Feed composition and feed additive, which comprise catechin and vitamin k

Abstract

The present application relates to: a feed composition or a feed additive, which comprise catechin and vitamin K; a method for inhibiting bile salt hydrolase (BSH) activity of an animal by using catechin and vitamin K or the feed composition comprising same; and a method for improving intestinal health of an animal.

Description

Feed composition and feed additive containing catechin and vitamin K

[0001] The present application relates to a feed composition or feed additive comprising catechin and vitamin K; and a method for inhibiting bile salt hydrolase (BSH) activity in an animal and a method for improving intestinal health in an animal using catechin and vitamin K, or a feed composition comprising them.

[0002]

[0003] Antibiotic growth promoters (AGPs) are commonly used to promote the growth of livestock. However, the misuse and overuse of antibiotics in domestic livestock farming has caused numerous problems, including reduced treatment efficacy in sick animals due to the emergence of drug-resistant bacteria, issues with residues in meat, and the potential transmission of drug-resistant bacteria from animals to humans. Countries around the world are pointing out the problems caused by the misuse of antibiotics in livestock farming and calling for the prudent use and regulation of antibiotics (Marshall BM, Levy SB., Clin Microbiol Rev 2011 Oct;24(4):718-33).

[0004] In this regard, Begley M et al. disclosed that inhibitors of bile salt hydrolase (BSH) can improve the growth performance of livestock as an alternative to antibiotic growth promoters (AGP) (Begley M et al., Appl Environ Microbiol 2006 Mar;72(3):1729-38). Meanwhile, Korean Patent Publication No. 10-2018-0092526 discloses a feed additive as an alternative to antibiotics and a method for producing the same, which uses a fermented product of oregano, cloves, and rice bran fermented with effective microorganisms (EM) as a main ingredient. Although research is being conducted to develop feed additives as an alternative to antibiotics, the results are still insufficient.

[0005] Therefore, there is a need to develop feed additives that can replace antibiotics while promoting livestock growth and increasing feed efficiency to maintain the productivity and sustainability of food animals.

[0006]

[0007] One object of the present application is to provide a feed composition comprising catechin and vitamin K.

[0008] Another object of the present application is to provide a feed additive comprising catechin and vitamin K.

[0009] Another object of the present application is to provide a method for inhibiting bile salt hydrolase (BSH) activity in an animal, comprising the step of feeding to the animal a feed composition comprising catechin and vitamin K, or the feed composition containing them as active ingredients.

[0010] Another object of the present application is to provide a method for improving intestinal health of an animal, comprising the step of feeding the animal a feed composition comprising catechin and vitamin K, or these as active ingredients.

[0011] Another object of the present application is to provide a use of a feed composition comprising catechin and vitamin K, or containing them as active ingredients, for inhibiting bile salt hydrolase (BSH) activity.

[0012] Another object of the present application is to provide a use of catechin and vitamin K, or a feed composition containing them as active ingredients, for improving intestinal health.

[0013]

[0014] As one embodiment to achieve the above purpose, the present application provides a feed composition comprising catechin and vitamin K.

[0015] In another aspect, the present application provides a feed additive comprising catechin and vitamin K.

[0016] In another aspect, the present application provides a method for inhibiting bile salt hydrolase (BSH) activity in an animal, comprising the step of feeding the animal a feed composition comprising catechin and vitamin K, or the same as active ingredients.

[0017] In another aspect, the present application provides a method for improving intestinal health of an animal, comprising the step of feeding the animal a feed composition comprising catechin and vitamin K, or the same as active ingredients.

[0018] In another aspect, the present application provides a use of a feed composition comprising catechin and vitamin K, or the feed composition comprising them as active ingredients, for inhibiting bile salt hydrolase (BSH) activity.

[0019] In another aspect, the present application provides a use of a feed composition comprising catechin and vitamin K, or the same as active ingredients, for improving intestinal health.

[0020]

[0021] The feed composition or feed additive of the present application contains catechin and vitamin K, thereby inhibiting bile salt hydrolase (BSH) activity, thereby improving intestinal health. Furthermore, it inhibits the growth of harmful Escherichia coli and exhibits antioxidant activity, thereby exhibiting excellent effects that can replace antibiotics while promoting livestock growth and increasing feed efficiency. Therefore, it can contribute to improving livestock productivity and quality.

[0022]

[0023] Figures 1 and 2 are diagrams showing the results of confirming the inhibitory effect of bile salt hydrolase (BSH) activity according to the combination of catechin and vitamin K of the present application.

[0024] Figure 3 is a diagram showing the results of confirming the efficacy of bile acids (GDCA, TDCA) in inhibiting the growth of harmful Escherichia coli in farms.

[0025] Figure 4 is a diagram showing the results of the antioxidant efficacy evaluation by material of catechin or vitamin K.

[0026] Figure 5 is a diagram showing the results of confirming the antioxidant efficacy according to the combination of catechin and vitamin K of the present application.

[0027] Figure 6 is a diagram showing the results of confirming the effect of reducing free bile acid in feces when feeding a weaned piglet with a combination of catechin and vitamin K of the present application.

[0028]

[0029] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0030] Furthermore, those skilled in the art will recognize or be able to ascertain, using only routine experimentation, numerous equivalents to the specific embodiments of the present application described herein. Furthermore, such equivalents are intended to be encompassed by this application.

