• Mycobiology
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• v.30 no.3
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• pp.160-165
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• 2002

Aspergillus niger has been used as a host to express many heterologous proteins. It has been known that the presence of an- abundant protease is a limiting factor to express a heterologous protein. The protease deficient mutant of A. niger was obtained using UV-irradiation. A total of $1{\times}10^5$ spores were irradiated with $10{\sim}20%$ survival dose of UV, 600 $J/m^2$ at 280 nm, and the resulting spores were screened on the casein-gelatin plates. Ten putative protease deficient mutants showing the reduced halo area around colonies were further analyzed to differentiate the protease deficient mutant from other mutant types. Among ten putative mutants, seven mutants showed significant growth defect on nutrient rich medium and two mutants appeared to be the secretory mutants, which resulted in the impaired secretion of extracellular proteins including proteases. A mutant $pro^--20$ showed reduced halo zone without any notable changes in growth rate. In addition, the starchdegrading and glucose oxidase activities in the culture filtrate of $pro^--20$ mutant showed the similar range as that of the parental strain, which suggested that the $pro^--20$ mutant ought to be the protease deficient mutant rather than a secretory mutant. The reduced proteolytic activity of the $pro^--20$ was demonstrated using SDS-fibrin zymography gel. The reduced extracellular proteolysis was quantified by casein degradation assay and, comparing with the parental strain, less than 30% residual extracellular protease activity was detected in the culture filtrate of the $pro^--20$ mutant. The bio-activity of an exogenously supplemented hGM-CSF(human Granulocyte-Macrophage Colony Stimulating Factor) in the culture filtrate of $pro^--20$ mutant was detected until eight times more diluted preparations than that of the parental strain.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.10
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• pp.1460-1468
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• 2003

An experiment was conducted to quantify the flow of soluble non-ammonia nitrogen (SNAN) in the liquid phase of ruminal (RD) and omasal digesta (OD), and to investigate diurnal pattern in SNAN flow in OD. Five ruminally cannulated Finnish-Ayrshire dairy cows in a $5{\times}5$ Latin square design consumed a basal diet of grass silage and barley grain, and that supplemented with four protein feeds (kg/d DM basis) as follows: skimmed milk powder (2.1), wet distiller' solubles (3.0), untreated rapeseed meal (2.1) and treated rapeseed meal (2.1). Ruminal digesta was sampled using a vacuum pump, whereas OD was collected using an omasal sampling system at 1.0 h interval during a 12 h feeding cycle. Both RD and OD were acidified, centrifuged to remove microbes and precipitated with trichloroacetic acid followed by centrifugation. The SNAN fractions (free amino acid (AA), peptide and soluble protein) in RD and OD were assessed using ninhydrin assay. Free AA, peptide and soluble protein averaged 60.0, 89.4 and 2.1 g/d, respectively, for RD, and 81.8, 121.5 and 2.5 g/d, respectively, for OD. Although free AA flow was relatively high, mean peptide flow was quantitatively the most important fraction of SNAN, indicating that degradation of peptide to AA rather than hydrolysis of soluble protein to peptide or deamination may be the most limiting step in rumen proteolysis. Diurnal pattern in flow of peptide including free AA in OD during a 12 h feeding cycle peaked 1 h post-feeding, decreased by 3 h post-feeding and was relatively constant thereafter. Protein supplementation showed higher flow of peptide including free AA immediately after feeding compared with no supplemented diet. There were no differences among protein supplements in diurnal pattern in flow of peptide including free AA in OD.

• Asian-Australasian Journal of Animal Sciences
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• v.5 no.4
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• pp.673-679
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• 1992

To investigate the effects of molasses addition at ensiling on nutritional quality of wilted napier grass, chemical quality and nutrient composition of the silages, digestibility and nitrogen retention at feeding trials were analysed using 4 goats in a cross over design. The results are as follows : 1. Molasses addition at ensiling decreased pH value (3.99) and ammonia nitrogen, and increased lactic acid content by 285% compared to non-additive silage (83.5 g/kg dry matter). 2. There were no differences in digestibilities of dry matter, crude protein, neutral detergent fiber, acid detergent fiber, hemicellulose and cellulose between the silage ensiled with molasses (MS silage) and the silage ensiled without molasses (WS silage). Urinary nitrogen excretion, however, significantly (p<0.05) decreased in goats fed the MS silage, and nitrogen retention was positive in goats fed the MS silages, but negative in goats fed the WS silage. 3. Acetic acid concentration in remained fluids in goats fed the MS silage was lower and propionic and butyric acid concentrations were higher than those in goats fed the WS silage. As water soluble carbohydrate content was higher in the MS silage than in the WS silage, a part of added molasses was still remained in the silage at the feeding trials and could be utilized for energy sources by the goats. Nitrogen may be also effectively utilized in goats fed the MS silage, because the silage were inhibited in proteolysis during ensiling.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.9
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• pp.1269-1275
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• 2012

