KR20030039730A - Feed additives containing citrus extracts and process for making the same - Google Patents

Abstract

PURPOSE: Provided is an animal feed additive containing a citrus extract instead of antibiotics. The feed additive is improved in its storageability, has enhanced digestion property and is capable of preventing the abuse of antibiotics. CONSTITUTION: The animal feed additive characteristically comprises peels of citrus fruits, by-products from citrus fruit processing, an extract of citrus fruits and limonene which is extracted from citrus fruits with an organic solvent or water.

Description

Feed additive containing citron extract and method for producing the same {Feed additives containing citrus extracts and process for making the same}

The present invention relates to a feed additive containing citron extract and a method for manufacturing the same, and more specifically, to replace the antibiotics for animal husbandry abused domestically and abroad, by using the antibacterial action of the compound extracted from the citron juice and citron shell It relates to a method used as a feed additive.

One of the problems to be solved in the livestock industry is that the economic loss caused by various diseases of the livestock due to the deterioration of the sanitary environment is large. In particular, as a countermeasure against diseases caused by pathogenic microorganisms, vaccination, addition of various antibiotics or synthetic antimicrobial substances is added to the feed, but except for special cases, it is a sufficiently effective countermeasure against many infectious diseases in the field. In addition, the administration of a large amount of the drug may cause a decrease in productivity due to its side effects, and in some cases, the product value of the livestock product may be lowered due to the remaining drug, which requires high safety and disease prevention measures to reduce labor. It's happening. In particular, it is almost impossible to respond with vaccines or antibiotics for all diseases in the field, and even if it is feasible, it requires a huge amount of drugs and their costs. There are many problems in terms of safety, such as side effects and residual in livestock products.

However, the resistance of bacteria to such antibiotics is also a big problem. It has been known that bacteria are resistant to antibiotics since the presence of antibiotics. Alexander Flaming, who discovered penicillin in 1928, warned against the resistance of bacteria to the development of antibiotics, and Watanabe, T., 1963. Infectiveheridity of multiple drug resistance in bacteria.Bacteriol. Rev. vol. 27 pp. 87 .) Said that antibiotic resistance could progress from a single bacterium and that antibiotic resistance problems progress faster than expected due to the increased presence of antibiotic resistant microorganisms in the field. For example, Salmonella is evidence that only 20% of antibiotic resistance was 5% 20 years ago.

The EU, made up of countries most susceptible to antibiotics and environmental pollutants, will completely ban animal use of antibiotics in the next five years, the EU said. Currently, only abliamine (Avliamycin) and flabomycin (Flavomycin) are used as antibiotics for promoting growth of livestock. As a result, many countries in Europe are striving to develop antibiotic substitutes and have already achieved 10,000 results after various experiments.

Kirk C. Klasing (2000) New vision of growth permitents permitents and the role of Immune Modulation. 2000 Drug-free Chicken Problems and Salutlons.Boi-mos Conf. Pp. 35. There is no controversy about the use of antibiotics, but it is expected that it will be a big hit in domestic livestock exports and domestic demand. Therefore, antibiotics and nature-friendly antibiotics will be used in combination.

However, in Korea and Asia, the use of antibiotics is on the rise, which may be attributed to the governments of each country where productivity and economics are important, and livestock farmers with weak competitiveness. However, some countries recognize the importance of antibiotic substitution in the near future, and these studies are being conducted intermittently.

In particular, despite the restrictions on the use of antibiotics in Korea, the abuse of antibiotics is not yesterday or today. In particular, in the case of poultry or pigs, the current address of domestic livestock farmers is that they are feeding or injecting antibiotics during the subconscious season.

Another problem with antibiotics is their residual in livestock. In the case of beef cattle using the least antibiotics from 1991 to 1998 when the harmful substances of livestock were tested in Korea, 402 of the 12.870 cases were tested for residues of antibiotics or synthetic antibiotics using biological and chemical analysis methods. Showed a positive response at 3.12%. As such, the domestic livestock industry is abusing antibiotics and antimicrobial agents, and this problem is expected to be a major obstacle to the international competitiveness of domestic livestock products in the future.

At present, the development of various antibiotic substitutes and bioactive substances using natural products has been actively researched and progressed by pharmaceutical companies and food companies and is already on the market. In addition, in the field of animal husbandry, the production of natural, hygienic and stable livestock food using natural products will be a key issue in the future. In particular, with the introduction of the Hazard Analysis and Critical Control System (HACCP) in the livestock product processing process, national interest in livestock hygiene has been increasing, so that the natural antimicrobial active substances in livestock feed are added to the existing food preparations. In addition to preventing shelf life, mastitis and various bacterial diseases, it will be possible to solve the safety issues in livestock products.

Domestic research on the development of such natural antimicrobial materials includes the development of antibiotic substitutes and feed additives using yucca extract or herbal medicines (Kwon, Myung-Sang, 1998. Natural antibiotics for the development of environment-friendly feed for livestock and pollution-free livestock production. Academic Symposium on Development, Korean Society of Experimental Zoology, Kwon, Myung-Sang et al., 1998), but the problem of importing raw materials and economics as a feed additive may be a problem.

On the other hand, the use of citrus fruits native to southern Korea and Jeju Island is likely to solve such problems.

