KR100195984B1 - Peptide compositions for inhibition of angiotensin converting enzyme and process for the preparation thereof - Google Patents

Peptide compositions for inhibition of angiotensin converting enzyme and process for the preparation thereof Download PDF

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KR100195984B1
KR100195984B1 KR1019960000412A KR19960000412A KR100195984B1 KR 100195984 B1 KR100195984 B1 KR 100195984B1 KR 1019960000412 A KR1019960000412 A KR 1019960000412A KR 19960000412 A KR19960000412 A KR 19960000412A KR 100195984 B1 KR100195984 B1 KR 100195984B1
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converting enzyme
angiotensin converting
hydrolysis
protein
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KR970058723A (en
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신재익
남희섭
안창원
안상원
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이상윤
주식회사농심
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    • A—HUMAN NECESSITIES
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects

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Abstract

본 발명은 안지오텐신 전환효소(Angiotensin Converting Enzyme, ACE)의 저해 활성이 높은 펩티드 혼합물을 효소 분해에 의하여 제조하는 방법 및 이에 의하여 제조되는 안지오텐신 전환 효소 저해 펩티드 혼합물에 관한 것이다.The present invention relates to a method for producing a peptide mixture with high inhibitory activity of Angiotensin Converting Enzyme (ACE) by enzymatic digestion and an angiotensin converting enzyme inhibitory peptide mixture prepared thereby.

본 발명에 의하면, 단백질을, 프로모드 278[아스퍼질루스 유래 엔도 펩티다아제 및 파파인], 또는 프로모드 278 및 프로모드 279(아스퍼질루스 유래 엑소펩티다아제) 효소에 의하여 가수분해도가 16~51%가 되도록 가수분해하여 얻어지는 안지오텐신 전환 효소 저해 펩티드 혼합물이 제공된다.According to the present invention, the protein is hydrolyzed by the promodal 278 (aspergillus-derived endopeptidase and papain), or the promodal 278 and promod 279 (aspergillus-derived exopeptidase) enzymes so that the degree of hydrolysis is 16 to 51%. An angiotensin converting enzyme inhibitory peptide mixture obtained by hydrolysis is provided.

본 발명에 의할 경우, 저농도의 가수분해 효소를 사용하여 일정한 범위의 가수분해도를 가지도록 단백질 기질을 가수분해함으로써 안지오텐신 전환 효소 저해 펩티드 혼합물을 얻을 수 있다.According to the present invention, an angiotensin converting enzyme inhibitor peptide mixture can be obtained by hydrolyzing a protein substrate to have a range of degree of hydrolysis using a low concentration of hydrolase.

Description

안지오텐신 전환효소 저해 펩티드 혼합물 및 이의 제조 방법Angiotensin converting enzyme inhibitory peptide mixture and preparation method thereof

제1도는 탈지 대두박의 가수분해도와 안지오텐신 전환효소 저해활성의 상관관계를 나타낸 그래프이고,1 is a graph showing the correlation between the hydrolysis of the skim soybean meal and angiotensin converting enzyme inhibitory activity,

제2도는 카제인의 가수분해도와 안지오텐신 전환효소 저해활성의 상관관계를 나타낸 그래프이다.2 is a graph showing the correlation between casein hydrolysis and angiotensin converting enzyme inhibitory activity.

본 발명은 안지오텐신 전환효소(Angiotensin Converting Enzyme, ACE)의 저해 활성이 높은 펩티드 혼합물을 단백질 효소분해에 의하여 제조하는 방법 및 이에 의하여 제조되는 안지오텐신 전환 효소 저해 펩티드 혼합물에 관한 것이다.The present invention relates to a method for preparing a mixture of peptides having high inhibitory activity of Angiotensin Converting Enzyme (ACE) by proteolytic digestion, and an angiotensin converting enzyme inhibiting peptide mixture prepared thereby.

