because it is cheaper than beef and contains high quality protein. On the other hand, with the recent increase in income level, consumers' meat preference is getting more attention on quality than quantity, and import liberalization of all livestock products is getting stronger and the world is becoming more and more single market. . As a result, it is urgent to differentiate production from cheap imported meat by improving the quality of production and quality of livestock products, and in order to survive the competition with imported meat, it is inexpensive, high quality, hygienic and safe brand meat. Production is urgently needed.

Green tea used in the present invention has not only been consumed as a symbol tea since about 2,700 BC in China, Korea, Japan, etc., and recently, the value of the green tea is gradually recognized as the pharmacological mechanism of various ingredients contained in green tea is gradually revealed. In particular, anti-tumor of polyphenols (EC, EgC, ECg, EgCg), which is one of the main ingredients in green tea, antioxidant prevention by antioxidant free radicals, lowering blood sugar, lowering cholesterol in blood and preventing food corruption It is known to have various functional functions in health. In addition, green tea is widely known as a nutritional and favorite food, such as environmental hormone inhibitory effect, E. coli O-157 and food poisoning inhibitory effect, diet effect, skin care effect, alcohol and tobacco detoxification effect. Green tea is a non-alcoholic beverage. It is not only a nutritional and favorite food, but also a food with a body control function from the viewpoint of physiological control and nutrition as well as prevention and treatment of human diseases. With the development of modern life science and technology, the pharmacological effects of green tea become more obvious. Various physiological activities of catechins have been studied for vascular strengthening. Among them, catechin activity, namely blood pressure lowering, antioxidant activity, cancer suppression effect, HIV reverse transcriptase inhibitory effect, cholesterol reuptake inhibition and caries prevention There are reports of various pharmacological effects, and the effects and efficacy of scientific reports such as the report of platelet aggregation inhibitory activity are scientifically proved, and thus it is highly applicable as a natural substance.

In addition, the beneficial lactic acid bacteria of Lactobacillus plan taryum (Lactobacillus plantarum) bacteria Lactobacillus Ecija utilized with the present invention the filler's (Lactobacillus acidophilus) bacteria and the like to help the digestion of the feed to produce a starch digestive enzyme, a proteolytic enzyme, Produces antibiotics (Bacteriocin) to prevent intestinal harmful bacteria from adhering to the digestive tract and settles, and is effective in preventing diseases through the production of organic acids, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) bacteria It has the effect of enhancing the source and palatability of the unknown growth factor (UGF) and promoting digestion utilization. Bacillus subtilis and Bacillus coagulans bacteria have excellent fermentation ability and enteric harmful bacteria. Its antagonistic activity and ability to produce large amounts of digestive enzymes are excellent.

Therefore, the inventors of the present invention, using the useful microorganisms in view of the above point to produce a functional fermented green tea microbial agent through a biological process, and by adding a fermented green tea microbial agent to a general formulated feed to breed pigs in the same breeding period When the weight gain and feed requirements are significantly improved, and the slaughtered pigs are fermented using the fermented green tea microorganisms, high protein content, low fat content, good meat color, and low cholesterol are obtained. I would have to.

It is an object of the present invention to provide a functional fermented green tea microbial agent having excellent anti-pathogenic and antibacterial properties using green tea and useful microorganisms and a method for producing the same.

Another object of the present invention is to add a fermented green tea microbial agent using green tea and useful microorganisms in the feed to feed the pigs to significantly improve the weight gain and feed efficiency during the same breeding period, to increase productivity It is to provide a method of breeding pigs using a fermented green tea microbial agent.

Another object of the present invention is to obtain a high protein content, low fat content and good meat color, low cholesterol, soft and tender pork from pigs raised using fermented green tea microorganisms using green tea and useful microorganisms will be.

In order to achieve the above object, the method for producing a functional fermented green tea microbial agent using green tea and useful microorganisms according to the present invention is pulverized and dried powdered green tea leaves, green tea powder and grain by-product degreasing rice and wheat bran Mixing at a weight ratio of 3: 5: 2, respectively;

Lactobacillus acidophilus Lactobacillus acidophilus KCTC 3111, Lactobacillus plantarum KCTC, a beneficial lactic acid bacterium, in a mixture of the above-mentioned green tea powder, skim bran, and wheat flakes in a weight ratio of 3: 5: 2, respectively. 3104 bacteria are sprayed and inoculated evenly at the level of 1 × 10 ¹⁰ / ml to obtain a water content of about 40% of the total mixture, followed by an anaerobic and aerobic lactic acid bacteria fermentation process at 37 ° C. for 3 days to produce a solid fermentation product. Wow;

Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) KCTC 7915 bacteria, Bacillus subtilis KCTC 3239 bacteria, Bacillus coagulans ( Bacillus coagulans ) KCTC 1015 bacteria 1 × 10 evenly sprayed with ¹² / ml level, inoculation by a step of two days and dried at 35~40 ℃ conditions.

In order to achieve the above another object, by using the fermented green tea microbial agent prepared by the manufacturing method to feed pigs an unlimited amount of mixed feed added to the commercial blended feed 0.1 to 1% by weight based on the total feed amount By breeding, it is possible to improve productivity by increasing the weight gain and feed rate significantly during the same breeding period, and to get high protein content, low fat content, good meat color, and low-cholesterol tender and tender pork. Have

Another object of the present invention is that the functional substances possessed by green tea and useful strains used for fermentation inhibit the enteric harmful bacteria and increase the cellular and humoral immunity of pigs.

