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Circular bacteriocins are known for their structural stability and effective antimicrobial properties, positioning them as potential natural food preservatives. However, their widespread application is impeded by restricted availability. This research developed a total biosynthesis platform for circular bacteriocins, with a focus on AS-48 by involving recombinant production of the linear precursor in Escherichia coli , followed by enzymatic cyclization of the precursor into cyclic AS-48 using the ligase butelase-1 in vitro . An important discovery is that, aside from fusion tags, the C-terminal motif LE and LEKKK also could affect the expression yield of the precursor. This biosynthesis platform is both versatile and high-yielding, achieving yields of 10-20 mg/L of AS-48. Importantly, the biosynthetic AS-48 exhibited a secondary structure and antimicrobial activities comparable to those of the native molecules. As such, this work proposes an effective synthetic approach for circular bacteriocins, facilitating their advancement and application in the food industry.

Applying a circular economy approach, this research explores the use of cheese whey permeate (CWP), by-product of whey ultrafiltration, as cheap substrate for the production of bacterial cellulose (BC) and Sakacin-A, to be used in an antimicrobial packaging material. BC from the acetic acid bacterium Komagataeibacter xylinus was boosted up to 6.77 g/L by supplementing CWP with β-galactosidase. BC was then reduced to nanocrystals (BCNCs, 70% conversion yield), which were then conjugated with Sakacin-A, an anti-Listeria bacteriocin produced by Lactobacillus sakei in a CWP based broth. Active conjugates (75 Activity Units (AU)/mg), an innovative solution for bacteriocin delivery, were then included in a coating mixture applied onto paper sheets at 25 AU/cm 2 . The obtained antimicrobial food package was found effective in reducing Listeria population in storage trials carried out on a fresh Italian soft cheese (named "stracchino") intentionally inoculated with Listeria. Production costs of the active material have been mainly found to be associated (90%) to the purification steps. Setting a maximum prudential 50% cost reduction during process up-scaling, conjugates coating formulation would cost around 0.89 €/A4 sheet. Results represent a practical example of a circular economy production procedure by using a food industry by-product to produce antimicrobials for food preservation.

Background: Sakacin-A due to its specific antimicrobial activity may represent a good candidate to develop active packaging solutions for food items supporting Listeria growth. In the present study a protein extract containing the bacteriocin sakacin-A, produced by Lactobacillus sakei Lb 706 in a low-cost culture medium containing deproteinized cheese whey, was adsorbed onto cellulose nanofibers (CNFs) to obtain an active material to be used as a mat (or a separator) in direct contact with foods., Results: The applied fermentation conditions allowed 4.51 g L -1 of freeze-dried protein extract to be obtained, characterized by an antimicrobial activity of near 16 700 AU g -1 , that was used for the preparation of the active material by casting. The active material was then characterized by infrared spectra and thermogravimetric analyses. Antimicrobial trials were carried out in vitro using Listeria innocua as indicator strain; results were also confirmed in vivo, employing smoked salmon fillets intentionally inoculated with Listeria innocua: its final population was reduced to about 2.5-3 Log cycles after 28 days of storage at 6 °C in presence of sakacin-A, compared with negative control mats produced without the bacteriocin extract., Conclusion: This study demonstrates the possibility of producing an antimicrobial active material containing sakacin-A absorbed onto CNFs to decrease Listeria population in smoked salmon, a ready-to eat-food product. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Background: Minimally processed ready-to-eat products are considered a high-risk food because of the possibility of contamination with pathogenic bacteria, including Listeria monocytogenes from the animal reservoir, and the minimal processing they undergo. In this study, a sakacin-A anti-Listeria active package was developed and tested on thin-cut veal meat slices (carpaccio)., Results: Enriched food-grade sakacin-A was obtained from a cell-free supernatant of a Lactobacillus sakei culture and applied (0.63 mg cm -2 ) onto the surface of polyethylene-coated paper sheets to obtain an active antimicrobial package. The coating retained antimicrobial features, indicating that the process did not affect sakacin-A functionality, as evidenced in tests carried out in vitro. Thin-cut veal meat slices inoculated with Listeria innocua (a surrogate of pathogenic L. monocytogenes) were laid on active paper sheets. After 48 h incubation at 4 °C, the Listeria population was found to be 1.5 log units lower with respect to controls (3.05 vs 4.46 log colony-forming units (CFU) g -1 )., Conclusion: This study demonstrates the possibility of using an antimicrobial coating containing sakacin-A to inhibit or decrease the Listeria population in ready-to-eat products, thus lowering the risk of food-related diseases. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)