KR100560064B1 - Manufacturing method for peptide containing immune-activating factor from black soy bean - Google Patents
Manufacturing method for peptide containing immune-activating factor from black soy bean Download PDFInfo
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- protein hydrolyzate
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- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 20
- 244000068988 Glycine max Species 0.000 title claims abstract description 20
- 238000004519 manufacturing process Methods 0.000 title abstract description 5
- 108090000765 processed proteins & peptides Proteins 0.000 title abstract description 4
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 239000000706 filtrate Substances 0.000 claims abstract description 9
- 239000012141 concentrate Substances 0.000 claims description 14
- 241001107116 Castanospermum australe Species 0.000 claims description 8
- 235000021279 black bean Nutrition 0.000 claims description 8
- 102000005593 Endopeptidases Human genes 0.000 claims description 6
- 108010059378 Endopeptidases Proteins 0.000 claims description 6
- 102000018389 Exopeptidases Human genes 0.000 claims description 6
- 108010091443 Exopeptidases Proteins 0.000 claims description 6
- 230000006862 enzymatic digestion Effects 0.000 claims description 6
- 102000035195 Peptidases Human genes 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 230000002255 enzymatic effect Effects 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 235000019833 protease Nutrition 0.000 claims description 2
- 239000003531 protein hydrolysate Substances 0.000 abstract description 22
- 230000005965 immune activity Effects 0.000 abstract description 20
- 108010073771 Soybean Proteins Proteins 0.000 abstract description 19
- 229940001941 soy protein Drugs 0.000 abstract description 11
- 235000019710 soybean protein Nutrition 0.000 abstract description 8
- 235000013305 food Nutrition 0.000 abstract description 6
- 239000007791 liquid phase Substances 0.000 abstract description 6
- 230000001900 immune effect Effects 0.000 abstract description 4
- 239000007790 solid phase Substances 0.000 abstract description 3
- 206010020751 Hypersensitivity Diseases 0.000 abstract 1
- 239000012190 activator Substances 0.000 abstract 1
- 230000007815 allergy Effects 0.000 abstract 1
- 231100000957 no side effect Toxicity 0.000 abstract 1
- 239000000463 material Substances 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 210000000265 leukocyte Anatomy 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 238000007664 blowing Methods 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 229940069765 bean extract Drugs 0.000 description 3
- 235000019711 black bean protein Nutrition 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 108010009736 Protein Hydrolysates Proteins 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 235000020712 soy bean extract Nutrition 0.000 description 2
- 125000003831 tetrazolyl group Chemical group 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 231100000750 In vitro toxicology Toxicity 0.000 description 1
- 235000015429 Mirabilis expansa Nutrition 0.000 description 1
- 244000294411 Mirabilis expansa Species 0.000 description 1
- 241000266501 Ormosia ormondii Species 0.000 description 1
- 108010064851 Plant Proteins Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 231100000480 WST assay Toxicity 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 230000003588 decontaminative effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000013536 miso Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 235000014102 seafood Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
- A23J1/148—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds by treatment involving enzymes or microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/14—Vegetable proteins
- A23J3/16—Vegetable proteins from soybean
-
- 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
- A23V2300/28—Hydrolysis, degree of hydrolysis
Abstract
본 발명은 검은콩 단백 가수 분해물의 제조방법에 관한 것으로, 더욱 상세하게는, 검은콩을 효소로 가수 분해하여 고체상과 액상으로 분리한 후, 상기 액상을 원심분리하고, 여과액을 분말화하여 얻어진 면역 활성이 강화된 검은콩 단백 가수 분해물의 제조방법에 관한 것이다. 본 발명에 의하면, 일반적인 면역 활성제와는 달리, 일반 식품으로 우리가 흔히 섭취하는 검은콩을 이용하여 상대적으로 알레르기 등의 부작용이 없고, 면역 활성을 강화해주는 인자들을 다량 함유한 검은콩 단백 가수 분해물을 얻을 수 있다.The present invention relates to a method for producing a black soybean protein hydrolyzate, and more specifically, black soybean is hydrolyzed by an enzyme and separated into a solid phase and a liquid phase, the liquid phase is centrifuged, and the filtrate is powdered. It relates to a method for producing a black soy protein hydrolyzate with enhanced immune activity. According to the present invention, unlike the general immunological activator, using black soybeans commonly consumed as a general food, there is relatively no side effects such as allergies, and the black soybean protein hydrolyzate containing a large amount of factors that enhance immune activity. You can get it.
