KR20230087856A - water-soluble Chicken breast hydrolysate with inhanced bioavailablity comprising increased specific imidazole peptides and preparation method for thereof - Google Patents
water-soluble Chicken breast hydrolysate with inhanced bioavailablity comprising increased specific imidazole peptides and preparation method for thereof Download PDFInfo
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- KR20230087856A KR20230087856A KR1020210176459A KR20210176459A KR20230087856A KR 20230087856 A KR20230087856 A KR 20230087856A KR 1020210176459 A KR1020210176459 A KR 1020210176459A KR 20210176459 A KR20210176459 A KR 20210176459A KR 20230087856 A KR20230087856 A KR 20230087856A
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- chicken breast
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
Description
본 발명은 닭가슴살을 가수분해 효소분해를 사용하여 MDM2 단백질과 P53-유사 펩타이드의 상호 작용을 억제하여 항증식 활성을 가지는 특정 이미다졸 펩타이드 함량이 증대 되고, 그 대사물들이 전부 포함된 수용성 닭가슴살 단백질 가수분해물에 관한 것으로, 상세하게는 닭가슴살을 pH 변동 없이 효소를 처리하여 수용성 단백질로의 전환하는 과정을 통해 특정 저분자 펩타이드 이미다졸 펩타이드를 포함하는 수용성단백질 가수분해물, 그 제조방법 및, 그 가수분해물의 용도에 관한 것이다.The present invention inhibits the interaction of MDM2 protein and P53-like peptide using hydrolysis of chicken breast to increase the content of a specific imidazole peptide having antiproliferative activity, and water-soluble chicken breast containing all of its metabolites It relates to a protein hydrolysate, and in detail, a water-soluble protein hydrolysate containing a specific low-molecular peptide imidazole peptide through the process of converting chicken breast meat into a water-soluble protein by treating enzymes without pH change, a method for preparing the same, and a hydrolyzate thereof It is about the use of the decomposition product.
본 발명 기술분야에서 단백질 가수분해물의 식품소재화를 위한 특허기술들은 국내 특허원 10-2008-7019149, 홍삼 가수분해물 10-2018-71954, 옥수수 글루텐 가수분해물 10-2009-44178, 동물식재료로서 수용성 돈혈 가수분해물은 10-2016-73727이 공지되었다. 본 발명이 목표로 하는 특정 단백질의 항증식 활성 펩타이드로서 아미다졸린 펩티드 화합물은 10-2008-7005155가 공지된 바 있다.In the technical field of the present invention, patented technologies for making protein hydrolysates into food materials are domestic patent application 10-2008-7019149, red ginseng hydrolysate 10-2018-71954, corn gluten hydrolysate 10-2009-44178, water-soluble pig blood as animal food material As for the hydrolyzate, 10-2016-73727 is known. An amidazoline peptide compound as an antiproliferative active peptide of a specific protein targeted by the present invention is known as 10-2008-7005155.
일반적으로 단백질은 음식을 통해 제공이되는 영양물질로 기원에 따라 식물성단백질, 동물성 단백질 및 곤충단백질등으로 분류된다. 단백질의 구조는 아미노산들이 펩타이드(peptide) 결합으로 결합된 거대 분자로 자연에 존재하는 단백질은 대략 20가지의 아미노산으로 구성되어 있다. 생물체 내에서 단백질은 만능이라 해도 좋을 정도로 다양하게 쓰인다. 생물체 내에서 일어나는 복잡한 화학반응을 일으키는 효소들은 대부분 단백질이다. 또한 근육과 같이 몸을 구성하는 역할 뿐만 아니라 면역에 중요한 항체도 단백질로 이루어졌으며, DNA 사슬을 감아 뭉치고 2차적 유전정보를 저장하는 히스톤도 단백질이고, 연골, 피부, 가죽, 털, 비늘 등을 이루는 주성분 콜라겐과 케라틴도 단백질이다. 또한 몇몇 호르몬까지도 모두 단백질이다. 따라서 단백질은 생물의 DNA 복제로부터 생물의 외형 형성에 이르기까지 생명의 정수(essence)이자 필수 요소, 생명체의 거의 모든 것으로 작용하는 물질. 생명 현상의 핵심으로 일컬어지는 센트럴 도그마가 단백질의 합성 과정이라는 부분에서부터 얼마나 중요한지 알 수 있는 생물학적 물질이다.In general, protein is a nutrient provided through food and is classified into vegetable protein, animal protein and insect protein according to its origin. The structure of a protein is a macromolecule in which amino acids are bound by peptide bonds, and proteins existing in nature are composed of about 20 amino acids. Within living organisms, proteins are used in so many ways that they can be said to be versatile. Enzymes that cause complex chemical reactions in living organisms are mostly proteins. In addition, antibodies that are important for immunity as well as the role of composing the body like muscles are made of proteins, and histones that wind and bind DNA chains and store secondary genetic information are also proteins. The main components collagen and keratin are also proteins. Also, even some hormones are all proteins. Therefore, protein is the essence and essential element of life, from the DNA replication of organisms to the formation of the external appearance of organisms, and substances that act as almost everything in life. It is a biological material that shows how important the central dogma, which is referred to as the core of life phenomena, is from the part of the protein synthesis process.
단백질이 다양한 기능을 발휘할 수 있는 이유는 20여 종의 다양한 아미노산들이 다양한 모양으로 엄청난 숫자가 모여서 무궁무진한 경우의 수로 다양한 결합을 하며 특이 구조를 다 만들어낼 수 있기 때문이다. 이렇게 만들어진 특수한 구조들은 일종의 나노머신과 같이 단백질의 특별한 기능을 수행한다. 단백질은 인체를 비롯하여 자연계의 모든 생물체를 구성하는 구성물질 중 필수성분이다.The reason why proteins can perform various functions is that a huge number of 20 kinds of amino acids in various shapes can be combined in an infinite number of cases and unique structures can be created. The special structures made in this way perform the special functions of proteins, like a kind of nanomachine. Protein is an essential component among the components that make up all organisms in the natural world, including the human body.
최근 질병치유를 위한 의약품이나 의료기술의 발달, 식품위생의 개선, 다양한 식품 영양소 섭취상태로 인간의 수명은 지속적으로 연장되고 있는 가운데, 특히 우리나라는 출산율의 저하와 함께 고령화가 빠른 속도로 진행되고 있다. 현재의 추세가 지속된다면 2029년에는 기대수명 90세 시대, 2045년에는 기대수명 100세 시대에 돌입할 것으로 전망되고, 고령화사회로의 진전은 노동생산성의 하락, 연금 및 건강보험의 고갈, 노인부양문제 등 다양한 사회적 이슈를 야기할 수 있으나, 고령친화식품산업에 있어서는 새로운 사업 기회가 될 수 있다. 특히 고령자를 대상으로 하는 건강기능식품 및 특수용도식품군에서 고령친화제품의 성장이 두드러질 것으로 예상되고 있다. 우리나라의 고령친화식품시장은 다양한 요인에 의하여 활성화되지 못하고 있어 정부에 의한 활성화 정책이 필요한 실정이다. 일반적으로 65세를 기점으로 탄수화물의 섭취량은 증가하며 단백질 섭취가 감소되고 있는 실정이다. 고령층 연령대가 증가할수록 에너지와 3대 영양소의 섭취량도 감소하였다. 영양소별 권장량 미만 섭취자 비율을 계산해 보니(65~69세기준), 단백질 22.2%, 칼슘 72.5%, 인 14.3%, 철 9.8%, 비타민A 49.2%, 티아민 22.0%, 리보플라빈 63.0%, 나이아신 40.4%, 비타민C 51.1% 등으로 나타나 영양섭취 부족/불균형 문제가 심각하다.Recently, with the development of medicines or medical technologies for disease treatment, improvement of food hygiene, and intake of various food nutrients, human lifespan is continuously extended. . If the current trend continues, it is expected to enter the age of 90 years of life expectancy in 2029 and 100 years of life expectancy in 2045. It can cause various social issues such as problems, but it can be a new business opportunity in the elderly-friendly food industry. In particular, the growth of senior-friendly products is expected to be remarkable in the health functional food and special purpose food groups targeting the elderly. The senior-friendly food market in Korea is not vitalized due to various factors, so the government's activation policy is needed. In general, from the age of 65, carbohydrate intake increases and protein intake decreases. As the age of the elderly increased, the intake of energy and three major nutrients also decreased. Calculating the proportion of people consuming less than the recommended amount for each nutrient (65-69 years of age), protein 22.2%, calcium 72.5%, phosphorus 14.3%, iron 9.8%, vitamin A 49.2%, thiamin 22.0%, riboflavin 63.0%, niacin 40.4% , vitamin C 51.1%, etc., and the problem of insufficient/imbalanced nutrition intake is serious.
