Anti-Listeria bacteriocin Sakacin-A: biotechnological and biochemical approaches for high-yield food-grade production

Cases of listeriosis, a serious food-borne infection, increased sharply between 2002 and 2006, and remained constant afterward, with severe illness and a 20% mortality reported. Food exceeding the safety criterion for L. monocytogenes was highest in Ready to Eat fishery products, followed by meat products and by fresh cheeses. Among antimicrobial agents that can be used to reduce food-related risks, bacteriocins are a class of small bacterial peptides with a greater potential. Sakacin-A is a class IIa bacteriocin, produced by the GRAS bacterium Lactobacillus sakei, showing a specific anti-Listeria activity. Its application as food preservative in active packaging - through appropriate devices - may offer many advantages, but requires low production cost and high conversion yield. Two biochemical approaches were followed: the investigation of L. sakei specific nutritional requirements as well as its overexpression and isolation in a food-grade form. HPLC analysis of free aminoacids present into culture supernatant showed arginine to be the only component totally depleted during L. sakei growth and sakacin-A production. Arginine supplementation helps maintaining culture pH levels around 6, thanks to the arginine deaminase pathway. Overexpression of sakacin-A under food-grade requirements was attempted in the GRAS yeast Kluyveromyces lactis. Competent wild-type strain of K. lactis were transformed by employing an integrative plasmid carrying the his-tagged sakacin-A gene optimized for codon-usage. The highest producer clone was select by RT-PCR. Practical applications of sakacin-A need to control the diffusion rate from the packaging to the food. Hydrophobic (polystyrene) and negatively charged (cellulose) nanoparticles are being tested, taking advantage of the amphipathic nature of sakacin-A. Preliminary results show a good adhesiveness of Sakacin-A onto hydrophobic nanoparticles, thus paving the way to the application of this natural antimicrobial in food packaging.