WO2017099475A1 - Rumen protecting peptide derivative and use thereof - Google Patents

Abstract

The present invention relates to a peptide derivative having two or more amino acids linked to each other, a method for preparing the peptide derivative, and a feed additive and feed composition comprising the peptide derivative. The peptide derivative of the present invention is slowly digested in the rumen, resulting in a high rumen bypass rate, and the peptide derivative can be absorbed by a ruminant after passing through the rumen due to the presence of amide bonds, and thus can be very favorably used as an amino acid source of a ruminant.

Description

Rumen protective peptide derivatives and uses thereof

The present invention relates to a peptide derivative wherein two or more amino acids are linked to each other, a method for producing the peptide derivative, and a feed additive and a feed composition comprising the peptide derivative.

The productivity of ruminants can be improved through the supply of energy and amino acids, and if the amino acids are sufficiently supplied for ruminants, the effects of animal breeding and meat quality improvement can be expected.

Animal by-products are advantageous as feed ingredients that contain high amounts of amino acids.However, unlike ruminants such as chickens and poultry such as chickens, ruminants are fed protein-rich animal by-products to ruminants due to concerns about mad cow disease. Things are limited all over the world. Therefore, there is a growing interest in plant protein sources capable of supplying essential amino acids sufficiently. However, plant protein sources have a short supply of amino acids compared to animal protein supplementation, and feeds such as corn and corn gluten meal, which are most commonly used for ruminant feed, are unable to meet sufficient supply of amino acids. none. Therefore, efforts were made to improve the properties such as weight gain and meat quality by directly feeding amino acids, which are essential nutrients to livestock. However, amino acids are easily degraded by the microorganisms living in the rumen and could not actually see their effects. Therefore, methods for suppressing rumen degradation of these amino acids have been steadily studied.

Known methods of protecting amino acids include amino acid-mineral chelate methods and encapsulation methods using pH-sensitive polymers. Commercially available products include Smartamine M coated with 2-vinylpyridine-co-styrene and stearic acid. ^TM , Mepron M85 coated with ethylcellulose and stearic acid, and METHIO-BY coated with lipid matrix. However, these products have the disadvantage that the use of excipients in the coating process and the price of the coating material increases. In addition, sugar, lysine, gum arabic, cellulose and the like are dissolved in water or a solvent using a dragee to prepare spherical lysine, and then cellulose, gums, sugars, calcium carbonate, The manufacture of protective lysine by spraying talc etc. is proposed. However, the method is difficult to mass production due to the long production time, and in order to feed, the solvent used during the process must be completely removed, and the coating material such as cellulose or gum is used as a nutrient source by the microbes in the rumen. It has a disadvantage of poor rumen protection. In addition, there is a product of the method of refrigeration spray by mixing amino acids with protective substances such as ethyl cellulose, hardened fat, wax, etc., but in this case, the site and equipment costs are high, and the cold air temperature used is below 60 degrees C. This increases energy costs and increases product production costs. In addition, since the product is produced by the instant cooling method, there is a possibility of forming macropores, bubbles, and cracks in the product, and operating conditions are difficult in producing the product. In addition, since the amino acid arranged on the surface of the prepared rumen protective amino acid does not protect, there is a disadvantage that the degradation proceeds through the amino acid located on the surface is reduced.

Therefore, there is an urgent need for the development of rumen protective amino acids that can be used by ruminants across the rumen while maintaining high stability against microbes inhabiting the rumen and water.

Amino acids have excellent effects such as weight gain and quality improvement of livestock as feed additives. However, in the case of ruminants, unlike other animals, peptides can be decomposed and used by microorganisms present in the rumen. There is difficulty.

The inventors have tried to prepare peptide derivatives that can bypass the rumen and break down in the gut. As a result of the preparation of the peptide derivative through an amide bond using an amide bond other than an amide bond, specifically, an amino acid side chain (R group), rather than a peptide bond, the digestion proceeds slowly in the rumen due to the different chemical structure from the peptide. Through this, the rumen bypass rate could be increased. In addition, it has an amide bond, such as a peptide bond, only the difference in degradation rate was absorbed and digested by ruminants, and could have an amino acid-like effect. Thus, the peptide derivative of the present invention was confirmed that can be usefully used as a component of a feed additive or feed composition of ruminants, and completed the present invention.

One object of the present invention is to provide a peptide derivative or salt thereof in which two or more amino acids are linked to each other.

Another object of the present invention is to provide a method for preparing the peptide derivative or salt thereof.

Still another object of the present invention is to provide a feed additive comprising the peptide derivative or a salt thereof.

Still another object of the present invention is to provide a feed composition comprising the feed additive.

Yet another object of the present invention is to provide a method of raising an animal, comprising feeding the feed composition to the animal.

Still another object of the present invention is to provide a rumen by-pass use of the peptide derivative or salt thereof.

Another object of the present invention is to provide a use of the peptide derivative or salt thereof as a feed additive.

Another object of the present invention is to provide the use of said peptide derivative or salt thereof for use in the preparation of a feed additive or feed composition.

Peptide derivatives of the present invention is a slow digestion in the rumen, high bypass rate (bypass) through this, and can be absorbed by ruminants after passing through the rumen with amide bonds, very useful as amino acid source of ruminants Can be used.

1 shows a dipeptide derivative using an omega amine of lysine.

2 shows a dipeptide derivative using an omega amine of arginine.