대체로 닭가슴살은 수분함량이 74% 지방함량이 1.2% 단백질 함량이 23%로 수분과 지방을 제외한 나머지 부분은 모두 단백질로 구성이 되어 있다. 환자 및 노약자의 경우 면역력 증가를 위하여 신체에 필요한 필수아미노산을 모두 함유하고 있는 양질의 육류 단백질 섭취가 권장된다. 그러나 암이나 당뇨 등과 같은 질병과 투병하는 환자 및 노약자의 경우 약화된 신체적 상태 때문에 소화능력이 정상인에 비하여 현저히 낮아 소화 및 흡수율이 매우 낮은 것으로 보고되어 있다. 육류를 고온에서 구운 형태로 섭취할 경우는 구울 때 발생하는 heterocyclic amine류나 연기에서 발생하는 polycyclic aromatic hydrocarbon류, 고기에 함유된 돌연변이 유발물질인 N-nitro 화합물이 발생한다고 알려져 있다. 단백질을 식사 이외에 추가로 섭취할 경우 근력증진효과 및 운동능력개선에 대한 효과가 보고 되고있으며, 이에 따라 다양한 형태의 육류단백질 제품이 출시되어 시장규모가 점진적으로 증가 되는 상황이다.In general, chicken breast has a moisture content of 74%, a fat content of 1.2%, and a protein content of 23%, and all other parts except for moisture and fat are composed of protein. In the case of patients and the elderly, it is recommended to consume high-quality meat protein containing all essential amino acids necessary for the body to increase immunity. However, it is reported that the digestion and absorption rates are very low in the case of patients suffering from diseases such as cancer or diabetes, and in the case of the elderly, whose digestive capacity is significantly lower than that of normal people due to their weakened physical condition. It is known that when meat is consumed in a roasted form at high temperatures, heterocyclic amines generated during roasting, polycyclic aromatic hydrocarbons generated from smoke, and N-nitro compounds, which are mutagenic substances contained in meat, are generated. When protein is additionally consumed in addition to meals, the effect of increasing muscle strength and improving exercise capacity has been reported, and accordingly, various types of meat protein products have been released and the market size is gradually increasing.
단백질 가수분해물의 소화 및 흡수에 관한 연구에서 10명의 남자 노인을 대상으로 가수분해 단백질과 원래의 원형 단백질을 비교한 결과, 가수분해 단백질은 원래의 원형단백질과 비교하여 장에서의 소화흡수력이 촉진되고 식후의 아미노산의 이용도와 골격근내의 식이 아미노산의 유입이 증가 되는 것으로 나타났다. 그러나 단백질이 함유된 식품재료라고 하여도 모두 똑같은 것은 아니다. 주로 문제되는 것은, 단백질 자체의 경우 필수 아미노산의 비율이며 전체 단백질에서 필수아미노산 함량이 얼마나 높냐가 단백질의 질을 좌우하게 된다. 인간은 포유류이므로 포유류에 가까운 생물일수록 인간이 몸에서 만들어야 하는 단백질의 아미노산 비율과 흡사한 아미노산 비율을 가진 고기를 만든다. 대체로 동물성 단백질이 대두를 제외한 콩류의 식물성 단백질 유래의 두부, 간장 등 가공식품 보다는 필수아미노산 비율이 높다.In a study on the digestion and absorption of protein hydrolysates, a comparison between hydrolyzed protein and original intact protein in 10 elderly males was conducted. Postprandial amino acid utilization and uptake of dietary amino acids into skeletal muscle have been shown to increase. However, not all food ingredients that contain protein are created equal. The main problem is the ratio of essential amino acids in the case of the protein itself, and how high the essential amino acid content in the total protein determines the quality of the protein. Humans are mammals, so animals closer to mammals make meat with amino acid ratios similar to those of protein that humans need to make in the body. In general, animal protein has a higher essential amino acid ratio than processed foods such as tofu and soy sauce derived from vegetable proteins of legumes, except for soybeans.
일반적으로 음식으로 섭취하는 단백질이 인체에 들어오면 소화되면서 아미노산 분자 형태로 작게 쪼개지기 때문에 '같은 양의 같은 아미노산'이라면 동물성이건 식물성이건 인체 활용도는 동일하다는 점에서 틀리다. 하지만 식물성 단백질일 경우 필수 아미노산이 동물성 단백질에 비해 부족해서 '같은 양의 같은 아미노산'을 공급하지 못한다는 점에서는 맞는 말이다. 한국인의 경우 대두를 이용한 식품이나 쌀 섭취를 통해 상당한 양의 식물성 단백질을 섭취하므로 동물성 단백질을 보충해 주는 것이 더 좋다. In general, when protein consumed as food enters the human body, it is digested and broken down into small amino acid molecules, so 'the same amount of amino acids' is different in that the utilization of the human body is the same whether it is animal or vegetable. However, in the case of vegetable protein, essential amino acids are insufficient compared to animal protein, so it is correct in that it cannot supply 'the same amino acid in the same amount'. In the case of Koreans, it is better to supplement with animal protein because they consume a significant amount of vegetable protein through consumption of soybean or rice.
앞서 언급한 대로 식품재료의 가수분해물은 다수 공지되어 있다. 그러나, 닭가슴살을 이용하여 단백질 가수분해 후 분말화를 하는데 있어서 단백질 가수분해는 크게 효소 분해 및 산 알칼리 가수분해 방법으로 얻을 수 있다. 전자의 효소가수분해 방법은 산업적 활용성이 가장 크다. 산 알칼리 가수 분해 방법은 폐수처리, 산, 알칼리 사용 시 남는 잔여물의 처리가 문제되고 있으며, 또한, 가열처리 과정을 거치게 되는데, 이 과정에서 단백질이 응고되어 단백질의 열변성에 따른 이화학적 품질 특성의 변화와 동시에 생산 수율이 현저히 낮아지는 단점이 있다.As mentioned above, many hydrolysates of food materials are known. However, in powdering after protein hydrolysis using chicken breast, protein hydrolysis can be largely obtained by enzymatic digestion and acid alkali hydrolysis. The former enzymatic hydrolysis method has the greatest industrial applicability. The acid-alkaline hydrolysis method has problems with wastewater treatment, treatment of residues left when using acids and alkalis, and also undergoes a heat treatment process. At the same time, there is a disadvantage that the production yield is significantly lowered.
