WO2016182403A1 - Feed additive comprising lysine derivative - Google Patents

Abstract

The present application relates to a feed additive comprising a lysine derivative, and a method for raising animals using same.

Description

Feed additives containing lysine derivatives

The present application relates to a feed additive comprising a lysine derivative and a method for raising animals using the same.

In order to sustain life, to form new tissues, or to continue the work of producing milk, meat, eggs, etc., animals must be supplied with adequate nutrients in vitro.

Therefore, efforts have been made to improve the characteristics such as weight gain and meat quality by directly feeding amino acids, which are essential nutrients to animals. However, most amino acids consumed by ruminants are consumed 60-70% by the digestion process of the microbes in the rumen, and only some less digestible amino acids are digested and absorbed in the small intestine. There is a growing need for new technologies and materials that have improved protection and / or bypass effects in the rumen because they must be avoided and absorbed by the microorganisms in the rumen and stably reached and absorbed in the small intestine.

Known methods of protecting amino acids are amino acid-mineral chelate method, encapsulation method using pH sensitive polymer, and coating treatment technology (Smartamine MTM, Mepron M85, METHIO-BY). However, the product to which the coating treatment technology is applied has a disadvantage of being affected by the use of excipients and the price of the coating material.

There is a method of manufacturing a protective lysine by spraying cellulose, gum, sugar, calcium carbonate, talc and the like, but mass production is difficult, and the coating material is used as a nutrient source of microorganisms, thereby decreasing efficiency. In addition, there is a product of the method of refrigeration spray by mixing amino acids with protective substances such as ethyl cellulose, hardening oil, wax, etc. In this case, the energy cost is high, and the degradation is progressed through the unprotected amino acids located on the surface. There is this. 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 microorganisms in the intestinal and rumen.

As a result of intensive efforts to develop rumen protective amino acids, the inventors have found that α-amino-ε-caprolactam (ACL) can be used as rumen protective amino acid derivatives. The ACL has a significantly low degree of degradation in ruminant microorganisms, bypasses the rumen and confirms that it can be decomposed and absorbed in the stomach or small intestine of ruminants, thus completing the present application.

One object of the present application is to provide a feed additive comprising α-amino-ε-caprolactam (ACL) or a salt thereof as an active ingredient.

Another object of the present application is to provide a feed composition comprising the feed additive.

Another object of the present application is to provide a method of raising an animal, comprising feeding the feed composition to the animal.

α-amino-ε-caprolactam (ACL) is a lysine derivative, which has a low rate of being degraded and absorbed by ruminant microorganisms, so that it can be absorbed in the stomach or small intestine of ruminants, resulting in an increase in ruminants and improvement of oil quality. .

1 is a diagram showing a scheme for generating α-amino-ε-caprolactam (ACL) from lysine.

Figure 2 is a diagram showing the total gas generation amount (ml) of each of the control, lysine, L-ACL, and D / L-ACL.

Fig. 3 is a diagram showing the results of confirming the ratios of cattle rumen bypass by lysine, L-ACL, and D / L-ACL.

One embodiment embodying the present application is a feed additive, comprising α-amino-ε-caprolactam (ACL) or a salt thereof as an active ingredient.

In the present application, the term "α-amino-ε-caprolactam (ACL)" means a compound represented by the following Chemical Formula 1. The ACL is one of the lysine derivatives, also named lysine lactams.

[Formula 1]

Preparation of the ACL can be carried out using a variety of methods known in the art, for example, it can be carried out by enzymatic synthesis or chemical synthesis. More specifically, it can be synthesized using a ring closure reaction for converting lysine to ACL, using a catalyst (catalyst) for converting lysine to ACL, or using an appropriate organic solvent for converting lysine to ACL. It can be prepared by, but is not limited to those described above.

In addition, the ACL may be included in the feed additive in the form of its salt. Specifically, the ACL may be provided as a salt in an acceptable form as a feed ingredient, which may be prepared by known methods. Specific examples include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid and sulfuric acid; Sulfonic acids such as methanesulfonic acid; Or acid addition salts formed by organic carbon acids such as oxalic acid, acetic acid, fumaric acid, malonic acid, maleic acid, malic acid, succinic acid, but are not limited thereto.

In addition, the ACL may be in the form of L-ACL, D-ACL or D / L-ACL, but is not limited thereto.

