Thermal stability of an extracellular proteinase from Pseudomonas fluorescens AFT 36

A metalloproteinase, isolated from a shaken milk culture of Pseudomonas fluorescens AFT 36 by chromatography in DEAE and CM-cellulose and Sephadex G-150, was very unstable in 0.1 M-phosphate buffer, pH 6.6, being completely denatured above 70 degrees C in 1 min. It was also unstable in a Ca-containing buffer (synthetic milk salts, SMS) between 50 and 60 degrees C (minimum at 55 degrees C), but stability was very high above 80 degrees C in this buffer. D-values were determined at 10 degrees C intervals in the range 70-150 degrees C in SMS from which a Z value of 31.9 degrees C and an Ea of 8.82 X 10(4) J mol-1 were calculated; the half-life at 150 degrees C was 9 s. Instability at 55 degrees C was due to autolysis as evidenced by gel electrophoresis, gel filtration and increase in 2,4,6-trinitrobenzenesulphonic acid-reactive amino groups. The extent of inactivation experienced at 80 degrees C was inversely related to the rate of heating to 80 degrees C, i.e. length of time spent in the neighbourhood of 55 degrees C. Addition of increasing concentrations of caseinate substrate reduced inactivation of the enzyme at 55 degrees C, presumably due to substrate binding. Attempts to stabilize the enzyme at 55 degrees C by addition of EDTA or by adjusting the reaction pH to 4.2, at which the enzyme has little proteolytic activity, were unsuccessful, although autolysis was prevented. Unlike the proteinase from Ps. fluorescens MC 60, AFT 36 proteinase did not inactivate itself on cooling to 55 degrees C from 80, 100 or 150 degrees C, but did regain autolytic activity on cooling to below 50 degrees C to an extent dependent on the duration of holding at the lower temperature. It is suggested that on heating to approximately 55 degrees C, a conformational change occurs which renders the enzyme susceptible to proteolysis by still active enzyme; at higher temperatures the enzyme, although susceptible to autolysis, is inactive; an active conformation is restored on cooling to below 50 degrees C.     

Effect of dissolved carbon dioxide on thermal inactivation of microorganisms in milk

Postpasteurization addition of CO2 inhibits growth of certain microorganisms in dairy products, but few workers have investigated the effect of CO2 on the thermal inactivation of microorganisms during pasteurization. Concentrations of CO2 ranging from 44 to 58 mM added to raw whole milk significantly (P < 0.05) reduced the number of surviving standard plate count (SPC) organisms in milk heated over the range of 67 to 93 degrees C. A decrease in thermal survival rates (D-values) for Pseudomonas fluorescens R1-232 and Bacillus cereus ATCC 14579 spores in milk was positively correlated with CO2 concentrations (1 to 36 mM). D(50 degrees C)-values for P. fluorescens significantly decreased (P < 0.05) in a linear fashion from 14.4 to 7.2 min. D(89 degrees C)-values for B. cereus spores were significantly (P < 0.05) decreased from 5.56 min in control milk to 5.29 min in milk containing 33 mM CO2. The Weibull function was used as a model to describe the thermal inactivation of P. fluorescens, B. cereus spores, and SPC organisms in raw milk. Nonlinear parameters for the Weibull function were estimated, and survival data fitted to this model had higher R2 values than when fitted to the linear model, further providing support that the thermal inactivation of bacteria does not always follow first-order reaction rate kinetics. These results suggest that CO2 could be used as a processing aid to enhance microbial inactivation during pasteurization.     

