Composition for reducing oxidative stress

Abstract

To provide a technology for early reduction of oxidative stress in animals, particularly lactating mammals, under high or low temperature conditions. [Solution] A composition for reducing oxidative stress in animals, containing difructose anhydride III (DFAIII), molasses, and betaine.

Description

Technical Field

[0001] The present invention provides a composition for reducing oxidative stress in animals such as livestock, particularly in lactating animals under high or low temperature environments.

Background Art

[0002] In the energy production process of animals, it is known that some oxygen is converted into reactive oxygen species. Reactive oxygen species are a type of free radical derived from oxygen molecules, which are very unstable, have a strong oxidizing power, and are oxygen species with strong chemical reactivity. Oxidation of biological substances by reactive oxygen species occurs daily, and the oxidation reaction by reactive oxygen species is an essential reaction for the living body, such as being used for sterilization in some immune systems. On the other hand, it is also known that reactive oxygen species cause damage to cells and tissues through the oxidation of biological substances.

[0003] In a healthy state, reactive oxygen species generated in the living body due to endogenous or exogenous factors are removed by the antioxidant system of the living body, thereby maintaining the redox state in the body so that it does not deviate towards the oxidation side. However, if the adjustment by this maintenance mechanism does not work well, cell membrane damage, protein denaturation, enzyme inactivation, DNA damage, etc. caused by reactive oxygen species frequently occur in the living body, which is said to be one of the major causes of aging and diseases. When reactive oxygen species are generated and accumulated beyond the removal ability of the antioxidant system of the living body and become excessive, the balance between oxidation and reduction that should be maintained in the living body collapses and deviates towards the oxidation side, which is the oxidative stress state. The oxidative stress state can cause various disorders in the living body.

[0004] The body's antioxidant system works to restore the balance between oxidation and reduction from an oxidative bias by inducing various antioxidant enzyme reactions and mobilizing reducing molecules, and antioxidants play a primary role in this process. In addition to antioxidants produced in the body, such as antioxidant enzymes and antioxidant peptides, antioxidants derived from ingested food also function in the body's antioxidant system. Known antioxidants include antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx), vitamins such as vitamin A, vitamin C, and vitamin E, polyphenols, carotenoids, glutathione, albumin, and bilirubin. For example, in cattle, vitamin C, glutathione, SOD, and GPx are produced in the body as antioxidants. On the other hand, cattle need to obtain vitamin E from outside the body, and vitamin A needs to be obtained in the form of β-carotene found in pasture grass, etc.

[0005] In dairy cows, oxidative stress increases during the periparturient period. This is thought to be due to increased energy requirements, i.e., oxygen requirements, resulting from physiological factors such as significant mammary gland growth and the initiation of milk synthesis and secretion during the periparturient period. In particular, high-yielding dairy cows experience even greater energy (oxygen) requirements associated with milk production, leading to a state of severe oxidative stress.

[0006] Furthermore, in biological tissues exposed to high or low thermal environments, increased oxidative stress due to heat stress or cold stress can occur, leading to various problems such as decreased metabolic activity, reduced productivity, decreased feed intake, and deterioration of reproductive performance, including lower conception rates. The optimal temperature range for the health and production activities of mammals varies depending on the animal species and growth stage. For example, it is said to be 4-24°C for dairy cows, and 10-20°C especially for lactating cows, and stress can occur outside of this temperature range. Similarly, the critical high temperature, at which body temperature homeostasis cannot be maintained by sweating or increased respiration alone, also varies depending on the animal species. For dairy cows, it is said to be 29-32°C, and 26-27°C especially for lactating cows. The critical low temperature, at which the body's heat dissipation prevention function becomes active to maintain a constant body temperature, is said to be -8°C for healthy lactating cows, and 7°C for unwell or malnourished cows, where cold tolerance decreases.

[0007] Heat stress is greatly influenced not only by temperature but also by humidity. The Temperature-Humidity Index (THI), calculated from temperature and humidity, is used as an indicator of heat stress. In cattle, a THI of 65-71 is considered mild stress, 72-81 is considered strong stress, and 82-92 is considered very strong stress. The Temperature-Humidity Index (THI) is calculated using the following formula. THI = 0.8 × dry-bulb temperature (°C) + 0.01 × relative humidity (%) × (dry-bulb temperature (°C) - 14.4) + 46.4

[0008] In Japan's Ministerial Ordinance on Milk and Dairy Products ("Ministerial Ordinance Concerning Standards for Ingredients of Milk and Dairy Products"), acidity is used as one of the quality indicators for raw milk, milk, etc., and the standard is set at 0.18 or less. Although no lower limit is set, generally milk with an acidity of less than 0.11% is considered low-acidity milk and is subject to disposal. While the acidity of milk decreases when the THI exceeds 65, it has also been reported that acidity decreases when the temperature drops below 5°C.

[0009] Dairy cows are known to exhibit symptoms caused by oxidative stress immediately after calving. In particular, when exposed to heat stress immediately before calving, feed intake decreases from 45 days before calving to 21 days after calving, milk production declines, and oxidative stress indicators worsen. Furthermore, in dairy cows, the concentration of the antioxidant glutathione in liver tissue, as well as the concentration of vitamins A and E in the blood, decreases by 7 days postpartum compared to 10 days before calving, indicating a tendency toward a decline in antioxidant capacity.

[0010] To alleviate oxidative stress in dairy cows, it is common practice to administer antioxidants. Patent Document 1 discloses a stress reduction technique for animals (cattle, pigs, dogs, cats, fish, etc.) by administering a stable L-ascorbic acid (vitamin C) derivative. Non-Patent Document 1 discloses that administering betaine to dairy cows under heat stress reduces the concentration of free fatty acids (NEFA) in the blood, which is an indicator of energy utilization in cattle, thereby improving energy metabolic efficiency and milk productivity, as well as suppressing the inhibition of the antioxidant enzyme GPx, and reducing the effects of heat stress. Non-Patent Document 2 shows that administering molasses to female fattening cattle increases the butyric acid concentration in the rumen, improving the gastrointestinal barrier function, and reduces serum haptoglobin and lipopolysaccharide-binding protein, thereby improving the growth of stressed cows. However, the examples in Patent Document 1 and Non-Patent Documents 1 and 2 involve administering antioxidants over a period of 60 days, and rapid effects have not been confirmed.

