• Journal of Animal Science and Technology
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• 제62권6호
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• pp.933-947
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• 2020

This study was conducted to develop an effective heparin extraction method by using low-cost and highly effective enzymes from six pig by-products (liver, lung, heart, stomach, small intestine, and large intestine), and analyze their bioavailability. Low-cost and highly effective enzymes (alkaline-AK and papain) and a common enzyme (trypsin) were used for the heparin extraction. The angiotensin I- converting enzyme (ACE) inhibitory activity and the antimicrobial activity of extracted heparin were analyzed to verify their bioavailability. The average amount of heparin extracted per kilogram of pig by-products was 439 mg from the liver, 127 mg from the lung, 398 mg from the heart, 261 mg from the stomach, 197 mg from the small intestine, and 239 mg from the large intestine. Various enzymes were used to extract heparin, and the amount of extracted heparin was similar. Based on 1 g of pig by-product, the enzymes trypsin, papain, and alkaline-AK could extract 1,718 mg, 1,697 mg, and 1,905 mg of heparin, respectively. Heparin extracted from pig by-products showed antihypertensive activity and antimicrobial activity against Staphylococcus aureus at low populations. These results indicated that heparin can be obtained from pig by-products at a low cost.

• 한국미생물·생명공학회지
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• 제10권4호
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• pp.275-281
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• 1982

Trypsin에 대한 강한 저해물질을 생성하는 Streptomyces속 균주 AS-707을 토양으로부터 얻어 그 배양액에서 Trypsin inhibitor를 분리정제하여 저해물질의 안정성과 여러가지의 protease에 대한 저해성 여부를 검토한 결과는 다음과 같다. 배양액을 Amberlite IRC-50에 흡착 methanol추출. 2차 Amberlite IRC-50, CM-cellulosecolumn chromatography로 정제하여 active amorphous powder를 얻었는데 이 때의 비율은 26%였다. 분리정제된 물질은 trypsin 이외에 papain, $\alpha$-chymotrypsin, Azotobacter vineiandi protease와 Bacillus subtilis protease 등에 대해서도 저해작용을 나타내었으며, 안정성은 비교적 커서 10$0^{\circ}C$에서 120분간 가열해도 잔존활성이 약90%였으며, pH처리에 대해서는 37$^{\circ}C$에서 처리하면 산에서 Alkali에 걸치는 대단히 넓은 pH범위 (pH 2.0~12.0)에서 안정하였으나 6$0^{\circ}C$에서 처리하면 산에서는 안정하였으나 Alkali에서는 불안정하였다.

• BMB Reports
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• 제33권2호
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• pp.112-119
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• 2000

Three kinds of serine protease inhibitors, members of the Bowman-Birk trypsin inhibitor, were purified from Dolichos lablab seeds and named Dolichos protease inhibitor 1, 2 and 3 (DI-1, DI-2 and DI-3), respectively. Each inhibitor showed a single band with gel mobility at around 15.9, 12.1 and 14.6 kDa on 20% SDS-PAGE under reducing conditions. To characterize inhibitory specificity, the inhibition constant (Ki) for these inhibitors was measured against several known serine proteases. All three Dolichos protease inhibitors (DI-1, DI-2 and DI-3) inhibited the activity of trypsin and plasmin, but had no effect on thrombin and kallikrein (either for human plasma kallikrein or for porcine pancreas kallikrein). DI-1 inhibited chymotrypsin most effectively (Ki = $3.6{\times}10^{-9}\;M$), while DI-2 displayed inhibitory activity for porcine pancreatic elastase (Ki = $6.2{\times}10^{-8}\;M$). Pre-treatment of the 33 mg/kg of DI-mixture (active fractions from $C_{18}$ open column chromatography that included DI-1, DI-2 and DI-3) inhibited the induction of pseudomonal elastase-induced septic hypotension and prevented an increase in bradykinin generation in pseudomonal elastase-treated guinea pig plasma. Also, the increase of kallikrein activity, by injection of pseudomonal elastase, was inhibited by the pretreatment of the DI-mixture in a guinea pig. Since the DI-mixture had no inhibitory effect on kallikrein activity when Z-Phe-Arg-MCA was used as a substrate in vitro, its inhibitory activity in the pseudomonal elastase-induced septic hypotension model might not be due to a direct inhibition of plasma kallikrein in the activation cascade of the Hageman factor and prekallikrein system. These results suggest that the Dolichos DI-mixture might be used as an inhibitor in pathogenic bacterial protease-induced septic shock.

