VO2max changes in English futsal players after a 6-week period of specific small-sided games training

Futsal is a high-intensity, intermittent sport where accelerations and short sprints are performed at maximal or sub-maximal intensity. These efforts are interspersed by brief recovery periods, during 2 halves of 20 minutes stopping clock. Aerobic endurance inevitably plays a key role in the players’ performance. The aim of this study was to analyse the VO2 max progression and the agility (with and without ball) of English futsal players during a 6-week period of small-sided games practice. Two teams volunteered to participate in this study; an experimental group (EG), which performed a specialised small-sided training regime and a control group (CG) (normal training regime). VO2max was estimated from the results of the 20-metre Multi-Stage Fitness Test. The VO2max of the futsal players in the EG improved significantly (58.73±2.41 ml/kg/min vs. 60.11 ± 2.99 ml/kg/min, p=0.04). The same player's agility and agility with ball performance did not report any significant changes in either group. The results showed that periodisation, training sessions and methods based on small-sided games, which implied a change in the number of players, the size of the pitch and the task constraints, were adequate to increase aerobic endurance

Exploiting the synergism among physical and chemical processes for improving food safety

This article provides an overview of recent published information on the subject of synergistic lethal effects that emerge from the combination of physical and/or chemical processes applied to enhance food safety. Despite important recent advances in non-thermal technologies, the greatest synergistic lethal effects emerge from combining them with traditional, relatively mild heat treatments. The combined application of antimicrobials has shown that their main constituents interact effectively, and great synergistic effects have been described with the capacity of either inhibiting or inactivating pathogens. Moreover, natural antimicrobials are more effective when pathogens are previously damaged sublethally by the application of physical technologies. Such combinations allow for a considerable reduction of treatment intensity and costs, along with a noticeable improvement in food quality and safety

Genetic Variants and Phenotypic Characteristics ofSalmonellaTyphimurium-Resistant Mutants after Exposure to Carvacrol

The emergence of antimicrobial resistance has raised questions about the safety of essential oils and their individual constituents as food preservatives and as disinfection agents. Further research is required to understand how and under what conditions stable genotypic resistance might occur in food pathogens. Evolution experiments onSalmonellaTyphimurium cyclically exposed to sublethal and lethal doses of carvacrol permitted the isolation of SeSCar and SeLCar strains, respectively. Both evolved strains showed a significant increase in carvacrol resistance, assessed by minimum inhibitory and bactericidal concentrations, the study of growth kinetics in the presence of carvacrol, and the evaluation of survival under lethal conditions. Moreover, antibiotic susceptibility tests revealed a development of SeLCar resistance to a wide range of antibiotics. Whole genome sequencing allowed the identification of single nucleotide variations in transcriptional regulators of oxidative stress-response:yfhPin SeSCar andsoxRin SeLCar, which could be responsible for the increased resistance by improving the response to carvacrol and preventing its accumulation inside the cell. This study demonstrates the emergence of S. Typhimurium-resistant mutants against carvacrol, which might pose a risk to food safety and should therefore be considered in the design of food preservation strategies, or of cleaning and disinfection treatments

Antimicrobial activity of suspensions and nanoemulsions of citral in combination with heat or pulsed electric fields

The application of essential oils in form of nanoemulsions has been proposed as a method to improve their solubility in aqueous solutions, and hence their antimicrobial activity. The objective of this study was to evaluate the antimicrobial activity of citral, applied directly or in combined treatments with heat or pulsed electric fields (PEF), as a function of the inoculation procedure assayed: (i) a simple, vigorous shaking method by vortex agitation (suspension of citral; s-citral) or (ii) the previous preparation of nanoemulsions by the emulsion phase inversion (EPI) method (nanoemulsion of citral; n-citral). n-Citral was more effective in either inhibiting or inactivating Escherichia coli O157:H7 Sakai than s-citral. However, when combined with heat, a greater synergistic effect was observed with s-citral rather than with n-citral, either in lab media (pH 7·0 and 4·0) or apple juice. For instance, while almost 5 log10 cell cycles were inactivated in apple juice after 15 min at 53°C in the presence of 0·1 µl ml-1 of s-citral, the use of n-citral required 30 min. The use of nanoemulsions did not modify the slight synergism observed when citral and mild PEF were combined (150 µs, 30 kV cm-1). Significance and Impact of the Study: The exploration of different delivery systems of antimicrobial compounds such as citral in aqueous food products aids in the establishment of successful combined treatments for food preservation. While at room temperature, citral in form of a nanoemulsion shows a higher antimicrobial activity; its combination with heat would imply a partial loss of the outstanding synergistic lethal effect achieved when added in suspension form. Therefore, the most suitable procedure to magnify the synergism between heat and citral when processing juices would merely require an intense homogenization step prior to the combined treatment

Incubation with a complex orange essential oil leads to evolved mutants with increased resistance and tolerance

Emergence of strains with increased resistance/tolerance to natural antimicrobials was evidenced after cyclic exposure to carvacrol, citral, and (+)-limonene oxide. However, no previous studies have reported the development of resistance and tolerance to complex essential oils (EOs). This study seeks to evaluate the occurrence of Staphylococcus aureus strains resistant and tolerant to a complex orange essential oil (OEO) after prolonged cyclic treatments at low concentrations. Phenotypic characterization of evolved strains revealed an increase of minimum inhibitory and bactericidal concentration for OEO, a better growth fitness in presence of OEO, and an enhanced survival to lethal treatments, compared to wild-type strain. However, no significant differences (p > 0.05) in cross-resistance to antibiotics were observed. Mutations in hepT and accA in evolved strains highlight the important role of oxidative stress in the cell response to OEO, as well as the relevance of the cell membrane in the cell response to these natural antimicrobials. This study demonstrates the emergence of S. aureus strains that are resistant and tolerant to EO (Citrus sinensis). This phenomenon should be taken into account to assure the efficacy of natural antimicrobials in the design of food preservation strategies, in cleaning and disinfection protocols, and in clinical applications against resistant bacteria