그러나 단백질의 효소분해 방법은 단백질 가수분해 효소를 육류단백질에 적용시켜 단백질을 용해 및 분해시켜 펩타이드 형태의 수용성 물질을 얻는 것으로, 이 과정에서 육류가 지니는 풍미를 증가 시키는 아미노산이 방출되기 때문에, 식품산업에서 향미증진제와 같은 기능성 소재로 평가되고 있다. 그러나 국내에 유통되는 닭가슴살 단백질 제품은 대부분 육류 가공 상태가 그대로 보존된 형태로 판매되고 있어, 섭취 시 거북스럽고 부드러운 식감을 주지 못하는 단점이 있다. 따라서 닭가슴살 단백질을 가수 분해하여 수용성이 증가된 형태로 소비자에게 전달 취식하게 한다면 체내 흡수도가 개선되어 생체 이용율이 소화, 흡수기능이 저하된 노년층에 적합한 단백질 급원 및 신규한 소재 제품으로의 활용이 가능하다 할 것이다.However, the enzymatic degradation method of protein is to apply proteolytic enzymes to meat proteins to dissolve and decompose the proteins to obtain water-soluble substances in the form of peptides. Since amino acids that increase the flavor of meat are released during this process, it is widely used in the food industry. has been evaluated as a functional material such as a flavor enhancer in However, most of the chicken breast protein products distributed in Korea are sold in a form in which the meat processing state is preserved, and there is a disadvantage in that it does not give a uncomfortable and soft texture when ingested. Therefore, if chicken breast protein is hydrolyzed and delivered to consumers in the form of increased water solubility, absorption in the body is improved, and bioavailability is reduced. it will be possible
따라서, 본 발명의 목적은 수용성이 증가되고 생체 이용율이 증진된 닭가슴살 가수분해물과 그 제조방법을 제공하는 데 있다.Accordingly, an object of the present invention is to provide a chicken breast hydrolyzate with increased water solubility and improved bioavailability and a manufacturing method thereof.
또 본 발명의 다른 목적은 닭가슴살을 활용하여 특정 펩타이드 이미다졸 펩타이드 함량이 증가된 수용성 닭가슴살 가수분해 단백질 분말을 제공한다.Another object of the present invention is to provide a water-soluble chicken breast hydrolyzed protein powder in which the content of a specific peptide imidazole peptide is increased by utilizing chicken breast meat.
본 발명의 또 다른 목적은 본 발명에 따라 수득한 수용성과 생체 이용율이 증진된 닭가슴살 가수분해물을 포함하는 첨가제, 고령 친화식품, 특정 기능성 보조식품 조성물등 신규한 소재의 특정용도 식품을 제공하는 데 있다.Another object of the present invention is to provide additives containing chicken breast hydrolyzate with improved water solubility and bioavailability obtained according to the present invention, age-friendly foods, and special functional food compositions of novel materials, such as food for specific uses. there is.
본 발명의 상기 목적은 본 명세서에 사용된 용어,“특정용도식품”또는“특수용도식품”은 영·유아, 병약자, 노약자, 비만자 또는 임산·수유부 등 특별한 영양관리가 필요한 특정 대상을 위하여 식품과 영양성분을 배합하는 등의 방법으로 제조·가공한 것으로 조제유류, 영아용 조제식, 성장기용 조제식, 영·유아용 이유식, 특수의료용도등식품, 체중조절용 조제식품, 임산·수유부용 식품을 말한다.The above object of the present invention, as used herein, the term “food for special use” or “food for special use” refers to food for a specific target requiring special nutritional management, such as infants/young children, the sick, the elderly, the obese, or pregnant/lactating women. It refers to milk formula, formula for infants, formula for growth, weaning food for infants and young children, food for special medical purposes, formula for weight control, and food for pregnant/lactating women.
본 발명의 일 실시예에 따르면, 노인들은 스스로 식품의 안전 및 영양관리를 하는데 어려움을 겪고 있는 취약 집단으로, 노인기에는 신체 노화가 진행되어 세포 수가 감소하고 식욕 및 소화 흡수 능력이 감소되며, 치아 소실로 저작능력이 감소되고, 약물 복용으로 인한 식욕 감퇴 및 소화기능장에 등의 문제가 발생될 수 있고 이는 영양 부족 현상으로 귀결된다. 따라서 균형 잡힌 용도의 식단을 제공받아 섭취할 경우 영양 부족으로 인한 당뇨, 고혈압, 심혈관계질환 등의 만성질환 및 골다공증 등의 만성 퇴행성 질환 발병을 예방 할 수 있는 장점이 있다.According to one embodiment of the present invention, the elderly are a vulnerable group having difficulty in managing food safety and nutrition by themselves. In the elderly, aging of the body progresses, the number of cells decreases, appetite and digestive absorption capacity decrease, and teeth Loss of masticatory ability decreases, and problems such as loss of appetite and digestive function due to drug intake may occur, which results in malnutrition. Therefore, when a balanced diet is provided and consumed, there is an advantage in preventing the occurrence of chronic diseases such as diabetes, hypertension, cardiovascular diseases, and chronic degenerative diseases such as osteoporosis due to lack of nutrition.
본 발명에 따르면, 특정의 신규 용도의 식품 조성물의 유효성분으로 사용된 ‘닭가슴살 가수분해물’의 식재료는 백웅계(白雄鷄)계통의 닭에서 적출한 늑골 근육을 사용함으로서 동의보감에서 정신질환을 치료하고 오장을 편안하게 하며 소갈을 멎게하며 소변을 잘 배설하게 한다고 기술되어 있으며, 본초강목에서는 단독丹毒을 치료한다고 알려져 있다. 그리고 건강기능식품 공전의 단백질 규격에 해당하며, 닭고기의 부위 중 단백질 함량이 현저히 많으며 지방 함량과 콜레스테롤이 거의 없어 대표적인 노인성 질환인 심장병, 동맥경화, 심근경색 등의 심혈관 질환들에게 좋은 식품 재료로 알려져 있다.According to the present invention, the ingredient of 'chicken breast hydrolyzate' used as an active ingredient of a food composition for a specific new purpose uses rib muscles extracted from chickens of the Baekwoong system, thereby preventing mental illness in Donguibogam. It is described as treating, relaxing the five viscera, stopping sore throat, and excreting urine well, and is known to treat solitary 丹毒 in herbal medicine. In addition, it corresponds to the protein standard of health functional food, and the protein content of chicken parts is significantly high, and there is almost no fat content and cholesterol. there is.
본 발명의 다른 실시예에 따르면, 상기‘닭가슴살 가수분해물’은 foodpro alkaline protease, prozyme, bromelain 또는 papain 및 이들 효소를 일정 비율로 배합한 혼합물을 적용하는데 그 특징이 있고 효소분해를 진행한 결과 목표(target)로 하는 특정 펩타이드 즉 이미다졸 펩타이드가 다량 포함된 수용성 닭가슴살의 단백질 가수분해물의 분말을 수득할 수 있다.According to another embodiment of the present invention, the 'chicken breast hydrolyzate' is characterized by applying a mixture of foodpro alkaline protease, prozyme, bromelain or papain and these enzymes in a certain ratio, and as a result of enzymatic degradation, the target It is possible to obtain a powder of a protein hydrolyzate of water-soluble chicken breast containing a large amount of a specific peptide as a target, that is, an imidazole peptide.
본 발명에 따르면, 예컨대, 효소분해 후에, 수득한 가수분해물을 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 이용하여 분리하거나, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의하여 분리하거나 하는 등, 추가적으로 실시한 다양한 정제 방법을 통해 얻어진 분획물도 본 발명의 수용성 닭가슴살 가수분해물에 포함된다. 또 본 발명에 따른 수용성 닭가슴살 가수분해물은 감압 증류 및 동결 건조 또는 분무 건조 등과 같은 추가적인 과정에 의해 분말 상태로 제조될 수 있음을 포함한다.According to the present invention, for example, after enzymatic digestion, the obtained hydrolyzate is separated using an ultrafiltration membrane having a certain molecular weight cut-off value, or various chromatography (designed for separation according to size, charge, hydrophobicity or affinity) Fractions obtained through various purification methods additionally performed, such as separation or separation by), are also included in the water-soluble chicken breast hydrolyzate of the present invention. In addition, the water-soluble chicken breast hydrolyzate according to the present invention includes that it can be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying.