[0031] As used in the specification and appended claims of this application, the singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless the context clearly dictates otherwise, the singular terms include the plural and the plural terms include the singular. In the specification and appended claims of this application, unless the context clearly dictates otherwise, the use of "or" is intended to include "and / or."

[0032]

[0033] In this application, the term "about" may be used before a specific numerical value. As used herein, the term "about" encompasses not only the exact number described after the term, but also a range that is or is nearly that number. Whether a number is or is nearly the specific number described can be determined based on the context in which it is presented. For example, the term "about" may refer to a range of -10% to +10% of a numerical value. In another example, the term "about" may refer to a range of -5% to +5% of a given numerical value. However, this is not a limitation.

[0034] In this application, the terms "first, second, third," "i), ii), iii),," or "(a), (b), (c), (d),," are used to distinguish similar configurations, and these terms do not imply that the steps are performed consecutively or in order. For example, when the terms are used in connection with steps of a method, use, or analysis, these steps may be performed simultaneously, or may be performed at intervals of seconds, minutes, hours, days, or months.

[0035] In this application, the term "comprising" means the presence of a feature, step, or component described below, and does not exclude the presence or addition of one or more features, steps, or components. The components or features described below "comprising" in this application may be essential or mandatory, but in some embodiments, other optional or non-essential components or features may be further included.

[0036]

[0037] One aspect of the present application provides a feed composition comprising catechin and vitamin K.

[0038] Another aspect of the present application provides a feed additive comprising catechin and vitamin K.

[0039]

[0040] In the present application, the term "catechin" is a type of polyphenol, and may include at least one selected from the group consisting of epigallocatechin (EGC), epicatechin (EC), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), gallocatechin (GC), catechin gallate (CG), gallocatechin gallate (GCG), and catechin (C), but is not limited thereto.

[0041] In this application, the term "vitamin K" refers to a fat-soluble vitamin known to be involved in blood coagulation. In this application, the vitamin K may include all of its derivatives, for example, phylloquinone (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), vitamin K4, and its active forms (hydroxy derivatives).

[0042] As an example of implementation, the vitamin K of the present application may be, but is not limited to, menadione.

[0043] As used herein, the term "feed composition" refers to feed fed to animals. The feed composition may be the final form of feed fed to animals. The feed composition refers to a substance that supplies organic or inorganic nutrients necessary for sustaining the life of an animal or producing meat, milk, etc. The feed composition may include the feed additive of the present application and may additionally include nutrients necessary for sustaining the life of an animal or producing meat, milk, etc.

[0044] For example, the feed composition of the present application may include the feed additive of the present application.

[0045] As used herein, the term "feed additive" refers to a substance added to a feed composition, which may be considered a supplementary feed under the Feed Management Act. The feed additive may be intended to enhance the productivity or health of animals or livestock, but is not limited thereto.

[0046]

[0047] Antibiotic growth promoters (AGPs) are commonly used to promote the growth of livestock. However, countries around the world are raising concerns about the misuse of antibiotics in livestock farming and are calling for the prudent use and regulation of antibiotics (Marshall BM, Levy SB., Clin Microbiol Rev 2011).

[0048] Therefore, there is a need to develop feed additives that can replace antibiotics while promoting livestock growth and increasing feed efficiency to maintain the productivity and sustainability of food animals.

[0049] The feed composition or feed additive of the present application contains both catechin and vitamin K as active ingredients, thereby improving intestinal health by inhibiting bile salt hydrolase (BSH) activity, inhibiting the growth of harmful Escherichia coli, and exhibiting antioxidant activity. In addition, the combination of catechin and vitamin K can exhibit a synergistic effect, and thus can exhibit an excellent effect of promoting the growth of livestock and increasing feed efficiency while replacing antibiotics. For example, the feed composition or feed additive of the present application can exhibit a bile salt hydrolase (BSH) inhibitory effect by containing catechin and vitamin K as active ingredients, and thus can be used for inhibiting bile salt hydrolase (BSH). In addition, the feed composition or feed additive of the present application can improve the intestinal health of animals and exhibit an effect of increasing body weight by containing catechin and vitamin K as active ingredients, and thus can be used for improving intestinal health and / or increasing body weight of animals.

[0050]

[0051] Specifically, the feed composition or feed additive of the present application may be for inhibiting bile salt hydrolase (BSH), but is not limited thereto.

[0052] Specifically, the feed composition or feed additive of the present application may be for improving intestinal health, but is not limited thereto.

[0053] Specifically, the feed composition or feed additive of the present application may exhibit one or more activities selected from the group consisting of antioxidant and animal growth, but is not limited thereto.

[0054]

[0055] In this application, the term "bile salt hydrolase (BSH)" refers to an enzyme whose primary function is to convert conjugated bile salts into unconjugated bile salts. The bile salt hydrolase may be produced by, but is not limited to, intestinal commensals. It has been disclosed that since bile salt hydrolase has a negative effect on fat digestion and utilization in a host animal, it is possible to promote animal growth by reducing the activity of bile salt hydrolase (Begley M et al., Appl Environ Microbiol 2006).

[0056] In one embodiment, the bile salt hydrolase of the present application may be derived from a gram-positive bacterium, specifically from the genus Bacteroides, more specifically from Bacteroides fragilis, or may be derived from a gram-negative bacterium, specifically from the genus Clostridium, more specifically from Clostridium perfringens, but is not limited thereto.

[0057] In one embodiment, the feed composition or feed additive of the present application may have a bile salt hydrolase (BSH) inhibitory activity, thereby reducing the activity of the enzyme and promoting the growth of the animal, but is not limited thereto.