Determination of thermal inactivation kinetics of microorganisms with a continuous microflow apparatus

Use of a continuous microflow submerged microcoil (CSMC) apparatus was compared with the capillary tube (CT) method for measuring the thermal inactivation kinetics of Pseudomonas fluorescens at 61 degrees C for 3 to 29 s. Inocula were continuously pumped through a microbore (< or = 0.0762 cm inside diameter) thin-walled stainless steel capillary tube submerged in a heated oil bath. The heating time was set by changing the flow rate, tube dimensions, or both. With the use of microthermo-couples, the time for the inocula to reach within 1 degree C of the set temperature was <3 s, and shorter than that with capillary tubes or vials. Inactivation curves (61 degrees C) for P. fluorescens prepared by the CSMC method were not different from curves prepared by the CT method, as determined by analysis of variance (P > 0.05). Inactivation of Bacillus cereus spores (105 degrees C) and native microflora found in raw milk (72 degrees C) over heating times of 3 to 42 s were determined by CSMC. CSMC can measure thermal inactivation kinetics of microorganisms efficiently and simply at high temperatures and in short times. Survivors can be enumerated in 1-ml volumes of heat-treated samples, making it useful for determining inactivation kinetics of low numbers of microorganisms, such as those found in high-quality raw milk. Inactivation kinetics were generally more accurately described by the Weibull function (R2 > or = 0.97) than the linear kinetic model.     

Effect of temperature history on the growth of Listeria monocytogenes Scott A at refrigeration temperatures

The effect of pre-inoculation temperature on the subsequent growth of Listeria monocytogenes Scott A at 5 degrees C was examined in microbiological medium, UHT milk, canned dog food, and raw ground beef (untreated and irradiation-sterilized). In microbiological medium, the duration of the lag phase was decreased when aerobic and anaerobic cultures were initially grown at less than or equal to 28 and less than or equal to 13 degrees C, respectively. Subsequent exponential growth rates and maximum population densities of the 5 degrees C cultures were not affected by temperature history. Differences in lag phase durations were also observed when L. monocytogenes initially cultured at 19 and 37 degrees C were grown at 5 degrees C in UHT milk and some of the canned dog food varieties. Growth of L. monocytogenes was not observed in either untreated or irradiation-sterilized raw ground beef. While temperature history can affect the growth kinetics of L. monocytogenes at 5 degrees C, it did not account for the lack of growth in raw meat, suggesting that there is an inhibitory condition or component in ground beef that is lost upon cooking.     

Effects of high pressure homogenisation of raw bovine milk on alkaline phosphatase and microbial inactivation. A comparison with continuous short-time thermal treatments

Raw whole milk of high microbial quality (58 degrees C), but markedly decreased above 200 MPa when Tin=24 degrees C (T2>60 degrees C). In contrast to inactivation induced by continuous short-time thermal treatments, ALP inactivation induced by HP homogenisation was clearly due to mechanical forces (shear, cavitation and/or impact) in the HP valve and not to the short (<1 s) residence time at temperature T2 in the same valve. Inactivation of the three exogenous microorganisms led to similar conclusions. Homogenisation at 250 MPa or 300 MPa (Tin=24 degrees C) induced a 2-3 log cycle reduction of the total endogenous milk flora and a 1.5-1.8 log cycle reduction of inoculated List. innocua. Higher reduction ratios (2-4 log cycles) were obtained for the two other microorganisms. The highest levels of ALP inactivation corresponded to the highest extents of microbial reduction. Running the milk twice or three times through the homogeniser (recycling), keeping temperature T1 approximately 29 degrees C and pressure=200 MPa, increased homogenisation efficiency.     

High pressure inactivation of microorganisms inoculated into ovine milk of different fat contents

High hydrostatic pressure inactivation of Escherichia coli, Pseudomonas fluorescens, Listeria innocua, Staphylococcus aureus, and Lactobacillus helveticus were studied. These microorganisms were inoculated at a concentration between 10(7) and 10(8) cfu/ml in Ringer solution and in ovine milk adjusted to 0, 6, and 50% fat content to evaluate the baroprotective effect of fat content on inactivation of microorganisms. Treatments of pressurization consisted of combinations of pressure (100 to 500 MPa) and temperature (4, 25, and 50 degrees C) for 15 min. Gram-negative microorganisms were more sensitive than were Gram-positive ones (more destruction P. fluorescens > E. coli > or = List. innocua > Lb. helveticus > S. aureus). Pressurizations at low temperature (4 degrees C) produced greater inactivation on P. fluorescens, List. innocua, and Lb. helveticus than at room temperature (25 degrees C), whereas for E. coli and S. aureus the results were opposite. Ovine milk per se (0% fat) showed a baroprotective effect on all microorganisms, but percentage of fat (6 and 50%) did not show a progressive baroprotective effect in all pressurization conditions or for all microorganisms.     

