Identification, activity and delivery of new LysFA67 endolysin to target cheese spoilage Clostridium tyrobutyricum

• DOI: 10.1016/j.fm.2023.104401
• Publication Date: 2023-10-10T09:06:24Z
• Authors: Carmen Sánchez; Sonia Garde; José María Landete; Javier Calzada; Dave J. Baker; Rhiannon Evans; Arjan Narbad; Melinda J. Mayer; Marta Ávila
• Keywords: Lysin; Clostridia; Lytic cycle

Bacteriophages and their endolysins are potential biocontrol agents for the anaerobic spoilage organism Clostridium tyrobutyricum, which causes cheese late blowing defect. This study sequenced compared genomes of eight bacteriophages from Spanish dairy farms that were active against C. to identify novel species phage proteins. Phages vB_CtyS-FA67 vB_CtyS-FA70 shared >94% intergenomic similarity each other but neither had significant ΦCTP1, unique tyrobutyricum date. Taxonomic analysis indicated both phages belong class Caudoviricetes related dsDNA viruses with long non-contractile tails. no close relatives encoded a endolysin, LysFA67, predicted glycoside hydrolase GH24 family. LysFA67 lysed 93% cells after 4 min in turbidity reduction assays, retaining lytic activity at pHs 4.2-8.1 30-45 °C. The endolysin remained stable 30 d storage 4, 12 25 °C, while its decreased -20 several clostridia species, common bacteria not affected. Lactococcus lactis INIA 437, used as starter, was engineered deliver red fluorescent LysFA67-mCherry products. We demonstrated these strains able maintain fluorescence without affecting technological properties milk.