본 발명에 따르면, 상기 분말형태의 제제는 과립제, 산제, 정제, 피복정, 캡슐제, 좌제, 액제, 시럽, 즙, 현탁제, 유제, 점적제 또는 주사 가능한 액제 등의 다양한 제형으로 제조될 수 있다. According to the present invention, the powdered formulation can be prepared in various formulations such as granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. there is.
본 발명의 실시예에 따르면, 본 발명 수용성 닭가슴살 단백질 가수분해물은 필수 아미노산의 함량이 고시기준에 충족되었으며, 당뇨 개선 효과, 콜레스테롤 감소, 근육량 증가, 인체 소화 흡수율을 개선할 수 있으므로 특정용도식품 조성물의 용도로 유용하게 사용될 수 있다. According to an embodiment of the present invention, the content of essential amino acids in the water-soluble chicken breast protein hydrolyzate of the present invention meets the notification standards, and can improve diabetes, cholesterol reduction, muscle mass increase, and human digestion and absorption rate, so it is a food composition for specific use. can be usefully used for
본 발명의 다른 일 실시예에 따르면, 본 발명 닭가슴살 가수분해물을 일정량 포함하는 조성물을 투여하여 특정용도 식품을 제공하기 위하여는 식이 방식, 환자, 적용자의 연령, 체중, 성, 병적 상태, 배설 속도 및 반응 감응성 등 다양한 요인들에 의해 그 배합비율을 결정할 수 있다.According to another embodiment of the present invention, in order to provide a food for a specific purpose by administering a composition containing a certain amount of the chicken breast hydrolyzate of the present invention, the diet, age, weight, sex, morbid condition, and excretion rate of the patient and patient And the mixing ratio can be determined by various factors such as reaction sensitivity.
또, 본 발명에 따른 특정용도의 식품 조성물은 상기 제제화 방식을 적용하여 다양한 기능성 식품으로 제공하거나, 각종 식품의 첨가제로 할 수 있다. 본 발명에 따른 식품의 조성물로 을 첨가할 수 있는 식품으로는 예를 들어, 음료류, 과자류, 젤리류, 다이어트바, 피자, 라면, 기타 면류, 건강보조식품류, 아이스크림류 등을 들 수 있다.In addition, the food composition for a specific purpose according to the present invention can be provided as various functional foods by applying the above formulation method, or can be used as additives for various foods. Foods to which can be added to the food composition according to the present invention include, for example, beverages, confectionery, jellies, diet bars, pizza, ramen, other noodles, health supplements, and ice cream.
이 밖에 본 발명에 따르면 본 발명의 식품 조성물은 수용성 닭가슴살 가수분해물을 포함하는 이외에도 식품 제조시에 통상적으로 첨가되는 성분을 더 포함할 수 있으며, 예를 들어, 탄수화물, 지방, 영양소, 조미제 및 향미제가 포함될 수 있다. 상기 탄수화물은 예를들면 모노사카라이드, 예컨대 포도당, 과당과 디사카라이드, 예컨대 말토스, 슈크로스, 올리고당과 폴리사카라이드, 예컨대 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당을 포함하고 이 밖에도 자일리톨, 소르비톨, 에리트리톨 등 당알콜류다.In addition, according to the present invention, the food composition of the present invention may further include ingredients commonly added during food production in addition to containing the water-soluble chicken breast hydrolyzate, for example, carbohydrates, fats, nutrients, seasonings and Flavoring agents may be included. The carbohydrates include, for example, monosaccharides such as glucose, fructose and disaccharides such as maltose, sucrose, oligosaccharides and polysaccharides such as dextrin, cyclodextrin, and the like, and in addition, xylitol, sorbitol , sugar alcohols such as erythritol.
향미제로서는 천연 향미제[타우마틴, 스테비아 추출물 예컨대 레바우 디오시드 A, 글리시르히진 등과 합성 향미제로 예컨대 사카린, 아스파르탐 등이 사용할 수 있다. 또, 본 발명의 식품 조성물이 드링크제 또는 음료류로 제조되는 경우에는 본 발명의 수용성 닭가슴살 가수분해물 외에도 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙, 및 또는 각종 동물, 식물 추출액 등이 추가로 더 포함할 수 있다.As the flavoring agent, natural flavoring agents such as thaumatin and stevia extract such as rebaudioside A and glycyrrhizin and synthetic flavoring agents such as saccharin and aspartame may be used. In addition, when the food composition of the present invention is prepared as a drink or beverage, in addition to the water-soluble chicken breast hydrolyzate of the present invention, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various animal and plant extracts are added. can be further included.
본 발명에 따르면 본 발명의 수용성 닭가슴살을 효소분해 하여 항증식 특정 성분인 이미다졸 펩타이드가 고함유된 단백질 가수분해물을 제공하는 효과가 있고, 상기 효소 가수분해를 통해 수용성이 증대되고 저분자화가 증진되어 생체 이용율이 증강된 단백질 가수분해물을 포함하는 식품 조성물은 당뇨 예방 및 개선 효과를 비롯하여 콜레스테롤 감소, 근육량 증가, 조골세포 분화능 증가, 다양한 건강 기능성 식품 또는 건강 보조식품을 제공하는 뛰어난 효과가 있다.According to the present invention, the water-soluble chicken breast of the present invention is enzymatically digested to provide a protein hydrolyzate containing a high content of imidazole peptide, which is an anti-proliferative specific component, and through the enzymatic hydrolysis, water solubility is increased and molecularization is promoted. Food compositions containing protein hydrolysates with enhanced bioavailability have excellent effects of preventing and improving diabetes, as well as reducing cholesterol, increasing muscle mass, increasing osteoblast differentiation potential, and providing various health functional foods or dietary supplements.
이밖에도 본 발명에 따라 제조된 수용성 닭가슴살 단백질 가수분해물은 특히 이미다졸 펩타이드를 포함하는 신규한 단백질 소재 식품의 가공 및 첨가물 식품제조 분야에서 매우 유용하게 사용할 수 있는 뛰어난 효과가 있다.In addition, the water-soluble chicken breast protein hydrolysate prepared according to the present invention has an excellent effect that can be used very usefully in the field of processing novel protein-based food products containing imidazole peptides and manufacturing food additives.
도 1은 이미다졸 펩타이드가 포함된 수용성 닭가슴살 단백질의 효소 가수분해물을 제조하는 조건을 예시한 것이다.
도 2는 도 1에 따라 생산된 수용성 닭가슴살 단백질의 가수분해물의 건조 분말 및 그 수용화를 도시한 도면이다.
도 3은 도 1에 따라 생산한 수용성 닭가슴살 단백질의 가수분해물의 분자량을 분석한 결과이다.
도 4는 도 1에 따라 생산한 수용성 닭가슴살 단백질의 가수분해물에 포함된 이미다졸 펩타이드 분석 및 그 대사산물에 대한 제조공정도이다.
도 5는 50주령의 고령 마우스의 혈청에서 분석한 고함량 닭가슴살 유래의 수용성 단백질 가수분해물의 공복 혈당 감소 결과를 보인 그래프,
도 6은 50주령의 고령 마우스의 혈청에서 분석한 고함량 닭가슴살 유래의 수용성 단백질 가수분해물의 토탈 콜레스테롤 감소 결과를 보인 그래프,
도 7은 50주령의 고령 마우스에 4주간 닭가슴살 유래의 수용성 단백질 가수분해물의 식이 결과 뒷다리 양쪽 근육량을 측정한 결과를 보인 막대그래프,
도 8은 50주령의 고령 마우스에 12주간 닭가슴살 유래 수용성 단백질 가수분해물을 식이한 결과, 다리 근육 무게, 두께, 근력 평가 및 근육내 지방 관련 지표 분석 결과를 보인 그래프들이다.
