KR20120056138A - Method for preparing fermented silkworm powder and composition comprising fermented silkworm powder for the preventing or treating of hyperlipidemia and fatty liver - Google Patents

Method for preparing fermented silkworm powder and composition comprising fermented silkworm powder for the preventing or treating of hyperlipidemia and fatty liver Download PDF

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KR20120056138A
KR20120056138A KR1020100117707A KR20100117707A KR20120056138A KR 20120056138 A KR20120056138 A KR 20120056138A KR 1020100117707 A KR1020100117707 A KR 1020100117707A KR 20100117707 A KR20100117707 A KR 20100117707A KR 20120056138 A KR20120056138 A KR 20120056138A
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조영수
차재영
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동아대학교 산학협력단
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Abstract

PURPOSE: A composition containing fermented silkworm powder is provided to prevent and treat various adult diseases such as hyperlipidemia and fatty liver. CONSTITUTION: A method for preparing fermented silkworm powder comprises: a step adding sterilized water to silkworm powder and sterilizing; and a step of adding Bacillus subtilis or an Aspergillus kawachii culture liquid; and a step of fermenting at 37°C for 3-12 days and drying. A pharmaceutical composition for preventing or treating hyperlipidemia and fatty liver contains the fermented silkworm powder as an active ingredient. A pharmaceutical composition for antioxidation and thrombolysis effects contains the fermented silkworm powder as an active ingredient.

Description

발효 누에 분말의 제조방법 및 발효 누에 분말을 포함하는 고지혈증 및 지방간 예방 또는 치료용 조성물{Method for preparing fermented silkworm powder and composition comprising fermented silkworm powder for the preventing or treating of hyperlipidemia and fatty liver}Method for preparing fermented silkworm powder and composition comprising fermented silkworm powder for the preventing or treating of hyperlipidemia and fatty liver}

본 발명은 누에 분말에 Bacillus subtilis(바실러스 서브틸리스) 또는 Aspergillus kawachii(아스페르길루스 카와치)균주를 첨가하여 발효시킨 발효 누에 분말 제조방법 및 발효 누에 분말을 포함하는 고지혈증 및 지방간과 같은 각종 성인질환의 예방 또는 치료용 조성물에 관한 것이다.The present invention Bacillus in silkworm powder A method for producing fermented silkworm powder fermented with the addition of subtilis (Bacillus subtilis ) or Aspergillus kawachii ( Aspergillus kawachi ) strain and a composition for preventing or treating various adult diseases such as hyperlipidemia and fatty liver including fermented silkworm powder It is about.

최근, 생활수준의 향상으로 인하여 식생활 형태의 변화 및 과도한 음주에 의해, 고지혈증 및 지방간 환자의 수가 급증하고 있는 것으로 보고되고 있으며, 특히 지방의 과다 섭취는 혈중 LDL 콜레스테롤을 상승시키고 체내 지방의 축적을 초래하며, 결국 동맥경화 등 심혈관계 질환을 유발한다. 따라서 고지혈증 및 지방간을 예방 및 치료하기 위하여, 새로운 물질을 개발하고자 하는 시도가 있어 왔다.Recently, the number of patients with hyperlipidemia and fatty liver has been reported to increase rapidly due to the change of diet and excessive drinking due to the improvement of living standard. In particular, excessive intake of fat increases blood LDL cholesterol and causes the accumulation of fat in the body. In the end, it causes cardiovascular diseases such as atherosclerosis. Thus, there have been attempts to develop new materials to prevent and treat hyperlipidemia and fatty liver.

새로운 생물자원 소재로서 곤충 관련 산업이 주목받고 있는 가운데, 바이오신소재 개발을 위한 새로운 천연 소재로서 누에 및 그 관련 부산물에 대한 관심이 고조되고 있다. Insect-related industries are attracting attention as new biomass materials, and interest in silkworms and related by-products is increasing as a new natural material for developing new biomaterials.

고지혈증을 유발시킨 실험용 쥐에 누에 번데기에서 추출한 불포화 지방산을 먹인 결과 동맥 경화 지수를 30 % 정도 감소시키는 효과를 얻었다고 연구 결과가 있으며, 또 피부의 기름기를 제거해 피부 건조증을 유발시킨 뒤 불포화 지방산을 바른 결과 피부 보습도가 바르지 않은 것에 비해 50 % 이상 향상된다는 보고가 된 바 있다. 그리고 수컷 누에 번데기의 성분에서 남성의 발기 촉진 성분의 하나로 알려진 cGMP의 합성을 촉진하는 단백질을 추출 분리해 그 구조를 구명하고 발기 촉진 활성을 강화하는 성분이라는 사실을 실험적으로 입증한바 있다. The study showed that feeding rats with hyperlipidemia induced unsaturated fatty acid extracted from silkworm pupa resulted in a 30% reduction in atherosclerosis index. As a result, it has been reported that the skin moisturization is improved by more than 50% compared to the non-corrected skin. And it has been experimentally proved that male silkworm chrysalis extracts and isolates a protein that promotes the synthesis of cGMP, which is known as one of the male's erectile promoting ingredients, to rescue its structure and enhance its erectile promoting activity.

한편, 누에의 먹이인 뽕잎의 주성분은 수분, 단백질, 탄수화물, 지방, 무기염류, 비타민 등으로 뽕잎의 생리활성으로는 혈당강하효과, 그리고 비만에 원인이 되고 있는 중성지질의 함량도 낮추어주고,과산화지질의 함량도 낮추어 준다고 보고 되었으며, 노화에 원인이 되고 있는 활성산소 생성을 약 20 % 억제하고 동시에 활성산소 제거효소의 활성을 약 14 % 높여주고 있어 노화억제에도 효과가 있을 것으로 보고 되었다. 또한 누에의 성분 중 필수 아미노산이 풍부하게 함유되어 있으며, 식품개발연구원 보고에 따르면 필수아미노산 중 알라닌은 알코올 대사를 촉진하여 숙취해소와 간기능 개선에 도움을 준다고 보고되어 있다.On the other hand, the main components of the mulberry leaf feeding silkworms are moisture, protein, carbohydrates, fats, inorganic salts, vitamins, etc., and the physiological activity of the mulberry leaves lowers the hypoglycemic effect and lowers the amount of neutral lipids that cause obesity. It has been reported to reduce the amount of, and inhibits the generation of active oxygen caused by aging about 20% and at the same time increases the activity of free radical scavenging enzyme by about 14% has been reported to be effective in inhibiting aging. In addition, silkworms contain abundant essential amino acids, and according to a report from the Korea Food Research Institute, it is reported that alanine in essential amino acids promotes alcohol metabolism to help hangover and improve liver function.

대한민국 등록특허 제10-0396986호는 누에분말로부터 식후 혈당상승에 대한 억제효과가 탁월한 혈당강하 물질을 제조하는 방법에 관한 것이고, 대한민국 등록특허 제10-0361085호는 누에분말의 에탄올추출물을 주성분으로하고, 상엽건조엑스, 가시오가피건조엑스, 실크푸로테인, 베타카로틴, 은행잎건조엑스, 비타민 B1, B6, C 및 E 그리고 달맞이 종자유를 보조성분으로 함유한 항당뇨효과를 갖는 건강보조식품의 조성물 및 그 제조방법에 관한 것이며, 대한민국 등록특허 제10-0721644호는 누에 번데기 열수추출물을 유효성분으로 포함하는 숙취해소용 건강 기능성 식품에 대하여 기재하고 있으나, 주로 항당뇨 효능 또는 숙취해소 효능에 관한 것으로, 누에의 지방간 및 고지혈증 예방 및 치료 효능에 대하여는 더 많은 연구가 이루어져야 될 것으로 보인다.
Republic of Korea Patent No. 10-0396986 relates to a method for producing a blood glucose lowering substance excellent in inhibiting the post-prandial blood sugar rise from silkworm powder, Republic of Korea Patent No. 10-0361085 is based on the ethanol extract of silkworm powder , Composition of the health supplements having anti-diabetic effect, containing the dry leaf extract extract, silk gourd dry extract extract, silk furotene, beta-carotene, ginkgo leaf extract extract, vitamin B1, B6, C and E and evening seed oil as supplementary ingredients It relates to a method, the Republic of Korea Patent No. 10-0721644 discloses a hangover rehabilitation health functional food comprising a silkworm pupa hot water extract as an active ingredient, but mainly relates to anti-diabetic effect or hangover efficacy, More research is needed to determine the efficacy of preventing and treating fatty liver and hyperlipidemia.

본 발명은 상기와 같은 점들을 감안하여 안출한 것으로, 누에 분말에 Bacillus subtilis(바실러스 서브틸리스) 또는 Aspergillus kawachii(아스페르길루스 카와치) 균주를 첨가하여 발효시킨 발효 누에 분말 제조방법 및 발효 누에 분말을 포함하는 고지혈증, 지방간과 같은 각종 성인질환의 예방 또는 치료용 조성물을 제공하는 것을 목적으로 한다.The present invention has been made in view of the above, fermented silkworm powder production method and fermented silkworm fermented by adding Bacillus subtilis ( Bacillus subtilis ) or Aspergillus kawachii ( Aspergillus Kawachi) strain to silkworm powder An object of the present invention is to provide a composition for preventing or treating various adult diseases such as hyperlipidemia and fatty liver containing powder.

본 발명의 상기 목적은 누에 분말에 Bacillus subtilis(바실러스 서브틸리스) 또는 Aspergillus kawachii(아스페르길루스 카와치) 균주를 첨가한 후 발효시켜 발효 누에 분말을 제조한 후 이의 구성 단백질 및 아미노산을 확인하고, 알코올 투여 동물의 간 및 혈중의 지질, 알코올, 알데하이드 등의 성분에 대한 상기 발효 누에 분말의 효과를 확인함으로써 달성하였다.
The object of the present invention is Bacillus subtilis ( Bacillus subtilis ) or Aspergillus in silkworm powder Fermented silkworm powder was prepared by adding kawachii (Aspergillus Kawachi) strain, followed by fermentation to identify its constituent proteins and amino acids, and for the components of liver, blood, lipids, alcohols, aldehydes, etc. It was achieved by confirming the effect of the fermented silkworm powder.

본 발명은 균주를 사용하여 누에 분말을 발효시킴으로써 기존의 비발효 누에 분말에 비해 개선된 고지혈증 및 지방간 예방 및 치료 효능을 나타내는 뛰어난 효과가 있다.The present invention has an excellent effect showing improved hyperlipidemia and fatty liver prevention and treatment effect compared to the existing non-fermented silkworm powder by fermenting the silkworm powder using the strain.

도 1은 Bacillus subtilisAspergillus kawachii 에 의해 발효된 누에 분말의 native-PAGE 전기영동 결과를 나타낸 것이다.
도 2는 Bacillus subtilisAspergillus kawachii 에 의해 발효된 누에 분말의 SDS-PAGE 전기영동 결과를 나타낸 것이다.
도 3은 Bacillus subtilisAspergillus kawachii 에 의해 발효된 누에 분말의 유리 아미노산 조성을 나타낸 것이다.
도 4는 Bacillus subtilisAspergillus kawachii 에 의해 발효된 누에 분말의 혈전용해 활성을 나타낸 것이다.
도 5는 Bacillus subtilisAspergillus kawachii 에 의해 발효된 누에 분말의 DPPH 자유 라디칼 소거능을 나타낸 것이다. BHT는 Butylated hydroxyl toluene의 약자이며 그외 약자들은 도 4와 동일하다. 데이터는 평균±SD (n=3)으로 나타냈다.
도 6은 Bacillus subtilisAspergillus kawachii 에 의해 발효된 누에 분말의 환원력을 나타낸 것이다. BHT는 Butylated hydroxyl toluene의 약자이고 AA는 Ascorbic acid의 약자이며, 그외 약자들은 도 4와 동일하다. 데이터는 평균±SD (n=3)으로 나타냈다.
도 7은 알코올 투여 랫의 간에서 ADH(alcohol dehydrogenase) 및 ALDH(acetaldehyde dehydrogenase)의 활성에 대한 SP, BFSP 및 AFSP의 효과를 나타낸 것이다. 다른 문자로 표시된 수치는 p〈 0.05에서 통계적으로 유의차가 있는 것이다. (평균±S.E., n=6).
도 8은 알코올 투여 랫의 간에서 ADH 및 ALDH 웨스턴 블롯 분석에 의한 SP, BFSP 및 AFSP의 효과를 나타낸 것이다.
도 9는 알코올 투여 랫의 간에서 Zn 및 Fe 농도에 대한 SP, BFSP 및 AFSP의 효과를 나타낸 것이다. 다른 문자로 표시된 수치는 p〈 0.05에서 통계적으로 유의차가 있는 것이다. (평균±S.E., n=6).
도 10은 알코올 투여 랫의 간 분획 및 혈청에서 TBARS에 대한 SP, BFSP 및 AFSP의 효과를 나타낸 것이다. 다른 문자로 표시된 수치는 p〈 0.05에서 통계적으로 유의차가 있는 것이다. (평균±S.E., n=6).
도 11은 알코올 투여 랫의 간 및 혈청에서 글루타치온 농도에 대한 SP, BFSP 및 AFSP의 효과를 나타낸 것이다. 다른 문자로 표시된 수치는 p〈 0.05에서 통계적으로 유의차가 있는 것이다. (평균±S.E., n=6).
도 12는 알코올 투여 랫의 간의 병리조직학적 변화에 대한 SP, BFSP 및 AFSP의 효과를 나타낸 것이다(magnification x 200). 간세포 염색을 H&E (hematoxylin and eosin) 염색 방법으로 실시하였다.
도 13은 알코올 투여 랫의 간의 병리조직학적 변화에 대한 SP, BFSP 및 AFSP의 효과를 나타낸 것이다(magnification x 200). 간세포 염색을 H&E 염색 방법으로 실시하였다.
1 is Bacillus subtilis and Aspergillus Native-PAGE electrophoresis of silkworm powder fermented by kawachii is shown.
2 is Bacillus subtilis and Aspergillus SDS-PAGE electrophoresis of silkworm powder fermented by kawachii is shown.
3 is Bacillus subtilis and Aspergillus It shows the free amino acid composition of silkworm powder fermented by kawachii .
4 is Bacillus subtilis and Aspergillus It shows the thrombolytic activity of silkworm powder fermented by kawachii .
5 is Bacillus subtilis and Aspergillus It shows DPPH free radical scavenging ability of silkworm powder fermented by kawachii . BHT stands for Butylated hydroxyl toluene and the other abbreviations are the same as in FIG. Data is shown as mean ± SD (n = 3).
6 is Bacillus subtilis and Aspergillus It shows the reducing power of silkworm powder fermented by kawachii . BHT stands for Butylated hydroxyl toluene and AA stands for Ascorbic acid. Other abbreviations are the same as in FIG. 4. Data is shown as mean ± SD (n = 3).
Figure 7 shows the effect of SP, BFSP and AFSP on the activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the liver of alcohol-administered rats. Figures in other letters are statistically significant at p <0.05. (Mean ± SE, n = 6).
Figure 8 shows the effect of SP, BFSP and AFSP by ADH and ALDH Western blot analysis in liver of alcohol administered rats.
9 shows the effects of SP, BFSP and AFSP on Zn and Fe concentrations in the liver of alcohol-administered rats. Figures in other letters are statistically significant at p <0.05. (Mean ± SE, n = 6).
10 shows the effect of SP, BFSP and AFSP on TBARS in liver fraction and serum of alcohol-administered rats. Figures in other letters are statistically significant at p <0.05. (Mean ± SE, n = 6).
FIG. 11 shows the effects of SP, BFSP and AFSP on glutathione concentrations in liver and serum of alcohol administered rats. Figures in other letters are statistically significant at p <0.05. (Mean ± SE, n = 6).
Figure 12 shows the effect of SP, BFSP and AFSP on the histopathologic changes of livers of alcohol-administered rats (magnification x 200). Hepatocyte staining was performed by H & E (hematoxylin and eosin) staining method.
Figure 13 shows the effect of SP, BFSP and AFSP on the histopathologic changes of liver of alcohol-administered rats (magnification x 200). Hepatocyte staining was performed by H & E staining method.

본 발명은 누에 분말에 Bacillus subtilis(바실러스 서브틸리스) 또는 Aspergillus kawachii(아스페르길루스 카와치)균주를 첨가하여 발효시킨 발효 누에 분말 제조방법 및 발효 누에 분말을 포함하는 조성물에 관한 것이다.The present invention Bacillus in silkworm powder The present invention relates to a method for producing fermented silkworm powder fermented by adding subtilis (Bacillus subtilis ) or Aspergillus kawachii ( Aspergillus kawachi ) strains, and a composition comprising fermented silkworm powder.

본 발명의 발효 누에 분말을 포함하는 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.The composition comprising the fermented silkworm powder of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

본 발명의 발효 누에 분말을 포함하는 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는, 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘, 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 사용할 수 있다.Carriers, excipients and diluents that may be included in the composition comprising the fermented silkworm powder of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium, phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used.

본 발명에 따른 발효 누에 분말을 포함하는 조성물은, 각각 통상의 방법에 따라 산제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구제제; 외용제; 좌제; 및 멸균 주사제의 형태로 제형화하여 사용될 수 있다. 발효 누에 분말의 사용량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으나, 10 내지 35 mg/㎏ 체중, 바람직하게는 15 내지 25 mg/kg 체중의 양을 일일 1회 내지 3회 투여할 수 있다. 추출물 및 분획물의 투여량은 질환의 종류 투여경로, 질병의 정도, 성별, 체중, 나이 등에 따라서 증감될 수 있다. 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.
The composition comprising the fermented silkworm powder according to the present invention may be prepared by oral preparations such as powders, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to a conventional method; External preparations; Suppositories; And in the form of sterile injectables. The amount of fermented silkworm powder may vary depending on the age, sex, and weight of the patient, but may be administered once to three times daily in an amount of 10 to 35 mg / kg body weight, preferably 15 to 25 mg / kg body weight. . The dosage of the extracts and fractions can be increased or decreased depending on the type of disease administration route, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로써, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

실험으로부터 얻어진 결과치는 one-way ANOVA 검정에 의한 평균치와 표준오차(mean± SE)로 표시하였으며, 각 실험군 간의 유의성 검증은 Duncan's multiple range test로 하였다.The results obtained from the experiments were expressed as mean and standard error (mean ± SE) by one-way ANOVA test, and the significance test between each experimental group was Duncan's multiple range test.

실시예Example 1. 발효 누에 분말의 제조 1. Preparation of Fermented Silkworm Powder

실험 재료인 누에 분말은 열풍 건조시킨 것으로 순천영농법인(순천, 전남)에서 구입하였다. 열풍 건조 누에 분말의 경시적인 발효를 위하여 Bacillus subtilis KACC 91157 및 Aspergillus kawachii KCCM 32819 균주를 한국미생물보존센터로부터 분양받아 사용하였다. 열풍 건조 누에 분말 각 50 g씩을 500 ml 비이커에 넣고 중량 대비 1.5배의 증류수를 가하여 분말에 모두 흡수시킨 후 121℃에서 15분간 멸균하였다. 실온이 되었을 때 전배양시킨 Bacillus subtilis 배양액을 누에 분말에 1% (v/w) 수준으로 잘 섞은 다음 37℃에서 최대 12일간 발효시켰다. 또한 전배양 한 Aspergillus kawachii 균사체도 누에 분말에 5% (v/w) 수준으로 섞은 다음 37℃에서 최대 12일간 발효시켰다. 이때 발효과정 중에 건조를 막기 위하여 멸균된 증류수를 일정량씩을 뿌려주면서 발효를 진행시켰다. 두 균주로 최장 12일까지 발효시키면서 3일 간격으로 시료를 하나씩 취하여 완전히 건조시킨 후 분석에 사용하였다.
Silkworm powder, an experimental material, was hot-air dried and purchased from Suncheon Farming Corporation (Suncheon, Jeonnam). Bacillus for Time-Based Fermentation of Hot Air Dried Silkworm Powder subtilis KACC 91157 and Aspergillus kawachii KCCM 32819 strain was used from the Korea Microorganism Conservation Center. 50 g of each hot-air dried silkworm powder was put in a 500 ml beaker, and 1.5 times distilled water was added to the powder to absorb all the powder, followed by sterilization at 121 ° C. for 15 minutes. Bacillus precultured at room temperature The subtilis culture was well mixed with silkworm powder at 1% (v / w) level and then fermented at 37 ° C. for up to 12 days. Also precultured Aspergillus The kawachii mycelium was also mixed with silkworm powder at 5% (v / w) level and then fermented at 37 ° C for up to 12 days. At this time, in order to prevent drying during the fermentation process, the sterilized distilled water was sprinkled with a predetermined amount to proceed with the fermentation. The samples were taken one by one every three days while fermenting up to 12 days with two strains, and then completely dried and used for analysis.

실시예Example 2. 경시적인 발효 누에의  2. Over time fermentation of silkworm pHpH 및 단백질 농도의 측정 And measurement of protein concentration

경시적인 발효 누에를 각각 증류수에 1% (w/v) 농도로 첨가하여 교반한 후 pH meter (Methrohm 691, Swiss)로 직접 pH를 측정하였다. 경시적인 발효 누에의 단백질 농도는 Lowry 방법(Lowry, O. H., N. J. Rosebrogh, A. L. Farr and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol . Chem . 193, 265-271)으로 540 nm에서 흡광도를 측정하여 우혈청 알부민을 표준품으로 하여 측정하였다.
The fermented silkworms over time were added to distilled water at a concentration of 1% (w / v) and stirred, and the pH was measured directly with a pH meter (Methrohm 691, Swiss). The protein concentration of fermented silkworms over time was low at 540 nm by Lowry method (Lowry, OH, NJ Rosebrogh, AL Farr and RJ Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol . Chem . 193, 265-271) . Absorbance was measured and measured using bovine serum albumin as a standard.

