• Bulletin of the Korean Chemical Society
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• 제32권2호
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• pp.599-604
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• 2011

In typical proteomic analysis, trypsin digestion is one of the most time-consuming steps. Conventional proteomic sample preparation methods use an overnight trypsin digestion method. In this study, we compared high-pressure cycling technology (PCT) during enzyme digestion for proteome analysis to the conventional method. We examined the effect of PCT on enzyme activity at temperatures of 25, 37, and $50^{\circ}C$. Although a fast digestion (1 h) was used for the standard protein mixture analysis, the PCT-assisted method with urea showed better results for protein sequence coverage and the number of peptides identified compared with the conventional method. There was no significant difference between temperatures for PCT-assisted digestion; however, we selected PCT-assisted digestion with urea at $25^{\circ}C$ as an optimized method for fast enzyme digestion, based on peptide carbamylation at these conditions. The optimized method was used for stem cell proteome analysis. We identified 233, 264 and 137 proteins using the conventional method with urea at $37^{\circ}C$ for 16h, the PCT-assisted digestion with urea at $25^{\circ}C$ for 1 h, and the non-PCT-assisted digestion with urea at $25^{\circ}C$ for 1 h, respectively. A comparison of these results suggests that PCT enhanced the enzyme digestion by permitting better access to cleavage sites on the proteins.

• Asian-Australasian Journal of Animal Sciences
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• 제15권11호
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• pp.1659-1676
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• 2002

Ruminant animals develop a diverse and sophisticated microbial ecosystem for digesting fibrous feedstuffs. Plant cell walls are complex and their structures are not fully understood, but it is generally believed that the chemical properties of some plant cell wall compounds and the cross-linked three-dimensional matrix of polysaccharides, lignin and phenolic compounds limit digestion of cell wall polysaccharides by ruminal microbes. Three adaptive strategies have been identified in the ruminal ecosystem for degrading plant cell walls: production of the full slate of enzymes required to cleave the numerous bonds within cell walls; attachment and colonization of feed particles; and synergetic interactions among ruminal species. Nonetheless, digestion of fibrous feeds remains incomplete, and numerous research attempts have been made to increase this extent of digestion. Exogenous fibrolytic enzymes (EFE) have been used successfully in monogastric animal production for some time. The possibility of adapting EFE as feed additives for ruminants is under intensive study. To date, animal responses to EFE supplements have varied greatly due to differences in enzyme source, application method, and types of diets and livestock. Currently available information suggests delivery of EFE by applying them to feed offers the best chance to increase ruminal digestion. The general tendency of EFE to increase rate, but not extent, of fibre digestion indicates that the products currently on the market for ruminants may not be introducing novel enzyme activities into the rumen. Recent research suggests that cleavage of esterified linkages (e.g., acetylesterase, ferulic acid esterase) within the plant cell wall matrix may be the key to increasing the extent of cell wall digestion in the rumen. Thus, a crucial ingredient in an effective enzyme additive for ruminants may be an as yet undetermined esterase that may not be included, quantified or listed in the majority of available enzyme preparations. Identifying these pivotal enzyme(s) and using biotechnology to enhance their production is necessary for long term improvements in feed digestion using EFE. Pretreating fibrous feeds with alkali in addition to EFE also shows promise for improving the efficacy of enzyme supplements.

• Asian-Australasian Journal of Animal Sciences
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• 제16권3호
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• pp.417-424
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• 2003