Citrus junos belongs to citrus fruits, and in Korea, it grows only in southern coastal areas such as Goheung, Geochang, Wando, Jangheung, Gangjin, Geoje, and Namhae. Kwon, Dong-Jin, Hwang, Jin-Bong, Cho, Yong-Jin, 1994. Quality Comparison of Citron Juice by Juice Extracting Method, Korean Society of Food Science and Technology, Vol26 (6), pp. 704-708. The domestic production was about 10,000 tons in 1989, but it is estimated to be about 15,000 tons in 1994. However, it is very insufficient in terms of available use and is mainly used for ornamental purposes. At present, only citron tea is made at home or through a small-scale processing process. However, Goheon, Jeonnam, has been operating the juice processing plant for citron processing for the first time in Korea since December 1993. (Jin Jin-woong, 1997. Development of technology for processing and storage of domestic citrons. Korea Food Research Institute Jeon Jin-woong, 1997 Korean studies on citron include composition of amino acids (Jeong-Hee Jeong, 1972. Chemical composition of citron and physicochemical properties of aquatic soils. Journal of the Korean Agricultural Chemistry, Vol. 15 pp. 169) Effect of Citrons on the Composition of Citron (Jeong-Hyun Jeong, 1972. A Study on Amino Acids of Citron. Journal of the Korean Agricultural Chemistry, Vol. 15 pp. 175), The Use of Sorbitol as a Sugar Substance in Yujacheong A Study on the Quality Stability of Citron Administration by Sugar Replacement Effect of Sorbitol.Korean Journal of Food and Nutrition.Vol. 19 pp. 13), Flavor Components of Citron (Hyun Yoo Kim, Young Myung Kim, Dong Hwa Shim, Seon Bong Kyu. 1987. Korea Flavor Compounds of Mountain Citrons, Vol. 19 pp. 361 Hyun-Yu Lee, 1987), A Study on the Change of Color during Ripening of Yuzu (Jeong Ji-Heun. 1974. Korean Journal of Agricultural Chemistry, Vol. 17 pp. 63).

On the other hand, in the case of Japan, the study on citron was carried out by factor (Nakanishi, M. 1988. On the qulity of Yuzu Juice Produced in Kochi Prefeture 高 知 工 試 告. Vol. 16 pp. 54.) Limonoid compounds (Hashinaga, F., Z. Herman and S. Hasegawa. 1990. Limonoids in seeds of yuzu.Nippon Shokuhin Kogyo Gakkaishi Vol. 37 pp. 380.) and the composition of carotenoids (Kon, M and R. Shimba. Seasonal Changes in color and carotenoid Composition of yuzu and lisbon lemon peel.Nippon Shokuhin Kogyo Gakkaishi.Vol. 34 pp. 28) Structure of flavonoid compounds and inhibitory effects on hypertension (Kumamoto, H., Y. Matsubara, Y. Ilzuka, 1987) K. Okamoto and K. Yokoi. 1985. Structure and hypotensive effect of flavonoid glycosides in yuzu peelings.Nippon Nogeikagaku Kaishi. Vol. 59 pp. 683.

In addition, studies on feed processing of dairy processed by-products include various studies as feed substitutes in livestock (Belibasakis, N. G and DT sirgogianni. 1996. Effects of dried citrus pulp on milk yield, milk composition and blood components of dairy cows. Ani.Feed.Sci.Tech. 60 pp. 87-92.) And a study on the palatability of livestock due to unique fragrance components (Provenza, FD, JJ Lynch and CD Cheney. 1995. Effects of a flavor and food restriction on the Citrus peel has been used mainly in response of sheep to novel foods.Applied Animal Behaviour Science Vol.43 (2) pp. 83-93.

The yuzu has a unique rich aroma and taste, which is said to be effective in the treatment and prevention of colds, neuralgia, and wind.The seeds are burned in corns and warts, mixed with rice, and attached to the affected part. Folk remedies such as are transmitted.

Thus, citron has been widely used as a medicinal fruit traditionally used in Korea, and the domestic paper as a medicinal fruit was examined for the components of amino acids, free sugars, organic acids, etc., which are general and special ingredients (Jeong Ji Hun. 1972. Yuzu) Studies on Amino Acids in Korea Vol. 15 pp. 175) Only three species, α-Pinene, D-Limonene, and p-Cymene, were identified as fragrance components. (Han Sang-sun, Yoil Jun. 1988. Antimicrobial Activity and Safety of Natural Naringin from Korea. Journal of Pharmacology of Korea. Vol. 16 (1). Pp. 33-40.) And Antimicrobial Properties of Pectin Degradants in Citrons (Mi-Yeon Park, Seung-Tae Choi, Dong-Suk Jang 1998. Antimicrobial Properties and Food Preservation Effects of Pectin Degradants J. Food Hyg.Safety Vol. 13 (2) pp. 99-105), but naringin and pectin degradates are not easy to extract and isolate. , Now at a high price Due to the fact that it is used only for laboratory research, there was a problem that the feed additive for livestock was not economical.

The present inventors obtained the extract from the citron shell and the citron liquid by the continuous distillation extraction method, and used it as an antibiotic substitute to measure the feed rot and aflatoxin production degree, as well as the shelf life of the feed such as ruminant In vitro experiments were conducted to investigate the nutrient digestibility, volatile fatty acid and ammonia production, and pH change of the feed. The results showed that the extract of citron containing limonene compound had a very good effect on the shelf life and digestibility of the feed. The present invention has been completed.

Accordingly, it is an object of the present invention to provide a feed composition or feed additive of livestock containing the yuzu extract.

Another object of the present invention is to provide a method for producing a feed composition or feed additive.

Other objects and advantages of the present invention will become more apparent by the following configuration. Hereinafter, the configuration of the present invention will be described in detail.

Figure 1 shows the process of extracting oil from the citron shell.

Figure 2 shows the E. coli growth inhibition effect by the tube dilution technique of citron oil extract.

Figure 3 shows the Salmonella growth inhibition effect by the tube dilution technique of citron oil extract.

4 shows the color L * change during the experiment (0-26 days).

5 shows the color a * change over the experimental period (0-26 days).

Figure 6 shows the color b * change over the experimental period (0-26 days).

7 shows the pH change during the experiment (0-26 days).

Figure 8 shows the difference in ammonia concentration of the rumen fluid.

