Residual alkaline phosphatase activity in pasteurized milk heated at various temperatures--measurement with the fluorophos and Scharer rapid phosphatase tests.

Milk containing three levels of milkfat (skim [0.5%], lowfat [2.0%], and whole [3.25%]) were heat treated at five different temperatures (59, 61, 63, 65, and 67 degrees C) using a laboratory scale, batch pasteurization method. Heated milk samples were removed at 5-min intervals, immediately cooled, and then assayed using the quantitative fluorometric method and the qualitative Scharer rapid test. Mean alkaline phosphatase (ALP) activity values as measured with the Fluorophos method decreased in all milk preparations as the time of sampling and temperature of heating increased. Samples representing the three fat levels and heat treated at 63 degrees C for 30 min, the minimum time/temperature allowed by the 1995 pasteurized milk ordinance (PMO), had ALP activity values <100 mU/liter. All values were below the 350 mU/liter standard for fluid milk products established by the Food and Drug Administration and cited in the 1995 PMO. Evaluation of the milks for adequacy of pasteurization with the Scharer rapid method indicated that those same milks were adequately pasteurized.

Survival of Selected Pathogenic Bacteria in White Pickled Cheese Made With Lactic Acid Bacteria or Antimicrobials.

Effect of lactic acid bacteria starter culture, nisin, hydrogen peroxide, or potassium sorbate on Listeria monocytogenes , Staphylococcus aureus , and Salmonella typhimurium in white pickled cheese made from pasteurized milk with 4% salt and preserved in 4% brine solution at 4°C for 60 d was studied. The starter culture inhibited all three pathogens while antimicrobials did not. Beyond day 50 in curd and day 30 in brine solution, L. monocytogenes was not detected by direct plating in cheese with added starter culture. S. aureus was not detected after day 30 in curd and day 20 in brine solution in the same cheese. S. typhimurium was not detected after day 30 in cheese curd and was not detected in brine solution at any time with lactic acid bacteria starter culture added. The pH of brine solution of starter treatment dropped below 4.7 in all experiments, while antimicrobial treatments all had a pH >5.5.

Chemical Composition of and Listeria monocytogenes Survival in White Pickled Cheese.

White pickled cheese was made from pasteurized milk with 8% salt and preserved in the whey at 4°C. About 5.0 log10 CFU/ml cells of Listeria monocytogenes were inoculated into milk, and the survival of the pathogen was studied during the storage period. The chemical composition of the cheese was also determined. L. monocytogenes were not inhibited by 8% salt, but lactic acid bacteria were unable to grow and produce acid to inhibit L. monocytogenes . During ripening, the pH never decreased below 6.0. While fat, protein, total solids, and ash contents decreased in curd during ripening, the acidity and salt did not change. In cheese whey, fat, protein, acidity, and salt showed a slight increase, while total solids and ash contents were unchanged.

Influence of Bovine Lactoferrin on the Growth of Listeria monocytogenes.

The influence of bovine lactoferrin (LF) and Apo-LF on growth of Listeria monocytogenes in Ultra-High Temperature (UHT) 2% fat milk was determined. The effect of LF was dependent upon both the degree of iron saturation and concentration. Before iron removal, LF was found to be approximately 52% saturated with iron; and at 23 and 46 mg/ml LF, minimal growth inhibition of L. monocytogenes was observed. Following dialysis, Apo-LF iron saturation was reduced to approximately 18%. At 15 and 30 mg/ml Apo-LF, a bacteriostatic effect against L. monocytogenes was observed. Inhibition of growth associated with Apo-LF was abolished when ferric ammonium citrate was added to saturate the iron binding sites of the Apo-LF.

Characterization of lipase of Pseudomonas fluorescens 27 based on fatty acid profiles.

