Isolation and characterization of heat stable proteinases from Pseudomonas isolate AFT 21

Pseudomonas strain AFT 21 produced three heat stable extracellular proteinases in milk and nutrient broth at 7 or 21 degrees C, but the proportions depended on medium and cultivation temperature. The three proteinases were EDTA- and o-phenanthroline-sensitive metalloenzymes and were not inhibited by N-ethylmaleimide or phosphoramidon. Proteinases I and II showed maximum activity at pH 7-7.5 and proteinase III at pH 8.5. All three enzymes showed maximum activity at 45-47.5 degrees C, but had relatively high (19-27% of maximum) activity at 4 degrees C. They were unstable at 55 degrees C in phosphate buffer, pH 6.6, or synthetic milk ultrafiltrate (SMUF) containing 12 mmol Ca2+, but were stabilized by short preheating at 100 degrees C. They were extremely heat stable in both phosphate buffer and SMUF, pH 6.6, at 70-150 degrees C. Their D-values at 140 degrees C were 69, 54 and 80 s respectively. The Z-values for Pseudomonas AFT 21 proteinase III in phosphate buffer and SMUF were 29.7 and 30.3 degrees C respectively; the corresponding activation energies for inactivation were 8.7 x 10(4) J mol-1 and 9.2 X 10(4) J mol-1.     

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.     

Proteolysis in milk: the significance of proteinases originating from milk leucocytes and a comparison of the action of leucocyte, bacterial and natural milk proteinases on casein

The caseinolytic activities at pH 6.8 of polymorphonuclear (PMN) and mononuclear leucocyte homogenates (equivalent to a level of 10(6) cells/ml milk) were less than the levels of natural milk proteinase activity found in milk from healthy cows. Bulk milks contained approximately 4 times more milk proteinase activity than the composite milks from individual healthy cows. Isolated blood leucocytes, when added to raw milk of good bacteriological quality and stored at 5 degrees C, did not readily degenerate and had no detectable effect on the milk proteins even when these cells were completely disrupted by homogenization of the milk. Pasteurization of milk which contained leucocytes caused loss of cell vitality. Extracellular proteinases of psychrotrophic bacteria growing in milk were not detected until the early stationary phase of growth. The total viable count at which this occurred varied greatly. Proteinase production by a pure culture of Pseudomonas fluorescens was not detected in milk stored at 5 degrees C until a viable count of approximately 10(9) colony forming units (c.f.u.)/ml was obtained, whilst normal bulk milks stored at 5 degrees C produced detectable levels of extracellular proteinase(s) when the psychrotrophic flora reached 10(7)-10(8) c.f.u./ml. Casein proteolysis by PMN and mononuclear leucocyte homogenates resulted in similar polypeptide maps, but plasmin and bacterial proteinase isolated from a strain of Serratia marcescens resulted in polypeptide maps different from each other and from that produced by the leucocyte proteinase(s). The rate of proteolysis of caseins by the different proteinase sources appeared to be in the order alpha s1- greater than beta- greater than greater than kappa-casein for the leucocyte extracts, beta- greater than alpha s1- greater than greater than greater than kappa-casein for bovine plasmin and beta- approximately kappa- greater than alpha s1-casein for for S. marcescens proteinase.     

Inhibition by chelating agents of the formation of active extracellular proteinase by Pseudomonas fluorescens 32A

The effect of chelating agents on extracellular proteinase production by Pseudomonas fluorescens 32A was examined. Increasing concentrations of orthophosphate slightly stimulated growth while inhibiting proteinase synthesis. Fifty per cent inhibition was found at 35 and 28 mM-orthophosphate at 5 and 20 degrees C respectively. Extracellular protein concentration was reduced by 30% when cells were grown with 100 mM-orthophosphate. Polyacrylamide gel electrophoresis of the cell-free supernatants suggested that reduced enzyme synthesis had taken place as evidenced by the decrease in staining intensity of the protein band corresponding to the proteinase. Other phosphate compounds could replace orthophosphate as an inhibitor. Extent of inhibition was related to chain length; polyphosphates with 4-6 or 13-18 phosphorus atoms were the most effective inhibitors. EDTA (0.5 mM) completely inhibited proteinase synthesis. This inhibition could be partly reversed by Ca2+ and, to a lesser extent, Mn2+. Proteinase production at 5 degrees C in skim milk was completely inhibited by phosphate glass (P13-P18). Control experiments showed that loss of activity with chelators was not due to inhibition of preformed enzyme. The results suggest a possible role for polyphosphates in controlling proteinase production in stored milk.     

Heat inactivation of exogenous proteinases from Pseudomonas fluorescens. I. Possibility of inactivation in milk

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.     

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.     