The present invention relates to a feed composition containing a citron extract and a method for producing the same, the above object of the present invention is to use the yuzu bark and citron liquid in order to use a natural antimicrobial-containing citron extract extracted from citron as an antibiotic replacement material of livestock The limonene compound was extracted by distillation, which was compared with Limonene standard for quantitative and qualitative analysis. After establishing the optimum antimicrobial activity condition using MIC of the compound, the shelf life effect of the feed as an antibiotic substitute was investigated. In order to confirm, it was achieved by measuring the decay of feed and the degree of aflatoxin production, and by examining the nutrient digestibility, volatile fatty acid and ammonia production, and pH change through in vitro experiments with ruminant fluid of ruminants.

According to the GC analysis of the citron extract, the present inventors found that the compound having excellent antibacterial activity against Gram-positive and Gram-negative bacteria was a limonene compound, which contained 70.10% in citron solution and 63.64% in citron shell. Based on this fact, the present inventors found that not only limonene but also limonene-containing citron extract was added to the feed as a functional substance that could replace antibiotics for animal husbandry, and the shelf life and digestive efficiency of the feed were improved.

In a preferred embodiment of the present invention, the antibacterial activity of the Gram-negative bacterium Escherichia coli ATCC 25922 and Salmonella ( Salmonala enteritidis IFO3313) with respect to the limonene compound, both showed growth inhibition at more than 1,500ppm E. coli In the case of Escherichia coli ATCC 25922, bacterial activity was restored after 24 hours or more, but in the case of Salmonala ( Salmonala enteritidis IFO3313) strain, the bacterial activity was increased from 0 to 72 hours with limonene. It was shown to completely inhibit (FIGS. 2, 3).

In another preferred embodiment of the present invention, as a result of experiments on the shelf life of the livestock feed containing the limonene-containing citron extract to the viable bacteria inhibitory activity against harmful microorganisms, DCP 2,000ppm in the apparent state of the sample After 21 days, some citrus odor was detected. The control group began to rot after 12 days of storage, and the AF group began to show signs of rot after 21 days of storage. As a result of color change by L * (Lightness), a * (redness), and b * (yrellowless), L * was not changed but b * of control was rapidly yellow after 12 days of storage. I noticed that I lost color. In addition, when comparing the color of the sample with a * and b *, the control and AF were changed to green and blue color as the sample decayed (Figs. 4, 5, 6).

On the other hand, as a result of pH measurement, the pH of the initial DCP treatment was the lowest, which was determined to be the effect of various acidic substances such as ascorbic acid contained in the citron shell. On the contrary, the pH of DCP treatment was increased after 16 days of storage, which is considered to be due to the use of microorganisms in organic acids contained in large amounts in the citron shell. The pH of the other treatments showed a tendency to slowly decrease as the storage period passed (Fig. 7).

In order to measure the aflatoxin production in the feed, samples of all treatments were collected at 16 and 26 days after the storage of the feed and analyzed for aflatoxin. The samples stored for 16 days were measured with 8 ppb and AF 4 ppb. In the samples stored for 26 days, control 8ppb and AF 8ppb were detected. However, aflatoxin was not detected in DCP and Lim treatments.

In another preferred embodiment of the present invention, as a result of investigating the effect of natural antibiotic substitutes on the digestibility of ruminants, the pH of the culture medium for each sample was 5.45 in control, Lim, AF and DCP treatment at 12 hours of incubation. , 5.46, 5.44, and 5.30 showed significant differences (P <0.05). After 48 hours of cultivation, the dry loss rate was found to be 72.59% in Lim, 69.09% in Control, 68.44% in DCP and 68.23% in AF. In addition, the digestibility of organic matter after 0-24 hours of cultivation showed the highest trend in the DCP treatment group, showing a significant difference (P <0.05). The reason why the DCP treatment showed high organic physiochemical digestion rate was because it contained a large amount of pectin component that was easily decomposed in rumen microorganisms during the mixing of the test sample. In the case of acetic acid production, VFA at 3 hours of culture was 27.18 in the Lim treatment group, which was significantly increased (P <0.05) compared to the other treatment groups. In general, the acetic acid production rate of Lim treatment was high at 3 hours, which is the beginning of culture, and 12 hours, but no significant difference was observed after 3 hours of culture. Propionate (propionate) showed similar tendency as acetic acid, but showed significant difference (P <0.05) from 3 hours to 6 hours in cultivation of Lim treatment group, and there was no significance afterwards, but this tendency persisted for 24 hours At the later 48 hours, VFA production increased significantly in the Lim, DCP, and AF treatments (P <0.05). Total VFA production was significantly different in Lim, DCP, Control and AF in order of 290.06, 273.28, 268.34 and 261.69 mM, respectively (P <0.05). On the other hand, no significant difference was found in the A / P ratio. The ammonia-N concentration (mg / 100ml) was relatively low in Lim, DCP, and AF treatments compared to the control, but the ammonia production rate was deteriorated due to the high VFA production (Fig. 8).

The citron extract of the present invention may be obtained by a general method of preparing plant essential oil extracts by grinding or by using citron or citron processing by-products, citron shell, citron liquid and the like, for example, citron extract is water or methanol, ethanol in the citron shell By adding an organic solvent, such as acetone, it can be obtained by homogenizing and distilling it. Alternatively, organic solvent extraction, distillation, filtration, etc. may be additionally performed to remove impurities and purify to higher purity. In addition, by using a supercritical extraction principle that a certain substance becomes a fluid state after a certain high pressure and a high temperature, it is also possible to separate and purify only the active ingredient which is beneficial to livestock without high toxicity or residual substances, specifically, a limonene compound Can be purified to high purity and used as a citron extract. Citron shells, etc., may be dried and stored at 15% by weight or less when used for a long period of time, and then crushed by a grinder. The citron extract may be used in feed additives in liquid form or in combination with other carriers, or in the form of powder.