Hereinafter, preferred embodiments of the present invention will be described in more detail.

Example 1

In order to select useful bacteria for the preparation of fermented green tea microorganisms in the present invention, the following experiment was performed.

[Table 1] Experimental design for selection of beneficial strains

In this experiment, it was necessary to maintain high viability in the intestine of livestock under the condition of feed additive microorganism. Therefore, the ability of acid resistance, bile acid, and heat resistance was evaluated for selection of strains resistant to gastric acid and bile acid.

In order to select useful bacteria for the preparation of the fermented green tea microorganism of Example 1, 11 strains and 15 strains possessed by the Department of Animal Resources, Sunchon National University, and 26 strains in total were selected as the public strains. Selected. The strains were selected based on their separation from pig and chicken intestine, release of antibiotics or microorganisms during growth, and production of strong digestive substances.

In the acid resistance test of Example 1, 26 strains of the target strains were inoculated with each bacterium in sterile 50 ml medium, and then cultured at 30 ° C. or 37 ° C. for 24 hours.

50 ml of MRS medium (Difco, USA), NB medium (Nutrient Broth, Difco), and YM broth medium (Difco) each adjusted to pH 4 using artificial gastric fluid (added 1,000 U pepsin / ml) As a control, 50 ml of MRS medium (Difco, USA), NB medium (Nutrient Broth, Difco), and YM broth medium (Difco) at pH 6-7 were prepared. 1 ml of the culture medium of 26 target strains was added to 50 ml of each of the experimental (pH 4) and control (pH 6 to 7) culture mediums in the prepared medium for Example 1, 30 ° C. or 37 for 2 hours. After shaking culture and stationary culture at ℃, 0.1 ml of this was taken and serially diluted with 0.85% NaCl and cultured in petridish to measure the number of viable cells. Preparation of artificial gastric juice was a modification of Kobayashi et al. (1974) method to adjust the pH of the culture medium to 4 with 1N HCI, pepsin was added to 1,000 U / ml.

In addition, each bacterium was inoculated into each sterilized 100 ml medium for resistance to bile acids, and incubated at 37 ° C. for 48 hours (30 ° C., 24 hours in the genus Saccharomyces ), and then 1 ml of strain culture was tested. As a control, 100 ml of NB medium containing 0.3% of sterile porcine bile acid (sigma) and 100 ml of NB medium without pork bile acid as control were inoculated respectively and incubated at 37 ° C (30 ° C for Saccharomyces genus) for 24 hours. Incubated. Thereafter, 1 ml was taken and serial dilution with 0.85% NaCl was used to measure the number of colonies grown on plate media (NA, petridish).

In addition, the heat resistance test was carried out heat resistance test for the strains confirmed acid resistance and bile resistance. For heat resistance experiment, Lactobacillus, Enterococcus genus were cultured in MRS broth, Bacillus genus was cultured in NB medium, Saccharomyces genus 100 ml was cultured in sterile test tubes, and 10 ml of Lactobacillus , Enterococcus spp. At 15 minutes, Saccharomyces genus strain was reacted for 5 minutes at 70 ℃. Thereafter, 1 ml was taken and 10-fold serial dilution with 0.85% NaCl was carried out to identify surviving heat resistant strains, and the number of colonies grown by plating on MRS, NA, and YM petri dishes was measured.

Experimental Example 1

Experimental Selection of Beneficial Strains for Preparation of Fermented Green Tea Microorganism of Example 1

As shown in Table 1, the selection test results of beneficial strains through acid resistance, bile acid, and heat resistance using a total of 26 strains including 14 kinds of lactic acid bacteria, 8 Bacillus strains, and 4 yeast strains are shown in Table 2 below. .

[Table 2] Acid resistance test results of lactic acid bacteria

[Table 3] Bile Acid Resistance Test Results of Lactic Acid Bacteria

[Table 4] Acid resistance test results of Bacillus and yeast

[Table 5] Bile Acid Resistance Test Results of Bacillus and Yeast

As can be seen from Table 2 to Table 5, KCTC 3111, KCTC 3146, KCTC 3150 strains of Lactobacillus acidophilus monarchs in 14 species of Lactobacillus acidophilus monarchs, respectively, 66.9%, 61.9%, 57.29% as a very strong strain to stomach acid The Lactobacillus casei KCTC 3109 strain had a survival rate of 62.5%, the Lactobacillus plantarum KCTC 3104 strain had a survival rate of 54.2%, and the Enterococcus faecium KCTC 2022 strain had a survival rate of 55.3%.

In addition, Lactobacillus acidophilus KCTC 3111, KCTC 3150 were strains beam a high survival rate, respectively to 43.2% and 44.6%, Lactobacillus casei KCTC 3109 strains survival 42.1%, Lactobacillus plantarum KCTC 3104 strains survival 41.3%, Entercococus faecium KCTC 2022 strain The survival rate was 41.0%, and the strain was evaluated as having a high ability to withstand bile acids. In Bacillus acid resistance test, Bacillus subtilis KCTC 3239 strain showed the highest survival rate of 42.4%, and Bacillus coagulans , KCTC 1015 strain was 42.98%.

Results of acid resistance test for yeast were 38.1% and 37.69% of Saccharomyces cerevisiae KCTC 7915 and KCTC 7928 strains, respectively.

