Salmonella enterica serovar Typhimurium genetic variants isolated after lethal treatment with Thymbra capitata essential oil (TCO) showed increased resistance to TCO in milk.

The high prevalence of Salmonella enterica in milk poses a risk of considerable concern in the preservation of certain dairy products, mainly those elaborated from raw milk. Essential oils (EOs) have been proposed as a promising food preservative for such products due to their strong antimicrobial properties. Additionally, these natural antimicrobials have been shown to be effective against multi-drug resistant strains. They can thus also be utilized to prevent the dissemination of antimicrobial resistances (AMR). However, recent evidence of the development of bacterial resistance under EO treatments may call their use into question. This study sought to assess the emergence of antimicrobial resistant genetic variants of S. enterica serovar Typhimurium from survivors after cyclic exposure to lethal doses (>5 log10 cycles of inactivation) of Thymbra capitata EO (TCO), in order to evaluate the impact that it could have on milk preservation, to ascertain whether cross-resistance to antibiotics occurs, and to identify the genomic changes responsible for their phenotype. Isolated strains by TCO (SeTCO) showed a two-fold increase in minimum inhibitory and bactericide concentrations (MIC and MBC) of TCO compared to Salmonella enterica serovar Typhimurium wild-type strain (SeWT) in laboratory growth medium, as well as a greater adaptation and growth rate in the presence of the EOs and a higher survival to TCO treatments in buffers of pH 4.0 and 7.0. The increased resistance of SeTCO was confirmed in skimmed milk: 300 µL/L TCO reduced only 1 log10 cycle of SeTCO population, whereas it inactivated more than 5 log10 cycles in SeWT. Moreover, SeTCO showed an increased cross-resistance against aminoglycosides, quinolones and tetracyclines. Whole genome sequencing revealed 5 mutations in SeTCO: 2 in genes involved in O-antigens synthesis (rfbV and rfbX), 2 in genes related to adaptation to the growing medium (trkA and glpK), and 1 in a redox-sensitive transcriptional regulator (soxR). The phenotypic characterization of a constructed SeWT strain with mutant soxRSeTCO demonstrated that the mutation of soxR was the main cause of the increased resistance and tolerance observed in SeTCO against TCO and antibiotics. The emergence of resistant strains against EOs might jeopardize their use as food preservatives. Further studies will thus be required to determine under which conditions such resistant strains might occur, and to assess the food risk they may pose, as well as to ascertain their impact on the spread of AMR.

Chitosan nanoemulsions of cold-pressed orange essential oil to preserve fruit juices.

Sweet orange essential oil is obtained from the peels of Citrus sinensis (CSEO) by cold pressing, and used as a valuable product by the food industry. Nanoencapsulation is known as a valid strategy to improve chemical stability, organoleptic properties, and delivery of EO-based products. In the present study we encapsulated CSEO using chitosan nanoemulsions (cn) as nanocarrier, and evaluated its antimicrobial activity in combination with mild heat, as well as its sensorial acceptability in orange and apple juices. CSEO composition was analyzed by GC-MS, and 19 components were identified, with limonene as the predominant constituent (95.1%). cn-CSEO was prepared under low shear conditions and characterized according to droplet size (<60 nm) and polydispersity index (<0.260 nm). Nanoemulsions were stable for at least 3 months at 4 ± 2 °C. cn-CSEO were compared with suspensions of CSEO (s-CSEO) (0.2 µL of CSEO/mL) in terms of antibacterial activity in combination with mild heat (52 °C) against Escherichia coli O157:H7 Sakai. cn-CSEO displayed a greater bactericidal activity than s-CSEO at pH 7.0 and pH 4.0. The validation in fruit juices showed an improved bactericidal effect of cn-CSEO in comparison with s-CSEO when combined with mild heat in apple juice, but not in orange juice. In both juices, the combination of CSEO and mild heat exerted synergistic lethal effects, reducing the treatment time to cause the inactivation of up to 5 Log10 cycles of E. coli O157:H7 Sakai cells. Finally, the sensory characteristics of both juices were acceptable either when using s-CSEO or CSEO nanoemulsified with chitosan. Therefore, as a promising carrier for lipophilic substances, the encapsulation of EOs with chitosan nanoemulsions might represent an advantageous alternative when combined with mild heat to preserve fruit juices.

Combination of mild heat and plant essential oil constituents to inactivate resistant variants of Escherichia coli in buffer and in coconut water.