Feed composition or feed additive containing the citron extract of the present invention can replace the antibiotic currently in use, characterized in that it contains a high-safety natural products that improve the clinical symptoms and weight gain effect caused by diseases of the livestock In addition, when supplemented with yuzu extract is fed to feed, there is no problem of antibiotic contamination, and the digestibility and weighting rate of livestock can be increased compared to the case of feeding commercial commercial feed, and feed additives containing citron extract of the present invention are used. By using discarded citron processed by-products as resources, not only can they contribute to increasing farm household income, but also have side effects that can prevent environmental pollution.

Meanwhile, the present invention citron extract-containing feed composition or feed additive may contain not only limonene or citron extract but also a solid form such as powdered or granulated ground dry citron peel itself. When the citron shell is stored for a long time, it is preferable to dry it to 15 wt% or less of moisture content, and to store it in a container that can block moisture such as a vinyl bag.

Feed composition or feed additive containing the present invention citron extract or citron shell grinding, etc. may be appropriately selected and used in consideration of the known feed or feed additives in consideration of the type and age of the subject animals to be fed or the weight and nutrition or palatability. Moreover, it is necessary to contain vitamins and minerals, and it can mix | blend or add like a well-known feed material. The physical form of the feed and feed additives may be any of powder, granular and liquid, and may be appropriately selected depending on the conditions of farming management or farming equipment. In one embodiment, it is easy to mix in the case of powdered feed when added to the feed, but in the case of pellets or crumbled feed it is not easy to mix evenly with the feed, so mixed with each feed material It is preferable to make from the complete mixed feed made.

Hereinafter, the configuration of the present invention in more detail through the following examples. In particular, the following examples show a case in which a ruminant is used with a feed additive containing purely purified limonene as an active ingredient from citron extract, but not only limonene component but also citron extract or citron shell or processed product thereof. It is obvious that the scope of the present invention is not limited to the limonene and livestock exemplified in the following examples, since the same results as in the following examples can be expected even if other livestock are used for breeding.

Example 1 Extraction and Analysis of Antimicrobial Substances

The citron used in this example is juiced at the citron juice factory in Goheung-gun, Jeollanam-do, and classified into citron juice and citron shell to prevent oxidation and to reduce volatilization. Used.

Experimental Example 1: Extraction of Antimicrobial-Containing Oil

The oil extraction of the citron was extracted by a continuous distillation extraction method to extract the essential oil components by collecting water vapor, the extraction process is as shown in FIG. In order to prevent oxidation of the extracted oil, it was stored in -35 ℃ freezer after nitrogen filling and used for quantitative analysis and antibacterial activity test.

Extraction of organic citron or distilled water was performed to extract the optimum citron day. As a result, only the citron raw material was homogenized and extracted. In general, oil extraction amount was about 5-10ml / 300g, and the extraction temperature and time were 80 ~ 90 ℃ for 50 minutes ~ 1 hour to be less affected by brown change.

Most of the oils extracted from citron are polar oils and are extracted with water, so after extraction, they must be separated from the water layer by using polar organic solvent and pure organic oil can be obtained only by separating organic solvent using rotary concentrator.

Experimental Example 2 Quantitative and Qualitative Analysis by GC

HP 6890 Gas Chromatography (1998) was carried out under the analysis conditions of Table 1. Columm used polarized HP-INNOWax (Ø0.32 mm × 30m) Crosslinked polyethylene Glycol and Limonene standard was purchased by using 100ml of (R)-(+)-Limonene 98% (Lot No: 18,316-4) of Aldrich. . The content of Limonene in the oil extracted from citron was analyzed by comparing the retention time of citron shell and citron liquid with those of the standard.

GC Analysis Conditions of Citron Extract device HP-6890 GC with auto sampler and chemstation data system column HP-INNOWax (0.32mm X 30m) Stationary phase Crosslinked polyethylene glycol detector FID Injection temperature 230 ℃ Detection temperature 250 ℃ Oven temperature 70 (2 min) -25.25 ° C / min-125 (2 min)-25.5 ° C / min-180 (10 min) post run temp. 240 ° C (3 minutes) Carrier gas helium

Limonene is a terpenoid hydrocarbon material. Terpenoid is a terpenoid, which is composed of two or more isoperne molecules CH ₂ = C (CH ₃ ) -CH = CH ₂ polymerized. Among them, group 1 of monoterpenoid geraniol, Limonene, α and β-pinene (Watanable, N., Nakajima, R., Watanabe, S., Moon, J., Inagaki, J., Sakata, K., A Yagi and K. Ina. 1994. Essential oils of some orange peels Agri. Bio.Cham.Vol. 34 (2) pp. 234.). Qualitative analysis of Aldrich's (R)-(+)-Limonene 98% (Lot No: 18,316-4) Standard and Limonene extracted from citron shell and citron liquid using gas chromatography (HP 6890, 1998 USA) , 19 minutes 8,83 seconds for Aldrich's (R)-(+)-Limonene 98% (Lot number: 18,316-4), 19 minutes 6,76 seconds for oil extracted from citron shell In the case of oil, it was found that 19 minutes, 7,24 seconds, etc. were found to contain a considerable amount of Limonene component in the oil extracted from citron shell and citron liquid.

Tables 2 to 4 show the quantitative analysis of Aldrich's Limonene Standard, oil extracted from citron shell and oil extracted from citron liquid under the same conditions as qualitative analysis. Aldrich's Limonene Standard is 98.97%, and Limonene content of oil extracted from citron shell is 63.64% and limonene content of oil extracted from citron juice contained 70.10%. As such, it was found that the citron contained a large amount of antibacterial natural Limonene. This is reported by Shinoda et al. (Shinoda, N., M. Shiga and K. Nishimura, 1970. Constituents of Yuzu ( Citrus Junos ) oil.Agri. Bio. Chem. Vol. 34 pp. 234.1970). Kusunose (Kusunose, H and M. Sawamura. 1980. Aroma constituents of some sour Citrus oils. J. of Japanese Society of Food Science Technology Vol. 27 (10) pp. 517. 1980) et al. 75.7%, Ohta (1983) It was 77.10%, and Limonene was extracted from the citron of this experiment and quantitatively analyzed by GC, which was about 60 ~ 70%. However, according to Shin (1987), the average content of Limonene in citron is 43.62%, and terpane hydrocarbons such as Limonene are converted to alcohol when storage period is longer (Shinoda et al., 1970). Care should be taken because errors in analysis may occur.