[0011] Patent Document 2 describes a feed improvement material for improving the taste quality and shelf life of meat, eggs, etc., comprising calcined char powder, polyphenol compounds which are antioxidants, wood vinegar components, seaweed powder, and tannin components. Patent Document 3 discloses a technique in which feeding castrated cattle a compound feed for beef cattle containing corn germ containing 18 mg / 100g or more of vitamin E suppresses meat oxidation and maintains good meat color. These techniques disclosed in Patent Documents 2 and 3 are aimed at preventing oxidation of products such as meat and eggs, rather than managing the health of livestock. Non-Patent Document 3 describes how feeding molasses to broilers increased body weight, promoted hematopoiesis, and reduced the effects of oxidative stress caused by heat stress.

[0012] Reducing oxidative stress in livestock is crucial for modern livestock farming, as it aims to improve the health of livestock and maintain and enhance productivity. In particular, for lactating livestock in high-temperature or low-temperature environments, early reduction of oxidative stress is strongly desired in order to ensure sufficient milk production during the limited lactation period. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] Japanese Patent Application Publication No. 10-175866 [Patent Document 2] Japanese Patent Publication No. 2005-341828 [Patent Document 3] Japanese Patent Publication No. 2006-49 [Non-patent literature]

[0014] [Non-Patent Document 1] Ali Mujtaba Shah,et.al.,Animals 2020,10(4),634-643 [Non-Patent Document 2] Breno C.Silva,et.al.,Journal of Animal Science,2023,101,1-11 [Non-Patent Document 3] Habibu et.al.,International Journal of Veterinary Science,2014,3(4),181-188 [Overview of the project] [Problems that the invention aims to solve]

[0015] The present invention aims to provide a technology for early reduction of oxidative stress in animals, particularly lactating mammals, under high or low temperature conditions. [Means for solving the problem]

[0016] As a result of diligent research to solve the above problems, the inventors of the present invention have found that by administering difructose anhydride III to animals in combination with betaine and molasses, an oxidative stress reduction effect can be obtained in a short period of time, and have completed the present invention.

[0017] In other words, the present invention encompasses the following:

[0018] [1] A composition for reducing oxidative stress in animals, comprising difructose anhydride III (DFAIII), molasses, and betaine. [2] The composition according to [1], further comprising at least one selected from the group consisting of calcium and magnesium. [3] The composition according to [1] or [2] above, containing DFAIII in an amount of 1 to 30% by weight. [4] The composition according to any one of [1] to [3] above, containing molasses in an amount of 20 to 70% by weight and betaine in an amount of 1 to 10% by weight. [5] A liquid composition according to any of [1] to [4] above. [6] A feed composition, the composition described in any of [1] to [5] above. [7] The composition according to any one of [1] to [5] above, which is a pharmaceutical composition. [8] The composition according to any one of [1] to [7] above, wherein the animal is a lactating mammal. [9] The composition according to any one of [1] to [8] above, wherein the animal is a lactating dairy cow.

[10] The composition according to [8] above, wherein the lactating mammal is under cold stress or heat stress.

[11] The composition according to any one of [1] to

[10] above, which is for reducing oxidative stress in an animal by administration or dosing over a period of 2 to 7 days.

[12] A method for reducing oxidative stress in an animal, comprising administering or dosing the animal with the composition according to any one of [1] to

[11] above.

[13] The method according to

[12] above, wherein the animal is a non-human mammal.

[14] The method according to

[12] or

[13] above, wherein the animal is a lactating non-human mammal.

[15] The method according to any one of

[12] to

[14] above, wherein the animal is a lactating dairy cow.

[16] The method according to

[14] or

[15] above, wherein the lactating non-human mammal is under cold stress or heat stress. [[ID=二十一]]

[17] The method according to any one of

[12] to

[16] above, wherein the composition is administered or dosed over a period of 2 to 7 days. [[ID=二十二]] [[ID=二十三]]

Advantages of the Invention

[0019] [[ID=二十七]] [[ID=二十八]]According to the present invention, oxidative stress in an animal can be reduced at an early stage. [[ID=二十九]] [[ID=三十]]

Brief Description of the Drawings

[0020] [[ID=三十四]] [[ID=三十五]] [Figure 1] [[ID=三十六]]Figure 1 is a graph comparing the d-ROMs (oxidative stress values) of lactating cows and dry cows. p < 0.01. [[ID=三十七]] [[ID=三十八]] [Figure 2] [[ID=三十九]]Figure 2 is a graph comparing the BAP (antioxidative capacity values) of lactating cows and dry cows. Mean value ± standard error (SE) of BAP. p < 0.1. [[ID=四十]] [[ID=四十一]] [Figure 3]Figure 3 is a graph comparing the OSI (relative oxidative stress index) of lactating cows and dry cows. p<0.05. [Figure 4] Figure 4 is a graph comparing the percentage increase or decrease in d-ROMs (oxidative stress levels) before and after feeding in lactating cows that were given the feed composition according to the present invention for three days, and in lactating cows that were not given the composition, showing the results of a test conducted in February during the coldest period of the year. Vertical axis: Mean ± standard error (SE) of the percentage increase or decrease in d-ROMs before and after feeding. p<0.05. [Figure 5] Figure 5 is a graph comparing the percentage increase or decrease in BAP (antioxidant capacity) before and after feeding in lactating cows that were given the feed composition according to the present invention for three days, and in lactating cows that were not given the composition, showing the results of a test conducted in February during the coldest period of the year. Vertical axis: Mean ± standard error (SE) of the percentage increase or decrease in BAP before and after feeding. [Figure 6] Figure 6 is a graph comparing the percentage increase or decrease in OSI (relative oxidative stress) before and after feeding in lactating cows that were given the feed composition according to the present invention for three days, and in lactating cows that were not given the composition, showing the results of a test conducted in February during the coldest period of the year. Vertical axis: Mean ± standard error (SE) of the percentage increase or decrease in OSI before and after feeding. p<0.05. [Figure 7] Figure 7 is a graph comparing d-ROMs (oxidative stress levels) on day 1 (before feeding began) and day 4 (the day after feeding ended) in lactating cows fed the feed composition according to the present invention for three days, showing the results of a test conducted in August during the hot season. Vertical axis: Mean value ± standard error (SE) of d-ROMs. [Figure 8] Figure 8 is a graph comparing the BAP (antioxidant capacity) values ​​on day 1 (before the start of feeding) and day 4 (the day after the end of feeding) in lactating cows fed the feed composition according to the present invention for three days. The results are from a test conducted in August during the hot season. Vertical axis: Mean value ± standard error (SE) of BAP. [Figure 9]Figure 9 is a graph comparing the OSI (relative oxidative stress) on day 1 (before the start of feeding) and day 4 (the day after the end of feeding) in lactating cows fed the feed composition according to the present invention for three days, showing the results of a test conducted in August during the hot season. Vertical axis: mean value ± standard error (SE) of OSI. p<0.01. [Figure 10] Figure 10 is a graph comparing the percentage change in d-ROMs (oxidative stress values) before and after feeding between lactating cows in Example 3 under cold stress and lactating cows in Example 4 under heat stress. Vertical axis: Mean ± standard error (SE) of the percentage change in d-ROMs before and after feeding. p<0.01. [Figure 11] Figure 11 is a graph comparing the percentage increase or decrease in BAP (antioxidant capacity) before and after feeding between lactating cows in Example 3 under cold stress and lactating cows in Example 4 under heat stress. Vertical axis: Mean ± standard error (SE) of the percentage increase or decrease in BAP before and after feeding. [Figure 12] Figure 12 is a graph comparing the percentage change in OSI (relative oxidative stress) before and after feeding between lactating cows in Example 3 (treated under cold stress) and lactating cows in Example 4 (treated under heat stress). Vertical axis: Mean ± standard error (SE) of the percentage change in OSI before and after feeding. p<0.05. [Modes for carrying out the invention]