본태성 고혈압은 고혈압의 약 90%를 차지하며, 안지오텐신 전환효소의 작용으로 안지오텐신-Ⅰ로부터 생성된 안지오텐신-Ⅱ가 혈압 상승을 일으켜 발생하는 것으로 알려져 있다. 따라서, 안지오텐신 전환효소 제해제인 캡토프릴 등이 개발되어 본태성 고혈압의 치료제로 사용되고 있다.Essential hypertension accounts for about 90% of hypertension, and it is known that angiotensin-II produced from angiotensin-I is caused by an increase in blood pressure due to the action of angiotensin converting enzyme. Therefore, captopril, which is an angiotensin converting enzyme inhibitor, has been developed and used as a therapeutic agent for essential hypertension.

최근에는 안전성이 높은 식품으로 부터 분리된 식품 유래 안지오텐신 전환 효소 저해 펩티드가 다수 보고되고 있으며, 식품 단백질을 가수분해하여 안지오텐신 전환 효소 저해 펩티드를 제조하는 연구도 활발히 진행되고 있다. 예를들어, 가격이 저렴하고 안정되게 공급 가능한 대두 단백질을 식품 단백질로 사용하여 단백 분해효소로 분해함으로써 안지오텐신 전환 효소 저해 활성을 가진 펩티드 화합물을 수종 분리한 바 있다. 젤라틴 및 카제인 효소분해물로 부터 유래한 안지오텐신 전환 효소저해 펩티드도 다수 보고 되어 있다(일본국 특허 공개 평 3-167198호, 소 62-270533호, 소 64-5497호, 소 64-83096호). 이러한 방법들은 모두 안지오텐신 전환 효소 저해 활성이 높은 펩티드 화합물을 순수하게 분리, 정제하는 방법이다.Recently, a large number of food-derived angiotensin converting enzyme inhibitor peptides separated from foods having high safety have been reported, and studies are being actively conducted to prepare angiotensin converting enzyme inhibitor peptides by hydrolyzing food proteins. For example, several types of peptide compounds having angiotensin converting enzyme inhibitory activity have been isolated by using soy protein, which is inexpensive and stable supply, as a food protein and digested with protease. A large number of angiotensin converting enzyme inhibitory peptides derived from gelatin and casein degradates have also been reported (Japanese Patent Laid-Open Nos. 3-167198, 62-270533, 64-6497, and 64-83096). All of these methods are methods for purely separating and purifying peptide compounds having high angiotensin converting enzyme inhibitory activity.

한편, 대두 단백질을 특정한 단백 분해효소(Bacillus속 protease)로 가수분해하여, 분자량이 300~1,500인 다양한 종류의 펩티드를 40%이상 포함하고 있는 가수분해 혼합물을 제조(일본 특허 공개 소 62-169732호)하여, 안지오텐신 전환 효소 저해제로 사용할 수 있다. 대두 단백질로부터 안지오텐신 전환 효소 저해 펩티드 혼합물을 얻는 다른 방법으로, 대두 단백질을 브로멜라닌(Bromelanin) 단백 분해효소로 분해율 50~70%로 분해하여 제조하는 방법(일본 특허 공개 평 4-299991호)이 있다. 그러나, 전자의 방법에서와 같이 안지오텐신 전환 효소 저해 활성이 높은 혼합물을 얻기 위하여 분자량이 300~1,500인 펩티드만을 제조할 경우 수율이 낮아지는 단점이 있으며, 후자의 방법에서와 같이 분해율을 50~70%로 하여 수율을 높이는 경우 분해율을 향상시키기 위하여 단백 분해효소를 상당량 투입하여야 하므로 경제성이 떨어진다는 문제가 있다. 후자의 방법에서, 분해율을 70%로 하기 위하여는 효소를 분해 기질 중량의 30%까지 첨가하여야 한다.On the other hand, the soy protein is hydrolyzed with a specific protease (Bacillus protease) to prepare a hydrolysis mixture containing 40% or more of various peptides having a molecular weight of 300-1,500 (Japanese Patent Publication No. 62-169732). Can be used as an angiotensin converting enzyme inhibitor. Another method of obtaining an angiotensin converting enzyme inhibitor peptide mixture from soy protein is a method of preparing soy protein by digesting it with a bromelain proteinase at a degradation rate of 50 to 70% (Japanese Patent Laid-Open No. 4-299991). . However, in order to obtain a mixture having a high angiotensin converting enzyme inhibitory activity as in the former method, only a peptide having a molecular weight of 300 to 1,500 has a disadvantage in that the yield is lowered, and the decomposition rate is 50 to 70% as in the latter method. In case of increasing the yield, a significant amount of protease should be added in order to improve the decomposition rate. In the latter method, the enzyme must be added up to 30% of the weight of the degradation substrate in order to achieve a degradation rate of 70%.