Inhibitory activities against angiotensin I-converting enzyme (ACE) of enzymatic hydrolysates of porcine skeletal muscle proteins were investigated. Myosin B, myosin, actin, tropomyosin, troponin and water-soluble proteins extracted from pork loin were digested by eight kinds of proteases, including pepsin, $\alpha$-chymotrypsin, and trypsin. After digestion, hydrolysates produced from all proteins showed ACE inhibitory activities, and the peptic hydrolysate showed the strongest activity. In the case of myosin B, the molar concentration of peptic hydrolysate required to inhibit 50% of the activity increased gradually as digestion proceeded. The hydrolysates produced by sequential digestion with pepsin and $\alpha$-chymotrypsin, pepsin and trypsin or pepsin and pancreatin showed weaker activities than those by pepsin alone, suggesting that ACE inhibitory peptides from peptic digestion might lose their active sequences after digestion by the second protease. However, the hydrolysates produced by sequential digestion showed stronger activities than those by $\alpha$-chymotrypsin, trypsin or pancreatin alone. These results suggested that the hydrolysates of porcine meat were able to show ACE inhibitory activity, even if they were digested in vivo, and that pork might be a useful source of physiologically functional factors.

• Mass Spectrometry Letters
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• 제5권2호
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• pp.52-56
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• 2014

Glycated hemoglobin (HbA1c) is used as an index of mean glycemia over prolonged periods. This study describes an optimization of enzyme digestion conditions for quantification of non-glycated hemoglobin (HbA0) and HbA1c as diagnostic markers of diabetes mellitus. Both HbA0 and HbA1c were quantitatively determined followed by enzyme digestion using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with synthesized N-terminal hexapeptides as standards and synthesized isotope labeled hexapeptides as internal standards. Prior to quantification, each peptide was additionally quantified by amino acid composition analysis using ID-LC-MS/MS via acid hydrolysis. Each parameter was considered strictly as a means to improve digestion efficiency and repeatability. Digestion of hemoglobin was optimized when using 100 mM ammonium acetate (pH 4.2) and a Glu-C-to-HbA1c ratio of 1:50 at $37^{\circ}C$ for 20 h. Quantification was satisfactorily reproducible with a 2.6% relative standard deviation. These conditions were recommended for a primary reference method of HbA1c quantification and for the certification of HbA1c reference material.

• Journal of Animal Science and Technology
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• 제65권1호
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• pp.32-56
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• 2023

This review explores the factors that improve meat protein digestibility and applies the findings to the development of home meal replacements with improved protein digestion rates in older adults. Various methods improve the digestion rate of proteins, such as heat, ultrasound, high pressure, or pulse electric field. In addition, probiotics aid in protein digestion by improving the function of digestive organs and secreting enzymes. Plant-derived proteases, such as papain, bromelain, ficin, actinidin, or zingibain, can also improve the protein digestion rate; however, the digestion rate is dependent on the plant enzyme used and protein characteristics. Sous vide processing improves the rate and extent of protein digestibility, but the protein digestion rate decreases with increasing temperature and heating time. Ultrasound, high pressure, or pulsed electric field treatments degrade the protein structure and increase the proteolytic enzyme contact area to improve the protein digestion rate.

• Mycobiology
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• 제39권1호
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• pp.67-69
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• 2011

A cell-free extract of Saccharomyces cerevisiae containing the angiotensin I-converting enzyme (ACE) inhibitory peptide was treated in a successive simulated gastric-intestinal bioreactor (step 1: amylase digestion, step 2: gastric fluid digestion, step 3: intestinal fluid digestion) to illustrate the absorption pattern of antihypertensive ACE inhibitory peptide, and the ACE inhibitory activities of each step were determined. Total ACE inhibitory activities of step 1, step 2, and step 3 were 55.96%, 80.09%, and 76.77%, respectively. The peptide sequence of each steps was analyzed by MS/MS spectrophotometry. Eleven kinds of representative peptide sequences were conserved in each step, and representative new peptides including RLPTESVPEPK were identified in step 3.