• 한국수산과학회지
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• 제44권5호
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• pp.456-463
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• 2011

We attempted to isolate a collagenase-1 inhibitory peptide from skate Dipturus chilensis skin protein. The protein from skate skin was digested by various enzymes (alcalase, ${\alpha}$-chymotrypsin, neutrase, papain, pepsin, and trypsin) to produce a collagenase-1 inhibitory peptide. The collagenase-1 inhibitory activity of the peptides obtained was measured by gelatin digestion assay. Among the six hydrolysates, pepsin hydrolysate exhibited the highest collagenase-1 inhibitory activity. The peptide showing strong collagenase-1 inhibitory activity was purified by Sephadex G-25 gel chromatography and HPLC using an octadecylsilyls (ODS) column. The amino acid sequence of purified collagenase-1 inhibitory peptide was identified to be Asn-Leu-Asp-Val -Leu-Glu-Val-Phe (961 Da) by quadrupole time of flight (Q-TOF) and electrospray ionization mass spectrometry (ESI-MS) mass spectroscopy. The $IC_{50}$ value of purified peptide was 87.0 ${\mu}M$. Moreover, the peptide did not exhibit cytotoxic effects on human dermal fibroblast cell lines.

• 한국수산과학회지
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• 제46권4호
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• pp.351-358
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• 2013

A protease inhibitor from fish eggs was fractionated using chromatographic methods. The fractionation efficiency was evaluated in terms of specific inhibitory activity (SIA, U/mg), purity (fold), total inhibitory activity (TIA, U), and recovery (%). The protease inhibitor (PI) from egg extracts of skipjack tuna (ST Katsuwonus pelamis), yellowfin tuna (YT Thunnus albacares) and Alaska pollock (AP Theragra chalcogramma) was fractionated using Sephadex G-50 gel filtration and DEAE-Sepharose CL-6B anion exchange chromatography based on protein size exclusion and net charge, respectively. Fractions exhibiting strong inhibitory activity were contained in the 30-50 kDa fraction on gel filtration and in the range of 0.4-0.7 M NaCl gradient fraction on anion exchange chromatography. The respective TIA and percent recovery of the fraction obtained with gel filtration toward trypsin and $N{\alpha}$-benzoyl-L-arginine-p-nitroanilide (BAPNA) were 2,758.7 U and 29.6% for ST, 1,005.5 U and 25.6% for YT, and 1,267.5 U and 26.0% for AP. Gel filtration chromatography was more effective at fractionating PI than using ion exchange chromatography. These results suggest that fish eggs act as serine protease inhibitors and might be useful for protease inhibition in foodstuffs.

• 한국식품영양과학회지
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• 제21권3호
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• pp.255-262
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• 1992

단백소화율에 미치는 식이 섬유소의 영향에 대하여 알아보기 위해, 채소류(상치, 깻잎, 고추. 다시마)로부터 추출한 식이 섬유소와 시판용 정제 식이 섬유소 (cellulose, pectin, sodium alginate, gum karaya)를 어류 단백질인 말쥐치 단백질(냉동건조육 및 myofibrils)에 첨가 반응시켜, 단백질 의 소화율에 어느 정도 영향을 미치는가에 대해 알아보았다. 각 시료의 중성세제 추출섬유소 (neutral detergent fiber) 함량은 24.21%(고추) 9.75%(다시마)의 범위였고, 산성세제 추출섬유소 (acid detergent fiber) 함량은 20.85%(고추) 11.97%(깻잎)의 범위였으며, 수용성 섬유소 함량은 13.79%(다시마) 4.41%(상치)의 범위였다. 말쥐치 단백질에 대한 식이 섬유소의 반응 비율을 1 : 1 (wt/wt)로 하고, 37$^{\circ}C$에서 2시간 동안 반응시켰을 때. 말쥐치 단백소화율은 정제 식이 섬유소 첨가의 경우, 1.52%(cellulose) 9.97%(pectin)가 감소되었고. 추출한 식이 섬유소 첨가의 경우, 5.15%(고추) 12.36%(다시마)가 감소되었다. 섬유소의 trypsin 활성저해능은 단백소화율이 감소함에 따라 증가하여, ANRC casein에 대한 soybean trypsin inhibitor 22mg/g (cellulose) 61.82mg/g(gum karaya), 49.75mg/g(고추) 171.52mg/g(상치)에 상응하는 것으로 나타났다. 정제 식이 섬유소에 의한 단백분해효소의 활성 변화는 sodium alginate를 제외하고는 거의 없어, 어류 단백소화율의 저하는 식이 섬유소가 단백질에 직접 결합하여 비소화성 물질을 형성한 결과가 주도하리라 생각되었다. 말쥐치 단백질과 섬유소를 반응시킨 것을 효소 가수분해시킨 후에 측정한 유리 필수 아미노산의 함량은 sodium alginate와 다시마 섬유소의 경우 현저하게 저하하였으며(75% 이상), isoleucine과 valine이 크게 영향을 받았다.