In the bile acid resistance test against Bacillus , Bacillus subtillis KCTC 3229 strain showed the highest survival rate of 34.25%. In Bacillus colagulans , KCTC 1015 strain showed 31.25% survival rate. Was evaluated as a strong strain against bile acids. In yeast, Saccharomyces cerevisiae KCTC 7915 and KCTC 7928 strains showed high survival rates of 34.71% and 34.52%, respectively.

In Experimental Example 1, a total of ten strains, including six lactic acid bacteria, two Bacillus bacteria and two yeast strains, were selected in the acid and bile acid resistance experiments using artificial gastric juice and bile acids to select strains resistant to gastric and bile acids in the animal body. It was.

The results of evaluation of heat resistance using the 10 selected strains are shown in Table 6 below.

For this experiment, Lactobacillus spp., Enterococcus spp. And Bacillus spp. Strains were reacted for 15 minutes at 80 ° C water bath and Saccharomyces spp. For 5 minutes at 70 ° C for survival.

[Table 6] Heat resistance test results of selected strains

As can be seen in Table 6, the heat survival rate of the selected strains through acid resistance and bile resistance was 35-54%, and in the case of Lactobacillus acidophilus bacteria, KCTC strain was 51.41%, the most excellent ability to withstand heat The Lactobacillus casei KCTC 3109 strain had a survival rate of 40.28%, the Lactobacillus planatarum KCTC 3014 strain had a survival rate of 44.63%, the Enterococcus faecium KCTC 2022 strain had a survival rate of 45.05%, the Bacillus subtilis KCTC 3239 strain had a survival rate of 54.44%, and the Bacillus coagulans strain KTCT 1015. In the case of Saccharomyces cerevisiae, the strains of KCTC 7915 and KCTC 7928 showed 50% and 54.31% survival rate, respectively, and selected strains selected by acid and bile resistance were excellent in heat resistance.

Example 2

The stability of the green tea component (green tea extract) was evaluated using 10 strains selected through acid resistance, bile acid resistance, and heat resistance experiments in Example 1, which was conducted to select useful microorganisms in the present invention.

In order to evaluate the stability of the green tea component of Example 2, the green tea material was used green tea grown in Boseong-gun, Jeonnam, green tea extract was taken by taking 100 mg of green tea sample and adding 100 ml of distilled water and warming it for 30 minutes in 80 ℃ water bath. It was. Extracted green tea solution was added to each of the sterilized MRS, NA, YM medium by 2 ml to prepare a plate medium (agar medium, Petridish). Ten strains of acid, bile, and heat resistances were inoculated on plate medium containing each green tea extract, and cultured at 37 ° C. for 48 hours (30 ° C. for 24 hours in Saccharomyces genus). After that, compared to the positive control on each flat medium without green tea extract, the growth is maintained and resistant (+) if the green tea extract is resistant, intermediate (W) if the growth is weak, and susceptible if the growth is suppressed. It was determined as (-).

Experimental Example 1

Resistance test against the green tea extract of the strain of Example 2-

As shown in Table 7, the results of resistance test against green tea extract were shown using a total of 10 selection strains, including 6 lactic acid bacteria, 2 Bacillus bacteria, and 2 yeast strains.

As can be seen in Table 7, five strains of the selected strains, including Lactobacillus acidophilus KCTC 3111 strain, Lactobacillus plantarum KCTC 3104 strain, Bacillus subtilis KCTC 3239 strain, Bacillus coagulans KCTC 1015 strain, and Saccharomyces cerevisiae KCTC 7915 strain. Was selected as resistant to green tea extract.

[Table 7] Results of resistance test against green tea extract of selected strains

Example 3

In order to determine the addition level of green tea for the production of fermented green tea microbial agent in the present invention was tested as follows.

100 ml of the sterilized glucose medium solution for Example 3 was mixed and inoculated with the selected five strains, and the green tea was added at 10%, 20%, 30%, 40%, and 50%, respectively. After culturing for 24 hours, samples were taken at intervals of 2 hours and analyzed over time for the proliferation of the total strains of each green tea addition level.

Experimental Example 1

Experiment of Determining the Optimal Green Tea Addition Level of Example 3

As shown in Table 8, using the five strains selected in Experiment 1 of Example 2 showed the proliferative results of the total strain according to the green tea addition level.

[Table 8] Proliferative test results of total strains by green tea addition

As can be seen in Table 8, the total number of bacteria by the green tea addition level showed a high growth rate in the green tea 10%, 20%, 30% treatment, the growth rate is significantly lowered at the green tea 40%, 50% added level Could see. After 24 hours of incubation, there was no significant difference in the growth of the composite strain over time.

The optimum level of green tea addition for the preparation of the fermented green tea additive of this experiment was determined to be 10% to 30%, and the level of 30% to which the maximum amount of green tea could be added was evaluated as an appropriate addition level.

Example 4

In order to determine the appropriate mixing ratio of green tea and excipients for the production of fermented green tea microbial agent in the present invention was tested as follows.

For Example 4, the proliferative properties of the complex strains by the appropriate combination of green tea and excipients were investigated using grain by-products degreasing rice bran and wheat bran. In order to determine the optimum mixing condition of green tea and excipients in this experiment, a treatment mixture containing 30% green tea weight and 70% degreasing rice, a treatment mixture containing 30% green tea and 70% wheat flour, and 30% green tea Treatment group blended with 50% skimmed rice and 20% wheat flour, 30% green tea, 20% skimmed wheat and 50% wheat flour, 30% green tea, 40% skimmed wheat and wheat Experimental design with 5 treatments with 30 %% of the weight was inoculated to inoculate 5 kinds of complex strains selected for each treatment, and anaerobic and aerobic fermentation were performed by standing and stirring for 3 days. It was.