[0058] In one embodiment, the feed composition or feed additive of the present application may exhibit, but is not limited to, an activity of improving the intestinal health of an animal by inhibiting the activity of bile salt hydrolase (BSH), thereby reducing the content of liberated bile acids.

[0059] In one embodiment, the feed composition or feed additive of the present application exhibits activity in improving the intestinal health of animals by inhibiting the growth of harmful E. coli, and can be usefully used as a growth promoter in place of antibiotics, but is not limited thereto.

[0060] In one embodiment, the feed composition or feed additive of the present application may exhibit antioxidant activity and / or weight gain activity in animals, but is not limited thereto. Weight gain in the present application may refer to an increase in the body weight of an animal after feeding the feed composition or feed additive of the present application compared to before feeding.

[0061]

[0062] In one embodiment, the feed composition of the present application contains the catechin in an amount of 2 to 1,000 ppm, specifically 2 to 500 ppm, 2 to 200 ppm, 2 to 150 ppm, 2 to 125 ppm, 2 to 100 ppm, 2 to 90 ppm, 2 to 88 ppm, 2 to 86 ppm, 2 to 80 ppm, 2 to 60 ppm, 2 to 50 ppm, 2 to 45 ppm, 2 to 44 ppm, 2 to 43 ppm, 2 to 32 ppm, 2 to 31.25 ppm, 2 to 31 ppm, 2 to 30 ppm, 2 to 22 ppm, 2 to 21 ppm, 22 to 1,000 ppm, 22 to 500 ppm, 22 to 200 ppm, 22 to 150 ppm, 22 to 125 ppm, 22 to 100 ppm, 22 to 90 ppm, 22 to 88 ppm, 22 to 86 ppm, 22 to 80 ppm, 22 to 60 ppm, 22 to 50 ppm, 22 to 45 ppm, 22 to 44 ppm, 22 to 43 ppm, 22 to 32 ppm, 22 to 31.25 ppm, 22 to 31 ppm, 31 to 1,000 ppm, 31 to 500 ppm, 31 to 200 ppm, 31 to 150 ppm, 31 to 125 ppm, 31 to 100 ppm, 31 to 90 ppm, 31 to 88 ppm, 31 to 86 ppm, 31 to 80 ppm, 31 to 60 ppm, 31 to 50 ppm, 31 to 45 ppm, 31 to 44 ppm, or 31 to 43 ppm, but is not limited thereto.

[0063] At the same time, the feed composition of the present application contains the vitamin K in an amount of 0.5 to 200 ppm, specifically 0.5 to 150 ppm, 0.5 to 125 ppm, 0.5 to 100 ppm, 0.5 to 50 ppm, 0.5 to 44 ppm, 0.5 to 32 ppm, 0.5 to 31.25 ppm, 0.5 to 31 ppm, 0.5 to 25 ppm, 0.5 to 22 ppm, 0.5 to 20 ppm, 0.5 to 15 ppm, 0.5 to 10 ppm, 0.5 to 9 ppm, 0.5 to 8 ppm, 1 to 200 ppm, specifically 1 to 150 ppm, 1 to 125 ppm, 1 to 100 ppm, 1 to 50 ppm, 1 to 44 ppm, 1 to 32 ppm, 1 to 31.25 ppm, 1 to 31 ppm, 1 to 25 ppm, 1 to 22 ppm, 1 to 20 ppm, 1 to 15 ppm, 1 to 10 ppm, 1 to 9 ppm, 1 to 8 ppm, 2 to 150 ppm, 2 to 125 ppm, 2 to 100 ppm, 2 to 50 ppm, 2 to 44 ppm, 2 to 32 ppm, 2 to 31.25 ppm, 2 to 31 ppm, 2 to 25 ppm, 2 to 22 ppm, 2 to 20 ppm, 2 to 15 ppm, 2 to 10 ppm, 2 to 9 ppm, or 2 to 8 May include, but is not limited to, ppm.

[0064] The feed composition may contain 2 to 200 ppm of the catechin and 0.5 to 32 ppm of the vitamin K based on the total weight of the feed when actually fed to an animal, but is not limited thereto.

[0065] Based on the total weight of the feed additive, the catechin may be 1,000 to 100,000 ppm, specifically 20,000 to 90,000 ppm, 20,000 to 88,000 ppm, or 22,000 to 88,000 ppm, and the vitamin K may be 100 to 10,000 ppm, specifically 1,000 to 8,000 ppm, or 2,000 to 8,000 ppm, but is not limited thereto.

[0066] The concentration of catechin and vitamin K included in the feed composition and feed additive described above can be appropriately adjusted depending on the type and age of livestock, application form, desired effect, etc.

[0067] In one embodiment, the feed composition or feed additive of the present application may include the catechin and vitamin K in a content ratio (weight ratio and mixture) of 1:1 to 1,000:1. The content ratio (weight ratio) of the above catechin and vitamin K may be, for example, 1:1 to 1,000:1, 1:1 to 500:1, 1:1 to 400:1, 1:1 to 300:1, 1:1 to 200:1, 1:1 to 100:1, 1:1 to 90:1, 1:1 to 80:1, 1:1 to 70:1, 1:1 to 60:1, 1:1 to 50:1, 1:1 to 40:1, 1:1 to 30:1, 1:1 to 20:1, 1:1 to 15:1, 1:1 to 13:1, 1:1 to 11:1, or 1:1 to 10:1, but is not limited thereto, depending on the type and age of the livestock, application It can be adjusted appropriately depending on the shape, desired effect, etc.