Figure 3 shows a dipeptide derivative using an omega amine of asparagine

Figure 4 shows a dipeptide derivative using an omega amine of glutamine.

Figure 5 shows a dipeptide derivative using a carboxyl group linked to the beta carbon of aspartate.

Figure 6 shows a dipeptide derivative using a carboxyl group linked to the gamma carbon of glutamate.

One embodiment embodying the present invention is a peptide derivative or salt thereof in which two or more amino acids are linked to each other, wherein the peptide derivative is

(i) at least one amino acid selected from the group consisting of lysine, arginine, asparagine, glutamine, aspartate, and glutamate,

(ii) an amide linkage between an amino acid to which lysine, arginine, asparagine, or glutamine is linked thereto, and an omega amine located in the side chain of the lysine, arginine, asparagine or glutamine (R group) and a carboxyl group of the amino acid linked thereto; Connected to each other,

Aspartate or glutamate is a peptide derivative or salt thereof, wherein the aspartate or glutamate is linked to each other by an amide bond between an amino acid linked thereto and a carboxyl group located on the R group of the aspartate or glutamate group and an amine group of the amino acid linked thereto.

Specifically, the peptide derivative or salt thereof is an oligopeptide derivative or salt thereof in which 2 to 10 amino acids are linked to each other, and the oligopeptide derivative is

(i) at least one amino acid selected from the group consisting of lysine, arginine, asparagine, glutamine, aspartate, and glutamate,

(ii) an amide linkage between an amino acid to which lysine, arginine, asparagine, or glutamine is linked thereto, and an omega amine located in the side chain of the lysine, arginine, asparagine or glutamine (R group) and a carboxyl group of the amino acid linked thereto; Connected to each other,

Aspartate or glutamate are oligopeptide derivatives or salts thereof comprising those linked to each other by an amide bond between an amino acid linked thereto and a carboxyl group located on the R group of the aspartate or glutamate group and an amine group of the amino acid linked thereto.

As used herein, the term "amino acid" refers to all molecules comprising an amino group and a carboxyl group. In the present invention, the amino acid includes both natural amino acids or artificial amino acids, specifically, natural amino acids, but is not limited thereto. Specifically, amino acids include amine groups, carboxyl groups and side chains (R groups) specific for each amino acid.

Examples of the amino acids include glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, aspartate, asparagine, glutamate, glutamine, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan or proline. Include.

Any amino acid linked with lysine, arginine, asparagine, or glutamine may comprise all kinds of amino acids, and any amino acid linked with aspartate or glutamate may include all kinds of amino acids except proline Can be.

The amino acids used in the present invention may be described as abbreviations as follows according to the IUPAC-IUB nomenclature.

Alanine A Arginine R

Asparagine N Aspartate D

Cysteine C Glutamate E

Glutamine Q Glycine G

Histidine H isoleucine I

Leucine L Lysine K

Methionine M-Phenylalanine F

Proline P Serine S

Threonine T Tryptophan W

Tyrosine Y Valine V

As used herein, the term “peptide derivative” refers to a polymer of two or more amino acids, wherein at least two amino acids are linked by an amide bond between an amine group or a carboxyl group located at the R group of one amino acid and a carboxyl group or an amine group of another amino acid. Says a polymer.

As used herein, the term “oligopeptide derivative” refers to a polymer of 2 to 10 amino acids, wherein at least two amino acids are amide bonds between an amine group or a carboxyl group located at the R group of one amino acid and a carboxyl group or an amine group of another amino acid. Refers to a polymer comprising a linked one. The oligopeptide derivatives may be more specifically polymers of two, three, four, five, six, seven, eight, nine, or ten amino acids.

Amide bond is an amine group (-NH ₂ ) and a carboxyl group (-COOH) is bonded as shown in the formula (1).

[Formula 1]

A peptide refers to a polymer of amino acids formed by an amide bond, ie a peptide bond, between the alpha amine of each amino acid constituting it and the carboxyl group linked to the alpha carbon. The peptide derivative or oligopeptide derivative of the present invention is characterized in that it contains at least one amide bond between an amine group or a carboxyl group located at the R group of an amino acid and a carboxyl group or an amine group of another amino acid, which is not a general peptide bond.

In addition, the peptide derivative of the present invention includes both polymers in which amino acids are linked to each other only by the above-described characteristic amide bonds, and polymers in which amino acids are linked to each other by peptide bonds, as well as the above-described characteristic amide bonds.

More specifically, an amide bond between the omega amine located at the R group of lysine, arginine, asparagine or glutamine and the carboxyl group of any other amino acid, or between the carboxyl group located at the R group of aspartate or glutamate and an amine group of any other amino acid It may comprise at least one of the amide bonds.

More specifically, when lysine, arginine, asparagine, or glutamine is linked to any amino acid, it is linked to the alpha carbon of the omega amine located at the R group of the lysine, arginine, asparagine or glutamine and the amino acid linked thereto. Amide bonds between carboxyl groups located at the carboxyl or R groups may be linked to each other.

In addition, when aspartate or glutamate is linked to any amino acid, an amide bond such as a carboxyl group located on the R group of the aspartate or glutamate group and an amine group linked to the alpha carbon of the amino acid linked thereto or an omega amine group located on the R group Can be connected.

The oligopeptide derivative may be a dipeptide derivative in which two amino acids are linked. The dipeptide derivative may be represented by the following Chemical Formula 2 or Chemical Formula 3.

