WO2017099475A1 - Dérivé peptidique protégeant le rumen et son utilisation - Google Patents

Dérivé peptidique protégeant le rumen et son utilisation Download PDF

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WO2017099475A1
WO2017099475A1 PCT/KR2016/014327 KR2016014327W WO2017099475A1 WO 2017099475 A1 WO2017099475 A1 WO 2017099475A1 KR 2016014327 W KR2016014327 W KR 2016014327W WO 2017099475 A1 WO2017099475 A1 WO 2017099475A1
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group
amino acid
amine
carboxyl
linked
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PCT/KR2016/014327
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English (en)
Korean (ko)
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홍국기
전진우
문준옥
김정현
박연희
박혜민
최수진
박진승
양영렬
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씨제이제일제당 (주)
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Publication of WO2017099475A1 publication Critical patent/WO2017099475A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • 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.
  • these products have the disadvantage that the use of excipients in the coating process and the price of the coating material increases.
  • Amino acids have excellent effects such as weight gain and quality improvement of livestock as feed additives.
  • 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.
  • the digestion proceeds slowly in the rumen due to the different chemical structure from the peptide.
  • the rumen bypass rate could be increased.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • amino acid refers to all molecules comprising an amino group and a carboxyl group.
  • the amino acid includes both natural amino acids or artificial amino acids, specifically, natural amino acids, but is not limited thereto.
  • amino acids include amine groups, carboxyl groups and side chains (R groups) specific for each amino acid.
  • amino acids examples 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.
  • amino acids used in the present invention may be described as abbreviations as follows according to the IUPAC-IUB nomenclature.
  • 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.
  • 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).
  • 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.
  • 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.
  • lysine, arginine, asparagine, or glutamine 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • protecting group refers to binding of a specific functional group of a compound to prevent the bound functional group from reacting
  • deprotection refers to removing the attached protecting group to return the functional group to its original state.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • composition for feed of the present invention is not particularly limited and may be applied in any form.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • N ⁇ -Boc-L- in order to protect the carboxylic acid functionality of the eggs with, N ⁇ -Boc-L- after the arginine (0.1 mol) was dissolved in methanol, tea from 0 °C 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 ⁇ -Boc-L- arginine methyl ester (N ⁇ -Boc-L-arginine 4-methylester) was obtained.
  • EA Ethyl acetate
  • Example 2 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 ⁇ -Boc-L-arginine 4-methyl ester (0.1 mol) prepared above were prepared using DMF (N, N-Diisopropylethylamine, 0.15 mol).
  • HBTU N, N, N ', N'-Tetramethyl-O- (1H-benzotriazol-1-yl) uronium hexafluorophosphate, 0.15 mol
  • the product was extracted with EA and crystallized.
  • 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).
  • N ⁇ -Boc-L- asparagine (0.1 mol) was dissolved in methanol, tea from 0 °C 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 ⁇ -Boc-L-asparagine 4-methyl ester (N ⁇ -Boc-L-asparagine). 4-methyl ester) was obtained.
  • Example 2 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 ⁇ -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).
  • N ⁇ -Boc-L- in order to protect the carboxylic acid functionality of Glutamine, N ⁇ -Boc-L- after glutamine (0.1 mol) was dissolved in methanol and slowly injected with thionyl chloride (thionyl chloride, 0.15 mol) at 0 °C 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 ⁇ -Boc-L- glutamine methyl ester (N ⁇ -Boc-L-glutamine 4-methyl ester) and extracted with Obtained.
  • EA Ethyl acetate
  • Example 2 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 ⁇ -Boc-L- glutamine methyl ester prepared above produced by the above method (N ⁇ -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.
  • Example 4 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).
  • 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 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.
  • EA Ethyl acetate
  • 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.
  • McDougall's buffer simulation solution is as shown in Table 1 below.
  • 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- ⁇ -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.
  • 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.
  • 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.
  • 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 (%).
  • methionine- ⁇ -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 8-1 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).
  • 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.
  • 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
  • the molecular weight of methionine is 149.25 g / L.
  • Example 8-3 Small intestine enzyme reaction
  • Methionine- ⁇ -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) 400 400 400 400 Final reaction volume ( ⁇ l) 1000 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.
  • 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).
  • Lys-Lys dipeptide was converted to lysine at about 89 mole% and Met-Met dipeptide was converted to methionine at about 89 mole%.
  • methionine- ⁇ -lysine dipeptide was confirmed to have digestion rate of about 83-84% based on Lys and Met concentrations. (Table 6).
  • methionine- ⁇ -lysine showed a slightly reduced level of digestion (mole%) compared to Lys-Lys dipeptide and Met-Met dipeptide.
  • the methionine- ⁇ -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.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Animal Husbandry (AREA)
  • Molecular Biology (AREA)
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  • Biochemistry (AREA)
  • Biophysics (AREA)
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Abstract

La présente invention concerne un dérivé peptidique présentant deux acides aminés ou plus liés les uns aux autres, un procédé de préparation du dérivé peptidique et un additif alimentaire et une composition alimentaire comprenant le dérivé peptidique. Le dérivé peptidique de la présente invention est digéré lentement dans le rumen, entraînant un taux élevé de dilution du rumen, et le dérivé peptidique peut être absorbé par un ruminant après le passage à travers le rumen en raison de la présence de liaisons amide et peut ainsi être utilisé très avantageusement comme une source d'acides aminés d'un ruminant.
PCT/KR2016/014327 2015-12-09 2016-12-07 Dérivé peptidique protégeant le rumen et son utilisation WO2017099475A1 (fr)

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KR10-2015-0174824 2015-12-09

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5167957A (en) * 1990-09-06 1992-12-01 Virginia Tech Intellectual Properties, Inc. Compositions and methods for the treatment of dietary deficiencies
US20150231194A1 (en) * 2008-06-13 2015-08-20 Westfälische Wilhelms-Universität Münster Biotechnological production of cyanophycin dipeptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5167957A (en) * 1990-09-06 1992-12-01 Virginia Tech Intellectual Properties, Inc. Compositions and methods for the treatment of dietary deficiencies
US20150231194A1 (en) * 2008-06-13 2015-08-20 Westfälische Wilhelms-Universität Münster Biotechnological production of cyanophycin dipeptides

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CSILLA: "Transition Metal Complexes of Amino Acid and Peptide Derivatives Containing Carboxylate and Imidazole Side Chains", PROPOSITIONS OF PHD THESIS, 2005, pages 1 - 10, XP055598822 *
MCGEE: "Efficient and Directed Peptide Bond Formation in the Gas Phase via Ion/Ion Reactions", PNAS, vol. 111, no. 4, 28 January 2014 (2014-01-28), pages 1288 - 1292, XP055598826 *
NUVOBNT: "Development of Feed Additives for Improving Milk-Protein by Rumen Preotected Amino Acid and Choline", DEVELOPMENT OF FEED ADD, 18 July 2014 (2014-07-18), pages 11 - 13 *

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KR102700159B1 (ko) 2024-08-30
KR20170069152A (ko) 2017-06-20

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