도 9는 조골세포 MC3T3-E를 3 내지 6일간 닭가슴살 유래 수용성 단백질 가수분해물을 처리한 결과, 대조군에 비해 250㎍/mL의 단백질 가수분해물 처리군에서 세포분화능이 유의하게 증가한 결과를 보인 그래프이다.
도 10은 조골세포 MC3T3-E를 3 내지 6일간 닭가슴살 유래 수용성 단백질 가수분해물을 처리하여 Alizarin red staining을 통해 미네랄 침착 정도를 확인한 결과, 조골세포에서의 미네랄 침착이 단백질 가수분해물에 의해 농도 의존적인 증가를 보인 그래프이다.
도 11a부터 11b는 단백질 표준품의 크로마토그래프이고, 도 11c는 단백질표준품을 이용하여 작성된 표준곡선이다.
도 12는 GPC를 이용하여 수용성 닭가슴살 단백질 가수분해물(K)의 분자량을 분석한 크로마토그래프이다.
도 13a는 이미다졸 펩타이드 anserine 표준품을 HPLC로 분석한 크로마토그래프이다.
도 13b는 이미다졸 펩타이드 L-carnosine 표준품을 HPLC로 분석한 크로마토그래프이다.
도 14는 HPLC를 이용하여 수용성 닭가슴살 단백질 가수분해물(K) 분석 크로마토그래프와 이미다졸 펩타이드 표준품 분석한 크로마토그래프이다.1 illustrates conditions for preparing an enzymatic hydrolyzate of water-soluble chicken breast protein containing an imidazole peptide.
FIG. 2 is a view showing a dry powder of a hydrolyzate of water-soluble chicken breast protein produced according to FIG. 1 and its water-solubility.
Figure 3 is the result of analyzing the molecular weight of the hydrolyzate of the water-soluble chicken breast protein produced according to Figure 1.
Figure 4 is a manufacturing process diagram for the analysis of imidazole peptides contained in the hydrolyzate of water-soluble chicken breast protein produced according to Figure 1 and its metabolites.
Figure 5 is a graph showing the results of fasting blood glucose reduction of water-soluble protein hydrolysates derived from high-content chicken breast meat analyzed in the serum of 50-week-old mice;
Figure 6 is a graph showing the total cholesterol reduction results of water-soluble protein hydrolysates derived from high-content chicken breast analyzed in the serum of 50-week-old mice;
Figure 7 is a bar graph showing the results of measuring the muscle mass of both hind limbs as a result of feeding a water-soluble protein hydrolyzate derived from chicken breast for 4 weeks to 50-week-old mice;
8 is graphs showing the results of leg muscle weight, thickness, muscle strength evaluation, and intramuscular fat-related index analysis results as a result of feeding 50-week-old mice with chicken breast-derived water-soluble protein hydrolysate for 12 weeks.
9 is a graph showing the results of treatment of osteoblast MC3T3-E with chicken breast-derived water-soluble protein hydrolysate for 3 to 6 days, and a significant increase in cell differentiation ability in the group treated with 250 μg/mL protein hydrolysate compared to the control group. .
10 is a result of confirming the degree of mineral deposition through Alizarin red staining by treating osteoblast MC3T3-E with water-soluble protein hydrolysate derived from chicken breast for 3 to 6 days. This is the graph showing the increase.
11a to 11b are chromatographs of protein standards, and FIG. 11c is a standard curve prepared using protein standards.
12 is a chromatograph analyzing the molecular weight of water-soluble chicken breast protein hydrolyzate (K) using GPC.
13a is a chromatograph obtained by analyzing an imidazole peptide anserine standard by HPLC.
13b is a chromatograph obtained by HPLC analysis of the standard imidazole peptide L-carnosine.
14 is a chromatograph for analyzing a water-soluble chicken breast protein hydrolyzate (K) and an imidazole peptide standard using HPLC.
이하, 본 발명에 따른 수용성 닭가슴살 단백질을 이용하여 이미다졸 펩타이드 및 저분자화된 단백질 가수분해물 및 이를 포함하는 식품 조성물을 도면을 참고하여 상세히 설명한다.Hereinafter, an imidazole peptide and a low-molecular-weight protein hydrolyzate using the water-soluble chicken breast protein according to the present invention and a food composition containing the same will be described in detail with reference to the drawings.
실시예 1. 본 발명 닭가슴살 단백질 가수분해 조건 및 최적화Example 1. Chicken breast protein hydrolysis conditions and optimization of the present invention
먼저 본 발명 닭가슴살 유래의 수용성단백질분말을 생산하기 위해 다양한 시판 단백질 가수분해 효소제품 중 최적 효소를 선정하는 작업이 필요하다. 본 발명에서는 여러 점들을 고려하여 foodpro alkaline protease, prozyme 2000P, bromelain, papain 효소를 공시재료로 선정하였다. pH 변화가 없는 효소분해를 유도하기 위한 전처리로 닭가슴살은 슬라이스하여 10 ~ 60분 동안 물(H2O)에 교반하며 진탕하여 방혈한 후, 2mm chopper를 이용해 분쇄하고 이어 효소를 첨가 단백질 가수분해를 진행하였다. 본 발명에 따라 가수분해를 위한 조건으로는 닭가슴살 분쇄물 총중량 1 ~ 3배의 물을 첨가한 후, 1차 foodpro alkaline protease를 0% ~ 1.0% 첨가한 후 55℃에서 4 ~ 6시간 반응시켰다. 다음 2차로 prozyme 2000P 0% ~ 1.0%, 3차로, bromelain 0 ~ 0.3%, 그리고 최종적으로 papain 0 ~ 0.3%를 넣고 4 ~ 6시간 순차 반응시켰다. 효소 반응이 끝난 후 남은 효소를 실활 시키기 위해서는 60℃ ~ 90℃에서 10 ~ 60분 처리하였다. 불순물의 냄새 성분의 제거 활성탄을 이용하여 여과한 후 Brix 15 ~ 20까지 농축하여 95℃에서 살균한 다음 분무건조를 진행하였다. 일반적으로 효소분해 과정에서는 pH의 변화는 일반적이나, 본 발명에서는 순차적 환처리 전과정에서 pH의 변화가 전혀 없이 효소의 최적화 조건을 찾아 수용화를 진행하는 것을 그 특징으로 하였다. 이밖에 본 발명에서는 전처리 공정에서 방혈 작업, 활성탄 처리 공정을 채택하여 최종 목표 가수분해산물의 염도, 취, 색감이 불쾌감을 최소화 하는데도 그 특징이 있다. 본 발명의 방혈공정과 이어서 활성탄 처리 공정을 통하여 최종산물인 수용성 단백질 가수분해물의 풍미와 색택이 현저히 증가되는 효과가 있다.First, in order to produce the water-soluble protein powder derived from chicken breast of the present invention, it is necessary to select an optimal enzyme among various commercially available proteolytic enzyme products. In the present invention, foodpro alkaline protease, prozyme 2000P, bromelain, and papain enzymes were selected as test materials in consideration of various points. As a pretreatment to induce enzymatic degradation without pH change, chicken breast is sliced, stirred and shaken in water (H 2 O) for 10 to 60 minutes to exsanguinate, then pulverized using a 2mm chopper, and then enzymes are added for protein hydrolysis. proceeded. As conditions for hydrolysis according to the present invention, after adding 1 to 3 times the total weight of chicken breast pulverized water, 0% to 1.0% of primary foodpro alkaline protease was added, followed by reaction at 55 ° C for 4 to 6 hours. . Next, 0% to 1.0% of prozyme 2000P was added, 0 to 0.3% of bromelain was added, and 0 to 0.3% of papain was added, followed by sequential reaction for 4 to 6 hours. After the end of the enzyme reaction, in order to inactivate the remaining enzyme, treatment was performed at 60 ° C to 90 ° C for 10 to 60 minutes. Removal of odor components of impurities After filtering using activated carbon, the mixture was concentrated to Brix 15-20, sterilized at 95 ° C, and then spray-dried. In general, changes in pH are common in the enzymatic degradation process, but in the present invention, it is characterized by finding optimized conditions for the enzyme and proceeding with water solubilization without any change in pH in the entire sequential ring treatment process. In addition, the present invention adopts the bloodletting operation and the activated carbon treatment process in the pretreatment process, thereby minimizing the unpleasant feeling of the final target hydrolyzate in terms of salinity, odor, and color. Through the bleeding process of the present invention followed by the activated carbon treatment process, the flavor and color of the final product, the water-soluble protein hydrolysate, is remarkably increased.