[Formula 2]

X ₁ -A

Wherein X ₁ is lysine, arginine, asparagine or glutamine,

A is any amino acid,

X ₁ -A is _{one in which} the omega amine of X ₁ and the carboxyl group of A are linked by an amide bond;

[Formula 3]

X ₂ -A

Wherein X ₂ is aspartate or glutamate,

A is any amino acid,

X ₂ -A is a carboxyl group located at the R group of X ₂ and an amine group of A connected by an amide bond.

Any of the above amino acids is a group consisting of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, aspartate, asparagine, glutamate, glutamine, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan and proline It may be a type selected from, but is not limited thereto.

In addition, the present invention includes salts of the peptide derivative or oligopeptide derivative. Preferred cations for salt preparations are sodium (Na ⁺ ), potassium (K ⁺ ), lithium (Li ⁺ ), magnesium (Mg ²⁺ ), calcium (Ca ^{2 +} ), barium (Ba ^{2 +} ), strontium (Sr ^{2) +} ) And ammonium (NH ^{4 +} ), but is not limited thereto. Salts can also be prepared from alkali or alkaline earth metals, but are not limited thereto. Salts of the peptide derivatives include all salt forms of suitable forms to be added to the feed, salts of the peptide derivatives can be easily prepared by those skilled in the art.

Another embodiment embodying the present invention is a method for producing the peptide derivative.

More specifically, the preparation method includes forming an amide bond between an amino acid having an amine group in the R group and a carboxylic acid of another amino acid, and / or forming an amide bond between an amino acid having a carboxyl group in the R group and an amine group of another amino acid. Include.

The amino acid having an amine group in the R group may be lysine, arginine, asparagine or glutamine, and the amino acid having a carboxyl group in the R group may be aspartate or glutamate.

The amino acid having an amine group in the R group is protected with amine groups and carboxyl groups other than the amine of the R group, the amino acid having a carboxyl group in the R group is protected with other carboxyl groups and amine groups other than the carboxyl group of the R group The amine group of the amino acid having an amine group in the R group, or the carboxyl group of another amino acid linked with the amino acid having a carboxyl group in the R group may be protected by a protecting group, but is not limited thereto.

In addition, the manufacturing method may include, but is not limited to, forming an amide bond using an amino acid having a protecting group, and then removing the attached protecting group, that is, deprotecting.

As used herein, the term "protecting group" refers to binding of a specific functional group of a compound to prevent the bound functional group from reacting, and "deprotection" refers to removing the attached protecting group to return the functional group to its original state.

In the present invention, the protecting group may be appropriately selected and used by those skilled in the art as long as it is a known protecting group for a specific functional group, specifically an amine group or a carboxyl group, and is not particularly limited to the kind. In addition, the deprotection procedure can vary depending on the protecting group used, and those skilled in the art can use any known method as appropriate, provided that the protecting group is removed and the functional group that was protected can be returned to its original state.

In particular, the protecting group for the amine in the present invention are carbonyl a benzyloxy group (carbobenzyloxy group), p - methoxybenzyl carbonyl group (p -methoxybenzyl carbonyl group), tert - butyl-oxy-carbonyl group (tert -butyloxycarbonyl group), 9- Floresta carbonyl methyloxy carbonyl group (9-fluorenylmethyloxycarbonyl group), acetyl group (acetyl group), a benzoyl group (benzoyl group), benzyl group (benzyl group), a carbamate group (carbamate group), p - methoxy Siebel chewy (p -methoxybenzyl group), 3,4- dimethoxybenzyl groups (3,4-dimethoxybenzyl group), p - methoxy may be a phenyl group (p -methoxyphenyl group), or a tosyl group (tosyl group), protecting groups for carboxyl groups are methyl ester (methyl ester), benzyl ester (benzyl ester), tert - butyl ester (tert -butyl ester), a silyl ester (silyl ester), an ortho ester (orthoester), or may be an oxazoline (oxazoline), that it is limited to no.

Another embodiment embodying the present invention is a feed additive comprising the peptide derivative or salt thereof.

The peptide derivative or salt thereof is as described above.

As used herein, the term "feed additive" means a substance added to a feed composition. The feed additive may be to improve productivity or health of the target animal, but is not limited thereto.

In the present invention, a feed additive including the peptide derivative or a salt thereof is used, and the feed additive may be used in addition to the peptide derivative or salt thereof, such as nucleotides, amino acids, calcium, phosphoric acid, organic acid, etc. It may further include nutrients, but is not limited thereto.

In the present invention, the term "feed composition" refers to the food to be given to the animal. The feed composition refers to a substance that supplies organic or inorganic nutrients necessary for maintaining the life of an animal or producing meat, milk, and the like. The feed composition may include a feed additive, and the feed additive of the present invention may correspond to a feed supplement on a feed management method.

The kind of the feed is not particularly limited, and may be used a feed commonly used in the art. Non-limiting examples of the feed may include plant feeds such as cereals, fruits, food processing by-products, algae, fibres, pharmaceutical by-products, oils, starches, gourds or grain by-products; Animal feeds such as proteins, minerals, fats, oils, minerals, single-cell proteins, zooplankton or foods. These may be used alone or in combination of two or more thereof.