Phenotypic and genotypic characterization of Pseudomonas fluorescens isolated from bulk tank milk.

Pseudomonas fluorescens isolates (n = 55) isolated from farm bulk tank milk (n = 55) from dairy herds in eastern South Dakota and western Minnesota were examined for phenotypic (biotype, proteolytic, and lipolytic profiles) and genotypic (plasmid profiles and 16S-23S PCR ribotypes) characteristics. The observed phenotypic and genotypic characteristics were used to conduct phylogenetic analysis. Pseudomonas fluorescens belonged to 28 API 20 NE biotypes and 14 proteolytic and lipolytic profiles. It was observed that 80, 91, and 58% of the isolates were proteolytic at 7, 22, and 32 degrees C, respectively. Only 7, 44, and 7% of the isolates were lipolytic at the same three temperatures. Pseudomonas fluorescens was more likely to produce proteinases at 7 and 22 degrees C and lipases at 22 degrees C. Only 9 of 55 isolates of P. fluorescens harbored plasmids. This small percentage of plasmid-bearing isolates provided insufficient data for inferences related to the distribution of plasmid-bearing clonal types. Based on 16S-23S PCR ribotyping, P. fluorescens belonged to 14 subtypes. The 16S-23S PCR ribotyping technique allowed differentiation between strains; however, it did not concur with the biotypes and proteolytic and lipolytic profiles. Use of biotypes in conjunction with proteolytic and lipolytic profiles might have practical value for conducting trace-back studies related to P. fluorescens. Based on phylogenetic analysis, it was inferred that for the given geographical area and time period, P. fluorescens isolated from farm bulk tank milk consists of a large heterogeneous group of organisms.     

Kinetics of the thermal inactivation of the Lactococcus lactis bacteriophage P008

The thermal resistance of the lactococcal bacteriophage P008 was investigated between 55 and 80 degrees C. Inactivation kinetics revealed an order of reaction above 1 and could be determined by a non-1st-order regression model. Phage inactivation was influenced by the medium (milk and Ca-M17-broth). Within the investigated temperature range, milk had a protective effect on phage P008. This was reflected in the rate constant and in the activation energy. Thermal phage inactivation studies reported in literature were re-analysed using non-1st-order regression. The obtained kinetic parameters showed that phage P008 belongs to the most heat resistant lactococcal phages investigated so far.     

Saccharomyces cerevisiae thermal inactivation kinetics combined with ultrasound.

Inactivation kinetics of Saccharomyces cerevisiae during thermal treatments at moderate temperatures (45.0, 47.5, 50.0, 52.5, or 55.0 degrees C) combined with application of 20 kHz of ultrasound were evaluated. S. cerevisiae inactivation under the combined effects of heat and ultrasound followed first-order reaction kinetics, with decimal reduction times (D) that varied from 22.3 to 0.8 min. D values in treatments that combined heat and ultrasound were significantly smaller (P < 0.05) than D values obtained for thermal treatments and were more noticeable at temperatures below 50 degrees C. The dependence of the D value on temperature had a significantly (P < 0.05) greater z value for combined treatments. Yeast heat inactivation kinetics revealed decreased thermal resistance caused by ultrasound.     