[0021] The present invention will be described in detail below.

[0022] This invention relates to a technique for effectively reducing oxidative stress in animals by using a combination of difructose anhydride III, molasses, and betaine. This invention particularly relates to a technique for effectively reducing elevated oxidative stress in lactating mammals under high or low temperature conditions. This invention also relates to the use of compositions containing difructose anhydride III, molasses, and betaine for reducing oxidative stress in animals. This invention provides compositions, such as feed compositions and pharmaceutical compositions, containing difructose anhydride III, molasses, and betaine. This invention also provides a method for reducing oxidative stress in animals, particularly lactating mammals under cold or heat stress, using difructose anhydride III, molasses, and betaine.

[0023] Difructose anhydride (DFA) is a general term for reduced disaccharide compounds in which the reducing ends of two fructose molecules are bonded to the hydroxyl groups other than the reducing end of the other fructose molecule. Difructose anhydride III (DFAIII), a type of difructose anhydride, is a disaccharide in which two fructose molecules are linked at the 1,2 and 2,3' ends, and is also written as di-D-fructofuranose 1,2:2,3' dianhydride. DFAIII is known to be found in caramel and honey, but industrially it is produced by enzymatically treating inulin derived from chicory, etc., with fructotransferase (EC 2.4.1.93) derived from Arthrobacter sp. H65-7 strain. However, the DFAIII usable in this invention is not limited to that obtained by a specific manufacturing method.

[0024] In this invention, by using DFAIII in combination with molasses and betaine, oxidative stress in animals can be reduced in a short period of time.

[0025] Molasses, a by-product of sugar production, typically contains 50-60% by weight of total sugars, including sucrose, as well as a variety of other nutrients. Molasses is highly palatable and can increase the appetite and feed intake of livestock, making it a popular feed ingredient. Molasses is broken down by microorganisms to produce butyric acid, which improves the digestive tract barrier function and contributes to health promotion (Non-Patent Literature 2). On the other hand, molasses is known to have antioxidant properties and is attracting attention as a functional food ingredient. Molasses contains brown substances produced by the Maillard reaction between nitrogen-containing components such as amino acids, peptides, and proteins and reducing sugars such as glucose or fructose, and among these brown substances, melanoidins have strong antioxidant properties. In this invention, "molasses" refers to a dark brown viscous liquid produced as a by-product (sugar production by-product) in the process of producing sugar from beets, sugarcane, etc. (hereinafter also referred to as "molasses as a sugar production by-product"), or sugar containing it or processed therefrom, and includes waste molasses and refined molasses. Molasses, a by-product of sugar production, that has been mixed with other sugar-containing materials, or molasses that has been processed by purification, dilution, drying, etc., is also included in the definition of "molasses" in this invention, as long as it contains molasses as its main component. In this invention, "containing molasses as its main component" means that it contains molasses as its main component by weight of 50% or more of the total weight. For example, a sugar solution (containing fructooligosaccharides, glucose, etc.) in a DFAIII-containing solution produced by treating inulin with fructotransferase, mixed with molasses as a by-product of sugar production, is also considered "molasses" in this invention. In this invention, w / w% is used interchangeably with w / w% [weight / weight%].

[0026] Betaine is an amino acid derivative in which three methyl groups are added to glycine, and is also called trimethylglycine. Betaine is known to have antioxidant properties.

[0027] The composition according to the present invention (for example, a feed composition or a pharmaceutical composition) contains DFAIII, molasses, and betaine. The composition according to the present invention may be an oxidative stress improving agent. The composition according to the present invention is preferably an orally ingestible composition.

[0028] The composition according to the present invention preferably contains DFAIII in an amount of 1.0% by weight or more, more preferably 3.0% by weight or more, even more preferably 5.0% by weight or more, with an upper limit of 30.0% by weight or less, more preferably 25.0% by weight or less, even more preferably 20.0% by weight or less, and particularly preferably 18.0% by weight or less. In one embodiment, the composition according to the present invention contains DFAIII in an amount of 1 to 30% by weight, 1 to 25% by weight, 1 to 10% by weight, or 5 to 20% by weight, based on the total weight of the composition (wet weight when raw materials are mixed). In one embodiment, the composition according to the present invention may include a DFAIII-containing liquid produced by treating inulin with fructotransferase.

[0029] The composition according to the present invention preferably contains molasses and betaine in a total amount of 1.0% by weight or more, more preferably 30% by weight or more, even more preferably 35% by weight or 45% by weight or more, and as an upper limit, preferably 80% by weight or less, more preferably 75% by weight or less or 70% by weight or less, and even more preferably 60% by weight or less. In one embodiment, the composition according to the present invention contains molasses and betaine in amounts of 1 to 80% by weight, 1 to 70% by weight, 35 to 75% by weight, or 45 to 60% by weight (total amount of molasses and betaine) based on the total weight of the composition (wet weight when raw materials are mixed).

