KR100423708B1 - Agent of immunological enhancement comprising colostral fractions as active ingredient, method of the preparation for the same and its use - Google Patents
Agent of immunological enhancement comprising colostral fractions as active ingredient, method of the preparation for the same and its use Download PDFInfo
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- KR100423708B1 KR100423708B1 KR10-2000-0056736A KR20000056736A KR100423708B1 KR 100423708 B1 KR100423708 B1 KR 100423708B1 KR 20000056736 A KR20000056736 A KR 20000056736A KR 100423708 B1 KR100423708 B1 KR 100423708B1
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- South Korea
- Prior art keywords
- colostrum
- bcwie
- cells
- molecular weight
- growth
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2300/00—Processes
- A23V2300/18—Fractionation
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Mycology (AREA)
- Pediatric Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
본 발명은 카세인과 지방이 제거된 소의 초유 유청으로부터 10 KD 초과 분자량을 갖는 펩타이드를 여과 제거하여 얻어진 초유분획을 유효성분으로 하는 면역증강제, 이의 제조방법 및 그 용도에 관한 것으로서, 초유 중에 포함된 면역억제성분을 제거하여 효과적인 면역증강제를 제공하고, 또한 상기 면역증강제를 포함하는 조제분유, 이유식, 특수영양식품, 위궤양 또는 장관궤양용 환자식이, 자양강장식품 등과 같은 식품, 음료, 또는 의약품등을 제공한다.The present invention relates to an immunopotentiator using the colostrum fraction obtained by filtering out a peptide having a molecular weight greater than 10 K from the colostrum whey of casein and fat removed, as an active ingredient, a method for preparing the same, and a use thereof, and the immunity contained in colostrum. Providing effective immune enhancers by removing the inhibitory ingredients, and also providing foods, beverages, or medicines, such as formulas, baby foods, special nutritional products, gastric or intestinal ulcer patient diets, nourishment tonics, etc. do.
Description
본 발명은 낙농분야에서 대부분 폐기되고 있는 소의 초유로부터 유용한 생리활성물질의 분리 및 이용에 관한 것으로서 초유의 효율적인 이용기술과 유효성분을 분리하고 분리된 성분의 이용에 관한 정보를 제공하기 위한 것으로 소의 초유 분획에 관한 것이다.The present invention relates to the separation and use of useful bioactive substances from bovine colostrum, which is mostly discarded in the dairy field, and to provide information on the efficient use of colostrum and the separation of active ingredients and the use of separated components. To fractions.
초유는 일반적으로 분만 후 3일까지 모체의 유선을 통해 분비되는 유즙 성분으로 알려져 있다. 소의 초유에는 정상유와는 달리 단백질의 함량이 높을 뿐만 아니라 유청단백질의 함량이 카세인보다 높은 특징이 있다. 그 이유는 유청단백질에는 다량의 면역글로불린이 함유되어 있기 때문이다. 면역글로불린은 새끼에게 수동면역기능을 제공하여 출생초기 동안 능동면역기능이 성숙하기 전까지 외부의 감염에 대한 저항성을 높여주는 역할을 한다. 최근 초유를 이용한 기능성식품의 개발이 활발히 진행되었으며 이용범위도 또한 다양하다. 초유를 섭취하면 면역력을 증가시킬 수 있는 것으로 알려지고 있으며 초유는 유아 또는 성인의 면역증강 소재로 이용되고 있으나, 초유 중에는 면역증강 성분뿐만 아니라 면역억제 성분도 함께 함유되어 있어, 면역증강 및 억제 성분에 의해 면역조절 기능을 가지고 있다.Colostrum is generally known as a milk component that is secreted through the mother's mammary gland until three days after delivery. Unlike normal milk, bovine colostrum is characterized by higher protein content and higher whey protein content than casein. This is because whey proteins contain large amounts of immunoglobulins. Immunoglobulins provide passive immunity to pups, increasing their resistance to external infections in the early stages of birth until active immune function matures. Recently, functional foods using colostrum have been actively developed, and the range of use is also diverse. It is known that ingestion of colostrum can increase immunity. Colostrum is used as an immune enhancing material for infants or adults. However, colostrum contains not only immune enhancing components but also immunosuppressive components. It has an immunomodulatory function.
Noda 등(1984)은 transforming growth factor(TGF)가 EL-4-NOB-1 murine thymoma 세포에 의한 인터루킨-2(IL-2) 생산을 억제한다고 보고하였다.(Gann(75):109-112, 1984). 그리고 초유 중에 함유되어 있는 체액성 인자(humoral factor)가 자연 살상(natural killer, NK) 세포 활성을 낮춘다는 보고도 있다(Baley and Schacter, J. Immunology (134):3042-3048, 1985; McDonald 등, Pediatr Res(31):376-380, 1992; Sanco 등, J. Pediatr(119):446-454, 1991; Engelhard 등, J. Pediatr(108):77-81, 1986). 그리고 최근 모유초유가 피토헤마글루틴(phytohemagglutinin(PHA))으로 자극한 말초혈액단핵구의 IL-2 생산과 NK 세포활성을 초유 투여량에 반비례하여 영향을 미쳤다(Sirota 등, Arch Dis Child(73):F99-F102, 1995)고 보고하였고 그리고 초유가 콘카나바린 A(Con A) 활성화에 의한 말초혈액단핵구의 IL-2 생산을 저해한다(Hooton 등, Clin Exp Immunol(86):520-524, 1991)고 보고되고 있다.Noda et al. (1984) reported that transforming growth factor (TGF) inhibits interleukin-2 (IL-2) production by EL-4-NOB-1 murine thymoma cells. (Gann (75): 109-112, 1984). In addition, humoral factors in colostrum have been reported to lower natural killer (NK) cell activity (Baley and Schacter, J. Immunology (134): 3042-3048, 1985; McDonald et al. Pediatr Res (31): 376-380, 1992; Sanco et al., J. Pediatr (119): 446-454, 1991; Engelhard et al., J. Pediatr (108): 77-81, 1986). Recently, breast milk colostrum influenced IL-2 production and NK cell activity of peripheral blood monocytes stimulated with phytohemagglutinin (PHA) in inverse proportion to colostrum dose (Sirota et al., Arch Dis Child (73)). : F99-F102, 1995) and colostrum inhibits IL-2 production of peripheral blood monocytes by activation of Concanavarin A (Con A) (Hooton et al., Clin Exp Immunol (86): 520-524, 1991).
