• Bulletin of the Korean Chemical Society
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• v.32 no.2
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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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• v.15 no.11
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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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• v.16 no.3
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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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• v.5 no.2
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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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• v.65 no.1
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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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• v.39 no.1
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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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• v.33 no.10
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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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• v.13 no.1
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• pp.29-34
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• 1970

In order to prepare digested Protein source for the Weanling Food from soybean, an attempt was made to decompose steamed soybean protein to amino acids and peptides by protease and cellulase produced from Aspergillus niger and Aspergillus sojae. In this paper, the optimum condition for digestion of soybean protein were studied and also investigated the effects of decolorization of it. As the results, followings were obtained; 1. As steaming conditions, a treatment under 15 lb of pressure and 10 minutes of heating shows most effective. 2. The optimum pH of Asp, sojae enzyme for the digestion of soybean protein is 6.0, while that of Asp. niger enzyme is 4.4. In successievly-decomposing with Asp. sojae and Asp. niger, it shows the most effective on ratio of water-soluble-nitrogen to total nitrogen and amino-nitrogen to total nitrogen than any other separate treatments. 3. The suitable amount of the enzyme solution to that of the soybean substrate paste, in volume, is 1 : 2. 4. Digestion ratio of soybean protein indicates the gradual and steady effects of increasing time of digestion, but 8 hour-digestion regarding to putrefaction was suitable. 5. The most effective decolorization was successively passed on culumns of active carbon and anion exchanger (Dowex 2-x-8) at room temperature. In separate treatments, the effective order of decolorization was as follows; (Dowex 2-x-8)>Active carborn>Amberite IR-120 6. The powder type of the soy protein source obtained by concentration below $60^{\circ}C$ contains 12.51% of moisture, 66.31% of protein, 4.25% of fat, 12.75% of carbohydrate, 4.18% of ash.

• Korean Journal of Food Science and Technology
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• v.10 no.4
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• pp.394-397
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• 1978

In vitro digestibility study using pepsin was conducted on round muscle previously treated with papain and the change in the amounts of liberated amino nitrogen was followed during the time course of digestion procedure. The obtained results were summarized as follows. 1. As compared with control, enzyme treatment groups had higher digestibility and curtailed digestion time. 2. All the enzyme treatment groups showed the highest digestibility between 6 and 10 hours in the course of digestion procedure. 3. The amounts of liberated amino nitrogen were increased similarly to digestibility and digestion time. 4. As compared with control, enzyme treatment groups showed considerably higher amounts of liberated amino nitrogen, the liberation rate being the highest between 2 and 8 hours of digestion time.

• Journal of the Korean Society of Food Science and Nutrition
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• v.13 no.1
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• pp.97-106
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• 1984

The Antarctic krill, Euphausia superba, is drawing attention over the world as the largest source of unutilized proteins in the ocean. For the use of krill as a human food, processing conditions of krill sauce by autolysis and/or commercial proteolytic enzyme digestion were examined. The krill was chopped and mixed with equal weight of water, and hydrolyzed by autolysis and/or commercial proteolytic enzyme digestion. The optimal conditions for hydrolysis of krill were $52.5^{\circ}C$, pH 7.0-7.5, 3 hours by autolysis, $52.5^{\circ}C$, pH 6.3, 3hours by bromelain (0.5 %) digestion, and $52.5^{\circ}C$, pH 7.0-7.5, 3 hours by commercial complex enzyme (5 %) digestion, respectively The maximum hydrolyzing rate of protein were 83.2 % by autolysis, 89.7 % by bromelain digestion, 92.7 % by commercial complex enzyme digestion. After krill meat hydrolyzed by autolysis at optimum condition, inactivated at $100^{\circ}C$ for 20 minutes and filtered with Buchner funnel. Two kinds of products were prepared with krill hydrolysate and preservatives: one contained 10 % of sodium chloride and 0.06 % of benzoic acid and the other 10 % of sodium chloride and 3 % of ethyl alcohol. These products were filled in the sterilized glass bottle and sealed. The pH, volatile basic nitrogen, amino nitrogen, color value (L, a and b values) and viable counts of bacteria were determined during storage at $37^{\circ}C$. The results showed that the products could be preserved in good condition during one month at $37^{\circ}C$. As a method to reduce the sodium level in krill sauce, it is convinced that sodium chloride could be replaced half in partially by potassium chloride. In the products prepared from krill by autolysis, bromelain or commercial complex enzyme digestion, hypoxanthine and 5'-IMP were abundant among the nucleotides and their related compounds as 15.3-20.4 ${\mu}mole/g$, dry solid, 2.2-2.5 ${\mu}mole/g$, dry solid, respectively. The abundant free amino acids were lysine, leucine, proline, alanine and valine. The contents of these amino acids were 67.4 %, 69.4 %, 69.8 % of the total free amino acids of each products. And TMAO, betaine and total creatinine were low in contents. The flavor of krill sauce prepared from krill by autolysis or enzyme digestion was not inferior to that of traditional Kerean soy sauce by sensory evaluation.