[0030] In one embodiment, the composition according to the present invention preferably contains molasses in an amount of 20% by weight or more, more preferably 30% by weight or more, even more preferably 35% by weight or more, particularly preferably 40% by weight or more, and as an upper limit, preferably 70% by weight or less, more preferably 60% by weight or less, and even more preferably 50% by weight or less. In one embodiment, the composition according to the present invention contains molasses in an amount of 20-70% by weight, 30-70% by weight, 20-60% by weight, 20-50% by weight, or 30-50% by weight, based on the total weight of the composition (wet weight when raw materials are mixed).

[0031] In one embodiment, the composition according to the present invention preferably contains betaine in an amount of 1.0% by weight or more, more preferably 2.0% by weight or more, even more preferably 3.0% by weight or more, and as an upper limit, preferably 10.0% by weight or less, more preferably 8.0% by weight or less, and even more preferably 7.0% by weight or less. In one embodiment, the composition according to the present invention contains betaine in an amount of 1 to 10% by weight, 1 to 7% by weight, or 2 to 5% by weight, based on the total weight of the composition (wet weight when raw materials are mixed).

[0032] In one embodiment, the composition according to the present invention contains molasses in an amount of 20 to 70% by weight (preferably 20 to 60% by weight) and betaine in an amount of 1 to 10% by weight (preferably 1 to 5% by weight) based on the total weight (wet weight when the raw materials are mixed) of the composition.

[0033] The composition according to the present invention may further contain added minerals and / or vitamins, etc. Examples of minerals include, but are not limited to, calcium, magnesium, iron, zinc, etc. In one embodiment, the composition according to the present invention further contains, in addition to difructose anhydride III, molasses, and betaine, at least one, preferably both, selected from the group consisting of calcium and magnesium. In one embodiment, the composition according to the present invention preferably further contains calcium in an amount of 0.01% by weight or more based on the total weight of the composition (wet weight when the raw materials are mixed), for example, calcium may be further contained in amounts of 0.01 to 10%, 0.1 to 10%, 0.01 to 5%, or 0.1 to 1% by weight. In one embodiment, the composition according to the present invention preferably further contains magnesium in an amount of 0.01% by weight or more based on the total weight of the composition (wet weight when the raw materials are mixed), for example, magnesium may be further contained in amounts of 0.01 to 10%, 0.1 to 10%, 0.01 to 5%, or 0.1 to 5% by weight. In one embodiment, the composition according to the present invention may further contain 0.01 to 10% by weight (e.g., 0.1 to 10% by weight) of calcium and / or 0.01 to 10% by weight (e.g., 0.1 to 10% by weight) of magnesium, based on the total weight (wet weight when the raw materials are mixed) of the composition.

[0034] The composition according to the present invention may contain alcohol. Examples of alcohol include, but are not limited to, ethanol (fermented ethanol, anhydrous ethanol, or aqueous ethanol), shochu, beer, sake, fruit wine (wine, etc.), and distilled spirits. The composition according to the present invention may contain an aqueous medium such as water. In one embodiment, the composition according to the present invention may contain alcohol and water. In one embodiment, the composition according to the present invention contains alcohol in an amount of 3 to 20% by weight, for example, 7 to 15% by weight, based on the total weight of the composition (wet weight when the raw materials are mixed), but is not limited thereto. The composition according to the present invention may also contain an aqueous medium such as water. In one embodiment, the composition according to the present invention contains an aqueous medium (for example, water) in an amount of 10 to 50% by weight, for example, 20 to 40% by weight, based on the total weight of the composition (wet weight when the raw materials are mixed), but is not limited thereto.

[0035] In one embodiment, the composition according to the present invention contains 1 to 30% by weight (preferably 1 to 10% by weight) of DFAIII, 20 to 70% by weight (preferably 20 to 60% by weight) of molasses, and 1 to 10% by weight (preferably 1 to 5% by weight) of betaine, based on the total weight (wet weight when the raw materials are mixed) of the composition. In another embodiment, the composition according to the present invention contains 1 to 25% by weight of DFAIII, 20 to 50% by weight of molasses, and 2 to 5% by weight of betaine, based on the total weight (wet weight when the raw materials are mixed) of the composition. In one embodiment, the composition according to the present invention contains, in addition to DFAIII, molasses, and betaine, calcium in an amount of 0.1 to 10% by weight (preferably 0.1 to 5% by weight), magnesium in an amount of 0.1 to 10% by weight (preferably 0.1 to 5% by weight), alcohol in an amount of 3 to 20% by weight (preferably 7 to 15% by weight), and an aqueous medium such as water in an amount of 10 to 40% by weight (preferably 20 to 30% by weight), based on the total weight (wet weight when the raw materials are mixed) of the composition. In one embodiment, the composition according to the present invention contains, based on the total weight (wet weight when the raw materials are mixed), DFAIII in amounts of 1 to 30% by weight (preferably 1 to 10% by weight), molasses in amounts of 20 to 70% by weight (preferably 30 to 60% by weight), betaine in amounts of 1 to 10% by weight (preferably 1 to 5% by weight), calcium in amounts of 0.1 to 10% by weight (preferably 0.1 to 5% by weight), magnesium in amounts of 0.1 to 10% by weight (preferably 0.1 to 5% by weight), alcohol in amounts of 3 to 20% by weight (preferably 7 to 15% by weight), and an aqueous medium (e.g., water) in amounts of 10 to 50% by weight (preferably 20 to 40% by weight).

[0036] The composition according to the present invention may be in any form, such as liquid, granular, powder, solid, semi-solid, sheet, or gel. When the composition according to the present invention is a liquid composition, it usually contains an aqueous medium such as water and / or alcohol.

