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The prevalence of disinfectant-resistant food-related microorganisms is of concern to the food industry. The Staphylococcus saprophyticus strain ST2H6 isolated from a poultry processing plant contained a 2.4-kb plasmid (p2H6) harbouring qacH, which encodes resistance to disinfectants based on quaternary ammonium compounds. The complete p2H6 nucleotide sequence revealed an open reading frame encoding a putative protein of 107 amino acid residues with strong similarity to members of the small multidrug resistance protein family. QacH also conferred high-level ethidium bromide resistance and low-level proflavine resistance and thus differed phenotypically from the similar proteins Smr and QacG. Fluorimetry indicated that the high-level ethidium bromide resistance was due to improved efflux energised by the proton motive force. Site-directed mutagenesis substituting the Asp-24 residue with Glu-24 had no effect on resistance characteristics. An additional open reading frame on p2H6 encoded a putative protein with similarity to rolling circle replication proteins.

Heparin can dissociate lipoprotein lipase from casein micelles, and addition of heparin enhances lipolysis in bovine but not in caprine milk. Heparin shortened the lag-time for binding of lipoprotein lipase to milk fat globules and for lipolysis. Heparin counteracted the inhibitory effects of skim milk on binding of lipase and on lipolysis. Heparin stimulated lipolysis in all bovine milk samples when added before cooling and in spontaneously lipolytic milk samples also when added after cooling. Heparin enhanced lipolysis of isolated milk fat globules. Hence, its effect is not solely due to dissociation of lipoprotein lipase from the casein micelles. Cooling of goat milk caused more marked changes in the distribution of lipase than cooling of bovine milk; the fraction of added 125I-labeled lipase that bound to cream increased from about 8 to 60%. In addition, caprine skim milk caused less inhibition of lipolysis than bovine skim milk. These observations provide an explanation for the high degree of cold storage lipolysis in goat milk. Heparin had only small effects on the distribution of lipoprotein lipase in caprine milk, which explains why heparin has so little effect on lipolysis in caprine milk. The distribution of 35S-labeled heparin in bovine milk was studied. In warm milk less than 10% bound to the cream fraction, but when milk was cooled, binding of heparin to cream increased to 45%.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of lipoprotein lipase among cream, casein, and milk serum can be evaluated by addition of a trace amount of 125I-labeled lipoprotein lipase to milk. Radioactive lipase was distributed in parallel to endogenous lipase under several conditions. In some milk samples, binding of lipase to cream increased when the milk was cooled. Correlation was good between bound labeled lipase and degree of cold-induced lipolysis in corresponding milk samples. Binding of lipase to cream or to casein was not saturable by addition of two-to threefold more lipase than is normally present in milk. In milk with a relatively high fraction of lipase bound to cream, a correspondingly lower fraction was associated with casein, whereas the fraction of lipase in milk serum was similar in all milk samples. Cold-induced binding of lipoprotein lipase to cream was not fully reversed when the milk was warmed again. Heparin released lipase from casein and increased the amount of lipase bound to cream after cooling.

Methods to quantitate factors in milk relevant to cold-induced lipolysis are described. Skim milk was incubated with milk fat globules isolated from a pool of normal milk and a fixed amount of purified lipoprotein lipase. The release of fatty acids in 24 h at 4 degrees C was determined. Most skim milk samples inhibited lipolysis, but the effect varied greatly. Skim milk from milk prone to spontaneous lipolysis was less inhibitory than skim milk from normal milk. In general, both the casein and the serum fractions of skim milk inhibited lipolysis. However, variation was greater in the effects of individual samples of milk serum. In a few extreme cases, with samples from milk with spontaneous lipolysis, the serum fraction actually stimulated lipolysis. Globules were isolated and then incubated with skim milk from normal milk and a fixed amount of purified lipoprotein lipase. This gave a measure of accessibility to lipolysis of milk fat globules in normal skim milk. There was a considerable variation in propensity toward lipolysis between milk fat globules from individual milk samples. Milk showing different levels of lipolysis obtained from five cows revealed that skim milk inhibition of lipolysis and the propensity of milk fat globules toward lipolysis were characteristic for each cow.

Extraction of membrane proteins from milk fat globules by GuHCl or by MgCl2 made the lipids more accessible to lipolysis by added lipoprotein lipase. The increase in lipolysis paralleled the loss of membrane proteins and was continuous up to 2.5 M GuHCl, which was the highest concentration used. About twice as much protein was extracted with 2.5 M GuHCl as with buffer only. The amount of protein lost was about 50% of total milk fat globule protein. Lipolysis of milk fat globules was inhibited by addition of the extracted protein. The extracted proteins also reduced lipolysis when added to whole milk. More protein was needed to inhibit lipolysis of milk fat globules treated with GuHCl compared with globules treated with buffer only. The inhibition by a given amount of protein decreased if more milk fat globules were used. Protein extracted with MgCl2 had similar effects as those extracted with GuHCl. The major components extracted with MgCl2 migrated in the 40 to 50-kdalton region on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By gel filtration chromatography, two protein fractions were obtained, which inhibited lipolysis more efficiently than the total extract. As has previously been found for inhibition of lipolysis by skim milk, the amount of extracted protein needed to inhibit lipolysis varied between preparations of milk fat globules. Milk with propensity to cold-induced ("spontaneous") lipolysis was normalized by addition of extracted proteins.