Characterisation of Lactococcus lactis isolates from herbs, fruits and vegetables for use as biopreservatives against Listeria monocytogenes in cheese

The control of Listeria monocytogenes in soft cheese manufacture remains a challenge due to its widespread occurrence, ability to grow at refrigerated temperatures and salt resistance. The application of antimicrobial compounds produced by lactic acid bacteria (LAB) as natural preservatives for improving cheese quality and safety has attracted significant research interest. The aim of this study was to select non-dairy origin LAB strains with anti-Listeria activity and to characterise their industrially relevant properties for potential roles as biopreservative adjunct cultures for use in cheese production. A total of 897 LAB isolated from herbs, fruits and vegetables were screened for their anti-Listeria activity using an MRS agar overlay assay. Fourteen isolates with strong activity were identified by 16S rRNA gene sequencing as Lactococcus lactis and all contained the nisin gene cluster. Of these fourteen isolates, four strains also showed detectable anti-Listeria activity in a milk medium. These wild Lc. lactis strains possessed slow lactose utilisation and acidification capacity in milk despite exhibiting high growth rates. This was likely due to the adoption of the mixed acid fermentation pathway since ethanol was a major metabolic end product. Analysis of volatiles in milk and laboratory scale cheese fermented with wild and industrial Lc. lactis strain combination using headspace solid phase microextraction (SPME) GC-MS identified up to a 300-400 fold increase in the production of 3-methylbutanal, an important cheese odorant with a malty flavour. Whole genome sequencing of four wild strains provided some genetic insights into their phenotypes. The wild Lc. lactis lack the lactose utilisation operon (lacABCDFEG) commonly found in industrial strains, but contain genes encoding a putative β-galactosidase and a previously characterised alternative lactose assimilation pathway with slow lactose uptake. Wild strains also contain genes encoding α-keto-acid decarboxylase (kdcA) required for 3-methylbutanal production. These results provide insight into technological properties of wild non-dairy Lc. lactis strains which could be potentially used as biopreservative adjunct cultures to control the growth of L. monocytogenes in cheese.