title: Binary combination of epsilon-poly-L-lysine and isoeugenol affect progression of spoilage microbiota in fresh turkey meat, and delay onset of spoilage in Pseudomonas putida challenged meat.
abstract: Proliferation of microbial population on fresh poultry meat over time elicits spoilage when reaching unacceptable levels, during which process slime production, microorganism colony formation, negative organoleptic impact and meat structure change are observed. Spoilage organisms in raw meat, especially Gram-negative bacteria can be difficult to combat due to their cell wall composition. In this study, the natural antimicrobial agents ε-poly-L-lysine (ε-PL) and isoeugenol were tested individually and in combinations for their activities against a selection of Gram-negative strains in vitro. All combinations resulted in additive interactions between ε-PL and isoeugenol towards the bacteria tested. The killing efficiency of different ratios of the two antimicrobial agents was further evaluated in vitro against Pseudomonas putida. Subsequently, the most efficient ratio was applied to a raw turkey meat model system which was incubated for 96 h at spoilage temperature. Half of the samples were challenged with P. putida, and the bacterial load and microbial community composition was followed over time. CFU counts revealed that the antimicrobial blend was able to lower the amount of viable Pseudomonas spp. by one log compared to untreated samples of challenged turkey meat, while the single compounds had no effect on the population. However, the compounds had no effect on Pseudomonas spp. CFU in unchallenged meat. Next-generation sequencing offered culture-independent insight into population diversity and changes in microbial composition of the meat during spoilage and in response to antimicrobial treatment. Spoilage of unchallenged turkey meat resulted in decreasing species diversity over time, regardless of whether the samples received antimicrobial treatment. The microbiota composition of untreated unchallenged meat progressed from a Pseudomonas spp. to a Pseudomonas spp., Photobacterium spp., and Brochothrix thermosphacta dominated food matrix on the expense of low abundance species. We observed a similar shift among the dominant species in meat treated with ε-PL or the antimicrobial blend, but the samples differed markedly in the composition of less abundant species. In contrast, the overall species diversity was constant during incubation of turkey meat challenged with P. putida although the microbiota composition did change over time. Untreated or ε-PL treated samples progressed from a Pseudomonas spp. to a Pseudomonas spp. and Enterobacteriaceae dominated food matrix, while treatment with the antimicrobial blend resulted in increased relative abundance of Hafnia spp., Enterococcaceae, and Photobacterium spp. We conclude that the blend delayed the onset of spoilage of challenged meat, and that all antimicrobial treatments of unchallenged or challenged meat affect the progression of the microbial community composition. Our study confirms that the antimicrobial effects observed in vitro can be extrapolated to a food matrix such as turkey meat. However, it also underlines the consequence of species-to-species variation in susceptibility to antimicrobials, namely that the microbial community change while the CFU remains the same. Addition of antimicrobials may thus prevent the growth of some microorganisms, allowing others to proliferate in their place.
authors.affiliation: Biofilm Group, Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark; Antimicrobial & Antioxidant Development, Nutrition & Health, DuPont, Edwin Rahrs Vej 38, 8220 Brabrand, Denmark. Electronic address: Morten.hyldgaard@dupont.com.
authors.forename: Morten
authors.initials: M
authors.lastname: Hyldgaard
authors.affiliation: Biofilm Group, Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark.
authors.forename: Rikke L
authors.initials: RL
authors.lastname: Meyer
authors.affiliation: Genomics and Ecology of Microbes, Nutrition & Health, DuPont, Waukesha, W227 N752 Westmound Drive, 53186 Wisconsin, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Peng
authors.affiliation: Genomics and Ecology of Microbes, Nutrition & Health, DuPont, Waukesha, W227 N752 Westmound Drive, 53186 Wisconsin, USA.
authors.forename: Ashley A
authors.initials: AA
authors.lastname: Hibberd
authors.affiliation: Antimicrobial & Antioxidant Development, Nutrition & Health, DuPont, Edwin Rahrs Vej 38, 8220 Brabrand, Denmark.
authors.forename: Jana
authors.initials: J
authors.lastname: Fischer
authors.affiliation: Antimicrobial & Antioxidant Development, Nutrition & Health, DuPont, Edwin Rahrs Vej 38, 8220 Brabrand, Denmark.
authors.forename: Arnar
authors.initials: A
authors.lastname: Sigmundsson
authors.affiliation: Antimicrobial & Antioxidant Development, Nutrition & Health, DuPont, Edwin Rahrs Vej 38, 8220 Brabrand, Denmark.
authors.forename: Tina
authors.initials: T
authors.lastname: Mygind
mesh_terms: D000818:Animals; D058491:Bacterial Load; D058640:Brochothrix / Q000187:drug effects / Q000254:growth & development; D004755:Enterobacteriaceae / Q000187:drug effects / Q000254:growth & development; D005054:Eugenol / Q000031:analogs & derivatives* / Q000494:pharmacology; D005516:Food Microbiology; D005519:Food Preservation / Q000379:methods*; D005520:Food Preservatives / Q000494:pharmacology*; D020595:Hafnia / Q000187:drug effects / Q000254:growth & development; D008460:Meat / Q000382:microbiology*; D008826:Microbial Sensitivity Tests; D064307:Microbiota / Q000187:drug effects; D010776:Photobacterium / Q000187:drug effects / Q000254:growth & development; D011107:Polylysine / Q000494:pharmacology*; D016958:Pseudomonas putida / Q000187:drug effects* / Q000254:growth & development; D014422:Turkeys / Q000382:microbiology*
chemical_list: D005520:Food Preservatives; D011107:Polylysine; D005054:Eugenol; C036643:isoeugenol
keywords: Ecology; Fractional inhibitory concentration; Isoeugenol; Microbiota; Sequencing; ε-Poly-l-lysine