Emergence of hyper-resistant Escherichia coli MG1655 derivative strains after applying sub-inhibitory doses of individual constituents of essential oils

The improvement of food preservation by using essential oils (EOs) and their individual constituents (ICs) is attracting enormous interest worldwide. Until now, researchers considered that treatments with such antimicrobial compounds did not induce bacterial resistance via a phenotypic (i.e., transient) response. Nevertheless, the emergence of genotypic (i.e., stable) resistance after treatment with these compounds had not been previously tested. Our results confirm that growth of Escherichia coli MG1655 in presence of sub-inhibitory concentrations of the ICs carvacrol, citral, and (+)-limonene oxide do not increase resistance to further treatments with either the same IC (direct resistance) or with other preservation treatments (cross-resistance) such as heat or pulsed electric fields (PEF). Bacterial mutation frequency was likewise lower when those IC''s were applied; however, after 10 days of re-culturing cells in presence of sub-inhibitory concentrations of the ICs, we were able to isolate several derivative strains (i.e., mutants) displaying an increased minimum inhibitory concentration to those ICs. Furthermore, when compared to the wild type (WT) strain, they also displayed direct resistance and cross-resistance. Derivative strains selected with carvacrol and citral also displayed morphological changes involving filamentation along with cell counts at late-stationary growth phase that were lower than the WT strain. In addition, co-cultures of each derivative strain with the WT strain resulted in a predominance of the original strain in absence of ICs, indicating that mutants would not out-compete WT cells under optimal growth conditions. Nevertheless, growth in the presence of ICs facilitated the selection of these resistant mutants. Thus, as a result, subsequent food preservation treatments of these bacterial cultures might be less effective than expected for WT cultures. In conclusion, this study recommends that treatment with ICs at sub-inhibitory concentrations should be generally avoided, since it could favor the emergence of hyper-resistant strains. To ascertain the true value of EOs and their ICs in the field of food preservation, further research thus needs to be conducted on the induction of increased transient and stable bacterial resistance via such antimicrobial compounds, as revealed in this study

Whole-genome sequencing and genetic analysis reveal novel stress responses to individual constituents of essential oils in Escherichia coli

Food preservation by the use of essential oils (EOs) is being extensively studied because of the antimicrobial properties of their individual constituents (ICs). Three resistant mutants (termed CAR, CIT, and LIM) of Escherichia coli MG1655 were selected by subculturing with the ICs carvacrol, citral, and (+)-limonene oxide, respectively. These derivative strains showed increased MIC values of ICs and concomitantly enhanced resistance to various antibiotics (ampicillin, trimethoprim, chloramphenicol, tetracycline, kanamycin, novobiocin, norfloxacin, cephalexin, and nalidixic acid) compared to those for the parental strain (wild type [WT]). Whole-genome sequencing (WGS) of these hyperresistant strains permitted the identification of single nucleotide polymorphisms (SNPs) and deletions in comparison to the WT. In order to analyze the contribution of these mutations to the increased antimicrobial resistance detected in hyperresistant strains, derivative strains were constructed by allelic reversion. A role of the SoxR D137Y missense mutation in CAR was confirmed by growth in the presence of some ICs and antibiotics and by its tolerance to ICs but not to lethal heat treatments. In CIT, increased resistance relied on contributions by several detected SNPs, resulting in a frameshift in MarR and an in-frame GyrB ¿G157 mutation. Finally, both the insertion resulting in an AcrR frameshift and large chromosomal deletions found in LIM were correlated with the hyperresistant phenotype of this strain. The nature of the obtained mutants suggests intriguing links to cellular defense mechanisms previously implicated in antibiotic resistance

Modified cyclodextrin type and dehydration methods exert a significant effect on the antimicrobial activity of encapsulated carvacrol and thymol

BACKGROUND The antimicrobial activity of essential oils and their constituents has led to increasing interest in using them as natural preservative agents. However, their high sensitivity to light and oxygen, their volatility and their low aqueous solubility are all obstacles to their application in the food, cosmetic or pharmaceutical industries. Encapsulation in cyclodextrins (CDs) is a solution for the application of such essential oils. RESULTS The complexation of carvacrol and thymol with hydroxypropyl (HP)‐α‐, HP‐β‐ and HP‐γ‐CD, the behavior of the solid complexes prepared by freeze‐drying and spray‐drying methods and the antibacterial activity of solid complexes were studied. Kc values of HP‐α‐ and HP‐γ‐CD complexes with carvacrol (118.4 and 365.7 L mol−1) and thymol (112.5 and 239.7 L mol −1) were far lower than those observed for HP‐β‐CD complexes with carvacrol (2268.2 L mol −1) and thymol (881.6 L mol −1). The lower stability of HP‐α‐ and HP‐γ‐CD complexes increased the release of compounds, thereby affecting the antimicrobial activity of carvacrol and thymol to a lesser extent than complexation with HP‐β‐CD, normally used in the encapsulation of carvacrol and thymol. HP‐β‐CD encapsulation of carvacrol and thymol markedly reduced their antimicrobial activity. The freeze‐drying method barely affected the antimicrobial activity of carvacrol and thymol after encapsulation, while spray drying could be considered for the production of solid complexes in combination with the appropriate CD. CONCLUSIONS It was thus demonstrated that HP‐α‐ and HP‐γ‐CD are very suitable alternatives for the encapsulation of carvacrol and thymol with the purpose of preserving their bacteriostatic and bactericidal activities