KR102571169B1 - Composition for promoting synthesis of milk protein comprising L-phenylalanine, L-tryptophan and acetate - Google Patents

Composition for promoting synthesis of milk protein comprising L-phenylalanine, L-tryptophan and acetate Download PDF

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KR102571169B1
KR102571169B1 KR1020200183747A KR20200183747A KR102571169B1 KR 102571169 B1 KR102571169 B1 KR 102571169B1 KR 1020200183747 A KR1020200183747 A KR 1020200183747A KR 20200183747 A KR20200183747 A KR 20200183747A KR 102571169 B1 KR102571169 B1 KR 102571169B1
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milk
tryptophan
composition
phenylalanine
amino acids
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KR20220092204A (en
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이홍구
이재성
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건국대학교 산학협력단
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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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K5/00—Feeding devices for stock or game ; Feeding wagons; Feeding stacks
    • 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
    • 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
    • A23K50/15—Feeding-stuffs specially adapted for particular animals for ruminants containing substances which are metabolically converted to proteins, e.g. ammonium salts or urea
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S426/00—Food or edible material: processes, compositions, and products
    • Y10S426/807—Poultry or ruminant feed

Abstract

본 발명은 L-페닐알라닌, L-트립토판 및 아세트산 포함하는 유단백 합성 촉진용 조성물에 관한 것이다.The present invention relates to a composition for promoting milk protein synthesis containing L-phenylalanine, L-tryptophan and acetic acid.

Description

L-페닐알라닌, L-트립토판 및 아세트산 포함하는 유단백 합성 촉진용 조성물 {Composition for promoting synthesis of milk protein comprising L-phenylalanine, L-tryptophan and acetate}Composition for promoting synthesis of milk protein comprising L-phenylalanine, L-tryptophan and acetic acid

본 발명은 L-페닐알라닌, L-트립토판 및 아세트산 포함하는 유단백 합성 촉진용 조성물에 관한 것이다.The present invention relates to a composition for promoting milk protein synthesis containing L-phenylalanine, L-tryptophan and acetic acid.

소비자들의 관심사 변화에 따라 고단백 저지방 우유, 우유 단백질이 포함된 화장품 및 치즈 수요가 증가하고 있으며, 유단백질 증가에 대한 관심도 증가하고 있다. 이에 따라 우리나라 원유가격산정체계는 2014년부터 유단백질 함량을 포함하는 쪽으로 변화하고 있다. 유단백질 양을 최대화하기 위해서는 사료 내 crude protein (CP) 함량이 22-23% 정도가 되어야 하지만 (NRC, 2001), 젖소가 CP 함량이 높은 사료 섭취 시 오줌으로 배출되는 질소 함량도 높아져 결국 질소로 인한 환경오염과 질소 이용효율 감소를 야기한다. 따라서 영양소를 제한하면서도 유단백질 양을 증가시킬 수 있는 방안을 모색해야 한다. 유단백질을 합성하는 기전에 필요한 것은 일반적으로 아미노산, 에너지원, 그리고 성장호르몬으로 알려져 있다 (Kim et al., 2013). 현재 우리나라는 젖소에 성장호르몬 사용을 금지하고 있기 때문에 아미노산과 에너지원에 주목하게 되었다.As consumers' interest changes, demand for high-protein, low-fat milk, cosmetics and cheese containing milk protein is increasing, and interest in increasing milk protein is also increasing. Accordingly, the crude oil price calculation system in Korea has been changing to include milk protein content since 2014. In order to maximize the amount of milk protein, the crude protein (CP) content in feed should be 22-23% (NRC, 2001), but when cows consume feed with high CP content, nitrogen excreted in urine also increases, resulting in nitrogen It causes environmental pollution and reduced nitrogen utilization efficiency. Therefore, it is necessary to find a way to increase the amount of milk protein while limiting nutrients. It is generally known that amino acids, energy sources, and growth hormones are required for the mechanism of milk protein synthesis (Kim et al., 2013). Since Korea is currently banning the use of growth hormone in dairy cows, attention has been paid to amino acids and energy sources.

아미노산은 단백질의 기본 단위일 뿐만 아니라 생리활성물질이다 (Kim et al., 2013). 아미노산 중 동물 체내에서 합성되지 않거나, 합성되더라도 적게 합성되어 섭취를 통해 보충해야 하는 것들을 필수아미노산이라고 하는데, 필수아미노산 중에서도 함량이 적어서 단백질 합성을 제한시키는 아미노산 (제한아미노산)에 대한 연구가 활발히 진행되어 왔다. Amino acids are not only the basic units of proteins but also bioactive substances (Kim et al., 2013). Among amino acids, amino acids that are not synthesized in the animal body or are synthesized in a small amount and need to be supplemented through ingestion are called essential amino acids. .

필수아미노산 첨가로 젖소의 유단백질을 늘리고자 하는 연구들이 많이 진행되었으나, 이는 몇몇 아미노산에 집중되어 있다. 본원 발명자들은 젖소의 유단백질의 함량을 증가하는 방법을 연구하던 중에, 젖소 유래 유선상피세포(MAC-T)에 트립토판 및 페닐알린, 또는 트립토판, 페닐알라닌 및 아세트산을 처리하면 유단백질의 생산이 증가하는 것을 확인하였고, 또한 젖소에 트립토판 및 페닐알린, 또는 트립토판, 페닐알라닌 및 아세트산을 급여한 결과 젖소의 사료 섭취량 및 유량을 변화하지 않으면서 유단백질량을 증가시키고 체세포 수를 감소시키는 것을 확인함으로써, 본원 발명을 완성하였다. Many studies have been conducted to increase cow's milk protein by adding essential amino acids, but these are concentrated on a few amino acids. While studying a method for increasing the content of cow's milk protein, the present inventors confirmed that the production of milk protein increased when cow-derived mammary epithelial cells (MAC-T) were treated with tryptophan and phenylalin, or tryptophan, phenylalanine and acetic acid. In addition, as a result of feeding tryptophan and phenylalin, or tryptophan, phenylalanine and acetic acid to cows, the present invention was completed by confirming that the amount of milk protein increased and the number of somatic cells decreased without changing the feed intake and flow rate of cows. .

필수아미노산 첨가로 젖소의 유단백질을 늘리고자 하는 연구들이 많이 진행되었으나, 이는 몇몇 아미노산에 집중되어 있다. 본원 발명자들은 젖소의 유단백질의 함량을 증가하는 방법을 연구하던 중에, 젖소 유래 유선상피세포(MAC-T)에 트립토판 및 페닐알린, 또는 트립토판, 페닐알라닌 및 아세트산을 처리하면 유단백질의 생산이 증가하는 것을 확인하였고, 또한 젖소에 트립토판 및 페닐알린, 또는 트립토판, 페닐알라닌 및 아세트산을 급여한 결과 젖소의 사료 섭취량 및 유량을 변화하지 않으면서 유단백질량을 증가시키고 체세포 수를 감소시키는 것을 확인함으로써, 본원 발명을 완성하였다. Many studies have been conducted to increase cow's milk protein by adding essential amino acids, but these are concentrated on a few amino acids. While studying a method for increasing the content of cow's milk protein, the present inventors confirmed that the production of milk protein increased when cow-derived mammary epithelial cells (MAC-T) were treated with tryptophan and phenylalin, or tryptophan, phenylalanine and acetic acid. In addition, as a result of feeding tryptophan and phenylalin, or tryptophan, phenylalanine and acetic acid to cows, the present invention was completed by confirming that the amount of milk protein increased and the number of somatic cells decreased without changing the feed intake and flow rate of cows. .