Limonene content of standard Aldrich limonene 97% (Lot No: 18,316-4) Peak number ret. time (minutes) Area (pA * s) Height (pA) area(%) One 5.75 4.87 1.16 0.02 2 17.21 24.39 2.90 0.11 3 18.57 43.78 4.32 0.19 4 (limonene) 19.88 2.19-e * 1501.57 98.97 5 20.32 63.67 6.90 0.28 6 22.25 24.47 2.71 0.12 7 23.88 7.80 8.27e-1 # 0.03 8 24.59 45.51 4.26 0.20 9 25.04 10.29 1.02 0.04

*: pAs> 10000, #: pA <1

Limonene Content in Citron Shells Peak number ret. time (minutes) Area (pA * s) Height (pA) area(%) One 5.75 4.26 1.01 0.05 2 8.08 140.46 22.88 1.75 3 8.91 477.44 63.53 5.97 4 11.61 65.68 8.93 0.82 5 14.48 4.99 6.52-e 0.06 6 14.94 24.96 3.00 0.31 7 15.15 13.13 1.56 0.16 8 17.21 141.82 16.26 1.77 9 17.76 73.96 8.21 0.92 10 18.54 169.14 16.36 2.12 11 (limonene) 19.67 5081.79 484.45 63.63 12 20.26 156.94 16.11 1.96 13 22.25 778.02 73.85 9.74 14 23.87 43.29 4.07 0.54 15 24.58 159.29 14.64 1.99 16 25.04 27.48 2.54 0.34 17 47.69 35.15 2.75 0.44 18 46.41 36.64 2.92 0.45 19 48.03 37.06 2.73 0.46 20 49.79 80.73 6.55 1.01 21 52.54 413.57 23.08 5.17 22 25.62 18.67 1.44 0.23

Limonene content in citron juice Peak number ret. time (minutes) Area (pA) Height (pA) area(%) One 8.92 382.00 48.15 4.22 2 11.63 109.09 14.18 1.20 3 14.97 20.23 2.40 0.22 45 15.18 27.03 3.02 0.29 6 17.24 167.50 18.77 1.85 7 17.80 81.21 8.82 0.89 8 (limonene) 18.58 123.25 11.98 1.36 9 19.72 6344.03 582.17 70.10 10 20.29 219.46 22.24 2.42 11 22.29 891.76 87.61 9.85 12 23.90 50.66 4.69 0.55 13 24.61 117.99 10.94 1.30 14 25.09 16.02 1.56 0.17 15 41.72 56.88 4.43 0.62 16 46.42 46.12 3.58 0.50 17 48.05 13.16 1.75 0.25 18 49.08 54.27 4.38 0.59 19 52.55 318.65 19.88 3.52

Example 2 Antibacterial Activity Test of Oil Extracted from Citron Fluid and Citron Shell (MinimumInhibitory Concentration)

Assays and media used in the antibacterial activity test of oil extracted from citron juice and citron shell are shown in Table 5. Antimicrobial activity was measured using Agar diffusion method (Paper disc) and tube dilution technique.

Strains and Media Used for Antimicrobial Testing microbe badge Gram-negative bacteria Escherichia coli ATCC 25922 Nutritional Agar & Liquid Medium Salmonala enteritidis IFO3313

Experimental Example 1: Agar Diffusion Method (Paper disc)

Each assay strain in the inoculated strains was plated on a nutrient agar medium (23 g / liter plate (87 × 15 mm)) and incubated at 37 ° C. for 24 hours, and used as an inoculum of the strain culture solution for the experiment.

Take 1 colony from the nutrient agar medium in which the strain was cultured, inoculate a test tube containing 10 ml of nutrient solution (8 g / liter) (3 per strain), and then live. These were put in a shaker incubator and shaken for 24 hours at 37 ℃ at a speed of 150rpm.

Only 1 ml was taken from each of the cultured strain cultures and mixed with 9 ml of agar (0.75%) and plated on previously prepared nutrient agar plates. At this time, 0.75% of agar should be cooled to about 45 ℃ and mixed with the bacterial solution so that the test bacteria are not killed.

The test sample was sterilized with a membrane filter (0.2 µm), and then a predetermined concentration (ppm) was prepared and diffused into 100 mm sterilized 8 mm paper disks. They were dried under UV for 30 minutes and then adhered to the inoculated plate surface and 0.85% physiological saline (50 μl) was diffused onto the disc attached to the plate surface. And the degree of antimicrobial activity was measured by the size of the growth inhibition ring (clear zone dermeter, mm) formed around the disk by culturing for 24 to 48 hours at the optimum growth temperature of the assay strain.

Experimental Example 2: Tube Dilution Technique

Each strain 1 colony was taken from the slant inoculated with the assay strain, inoculated into 10 ml of nutrient liquid medium, and incubated for 24 to 48 hours at the optimum temperature (39 ° C) of each strain. 0.1 ml of this strain culture solution was inoculated again to 10 mm of nutrient liquid medium and cultured once again under the same conditions. Samples sterilized with a membrane filter (0.2 μm) in 0.1 mm of the culture medium thus cultured were added at a constant concentration (ppm) and incubated for 72 hours, followed by turbidity at 620 nm (SHIMADZU, UV-160A, 1996) at 12 hour intervals. Was measured to compare the effect of inhibiting proliferation. In this case, in order to exclude the antimicrobial activity of the solvent in which the sample was dissolved, all experiments set a control group to which the same solvent as the sample was added.