Fluidised bed drying of soybeans

The fluidised bed drying characteristics of soybeans at high temperatures (110-140 degrees C) and moisture contents, 31-49% dry basis, were modelled using drying equations from the literature. Air speeds of 2.4-4.1 m/s and bed depths from 10 to 15 cm were used. The minimum fluidised bed velocity was 1.9 m/s. From a quality point of view, fluidised bed drying was found to reduce the level of urease activity which is an indirect measure of trypsin inhibitor, with 120 degrees C being the minimum required to reduce the urease activity to an acceptable level. Increased air temperatures caused increased cracking and breakage, with temperatures below 140 degrees C giving an acceptable level for the animal feed industry in Thailand. The protein level was not significantly reduced in this temperature range. The drying rate equations and quality models were then combined to develop optimum strategies for fluidised bed drying, based on quality criteria, drying capacity, energy consumption and drying cost. The results showed that from 33.3% dry basis, soybean should not be dried below 23.5% dry basis in the fluidised bed dryer, to avoid excessive grain cracking. The optimum conditions for minimum cost, minimum energy and maximum capacity coincided at a drying temperature of 140 degrees C, bed depth of 18 cm, air velocity of 2.9 m/s and fraction of air recirculated of 0.9. These conditions resulted in 27% cracking, 1.7% breakage and an energy consumption of 6.8 MJ/kg water evaporated.     

Autolysis of the proteinase from Pseudomonas fluorescens.

The gene encoding the proteinase from Pseudomonas fluorescens was cloned and sequenced in an effort to identify the cleavage sites involved in its autolysis at 50 degrees C. A single open reading frame consisting of 1449 nucleotides, encoding a protein of 482 amino acids, was found. Analysis of the N-terminal amino acid sequence of the purified proteinase indicated the presence of a prosequence consisting of 13 amino acid residues. The molecular weight of the mature protein was calculated as 48,900 based on the deduced amino acid sequence, which was consistent with that of the purified proteinase as determined by sodium dodecylsulfate-PAGE. Greater than 90% loss of proteolytic activity was observed upon heating at 50 degrees C for 2 min compared with the unheated enzyme. Incubation of the proteinase at 50 degrees C led to disappearance of the intact enzyme, as shown by sodium dodecyl sulfate-PAGE, whereas it was stable in the presence of the protease inhibitor o-phenanthroline. Autolytic fragments were fractionated by reverse-phase HPLC and subjected to N-terminal amino acid sequence analysis in an effort to determine the cleavage sites. The cleavage profile was not definitive; however, amino acid residues with small side chain groups, such as glycine or alanine, were frequently found adjacent to the cleavage sites.     

Some biochemical properties of trypsin inhibitor type antinutrients derived from extracts of wheat grain, Beta variety

Trypsin inhibitors were isolated and partially purified from wheat grain, Beta variety. The procedure for determination of the inhibitory activity was simplified. This pertains shortening of the reaction time as well as quantitative decrease of components in the incubation mixture. The inhibitory fraction was salted out at 30-65% ammonium sulfate saturation. The experimental material has not been initially defatted. For isolation of the inhibitors pH 4.4 was demonstrated to be optimal. The trypsin inhibitor was characterized by relatively low activity against trypsin (1.5% of soya inhibitory activity). Preparations showing inhibitory properties when stored at -18 degrees C retained their original activity for 40 days, whereas at 4 degrees C only for 10 days, respectively. Storage at 18 degrees C for 10 days resulted in 50% loss of the original activity. Among various factors stabilizing the inhibitory activity being studied, 2-mercaptoethanol at 0.01% final concentration was found to be most effective. Study on the effect of temperature on the antitrypsin activity revealed that the preparation retained its initial activity up to 80 degrees C. It has been demonstrated by wheat proteins fractionation that both albumin and globulin fractions were accompanied by the antitrypsin activity. Moreover gluten was also shown to exhibit some inhibitory activity. Variations in the inhibitory activity were evidenced during germination of wheat grain. After 2 days period of germination it tended to decrease, disappearing completely at the fifth day, respectively. The inhibitory activity appeared in coleoptile and root at fourth day of germination, being higher in coleoptile than in the roots.     

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.     