본 발명의 목적은 페닐알라닌, 트립토판 및 아세트산을 포함하는 착유 반추동물의 유중 단백질 합성 촉진용 조성물을 제공하는 것이다. An object of the present invention is to provide a composition for promoting protein synthesis in the milk of milking ruminants containing phenylalanine, tryptophan and acetic acid.

본 발명의 또 다른 목적은 상기 서술한 조성물을 포함하는 착유 반추동물의 유중 단백질 합성 촉진용 사료 첨가제를 제공하는 것이다. Another object of the present invention is to provide a feed additive for promoting protein synthesis in milk of milking ruminants comprising the above-described composition.

본 발명의 다른 목적은 또한 상기 서술한 조성물을 포함하는 착유 반추동물의 유중 단백질 합성 촉진용 사료 조성물을 제공하는 것이다. Another object of the present invention is to provide a feed composition for promoting protein synthesis in milk of milking ruminants comprising the above-described composition.

본 발명의 다른 목적은 상기 서술한 조성물을 젖소에 급여하는 단계를 포함하는 젖소의 유중 단백질의 함량을 증가시키는 방법을 제공하는 것이다. Another object of the present invention is to provide a method for increasing the protein content in cow's milk, comprising the step of feeding the above-described composition to the cow.

본 발명은 페닐알라닌, 트립토판 및 아세트산을 포함하는 착유 반추동물의 유중 단백질 합성 촉진용 조성물을 제공할 수 있다. The present invention can provide a composition for promoting protein synthesis in the milk of a milking ruminant containing phenylalanine, tryptophan and acetic acid.

상기 유단백질은 베타-카제인(beta-casein)일 수 있다. The milk protein may be beta-casein.

본 발명은 또한 상기 서술한 조성물을 포함하는 착유 반추동물의 유중 단백질 합성 촉진용 사료 첨가제를 제공할 수 있다. The present invention can also provide a feed additive for promoting protein synthesis in milk of milking ruminants comprising the above-described composition.

본 발명은 또한 상기 서술한 조성물을 포함하는 젖소의 유중 단백질 합성 촉진용 사료 조성물을 제공할 수 있다. The present invention can also provide a feed composition for promoting protein synthesis in milk of cows comprising the above-described composition.

본 발명은 또한 상기 서술한 조성물을 젖소에 급여하는 단계를 포함하는 젖소의 유중 단백질의 함량을 증가시키는 방법을 제공할 수 있다. The present invention can also provide a method for increasing the protein content in cow's milk, comprising the step of feeding the above-described composition to the cow.

본 발명의 페닐알라닌, 트립토판 및 아세트산의 조합은 젖소의 사료 섭취량 및 유량을 변화시키지 않으면서 유단백질량을 증가시키는 효과가 있다. 또한 대조구 대비 모든 처리구에서 항염증 효과로 체세포 수를 감소하는 효과가 있다. The combination of phenylalanine, tryptophan and acetic acid of the present invention has the effect of increasing the amount of milk protein without changing the feed intake and milk yield of cows. In addition, there is an effect of reducing the number of somatic cells due to the anti-inflammatory effect in all treatment groups compared to the control group.

도 1은 72시간 동안 대사촉진 물질 및 다양한 아미노산을 처리할 때 MAC-T 세포에서 상대적인 세포 외 단백질 농도를 확인한 결과이다.
도 2는 72시간 동안 대사촉진 물질 및 다양한 아미노산을 처리할 때 MAC-T 세포에서 상대적인 β- 카제인 mRNA 발현을 확인한 결과이다. 1 is a result of confirming the relative extracellular protein concentration in MAC-T cells when metabolites and various amino acids were treated for 72 hours.
Figure 2 is a result of confirming the relative β- casein mRNA expression in MAC-T cells when the metabolite and various amino acids were treated for 72 hours.

이하, 본 발명을 상세하게 설명한다. Hereinafter, the present invention will be described in detail.

필수아미노산 첨가로 젖소의 유단백질을 늘리고자 하는 연구들이 많이 진행되었으나, 이는 몇몇 아미노산에 집중되어 있다. 본원 발명자들은 젖소의 유단백질의 함량을 증가하는 방법을 연구하던 중에, 젖소 유래 유선상피세포(MAC-T)에 트립토판 및 페닐알린, 또는 트립토판, 페닐알라닌 및 아세트산을 처리하면 유단백질의 생산이 증가하는 것을 확인하였고, 또한 젖소에 트립토판 및 페닐알린, 또는 트립토판, 페닐알라닌 및 아세트산을 급여한 결과 젖소의 사료 섭취량 및 유량을 변화하지 않으면서 유단백질량을 증가시키고 체세포 수를 감소시키는 것을 확인함으로써, 본원 발명을 완성하였다. Many studies have been conducted to increase cow's milk protein by adding essential amino acids, but these are concentrated on a few amino acids. While studying a method for increasing the content of cow's milk protein, the present inventors confirmed that the production of milk protein increased when cow-derived mammary epithelial cells (MAC-T) were treated with tryptophan and phenylalin, or tryptophan, phenylalanine and acetic acid. In addition, as a result of feeding tryptophan and phenylalin, or tryptophan, phenylalanine and acetic acid to cows, the present invention was completed by confirming that the amount of milk protein increased and the number of somatic cells decreased without changing the feed intake and flow rate of cows. .

본 발명은 페닐알라닌, 트립토판 및 아세트산을 포함하는 착유 반추동물의 유중 단백질 합성 촉진용 조성물을 제공할 수 있다. The present invention can provide a composition for promoting protein synthesis in the milk of a milking ruminant containing phenylalanine, tryptophan and acetic acid.

상기 페닐알라닌 및 트립토판은 L-페닐알라닌 및 L-트립토판 일 수 있으며, 페닐알라닌(phenylalanine; Phe) 및 트립토판(tryptophan; Trp)은 필수 아미노산으로 동물조직에 의해 합성 될 수 없으며, 설사 합성된다 하더라도 요구량만큼 충분히 합성되지 못한다. 유선상피세포에 트립토판 및 페닐알라닌을 처리한 결과 Extra cellular 단백질 농도가 크게 증가하는 것을 확인하였다 (도 1). 페닐알라닌, 트립토판 및 아세트산을 처리한 경우 β-casein 발현량 증가하였다 (도 2). 따라서 MAC-T cell에서 가장 효율적으로 유단백질을 생산하는 조합은 트립토판 및 페닐알라닌과 트리토판, 페닐알라니 및 아세트산인 것을 확인하였다. The phenylalanine and tryptophan may be L-phenylalanine and L-tryptophan, and phenylalanine (Phe) and tryptophan (Trp) are essential amino acids and cannot be synthesized by animal tissue, and even if synthesized, they are sufficiently synthesized as required. can't be As a result of treating mammary epithelial cells with tryptophan and phenylalanine, it was confirmed that the concentration of extra cellular protein significantly increased (FIG. 1). When treated with phenylalanine, tryptophan, and acetic acid, the expression level of β-casein increased (FIG. 2). Therefore, it was confirmed that the combinations that produced milk protein most efficiently in MAC-T cells were tryptophan and phenylalanine, and tryptophan, phenylalanine and acetic acid.

상기 트립토판은 0.9 mM의 농도로 포함할 수 있다. The tryptophan may be included at a concentration of 0.9 mM.

상기 페닐알라닌은 0.6 mM의 농도로 포함할 수 있다. The phenylalanine may be included at a concentration of 0.6 mM.

상기 아세트산은 0.3 mM의 농도로 포함할 수 있다. The acetic acid may be included at a concentration of 0.3 mM.

상기 유단백질은 베타-카제인(beta-casein)일 수 있다. The milk protein may be beta-casein.

