No abstract

Several peptides sharing high sequence homology with lactoferricin B (Lf-cin B) were generated from bovine lactoferrin (Lf) with recombinant chymosin. Two peptides were copurified, one identical to Lf-cin B and another differing from Lf-cin B by the inclusion of a C-terminal alanine (lactoferricin). Two other peptides were copurified from chymosin-hydrolyzed Lf, one differing from Lf-cin B by the inclusion of C-terminal alanyl-leucine and the other being a heterodimer linked by a disulfide bond. These peptides were isolated in a single step from chymosin-hydrolyzed Lf by membrane ion-exchange chromatography and were purified by reverse-phase high-pressure liquid chromatography (HPLC). They were characterized by N-terminal Edman sequencing, mass spectrometry, and antibacterial activity determination. Pure lactoferricin, prepared from pepsin-hydrolyzed Lf, was purified by standard chromatography techniques. This peptide was analyzed against a number of gram-positive and gram-negative bacteria before and after reduction of its disulfide bond or cleavage after its single methionine residue and was found to inhibit the growth of all the test bacteria at a concentration of 8 microM or less. Subfragments of lactoferricin were isolated from reduced and cleaved peptide by reverse-phase HPLC. Subfragment 1 (residues 1 to 10) was active against most of the test microorganisms at concentrations of 10 to 50 microM. Subfragment 2 (residues 11 to 26) was active against only a few microorganisms at concentrations up to 100 microM. These antibacterial studies indicate that the activity of lactoferricin is mainly, but not wholly, due to its N-terminal region.

Management of dairy whey has often involved implementation of the most economical disposal methods, including discharge into waterways and onto fields or simple processing into low value commodity powders. These methods have been, and continue to be, restricted by environmental regulations and the cyclical variations in price associated with commodity products. In any modern regimen for whey management, the focus must therefore be on maximizing the value of available whey solids through greater and more varied utilization of the whey components. The whey protein constituents offer tremendous opportunities. Although whey represents a rich source of proteins with diverse food properties for nutritional, biological, and functional applications, commercial exploitation of these proteins has not been widespread because of a restricted applications base, a lack of viable industrial technologies for protein fractionation, and inconsistency in product quality. These shortcomings are being addressed through the development of novel and commercially relevant whey processing technologies, the preparation of new whey protein fractions, and the exploitation of the properties of these fractions in food and in nontraditional applications. Examples include the following developments: 1) whey proteins as physiologically functional food ingredients, 2) alpha-lactalbumin and beta-lactoglobulin as nutritional and specialized physically functional food ingredients, and 3) minor protein components as specialized food ingredients and an important biotechnological reagents. Specific examples include the isolation and utilization of lactoferrin and the replacement of fetal bovine serum in tissue cell culture applications with a growth factor extract isolated from whey.

SUMMARYThe caseinolytic activities at pH 6·8 of polymorphonuclear (PMN) and mononuclear leucocyte homogenates (equivalent to a level of 106cells/ml milk) were less than the levels of natural milk proteinase activity found in milk from healthy cows. Bulk milks contained ∼ 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 °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 ofPseudomonas fluorescenswas not detected in milk stored at 5 °C until a viable count of ∼ 109colony forming units (c.f.u.)/ml was obtained, whilst normal bulk milks stored at 5 °C produced detectable levels of extracellular proteinase(s) when the psychrotrophic flora reached 107–108c.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 ofSerratia marcescensresulted 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 αsl- > β- > > κ-casein for the leucocyte extracts, β- > αsl- > > > κ-casein for bovine plasmin and β- ≈ κ- > αsl-casein forS. marcescensproteinase.

The antibacterial effects of various forms of lactoferrin on enterotoxigenic strains of Escherichia coli were tested in vitro using a microassay for bacterial growth. Native and apo-lactoferrin exhibited variable activity against 19 strains, whereas holo-lactoferrin had no effect. At a concentration of .2 mg/ml of apo-lactoferrin, strains could be distinguished as either sensitive or resistant to inhibition. Zinc-saturated lactoferrin was as inhibitory as apo-lactoferrin when sensitive and resistant strains were tested over the concentration range .04 to 1.0 mg/ml of lactoferrin. A bactericidal effect was observed for native, apo-, and Zn-saturated lactoferrin against some sensitive strains. The antibacterial activity of apo-lactoferrin depended on bacterial inoculum size and was not enhanced by the addition of lysozyme. Addition of holo-lactoferrin or cytochrome c diminished the antibacterial effect of apo-lactoferrin, whereas addition of BSA had no effect. Resistance to inhibition by lactoferrin was not related to the production of bacterial siderophores.