검은콩 (흑두), 면역활성, 펩티드Black Soybean, Immune Activity, Peptide
Description
본 발명은 검은콩 단백 가수 분해물의 제조방법에 관한 것으로, 더욱 상세하게는, 검은콩을 효소로 가수 분해하여 고체상과 액상으로 분리한 후, 상기 액상을 원심분리하고, 여과액을 분말화하여 얻어진 면역 활성이 강화된 검은콩 단백 가수 분해물에 관한 것이다.The present invention relates to a method for producing a black soybean protein hydrolyzate, and more specifically, black soybean is hydrolyzed by an enzyme and separated into a solid phase and a liquid phase, the liquid phase is centrifuged, and the filtrate is powdered. It relates to a black soybean protein hydrolysate with enhanced immune activity.
인간의 항상성(homeostasis)을 유지하기 위해 조직이나 기관에 손상을 줄 수 있는 모든 병원체나 독소에 저항할 수 있는 능력을 면역이라 부른다. 이들 면역에 관여하는 세포들 중에서 가장 흔하게 발견되는 세포는 백혈구(leukocyte, white blood cell, WBC)이다. 특히, 이들 백혈구 중 티 세포(T cell)는 항체를 생산하는 비 세포(B cell)에 작용하여 항체를 만들도록 하며, 대식세포에 작용하여 대식세포를 활성화시키기도 하고, 씨티엘(CTL, Cytotoxic T Lymphocyte)에 작용하여 그들을 활성화시키기도 한다(김세종 : 면역학, 고려의학, 1-112 (2000)). The ability to resist any pathogen or toxin that can damage tissues or organs to maintain human homeostasis is called immunity. Among the cells involved in these immunity, the most commonly found are leukocytes (leukocytes, white blood cells, WBC). In particular, T cells in these leukocytes act on non-cells (B cells) that produce antibodies, and also act on macrophages to activate macrophages. ) To activate them (Kim Sejong: Immunology, Korea Medicine, 1-112 (2000)).
지금까지 이러한 면역기능을 위한 연구에 이용되는 식품 및 식품소재로는, 일반 식물성 소재, 해산물 소재, 버섯 소재, 유산균 등 미생물 소재, 키토산 소재 등이 이용되어 왔다(임중선, 천연소재 활용한 활성화 규명 필요, 월간식품, 2, 11, 90-96(2003)). Until now, as a food and food material used for such immune function research, general vegetable material, seafood material, mushroom material, microbial material such as lactic acid bacteria, chitosan material, etc. have been used (Improvement of activation using forests and natural materials) Necessary, Monthly Food, 2, 11, 90-96 (2003)).
그러나, 상기의 소재를 활용한 면역 활성 식품 소재는 효능이 기대에 미치지 못하거나 독성의 문제 그리고 산업적 경제성이 상대적으로 낮다는 문제점이 있었다. However, the immuno-active food material using the above-described material has a problem that the efficacy does not meet expectations or the toxicity and industrial economics are relatively low.
한편, 최근 관심이 증가되고 있는 검은콩의 껍질 표면에는 일반 황색 콩에 없는 글리시테인이라는 물질이 많이 함유되어 있어 면역 증강과 항 바이러스에 효과(Yamai, M. et, al. : Biosci. Biotechnol. Biochem., 67, 5, 1071-1079 (2003))가 있다고 알려져 있다. On the other hand, the surface of black soybeans, which has recently been gaining attention, contains a lot of a substance called glycidin, which is not found in ordinary yellow soybeans, and thus is effective in enhancing immunity and antiviral (Yamai, M. et, al .: Biosci. Biotechnol. Biochem., 67, 5, 1071-1079 (2003)).