한편 소의 초유성분은 래트의 장관상피세포(intestinal epithelial 세포(RIE)와 사람의 결장암세포인 HT-29 세포의 성장을 증진시키는데, 이러한 효과는 주로 초유중 분자량이 30 KD 이상인 성분에서 기인한다는 연구결과가 보고된바 있다(Playford 등, Gut(44):653-658, 1999). 지금까지 주로 소의 초유중에 들어있는 생리활성 펩타이드는 분자량 30 KD 이상의 단백질에 관심이 집중되어 왔다. 위궤양 모델을 이용한 장관세포의 재생효과는 분자량 30 KD 이상의 단백질에서 기인하며 그 중에서 TGF-β가 주요 성분이라고 밝혀진 바 있다(Howarth 등, J. Nutr(26):2519-2530, 1996).The colostrum of bovine colostrum enhances the growth of rat intestinal epithelial cells (RIEs) and human colon cancer cells, HT-29 cells. (Playford et al., Gut (44): 653-658, 1999). Until now, bioactive peptides mainly in bovine colostrum have been focused on proteins with molecular weights above 30 KD. The regenerative effect of cells is attributed to proteins with molecular weight of 30 KD or more, and TGF-β has been found to be a major component (Howarth et al., J. Nutr (26): 2519-2530, 1996).
그러나 소의 초유에 함유되어 있는 단백질 중에는 면역력을 억제할 수 있는 단백질과 펩타이드도 포함되어 있다. 면역글로불린이 다량 함유된 초유는 신생아에게 정맥주사 했을 때 NK세포 활성을 억제한다고 한다(J. Pediatrics (117):465-466, 1990). 그리고 소의 초유 중에 함유되어 있는 성장인자인 TGF-β는 EL-4-NOB murine thymoma cell에 의한 IL-2 생산을 억제하는 효과가 있다(Noda 등, Gann(75):109-112, 1984). 또한 모유에 함유되어 있는 락토페린은 미토젠이나 동종항원(alloantigen)에 대한 사람의 림프구 증식을 억제한다고 보고된바 있다(Richie 등, J. Reprod. Immunol(12):137-148, 1987). 사람 초유 중에 함유되어 있는 단백질에 의한 면역억제 활성은 신생아에 있어서 항원에 대한 면역반응을 억제하는 T 억제세포(T suppress cell)의 활성을 유도하여 그 이후 항원에 대한 과민반응을 예방할 수 있다. 그러나 이와 같은 활성은 신생아의 장관에서 주로 발휘되어 장관벽림프구(intestinal wall lymphocytes)의 반응을 억제한다. 이러한 과정은 장관내의 항원유입에 의한 과민반응을 예방하여 알레르기의 발생을 억제하는 기작으로 제안된 바 있다(Mincheva-Nilsson 등 Clin. Exp. Immunol(79):463-469, 1990). 그러나 Hooton 등(1991)은 모유 초유중에 함유되어 있는 어떤 성분은 IL-2의 생산을 저해한다고 하였다(Hooton 등, Clin. Exp. Immunol(86):520-524, 1991). IL-2는 면역기능 중에서 세포면역의 중추적 역할을 하는 T 세포의 성장인자로서 체액성면역(humoral immunity)의 발달에도 많은 영향을 줄 수 있다. 위와 같은 초유의 면역억제 기능은 대부분 분자량 10 KD 이상의 단백질에서 기인한다.However, protein contained in bovine colostrum also contains proteins and peptides that can suppress immunity. Colostrum containing high levels of immunoglobulin inhibits NK cell activity when injected intravenously in newborns (J. Pediatrics (117): 465-466, 1990). TGF-β, a growth factor contained in bovine colostrum, has the effect of inhibiting IL-2 production by EL-4-NOB murine thymoma cells (Noda et al., Gann (75): 109-112, 1984). It has also been reported that lactoferrin contained in breast milk inhibits human lymphocyte proliferation against mitogen or allogenegen (Richie et al., J. Reprod. Immunol (12): 137-148, 1987). Immunosuppressive activity by proteins contained in human colostrum can induce the activity of a T suppress cell that inhibits the immune response to the antigen in the newborn, thereby preventing hypersensitivity to the antigen. However, this activity is mainly exerted in the intestinal tract of newborns and inhibits the response of intestinal wall lymphocytes. This process has been proposed as a mechanism to suppress the development of allergies by preventing hypersensitivity reactions by intestinal antigen influx (Mincheva-Nilsson et al. Clin. Exp. Immunol (79): 463-469, 1990). However, Hooton et al. (1991) found that certain components contained in colostrum inhibit the production of IL-2 (Hooton et al., Clin. Exp. Immunol (86): 520-524, 1991). IL-2 is a growth factor of T cells that plays a pivotal role in cellular immunity in immune function, and can also have a significant effect on the development of humoral immunity. The above immunosuppressive function of colostrum is mostly due to the protein of 10 KD or more molecular weight.
따라서 면역기능을 저해하는 10 KD 초과 단백질 혹은 폴리펩타이드를 선택적으로 제거하여 면역억제 효과가 전혀 없이 면역증진효과만을 갖는 성분으로 이루어지는 면역증강제가 필요하게 되었다. 특히, 유아식에 이용하는 경우 장관면역과 전신면역의 성숙과 발달을 촉진하여 능동면역의 발달을 증진할 수 있다. 그리고 면역기능이 저하된 환자나 혹은 노약자의 경우에도 면역기능을 증진시킬 수 있는 식품의 소재로 이용될 수 있는 면역증강제가 필요하다.Therefore, there is a need for an immunopotentiator consisting of a component having only an immune enhancing effect without any immunosuppressive effect by selectively removing a protein or polypeptide greater than 10 KD that inhibits immune function. In particular, when used in baby food it can promote the development of active immunity by promoting the maturation and development of intestinal and systemic immunity. In addition, even if the immune function is reduced or elderly people need an immune booster that can be used as a material of food that can enhance the immune function.
본 발명의 또다른 목적은 1 KD 내지 10 KD의 분자량을 갖는 초유분획과 1 KD 미만의 분자량을 갖는 초유분획으로 이루어진 면역증강제을 제공하고자 한다.It is another object of the present invention to provide an immunopotentiator comprising a colostrum fraction having a molecular weight of 1 KD to 10 KD and a colostrum fraction having a molecular weight of less than 1 KD.
본발명의 또다른 목적은 상기 초유분획 면역증강제를 식품 또는 음료에 사용하는 방법을 제공하고자 한다.It is another object of the present invention to provide a method of using the colostrum fractional immunopotentiator in foods or beverages.
본발명의 또 다른 목적은 소의 초유에서 지방을 제거하고, pH를 4.5-4.6으로 조절하여 카세인을 분리 제거한 다음, 얻어진 유청으로부터 10 KD 초과 분자량을 갖는 물질을 제거하여, 10 KD 초과 분자량을 갖는 펩타이드가 제거된 초유분획을 얻는 방법을 제공하고자 한다.Another object of the present invention is to remove the fat from bovine colostrum, remove the casein by adjusting the pH to 4.5-4.6, and then remove the substance having a molecular weight greater than 10 KD from the obtained whey, thereby obtaining a peptide having a molecular weight greater than 10 KD. It is intended to provide a method for obtaining colostrum fraction from which is removed.