[0037] In one embodiment, the composition according to the present invention may be a feed composition. The feed composition according to the present invention may further contain, or may not contain, any feed raw materials or feed products such as grains, oilseed meals, bran, and / or roughage. The feed composition according to the present invention may further contain any additives (feed additives) that are permissible in feed production, such as inert carriers (solid carriers or liquid carriers), excipients, diluents, binders, disintegrants, emulsifiers, lubricants, stabilizers, surfactants, preservatives, antimicrobial agents, thickeners, chelating agents, solubilizers, suspending agents, coating agents, flavorings, colorings, tasters, flavoring and odor-correcting agents, buffering agents, pH adjusters, defoaming agents, foaming agents, etc. The feed composition according to the present invention may also further contain other physiologically active substances and / or other substances such as antibiotics.

[0038] The feed composition according to the present invention may be in any form, but may be in liquid form (liquid composition). The liquid feed composition is suitably used as a liquid mixed feed for feeding to non-human animals (e.g., livestock). The liquid mixed feed can be easily mixed with or added to feeds such as compound feed or silage, and can be easily consumed by non-human mammals with a good appetite (e.g., livestock such as dairy cows) by allowing them to eat it freely. On the other hand, for non-human mammals with poor appetite (e.g., livestock such as dairy cows), the liquid mixed feed can be put in a bottle or the like and given or administered directly into the mouth to ensure that the feed is consumed. The feed composition according to the present invention may also be a nutritional supplement. The feed composition according to the present invention may be added to other feeds such as compound feed. The amount of the feed composition according to the present invention added to other feeds is not particularly limited and can be appropriately selected depending on the feeding method and the type of animal. For example, the feed composition according to the present invention may be added in amounts of 0.5 to 90% by weight, 5.0 to 80% by weight, 10 to 60% by weight, or 15 to 50% by weight (wet weight) relative to the total weight (wet weight) of the feed composition according to the present invention and other feeds. The feed composition according to the present invention can be prepared by mixing the raw materials by conventional methods and, if necessary, molding and / or packaging.

[0039] In one embodiment, the composition according to the present invention may be a pharmaceutical composition. The pharmaceutical composition according to the present invention may be for human use (human pharmaceutical) or for non-human animal use (veterinary pharmaceutical). The composition according to the present invention may further contain any pharmaceutically permissible additives (pharmaceutical additives), such as inert carriers (solid or liquid carriers), excipients, diluents, binders, disintegrants, emulsifiers, lubricants, stabilizers, surfactants, preservatives, antimicrobial agents, thickeners, chelating agents, solubilizers, suspending agents, coating agents, fragrances, colorants, flavorings, flavoring and deodorizing agents, buffers, pH adjusters, defoaming agents, foaming agents, etc. The pharmaceutical composition according to the present invention may further contain other physiologically active substances and / or other substances such as antibiotics. The pharmaceutical composition according to the present invention may be formulated into any dosage form such as solid formulations such as tablets, granules, powders, pills, capsules, pellets, liquid formulations such as liquids, suspensions, syrups, pastes, gels, aerosols, etc. The pharmaceutical composition according to the present invention can be manufactured in any dosage form by mixing raw materials using conventional methods.

[0040] A composition according to the present invention (for example, a feed composition or a pharmaceutical composition) containing DFAIII, molasses, and betaine can effectively reduce oxidative stress in animals. In particular, the composition according to the present invention can effectively reduce elevated oxidative stress in lactating animals. By feeding or administering the composition according to the present invention to animals, oxidative stress in animals can be reduced at an early stage. In a more preferred embodiment, the composition according to the present invention can effectively and quickly reduce oxidative stress in animals under cold stress or heat stress, in particular lactating animals (mammals) under cold stress or heat stress.

[0041] In the present invention, the animal (target) for which oxidative stress is reduced may be a human or a non-human animal. In one embodiment, the target for oxidative stress reduction in the present invention is preferably a mammal, and may be, for example, a non-human mammal. The target may be any non-human mammal, including, for example, livestock such as cattle, horses, camels, pigs, sheep, and goats; pets such as dogs and cats; laboratory animals such as monkeys, mice, rats, and guinea pigs; animals kept in zoos such as lions, giraffes, and hippos; protected wild animals; and racing animals such as racehorses. The target may also be primates such as humans, chimpanzees, and gorillas; rodents such as mice, rats, guinea pigs, and hamsters; ungulates such as cattle, goats, sheep, deer, giraffes, pigs, and wild boars; and carnivores such as dogs, cats, bears, ferrets, and otters. In one embodiment, the target in the present invention may be an even-toed or odd-toed ungulate belonging to the ungulates, and may be a ruminant belonging to the even-toed ungulates. Examples of ruminants include, but are not limited to, cattle, sheep, and goats. In the present invention, cattle, and especially dairy cows, are particularly preferred as targets for reducing oxidative stress.

[0042] Furthermore, the target of oxidative stress reduction in this invention is preferably lactating mammals, for example, lactating non-human mammals (e.g., livestock). For mammals in general, the lactating period refers to the period after calving when female individuals produce and secrete milk. Particularly preferred targets are lactating ruminants, for example, lactating dairy cows. In one embodiment, the target of this invention is a lactating high-yielding cow. In this invention, "dairy cow" refers to a cow raised as livestock for milk production. In this invention, a high-yielding cow refers to a dairy cow whose milk production is higher than average. In dairy farming, dairy cows that have become pregnant and given birth through insemination (artificial insemination) are milked daily for approximately 280 to 300 days after calving. For dairy cows, this milking period is called the "lactation period," and dairy cows in the lactation period are called lactating cows. The period from 50 to 110 days after calving is the time when dairy cows produce the most milk (peak lactation period), after which milk production gradually decreases. Milking is usually stopped around 280 to 300 days after calving, and the cows are allowed to rest for a predetermined period (typically 60 to 90 days) in preparation for the next calving (dry period). Dairy cows in the dry period are called dry cows. Oxidative stress is particularly heightened in lactating mammals (e.g., dairy cows). In a preferred embodiment, the subject of the present invention is a lactating animal (mammal) under cold stress or heat stress, for example, a lactating cow under cold stress or heat stress. The present invention can provide a particularly effective reduction effect on the heightened oxidative stress in lactating mammals in such a state of oxidative stress.