상기 반추동물은 소, 젖소, 양, 사슴, 기린 또는 낙타일 수 있으며, 바람직하게는 젖소일 수 있으나, 이에 제한되지 않는다. The ruminant may be a cow, cow, sheep, deer, giraffe or camel, preferably a cow, but is not limited thereto.

일반적으로 반추위를 가진 반추동물의 영양소 소화과정은 반추위 내에서 미생물에 의한 제1단계 발효 과정 및 하부 소화장기(제4위, 소장 및 대장 등)에서 소화효소에 의한 제2단계 소화과정으로 이루어진다. 반추동물은 반추위에 존재하는 미생물의 원료로 아미노산이 소비되는 점과 고능력우 또는 착유우의 경우에는 필요한 영양소가 부족한 점이 있어서 사료를 통한 공급이 필요하다. 또한, 반추동물은 반추위 구조상 합성 아미노산 급이시 반추위 내에서 반추미생물에 의해 분해되어 반추미생물의 영양원으로 거의 모두 이용된다. 결국 급이된 합성아미노산 중 최대 20% 정도만 소장으로 이전된다. 따라서 반추 동물에 있어서는 사료 공급시 반추위에서의 소화 흡수율 및 흡수량 뿐만 아니라 반추위를 우회하여 소장에 도달하는 영양소의 양 및 소장에서 효소에 의하여 흡수되는 흡수율 등을 모두 고려하여 사료를 공급해야 할 필요성이 있다. 따라서 반추동물의 생산능력을 최대한 발휘하기 위해서는 제1위에서 최대한 미생물체 단백질을 많이 생산할 수 있는 환경을 만들어 주고 동시에 반추미생물의 분해작용으로부터 보호된 단백질을 적절히 급이해야 한다. 그런데 미생물체 단백질은 반추동물의 능력을 최대로 발휘하기 위하여 필요한 아미노산의 양을 충족할 수 없으므로, 반추미생물에 의해 분해되지 않고 소장으로 내려가 흡수 이용될 수 있는 사료 단백질의 급이가 필요하다.In general, the nutrient digestion process of ruminants having a rumen consists of a first stage fermentation process by microorganisms in the rumen and a second stage digestion process by digestive enzymes in the lower digestive organs (4th stomach, small intestine and large intestine, etc.). In ruminants, amino acids are consumed as a raw material for microorganisms present in the rumen, and in the case of high-capacity cows or milking cows, necessary nutrients are insufficient, so supply through feed is necessary. In addition, ruminants are decomposed by ruminant microorganisms in the rumen when feeding synthetic amino acids due to the structure of the rumen, and are almost all used as a nutrient source for the rumen microorganisms. Ultimately, only up to 20% of the synthetic amino acids fed are transferred to the small intestine. Therefore, in ruminant animals, when supplying feed, it is necessary to supply feed in consideration of not only the digestion and absorption rate in the rumen, but also the amount of nutrients reaching the small intestine by bypassing the rumen and the absorption rate absorbed by enzymes in the small intestine. . Therefore, in order to maximize the production capacity of ruminants, it is necessary to create an environment that can produce as much microbial protein as possible in the first place and at the same time to properly feed the protein protected from the decomposition of ruminant microorganisms. However, since microbial protein cannot satisfy the amount of amino acids required to maximize the ability of ruminants, it is necessary to feed protein that can be absorbed and used by going down to the small intestine without being decomposed by ruminant microorganisms.

본 발명은 또한 상기 서술한 조성물을 포함하는 착유 반추동물의 유중 단백질 합성 촉진용 사료 첨가제를 제공할 수 있다. The present invention can also provide a feed additive for promoting protein synthesis in milk of milking ruminants comprising the above-described composition.

본 발명은 또한 상기 서술한 조성물을 포함하는 젖소의 유중 단백질 합성 촉진용 사료 조성물을 제공할 수 있다. The present invention can also provide a feed composition for promoting protein synthesis in milk of cows comprising the above-described composition.

"사료첨가제"는 사료에 첨가되는 물질을 의미하며, 상기 사료첨가제는 대상 개체의 생산성 향상이나 건강을 증진시키기 위한 것일 수 있으나, 이에 제한되는 것은 아니다. 또한, 상기 사료첨가제는 사료 관리법상의 보조사료에 해당할 수 있다."Feed additive" refers to a substance added to feed, and the feed additive may be used to improve productivity or improve health of a subject, but is not limited thereto. In addition, the feed additive may correspond to supplementary feed under the Feed Management Act.

본 발명의 젖소의 유중 단백질 합성 촉진용 사료 조성물의 사료첨가제형태는 페닐알라닌, 트립토판 및 아세트산 이외에도 대상 개체의 생산성 또는 건강 증진을 위한 뉴클레오티드류, 아미노산, 칼슘, 인산, 유기산 등의 영양소를 추가로 포함할 수 있으나, 이에 제한되지 않는다.The feed additive form of the feed composition for promoting protein-in-oil synthesis of dairy cows of the present invention may further include nutrients such as nucleotides, amino acids, calcium, phosphoric acid, organic acids, etc. It can, but is not limited thereto.

본 발명의 페닐알라니, 트립토판 및 아세트산은 급여 대상, 급여 대상의 종, 체중, 급여 시기, 급여 사료의 종류, 급여 목적등을 고려하여 당업자가 그 함량을 결정할 수 있다.Phenylalani, tryptophan and acetic acid of the present invention can be determined by those skilled in the art in consideration of the target to be fed, the species to be fed, the weight, the timing of feeding, the type of feed to be fed, and the purpose of feeding.

본 출원의 용어, "사료"는 개체가 먹고, 섭취하며, 소화시키기 위한 또는 이에 적당한 임의의 천연 또는 인공 규정식, 한끼식 등 또는 상기 한끼식의 성분을 의미한다. 상기 사료의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용되는 사료를 사용할 수 있다. 상기 사료의 비제한적인 예로는, 곡물류, 근과류, 식품 가공 부산물류, 조류, 섬유질류, 제약 부산물류, 유지류, 전분류, 박 류 또는 곡물 부산물류 등과 같은 식물성 사료; 단백질류, 무기물류, 유지류, 광물성류, 유지류, 단세포 단백질류, 동물성 플랑크톤류 또는 음식물 등과 같은 동물성 사료를 들 수 있다. 이들은 단독으로 사용되거나 2종 이상을 혼합하여 사용될 수 있다.As used herein, the term “feed” refers to any natural or artificial diet, meal plan, etc., or component of said meal plan, intended for or suitable for eating, ingestion and digestion by an individual. The type of feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products; Animal feed such as proteins, inorganic materials, oils, mineral oils, oils, single cell proteins, zooplankton, or food may be mentioned. These may be used alone or in combination of two or more.

본 발명은 또한 상기 서술한 조성물을 젖소에 급여하는 단계를 포함하는 젖소의 유중 단백질의 함량을 증가시키는 방법을 제공할 수 있다. The present invention can also provide a method for increasing the protein content in cow's milk, comprising the step of feeding the above-described composition to the cow.

이하, 실시예를 통하여 본 발명을 보다 상세하게 설명한다. Hereinafter, the present invention will be described in more detail through examples.