Citron extract of the Limonene coli the Agar Diffusion Method and Tube dilution technique how the antimicrobial search for black strains (Escherichia coli ATCC 25922) and Salmonella, the bacteria of the results, such as Escherichiacoli or Salmonella conducted for (Salmonella enteritidis IFO3313) strains Limonene concentration 1,500 Growth inhibition was observed at more than ppm, but Escherichia coli was found to decrease the clear zone by restoring the activity of bacteria as time elapsed after 24 hours of culture. Salmonella, on the other hand, was found to exhibit persistent antimicrobial activity regardless of incubation period. Table 6 shows the antimicrobial activity of the Limonene component as a clear zone diameter / mm showed the inhibition of bacteria according to each concentration.

Antibacterial Activity of Citron Extracts by Agar Diffusion Method Citron shell Citron amount 1500 ppm 2000 ppm 1500 ppm 2000 ppm E.coli + + + ++ Salmonella + ++ + ++

Reports on the antimicrobial activity of Limonene can be found in Duccio (Dccio, RL, G. Monica, DM Bionid, A. Renda and G. Ruberto. 1998. Relationship between volatile components of citrus fruit essential oils and antimicrobial action on Penicillium digitatum and Penicillium italicum.International J. Food Micro.Vol. 43 PP.73-79.1998) reported on blue mold of Penicillium strain, but there was no report on gram-negative bacteria, but we have antimicrobial activity against gram-negative bacteria that Limonene has not been reported to date. It turns out that there is.

Figures 2 and 3 show the effect on the activity of Eshericaia coli and Salmonella according to the oil extract concentration in the tube dilution technique in the antimicrobial activity test with Esherichia coli strain of Figure 2 was produced at 2,000ppm 24 Over time, the activity of Esherichia coli is again active. These results were also shown in the Agar Diffusion Method. The reason for this is that Esherichia coli is resistant to Terpernoid substance or Limonene is a kind of carbon compound, so Escherichia coli can be used as carbon and energy source (Frederick, C., JL Neidhardt, K). lngaham, Brooks Magasanik, M. Schaechter, and H. Edwin Umbarger. 1987. Escherichia coli and Salmonella typhimurium.vol. 1 pp. 3-104. Frederick et al., 1987).

On the other hand, 2,000ppm Limonene addition of Figure 3 was shown to completely inhibit the activity of Salmonella from 0 hours to 72 periods. Based on the results of the Agar diffusion method and tube dilution technique, the extract of citron fluid or citron shell showed antibacterial activity against bacteria such as Escherichia coli and Salmonalla at 1,500ppm or 2,000ppm concentration.

Example 3 Shelf-life Experiment of Feed for Livestock Added with Citron Extract

In order to investigate the effect of the yuzu extract extracted in Example 1 on the shelf life of the feed, the raised pig feed was directly prepared based on corn and soybean meal. The prepared feed did not use any feed additives (antibiotic, antifungal, probiotic, etc.) added to commercial feeds, and the raw material blending ratio of the test feed is shown in Table 7, and the chemical composition of the test feed. Is shown in Table 8.

Raw material mix ratio of test feed Composition content(%) yellow corn 61.17 rice hull 8.00 soybean meal 19.00 lupin seed 3.20 animal fat 3.00 molasses 3.00 limestone 0.90 TCP 0.70 salt 0.30 vit-min. premix 0.28 newtracell 0.02 methionine 0.01 lysine 0.04

Chemical composition of the test feed (% of DM basis) dry matter crude fat crude protein crude fiber ash 87.66 6.95 17.47 4.05 5.27

In addition, in order to exclude microbial contamination of the test feed, it was completely sterilized at 120 ° C / 1.2 atm for 15 to 20 minutes, and this was used as a control.Anti-fungal agent (0.05%) and citron extract Limonene (LIM 500 (0.028%), 1000 (0.056%), 2000 (0.112%) ppm) and dried citron shells (DCP500 (5.55%), 1000 (11.11%), 2000 (22.22%) ppm) were quantitated in petri dishes by 15g and the temperature was 31 The Limonene content in each DCP treatment was calculated to be the same as that of the Lim treatment by calculating the Limnene content in the crude husk shell. In addition, the humidity was maintained at 75-79% and then cultured to see the inhibitory effect on the microorganisms of the natural antibiotic extract. LIM was mixed with CH ₂ CL ₂ by concentration and sprayed directly on the sample, and DCP was used as a treatment tool after mixing with each sample and concentration after grinding.

1) Survey items and analysis method

(1) Examination of external condition of sample (odor, decay rate, chromaticity)

After the sample was collected, the appearance of the sample, such as smell and color, was examined for each treatment group according to the stored dates. Odor was characterized by the characteristic of odor that appeared after storage period, and color was measured by using Spectro Pilotometer (Minolta CM5081, CIE L *, a *, b * Japan) by treatment and storage period. (redness), b * (yellow

ness) values were measured.

Compared with the control, the samples by each treatment showed a unique aroma of citron, but the fragrance gradually decreased as the storage period increased. However, in case of 2,000ppm of dried citron shell (DCP), it was found that even after 21 days of experiment, some citrus flavor appeared. In addition, the control group began to rot after 12 days of storage and mold spores began to form. In the case of 0.05% antifungal drug (AF), the storage period started to show signs of decay after 21 days, and DCP 500ppm began to decay after 26 days, but all other treatments, DCP1, 000 ppm (11.11%) DCP 2,000ppm (22.22%), Limonene (Lim) 500ppm (0.028%), Lim 1,000ppm (0.056%), Lim 2,000ppm (0.112%) showed no signs of decay until the 26th day of the experiment. Did not find.

The results of the chromaticity change during the storage period of each treatment section are shown in FIG. 4 (Lightness), FIG. 5 (redness), and FIG. 6 (yellowness). The color of L * (Lightness), a * (redness), b * (yellowness) among test colors of each treatment group showed the change of color. Significant changes occurred over 12 days. This is a change in the color of the sample appears as the sample decay, it can be seen at a glance the degree of decay of the control compared to the other treatment.