As used herein, the term “lysine” is one of the basic α-amino acids, which, together with methionine and cysteine, belong to the first limited amino acid of ruminants, and lysine is biosynthesized from oxalacetic acid via the lysine biosynthetic pathway.

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. The feed additive may correspond to auxiliary feed in the feed management method.

In addition to the ACL or salts thereof, the feed additive may further include nutrients such as nucleotides, amino acids, calcium, phosphoric acid, organic acids, etc. to increase productivity or health of the target animal, but is not limited thereto.

As used herein, the term "feed composition" refers to food to be given to animals. 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 may further include nutritional ingredients necessary for maintaining the life of the animal, or producing meat, milk, and the like.

The individual to which the feed additive or feed composition including the same is applicable is not particularly limited, and any form may be applied. For example, the present invention may be applied to animals such as cattle, sheep, giraffes, camels, deer, goats, and the like without limitation, and specifically, but not limited to ruminants having rumen.

In the present application, 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 food into the mouth again to chew well swallow swallowing, this rumen is called the stomach to enable rumination. 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, which includes the animals of the family Camel, Baby 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. Specifically, bovine animals include, for example, cattle, goats, black goats, sheep, and the like, and more specifically, cattle. However, it is not limited thereto.

In addition, the feed additive according to the present application 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.

Another embodiment embodying the present application is a feed composition comprising the feed additive.

The feed additive and feed composition are as described above.

The feed composition may be paid according to a general specification management method, may be paid in the morning and afternoon, but is not limited thereto. In addition, the amount of salary is not particularly limited.

Another aspect of embodying the present application is a method of raising an animal, comprising feeding the feed additive or feed composition to the animal.

Specifically, the method may be a method comprising feeding the feed composition to a ruminant. In addition, the method may be paid according to a general specification management method, and may be paid in the morning and afternoon, but is not limited thereto. In addition, the amount of salary is not particularly limited.

Hereinafter, the present application will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present application is not limited to these examples.

Example  1. In vitro rumen fermentation ACL  Bypass efficiency measurement experiment

1-1. Rumen juice  Collection

Holstein (Holstein) castor with rumen cannula (with weight of 630 ~ 650kg) was released and two dogs were fed twice a day (7:30 am, 3:00 pm). Commercial feed (CJ CheilJedang, MilkzenTM) And rice straw was fed by breeding.

The rumen fluid was collected around 10 am on the day of the experiment, and the contents of the rumen were taken out through the cannula, squeezed out with gastric fluid with gauze, and then packed in a thermos bubbling with CO ₂ and transported to the laboratory in a state of blocking oxygen invasion. It was used later. It took less than an hour to transport to the lab.

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 simulations is shown in Table 1.

Test feed was used as a commercial feed (CJ Cheil Jedang, Milk JenTM) as a basic feed, and the test material was prepared by mixing the test material with the basic feed. Test materials were used as L-ACL and D / L-ACL, and experimental group 1 using L-ACL as test substance and experimental group 2 using D / L-ACL as test substance. Control 1 consisting of only basic feed without test substance and control 2 using L-lysine as test substance were compared together. In each experimental group, the culture was carried out in three repetitions. The basic feed and the test substance were mixed in a ratio of 4: 1 (basic feed 0.4 g, test substance 0.1 g, except for control 1, 0.5 g only basic feed), and 0.5 g of the mixed test feed was added to a 125 ml culture bottle. After mixing 50 ml of the prepared anaerobic broth, the mixture was sealed and left in a 39 ° C. incubator to start culture.

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

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
Casein	2.5 g
Resazulin (0.1%)	1.5 ml

1-3. Sampling and Test Substance Remaining Analysis Results

1-3-1. Total gas generation measurement

The culture was finally carried out for 72 hours, and culture sampling was conducted at 0h, 48h, 72h after the start of the culture. In order to confirm the culture state, the total gas generation amount of the culture solution sample was primarily measured.

In the rumen, microbial symbiosis has the ability to decompose and energize the cellulose of plants that cannot be digested by ordinary animals. In this process, the microbes in the rumen produce a gas, most of which is volatile fatty acids (VFA). Volatile fatty acids are generally generated at 65% acetic acid, 20% propionic acid, and 15% butyric acid, and these volatile fatty acids are used as ruminant energy sources. Journal of Biotechnology Vol. 9 (38), pp. 6229-6232). Therefore, the increase or decrease of volatile fatty acid production by the microorganisms in the rumen can be used as an indicator of the degradation of feed or feed additives, thereby inferring the link between the protective effect of the feed additives in the rumen and the reduction of volatile fatty acid production. have.

