From cheese whey permeate to Sakacin-A/bacterial cellulose nanocrystal conjugates for antimicrobial food packaging applications: a circular economy case study

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/cm2. 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.

From Cheese Whey Permeate to Sakacin A: A Circular Economy Approach for the Food-Grade Biotechnological Production of an Anti-Listeria Bacteriocin

Cheese Whey Permeate (CWP) is the by-product of whey ultrafiltration for protein recovery. It is highly perishable with substantial disposal costs and has serious environmental impact. The aim of the present study was to develop a novel and cheap CWP-based culture medium for Lactobacillus sakei to produce the food-grade sakacin A, a bacteriocin exhibiting a specific antilisterial activity. Growth conditions, nutrient supplementation and bacteriocin yield were optimized through an experimental design in which the standard medium de Man, Rogosa and Sharpe (MRS) was taken as benchmark. The most convenient formulation was liquid CWP supplemented with meat extract (4 g/L) and yeast extract (8 g/L). Although, arginine (0.5 g/L) among free amino acids was depleted in all conditions, its supplementation did not increase process yield. The results demonstrate the feasibility of producing sakacin A from CWP. Cost of the novel medium was 1.53 €/L and that of obtaining sakacin A 5.67 €/106 AU, with a significant 70% reduction compared to the corresponding costs with MRS (5.40 €/L, 18.00 €/106 AU). Taking into account that the limited use of bacteriocins for food application is mainly due to the high production cost, the obtained reduction may contribute to widening the range of applications of sakacin A as antilisterial agent.

Mechanism for temperature-dependent production of piscicolin 126

Piscicolin 126 is a class 2a bacteriocin produced by Carnobacterium maltaromaticum strains UAL26 and JG126. Whilst strain UAL26 shows temperature-dependent piscicolin 126 production, strain JG126 produces bacteriocin at any growth temperature. Several clones containing combinations of the ATP-binding cassette transporter (pisT) and transporter accessory (pisE) genes from JG126 and UAL26 were created and tested for bacteriocin production. Bacteriocin production at 25 °C was observed only for a clone containing both pisT and pisE from JG126 (U-TJEJ) and a clone containing pisT from UAL26 and pisE from JG126 (U-BamTUEJ). Therefore, the deletion of a single CG base pair located on pisE of UAL26 that results in a frameshift and truncation of PisE causes the temperature-dependent piscicolin 126 production. Bacteriocin production of UAL26 was induced at 25 °C by the addition of supernatant containing the autoinducer peptide (AIP); however, the antimicrobial activity was lost after two subsequent overnight cultivations due to the presumed lack of the AIP. Changes in membrane fluidity due to changes in temperature or the presence of 2-phenylethanol (PHE) affected bacteriocin production of UAL26, but not of clones U-TJEJ or U-BamTUEJ. Similarly, increased membrane fluidity due to PHE addition reduced production of sakacin A in Lactobacillus sakei Lb706 and Lactobacillus curvatus LTH 1174. The mechanism involved in the temperature-dependent piscicolin 126 production was described. Due to the conformational change in PisE at 25 °C, the transport machinery was not able to translocate AIP. To the best of our knowledge, this is the first report that links membrane fluidity with the regulation of bacteriocin production.