The individual to which the composition for feed of the present invention can be applied is not particularly limited and may be applied in any form. For example, it is applicable without limitation to animals such as cows, sheep, giraffes, camels, deer, goats, etc., and particularly preferably applicable to ruminants having a rumen, a representative example may include cattle, but is not limited thereto. Do not.

According to one embodiment of the present invention, the peptide derivative or salt thereof may be used as a rumen protective peptide derivative because the degree of degradation by the rumen microorganism is low. Therefore, the peptide derivative or salt thereof may be effectively used as a feed additive for ruminants, but is not limited thereto.

As used herein, the term "ruminant" is a special digestive tract found in some animals of mammalian joiners, and is divided into four rooms, called hump, honeycomb, folds, and wrinkles, for the purpose of rubbing. Also known as ruminwiwi, once swallowed foods in the forest again vomiting and swallowing well called swallowing, this rumen is called stomach rubbing is possible. In the rumen, microbial symbiosis has the ability to decompose and energize the cellulose of plants that cannot be digested by ordinary animals.

As used herein, the term "ruminant" refers to an animal having the rumen described above, and includes the animals of the family Camel, Deer, Deer, Giraffe and Bovine. However, the camel family and the baby deer are known to have a rumen consisting of three rooms because the folds and wrinkles are not completely differentiated.

The feed additive may be added to the feed composition to include the peptide derivative or salt thereof in an amount of 0.01 to 90% by weight based on the total weight of the feed, but is not limited thereto.

In addition, the feed additive according to the present invention may be used individually, may be used in combination with a conventionally known feed additive, and may be used sequentially or simultaneously with the conventional feed additive. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

Another aspect of embodying the present invention is a method of breeding an animal, comprising feeding the animal a feed composition.

The feed composition is as described above.

Animal breeding method of the present invention in detail (a) mixing the feed composition for animal feed; And (b) feeding the feed to the animal.

Step (a) according to the present invention is a step of mixing the feed composition comprising the peptide derivative of the present invention or a salt thereof in a general feed for livestock, 0.01 to 20% by weight, preferably 0.1 to 10% by weight in the mixed feed You can pay in percentage.

Step (b) according to the present invention is a step of feeding the animal prepared in step (a) to the animal, the feedable livestock is not particularly limited as described above, in particular may be a ruminant.

When feeding the feed composition according to the present invention to livestock, excellent effects such as weight gain and meat quality improvement of the livestock can be expected.

Another embodiment embodying the present invention is the use of a bypass of the peptide derivative or salt thereof.

The peptide derivative or its salt, and the use of the rumen bypass is as described above.

Another embodiment embodying the present invention is the use of said peptide derivative or salt thereof as a feed additive.

The peptide derivative or its salt, and feed additive use are as described above.

Another embodiment embodying the present invention is the use of said peptide derivative or salt thereof for use in the preparation of a feed additive or feed composition.

The peptide derivatives or salts thereof, feed additives, and feed compositions are the same as described above.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only intended to illustrate the invention and are not to be construed as limiting the scope of the invention by these examples.

Example  One. Lysine  omega Amines  Used Dipeptide  ( dipeptide ) Preparation of Derivatives

To prepare a Lysine-copper complex, lysine hydrochloride and CuCO _{3 ·} Cu (OH) ₂ The aqueous solution was heated to reflux for 2 hours, and then the reaction solution was filtered at the same temperature and the filtrate was cooled to 20 ° C. After a small amount of acetone was added to the filtrate, the product precipitated at 4 ° C. overnight when crystals began to form. The precipitate was filtered off and dried and then crystallized in acetone-water. Meanwhile, in order to protect the amine functional group of the amino acid linked with lysine with tert- Boc (Butyloxycarbonyl), the amino acid was dissolved in THF (tetrahydrofuran) and 2 hours after the addition of tert -butyl dicarbonate and TEA (triethylamine). Stirred at room temperature. The resulting Boc-amino acid (0.1 mol) was dissolved in 20-30 ml of THF with TEA (0.01 mol) and then cooled to -10 ° C, followed by addition of 0.01 mol of ethyl chlorocarbonate for 5 minutes. Stirred. 10 ml of the 0.01 mol lysine-copper composite aqueous solution prepared above was added to the mixed solution, followed by reaction, and then 200 to 300 ml of water was added to precipitate the precipitated product. The obtained precipitate was dissolved in 100 ml of water and then hydrogen sulfide gas was injected and heated with stirring for 3 hours. At this time, the produced copper sulfate was filtered, and the remaining target compound was crystallized twice in hot water. Then, in order to remove the protecting group ( tert- Boc) from the dipeptide derivative using lysine, the derivative was dissolved in methylene chloride (30% (v / v) trifluoroacetic acid). 200 ml), and then reacted for 6 hours to obtain the desired product (FIG. 1).