본 발명자들은 이러한 문제점을 해결하기 위하여 저농도의 효소처리로 높은 안지오텐신 전환 효소 저해 활성을 가지는 펩티드 혼합물을 고수율로 제조하는 방법을 연구한 결과, 단백질에 프로모드(Promod)계 단백 분해효소를 저농도로 첨가하여 가수분해도가 16~51%가 되도록 분해함으로써 안지오텐신 전환 효소 저해 활성이 높은 펩티드 혼합물을 얻을 수 있음을 알아내어 본 발명을 완성하게 되었다.In order to solve this problem, the present inventors have studied a method for producing a peptide mixture having a high angiotensin converting enzyme inhibitory activity by a low concentration of enzyme treatment in a high yield, and a low concentration of promodal protease in protein. The present invention was completed by finding that a peptide mixture having high angiotensin converting enzyme inhibitory activity can be obtained by adding and degrading the hydrolysis degree to 16 to 51%.

따라서, 본 발명의 목적은 식품 단백질을 저농도의 효소로 분해하여 안지오텐신 전환 효소 저해 활성이 높은 펩티드 혼합물을 제공하는 데에 있다.Accordingly, it is an object of the present invention to provide a peptide mixture having high angiotensin converting enzyme inhibitory activity by digesting food proteins with low concentrations of enzymes.

이러한 목적을 달성하기 위하여, 본 발명의 제 1 실시 형태에 의하면 단백질 기질을 프로모드 278[Promod 278, Biocatalyst사; 아스퍼질루스(Aspergillus) 유래 엔도펩티다아제 및 파파인(papain), 이하 같음] 효소를 첨가하여 가수분해도가 16~31%가 되도록 가수분해하여 얻어지는 안지오텐신 전환 효소 저해 펩티드 혼합물이 제공된다.In order to achieve this object, according to the first embodiment of the present invention, a protein substrate is prepared using Promod 278 [Promod 278, Biocatalyst; An angiotensin converting enzyme-inhibiting peptide mixture obtained by hydrolysis such that the aspergillus-derived endopeptidase and papain, hereinafter is equal to 16 to 31% by hydrolysis is provided.

본 발명의 다른 실시형태에 의하면, 단백질 기질에 프로모드 278 효소를 첨가하여 가수분해도가 16~31%가 되도록 1차 가수분해한 후, 얻어진 가수분해물에 프로모드 279(Promod 279, Biocatalyst사; 아스퍼질루스 유래 엑소펩티다아제, 이하 같음) 효소를 첨가하여 가수분해도가 31~51%가 되도록 2차 가수분해하여 얻어지는 안지오텐신 전환 효소 저해 펩티드 혼합물이 제공된다.According to another embodiment of the present invention, by adding promod 278 enzyme to the protein substrate and subjecting the first hydrolysis to have a degree of hydrolysis of 16 to 31%, the obtained hydrolyzate is subjected to promod 279 (Promod 279, Biocatalyst; An angiotensin converting enzyme inhibiting peptide mixture obtained by secondary hydrolysis to obtain a degree of hydrolysis of 31 to 51% by adding an enzyme derived from Pergillus, hereinafter) is provided.