그러나, 검은콩 하나 당 껍질이 차지하는 양은 극히 소량일 뿐만 아니라, 이들 검은콩 껍질 표면에 존재하는 면역 증강에 효과가 있는 글리시테인을 추출하기 위하여는 1-2개월에 걸쳐 된장을 제조하여야 하며, 이 된장을 냉수 조건에서 추출하고, 추출액을 1차로 한외여과 공정을 거쳐 여과한 후 3차로 제염을 위한 역삼투여과를 하여야 하므로, 공정이 비교적 복잡하고 경제성이 상대적으로 낮다는 단점이 있다. However, the amount of skin per black bean is extremely small, and soybean paste should be prepared for 1-2 months in order to extract glycine, which is effective in enhancing the immunity present on the surface of these black bean skins. This miso is extracted under cold water conditions, the extract is first filtered through an ultrafiltration process, and then reverse osmosis for decontamination in the third step has a disadvantage in that the process is relatively complicated and economical is relatively low.
따라서, 본 발명자는 검은콩 단백의 가수 분해물에서도 면역 활성을 강화시킬 수 있는 펩티드가 함유되어 있을 것으로 판단하여 일련의 실험을 행한 결과, 일반 황색 콩 단백 가수물과 검은콩 추출물보다 면역 활성이 우수한 검은콩 단백 가수 분해물의 제조방법을 발견하여 본 발명을 완성하였다.Therefore, the present inventors have determined that the hydrolyzate of black soybean protein may contain a peptide that can enhance immune activity. A method of preparing soy protein hydrolyzate was found to complete the present invention.
본 발명의 목적은 우리가 일상에서 섭취하는 식물 단백질원인 검은콩을 사용하여 면역 활성을 강화하는 인자들이 다량 함유된 검은콩 단백 가수 분해물을 경제적으로 제공하는데 있다. It is an object of the present invention to economically provide a black soy protein hydrolyzate containing a large amount of factors that enhance immune activity using black soybean, which is a plant protein source that we consume everyday.
본 발명의 다른 목적 및 장점들은 하기에 설명될 것이며, 본 발명의 실시에 의해 알게 될 것이다. 또한, 본 발명의 목적 및 장점들은 특허 청구 범위에 나타낸 수단 및 조합에 의해 실현될 수 있다. Other objects and advantages of the invention will be described below and will be appreciated by the practice of the invention. In addition, the objects and advantages of the present invention can be realized by means and combinations indicated in the claims.
상기 목적을 달성하기 위하여, 분쇄된 검은콩에 펩티다아제를 첨가하는 1차 효소 분해 단계; 상기 1차 효소 분해된 용액을 가열하여 효소를 실활시킨 후 여과한 여과액을 농축하여 검은콩 농축액을 얻는 단계; 상기 검은콩 농축액에 엔도펩티다아제를 첨가하는 2차 효소 분해 단계; 상기 2차 효소 분해된 농축액에 엑소펩티다아제를 첨가하는 3차 효소 분해 단계; 및 상기 3차 효소 가수분해된 농축액을 여과한 후 여과액을 농축하여 분말화하는 단계;를 통하여 얻어진 면역 활성이 강화된 검은콩 단백 가수분해물의 제조방법이 제공된다. In order to achieve the above object, the first enzymatic digestion step of adding a peptidase to the ground black beans; Heating the first enzymatically digested solution to deactivate the enzyme, and then concentrating the filtrate to obtain black soybean concentrate; Secondary enzymatic digestion step of adding endopeptidase to the black soybean concentrate; A third enzymatic digestion step of adding exopeptidase to the secondary enzymatic digested concentrate; And filtering the tertiary enzymatic hydrolyzed concentrate, and then filtrating the filtrate by concentrating the filtrate. There is provided a method for producing a black soy protein hydrolyzate having enhanced immune activity.
삭제delete
본 발명의 내용을 상세히 설명하면 다음과 같다.The content of the present invention will be described in detail below.