Example  2. Arginine  omega Amines  Used Dipeptide  ( dipeptide ) Preparation of Derivatives

N ^α -Boc-L- in order to protect the carboxylic acid functionality of the eggs with, N ^α -Boc-L- after the arginine (0.1 mol) was dissolved in methanol, tea from 0 ℃ chloride (thionyl chloride, 0.15 mol) of Inject slowly and stir for 15 minutes. After reacting for 3 hours, the mixed solution at room temperature, the reaction was complete, EA (Ethyl acetate), and then concentrated under reduced pressure and extracted with N ^α -Boc-L- arginine methyl ester (N ^α -Boc-L-arginine 4-methylester) was obtained. On the other hand, the same method as in Example 1 was used to protect the amine functional group of the amino acid linked to the omega amine of arginine with tert- Boc. The Boc-protected amino acid (0.1 mol) produced by the above method and the N ^α -Boc-L-arginine 4-methyl ester (0.1 mol) prepared above were prepared using DMF (N, N-Diisopropylethylamine, 0.15 mol). After dissolving in N, N-Dimethylmethanamid), HBTU (N, N, N ', N'-Tetramethyl-O- (1H-benzotriazol-1-yl) uronium hexafluorophosphate, 0.15 mol) was added and reacted at room temperature for 4 hours. After the reaction was completed, the product was extracted with EA and crystallized. Then, the same reaction as in Example 1 was used to remove the tert- Boc protecting group from the dipeptide derivative, and the target product was obtained after removing the ester protecting group of the arginine using an aqueous NaOH solution (FIG. 2).

Example  3. Asparagine  omega Amines  Used Dipeptide ( dipeptide ) Preparation of Derivatives

N ^α -Boc-L- in order to protect the carboxylic acid functional groups of the asparagine, N ^α -Boc-L- asparagine (0.1 mol) was dissolved in methanol, tea from 0 ℃ chloride (thionyl chloride, 0.15 mol) of Inject slowly and stir for 15 minutes. After reacting the mixed solution for 3 hours at room temperature, the reaction solution was extracted with EA (Ethyl acetate) and concentrated under reduced pressure to obtain N ^α -Boc-L-asparagine 4-methyl ester (N ^α -Boc-L-asparagine). 4-methyl ester) was obtained. On the other hand, the same procedure as in Example 1 was used to protect the amine functionality of the amino acid that is linked to asparagine in tert -Boc. Bom-protected amino acid (0.1 mol) produced by the above method and the above prepared N ^α -Boc-L-asparagine 4-methyl ester (0.1 mol) DMF (N, N-Dimethylmethanamid) containing DIPEA (0.15 mol) HBTU (0.15 mol) was added to the solution dissolved in the reaction and reacted at room temperature for 4 hours, after which the product was extracted with EA and crystallized. Then, the same reaction as in Example 1 was used to remove the tert- Boc protecting group from the dipeptide derivative, and the target product was obtained after removing the ester protecting group of asparagine using an aqueous NaOH solution (FIG. 3).

Example  4. Glutamine Omega Amines  Used Dipeptide ( dipeptide ) Preparation of Derivatives

N ^α -Boc-L- in order to protect the carboxylic acid functionality of Glutamine, N ^α -Boc-L- after glutamine (0.1 mol) was dissolved in methanol and slowly injected with thionyl chloride (thionyl chloride, 0.15 mol) at 0 ℃ Then stirred for 15 minutes. After reacting for 3 hours, the mixed solution at room temperature, EA (Ethyl acetate), and then concentrated under reduced pressure to N ^α -Boc-L- glutamine methyl ester ^{(N α -Boc-L-glutamine} 4-methyl ester) and extracted with Obtained. On the other hand, the same method as in Example 1 was used to protect the amine functional group of the amino acid linked with glutamine with tert- Boc. The Boc- protected amino acid (0.1 mol) and the N ^α -Boc-L- glutamine methyl ester prepared above produced by the above method ^{(N α -Boc-L-glutamine} 4-methylester, 0.1 mol) of DIPEA (0.15 mol HBTU (0.15 mol) was added to a solution dissolved in DMF (N, N-Dimethylmethanamid) containing 4 h) and reacted at room temperature for 4 hours, followed by crystallization by extracting the product with EA. Then, the same reaction as in Example 1 was used to remove the tert-Boc protecting group from the dipeptide derivative, and the desired product was obtained after removing the carboxyl protecting group (methylester) of glutamine using an aqueous NaOH solution (FIG. 4).

Example  5. Aspartate Residue Carboxyl group  Used Dipeptide  of dipeptide derivatives

The amino acid (0.1 mol) linked to the carboxyl group of the aspartate residue was dissolved in methanol, and then slowly injected with thionyl chloride (0.15 mol) at 0 ° C., followed by stirring for 15 minutes. The mixed solution was reacted at room temperature for 3 hours, and when the reaction was completed, the mixture was extracted with EA (Ethyl acetate) and concentrated under reduced pressure to obtain an amino acid having a carboxyl group protected with methyl ester. The compound (0.1 mol) and N-Boc-L-aspartic acid 1-benzyl ester (0.1 mol) were converted into DMF (N, HBTU (0.15 mol) was added to the solution dissolved in N-Dimethylmethanamid) and reacted at room temperature for 3-4 hours. After the reaction, the product was extracted / crystallized with EA, and the same reaction as in Example 1 was used to remove the tert-Boc protecting group from the dipeptide derivative, and the carboxyl protecting group (benzyl ester) of aspartate was removed using an aqueous NaOH solution. The desired product was obtained later (FIG. 5).