가잠 유래의 천연 단백질인 실크 피브로인과 누에 실크 유래의 세리신 단백질은 알코올성 간 독성 개선효과와 항산화 효과가 높은 것으로 보고되었다. 이전의 실험에서 고단백 식품소재로 각광받고 있는 누에를 단백질 분해력이 뛰어난 Bacillus Aspergillus 속 미생물로 발효시켜 얻은 발효 누에에서 단백질 함량 증가, 항산화 작용, 혈전 용해 작용, 티로시나제 활성 저해와 같은 생리활성작용이 발효 전 누에보다 증가하는 경향을 보였다[Rahama, E. H. and M. S. Narasinga Rao. 1983. Effect of limitted proteolysis on the functional properties of cottonseed flour. J. Agric . Food Chem . 31, 356-361; Sekul, A. A., C. H. Vinnett and R. L. Ory. 2000. Some functional properties of peanut protein partially hydrolyzed with papain. J. Agric . Food Chem . 26, 855-859]. 또한 SDS-PAGE 상의 단백질 분해 패턴 분석에서도 97-66 kDa 정도 크기의 단백질이 분해되어 밴드상의 차이를 보이고 있어 단백질 분해 효소의 작용에 의해 아미노산과 펩타이드 조성에 영향을 미쳐 생리활성 작용에 차이를 보인 것으로 시사한 바 있다[Rahama et al., 1983; Sekul et al., 2000]. Silk fibroin, a natural protein derived from gajam, and sericin protein from silkworm silk, have been reported to have high alcoholic liver toxicity and antioxidant effects. Bacillus and Aspergillus, which has a high protein breakdown, has been used as a high protein food material in previous experiments. In fermented silkworms obtained by fermentation of the genus microorganisms, physiological activities such as increased protein content, antioxidant activity, thrombolytic action, and tyrosinase activity inhibition tended to be higher than those before fermentation [Rahama, EH and MS Narasinga Rao. 1983. Effect of limitted proteolysis on the functional properties of cottonseed flour. J. Agric . Food Chem . 31, 356-361; Sekul, AA, CH Vinnett and RL Ory. 2000. Some functional properties of peanut protein partially hydrolyzed with papain. J. Agric . Food Chem . 26, 855-859. In addition, in the analysis of proteolytic pattern on SDS-PAGE, the protein of about 97-66 kDa was decomposed and showed the difference in band, which showed the difference in physiological activity by affecting amino acid and peptide composition by the action of proteolytic enzymes. It has been suggested [Rahama et al., 1983; Sekul et al., 2000].

비발효 누에의 단백질 농도는 각각 48.6±0.28% 및 47.0±0.08% 이었다(표 1). Bacillus 균주에 의한 발효 누에의 단백질 농도는 발효 경과 시간과 더불어 지속적으로 높아지는 경향을 보였는데, 발효 12일째 55.6%로 최대 함량을 보였다. Bacillus sp. b01 균주를 이용한 청국장에서의 protease 효소 활성은 발효 9일까지 완만한 증가를 보였다는 실험 결과도 보고된 바 있다(In, J. P., S. K. Lee, B. K. Ahn, I. M. Chung and C. H. Jang. 2002. Flavor improvement of cheonggkukjang by addition of Yucca (Yucca shidigera) extract. Korean J. Food Sci Technol . 34, 57-64). 또한 Aspergillus 균주에 의한 발효 누에의 단백질 농도는 발효 9일째까지 큰 변화가 없다가 12일째 52.3%로 최대 함량을 보였으나, Bacillus 균주에 의한 발효 누에의 단백질 농도보다는 낮은 값을 나타내었다.
The protein concentrations of nonfermented silkworms were 48.6 ± 0.28% and 47.0 ± 0.08%, respectively (Table 1). Bacillus Protein concentration of fermented silkworms by strains showed a tendency to increase continuously with the time of fermentation, with a maximum content of 55.6% at 12 days of fermentation. Bacillus sp. Experimental results have also been reported to increase the protease enzyme activity in Cheonggukjang using b01 strain until 9 days of fermentation (In, JP, SK Lee, BK Ahn, IM Chung and CH Jang. 2002. Flavor improvement of cheonggkukjang by addition of Yucca (Yucca shidigera ) extract. Korean J. Food Sci Technol . 34, 57-64). Aspergillus The protein concentration of the fermented silkworm by the strain showed no significant change until the 9th day of fermentation, but the maximum content was 52.3% on the 12th day, but was lower than the protein concentration of the fermented silkworm by the Bacillus strain.

Bacillus sp. JH-209 균주 유래 단백질 분해 효소로 누에 번데기에 함유되어 있는 불용성 단백질을 가용성 단백질로 추출하기 위한 실험에서 pH 5.0에서 최저값을 보이다가 pH가 높아짐에 따라 단백질 추출량이 약간씩 증가하는 경향을 보였다고 하였다(Kwon, H. J., K. H. Lee, J. H. Kim, S. S. Chun, Y. J. Cho and W. S. Cha. 2006. Effect of protease on the extraction and properties of the protein from silkworm pupa. J. Korean Soc . Appl . Biol . Chem . 49, 304-308). 본 실험에서도 Bacillus 균주에 의한 발효에서 경시적으로 pH 값이 차츰 증가하면서 단백질 분해 효소의 작용이 활발하게 이루어져 단백질 함량이 경시적으로 증가한 것으로 보여진다(표 1). 또한 Aspergillus 균주에 의한 발효에서는 9일째까지 pH 값에서 큰 변화가 없다가 12일째 증가하면서 동시에 단백질 농도도 증가한 것으로 나타나 누에 번데기 단백질 농도 변화와 유사한 것으로 나타났다.
Bacillus sp. In the experiment for extracting insoluble protein contained in silkworm pupa with soluble protein using JH-209 strain, it showed the lowest value at pH 5.0 and showed a tendency to increase the amount of protein extraction as the pH increased. kwon, HJ, KH Lee, JH Kim, SS Chun, YJ Cho and WS Cha. 2006. Effect of protease on the extraction and properties of the protein from silkworm pupa. J. Korean Soc . Appl . Biol . Chem . 49, 304-308). In this experiment, the protein content increased over time as the pH value gradually increased during fermentation by Bacillus strains, and the activity of proteolytic enzymes became active (Table 1). In the fermentation with Aspergillus strain, there was no significant change in pH value until day 9, but increased protein concentration at day 12, which was similar to silkworm pupa protein concentration.

발효 시간에 따른 바실러스 서브틸리스 및 아스페르길루스 카와치에 의해 발효된 누에의 pH 및 단백질 농도PH and Protein Concentration of Silkworms Fermented by Bacillus Subtilis and Aspergillus Kawachi with Different Fermentation Times SampleSample TimeTime pHpH Protein content (%)Protein content (%) Silkworm powder (SP)Silkworm powder (SP) 0 day0 day 7.117.11 48.60±0.2848.60 ± 0.28 BacillusBacillus subtilissubtilis 3 day3 day 7.277.27 51.40±0.3851.40 ± 0.38 6 day6 day 7.437.43 52.51±0.1252.51 ± 0.12 9 day9 day 7.537.53 53.53±0.1853.53 ± 0.18 12 day12 day 7.977.97 55.60±0.0855.60 ± 0.08 AspergillusAspergillus kawachiikawachii 3 day3 day 6.556.55 49.60±0.2649.60 ± 0.26 6 day6 day 6.566.56 49.60±0.2849.60 ± 0.28 9 day9 day 6.746.74 50.10±0.3650.10 ± 0.36 12 day12 day 7.267.26 52.32±0.3852.32 ± 0.38

실시예Example 3. 경시적인 3. Over time 발효Fermentation 누에의 Silkworm NativeNative -- PAGEPAGE 에 의한 단백질 패턴 비교Protein pattern comparison

경시적인 발효 누에의 단백질 패턴은 Davis 방법[7]에 따라 slab-type native-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)의 겔 농도는 7.5%를 사용하였다. 즉 분리 겔은 30% 아크릴아미드, 0.8% bis-아크릴아미드, 1.5 M Tris-HCl 완충용액 (pH 8.8), 0.05% TEMED 및 10% 과산화암모늄을 혼합하여 1 mm 두께로 만들어 사용하였다. 분리 겔 위에는 5% 농축 겔 [30% 아크릴아미드, 0.8% bis-아크릴아미드, 0.5 M Tris-HCl 완충용액 (pH 6.8)을 만들어 5 × 샘플 완충용액 (312.5 mM Tris-HCl, pH 6.8), 50% 글리세롤, 0.05% bromophenol blue] 용액에 혼합한 시료를 농축 겔내에 성형된 wall에 일정량씩 주입하였다. 전기영동은 Tris-글리신 완충액 (25 mM Tris, 192 mM 글리신, pH 8.8) 하에서 1.5 mA/겔 (cm)의 정전류를 통하여 행하고, 4℃ 저온에서 2시간 통전시켰다. 전기영동이 완료된 겔은 7% 아세트산액에 용해한 1% 쿠마시 브릴리언트 블루(coomassie brilliant blue) R-250으로 염색한 후 7% 아세트산액으로 충분히 탈색시켜 건조 및 사진촬영을 행하였다.
As the protein pattern of fermented silkworms over time, the gel concentration of slab-type native-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was 7.5% according to Davis method [7]. In other words, the separation gel was mixed with 30% acrylamide, 0.8% bis-acrylamide, 1.5 M Tris-HCl buffer (pH 8.8), 0.05% TEMED and 10% ammonium peroxide to make 1 mm thick. On a separation gel was prepared a 5% concentrated gel [30% acrylamide, 0.8% bis-acrylamide, 0.5 M Tris-HCl buffer (pH 6.8) to give 5 × sample buffer (312.5 mM Tris-HCl, pH 6.8), 50 % Glycerol, 0.05% bromophenol blue] solution was injected in a predetermined amount into the wall formed in the concentrated gel. Electrophoresis was performed through a constant current of 1.5 mA / gel (cm) under Tris-glycine buffer (25 mM Tris, 192 mM glycine, pH 8.8) and energized for 2 hours at 4 ° C. low temperature. After the electrophoresis was completed, the gel was stained with 1% Coomassie brilliant blue R-250 dissolved in 7% acetic acid, and then decolorized with 7% acetic acid, and dried and photographed.

경시적인 누에 단백질을 native-PAGE로 전기영동 하여 비교분석 한 결과 비발효 누에 단백질 상단부의 97-66 kDa 크기 밴드 농도가 짙은 것에 비해 Bacillus subtilis 발효 누에에서는 발효 3일째부터 대부분 분해되어 저분자화됨으로서 밴드의 윤곽이 잘 보이지 않는 것으로 나타났다(도 1). 그러나 Aspergillus kawachii 균주에 의한 발효 누에 단백질 밴드는 발효 시간이 경과하면서 밴드의 농도가 차츰 연해지는 경향을 보이고 있어 두 균주간에 큰 차이를 보였다. Kang 등의 문헌에서는 누에 장면과 단명 품종의 유충 체액 단백질(Major hemolymph protein)을 native-PAGE로 비교한 결과 5령 초기에 많은 종류의 밴드가 관찰되었고, 5령 일수가 증가할수록 새로운 단백질의 출현뿐만 아니라 전체적인 단백질량의 증대가 뚜렷하다고 하였다(Kang, P. D., H. J. Yoon, K. S. Ryu, B. H. Sohn and H. D. Sohn. 1999. Electrophoretic patterns of hemolymph proteins of varieties with long and short life span in the silkworm Bombyx mori L. Korean J. Seric . Sci . 41, 1-8).
As a result of comparative analysis by electrophoresis of silkworm protein over time with native-PAGE, Bacillus subtilis fermented silkworms were mostly decomposed and low-molecularized from day 3 of fermentation silkworm compared to the high concentration of 97-66 kDa band at the top of non-fermented silkworm protein. It appears that the outline is hard to see (FIG. 1). But Aspergillus Fermented silkworm protein bands by kawachii strains showed a tendency to gradually soften as the fermentation time elapsed, resulting in a significant difference between the two strains. In Kang et al., Native-PAGE comparisons of silkworm scenes and major hemolymph proteins of short-lived varieties showed a large number of bands at the beginning of 5 years of age. However, the overall increase in protein content is apparent (Kang, PD, HJ Yoon, KS Ryu, BH Sohn and HD Sohn. 1999. Electrophoretic patterns of hemolymph proteins of varieties with long and short life span in the silkworm Bombyx mori L. Korean J. Seric . Sci . 41, 1-8).

실시예Example 4. 경시적인 발효 누에의  4. Over time fermented silkworm SDSSDS -- PAGEPAGE 에 의한 단백질 패턴비교Protein pattern comparison by

경시적인 발효 누에의 단백질 패턴 분석은 Weber 및 Osbom 방법(Weber, K. and M. Osborn. 1969. The reliability of molecular weight determination by sodium dodesyl sulphate-polyacrylamide gel electrophoresis. J. Biol . Chem . 244, 4406-4412)에 따라 실행하였는데, SDS-PAGE의 조성은 10% 아크릴아미드, 1.5 M Tris-HCl 완충용액 (pH 8.8), 0.4% SDS(Sodium dodecyl sulfate), 10% 과산화암모늄 및 0.05% TEMED로 만들어 사용하였다. 농축 겔의 조성은 5% 아크릴아미드, 0.5M Tris-HCl 완충용액 (pH 6.8), 0.4% SDS, 0.05% TEMED 였다. 시료단백질의 SDS 처리는 최종농도 2% SDS, 14.4 mM β-머캅토에탄올, 60 mM Tris-HCl 완충용액 (pH 6.8), 25% 글리세롤 및 0.1% BPB 혼합액에서 100℃로 5분간 열처리 하였다. 전기영동은 Tris-글리신 완충액 (25 mM Tris, 192 mM 글리신, pH 8.3) 하에서 130-150 mA/gel (cm)의 정전류를 통하여 1시간 30분간 하였다. 전기영동 후 겔은 고정액 (메탄올 : 아세트산 : 물 = 10 : 10 : 80)에 1% 쿠마시 브릴리언트 블루 R-250을 첨가하고 10시간 염색한 후 탈색액(메탄올 : 아세트산 : 물 = 10 : 10 : 80)으로 충분히 탈색시켜 건조 및 사진촬영을 행하였다.
Over time fermented silkworm Protein pattern analysis was performed according to Weber and K. Osbom method (Weber, K. and M. Osborn. 1969. The reliability of molecular weight determination by sodium dodesyl sulphate-polyacrylamide gel electrophoresis. J. Biol . Chem . 244, 4406-4412) . The composition of SDS-PAGE was made of 10% acrylamide, 1.5 M Tris-HCl buffer (pH 8.8), 0.4% Sodium dodecyl sulfate (SDS), 10% ammonium peroxide and 0.05% TEMED. The composition of the concentrated gel was 5% acrylamide, 0.5M Tris-HCl buffer (pH 6.8), 0.4% SDS, 0.05% TEMED. SDS treatment of the sample protein was heat-treated at 100 ° C. for 5 minutes in a final concentration of 2% SDS, 14.4 mM β-mercaptoethanol, 60 mM Tris-HCl buffer (pH 6.8), 25% glycerol and 0.1% BPB. Electrophoresis was performed for 1 hour and 30 minutes through a constant current of 130-150 mA / gel (cm) under Tris-glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3). After electrophoresis, the gel was added with 1% Coomassie Brilliant Blue R-250 to a fixed solution (methanol: acetic acid: water = 10: 10: 80) and stained for 10 hours, followed by a decolorizing solution (methanol: acetic acid: water = 10: 10: 80) was sufficiently decolored and dried and photographed.

경시적인 발효 누에 단백질을 SDS-PAGE상의 전기영동 패턴을 비교분석 한 결과(도 2), 발효 시작 전 97-66 kDa 크기의 누에 단백질 밴드가 뚜렷이 있는 것에 비해 Bacillus subtilis 균주에 의한 발효 누에의 단백질에서는 발효 3일째부터 전혀 없는 것으로 보아 단백질 분해 효소에 의해 대부분 분해된 것으로 보여진다. 그러나 Aspergillus kawachii 균주에 의한 발효 누에의 97-66 kDa 크기의 단백질 밴드는 발효 시간이 경과하면서 차츰 없어지면서 최장 12일째에는 대부분이 분해가 일어난 것으로 보여 분해에 상당한 시간이 필요한 것으로 생각되어 진다. 따라서 누에 단백질로부터 아미노산이나 펩타이드와 같은 생리활성 물질을 산업적으로 얻기 위해서는 Aspergillus kawachii 균주에 의한 발효보다는 Bacillus subtilis 균주에 의한 발효가 더 경제적일 것으로 사료되어 진다.
As a result of comparing the electrophoresis pattern of fermented silkworm protein over time on SDS-PAGE (Fig. 2), Bacillus compared to the clear silkworm protein band of 97-66 kDa size before fermentation started. In the protein of fermented silkworm by the subtilis strain, since it was absent from the 3rd day of fermentation, it was mostly degraded by proteolytic enzyme. But Aspergillus Protein bands of 97-66 kDa size in fermented silkworms by kawachii strains gradually disappeared as fermentation time passed, and most of them appeared to be degraded up to 12 days. Therefore, in order to obtain bioactive substances such as amino acids or peptides from silkworm protein in industry, Aspergillus Bacillus rather than fermentation by kawachii strain Fermentation by subtilis strains may be more economical.

실시예Example 5. 발효 누에의 구성 및 유리 아미노산 측정 5. Composition and Free Amino Acid Measurement of Fermented Silkworms

구성 아미노산 분석은 12일째 발효 누에 시료 0.2 g에 15 ml 과산화포름산, 6 N HCl 15 ml를 가하여, 110℃ dry oven에서 24시간 이상 동안 산 가수분해시켰다. 분해된 시료를 55℃ Water bath에서 감압농축 한 후 pH 2.20 구연산 희석 완충용액로 25 ml에 volumetric flask에 정용하여 0.2 μm membrane filter로 여과시킨 시료 일정량을 아미노산 자동분석기 (Biochrom 30, Amersham Biosciences Ltd., Cambridge, UK)로 분석하였다. 유리 아미노산 분석은 12일째 발효 누에 시료를 이용하여 아미노산 자동분석기 (Biochrom 30, Amersham Biosciences Ltd., Cambridge, UK)로 분석하였다.
Constitutive amino acid analysis was performed by adding 15 ml of formic acid peroxide and 15 ml of 6N HCl to 0.2 g of the fermented silkworm sample on day 12, and acid hydrolyzed for more than 24 hours in a 110 ℃ dry oven. The decomposed sample was concentrated under reduced pressure in a 55 ° C water bath, and then a predetermined amount of the sample was filtered through a 0.2 μm membrane filter by applying a volumetric flask to 25 ml with a pH 2.20 citric acid dilution buffer (Biochrom 30, Amersham Biosciences Ltd., Cambridge, UK). Free amino acid analysis was carried out using fermented silkworm samples on day 12 with an amino acid autoanalyzer (Biochrom 30, Amersham Biosciences Ltd., Cambridge, UK).

구성 아미노산의 총 함량은 비발효 누에의 354,610 ppm에 비해 Aspergillus kawachii 균주 발효 누에 661,692 ppm 및 Bacillus subtilis 균주 발효 누에 679,708 ppm으로 상당히 많은 량이 검출되었다(표 2). 비발효 누에의 구성 아미노산 농도는 글리신(48,519 ppm), 알라닌(46,349 ppm), 글루탐산(42,402 ppm), ㅇ아스파트산(32,798 ppm) 및 세린(29,882 ppm) 순으로 분석되었다[3]. 그러나 본 실험에서 이들 두 균주에 의한 발효누에의 구성 아미노산 중에서 가장 많이 함유된 아미노산은 글루탐산로 63.25%-63.51%를 차지하여 발효전과 다른 양상을 보였다. Aspergillus kawachii 발효 누에의 구성 아미노산 조성은 글루탐산 418,536, 아스파트산 32,335, 세린 30,190, 티로신 21,848, 루이신 21,145 ppm 순으로 나타났다. Bacillus subtilis 발효 누에의 구성 아미노산 조성은 글루탐산 431,660, 아스파트산 32,135, 세린 29,195, 리신 24,270, 히스티딘 23,857 ppm 순으로 나타나 두 균주에 의한 발효 누에의 구성 아미노산 조성에서도 차이가 있는 것으로 관찰되었다. 특히 비발효 누에의 글루탐산, 프롤린, 히스티딘은 두 균주의 발효에 의해 현저히 증가한 반면, 글리신, 알라닌, 발린은 현저히 감소한 것으로 나타났다. 지금까지 알코올성 간 독성 관련 연구에서 글리신, 글루탐산, 아르기닌, 세린, 아스파트산과 같은 단일 아미노산 처리에 의해서도 개선효과가 보고 되었다(Lee, J. H., N. K. Kim, D. Y. Lee and C. H. Lee. 1999. Protective effect of selected amino acids and food extracts on ethanol toxicity determent in rat liver. Korean J. Food Sci . Technol . 31, 802-808; Yin, M., K. Ikejima, G. E. Arteel, V. Seabra, B. U. Bradford, H. Kono and I. Rusyn and R. G. Thurman. 1998. Glycine accelerates recovery from alcohol-induced liver injury. J. Pharacol . Exp . Ther. 286, 1014-1019). 한편, 숙취해소에 널리 사용되고 있는 콩나물의 구성 아미노산 조성에서도 아스파트산, 글루탐산, 아르기닌이 많이 함유되어 있어 역시 간 보호 효과가 높았다고 하였다(Lee et al., 1999). 한편 세리신 단백질의 구성 아미노산 조성에서도 세린 31%, 아스파트산 17.8% 및 글리신이 19.1%를 차지하고 있었으며, 또한 알코올성 간 독성 개선효과가 보고된 바 있다(Kato, N., S. Sato, A. Yamanaka, H. Yamada, N. Fuwa and M. Nomura. 1998. Silk protein, sericin, inhibits lipid peroxidation and tyrosinase activity. Biosci . Biotechnol . Biochem . 62, 145-147). 따라서 간 보호 효과가 있는 글루탐산, 세린 및 아스파트산과 같은 아미노산을 많이 함유하고 있어 간 기능개선 건강보조식품 소재로서의 활용가치가 기대된다. The total content of constituent amino acids was 661,692 ppm and Bacillus of Aspergillus kawachii strain fermented silkworm compared to 354,610 ppm of unfermented silkworm Subtilis strain fermented silkworms were detected in significant amounts of 679,708 ppm (Table 2). Constituent amino acid concentrations of unfermented silkworms were analyzed in order of glycine (48,519 ppm), alanine (46,349 ppm), glutamic acid (42,402 ppm), aspartic acid (32,798 ppm), and serine (29,882 ppm) [3]. However, in this experiment, the most amino acid among the constituent amino acids of fermented silkworm by these two strains accounted for 63.25% -63.51% with glutamic acid, which was different from before fermentation. The constituent amino acid composition of Aspergillus kawachii fermented silkworms was as follows: glutamic acid 418,536, aspartic acid 32,335, serine 30,190, tyrosine 21,848, and leucine 21,145 ppm. Constituent amino acid composition of Bacillus subtilis fermented silkworm was found in the order of glutamic acid 431,660, aspartic acid 32,135, serine 29,195, lysine 24,270, histidine 23,857 ppm, and it was observed that there was a difference in the constituent amino acid composition of fermented silkworm by the two strains. In particular, glutamic acid, proline and histidine of non-fermented silkworms were significantly increased by fermentation of two strains, while glycine, alanine and valine were significantly decreased. Up to now, studies on alcoholic liver toxicity have been reported by single amino acid treatment such as glycine, glutamic acid, arginine, serine, aspartic acid (Lee, JH, NK Kim, DY Lee and CH Lee. 1999. Protective effect of selected amino acids and food extracts on ethanol toxicity determent in rat liver. Korean J. Food Sci . Technol . 31, 802-808; Yin, M., K. Ikejima, GE Arteel, V. Seabra, BU Bradford, H. Kono and I. Rusyn and RG Thurman. Glycine accelerates recovery from alcohol-induced liver injury. J. Pharacol . Exp . Ther. 286, 1014-1019). On the other hand, the amino acid composition of soybean sprouts, which are widely used to relieve hangovers, contained aspartic acid, glutamic acid, and arginine, which also showed high liver protection effects (Lee et al., 1999). Meanwhile, serine 31%, aspartic acid 17.8% and glycine accounted for 19.1% in the constituent amino acid composition of sericin protein, and alcoholic liver toxicity has been reported (Kato, N., S. Sato, A. Yamanaka). , H. Yamada, N. Fuwa and M. Nomura. 1998. Silk protein, sericin, inhibits lipid peroxidation and tyrosinase activity. Biosci . Biotechnol . Biochem . 62, 145-147). Therefore, since it contains many amino acids such as glutamic acid, serine and aspartic acid, which are effective for protecting the liver, it is expected to be useful as a health supplement material for improving liver function.