The growing demand for minimally processed foods with clean labels has stimulated research into mild processing methods and natural antimicrobials to replace intensive heating and conventional preservatives, respectively. However, we have previously demonstrated that repetitive exposure of some bacteria to mild heat or subinhibitory concentrations of essential oil constituents (EOCs) may induce the emergence of mutants with increased resistance to these treatments. Since the combination of mild heat with some EOCs has a synergistic effect on microbial inactivation, we evaluated the potential of such combinations against our resistant E. coli mutants. While citral, carvacrol and t-cinnamaldehyde synergistically increased heat inactivation (53.0 °C, 10 min) of the wild-type MG1655 suspended in buffer, only the combination with carvacrol (200 µl/l) was able to mitigate the increased resistance of all the mutants. Moreover, the combination of heat and carvacrol acted synergistically inactivating heat-resistant variants of E. coli O157:H7 (ATCC 43888). This combined treatment could synergistically achieve more than 5 log10 reductions of the most resistant mutants in coconut water, although the temperature had to be raised to 57.0 °C. Therefore, the combination of mild heat with carvacrol appears to hold promise for mild processing, and it is expected to counteract the development of heat resistance.

Control of Autochthonous Spoilage Lactic Acid Bacteria in Apple and Orange Juices by Sensorially Accepted Doses of Citrus Spp. Essential Oils Combined with Mild Heat Treatments.

This study assessed the compromised acceptance threshold (CAT) and rejection threshold (RT) of Citrus lemon (CLEO) and Citrus reticulata essential oil (CREO) in apple and orange juices. The efficacy of CLEO and CREO concentrations below the RT were evaluated alone and combined with mild heat treatment (MHT) (54 °C, up to 12 min) to inactivate the autochthonous spoilage bacteria Lactobacillus brevis, Lactobacillus plantarum, and Leuconostoc mesenteroides in apple and orange juices. The CAT of CLEO and CREO varied from 0.15 to 0.17 µL/mL in orange and apple juices. The RT of CLEO was approximately 0.58 µL/mL in apple and orange juices, and the RT of CREO was 0.68 µL/mL in both juices. When CLEO and CREO were assayed alone, the highest concentration (0.50 µL/mL) decreased counts of all strains approximately 2 log10 CFU/mL after 12 min of exposure to 54 °C. All concentrations of CLEO or CREO in combination with MHT acted synergistically against L. brevis, L. plantarum, and L. mesenteroides. Decreases in counts varied with the strain, CLEO and CREO concentrations, juice type, and exposure time to the combined treatment. CREO was more effective than CLEO in combination with MHT against the strains in apple and orange juices. Effective combinations of CLEO or CREO with MHT to control the autochthonous spoilage bacteria did not compromise the quality parameters (°Brix, pH, and titratable acidity) that characterize unsweetened juices. These results indicate CLEO or CREO at concentrations below the sensory RT in combination with MHT as a feasible technology to control autochthonous spoilage bacteria in fresh fruit juices. PRACTICAL APPLICATION: The present study provides novel information concerning the efficacy of sensorially accepted doses of CLEO and CREO combined with MHT against autochthonous spoilers in fruit juice. The valuable synergistic effects that can be observed when combining CLEO and CREO with MHT reveal a feasible preservation technology and alternative to traditional treatments that are successful because they help reduce treatment intensity, thereby avoiding adverse effects on the sensory, physicochemical, and nutritional properties of these products.

Bioactive properties of a propolis-based dietary supplement and its use in combination with mild heat for apple juice preservation.

This study characterizes the antioxidant and antibacterial properties of a propolis-based dietary supplement (PDS) and investigates its incorporation into apple juice to decrease the intensity of the heat treatment required to inactivate 5 log10 cycles of Escherichia coli O157:H7. As the source of propolis, we used a PDS containing 0.2 mg/µL of propylene glycol-extracted propolis (propolis). The total phenolic content and antioxidant activity (IC50) of the PDS were 82.15±3.53 mg/g and 0.055±0.003 mg/mL, respectively. Regarding antimicrobial activity, propolis (0.2 mg/mL) was very effective under acidic pH against Listeria monocytogenes EGD-e, inactivating more than 5 log10 cell cycles in 1h, but hardly inactivated or sub-lethally injured E. coli O157:H7 Sakai. However, incorporating propolis (0.2 mg/mL) into acidic buffer decreased the time needed to inactivate 5 log10 cycles of E. coli O157:H7 Sakai at 51 °C by more than 40 times. Moreover, when combined with heat in apple juice, propolis (0.1mg/mL) reduced the thermal treatment time and temperature needed to inactivate 5 log10 cycles of E. coli by 75% and 3 °C, respectively. The corresponding PDS concentration did not decrease the organoleptic properties of the apple juice, which implies the possibility of obtaining a sensorially appealing, low-pasteurized apple juice with the functional properties provided by propolis.