Experimental Example 1

Experiment of Determination of Optimal Formulation of Excipients for Preparation of Fermented Green Tea Microorganism of Example 4

As shown in Table 9, the results of the proliferation of the strain according to the blending ratio in order to determine the appropriate mixing ratio of green tea and excipients.

[Table 9] Experimental Results of Optimal Formulation of Excipients

As can be seen in Table 9, the proliferative investigation of the complex strain to determine the optimal mixing condition of green tea and excipients was 30% green tea, 50% degreasing steel, 20% wheat flour and 30% green tea and The total bacterial count of the mixed strain was the highest in the treated group of 40% of skim steel and 30% of the wheat bran, and the lowest of the treated group of 30% of the green tea and 70% of the wheat bran. In this experiment, the higher the blending ratio of wheat vegetation, the lower the proliferative potential of the complex strain.

Example 5

The present inventors prepared fermented green tea microorganisms based on the results obtained in Examples 1, 2, 3 and 4.

30 kg of green tea powder pulverized with dried green tea leaves, 50 kg of degreasing rice, and 20 kg of bovine skin are uniformly mixed, and Lactobacillus acidophilus KCTC 3111, Lactobacillus plan Tactile ( Lactobacillus plantarum ) KCTC 3104 bacteria were diluted to 1 × 10 ¹⁰ / ml, respectively, and evenly sprayed and inoculated to the mixture to obtain a water content of about 40% of the total mixture for 3 days in a fermentor controlled at 37 ° C. The fermentation process of anaerobic and aerobic lactic acid bacteria is carried out to produce a reddish brown culture with sweet and sour flavor.

In addition, Saccharomyces cerevisiae KCTC 7915 bacteria, Bacillus subtilis KCTC 3239 bacteria, Bacillus coagulans KCTC 1015 bacteria, respectively, were prepared in the fermented green tea culture. Evenly sprayed and inoculated at a level of 1 × 10 ¹² / ml and dried at 35-40 ° C. for 2 days to prepare a fermented green tea microorganism having a moisture content of about 15%.

The present inventors prepared 100 kg of the fermented green tea microbial agent in the above examples.

Example 6

In order to investigate whether the fermented green tea microbial agent prepared in Example 5 has a propagation inhibitory activity against bacterial pathogens, inhibition of E. coli KCTC 2618, which is a dominant bacterium that causes diarrhea in livestock, was evaluated.

E. coli KCTC 2618 was incubated in Nutrient Broth (Difco) medium for 24 hours at 37 ° C. Then, 1 ml of the culture medium and 1 g of the fermented green tea microorganism prepared in Example 5 were taken to obtain a new sterilized Nutrient Broth (Difco). 100 ml of the medium was inoculated and shaken incubated at 37 ° C. for 24 hours, and the density of E. coli KCTC 2618 in the culture medium was analyzed at 2 hour intervals.

As a selective medium for E. coli density analysis, a petrifilm plate for E. coli (3M, St. Paul USA) was used.

Experimental Example 1

E. coli propagation inhibitory test of fermented green tea microbial agent of Example 6-

The results of the propagation inhibitory test of E. coli KCTC 2618 of the fermented green tea microbial agent are shown in Table 10 below.

[Table 10] Test Result of Escherichia Coli Reproduction of Fermented Green Tea Microorganisms

As can be seen in Table 10, the mixed culture of E. coli KCTC 2618 strain of pig E. coli KCTC 2618 and fermented green tea microorganisms in NB medium to examine the number of bacteria of E. coli according to the culture time, after the start of the mixed culture, 4 By the time the number of Escherichia coli increased, but rapidly decreased after 6 hours, the survival rate of E. coli was suppressed more than 99% after 10 hours of culture. In addition, in the control culture cultured only E. coli , the number of E. coli KCTC 2618 strains increased rapidly over time. As a result, when fermented green tea microbial agent is added to the feed of livestock, it is considered that it can significantly reduce the diseases such as diarrhea of livestock.

Example 7

Pig specification test was performed using the fermented green tea microbial agent prepared in Example 5.

The pigs used in this study were 90 heads of two-way hybrid Landrace (L) x Yorkshire (Y) finishing pigs with an average body weight of 70-75 kg. The pigs were divided into 15 pigs of 6 heads each, and the control (3 pigs) and antibiotics were used. 5 treatments containing 3 (3 pigs), 0.1% (3 pigs) fermented green tea microorganisms (3 pigs), 0.5% (3 pigs) fermented green tea microorganisms (3 pigs) Specification test was carried out.

The test feed of this test was formulated with the control diet according to the nutritional requirements of NRC (1994) without adding antibiotics as shown in Table 11. Antibiotics were added to the control diet by adding chlotetracline 0.003%, and fermented green tea microorganisms were added by adding 0.1%, 0.5% and 1.0% to the control diet, respectively. During the entire test period, the diet was allowed to have unlimited feed and free water.

Table 11 Components of Test Feed

The test period of this test was carried out for six weeks from October 29 to December 10, 2005 at a finishing farm in the Sungyang Pig Farming Corporation in Sinwol-ri, Joseong-myeon, Boseong-gun, Jeonnam.