12일의 발효 기간 동안 Bacillus subtilisAspergillus kawachii 에 의해 발효된 누에의 아미노산 조성 (ppm) Bacillus for 12 days of fermentation subtilis and Aspergillus Amino Acid Composition of Silkworms Fermented by kawachii (ppm) Amino acidsAmino acids SilkwormSilkworm BacillusBacillus subtilissubtilis AspergillusAspergillus kawachiikawachii L-Aspartic acidL-Aspartic acid 32,49832,498 32,33532,335 32,13532,135 L-ThreonineL-Threonine 15,93015,930 16,34116,341 15,10815,108 L-SerineL-Serine 29,88229,882 30,19030,190 29,19529,195 L-Glutamic acidL-Glutamic acid 42,40242,402 418,536418,536 431,660431,660 L-ProlineL-Proline 9,3599,359 13,83113,831 13,62913,629 L-GlycineL-Glycine 48,51948,519 8,2708,270 9,2569,256 L-AlanineL-Alanine 46,34946,349 7,8187,818 8,0218,021 L-ValineL-Valine 17,02717,027 3,0593,059 3,4933,493 L-IsoleucineL-Isoleucine 13,10313,103 14,25614,256 13,82213,822 L-LeucineL-Leucine 18,82018,820 21,14521,145 20,36920,369 L-TyrosineL-Tyrosine 20,11420,114 21,84821,848 23,55723,557 L-PhenylalanineL-Phenylalanine 13,76913,769 14,92614,926 15,34915,349 L-HistidineL-Histidine 9,3739,373 20,70820,708 23,85723,857 L-LysineL-Lysine 19,61319,613 20,52620,526 24,27024,270 L-ArginineL-Arginine 17,55317,553 17,90517,905 15,98715,987 TotalTotal 354,610354,610 661,692661,692 679,708679,708

본 실험에서 Bacillus subtilisAspergillus kawachii 두 균주에 의한 발효 누에의 유리 아미노산 중에서 가장 많이 함유된 아미노산은 알라닌으로 각각 16.257% 및 16.198%를 함유하였으며, 그 다음으로 글루탐산이 12.540% 및 12.678%를 각각 함유하였다 (표 3 및 도 3). 그 다음으로 글리신, 발린, 루이신 및 이소루이신 순으로 많이 함유되었다. 그러나 두 균주의 발효에 의한 유리 아미노산 조성상의 큰 차이는 없는 것으로 나타났다. Bacillus in this experiment subtilis and Aspergillus kawachii Among the free amino acids of fermented silkworms by the two strains, alanine contained 16.257% and 16.198%, respectively, followed by glutamic acid 12.540% and 12.678%, respectively (Table 3 and FIG. 3). Next, glycine, valine, leucine, and isoleucine were found in order. However, there was no significant difference in free amino acid composition by fermentation of the two strains.

12일의 발효 기간 동안 Bacillus subtilisAspergillus kawachii 에 의해 발효된 누에의 유리 아미노산 Bacillus for 12 days of fermentation subtilis and Aspergillus Free amino acids of silkworms fermented by kawachii
Free amino acids

Free amino acids
Retention timeRetention time Bacillus subtilis
(%)
Bacillus subtilis
(%)
Aspergillus kawachii
(%)
Aspergillus kawachii
(%)
PhosphoserinePhosphoserine 4.3334.333 0.470.47 0.480.48 TaurineTaurine 6.9336.933 0.360.36 0.750.75 UreaUrea 10.56610.566 0.0520.052 0.0520.052 Aspartic acidAspartic acid 20.79920.799 4.0284.028 3.5803.580 ThreonineThroneine 26.66526.665 4.9354.935 4.9214.921 SerineSerine 28.63228.632 5.2835.283 5.7815.781 Glutamic acidGlutamic acid 34.26434.264 12.54012.540 12.67812.678 AAAA(α-Aminoadipic acid)AAAA (α-Aminoadipic acid) 42.26442.264 0.0850.085 NDND ProlineProline 44.16444.164 0.1470.147 0.1480.148 GlycineGlycine 45.96445.964 8.0488.048 8.5338.533 AlanineAlanine 47.46447.464 16.25816.258 16.19716.197 CitrullineCitrulline 48.99748.997 1.631.63 0.790.79 AABA (α-Aminobutyric acid)AABA (α-Aminobutyric acid) 50.83050.830 0.1640.164 0.0630.063 ValineValine 53.69753.697 6.4706.470 6.6116.611 CystienCystien 58.08358.083 0.0650.065 0.0640.064 MethionineMethionine 60.56360.563 0.6240.624 0.7760.776 CystachionineCystachionine 63.16363.163 0.2470.247 0.1770.177 IsoleucineIsoleucine 65.29665.296 4.1374.137 4.0964.096 LeucineLeucine 67.06967.069 6.8176.817 6.8176.817 TyrosineTyrosine 70.19670.196 2.7932.793 2.7502.750 β-Alanineβ-Alanine 72.06272.062 NDND NDND PhenylalaninePhenylalanine 73.42973.429 3.7623.762 3.6563.656 β-Aminoisobutylic acidβ-Aminoisobutylic acid 75.22975.229 NDND NDND HomocysteineHomocysteine 77.39577.395 NDND NDND GABA(γ-Aminobutyric acid)Γ-Aminobutyric acid (GABA) 80.06280.062 0.9300.930 0.9060.906 EthanolamineEthanolamine 83.76183.761 0.4990.499 0.3890.389 AmmoniaAmmonia 87.12887.128 8.0268.026 6.9586.958 OrnithineOrnithine 94.42794.427 0.8220.822 0.9390.939 LysineLysine 97.36097.360 4.3984.398 4.5104.510 3-Methyl-histidine3-Methyl-histidine 99.12799.127 0.0470.047 NDND HistidineHistidine 100.560100.560 2.3382.338 2.4602.460 ArginineArginine 114.193114.193 3.1773.177 3.2833.283

실시예Example 6. 경시적인 발효 6. Over time fermentation 누에의 혈전용해 효소 활성 측정Measurement of thrombolytic enzyme activity of silkworm

경시적인 발효 누에의 혈전용해 효소 활성은 fibrin plate 법(Astrup, T. and S. Mullertz. 1991. The fibrin plate method for estimating fibrinolytic activity. Arch . Biochem . Biophys 40, 346-351)을 변형하여 lysed zone으로 측정하였다. 혈전용해 활성 측정에 사용된 피브린, 피브리노겐, 트롬빈은 Sigma Co. (St. Louis MO, USA) 제품을 구입하여 사용하였다. 피브린 플레이트는 0.06% 피ㅂ브리노겐을 0.2 M 브롬산염 완충용액 (pH 7.5)에 용해시킨 후 패트리 디쉬에 10 ml씩 분주하고 트롬빈 (5,000 unit) 40 unit를 균일하게 섞이도록 가하면서 균일한 두께의 피브린 클롯(clot) 형성시킨 후 실온에서 30분간 방치한 후 사용하였다. 시료를 증류수에 1% 농도로 용출시킨 후 여과(Whatman No. 2)하여 피브린 플레이트 상에 50 μl씩 점적하여 37℃에서 3시간 동안 반응시킨 후 생성된 투명환 부위의 직경을 측정하였다. 직경은 서로 수직인 두 개의 지름을 측정하여 투명대의 면적을 구하여 unit/ml로 표시하였다.
Fibrinolytic activity over time of fermentation silkworm is fibrin plate method to transform the (Astrup, T. and S. Mullertz. 1991. The fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. Biophys 40, 346-351) lysed zone Measured by. Fibrin, fibrinogen, and thrombin used to measure thrombolytic activity are determined by Sigma Co. (St. Louis MO, USA) was purchased and used. Fibrin plates were dissolved in 0.06% fibrinogen in 0.2 M bromide buffer (pH 7.5), then dispensed in a 10 ml aliquot of Petri dishes and uniformly mixed with 40 units of thrombin (5,000 units). After forming (clot) and used for 30 minutes at room temperature. The sample was eluted with distilled water at a concentration of 1%, filtered (Whatman No. 2), and 50 μl was added dropwise onto the fibrin plate and reacted at 37 ° C. for 3 hours, and the diameter of the resulting transparent ring site was measured. The diameter was measured in two perpendicular to each other to obtain the area of the transparent zone and expressed in unit / ml.

혈관에 혈전이 만들어지게 되면 혈액순환을 방해하여 고혈압, 동맥경화, 뇌졸중, 협심증 등을 일으키는데, 이 때 혈전 용해 효소가 활성화되면 혈전 생성이 억제되어 각종 순환기계 질병을 예방하는데 효과가 있다고 알려져 있다. 자연계에 존재하는 Bacillus subtilis , Fusarium pallidoroserum , Katsuwonus pelamis , Streptococcus aureus 미생물 유래나 청국장, 된장, 멸치젓갈, natto (nattokinase) 및 shiokara (katauwokinase)와 같은 전통 발효 식품 유래의 혈전 용해 효소의 활성을 위한 소재로 많이 연구개발되었으며, 주로 세린 프로테아제로 분류되고 있다. 또한 국내 자생곤충 76종으로부터 304종의 추출물 중에서 방아깨비, 왕잠자리, 비단노린재 등 10종에서 우수한 항혈전 활성이 있다고 할 정도로 극히 일부의 곤충에서만 관찰되는 것으로 보고되고 있다(Ryu, H. Y., J. C. Heo, J. S. Hwang, S. W. Kang, C. Y. Yun, S. H. Lee and H. Y. Sohn. 2008. Screening of thrombin inhibitor and its DPPH radical scavenging activity from wide insects. J. Life Sci . 18, 363-368). 냉동건조 누에분말의 70% 메탄올 추출물에서도 혈전용해 활성이 약간 있는 반면 열풍 건조 누에 추출물에서는 활성이 전혀 없는 것으로 나타나 누에 유충의 건조 방법에 따라 달라지는 것으로 관찰되었다(Cha, J. Y., Y. S. Kim, H. Y. Ahn, K. E. Eom, B. K. Park and Y. S. Cho. 2009. Biological activity of fermented silkworm powder. J. Life Sci . 19, 1468-1477). 이전의 실험에서 열풍 건조 누에에 B. subtilis 균주를 1% 접종하여 2일간 발효시킨 발효 누에에서 혈전용해 활성이 2.2 unit 였으나, A. kawachii 균주 5% 접종으로 12일간 발효한 발효 누에에서는 3.4 unit로 약간 높은 혈전용해 활성이 관찰되었다(Cha, J. Y., Y. S. Kim, P. D. Kang, H. Y. Ahn, K. E. Eom and Y. S. Cho. 2010. Biological activity and chemical characteritics of fermented silkworm powder by mold. J. Life Sci . 20, 237-244). 본 발명에서도 열풍 건조 누에에 B. subtilis 균주를 1% 접종하여 경시적인 혈전용해 활성을 조사한 결과 3일째 2.79 uint였으며, A. kawachii 균주 5% 접종으로 12일간 발효한 발효 누에에서는 5.83 unit로 이전의 발효 누에 결과와 비슷한 결과가 관찰되었다(도 4). 그러나 두 균주로 발효시킨 발효누에의 혈전용해 활성은 6일째 최대 활성을 나타내다가 이후부터 감소하는 경향을 보였다. Soybean grit를 이용한 B. subtilis HA 균주 발효에서 5일째 가장 높은 혈전용해 효소 활성을 보여 발효 기간에 따라 효소 활성에 영향을 미치는 것으로 보고되었다[Kim, J. E. and S. B. Lee. 2009. Production of bioactive components and anti-oxidative activity of soybean grit fermented with Bacillus subtilis HA according to fermentation time. Korean J. Food Sci . Technol. 41, 179-185]. 따라서 A. kawachii 균주로 발효시킨 발효 누에의 항혈전 작용은 발효 6일째 15.02 uint로 최대 활성을 보여 발효 누에의 혈전용해 효소 활성을 갖는 발효산물을 얻기 위해서는 5-6일 정도의 발효가 적절한 시간으로 사료된다.
When blood clots are made in blood vessels, it interferes with blood circulation, causing high blood pressure, arteriosclerosis, stroke, and angina pectoris. At this time, thrombolytic enzymes are known to be effective in preventing various circulatory diseases by inhibiting blood clot production. Bacillus in Nature subtilis , Fusarium pallidoroserum , Katsuwonus pelamis , Streptococcus It has been extensively researched and developed for the activity of thrombolytic enzymes derived from aureus microorganisms and traditional fermented foods such as cheonggukjang, soybean paste, anchovy salt, natto (nattokinase) and shiokara (katauwokinase). In addition, it has been reported that only a few insects are found in 76 species of Korean native insects, which have excellent antithrombotic activity in 10 kinds of extracts, including worms, dragonflies, and silkworms (Ryu, HY, JC Heo, JS). hwang, SW Kang, CY Yun, SH Lee and HY Sohn. 2008. Screening of thrombin inhibitor and its DPPH radical scavenging activity from wide insects. J. Life Sci . 18, 363-368). The 70% methanolic extract of lyophilized silkworm powder showed some thrombolytic activity, while the hot-air dried silkworm extract showed no activity. It was observed that it depends on the method of drying silkworm larvae. KE Eom, BK Park and YS Cho . 2009. Biological activity of fermented silkworm powder. J. Life Sci . 19, 1468-1477). In the previous experiment, the thrombolytic activity of fermented silkworms fermented with 1% inoculation of B. subtilis strains on hot air dried silkworms and fermented for 2 days was 2.2 unit, but it was 3.4 units for fermented silkworms fermented for 12 days with 5% inoculation of A. kawachii strain. some high fibrinolytic activity was observed (Cha, JY, Kim YS, PD Kang, HY Ahn, KE Eom and Cho YS. 2010. Biological activity and chemical characteritics of fermented silkworm powder by mold. J. Life Sci . 20, 237-244). In the present invention, hot air dried silkworms were inoculated with 1% of B. subtilis strains and examined for thrombolytic activity over time. The fermented silkworms fermented 12 days with 5% inoculation of A. kawachii strains were 5.83 units. Results similar to those of the fermented silkworms were observed (FIG. 4). However, the thrombolytic activity of fermented silkworms fermented with both strains showed a maximum activity at 6 days and then decreased. B. subtilis using Soybean grit The highest thrombolytic enzyme activity at 5 days in HA strain fermentation was reported to affect the enzyme activity according to fermentation period [Kim, JE and SB Lee. 2009.Production of bioactive components and anti-oxidative activity of soybean grit fermented with Bacillus subtilis HA according to fermentation time. Korean J. Food Sci . Technol. 41, 179-185]. Therefore, the antithrombotic activity of fermented silkworm fermented with A. kawachii strain was 15.02 uint on the 6th day of fermentation, and the fermentation of about 5-6 days was necessary for obtaining fermentation products with thrombolytic enzyme activity of fermented silkworm. It is feed.

실시예Example 7. 경시적인 발효 누에의  7. Over time fermentation of silkworms DPPHDPPH 법에 의한 항산화 활성 측정 Determination of antioxidant activity by the method

경시적인 발효 누에의 항산화 활성은 Blois의 방법에 따라 측정하였다(Blois, M. S. 1958. Antioxidant determination by the use of a stable free radical. Nature 26, 1199-1204). 즉 DPPH(α,α'-diphenyl-β-picrylhydrazyl) 용액은 100 ml 에탄올에 DPPH 16 ㎎을 녹인 후 증류수 100 ml를 혼합하여 여과지(Whatman filter paper NO. 2)로 여과시켜 만들었다. DPPH 용액 5 ml에 0.1% (1 mg/ml) 시료 용액 1 ml을 혼합하여 실온에서 30분간 반응시킨 후 528 nm에서 흡광도의 감소를 측정하였다. 대조구인 시판항산화 BHT는 0.05%로 첨가하여 상기와 동일한 방법으로 흡광도 감소를 측정하였다. DPPH 자유 라디칼 소거능은 시료 첨가구와 무첨가구의 흡광도차를 백분율(%)로 표시하였다.Over time fermented silkworm Antioxidant activity was determined according to the method of Blois (Blois, MS 1958. Antioxidant determination by the use of a stable free radical. Nature 26, 1199-1204). That is, DPPH (α, α'-diphenyl-β-picrylhydrazyl) solution was prepared by dissolving DPPH 16 mg in 100 ml ethanol and then mixing 100 ml of distilled water and filtering with filter paper (Whatman filter paper NO. 2). 1 ml of a 0.1% (1 mg / ml) sample solution was mixed with 5 ml of DPPH solution for 30 minutes at room temperature, and then the absorbance was measured at 528 nm. As a control, commercial antioxidant BHT was added at 0.05%, and the absorbance decrease was measured in the same manner as above. DPPH free radical scavenging ability was expressed as a percentage (%) of the absorbance difference between the sample addition and no addition.