Example  6. Glutamate Residue Carboxyl group  Used Dipeptide ( dipeptide ) Preparation of Derivatives

The amino acid (0.1 mol) linked to the carboxyl group of the glutamate residue was dissolved in methanol, and then slowly injected with thionyl chloride (0.15 mol) at 0 ° C., followed by stirring for 15 minutes. After reacting the mixed solution for 3 hours at room temperature, the reaction solution was extracted with EA (Ethyl acetate) and concentrated under reduced pressure to obtain an amino acid in which the carboxyl group was protected with methyl ester. 0.1 mol of the compound and N-Boc-L-glutamic acid 1-benzyl ester (0.1 mol) were converted into DMF (N, HBTU (0.15 mol) was added to the solution dissolved in N-Dimethyl methanamide) and reacted at room temperature for 3-4 hours. After the reaction, the product was extracted / crystallized with EA, the same reaction as in Example 1 was used to remove the tert- Boc protecting group from the dipeptide derivative, and the carboxyl protecting group (benzyl ester) of glutamate was removed using an aqueous NaOH solution. The desired product was obtained (FIG. 6).

Example  7. Dipeptide  ( dipeptide Evaluation of Bypass and Digestion Degradation of Derivatives

7-1. Rumen bypass evaluation

7-1-1. Rumen juice collection

Holstein cows with rumen cannula (Weighing between 630 and 650 kg) 1 dog and 2 dogs per day (7:30 am, 3 pm). And rice straw was fed by breeding.

The rumen fluid was collected at about 10 am on the day of the experiment, and the contents of the rumen were taken out through the cannula, squeezed out of the gastric fluid with gauze, and then bubbled into a thermos bubbled with CO ₂ to block the invasion of oxygen. Used after transporting to the laboratory. It took approximately 1 hour to transport to the lab.

7-1-2. Anaerobic Culture Progress

The rumen solution transported to the laboratory was mixed with a simulated solution of McDougall's buffer (Troelsen and Donna, 1966), which is generally used in rumen experiments after filtration with two layers of gauze, and used as an anaerobic culture solution. The composition of McDougall's buffer simulation solution is as shown in Table 1 below.

Composition of McDougall's Buffer Simulation

buffer (based on 1L)
NaH 2 PO 4 · 2H 2 O	9.3 g
NaHCO 3	9.8 g
NaCl	0.47 g
KCl	0.57 g
MgCl 2	0.256 g
CaCl 2	0.106 g
EZMIX N-ZAMIN	2.5 g
resazulin (0.1%)	1.5 ml

Milk feed ^TM (CJ Cheil Jedang Sugar) was used as the basic feed, and the test feed was prepared by mixing the test material with the basic feed. The test substance was methionine-ε-lysine produced by the amide bond between the omega amine located in the side chain of lysine and the carboxyl group of methionine. Lys-Lys dipeptide and Met-Met dipeptide generated by the binding of the carboxyl group of one amino acid and the amino group of the other amino acid were compared with the control groups 1 and 2 using the test substance. In each experimental group, the culture was carried out in three replicates.

The basic feed and the test substance were mixed at a ratio of 4: 1 (based feed 0.4 g, test substance 0.1 g), 0.5 g of the mixed test feed was added to a 125 ml culture bottle, and 50 ml of the prepared anaerobic culture solution were mixed. Incubation was started by standing in a 40 degreeC incubator sealed.

During the whole process up to the start of the culture including the dilution of the rumen liquid, O ₂ free CO ₂ was injected to maintain the anaerobic state so that the rumen liquid was not exposed to oxygen.

7-1-3. Sampling and Bypass Efficiency Measurements of Test Substances

The culture was finally performed for 24 hours, and the culture sample was sampled two times (0 hour and 24 hours) after the start of the culture. At each sampling, the specimen left in a 40 ° C. incubator was taken out, the lid was opened, the culture was centrifuged (4000 rpm, 10 minutes), and the amount of test substance present in the supernatant was measured.

The rumen bypass rate (%) was calculated based on the LC quantitative analysis of the test material, the value is shown in Table 2 below.

 Rumen bypass rate of methionine-ε-lysine (%)

Rumen bypass rate (%)
	0h					24h
	One	2	3	medium	SD	One	2	3	medium	SD
Control group 1	100.0	100.0	100.0	100.0	0.0	0.0	0.0	0.0	0.0	0.0
(Lys-Lys Dipeptide)
Control 2	100.0	100.0	100.0	100.0	0.0	-	8.07	6.16	7.1	1.4
(Met-Met Dipeptide)
Experimental group 1	100.0	100.0	100.0	100.0	0.0	71.8	69.8	68.1	69.9	1.9
(Methionine-ε-lysine)

The rumen bypass rate (%) was expressed as the relative residual percentage (%) of the 24-hour specimens when the residual amount of test substance at the time of zero hour sampling was converted to 100%. Figure 7 graphically shows the rumen bypass rate (%).

As a result, the control group Lys-Lys dipeptide and Met-Met dipeptide were 0%, and the rumen bypass rate (%) after 24 hours was 0%, which was almost digested by rumen microorganisms. Phosphorus methionine-ε-lysine showed lysine bypass rate (%) of 69.9% at 24 hours compared to 0 hours.

Thus, methionine-ε-lysine is relatively less degraded by the microorganisms in the rumen compared to Lys-Lys dipeptide and Met-Met dipeptide, which is a general peptide bond, it can be determined that the rumen bypass rate (%) is high.

