Analysis of the effect of high hydrostatic pressure treatment and enterocin AS-48 addition on the bacterial communities of cherimoya pulp.

In the present study, pulp obtained from cherimoya pulp (Annona cherimola) was inoculated with epiphytic microbiota collected from cherimoya fruits, and supplemented or not with the circular bacteriocin enterocin AS-48 (50µg/g) and then packed under vacuum. Samples supplemented or not with enterocin were treated by high hydrostatic pressure (600MPa, 8min) and then stored at 5°C for 30days. The single AS-48 treatment only delayed microbial growth non-significantly (p>0.05). HHP treatment reduced microbial counts by five log cycles, but it did not prevent further growth of survivors by day 7. The combined treatment (AS-48+HHP) was the most effective, keeping bacterial cell densities at ≤1.5 log CFU/g for up to 15days. 16S rRNA gene pyrosequencing analysis was done on amplicon libraries from the growth on TSA plates seeded with ten-fold dilutions of pulp suspensions and incubated at 22°C for 24h. The results obtained are limited by the experimental conditions used in the study, and only concern the bacterial fraction that was selected by the TSA and growth conditions used. Pantoea (Pantoea agglomerans, Pantoea vagans) were the operational taxonomic units (OTUs) detected at highest relative abundance in bacterial biomass grown from control samples for the first 7days of storage, followed by Enterococcus gallinarum and Leuconostoc mesenteroides during late storage. The single HHP treatment significantly reduced the relative abundance of OTUs belonging to Pantoea and strongly increased that of endosporeformers (mainly Bacillus firmus and Bacillus stratosphericus) early after treatment, although Pantoea became again the predominant OTUs during storage. Samples singly treated with enterocin AS-48 revealed a strong inhibition of E. gallinarum as well as an early decrease in the relative abundance of Pantoea and an increased relative abundance of OTUs belonging to other Gram-negative species (mainly from genera Serratia and Pseudomonas). The strong microbial inactivation achieved by the combined treatment with enterocin and HHP reduced the levels of viable cells below detectable limits at days 0 and 1, and survivors recovered on TSA at day 7 were represented in >99% by B. firmus OTU. OTUs from endosporeformers were no longer detected during prolonged incubation, displaced by Pantoea spp., Erwinia billingiae and leuconostocs. Results from the present study indicate that HHP in combination with enterocin AS-48 is more effective in preserving the microbiological quality of cherimoya pulp during storage than the single HHP treatment.

Combined treatments of enterocin AS-48 with biocides to improve the inactivation of methicillin-sensitive and methicillin-resistant Staphylococcus aureus planktonic and sessile cells.

Control of staphylococci during cleaning and disinfection is important to the food industry. Broad-spectrum bacteriocins with proved anti-staphylococcal activity, such as enterocin AS-48, could open new possibilities for disinfection in combination with biocides. In the present study, enterocin AS-48 was tested singly or in combination with biocides against a cocktail of six Staphylococcus aureus strains (including three methicillin-resistant strains) in planktonic state as well as in biofilms formed on polystyrene microtiter plates. Cells were challenged with enterocin, biocides or enterocin/biocide combinations. Inactivation of planktonic cells increased significantly (p<0.05) when enterocin AS-48 (25mg/l) was tested in combination with benzalkonium chloride (BC), cetrimide (CT) and hexadecylpyridinium chloride (HDP), and non-significantly in combination with didecyldimethylammonium bromide (AB), triclosan (TC), hexachlorophene (CF), polyhexamethylen guanidinium chloride (PHMG), chlorhexidine (CH) or P3-oxonia (OX). In the sessile state (24h biofilms), staphylococci required higher biocide concentrations in most cases, except for OX. Inactivation of sessile staphylococci increased remarkably when biocides were applied in combination with enterocin AS-48, especially when the bacteriocin was added at 50mg/l. During storage, the concentrations of sessile as well as planktonic cells in the treated samples decreased remarkably for BC, TC and PHMG, but OX failed to inhibit proliferation of the treated biofilms as well as growth of planktonic cells. The observed inhibitory effects during storage were potentiated when the biocides were combined with 50 mg/l enterocin AS-48. Results from this study suggest that selected combinations of enterocin AS-48 and biocides offer potential use against planktonic and sessile, methicillin-sensitive and methicillin-resistant S. aureus.