[0068]

[0069] The type of the above feed is not particularly limited, and feed commonly used in the relevant technical field can be used. Non-limiting examples of the above feed include plant-based feed such as grains, roots, food processing by-products, algae, fiber, pharmaceutical by-products, oils, starches, meal, or grain by-products; and animal-based feed such as proteins, inorganic substances, oils, minerals, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more types.

[0070] The feed composition or feed additive of the present application may be manufactured in a powder or granular form, and may further include, if necessary, one or more of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate; and natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid. The feed composition or feed additive of the present application may be formulated in the form of a conventional feed, and may include conventional feed ingredients together.

[0071] The feed composition or feed additive of the present application may further include grains, such as ground or crushed wheat, oats, barley, corn, and rice; plant-based protein feeds, such as feeds mainly composed of rapeseed, soybeans, and sunflower; animal-based protein feeds, such as blood meal, meat meal, bone meal, and fish meal; dry ingredients composed of sugars and dairy products, such as various types of milk powder and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.

[0072] The feed composition or feed additive of the present application may be administered to animals alone or in combination with other feed additives in an edible carrier. Furthermore, the feed additive may be readily administered to animals as a top dressing, by mixing it directly into the feed, or in an oral formulation separate from the feed. When the feed composition or feed additive is administered separately from the feed, it may be prepared as an immediate-release or sustained-release formulation by combining it with a pharmaceutically acceptable edible carrier, as is well known in the art. Such edible carriers may be solid or liquid, such as cornstarch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, the feed additive may be in the form of a tablet, capsule, powder, troche, or saccharide tablet, or a top dressing in a microdispersible form. When a liquid carrier is used, the feed additive may be in the form of a gelatin soft capsule, or a syrup, suspension, emulsion, or solution.

[0073] The feed composition or feed additive of the present application may contain, for example, a preservative, a stabilizer, a wetting agent or emulsifier, a solution accelerator, etc. The feed additive may be added to animal feed by injecting, spraying, or mixing.

[0074] The feed composition or feed additive of the present application may be used by mixing one or more of organic acids such as citric acid, fumaric acid, adipic acid, and lactic acid; phosphates such as potassium phosphate, sodium phosphate, and polymeric phosphate; and natural antioxidants such as polyphenol, catechin, tocopherol, vitamin C, green tea extract, chitosan, and tannic acid for administration, and other conventional additives such as anti-influenza agents, buffers, and bacteriostatic agents may be added as needed. In addition, the feed composition of the present application may be formulated into an injectable formulation such as an aqueous solution, suspension, or emulsion, capsule, granule, or tablet by additionally adding a diluent, dispersant, surfactant, binder, or lubricant. In addition, the feed composition or feed additive of the present application may be used together with various auxiliary ingredients such as amino acids, inorganic salts, vitamins, antioxidants, antifungals, antibacterial agents, etc., and plant-based protein feed such as ground or crushed wheat, barley, corn, etc., animal-based protein feed such as blood meal, meat meal, fish meal, etc., animal fat, and plant-based fat, in addition to the main ingredients such as nutritional supplements, growth promoters, digestion and absorption promoters, and disease preventive agents.

[0075] The feed composition or feed additive of the present application can be mixed into livestock feed in an amount of about 10 to 500 g per kg on a dry weight basis, for example, 10 to 100 g, and after being thoroughly mixed, can be supplied as a mash or can be subjected to a pelletizing, expansion or extrusion process through an additional processing process.

[0076] The content of the feed additive in the feed composition of the present application can be appropriately adjusted depending on the type and age of the livestock to be applied, the form of application, the desired effect, etc., and can be used, for example, at 0.01 to 90 wt%.

[0077] Furthermore, the feed additive according to the present application can be used individually, in combination with conventionally known feed additives, or sequentially or simultaneously with conventional feed additives. It can be administered singly or in multiple doses. Taking all of the above factors into account, it is important to administer the amount that achieves maximum effect with the minimum amount possible without causing side effects, a determination readily made by those skilled in the art.

[0078]

[0079] The feed composition or feed additive of the present application may be for animals, but is not limited to any species that can be fed the feed.

[0080] The feed composition or feed additive of the present invention can be applied to animal diets. The animals may be, but are not particularly limited to, humans, and may include a variety of animals, including mammals and poultry. Examples of mammals include pigs, cattle, sheep, goats, laboratory rodents, and companion animals (e.g., dogs and cats). Examples of poultry include chickens, turkeys, ducks, geese, pheasants, and quail. The feeding method is not limited.

[0081]

[0082] Another aspect of the present application provides a method for inhibiting bile salt hydrolase (BSH) activity in an animal, comprising the step of feeding the animal a feed composition comprising catechin and vitamin K, or the same as active ingredients.

[0083] Another aspect of the present application provides a method for improving intestinal health of an animal, comprising the step of feeding the animal a feed composition comprising catechin and vitamin K, or the feed composition comprising them as active ingredients.

[0084] The feed composition, feed additives, and animals are as described above.

[0085]

[0086] The method for inhibiting the activity of bile salt hydrolase (BSH) in the animal or the method for improving the intestinal health of the animal may specifically include the steps of (a) mixing the feed additive or feed composition into animal feed; and (b) feeding the feed to the animal.