본발명은 초유중에 포함된 면역억제성분을 제거하여 효과적인 면역증강제를 제공하고자, 카세인 및 지방이 제거된 소의 초유 유청으로부터 얻어진 10 KD 초과 분자량을 갖는 펩타이드가 제거된 초유분획을 유효성분으로 하는 면역증강제, 이의 제조방법 및 그 용도에 관한 것이다.The present invention is to provide an effective adjuvant by removing the immunosuppressive components contained in colostrum, an immunopotentiator using a colostrum fraction with a peptide having a molecular weight greater than 10 KD obtained from casein and fat-derived bovine colostrum whey. It relates to a preparation method thereof and its use.
본발명은 초유 분획을 유효성분으로 하는 면역증강제의 제조방법에 관한 것이다. 본 발명에 의한 소의 초유 중 면역력을 증진하는 생리활성 펩타이드 혼합물의 제조방법은 소의 초유의 불순물을 제거하고, 지방을 분리하고 카세인과 유청을 분리하고, 얻어진 유청에서부터 한외여과법(이하 UF라 칭함)과 겔여과법 등의 분리법을 사용하여 10 KD 초과의 분자량을 갖는 펩타이드를 제거하여 얻어진 초유분획 및 이의 분리방법에 관한 것이다.The present invention relates to a method for producing an immunopotentiator using colostrum fraction as an active ingredient. The method for preparing a bioactive peptide mixture for enhancing immunity in bovine colostrum according to the present invention is to remove impurities of bovine colostrum, to separate fat, to separate casein and whey, and to obtain ultrafiltration (hereinafter referred to as UF) and The present invention relates to a colostrum fraction obtained by removing a peptide having a molecular weight of more than 10 KD using a separation method such as gel filtration and a separation method thereof.
본발명의 일례에서, 소의 초유에서 지방을 제거하고, pH를 4.5-4.6으로 조절하여 카세인을 분리 제거한 다음, 얻어진 유청으로부터 10 KD 초과 분자량을 갖는 물질을 제거하여 얻어진다. 추가로, 1 KD 이상의 분자량을 갖는 물질을 제거하는 단계를 수행함으로써 1 KD 내지 10 KD 범위의 분자량을 갖는 펩타이드를 주성분으로 하는 초유분획을 얻을 수 있다. 상기 방법에서 특정 분자량을 갖는 펩타이드를 선별적으로 제거하는 방법으로는 단백질등의 분자량의 크기에 기초를 둔 모든 알려진 분리방법을 이용할 수 있으며, 바람직하게는 한외여과 또는 겔여과법을 사용한다. 본발명의 바람직한 일례에서, 1차 10 KD 막을 이용한 한외여과를 행하여 얻어진 여액을 다시, 1 KD 막을 이용하여 여과하여 얻어진 여액에 존재하는 초유분획(이하 BCWIE-III라고 칭함)을 얻을 수 있다. 이 결과 10 KD 초과의 단백질과 펩타이드 분획(이하 BCWIE-I라고 칭함)에 대한 BCWIE-II 분획량의 비율은 15:1에서 20:1 정도이다. 본 발명의 제조과정에서 얻은 BCWIE-II 분획이 세포면역 및 체액성면역을 증진시킬 수 있는 면역증진 생리활성펩타이드이다.In one example of the present invention, it is obtained by removing fat from bovine colostrum, separating the casein by adjusting the pH to 4.5-4.6, and then removing the substance having a molecular weight greater than 10 KD from the obtained whey. In addition, by performing the step of removing a substance having a molecular weight of 1 KD or more, it is possible to obtain a colostrum fraction mainly composed of a peptide having a molecular weight in the range of 1 KD to 10 KD. As a method for selectively removing a peptide having a specific molecular weight in the above method, all known separation methods based on the size of the molecular weight, such as protein, may be used. Preferably, ultrafiltration or gel filtration is used. In a preferred example of the present invention, the filtrate obtained by performing ultrafiltration using a primary 10 KD membrane can be obtained again with a colostrum fraction (hereinafter referred to as BCWIE-III) present in the filtrate obtained by filtration using a 1 KD membrane. As a result, the ratio of the BCWIE-II fraction amount to the protein and peptide fraction (hereinafter referred to as BCWIE-I) of more than 10 KD is about 15: 1 to 20: 1. The BCWIE-II fraction obtained in the production process of the present invention is an immunostimulating bioactive peptide capable of enhancing cell immunity and humoral immunity.
본 발명의 초유분획은 면역세포인 Ts 세포, Th 세포, 또는 B 세포의 성장 또는 기능을 촉진시켜 면역증진효과를 나타낸다. 세포성 면역을 구성하고 있는 T 세포는 주로 감염에 대한 저항성과 B 세포의 성장과 분화를 조절하는 기능이 있다. Ts 세포는 면역기능의 억제기능이 있는 T 세포이며 주로 알레르기와 같은 과잉반응을 적절히 제어하는 역할을 한다. 그리고 또 다른 T 세포는 Th 세포이다. Th 세포는 Th1 세포와 Th2 세포의 두가지로 분류하는데 이 두가지 세포는 각각 감염에 대한 억제기능과 체액성면역을 증진시키는 기능을 가지고 있다. 그리고 B 세포는 항체를 만드는 기능을 가지고 있다. 위와 같이 각각의 면역세포는 제각기 다른 기능을 가지고 있다. 그리고 이와같이 면역세포에서 생산되어 세포외로 분비되는 물질들은 신체의 방어에 중요한 역할을 수행하고 있다. 각각의 세포에서 생산되는 분비물들은 면역조절 기능과 생체방어기능을 수행하며 이러한 물질의 생산량은 면역세포의 수에 정비례한다. 따라서 면역세포의 성장증진효과는 곧 면역증진효과라고 할 수 있다.The colostrum fraction of the present invention promotes the growth or function of immune cells, Ts cells, Th cells, or B cells, thereby exhibiting an immunostimulating effect. T cells, which make up cellular immunity, are primarily resistant to infection and regulate the growth and differentiation of B cells. Ts cells are T cells that suppress immune function and play a role in properly controlling overreaction such as allergies. And another T cell is a Th cell. Th cells are classified into two types, Th1 cells and Th2 cells, each of which has a function of inhibiting infection and enhancing humoral immunity. B cells have the function of making antibodies. As above, each immune cell has a different function. And the substances produced by the immune cells and secreted to the extracellular play an important role in the body's defense. The secretions produced by each cell perform immunomodulatory and bioprotective functions and the production of these substances is directly proportional to the number of immune cells. Therefore, the growth promoting effect of immune cells can be said to be an immune promoting effect.