The objectives of this research were to isolate lipase from Pseudomonas fluorescens 27, to compare the purity of the partially purified lipase preparation with crude extract, and to determine if bands of lipase activity revealed by disc gel electrophoresis liberated different free fatty acids from milk fat. Lipases were isolated from a shaken skim milk culture of P. fluorescens 27 by using ion-exchange chromatography on DEAE cellulose (Whatman DE 32) and gel filtration on Sephadex G-150. The principal lipase-rich fractions from gel filtration represented 6.2% of total lipolytic activity. Disc gel electrophoresis of partially purified enzyme revealed two protein bands. These protein bands were cut from disc electrophoresis gels and used as an enzyme source for reaction with butter oil. Free fatty acids were isolated from the assay mixture, separated, and quantified by gas chromatography. Data from gas chromatographic analysis indicated that P. fluorescens 27 produces at least two different lipases.

A rapid method for measuring protease activity in milk using radiolabeled casein.

A rapid means to detect the presence of protease activity in raw milk could be useful in predicting keeping ability of products made from that milk. A 30-min assay has been developed and compared with three other methods of detecting protease. Casein, [methyl-14C]-methylated-alpha was purchased from a radioisotope supplier. Concentrations of substrate from 2 to 20 nCi gave counts per minute, which increased linearly when counted with the Charm analyzer. There was not a significant difference in counting times of 10, 20, or 30 min. A mixture of sodium acetate and acetic acid precipitated nonhydrolyzed substrate with an efficiency of 97%. Comparison of the [14C] casein assay, a casein fluorescein isothiocyanate assay, trinitrobenzenesulfonic acid procedure, and the Hull procedure using protease from psychrotrophic bacteria revealed that the [14C] casein and casein fluorescein isothiocyanate methods were roughly equivalent and that the radiometric procedure was 10 times more sensitive than the trinitrobenzenesulfonic acid assay. The radiometric procedure was approximately 10(4) times more sensitive than the Hull procedure. The [14C] casein and casein fluorescein isothiocyanate methods were similar in time required, about 30 min, while the trinitrobenzenesulfonic acid assay and Hull method required about 1 h plus reagent preparation time. The [14C] casein procedure was most expensive per test; the other three were cheaper and similar to each other in cost.

Comparison of the heat resistance of bacterial lipases and proteases and the effect on ultra-high temperature milk quality.

Thirty-four of 49 isolates of psychrotrophic bacteria produced extracellular lipase or protease when grown in rehydrated nonfat dry milk. The cell-free crude preparation from 50% of these had either heat-resistant lipase or protease; in 30% both enzymes were heat resistant. Eight isolates were selected for further evaluation of effect on ultra-high temperature processed milk. Free fatty acids and free amino groups of milk precultured with the bacteria increased at different rates depending on the isolate. Partially purified lipase from one of these bacteria (Acinetobacter sp.) caused free fatty acids to increase following ultra-high temperature processing when the milk was stored at 10, 21, or 32 degrees C for 4 wk. The increase was temperature dependent. Lipase activity of .0012 units/ml added prior to processing caused significant increases in free fatty acid at 21 and 32 degrees C in 4 wk.

Measurement of Microbial Protease Activity Using a pH-Stat Titration.

The activity of purified and crude microbial proteases was measured by titration at pH 9 using an automatic pH-stat instrument. The ability of the pH-stat titration method and the trinitrobenzenesulfonic acid (TNBS) method to detect protease activity was compared. The pH-stat titration produced higher measurements of activity than the TNBS method when purified protease from Bacillus amyloliquefaciens was tested. Protease activity determination using the two methods resulted in a linear correlation of R2 = 0.985 and the same repeatability (C.V.=2.6%). The pH-stat titration method was more sensitive than the TNBS method in measuring activity of the purified protease, and it indicated greater proteolytic activity than the TNBS method when culture filtrates from five Pseudomonas spp. which produced proteolytic enzyme with greater activity at pH 9 than pH 7.5 were tested. When culture filtrates from four Pseudomonas spp. with similar but low proteolytic activity at pH 9 and 7.5 were tested, the two methods produced similar results. For proteases with optimum activity at or above pH 9, the pH-stat titration method was simplier, faster, and more sensitive than the TNBS method in determining activity.