[0087] The step (a) according to the present application is a step of mixing the feed composition or feed additive containing catechin and vitamin K of the present application into general feed for livestock, and can be fed by mixing it in the mixed feed at 0.01 to 90 wt%, preferably 0.1 to 10 wt%.

[0088] The step (b) according to the present application is a step of feeding the feed manufactured in the step (a) to an animal, and the method thereof is not limited. The animals to which the feed can be fed are not particularly limited as described above, and the feed can be applied to a number of animal diets, including mammals and poultry. The mammals include pigs, cows, sheep, goats, laboratory rodents, and laboratory rodents, as well as pets (e.g., dogs, cats), and the like, and the poultry includes chickens, turkeys, ducks, geese, pheasants, and quail.

[0089] When the feed additive or feed composition according to the present application is fed to animals, it is expected to have excellent effects of improving intestinal health by inhibiting bile salt hydrolase (BSH) activity, inhibiting the growth of harmful Escherichia coli, and exhibiting antioxidant activity, thereby promoting the growth of livestock and increasing feed efficiency while replacing antibiotics.

[0090]

[0091] Another aspect of the present application provides a use of the feed composition or feed additive of the present application for inhibiting bile salt hydrolase, improving intestinal health of animals, inhibiting growth of harmful Escherichia coli, antioxidant use, and increasing body weight of animals.

[0092] The feed composition, feed additive, bile salt hydrolase, etc. are as described above.

[0093]

[0094] Hereinafter, this application will be described in more detail through examples. However, the following examples are merely preferred embodiments intended to illustrate the present application and are therefore not intended to limit the scope of the present application. Furthermore, technical details not described in this specification can be readily understood and implemented by those skilled in the technical field of this application or similar technical fields.

[0095]

[0096] Example 1. Evaluation of the inhibitory effect on bile salt hydrolase (BSH) activity

[0097]

[0098] Example 1-1. Comparison of the inhibitory effects of bile salt hydrolase (BSH) activity by material

[0099]

[0100] The purpose of this study was to evaluate the inhibitory efficacy of each ingredient, epicatechin gallate (ECG), epigallocatechin gallate (EGCG), epicatechin (EC), epigallocatechin (EGC), vitamin K, and oxytetracycline, on bile salt hydrolase (BSH) activity.

[0101] Specifically, Bacteroides fragilis (KCTC 5013) or Clostridium perfringens (KCTC 5014) strains were inoculated into liquid or solid media prepared with Reinforced clostridial medium (BD DIFCO 218081) or brain heart infusion powder (BD DIFCO 237500), and then cultured anaerobically and statically at 37°C for 2 days, respectively. The strain cultures were diluted with 0.1 M sodium phosphate buffer (pH 6.0) containing DL-dithiothreitol (DTT) to an absorbance (600 nm) of 0.3. The substrate was prepared with 100 mM glycodeoxycholic acid (GDCA, Sigma G9910) and taurodeoxycholic acid (TDCA, Sigma T0875) in dimethyl sulfoxide (DMSO, Sigma 276855) and stored in a refrigerator. The strain information and culture medium mentioned above are shown in Table 1 below, and the information on each evaluation material is shown in Table 2 below.

[0102]

[0103] Strain name Strain number Medium Gram (-)Bacteroides fragilisKCTC 5013Reinforced clostridial mediumGram (+)Clostridium perfringensKCTC 5014Brain heart infusion

[0104] Classification Evaluation Material Information Auxiliary / Extract Active Ingredients Epicatechin gallate (ECG) Sigma, E3893 Epigallocatechin gallate (EGCG) Sigma, PHR1333 Epicatechin (EC) Sigma, E1753 Epigallocatechin (EGC) Sigma, E3768 Vitamins Vitamin K China, Hubei Zhenhua Chemical Co. Antibiotics Oxytetracycline Sigma, O4636

[0105]

[0106] Next, ECG, EGCG, EC, EGC, vitamin K, and oxytetracycline were diluted using 0.1 M sodium phosphate buffer containing 5 mM DTT and prepared according to each evaluation concentration. After reacting the prepared materials and the BSH-producing strain culture medium at 37°C for 30 minutes, the substrates glycodeoxycholic acid and taurodeoxycholic acid were added. The reaction was terminated when the absorbance (OD 600 nm) of the reaction solution including the substrate and strain was 0.5 or higher. The BSH inhibition rate (%) was expressed as '% inhibition = (OD 소재처리 - OD 균주 ) / (OD 균주 / 기질 - OD 균주 ) X 100(%)', the BSH inhibitory efficacy of each material was compared. At this time, OD material treatment = absorbance after reaction of material, strain, and substrate; OD strain = absorbance according to strain treatment alone; and OD 균주 / 기질 = The absorbance was measured after the reaction between the strain and the substrate.

[0107] The results of the BSH inhibition test produced from Gram (+)C. perfringens are shown in Table 3 below, and the results of the BSH inhibition test produced from Gram (-)B. fragilis are shown in Table 4 below.