Experimental Example 1

-Measurement of weight gain, feed intake and feed demand rate of pigs belonging to each treatment group of Example 7

In the test, body weight was measured by total weight for each treatment every two weeks from the start to the end of the test. Feed intake was determined by measuring the remaining amount of feed before weighing every treatment group from the beginning to the end of the test. The feed intake rate was obtained by dividing the feed intake by the weight gain.

The results for the weight gain, feed intake and feed rate of the above hog test are shown in Table 12 below.

[Table 12] Weight gain, feed intake, feed rate

As can be seen in Table 12, the weight gain of each treatment group of 0 ~ 2 weeks of age was the highest with 0.183 addition of fermented green tea microorganisms, 14.83 kg, the control was the lowest as 12.28 kg, fermentation at 2-4 weeks of age The 0.5% green tea microorganism added group was the highest with 16.44 kg and the control group was the lowest with 13.28 kg (P <0.05). The weight gain during the 0-6 week-old test was highest in antibiotics and 0.1% fermented green tea microorganisms, and the control was lowest, but there was no statistically significant difference.

At 4 to 6 weeks of age, antibiotics added the highest amount of antibiotics at 56.67 kg, and 0.5% added fermented green tea microorganisms showed the lowest value at 48.89 kg (P <0.05). Feed intake between 0 and 6 weeks, which was the period of transcription test, was the highest with 152.06 kg for antibiotics and the lowest with 139.44 kg, but there was no statistical difference.

In the feed demand rate, the control group showed the highest value of 3.35 at 0 ~ 2 weeks of age, and the 0.1% addition of fermented green tea microbial agent was the lowest at 2.88, but there was no statistical difference. At 2 to 4 weeks of age, the control was the highest with 3.56 and the 0.5% addition of fermented green tea microorganism was the lowest with 3.16, but there was no statistically significant difference. At 4-6 weeks of age, 0.1% of fermented green tea microorganisms was the highest at 4.59, and the control was the lowest at 3.76. The feed demand for the 0-6 weeks of age was highest in the addition of antibiotics (3.60) and lowest in the fermented green tea microorganisms (0.5%) of 3.42.

Therefore, in view of the productivity, the present inventors evaluated that fermented green tea microorganisms significantly improved the weight gain and feed demand of pigs when 0.5 to 1.0% by weight of the feed was added to commercial feed for pigs.

Experimental Example 2

-Analysis of moisture, crude protein, crude fat and crude ash of pork belonging to each treatment of Example 7

In the analysis, immediately after the end of the specification test, 15 pigs were selected, with 3 heads per repetition in pigs near the average weight in each treatment group. The sirloin was extracted from the selected pigs and ground in a growing machine, and analyzed according to AOAC (1995) method such as moisture, crude protein, crude fat and crude ash.

Table 13 shows the analysis results of water, crude protein, crude fat, and crude ash of pork by each treatment.

As can be seen from Table 13, crude protein showed the highest level of fermented green tea microorganism with 1.0% added group (22.58%) and showed statistically significant difference between treatments (P <0.05). The control group had the highest control (1.87%) and the lowest fermented green tea microorganism (1.0%) showed 1.27% (P <0.05). Crude fat was the highest with the addition of antibiotics (2.87%), and fermented green tea microorganism with the 1.0% addition was the lowest (1.75%), but there was no statistical difference.

[Table 13] Analysis of moisture, crude protein, crude fat and crude ash of pork by test group (%)

Experimental Example 3

Analysis of LBA (TBA) of Pork in Each Treatment of Example 7

In the above analysis, acidity was mixed with 2 g phosphoric acid and 20% trichloroacetic acid in 50 ml of 20 g of analyte, and the slurry for the extracted mixture was diluted with 40 ml of distilled water (DW) and shaken to homogenize. Filter 50 ml of Whatman NO 1. Filter paper, then transfer 5 ml of the filtrate to a test tube and add 5 ml of 2-thiobarbituric acid (0.005 M in DW). The tube was then set up and the mixture was mixed by conduction, kept at room temperature for 15 hours in the dark, and the resulting color was analyzed by measuring the absorbance at 530 nm using spectronic -20D ⁺ (Vernon et al. , 1970).

The results of the TBA analysis of pork in each treatment group are shown in Table 14 below.

[Table 14] Result of rancidity (TBA) analysis of pork by test group (μmol / 100g)

As can be seen in Table 14, the acidity of fresh pork was the highest as 1.42 μmol / 100g in 0.1% added fermented green tea microorganisms, and the control was the lowest as 1.02 μmol / 100g, but there was no statistically significant difference. . At 3 weeks of treatment, the degree of rancidity was the highest in the control group (4.46 μmol / 100g) and the lowest in 0.5% fermented green tea (3.94 μmol / 100g).

Experimental Example 4

-Meat color analysis of pork belonging to each treatment tool of Example 7-

In the above analysis, L value indicating brightness, a value indicating redness and b value indicating yellowness were measured on the surface of the fillet using a Croma meter (Minolta Co. Cr 301, Japan). At this time, a white standard plate having an L-value of 97.10, an a-value of -0.17, and a b-value of 1.99 was used.

The meat color analysis results of the pork by each treatment section are shown in Table 15 below.