DPPH free radical scavenging activity(%) = 1 - (Abs/Abc) × 100DPPH free radical scavenging activity (%) = 1-(Abs / Abc) × 100

Abc : Absorbance of control treatment at 528nmAbc: Absorbance of control treatment at 528nm

Abs : Absorbance of sample treatment at 528nm
Abs: Absorbance of sample treatment at 528nm

식품에 함유되어 있는 폴리페놀계 물질은 히드록시기를 하나 이상 함유하고 있는 환구조와 메틸기 또는 비극성 탄화수소 사슬 구조와 같은 비극성기를 가진 분자구조적 특징을 가진 수소공여 항산화 물질로 알려져 있으며, 이를 측정하는 대표적인 방법이 DPPH 자유 라디칼 소거 항산화 활성 측정법이다(Blois, 1958). DPPH는 자유 라디칼(free radical)에서 특유의 색을 나타내다가 전자나 수소원자에 의해 전자가 쌍을 이루게 되면 비라디칼 형성으로 특유의 색이 사라지는 색도상의 차이를 측정하는 원리를 이용한 가장 일반적인 항산화 활성 측정 방법으로 실제 천연물의 항산화 활성과도 매우 연관성이 높은 것으로 잘 알려져 있다. 항산화 활성은 비발효 누에 물추출물(1 mg/ml)에서 42.12%를 나타내었고, 대조구로 사용한 시판 항산화제 BHT 1 mg/ml 농도에서는 95.96%의 높은 활성을 보였다(도 5). B. subtilis 발효 누에 물추출물(1 mg/ml)의 항산화 활성은 발효 기간이 길어지면서 비례적으로 증가하는 것으로 나타났다. 발효 전 42.12%에서 발효 9일째 75.90% 이상의 활성을 보인 이 후 완만하게 증가하는 경향을 나타내었다. A. kawachii 발효 누에 물추출물의 항산화 활성은 발효 9일째까지 큰 변화를 보이지 않다가 발효 12일째 73.99%의 높은 활성을 보였다(도 5). 우리나라 고유의 전통식품인 된장, 일본 Okara koji, 인도네시아 thempeh에서 발효과정 중에 단백질 분해가 일어나 이 때 생성되는 대사산물인 펩타이드나 아미노산 등의 화합물에 의해 항산화 활성이 증가하였다는 보고가 있었다(Matsuo, M. 1997. In vitro antioxidant activity of Okara koli, a fermented Okara, by Aspergillus oryzae. Biothechol . Biochem. Bioch . 61, 1968-1972; Matsuo, M. N. Nakamura, Y. Shidoji and T. Osawa. 1997. Antioxidative mechanism and apoptosis induction by 3-hydroxy anthranilic acid, an antioxidant in Indonesian food, tempeh, in the hyamn hepatoma derived cell line, HuH-7. J. Nutr . Sci . Vitaminol . 43, 249-259; Shon, M. Y., J. Lee, J. H. Choi and S. Y. Park. 2007. Antioxidant and free radical scavenging activity of methanol extract of chenoggkukjang. J. Food Compos . Anal. 20, 113-118). Marcuse의 문헌에서는 cystein을 제외한 대부분의 아미노산에서 항산화 활성이 있었다고 보고하였다(Marcuse, R. 1962. The effect of some amino acids on the oxidation of linoleic acid and its methyl ester. J. Am . Oil Chem . 39, 97-103). 발효 전 누에 물추출물에 비해 발효 누에의 물추출물에서 항산화 활성이 증가한 것은 발효 과정 중에 생성되는 대사산물에 의한 효과로 사료된다. 한편, 국내 자생곤충 76종의 304종 추출물 중 DPPH 소거능으로 항산화 활성을 측정한 결과 방아깨비, 가시길쭉바구미, 알락수엽노린재, 송장벌레과 유충 4종에서만 항산화 활성이 있었다고 하였다(Park, Y. J., J. C. Heo, S. M. An, E. Y. Yun, S. M. Han, J. S. Hwang, S. W. Kang, C. Y. Yun and S. H. Lee. 2005. High throughput-compatible screening of antioxidative substances by insect extract library. Korean J. Food Preserv . 12, 482-488). 이처럼 극히 일부 곤충에서만 항산화 활성이 있는 것에 비해 누에 자체에 항산화 활성이 있으면서 미생물 발효에 의해 더욱더 활성이 증가함으로서 누에의 생리활성 작용을 이용한 건강기능식품으로서 활용가치가 높은 것으로 사료된다.
Polyphenol-based materials in foods are known as hydrogen donor antioxidants that have molecular structural characteristics including ring structures containing at least one hydroxy group and nonpolar groups such as methyl groups or nonpolar hydrocarbon chain structures. DPPH free radical scavenging antioxidant activity assay (Blois, 1958). DPPH is the most common antioxidant activity measurement based on the principle of measuring the difference in chromaticity when the specific color disappears due to non-radical formation when electrons are paired by electrons or hydrogen atoms. It is well known that the method is highly related to the antioxidant activity of natural products. Antioxidant activity was 42.12% in non-fermented silkworm water extract (1 mg / ml), and showed a high activity of 95.96% at the concentration of 1 mg / ml of the commercial antioxidant BHT used as a control (FIG. 5). Antioxidant activity of B. subtilis fermented silkworm water extract (1 mg / ml) was found to increase proportionally with longer fermentation period. It showed a tendency to increase slowly after 42.12% of fermentation showed more than 75.90% of activity on 9th day of fermentation. Antioxidant activity of A. kawachii fermented silkworm water extract showed no significant change until day 9 of fermentation, but showed a high activity of 73.99% at day 12 of fermentation (FIG. 5). Antioxidant activity has been reported to increase the antioxidant activity by compounds such as peptides and amino acids, which are metabolites produced during fermentation in the Korean traditional food, Doenjang, Okara koji and Indonesia thempeh (Matsuo, M). . 1997. In vitro antioxidant activity of Okara koli, a fermented Okara, by Aspergillus oryzae. Biothechol . Biochem. Bioch . 61, 1968-1972; Matsuo, MN Nakamura, Y. Shidoji and T. Osawa. 1997. Antioxidative mechanism and apoptosis induction by 3-hydroxy anthranilic acid, an antioxidant in Indonesian food, tempeh, in the hyamn hepatoma derived cell line, HuH-7. J. Nutr . Sci . Vitaminol . 43, 249-259; Shon, MY, J. Lee, JH Choi and SY Park. 2007. Antioxidant and free radical scavenging activity of methanol extract of chenoggkukjang . J. Food Compos . Anal. 20, 113-118). Marcuse's literature reported antioxidant activity in most amino acids except cystein (Marcuse, R. 1962. The effect of some amino acids on the oxidation of linoleic acid and its methyl ester. J. Am . Oil Chem . 39, 97-103). The increase of antioxidant activity in water extract of fermented silkworm compared to silkworm extract before fermentation is considered to be due to the metabolites produced during fermentation. Antioxidant activity was measured by DPPH scavenging activity among the 304 extracts of 76 Korean native insects, which were found to have antioxidant activity only in four species of worms, thorns, weeds, and leaves and pine larvae (Park, YJ, JC Heo, SM An, EY Yun, SM Han, JS Hwang, SW Kang, CY Yun and SH Lee. 2005. High throughput-compatible screening of antioxidative substances by insect extract library. Korean J. Food Preserv . 12, 482-488). As such, only a few insects have antioxidant activity, and the silkworm itself has an antioxidant activity and is more active by microbial fermentation.

실시예Example 8. 경시적인 발효 누에의  8. Over time fermentation of silkworm FeFe 환원력 측정 Reducing power measurement

경시적인 발효 누에의 Fe-환원력 측정은 Zhu 등의 방법[31]에 따라 측정하였다. 발효 누에 0.1% 물추출물 0.75 ml을 취하고, 0.2 M sodium phosphate 완충용액 (pH 6.6) 1.25 ml 및 1% (w/v) potassium ferricyanide [K3Fe(CN)6] 1.25 ml을 혼합하여 50℃에서 20분간 진탕반응시켰다. 이 반응액에 10% 트리클로로아세트산 (w/v)를 1.25 ml 가하여 산성화시키고, 3,000 rpm에서 20분간 원심분리시켰다. 상층액 2.5 ml를 취하고 증류수 2.5 ml 및 0.5% 염화제이철 (FeCl3) 0.5 ml를 혼합한 후 실온에서 10분간 반응시켜 Spectrophotometer (HITACHI U-2900)의 700 nm에서 흡광도를 측정하였다. 이때 환원력 비교를 위하여 시판 항산화제로 많이 사용되고 있는 아스코르브산, BHT를 각각 시료와 동일 농도로 만들어 동일한 방법에 의해 흡광도를 측정하였다. 환원력은 시료 반응액에서 흡광도가 증가된 만큼 강한 환원력을 나타내어 준다. Fe-reduction of fermented silkworms over time was measured according to the method of Zhu et al. [31]. Take 0.75 ml of 0.1% water extract from fermented silkworm, mix 1.25 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 1.25 ml of 1% (w / v) potassium ferricyanide [K 3 Fe (CN) 6 ] at 50 ° C. The reaction was shaken for 20 minutes. The reaction solution was acidified by adding 1.25 ml of 10% trichloroacetic acid (w / v), and centrifuged at 3,000 rpm for 20 minutes. 2.5 ml of the supernatant was taken, and 2.5 ml of distilled water and 0.5 ml of 0.5% ferric chloride (FeCl 3 ) were mixed, and reacted at room temperature for 10 minutes to measure absorbance at 700 nm of a Spectrophotometer (HITACHI U-2900). At this time, ascorbic acid and BHT, which are widely used as commercial antioxidants, were compared with the sample to measure reducing power, and the absorbance was measured by the same method. Reducing power shows strong reducing power as the absorbance is increased in the sample reaction solution.

Fe 환원력(reducing power) 활성 정도는 수소 원자를 제공하는 자유 라디칼의 연쇄 반응으로 금속 이온을 환원시키는 정도를 흡광도 값으로 나타내는 것으로, 환원력의 효과는 반응 물질의 흡광도 수치가 높을수록 항산화 활성이 높다는 것을 나타내어 준다. Fe-환원력은 대조구로 사용한 시판 항산제인 BHT 및 ascorbic acid 0.1%의 농도에서 1.29 및 1.21의 흡광도를 나타내었다(도 6). 비발효 누에 물추출물(1 mg/ml)의 환원력은 0.41로 대조구로 사용한 시판 항산화제 BHT 또는 ascorbic acid 흡광도 보다 3배 정도 낮은 값을 보였다(도 6). 그러나 B. subtilis 발효 누에 물추출물(1 mg/ml)에서는 발효 6일째 0.56으로 증가하다가 차츰 낮아지는 경향을 보였으며, A. kawachii 발효 누에 물추출물(1 mg/ml)에서는 발효 9일째까지 큰 변화를 보이지 않다가 발효 12일째 0.47로 가장 높은 활성을 보였다. 더덕의 경우 열수 추출물(1 mg/ml)에서 환원력으로 측정한 흡광도는 0.34인데 비해 발효 더덕의 경우 0.65으로 발효에 의해 활성이 증가된 것으로 나타나 본 발명의 결과와는 일치하는 것으로 나타났다(Park, S. J., S. W. Song, D. H. Seong, D. S. Park, S. S. Kim, J. Gou, J. H. Ahn, W. B. Yoon and H. Y. Lee. 2009. Biological activities in the extract if fermented Codonopsis lanceolata. J. Korean Soc. Food Sci. Nutr. 38, 983-988). The degree of Fe reducing power activity is the absorbance value indicating the reduction of metal ions by the chain reaction of free radicals providing hydrogen atoms. The effect of reducing power is that the higher the absorbance value of the reactant, the higher the antioxidant activity. To indicate. Fe-reducing power showed absorbances of 1.29 and 1.21 at concentrations of 0.1% of BHT and ascorbic acid, which are commercial antioxidants used as controls (FIG. 6). The reducing power of the non-fermented silkworm water extract (1 mg / ml) was 0.41, which was three times lower than the absorbance of commercial antioxidant BHT or ascorbic acid used as a control (FIG. 6). However the B. subtilis fermentation silkworm water extract (1 mg / ml) showed a tendency to decrease gradually while increasing the fermentation day 6 0.56, A. kawachii fermentation silkworm water extract (1 mg / ml) in a large change in fermentation to 9 days After 12 days of fermentation, the highest activity was 0.47. The absorbance measured by reducing power in hot water extract (1 mg / ml) was 0.34, whereas the activity was increased by 0.65 in fermentation, which is consistent with the results of the present invention (Park, SJ). , SW Song, DH Seong, DS Park, SS Kim, J. Gou, JH Ahn, WB Yoon and HY Lee.2009.Biological activities in the extract if fermented Codonopsis lanceolata.J . Korean Soc.Food Sci.Nutr. 38, 983-988).

이상의 실험에서 B. subtilis 발효 누에에서 환원력과 혈전 분해 활성은 발효 6일째에 항산화 활성과 단백질 농도는 발효 12일째에 가장 높았고, SDS-PAGE상의 전기영동 분석에서는 발효 초기인 3일째 96-66 kDa 크기의 단백질이 대부분 분해가 일어났었다. A. kawachii 발효 누에의 환원력, 항산화 활성 및 단백질 농도는 발효 12일째에 가장 높았고, SDS-PAGE상의 전기영동 분석에서 96-66 kDa 크기의 단백질은 발효 3일째부터 차츰 분해되기 시작하여 12일째 분해가 대부분 이루어졌다. 따라서 미생물을 이용한 발효 누에에서 특정의 생리활성 작용을 기대하기 위해서는 사용하는 균종에 따라 발효 시간이 달라져야 한다는 것을 시사하였다.
In the above experiments, the reducing power and thrombolytic activity of B. subtilis fermented silkworms were highest at 6 days of fermentation and the highest at 12 days of fermentation, and the electrophoretic analysis on SDS-PAGE showed 96-66 kDa size at 3 days of fermentation. Most of the protein had been degraded. The reducing power, antioxidant activity and protein concentration of A. kawachii fermented silkworms were highest at day 12 of fermentation.In the electrophoresis analysis of SDS-PAGE, 96-66 kDa-sized proteins began to degrade gradually from day 3 of fermentation. Mostly done. Therefore, it was suggested that the fermentation time should be changed according to the species used in order to expect a specific physiological activity in the fermented silkworm using microorganisms.

실시예Example 9. 동물 실험  9. Animal Experiment

실험재료Experimental material

전배양시킨 Bacillus subtilis 균주를 살균한 열풍건조 누에분말에 접종한 후 37℃에서 48시간 발효시킨 후 6시간 동안 열풍건조시켜 실험동물에 사용하였다. 또한 PD 액체배지에서 150 rpm, 30℃ 조건에서 72시간 전배양시킨 Aspergillus kawachi 균주도 살균한 열풍건조 누에분말에 접종하여 30℃에서 12일간 발효시킨 후 6시간 동안 열풍건조시켜 얻은 발효 누에분말 시료를 동물실험에 사용하였다.
Precultured Bacillus subtilis The strain was inoculated into sterilized hot air dried silkworm powder, fermented at 37 ° C. for 48 hours, and then hot air dried for 6 hours to be used in experimental animals. Aspergillus kawachi strains were also inoculated in sterilized hot-air dried silkworm powder for 72 hours at 150 rpm and 30 ℃ in PD liquid medium, fermented at 30 ℃ for 12 days, and then fermented silkworm powder sample obtained by hot-air drying for 6 hours. Used for animal experiments.

식이조성Diet  And 실험군Experimental group

식이 조성은 표 4와 같으며, 정상군 (N), 알코올 투여 대조군 (C), 알코올+열풍건조 누에분말 투여군 (SP), 알코올+Bacillus subtilis 발효 누에분말 투여군 (BFSP), 알코올+Aspergillus kawachi 발효 누에분말 투여군 (AFSP)으로 나누어 실험을 진행하였다. 발효 누에분말의 식이 중 첨가량 결정은 Yoon 등의 문헌[Yoon JW, Rhee SK, Lee KB. Effects of silkworm extract powder on plasma lipids and glucose in rats. Korean J Food Nutr 18: 140-145 (2005)]의 방법에 준하였으며, 이때 식이 중에 첨가한 발효 누에분말은 단백질원인 카제인과 당질원인 설탕으로 대체하여 첨가하였다. Dietary composition is shown in Table 4, normal group (N), alcohol administration control group (C), alcohol + hot air drying silkworm powder administration group (SP), alcohol + Bacillus subtilis Fermented silkworm powder group (BFSP), alcohol + Aspergillus kawachi The experiment was divided into fermented silkworm powder administration group (AFSP). Determination of the amount of fermented silkworm powder in the diet is described by Yoon et al. In Yoon JW, Rhee SK, Lee KB. Effects of silkworm extract powder on plasma lipids and glucose in rats. Korean J Food Nutr 18: 140-145 (2005)], wherein the fermented silkworm powder added in the diet was replaced with casein and sugar as a protein source.

실험 식이 조성 (%)Experimental dietary composition (%) IngredientsIngredients NormalNormal Alcohol Alcohol ControlControl SP1 ) SP 1 ) BFSP2 ) BFSP 2 ) AFSP3 ) AFSP 3 ) CaseinCasein 2020 2020 1717 1717 1717 Corn starchCorn starch 1515 1515 1515 1515 1515 SucroseSucrose 5555 5555 5353 5353 5353 CelluloseCellulose 55 55 55 55 55 Corn oilCorn oil 1010 1010 1010 1010 1010 Mineral mixture4 ) Mineral mixture 4 ) 3.53.5 3.53.5 3.53.5 3.53.5 3.53.5 Vitamin mixture5 ) Vitamin mixture 5 ) 1One 1One 1One 1One 1One Choline bitartrateCholine bitartrate 0.20.2 0.20.2 0.20.2 0.20.2 0.20.2 DL-MethionineDL-Methionine 0.30.3 0.30.3 0.30.3 0.30.3 0.30.3 SPSP 00 00 55 00 00 BFSPBFSP 00 00 00 55 00 AFSPAFSP 00 00 00 00 55

1)SP: Silkworm powder. 1) SP: Silkworm powder.

2)BFSP: Bacillus subtillis fermented silkworm powder. 2) BFSP: Bacillus subtillis fermented silkworm powder.

3)AFSP: Aspergillus kawachii fermented silkworm powder. 3) AFSP: Aspergillus kawachii fermented silkworm powder.

4)AIN 93 M-MX mineral mix, MP Biomedicals, Illkirch, France. 4) AIN 93 M-MX mineral mix, MP Biomedicals, Illkirch, France.

5)AIN 93 VX vitamin mix, MP Biomedicals, Illkirch, France.
5) AIN 93 VX vitamin mix, MP Biomedicals, Illkirch, France.

실험동물 및 사육조건Experimental Animals and Breeding Conditions

실험동물은 6주령의 Sprague-Dawley계 수컷 흰쥐를 구입하여 일주일간 시판 고형사료를 급여하면서 환경에 적응시킨 후 본 실험에 사용하였다. 본 실험은 체중이 동일하게 난괴법(randomized complete block design)으로 분류하여 사육실 온도(22±2℃)와 습도(50±5%) 및 명암주기(명주기: 07:00?19:00)가 조절되는 동물 사육실에서 사육하였다. 사육 기간 중 식이 섭취량은 매일 측정하였고, 체중은 매주 한번씩 일정한 시간에 측정하였다. 이 때 알코올 투여는 실험동물에서 알코올에 의한 급성독성을 막기 위하여 알코올 농도를 처음 10%로 시작해서 최종 30% 농도로 1주일마다 단계적으로 높이면서 본 실험을 시작하면서 시료를 함께 투여하였다.
The experimental animals were purchased for 6 weeks old Sprague-Dawley male rats. In this experiment, body weight was equally classified into randomized complete block design, and the room temperature (22 ± 2 ℃), humidity (50 ± 5%) and contrast cycle (light cycle: 07: 00 ~ 19: 00) It was raised in a controlled animal breeding room. Dietary intake was measured every day during the breeding period, and body weight was measured at a fixed time once a week. At this time, the alcohol administration was administered together with the sample at the beginning of the experiment, starting with the first 10% alcohol concentration step by step to a final 30% concentration to prevent acute toxicity by alcohol in the experimental animals.

동물실험, 시료 채취 및 분석시료 조제Animal Testing, Sampling and Analytical Sample Preparation

동물실험은 4주간 각 군별로 조제사료를 급여하면서 사육한 후, 실험 최종일 12시간 이상 절식시킨 후 에테르로 가볍게 마취시켜 해부하였다. 개복 후 복부 대동맥으로부터 채혈하여 혈액을 채취하고, 약 30분간 실온에 방치시킨 후 3,000 rpm에서 20분간 원심분리 하여 얻은 혈청을 혈청 생화학적 분석에 제공하였다. 채혈 후 각 조직을 적출하여 차가운 0.9% 생리식염수로 세척하고 여과지로 물기를 제거한 후 무게를 측정하고 분석시료로 제공하였다.
Animal experiments were reared for four weeks with feeding for each group, followed by fasting for more than 12 hours on the last day of the experiment, and dissected by light anesthesia with ether. Blood was collected from the abdominal aorta after laparotomy, blood was collected, and the serum obtained by centrifugation at 3,000 rpm for 20 minutes after being left at room temperature for about 30 minutes was used for serum biochemical analysis. After blood collection, each tissue was extracted, washed with cold 0.9% saline solution, dried with filter paper, weighed, and provided as an analytical sample.

실시예Example 10. 동물실험 결과 평가 10. Evaluation of animal test results

체중, 식이 및 음료 섭취량 변화Changes in weight, diet and drink intake

4주 동안 알코올 급여에 의해 체중 증가량이 정상군에 비해 많이 증가하지 않은 것으로 보아 알코올에 의해 성장에 영향을 미친 것으로 보여지며, 식이 섭취량도 동시에 많이 줄어든 것으로 나타났다(표 5). 한편 알코올은 소화흡수 기관인 소장에서 영양소나 식이성분의 흡수를 떨어트려 영향불균형을 초래함으로서 체중 증가량의 감소를 가져온다. 그러나 알코올 급여 흰쥐에 SP, BFSP, AFSP의 첨가식이 투여는 식이섭취량의 증가에 의해 체중이 증가하는 것으로 나타났다(표 5). It was shown that the weight gain did not increase much by the alcohol intake for 4 weeks compared with the normal group, and that the growth was influenced by the alcohol, and the dietary intake was also decreased at the same time (Table 5). On the other hand, alcohol decreases the absorption of nutrients or dietary components in the small intestine, which is a digestive organ, resulting in an effect imbalance, leading to a decrease in weight gain. However, dietary supplementation of SP, BFSP, and AFSP in alcohol-fed rats showed an increase in body weight due to increased dietary intake (Table 5).

알코올 투여 랫에서 체중, 식이 및 음료 섭취 및 상대적인 조직 무게에 대한 SP, BFSP 및 AFSP의 효과 Effects of SP, BFSP and AFSP on Body Weight, Diet and Drink Intake, and Relative Tissue Weight in Alcoholic Rats GroupGroup Body weight gain
(g)
Body weight gain
(g)
Food intake
(g/day)
Food intake
(g / day)
Water consumption
(mL/day)
Water consumption
(mL / day)
NormalNormal 212.19±7.55a 212.19 ± 7.55 a 19.83±0.17a 19.83 ± 0.17 a 35.50±1.28a 35.50 ± 1.28 a ControlControl 99.04±17.17b 99.04 ± 17.17 b 13.00±0.63b 13.00 ± 0.63 b 14.50±0.67b 14.50 ± 0.67 b SPSP 119.92±18.90b 119.92 ± 18.90 b 12.83±0.40b 12.83 ± 0.40 b 15.67±0.92b 15.67 ± 0.92 b BFSPBFSP 116.34±13.13b 116.34 ± 13.13 b 12.83±0.54b 12.83 ± 0.54 b 17.67±0.92b 17.67 ± 0.92 b AFSPAFSP 115.63±13.46b 115.63 ± 13.46 b 12.50±0.43b 12.50 ± 0.43 b 16.83±1.47b 16.83 ± 1.47 b

SP: Silkworm powder.SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder. AFSP: Aspergillus kawachii fermented silkworm powder.

Values with different letters are significantly different at p〈 0.05. (mean±S.E., n=6). Values with different letters are significantly different at p <0.05. (mean ± S.E., n = 6).

NS: Not significant different.
NS: Not significant different.

각 장기의 절대 및 상대중량Absolute and relative weight of each organ

체중에 대한 각 장기의 절대증량과 상대중량을 각각 표 6과 표 7에 나타내었다. 절대 중량에서는 정상군에 비해 모든 실험+군에서 유의적인 차이를 나타내었다(표 6).The absolute increase and relative weight of each organ with respect to body weight are shown in Table 6 and Table 7, respectively. Absolute weight showed a significant difference in all experiments + group compared to normal group (Table 6).