[0108]

[0109] Classification Material Material 200 ppm standard BSH inhibition rate (%) Average * 2 mM GDCA 2 mM TDCA Auxiliary / extract active ingredient ECG494455 EGCG645374 EC15723 EGC352 Vitamin Vitamin K9898100 Antibiotic Oxytetracycline817785

[0110] * Average: Average of BSH inhibition rate (%) using 2 mM GDCA as a substrate and BSH inhibition rate (%) using 2 mM TDCA as a substrate

[0111] Classification Material Material 125 ppm standard BSH inhibition rate (%) Average * 2 mM GDCA 2 mM TDCA Auxiliary / extract active ingredient ECG604674 EGCG766586 EC342048 EGC807388 Vitamin Vitamin K665578 Antibiotic Oxytetracycline 100 100 100

[0112] * Same as the average calculation method in Table 3

[0113]

[0114] As a result, as shown in Table 3 above, the average inhibition rate of BSH produced by Gram (+) C. perfringens was confirmed for each material, and the positive control, oxytetracycline, was 81%, the auxiliary / extract active ingredient, ECG, was 49%, EGCG was 64%, EC was 15%, and EGC was 3%, and vitamin K was confirmed to have a BSH inhibition rate of 98%.

[0115]

[0116] In addition, as shown in Table 4 above, the average inhibition rate of BSH produced from Gram (-) B. fragilis was confirmed for each material, and the positive control, oxytetracycline, was 100%, the auxiliary / extract active ingredient, ECG, was 60%, EGCG was 76%, EC was 34%, and EGC was 80%, and vitamin K was confirmed to have a BSH inhibition rate of 66%.

[0117]

[0118] Example 1-2. Evaluation of the synergistic effect of catechin and vitamin K combinations on the inhibition of bile salt hydrolase (BSH) activity.

[0119]

[0120] The synergistic effect of the combination of catechin and vitamin K on the inhibition of bile salt hydrolase (BSH) activity was examined using the method of Example 1-1. BSH derived from B. fragilis was used as a representative example, and the evaluation materials and treatment concentrations are shown in Tables 5 and 6 below. The evaluation results are shown in Figures 1 and 2, respectively.

[0121]

[0122] Evaluation Material Treatment Concentration (ppm)ECG31.25EGCEGCGECCinflammatories KECG + Vitamin K31.25EGC + Vitamin KEGCG + Vitamin KEC + Vitamin K

[0123]

[0124] As a result, as shown in Fig. 1, when EGCG and vitamin K were treated alone at 31.25 ppm each, the BSH inhibition rates were 37.55% and 20.82%, respectively, but when EGCG and vitamin K were treated in combination at 31.25 ppm each, the inhibition rate was 90.82%, confirming that it was 156% (32.45% synergy) superior to the combined efficacy of 58.37% of the single substances.

[0125] In addition, when EGC and vitamin K were treated alone at 31.25 ppm, the BSH inhibition rates were 25.10% and 20.82%, respectively, but when EGC and vitamin K were treated in combination, the inhibition rate was 91.22%, indicating a synergistic effect of 45.3%. In the same way, ECG and vitamin K confirmed a synergistic effect of 29.38% when treated in combination compared to when treated alone, and EC and vitamin K confirmed a synergistic effect of 55.91% when treated in combination compared to when treated alone.

[0126]

[0127] In addition, the BSH inhibition rate was evaluated as shown in Table 6 below.

[0128]

[0129] Evaluation Material Treatment Concentration (ppm) Total catechin 22 Vitamin K 2 Total catechin + Vitamin K 22 + 2 each

[0130] As a result, as shown in Fig. 2, when catechin (total catechin) and vitamin K were treated alone at 22 ppm and 2 ppm, respectively, BSH inhibition efficiencies of 58.5% and 0% were confirmed, but when they were treated in combination, an inhibition rate of 80.5% was observed, confirming a synergistic effect of 22.5%.

[0131]

[0132] Example 2. Evaluation of the efficacy of suppressing the growth of harmful Escherichia coli in farms by inhibiting bile acids (GDCA, TDCA).

[0133]

[0134] The present application was conducted to confirm the efficacy of the feed composition or feed additive in inhibiting the growth of harmful Escherichia coli in farms.

[0135] Specifically, Escherichia coli (ETEC (Enterotoxigenic E. coli) (F18, Sta) isolated from a pig farm in Gyeonggi-do, South Korea, was cultured in liquid medium. Culture medium prepared with an absorbance of 0.3 (OD 600 nm) and 0, 0.5, 1, 2, and 4 mM glycodeoxycholic acid and taurodeoxycholic acid were prepared, reacted overnight at 37°C, and then the absorbance was measured.

[0136] As a result, as shown in Fig. 3, in the 0 mM treatment group without bile acid treatment, the absorbance was confirmed to be 0.45 or higher, but in the 0.5 to 4 mM treatment group to which 0.5 mM or more of glycodeoxycholic acid and taurodeoxycholic acid were added, the absorbance decreased. Therefore, it was confirmed that the bile acids glycodeoxycholic acid and taurodeoxycholic acid inhibit the growth of ETEC strains.

[0137] A decrease in the OD value indicates inhibition of E. coli growth, and since, according to Figure 3, when the substrate of bile salt hydrolase increases (i.e., corresponding to a decrease in the activity of bile salt hydrolase), the growth of E. coli is inhibited, these results suggest that inhibition of bile salt hydrolysis can inhibit E. coli growth.

[0138] This suggests that the feed composition or feed additive of the present application exhibits bile salt hydrolase (BSH) inhibitory activity, thereby reducing the bile acid content liberated by bile salt hydrolase, and ultimately inhibiting the growth of ETEC strains, thereby improving intestinal health.