In addition, the a * of FIG. 5 also shows a decrease in red color after 12 days of storage in the control. In the case of b * of FIG. 6, the control shows a similar tendency to other treatments until 12 days after storage. You can see that the yellow color is suddenly lost with the peak. When the colors of a * and b * samples were compared, they changed to green and blue colors. The 0.05% of AF began to rot 16 days after storage.

(2) pH concentration measurement

First, 5g of samples were collected for each treatment period, and 50ml of distilled water with pH adjusted to 6.2 was added, followed by homogenization with a homogenizer (Ace AM-11, Japan) for 10 minutes at 10,000rpm. The extract was filtered. 40 ml of the above extract was taken and measured for pH (Park, Bu-Ha, Ha-Ki, Bum-Young Park. 1996. Effect of Packaging Method on Physicochemical Properties of Frozen Chicken. Kor. J. Poult. Sci. 23 (4): 193-201).

Figure 7 shows the change in pH according to the storage period of each treatment and control, the lowest pH of the DCP 2,000ppm (22.22%) and DCP 1,000ppm11.11%) treatment. The reason is thought to be due to the influence of various acidic strong substances such as ascorbic acid contained in DCP. However, after 16 days of storage, the pH of each DCP treatment group tended to increase significantly. These results cannot be explained precisely because the concentrations of organic acids and microorganisms in the sample were not investigated. It is believed that the pH increased again due to the use of organic acids such as citric acid, which is contained in large amounts in the shell (Kim, et al., 1999). The pH of the control and AF treatments was highest at the beginning of storage, but showed a downward trend after 12 days of storage, and the pH tended to decrease slowly in all treatments except DCP treatment.

(3) Determination of Aflatoxin Formation

Aflatoxin measurement was performed by using Aflatoxin kits (Sigma, A-6636, A-9887) at the end of the experiment and at the end of the experiment. (Anonymous. 1972. A rapid qualitative test for aflatoxin. Feedstuffe. Nov 27 Anonymous, 1972).

In Table 9, samples were taken on the 16th day when the decay of the sample peaked after storage and 26 days on the end of the experiment. Aflatoxin was detected only in the control and 0.05% AF. However, Aflatoxin was not detected in Lim and DCP treatments. The control group showed 8 ppb regardless of the measurement date, but Aflatoxin production was doubled in AF% 0.05%. Therefore, it is believed that citron has antifungal effect.

Aflatoxin content in samples irradiated at 16 and 26 days, respectively, during the trial period (0-26 days) Days Control AF0.05 Lim0.028 Lim0.056 Lim0.112 DCP5.55 DCP11.11 DCP22.22 16 8 ppb 4 ppb ND ND ND ND ND ND 26 8 ppb 8 ppb ND ND ND ND ND ND

-ND: not detected

Example 4 Effects of Natural Antibiotic Substances on In vitro Digestibility of Ruminants

Inhibition of the extract based on the concentration obtained in the experiment Lim organic solvent (CH₂Cl₂) And mixed by concentration, and sprayed directly on the sample, DCP was pulverized and quantitated about 4g by sample and concentration, and then mixed with the amount of rumen and buffer in a 1: 1 mixture and incubated in artificial rumen.

In vitro digestibility was determined by using a batch culture system, and the amount of raw material of the test sample was shown in Table 10 and the chemical composition is shown in Table 11, and each culture was analyzed according to the analysis items below after incubation for a predetermined time.

Chemical composition of the test feed Composition content(%) wheat 3.1 wheat bran 6.9 soy bean 10.6 corn gluten feed 4.4 cotton seed meal 1.3 coconut meal 1.1 soybean meal 7.3 limestone 1.2 lupin 1.2 corn flaked 30.3 salts 0.6 cotten 2.4 cotten hull 6.2 alfalfa cube 2.4 rice straw 5.0 alfalfa pellet 2.2 beet pulp 7.3 alfalfa hay 6.1 SUM 100

Chemical composition of the test feed (% of DM basis) process dry matter crude fat crude protein crude fiber ash Control 88.9 3.73 16.6 18.57 8.06 DCP 22.22% 98.57 4.23 16.19 18.45 8.38

1) Survey items and analysis method

(1) chemical analysis (pH, NH ₃ , DM digestion rate, OM digestion rate)

In the pH measurement, each sample was taken out for each treatment time and put in a refrigerator at 4 ° C. or less for about 10 minutes in order to suppress the activity of microorganisms, and then the pH was measured by a digital pH meter (ORION 290A). In VFA analysis, 5 ml of the supernatant was diluted with 25% Metaphosphoric Acid, put in a vial, stored in a refrigerator at -30 ° C, and thawed slowly at low temperature for analysis. Centrifugation was performed at 14,000 rpm for 10 minutes, followed by gas chromatography (HP 6890). USA, 1998) were analyzed for the contents of acetate, propionate, iso-butyrate, butyrate, iso-valerate, valerate according to the analysis conditions of Table 12.

GC Analysis Condition of Volatile Fatty Acids device HP-6890 GC with auto sampler and chemstation data system column HP-FFAP (0.53mm X 30m) Stationary phase Crosslinked polyethylene glycol detector FID Injection temperature 240 ℃ Detection temperature 265 ℃ Oven temperature 120 ° C (1 minute) -22.75 ° C (minutes)-165 (3 minutes) post run temp. 300 ° C (3 minutes) Carrier gas helium

NH ₃ was analyzed according to Chaney and Marbach (Chaney, A. L and EP Marbach. 1962. Modified reagents for determination of urea and ammonia. Clinical Chemistry Vol. 8 pp. 130-132). First, 5 ml of the supernatant was mixed with 0.1N HCl, stored in a vial, stored in a refrigerator at -30 ° C., and thawed slowly at low temperature for analysis. After centrifugation at 14,000 rpm for 10 minutes, the supernatant was taken and used for UV (SHIMAD-ZU, UV- 160A, 1996), the wave-length of the spectrophotometer was set to 630 nm, and the blank was set to 0. Then, the OD of the standard ammonia-nitrogen solution and the sample was measured. The building fire extinguishing rate and the OM loss rate were analyzed according to the AOAC (Official Methods of Analysis (15th ed ..) Association of Official Agricutural Chemists Washington, DC 13th ed., (1980). 1990).