알코올 투여 랫의 조직 무게에서 SP, BFSP 및 AFSP의 효과 Effects of SP, BFSP and AFSP on Tissue Weight in Alcohol-administered Rats GroupGroup LiverLiver KidneyKidney SpleenSpleen HeartHeart TestisTestis Epididymal
WAT
Epididymal
WAT
Perirenal
WAT
Perirenal
WAT
NormalNormal 11.48
±0.22a
11.48
± 0.22 a
3.31
±0.10a
3.31
± 0.10 a
0.79
±0.05a
0.79
± 0.05 a
1.57
±0.04a
1.57
± 0.04 a
3.32
±0.13a
3.32
± 0.13 a
8.12
±0.39a
8.12
± 0.39 a
8.54
±0.40a
8.54
± 0.40 a
ControlControl 8.89
±0.65b
8.89
± 0.65 b
2.84
±0.09b
2.84
± 0.09 b
0.59
±0.04b
0.59
± 0.04 b
1.31
±0.05b
1.31
± 0.05 b
2.96
±0.09b
2.96
± 0.09 b
4.57
±0.66b
4.57
± 0.66 b
3.96
±0.89b
3.96
± 0.89 b
SPSP 9.04
±0.45b
9.04
± 0.45 b
2.63
±0.05b
2.63
± 0.05 b
0.51
±0.04b
0.51
± 0.04 b
1.23
±0.06b
1.23
± 0.06 b
3.10
±0.05ab
3.10
± 0.05 ab
4.90
±0.57b
4.90
± 0.57 b
4.10
±0.75b
4.10
± 0.75 b
BFSPBFSP 9.71
±0.60b
9.71
± 0.60 b
2.86
±0.20b
2.86
± 0.20 b
0.56
±0.03b
0.56
± 0.03 b
1.17
±0.06b
1.17
± 0.06 b
3.10
±0.06b
3.10
± 0.06 b
4.94
±0.42b
4.94
± 0.42 b
3.97
±0.53b
3.97
± 0.53 b
AFSPAFSP 9.23
±0.37b
9.23
± 0.37 b
2.67
±0.07b
2.67
± 0.07 b
0.54
±0.03b
0.54
± 0.03 b
1.23
±0.04b
1.23
± 0.04 b
3.01
±0.10ab
3.01
± 0.10 ab
4.64
±0.27b
4.64
± 0.27 b
3.82
±0.55b
3.82
± 0.55 b

SP: Silkworm powder.SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder. AFSP: Aspergillus kawachii fermented silkworm powder.

Values with different letters are significantly different at p〈 0.05. (mean±S.E., n=6).
Values with different letters are significantly different at p <0.05. (mean ± SE, n = 6).

또한 체중에 대한 각 장기의 상대중량은 간, 신장, 심장, 고환에서는 각 실험군간에 유의적인 차이가 없었으나, 비장, 신장주변 지방조직 및 고환주변 지방조직의 경우 군간에 약간의 차이를 보였다(표 7).
In addition, the relative weight of each organ to body weight was not significantly different among the experimental groups in the liver, kidney, heart, and testicles, but there were slight differences among the groups in the spleen, the surrounding adipose tissue and the surrounding testicular adipose tissue. 7).

알코올 투여 랫의 상대적인 조직 무게에서 SP, BFSP 및 AFSP의 효과 Effect of SP, BFSP and AFSP on Relative Tissue Weight of Alcohol-administered Rats GroupGroup LiverLiver KidneyKidney SpleenSpleen HeartHeart TestisTestis Epididymal
WAT
Epididymal
WAT
Perirenal
WAT
Perirenal
WAT
NormalNormal 2.65
±0.04NS
2.65
± 0.04 NS
0.76
±0.03NS
0.76
± 0.03 NS
0.18
±0.01a
0.18
± 0.01 a
0.75
±0.40NS
0.75
± 0.40 NS
0.78
±003NS
0.78
± 003 NS
1.87
±0.08a
1.87
± 0.08 a
1.97
±0.09a
1.97
± 0.09 a
ControlControl 2.62
±0.07
2.62
± 0.07
0.85
±0.03
0.85
± 0.03
0.18
±0.01a
0.18
± 0.01 a
0.39
±0.01
0.39
± 0.01
0.88
±0.07
0.88
± 0.07
1.33
±0.14b
1.33
± 0.14 b
1.13
±0.21b
1.13
± 0.21 b
SPSP 2.57
±0.09
2.57
± 0.09
0.76
±0.05
0.76
± 0.05
0.14
±0.01b
0.14
± 0.01 b
0.35
±0.03
0.35
± 0.03
0.92
±0.06
0.92
± 0.06
1.37
±0.11b
1.37
± 0.11 b
1.13
±0.17b
1.13
± 0.17 b
BFSPBFSP 2.76
±0.07
2.76
± 0.07
0.82
±0.04
0.82
± 0.04
0.16
±0.01ab
0.16
± 0.01 ab
0.33
±0.02
0.33
± 0.02
0.91
±0.02
0.91
± 0.02
1.40
±0.07b
1.40
± 0.07 b
1.12
±0.13b
1.12
± 0.13 b
AFSPAFSP 2.66
±0.05
2.66
± 0.05
0.77
±0.03
0.77
± 0.03
0.16
±0.01a b
0.16
± 0.01 a b
0.36
±0.01
0.36
± 0.01
0.85
±0.06
0.85
± 0.06
1.34
±0.08b
1.34
± 0.08 b
1.08
±0.13b
1.08
± 0.13 b

Relative tissue weight means the percent of the liver weight in the body weight.Relative tissue weight means the percent of the liver weight in the body weight.

SP: Silkworm powder.SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder. AFSP: Aspergillus kawachii fermented silkworm powder.

Values with different letters are significantly different at p〈 0.05. (mean±S.E., n=6). Values with different letters are significantly different at p <0.05. (mean ± S.E., n = 6).

NS: Not significant different.
NS: Not significant different.

실시예Example 11. 간 조직 및 혈청 지질농도 및 생화학적 지표분석 11. Liver tissue and serum lipid concentration and biochemical indicator analysis

간 조직으로부터 균질액 분획 조제는 간을 일정량 취해 ice-cold 인산칼륨 완충용액 용액(1 mmol/L sodium EDTA 및 1 mmol/L 디티오트레이톨을 포함하는 0.1 mol/L 인산칼륨, pH 7.4)을 9배량 첨가하여 IKA-ULTRA-TURRAX T25 basic homogenizer (IKA-WERKE GMBH & CO., KG, Staufen, Germany)를 이용하여 균질액을 제조하였다. 균질화 용액을 먼저 800 x g에서 10분간 원심분리하고, 상등액을 다시 고속원심분리기(VS-24SMTi, Vision Scientific Co., Ltd.)에서 10,000 × g로 20분간 원심분리 하여 침전된 펠렛은 미토콘드리아 분획으로 하였다. 이때 마이크로솜이 함유된 상층액을 105,000 x g에서 60분간 초원심분리하여 상층의 cytosol 분획과 침전된 마이크로솜 분획을 얻어 실험에 제공하였다. 균질화액, 미토콘드리아 마이크로솜 분획의 단백질 농도는 Lowry 방법에 준하여 우혈청 알부민을 표준품으로 하여 측정하였다(Lowry OH, Rosebrough NJ, Farr AL, Randall RS. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275 (1951)).The homogenate fraction preparation from the liver tissue was prepared by taking an amount of liver and adding ice-cold potassium phosphate buffer solution (0.1 mol / L potassium phosphate, pH 7.4 containing 1 mmol / L sodium EDTA and 1 mmol / L dithiothreitol). A homogenate was prepared by adding 9 times the amount using an IKA-ULTRA-TURRAX T25 basic homogenizer (IKA-WERKE GMBH & CO., KG, Staufen, Germany). The homogenization solution was first centrifuged at 800 xg for 10 minutes, and the supernatant was centrifuged at 10,000 x g for 20 minutes in a high-speed centrifuge (VS-24SMTi, Vision Scientific Co., Ltd.) to precipitate the pellet as a mitochondrial fraction. . At this time, the supernatant containing microsomes was ultracentrifuged at 105,000 xg for 60 minutes to obtain the cytosol fraction and precipitated microsomal fraction of the upper layer. Homogenizer, Mitochondria And Protein concentration of the microsomal fraction was measured using bovine serum albumin as a standard according to the Lowry method (Lowry OH, Rosebrough NJ, Farr AL, Randall RS. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275 ( 1951)).

혈청 중의 총 지질, 중성지질, 총-콜레스테롤, HDL-콜레스테롤, 유리지방산, 총 단백질, 알부민 농도 및 ALT, AST, γ-GTP, LDH 활성은 의료전문수탁검사기관인 네오딘의학연구소(서울, 한국)에 의뢰하여 분석하였다.
Serum total lipid, triglyceride, total cholesterol, HDL-cholesterol, free fatty acid, total protein, albumin concentration, and ALT, AST, γ-GTP, LDH activity were investigated at Neodin Medical Research Institute (Seoul, Korea). Requested analysis.

간 조직 및 혈중의 중성지질 농도Neutral lipid concentration in liver tissue and blood

간은 많은 지질과 지질대사에 관련된 중요한 조직으로 알려져 있으며, 혈중 중성지질 농도는 간 중성지질 대사와 밀접한 관련성이 있다. 특히 간 지질 중에서 중성지질 축적은 비 알코올성 또는 알코올성 지방간 유발 원인 물질로 잘 알려져 있다. 본 발명에서 알코올 급여에 의해 간 중성지질 농도가 현저히 증가하여 알코올성 지방간의 특징을 잘 나타내어(표 8) 이전의 실험 결과와 일치하였다(Oliva L, Beauge F, Choquart D, Montet AM, Guitaoui M, Montet JC. Ursodeoxycholate alleviates alcoholic fatty liver damage in rats. Alcohol Clin Exp Res 22: 1538-1543 (1998)). 흰쥐에서 간 독성에 의한 간 지질의 축적은 글리신, 아르기닌, 타우린, 시스테인, 알라닌 등과 같은 항산화 및 간독성 개선 효과를 가지는 아미노산에 의해 어느 정도 개선되는 것으로 알려져 있다. 높은 항산화 활성을 가지고 있는 BFSP 투여에 의해서도 알코올에 의한 간 중성지질 축적을 현저하게 경감시키는 효능이 있는 것으로 밝혀졌으며(표 8), 특히 BFSP 투여군의 간 중성지질 수준이 정상군 수준까지 감소하는 것으로 나타났다. 한편 알코올 섭취는 혈중 중성지질 수준을 증가시키는 것으로 많이 보고되었으나(Oliva L, Beauge F, Choquart D, Montet AM, Guitaoui M, Montet JC. Ursodeoxycholate alleviates alcoholic fatty liver damage in rats. Alcohol Clin Exp Res 22: 1538-1543 (1998)), 본 발명의 결과에서는 이들 결과와 일치하지 않았다(표 8). The liver is known to be an important tissue involved in many lipids and lipid metabolism, and blood triglyceride levels are closely related to hepatic triglyceride metabolism. In particular, triglyceride accumulation among liver lipids is well known as a causative agent for non-alcoholic or alcoholic fatty liver. In the present invention, the alcohol triglyceride concentration was significantly increased and the characteristics of alcoholic fatty liver were well represented (Table 8), which is consistent with the previous experimental results (Oliva L, Beauge F, Choquart D, Montet AM, Guitaoui M, Montet). JC.Usodeoxycholate alleviates alcoholic fatty liver damage in rats.Alcohol Clin Exp Res 22: 1538-1543 (1998)). Accumulation of hepatic lipids by hepatotoxicity in rats is known to be somewhat improved by amino acids with antioxidant and hepatotoxicity-improving effects such as glycine, arginine, taurine, cysteine and alanine. BFSP administration, which has high antioxidant activity, has been shown to significantly reduce the accumulation of hepatic triglycerides by alcohol (Table 8). In particular, hepatic triglyceride levels in the BFSP-administered group were reduced to normal levels. . Alcohol consumption has been reported to increase blood triglyceride levels (Oliva L, Beauge F, Choquart D, Montet AM, Guitaoui M, Montet JC. Ursodeoxycholate alleviates alcoholic fatty liver damage in rats.Alcohol Clin Exp Res 22: 1538 -1543 (1998)), the results of the present invention were inconsistent with these results (Table 8).

혈중 중성지질 농도가 정상군과 알코올 투여 대조군 사이에 큰 유의적인 차이가 없었다. AFSP 투여군에서 혈중 중성지질 농도는 감소경향을 보였고, BFSP 투여군에서는 현저한 감소를 보여 발효시 사용한 균주 간에 차이를 나타내었다. 이러한 BFSP 투여에 의한 혈중 중성지질의 감소는 유리지방산 감소에 기인하는 것으로 사료되어 진다.There was no significant difference in serum triglyceride levels between the normal group and the alcohol control group. Serum triglyceride concentrations tended to decrease in the AFSP-treated group and markedly decreased in the BFSP-administered group, showing differences among the strains used during fermentation. The decrease of triglyceride in blood by the administration of BFSP is thought to be due to the reduction of free fatty acid.

알코올 투여 랫의 간 및 혈청의 중성지질 농도에서의 SP, BFSP 및 AFSP 효과 Effects of SP, BFSP, and AFSP on Neutral Lipid Concentrations in Liver and Serum of Alcoholic Rats Serum Triglyceride
(mg/100 mL)
Serum Triglyceride
(mg / 100 mL)
Liver Triglyceride
(mg/g)
Liver triglyceride
(mg / g)
46.67±3.38a 46.67 ± 3.38 a 19.71±1.38a 19.71 ± 1.38 a 51.33±9.74a 51.33 ± 9.74 a 53.13±3.54b 53.13 ± 3.54 b 52.80±13.14a 52.80 ± 13.14 a 31.35±3.14ab 31.35 ± 3.14 ab 35.50±8.01ab 35.50 ± 8.01 ab 33.50±8.01ab 33.50 ± 8.01 ab 19.00±2.22b 19.00 ± 2.22 b 21.10±1.22b 21.10 ± 1.22 b

SP: Silkworm powder.SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder.
AFSP: Aspergillus kawachii fermented silkworm powder.

혈중 지질 농도 변화Changes in blood lipid concentration

혈중 지질 농도는 동맥경화, 고혈압, 심장병, 고지혈증과 같은 심혈관계 질환인의 진단지표로 사용되고 있는 가운데, 고콜레스테롤혈증, 고중성지혈증, 저HDL-콜레스테롤혈증도 이들 질환의 위험 인자로 주목받게 되면서 유럽과 미국 등에서 새로운 임상 지침이 설정되었다. 최근 천연 식물자원을 대상으로 혈중 콜레스테롤 농도뿐만 아니라 중성지질 농도를 감소시키고 HDL-콜레스테롤 농도를 증가시키려는 시도가 활발하게 전개되고 있다. 한편 혈청 HDL-콜레스테롤 농도는 항 동맥경화의 지표로서 주로 간장에서 합성되고 다른 지단백질과는 달리 혈관 벽에 침착되어 있는 LDL-콜레스테롤을 분리해내어 간장으로 운반하여 에너지로 이용하거나 체외 배설을 촉진하는 작용을 함으로써 심혈관계 질환의 유발 위험성을 감소시킬 수 있는 유익한 콜레스테롤로 널리 알려져 있다. 혈중 총 콜레스테롤 농도의 증가는 알코올 투여 동물에서 나타나는 일반적인 현상 중의 하나로 받아들여지고 있다. 본 실험에서도 혈중 총 콜레스테롤 및 유리 지방산 농도가 정상군에 비해 알코올 투여군에서 유의적으로 증가하였으나, 혈중 총 지질 및 HDL-콜레스테롤 농도는 각 실험군간에 유의적인 차이는 관찰되지 않았다(표 9). 그러나 혈중 총 지질 농도는 알코올 대조군에 비해 누에분말 및 발효 누에분말 투여군에서 현저히 감소된 것으로 나타났다. 혈중 유리 지방산 농도는 BFSP 투여에 의해서는 유일하게 감소되었다. 따라서 발효 누에분말은 알코올에 의한 혈중 총 콜레스테롤 농도를 감소시키는 효과가 강한 것으로 관찰되었다. Blood lipid levels are used as diagnostic indicators of cardiovascular diseases such as atherosclerosis, hypertension, heart disease, and hyperlipidemia. And new clinical guidelines have been established in the US and elsewhere. Recently, attempts have been actively made to reduce blood cholesterol levels as well as neutral lipid concentrations and increase HDL-cholesterol levels in natural plant resources. Serum HDL-cholesterol concentration is an indicator of anti-arteriosclerosis. Unlike other lipoproteins, serum HDL-cholesterol concentration separates and transports LDL-cholesterol deposited on the walls of blood vessels to the liver for energy use or to promote excretion in vitro. It is widely known as a beneficial cholesterol that can reduce the risk of causing cardiovascular disease. Increasing total cholesterol levels in blood is one of the common phenomena seen in alcoholic animals. In this experiment, the total cholesterol and free fatty acid concentrations were significantly increased in the alcohol-administered group compared to the normal group. However, the total lipid concentration in blood was significantly decreased in silkworm powder and fermented silkworm powder administration group compared to alcohol control. Blood free fatty acid concentrations were only reduced by BFSP administration. Therefore, fermented silkworm powder was observed to have a strong effect of reducing the total cholesterol concentration by alcohol.

알코올 투여 랫의 혈청 지질에서 SP, BFSP 및 AFSP의 효과 Effects of SP, BFSP and AFSP on Serum Lipids in Alcohol-administered Rats GroupGroup Total lipid
(mg/100 mL)
Total lipid
(mg / 100 mL)
Free fatty acid
(nmol/L)
Free fatty acid
(nmol / L)
Cholesterol
(mg/100 mL)
Cholesterol
(mg / 100 mL)
HDL
-cholsterol
(mg/100 mL)
HDL
-cholsterol
(mg / 100 mL)
NormalNormal 216.8±7.9NS 216.8 ± 7.9 NS 0.82±0.09a 0.82 ± 0.09 a 52.00±1.93a 52.00 ± 1.93 a 21.00±0.89NS 21.00 ± 0.89 NS ControlControl 249.0±21.7249.0 ± 21.7 1.12±0.12b 1.12 ± 0.12 b 64.67±1.38b 64.67 ± 1.38 b 21.33±0.7621.33 ± 0.76 SPSP 234.0±27.0234.0 ± 27.0 1.30±0.05b 1.30 ± 0.05 b 51.00±4.85a 51.00 ± 4.85 a 21.60±1.7621.60 ± 1.76 BFSPBFSP 211.0±24.4211.0 ± 24.4 0.87±0.06a 0.87 ± 0.06 a 50.33±4.59a 50.33 ± 4.59 a 21.00±1.3921.00 ± 1.39 AFSPAFSP 222.5±19.3222.5 ± 19.3 1.15±0.08b 1.15 ± 0.08 b 48.83±1.78a 48.83 ± 1.78 a 21.50±0.7221.50 ± 0.72

SP: Silkworm powder.SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder. AFSP: Aspergillus kawachii fermented silkworm powder.

Values with different letters are the significantly different statistically at p〈 0.05. (mean±S.E., n=6). Values with different letters are the significantly different statistically at p 〈0.05. (mean ± SE, n = 6).

NS: Not significant different.NS: Not significant different.

혈중 Blood ALTALT , , ASTAST , γ-, γ- GTPGTP  And LDHLDH 활성 변화 Active change

간 손상의 임상적 지표로 사용되고 있는 ALT, AST, γ-GTP 및 LDH 활성 측정을 통하여 간 기능 효능을 검증한 결과를 표 10에 나타내었다. 혈청 ALT 및 AST 활성은 간 손상으로 인한 간세포의 괴사와 간 조직의 파괴가 진행됨에 따라 transaminase가 혈중으로 유리되어 높은 활성을 나타내는 것으로 간 손상 지표의 중요한 단서가 된다. Table 10 shows the results of verifying liver function efficacy by measuring ALT, AST, γ-GTP, and LDH activity, which are used as clinical indicators of liver damage. Serum ALT and AST activity is an important clue of liver damage indices because transaminase is released into the blood as hepatic necrosis and hepatic tissue destruction progress due to liver injury.

본 실험에서 이전의 실험 결과[Cha JY, Heo JS, Cho YS. Effect of zinc-enriched yeast FF-10 strain on the alcoholic hepatotoxicity in alcohol feeding rats. Food Sci Biotechnol 17: 1207-1213 (2008); Cha JY, Kim HS, Kang SC, Cho YS. Alcoholic hepatotoxicity suppression in alcohol fed rats by glutathione-enriched yeast FF-8 strain. Food Sci Biotechnol 18: 1411-1416 (2009)]와 같이 AST, γ-GTP 활성은 알코올 투여군에서 유의적으로 증가하였으나, ALT 활성은 통계상의 유의적인 차이 없이 증가하는 경향을 보였다(표 10). 알코올 급여 대조군에서의 AST 및 γ-GTP 활성 증가는 BFSP 및 AFSP 투여에 의해 현저히 감소하는 것으로 나타났다(표 10). 이러한 효과는 AFSP 보다 BFSP 투여에 의해 간 보호 효과가 더욱 우수하다는 것을 암시하고 있다. Previous experiments in this experiment [Cha JY, Heo JS, Cho YS. Effect of zinc-enriched yeast FF-10 strain on the alcoholic hepatotoxicity in alcohol feeding rats. Food Sci Biotechnol 17: 1207-1213 (2008); Cha JY, Kim HS, Kang SC, Cho YS. Alcoholic hepatotoxicity suppression in alcohol fed rats by glutathione-enriched yeast FF-8 strain. Food Sci Biotechnol 18: 1411-1416 (2009)], AST, γ-GTP activity was significantly increased in the alcohol-administered group, but ALT activity tended to increase without statistically significant difference (Table 10). The increase in AST and γ-GTP activity in the alcohol-fed control group was found to be markedly decreased by BFSP and AFSP administration (Table 10). This effect suggests that liver protection effect is better by BFSP administration than AFSP.