[Table 15] Meat color analysis results of pork by test section

As can be seen in Table 15, the lightness (L) was the highest with the addition of 0.5% fermented green tea microorganisms 55.56, the lowest with the addition of antibiotics was 49.03, red (a) is 0.5% of fermented green tea microorganisms The highest value was added with 10.18 and the lowest value with antibiotic added was 8.72. The yellowness (b) was the highest in the fermented green tea microorganism 0.5% addition group was 5.71, the lowest in the antibiotic addition group was 4.00.

Experimental Example 5

Analysis of cholesterol content of pork in each treatment group of Example 7

Table 16 shows the results of analyzing the cholesterol content of the pork by each treatment.

As can be seen in Table 16, the 0.5% added fermented green tea microorganisms were the lowest as 78.02 μg / g compared to the control and antibiotic added groups.

[Table 16] Analysis of cholesterol content of pork by test group (㎍ / g)

Experimental Example 6

-Sensory evaluation of pork belonging to each treatment section of Example 7-

In the above test, Kwak et al. (1992) conducted a method of cooking meats in front of the school by dividing the sirloin parts at regular intervals for students at Sunchon National University. Juicyness, age, and flavor were examined by a five-point measurement method with a score of (1: very bad, 2: bad, 3: moderate, 4: good, 5: very good).

The meat color analysis results of the pork by each treatment section are shown in Table 17 below.

[Table 17] Sensory Test Results of Pork

As can be seen in Table 17, the juiciness of fermented green tea microorganisms 1.0% added highest was 4.47, the control was the lowest to 3.50 showed a statistically significant difference between treatments (P <0.05). The year of antibiotics added was highest at 5.00, the control was lowest at 3.73, and the flavor was highest at 4.50 with 1.0% fermented green tea microorganisms, and the control was lowest at 3.77. (P <0.05).

Therefore, when the pigs were fermented using fermented green tea microorganisms, it was evaluated that low-cholesterol, low-cholesterol, low-cholesterol, low-cholesterol, low-cholesterol, soft, tasty high-quality meat could be produced.

Experimental Example 7

Determination of the Spleen Weight of Pigs in Each Treatment Zone of Example 7

In the above experiment, after the end of the specification test, six pigs of each treatment group were slaughtered, the spleen was extracted from the slaughtered carcass, and the weight of the spleen was measured using an electronic balance. The mean and standard error of the measured values were calculated.

In the above experiments, the spleen is a peripheral lymphoid organ where lymphocytes are collected, and the spleen recognizes, captures, and delivers pathogens to the lymphocytes. Lymphocytes in the spleen leave the spleen and circulate lymph and blood. That is, if the number of lymphocytes circulating away from the spleen is one of the reasons that the weight of the spleen decreases. As lymphocytes and lymphocytes circulate in the blood increase, the externally invading pathogens can be recognized more quickly and induce an appropriate immune response.

The results of measuring the spleen weight of the pigs for each treatment group are shown in Table 18 below.

[Table 18] Spleen weight measurement results of pigs by test

As can be seen in Table 18, the weight of the spleen weight was decreased in fermented green tea microbial agent 0.1%, fermented green tea microbial agent 0.5%, fermented green tea microbial agent 1.0% compared to the control and antibiotic addition. Therefore, the number of circulating lymphocytes increased with the addition of fermented green tea microbial agent.

Therefore, when fermented green tea microorganisms were added and reared, it was evaluated that an appropriate immune response could be induced against externally invading pathogens.

Experimental Example 8

Induction experiment of spleen cell proliferation of pigs belonging to each treatment group of Example 7

In the above experiment, spleens extracted in Experimental Example 7 were made into single cells using tweezers, and then dead cells and erythrocytes were removed using NycoPrep ™ 1.077A, and viable cell counts were measured using a hemocytometer using Trypan blue reagent. . Splenocytes (5 × 10 ⁵ cells) were dispensed into 96 well microplates, and LPS (1, 3 and 10 μg / ml) and Con A (0.1, 0.3 and 1.0 μg / ml) were added to each concentration. After the ul was incubated in a 5% CO ₂ incubator for 3 days, the degree of cell proliferation was measured. Cell Titer 96 ^® Aqueous One Solution Cell Proliferation Assay was used. Discard 100 μl of the culture solution and add 15 μl of cell titer to 100 μl of the remaining culture and incubate for 4-8 hours, followed by Microplate reader (OPTImax, Molecular Devices, USA) and the absorbance at 490 nm.

In this experiment, the pathogen invading the living body is circulated along the blood vessel entering the blood, and is captured by antigen-presenting cells such as macrophages in the spleen. The spleen is a peripheral lymphoid organ mainly composed of T cells, B cells and macrophages, and antigen-presenting cells induce cellular and humoral immune responses by informing T cells and B cells of antigen invasion (Ezekowitz Et al., 1998). Therefore, it is necessary to search how fermented green tea microorganisms affect spleen cells involved in cellular and humoral immune responses.

Since the initial start of each immune response that recognizes a pathogen begins with a cell proliferation reaction, the effect of fermented green tea microbial agent was measured by the proliferation-induced response of splenocytes.

The results of the spleen cell proliferation induction test of pigs for each treatment group are shown in Table 19 and Table 20 below.

[Table 19] T cell proliferation test results in spleen cells of pigs by test (0.D. at 490 nm)

Since T cells are important cells responsible for cellular immune responses in the immune response, the effect of the addition of fermented green tea microbial agent on the cellular immune enhancing effect can be seen.

