Streptococcal cysteine proteinase releases kinins: a virulence mechanism.

Previous work has indicated a crucial role for the extracellular cysteine proteinase of Streptococcus pyogenes in the pathogenicity and virulence of this important human pathogen. Here we find that the purified streptococcal cysteine proteinase releases biologically active kinins from their purified precursor protein, H-kininogen, in vitro, and from kininogens present in the human plasma, ex vivo. Kinin liberation in the plasma is due to the direct action of the streptococcal proteinase on the kininogens, and does not involve the previous activation of plasma prekallikrein, the physiological plasma kininogenase. Judged from the amount of released plasma kinins the bacterial proteinase is highly efficient in its action. This is also the case in vivo. Injection of the purified cysteine proteinase into the peritoneal cavity of mice resulted in a progressive cleavage of plasma kininogens and the concomitant release of kinins over a period of 5 h. No kininogen degradation was seen in mice when the cysteine proteinase was inactivated by the specific inhibitor, Z-Leu-Val-Gly-CHN2, before administration. Intraperitoneal administration into mice of living S. pyogenes bacteria producing the cysteine proteinase induced a rapid breakdown of endogenous plasma kininogens and release of kinins. Kinins are hypotensive, they increase vascular permeability, contract smooth muscle, and induce fever and pain. The release of kinins by the cysteine proteinase of S. pyogenes could therefore represent an important and previously unknown virulence mechanism in S. pyogenes infections.

Cheddar cheesemaking from whole milk concentrated by ultrafiltration and heated to 90 °C

SummaryCheddar cheeses were made from milks concentrated approximately 2·3-and 3·4-fold by ultrafiltration, then heated at 90 °C for 15 s. They were compared with cheeses made from pasteurized unconcentrated milks and unheated 5·3-fold concentrates. The recoveries of fat and protein were comparable in the cheeses from heated 2·3-fold concentrates and unheated 5·3-fold concentrates and both were higher than the controls. However, fat losses were high in curds from 3·4-fold heated concentrates. The use of paired fast and slow starter strains and coagulation at 26 °C optimized the curd composition. Proteolysis and flavour development were only slightly reduced in all the cheeses from concentrated milks, perhaps aided by higher starter levels in the curd than in the controls. The structures of the curds from the heated concentrated milks were coarser than those of the controls and the curd particles fused poorly. This may explain the grainy, dry, crumbly texture in the cheeses from the heated concentrates. The texture was not improved by adding a bacterial proteinase to the curd.

Laboratory simulations of a commercial UHT treatment using an oil bath and a programmable, electronically temperature-controlled oven

SummaryHeat stability of secreted bacterial proteinase is normally assessed using an oil bath test in which the sample is heated at a temperature and time representing the commercial UHT treatment of milk. The temperature/time (T/t) profiles of the oil bath and the commercial UHT process being represented are not taken into account. Two laboratory procedures were used to simulate a commercial UHT process in which milk was heated to 140°C and held for 3 s in a Spiroflo heat exchanger. They consisted of a conventional oil bath test and the use of a programmable, electronically temperature-controlled oven.T/tprofiles were established for each heat treatment. TheT/tprofile of the oil bath test was predicted accurately using the governing heat transfer equation.B*andC*values, which measure the severity of a heat process, were calculated from theT/tprofiles and used to compare the three different heat treatments.B*andC*values of 4·95 and 1·92 respectively were calculated for the Spiroflo heat exchanger. An oil bath test, in which the sample was immersed in a bath at 136°C for 73 s, gave approximately the sameB*andC*values as calculated for the Spiroflo heat exchanger.B*andC*values of 5·01 and 1·73 respectively were calculated for the oven procedure. The oven test gave the better laboratory simulation of the Spiroflo UHT treatment. Despite the slight difference in each of theB*andC*values between the oven and the Spiroflo, theT/tprofile of the oven test closely resembled that of the Spiroflo. TheT/tprofile of the oil bath was completely dissimilar. Although it was possible to use an oil bath test to replicate simultaneously both theB*andC*values of this particular UHT treatment, this would not be the case for other commercial processes.

Production of extracellular lipases and proteinases during prolonged growth of strains of psychrotrophic bacteria in whole milk

SummaryThree strains of psychrotrophicPseudomonasspp., each with different lipolytic and proteolytic phenotypes (including a proteinase-deficient mutant), were cultured separately in whole milk at 7°C. Growth rates were the same during logarithmic growth phase, but during early stationary phase the cell densities were related to the activities of lipase and proteinase in the cultures. Only one strain underwent pronounced death phase. Proteinase activity was not detected in the culture of the proteinase-deficient mutant, but in those of the other strains it increased to a plateau, or continued to increase linearly. Lipase activity of the culture of each strain reached a peak in early stationary phase; in late stationary phase activity was highest for the proteinase-deficient mutant strain where degradation of lipase by bacterial proteinase would have been least. The ability of psychrotrophic bacteria both to survive for long periods and to produce high levels of proteinases and lipases on prolonged incubation in milk emphasizes the spoilage potential arising from psychrotrophic bacteria in inadequately cleaned dairy equipment.

Effect of milk pretreatment and making conditions on the properties of Cheddar cheese from milk concentrated by ultrafiltration

SUMMARYCheddar cheeses were made from milks concentrated 3- to 6-fold by ultrafiltration. Modifications to the conventional cheesemaking process were designed to obtain the correct composition and pH value in the final curd, improve the fat and moisture retention and cheese texture, and increase the ripening rate. The compositions of all cheeses were similar to those of controls made with unconcentrated pasteurized milk. The curd and cheese structures and the textural properties of the cheeses were closer to those of the controls when made from milk concentrated 5-fold than when made from milk concentrated 3-fold. Light homogenization of the milk before concentration led to improved fat recovery and decreased graininess and crumbliness in the cheese. Lowering the coagulation temperature of concentrated milks gave improved curd structure and less grainy and more sticky cheeses. Addition of the bacterial proteinase, Neutrase, with the salt, stimulated proteolysis and flavour production in the cheeses and made them more sticky. Neither reduction of the mineral levels in the milk by acidification before concentration nor reduction of the lactose concentration by diafiltration had any significant effect on the composition or textural properties of the cheeses.