[0139]

[0140] Example 3. Evaluation of antioxidant efficacy

[0141]

[0142] Example 3-1. Evaluation of antioxidant efficacy of catechin or vitamin K by material

[0143]

[0144] We aimed to confirm the antioxidant efficacy of each material, catechin or vitamin K.

[0145] Specifically, to extract catechin from natural products containing more than 10% of total catechin, ethanol was added at a concentration of 5 mg / ml and shaken. After filtering using a 0.2 μm syringe filter, high performance liquid chromatography (HPLC) analysis confirmed that the natural products contained 3% EGC, 7.8% EGCG, 1.6% ECG, and 0.9% EC. Vitamin K products containing 50% menadione were dissolved by adding DMSO at a concentration of 100 mg / ml and then stored in the refrigerator. Information on the evaluated materials is shown in Table 7 below.

[0146]

[0147] Classification Evaluation Material Information Auxiliary / Extract Effective Ingredients: Plant extracts containing catechin (EGC: 3%, EGCG: 7.8%, ECG: 1.6%, EC: 0.9%) Korea Vitamin Ryu Vitamin K China, Brother Co., Ltd.

[0148]

[0149] Next, the ROS reduction rate was evaluated. Specifically, the porcine intestinal epithelial cell line IPEC-J2 (DSMZ No. ACC701) was cultured (37°C, 5% CO2) in a 96-well plate using culture medium (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12, 11320033) containing 1% penicillin / streptomycin (Gibco, 15140-122) and 10% FBS (Hyclone, SV30207.02).

[0150] Before material treatment, the prepared materials were washed with phosphate-buffered saline (PBS) and diluted in the medium. The prepared porcine intestinal epithelial cell lines were treated with the prepared materials and cultured overnight. Subsequently, the culture medium was removed and washed with Hank's Balanced Salt Solution (Gibco, 14175095). Next, a 10 μM DCF-DA (Sigma-Aldrich, D6883) solution diluted in HBSS was added to each well and incubated in the dark for 30 minutes in a 37℃ incubator. After the reaction, the culture medium was removed, a 1 mM H2O2 solution was added to each well, and the reaction was again performed at 37℃ for 30 minutes. The ROS reduction rate was compared by measuring the fluorescence intensity (Extinction 485 nm, emission 530 nm) using a microplate spectrophotometer (Microplate spectrophotometer, BIOTEK, EPOCH2NS, SYNERGY H1).

[0151] As a result, as shown in Fig. 4, it was confirmed that the plant extract including catechin had ROS reduction rates of 8.28%, 15.24%, and 28% at concentrations of 25 ppm, 50 ppm, and 100 ppm, respectively, and vitamin K had ROS reduction rates of 0%, 5.66%, and 9.47% at concentrations of 0.1 ppm, 0.25 ppm, and 0.5 ppm, respectively.

[0152]

[0153] Example 3-2. Evaluation of antioxidant efficacy according to the combination of catechin and vitamin K.

[0154]

[0155] The antioxidant efficacy of the combination of catechin and vitamin K of the present application was confirmed using the same method as in Example 3-1 above.

[0156] Specifically, a ROS reduction efficacy test was conducted using a combination of 100 ppm of plant extract containing catechin and 0.5 ppm of vitamin K.

[0157]

[0158] As a result, as shown in Fig. 5, when 100 ppm of plant extract containing catechin and 0.5 ppm of vitamin K were treated alone, the combined treatment showed a 45.31% ROS reduction rate, which was 7.8% higher than the simple sum of the ROS reduction rates, confirming the existence of a synergistic effect.

[0159]

[0160] Example 4. Evaluation of the efficacy of combinations of catechin and vitamin K on improving the growth performance of weaned piglets (in vivo evaluation)

[0161]

[0162] The purpose of this study was to determine the effect of adding the combination of catechin and vitamin K of this application to weaning piglet feed on the productivity of weaning piglets.

[0163] Specifically, an in vivo weaned piglet feeding experiment was conducted at an animal testing facility in Gyeonggi-do, South Korea. Three-week-old weaned piglets were individually weighed and randomly assigned to groups for the experiment. The experimental design and conditions are described in Table 8. The breeding pens were managed in accordance with the Korean Pig Feeding and Management Guidelines. Pens and breeding pens were cleaned and disinfected before the start of the experiment. The temperature in the breeding pens was maintained at 28–31°C and humidity at 50%, and the animals were continuously monitored.

[0164]

[0165] Category Experimental Variables Housing Type Pen Animal Weaned Piglets 3-way Crossbreed (LxY) x D Age of Weaned Piglets 3 weeks Total Experimental Period 28 days Number of Weaned Piglets per Pen 4 Number of Repetitions by Treatment Group 8 Number of Repetitions by Treatment Group 4 Total Number of Weaned Piglets 128

[0166]

[0167] 400 mesh green tea powder containing more than 10% catechin was procured from Gyeongsang-do Agricultural Cooperative Association, Republic of Korea, and vitamin K was procured from Dawon Chemical (Gyeonggi-do, Republic of Korea). The test feed was a corn-soybean meal-based feed mixed according to the NRC (2012) requirements. The feed was mixed and prepared using a mixer located in the test farm and was fed ad libitum. Water was regulated to be available ad libitum using an automatic waterer. Antibiotics and zinc oxide (ZnO) were not added to the regular feed and the mixed feed, and the mixed feed prepared above was separately consumed by the control or test groups. The feed compositions administered to the control and test groups are shown in Table 9 below.