실시예 1. 유선상피세포에서 대사촉진 물질과 필수아미노산의 조합이 유단백질 합성에 미치는 영향의 확인 Example 1. Confirmation of the effect of a combination of a metabolism promoting substance and an essential amino acid on milk protein synthesis in mammary epithelial cells

실시예 1-1. 유선상피세포에서 대사촉진 물질과 필수아미노산의 조합이 Extra cellular 단백질 농도에 미치는 영향의 확인Example 1-1. Identification of the Effects of Combinations of Metabolites and Essential Amino Acids on Extra Cellular Protein Concentrations in Mammary Epithelial Cells

유선상피세포에서 대사촉진 물질과 필수아미노산의 조합이 유단백질 합성에 미치는 영향을 확인하기 위해, 다양한 필수아미노산, 대사촉진 물질 및 이들의 조합을 유선상피세포(immortalized bovine mammary epithelial cell line; MAC-T (University of Vermont, Burlington, VT, USA))에 처리하여 Extra cellular 단백질 농도를 확인하였다. 유선상피세포(MAC-T)를 성장 배지(10% FBS, 100 units/mL penicillin/streptomycin, 50 μg/mL gentamycin, 5 μg/mL insulin 및 1 μg/mL hydrocortisone를 포함하는 DMEM/F12 배지)에서 배양한 후, plate의 80% 정도 성장하면, 다른 plate로 계대배양 하였다. Extra cellular 단백질 농도의 변화를 시험하기 위해 이를 6 well에 계대배양 한 후, MAC-T cell이 100% confluence 상태로 성장했을 때, 대조구로서 분화 미디어(DMEM/F12 (Gibco) + 10% Fetal bovine serum (Gibco) + 1% Penicillin/streptomycin (Hyclone) + 0.1% Gentamycin (Sigma-aldrich) + 0.1% Hydrocortisone (Sigma-aldrich) + 0.1% Insulin (Sigma-aldrich) + 0.1% Prolactin (Sigma-aldrich))와 처리구로서 각 필수아미노산이 농도별 첨가된 분화 미디어를 분주 후 72시간 동안 배양하였다. 상기 처리군은 하기와 같았다: 0.3 mM (acetate); 0.6 mM (Met, Lys, Phe), 0.9 mM (Trp, glucose), 1.5 mM (Ile, t-10, c-12 CLA). 72시간 배양 후 세포 외 단백질을 추출하고, BCA protein assay kit (Thermo Scientific, South Logan, UT, USA)를 이용하여 분석하였다. 데이터 평균값에 따른 통계적 유의차는 SPSS 통계 소프트웨어(SPSS Inc., Chicago, IL, USA)의 Tucky's HSD test로 실시하였다. 모든 실험은 3 반복으로 진행되었으며, p < 0.05에서 유의하다고 판단하였다. 도 1과 같이 Extracellular 단백질 농도는 모든 처리구 중 Trp-Phe 처리구에서 가장 증가하는 것으로 나타났다. In order to examine the effect of the combination of metabolites and essential amino acids on milk protein synthesis in mammary epithelial cells, various essential amino acids, metabolites and their combinations were tested in mammary epithelial cells (immortalized bovine mammary epithelial cell line; MAC-T ( University of Vermont, Burlington, VT, USA)) to confirm the extra cellular protein concentration. Mammary epithelial cells (MAC-T) were cultured in growth medium (DMEM/F12 medium containing 10% FBS, 100 units/mL penicillin/streptomycin, 50 µg/mL gentamycin, 5 µg/mL insulin, and 1 µg/mL hydrocortisone). After culturing, when about 80% of the plate had grown, it was subcultured to another plate. To test the change in extra cellular protein concentration, after subculturing them in 6 wells, when MAC-T cells were grown to 100% confluence, differentiation media (DMEM/F12 (Gibco) + 10% Fetal bovine serum as a control) (Gibco) + 1% Penicillin/streptomycin (Hyclone) + 0.1% Gentamycin (Sigma-aldrich) + 0.1% Hydrocortisone (Sigma-aldrich) + 0.1% Insulin (Sigma-aldrich) + 0.1% Prolactin (Sigma-aldrich)) and As a treatment group, differentiation media in which each essential amino acid was added at different concentrations were cultured for 72 hours after dispensing. The treatment groups were as follows: 0.3 mM (acetate); 0.6 mM (Met, Lys, Phe), 0.9 mM (Trp, glucose), 1.5 mM (Ile, t-10, c-12 CLA). After 72 hours of culture, extracellular proteins were extracted and analyzed using BCA protein assay kit (Thermo Scientific, South Logan, UT, USA). Statistically significant differences according to the mean values of the data were performed by Tucky's HSD test of SPSS statistical software (SPSS Inc., Chicago, IL, USA). All experiments were conducted in 3 repetitions, and it was judged to be significant at p < 0.05. As shown in Figure 1, the extracellular protein concentration was found to increase the most in the Trp-Phe treatment group among all treatment groups.

실시예 1-2. 유선상피세포에서 대사촉진 물질과 필수아미노산의 조합이 β-casein 발현량의 효과 확인 Example 1-2. Confirmation of the effect of β-casein expression level of the combination of metabolism promoting substances and essential amino acids in mammary epithelial cells