The effect of nisin on the keeping quality of reduced heat-treated milks

Milk was subjected to a combination process involving reduced heat treatment (RHT) of 117 degrees C for 2 s and nisin (75 and 150 IU ml(-1)). The microbial activity and other quality aspects were compared with a RHT control (without nisin) and with a ultrahigh temperature (UHT) milk processed at 142 degrees C for 2 s. Nisin was found to inhibit microbial growth for products stored without refrigeration, and RHT-nisin samples stored at 30 degrees C showed very low spoilage rates during 150 days, although not low enough to satisfy requirements for commercial sterility. RHT-nisin samples could be distinguished from and were preferred to the UHT control. Significant browning occurred during storage at 30 degrees C and above but was less in the RHT-nisin milk samples compared with the UHT milk. In RHT-nisin milk samples stored at 20 and 10 degrees C, no microbial activity could be detected in most samples after storage for 1 year. The effectiveness of this combination of RHT, nisin, and low storage temperatures against gram-positive spore-forming bacteria suggests potential for use of nisin in extended shelf life products.     

INACTIVATION OF SELECTED MICROORGANISMS AND PROPERTIES OF PULSED ELECTRIC FIELD PROCESSED MILK

Raw skim milk and ultra-high-temperature (UHT) skim milk inoculated with Pseudomonas fluorescens, Lactococcus lactis and Bacillus cereus were processed by pulsed electric field (PEF) treatment. A continuous PEF bench scale system was set to deliver 35 kV/cm field strength with 64 pulses of bipolar square wave for 188 μs. The flow rate of milk was 1 mL/s. Milk temperature was raised to 52C and cooled to 22C during PEF treatment. Pasteurization at 73C for 30 s was used for comparison. Microbial inactivation and cell morphology were investigated. Analyses for protein, total solids, color, pH, particle size, density and electrical conductivity showed no significant effects (P > 0.05) in the PEF processed milk. PEF treatment accomplished a 0.3 to 3.0 log reduction of P. fluorescens, L. lactis and B. cereus in UHT milk and of total microorganisms in raw milk.     

Changes in phosphoglyceride composition during storage of ultrahigh-temperature milk, as assessed by 31P-nuclear magnetic resonance: possible involvement of thermoresistant microbial enzymes

Soluble phosphoglycerides were studied in ultrahigh-temperature (UHT) milk by 31P-nuclear magnetic resonance. It was shown that, during storage of UHT milk, manufactured from raw milk with poor microbial quality, glycerophosphocholine and glycerophosphoethanolamine disappeared in parallel with an increase in alpha-glycerophosphate (GP). Storage at 10, 20, and 30 degrees C showed a faster transformation as the temperature increased. UHT milk samples manufactured from raw milks with better microbial quality and submitted to severe heat processes did not display changes in phosphoglycerides during storage. Screening of commercial UHT milks showed variations regarding the presence of GP, while in pasteurized milk samples, the appearance of GP occurred when the commercial life had been exceeded. Inoculation of sterile milk with Pseudomonas fluorescens NCIB9046 and incubation at 10 degrees C supported that changes in phosphoglycerides could be the consequence of a phosphodiesterase activity of bacterial origin, able to survive UHT processing. A similar behavior was observed between this activity and proteolytic activity. The potential application of the detection of these compounds as spoilage predictor indices is discussed.     

Enhanced inactivation of bacterial lipases and proteinases in whole milk by a modified ultra high temperature treatment

Cultures of Pseudomonas spp. strains P10, P12 and P15 grown in whole milk which contained approximately 1 x 10(8) viable bacteria ml-1 demonstrated near linear increases in the concentration of short-chain free fatty acids and trichloroacetic acid soluble free amino groups at 20 degrees C, following either ultra high temperature (UHT) treatment (140 degrees C for 5 s) or dual heat treatments (140 degrees C followed by either 57, 60 or 65 degrees C). The dual heat treatments reduced the rates of lipolysis and proteolysis compared to the UHT treatment by up to 25-fold. The dual heat treatment utilizing 60 degrees C for 5 min also effectively limited both lipase and proteinase activities in raw milk culture samples which had contained either 6 x 10(6), 5 x 10(7) or 1 x 10(8) viable bacteria ml-1. In this system enzyme activities were reduced by up to 10-fold following dual heat treatment compared to UHT treatment alone.     