이하 본 발명을 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.

탈지 대두박, 카제인 또는 이들의 혼합물에 물을 가하여 15 w/v% 용액으로 한 후 100°C에서 15분간 살균하여 단백 가수분해물의 기질로 사용한다. 60°C 항온조에서, 수산화나트륨으로 pH 8.0으로 조정하여 프로모드 278 효소를 단백질 기준으로 0.4~0.8w/v% 범위의 농도로 첨가한 후 2시간 반응시킨다. 반응후 염산을 첨가하여 pH를 5.0으로 낮추어 효소분해를 종료시킨다. 효소분해물을 원심분리하여 상층액만을 회수하여 효소분해물을 얻는다. 얻어진 효소분해물의 가수분해도는 16~31%이며 안지오텐신 전환 효소 저해 활성은 IC50=40~100μg/㎖이다.Degreased soybean meal, casein or a mixture thereof is added to water to make a 15 w / v% solution, and then sterilized at 100 ° C. for 15 minutes to be used as a substrate of protein hydrolysate. In a 60 ° C. incubator, adjust pH to 8.0 with sodium hydroxide, add Promodal 278 enzyme at a concentration in the range of 0.4 to 0.8 w / v% based on protein, and then react for 2 hours. After the reaction, hydrochloric acid was added to lower the pH to 5.0 to terminate enzymatic digestion. Centrifugation of the enzyme digestion to recover only the supernatant to obtain an enzyme digest. The degree of hydrolysis of the obtained enzymatic degradation product was 16 to 31% and the angiotensin converting enzyme inhibitory activity was IC 50 = 40 to 100 µg / ml.

또는, 프로모드 278로 분해한 가수분해물에 프로모드 279 효소를 단백질 기준으로 0.05~1.0w/v%범위의 농도로 첨가한 후 55°C, pH 5.0 에서 4시간 가수분해한다. 100°C에서 15분간 가열하여 효소를 불활성화시켜 반응을 종료시키며, 가수분해물을 원심분리하여 상층액만을 얻는다. 얻어진 가수분해물의 가수분해도는 31~51%이며, 안지오텐신 전환 효소 저해 활성은 IC50=40~100μg/㎖이다.Or, to the hydrolyzate decomposed with the promode 278, the promode 279 enzyme is added at a concentration ranging from 0.05 to 1.0 w / v% based on protein, and then hydrolyzed at 55 ° C. and pH 5.0 for 4 hours. The reaction is terminated by inactivating the enzyme by heating at 100 ° C. for 15 minutes and centrifuging the hydrolyzate to obtain only the supernatant. The degree of hydrolysis of the obtained hydrolyzate is 31 to 51%, and the angiotensin converting enzyme inhibitory activity is IC 50 = 40 to 100 µg / ml.

효소의 농도에 따른 가수분해도(Degree of Hydrolysis)와 안지오텐신 전환 효소활성의 저해도의 상관 관계는 제1도 및 제2도에 나타낸 바와 같으며, 가수분해도 16~51%에서 최대의 안지오텐신 전환 효소 저해 활성을 보인다. 가수분해도 16% 이하에서는 단백질의 가수분해가 미진하여 생성되는 안지오텐신 전환 효소 저해 펩티드의 양이 적어 전체 안지오텐신 전환 효소 저해 활성이 낮으며, 가수분해도 51% 이상에서는 오히려 생성된 안지오텐신 전환 효소 저해 펩티드가 유리 아미노산 형태로 분해되어 전체 안지오텐신 전환 효소 저해 활성이 낮아지며, 가수분해도를 높이기 위하여 소모되는 효소의 양이 많아지게 되므로 제조 공정면에서 비경제적이다.The correlation between degree of hydrolysis and inhibition of angiotensin-converting enzyme activity according to enzyme concentration is shown in FIGS. 1 and 2, and the maximum angiotensin-converting enzyme inhibition at 16-51% hydrolysis degree. Active The angiotensin converting enzyme inhibitory peptide was less than 16% of the hydrolysis, resulting in low angiotensin converting enzyme inhibitory peptide. Decomposition in amino acid form lowers the total angiotensin converting enzyme inhibitory activity, and the amount of enzyme consumed to increase the degree of hydrolysis is uneconomical in terms of manufacturing process.