구체적으로, 상기와 같이, 본 발명에서 제시한 방법으로 분리한 소의 초유중 BCWIE-II과 BCWIE-III는 다른 초유성분보다 모든 면역세포의 성장을 증진하는 효과가 탁월하다는 사실이 증명되었고 그리고 이 성분은 다량 투여할 경우에도 면역세포의 성장을 억제하는 특성을 전혀 나타내지 않았다. 따라서 본 발명에서 제시한 BCWIE-II과 BCWIE-III는 Ts 세포, Th 세포 그리고 B 세포의 성장을 증진시키는 효과가 있어 면역기능이 성숙되지 않은 유아와 면역기능이 저하된 성인의 면역증강 소재로 적합할 것으로 믿어진다.Specifically, as described above, BCWIE-II and BCWIE-III in colostrum of bovine colostrum isolated by the method proposed in the present invention have been proved to be superior in promoting the growth of all immune cells than other colostrum components and this component Even when administered in large doses, it did not show any property of inhibiting the growth of immune cells. Therefore, BCWIE-II and BCWIE-III presented in the present invention have the effect of enhancing the growth of Ts cells, Th cells and B cells, and thus are suitable as immune enhancing materials for infants with immature immune function and adults with reduced immune function. It is believed that
아울러 이들 성분과 비슷한 분자량의 성분들이 장관발달을 촉진시킨다는 연구보고(Playford 등, Am. J. Clin Nutr(72):5-14, 2000)와 세포면역기능과 체액성면역기능을 발휘하는 신체의 림프조직 중에서 50% 이상이 외부 항원이 유입되는 점막조직에 존재하지만 주로 장관림프조직(Gut-associated lymphoid tissue, GALT)에 집중되어 있다는 사실(Riott 등, Immunology, Mosby, 5th edition)과 염증성 장질환이나 소화장관이 손상되면 관강내의 성장인자 수용체가 관강막(luminal membrane)의 선단에 분포하게 되고 그리고 경구섭취한 성장인자가 혈류로 흡수되어 전신면역 증진을 유도하기 때문에 성장인자 성분이 장관면역의 발달하게 하고 그리고 경구섭취한 성장인자가 혈류로 흡수되기 때문에 전신면역을 발휘한다.In addition, studies of molecular weights similar to those of these compounds promote intestinal development (Playford et al., Am. J. Clin Nutr (72): 5-14, 2000). More than 50% of lymphoid tissues are present in mucosal tissue to which foreign antigens are introduced but are mainly concentrated in gut-associated lymphoid tissue (GALT) (Riott et al., Immunology, Mosby, 5th edition) and inflammatory bowel disease If the digestive tract is injured, the growth factor receptors in the lumen are distributed at the tip of the luminal membrane, and the growth factor is absorbed by the bloodstream and induces systemic immunity. It develops and exerts systemic immunity because orally ingested growth factors are absorbed into the bloodstream.
본 발명에서 제공하는 면역증강제의 면역증진 효과는 in vitro 상에서 1.0mg/ml 이상의 함량에서 유효하게 나타났으며, 과량투여에 의해서 면역억제효과가 나타나지 않았다. 이러한 결과를 지금까지 연구된 결과들과 종합해서 검토하면 성장인자 성분의 경구섭취후의 생체 내에서 거동이 장관에서 성장인자 수용체에 의해서 흡수되며 혈류 내로 흡수되기 때문에 장관에서의 국소면역 혹은 전신면역 증진에 효과를 발휘할 것으로 판단된다.The immunostimulating effect of the immunopotentiator provided in the present invention was shown to be effective at a content of 1.0 mg / ml or more in vitro, and did not show an immunosuppressive effect by overdose. These results, combined with the results studied so far, suggest that in vivo after oral ingestion of growth factor components are absorbed by the growth factor receptors in the intestine and are absorbed into the bloodstream. It will be effective.
따라서 본 발명에서 얻은 결과를 인체에 적용한다면 면역증진 유효량은 체중 1kg 당 1일 340mg 이상으로 사용가능하다. Regester 등(1997)은 치즈유청에서 분리한 유청 성장인자 추출물(whey growth factor extract) 0.6 내지 2.5mg/ml이 섬유아세포의 성장을 각각 약 100배 내지 200배 증진시켰으나 유전자재조합 기술에 의해서 생산된 IGF와 FGF는 약 20% 미만 그리고 TGF-β는 약 50% 정도 성장을 촉진한다고 하였다(Regester 등, Proceedings of the second international whey conference, Chicago) 그리고 Howarth 등(1996)은 Regester 등(1997)의 연구에서 사용한 것과 동일한 유청 성장인자 추출물로 동물실험한 결과 1.5kg의 랫트에게 1일 500mg을 경구투여하면 항암제인 메토트렉세이트 투여에 의해서 손상된 장관세포의 회복을 증진한다(Howarth 등, J. Nutr(126):2519-2530, 1996)는 연구결과를 기초로 체중환산법으로 추정하였다. 따라서 본 발명에서 얻은 BCWIE-II도 Howarth 등(1996)의 연구와 비슷한 결과를 나타낼 것이다.Therefore, if the result obtained in the present invention is applied to the human body, the effective amount of immunostimulation can be used at 340 mg or more per day per 1 kg of body weight. Regester et al. (1997) found that Whey growth factor extract 0.6 to 2.5 mg / ml from cheese whey increased fibroblast growth about 100 to 200 fold, respectively, but IGF produced by genetic recombination technology. And FGF less than 20% and TGF-β promotes growth by about 50% (Regester et al., Proceedings of the second international whey conference, Chicago) and Howarth et al. (1996) in a study by Regester et al. (1997). Animal testing with the same whey growth factor extract as used showed that oral administration of 500 mg daily to 1.5 kg rats promotes the recovery of intestinal cells damaged by the administration of the anticancer drug methotrexate (Howarth et al., J. Nutr (126): 2519 -2530, 1996) estimated the weight conversion method based on the study results. Therefore, BCWIE-II obtained in the present invention will show similar results to the study of Howarth et al. (1996).