Example 8 Digestion Test of Dipeptide Derivatives

Example 8-1. Small intestine enzyme group

A 40-meter collection of Hanwoo (History: KOR005078680400) slaughtered at the Bucheon Livestock Products Fair of the NACF was purchased. After cutting the small intestine to about 1 m, 5 ml of 20 mM sodium phosphate buffer (pH 7.4) was placed in the cut small intestine. Then, both ends of the small intestine were held, and the small intestine was moved from side to side to allow the enzyme in the small intestine to dissolve in sodium phosphate buffer. Approximately 200 ml of the small intestine enzyme solution was obtained at 40 m of the small intestine, and the supernatant was obtained by centrifugation (14000 rpm, 10 minutes) at 4 ° C. It was used diluted twice in 20 mM sodium phosphate buffer (pH 7.4).

Example 8-2. Evaluation of Small Intestine Enzyme Capacity

To determine whether the enzyme group of the small intestine is secured, the acylease I (sigma A3010) extracted from the pig kidney was used as a positive control, and it was decomposed into methionine using N-acetyl-L-methionine (TCI America A2056). It was confirmed. Experimental conditions are shown in Table 3 below, and the reaction proceeded for 24 hours while standing at 37 ° C.

	Acylate I (1 mg / ml)	Small intestine enzyme group
N-acetyl-L-methionine (8 g / L)	500	500
Acylate I (1 mg / ml)	100
Small intestine enzyme group (two-fold dilution)		100
20 mM sodium phosphate buffer (pH 7.4)	400	400
Final reaction volume (μl)	1000	1000

Perchloric acid (DEA JUNG, purity 60-62%) was used to remove the trimmed protein of the sample after the reaction was completed.The reaction sample was diluted 10-fold with 0.5% perchloric acid and mixed, followed by centrifugation (14000 rpm, 10 The concentrations of N-acetyl-L-methionine and methionine present in the supernatant were measured by LC quantitation. The conversion (%) was obtained by calculating the concentration of N-acetyl-L-methionine (mM) before the reaction and calculating the concentration of methionine after the reaction, in terms of mole%. The molecular weight of N-acetyl-L-methionine is 191.25 g / L, and the molecular weight of methionine is 149.25 g / L.

As a result, it was confirmed that N-acetyl-L-methionine was converted to methionine by 100% in the enzyme reaction using the small intestine enzyme group as in the positive control group Asilase I, and secured through Example 8-1. One group of small intestine enzymes was found to have a suitable activity for the digestion of dipeptides (Table 4).

	Asilase I
	N-acetyl-L-methionine (g / L)	Methionine (g / L)	% Conversion
Before the reaction	3.85	0.00
After the reaction	0.00	3.13	100
	Small intestine
	N-acetyl-L-methionine (g / L)	Methionine (g / L)	% Conversion
Before the reaction	3.85	0.00
After the reaction	0.00	3.14	100

* Conversion rate: Method (methionine) concentration (mM) / Substrate (N-acetyl-L-methionine) concentration (mM) × 100

Example 8-3. Small intestine enzyme reaction

By the small intestine enzyme group to compare and confirm the degree of digestion and digestion of the small intestine of Lys-Lys or Met-Met dipeptide made of the same amino acid as the methionine-ε-lysine dipeptide prepared through the procedures of Examples 1 to 6 above. Digestion digestion experiments were conducted. The concentration of dipeptide used for the evaluation is specified in the LC quantification results (g / L) of Examples 8-4 below. And the final volume of the reaction is 1000 μl, the amount used the substrate, small intestine, buffer is shown in Table 5 below.

	Methionine-ε-Lysine	Lys-lys	Met-met
Substrate (Storage Concentration 4 g / L)	500	500	500
Small intestine enzyme group (two-fold dilution)	100	100	100
20 mM Sodium Phosphate Buffer (pH 7.4)	400	400	400
Final reaction volume (μl)	1000	1000	1000

Example 8-4. Small intestine enzyme reaction result

The enzymatic reaction was carried out by conducting one sampling (24 hours) after the start of the reaction. In the same manner as described in Example 8-2, 5% perchloric acid was used to remove the cutting bag, and the amount of lysine and methionine to be the amount of the remaining substrate and the product at 24 hours was quantified by LC and converted into mM. It was. The conversion (mole%) was quantified in the same manner as described in Example 8-2 (conversion (mole%): product concentration (mM) / substrate concentration (mM) x 100). When Lys-Lys and Met-Met dipeptides are decomposed, they become Met 2 mole and Lys 2 mole. Therefore, after converting to mole concentration, the value of 2 divided by the mM concentration is calculated and the conversion rate (mole%) is calculated. Calculated.

Lys-Lys dipeptide was converted to lysine at about 89 mole% and Met-Met dipeptide was converted to methionine at about 89 mole%. On the other hand, methionine-ε-lysine dipeptide was confirmed to have digestion rate of about 83-84% based on Lys and Met concentrations. (Table 6).