[0043] In this invention, "heat stress" refers to the physiological stress that animals experience in high-temperature environments. "Animals under heat stress" refers to animals that are experiencing heat stress. In relation to this invention, "high temperature" refers to a temperature that exceeds the optimal temperature range for animals receiving the composition according to this invention. This temperature varies depending on the type of animal and its growth stage, but for example, it is a temperature above 24°C for dairy cows and a temperature above 20°C for lactating dairy cows. In high-temperature environments, heat stress typically increases as the air temperature rises, and for example, it becomes particularly severe in dairy cows at temperatures above the critical high temperature of 29°C, and in lactating dairy cows at temperatures above 26°C. Alternatively, for example, if the temperature and humidity index (THI) of the environment in which an animal is placed is 65 or higher, it can be said that the animal is under at least mild heat stress; if it is 72 or higher, it is under at least severe heat stress; and if it is 82 or higher, it is under very severe heat stress.

[0044] In this invention, "cold stress" refers to the physiological stress that animals experience in low-temperature environments. "Animals under cold stress" refers to animals that are experiencing cold stress. In relation to this invention, "low temperature" refers to a temperature below the optimal temperature range for animals receiving the composition according to this invention. This varies depending on the animal species and growth stage, but for example, it is a temperature below 4°C for dairy cows and a temperature below 10°C for lactating dairy cows. In low-temperature environments, cold stress typically increases as the temperature decreases. For example, in dairy cows, it becomes particularly severe at temperatures below -8°C for healthy lactating cows, and below 7°C for sick or malnourished cows with reduced cold tolerance.

[0045] Oxidative stress can be reduced by feeding or administering the composition according to the present invention to the above-mentioned animals that are targeted for reduction of oxidative stress. In the present invention, the composition according to the present invention (for example, a feed composition) may be given to the subject by methods such as free feeding (ad libitum intake) or force-feeding. Alternatively, the composition according to the present invention (for example, a pharmaceutical composition) may be administered to the subject by methods such as oral administration or enteral administration (administration through a nasogastric or oral tube, or gastrostomy). The present invention also provides a method for reducing oxidative stress in animals, which includes feeding or administering the composition according to the present invention to the above-mentioned animals that are targeted for reduction of oxidative stress. The present invention also provides a method for reducing oxidative stress in animals to mitigate the effects of cold stress or heat stress, which includes feeding or administering the composition according to the present invention to the above-mentioned animals that are targeted for reduction of oxidative stress.

[0046] The composition according to the present invention may be administered at any time, frequency, and number of times, but it is preferable to administer it multiple times, and it is preferable to administer it continuously (for example, daily or every other day, once or more times a day, or by free feeding), and it is more preferable to administer it daily.

[0047] The compositions according to the present invention can effectively reduce oxidative stress in animals, even when administered for a short period or in a small total number of doses. In one embodiment, the compositions according to the present invention can reduce oxidative stress by administering them to a subject once or more times a day for two or more days, preferably three or more days, for example, two to seven days or three to seven days. In one embodiment, the compositions according to the present invention can reduce oxidative stress in animals by administering them to a subject once or more times a day and / or in total three or more doses over two or more days, preferably three or more days, for example, two to seven days, three to seven days, two to five days, three to five days, two to four days or three to four days.

[0048] The oxidative stress reduction effect of the composition according to the present invention in animals can be evaluated based on changes in oxidative stress indicators in animals that have been fed or administered the composition according to the present invention. Any oxidative stress indicator can be used to evaluate the oxidative stress reduction effect in the present invention, and it is preferable to use the concentration of oxidative stress markers in the blood. Blood samples are preferred as specimens for testing oxidative stress indicators, and typically serum or plasma samples are used. For evaluating the oxidative stress reduction effect in the present invention, it is preferable to use at least one selected from the group consisting of oxidative stress values ​​d-ROMs (Reactive Oxygen Metabolites-derived compounds), antioxidant capacity values ​​BAP (Biological Antioxidant Potential), and relative oxidative stress index (OSI), and it is more preferable to use at least the relative oxidative stress index (OSI), which indicates the balance ratio between oxidation and antioxidant activity. The relative oxidative stress index (OSI) is calculated based on the oxidative stress values ​​d-ROMs and antioxidant capacity values ​​BAP. d-ROMs is a quantitative value measured by the blood concentration of peroxides, including hydroperoxides, which are metabolites produced during the oxidation of endogenous substances by free radicals such as reactive oxygen species. It represents the overall degree of oxidation in the blood (measurement unit: U.CARR = 0.08 mg H2O2 / dL). The higher the d-ROMs, the higher the degree of oxidation. BAP is a value measured by the reduction level from trivalent iron ions to divalent iron ions due to the reducing action of antioxidants in a blood sample (not limited to, but including albumin, bilirubin, reduced glutathione, uric acid, vitamins C and E, polyphenols, etc.). It represents the overall antioxidant capacity in the blood (measurement unit: μmol / L). The higher the BAP, the higher the antioxidant capacity. d-ROMs and BAP can be measured by conventional methods, for example, using a commercially available free radical analyzer. In this invention, the relative oxidative stress (OSI) can be calculated according to the following formula: OSI = d-ROMs / BAP × 8.85. A higher relative oxidative stress index (OSI) indicates stronger oxidative stress (higher), while a lower OSI indicates milder oxidative stress (lower).For example, it is known that in dairy cows that develop ketosis after calving, d-ROMs increase compared to before calving, and are higher than in cows that do not develop ketosis after calving. It is also known that BAP decreases in dairy cows on the day after calving compared to before calving. In the present invention, oxidative stress can be evaluated by comparing at least one, two, or all of the group consisting of d-ROMs, BAP, and OSI in animals that are fed or administered the composition according to the present invention with animals that are not fed or administered the composition according to the present invention (control). If OSI decreases in animals that are fed or administered the composition according to the present invention compared with a control that is not fed or administered the composition according to the present invention, it can be determined that oxidative stress has been reduced in those animals. Preferably, animals that are fed or administered the composition according to the present invention show a decrease in d-ROMs, maintenance or increase in BAP, and a decrease in OSI compared with a control that is not fed or administered the composition according to the present invention, indicating that oxidative stress has been reduced due to an improvement in the balance of oxidation and antioxidant capacity in the animals.

[0049] The reduction of oxidative stress by the composition according to the present invention can lead to improvement (reduction or elimination of the degree or type of symptoms, or prevention) of various symptoms of oxidative stress in animals (for example, decreased milk yield during lactation). [Examples]

[0050] The present invention will be specifically described below using examples, but the technical scope of the present invention is not limited to these examples.