The effects of growth temperature and growth phase on the inactivation of Listeria monocytogenes in whole milk subject to high pressure processing

The aim of this study was to explore the effect of a wide range of growth temperatures, growth phases and plating media on the inactivation of Listeria monocytogenes by high pressure processing (HPP). In part one, L. monocytogenes was grown to mid-stationary phase at 4, 15, 25, 35 or 43 degrees C, inoculated into whole UHT milk at approximately 10(7) CFU/ml and high pressure processed at 400 MPa at room temperature (20-25 degrees C). Afterward, the HPP milk was plated on Tryptic Soy Yeast Extract Agar (TSYEA) and Modified Oxford Agar (MOX) to determine the degree of injury. For part two, cells were grown to mid-exponential, late-exponential or mid-stationary phase at 15 or 43 degrees C and processed in the same way. Time to reach a 5-log reduction was determined and data were analysed by ANOVA. The results from part one showed that both growth temperature and plating medium had a significant effect (P < 0.001) on the inactivation of stationary phase L. monocytogenes by HPP. Tukey's pairwise comparisons revealed that the effects of all temperatures, except 35 and 43 degrees C, were significantly different (P < 0.05). Cells grown at 15 degrees C were most sensitive to HPP, followed by cells grown at 4, 25 or 35 degrees C, with cells grown at 43 degrees C appearing to be the most resistant. Inactivation of cells grown at 4, 15 or 25 degrees C followed first order kinetics, whereas cells grown at 35 or 43 degrees C displayed non-linear inactivation kinetics due to tailing. In part two, both growth phase and plating medium had significant effects on the inactivation (P < or = 0.001) of L. monocytogenes by HPP. Cells grown at 15 degrees C to mid-stationary phase were the most pressure-resistant when tested on both media, and were significantly more resistant (P < 0.05) than cells grown at the same temperature to the other two phases of growth. There was no significant difference between mid- and late-exponential phase cells grown at 15 degrees C. When cells were grown at 43 degrees C, mid-exponential phase cells were significantly more sensitive (P < 0.05) than either late-exponential or mid-stationary phase cells, with no difference between late-exponential or mid-stationary phase cells. It was postulated that membrane composition, stationary phase proteins and/or stress proteins may affect pressure resistance.     

Manothermosonication of heat-resistant lipase and protease from Pseudomonas fluorescens: effect of pH and sonication parameters

The effect of different parameters (pH, ultrasonic amplitude and pressure) on the resistance to heat and manothermosonication (MTS) treatments of heat resistant lipase and protease produced by Pseudomonas fluorescens B52 and NCDO 2085, respectively, were studied. Lipase B52 thermoresistance decreases with an increase of pH. However, inactivation by MTS seems to be pH independent. There were only slight increases in the MTS efficiency when increasing pressure at UHT temperatures and the effect of amplitude was different depending on treatment temperature. Protease NCDO 2085, which was very resistant to MTS at 30 degrees C. was very sensitive to MTS at 76 degrees C. Increases in applied pressure had no effect on MTS efficiency at 140 degrees C and its inactivation by MTS was almost temperature independent between 76-109 degrees C. Data obtained are compared with previous published data and inactivation mechanisms are discussed.     

The effect of growth stage and growth temperature on high hydrostatic pressure inactivation of some psychrotrophic bacteria in milk