• Bulletin of the Korean Chemical Society
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• 제33권10호
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• pp.3233-3240
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• 2012

We demonstrated the combined applications of online protein digestion using trypsin immobilized enzyme reactor (IMER) and dual tandem mass spectrometry with collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) for tryptic peptides eluted through the trypsin-IMER. For the trypsin-IMER, the organic and inorganic hybrid monolithic material was used. By employing the trypsin-IMER, the long digestion time could be saved with little or no sacrifice of the digestion efficiency, which was demonstrated for standard protein samples. For three model proteins (cytochrome c, carbonic anhydrase, and bovine serum albumin), the tryptic peptides digested by the IMER were analyzed using LC-MS/MS with the dual application of CAD and ETD. As previously shown by others, the dual application of CAD and ETD increased the sequence coverage in comparison with CAD application only. In particular, ETD was very useful for the analysis of highly-protontated peptide cations, e.g., ${\geq}3+$. The combination approach provided the advantages of both trypsin-IMER and CAD/ETD dual tandem mass spectrometry applications, which are rapid digestion (i.e., 10 min), good digestion efficiency, online coupling of trypsin-IMER and liquid chromatography, and high sequence coverage.

• Applied Biological Chemistry
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• 제13권1호
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• pp.29-34
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• 1970

우수한 영양가를 가진 대두를 이용하여 이유식을 제조하기 위하여 가압증자한 대두에 Protease및 Cellulase 역가가 비교적 높은 Asp. niger 및 Asp. sojae균의 피국 추출조효소액을 작용시켜 대두 단백질을 아미노산 내지 Peptide 태로 분해시키는 최적조건을 결정하고 여기서 얻은 분해물을 탈색 농축시키는 효과에 관하여 실험하여 다음과 같은 결과를 얻었다. 1. 대두의 가압증자는 15Ib에서 10분간 처리함이 가장 높은 단백응해율 및 단백분해율을 나타냈다. 2. Asp. sojae enzyme은 pH 6.0, Asp. niger enzyme은 pH 4.4에서 가장 높은 단백용해율과 단백분해율을 나타냈다. Asp. sojae enzyme을 처리한 다음 Asp. niger emzyme을 작용 분해시킨 것이 각 효소 단독으로처리했을때 보다 높은 단백응해율(62.3%) 및 단백분해율을 (56.4%) 나타냈다. 3. 기질에 대한 효소액 첨가량은 원료 대두에 10배의 물을 가한 마쇄기질액에 피국에 대하여 10배의 물로 추출한 효소액 1/2에 해당하는 양 (Asp. sojae enzyme와 Asp. niger emzyme 총량)을 가하는것이 가장 실용적이었다. 4. 분해시간이 길 수록 단백응해율 및 단백분해율이 높아지나 부패 등을 고려할 때 실용분해 시간은 8시간 정도가 적당하다. 5. 탈색 효과는 활성탄으로 처리한 후 음이온교환수지 (Dowex 2-x-8)을 처리한 것이 가장 좋고 단독처리로는 음이온교환수지, 활성탄, 양이온교환 수지(Amberite)의 순으로 효과가 적었다. 6. 이상의 최적 조건으로 대두단백질을 분해하고 $60^{\circ}C$ 이하에서 감압농축하여 얻은 제품은 수분 12.51%, 단백질 66.31%, 지방 4.25%, 탄수화물 12.75%,인 건조분말을 얻었다. 이 연구는 1969년도 문교부 학술연구 조성비로 이루워진 것이며 본 연구에 헌신적인 보조를 아끼지 않았던 박 관화 군에게 감사하는 바이다.

• 한국식품과학회지
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• 제10권4호
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• pp.394-397
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• 1978

한우(韓牛)의 round muscle에 papain으로 일차(一次) 분해(分解)시킨 후 pepsin에 의한 in vitro소화율(消化率)과 소화과정중(消化過程中) 생성(生成)되는 amino 태(態)-N의 경분적(經分的)인 변화(變化)를 실험(實驗)한 결과(結果)는 다음과 같다. 1. 효소처리구(酵素處理區)가 대조구(對照區)에 비(比)하여 소화율(消化率)도 높았으며 소화시간(消化時間)도 단축되었다. 2. 효소처리구(酵素處理區)는 소화과정중(消化過程中) $6{\sim}10$분간(分間)사이에 소화율(消化率)이 현저하게 증가(增加)하였다. 3. amino태(態)-N는 소화율(消化率)과 소화시간(消化時間)에 거의 비슷하게 증가(增加)하였다. 4. 대조구(對照區)에 비(比)하여 효소처리구(酵素處理區)가 amino태(態)-N 생성량(生成量)이 현저하게 높았으며$2{\sim}8$시간(時間)사이에서 그 증가율(增加率)이 가장 높았다.

