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Efficacy of bioengineered nisin derivatives H27/31K in combination with phage endolysin PlyP100 to eliminate Listeria monocytogenes in queso fresco.

L. A. Ibarra-Sánchez

Abstract:

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Efficacy of bioengineered nisin derivatives H27/31K in combination with phage endolysin PlyP100 to eliminate Listeria monocytogenes in queso fresco.
L. A. Ibarra-Sánchez*, W. Kong, T. Lu, M. J. Miller. University of Illinois at Urbana-Champaign Urbana, IL.

Listeria monocytogenes is a food-borne pathogen of concern in fresh Hispanic-style cheeses, such as queso fresco (QF). Nisin's ability to inhibit L. monocytogenes is well-known, but its activity is limited in QF. The objective of this study was to evaluate the antilisterial efficacy of bioengineered nisin H27/31K, alone and in combination with endolysin PlyP100 in QF. Nisin H27/31K (histidine at position 27 and 31 replaced with lysine) was produced in Lactococcus lactis MG1363, an engineered strain having the nisin biosynthetic pathway. Commercial nisin (nisin A) and H27/31K were evaluated to determine their minimum inhibitory concentration (MIC) and residual nisin after 24 h exposure to pH 7 + 22% milk fat at 37°C. His-tagged PlyP100 was overexpressed in Escherichia coli and subsequently purified. Nisin A, H27/31K and PlyP100 were added to QF at the following concentrations: 250 µg/g H27/31K with or without 2.5 U/g PlyP100, and 250 µg/g nisin A. Cheese curds were inoculated with ~3.5 log cfu/g of L. monocytogenes cocktail of 5 different foodborne outbreak-associated strains. L. monocytogenes cells were enumerated by spread plating on PALCAM (polymyxin-acriflavine-LiCl-ceftazidime-esculin-mannitol) agar supplemented with ceftazidime, across 28 d of storage at 4°C. All experiments were repeated 2 times with duplicated samples. H27/31K showed reduced antilisterial activity (MIC range: 12.5 - 50 µg/mL) compared with nisin A (MIC range: 1.56 — 6.25 µg/mL). After 24 h of exposure to pH 7 + 22% milk fat, > 96% residual nisin H27/31K was observed, but residual nisin A was not detected. H27/31K reduced initial viable counts of L. monocytogenes in QF by up to ~1.5 log cfu/g. PlyP100 exhibited a strong listeriostatic effect in QF over 28 d of cold storage. The treatment combining H27/31K and PlyP100 in QF achieved L. monocytogenes reduction below the detection limit of plating, and, the pathogen was not recovered after enrichment in all QF samples treated with that antimicrobial mixture. In conclusion, our results demonstrate that nisin H27/31K combined with PlyP100 can be used to eradicate Listeria in QF.

Keywords: queso fresco, Listeria monocytogenes, nisin bioengineering.