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Differential Scanning Calorimetry and Fluorescence Spectroscopy as Indicators of Alkaline Inactivation of Porcine Pepsin
DIFFERENTIAL SCANNING CALORIMETRY AND FLUORESCENCE SPECTROSCOPY AS INDICATORS OF ALKALINE INACTIVATION OF PORCINE PEPSIN. M.G. Wynne* and R.Y. Yada, Department of Food Science, University of Guelph, Guelph, Ontario NIG 2WI. . Critical to the use of pepsin as a milk-clotting enzyme is a knowledge of its denaturation characteristics. The activity, fluorescence scans and thermal properties of porcine pepsin were assessed over the pH range 5.5-7.0. Enzymatic activity decreased significantly (p<0.05) with increased pH. Fluorescence emission scans exhibited significant increases (p<0.05) in maximum wavelength and decreases (p < 0.05) in fluorescence intensity with increased pH. Differential scanning calorimetry showed that the temperature of denaturation and enthalpy both decreased over the pH values 5.5, 6.0 and 6.5, but increased at pH 7.0. The results were attributed to pH-induced conformational changes in pepsin. STUDIES ON OXIDATIVE DEAMINASE ACTIVITY OF FRENCH BEAN (PHASEOLUS VULGARIS) SEEDS. S. Kermasha*, I. Alii and M. Metche, Department of Food Science and Agricultural Chemistry, Macdonald College of McGiII University, Ste. Anne de Bellevue, Quebec H9X lCO. An active oxidative deaminase enzyme was extracted from seeds of the French bean (Phaseolus vulgaris). The enzyme was purified by precipitation with ammonium sulfate (35-65070 saturation) then with ethanol (10-36%) before being chromatographed on a phosphocellulose column. Electrophoresis of purified enzyme protein showed that the enzyme separated into one major and one minor band. A chromogenic reaction on the polyacrylamide gel demonstrated that the enzyme activity is located on the major band. The purified enzyme demonstrated higher specificity for amino acid than for monoamine. The pH for optimum activity of the enzyme was 7.4. Oxidative deaminase activity was determined in French bean seed prior to germination and 2, 4, 6, 50, and 80 days after germination. The enzyme activity was absent in the ungerminated mature dry seed, but it increased dramatically during the early stages of germination then decreased with time.
THE EFFECT OF TEMPERING ON THE PHYSICAL PROPER· TIES OF SHORTENING. C. Moziar*, J.M. deMan and L. deMan, Department of Food Science, University of Guelph, Guelph, Ontario NIG 2WI. A study was conducted to determine physical changes in shortening due to tempering. Shortening was tempered at 23°C, 26.5°C, 30°C, and 10°C for 2 days and 9 days, and then stored at 23°C. Solid fat content was measured using pulse NMR. Polarized light microscopy was used to monitor crystal growth. The polymorphic transitions were followed by x-ray diffraction and quantified using Soft Laser Scanning Densitometry. The change in melting behaviour was evaluated by Differential Scanning Calorimetry. Results indicate that transitions of (3' to (3-polymorphic forms can be delayed as a result of tempering. Firmness and hardness were evaluated using Instron and cone penetrometer, respectively. Tempering appears to influence the degree to which firmness or hardness increases.
CATALYSIS OF LIPID OXIDATION BY POULTRY MEAT EXTRACTS. J.W. MacNeill, G. Mahaffy and Y. Kakuda*, Department of Food Science. University of Guelph, Guelph, Ontario NIG 2WI. A model system consisting of a monolayer of linoleic acid adsorbed to silica was developed to study the catalytic properties of chicken meat extracts. The extracts were prepared using mechanically separated meats from necks and backs, frames, hand deboned whole breast and thigh. Oxidation rates were monitored by measuring the depletion of oxygen in the silica mixture with a Gilson Oxygraph. Log % oxygen remaining vs time data produced linear plots and first order rate constants were calculated. The extracts from mechanically separated meats (frames, necks/back) had greater catalytic activities than those extracts prepared from hand deboned and ground meats. Total heme was found to strongly correlate with the monolayer oxidation rate. Activation energies were found to range from 17 to 20 Kcal/mole. Heat treatment of the extract decreased the oxidation rate by 71 % compared to 64% for pure myoglobin heated in a similar manner. Treatment of the extracts with saturating levels of CO had no effect on their catalytic activities while the addition of a commercial antioxidant preparation reduced the rates by 80%. The addition of free iron had no effect on the rates at levels below 60 ppm.
