Antibacterial activity in bovine lactoferrin-derived peptides.

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.

New opportunities from the isolation and utilization of whey proteins.

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.

Forms of lactoferrin: their antibacterial effect on enterotoxigenic Escherichia coli.

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.

Analytical methods for differentiating minor sequence variations in related peptides.

A proline-rich peptide was isolated and purified to homogeneity from an extract of bovine neutrophil granules using semi-preparative RP-HPLC. The relative molecular mass of the peptide (called Bac-X) was determined by ionspray MS to be 5149 +/- 0.5. The amino acid composition of the peptide was characterized by its limited number of amino acid types, which included a high proline (43.3%) and arginine content (20.3%), and hydrophobic residues. Bac-X had similar characteristics to Bac-5, a previously characterised bactenecin of bovine neutrophil granules, with respect to its proline, arginine and hydrophobic amino acid content, molecular mass and antibacterial specificity. Tryptic and N-bromosuccinimide digestion of Bac-X produced fragments with masses (M(r) 785 and 4224 and 3100 respectively) consistent with those expected from a peptide with the reported sequence of Bac-5. Bac-X differed from Bac-5 in the number of amino acid residues (43 for Bac-X versus 42 for Bac-5) and contained glycine which Bac-5 did not. However, the calculated molecular mass of the peptide, based on the amino acid compositional data, did not match the experimental value. The purified peptide could not be sequenced by Edman degradation due to apparent blockage of the N-terminus. Partial sequence information, obtained by LC-MS and collision induced dissociation MS-MS analysis of a M(r) 785 tryptic fragment of Bac-X, showed that this peptide contained a six residue sequence (-RFPPIR-) not found in Bac-5 which, based on its reported sequence, contained a M(r) 785 tryptic fragment with the sequence -FRPPIR-. This difference in sequence of Bac-X compared with Bac-5 illustrates the application of electrospray (ionspray) MS techniques to the detection and identification of minor differences in related protein/peptide forms.

In vitro antibacterial activity of the lactoperoxidase system towards enterotoxigenic strains of Escherichia coli.

The lactoperoxidase-thiocyanate-hydrogen peroxide (LP) system inhibited the growth of enterotoxigenic Escherichia coli strains responsible for scouring in neonatal and post-weaning piglets. An enzymatic system for hydrogen peroxide generation (glucose oxidase, GO; 0.1 U/ml) and a chemical source (sodium carbonate peroxyhydrate, SCP; 90 mg/l) were used in the LP system to test 19 strains in a 6-h growth assay at 37 degrees C. Only three strains were highly sensitive to the LP/GO system, while all exhibited significant growth inhibition with the LP/SCP system. Hydrogen peroxide alone had less effect than the complete system. The bactericidal activity of the LP/GO system towards a previously resistant strain was greatly increased by increasing the level of glucose oxidase in the system by three- or five-fold.

Studies on the lactoperoxidase system: reaction kinetics and antibacterial activity using two methods for hydrogen peroxide generation.

Components of the lactoperoxidase system were measured during incubation in Isosensitest broth, with enzymatic (glucose oxidase, GO) or chemical (sodium carbonate peroxyhydrate, SCP) means to generate H2O2. When low levels of thiocyanate (SCN-) were used in the GO system, H2O2 was detected and lactoperoxidase (LP) was inactivated when SCN- was depleted. With 10-fold higher SCN-, LP remained active and H2O2 was not detectable. The oxidation product of the LP reaction, most likely hypothiocyanite, was present in low concentrations. When SCP was used for the immediate generation of H2O2 in a system employing low SCN-, half the LP activity was lost within minutes but thereafter it remained stable. Low concentrations of oxidation product were measured and H2O2 was not detected during the course of the experiment. At high SCN- levels, relatively high concentrations of oxidation product were produced immediately, with H2O2 undetectable. The results suggest that the final product of the LP reaction depends on the method of H2O2 generation and the relative proportions of the substrates. Antibacterial activity of the two LPS was tested against an enterotoxigenic strain of Escherichia coli. Both systems showed bactericidal activity within 4 h incubation at 37 degrees C.

Non-oxidative antibacterial activity of bovine neutrophil granule proteins towards mastitis pathogens.

Acid extracts of bovine neutrophil granules displayed potent antibacterial activity towards a number of mastitis pathogens in vitro. Killing of pathogens by acid extractable granule protein was dependent on incubation time, protein concentration, bacterial cell load, pH and ionic strength. Gram-negative and Gram-positive organisms showed variable sensitivity to granule extract. Strains of Staphylococcus aureus were the most resistant of tested organisms to granule extract. Gram-negative organisms were neither consistently more nor less sensitive than Gram-positive organisms. Maximal killing of Gram-positive pathogens, after 30 minutes exposure to granule extract at 37 degrees C, occurred between pH 7.0 and 8.0. The Gram-negative organism Escherichia coli B117 was more sensitive to neutrophil granule extract at pH 5.0.

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.

Fine structural changes in cat L7 ventral horn neurones after chronic sub LD50 DFP.

The fine structural changes in the ventral anterior horn of spinal segment L7, have been studied in adult cats after single and chronic sub LD50 (0.1 to 0.75 mg/kg SC with a cumulative range of 1.3 mg/kg to 10.5 mg/kg) low dose exposure to diisopropylfluorophosphate (DFP). Only the motoneurons of the chronically treated animals show an increase in the number of lysosomes, neurofilaments and vesicle-like structures. A large number of coated vesicles is observed within axons and axon terminals of both acute and chronically treated animals. Morphological evidence of axon and terminal degeneration is seen only in chronically treated animals. The present study shows that chronic sub LD50 low dose administration of DFP over periods from 5 to 21 days results in degenerative changes of presynaptic terminals and axons, with the severity of the changes being dependent on dose and duration of treatment. The data are interpreted by comparison with single high dose exposure reported in the literature with a discussion of acute and delayed neurotoxic effects of DFP, on the central nervous system.