상기 공정을 통해 수용화한 단백질은 가수분해물을 목표로 하는 이미다졸 펩타이드를 포함하고 그 함량도 현저히 증가시키는 효과가 있으며, 그 결과 소화흡수율을 증가시켜 생체 이용율을 현저히 증가시킬 수 있었다.The protein solubilized through the above process contains an imidazole peptide targeting the hydrolyzate and has an effect of significantly increasing its content, and as a result, the bioavailability can be significantly increased by increasing the digestion and absorption rate.
이미다졸 펩타이드의 일종인 안세린 카르노신은 근육량 증강, 당뇨예방 및 개선, 심혈관계 질환 예방 및 개선, 골다공증 예방 및 개선 기타 소화 흡수율 증진에 도움을 줄 수 있다고 사료된다. Anserine carnosine, a type of imidazole peptide, is thought to help increase muscle mass, prevent and improve diabetes, prevent and improve cardiovascular diseases, prevent and improve osteoporosis, and improve other digestion and absorption rates.
본 발명에 따르면 상기 이미다졸 펩타이드가 포함된 닭가슴살 유래의 수용성 단백질 가수분해물에는 아미노산 대사물질들을 포함하고 있는 조추출물이므로 이들 수용성단백질 펩타이드류를 이용하여, 젤리, 음료, 단백질바 등에 적용이 가능하며 따라서 다양한 단백질 펩타이드 식품의 제조에 활용이 가능하다. According to the present invention, since the water-soluble protein hydrolyzate derived from chicken breast containing the imidazole peptide is a crude extract containing amino acid metabolites, these water-soluble protein peptides can be used for jelly, beverages, protein bars, etc. Therefore, it can be used in the manufacture of various protein peptide foods.
이밖에도, 본 발명에 따르면 상기 이미다졸 펩타이드를 포함하는 닭가슴살 유래의 수용성단백질 가수분해물 및 이를 포함하는 식품 조성물은 향신료 등 식품 첨가제 및 기능성식품 단백질 소재 원료를 제공할 수 있다. 본 발명에 따른 수용성단백질 가수분해물은 기능성 식품 또는 보조 식품 제조에 활용할 수 있다. 또한, 본 발명의 수용성단백질 가수분해물은 건강 기능성 식품 또는 식품 제조에 유용한 첨가제로서 용이하게 활용될 수 있다.In addition, according to the present invention, the water-soluble protein hydrolyzate derived from chicken breast containing the imidazole peptide and the food composition containing the same can provide food additives such as spices and functional food protein material raw materials. The water-soluble protein hydrolyzate according to the present invention can be used for manufacturing functional foods or supplements. In addition, the water-soluble protein hydrolyzate of the present invention can be easily utilized as a health functional food or a useful additive for food production.
이하, 본 발명을 다른 실시예를 통하여 더욱 상세히 설명한다. 그러나, 하기 실시예는 본 발명을 예시하기 위한 것으로, 본 발명의 권리범위가 이에 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through other embodiments. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.
실시예 2. 본 발명 수용성 닭가슴살 단백질 가수분해물(K)의 분자량 분석 조건Example 2. Molecular weight analysis conditions of water-soluble chicken breast protein hydrolyzate (K) of the present invention
분해도를 측정하기 위하여 Gel Permeation Chromatography (GPC)를 통해 평균 분자량을 산출하여, 식품의 기준 및 규격 > 제8.일반시험법 > 2. 식품성분시험법 > 2.1 일반성분시험법 > 2.1.4 탄수화물 > 2.1.4.1 당류 > 2.1.4.1.4 기기분석법에 의한 당류의 정량 > 액체크로마토그래프-시차굴절계검출기(RI)법에 따라 시험을 진행하였다.In order to measure the degree of degradation, the average molecular weight is calculated through Gel Permeation Chromatography (GPC), and food standards and specifications > 8. General test methods > 2. Food ingredient test methods > 2.1 General ingredient test method > 2.1.4 Carbohydrates > 2.1.4.1 Sugars > 2.1.4.1.4 Quantification of sugars by instrumental analysis > The test was conducted according to the liquid chromatograph-differential refractometer (RI) method.
[검액 조제][Preparation of sample solution]
1. 25ml volumetric flask에 3차수 10ml을 넣는다.1. Put 10ml of tertiary water into a 25ml volumetric flask.
2. 검체 100mg을 칭량하여 1)의 volumetric flack에 넣고 10분간 초음파 추출한다.2. Weigh 100mg of the sample, put it in the volumetric flack of 1), and ultrasonically extract for 10 minutes.
3. Flask를 식힌 후 3차수로 정용하여 4mg/ml이 되도록 한다.3. After cooling the flask, apply it 3 times to make it 4mg/ml.
4. 이후 PVDF syringe filter (0.45㎛)를 이용하여 여과하여 분석 시료로 사용한다.4. Afterwards, filter using PVDF syringe filter (0.45㎛) and use as analysis sample.
[표준액 조제][Standard solution preparation]
1. 표준물질1. Standard material
Agilent Easivial PEG (Part No. PL2070-0201)Agilent Easivial PEG (Part No. PL2070-0201)
2. 표준액 조제2. Standard solution preparation
3개의 표준물질에 각 1.8ml의 3차수를 이용하여 용해 후 분석에 사용한다.It is used for analysis after dissolution using 1.8 ml of tertiary water for each of the three standard materials.