이상의 본 발명의 방법에 의하면 저농도의 가수분해 효소를 사용하여 일정한 범위의 가수분해도를 가지도록 단백질 기질을 가수분해함으로써 안지오텐신 전환 효소 저해 활성이 높은 안지오텐신 전환 효소 저해 펩티드 혼합물을 경제적으로 제조할 수 있다.According to the above method of the present invention, an angiotensin converting enzyme inhibitory peptide mixture having high angiotensin converting enzyme inhibitory activity can be economically prepared by hydrolyzing the protein substrate to have a range of degree of hydrolysis using a low concentration of hydrolase.

이하 본 발명을 하기에 실시예에 의하여 더욱 상세히 설명한다. 이 들 실시예는 본 발명의 이해를 돕기 위하여 제공되는 것일 뿐 본 발명의 범위가 이에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are provided only to assist in understanding the present invention, but the scope of the present invention is not limited thereto.

[실시예 1]Example 1

탈지 대두박 150g을 2.0ℓ 삼각플라스크에 넣은 후 물 1.0ℓ를 첨가한 후 100°C에서 15분간 살균하였다. 이를 200㎖ 삼각 플라스크에 각각 100㎖씩 분주한 후 냉각하였다. 60°C까지 냉각 후 60°C 항온 교반 수조(Shaking water bath)에서 5.0 M 수산화나트륨으로 pH를 8.0으로 조정하고, 프로모드 278 효소를 단백질 기준으로 각각 0.0w/v%, 0.05w/v%, 0.1w/v%, 0.2w/v%, 0.3w/v%, 0.4w/v%, 0.6w/v%, 0.8w/v% 씩 첨가한 후 2시간 반응시켰다. 반응이 완료된 후 5.0 M 염산을 첨가하여 pH를 5.0으로 조정함으로써 효소를 불환성시켰다. 반응물을 12,000rpm에서 원심분리하여 상층액을 분리하여, 상층액의 가수분해도 및 안지오텐신 전환 효소 저해활성과 단백질 회수율을 구하였다(표1).150 g of skim soybean meal was added to a 2.0 L Erlenmeyer flask, and then 1.0 L of water was added and sterilized at 100 ° C. for 15 minutes. 100 ml of each of them was dispensed into a 200 ml Erlenmeyer flask and cooled. After cooling to 60 ° C, pH was adjusted to 8.0 with 5.0 M sodium hydroxide in a 60 ° C constant temperature shaking water bath, and 0.0w / v% and 0.05w / v% of Promod 278 enzyme based on protein, respectively. , 0.1w / v%, 0.2w / v%, 0.3w / v%, 0.4w / v%, 0.6w / v%, 0.8w / v% of each were added and reacted for 2 hours. After the reaction was completed, the enzyme was refluxed by adjusting the pH to 5.0 by adding 5.0 M hydrochloric acid. The reaction was centrifuged at 12,000 rpm to separate the supernatant, and the degree of hydrolysis, angiotensin converting enzyme inhibitory activity and protein recovery of the supernatant were determined (Table 1).

