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Use of lysozymes in combination with high pressure (HP) is a promising example of hurdle technology, capable of achieving safety and shelf-life extension of foods, while meeting consumer preference for fresh-like food products. As a step towards harnessing this potential, this work investigated a wide range of lysozymes for their potential as food bio-preservatives. First, the antibacterial working range of six lysozymes was tested in a buffer system under ambient pressure and HP, on a panel five gram-positive ( Enterococcus faecalis, Bacillus subtilis, Listeria innocua, Staphylococcus aureus and Micrococcus lysodeikticus ) and five gram-negative bacteria ( Yersinia enterocolitica, Shigella flexneri, Escherichia coli O157:H7, Pseudomonas aeruginosa and Salmonella Typhimurium). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage l lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). T4L, LaL and GEWL were highly pure as evaluated by silver staining of SDS-PAGE gels and zymogram analysis while CFL was only partially pure. At ambient pressure, each gram-positive test organism displayed a specific pattern of sensitivity to the six lysozymes, but none of the gram-negative bacteria was sensitive to any of the lysozymes. HP treatment (130 - 300 MPa, 25°C, 15 min) sensitized several gram-positive and gram-negative bacteria for one or more lysozymes. M. lysodeikticus and P. aeruginosa became sensitive to all lysozymes under high pressure, S. Typhimurium remained completely insensitive to all lysozymes, and the other bacteria showed sensitization to some of the lysozymes. Overall, HEWL and LaL displayed the highest efficacy and widest spectra. Next, the effect of HEWL and LaL in combination with HP treatment on the inactivation of four gram-negative bacteria ( Escherichia coli O157:H7, Shigella flexneri , Yersinia enterocolitica and Salmonella Typhimurium), was studied in skim milk (pH 6.8; aw 0.997) and in banana juice (pH 3.8; aw 0.971). In the absence of lysozymes, S. flexneri was more sensitive to HP in milk than in banana juice, while the opposite was observed for the other three bacteria. In combination with HP treatment, LaL was more effective than HEWL on all bacteria in both milk and banana juice. Depending on the bacteria, inactivation levels in banana juice were increased from 0.4 - 2.7 log units by HP treatment alone to 3.6 - 6.5 log units in the presence of 224 U/ml LaL. Bacterial inactivation in milk was also enhanced by LaL but only by 0.5 - 2.1 log units. The higher effectiveness of LaL in banana juice than in milk, in combination with HP, could be ascribed to the low pH of the banana juice since LaL was also more effective on E. coli in buffer at pH 3.8 than at pH 6.8. The same pH effect was seen with HEWL, but the latter enzyme was generally less effective than LaL. Since neither LaL nor HEWL are enzymatically active at pH 3.8, we analyzed bacterial lysis after HP treatment in the presence of these enzymes, and found that inactivation proceeds through a non-lytic mechanism at pH 3.8 and a lytic mechanism at pH 6.8. Based on these results, we conclude that LaL may offer interesting perspectives for use as an extra hurdle in HP food preservation. Finally, we investigated the specificity of the six lysozymes (HEWL, M1L, LaL, T4L, GEWL and CFL) for peptidoglycan (PG) substrates obtained by chloroform extraction from a number of gram-negative bacteria and from Micrococcus lysodeikticus. HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. The gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella T yphimurium . These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that extraction with chloroform containing Tris-HCl buffer effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform containing Tris-HCl buffer extracts of S . Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa , suggesting that other lysozyme inhibitors than Ivy may exist and be widespread in gram-negative bacteria. Lysozymes are enzymes that hydrolyse peptidoglycan (PG), the major cell wall polysaccharide in bacteria. They are produced by a wide variety of organisms, where they play a role in antibacterial defense. Hen egg white lysozyme (HEWL) is permitted as a food additive but its efficacy is limited due to inaccessible or resistant PG. Lysozyme-resistant bacteria can often be sensitized by simultaneous treatment with specific chemical or physical stresses such as high pressure (HP). The objective of this work was therefore to explore the potential of a variety of lysozymes as antibacterial food bio-preservatives, either or not in combination with HP. First, the antibacterial working range of six lysozymes on a panel of ten gram-negative (G-) and gram-positive (G+) bacteria was studied in buffer. The lysozymes included two that are commercially available (HEWL and mutanolysin from Streptomyces globisporus, designated M1L), and four that were chromatographically purified (bacteriophage l lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). At ambient pressure, each G+ test organism displayed a specific pattern of sensitivity to the lysozymes, but none of the G- bacteria was sensitive to any of the lysozymes. HP treatment (130 - 300 MPa, 25°C, 15 min) sensitized several bacteria for one or more lysozymes. Overall, HEWL and LaL displayed the highest efficacy and widest working range. Next, the efficacy of the latter two enzymes against G- bacteria was studied in two foods, skim milk (pH 6.8; aw 0.997) and banana juice (pH 3.8; aw 0.971). The bacteria were sensitized to both lysozymes by HP but LaL was more effective than HEWL on all bacteria in both milk and banana juice in combination with HP and also more effective in banana juice than in milk. The latter observation could be ascribed to the low pH of the banana juice since LaL was also more effective on E. coli in buffer at pH 3.8 than at pH 6.8. The same pH effect was seen with HEWL, but the latter enzyme was generally less effective than LaL. Neither LaL nor HEWL are enzymatically active at pH 3.8, and inactivation proceeded through a non-lytic mechanism at pH 3.8 and a lytic mechanism at pH 6.8. Thus, LaL may offer interesting perspectives for use as an extra hurdle in HP food preservation. Finally, the specificity of the six lysozymes for PG substrates obtained by extraction with chloroform containing buffer from a number of G- bacteria and from Micrococcus lysodeikticus was investigated. HEWL was much more effective on M. lysodeikticus than on any of the G- cell walls, while the opposite was found for LaL. The G- cell walls showed differences in susceptibility to the different lysozymes, which could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that the extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa , suggesting that other lysozyme inhibitors than Ivy may exist and be widespread in G- bacteria.