유선상피세포에서 대사촉진 물질과 필수아미노산의 조합이 유단백질 합성에 미치는 영향을 확인하기 위해, 다양한 필수아미노산, 대사촉진 물질 및 이들의 조합을 유선상피세포(immortalized bovine mammary epithelial cell line; MAC-T (University of Vermont, Burlington, VT, USA))에 처리하여 β-casein 발현량를 확인하였다. 유선상피세포(MAC-T)를 성장 배지(10% FBS, 100 units/mL penicillin/streptomycin, 50 μg/mL gentamycin, 5 μg/mL insulin 및 1 μg/mL hydrocortisone를 포함하는 DMEM/F12 배지)에서 배양한 후, plate의 80% 정도 성장하면, 다른 plate로 계대배양 하였다. Extracellular 단백질 농도의 변화를 시험하기 위해 이를 6 well에 계대배양 한 후, MAC-T cell이 100% confluence 상태로 성장했을 때, 대조구로서 분화 미디어(DMEM/F12 (Gibco) + 10% Fetal bovine serum (Gibco) + 1% Penicillin/streptomycin (Hyclone) + 0.1% Gentamycin (Sigma-aldrich) + 0.1% Hydrocortisone (Sigma-aldrich) + 0.1% Insulin (Sigma-aldrich) + 0.1% Prolactin (Sigma-aldrich))와 처리구로서 각 필수아미노산이 농도별 첨가된 분화 미디어를 분주 후 72시간 동안 배양하였다. 상기 처리군은 하기와 같았다: 0.3 mM (acetate); 0.6 mM (Met, Lys, Phe), 0.9 mM (Trp, glucose), 1.5 mM (Ile, t-10, c-12 CLA). 72시간 배양 후 MAC-T 세포에 TRI reagent(MRC), chloroform, isopropanol, 80% ethanol, 100% ethanol을 차례로 처리하여 RNA를 추출하였고, 추출한 RNA에서 cDNA를 합성하였다. cDNA를 이용하여 beta-casein 합성 유전자인 bCSNB의 발현을 확인하기 위해 AccuPower 2X GreenStar qPCR MasterMix(Bioneer)로 RT-PCR을 수행하였다. 이때 사용한 bCSNB 프라이머는 forward(5'-GAGCCTGACTCTCACTGATGTTGAA-3'), reverse(5'-GACAGCACGGACTGAGGAGGAA-3')이고, bBActin 프라이머는 forward(5'-GCATGGAATCCTGCGGC-3'), reverse(5'-GTAGAGGTCCTTGCGGATGT-3')이며, bUXT 프라이머는 forward(5'-GCGCTACGAGGCTTTCATCT-3'), reverse(3'-CCAAGGGCCACATAGATCCG-5')이다. RT-PCR은 95℃에서 3분간 변성시킨 후 95℃에서 10초 반응시키는 조건으로 40회 수행하고, 55℃ 내지 65℃에서 30초, 72℃에서 30초간 반응시켰다. β-casein 발현량은 모든 처리구 중 Acetate-Trp-Phe 처리구에서 가장 높은 발현량을 보였다 (도 2). 따라서 MAC-T cell에서 가장 효율적으로 유단백질을 생산하는 조합은 Trp-Phe와 Acetate-Trp-Phe으로 이들 조합을 in vivo 실험을 통하여 확인하였다. In order to examine the effect of the combination of metabolites and essential amino acids on milk protein synthesis in mammary epithelial cells, various essential amino acids, metabolites and their combinations were tested in mammary epithelial cells (immortalized bovine mammary epithelial cell line; MAC-T ( University of Vermont, Burlington, VT, USA)) to confirm the β-casein expression level. Mammary epithelial cells (MAC-T) were cultured in growth medium (DMEM/F12 medium containing 10% FBS, 100 units/mL penicillin/streptomycin, 50 µg/mL gentamycin, 5 µg/mL insulin, and 1 µg/mL hydrocortisone). After culturing, when about 80% of the plate had grown, it was subcultured to another plate. To test the change in extracellular protein concentration, after subculturing them in 6 wells, when MAC-T cells were grown to 100% confluence, differentiation media (DMEM/F12 (Gibco) + 10% Fetal bovine serum ( Gibco) + 1% Penicillin/streptomycin (Hyclone) + 0.1% Gentamycin (Sigma-aldrich) + 0.1% Hydrocortisone (Sigma-aldrich) + 0.1% Insulin (Sigma-aldrich) + 0.1% Prolactin (Sigma-aldrich)) and treatment Differentiation media in which each essential amino acid was added at different concentrations were cultured for 72 hours after dispensing. The treatment groups were as follows: 0.3 mM (acetate); 0.6 mM (Met, Lys, Phe), 0.9 mM (Trp, glucose), 1.5 mM (Ile, t-10, c-12 CLA). After 72 hours of culture, MAC-T cells were sequentially treated with TRI reagent (MRC), chloroform, isopropanol, 80% ethanol, and 100% ethanol to extract RNA, and cDNA was synthesized from the extracted RNA. RT-PCR was performed with AccuPower 2X GreenStar qPCR MasterMix (Bioneer) to confirm the expression of bCSNB, a beta-casein synthesis gene, using cDNA. The bCSNB primers used at this time were forward (5'-GAGCTGACTCTCACTGATGTTGAA-3') and reverse (5'-GACAGCACGGACTGAGGAGGAA-3'), and the bBActin primers were forward (5'-GCATGGAATCCTGCGGC-3') and reverse (5'-GTAGAGGTCCTTGCGGATGT-3 '), and bUXT primers are forward (5'-GCGCTACGAGGCTTTCATCT-3') and reverse (3'-CCAAGGGCCACATAGATCCG-5'). RT-PCR was denatured at 95 ° C. for 3 minutes and then reacted 40 times at 95 ° C. for 10 seconds, followed by reaction at 55 ° C. to 65 ° C. for 30 seconds and at 72 ° C. for 30 seconds. Among all treatment groups, the expression level of β-casein was the highest in the Acetate-Trp-Phe treatment group (FIG. 2). Therefore, Trp-Phe and Acetate-Trp-Phe were the most efficient milk protein-producing combinations in MAC-T cells, and these combinations were confirmed through in vivo experiments.

실시예 2. Example 2. 젖소의 경정맥으로 주입된 Tryptophan(Trp), Phenylalanine(Phe) 및 Acetate의 조합의 유단백질 합성 효과의 확인 Confirmation of milk protein synthesis effect of the combination of Tryptophan (Trp), Phenylalanine (Phe) and Acetate injected into the jugular vein of cows

in vitro 결과로 Trp-Phe 및 Acetate-Trp-Phe 조합이 유단백 합성에 가장 효과적인 조합인 것으로 결정되어 이를 실제 젖소 착유우에게 적용하여 유단백 증진에의 효과를 확인하였다. 본 연구는 건국대학교 동물실험윤리위원회에 승인 하에 실시하였다. 홀스타인 (Holstein-Friesian) 착유우 젖소 중 5두를 사양 실험에 사용하였다. Total mixed ration (TMR), concentrate, hay를 NRC (2001)의 에너지 및 단백질 요구량에 맞춰 하루 두 번 (0830 and 1700 h) 급여하였다 [표 1].As a result of the in vitro results, it was determined that the Trp-Phe and Acetate-Trp-Phe combinations were the most effective combinations for milk protein synthesis, and this was applied to actual milking cows to confirm the effect of milk protein enhancement. This study was conducted under the approval of the Animal Research Ethics Committee of Konkuk University. Five Holstein-Friesian milking cows were used for feeding experiments. Total mixed ration (TMR), concentrate, and hay were fed twice a day (0830 and 1700 h) according to the energy and protein requirements of NRC (2001) [Table 1].

사료의 화학 성분 Chemical composition of feed Chemical compositions, g/d of DMChemical compositions, g/d of DM TMRTMR Concentrates Concentrates Roughage Roughage DM (%)DM (%) 61.9161.91 88.8488.84 85.8885.88 ― % of DM ― ― % of DM ― Crude proteinCrude protein 9.799.79 20.3320.33 9.619.61 Ether extractEther extract 1.791.79 3.103.10 3.713.71 Crude fiber Crude fiber 13.0213.02 7.257.25 15.6815.68 Crude ashCrude ash 5.095.09 6.946.94 4.864.86 NDFNDF 34.7834.78 24.3324.33 29.5229.52 ADFADF 18.2518.25 12.0512.05 18.0818.08 Ca Ca 0.590.59 0.690.69 0.520.52 P P 0.280.28 0.560.56 0.270.27 NEL (Mcal/kg of DM) 3 NE L (Mcal/kg of DM) 3 2.152.15 2.102.10 2.152.15 Amino acids Amino acids TryptophanTryptophan 0.100.10 0.210.21 0.15* 0.15 * ThreonineThreonine 0.360.36 0.670.67 0.24* 0.24 * SerineSerine 0.420.42 0.870.87 -- ProlineProline 0.530.53 1.061.06 -- ValineValine 0.430.43 0.790.79 0.26* 0.26 * IsoleucineIsoleucine 0.270.27 0.570.57 0.30* 0.30 * LeucineLeucine 0.600.60 1.421.42 0.43* 0.43 * TyrosineTyrosine 0.160.16 0.440.44 -- MethionineMethionine 0.090.09 0.200.20 0.25* 0.25 * CysteineCysteine 0.210.21 0.460.46 0.06* 0.06 * LysineLysine 0.400.40 0.590.59 0.34* 0.34 * GlycineGlycine 0.390.39 0.800.80 -- AlanineAlanine 0.480.48 0.940.94 -- ArginineArginine 0.530.53 1.141.14 0.28* 0.28 * Glutamic acidGlutamic acid 1.351.35 3.193.19 -- Aspartic acidAspartic acid 0.790.79 1.511.51 -- HistidineHistidine 0.160.16 0.370.37 0.21* 0.21 * PhenylalaninePhenylalanine 0.360.36 0.760.76 0.46* 0.46 * *Book Value (from NRC, 2001)*Book Value (from NRC, 2001)