TRYPTIC HYDROLYSIS OF CRYSTALLINE AND NONCRYSTALLINE PROTEINS FROM PHASEOLUS BEANS. I. AIIi*, Z. Li and S. Kermasha, Department of Food Science and Agricultural Chemistry, Macdonald College of McGiII University, Ste. Anne de Bellevue, Quebec H9X ICO. Crystalline proteins isolated from citric acid extracts and noncrystalline proteins from sodium hydroxide extracts of Phaseolus beans were subjected to in vitro tryptic hydrolysis. There was no relationship between the degree of hydrolysis as indicated by the liberation of amino acids, and the microstructure of the proteins. However, when benzoyl DL-arginine-p-nitroanilide was used as substrate for trypsin inhibitory activity, the non-crystalline proteins showed considerably higher tryptic inhibitory activity than the crystalline proteins. Sodium dodecyl sulphate-polyacrylamide disc gel electrophoresis also indicated differences in the behaviour during tryptic hydrolysis between the crystalline and non-crystalline proteins. The results suggest a relationship between the biological properties of the proteins and the microstructure of the proteins.
KINETIC MODELLING OF THE HARDENING PHENOMENA IN BEANS. A. Hohlberg* and J.M. Aguilera, Departamento de Ingenieria Quimica, Pontificia Universidad Cat61ica de Chile, P.O. Box 6177, Santiago-Chile. Hardening is a serious defect that happens in pulses stored for prolonged periods at high humidities and temperatures. It is caused by an increase in the resistance of the middle lamella to soften upon cooking. Several mechanisms have been proposed to explain hardening, most of them due to a combination of enzymatic reactions, diffusion processes and polymerization. Kinetic models were developed for these mechanisms including the effect of the temperature and water activity. Models were adjusted with data obtained from black beans stored for more than a year at temperatures between 4 and 40°C and water activities from 0.4 to 0.7.
AN ELECTRON PARAMAGNETIC RESONANCE AND DIFFERENTIAL SCANNING CALORIMETRY STUDY OF THE THERMOSTABILlTY OF SOME FUNGAL RENNETS. E.D. Brown* and R.Y. Yada, Department of Food Science, University of Guelph, Guelph, Ontario NIG 2WI. The technological suitability of an aspartic proteinase to the cheese-making process is determined, in part, by the thermostability of the enzyme. Spin labelling electron paramagnetic resonance (EPR) and differential scanning calorimetry (DSC) were employed to study the thermostability of proteinases from Endothia parasitica (EP) and Mucor miehei (MM). DSC revealed peak denaturation temperatures of 70° and 81°C and activation energies of 252 kJ/mo and 354 kJ/mol for EP and MM, respectively. Spin labelling experiments to study the thermally induced unfolding of these enzymes confirmed DSC work, the proteinase from MM demonstrating substantially greater stability than that from EP.
USE OF FISHERIES BYPRODUCTS AS NUTRIENT SUPPLEMENTS IN FERMENTATION PROCESSES. A.M. Martin* and E.C. Bassler, Food Science Program, Department of Biochemistry, Memorial University of Newfoundland, SI. John's, Newfoundland AIB 3X9. Fish meal and fish oil were utilized to supplement the nutrient content of acid-extracts from peat employed as substrate in the submerged culture of the edible mushroom mycelium Pleurotus ostreaIus. The operating conditions employed were: a pH of 5.0, a temperature of 28°C, an agitation speed of 150 rpm, an inoculum ratio of 5% (v/v). In eight days of fermentation, the best results were obtained with peat extract supplemented with yeast extract and fish oil. The results are reported in terms of dry biomass concentration, yield (g dry biomass concentration per g of substrate consumed), and efficiency (g of biomass concentration per g of initial substrate).
368 / Abstract
J. Inst. Can. Sci. Technol. Aliment. Vol. 21, No. 4, 1988
THE EFFECT OF TEMPERING ON THE PHYSICAL PROPER· TIES OF SHORTENING. C. Moziar*, J.M. deMan and L. deMan, Department of Food Science, University of Guelph, Guelph, Ontario NIG 2WI. A study was conducted to determine physical changes in shortening due to tempering. Shortening was tempered at 23°C, 26.5°C, 30°C, and 10°C for 2 days and 9 days, and then stored at 23°C. Solid fat content was measured using pulse NMR. Polarized light microscopy was used to monitor crystal growth. The polymorphic transitions were followed by x-ray diffraction and quantified using Soft Laser Scanning Densitometry. The change in melting behaviour was evaluated by Differential Scanning Calorimetry. Results indicate that transitions of (3' to (3-polymorphic forms can be delayed as a result of tempering. Firmness and hardness were evaluated using Instron and cone penetrometer, respectively. Tempering appears to influence the degree to which firmness or hardness increases.