[0051] [Example 1] Comparison of oxidative stress levels between lactating cows and dry cows Blood samples were taken from the top six high-yielding Holstein dairy cows (lactation group) and the six dry cows (dry group) at the Kiyokawa Farm of Tokachi Railway Co., Ltd. (Obihiro City, Hokkaido).

[0052] The d-ROMs (oxidative stress levels), BAP (antioxidant capacity), and OSI (relative oxidative stress level) of the obtained blood-derived serum samples were determined. d-ROMs, BAP, and OSI are indicators of oxidative stress.

[0053] Specifically, d-ROMs and BAP in serum samples were measured using a free radical analyzer (FREE Carrio Duo, Wismar), an oxidative stress measurement test kit (d-ROMs test, Wismar), and an antioxidant capacity measurement test kit (BAP test, Wismar). OSI was calculated according to the following formula: OSI = d-ROMs / BAP × 8.85. For statistical analysis, the Wilcoxon two-sample test was used for d-ROMs and OSI, and Welch's test was used for BAP.

[0054] The results are shown in Figures 1-3. Both the average values ​​of d-ROMs (Figure 1) and OSI (Figure 3) were statistically significantly higher in the lactating group compared to the dry group, and the average value of BAP (Figure 2) tended to be higher in the lactating group compared to the dry group. This indicates that lactating cows are subjected to a higher level of stress than dry cows.

[0055] [Example 2] Preparation of liquid feed composition Liquid molasses feed was prepared by mixing molasses and alcohol (fermented ethanol) in a weight ratio of 4:1. In addition, a powder mixture was prepared by blending difructose anhydride III (DFAIII), betaine, calcium, and magnesium chloride in a weight ratio of 55:24:1:20. A raw material solution was prepared by mixing this powder mixture with water as a solvent in a weight ratio of 1:1.

[0056] Next, a liquid feed composition was prepared by mixing the prepared liquid molasses feed with the raw material solution in a weight ratio of 6:4. The content of DFAIII, betaine, molasses, alcohol, calcium, and magnesium in this liquid feed composition was 9.81% by weight, 4.28% by weight, 42.8% by weight, 10.7% by weight, 0.18% by weight, and 3.21% by weight, respectively. This liquid feed composition was used as the feed composition according to the present invention in the following examples.

[0057] [Example 3] Administration (feeding) test of liquid feed composition to multiparous cows (during the coldest period) At the Kiyokawa Farm of Tokachi Railway Co., Ltd., the top 12 high-yielding Holstein dairy cows were divided into two groups: one group fed the feed composition according to the present invention (feeding group; 6 cows (odd-numbered ranking)) and the other group not fed the feed composition according to the present invention (control group; 6 cows (even-numbered ranking)). This administration test was conducted in February (minimum temperature during the test period: -14.2℃). The temperature inside the barn in February was approximately 2 to 15℃ higher than the outside temperature (air temperature).

[0058] Milking of dairy cows in both sections was performed using a milking robot (Astronaut A5; manufactured by Relly). The dairy cows in both sections were allowed to freely feed a PMR feed (Partial Mixed Feed) consisting of corn silage, grass silage, compound feed (Nikilon and Easter 18, manufactured by Nippon Beet Sugar Co., Ltd.), beet pulp mixed feed (Base Pellets, manufactured by Nippon Beet Sugar Co., Ltd.), molasses burger (manufactured by Nippon Beet Sugar Co., Ltd.), and probiotic (bobactin, manufactured by Miyarisan Pharmaceutical Co., Ltd.) in the amounts shown in Table 1. In addition, the milking robot-specific compound feed (MR Pellets, manufactured by Nippon Beet Sugar Co., Ltd.) shown in Table 1 was fed to the cows inside the milking robot. The composition of the feeds given is shown in Table 2.

[0059] [Table 1]

[0060] [Table 2]

[0061] Furthermore, the dairy cows in the feeding group were directly given 600 ml of the feed composition according to the present invention, prepared in Example 2, once a day for three days, in a bottle.

[0062] Blood samples were collected from dairy cows in the feeding group before the first day of feeding with the feed composition according to the present invention ("before feeding") and the day after the third day of feeding ("after feeding"). Blood samples were also collected from dairy cows in the control group at the same timing as the feeding group. Oxidative stress levels (d-ROMs), antioxidant capacity (BAP), and relative oxidative stress levels (OSI) were determined from the obtained blood-derived serum samples using the same method as in Example 1, and the increase / decrease ratio before and after feeding (= after feeding / before feeding) was calculated. Student's test was used for statistical analysis of all indicators. The results are shown in Figures 4-6.

[0063] As shown in Figure 4, d-ROMs, which indicate oxidative stress levels, decreased in the feeding group after feeding with the feed composition according to the present invention, and this decrease in the feeding group was statistically significant compared to the control group. This indicates that the feed composition according to the present invention reduced oxidative stress levels during lactation under low-temperature conditions.

[0064] Furthermore, as shown in Figure 5, BAP, which indicates antioxidant capacity, increased after feeding in the feeding group. The feed composition according to the present invention showed a tendency to increase antioxidant capacity during lactation under low-temperature conditions.

[0065] As shown in Figure 6, the OSI, which indicates the relative oxidative stress level, decreased in the feeding group after feeding with the feed composition according to the present invention, and this decrease in the feeding group was statistically significant compared to the control group. This indicates that the feed composition according to the present invention reduced the relative oxidative stress level during lactation under low-temperature conditions.

[0066] OSI is an index that shows the balance between d-ROMs (oxidative) and BAP (antioxidant), and represents the overall oxidative stress level. These results demonstrate that the feed composition according to the present invention can reduce oxidative stress levels during lactation, which are further enhanced under cold stress caused by low-temperature environments. This means that the effects of cold stress are significantly mitigated. Furthermore, it was shown that the feed composition according to the present invention can produce an early oxidative stress reduction effect with a short feeding period of three days.