5X5 Latin square design을 이용하여 휴지기 5일과 실험 6일을 반복하였다. control(saline), Trp, Phe, Trp-Phe, Acetate-Trp-Phe의 총 5개 시험구로 시험을 수행하였다. In vitro 연구에서 결정된 0.9 mM의 Trp, 0.6 mM의 Phe, 0.3 mM의 acetate를 분자량 대비 gram으로 환산하여 계산하였다. 아미노산과 acetate powder를 삼투압 및 pH 영향을 받지 않도록 조절하여 생리식염수에 첨가하였다. treatment solution은 10:00h에 경정맥 카테터를 이용하여 천천히 주입하였다. 공시 동물(총 5두)의 개체별 오전(03:00 h) 및 오후(15:00 h)에 착유된 유량을 매일 기록하였다. 공시 동물(총 5두)의 실험 기간 매일 오전(0300 h) 및 오후(1500 h)에 착유 된 원유를 pooling하여 우유 내 일반성분[milk fat, milk protein, lactose, solid-not fat(SnF), somatic cell count(SCC), milk urea nitrogen(MUN), acetone, beta-hydroxybutyrate(BHB), beta-casein, Mono- and Poly-unsaturated fatty acids, Saturated fatty acid]을 분석하였다. 사료 섭취량 분석은 매일 아침 사료 급이 전 남은 사료의 양을 기록하여 일당 섭취량을 분석하였다. 경정맥 카테터를 통해 실험 1, 3, 5일째에 treatment solution 주입 전 혈액을 채취하였다. 각 분석을 통해 얻어진 일별 성적들은 기간별로 평균값을 계산하였다. 데이터 평균값에 따른 통계적 유의차는 SAS 소프트웨어 v. 9.4 (SAS Institute, Cary, NC, USA)의 one-way ANOVA를 사용하였다. 표 2는 대사 촉진 물질 및 아미노산 급여에 의한 젖소의 우유 생산량 및 우유의 구성의 영향을 나타낸 것이다. 착유우의 경정맥 카테터를 이용해 아미노산 및 아세트산을 처리한 결과, 사료 섭취량과 유량에는 변화가 없었다 [표 2]. 유단백질량 (milk protein yield)이 처리구에서 유의적으로 증가하였다. Trp 단독 처리구가 모든 시험구를 통틀어 가장 증가했으며, 이후로 Acetate-Trp-Phe, Trp-Phe, Control, Phe 순으로 증가하였다. 유중 체세포 수는 모든 처리구에서 대조구 대비 감소함을 보여, 첨가물들이 동물 체내에서 염증 반응을 억제시킨 것으로 판단되었다. Using a 5X5 Latin square design, 5 days of resting period and 6 days of experiment were repeated. The test was performed with a total of 5 test plots: control (saline), Trp, Phe, Trp-Phe, and Acetate-Trp-Phe. The 0.9 mM Trp, 0.6 mM Phe, and 0.3 mM acetate determined in the in vitro study were calculated by converting the molecular weight to grams. Amino acids and acetate powder were added to the physiological saline solution after being adjusted so as not to be affected by osmotic pressure and pH. The treatment solution was slowly injected using a jugular vein catheter at 10:00 h. Milking yields were recorded daily in the morning (03:00 h) and afternoon (15:00 h) of each animal tested (5 heads in total). Common components in milk [milk fat, milk protein, lactose, solid-not fat (SnF), solid-not fat (SnF), somatic cell count (SCC), milk urea nitrogen (MUN), acetone, beta-hydroxybutyrate (BHB), beta-casein, mono- and poly-unsaturated fatty acids, and saturated fatty acids] were analyzed. For the analysis of feed intake, the amount of feed remaining before feeding was recorded every morning to analyze daily intake. Blood was collected through a jugular vein catheter before injection of the treatment solution on the 1st, 3rd, and 5th days of the experiment. The daily scores obtained through each analysis were averaged by period. Statistically significant differences according to data mean values were determined using SAS software v. One-way ANOVA of 9.4 (SAS Institute, Cary, NC, USA) was used. Table 2 shows the effect of milk yield and milk composition of dairy cows by feeding metabolites and amino acids. As a result of amino acid and acetic acid treatment using the jugular vein catheter of milking cows, there was no change in feed intake and milk yield [Table 2]. Milk protein yield was significantly increased in the treatment group. The Trp-only treatment group increased the most among all test groups, followed by an increase in the order of Acetate-Trp-Phe, Trp-Phe, Control, and Phe. The number of somatic cells in oil showed a decrease compared to the control group in all treatment groups, and it was judged that the additives suppressed the inflammatory response in the animal body.

표 3은 1, 3 및 5 일에 아미노산의 급여에 젖소의 CBC에 미치는 영향을 나타낸 것이다. 경정맥을 통한 아미노산 및 아세트산의 첨가는 혈액 성상에 아무런 영향을 보이지 않았다 [표 3]. 시험축들의 건강 상태에는 문제가 없는 것으로 확인되었다. Table 3 shows the effect on CBC of cows fed with amino acids on days 1, 3 and 5. Addition of amino acids and acetic acid via jugular vein did not show any effect on blood properties [Table 3]. It was confirmed that there was no problem with the health condition of the test axes.

경정맥을 통해 아미노산과 아세트산을 주입한 24시간 후 혈중 아미노산 함량은 표 4 및 표 5와 같다. 시험 기간 동안 처리구 간 아미노산 조성에는 유의적인 변화가 없었다 (표 4). 이는 경정맥 주입 24시간 후의 결과이므로 주입된 아미노산들이 이미 단백질 합성 및 체내 대사에 이용된 것으로 사료된다. 각 처리구별 실험 1, 3, 5일 째의 아미노산 함량에는 큰 차이가 없었다 (표 5). 대조구에서 Ser, Trp-Phe 처리구에서 Val에 유의적 차이가 있었고, Phe 처리구에서 Asp, Acetate-Trp-Phe 처리구에서 Trp에 변화하는 경향이 보였다. Phe 처리구에서 Asp는 day 3에 증가했다가 day 5에 다시 감소하는 패턴을 보였다. Phe가 amide를 포함한 아미노산의 장관 이동을 증가시키기 때문인 것으로 사료된다. Val은 Trp-Phe 처리구에 의해 day 3에 감소했다가 day 5에 증가하는 패턴을 보이는데 (p = 0.02), 이는 Trp, Phe, Val이 같은 amino acid transporter (LAT1)을 경유하기 때문에 경쟁에 의한 일부 효과일 수 있다. 혈중 Trp는 day 3에 증가했다가 day 5에 다시 감소하는 경향을 나타내었다. 아미노산의 수송은 에너지를 필요로 하는데, 혈중으로 추가된 acetate로부터 에너지를 만들어 이용하는 시간상의 문제가 이런 변화를 나타낸 것으로 사료된다. Tables 4 and 5 show amino acid contents in blood 24 hours after amino acid and acetic acid injection through jugular vein. There was no significant change in amino acid composition between treatments during the test period (Table 4). Since this is the result after 24 hours of intravenous injection, it is considered that the injected amino acids have already been used for protein synthesis and metabolism in the body. There was no significant difference in amino acid content on the 1st, 3rd, and 5th days of each treatment group (Table 5). There was a significant difference in Val between Ser and Trp-Phe treatments in the control group, Asp in the Phe treatment group, and a change in Trp in the Acetate-Trp-Phe treatment group. In the Phe treatment group, Asp increased on day 3 and decreased again on day 5. This is thought to be because Phe increases intestinal transport of amino acids, including amides. Val showed a pattern of decreasing on day 3 and increasing on day 5 by Trp-Phe treatment (p = 0.02), which is partly due to competition because Trp, Phe, and Val pass through the same amino acid transporter (LAT1). could be an effect. Blood Trp increased on day 3 and then decreased again on day 5. The transportation of amino acids requires energy, and it is thought that the time problem of making and using energy from acetate added to the blood showed this change.