검은콩 농축액은 검은콩을 분쇄하고 3배 ∼ 15배의 물을 가수한 후 단백 분해 효소(protease)를 이용하여 45℃ ~ 55℃에서 2 ~ 5시간 동안 효소분해 한 후 90℃ ~ 100℃에서 10 ~ 20분 동안 가열하여 효소를 실활 시킨 후 60 ~ 120 메쉬 (mesh)에서 여과 후 진공 농축기를 이용하여 45 ~ 55 브릭스(Brix)까지 농축하여 얻었다. 이 검은콩 농축액의 전처리를 위해서, 반응조에 넣고 검은콩 농축액의 5배 내지 10배의 물을 가하고, 교반하며 온도를 100℃로 상승시켜 5분 동안 유지하여 단백질을 변성시킨 후, 온도를 다시 60℃로 조정한다.Black soybean concentrate was pulverized black soybean, hydrolyzed 3 to 15 times of water, and then enzymatically decomposed at 45 ° C. to 55 ° C. for 2 to 5 hours using protease and then at 90 ° C. to 100 ° C. The enzyme was inactivated by heating for 10 to 20 minutes, filtered through 60 to 120 mesh, and concentrated to 45 to 55 Brix using a vacuum concentrator. For the pretreatment of the black soybean concentrate, it was placed in a reaction vessel, and 5-10 times the water of black soybean concentrate was added, stirred, and the temperature was raised to 100 ° C. for 5 minutes to denature the protein, and then the temperature was again 60. Adjust to ° C.
이에 5 노르말 수산화나트륨을 서서히 첨가하여 pH를 8.0으로 조정한 후 효소인 엔도펩티다아제(endopeptidase)를 원료 단백질의 0.4 ~ 0.8중량% 첨가하여 2시간 동안 교반하여 반응시킨다. 반응이 완료되면 온도를 55℃로 조정한 후, 6 노르말 염산을 첨가하여 pH를 5.0으로 조정한다. 이어 효소 엑소펩티다아제 (exopeptidase)를 원료 단백질의 0.4 ~ 0.8중량% 첨가하고 4시간 교반하여 반응시킨다. 반응이 완료되면 온도를 100℃로 상승시켜 5분간 유지하여 효소를 불활성화 시킨다. 생성된 효소반응물을 원심분리기를 사용하여 고체상과 액체상으로 분리한 후, 메쉬를 이용하여 직경 70 ㎛ 이하의 입자가 포함되어 있는 액상을 분리 및 회수하고 여과하여 이 여과액을 농축기로 이송하여 50±5℃에서 고형분 함량 40 중량%까지 농축한 후 분말화하였다. 분말화 조건은 송풍 온도 165℃, 배풍 온도 95℃로 하였으며, 아토마이저(atomizer)는 9,000 rpm으로 하였다. 얻어진 분말의 면역 활성은 티 세포(T cell)의 증식능(proliferation)으로 대조군에 대한 상대적인 값으로 하였다. After slowly adding 5 normal sodium hydroxide to adjust the pH to 8.0, 0.4 to 0.8% by weight of an enzyme, an endopeptidase, was added to the protein, followed by stirring for 2 hours. After the reaction was completed, the temperature was adjusted to 55 ° C., and then pH was adjusted to 5.0 by adding 6 normal hydrochloric acid. Then, the enzyme exopeptidase (0.4-0.8% by weight) of the raw protein is added and stirred for 4 hours to react. After the reaction is completed, the temperature is raised to 100 ° C. and maintained for 5 minutes to inactivate the enzyme. The resulting enzyme reaction was separated into a solid phase and a liquid phase using a centrifuge, and then a liquid phase containing particles having a diameter of 70 μm or less was separated, recovered, and filtered using a mesh, and the filtrate was transferred to a concentrator. It was concentrated to a solid content of 40 wt% at 5 ° C. and then powdered. The powdering conditions were the blowing temperature of 165 ° C, the blowing temperature of 95 ° C, and the atomizer of 9,000 rpm. The immune activity of the obtained powder was set as a relative value to the control group as the proliferation of T cells.