또한, 본 발명에서 기대되는 가장 큰 효과는 초유에서 부터 면역억제 성분을 효과적으로 제거할 수 있다는 것이다. 따라서 면역증강효과만 보유하는 초유성분을 선택적으로 분리하기 때문에 면역증강 소재로서 면역억제 부작용 없이 식품이나 음료 등에 이용할 수 있는 장점이 있다. 특히 면역기능이 미숙한 유아를 위한 유아식에 이용하는 경우 장관면역과 전신면역의 성숙과 발달을 촉진하여 능동면역의 발달을 증진할 수 있다. 그리고 면역기능이 저하된 환자나 혹은 노약자의 경우에도 면역기능을 증진시킬 수 있는 식품의 소재로 이용될 수 있다. 따라서, 본발명은 상기 초유분획, 1 KD 미만의 분자량을 갖는 펩타이드를 주성분으로 하는 초유분획과, 1 내지 10 KD 분자량을 갖는 펩타이드를 주성분으로 하는초유분획, 또는 이들의 혼합물을 유효성분으로 하는 면역증강제를 식품, 음료, 또는 의약품등에 추가성분으로 포함시킬 수 있다. 이때 면역증강제를 1 mg/ml이상을 사용하는 것이 면역증강 효과면에서 바람직하다. 상기 식품이 조제분유, 이유식, 특수영양식품, 위궤양 또는 장관궤양용 환자식이, 자양강장식품일 수 있다.In addition, the greatest effect expected in the present invention is that it can effectively remove the immunosuppressive components from colostrum. Therefore, because it selectively separates the colostrum component having only the immune enhancing effect, there is an advantage that can be used as food or drink without immunosuppressive side effects as an immune enhancing material. In particular, when used in infant food for infants with immature immune function, it is possible to promote the development of active immunity by promoting the maturation and development of intestinal and systemic immunity. And it can be used as a material for foods that can enhance immune function in the case of patients with weakened immune function or the elderly. Accordingly, the present invention is the colostrum fraction containing the colostrum fraction, the peptide having a molecular weight of less than 1 KD as a main component, the colostrum fraction containing the peptide having a molecular weight of 1 to 10 KD as an active ingredient, or an immunity comprising a mixture thereof as an active ingredient. Enhancers may be included as additional ingredients in food, beverages, or pharmaceuticals. At this time, it is preferable to use an adjuvant of 1 mg / ml or more in terms of the immune enhancing effect. The food may be formula, baby food, special nutrition, gastric ulcer or intestinal ulcer patient diet, nourishing tonic food.
본 발명의 면역증강제는 통상의 수용체의 조건, 나이 등에 기초하여 본 기술분야의 통상의 전문가가 결정할 수 있다. 또한 이러한 면역증강제는 다양한 형태로 제제화할 수 있으며 단위-투여량 또는 다-투여량 용기로 제공될 수도 있다. 또한 투여경로는 주로 경구로 섭취하는 가장 바람직하며, 이에 한정되지 않는 적합한 경로로 투여할 수 있다.The immunopotentiators of the present invention can be determined by one of ordinary skill in the art based on conditions, age, etc. of conventional receptors. Such adjuvant may also be formulated in a variety of forms and may be presented in unit-dose or multi-dose containers. In addition, the route of administration is most preferably ingested orally, and may be administered by a suitable route without being limited thereto.
초유에서 분리한 성장인자 혼합성분이 유전자 재조합기술로서 제조한 성장인자 보다도 위 또는 소화장관에서 단백질 분해효소에 대한 저항력이 더 높기 때문에 경구를 통해 투여하더라도 위나 장관에서 분해되지 않고 생리활성을 유지할 수 있다는 장점이 있다(Rao 등, Peptides(19):495-504, 1998; Regester 등, Proceedings of the second international whey conference, Chicago, 1997). 유즙에 함유된 성장인자 성분 중에서 모유의 경우 EGF가 주성분이지만 우유의 경우 IGF가 주요 성분이다. 모유나 우유에 존재하는 성장인자는 유아의 장관 상피에 도달하여 영양효과(trophic effect)를 나타내는 것으로 알려지고 있다(Oka 등, Endocronol(112):940-944, 1983; Malo와 Menard, Gastroenterol(83):28-35, 1982). Pollack 등(1987)은 쥐에게 16㎍/kg/day의 EGF를 경구투여한 실험에서 EGF가 소화장관의 발달을 증진한다고 하였다(Pollack 등, Regulatory peptides(17):124-132, 1987).The growth factor mixed components isolated from colostrum have higher resistance to proteolytic enzymes in the stomach or digestive tract than the growth factors prepared by genetic recombination technology, so that oral administration can maintain physiological activity without degradation in the stomach or intestines. (Rao et al., Peptides (19): 495-504, 1998; Regester et al., Proceedings of the second international whey conference, Chicago, 1997). Among the growth factors in milk, EGF is the main ingredient for breast milk, but IGF is the main ingredient for milk. Growth factors present in breast milk or milk are known to reach the intestinal epithelium of infants and have a trophic effect (Oka et al., Endocronol (112): 940-944, 1983; Malo, Menard, Gastroenterol (83)). ): 28-35, 1982). Pollack et al. (1987) found that EGF enhances the development of the gut, in an oral dose of 16 μg / kg / day to rats (Pollack et al., Regulatory peptides (17): 124-132, 1987).
알카리성 영역의 등전점을 가지는 성장인자는 경구투여시에 장관 점막염증을 개선한다고 하였다(Howarth 등, J. Nutr(126):2519-2530, 1996; McKenna 등, Sugery(115):626-632, 1994). 따라서 산성영역의 등전점의 펩타이드인 EGF 뿐만아니라 IGF를 함유하는 알카리영역 등전점 펩타이드가 장관세포의 성장과 발달에 많은 영향을 준다고 할 수 있다(Houle 등, Proc.Endocrine Soc. USA, 77th annual meeting. p 518; Kontura 등, Gut(34):881-887, 1998; Puolakkainen 등, Gastroenterol(107):1319-1326, 1994). 알카리성 영역의 등전점 성장인자로서 우유에 기장 많이 함유되어 있는 IGF는 선천성면역(innate immunity)과 후천성면역(acquired immunity)를 증진시킨다. T 세포, B 세포, 말초혈액단핵세포 그리고 NK세포는 IGF 수용체(IGF receptor)를 가지고 있으며 IGF에 대해서 아주 민감하게 작용한다(Kooijman 등, Endocrinol(31):244-250, 1992; Badolato 등, J. Clin. Endocrinol. Metab(79):984-990, 1994). 면역능력이 있는 세포와 IGF-1과의 상호작용에 의한 선천성면역 조절은 항원에 대한 성숙 면역세포 반응성에 영향을 줄 수 있다. 