[0067] [Example 4] Administration (feeding) test of liquid feed composition to multiparous cows (during hot weather) Twelve high-yielding Holstein dairy cows, raised at Tokachi Railway Co., Ltd.'s Kiyokawa Farm, were divided into two groups: one receiving the feed composition according to the present invention (feeding group; 6 cows (odd-numbered ranking)) and the other not receiving the feed composition according to the present invention (control group; 6 cows (even-numbered ranking)). This administration trial was conducted in August (maximum temperature during the trial period: 32.0°C, THI at 10:00: 81.7 (maximum value of the day)). The temperature inside the barn in August was approximately ±0~2°C of the outside temperature (air temperature).

[0068] Except for using a PMR feed consisting of corn silage, grass silage, compound feed (Nikilon and TM18, manufactured by Nippon Beet Sugar Manufacturing Co., Ltd.), beet pulp mixed feed (Base Pellets, manufactured by Nippon Beet Sugar Manufacturing Co., Ltd.), probiotic (Bobactin, manufactured by Miyarisan Pharmaceutical Co., Ltd.), and calcium carbonate mixed in the amounts shown in Table 3, the experiment was conducted using the same feeding and milking methods as in Example 3. The compound feed for the milking robot shown in Table 3 was also fed in the milking robot in the same manner as in Example 3. The composition of the fed feed is shown in Table 4.

[0069] [Table 3]

[0070] [Table 4]

[0071] Furthermore, the dairy cows in the feeding group were directly given 600 ml of the feed composition according to the present invention, prepared in Example 2, once a day for three days, in a bottle.

[0072] Blood samples were collected from dairy cows in the feeding group before feeding the feed composition according to the present invention on day 1 ("before feeding") and on day 4 ("after feeding"), the day after feeding on day 3. Blood samples were also collected from dairy cows in the control group at the same timing as in the feeding group. Oxidative stress (d-ROMs), antioxidant capacity (BAP), and relative oxidative stress (OSI) were determined from the obtained blood-derived serum samples using the same method as in Example 1, and the mean values ​​before and after feeding, as well as the percentage increase or decrease (%) before and after feeding (= after feeding / before feeding × 100), were calculated. Student's test was used for statistical analysis of all indicators. The measurement results of oxidative stress (d-ROMs), antioxidant capacity (BAP), and relative oxidative stress (OSI) in the feeding group before feeding (day 1) and after feeding (day 4) are shown in Figures 7 to 9.

[0073] As shown in Figure 7, d-ROMs, which indicate oxidative stress levels, were statistically significantly lower after feeding the feed composition according to the present invention compared to before feeding in the feeding group. This demonstrates that the feed composition according to the present invention reduced oxidative stress levels during lactation under high-temperature conditions.

[0074] Furthermore, as shown in Figure 8, BAP, which indicates antioxidant capacity, showed high levels after feeding in the feeding group. This demonstrates that the feed composition according to the present invention increased antioxidant capacity during lactation under high-temperature conditions.

[0075] As shown in Figure 9, the OSI, which indicates the relative oxidative stress level, was statistically significantly lower after feeding compared to before feeding. In the feeding group, the OSI was statistically significantly lower after feeding the feed composition according to the present invention compared to before feeding. This indicates that the feed composition according to the present invention reduced the relative oxidative stress level during lactation under high-temperature conditions.

[0076] These results demonstrate that the feed composition according to the present invention can reduce oxidative stress levels during lactation, which are further enhanced under heat stress caused by high-temperature environments. This means that the effects of heat stress are significantly mitigated. Furthermore, it was shown that the feed composition according to the present invention can produce an oxidative stress reduction effect quickly with a short feeding period of three days.

[0077] Next, the percentage increase or decrease (%) of oxidative stress index measurements in lactating cows under heat stress due to high temperatures in August, obtained above, was compared with the percentage increase or decrease (%) of oxidative stress index measurements in lactating cows under cold stress due to low temperatures in February, obtained in Example 3 (both percentage increases or decreases (%) were calculated using "after / before × 100"). Student's test was used for statistical analysis of all indicators. The results are shown in Figures 10-12.

[0078] As shown in Figure 10, the decrease in d-ROMs, an indicator of oxidative stress, was statistically significantly greater in lactating cows under heat stress compared to lactating cows under cold stress.

[0079] Furthermore, as shown in Figure 11, the increase in BAP, an indicator of antioxidant capacity, was shown to be greater in lactating cows under heat stress compared to lactating cows under cold stress.

[0080] As shown in Figure 12, the decrease in OSI, which indicates the degree of relative oxidative stress, was statistically significantly greater in lactating cows under heat stress compared to lactating cows under cold stress.

[0081] These results demonstrate that the feed composition according to the present invention can mitigate the effects of both cold stress and heat stress, and in particular, can more significantly reduce the oxidative stress level of animals under heat stress (high temperature environment) compared to those under cold stress (low temperature environment). [Industrial applicability]

[0082] The present invention can be advantageously used to mitigate the effects of heat stress or cold stress, promote early recovery from oxidative stress, and accelerate the recovery and improvement of biological functions in mammals, particularly lactating mammals (especially dairy cows), that are in a state of heightened oxidative stress due to an imbalance between oxidation and antioxidant reactions within the body. In particular, the present invention can maintain and improve the health and productivity of livestock, suppress the onset of diseases such as infectious diseases, and bring benefits to livestock farming.

Claims

1. A composition for reducing oxidative stress in dairy cows, containing difructose anhydride III (DFAIII), molasses, betaine, calcium lactate, and magnesium chloride.

2. The composition according to claim 1, comprising 1 to 30% by weight of DFAIII.

3. The composition according to claim 1, comprising 20 to 70% by weight of molasses and 1 to 10% by weight of betaine.

4. The composition according to claim 1, which is liquid.

5. The composition according to claim 1, which is a feed composition.

6. The composition according to claim 1, which is a pharmaceutical composition.

7. The composition according to claim 1, wherein the dairy cow is a lactating dairy cow.

8. The composition according to claim 1 or 7, wherein the dairy cow is under cold stress or heat stress.

9. The composition according to claim 1 for reducing oxidative stress in dairy cows by feeding or administering over a period of 2 to 7 days.

10. A method for reducing oxidative stress in dairy cows, comprising feeding or administering to dairy cows the composition described in any one of claims 1 to 6.

11. The method according to claim 10, wherein the dairy cow is a lactating dairy cow.

12. The method according to claim 10, wherein the dairy cow is under cold stress or heat stress.

13. The method according to claim 10, wherein the composition is administered or given over a period of 2 to 7 days.

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