As can be seen in Table 19, as a result of measuring the proliferative response of splenocytes by stimulating with Con A (Concanavalin A), which specifically proliferates only T cells among splenocytes, the control, when not stimulated with Con A, There was no significant increase in the addition of antibiotics, 0.1% of fermented green tea microorganisms, and 0.5% of fermented green tea microorganisms, but 0.69 ± 0.14 0.D. The proliferative response of splenocytes was reduced statistically significantly at 490 nm (P <0.05).

When stimulated with 0.1 μg / ml Con A, 1.0% of the fermented green tea microorganisms added 1.02 ± 0.21 0.1 D. The proliferative response of splenocytes was increased statistically at 490 nm (P <0.05).

When stimulated with 0.3 μg / ml Con A, the control was 0.88 ± 0.08 0.D. Compared to at 490 nm, 0.97 ± 0.19 0.D. There was a statistically significant increase at 490 nm (P <0.05).

When stimulated with 1.0 [mu] g / ml Con A, the control group had a 0.1% addition of fermented green tea microorganisms as compared to 0.92 ± 0.07 0.D at 490 nm. at 490 nm, fermented green tea microorganism 0.5% addition 1.11 ± 0.11 0.D at 490 nm and fermented green tea microorganism 1.0% addition 1.03 ± 0.12 0.D. Splenocyte proliferation was significantly increased at 490 nm (P <0.05).

Therefore, the addition of fermented green tea microbial agent was evaluated as having an effect on increasing cellular immune immunity in swine cells.

In addition, since B cells are important cells responsible for humoral immune responses that produce antibodies in the immune response, the effect of addition of fermented green tea microbial agents on the humoral immune enhancing effect can be seen.

[Table 20] B cell proliferation test results in splenocytes of pigs by test (0.D. at 490 nm)

As can be seen in Table 20, by stimulating with LPS (Lipopolysaccharide) to specifically proliferate only B cells to measure the effect on the splenocyte proliferation response of fermented green tea microbial agent, control, when not stimulated with LPS, There was no significant increase in the addition of antibiotics, 0.1% of fermented green tea microorganisms, and 0.5% of fermented green tea microorganisms, but 0.69 ± 0.14 0.D. The proliferative response of splenocytes was reduced statistically significantly at 490 nm (P <0.05).

When stimulated with 1.0 μg / ml LPS, the control group had 0.88 ± 0.13 0.D. Compared with at 490 nm, 0.5% addition of fermented green tea microorganism was 1.02 ± 0.12 0.D. The proliferative response of splenocytes was increased statistically at 490 nm (P <0.05).

When stimulated with 3.0 µg / ml and 10.0 µg / ml LPS, the proliferative response of splenocytes increased significantly in the 0.1%, 0.5%, and 1.0% fermented green tea microorganisms compared to the control and antibiotic treatments (P <0.05).

Therefore, the addition of fermented green tea microbial agent was evaluated to have a statistically significant increase in the proliferative response to LPS stimulation of splenocytes compared to the control group.

Experimental Example 9

Experiment for measuring cytokine (IL-6, TNF-α) secretion in spleen cells of pigs belonging to each treatment group of Example 7

In the above experiments, pig spleen cells were cultured for 24 hours by adding LPS (10 μg / ml) and con A (1.0 μg / ml), and then the culture supernatant was collected and IL-6, TNF contained in the supernatant. The amount of α was measured using an enzyme-linked immunsorbent assay (ELISA).

In other words, dilute the primary antibody (capture Ab) in PBS, put 100 ㎕ of each plate on the plate overnight at 4 ℃ and wash with washing solution (0.05% Tween 20 / PBS), then block solution (1% BSA, 5% sucrose) , 0.05% NaN ₃ ) was blocked for 2 hours. Then, the culture supernatant was added, and then washed with a washing solution after 3 hours, and a secondary antibody (dection Ab) was added thereto. After 2 hours, the solution was washed with washing solution, and then Streptavidin-HRP was added.

After an hour, washed again with washing solution, and then added the substrate (2, 2'-azino-bis, 0.1M citric acid, H ₂ O ₂ ) to develop the color and then measured the absorbance at 405 nm using a microplate reader. Converted using. At this time, the measurement limit of each cytokine was 7.8 pg / ml.

In Experimental Example 8, when a proliferative response of splenocytes to Con A or LPS stimulation occurs, the splenocytes secrete various types of cytokines.

Therefore, by analyzing the cytokines secreted when the proliferative response of splenocytes occurs, the immune response can be distinguished.

Among the cytokines secreted by splenocytes, IL-6 induces hepatocytes to synthesize some plasma proteins such as fibrinogen and acts as a B cell growth factor that activates B cells to increase antibody activity (Devries, 1999).

In addition, when the proliferative response of splenocytes occurs, another cytokine secreted by the splenocytes, TNF-α, expresses a new receptor for endothelial cells, which makes leukocytes adhere and activates neutrophils (Schall). Et al., 1994).

It is an endogenous pyrogen that acts on the hypothalamus of the brain to produce fever, acts on hepatocytes to stimulate the synthesis of serum proteins, and inhibits the division of bone marrow hepatocytes (Arai 1990; Beuther 1995).

Therefore, we measured the secretion of IL-6 and TNF-α secreted by splenocytes to investigate the effect on the secretion of IL-6 and TNF-α secreted by the growth of pig splenocytes following fermented green tea microbial agent. The experiment was conducted.

The results of measuring the cytokine (IL-6, TNF-α) secretion of porcine spleen cells of each treatment group are shown in Table 21 and Table 22 below.