이하, 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 이에 앞서, 본 명세서 및 청구범위에 사용된 용어나 단어는 통상적이거나 사전적인 의미로 한정해서 해석되어서는 아니 되며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다. Hereinafter, preferred embodiments of the present invention will be described in detail. Prior to this, terms or words used in the present specification and claims should not be construed as being limited to the common or dictionary meanings, and the inventors should properly explain the concept of terms in order to best explain their own invention. Based on the principle that can be defined, it should be interpreted as meaning and concept corresponding to the technical idea of the present invention.
따라서, 본 명세서에 기재된 실시예에 도시된 구성은 본 발명의 가장 바람직 한 실시예에 불과할 뿐이고 본 발명의 기술적 사상을 모두 대변하는 것은 아니므로, 본 출원 시점에 있어서 이들을 대체할 수 있는 다양한 균등물과 변형예들이 있을 수 있음을 이해하여야 한다. Therefore, the configurations shown in the embodiments described herein are only the most preferred embodiments of the present invention and do not represent all of the technical idea of the present invention, and various equivalents may be substituted for them at the time of the present application. It should be understood that there may be variations and variations.
(실시예 1)(Example 1)
검은콩 1kg을 분쇄하고 10 L 용량의 반응조에서 물 6 L를 가한 후 단백 분해 효소 (protease)를 1.0 g 가한 후 50℃에서 3시간 동안 효소분해 한 후 100℃에서 5분 동안 가열하여 효소를 실활 시킨 후 60 메쉬 (mesh)에서 여과 후 진공 농축기를 이용하여 50 브릭스(Brix)까지 농축하여 검은콩 농축액을 얻었다. 이 검은 농축액의 전처리10 L 용량의 반응조에 검은콩 농축액 (50 Brix)을 375 g을 넣고 물 2 L를 가한 후, 교반하여 온도를 100℃로 상승시켰다. 5분간 온도를 유지한 후 냉각하여 온도를 60℃로 맞추고 이어서 5 노르말 수산화나트륨 45 mL을 첨가하여 pH를 8.0으로 조정하였다. 여기에, 엔도펩티다아제 [Promod 278] 효소 1.5 g을 수용액으로 만들어 첨가한 후 2시간 동안 교반하며 반응시켰다. 반응이 완료된 후, 온도를 다시 55℃로 낮추고 6 노르말 염산 65 mL을 첨가하여 pH를 5.0으로 조정하였다. 여기에, 엑소펩티다아제 [Promod 279] 효소 3.0 g을 수용액으로 만들어 첨가한 후 다시 4시간 동안 교반하며 반응시켰다. 반응이 완료된 후, 온도를 100℃로 상승시켜 5분간 유지시킴으로써 효소를 불활성화 시켰다. 반응조의 제조물을 120메쉬로 여과한 후 여과액을 농축기로 이송하여 50±5℃로 유지하면서 고형분 함량이 40 중량%까지 농축한 후 분말화 하였다. 분말화 조건은 송풍 온도 165℃, 배풍 온도 95℃로 하였으며, 아토마이저(atomizer)는 10,000 rpm으로 하였다. 1 kg of black soybeans were ground, 6 L of water was added in a 10 L reactor, 1.0 g of protease was added, followed by enzymatic degradation at 50 ° C. for 3 hours, followed by heating at 100 ° C. for 5 minutes to deactivate the enzyme. After filtration in 60 mesh (mesh) and concentrated to 50 Brix (Brix) using a vacuum concentrator to obtain a black bean concentrate. Pretreatment of this black concentrate The 375 g of black bean concentrate (50 Brix) was added to a 10 L reaction tank, and 2 L of water was added, followed by stirring to raise the temperature to 100 ° C. The temperature was kept for 5 minutes, then cooled to adjust the temperature to 60 ° C., and then the pH was adjusted to 8.0 by the addition of 45 mL of 5 normal sodium hydroxide. To this, 1.5 g of an endopeptidase [Promod 278] enzyme was added into an aqueous solution, and then reacted with stirring for 2 hours. After the reaction was completed, the temperature was lowered to 55 ° C. and the pH was adjusted to 5.0 by adding 65 mL of 6 normal hydrochloric acid. Here, 3.0 g of an exopeptidase [Promod 279] enzyme was added into an aqueous solution, and then reacted with stirring for 4 hours. After the reaction was completed, the enzyme was inactivated by raising the temperature to 100 ° C. and maintaining it for 5 minutes. The product of the reactor was filtered with 120 mesh, and then the filtrate was transferred to a concentrator and the powder was concentrated to a solid content of 40 wt% while maintaining at 50 ± 5 ° C. The powdering conditions were the blowing temperature of 165 ° C, the blowing temperature of 95 ° C, and an atomizer of 10,000 rpm.