예를들면 NK 세포의 활성은 IGF-1의 자극에 의해서 증진되며(Kooijman 등, Endocrinol(31):244-250, 1992)IGF-1은 마크로파지 뿐만아니라 호중구에 의한 superoxide 생산을 직접 자극하는 것으로 보고되고 있다(Edward 등, Science(239):769-771). 후천적면역(acquired immunity)에 대하여 IGF는 전구세포가 2차림프기관으로 이동하기 전, 후의 림프구형성(lymphopoiesis)을 조절하는 역할을 한다(Geffner, Acta Paediatrica 86(Suppl. 423):76-79, 1997) 그리고 in vitro 실험에서 IGF가 T 세포와 B 세포에 특이적으로 결합하며(Stuart 등, J. Clin. Endocrinol. Metab(72):950-957, 1988) T 세포에서 IGF-1 수용체의 수는 T 세포에 대한 미토젠 자극에 의해 증가된다고 하였다(Xu 등, Immunol(85):394-399, 1995). In vitro에서 IGF-1을 전처리한 렛트의 비장과 림프절에서 분리한 T 세포와B 세포는 미토젠이나 항원자극에 의한 반응성이 더 높았으며(Clark 등, J. Clin. Invest(92):540-548, 1993) IGF를 전처리한 렛트의 항체생산능력이 증가한다고 보고하였다(Robins 등, Clin Exp. Immunol(95):337-342, 1994). 위의 연구결과는 1차와 2차 림프기관에서 성숙하였거나 성숙중인 림프구에 대한 IGF의 증식효과, 분화효과 그리고 기능적효과에 대해서 잘 설명해 주고 있다. 경구투여한 IGF는 분해되지 않고 소화장관에 결합하여 IGF 수용체의 수를 증가시키고 그리고 세포의 성장을 촉진한다(Baumrucker와 Blum, Livestock Prod(35):49-72, 1993). 그리고 경구섭취한 IGF는 혈액내로 흡수 될 수 있다(Donovan과 Odle, Ann. Rev. Nutr(14):147-167, 1994). 그리고 투여한 growth factor에 의한 장관면역 증진효과는 growth factor의 수용체를 통해서 이루어진다. 전술한 바와 같이 면역세포의 표면에는 IGF 수용체가 존재한다. 그리고 Wright 등(1993)은 염증성장질환과 같이 장관이 손상된 조건하에서는 growth factor의 수용체가 관강막(luminal membrane)의 선단에 분포한다고 보고하였다(Wright 등, Gastroenterol(104):12-20, 1993).Growth factors with isoelectric points in the alkaline region improve intestinal mucosal inflammation upon oral administration (Howarth et al., J. Nutr (126): 2519-2530, 1996; McKenna et al., Sugery (115): 626-632, 1994). ). Therefore, not only EGF, which is an isoelectric peptide in the acidic region, but also an alkaline region isoelectric peptide, which contains IGF, can affect the growth and development of intestinal cells (Houle et al., Proc. Endocrine Soc. USA, 77th annual meeting.p. 518; Kontura et al., Gut (34): 881-887, 1998; Puolakkainen et al., Gastroenterol (107): 1319-1326, 1994). IGF, which is an isoelectric point growth factor in the alkaline region, contains a lot of millet in milk, enhances innate immunity and acquired immunity. T cells, B cells, peripheral blood mononuclear cells, and NK cells have IGF receptors and are very sensitive to IGF (Kooijman et al., Endocrinol (31): 244-250, 1992; Badolato et al., J.). Clin Endocrinol Metab (79): 984-990, 1994). Innate immune regulation by the interaction of immune cells with IGF-1 may affect mature immune cell responsiveness to antigen. For example, NK cell activity is enhanced by stimulation of IGF-1 (Kooijman et al., Endocrinol (31): 244-250, 1992) and IGF-1 is reported to directly stimulate superoxide production by neutrophils as well as macrophages. (Edward et al., Science (239): 769-771). On acquired immunity, IGF regulates lymphocyte formation after progenitor cells migrate to secondary lymphoid organs (Geffner, Acta Paediatrica 86 (Suppl. 423): 76-79, 1997) and IGF binds specifically to T and B cells in vitro (Stuart et al., J. Clin. Endocrinol. Metab (72): 950-957, 1988) and the number of IGF-1 receptors in T cells. Is increased by mitogen stimulation on T cells (Xu et al., Immunol (85): 394-399, 1995). T cells and B cells isolated from spleen and lymph nodes of rats pretreated with IGF-1 were more responsive to mitogen or antigenic stimulation (Clark et al., J. Clin. Invest (92): 540-). 548, 1993). The production of IGF-prepared rats was reported to increase (Robins et al., Clin Exp. Immunol (95): 337-342, 1994). The above results explain the proliferative, differentiating and functional effects of IGF on mature or mature lymphocytes in primary and secondary lymphoid organs. Orally administered IGF binds to the digestive tract without degradation and increases the number of IGF receptors and promotes cell growth (Baumrucker and Blum, Livestock Prod (35): 49-72, 1993). And orally ingested IGF can be absorbed into the blood (Donovan and Odle, Ann. Rev. Nutr (14): 147-167, 1994). Intestinal immunity-promoting effects by the growth factor administered are achieved through receptors on the growth factor. As described above, IGF receptors are present on the surface of immune cells. Wright et al. (1993) reported that receptors for growth factor are distributed at the tip of the luminal membrane under conditions such as inflammatory bowel disease (Wright et al., Gastroenterol (104): 12-20, 1993). .
따라서 경구 섭취한 성장인자는 장관의 관강에 존재하는 수용체에 결합하여 장관면역증진을 유도하게 되며 더 나아가서 경구섭취한 성장인자가 혈류내로 흡수되어 전신면역(systemic immunity)증진을 유도하게 된다. 따라서 알카리영역 등전점 성장인자는 장관발달이 미숙한 유아 또는 장관이 손상된 잘환을 가지고 있는 환자 그리고 장관기능이 쇠퇴한 노인의 장관의 발달과 장관 및 전신면역의 발달을 증진할 수 있다.Therefore, oral growth factors bind to receptors in the lumen of the intestinal tract to induce intestinal immunity, and furthermore, oral ingestion of growth factors is absorbed into the bloodstream to induce systemic immunity. Thus, the isoelectric growth factor of the alkaline domain can enhance the development of the intestinal tract and the intestinal and systemic immunity in infants with immature intestinal development or patients with impaired intestinal tract and intestinal dysfunction.