(2) statistical analysis

Statistical analysis on the data obtained as a result of this experiment was carried out by variance analysis using SAS (Statistial analysis system, 1996) / PC system, and Duncan's multiple range test (Duncan, 1955) was performed for items with significant differences. .

The pH change in the fermentation broth according to the fermentation time is shown in Table 13. Lim and DCP treatments showed significant differences (P <0.05) at 6 h incubation compared to the other treatments, and pH for each 12 h culture was 5.45, 5.46, 5.44 and 5.30 in Control, Lim, AF and DCP treatments. Significant difference (P <0.05) was shown in Lim treatment group, which showed the highest pH, and DCP treatment group showed the lowest pH (P <0.05). After 24 hours of fermentation, the pH of Lim treatment was gradually lowered compared to other treatments. At 48 hours, the pH was lowest compared to other treatments, but there was no statistical difference.

PH change in fermentation broth with fermentation time Control AF0.05% Lim 0.112% DCP 22.2% 0 (hours) 6.32 6.34 6.36 6.36 3 5.70 5.74 5.67 5.47 6 5.58 5.56 5.47 5.47 9 5.43 5.50 5.49 5.40 12 5.45 5.44 5.46 5.30 24 5.39 5.38 5.34 5.31 48 5.45 5.45 5.29 5.35

Table 14 shows the dry matter loss rate in the artificial rumen by incubation time, and no significant difference was found between treatments until 24 hours.However, the dry loss rate after 48 hours incubation was 72.59% in control, 69.09% in control and 68.44% in DCP. It was significantly (P <0.05) higher than AF 68.23% addition group.

Building loss rate in artificial rumen by incubation time Control AF 0.05% Lim 0.112% DCP 22.22% 0 (hours) 29.59 26.91 41.19 32.06 3 43.48 38.11 42.72 41.43 6 36.68 43.70 52.07 42.71 9 47.50 54.64 58.52 48.81 12 55.12 53.15 57.73 54.95 24 59.57 62.05 62.14 61.46 48 69.09 68.23 72.59 68.44

Organic digestibility according to incubation time for each treatment is shown in Table 15. After 48 hours of incubation, organic digestibility was 58.85% AF, 59.09% Lim, 61.28% control. There was no significant difference between the treatments in the order of 67.11% of DCP, but the organic physiotherapy rate was the highest in the DCP treatment group after 0-24 hours of cultivation, showing a significant difference (P <0.01). The reason why DCP treatment showed high organic physicochemical digestion rate was because the rumen microorganisms contained a large amount of pectin, which was easily decomposed in the test sample formulation.

Digestion rate of organic matter according to incubation time by treatment Control AF 0.05% Lim 0.112% DCP 22.2% 0 (hours) 1.50 1.22 4.57 18.51 3 15.56 15.34 19.34 29.93 6 22.68 20.24 22.98 37.45 9 29.77 35.14 29.11 45.14 12 41.16 39.96 39.42 59.35 24 48.80 53.81 50.29 57.67 48 61.28 58.85 59.09 67.11

In the VFA production rate of each treatment and incubation time, the amount of Acetate production was significantly increased (P <0.05) compared to other treatments as 27.18 in the Lim treatment treatment. Overall, the rate of Acetate production was high in Lim treatment, but not significant. This tendency lasted up to 24 hours and at 48 hours was higher in the other treatments than the control.

Propionate showed similar tendency as Acetate, but increased in Lim treatment with significant significance (P <0.01) from 3 hours to 6 hours. There was no significance afterwards, but this tendency lasted up to 24 hours, and by 48 hours, the late Lim and DCP AF groups showed significantly higher production rate (P <0.05) than the control group. In particular, the increase of propionate production in the DCP added group was thought to have influenced on the rumen fermentation due to the large amount of organic acids such as citric acid. Total VFA production amount was 290.6, 273.28, 268.34, and 261.96mM in the order of Lim, DCP, Control and AF from 0 to 48 hours, respectively (P <0.05). This trend lasted up to 24 hours. On the other hand, no significant difference was found in the A / P ratio.

The results for ammonia-N concentration (mg / 100ml) of the rumen fluid are shown in FIG. 8. Compared with the control (36.77 mg / 100ml, 48h), the Lim (32.65mg / 100ml, 48h), DCP (33.73mg / 100ml, 48h) and AF (30.61mg / 100ml, 48h) treatments showed relatively low values. It is thought that ammonia production rate was relatively lowered due to VFA production. Although the production rate was relatively similar up to 6 hours after incubation, high ammonia production rate appeared in control after 6 hours, but there was no significant difference between treatments.

As described above, the feed composition or feed additive containing the present invention extract replaces antibiotics for animal husbandry, which is currently a problem due to abuse of antibiotics, to improve feed storage, as well as to enhance feed digestibility, thereby converting raw milk waste into livestock feed. Not only does it solve the problem of antibiotic contamination, it also has a very good effect of improving livestock growth and growth rate.

Claims (3)

A feed composition for livestock feed, wherein the feed composition for a livestock feed contains a citron shell crushed product, a citrus by-product or a citron extract. A feed composition for livestock feed, wherein the feed composition for livestock contains limonene separated from citron shell mill, citron by-product or citron extract. The feed composition for livestock according to claim 1 or 2, wherein the citron extract is extracted from citron, citron shell or citron powder using water or an organic solvent.