(실시예 2)(Example 2)
면역 세포 티 세포 (T cell)의 활성은 더블유에스티(WST assay)법으로 측정하였다 (Ishima, H., et al. : Novel cell proliferation and cytotoxicity assays using a tetrazolium salt that produces a water-soluble formazen dye, In Vitro Toxicology, 8, 2, 187-190 (1995)). 먼저, 일정하게 세포 수를 유지한 세포 부유액을 96-웰 플레이트 (well plate)에 90 uL씩 넣어주고 여기에 대조군은 10 uL의 설라인 (saline), 시험군에는 각각의 시료를 10 uL씩 적당한 농도로 3배수로 넣고 탄산가스 배양기 (CO2 incubator, 36.5℃, 5% CO2)에서 3일 동안 배양하였다. 이렇게 3일 동안 배양한 후, 더블유에스티(Water Soluble Tetrazolium) 시약을 각각의 웰(well)에 10 uL씩 넣고 배양기에서 4시간 동안 반응시켰다. 그리고 4시간 후 각 웰을 꺼내고 1분 동안 천천히 흔들어 고르게 한 후, 마이크로플레이드 리더 (microplate reader 550)으로 450 nm에서 흡광도를 측정하였다. 실시예 1에서 얻어진 분말을 이용한 면역활성을 검증한 결과, 무첨가군에 비해 면역 활성이 2배 이상 높았다 (표 1 참조).The activity of immune cells T cells was measured by WST assay (Ishima, H., et al .: Novel cell proliferation and cytotoxicity assays using a tetrazolium salt that produces a water-soluble formazen dye, In Vitro Toxicology, 8, 2, 187-190 (1995)). First, put 90 μL of the cell suspension with constant cell count in a 96-well plate, and 10 μL of saline for the control group and 10 μL of each sample for the test group. 3 times the concentration was incubated for 3 days in a carbon dioxide incubator (CO 2 incubator, 36.5 ℃, 5% CO 2 ). After culturing for 3 days, 10 uL of Water Soluble Tetrazolium reagent was added to each well and reacted for 4 hours in an incubator. After 4 hours, each well was removed, shaken slowly for 1 minute, and then absorbed at 450 nm with a microplate reader 550. As a result of verifying the immunological activity using the powder obtained in Example 1, the immunological activity was more than two times higher than the no addition group (see Table 1).
(비교예 1)(Comparative Example 1)
본 발명에서는 황색 콩을 상기의 실시예 1과 동일하게 제조한 것, 검은콩추출물과 검은콩 단백 가수 분해물 첨가군의 면역 활성을 비교하였다. 하기 표 1에서의 수치는 무첨가군의 면역 활성을 100을 기준으로 하여 나타낸 것이다.In the present invention, yellow beans were prepared in the same manner as in Example 1, and the immune activity of the black soybean extract and the black soy protein hydrolyzate addition group was compared. Table 1 below shows the immune activity of the no additive group based on 100.