실시예Example
실시예 1: 생리활성펩타이드의 분리Example 1 Isolation of Bioactive Peptides
소의 초유에서 면역력을 증진하는 생리활성 펩타이드를 제조하기 위해서, 분만후 48시간동안 분비되는 소의 초유를 수집하여 약 40℃로 가온한 다음 크림분리기를 이용하여 크림층을 제거하여 0.1% 이하의 지방이 함유된 탈지초유를 만든다. 탈지초유에 1N 염산을 이용하여 pH를 4.5-4.6으로 조절하고 약 30분간 상온에서 정치한 다음 카세인이 침전할 때까지 물을 가한다. 응고된 카세인과 유청은 원심분리기를 이용하여 분리한다. 분리된 유청은 1N NaOH를 이용하여 pH를 7.0으로 조절한다. 유청은 한외여과 또는 겔여과로 분자량별로 분리한다. 더욱 구체적으로, 30 KD 한외여과막이 장착된 Minitan UF Unit(Millipore)를 통과시켜서 여과액과 잔류물을 얻었다. 이중에서 여과물은 10 KD spiral wound 한외여과막으로 여과하여 얻어진 여과액을 1 KD 막을 이용하여 또다시 여과하여 1 KD 미만의 분자량을 가진 여액을 얻었다. 이 결과 10 KD 초과의 단백질과 펩타이드 분획(이하 BCWIE-I라고 칭함), 1 KD 내지 10 KD의 분자량을 갖는 성분(이하 BCWIE-II라고 칭함), 및 1 KD 미만의 분자량을 갖는 성분(이하 BCWIE-III라고 칭함)을 얻었으며, BCWIE-I과 BCWIE-II 분획량의 비율을 15:1에서 20:1 정도였다.In order to produce a biologically active peptide that enhances immunity in bovine colostrum, bovine colostrum secreted for 48 hours after delivery is collected and warmed to about 40 ℃, and the cream layer is removed using a cream separator to remove less than 0.1% of fat. Make skim colostrum containing. Adjust the pH to 4.5-4.6 with 1N hydrochloric acid in skim colostrum, allow to stand at room temperature for about 30 minutes, and then add water until casein precipitates. Solidified casein and whey are separated using a centrifuge. The separated whey is adjusted to pH 7.0 with 1N NaOH. Whey is separated by molecular weight by ultrafiltration or gel filtration. More specifically, the filtrate and residue were obtained by passing through a Minitan UF Unit (Millipore) equipped with a 30 KD ultrafiltration membrane. Among the filtrates, the filtrate obtained by filtration with a 10 KD spiral wound ultrafiltration membrane was filtered again using a 1 KD membrane to obtain a filtrate having a molecular weight of less than 1 KD. As a result, a protein and peptide fraction greater than 10 KD (hereinafter referred to as BCWIE-I), a component having a molecular weight of 1 KD to 10 KD (hereinafter referred to as BCWIE-II), and a component having a molecular weight of less than 1 KD (hereinafter referred to as BCWIE) -III), and the ratio of BCWIE-I and BCWIE-II fractions ranged from 15: 1 to 20: 1.
실시예 2: BCWIE-II의 면역증진효과Example 2: Immunostimulating Effect of BCWIE-II
혈청을 함유하지 않은 DMEM(Dulbecco's modified Eagle's medium)에 쥐의 T 림프세포주(EL-4 세포)을 분산시켜 96-웰 플레이트에 최종농도가 105세포/ml이 되도록 분주하였다. 그리고 실시예 1에서 얻어진 초유분획을 1mg/ml의 농도로 첨가하여 48시간 동안 37 ℃의 CO2배양기에서 배양하였다. 세포의 성장을 측정하기 위해서 48시간 이후에 3-(4,5-디메틸티아졸-2-일)-2,5-디페닐-테트라졸륨 브로마이드(MTT) 주입하고 4시간 후 각 웰당 100㎕의 20 % SDS 용액을 주입하여 MTT-포르마잔을 가용화하였다. 그리고 16시간 더 배양한 다음, 540nm에서 흡광도를 측정하여 EL-4 세포의 성장을 측정하였다. EL-4 세포의 증식효과는 초유성분을 함유하지 않은 조건에서 성장한 EL-4 세포의 성장에 대한 백분율로 나타냈다. EL-4 세포의 성장에 대한 효과는 열처리전의 BCWIE-II는 226.54%의 증식효과가 있었고 BCWIE-I 보다는 약 3배 정도의 성장을 증진시켰다. 그리고 열처리 후의 EL-4 세포 증식효과는 초유유청이 97.58%인데 비해서 BCWIE-II는 200.81%로 나타났다. BCWIE-I은 열처리 후에 오히려 EL-4 세포 증식효과가 높아지는 것으로 나타났다(표 1).Rat T lymphocyte lines (EL-4 cells) were dispersed in Dulbecco's modified Eagle's medium (DMEM) containing no serum and aliquoted in 96-well plates to a final concentration of 10 5 cells / ml. The colostrum fraction obtained in Example 1 was added at a concentration of 1 mg / ml and incubated in a CO 2 incubator at 37 ° C. for 48 hours. To measure the growth of cells, 48 μl of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) was injected after 48 hours and 100 μl of each well after 4 hours. MTT-formazan was solubilized by injection of 20% SDS solution. After 16 hours of further incubation, the absorbance was measured at 540 nm to measure the growth of EL-4 cells. The proliferative effect of EL-4 cells was expressed as a percentage of the growth of EL-4 cells grown in the absence of colostrum. The effect on the growth of EL-4 cells, BCWIE-II before heat treatment had a proliferative effect of 226.54% and enhanced the growth of about three times than BCWIE-I. The ELW cell proliferation effect after heat treatment was 97.58% for colostrum and 200.81% for BCWIE-II. BCWIE-I was found to increase EL-4 cell proliferation effect after heat treatment (Table 1).
실시예 3: BCWIE-II의 투여량에 따른 면역증진효과Example 3: Immunostimulating Effect According to the Dose of BCWIE-II
상기의 실시예 2와 동일한 방법으로 BCWIE-II의 투여량에 따른 실험을 실시한 결과 세포성장 증진효과는 BCWIE-II를 10-6- 10mg/ml 범위의 농도에서 실험한 결과 투여량에 정비례하여 세포의 성장을 증진하는 것으로 나타났다. 그리고 1 mg/ml의 BCWIE-II 투여시에는 138.50%의 세포성장 증진효과가 있었고 10 mg/ml의 경우에는 309.62%의 EL-4 세포 증식효과가 있는 것으로 나타났다(표 2).The enhancement of the embodiment example 2, the same procedure as a result of the experiment according to the dose of BCWIE-II cell growth and the effect is the BCWIE-II 10 -6 - in direct proportion to the dose resulting in a test concentration of 10mg / ml range cell Has been shown to promote growth. BCWIE-II administration at 1 mg / ml showed 138.50% cell growth and 309.62% EL-4 cell proliferation at 10 mg / ml (Table 2).