글루타치온, S-아데노실메티오닌, 아미노산, 아연을 많이 함유한 효모를 급여한 실험에서 혈중 ALT, AST, γ-GTP 활성을 감소시켜 강력한 간 보호효과를 나타내는 실험들이 수행되었다[Cha et al., 2008; Cha et al., 2009; Izu H, Shobayashi M, Manabe Y, Goto K, Iefuji H. S-adenosylmethionine (SAM)-accumulating sake yeast suppresses acute alcohol-induced liver injury in mice. Biosci Biotechnol Biochem 70: 2982-2989 (2006)]. 또한 글루타치온 고함유 효모 S. cerevisiae를 급여한 사염화탄소 유발 간독성 모델에서도 ALT 및 AST 활성을 감소시키는 결과가 이전 실험에서도 보고된 바 있다[40]. Sugimura 및 Yamamoto의 문헌[Sugiyama Y, Yamamoto K. The protective effect of glutathione-enriched yeast extract on acetaminophen-induced liver damage in rats. J. Jpn. Soc. Nutr. Food 51: 189-193 (1998)]에서는 아세트아미노펜 유발 간 독성 실험에서 glutathione 고함유 효모의 투여에 의해 AST 및 ALT 활성이 농도 의존적으로 감소되었으나, glutathione 저함유 빵효모 투여에 의해서는 이러한 효과가 없었다고 하였다. 이러한 효과는 glutathione 고함유 효모의 투여에 의해 간 세포내 글루타치온 농도가 높아지면서 간독성 개선효과가 나타난 것으로 보여지며, 본 실험에서도 발효 누에분말 투여에 의해 간 세포내 글루타치온 농도가 높아지는 결과를 얻음으로서 이를 뒷받침 하고 있다(도 10). In experiments fed glutathione, S-adenosylmethionine, amino acids, and zinc-rich yeast, experiments showing strong hepatoprotective effects by reducing blood ALT, AST and γ-GTP activity were performed [Cha et al., 2008 ; Cha et al., 2009; Izu H, Shobayashi M, Manabe Y, Goto K, Iefuji H. S-adenosylmethionine (SAM) -accumulating sake yeast suppresses acute alcohol-induced liver injury in mice. Biosci Biotechnol Biochem 70: 2982-2989 (2006). In addition, carbon tetrachloride-induced hepatotoxicity model supplemented with glutathione-containing yeast S. cerevisiae has been reported to reduce ALT and AST activity in previous experiments [40]. Sugimura and Yamamoto, Sugiyama Y, Yamamoto K. The protective effect of glutathione-enriched yeast extract on acetaminophen-induced liver damage in rats. J. Jpn. Soc. Nutr. Food 51: 189-193 (1998)] showed that AST and ALT activities were reduced in a concentration-dependent manner by the administration of glutathione-containing yeast in acetaminophen-induced hepatotoxicity experiments. It was. This effect is shown to improve liver toxicity by increasing glutathione concentration in liver cells by administration of glutathione-containing yeast, and this study was also supported by the result that the intracellular glutathione concentration was increased by fermented silkworm powder administration. (FIG. 10).

간 기능적 임상지표로 사용되고 있는 혈중 LDH 및 ALP 활성도 알코올 섭취에 의해 증가됨으로서 알코올성 간 독성 유발과 깊은 관련성을 가진 것으로 보고되고 있다[Cha et al., 2008; Sugiyama et al., 1998]. 본 실험에서도 LDH 활성은 알코올 섭취군에서 증가 경향을 보였으나, 이러한 알코올에 의한 증가는 BFSP 및 AFSP 투여에 의해 유의적으로 감소하였고 누에분말 투여에 의해서는 감소하는 경향을 나타내었다(Table 표 10). 따라서 BFSP 투여는 알코올에 의해 증가된 ALT, γ-GTP 및 LDH 활성을 유의적으로 감소시킴으로서 알코올성 간 질환을 개선할 수 있는 건강기능식품 소재로 활용할 수 있는 것으로 사료되어진다.It has been reported that blood LDH and ALP activity, which is used as a clinical functional indicator of liver, is increased by alcohol intake and has a close correlation with the induction of alcoholic liver toxicity [Cha et al., 2008; Sugiyama et al., 1998]. In this experiment, LDH activity was also increased in the alcohol intake group, but this increase by alcohol was significantly decreased by BFSP and AFSP administration and decreased by silkworm powder administration (Table Table 10). . Therefore, BFSP can be used as a dietary supplement for improving alcoholic liver disease by significantly reducing ALT, γ-GTP, and LDH activity.

알코올 투여 랫에서 AST, ALT, γ-GTP 및 LDH 활성에서의 SP, BFSP 및 AFSP의 효과 Effect of SP, BFSP and AFSP on AST, ALT, γ-GTP and LDH Activity in Alcohol-administered Rats GroupGroup AST
(IU/L)
AST
(IU / L)
ALT
(IU/L)
ALT
(IU / L)
γ-GTP
(IU/L)
γ-GTP
(IU / L)
LDH
(IU/L)
LDH
(IU / L)
NormalNormal 123.17±5.80a 123.17 ± 5.80 a 36.50±1.93NS 36.50 ± 1.93 NS 0.28±0.05a 0.28 ± 0.05 a 3191±348a 3191 ± 348 a Alcohol Alcohol 149.00±7.75b 149.00 ± 7.75 b 42.33±3.6642.33 ± 3.66 0.60±0.09b 0.60 ± 0.09 b 4243±532b 4243 ± 532 b SPSP 118.97±6.16a 118.97 ± 6.16 a 38.00±5.0538.00 ± 5.05 0.38±0.07ab 0.38 ± 0.07 ab 3096±183a 3096 ± 183 a BFSPBFSP 103.33±9.58a 103.33 ± 9.58 a 34.00±2.0334.00 ± 2.03 0.30±0.05a 0.30 ± 0.05 a 1407±461c 1407 ± 461 c AFSPAFSP 111.67±12.86a 111.67 ± 12.86 a 40.00±3.3040.00 ± 3.30 0.65±0.30b 0.65 ± 0.30 b 2100±613c 2100 ± 613 c

SP: Silkworm powder.SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder. AFSP: Aspergillus kawachii fermented silkworm powder.

AST: aspartate aminotransferase, ALT: alanine aminotransferase, γ-GTP: γ-glutamyltranspeptidase, LDH: lactate dehydrogenase. AST: aspartate aminotransferase, ALT: alanine aminotransferase, γ-GTP: γ-glutamyltranspeptidase, LDH: lactate dehydrogenase.

Values with different letters are the significantly different statistically at p〈 0.05. (mean±S.E., n=6). Values with different letters are the significantly different statistically at p 〈0.05. (mean ± SE, n = 6).

NS: Not significant different.
NS: Not significant different.

혈중 단백질, 알부민 및 Blood proteins, albumin and 글로부린Globulin 농도 변화 Concentration change

혈중 총 단백질 농도는 만성 감염증, 만성 간장애, 간경변 등에서 증가하는데, 정상군에 비해 알코올 투여 모든 실험군에서 유의적으로 감소하였다(표 11). 알부민은 간에서 합성되는 혈액 단백질로 혈장 총 단백의 60% 정도를 차지할 정도로 중요하며, 중증 간질환에서 감소되는 중요 간 질환 기능지표로 이용되고 있다. 본 실험에서 알부민 농도는 정상군에 비해 알코올 투여 모든 실험군에서 유의적으로 감소하였다(표 11). 또한 혈중 글로불린 농도는 혈중에서 알부민을 제외한 혈청 단백질의 대부분을 차지하며, 감염증, 염증성질환 및 악성종양 등에서 증가하는데, 정상군에 비해 알코올 대조군 및 누에분말과 발효 누에분말 투여군에서 유의적인 감소 또는 감소경향을 보이는 결과를 얻었다(표 11). Serum total protein concentration increased in chronic infections, chronic liver disorders, cirrhosis, etc., but significantly decreased in all experimental groups treated with alcohol compared to the normal group (Table 11). Albumin is a blood protein synthesized in the liver, which is important to account for about 60% of the total plasma protein, and is used as an important liver disease indicator that is reduced in severe liver disease. In this experiment, albumin concentrations were significantly decreased in all groups treated with alcohol compared to the normal group (Table 11). In addition, blood globulin concentration accounts for most of serum proteins except albumin in blood, and increases in infectious diseases, inflammatory diseases and malignancies. Significant decrease or decrease in alcohol control group and silkworm powder and fermented silkworm powder administration group compared to normal group Results were obtained (Table 11).

알코올 투여 랫의 혈청에서 총 단백질, 알부민 및 글로불린의 농도에 대한 SP, BFSP 및 AFSP의 효과 Effects of SP, BFSP, and AFSP on Concentrations of Total Protein, Albumin, and Globulin in Serum of Alcoholic Rats GroupGroup Protein
(g/100 mL)
Protein
(g / 100 mL)
Albumin
(g/100 mL)
Albumin
(g / 100 mL)
Globulin
(g/100 mL)
Globulin
(g / 100 mL)
NormalNormal 6.02±0.13a 6.02 ± 0.13 a 2.83±0.09a 2.83 ± 0.09 a 3.18±0.05a 3.18 ± 0.05 a ControlControl 5.57±0.12b 5.57 ± 0.12 b 2.57±0.05b 2.57 ± 0.05 b 3.00±0.08ab 3.00 ± 0.08 ab SPSP 5.37±0.07b 5.37 ± 0.07 b 2.48±0.03b 2.48 ± 0.03 b 2.88±0.04b 2.88 ± 0.04 b BFSPBFSP 5.57±0.08b 5.57 ± 0.08 b 2.57±0.03b 2.57 ± 0.03 b 3.00±0.05ab 3.00 ± 0.05 ab KWSPKWSP 5.30±0.09b 5.30 ± 0.09 b 2.43±0.03b 2.43 ± 0.03 b 2.87±0.08b 2.87 ± 0.08 b

SP: Silkworm powder.SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder. AFSP: Aspergillus kawachii fermented silkworm powder.

Values with different letters are the significantly different statistically at p〈 0.05. (mean±S.E., n=6).
Values with different letters are the significantly different statistically at p 〈0.05. (mean ± SE, n = 6).

실시예Example 12. 혈중 알코올 및  12. Blood alcohol and 아세트알데하이드Acetaldehyde 농도 측정 Concentration measurement

혈청에 함유된 알코올 및 아세트알데하이드 농도는 시판용 R-Biopharm사의 UV-Test kit (Darmstadt, Germany)를 이용하여 측정하였다. 즉 혈중 알코올 농도는 NAD와 인산염 완충용액(pH 9.0) 3 mL 혼합액에 혈청 0.1 mL를 첨가한 후 약 3분간 반응하여 340 nm 흡광도를 측정하고, 혼합액에 ADH 0.05 mL를 첨가하여 20℃에서 10분간 항온하고 흡광도 340 nm에서 생성된 NADH의 농도에 따른 흡광도의 변화를 관찰하였다. 이때 혈액중의 알코올 농도(%)는 표준용액을 이용하여 계산하였다. Alcohol and acetaldehyde concentrations in serum were measured using a commercial UV-Test kit (Darmstadt, Germany) of R-Biopharm. In other words, the blood alcohol concentration was measured by adding 0.1 mL of serum to a 3 mL mixture of NAD and phosphate buffer solution (pH 9.0), reacting for about 3 minutes, measuring absorbance at 340 nm, and adding 0.05 mL of ADH to the mixture for 10 minutes at 20 ° C. The change in absorbance according to the concentration of NADH produced at room temperature and absorbance at 340 nm was observed. The alcohol concentration in blood (%) was calculated using a standard solution.

혈청 아세트알데하이드 농도는 NAD와 인산염 완충용액(pH 9.0) 3 mL 혼합액에 혈청 0.2 mL를 첨가한 후 약 3분간 반응하여 340 nm 흡광도를 측정하고, 혼합액에 ALDH 0.05 mL를 첨가하여 20?25℃에서 5분간 반응시켜 흡광도 340 nm에서 생성된 NADH의 농도에 따른 흡광도의 변화를 관찰하였다. 이때 혈액 중의 아세트알데하이드 농도는 표준용액을 이용하여 계산하였다.
Serum acetaldehyde concentration was measured by adding 0.2 mL of serum to a 3 mL mixture of NAD and phosphate buffer (pH 9.0), reacting for about 3 minutes, measuring absorbance at 340 nm, and adding 0.05 mL of ALDH to the mixture at After reacting for 5 minutes, the change in absorbance according to the concentration of NADH produced at absorbance 340 nm was observed. At this time, the acetaldehyde concentration in the blood was calculated using a standard solution.

알코올과 그 일차 대사산물인 아세트알데하이드는 음주 후 숙취를 경험하게 하는 주요 원인 물질로서 강력한 독성물질로서 생체 내 활성 아민류와 축합반응을 거쳐 간 손상의 주요 매개체로 작용하는 동시에 숙취에 영향을 미치는데, ALDH에 의해 대사되어 아세트산을 형성한 후 이산화탄소와 물로 가수분해되어 완전히 분해과정을 거치게 된다. 아세트알데하이드의 독성으로는 미토콘드리아 기능저해로 인한 간경변과 심장 및 뇌 기능 저해 등이 보고되어 있다. 일반적으로 알코올에 과일즙이나 향신료, 그리고 여러 한방 약재를 혼합하여 음용시 취기가 다르게 나타나는데, 이는 알코올에 함유된 여러 물질들에 의해 알코올 대사가 촉진되었다고 고려 해 볼 수 있다. 최근에 알코올을 투여한 동물실험에서 홍국 추출물이 알코올 대사를 촉진시킨다는 보고가 있으며, 홍삼을 홍국으로 발효시킨 발효홍삼에서도 숙취 해소 효과가 확인되었다. Alcohol and its primary metabolite, acetaldehyde, are the main causative agents of experiencing hangover after drinking. They are potent toxins that act as a major mediator of liver damage through condensation reactions with active amines in vivo. It is metabolized by ALDH to form acetic acid and then hydrolyzed with carbon dioxide and water to undergo a complete decomposition process. Toxicity of acetaldehyde has been reported to impair cirrhosis and heart and brain function due to mitochondrial impairment. In general, alcohol and fruit juices, spices, and various herbal medicines appear to have different odors when drinking, which can be considered that alcohol metabolism was promoted by various substances in alcohol. Recently, it has been reported that red ginseng extract promotes alcohol metabolism in animal experiments with alcohol. Also, fermented red ginseng fermented red ginseng was confirmed to have a hangover effect.

혈중 알코올 농도는 정상 대조군의 혈중에서는 검출되지 않았으나, 알코올을 투여한 모든 실험군에서는 혈중 알코올이 검출되었다(표 12). 혈중 알코올 농도는 정상군에 비해 알코올 대조군에서 유의적으로 증가하였고, 이러한 증가는 SP, BFSP, AFSP 투여군에서 동일한 농도로 유의적인 감소를 나타내었다. 혈중 아세트알데히드 농도는 정상군에 비해 알코올 대조군에서 유의적으로 증가하였으며, 이러한 알코올군에서의 증가는 누에분말 및 발효 누에분말 투여군에서 각각 정상군과 큰 차이없이 감소하는 경향을 보였다(표 12). 발효 누에분말은 혈중의 알코올 농도뿐만 아니라 아세트알데히드 농도도 감소시키는 효능을 가진 것으로 보여지는데, 이는 간 조직속의 알코올 대사 관련 효소 ADH 및 ALDH 활성 증가에 의한 것으로 나타났다. 이전의 연구에서 B. breve 균주로 발효시킨 두유 급여에 의해 ALDH 활성 증가로 아세트알데하이드 농도가 낮아지면서 알코올성 간독성을 개선시키는 것으로 보고된 바 있다(Kano M, Ishikawa F, Matsubara S, Kikuchi-Hayakawa H, Shimakawa Y. Soymilk products affect ethanol absorption and metabolism in rats during acute and chronic ethanol intake. J Nutr 132: 238-244 (2002)). 본 실험에서도 누에분말 또는 Aspergillus 발효 누에분말 보다는 Bacillus 발효 누에분말 투여에 의해 알코올성 간독성이 더욱 개선되는 것으로 나타나 특히 Bacillus 균주의 누에 발효에 의해 간 독성 개선 효능이 증가되는 것으로 나타났다. Blood alcohol concentration was not detected in blood of the normal control group, but blood alcohol was detected in all experimental groups to which alcohol was administered (Table 12). The blood alcohol concentration was significantly increased in the alcohol control group compared to the normal group, and this increase was significantly decreased at the same concentration in the SP, BFSP, and AFSP groups. Plasma acetaldehyde concentration was significantly increased in the alcohol control group compared to the normal group, and the increase in the alcohol group tended to decrease in the silkworm powder and fermented silkworm powder administration groups without significant difference, respectively (Table 12). Fermented silkworm powder has been shown to have an effect of decreasing acetaldehyde concentration as well as blood alcohol concentration, which was shown to be due to the increase in the activity of alcohol-related enzymes ADH and ALDH in liver tissue. Previous studies have reported that soymilk supplemented with B. breve strains improves alcoholic hepatotoxicity due to a decrease in acetaldehyde concentration due to increased ALDH activity (Kano M, Ishikawa F, Matsubara S, Kikuchi-Hayakawa H, Shimakawa Y. Soymilk products affect ethanol absorption and metabolism in rats during acute and chronic ethanol intake.J Nutr 132: 238-244 (2002)). Silkworm powder or Aspergillus in this experiment Bacillus rather than fermented silkworm powder Fermented silkworm powder was administered to improve alcoholic hepatotoxicity, especially Bacillus The silkworm fermentation of the strain was shown to increase the efficacy of improving liver toxicity.

알코올 투여 랫에서 알코올 및 아세트알데하이드의 혈청 내 농도에서의 SP, BFSP 및 AFSP의 효과 Effects of SP, BFSP, and AFSP on Serum Concentrations of Alcohol and Acetaldehyde in Alcohol Administration Rats GroupGroup Serum alcohol
(mg/100 mL)
Serum alcohol
(mg / 100 mL)
Serum acetaldehyde
(mg/100 mL)
Serum acetaldehyde
(mg / 100 mL)
NormalNormal 0.000±0.00a 0.000 ± 0.00 a 2.63±0.44a 2.63 ± 0.44 a ControlControl 0.055±0.01b 0.055 ± 0.01 b 5.68±0.74b 5.68 ± 0.74 b SPSP 0.037±0.00c 0.037 ± 0.00 c 3.03±0.56a 3.03 ± 0.56 a BFSPBFSP 0.037±0.00c 0.037 ± 0.00 c 2.76±0.40a 2.76 ± 0.40 a AFSPAFSP 0.037±0.00c 0.037 ± 0.00 c 2.84±0.39a 2.84 ± 0.39 a

SP: Silkworm powder.SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder. AFSP: Aspergillus kawachii fermented silkworm powder.

Values with different letters are the significantly different statistically at p〈 0.05. (mean±S.E., n=6).
Values with different letters are the significantly different statistically at p 〈0.05. (mean ± SE, n = 6).

실시예Example 13.  13. ADHADH  And ALDHALDH 효소 활성 측정 Enzyme Activity Measurement

간 조직 유래의 ADH 활성 측정은 Bergmeyer's의 방법(Bergmeyer HU. Methods of Enzymatic Analysis. Academic Press, New York, pp. 28, (1974))을 약간 변형하여 흡광도 340 nm에서 NADH의 생성속도를 측정하였다. 즉, 반응액 조성은 증류수 1.5 ml, 1.0 M Tris-HCl 완충용액 (pH 8.8) 0.75 ml, 20 mM NAD+ 0.3 ml, ethanol 0.3 ml, 효소원 0.15 ml를 cuvette에 넣고 총 3 ml이 되도록 조절하여 30℃에서 5분간 전배양시킨 후 5분 동안 340 nm에서 흡광도의 변화를 측정하였다. Determination of ADH activity from liver tissue was performed by Bergmeyer's method (Bergmeyer HU.Methods of Enzymatic Analysis. Academic Press, New York, pp. 28, (1974)) to measure the rate of NADH production at absorbance 340 nm. In other words, the composition of the reaction solution was adjusted to 1.5 ml of distilled water, 0.75 ml of 1.0 M Tris-HCl buffer (pH 8.8), 20 mM NAD + 0.3 ml, 0.3 ml of ethanol, and 0.15 ml of enzyme source in a cuvette to a total of 3 ml. After 5 minutes of preincubation at 30 ° C., the change in absorbance at 340 nm was measured for 5 minutes.

간 조직 유래의 ALDH 활성 측정은 Koivula 등의 방법(Koivula T, Koivusalo M, Lindros KO. Liver aldehyde and alcohol dehydrogenase activities in rat strains genetically selected for their ethanol preference. Biochem Pharmacol 24: 1807-1811 (1975))을 약간 변형하여 아세트알데하이드에서 아세트산염을 생성하는 효소로 NAD로부터 NADH를 생성하는 원리를 이용하였다. 즉 반응액의 조성은 증류수 2.2 ml, 1.0 M Tris-HCl 완충용액 (pH 8.0) 0.3 ml, 20 mM NAD+ 0.1 ml, 0.1 M 아세트알데하이드 0.1 ml, 3.0 M KCl 0.1 mL, 0.33 M 2-머캅토에탄올 0.1 ml, 효소원 0.1 ml를 총 3 ml이 되도록 조절하여 cuvette에 넣고 30℃에서 5분간 전배양한 후, 5분 동안 340 nm에서 흡광도의 변화를 측정하였다.
Determination of ALDH activity from liver tissue was performed using Koivula et al. (Koivula T, Koivusalo M, Lindros KO.Liver aldehyde and alcohol dehydrogenase activities in rat strains genetically selected for their ethanol preference.Biochem Pharmacol 24: 1807-1811 (1975)). A slight modification was used to generate NADH from NAD as an enzyme that produces acetate in acetaldehyde. That is, the composition of the reaction solution was distilled water 2.2 ml, 1.0 M Tris-HCl buffer (pH 8.0) 0.3 ml, 20 mM NAD + 0.1 ml, 0.1 M acetaldehyde 0.1 ml, 3.0 M KCl 0.1 mL, 0.33 M 2-mercapto 0.1 ml of ethanol and 0.1 ml of enzyme source were adjusted to a total of 3 ml, placed in a cuvette, preincubated at 30 ° C. for 5 minutes, and then the change in absorbance at 340 nm was measured for 5 minutes.