검은콩 단백 가수 분해물은 검은콩 추출액의 1.17배보다 훨씬 높은 2.06배의 면역 활성을 보였다. 이는, 무첨가군의 면역 세포 수는 3.0 × 106이고 검은콩 추출액은 3.21 × 106 그리고 검은콩 단백 가수 분해물은 6.18 × 106인 것으로부터 알 수 있었다. 즉, 검은콩 단백 가수 분해물은 검은콩 추출액 보다 2.97 × 106 의 면역 세포 수를 증가시켰다.The black soy protein hydrolyzate showed 2.06 times higher immune activity than the 1.17 times black bean extract. This can be seen that the immune cell number of the no-added group was 3.0 × 10 6 , the black bean extract was 3.21 × 10 6, and the black bean protein hydrolyzate was 6.18 × 10 6 . In other words, the black soy protein hydrolyzate increased the number of immune cells 2.97 × 10 6 than the black soybean extract.
따라서, 본 발명은 검은콩에서의 면역 활성을 강화하는 인자를 포함하는 단백 가수 분해물의 면역 활성이 아주 우수하다는 것을 알 수 있다. Therefore, the present invention can be seen that the immune activity of the protein hydrolyzate including the factor that enhances the immune activity in black soybean is very excellent.
(비교예 2)(Comparative Example 2)
상기의 실시예 1에서 효소의 종류를 달리하여 제조된 검은콩 단백 가수분해물의 면역 활성을 비교하였다. 먼저, 엔도펩티다아제 (Promod 278)를 단독으로 원료 단백질의 0.5중량%를 첨가하여 실시예 1과 동일하게 제조하였고, 또, 엑소펩티다이제 (Promod 279)를 단독으로 원료 단백질의 0.5중량%를 첨가하여 실시예 1과 동일하게 검은콩 단백 가수 분해물을 각각 제조한 후, 실시예 1에서 제조한 검은콩 단백 가수 분해물과의 면역 활성을 각각 비교하였다 (표 2). In Example 1, the immune activity of the black soybean protein hydrolysates prepared by different enzymes was compared. First, endopeptidase (Promod 278) alone was added in the same manner as in Example 1 by adding 0.5% by weight of the source protein, and exopeptidase (Promod 279) alone was added 0.5% by weight of the source protein. By preparing the black soybean protein hydrolyzate in the same manner as in Example 1, and compared the immune activity with the black soybean protein hydrolyzate prepared in Example 1 (Table 2).
상기의 표 2에서 보듯이, 각각의 효소를 단독으로 사용하였을 경우보다 두 효소를 동시에 사용하였을 경우가 면역 활성이 1.5배 이상 더 높게 나타났다. 따라서, 본 발명은 두 엔도펩티다아제 (Promod 278)와 엑소펩티다아제 (Promod 279)를 동시에 사용함으로써 면역 활성을 가지는 인자를 더욱 많이 함유하는 단백 가수 분해물을 얻을 수 있다.As shown in Table 2, the use of both enzymes at the same time than the case of using each enzyme alone, the immune activity was 1.5 times higher. Therefore, the present invention can obtain a protein hydrolyzate containing more factors having immune activity by using two endopeptidase (Promod 278) and exopeptidase (Promod 279) at the same time.
이상과 같이, 본 발명은 비록 한정된 실시예에 의해 설명되었으나, 본 발명은 이것에 의해 한정되지 않으며 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 본 발명의 기술 사상과 아래에 기재될 특허 청구범위의 균등 범위 내에서 다양한 수정 및 변형이 가능함은 물론이다. As mentioned above, although this invention was demonstrated by the limited embodiment, this invention is not limited by this and it will be described below by the person of ordinary skill in the art, and the following. Of course, various modifications and variations are possible within the scope of the claims.
본 발명에 의하면, 일반적인 면역 활성제와는 달리, 일반 식품으로서 일상적으로 섭취할 수 있고 상대적으로 면역활성이 뛰어난 면역 활성 펩티드 인자들을 다량 함유한 검은콩 단백 가수 분해물을 얻을 수 있다. According to the present invention, unlike general immunoactive agents, black soy protein hydrolyzate containing a large amount of immunoactive peptide factors which can be routinely consumed as a general food and has relatively high immunological activity can be obtained.
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