[가수분해도 측정][Measuring Hydrolysis Degree]

단백질이나 펩티드의 결합이 가수분해되면서 유리되는 카르복실기나 아미노기에 의해 pH가 감소하고, 감소한 pH를 염기로 계속적으로 적정하여 일정한 pH를 유지시킬 때 [pH stat 이용] 소모된 염기량은 가수분해 과정 중 유리되는 펩티드 결합수에 비례하므로, 소모된 염기량으로부터 가수분해도를 다음의 식에서 구하였다.When the protein or peptide bond is hydrolyzed, the pH decreases due to the free carboxyl or amino group, and when the reduced pH is continuously titrated with a base to maintain a constant pH [using pH stat], the amount of base consumed is hydrolyzed during hydrolysis. Since it is proportional to the number of free peptide bonds, the degree of hydrolysis was calculated from the amount of base consumed by the following equation.

안지오텐신 전환 효소 저해 활성Angiotensin Converting Enzyme Inhibitory Activity

쿠시만과 쳉(Cushman cheung)의 방법에 따라 측정하였다. 즉, 안지오텐신 전환 효소와 기질을 반응시키기 전에 일정농도의 시료 또는 완충액(대조군)으로 전처리 반응(pre-incubation)시킨 후 일정시간 효소-기질반응을 진행시켰다. 반응 완료 후 반응생성물의 양을 측정하여, 시로 대신 완충액을 사용한 대조군의 생성물의 양과 비교함으로써 시료에 의한 안지오텐신 전환효소작용의 저해도를 구하였다. 이때, IC50은 안지오텐신 전환 효소를 50% 저해하는 효소분해물의 질소농도를 의미한다. IC50의 값이 작을수록 효소 분해물의 안지오텐신 전환 효소 저해활성이 높다는 것을 의미한다.It was measured according to the method of Cushman cheung. In other words, before reacting the angiotensin converting enzyme and the substrate, the enzyme-substrate reaction was performed for a predetermined time after pre-incubation with a predetermined concentration of sample or buffer (control). After completion of the reaction, the amount of the reaction product was measured, and the degree of inhibition of angiotensin converting enzyme activity by the sample was determined by comparing the amount of the product of the control group with the buffer instead of the siro. In this case, IC 50 refers to the nitrogen concentration of the enzymatic degradation product that inhibits angiotensin converting enzyme by 50%. The smaller the IC 50 value, the higher the angiotensin converting enzyme inhibitory activity of the enzymatic degradation product.

[단백질 회수율]Protein Recovery

효소분해 후 얻은 상층액 중 총단백질 양을 사용한 대두박의 총단백질 양으로 나누어 구하였다.The total protein amount of the supernatant obtained after enzymatic digestion was calculated by dividing the total protein amount of soybean meal.

[실시예 2]Example 2

실시예 1에서와 같은 방법으로 프로모드 278 효소를 0.4w/v% 첨가하여 2시간 효소 분해하여 얻어진 혼합물을, 5.0M 염산으로 pH를 5.0으로 조정하고, 55°C 항온 교반 수조(shaking water bath)에서 프로모드 279 효소를 단백질 기준으로 0.0w/v%, 0.05w/v%, 0.1w/v%, 0.2w/v%, 0.3w/v%, 0.5w/v%, 1.0w/v%, 1.5w/v%, 3.0w/v%, 5.0w/v% 씩 첨가하여 4시간 반응시켰다. 반응후 12,000rpm에서 원심분리하여 상충액만을 회수하여, 실시예 1에서와 같이 가수분해도, 안지오텐신 전환 효소 저해활성 및 단백질 회수율을 구하였다.(표 1).A mixture obtained by enzymatic digestion for 2 hours by adding 0.4 w / v% of promod 278 enzyme in the same manner as in Example 1 was adjusted to pH 5.0 with 5.0 M hydrochloric acid, and a 55 ° C shaking water bath. ), Promo 279 enzyme based on protein 0.0w / v%, 0.05w / v%, 0.1w / v%, 0.2w / v%, 0.3w / v%, 0.5w / v%, 1.0w / v %, 1.5w / v%, 3.0w / v% and 5.0w / v% were added and reacted for 4 hours. After the reaction, only the supernatant was recovered by centrifugation at 12,000 rpm, and the degree of hydrolysis, angiotensin converting enzyme inhibitory activity, and protein recovery were determined as in Example 1. (Table 1).