3. GPC 측정조건3. GPC measurement conditions
(1) 분석기기 : HPLC (Waters, 관리번호 : DHUB-093)(1) Analytical instrument: HPLC (Waters, control number: DHUB-093)
(2) Cloumn : Shodex OHpack SB-G 6B(2) Cloumn : Shodex OHpack SB-G 6B
Shodex OHpack SB-802 HQShodex OHpack SB-802HQ
Shodex OHpack SB-802.5 HQShodex OHpack SB-802.5 HQ
Shodex OHpack SB-803 HQShodex OHpack SB-803 HQ
(3) Detector : RI-detector(3) Detector : RI-detector
(4) Mobile phase : 0.1M NaNO3 (4) Mobile phase : 0.1M NaNO 3
(5) Flow rate : 0.8 mL/min(5) Flow rate: 0.8 mL/min
(6) Injection volumn : 80.0μL(6) Injection volume: 80.0 μL
닭가슴살 가수분해 단백질 GPC 분석 결과 : 평균MW. 688Results of GPC analysis of chicken breast hydrolyzed protein: average MW. 688
실시예 3. 이미다졸 펩타이드 및 그 대사산물이 함유된 수용성 닭가슴살Example 3. Water-soluble chicken breast meat containing imidazole peptide and its metabolites
가수분해물의 분석 Analysis of hydrolysates
본 발명 수용성 닭가슴살 단백질 가수분해물내 함유된 이미다졸 펩타이드 분석은 HPLC를 이용하여 실시하였다. 본 발명 단백질 가수분해물 시료 약 0.5 g을 정밀하게 달아 이동상(A) 50 mL를 넣어 10분간 초음파추출기를 이용하여 추출하였다. Syringe filter (0.45 um, PVDF)를 이용해 여과하여 HPLC 분석용 시료로 사용하였다. 이미다졸 펩타이드의 지표성분으로는 Anserine 및 Carnosine을 설정하여 Anserine 및 carnosine(Sigma)은 HPLC(Agilent 1260 infinity Ⅱ, Agilent)에 Shodex Asahipak NH2P-50 4E column (4.6 mm × 250 mm)을 사용하여 분석하였고, column의 온도는 40℃, 파장은 210 nm에서 측정하였다. 각각의 표준품은 1 mg을 정밀하게 달아 1 mL의 이동상에 녹여 1000 ppm으로 만들었고, 이동상으로 희석해 200 ppm으로 만들어 표준액으로 사용하였다.Analysis of the imidazole peptide contained in the water-soluble chicken breast protein hydrolyzate of the present invention was performed using HPLC. About 0.5 g of a sample of the protein hydrolyzate of the present invention was precisely weighed and extracted using an ultrasonic extractor for 10 minutes by adding 50 mL of the mobile phase (A). It was filtered using a syringe filter (0.45 um, PVDF) and used as a sample for HPLC analysis. Anserine and Carnosine were set as the index components of the imidazole peptide, and Anserine and carnosine (Sigma) were analyzed using a Shodex Asahipak NH2P-50 4E column (4.6 mm × 250 mm) in HPLC (Agilent 1260 infinity II, Agilent) , The temperature of the column was measured at 40 ℃, and the wavelength was measured at 210 nm. 1 mg of each standard was precisely weighed and dissolved in 1 mL of mobile phase to make 1000 ppm, which was diluted with the mobile phase to make 200 ppm and used as a standard solution.
이동상(A)은 50mM Sodium dihydrogen phosphate (Anhydrous) aq./ACN (40/60)을 이용 하고, 이동상(B)는 Distilled water를 이용하여 분석하였으며 이동상 조건은 아래 표 1 및 도 13a ∼ 도 13b와 같다 실험 결과는 표 2 및 도 14와 같다.The mobile phase (A) was analyzed using 50mM Sodium dihydrogen phosphate (Anhydrous) aq./ACN (40/60), and the mobile phase (B) was analyzed using distilled water. The same experimental results are shown in Table 2 and FIG. 14.
또, 본 발명에 따르면 함유된 필수 아미노산 분석결과 및 아미노산 스코어 지수는 표 3 및 표 4와 같다.In addition, the essential amino acid analysis results and amino acid score indices contained according to the present invention are shown in Tables 3 and 4.
+CysteinMethionine
+
실시예 4. 본 발명 수용성 닭가슴살 단백질 가수분해물(K)의 기능성 평가Example 4. Functional evaluation of water-soluble chicken breast protein hydrolyzate (K) of the present invention
4-1 실험 동물 및 실험 방법4-1 Experimental Animals and Experimental Methods
소화 흡수율 연구를 위한 실험동물은 3~4주령의 수컷 Weanling SD Rat (70 내지 80g)으로, 실험동물 사육장에서 4주일 동안 적응시킨 후, AIN-93G 식이 사료를 기반으로 하기 표에 제시된 함량으로 제조된 고형 사료를 5주간 식이하였다. 실험기간 중 물은 자유로이 섭취시켰으며 사육실의 온도(22±2℃), 상대습도(60±5%)와 명암은 12시간 주기를 유지하였다.Experimental animals for digestion and absorption rate studies were male Weanling SD Rats (70 to 80g) aged 3 to 4 weeks, adapted for 4 weeks in the laboratory animal breeding farm, and then prepared with the contents shown in the table below based on AIN-93G dietary feed The solid feed was fed for 5 weeks. During the experiment, water was freely consumed, and the temperature (22±2℃), relative humidity (60±5%), and light/darkness of the breeding room were maintained at a 12-hour cycle.
당뇨 및 심혈관계 질환과 근력 개선 연구를 위한 실험동물은 C57BL/6J mouse 8주령의 수컷(18 내지 20g)과 50주령의 수컷(30 내지 32g)으로, 실험동물 사육장에서 2주일 동안 적응시킨 후, AIN-93G 식이 사료를 기반으로 제조된 고형 사료를 8주간 식이 하였다. 실험기간 중 물은 자유로이 섭취시켰으며 사육실의 온도(22±2℃), 상대습도(60±5%)와 명암은 12시간 주기를 유지하였다.Experimental animals for research on diabetes and cardiovascular disease and muscle strength improvement were C57BL/6J mice, 8-week-old males (18 to 20 g) and 50-week-old males (30 to 32 g), after being adapted for 2 weeks in the laboratory animal breeding house, The solid feed prepared based on the AIN-93G dietary feed was fed for 8 weeks. During the experiment, water was freely consumed, and the temperature (22±2℃), relative humidity (60±5%), and light/darkness of the breeding room were maintained at a 12-hour cycle.
4-2 실험세포 및 실험 방법4-2 Experimental Cells and Experimental Methods
골다공증 개선 연구를 위한 실험 세포주는 조골세포 MC3T3-E1 subclone 4 (high osteoblast differentiation)로 37℃의 온도와 95%의 공기, 5% CO2가 유지되는 세포배양기에서 10% FBS, 1mM sodium pyruvate, 1% penicillin과 streptomycin이 포함된 α-MEM 배양 배지에서 배양 되었다. 조골세포의 분화는 80% confluence 상태의 세포에 10mmol/L β-glycerolphosphate 와 50μg/mL L-ascorbic acid 가 포함된 osteoblastic differentiation 배지에서 유도 되었다.The experimental cell line for osteoporosis improvement research is osteoblast MC3T3-E1 subclone 4 (high osteoblast differentiation), which is maintained in a cell culture medium maintained at a temperature of 37°C, 95% air, and 5% CO2 in 10% FBS, 1mM sodium pyruvate, 1% It was cultured in α-MEM culture medium containing penicillin and streptomycin. Osteoblast differentiation was induced in an osteoblastic differentiation medium containing 10 mmol/L β-glycerolphosphate and 50 μg/mL L-ascorbic acid in 80% confluence cells.
Alizarin red staining은 조골세포를 3 내지 6일 분화를 유도한 뒤, 배양된 세포의 배지를 제거하고, PBS로 2회 수세한 후, 70% EtOH로 10분간 고정하였다. 고정된 세포를 증류수로 세척 후 alizarin red staining (pH4.2) 용액으로 10분간 염색하였다. 염색 수준의 정량화를 위하여 10% cetylpyridinium chloride (CPC)를 이용하여 염색된 미네랄 입자를 용출시켰다. 흡광도는 ELISA reader를 이용하여 570 nm에서 측정하였다.Alizarin red staining after inducing differentiation of osteoblasts for 3 to 6 days, removing the medium of the cultured cells, washing twice with PBS, and fixing with 70% EtOH for 10 minutes. After washing the fixed cells with distilled water, they were stained with alizarin red staining (pH4.2) solution for 10 minutes. To quantify the level of staining, dyed mineral particles were eluted using 10% cetylpyridinium chloride (CPC). Absorbance was measured at 570 nm using an ELISA reader.