An experiment was conducted to study the effect of soluble protein supplements on concentration of soluble non-ammonia nitrogen (SNAN) in the liquid phase of ruminal (RD) and omasal digesta (OD) of Korean native steers, and to investigate diurnal pattern in SNAN concentration in RD and OD. Three ruminally cannulated Korean native steers in a $3{\times}3$ Latin square design consumed a basal diet of rice straw and corn-based concentrate (control), and that supplemented (kg/d DM basis) with intact casein (0.24; IC) or acid hydrolyzed casein (0.46; AHC). Ruminal digesta was sampled using a vacuum pump, whereas OD was collected using an omasal sampling system at 2.0 h intervals after a morning feeding. The SNAN fractions (free amino acid (AA), peptide and soluble protein) in RD and OD were assessed using the ninhydrin assay. Concentrations of free AA and total SNAN in RD were significantly (p<0.05) lower than those in OD. Although free AA concentration was relatively high, mean peptide was quantitatively the most important fraction of total SNAN in both RD and OD, indicating that degradation of peptide to AA rather than hydrolysis of soluble protein to peptide or deamination may be the most limiting step in rumen proteolysis of Korean native steers. Diurnal variation in peptide concentration in OD for the soluble protein supplemented diets during the feeding cycle peaked 2 h post-feeding and decreased thereafter whereas that for the control was relatively constant during the entire feeding cycle. Diurnal variation in peptide concentration was rather similar between RD and OD.

• Food Science of Animal Resources
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• v.37 no.5
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• pp.682-689
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• 2017

The aim of this study was to investigate the digestibility of different Korean Hanwoo beef cuts using an in vitro digestion model, in vitro physicochemical upper gastrointestinal system (IPUGS). The four most commonly consumed cuts - tenderloin, sirloin, brisket and flank, and bottom round - were chosen for this study. Beef samples (75 g) were cooked and ingested into IPUGS, which was composed of mouth, esophagus, and stomach, thereby simulating the digestion conditions of humans. Digested samples were collected every 15 min for 4 h of simulation and their pH monitored. Samples were visualized under a scanning electron microscope (SEM) to examine changes in the smoothness of the surface after digestion. Analysis of the amino acid composition and molecular weight (MW) of peptides was performed using reverse-phase high performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively. Following proteolysis by the gastric pepsin, beef proteins were digested into peptides. The amount of peptides with higher MW decreased over the course of digestion. SEM results revealed that the surface of the digested samples became visibly smoother. Total indispensable and dispensable amino acids were the highest for the bottom round cut prior to digestion simulation. However, the total amount of indispensable amino acids were maximum for the tenderloin cut after digestion. These results may provide guidelines for the elderly population to choose easily digestible meat cuts and products to improve their nutritional and health status.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.8
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• pp.1168-1174
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• 2017

Objective: The aim of the study was to evaluate the effects of different nitrate levels (150, 300, 450, and 600 ppm $KNO_3$) on the volatile compounds and some other properties of pastırma. Methods: Pastırma samples were produced under the controlled condition and analyses of volatile compounds, and thiobarbituric acid reactive substances (TBARS) as an indicator of lipid oxidation, non-protein nitrogenous matter content as an indicator of proteolysis, color and residual nitrite were carried out on the final product. The profile of volatile compounds of pastırma samples was analyzed by gas chromatography/mass spectrometry using a solid phase microextraction. Results: Nitrate level had a significant effect on pH value (p<0.05) and a very significant effect on TBARS value (p<0.01). No significant differences were determined in terms of $a_w$ value, non-protein nitrogenous substance content, color and residual nitrite between pastırma groups produced by using different nitrate levels. Nitrate level had a significant (p<0.05) or a very significant (p<0.01) effect on some volatile compounds. It was determined that the amounts and counts of volatile compounds were lower in the 450 and especially 600 ppm nitrate levels than 150 and 300 ppm nitrate levels (p<0.05). While the use of 600 ppm nitrate did not cause an increase in residual nitrite levels, the use of 150 ppm nitrate did not negatively affect the color of pastırma. However, the levels of volatile compounds decreased with an increasing level of nitrate. Conclusion: The use of 600 ppm nitrate is not a risk in terms of residual nitrite in pastırma produced under controlled condition, however, this level is not suitable due to decrease in the amount of volatile compounds.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.9
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• pp.1278-1284
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• 2017