Gas generation measured through this experiment is as shown in Table 2 and FIG. In the total gas generation amount, the total gas generation amount was decreased in the control group 2 using L-lysine as the test substance, and the control group 1, which used L-ACL and D / L-ACL as the test substance, compared to the control group 1 consisting of the basic feed without the test substance. 2, the total gas generation decreased compared to the control 1, 2. This is because the L-ACL, D / L-ACL is not consumed by the microorganisms in the rumen liquid, so that the total amount of gas produced by the microorganisms is reduced compared to the control group. As a result, the decrease in total gas generation indirectly means an increase in the rumen bypass rate, and it can be estimated that L-ACL and D / L-ACL have a higher rumen bypass rate than L-lysine.

Total gas generation amount (ml)

		Total gas generation amount (ml)
		48h					72h
		One	2	3	medium	SD	One	2	3	medium	SD
Control group 1	control	135	144	138	139	4.9	146	146	145	145	0.8
Control 2	L-Lys	126	128	126	127	1.2	129	129	130	129	0.1
Experiment group 1	L-ACL	113	115	113	113	1.1	117	115	118	117	1.5
Experiment group 2	D / L-ACL	114	114	113	114	0.8	117	119	117	118	1.5

1-3-2. Rumen bypass rate measurement

After measuring the total gas generation amount of the culture medium sample, the culture medium was centrifuged (4000rpm, 10 minutes) to measure the amount of test material present in the supernatant, and the rumen bypass rate was calculated based on the remaining amount of test material for each sampling time zone. As a result of LC analysis, the remaining amount of test substance is as shown in Table 3. FIG. 3 is a graph showing the rumen bypass rate (%), which compares the relative residual amount (%) of 24h and 48h samples when the residual amount of 0h sample is converted to 100%.

As a result, L-lysine was digested by rumen microorganisms at 0% and 48h after 48h, and L-ACL was digested by rumen microorganisms at 96% at 48h and 90% at 72h. Seemed. Similarly to L-ACL, D / L-ACL showed a rumen bypass rate of 102% at 48h and 91% at 72h. Considering the error range, it can be determined that L-ACL and D / L-ACL are bypassed by almost no degradation by the microorganisms in the rumen until 72h.

Residual amount of test substance in the culture supernatant (g / L)

	0h					48h					72h
	One	2	3	medium	SD	One	2	3	medium	SD	One	2	3	medium	SD
Control 2	2.23	2.37	2.06	2.22	0.16	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Experiment group 1	2.51	2.47	2.61	2.53	0.07	2.48	2.43	2.40	2.43	0.04	2.19	2.36	2.30	2.28	0.09
Experiment group 2	2.50	2.34	1.58	2.42	0.12	2.32	2.55	2.50	2.46	0.12	2.08	2.24	2.31	2.21	0.12

Example  2. ACL  Digestion test

Nutrients not broken down by the microbes in the rumen are absorbed in the small intestine and used for protein synthesis and energy metabolism. In the case of ACL, it is expected that most of it will be delivered to the small intestine due to the high bypass efficiency, and that the amide binding site of the ACL can be converted into lysine form when the amide binding site of the ACL is cleaved by the application of the degrading enzymes present in the small and liver. Absorption will be used. In order to confirm that the bypassed ACL is restored to lysine by the degrading enzyme, the following experiment was conducted.

In general, cleavage of amide bonds (or peptide bonds) is possible by digestive enzymes such as trypsin and pepsin, and can also be cleaved by hydrolytic enzymes such as asilase. In addition, in the small intestine responsible for digestion in vivo, most proteins having peptide bonds by digestive enzymes such as aminopeptidase, carboxypeptidase, endopeptidase, and dipeptidase, which are located in the mucosa, Digested into amino acids and some dipeptide forms, some undigested dipeptides are also absorbed into cells, hydrolyzed to amino acids by dipeptidase and then transported into the bloodstream. Numerous nutrients in a form that cannot be used directly are absorbed by the small intestine and then moved along the bloodstream to the liver, where they are converted into substances that can be metabolized in the body through various decomposition processes.

For this reason, this study confirmed the possibility of digestion and digestion of ACL against enzymes in small intestine and liver.