• Preventive Nutrition and Food Science
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• 제10권3호
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• pp.239-243
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• 2005

Angiotensin I-converting enzyme (ACE) inhibitory activities of elk antler hydrolysates prepared with three kinds of proteases, pepsin, trypsin and $\alpha-chymotrypsin$, were investigated. The ACE inhibitory activity of the pepsinolytic hydrolysate was the highest with an $IC_{50}$ value of $9.3\mu g/mL.$ In addition, three kinds of pepsinolytic hydrolysates with relatively high molecular weights (over 10,000 Da), medium molecular weights (5,000 to 10,000 Da), and low molecular weights (below 5,000 Da) were fractionated using an ultrafiltration membrane system. The below 5,000 Da hydrolysate exhibited the highest ACE inhibitory activity. These results indicate that the pepsinolytic hydrolysates of elk velvet antler could be a good source of peptides with ACE inhibitory activity.

• Food Science and Biotechnology
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• 제14권6호
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• pp.732-737
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• 2005

The combinational effect of gamma irradiation and moist heating on the trypsin inhibitor activity (TIA) in soaked and dried soybeans was evaluated by measuring the inhibition using N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. Gamma irradiation significantly decreased the TIA level in soybean at doses above 5 kGy, and the $ID_{50}$ (the gamma irradiation dose required to reach 50% inhibition) value for TIA was 13.53 kGy. Soaking prior to gamma irradiation significantly lowered the $ID_{50}$ to 8.44 kGy, and the soaking process enhanced the efficiency to inactivate TIA by as much as 48%. When soaking prior to gamma irradiation was followed by subsequent mild heating ($60^{\circ}C$) process, the $IT_{50}$ (heating time required to reach the 50% inhibition of TIA) value at even 1 kGy (5.28 min) was greatly reduced by over 50% compared to the level for the no-soaking process. In addition, the activation energy of soaking prior to gamma irradiation at 1 kGy was 2.45 kcal/mole, which was also about 50% lower than the 5.10 kcal/mole of dried soybean gamma-irradiated. Based on these results, soaking prior to gamma irradiation is an effective method for TIA inhibition. Furthermore, a combination of two or more processing methods such as soaking, heating and gamma irradiation is much more effective than any single processing method.

• 한국수산과학회지
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• 제46권2호
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• pp.119-128
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• 2013

A protease inhibitor was fractionated from fish eggs using methods based on protein solubility. Fractionation efficiency was evaluated with regard to percent recovery and total inhibitory activity (U). The fractionation of protease inhibitor (PI) from egg extracts of skipjack tuna (ST, Katsuwonus pelamis), yellowfin tuna (YT, Thunnus albacores), and Alaska pollock (AP, Theragra chalcogramma) was performed by precipitation with cold acetone or ammonium sulfate (AS). Fractions exhibiting the strongest inhibitory activity contained 20-40% (v/v) cold acetone or 40-60% saturated AS fractions. AS fractionation was more effective in isolating PI than was precipitation with acetone. The total inhibitory activity and percent recovery of fraction obtained with AS 40-60% toward trypsin and $N{\alpha}$-benzoyl-L-arginine-p-nitroanilide (BAPNA) were 4,976 U and 24.2% for ST, 3,331 U and 38.1% for YT, and 4,750 U and 43.8% for AP, respectively. In comparisons against six commercial proteases, 40-80% AS fractions, made by combining the 40-60% and 60-80% AS fractions from fish egg extract, exhibited the strongest inhibition of trypsin when using a casein substrate. These results suggest that fish eggs act as serine protease inhibitors and may be useful for protease inhibition in foodstuffs.

• Preventive Nutrition and Food Science
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• 제10권1호
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• pp.6-10
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• 2005

The trypsin inhibitor activity (TIA) of various soybean cultivars was evaluated by measuring the inhibition of trypsin activity using N-benzoyl-DL-arginine-p-nitro-anilide (BAPNA) as the substrate. The TIA values of eleven white shelled soybean cultivars including a glyphosate-tolerant soybean (16.58 to 17.90㎎/g) were not significantly different among cultivars. Black shelled soybeans had higher TIA values, ranging from 40.09 to 52.11㎎/g, compared to white shelled soybeans (p<0.05). When the TIA of commercially processed soybean foods were determined, no TIA was detected in soysauce, tofu and soybean paste. During conventional moist heating, the IT/sub 50/ (Time required to reach 50% inhibition of TIA) values were decreased as heating temperature and cooking pressure increased. The IT/sub 50/ values of moist heating were estimated to be 91.68, 37.71 and 19.50 min at 60, 80 and 100℃, respectively. The IT/sub 50/ value of microwave cooking was 4.75 min at medium heat, while that of the pressure cooking at 120℃ was only 2.62min. Moreover, there was a negative relationship between temperature and IT/sub 50/ values (R=0.92, p<0.01). The TIA of soybean sprouts was completely inactivated after heating at 100℃ for 5 min, although fresh soybean sprouts showed one fifth of the TIA value of white shelled soybeans. Based on our results, pressure cooking is the most effective cooking method to reduce TIA in soybeans.