Antimicrobial activity of recombinant bovine lactoferrin and lactoferricin-B and gylcosylated human lysozyme By El-Sayed, E. M., Mahfouz, M. B.*, Abdel-Salam, A. M*., Abd El Gawad, I. A,Naim, H.* and Gerlach, G.*** Department of Dairy Science, Faculty of Agriculture, Cairo University, Giza, Egypt. * Department of Food Technology and Dairying, National Research Centre, Giza, Egypt **Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Germany. *** Department of Microbiology and infectious diseases, School of Veterinary Medicine Hannover, Germany Antimicrobial activity of recombinant bovine lactoferrin and lactoferricin-B and gylcosylated human lysozyme By El-Sayed, E. M., Mahfouz, M. B.*, Abdel-Salam, A. M*., Abd El Gawad, I. A,Naim, H.* and Gerlach, G.*** Department of Dairy Science, Faculty of Agriculture, Cairo University, Giza, Egypt. * Department of Food Technology and Dairying, National Research Centre, Giza, Egypt **Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Germany. *** Department of Microbiology and infectious diseases, School of Veterinary Medicine Hannover, Germany

Summary Recombinant bovine lactoferrin demonstrated weak inhibitory activity against Staphylococcus aureus, Listeria monocytogenes and Escherichia coli at a concentration of 1mg/ml, and a strong inhibitory activity at a concentration of 2.0 mg/ml. However, recombinant lactoferrin showed no inhibitory effect against the Salmonella strains at these concentrations. The bactericidal activity of recombinant bovine lactoferrin digest (lactoferricin B) showed that lactoferrin digest alone or in combination with lysozyme had strongly antibacterial action against Staphylococcus aureus, Listeria monocytogenes and Escherichia coli when EDTA was added ( 30 µg/ml for lactoferricin, ~0.1 mg/ lysozyme and 0.25mM EDTA). Human gylcosylated lysozyme mutant demonstrated some bactericidal activity against pathogenic bacteria strains with minimal inhibitory concentrations (MICs) ranging from 0.8 mg/ml for Staphylococcus aureus, Listeria monocytogenes and Escherichia coli strain to greater than ~ 2,0 mg/ml for Salmonella strains.

Introduction Lactoferrin is a natural milk glycoprotein belonging to the cytokines family This glycoprotein has bacteriostatic and bactericidal properties. Besides the anti-microbial activity, there is solid evidence that lactoferrin modulates host defence responses, acting through multiple mechanisms which can be summarized as follow: inhibition of hydroxyl- radical; pro-and anti- coagulant effects; modulation of complement activation; immuno-modulation, inhibition of cytokine production; natural killer cell activation, and anti-tumer effects. Recently, it has been reported that peptides obtained by pepsin digestion of bovine or human lactoferrin have a strong bactericidal activity against a variety of Gram-positive and Gram-negative bacteria. Lactoferricin B shows brood spectrum anti-microbial activity not only toward Gram-positive and – negative bacteria, but also toward yeasts and moulds. Lysozyme is another antibacterial agent that causes lysis of some bacteria by enzymatic hydrolysis of peptidoglycans found in the cell wall of both Gram-positive and Gram-negative bacteria.

Methods Creation of recombinat bovine lactoferrin and human glycosylated lysozyme The cDNAs encoding bovine lactoferrin and human glycosylated lysozyme mutant Melcher et al., 2000) were used to generate MDCK cell lines expressing high quantities of recombinant bovine lactoferrin and human lysozyme. Generation of lactoferricin using pepsin digestion of recombinant of bovine lactoferrin Generation of lactoferricin using pepsin digestion of recombinant of bovine lactoferrin was carried out according to the method described by Michel and Brent (1996) using the native of bovine lactoferrin. Inhibition zone diffusion as a detection method for the antibacterial activity Microorganisms were grown overight in 12.5% milk-free fat medium at 37°C. One experiments carried out using this bacteria cultures and were plated on LB-medium plate agar and the another experiments carried out by making dilution of bacteria cultures (1:1000 and 1:100) in 1% Bacto-Trypton, 0.5 % Bacto Yeast-Extract, 1% NaCl and were plated on LB- medium plate agar.

Results and Discussion Lactoferrin demonstrated weak inhibitory activity against Staphylococcus aureus, Listeria monocytogenes and Escherichia coli when used at a concentration of 1mg/ml, and a strong inhibitory activity at a concentration of 2.0 mg/ml. However, recombinant lactoferrin showed no inhibitory effect against the two Sallmonella strains at these concentrations. Lactoferrin digest alone or in combination with lysozyme had little antibacterial action against Staphylococcus aureus, Listeria monocytogenes and Escherichia coli and no antibacterial action against all bacterial strains grown in fat-free milk medium at the used concentrations ( 30 µ g/ml for lactoferricin and ~0.1 mg/ lysozyme), while this acivity can be easily be demonstrated in 1% peptone or other simple growth medium. Human gylcosylated lysozyme mutant demonstrated some bactericidal activity against pathogenic bacteria strains with minimal inhibitory concentrations (MICs) ranging from 0.8 mg/ml for Staphylococcus aureus, Listeria monocytogenes and Escherichia coli strain to greater than ~ 2.0 mg/ml for Salmonella strains.

rbLF rbLfcin rhlyso rbLfcin + rhLyso Listeria monocytogenes St. aureus rbLf rbLfcin + rhLyso rbLfcin rhLyso E. coli rbLF rbLfcin rhlyso+ rLfcin rhLys o rbLf rbLfcin + rhLyso rbLfcin rhLyso Salmonella enteritidis Fig. (1) The antibacterial actiivity of recombinant bovine lactoferrin, recombinant bovine lactoferrin digest (lactoferricin B) and recombinant glycosylated human lysozyme alone or together with 0,25 mMEDTA against Staphylococcus aureus, Listeria monocytogenes, E. coli DH5  F and Salmonella enteritidis.

Conclusion These results concluded that: -Investigation of the effect of lactoferrin and lactoferricin on some selected pathogenic bacteria have been useful in employing these proteins as control agents for bacterial contamination during processing of dairy products. - Consequently, the information will be important in determining how and at what concentration lactoferrin and lactoferricin could be added to foods especially infants formulas.