대사 촉진 물질 및 아미노산의 급여에 따른 젖소의 혈액 아미노산 조성의 분석 Analysis of the blood amino acid composition of dairy cows according to the feeding of metabolites and amino acids Amino acidsAmino acids ControlControl TrpTrp PhePhe Trp-PheTrp-Phe AcetateAcetate
+ +
Trp-PheTrp-Phe SEMSEM pp -value-value Aspartic AcidAspartic Acid 12.4612.46 15.1415.14 14.7314.73 13.6513.65 18.1518.15 7.927.92 0.9900.990 ThreonineThreonine 102.48102.48 77.3277.32 85.1885.18 71.4371.43 95.9995.99 19.7519.75 0.7910.791 SerineSerine 105.72105.72 61.0061.00 100.46100.46 109.80109.80 91.8391.83 14.9914.99 0.1910.191 AsparagineAsparagine 105.52105.52 132.08132.08 67.5267.52 134.88134.88 104.05104.05 30.0430.04 0.5280.528 Glutamic AcidGlutamic Acid 289.54289.54 258.28258.28 270.92270.92 267.23267.23 259.85259.85 24.8524.85 0.9040.904 GlycineGlycine 345.54345.54 350.85350.85 353.29353.29 322.84322.84 352.12352.12 21.7921.79 0.8480.848 AlanineAlanine 289.58289.58 279.12279.12 277.24277.24 266.20266.20 280.07280.07 19.7619.76 0.9470.947 ValineValine 14.5114.51 14.3014.30 17.5317.53 22.9122.91 13.9613.96 3.393.39 0.3220.322 CystineCystine 41.4641.46 37.5937.59 41.8441.84 42.4842.48 40.1940.19 7.857.85 0.9930.993 MethionineMethionine 99.2099.20 100.70100.70 98.2198.21 75.3275.32 102.24102.24 22.6622.66 0.9100.910 PhenylalaninePhenylalanine 95.7995.79 91.0891.08 99.3599.35 95.6795.67 93.6593.65 22.2322.23 0.9990.999 IsoleucineIsoleucine 25.5725.57 28.3428.34 30.7630.76 26.9226.92 27.2827.28 8.198.19 0.9940.994 LeucineLeucine 20.6420.64 30.7330.73 28.1228.12 23.6023.60 20.0820.08 6.436.43 0.7180.718 TyrosineTyrosine 16.3016.30 21.2221.22 21.2121.21 24.7124.71 21.9321.93 5.595.59 0.8780.878 HistidineHistidine 55.6555.65 57.7457.74 56.3556.35 48.1748.17 61.9361.93 6.346.34 0.6530.653 TryptophanTryptophan 10.3710.37 18.5518.55 19.6619.66 17.5917.59 14.0414.04 4.834.83 0.6520.652 LysineLysine 83.1283.12 81.3581.35 83.8183.81 77.4777.47 74.7874.78 4.114.11 0.4910.491 ArginineArginine 105.36105.36 99.3699.36 104.95104.95 127.21127.21 105.02105.02 14.7614.76 0.7100.710