웨스턴 블롯 분석은 알코올성 간 질환에서 ADH 및 ALDH 효소 단백질의 발현에 대한 발효 누에분말 투여의 효과를 보기 위하여 실험하였다. 정량된 ADH 및 ALDH 효소 단백질을 10% SDS-폴리아크릴아미드 겔의 각 웰 당 각각 20 및 100 μg을 전보와 같이 전기영동 하였다(Koivisto T, Eriksson CJ. Hepatic aldehyde and alcohol dehydrogenases in alcohol-preferring and alcohol-avoiding rat lines. Biochem Pharmacol 48: 1551-8155 (1994)). 분리된 단백질은 300/240 mA/cm2 조건으로 4℃에서 16시간 동안 니트로셀룰로오스 맴브레인으로 이동시켰다. 이 맴브레인을을 차단제인 10% 무지방 우유가 함유된 TBST 완충용액 (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20)로 1시간 동안 실온에서 비-특이 결합 단백질을 차단한 후, TBST 완충용액로 5분 동안 세척하였다. 다시 차단시킨 맴브레인을 실온에서 1시간 동안 일차 및 이차 항체로 반응시킨 후 TBST 완충용액로 세척하였다. 세척된 맴브레인에 토끼 ADH 또는 ALDH1A1 접합 HRP 항체(1:5,000로 희석됨, Abcam, England)로 4℃에서 overnight하였다. 다시 TBST 완충용액로 세척시킨 후 goat anti-mouse IgG-HRP (diluted 1:2,000, DB USA)로 incuvation시켰다. Nitrocellulose membrane 상의 단백질은 SuperSignal West Pico Chemiluminescent Substrate의 image analysis 방법으로 검출하였다. 각각 반응된 ADH 또는 ALDH 단백질 밴드의 상대적 밀도는 densitometer (Lumi-Imager F1, Roche, Switzerland)로 정량하였다.
Western blot analysis was conducted to see the effect of fermented silkworm powder administration on the expression of ADH and ALDH enzyme proteins in alcoholic liver disease. Quantified ADH and ALDH enzyme proteins were electrophoresed 20 and 100 μg for each well of 10% SDS-polyacrylamide gel as well (Koivisto T, Eriksson CJ. Hepatic aldehyde and alcohol dehydrogenases in alcohol-preferring and alcohol). -avoiding rat lines.Biochem Pharmacol 48: 1551-8155 (1994)). The isolated protein was transferred to nitrocellulose membrane for 16 hours at 4 ° C. under 300/240 mA / cm 2 condition. The membrane was blocked with non-specific binding protein for 1 hour at room temperature with TBST buffer solution (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20) containing 10% nonfat milk as a blocker. Wash for 5 minutes with buffer. The blocked membrane was reacted with primary and secondary antibodies for 1 hour at room temperature and then washed with TBST buffer. The washed membranes were overnight at 4 ° C. with rabbit ADH or ALDH1A1 conjugated HRP antibody (diluted at 1: 5,000, Abcam, England). Again washed with TBST buffer and incuvation with goat anti-mouse IgG-HRP (diluted 1: 2,000, DB USA). Proteins on the nitrocellulose membrane were detected by image analysis of SuperSignal West Pico Chemiluminescent Substrate. Relative densities of the reacted ADH or ALDH protein bands, respectively, were quantified by densitometer (Lumi-Imager F1, Roche, Switzerland).

체내로 흡수된 알코올은 대부분 위장과 소장에서 흡수되어 간으로 이동된 후 대사과정을 거쳐 분해되는데, 만성적인 알코올 섭취는 영양소의 흡수장애와 간 기능 손상을 초래할 수 있다. 간에서 대사되는 알코올 분해는 먼저 alcohol dehydrogenase(ADH)와 cytochrom P-450에 의해 알코올이 아세트알데하이드로 전환되는데, 이때 만성적 알코올 섭취로 인한 중독시에는 MEOS(microsomal ethanol oxidizing system)에 의해 아세트알데하이드가 증가하게 된다. 주로 만성적 알코올 섭취 후 간 조직에 축적되는 아세트알데하이드는 ADH 및 ALDH 활성에 의해 영향을 받게 된다. 이들 ADH 및 ALDH 효소 활성은 체내 알코올 대사에서 필수적인 역할을 하기 때문에 천연 생리활성 성분에 의해 활성증가를 기대할 수 있는 가능성이 제기되고 있다. 실험동물에서 만성적 알코올 투여시 ADH 활성은 증가하고 ALDH 활성은 오히려 감소하는데, 이는 알코올의 장기간 섭취에 의해 생체조직 내에 아세트알데하이드의 축적현상이 초래될 가능성을 시사해주고 있다. 본 동물 실험에서 간 조직 중의 ADH 활성은 정상군에 비해 알코올 대조군에서 약간의 증가 경향을 보였으나, SP, BFSP, AFSP 투여에 의해서는 유의적으로 활성이 증가하였다(도 7). ALDH 활성은 정상군과 알코올 대조군 사이에서는 차이가 없었으나, SP, BFSP, AFSP 투여군에서 모두 활성이 증가된 것으로 나타났다(도 7). 이러한 결과는 발효 누에분말이 알코올 투여 흰쥐의 혈중 알코올 및 아세트알데하이드 농도를 효과적으로 감소시키는 것은 간 조직 중의 알코올 대사 효소 ADH 및 ALDH 활성을 촉진시키기 때문인 것으로 나타났다. 특히 BFSP 투여군에서 ADH 및 ALDH 활성 모두 가장 높은 것으로 나타나 이러한 효소 활성의 증가로 체내 알코올이 기질로 사용됨으로서 혈중의 알코올 및 아세트알데하이드 농도가 감소한 것으로 보여진다.
Most of the alcohol absorbed into the body is absorbed by the stomach and small intestine, moved to the liver and then metabolized and broken down. Chronic alcohol intake can cause nutrient absorption and impair liver function. Alcohol metabolism in the liver is first converted to acetaldehyde by alcohol dehydrogenase (ADH) and cytochrom P-450, which increases acetaldehyde by MEOS (microsomal ethanol oxidizing system) during chronic alcohol intoxication. Done. Acetaldehyde, which accumulates in liver tissue mainly after chronic alcohol intake, is affected by ADH and ALDH activity. Since these ADH and ALDH enzyme activities play an essential role in the body's alcohol metabolism, the possibility of increasing the activity by natural bioactive components has been raised. Chronic alcohol administration in experimental animals increases ADH activity and decreases ALDH activity, suggesting the possibility of acetaldehyde accumulation in biological tissues by prolonged ingestion of alcohol. In the animal experiments, ADH activity in liver tissue showed a tendency to increase slightly in the alcohol control group compared to the normal group, but was significantly increased by the administration of SP, BFSP, AFSP (Fig. 7). ALDH activity was not different between the normal group and the alcohol control group, but the SP, BFSP, AFSP administration group was found to increase the activity (Fig. 7). These results show that fermented silkworm powder effectively decreases the blood alcohol and acetaldehyde concentrations in alcohol-administered rats because it promotes alcohol metabolizing enzymes ADH and ALDH activity in liver tissue. In particular, the ADH and ALDH activity in the BFSP-administered group was the highest, indicating that the alcohol and acetaldehyde concentration in the blood was reduced by using the alcohol in the body as a substrate.

ADH 및 ALDH 단백질 발현량은 도 8에서 보여주고 있다. BFSP 투여군에서 가장 높은 ADH 발현량을 보이고 있으며, AFSP 투여군에서도 비교적 높은 ADH 발현량을 보이고 있다. 이러한 결과는 ADH 효소 활성과 단백질 발현량에서 높은 정의 상관관계를 보여주는 것이다. 그러나 BFSP 투여군에서는 다른 알코올 대사관련 효소인 ALDH 발현량에는 큰 영향을 미치지 않는 것으로 나타나 효소활성과 단백질 발현량에서 반드시 일치하지 않는다는 것을 시사해준다. 이전의 결과에서와 같이 ADH 효소 활성과 단백질 발현량은 알코올이나 다른 식이성분에 의해 크게 변화되는 영향을 받는 것으로 보고되고 있으나, ALDH 효소의 경우는 알코올이나 다른 식이성분에 의해 크게 영향을 받지 않고 약간씩 변화되는 것으로 보고되었. 따라서 알코올 급여 흰쥐에서 BFSP 동시 투여는 알코올 대사에 관련된 ADH 효소 활성과 단백질 발현량에서는 누에분말 또는 AFSP 보다 많은 영향을 미친 것으로 나타났으나, ALDH 단백질 발현량에 대해서는 큰 영향을 미치지 못하는 것으로 나타났다.
ADH and ALDH protein expression levels are shown in FIG. 8. BFSP-administered group showed the highest ADH expression and AFSP-administered group showed relatively high ADH expression. These results show a high positive correlation in ADH enzyme activity and protein expression. However, the BFSP-administered group did not appear to have a significant effect on the ALDH expression level, which is another alcohol metabolism-related enzyme, suggesting that the BFSP did not necessarily match the enzyme activity and protein expression level. As in the previous results, it is reported that ADH enzyme activity and protein expression are greatly affected by alcohol and other dietary components. However, ALDH enzyme is slightly unaffected by alcohol and other dietary components. Reported to change gradually. Therefore, simultaneous administration of BFSP in alcohol-fed rats had more effect on ADH enzyme activity and protein expression related to alcohol metabolism than silkworm powder or AFSP, but not on ALDH protein expression.

아연(Zinc)은 필수 미량성분으로 생리학적으로나 생물학적으로 중요한 역할을 수행하는 식이성분으로 인식되고 있고, 알코올성 간 독성에 대해서도 개선효과가 있는 것으로 알려져 있다. 아연은 많은 효소에 결합된 미네랄 성분으로 ADH 효소도 아연에 대해 직접적인 의존성을 가진 것으로 알려져 있다. 이전의 실험에서 알코올 투여 흰쥐에 ADH 효소의 촉매 원소로서의 아연을 고함유한 효모를 투여한 결과 ADH 효소 활성이 촉진되는 결과를 얻은 봐 있다(Cha et al., 2008). 본 실험에서 이러한 이유 때문에 간 조직 중의 아연 농도를 측정한 결과, BFSP 투여군에서 통계상의 유의적인 차이는 없었지만 가장 높은 아연 농도를 나타내어 ADH 효소 활성과 높은 상관관계를 보였다(도 9).
Zinc is recognized as a dietary ingredient that plays an important physiological and biological role as an essential trace ingredient, and is known to have an effect on improvement of alcoholic liver toxicity. Zinc is a mineral component bound to many enzymes, and ADH enzymes are known to have a direct dependency on zinc. In a previous experiment, the administration of zinc-rich yeast as a catalytic element of ADH enzymes in alcohol-administered rats resulted in the promotion of ADH enzyme activity (Cha et al., 2008). For this reason, the concentration of zinc in the liver tissues was measured for this reason, but there was no statistically significant difference in the BFSP-administered group, indicating the highest zinc concentration, which was highly correlated with ADH enzyme activity (FIG. 9).

알코올 대사에 관련한 ADH 및 ALDH 활성에 영향을 미치는 요인으로 미네랄 성분 이외도 아스파라긴, 아스파르트산, 아르기닌 등의 아미노산 성분도 시사되어 있다. 숙취해소 음료의 주요 소재로 사용되어지고 있는 매실즙과 그 주요 구성 아미노산인 아스파트산을 첨가하였을 경우 ADH 및 ALDH 활성이 많이 증가하였다(Hwang JY, Ham JW, Nam SH. Effect of Maesil (Prunus mume) juice on the alcohol metabolizing enzyme avtivities. Korean J Food Sci Technol 36: 329-332 (2004)). 이 실험에서도 매실즙은 ADH 활성을 증가시켜 알코올 분해를 촉진시킬 뿐 만 아니라 ALDH 활성 또한 증가시킴으로써 알코올 분해로 생성되는 아세트알데하이드의 분해를 촉진시키는 결과를 얻었으며, 매실즙의 주요 구성 아미노산인 아스파트산에 의해서도 비슷한 좋은 효과를 얻었다고 하였다. 특히 아미노산 중 아스파트산와 아르기닌은 NAD의 재생을 촉진시킴으로써 ADH 활성을 높여 알코올 분해를 촉진시켜 알코올의 독성을 낮추고, 아스파라긴은 아세트알데하이드와 반응하여 부가물을 생성하면서 아세트알데하이드의 농도를 낮추어 생물학적 독성을 약화시키는 것으로 시사되어 있다. 본 실험에서도 누에분말 또는 발효 누에분말에 이들 아미노산을 많이 함유하고 있어서 알코올성 간독성의 개선효과를 가질 가능성이 있으며, 특히 누에분말에 없는 황함유 아미노산이면서 혈중 알코올 농도를 낮추어 지방간 및 간독성 개선작용과 산화스트레스 억제작용을 가진 타우린, 시스테인, 메티오닌을 발효 누에분말에 많이 함유하고 있어서 이러한 가능성이 더욱 높아 보인다(표 13).
In addition to the mineral components, amino acid components such as asparagine, aspartic acid and arginine are also suggested as factors influencing ADH and ALDH activity related to alcohol metabolism. Addition of plum juice, a major component of hangover, and aspartic acid, its major constituent amino acids, increased ADH and ALDH activities (Hwang JY, Ham JW, Nam SH.Effect of Maesil ( Prunus mume) ) juice on the alcohol metabolizing enzyme avtivities.Korean J Food Sci Technol 36: 329-332 (2004)). In this experiment, plum juice promoted the decomposition of acetaldehyde produced by alcohol decomposition by increasing ADH activity and promoting alcohol degradation as well as ALDH activity. Aspart, the major constituent amino acid of plum juice Similar good effects were obtained with acid. In particular, aspartic acid and arginine among the amino acids promote the regeneration of NAD, which promotes alcohol degradation by promoting ADH activity, thereby lowering the toxicity of alcohol, and asparagine reacting with acetaldehyde to produce adducts while lowering the concentration of acetaldehyde, thereby reducing biological toxicity. It is suggested to weaken. In this experiment, the silkworm powder or fermented silkworm powder contains many of these amino acids, which may have the effect of improving alcoholic hepatotoxicity. This possibility appears to be higher because fermented silkworm powder contains taurine, cysteine and methionine which have inhibitory activity (Table 13).

SP, BFSP 및 AFSP에서의 유리 아미노산 함량 (ppm)Free Amino Acid Content (ppm) in SP, BFSP and AFSP Amino acidsAmino acids SPSP BSSPBSSP AKSPAKSP PhosphoserinePhosphoserine 183183 9696 8080 TaurineTaurine -- 7474 125125 PhosphoethanolaminePhosphoethanolamine 579579 -- -- UreaUrea -- 66 55 L-Aspartic acidL-Aspartic acid 477477 522522 237237 L-ThreonineL-Threonine 19881988 272272 768768 L-SerineL-Serine 20032003 970970 496496 AsparagineAsparagine 16441644 -- -- L-Glutamic acidL-Glutamic acid 62946294 26422642 30563056 L-α-Aminoadipic acidL-α-Aminoadipic acid -- 105105 5757 L-ProlineL-Proline 774774 608608 380380 L-GlycineL-Glycine 19041904 583583 426426 L-AlanineL-Alanine 66026602 18231823 17511751 L-CitrullineL-Citrulline -- 334334 132132 L-α-Aminobutyric acidL-α-Aminobutyric acid 4242 -- -- L-ValineL-Valine 29942994 21742174 13141314 L-CystineL-Cystine -- 7474 3737 L-MethionineL-Methionine -- 209209 1111 L-IsoleucineL-Isoleucine 23422342 15491549 10891089 L-LeucineL-Leucine 34073407 23682368 14211421 L-TyrosineL-Tyrosine 22712271 14771477 925925 L-PhenylalanineL-Phenylalanine 27242724 21632163 13921392 β-Alanineβ-Alanine -- -- -- DL-β-Aminoisobutyric acidDL-β-Aminoisobutyric acid -- -- -- GABA(γ-Aminobutyric acid)Γ-Aminobutyric acid (GABA) 4040 110110 8484 L-OrinithineL-Orinithine 218218 670670 22692269 L-LysineL-Lysine 10711071 337337 4646 1-Methyl-L-Histidine1-Methyl-L-Histidine -- 10491049 293293 L-HistidineL-Histidine 11021102 277277 227227 3-Methyl-L-Histidine3-Methyl-L-Histidine -- -- -- L-CarnosineL-Carnosine -- -- -- L-ArginineL-Arginine 31903190 7171 7373 TotalTotal 41,80941,809 20,44720,447 16,60616,606

-: Not detected.
-: Not detected.

실시예Example 15. 과산화지질 및 항산화 관련 물질 농도 변화 15. Changes in the concentration of lipid peroxides and antioxidants

과산화지질 측정Lipid peroxide measurement

분획한 균질액과 마이크로솜 생체막의 과산화지질 함량은 전보의 방법에 준하여 정량하였다(Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95: 351-358 (1979)). 즉, 단백질 량으로 1 ㎎을 함유한 분획 용액 1 ml에 각각 TBA(thiobarbituric acid) 시약 2 ml을 가하여 잘 혼합하고, 끓는 물에서 30분간 반응시킨 후 실온에서 방냉하여 3,000 rpm으로 10분간 원심분리 한 상등액을 535 nm에서 흡광도를 측정하였다. 과산화지질 함량은 말론디알데하이드를 nmol/g으로 나타내었다.
Lipid peroxide content of the homogenized and microsomal biofilm fractions was quantified according to the Telegram method (Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.Anal Biochem 95: 351-358 ( 1979)). In other words, 2 ml of TBA (thiobarbituric acid) reagent was added to 1 ml of a fraction solution containing 1 mg of protein, and the mixture was mixed well, and reacted in boiling water for 30 minutes, cooled at room temperature, and centrifuged at 3,000 rpm for 10 minutes. The supernatant was measured for absorbance at 535 nm. Lipid peroxide content was expressed in nmol / g of malondialdehyde.

미네랄 함량 측정Mineral content measurement

간조직의 미네랄 함량은 A.O.A.C. 분석 방법에 준하여 측정하였다(A.O.A.C. Official methods of analysis. 12th ed., Association of official analytical chemists. Washington, D.C., U.S.A. (1975)). 즉, 간 조직 1 g을 각 550℃ 회화로에서 3시간 회화시킨 후 6 N HCl에 용해시켜 완전히 산분해시켜 수욕(water bath)상에서 산을 완전히 제거하고, 여기에 3 N HCl를 가하여 Whatman No. 4 여과지로 여과하여 원소 종류에 따라 각각 일정비율로 희석하여 원자흡광 분광광도계(AAnalyst 300, Perkin Elmer, Norwalk CT, USA)를 이용하여 측정하였다.
The mineral content of liver tissue was determined according to the AOAC analysis method (AOAC Official methods of analysis. 12th ed., Association of official analytical chemists. Washington, DC, USA (1975)). That is, 1 g of liver tissue was incubated for 3 hours in each 550 ° C. incineration furnace, dissolved in 6 N HCl, and completely acid-decomposed to completely remove the acid in a water bath, followed by addition of 3 N HCl to Whatman No. The filter was filtered through 4 filter papers and diluted at a constant ratio according to the type of element, and measured using an atomic absorption spectrophotometer (AAnalyst 300, Perkin Elmer, Norwalk CT, USA).

글루타치온Glutathione 함량 측정 Content measurement

간조직의 글루타치온 함량은 간 균질액 분획 0.2 ml에 3차 증류수 0.3 ml과 0.4% 술포살리실산 0.5 ml를 가하여 혼합하고 원심분리시킨 뒤 상등액 0.3 ml에 DTNB(5,5'-dithiobis(2-nitrobenzoic acid)) 발색시약을 첨가하여 412 nm 흡광도에서 측정하여 글루타치온의 표준 검량 곡선에 의해 함량을 산출하였으며 간 조직 g당 mg으로 표시하였다(Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 61: 882-888 (1963)).
The glutathione content of liver tissue was mixed by adding 0.2 ml of tertiary distilled water and 0.5 ml of 0.4% sulfosalicylic acid to 0.2 ml of liver homogenate fraction, centrifuging, and then adding DTNB (5,5'-dithiobis (2-nitrobenzoic acid) Content was calculated by the standard calibration curve of glutathione, measured at 412 nm absorbance with the addition of a coloring reagent and expressed in mg per gram of liver tissue (Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione). J Lab Clin Med 61: 882-888 (1963).

초기 급성 간독성의 정도를 나타내는 지표인 ALT 및 AST 활성과 같이 간 조직 중의 과산화지질 농도도 간 독성의 지표로 사용되고 있다. 간 조직에서 산화스트레스의 측정 지표로 사용되고 있는 과산화지질 농도는 만성적인 알코올 섭취와도 밀접한 관련성을 가지는 것으로 알려져 있다. 만성적인 알코올 섭취는 조직 중의 과산화지질 및 자유 라디칼의 과잉 생산과 체내 항산화 물질인 글루타치온 농도의 감소를 가져 오는 것으로 알려져 있다. 이러한 결과는 알코올 섭취 후 간 조직의 균질액, 미토콘드리아 및 마이크로솜 분획에서 과산화지질 농도가 증가한 본 발명의 실험에서도 확인되었다(도 11). Like ALT and AST activity, which are indicators of early acute hepatotoxicity, lipid peroxide concentrations in liver tissues are also used as indicators of hepatotoxicity. Lipid peroxide concentration, a measure of oxidative stress in liver tissue, is known to be closely associated with chronic alcohol intake. Chronic alcohol intake is known to result in excess production of lipid peroxide and free radicals in tissues and a decrease in the concentration of glutathione, an antioxidant in the body. This result was also confirmed in the experiment of the present invention in which the lipid peroxide concentration was increased in the homogenate, mitochondria and microsomal fractions of liver tissue after alcohol intake (FIG. 11).

알코올 섭취 흰쥐에 BFSP 투여로 간 조직의 분획들에서 과산화지질 농도가 현저히 감소하였고, AFSP 투여에 의해서는 미토콘드리아 및 마이크로솜 분획에서는 유의적으로 감소하였으나 균질액 분획에서는 감소하는 경향을 보였으며, 이러한 결과는 발효 누에분말에 간 조직에서 항산화 효과가 있다는 것을 의미하는 것이다. 혈중 과산화지질 농도 역시 알코올 섭취에 의해 현저히 증가하였는데, 발효 누에분말 투여에 의해 유의적으로 감소하는 것으로 나타났다(도 11).
Lipid peroxide levels were significantly decreased in liver tissue fractions by administration of BFSP in alcohol-ingested rats, and significantly decreased in mitochondrial and microsomal fractions by AFSP administration, but decreased in homogenate fractions. Means that fermented silkworm powder has antioxidant effect in liver tissue. Serum lipid peroxide concentration was also significantly increased by alcohol intake, significantly decreased by fermented silkworm powder administration (FIG. 11).