[실시예 3 및 4]EXAMPLES 3 AND 4

탈지 대두박 대신에 카제인을 사용한 것을 제외하고는 실시예 1, 2와 같이 실시하여 가수 분해도와 안지오텐신 전환효소 저해활성(IC)을 구하였다(표2).Hydrolysis and angiotensin converting enzyme inhibitory activity (IC) were obtained in the same manner as in Examples 1 and 2, except that casein was used instead of skim soybean meal (Table 2).

Claims (5)

탈지 대두박, 카제인 또는 이들의 혼합물을, 프로모드 278[아스퍼질루스(Aspergillus) 유래 엔도펩티다아제 및 파파인], 또는 프로모드 278 및 프로모드 279(아스퍼질루스 유래 엑소펩티다아제) 효소에 의하여 가수분해도가 16~51%가 되도록 가수분해하여 얻어지는 펩티드 혼합물.Degreased soybean meal, casein or mixtures thereof were hydrolyzed by Promodal 278 (Aspergillus-derived endopeptidase and papain), or Promodal 278 and Promod 279 (Aspergillus-derived exopeptidase) enzyme. Peptide mixture obtained by hydrolysis to ˜51%. 탈지 대두박, 카제인 또는 이들의 혼합물을 프로모드 278[아스퍼질루스 유래 엔도펩티다아제 및 파파인]에 의하여 가수분해하여, 가수분해도가 16~31%인 펩티드 혼합물을 제조하는 방법.A method of producing a peptide mixture having a degree of hydrolysis of 16 to 31% by hydrolyzing skim soybean meal, casein or a mixture thereof by Promod 278 [aspergillus-derived endopeptidase and papain]. 제2항에 있어서, 상기 프로모드 278[아스퍼질루스 유래 엔도펩티다아제 및 파파인]이 단백질 기준으로 0.4~0.8w/v% 범위의 농도로 사용되는 것을 특징으로 하는 방법.The method according to claim 2, wherein the promod 278 [aspergillus-derived endopeptidase and papain] is used at a concentration of 0.4 to 0.8 w / v% based on the protein. 탈지 대두박, 카제인 또는 이들의 혼합물을 프로모드 278[아스퍼질루스 유래 엔도펩티다아제 및 파파인] 효소에 의하여 가수분해도가 16~31%가 되도록 1차 가수분해한 후, 얻어진 가수분해물을 프로모드 279(아스퍼질루스 유래 엑소펩티다아제) 효소에 의하여 2차 가수분해하여 가수분해도가 31~51%인 펩티드 혼합물을 제조하는 방법.The dehydrated soybean meal, casein, or mixtures thereof were first hydrolyzed by the Promod 278 [aspergillus-derived endopeptidase and papain] enzyme so as to have a degree of hydrolysis of 16 to 31%. A method for producing a peptide mixture having a degree of hydrolysis of 31 to 51% by secondary hydrolysis by a perzylus-derived exopeptidase) enzyme. 제4항에 있어서, 상기 프로모드 278[아스퍼질루스 유래 엔도펩티다아제 및 파파인]이 단백질 기준으로 0.4~0.8 w/v% 범위로 농도로 사용되고 상기 프로모드 279(아스퍼질루스 유래 엑소펩티다아제)가 단백질 기준으로 0.05~1.0w/v%범위의 농도로 사용되는 것을 특징으로 하는 방법.The method according to claim 4, wherein the promod 278 [aspergillus-derived endopeptidase and papain] is used at a concentration in the range of 0.4-0.8 w / v% based on the protein, and the promod 279 (aspergillus-derived exopeptidase) is a protein. The method characterized in that used as a concentration in the range of 0.05 ~ 1.0w / v%.
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