4-3 본 발명 수용성 닭가슴살 단백질 가수분해물(K)의 소화 흡수율4-3 Digestion and absorption rate of water-soluble chicken breast protein hydrolyzate (K) of the present invention
Rat을 각 그룹당 8마리씩 정상군 (NC; normal control), 자유 단백질군 (PFC; protein free control), 콩 단백질 식이군(SP; soy protein), 유청 단백질 식이군 (WP; whey protein), 닭가슴살 수용성 단백질 식이군(KP; kyochon protein), 닭가슴살 수용성 단백질과 콩 단백질 혼합 식이군 (KSP; kyochon soy protein)으로 나누고, 정상군에는 AIN-93G 기본 사료를 식이 하고 자유 단백질군에는 AIN-93G 식이에 10% casein protein을 첨가한 사료를 식이 하였다. 그 밖에 단백질 식이군은 해당 단백질을 AIN-93G 고형 사료에 10%씩 첨가하여 식이 하였다.Rats were 8 rats per group, normal control (NC; normal control), free protein group (PFC; protein free control), soy protein diet group (SP; soy protein), whey protein diet group (WP; whey protein), chicken breast Divided into water soluble protein diet group (KP; kyochon protein) and chicken breast water soluble protein and soy protein mixed diet group (KSP; kyochon soy protein), normal group fed AIN-93G basic feed, and free protein group fed AIN-93G diet Feed supplemented with 10% casein protein was fed. In addition, the protein diet group was fed by adding 10% of the corresponding protein to AIN-93G solid feed.
5주간 상기 제시한 조건으로 식이한 Rat의 단백질 소화 흡수율(TPD; True protein digestibility)과 아미노산 흡수율(PDCAAS; protein digestibility corrected amino acid score)을 분석 하였다. 그 결과, 하기 표 5에 개시된 바와 같이 닭 가슴살 수용성 단백질 식이군의 단백질과 필수 아미노산 흡수율이 다른 단백질 식이군과 비교할 때 더욱 우수한 소화 흡수율을 보여주는 것을 확인 할 수 있었다.TPD (True protein digestibility) and amino acid absorption rate (PDCAAS; protein digestibility corrected amino acid score) of rats fed under the above conditions for 5 weeks were analyzed. As a result, as shown in Table 5 below, it was confirmed that the protein and essential amino acid absorption rate of the chicken breast water-soluble protein diet group showed a better digestion and absorption rate compared to other protein diet groups.
Data are mean±S.E.Data are mean±S.E.
PDCAAS % = TPD % x AASPDCAAS % = TPD % x AAS
TPD%=((Nitrogen Intake-(Fecal Nitrogen loss-Metabolic Nitrogen loss))/TPD%=((Nitrogen Intake-(Fecal Nitrogen loss-Metabolic Nitrogen loss))/
Nitrogen Intake*100Nitrogen Intake*100
4-4 본 발명 수용성 닭가슴살 단백질 가수분해물(K)의 혈당과 콜레스테롤 개선 효과4-4 Blood sugar and cholesterol improvement effect of the water-soluble chicken breast protein hydrolyzate (K) of the present invention
C57BL/6J mouse를 각 그룹당 8마리씩 어린군 (YC; young control(8week)), r고령군 (NC; normal control(50week)), 닭 가슴살 수용성 단백질 저함량 식이군 (LP; low protein(50week)), 닭 가슴살 수용성 단백질 고함량 식이군 (HP; high protein(50week))으로 나누고, 어린군과 고령군에는 AIN-93G 기본 사료를 식이 하고 LP군에는 AIN-93G 사료에 20%의 닭 가슴살 수용성 단백질을 첨가한 사료를 식이하고 HP군에는 AIN-93G 사료에 30%의 닭 가슴살 수용성 단백질을 첨가한 사료를 식이하였다.C57BL/6J mice were 8 in each group: young group (YC; young control (8week)), elderly group (NC; normal control (50week)), chicken breast water-soluble protein low content diet group (LP; low protein (50week)), Divided into chicken breast water-soluble protein high content diet group (HP; high protein (50 weeks)), AIN-93G basic feed was fed to young and elderly groups, and 20% chicken breast water-soluble protein was added to AIN-93G feed to LP group. One feed was fed, and the HP group was fed the AIN-93G feed with 30% chicken breast soluble protein added.
8주간 식이한 후, 각 그룹의 mouse를 희생하여 혈장을 분리해 공복 혈당과 콜레스테롤 수치를 분석하였다. 그 결과 도 5와 도 6에 도시한 바와 같이 고함량의 닭 가슴살 수용성 단백질을 식이한 고령 마우스의 공복 혈당과 콜레스테롤의 수치가 유의하게 감소하고 있음이 확인 되었다.After feeding for 8 weeks, mice in each group were sacrificed, plasma was separated, and fasting blood glucose and cholesterol levels were analyzed. As a result, as shown in FIGS. 5 and 6, it was confirmed that the fasting blood glucose and cholesterol levels of the aged mice fed a high content of chicken breast water-soluble protein were significantly reduced.
4-5 본 발명 수용성 닭가슴살 단백질 가수분해물(K)의 근육량과 근력 개선 효과4-5 Muscle mass and strength improvement effect of the water-soluble chicken breast protein hydrolyzate (K) of the present invention
닭 가슴살 수용성 단백질을 저함량/고함량 첨가된 사료를 8주간 식이한 마우스에서 근력(grip strength, g)을 확인 하였다. 이후, 개체를 희생하여 양쪽 뒷다리의 근육 두께 및 무게를 측정하고 근육내 지방 관련 지표를 확인 하였다.Grip strength (g) was confirmed in mice fed a diet supplemented with low/high content of chicken breast water-soluble protein for 8 weeks. Thereafter, the individual was sacrificed to measure muscle thickness and weight of both hind limbs, and intramuscular fat-related indicators were confirmed.
그 결과, 도 7 과 도 8에 도시한 바와 같이 기본 사료를 식이한 고령 마우스와 비교해 저함량/고함량의 닭 가슴살 수용성 단백질을 식이한 마우스의 근력과 근육 두께 및 무게가 유의하게 증가함을 확인 되었고, 근육내 지방 관련 지표 또한 유의하게 감소함이 확인 되었다.As a result, as shown in FIGS. 7 and 8, it was confirmed that the muscle strength, muscle thickness and weight of mice fed low/high content chicken breast water-soluble protein significantly increased compared to aged mice fed basic feed. , it was confirmed that intramuscular fat-related indicators also significantly decreased.
4-6 본 발명 수용성 닭가슴살 단백질 가수분해물(K)의 골다공증 개선 효과4-6 Osteoporosis improvement effect of the water-soluble chicken breast protein hydrolyzate (K) of the present invention
닭 가슴살 수용성 단백질을 조골세포 분화 유도 배지에 0, 10, 50, 250μg/mL을 처리하여 3 내지 6일 동안 분화 시켜 조성물에 의한 조골세포의 생장 능력과 미네랄 형성정도를 확인 하였다. 그 결과, 도 9에 도시한 바와 같이 조성물에 의해 농도의존적으로 세포 생장이 유의하게 증가함을 확인 되었고 도 10에 도시한 바와 같이 미네랄 형성 또한 조성물의 농도 의존적으로 유의하게 증가함이 확인 되었다.Chicken breast soluble protein was treated with 0, 10, 50, and 250 μg/mL in an osteoblast differentiation induction medium and differentiated for 3 to 6 days to confirm the growth ability and mineral formation of osteoblasts by the composition. As a result, as shown in FIG. 9, it was confirmed that cell growth was significantly increased by the composition in a concentration-dependent manner, and as shown in FIG. 10, mineral formation was also significantly increased in a concentration-dependent manner of the composition.
이상 본 발명에 대한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 여기에 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 권리범위는 전술한 설명 외에도, 특히 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 균등물이나 단순한 수치 변경은 본 발명의 권리범위에 포함된 것으로 해석되어야 한다.Above, the examples of the present invention were mainly examined. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. The embodiments disclosed herein are to be considered in an illustrative rather than a limiting sense. In addition to the above description, the scope of the present invention is particularly shown in the claims, and equivalents or simple numerical changes within the same range should be construed as being included in the scope of the present invention.
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