[0168]

[0169] Group treatment: General diet feeding Control group: Basal diet Experimental group: 121.5 ppm catechin and 2 ppm vitamin K + Basal diet Experimental group: 243 ppm catechin and 4 ppm vitamin K + Basal diet Experimental group: 386 ppm catechin and 8 ppm vitamin K + Basal diet

[0170]

[0171] Daily gain, daily feed intake, and feed efficiency were measured for each individual at the beginning of the experiment and at 1 and 2 weeks after the start. Feed intake was calculated by subtracting the residual feed from the feed provided at the time of body weight measurement, and feed efficiency was calculated by dividing the daily gain by the feed intake. All data were processed using the General Linear Model procedure of SAS (2013) and Duncan's multiple range test (Duncan, 1955) to test the significance of the mean values. After the end of the experiment, feces from 15 heads each from the control group and experimental group 2 were randomly sampled and analyzed for residual free bile acids. 0.1 g of feces was weighed into a homogenization tube containing 5 beads, and 900 ml of cold 50% MeOH was added to it, followed by homogenization at 2,500 rpm for 10 seconds, repeated three times. After centrifugation at 13,000 rpm for 10 minutes, 500 μl of the supernatant was taken and diluted with 2 ml 50% MeOH. After filtration through 0.2 μm PVDF, it was stored in an LC vial and analyzed for free bile acids using LC-MS.

[0172] The results are shown in Table 10.

[0173]

[0174] ItemsNC Experimental Group 1 (TRT 1) Experimental Group 2 (TRT 2) Experimental Group 3 (TRT 3) Body weight, kgInitial6.466.476.476.47Week18.148.158.198.19Week210.65b10.69ab10.78ab10.8 0abWeek313.26c13.48bc13.58ab13.64abWeek416.17b16.61ab16.73a16.80aWeek0-1ADG, g239241245246ADFI, g268270273274G:F0.8940.8950.8960.897Week1-2ADG, g359362370372ADFI, g443446455457G:F0.8110.8120.8130.814Week2-3ADG, g373b398a400a406aADFI, g500511513520G:F0.7470.7800.7800.781Week3-4ADG, g415b448ab451ab452abADFI, g632646650651G:F0.6570.6930.6930.695OverallADG, g347b362ab366a369aADFI, g461468473475G:F0.7520.7740.7750.776a,b,c Means in the same row with different superscripts differ (P < 0.05)

[0175]

[0176] As a result, as shown in Table 10 above, it was confirmed that the growth performance of the experimental group was significantly improved compared to the control group fed regular feed in terms of the body weight of weaned piglets in the 3rd and 4th weeks of the test.

[0177] Next, in order to confirm whether the BSH inhibitory effect actually occurred by adding the combination of catechin and vitamin K of the present application, the feces of experimental group 2 were examined as a representative example.

[0178]

[0179] As a result, as shown in Fig. 6, it was confirmed that lithocholic acid (LCA), a free bile acid in the feces, was reduced in experimental group 2 (TRT 2; fed 43 ppm catechin and 4 ppm vitamin K + Basal diet) compared to the control group fed regular feed (p=0.112, n=15). This confirmed that feeding a combination of catechin and vitamin K through feed can improve intestinal health by suppressing BSH in the intestinal tract of weaned piglets, thereby reducing the content of free bile acids.

[0180]

[0181] From the above results, it was confirmed that catechin and vitamin K have a synergistic effect (e.g., BSH inhibition and antioxidant activity), suggesting that the present application has excellent effects on animal weight gain, improvement of intestinal health, and antioxidant activity.

[0182]

[0183] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

Claims

1. Feed composition containing catechin and vitamin K.

2. A feed composition according to claim 1, wherein the composition is for inhibiting bile salt hydrolase (BSH).

3. A feed composition according to claim 1, wherein the composition is for improving intestinal health.

4. A feed composition according to claim 1, wherein the composition exhibits at least one activity selected from the group consisting of antioxidant and animal growth.

5. A feed composition according to claim 1, comprising 2 to 200 ppm of the catechin and 0.5 to 32 ppm of the vitamin K based on the entire feed composition.

6. A feed composition according to claim 1, wherein catechin and vitamin K are included in a weight ratio of 1:1 to 200:1 based on the entire feed composition.

7. A feed composition according to claim 1, wherein the catechin comprises at least one selected from the group consisting of epicatechin gallate (ECG), epigallocatechin gallate (EGCG), epicatechin (EC), epigallocatechin (EGC), gallocatechin (GC), catechin gallate (CG), gallocatechin gallate (GCG), and catechin (C).

8. Feed additive containing catechin and vitamin K.

9. A feed additive according to paragraph 8, wherein the feed additive is for inhibiting bile salt hydrolase (BSH).

10. A feed additive according to claim 8, wherein the feed additive exhibits at least one activity selected from the group consisting of antioxidant and animal growth.

11. A method for inhibiting bile salt hydrolase (BSH) activity in an animal, comprising the step of feeding a feed composition containing catechin and vitamin K, or these as active ingredients, to an animal other than a human.

12. A method for improving intestinal health of an animal, comprising the step of feeding a feed composition containing catechin and vitamin K, or these as active ingredients, to an animal other than a human.

13. Use of catechin and vitamin K, or a feed composition containing them as active ingredients, for inhibiting bile salt hydrolase (BSH) activity.

14. Use of catechin and vitamin K, or a feed composition containing them as active ingredients, for improving intestinal health.

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