CATALYSIS OF LIPID OXIDATION BY POULTRY MEAT EXTRACTS. J.W. MacNeill, G. Mahaffy and Y. Kakuda*, Department of Food Science. University of Guelph, Guelph, Ontario NIG 2WI. A model system consisting of a monolayer of linoleic acid adsorbed to silica was developed to study the catalytic properties of chicken meat extracts. The extracts were prepared using mechanically separated meats from necks and backs, frames, hand deboned whole breast and thigh. Oxidation rates were monitored by measuring the depletion of oxygen in the silica mixture with a Gilson Oxygraph. Log % oxygen remaining vs time data produced linear plots and first order rate constants were calculated. The extracts from mechanically separated meats (frames, necks/back) had greater catalytic activities than those extracts prepared from hand deboned and ground meats. Total heme was found to strongly correlate with the monolayer oxidation rate. Activation energies were found to range from 17 to 20 Kcal/mole. Heat treatment of the extract decreased the oxidation rate by 71 % compared to 64% for pure myoglobin heated in a similar manner. Treatment of the extracts with saturating levels of CO had no effect on their catalytic activities while the addition of a commercial antioxidant preparation reduced the rates by 80%. The addition of free iron had no effect on the rates at levels below 60 ppm.
TRYPTIC HYDROLYSIS OF CRYSTALLINE AND NONCRYSTALLINE PROTEINS FROM PHASEOLUS BEANS. I. AIIi*, Z. Li and S. Kermasha, Department of Food Science and Agricultural Chemistry, Macdonald College of McGiII University, Ste. Anne de Bellevue, Quebec H9X ICO. Crystalline proteins isolated from citric acid extracts and noncrystalline proteins from sodium hydroxide extracts of Phaseolus beans were subjected to in vitro tryptic hydrolysis. There was no relationship between the degree of hydrolysis as indicated by the liberation of amino acids, and the microstructure of the proteins. However, when benzoyl DL-arginine-p-nitroanilide was used as substrate for trypsin inhibitory activity, the non-crystalline proteins showed considerably higher tryptic inhibitory activity than the crystalline proteins. Sodium dodecyl sulphate-polyacrylamide disc gel electrophoresis also indicated differences in the behaviour during tryptic hydrolysis between the crystalline and non-crystalline proteins. The results suggest a relationship between the biological properties of the proteins and the microstructure of the proteins.
KINETIC MODELLING OF THE HARDENING PHENOMENA IN BEANS. A. Hohlberg* and J.M. Aguilera, Departamento de Ingenieria Quimica, Pontificia Universidad Cat61ica de Chile, P.O. Box 6177, Santiago-Chile. Hardening is a serious defect that happens in pulses stored for prolonged periods at high humidities and temperatures. It is caused by an increase in the resistance of the middle lamella to soften upon cooking. Several mechanisms have been proposed to explain hardening, most of them due to a combination of enzymatic reactions, diffusion processes and polymerization. Kinetic models were developed for these mechanisms including the effect of the temperature and water activity. Models were adjusted with data obtained from black beans stored for more than a year at temperatures between 4 and 40°C and water activities from 0.4 to 0.7.
AN ELECTRON PARAMAGNETIC RESONANCE AND DIFFERENTIAL SCANNING CALORIMETRY STUDY OF THE THERMOSTABILlTY OF SOME FUNGAL RENNETS. E.D. Brown* and R.Y. Yada, Department of Food Science, University of Guelph, Guelph, Ontario NIG 2WI. The technological suitability of an aspartic proteinase to the cheese-making process is determined, in part, by the thermostability of the enzyme. Spin labelling electron paramagnetic resonance (EPR) and differential scanning calorimetry (DSC) were employed to study the thermostability of proteinases from Endothia parasitica (EP) and Mucor miehei (MM). DSC revealed peak denaturation temperatures of 70° and 81°C and activation energies of 252 kJ/mo and 354 kJ/mol for EP and MM, respectively. Spin labelling experiments to study the thermally induced unfolding of these enzymes confirmed DSC work, the proteinase from MM demonstrating substantially greater stability than that from EP.
USE OF FISHERIES BYPRODUCTS AS NUTRIENT SUPPLEMENTS IN FERMENTATION PROCESSES. A.M. Martin* and E.C. Bassler, Food Science Program, Department of Biochemistry, Memorial University of Newfoundland, SI. John's, Newfoundland AIB 3X9. Fish meal and fish oil were utilized to supplement the nutrient content of acid-extracts from peat employed as substrate in the submerged culture of the edible mushroom mycelium Pleurotus ostreaIus. The operating conditions employed were: a pH of 5.0, a temperature of 28°C, an agitation speed of 150 rpm, an inoculum ratio of 5% (v/v). In eight days of fermentation, the best results were obtained with peat extract supplemented with yeast extract and fish oil. The results are reported in terms of dry biomass concentration, yield (g dry biomass concentration per g of substrate consumed), and efficiency (g of biomass concentration per g of initial substrate).
368 / Abstract
J. Inst. Can. Sci. Technol. Aliment. Vol. 21, No. 4, 1988