실시예 4: BCWIE-II의 마우스 비장세포 성장 증진효과Example 4: BCWIE-II mouse splenocyte growth promoting effect
5주령의 암컷 Balb/c 마우스의 비장을 무균적으로 분리하여 단일세포를 만들어 BCWIE-II에 의한 비장세포(splenocyte)의 성장 효과에 대한 실험을 수행하였다. 2X106세포/웰 밀도의 비장세포를 FCS(Fetal calf serum)를 함유하지 않는 RPMI에 분산시킨 다음 96-웰 플레이트에 주입하고 1mg/ml의 농도로 BCWIE-II를 첨가하였다. 실시예 2와 같이 세포의 증식에 효과를 측정하였다. 비장세포의 증식효과는 초유성분을 함유하지 않은 조건에서 성장한 비장세포성장에 대한 백분율로 나타내었다. 그 결과 열처리 전의 BCWIE-I은 139.05%, BCWIE-II는 305.75% 그리고 BCWIE-III는 200.79%의 성장효과를 보였으나 100℃에서 10분간 열처리한 분획들의 비장세포 증식효과는 BCWIE-II가 146.90%로 증가한 반면에 다른 분획들은 증식효과가 낮아졌다.The spleen of five-week-old female Balb / c mice was aseptically isolated to make single cells, and experiments were performed on the growth effect of splenocytes by BCWIE-II. Splenocytes of 2 × 10 6 cells / well density were dispersed in RPMI without Fecal calf serum (FCS), then injected into 96-well plates and BCWIE-II was added at a concentration of 1 mg / ml. The effect on the proliferation of the cells was measured as in Example 2. The proliferative effect of splenocytes is expressed as a percentage of splenocyte growth grown in the absence of colostrum. As a result, BCWIE-I showed 139.05% of BCWIE-I, 305.75% of BCWIE-II, and 200.79% of BCWIE-III, but BCWIE-II showed 146.90% While other fractions had lower proliferative effect.
실시예 5: BCWIE-II의 투여량에 따른 비장세포 성장 증진효과Example 5: Splenocyte Growth Enhancement Effect of BCWIE-II
5 주령 암컷 Balb/c 마우스의 비장세포에 대한 BCWIE-II의 투여량에 따른 세포성장효과에 대해서 실험하기 위해서, BCWIE-II의 투여량이 10-3mg/ml - 10 mg/ml의 범위인 것을 제외하고는 실시예 4와 동일하여 수행하였다. 그 결과, BCWIE-II의 투여량에 정비례하게 비장세포의 성장이 증가하였다.In order to examine the effect of BCWIE-II on the growth of cells on splenocytes of 5 week-old female Balb / c mice, the dose of BCWIE-II was in the range of 10 -3 mg / ml-10 mg / ml. Except that was carried out in the same manner as in Example 4. As a result, the growth of splenocytes increased directly with the dose of BCWIE-II.
실시예 6: 억제T 세포 미토젠으로 자극한 비장세포에 대한 소의 초유성분의 증진효과Example 6: Enhancement effect of bovine colostrum on splenocytes stimulated with inhibitory T cell mitogen
소의 초유성분의 억제 T 세포 증식효과에 대해서 시험하기 위해서 미토젠으로서 Con A를 1㎍/ml 수준으로 첨가했을 때 BCWIE-II의 비장세포 증식효과는 BCWIE-I은 137.27%, BCWIE-II는 420.87% 그리고 BCWIE-III의 경우에는 360.66%의 증식효과가 있었다. 그리고 BCWIE-II의 비장세포 증식효과는 BCWIE-I 보다는 약 3배 정도 높았다.In order to test the inhibitory T cell proliferation effect of bovine colostrum, the splenocyte proliferation effect of BCWIE-II was 137.27% for BCWIE-I, and 420.87 for BCWIE-II when Con A was added at 1 µg / ml level as a mitogen. % And in the case of BCWIE-III there was a proliferation effect of 360.66%. And BCWIE-II's splenocyte proliferation effect was about 3 times higher than BCWIE-I's.
실시예 7: Th 세포 미토젠으로 자극한 비장세포에 대한 BCWIE-II의 증식효과Example 7: Proliferation of BCWIE-II on Th Cell Mitogen-stimulated Spleen Cells
소의 초유성분의 T 헬퍼(Th) 세포의 증식효과에 대해서 시험하기 위해서 미토젠으로서 PHA를 1㎍/ml 수준으로 첨가했을 때, 비장세포 증식효과는 초유유청이 96.34%, BCWIE-I이 108.25%, BCWIE-II가 214.74% 그리고 BCWIE-III가 99.98%로 나타났고 포볼 12-미리스테이트 13-아세테이트(PMA) 10ng/ml 첨가하였을 때 비장세포 증식효과는 초유유청 116.48%, BCWIE-I 138.25%, BCWIE-II 347.61%, BCWIE-III 218.51%로 나타났다.To test for the proliferative effect of bovine colostrum T-helper (Th) cells, when PHA was added at the level of 1 µg / ml as mitogen, splenocyte proliferation was 96.34% for colostrum and 108.25% for BCWIE-I. , BCWIE-II and 214.74% BCWIE-III and 99.98% BCWIE-III were added. BCWIE-II 347.61%, BCWIE-III 218.51%.
실시예 8: B 세포 미토젠으로 자극한 비장세포에 대한 BCWIE-II의 증식효과Example 8: Proliferation Effect of BCWIE-II on B Cell Mitogen Stimulated Splenocytes
소의 초유성분의 B 세포 증식효과에 대해서 시험하기 위해서, 미토젠으로서리포폴리사카라이드(LPS)를 1㎍/ml 첨가했을 때 비장세포의 증식효과는 초유유청과 BCWIE-I이 각각 151.66%와 121.03%인 반면에 BCWIE-II와 BCWIE-III의 경우에는 각각 454.19%와 269.51%로 나타났다.In order to test the B cell proliferation effect of bovine colostrum, the proliferation effect of splenocytes when lipopolysaccharide (LPS) was added as mitogen was 151.66% and 121.03% for colostrum and BCWIE-I, respectively. %, While BCWIE-II and BCWIE-III were 454.19% and 269.51%, respectively.
본 발명은 카세인과 지방이 제거된 소의 초유 유청으로부터 10 KD 초과 분자량을 갖는 펩타이드를 제거하고 얻어진 초유분획을 유효성분으로 하는 면역증강제, 이의 제조방법 및 그 용도에 관한 것으로서, 초유 중에 포함된 면역억제성분을 제거하여 효과적인 면역증강제를 제공하는 장점이 있다. 특히 면역기능이 미숙한 유아를 위한 유아식에 이용하는 경우 장관면역과 전신면역의 성숙과 발달을 촉진하여 능동면역의 발달을 증진할 수 있다. 그리고 면역기능이 저하된 환자나 혹은 노약자의 경우에도 면역기능을 증진시킬 수 있는 식품의 소재로 이용될 수 있다.The present invention relates to an immunopotentiator, a method for preparing the same, and a method of using the colostrum fraction obtained by removing a peptide having a molecular weight greater than 10 KD from colostrum whey of cows from which casein and fat have been removed. Removing the ingredients has the advantage of providing an effective adjuvant. In particular, when used in infant food for infants with immature immune function, it is possible to promote the development of active immunity by promoting the maturation and development of intestinal and systemic immunity. And it can be used as a material for foods that can enhance immune function in the case of patients with weakened immune function or the elderly.
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