• 한국식품영양과학회지
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• 제13권1호
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• pp.97-106
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• 1984

남대양(南大洋)에 다량 서식하고 있는 중요한 단백질자원(蛋白質資源)인 크릴을 보다 유효하게 식량(食糧)으로 이용하기 위한 방안의 하나로 크릴을 원료로 하여 그 자체에 존재하는 활성이 높은 자가소화효소(自家消化酵素)나 단백질분해효소(蛋白質分解酵素)로써 크릴간장 제조(製造)를 시도(試圖)하였다. 마쇄한 크릴에 동량(同量)의 물을 첨가하여 가수분해(加水分解)시킬 때의 최적가수분해조건(最適加水分解條件) 및 저장안정성(貯藏安定性)을 검토(檢討)하고 아울려 제품의 정미성분(呈味成分)을 분석(分析)하였다. 자가소화(自家消化)에 의한 경우와 bromelain, complex enzyme을 첨가한 시료(試料) 모두 $52.5^{\circ}C$에서 최대활성을 나타내었고, 분해시간(分解時間)은 3시간(時間)이 적합하였으며, 효소농도(醴素濃度)는 bromelain의 경우 0.5 %, complex enzyme은 5%가 가장 좋았다. 그리고 pH는 자가소화(自家消化)나 complex enzyme을 첨가하여 분해(分解)하였을 경우는 7.0${\sim}$7.5, bromelain을 첨가하여 분해(分解)하였을 때는 6.5부근에서 가장 활성이 높았다. 이와 같은 최적가수부해조건(最適加水分解條件)에서 크릴을 자가소화(自家消化)시킨 후 $100^{\circ}C$, 20 분간(分間) 불활성화한 다음 여과한 가수분해물(加水分解物)에 식염(食鹽)(10 %)과 벤조산(0.06%)이나 알코올(3 %)을 첨가하여 멸균한 유리병에 밀봉(貯減)한 결과 $37^{\circ}C$에서 한달간 저장(貯藏)하여도 화학적, 미생물적 및 관능적으로 안정하였다. 또한 관능적인 맛으로 보아 식염(食鹽) 10 % 중 5 % 정도까지는 나트륨염(鹽) 대신 칼륨염(鹽)을 대체 첨가할 수 있었다. 크릴간장의 단백질가수분해율(蛋白質加水分解率)은 자가소화법(自家消火法)인 경우 83.2%, bromelain 첨가구는 89.7%, complex enzyme 첨가구는 92.7 %이었다. 자가소화법(自家消化法), bromelain 또는 complex enzyme첨가에 의해 제조(製造) 된 크릴간장의 유리(遊離)아미노산(酸) 중 함량이 많은 것은 lysine, arginine, leucine, proline, alanine 및 valine으로서 전 유리(遊離)아미노산(酸)에 대해 각각 58.8 %, 56.0 %, 55.3 %를 차지하였다. 그리고 전엑스분망(分望) 소(素)에 대하여 유리(遊離)아미노산(酸)이 차지하는 비율은 각각 67.4 %,69.4%, 69.8 %이었다. 핵산관련물질(核酸關聯物質) 중 함량이 가장 많은 것은 hypoxanthine이었고, 다음이 5’-IMP였다. TMAO, betaine, 총 creatinine은 함량이 적었다. 관능검사결과(官能檢査結果) 자가소화(自家消化)시킨 크릴간장은 효소(酵素)처리한 것이나 재래식 콩간장에 비하여 품질 면에서 손색이 없고 저장성(貯藏性)이 좋은 크릴간장을 제조(製造)할 수 있다는 결론을 얻었다.