1일 내지 5일 동안 대사 촉진 물질 및 아니노산을 급여한 젖소의 혈액 아미노산 조성의 확인Identification of blood amino acid composition of dairy cows fed with metabolites and aninoic acid for 1 to 5 days Amino acidsAmino acids ControlControl TrpTrp PhePhe Trp-PheTrp-Phe Acetate+Trp-PheAcetate+Trp-Phe Aspartic Acid Aspartic Acid D1D1 5.705.70 2.602.60 7.557.55 30.1330.13 15.1315.13 D3D3 26.2326.23 36.5036.50 28.7228.72 4.194.19 43.1043.10 D5D5 5.445.44 6.336.33 7.927.92 8.248.24 5.215.21 SEMSEM 7.137.13 8.208.20 7.367.36 8.788.78 8.098.09 pp -value-value 0.420.42 0.190.19 0.440.44 0.450.45 0.370.37 ThreonineThreonine D1D1 83.1783.17 63.5563.55 84.0584.05 65.9265.92 80.2780.27 D3D3 108.82108.82 93.0593.05 96.6596.65 51.4951.49 117.98117.98 D5D5 115.46115.46 75.3575.35 74.8474.84 58.5858.58 118.47118.47 SEMSEM 16.2416.24 16.1116.11 16.5716.57 14.2814.28 16.6116.61 pp -Value-Value 0.720.72 0.780.78 0.880.88 0.930.93 0.540.54 SerineSerine D1D1 151.90a 151.90 a 101.23101.23 112.17112.17 77.3777.37 135.91135.91 D3D3 61.33b 61.33b 25.4725.47 120.32120.32 161.99161.99 43.3743.37 D5D5 103.93ab 103.93ab 56.2956.29 68.8768.87 75.2575.25 106.04106.04 SEMSEM 15.3215.32 13.8713.87 17.2817.28 21.2121.21 15.5515.55 pp -Value-Value 0.040.04 0.690.69 0.460.46 0.180.18 0.190.19 AsparagineAsparagine D1D1 90.5090.50 121.60121.60 63.8363.83 216.74216.74 101.20101.20 D3D3 136.33136.33 64.9364.93 105.24105.24 74.7574.75 80.7180.71 D5D5 89.7489.74 209.70209.70 33.4833.48 129.11129.11 81.1381.13 SEMSEM 27.5327.53 30.0730.07 13.6813.68 43.1243.12 23.3623.36 pp -Value-Value 0.760.76 0.140.14 0.090.09 0.420.42 0.390.39 Glutamic AcidGlutamic Acid D1D1 311.39311.39 239.85239.85 259.07259.07 265.13265.13 286.44286.44 D3D3 259.50259.50 307.62307.62 331.16331.16 266.91266.91 216.60216.60 D5D5 297.74297.74 227.38227.38 222.54222.54 253.36253.36 303.02303.02 SEMSEM 12.2712.27 20.5820.58 28.0728.07 31.9031.90 23.4123.41 pp -Value-Value 0.210.21 0.240.24 0.290.29 0.990.99 0.540.54 GlycineGlycine D1D1 356.13356.13 375.76375.76 402.90402.90 382.07382.07 362.08362.08 D3D3 341.44341.44 355.66355.66 392.27392.27 287.92287.92 384.06384.06 D5D5 339.05339.05 321.13321.13 264.71264.71 264.96264.96 328.79328.79 SEMSEM 16.7516.75 20.1920.19 30.0830.08 36.3536.35 19.8019.80 pp -Value-Value 0.920.92 0.570.57 0.110.11 0.390.39 0.110.11 AlanineAlanine D1D1 317.49317.49 295.04295.04 303.87303.87 304.35304.35 299.61299.61 D3D3 278.16278.16 266.32266.32 302.66302.66 243.53243.53 291.13291.13 D5D5 273.09273.09 275.98275.98 225.19225.19 231.03231.03 264.11264.11 SEMSEM 12.6312.63 14.0714.07 18.9718.97 29.5029.50 15.5415.54 pp -Value-Value 0.310.31 0.730.73 0.150.15 0.580.58 0.130.13 ValineValine D1D1 12.4112.41 13.2013.20 7.777.77 22.86ab 22.86 ab 8.418.41 D3D3 13.9013.90 11.1611.16 19.3519.35 8.02b 8.02b 15.3415.34 D5D5 17.2217.22 18.5218.52 25.4525.45 43.04a 43.04a 12.7012.70 SEMSEM 3.173.17 2.482.48 3.623.62 5.505.50 2.282.28 pp -Value-Value 0.840.84 0.490.49 0.130.13 0.020.02 0.410.41 CystineCystine D1D1 41.5741.57 33.9733.97 28.4128.41 41.9741.97 34.4034.40 D3D3 34.9934.99 36.6036.60 51.5951.59 43.1643.16 40.6040.60 D5D5 47.8247.82 42.2042.20 45.5245.52 55.7855.78 34.9134.91 SEMSEM 5.985.98 5.285.28 5.155.15 5.775.77 4.744.74 pp -Value-Value 0.710.71 0.830.83 0.170.17 0.610.61 0.960.96 MethionineMethionine D1D1 92.1492.14 81.0381.03 95.6595.65 64.6864.68 107.12107.12 D3D3 117.37117.37 111.96111.96 125.20125.20 78.6678.66 148.86148.86 D5D5 88.0988.09 109.12109.12 73.7973.79 60.9260.92 99.8699.86 SEMSEM 14.1914.19 16.4616.46 15.0015.00 14.4014.40 15.0915.09 pp -Value-Value 0.690.69 0.730.73 0.400.40 0.890.89 0.650.65 PhenylalaninePhenylalanine D1D1 81.8681.86 86.6186.61 86.0086.00 88.4588.45 57.2257.22 D3D3 91.9391.93 96.1496.14 101.19101.19 87.0087.00 78.1578.15 D5D5 113.59113.59 90.5090.50 110.86110.86 135.83135.83 95.9895.98 SEMSEM 14.1514.15 17.3017.30 11.1511.15 16.7216.72 12.1412.14 pp -Value-Value 0.680.68 0.980.98 0.790.79 0.450.45 0.780.78 IsoleucineIsoleucine D1D1 28.2228.22 22.3722.37 14.3214.32 26.2726.27 10.4310.43 D3D3 21.3621.36 29.2829.28 33.9633.96 18.8118.81 26.0226.02 D5D5 27.1227.12 33.3933.39 43.9943.99 47.8447.84 29.1029.10 SEMSEM 6.176.17 5.855.85 20.8120.81 7.587.58 5.435.43 pp -Value-Value 0.900.90 0.770.77 0.590.59 0.320.32 0.120.12 LeucineLeucine D1D1 26.3426.34 31.3831.38 17.0517.05 25.4025.40 12.3712.37 D3D3 13.1913.19 21.2821.28 37.7037.70 15.0215.02 34.4934.49 D5D5 22.4022.40 39.5239.52 29.6229.62 40.0740.07 15.0715.07 SEMSEM 5.375.37 6.306.30 5.275.27 8.218.21 4.894.89 pp -Value-Value 0.110.11 0.530.53 0.290.29 0.530.53 0.210.21 TyrosineTyrosine D1 D1 20.6320.63 24.0824.08 13.8813.88 25.0825.08 24.4624.46 D3 D3 15.9315.93 17.9617.96 19.3319.33 16.6016.60 26.3526.35 D5 D5 12.3312.33 21.6321.63 30.4230.42 31.4131.41 21.2121.21 SEMSEM 3.203.20 4.354.35 4.854.85 5.605.60 3.323.32 pp -Value-Value 0.610.61 0.870.87 0.390.39 0.620.62 0.790.79 HistidineHistidine D1D1 62.7662.76 61.6461.64 66.4566.45 55.2255.22 62.0662.06 D3D3 49.2149.21 55.6855.68 50.1150.11 51.1751.17 60.4860.48 D5D5 54.9954.99 55.9055.90 52.5152.51 51.8451.84 59.0959.09 SEMSEM 4.314.31 2.652.65 5.575.57 7.687.68 6.326.32 pp -Value-Value 0.470.47 0.620.62 0.470.47 0.980.98 0.260.26 TryptophanTryptophan D1D1 11.16 11.16 10.9310.93 14.4114.41 13.8313.83 13.8713.87 D3D3 10.2710.27 15.7715.77 18.4918.49 12.7512.75 21.9321.93 D5D5 9.679.67 28.9628.96 26.0826.08 31.1231.12 10.4810.48 SEMSEM 1.111.11 5.195.19 5.265.26 5.995.99 2.082.08 pp -Value-Value 0.880.88 0.370.37 0.690.69 0.430.43 0.080.08 LysineLysine D1D1 85.4785.47 82.9582.95 85.0385.03 83.1983.19 74.8074.80 D3D3 81.9881.98 85.7285.72 88.0188.01 61.1861.18 76.1076.10 D5D5 81.8981.89 75.3775.37 78.4078.40 83.8883.88 76.8476.84 SEMSEM 4.044.04 6.006.00 4.184.18 5.645.64 4.814.81 pp -Value-Value 0.930.93 0.790.79 0.660.66 0.200.20 0.560.56 ArginineArginine D1D1 114.88114.88 97.1597.15 101.01101.01 115.58115.58 108.10108.10 D3D3 92.7592.75 98.7098.70 111.37111.37 110.59110.59 99.4099.40 D5D5 108.46108.46 102.22102.22 102.48102.48 105.38105.38 123.90123.90 SEMSEM 6.166.16 6.416.41 6.876.87 13.9513.95 7.747.74 pp -Value-Value 0.340.34 0.950.95 0.820.82 0.960.96 0.940.94

in vitro 연구를 통해 가장 효율적으로 유단백질을 생산하는 조합은 Trp-Phe와 Acetate-Trp-Phe로 확인되었다. 상기 결과를 바탕으로 in vivo 실험을 수행하였다. in vivo를 통해 경정맥 카테터를 통한 착유우에 주입된 아미노산 및 아세트산은 젖소의 사료 섭취량 및 유량을 변화시키지 않으면서 유단백질량을 증가시켰다. 또한 대조구 대비 모든 처리구에서 체세포 수를 감소시켰으며, 이는 항염증 효과가 있는 것으로 사료된다. 혈중 아미노산 조성은 아미노산 첨가에 따라 큰 변화를 보이지 않았다. In vitro 세포 실험 결과를 in vivo에 적용하여 유단백질이 증가하는 것을 확인하였다.Through in vitro studies, the most efficient combination of milk protein production was identified as Trp-Phe and Acetate-Trp-Phe. Based on the above results, an in vivo experiment was performed. Amino acids and acetic acid injected in vivo into milking cows via jugular catheter increased milk protein content without altering cows' feed intake and yield. In addition, the number of somatic cells was reduced in all treatment groups compared to the control group, which is considered to have an anti-inflammatory effect. The amino acid composition in blood did not show a significant change according to the addition of amino acids. It was confirmed that milk protein increased by applying the in vitro cell test results to in vivo.

Claims (5)

페닐알라닌, 트립토판 및 아세트산을 2:3:1의 몰비로 포함하는, 착유 반추동물의 유중 단백질 합성 촉진용 조성물.A composition for promoting protein synthesis in the milk of milking ruminants, comprising phenylalanine, tryptophan and acetic acid in a molar ratio of 2:3:1. 제 1항에 있어서,
상기 유단백질은 베타-카제인(beta-casein)인, 착유 반추동물의 유중 단백질 합성 촉진용 조성물.According to claim 1,
The milk protein is beta-casein (beta-casein), a composition for promoting protein synthesis in the milk of milking ruminants. 제 1항 또는 제 2항의 조성물을 포함하는, 착유 반추동물의 유중 단백질 합성 촉진용 사료 첨가제. A feed additive for promoting protein synthesis in milk of milking ruminants, comprising the composition of claim 1 or 2. 제 1항 또는 제 2항의 조성물을 포함하는, 착유 반추동물의 유중 단백질 합성 촉진용 사료 조성물. A feed composition for promoting protein synthesis in milk of milking ruminants, comprising the composition of claim 1 or 2. 제 1항 또는 제 2항의 조성물을 젖소에 급여하는 단계; 를 포함하는, 젖소의 유중 단백질의 함량을 증가시키는 방법.Feeding the composition of claim 1 or 2 to cows; A method for increasing the content of protein in cow's milk, comprising a.
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