Heat inactivation of beta-lactam antibiotics in milk

The presence of residues of antimicrobial substances in milk is one of the main concerns of the milk industry, as it poses a risk of toxicity to public health, and can seriously influence the technological properties of milk and dairy products. Moreover, the information available on the thermostability characteristics of these residues, particularly regarding the heat treatments used in control laboratories and the dairy industry, is very scarce. The aim of the study was, therefore, to analyze the effect of different heat treatments (40 degrees C for 10 min, 60 degrees C for 30 min, 83 degrees C for 10 min, 120 degrees C for 20 min, and 140 degrees C for 10 s) on milk samples fortified with three concentrations of nine beta-lactam antibiotics (penicillin G: 3, 6, and 12 microg/liter; ampicillin: 4, 8, and 16 microg/liter; amoxicillin: 4, 8, and 16 microg/liter; cloxacillin: 60, 120, and 240 microg/liter; cefoperazone: 55, 110, and 220 microg/liter; cefquinome: 100, 200, and 400 microg/liter; cefuroxime: 65, 130, and 260 microg/liter; cephalexin: 80, 160, and 220 microg/ liter; and cephalonium: 15, 30, and 60 microg/liter). The method used was a bioassay based on the inhibition of Geobacillus stearothermophilus var. calidolactis. The results showed that heating milk samples at 40 degrees C for 10 min hardly produced any heat inactivation at all, while the treatment at 83 degrees C for 10 min caused a 20% loss in penicillin G, 27% in cephalexin, and 35% in cefuroxime. Of the three dairy industry heat treatments studied in this work, low pasteurization (60 degrees C for 30 min) and treatment at 140 degrees C for 10 s only caused a small loss of antimicrobial activity, whereas classic sterilization (120 degrees C for 20 min) showed a high level of heat inactivation of over 65% for penicillins and 90% for cephalosporins.     

Tetramethylsuccinonitrile in polyvinyl chloride products for food and its release into food-simulating solvents

The content of tetramethylsuccinonitrile (TMSN), the main decomposition product of 2,2'-azobis-isobutyronitrile, in polyvinyl chloride (PVC) products used for food packaging, were examined as well as food-simulating solvents. The TMSN concentration in 17 PVC products ranged from below the detection limit, 0.05 mg/kg, up to 523 mg/kg. The release of TMSN from two PVC products into five kinds of food-simulating solvents at 60 degrees C for 30 min was observed, except for 1 +/- 1 micrograms/kg of TMSN in n-heptane from a PVC bottle containing 523 +/- 30.4 mg/kg of TMSN. The detection limit of TMSN in the food-simulating solvents was 1 micrograms/kg. When pieces of the bottle were stored in olive oil at 40 degrees C for 120 days, 5 +/- 1 micrograms/kg of TMSN was detected in the oil. The release of TMSN from the pieces of the bottle into olive oil between 80 and 140 degrees C depended on the formula ln y = 0.08786x-5.696, r = 0.9927, where y is the concentration (microgram/kg) of TMSN in olive oil, x is the temperature (degrees C), and r is the correlation coefficient.     

A modified ultra high temperature treatment for reducing microbial lipolysis in stored milk

Cultures of Pseudomonas P46 grown in whole milk to contain approximately 2 X 10(7) or 1 X 10(8) viable cells ml-1 before ultra high temperature (UHT) treatment (140 degrees C for 5 s) demonstrated near linear increases in the concentration of short-chain free fatty acids (FFA) during storage at 20 degrees C. However with 5 X 10(6) cells ml-1 before UHT heat treatment there was no detectable increase in these FFA levels over a 6-month storage period. A novel heat treatment (140 degrees C for 5 s followed by 60 degrees C for 5 min) reduced the rate of production of volatile FFA to less than 10% of the rates achieved after the normal UHT treatment.     

Inhibitory activity of Thai condiments on pandemic strain of Vibrio parahaemolyticus

Antibacterial activity of 13 condiments used in Thai cooking was investigated with a pandemic strain of Vibrio parahaemolyticus. Using a disk diffusion technique, freshly squeezed extracts from galangal, garlic and lemon, at a concentration of 10 microl/disk produced a clear zone of 13.6+/-0.5, 11.6+/-0.5 and 8.6+/-1.2mm, respectively. The inhibitory activity of these 3 condiments on pandemic strains was not significantly different from that on non-pandemic strains of V. parahaemolyticus. Because of its popularity in seafood cooking, galangal was subjected to further investigation. Only a chloroform extract of galangal inhibited growth of V. parahaemolyticus producing a clear zone of 9.5+/-0.5, 12.0+/-0 and 13.5+/-0.5mm diameter at concentrations of 25, 50 and 100 microg/disk, respectively. One active component is identified as 1'-acetoxychavicol acetate. The activity of galangal was not reduced at pH 3 or in the presence of 0.15% bile salt but was reduced by freeze and spray drying. Heating a fresh preparation of galangal to 100 degrees C but not 50 degrees C for 30 min also reduced growth inhibition. Therefore, using fresh galangal in cooking was recommended. The MIC and MBC of a freshly squeezed preparation of galangal were 1:16 and 1:16, respectively. This is the first report of an inhibitory activity of a Thai medicinal plant, galangal that is used in Thai cooking, on the pandemic strain of V. parahaemolyticus.     

Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

Release of formaldehyde and melamine from tableware made of melamine-formaldehyde resin

The relationship between the concentrations of formaldehyde and melamine released into 4% acetic acid from dishes and bowls made of melamine-formaldehyde resin was determined. The average concentrations in the migration solution after the sample had been treated at 60, 80, and 95 degrees C for 30 min with 4% acetic acid were 0.0 +/- 0.1, 0.5 +/- 0.4 and 3.0 +/- 2.2 ppm, respectively for formaldehyde and 0.04 +/- 0.07, 0.21 +/- 0.20 and 1.19 +/- 1.18 ppm, respectively for melamine. The correlation between the concentrations of formaldehyde and melamine released at 95 degrees C was y=0.4858x-0.2728 (r=0.8860), where y is melamine concentration (ppm), x is formaldehyde concentration (ppm) and r is the correlation coefficient. The molar concentration ratios of formaldehyde to melamine (F/M ratio) were 15.4 +/- 11.6 at 80 degrees C and 14.9 +/- 10.1 at 95 degrees C. Hence the release of both migrants was affected by temperature but the F/M ratio was not affected. The release of both compounds was was increased on repetition of the migration test at 95 degrees C but their concentrations remained constant after the tenth and seventeenth repetitions of the treatment. During this period, the F/M ratio decreased according to the equation 1n y=-1.4344 1n x+3.7814 (r=-0.9984) for a sample before the tenth repetition of the treatment and remained between 1.7 and 1.9 after the twelfth repetition, where y is the F/M ratio and x is the number of repetitions of the treatment.     

Effect of irradiation temperature on inactivation of Escherichia coli O157:H7 and Staphylococcus aureus

The resistance of Escherichia coli O157:H7 and Staphylococcus aureus in ground beef to gamma radiation was significantly (P < 0.05) higher at subfreezing temperatures than above freezing. Ground beef was inoculated (ca. 2 x 10(8) CFU/g) with five isolates of either E. coli O157:H7 or S. aureus and subdivided into 25-g samples, vacuum packaged in barrier pouches, and tempered to 20, 12, 4, 0, -4, -12, -20, -30, -40, or -76 degrees C before gamma irradiation. The studies were repeated twice. The D10-values for both of these pathogens increased significantly at subfreezing temperatures, reaching maxima at approximately -20 degrees C. The D10-values for E. coli O157:H7 at 4 and -20 degrees C were 0.39 +/- 0.04 and 0.98 +/- 0.23 kGy, respectively. The D10-values for S. aureus at 0 and -20 degrees C were 0.51 degrees 0.02 and 0.88 +/- 0.05 kGy, respectively.     

Purification and characterization of three extracellular proteinases produced by Pseudomonas fluorescens INIA 745, an isolate from ewe's milk.

Three proteinases were isolated from culture medium of Pseudomonas fluorescens INIA 745 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, and Sephadex G-100 chromatography. Optimal temperature for enzymatic activity was 45 degrees C for all three proteinases. The pH optimum of proteinases I and II was found to be 7.0, while that of proteinase III was 8.0. Divalent metal ions like Cu2+, Co2+, Zn2+, Fe2+, and Hg2+ were inhibitory to proteinase activity while Ca2+, Mg2+, and Mn2+ had little or no inhibitory effect. The three enzymes were strongly inhibited by EDTA and 1,10-phenantroline and partially by cysteine. The three enzymes are metalloproteinases since they were inhibited by chelators and reactivated by Co2+, Mn2+, Cu2+, and Zn2+. The Km values of proteinases I, II, and III for casein were calculated to be 3.2, 2.6, and 5.2 mg/ml, respectively. Proteinases II and III rapidly degraded beta-casein, with preference to alphas1-casein, whereas proteinase I hydrolyzed both casein fractions at a slow rate.     

Enhanced deactivation of bacterial lipases by a modified UHT treatment

Lipase was produced by three non-fluorescent pseudomonads during culturing in resterilized UHT whole milk at 10°C. The enzymes exhibited pronounced thermostability in milk with 85–88 and 82–89% of the original activities retained after heat treatments at 140°C for 5 sec and 80°C for 10 min, respectively. Also, after a single treatment at 60°C for 10 min approximately 95% of the untreated activities remained.
Double heat treatments consisting of either 130 or 140°C for 5 sec followed by 60–80°C for 3 min enhanced lipase deactivation by as much as 40% compared with the combined effect of both higher and lower temperature treatments performed on separate enzyme samples. No significant enhancement of deactivation was noted when samples were heated at 60°C for 3 min followed by 140°C for 5 sec compared with the latter treatment alone.
Lipase deactivation was non-linear at 60°C following treatment at 140°C for 5 sec; no substantial additional loss of activity occurred at the lower temperature between 5 and 10 min.