Objective: To assess the potency of calcium propionate (CAP) used as silage additive, an experiment was carried out to evaluate the effect of CAP on the nitrogen transformation, fermentation quality and aerobic stability of alfalfa silages. Methods: Alfalfa was ensiled with four levels of CAP (5, 10, 15, and 20 g/kg of fresh weight [FW]) in laboratory silos for 30 days. After opening, the silages were analyzed for the chemical and microbiological characteristics, and subjected to an aerobic stability test. Results: The increasing proportion of CAP did not affect pH, lactic acid (LA) concentrations and yeast counts, while linearly decreased counts of enterobacteria (p = 0.029), molds (p<0.001) and clostridia (p<0.001), and concentrations of acetic acid (p<0.001), propionic acid (p<0.001), butyric acid (p<0.001), and ethanol (p = 0.007), and quadratically (p = 0.001) increased lactic acid bacteria counts. With increasing the proportion of CAP, the dry matter (DM) loss (p<0.001), free amino acid N (p<0.001), ammonia N (p = 0.004), and non-protein N (p<0.001) contents were linearly reduced, whereas DM (p = 0.048), water soluble carbohydrate (p<0.001) and peptide N (p<0.001) contents were linearly increased. The highest Flieg's point was found in CAP10 (75.9), represented the best fermentation quality. All silages treated with CAP improved aerobic stability as indicated by increased stable hours compared with control. Conclusion: The addition of CAP can suppress the undesirable microorganisms during ensiling and exposure to air, thereby improving the fermentation quality and aerobic stability as well as retarding the proteolysis of alfalfa silage. It is suggested that CAP used as an additive is recommended at a level of 10 g/kg FW.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.1
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• pp.139-147
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• 1999

Peptides are formed in the rumen as the result of microbial proteinase activity. The predominant type of activity is cysteine ptoteinase, but others, such as serine proteinases, are also present. Many species of protozoa, bacteria and fungi are involved in ptoteolysis; large animal-to-animal variability is found when proteinase activities in different animals are compared. The peptides formed from proteolysis are broken down to amino acids by peptidases. Different peptides are broken down at different rates, depending on their chemical composition and particularly their N-terminal structure. Indeed, chemical addition to the N-terminus of small peptides, such as by acetylation, causes the peptides to become stable to breakdown by the rumen microbial population; the microorganisms do not appear to adapt to hydrolyse acetylated peptides even after several weeks exposure to dietary acetylated peptides, and the amino acids present in acetylated peptides are absorbed from the small intestine. The amino acids present in some acetylated peptides remain available in nutritional trials with rats, but the nutritive value of the whole amino acid mixture is decreased by acetylation. The genus Prevotella is responsible for most of the catabolic peptidase activity in the rumen, via its dipeptidyl peptidase activities, which release dipeptides rather than free amino acids from the N-terminus of oligopeptides. Studies with dipeptidyl peptidase mutants of Prevotella suggest that it may be possible to slow the rate of peptide hydrolysis by the mixed rumen microbial population by inhibiting dipeptidyl peptidase activity of Prevotella or the rate of peptide uptake by this genus. Peptides and amino acids also stimulate the growth of rumen microorganisms, and are necessary for optimal growth rates of many species growing on tapidly fermented substrates; in rich medium, most bacteria use pre-formed amino acids for more than 90% of their amino acid requirements. Cellulolytic species are exceptional in this respect, but they still incorporate about half of their cell N from pre-formed amino acids in rich medium. However, the extent to which bacteria use ammonia vs. peptides and amino acids for protein synthesis also depends on the concentrations of each, such that preformed amino acids and peptides are probably used to a much lesser extent in vivo than many in vitro experiments might suggest.

• KSBB Journal
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• v.16 no.3
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• pp.237-240
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• 2001

Epithelial cell migration plays an important role in many physiological processes such as morphogenesis and wound healing, and cell mobility requires the release of the cell from its adhesion site. This is directed, at least in part, by limited proteolysis of matrix molecules by matrix metalloproteinases (MMPs). MMPs are zinc-dependent proteases produced by a variety of cell types, and have a fundamental role in tissue remodelling, tumour invasion and metastasis. In addition, the ability of cells to mediate fibrinolytic agent, plasmin. The purpose of this study was to test if vascular endothlial growth factor (VEGF) can regulate the production of MMPs and plasmin by keratinocyte cells. Supernatants from a human keratinocyte cell line grown in the presence or absence of VEGF (10ng/mL) produced ?2.5 fold increases in cell proliferation, and ?3.0 fold increses in MMP-2 and plasmin levels. Our results suggest that VEGF may modulate keratinocyte cell proliferating activity by increasing the abundance of MMP-2 and plasmin, and indicates a role for VEGF in the regulation of keratinocyte behaviour in wound healing and tissue remodelling.

• Journal of the Korean Society of Food Science and Nutrition
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• v.22 no.3
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• pp.334-339
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• 1993

Antitumor activities of tannin extract and chloroform fraction extract from the persimmon leaves, and 2, 4-decadienal identified as an antimutagenic compound in persimmon leaves were examined in sarcoma 180 implanted tumor in mice by using both light and transmission electron microscopes. Among them, tannin extracted from the persimmon leaves delayed the progression of malignant tumor but the other two did not show any noticeable effect. The antitumorigenic activity of tannin extract might not come from the selective cytotoxicity against tumor cells, but might be, in an anaerobic environment, from the inhibitory action against oncogenic protein synthesis or from the proteolysis of the preformed oncogenic proteins by autophagocytic granules. therefore, the tannin from persimmon leaves might protect cells from fast progression of malignant tumorigensis.