1.Methionine-ε-Lysine	Molecular Weight	Before reaction		After the reaction			Conversion rate (mole%)
		g / L	mM	g / L		mM	Product (mM) / Substrate (mM) * 100
Methionine-ε-Lysine	277.4	1.67	6.02
LYS	146.2			0.731		5.00	83.1
Met	149.2			0.753		5.05	83.8
2. Lys-Lys	Molecular Weight	Before reaction		After the reaction			Conversion rate (mole%)
		g / L	mM	g / L	mM	Considering Lys double factor in substrate (mM)	Product (mM) / Substrate (mM) * 100
Lys-lys	274.4	2.38	8.67
LYS	146.2			2.25	15.39	7.69	88.8
3. Met-Met	Molecular Weight	Before reaction		After the reaction			Conversion rate (mole%)
		g / L	mM	g / L	mM		Considering Met 2 fold in substrate (mM)	Product (mM) / Substrate (mM) * 100
Met-met	280.4	1.99	7.10
Met	149.2			1.89	12.67	6.33	89.2

In conclusion, methionine-ε-lysine showed a slightly reduced level of digestion (mole%) compared to Lys-Lys dipeptide and Met-Met dipeptide. However, when judging together with the rumen bypass rate result, it was confirmed that the methionine-ε-lysine dipeptide that passed through the rumen can be absorbed based on the high digestion rate after reaching the small intestine. It was confirmed that it can be applied as an additive.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (16)

1. Peptide derivatives or salts thereof in which two or more amino acids are linked to each other,

   The peptide derivative is

   (i) at least one amino acid selected from the group consisting of lysine, arginine, asparagine, glutamine, aspartate, and glutamate,

   (ii) an amide linkage between an amino acid to which lysine, arginine, asparagine, or glutamine is linked thereto, and an omega amine located in the side chain of the lysine, arginine, asparagine or glutamine (R group) and a carboxyl group of the amino acid linked thereto; Connected to each other,

   Comprising aspartate or glutamate linked to each other by an amide bond between an amino acid linked thereto and a carboxyl group located on the R group of aspartate or glutamate and an amine group of the amino acid linked thereto;

   Peptide derivatives or salts thereof.
2. The peptide derivative or salt thereof according to claim 1, wherein the oligopeptide derivative is an oligopeptide derivative having 2 to 10 amino acids linked to each other.
3. The peptide derivative or salt thereof according to claim 1, wherein the peptide derivative is a dipeptide derivative represented by Formula 2 or Formula 3 below:

   [Formula 2]

   X ₁ -A

   Wherein X ₁ is lysine, arginine, asparagine or glutamine,

   A is any amino acid,

   X ₁ -A are linked to each other by an amide bond between an omega amine located in the R group of X ₁ and a carboxyl group of A;

   [Formula 3]

   X ₂ -A

   Wherein X ₂ is aspartate or glutamate,

   A is any amino acid,

   X ₂ -A is connected to each other by an amide bond between the carboxyl group located at the R group of X ₂ and the amine group of A.
4. The peptide derivative or salt thereof according to claim 1, wherein the peptide derivative or salt thereof is for bypass of the rumen.
5. The peptide derivative or salt thereof of claim 1, wherein the peptide derivative or salt thereof is for feed additive.
6. Forming an amide bond between the amine group of the R group of an amino acid having an amine group in the R group and the carboxyl group of another amino acid, or forming an amide bond between the carboxyl group of the R group of the amino acid having a carboxyl group in the R Group and an amine group of another amino acid A method of preparing a peptide derivative or salt thereof according to claim 1,

   The amino acid having an amine group in the R group is selected from the group consisting of lysine, arginine, asparagine, and glutamine,

   The amino acid having a carboxyl group in the R group is aspartate or glutamate.
7. The method of claim 6,

   An amino acid having an amine group in the R group is protected by a protecting group with an amine group and a carboxyl group other than the amine of the R group,

   The amino acid which has a carboxyl group in R group is a carboxyl group and amine group other than the carboxyl group of R group protected by a protecting group.
8. The method of claim 6,

   An amine group of another amino acid linked to an amino acid having an amine group in the R group is protected with a protecting group,

   The carboxyl group of another amino acid linked with the amino acid which has a carboxyl group in R group is protected by a protecting group.
9. The method of claim 7, wherein

   After the step of forming an amide bond of the amine group of the R group of the amino acid having an amine group in the R group with another amino acid, or an amide bond of the carboxyl group of the R group of the amino acid having a carboxyl group in the R group with an amine group of another amino acid , Deprotecting the protecting group.
10. The method according to any one of claims 7 to 9,

    Protecting groups for amines include carbonyl a benzyloxy group (carbobenzyloxy group), p - methoxybenzyl carbonyl group (p -methoxybenzyl carbonyl group), tert - butyl-oxy-carbonyl group (tert -butyloxycarbonyl group), 9- Floresta carbonyl methyloxy carbonyl group (9-fluorenylmethyloxycarbonyl group), acetyl group (acetyl group), a benzoyl group (benzoyl group), benzyl group (benzyl group), a carbamate group (carbamate group), p - methoxy Siebel chewy (p -methoxybenzyl group), 3, 4-dimethoxybenzyl group (3,4-dimethoxybenzyl group), p - is methoxy group (p -methoxyphenyl group), and a tosyl group (tosyl group) one or more selected from the group consisting of,

    Protecting groups for carboxyl groups are methyl ester (methyl ester), benzyl ester (benzyl ester), tert - butyl ester with (tert -butyl ester), a silyl ester (silyl ester), an ortho ester (orthoester), and oxazoline (oxazoline) It is one or more selected from the group consisting of, manufacturing method.
11. A feed additive comprising the peptide derivative of any one of claims 1 to 5 or a salt thereof.
12. The feed additive of claim 11, wherein the feed additive is for ruminants.
13. A feed composition comprising the feed additive of claim 11.
14. The feed composition of claim 13, wherein the feed composition is for ruminants.
15. A method of breeding an animal, the method comprising feeding the feed composition of claim 13 to the animal.
16. The method of claim 15, wherein the animal is a ruminant.

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