필수 미량원소인 철은 지방산화를 촉진시키는 물질로 알려져 있으며, 체내 H2O2를 제거하는 카탈라아제의 구성 성분으로 체내의 비타민 C의 함량과 H2O2의 농도 차에 의해서 과산화지질 반응에 영향을 미침으로서 생체 내 과산화지질 반응을 조사하는데 있어서 비헴철 함량 측정은 중요한 요인으로 시사되어 있다. 간 조직 중의 비헴철 함량은 정상군에 비해 알코올 투여군에서 유의적으로 증가하였으나 BFSP 투여군에서는 유의적으로 감소하였다(도 9). 한편, 생체 내 항산화 물질로 잘 알려져 있는 간 조직 중의 아연 농도(Goel A, Dani V, Dhawan DK. Protective effects of zinc on lipid peroxidation, antioxidant enzymes and hepatic histoarchitecture in chlorpyrifos-induced toxicity. Chem Biol Interact 156: 131-140 (2005))는 각 실험 군 간에 통계상의 유의적인 차이는 없었지만 BFSP 투여군에서 약간 높게 나왔다(도 9). Iron, an essential trace element, is known as a substance that promotes fatty acid formation. It is a component of catalase that removes H 2 O 2 from the body and affects lipid peroxide reaction by the difference in the concentration of vitamin C in the body and the concentration of H 2 O 2 . In this study, the measurement of non-heme iron content is suggested as an important factor in the investigation of lipid peroxide reaction in vivo. The heme iron content in the liver tissue was significantly increased in the alcohol group compared to the normal group, but significantly decreased in the BFSP group (Fig. 9). Meanwhile, zinc concentrations in liver tissues (Goel A, Dani V, Dhawan DK.Protective effects of zinc on lipid peroxidation, antioxidant enzymes and hepatic histoarchitecture in chlorpyrifos-induced toxicity.Chem Biol Interact 156: 131) -140 (2005)) was slightly higher in the BFSP-administered group, although there was no statistically significant difference between the experimental groups (FIG. 9).

간은 알코올 대사에서 중심적인 역할을 수행하고, 유리기 생산에 의해 조직 손상을 일으키는 동시에 세포내 항산화 방어 시스템이 작동함으로서 이를 경감시키는 작용도 함께 일어나는 조직이다. 간 조직에서 글루타치온은 L-글루타메이트, L-시스테인및 글리신으로 구성된 비단백성 thiol tripeptide의 항산화 물질로 최근의 연구에서 동물 간 조직에서 독성제거 반응을 통한 항산화 방어계에서 중요한 역할을 하는 것으로 잘 알려져 있다. 조직 내 글루타치온 수준은 지질과산화 농도, 활성 유리기 및 다른 산화기의 독성 작용으로부터 세포를 방어하는 항산화 시스템과 밀접하게 관련되어 있다. 흰쥐에서 알코올 섭취는 정상군에 비해 간 조직 및 혈중 글루타치온 농도를 낮춘다(도 10). 그러나 알코올 섭취에 의한 간 조직 및 혈중 글루타치온 농도의 감소는 BFSP 투여에 의해 정상군 수준으로 회복되는 것으로 나타났다(도 10). 이전의 연구에서도 글루타치온 고함유 효모 투여가 사염화탄소 및 알코올 투여로 인한 간 손상을 효과적으로 막는 것으로 보고한 봐 있다[Cha et al., 2009; Shon MH, Cha JY, Lee CH, Park SH, Cho YS. Protective effect of administrated glutathione-enriched Saccharomyces cerevisiae FF-8 against carbon tetrachloride (CCl4)-induced hepatotoxicity and oxidative stress in rats. Food Sci Biochenol 16: 967-974 (2007)]. 알코올 섭취 흰쥐에 BFSP 투여로 인한 과산화지질 농도의 감소는 항산화 활성의 증가와 밀접한 관련성을 가지는 것으로 보였다. 비발효 누에 분말에 의한 간 보호 효과도 역시 아미노산과 같은 그의 구성 성분에 기인한 것으로 여겨진다. 간 조직에서 글루타치온 대사의 전구체로서 메티오닌 또는 시스테인은 내인성 글루타치온 합성에 중요한 역할을 수행하는 것으로 최근 보고되었으며[Peters TJ. Ethanol metabolism. Bri Med Bull 38: 17-20 (1982); Stipanuk MH, Coloso RM, Garcia RAG, Banks MF. Cysteine concentration regulates cysteine metabolism to glutathione, sulfate and taurine in rat hepatocytes. J Nutr 122: 420-427 (1992)], 시스테인 및 시스테인 함유 구성성분의 투여에 의해 간 및 신장 조직에 있어서 글루타치온 농도를 증가시키는 것으로 보고되었다[Hsu CC, Hiang CN, Hung YC, Yin MC. Five cysteine-containing compounds have antioxidative activity in Balb/cA mice. J Nutr 134: 149-152 (2004)].
The liver plays a central role in alcohol metabolism and causes tissue damage by the production of free radicals, and at the same time mitigates by acting as an intracellular antioxidant defense system. Glutathione in liver tissues is an antioxidant of nonprotein thiol tripeptide consisting of L-glutamate, L-cysteine and glycine and is known to play an important role in the antioxidant defense system through detoxification reactions in animal liver tissues in recent studies. Glutathione levels in tissues are closely associated with antioxidant systems that defend cells from the toxic effects of lipid peroxidation concentrations, active free radicals and other oxidative groups. Alcohol intake in rats lowers liver tissue and blood glutathione concentrations compared to normal group (FIG. 10). However, the decrease in liver tissue and blood glutathione concentration by alcohol intake was found to be restored to the normal group level by administration of BFSP (FIG. 10). Previous studies have reported that high levels of glutathione-containing yeast effectively prevent liver damage from carbon tetrachloride and alcohol administration [Cha et al., 2009; Shon MH, Cha JY, Lee CH, Park SH, Cho YS. Protective effect of administrated glutathione-enriched S accharomyces cerevisiae FF-8 against carbon tetrachloride (CCl 4 ) -induced hepatotoxicity and oxidative stress in rats. Food Sci Biochenol 16: 967-974 (2007). Decreased lipid peroxide levels due to BFSP administration in alcohol-fed rats seemed to be closely associated with increased antioxidant activity. The hepatoprotective effect of unfermented silkworm powder is also believed to be due to its constituents such as amino acids. Methionine or cysteine as a precursor of glutathione metabolism in liver tissue has recently been reported to play an important role in endogenous glutathione synthesis [Peters TJ. Ethanol metabolism. Bri Med Bull 38: 17-20 (1982); Stipanuk MH, Coloso RM, Garcia RAG, Banks MF. Cysteine concentration regulates cysteine metabolism to glutathione, sulfate and taurine in rat hepatocytes. J Nutr 122: 420-427 (1992)], reported to increase glutathione concentrations in liver and kidney tissues by administration of cysteine and cysteine containing components [Hsu CC, Hiang CN, Hung YC, Yin MC. Five cysteine-containing compounds have antioxidative activity in Balb / cA mice. J Nutr 134: 149-152 (2004).

최근 42% 글리신 및 32% 알라닌의 아미노산으로 구성된 실크 피브로인은 알코올성 간독성을 개선시키는 효과가 있다고 보고되었다[Kang GD, Lee KH, Do SG, Kim CS, Suh JG, Oh YS, Nham JH. Effect of silk fibroin on the protection of alcoholic hepatotoxicity in the liver of alcohol preference mouse. Int. J Indust Entomol 2: 15-18 (2001)]. 세린, 글리신, 히스티딘, 티로신과 같은 아미노산들도 갈락토스아민- 및 알코올-유발 간독성에 대해 개선효과가 있다고 알려져 있다[Lee JH, Kim NK, Lee DY, Lee CH. Protective effect of selected amino acids and food extracts on ethanol toxicity determent in rat liver. Korean J Food Sci Technol 31: 802-808 (1999); Ming Y, Ikejima K, Arteel GE, Seabra V, Bradford BU, Kono H, Rusyn I, Thurman RG. Glycine accelerates recovery from alcohol-induced liver injury. J Pharmacol Exp Ther 286: 1014-1019 (1998)]. 본 실험에서 Aspergillus- 및 Bacillus-발효 누에분말의 유리 아미노산 조성을 보면 주로 글루탐산 (3056~2642 ppm), 루이신 (1421~2368 ppm), 발린 (1314~2174 ppm), 알라닌 (1751~1823 ppm), 및 티로신(925~1477 ppm)으로 각각 구성되어 있으며(표 13), 이처럼 주로 이들 아미노산으로 구성된 발효 누에분말은 알코올 유발 산화스트레스를 감소시킬 가능성이 높은 것으로 사료되어 진다. 아미노산 L-세린, L-글리신 및 L-알라닌으로 구성된 세리신 단백질도 자유 라디칼에 의해 유발되는 간세포 독성을 경감시키는 것으로 보고되었다 [Ming Y, Ikejima K, Arteel GE, Seabra V, Bradford BU, Kono H, Rusyn I, Thurman RG. Glycine accelerates recovery from alcohol-induced liver injury. J Pharmacol Exp Ther 286: 1014-1019 (1998); Inkeles S, Eisenberg D. Hyperlipidemia and coronary atherosclerosis. Medicine (Baltimore) 60: 110-123 (1981); Petushok N. E., Miskevich D. The influence of the some amino acids on the antioxidant status in the liver of rats. Toxicology Letters 144: S116(2003)]. 한편 아스코르브산과 아르기닌, 히스티딘, 타우린, 리신 아미노산과의 조합은 간 조직 균질화 분획에서 글루타치온 농도가 70% 증가하였으며, 글루타치온 시스템 (글루타치온 리덕타아제/퍼옥시다아제) 활성도 거의 두 배 정도 증가하는 것으로 나타나 흰쥐 간 조직의 항산화 시스템을 크게 증가시키는 작용이 있는 것으로 드러났다(Petushok et al., 2003). Recently, silk fibroin consisting of amino acids of 42% glycine and 32% alanine has been reported to improve alcoholic hepatotoxicity [Kang GD, Lee KH, Do SG, Kim CS, Suh JG, Oh YS, Nham JH. Effect of silk fibroin on the protection of alcoholic hepatotoxicity in the liver of alcohol preference mouse. Int. J Indust Entomol 2: 15-18 (2001). Amino acids such as serine, glycine, histidine and tyrosine are also known to improve galactosamine- and alcohol-induced hepatotoxicity [Lee JH, Kim NK, Lee DY, Lee CH. Protective effect of selected amino acids and food extracts on ethanol toxicity determent in rat liver. Korean J Food Sci Technol 31: 802-808 (1999); Ming Y, Ikejima K, Arteel GE, Seabra V, Bradford BU, Kono H, Rusyn I, Thurman RG. Glycine accelerates recovery from alcohol-induced liver injury. J Pharmacol Exp Ther 286: 1014-1019 (1998). The free amino acid composition of Aspergillus- and Bacillus -fermented silkworm powders in this experiment were mainly glutamic acid (3056 ~ 2642 ppm), leucine (1421 ~ 2368 ppm), valine (1314 ~ 2174 ppm), alanine (1751-1823 ppm), And tyrosine (925-1477 ppm), respectively (Table 13), and fermented silkworm powder mainly composed of these amino acids is considered to have a high possibility of reducing alcohol-induced oxidative stress. Sericin proteins consisting of amino acids L-serine, L-glycine and L-alanine have also been reported to reduce hepatotoxicity induced by free radicals [Ming Y, Ikejima K, Arteel GE, Seabra V, Bradford BU, Kono H, Rusyn I, Thurman RG. Glycine accelerates recovery from alcohol-induced liver injury. J Pharmacol Exp Ther 286: 1014-1019 (1998); Inkeles S, Eisenberg D. Hyperlipidemia and coronary atherosclerosis. Medicine Baltimore 60: 110-123 (1981); Petushok NE, Miskevich D. The influence of the some amino acids on the antioxidant status in the liver of rats. Toxicology Letters 144: S116 (2003). Combination of ascorbic acid with arginine, histidine, taurine, and lysine amino acids resulted in a 70% increase in glutathione concentrations in liver tissue homogenization fractions, and a nearly double increase in glutathione system (glutathione reductase / peroxidase) activity in rat liver. It has been shown to act to significantly increase the antioxidant system of the tissue (Petushok et al., 2003).

또한 세린 및 트레오닌과 같은 히드록시 아미노산들은 구리나 철과 같은 미량원소와 chelating을 형성함으로서 항산화 작용을 나타내는 것으로 알려져 있다[Kato N, Sato S, Yamanaka A, Yamada H, Fuwa N, Nomura M. Silk protein, sericin, inhibits lipid peroxidation and tyrosinase activity. Biosci Biotechnol Biochem 62: 145-147 (1998)]. 본 실험에서 Aspergillus-발효 누에분말 보다는 Bacillus-발효 누에분말에서 세린, 글리신, 알라닌, 티로신 및 히스티딘과 같은 아미노산을 보다 많이 함유함으로서 강한 항산화 작용을 가지는 것과 무관하지 않을 가능성이 높아 보인다(표 2). 따라서 본 실험에서는 Bacillus-발효 누에분말에서 항산화 작용과 관련성이 높은 아미노산을 많이 함유함으로서 간 조직과 혈중의 과산화지질 작용에 대한 보호 효과가 누에분말이나 Aspergillus-발효 누에분말보다 큰 것으로 보여진다.
In addition, hydroxy amino acids such as serine and threonine are known to exhibit antioxidant activity by forming chelating with trace elements such as copper and iron [Kato N, Sato S, Yamanaka A, Yamada H, Fuwa N, Nomura M. Silk protein , sericin, inhibits lipid peroxidation and tyrosinase activity. Biosci Biotechnol Biochem 62: 145-147 (1998). In this experiment, the Bacillus -fermented silkworm powder contains more amino acids such as serine, glycine, alanine, tyrosine and histidine, rather than Aspergillus -fermented silkworm powder, which may not be related to having strong antioxidant activity (Table 2). Therefore, in this experiment, Bacillus -fermented silkworm powder contains more amino acids that are highly related to antioxidant activity, so that the protective effect against lipid peroxidation in liver tissue and blood is greater than silkworm powder or Aspergillus -fermented silkworm powder.

실시예Example 16.  16. 간조직의Liver tissue 병리조직학적 관찰 Histopathological observation

동물해부 직후 적출한 간을 냉각 생리식염수로 관류하여 혈액을 제거시킨 상태에서 조직의 일정한 부위의 일부를 취하여 10% 중성포르말린 용액에 고정하여 통상적인 조직처리인 파라핀 포매 과정을 거쳐 3~4 ㎛ 두께로 절편하여 H&E(hematoxylin and eosin) 염색 한 후 광학현미경(Olympus BX41, Olympus Co., Tokyo, Japan)으로 관찰하였다[Cha JY, Jun BS, Cho YS. Prevention of orotic acid-induced fatty liver in rats by capsaicin. Food Sci Biotechnol 13: 597-602 (2004)].
Immediately after animal dissection, the extracted liver was perfused with cold saline to remove blood, and a portion of the tissue was removed and fixed in a 10% neutral formalin solution, followed by paraffin embedding, which is a conventional tissue treatment. H & E (hematoxylin and eosin) staining was performed and observed with an optical microscope (Olympus BX41, Olympus Co., Tokyo, Japan) [Cha JY, Jun BS, Cho YS. Prevention of orotic acid-induced fatty liver in rats by capsaicin. Food Sci Biotechnol 13: 597-602 (2004)].

지방간은 과잉의 알코올 섭취로 인한 간 독성 유발의 시발점으로서 간 세포에 산소나 영양적인 측면의 불균형을 초래하는 것으로 알려져 있다. 간 세포에서의 지질의 축적인 지방간은 알코올성 간 독성 초기에 가장 흔하게 나타나는 증상이다. 실험동물을 해부한 직후 간 조직을 적출하여 조직학적 검사를 실시한 결과 대조군에서는 혈관을 중심으로 간 소엽 구조가 잘 유지되었으며, 간세포들은 풍부한 호산성 세포질과 둥근핵을 가지고 있었고 간세포판은 대부분 한 층으로 잘 유지되어 있었다(도 12). 발효 누에분말 투여군에서는 혈중 ALT 및 AST 활성의 감소와 간 조직의 과산화지질 농도의 감소에 의해 알코올성 간 독성이 개선되는 효과로 인해 조직학적 검사 결과에서도 정상군과 비슷한 모양을 보였다.Fatty liver is the starting point of liver toxicity caused by excessive alcohol intake and is known to cause oxygen or nutritional imbalance in liver cells. Accumulation of Lipids in Liver Cells Fatty liver is the most common symptom of alcoholic liver toxicity early in life. The liver tissues were removed and histologic examination immediately after dissection of the animals. The control group maintained well the hepatic lobule structure centered on blood vessels. It was well maintained (FIG. 12). In the fermented silkworm powder group, the alcoholic liver toxicity was improved by decreasing blood ALT and AST activity and decreased lipid peroxide concentration in liver tissue.

알코올 투여에 의해 대부분의 간 세포 내에 지방구의 증가에 의한 지방간 유발이 본 실험에서도 관찰되었으며, 이러한 모양은 전형적인 알코올성 지방간의 모습을 나타내는 것이다. 알코올은 탄수화물이나 단백질과 같은 영양소는 없으면서도 칼로리는 높아 결국은 간 조직에서 지방 축적의 원인이 될 수 있다. 한편 발효 누에분말 투여군들에서 알코올에 의한 간세포 내의 지방 축적은 개선되었으며, 간 조직학적 관찰에서도 정상군과 매우 유사한 모습을 보여 발효 누에분말의 간세포 보호 효과를 나타내는 증거가 되는 것이다(도 13).Fatty liver induction due to the increase of fat globules in most liver cells by alcohol administration was also observed in this experiment. Alcohol is high in calories without nutrients like carbohydrates and proteins, which can eventually lead to fat accumulation in liver tissue. Meanwhile, in the fermented silkworm powder-administered groups, fat accumulation in hepatocytes by alcohol was improved, and liver histological observation showed a very similar appearance to the normal group, which is evidence of the hepatocellular protective effect of fermented silkworm powder (FIG. 13).

Claims (11)

누에 분말에 증류수를 가한 후 멸균시키는 단계; 및
Bacillus subtilis 배양액 또는 Aspergillus kawachii 배양액을 첨가한 후 37℃에서 3일 내지 12일간 발효시킨 후 건조시키는 단계
를 포함하여 제조되는 발효 누에 분말 제조방법.
Adding distilled water to the silkworm powder and sterilizing it; And
Bacillus subtilis culture or Aspergillus fermentation at 37 ° C. for 3 to 12 days after the addition of kawachii culture, followed by drying
Fermented silkworm powder manufacturing method prepared including.
제1항에 있어서, 누에 분말에 대하여 1%(v/w)의 Bacillus subtilis 배양액을 첨가함을 특징으로 하는 발효 누에 분말 제조방법.The method of claim 1, wherein 1% (v / w) Bacillus relative to silkworm powder subtilis Culture medium Fermented silkworm powder production method, characterized in that the addition. 제1항에 있어서, 누에 분말에 대하여 5%(v/w)의 spergillus kawachii 배양액을 첨가함을 특징으로 하는 발효 누에 분말 제조방법.The method of claim 1, wherein 5% (v / w) of spergillus relative to silkworm powder kawachii Culture medium Fermented silkworm powder production method, characterized in that the addition. 제1항에 있어서, 6일 내지 12일간 발효시키는 것을 특징으로 하는 발효 누에 분말 제조방법. The method for producing fermented silkworm powder according to claim 1, wherein the silkworm is fermented for 6 days to 12 days. 제1항의 방법에 의해 제조된 발효 누에 분말.Fermented silkworm powder prepared by the method of claim 1. 제5항의 발효 누에 분말을 유효성분으로 포함하는 고지혈증 및 지방간 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating hyperlipidemia and fatty liver, comprising the fermented silkworm powder of claim 5 as an active ingredient. 제5항의 발효 누에 분말을 유효성분으로 포함하는 항산화 효능을 갖는 약학 조성물.A pharmaceutical composition having antioxidant efficacy, comprising the fermented silkworm powder of claim 5 as an active ingredient. 제5항의 발효 누에 분말을 유효성분으로 포함하는 혈전용해 효능을 갖는 약학 조성물.A pharmaceutical composition having a thrombolytic effect comprising the fermented silkworm powder of claim 5 as an active ingredient. 제5항의 발효 누에 분말을 포함하는 고지혈증 및 지방간 개선을 위한 건강 보조 식품 조성물.A dietary supplement for improving hyperlipidemia and fatty liver comprising the fermented silkworm powder of claim 5. 제5항의 발효 누에 분말을 포함하는 항산화 효능을 갖는 건강 보조 식품 조성물.A health supplement composition having antioxidant efficacy, comprising the fermented silkworm powder of claim 5. 제5항의 발효 누에 분말을 포함하는 혈전용해 효능을 갖는 건강 보조 식품 조성물.A dietary supplement having a thrombolytic effect comprising the fermented silkworm powder of claim 5.
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WO2020141778A1 (en) * 2018-12-31 2020-07-09 (주)엔아이앤팜 Composition comprising sericin for treating, preventing, or alleviating fatty liver, and method of preparing same
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KR101425486B1 (en) * 2012-09-26 2014-08-29 세종대학교산학협력단 Extract of Smilax china leaf with Aspergillus species, method for preparing the same and use of the same
KR101373940B1 (en) * 2012-10-17 2014-03-12 이성권 Method of preparing fermented and enzyme treated silkworm segment extract having high bioactive substances, the silkworm extract obtained thereby, and the use of the silkworm extract having antiinflammatory efficacy
WO2020141778A1 (en) * 2018-12-31 2020-07-09 (주)엔아이앤팜 Composition comprising sericin for treating, preventing, or alleviating fatty liver, and method of preparing same
KR20230098939A (en) 2021-12-27 2023-07-04 대한민국(농촌진흥청장) Composition for Anti- Inflammation Comprising Cocoon Hydrolysate Produced by Enzyme Treatment

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