Example 2 -One. Small intestine enzyme group

40m of Korean cattle (History: KOR005078680400) slaughtered at the Bucheon Livestock Products Market of the National Agricultural Cooperative Federation were purchased. 5m of 20mM sodium phosphate buffer (pH7.4) was added to the small intestine of about 1m and then digested so that the enzymes in the small intestine could be extracted into sodium phosphate buffer. About 200m of small intestine enzyme solution was obtained for small intestine with a total length of 40m. The supernatant was obtained by centrifugation (14000rpm, 10 minutes) at 4 ℃, and then diluted twice in 20mM sodium phosphate buffer (pH 7.4). This experiment was used.

Example 2 -2. Liver tissue  Enzyme group

Mix 0.125g of liver tissue and 1ml of 20mM sodium phosphate buffer (pH7.4) in the liver of Hanwoo (History: KOR005078680400) slaughtered at NACF Bucheon Livestock Products Market, and then glass bead (Sigma G1145) Hepatic tissue was disrupted via beadbeater (MP ™ FastPrep®-24) by adding (about 1/10 volume) (20 seconds, 3 times). The hepatic crushing fluid thus obtained was centrifuged at 4 ° C. (14000 rpm, 10 minutes) to secure the supernatant and used in this experiment.

Example 2 -3. Enzyme Reaction in the Small Intestine and Liver

In Example 1, in vitro digestion experiments were conducted using enzyme groups of small intestine and liver tissues to determine whether ACLs having a protective function (bypass) in the rumen can be digested and digested in the small intestine. The reaction proceeded at a concentration of about 2 g / L ACL, and the exact concentration is specified in the LC quantification results (g / L) of Example 2-4. The final volume of the reaction is 1000 μl and the composition of the substrate, small intestine, and buffer used is specified in Table 4 below.

In vitro digestion reaction composition

Substrate (ACL; stock concentration 4 g / L)	500
Small intestine enzyme group	100
20 mM sodium phosphate buffer (pH 7.4)	400
Final reaction volume (μl)	1000

Example  2-4. Small intestine enzyme reaction result

Sample analysis was performed after enzyme reaction for 24 hours at 37 ℃ reaction conditions. After the reaction was completed, the solution was diluted 10-fold by adding 0.5% perchloric acid (DEA JUNG, purity 60-62%), and then centrifuged (14000 rpm, 10 minutes) to remove the protein in the supernatant. The amount and product of lysine concentration was quantified by HPLC and converted to mM. The conversion rate (mole%) was converted from the molar concentration (mM) of the sample before the reaction and the molar concentration (mM) after the reaction as a percentage (%) (ACL molecular weight: 128.18g / L, lysine molecular weight: 146.19g / L) .

As a result of the reaction for 24 hours intestine enzyme group, the conversion (degradation rate) from ACL to lysine was 50.5 mole%, and the conversion rate (decomposition rate) of 18.9 mole% was confirmed in the liver tissue enzyme group (Table 5).

ACL Absorption Rate for Small Intestine and Liver Enzymes

Small intestine enzyme reaction	Before the reaction		After the reaction		Conversion rate (mole%)
	g / L	mM	g / L	mM
ACL	2.55	19.87
LYS			1.47	10.03	50.5
Liver tissue  Enzyme group reaction	Before the reaction		After the reaction		Conversion rate (mole%)
	g / L	mM	g / L	mM
ACL	2.55	19.87
LYS			0.549	3.75	18.9

In conclusion, ACL was converted to lysine in the in vitro reaction of small intestine and hepatic enzyme family. From these results, it was confirmed that when the ACL reaches the small intestine through the rumen, it can be converted into lysine by digestive enzymes in the small intestine. The possibility was confirmed. This means that the ACL provided as a feed additive can be utilized directly as lysine in substantially amino acid form in the body of ruminants.

From the above description, those skilled in the art will appreciate that the present application can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present application 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 application.

Claims (8)

1. α-amino-ε-caprolactam (ACL) or a salt thereof as an active ingredient, feed additive.
2. The feed additive of claim 1, wherein the feed additive is for ruminants.
3. The feed additive of claim 2 wherein the ruminant is a bovine.
4. A method of raising an animal, comprising feeding the animal the feed additive of any one of claims 1 to 3.
5. The method of claim 4, wherein the animal is a ruminant.
6. A feed composition comprising the feed additive of any one of claims 1 to 3.
7. A method of raising an animal, comprising feeding the animal the feed composition of claim 6.
8. 8. The method of claim 7, wherein said animal is a ruminant.

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