The effect of high hydrostatic pressure on the survival of the psychrotrophic organisms Listeria monocytogenes, Bacillus cereus, and Pseudomonas fluorescens was investigated in ultrahigh-temperature milk. Variation in pressure resistance between two strains of each organism were studied. The effect of growth stage (exponential and stationary phase), growth temperature (8 and 30 degrees C) on pressure resistance, and sublethal pressure injury were investigated. Exponential-phase cells were significantly less resistant to pressure than stationary-phase cells for all of the three species studied (P < 0.05). Growth temperature was found to have a significant effect at the two growth stages studied. Exponential cells grown at 8 degrees C were more resistant than those grown at 30 degrees C, but for stationary-phase cells the reverse was true. B. cereus stationary-phase cells grown at 30 degrees C were the most pressure resistant studied. L. monocytogenes showed the most sublethal damage compared to B. cereus and P. fluorescens. B. cereus spores were more resistant to pressure than vegetative cells. Pressure treatment at 400 MPa for 25 min at 30 degrees C gave a 0.45-log inactivation. Pressure treatment at 8 degrees C induced significantly less spore germination than at 30 degrees C. This study indicates the importance of the history of a bacterial culture prior to pressure treatment and that bacterial spores require more severe pressure treatments, probably in combination with other preservation techniques, to ensure inactivation.     

Thermal inactivation kinetics of bovine cathepsin D

Cathepsin D, the principal indigenous acid proteinase in bovine milk, is a lysosomal proteinase, which exists in milk in four forms, including the inactive zymogen procathepsin D. The thermal inactivation kinetics of bovine cathepsin D, isolated from spleen and milk, were studied under isothermal conditions, using a specific HPLC assay to determine residual activity. Inactivation of the blood enzyme preparation followed first order kinetics, with z-values in phosphate buffer (pH 6.7) and skimmed milk of 6.5 and 7.6 degrees C, respectively, the enzyme being far more stable in the latter environment. Inactivation kinetics of the enzyme purified from milk were more complex, and could be best approximated by a double exponential model. Again, stability was higher in milk than in buffer. The double exponential model may indicate differing heat stabilities of isoforms of the enzyme, or stabilization of the enzyme by some milk constituent. It is clear that the enzyme can survive, at least partially, processes such as heating at 55 degrees C for 30 min during manufacture of high-cook cheese varieties (45% survival), and HTST pasteurization (8% survival), and thus may contribute to proteolysis in a range of dairy products.     

Formation of lysinoalanine during heat treatment of milk

The heat-induced formation of lysinoalanine (LAL) was studied in raw skim milk that had been subjected to heat treatments as is usual in milk technology. Preheat treatment of milk at temperatures below 100 degrees C up to 20 min resulted in neglectable LAL amounts below 10 ppm i.p. (i.p.=in the protein) if at all. Tests with an UHT pilot plant showed that there was no proven formation of LAL with direct UHT-treatment at 110-130 degrees C for 10-25 min. In indirect UHT-treated milk vert small LAL amouints up to 50 ppm i.p. were detected only in those milk samples that were treated at temperatures high than 145 degrees C and holding times longer than 10 s. Autoclave sterilization in the range of 110-129 degrees C/10-25 min induced LAL amounts of 110 to 710 ppm i.p.. LAL formation in autoclave sterilized milk was almost directly dependent on heating temperature and time. Pre-treatment at temperatures below 100 degrees C induced no further LAL formation in any sterilization processes subsequent to preheating. In the pH range 6,50-6,90 LAL amounts in autoclave-sterilized milk increased directly with higher pH values at all temperatures. The varying degree of LAL formation with pH value was substantially more noticeable at higher than at lower temperatures. Increasing of lactose concentration caused only an insignificant decrease in LAL formation in autoclave-sterilized milk.     

Isolation and some properties of extracellular heat-stable lipases from Pseudomonas fluorescens strain AFT 36

Aeration increased the growth and lipase production in milk by Pseudomonas fluorescens strain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented approximately 71% of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8.0 and 35 degrees C; it had a Km on tributyrin of 3.65 mM and was inhibited by concentrations of substrate greater than approximately 17 mM. The enzyme was very stable over the pH range 6-9; it was relatively heat-labile in phosphate buffer in the temperature range 60-80 degrees C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100-150 degrees C: the D values at 150 degrees C were approximately 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the corresponding Z values in the temperature range 100-150 degrees C were approximately 40 and approximately 42 degrees C and the Ea for inactivation were 7.65 X 10(4) J mol-1 and 6.97 X 10(4) J mol-1 respectively.