Table 21-Results of measurement of IL-6 secretion of spleen cells of pigs by test group (pg / mL)

As can be seen in Table 21, the result of measuring the secretion amount of IL-6 secreted by the spleen cells to determine the effect on the secretion of IL-6 secreted when the splenocytes are added by fermented green tea microbial agent In case of non-stimulation with, Con A or LPS, the control and antibiotic addition groups showed no significant increase, but the 0.1% addition of fermented green tea microorganisms 22.88 ± 9.45 pg / ml and the 0.5% addition of fermented green tea microorganisms 18.50 ± 18.13 pg / ml The production of IL-6 was increased statistically to 16.00 ± 11.65 pg / ml with 1.0% addition of fermented green tea microbial agent (P <0.05).

In addition, when stimulated with 1.0 μg / ml Con A, which induces the most proliferative response of splenocytes, the control group showed IL-6 at 125.38 ± 85.71 pg / ml with 0.5% addition of fermented green tea microorganism compared to 36.63 ± 21.29 pg / ml. The secretion of was increased significantly (P <0.05).

In addition, when stimulated with LPS 10.0 μg / ml, which induces the most proliferative response of splenocytes, 0.1% added fermented green tea microorganisms 88.50 ± 37.42pg / ml and 1.0% added fermented green tea microorganisms compared to the control and antibiotic added groups. The secretion amount of IL-6 was increased to 81.00 ± 59.46 pg / ml (P <0.05).

Therefore, it was evaluated that the secretion amount of IL-6 secreted when the proliferation of splenocytes to Con A and LPS stimulation with the addition of fermented green tea microbial agent occurred.

Table 22: Result of measurement of THF-α secretion in pig splenocytes by test group (pg / mL)

As can be seen in Table 22, in order to determine the effect on the secretion amount of TNF-α secreted when the splenocytes are added by fermented green tea microbial agent, the secretion amount of TNF-α secreted by the splenocytes is measured When not stimulated with Con A or LPS, antibiotics added 97.50 ± 79.24 pg / ml and fermented green tea microorganisms 1.0% added 91.25 ± 42.07 pg / ml.

In addition, when stimulated with Con A 1.0 μg / ml, which induces the most proliferative response of splenocytes, TNF-α secretion was increased significantly in all fermented green tea microbial supplements compared to the control and antibiotic supplements. (P <0.05).

In addition, when stimulated with LPS 10.0 ㎍ / ml, which induces the most proliferative response of splenocytes, the amount of TNF-α secretion increased significantly in all fermented green tea microbial supplements compared to the control (P < 0.05).

Therefore, the fermented green tea was evaluated to increase the secretion of TNF-α secreted when the splenocyte proliferation response to Con A and LPS stimulation.

Therefore, when pigs are fermented with fermented green tea microbial agents, the secretion of cytokines (IL-6, TNF-α) in pig splenocytes increases, resulting in increased plasma protein synthesis, increased antibody activity, and activation of leukocytes and neutrophils. It was evaluated to be improved.

As such, when the present invention is applied to a pig using fermented green tea microorganisms, the pig's weight gain and feed demand are remarkably improved to increase the productivity of the pig, and the protein content is high and the fat content is low. It has a good color and is effective in improving low cholesterol and meat year.

In addition, the functional substances possessed by green tea and useful strains used for fermentation inhibit the intestinal harmful bacteria and increase the cellular and humoral immunity of pigs.

In addition, the cytokine (IL-6, TNF-α) secretion of porcine spleen cells is increased, thereby improving the immunity of pigs by plasma protein synthesis, increased antibody activity, activation of leukocytes and neutrophils.

Claims (4)

Pulverizing and drying the dried green tea leaves, and mixing green tea powder and grain by-product degreasing rice and wheat bran at a weight ratio of 3: 5: 2, respectively; Lactobacillus acidophilus Lactobacillus acidophilus KCTC 3111, Lactobacillus plantarum Lactobacillus plantarum KCTC 3104 Injecting and inoculating the bacteria evenly to the level of 1 × 10 ¹⁰ / ml to obtain a water content of about 40% of the total mixture to proceed to anaerobic and aerobic lactic acid bacteria for 3 days at 37 ℃ to produce a solid fermentation product and ; Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) KCTC 7915 bacteria, Bacillus subtillis KCTC 3239 bacteria, Bacillus coagulans KCTC 1015 bacteria 1 Method for producing a functional fermented green tea microorganism comprising the step of spraying and inoculated evenly at a level of × 10 ¹² / ml and drying for 2 days at 35 ~ 40 ℃ conditions. Functional fermented green tea microorganism obtained by the manufacturing method of claim 1. Increased body weight and feed rate during the same breeding period by feeding the fermented green tea microbial agent prepared according to claim 1 to the pigs in a fixed breeding period with an unlimited feed of 0.1 to 1% by weight based on the total feed. The pig breeding method using a functional fermented green tea microbial agent, which significantly improves and improves cellular immunity and humoral immunity of pigs to increase productivity. The functional fermented green tea microbial agent prepared according to claim 1 is raised in an unlimited manner to feed pigs added with 0.1-1% by weight of the commercially prepared feed based on the total feed amount to the pigs for a predetermined breeding period, so that the protein content is 19.7-22.5. Pork obtained by breeding with a functional fermented green tea microbial agent, characterized in that% / g, fat content is 1.7 ~ 2.7% / g, cholesterol content is 78 ~ 81.8 μg / g.

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