The inactivation reaction of the proteinase of a P. fluorescens strain of biotype I in milk was investigated at 130-150 degrees C, also in milk and in buffer with and without added CaCl2 at temperatures below 100 degrees C. The decline in activity corresponded to first order kinetics in the UHT region; Ea = 115 kJ/mol. D values were 290 (130 degrees C), 124 (140 degrees C) and 54 s (150 degrees C); therefore, the usual temperature time combinations of UHT treatment are not sufficient to achieve the required rates of inactivation. At temperatures below 80 degrees C, inactivation corresponded increasingly to second order kinetics with considerably higher reaction rates; at 55 degrees C, an inactivation reaction corresponding to that induced by UHT treatment could be achieved at a thermal stress lower by a factor of 500. This "low temperature inactivation" was observed in a further 20 strains representing the spectrum of P. fluorescens. The average rates of inactivation following heat treatment in milk for 20 min are 47% at 55 degrees C and 44% at 60 degrees C. This can be regarded as the most effective temperature range for the inactivation of the proteinases in milk. Clear connections can be seen between the biotype groups and the optimum temperature for inactivation: biotype group I ca. 55 degrees C, group II (with a few exceptions) less than or equal to 50 degrees C and group III greater than or equal to 60 degrees C. The inactivation reaction is systematically influenced by the proteins and Ca++ ions present in milk.
Heat inactivation of a metalloproteinase, isolated from Pseudomonas fluorescens biotype I strain 112, was investigated in the temperature ranges 50-60 °C and 90-140 °C. At 90 °C the denaturation of the enzyme followed first-order kinetics with a decimal reduction time of 110 min and a velocity constant K of 35 x 10~4 s" 1 . Activation energy E a was 100 kJ/mol for this temperature range. In the 50-60 °C region the proteinase was inactivated by autolysis, as shown by electrophoresis and gel filtration. At 55 °C the decimal reduction time was ~ 22 s, at 57 °C it was 8 s. Rapid inactivation at 55 °C was only possible if the enzyme was heated from lower temperatures, but not if cooled down from 90 °C. This is due to a conformational change of the protein at this temperature. A model for the description of heat inactivation in the two temperature ranges is proposed.
The inactivation of a metalloproteinase from Pseudomonas fluorescens Biotype I with EDTA was investigated at 22 degrees C and 37 degrees C. At 22 degrees C proteolytic activity decreases linearly with time and an inactive apoenzyme is obtained by dialysis. Proteolytic activity can be restored with several metal-ions, Ca2+, Zn2+, Mg2+, Sr2+ and co2+ give the best results. Activity and substrate specificity are influenced by the metal-ions. Reactivation depends on the concentration of the metal-ions, optimum concentration is 1 mM for Ca2+ and 50 microM for Zn2+. The isoelectric point of the apoenzyme is around 8.0, this is about 0.3 pH-units lower than the isoelectric point of the native proteinase. At 37 degrees C inactivation follows first order kinetics and is irreversible because of autolysis as shown by a gel filtration-experiment.
Proteolytic activity of psychotrophie bacteria in milk can be measured in a practical and comparatively sensitive manner using Azocasein as a substrate. The necessary parameters were exemplarily developed and demonstrated with a typical, strongly proteolytic strain of Pseudomonas fluorescens (No. 112). Comparison of the improved Azocasein method with a modified version of the HPA method of Cliffe and Law--they had proposed it for predicting the stability of UHT milk--showed nearly the same sensitivity with six test strains of Pseudomonas fluorescens. The first significant detection of proteolytic activity could be made approximately at the same time except with one strain of the biotype II (miscellaneous strains). It is discussed, whether the demonstrated methods can be judged (by the bacterial counts of detection) as sufficiently sensitive for to prediction of term spoilage during storage of UHT products.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.