Evolutionary trade-off between heat shock resistance, growth at high temperature, and virulence expression in Salmonella Typhimurium.

Understanding the evolutionary dynamics of foodborne pathogens throughout our food production chain is of utmost importance. In this study, we reveal that Salmonella Typhimurium can readily and reproducibly acquire vastly increased heat shock resistance upon repeated exposure to heat shock. Counterintuitively, this boost in heat shock resistance was invariantly acquired through loss-of-function mutations in the dnaJ gene, encoding a heat shock protein that acts as a molecular co-chaperone of DnaK and enables its role in protein folding and disaggregation. As a trade-off, however, the acquisition of heat shock resistance inevitably led to attenuated growth at 37°C and higher temperatures. Interestingly, loss of DnaJ also downregulated the activity of the master virulence regulator HilD, thereby lowering the fraction of virulence-expressing cells within the population and attenuating virulence in mice. By connecting heat shock resistance evolution to attenuation of HilD activity, our results confirm the complex interplay between stress resistance and virulence in Salmonella Typhimurium. IMPORTANCE: Bacterial pathogens such as Salmonella Typhimurium are equipped with both stress response and virulence features in order to navigate across a variety of complex inhospitable environments that range from food-processing plants up to the gastrointestinal tract of its animal host. In this context, however, it remains obscure whether and how adaptation to one environment would obstruct fitness in another. In this study, we reveal that severe heat stress counterintuitively, but invariantly, led to the selection of S. Typhimurium mutants that are compromised in the activity of the DnaJ heat shock protein. While these mutants obtained massively increased heat resistance, their virulence became greatly attenuated. Our observations, therefore, reveal a delicate balance between optimal tuning of stress response and virulence features in bacterial pathogens.

Adaptive evolution of Salmonella Typhimurium LT2 exposed to carvacrol lacks a uniform pattern.

Emergence of genetic variants with increased resistance/tolerance to natural antimicrobials, such as essential oils, has been previously evidenced; however, it is unknown whether mutagenesis follows a general or a specific pattern. For this purpose, we carried out four adaptive laboratory evolutions (ALE) in parallel of Salmonella enterica Typhimurium with carvacrol. After 10 evolution steps, we selected and characterized one colony from each lineage (SeCarA, SeCarB, SeCarC, and SeCarD). Phenotypic characterization of the four evolved strains revealed enhanced survival to lethal treatments; two of them (SeCarA and SeCarB) showed an increase of minimum inhibitory concentration of carvacrol and a better growth fitness in the presence of carvacrol compared to wild-type strain. Whole genome sequencing revealed 10 mutations, of which four (rrsH, sseG, wbaV, and flhA) were present in more than one strain, whereas six (nirC, fliH, lon, rob, upstream yfhP, and upstream argR) were unique to individual strains. Single-mutation genetic constructs in SeWT confirmed lon and rob as responsible for the increased resistance to carvacrol as well as to antibiotics (ampicillin, ciprofloxacin, chloramphenicol, nalidixic acid, rifampicin, tetracycline, and trimethoprim). wbaV played an important role in increased tolerance against carvacrol and chloramphenicol, and flhA in cross-tolerance to heat treatments. As a conclusion, no common phenotypical or genotypical pattern was observed in the isolated resistant variants of Salmonella Typhimurium emerged under carvacrol stress. Furthermore, the demonstration of cross-resistance against heat and antibiotics exhibited by resistant variants raises concerns regarding food safety. KEY POINTS: • Stable resistant variants of Salmonella Typhimurium emerged under carvacrol stress • No common pattern of mutagenesis after cyclic exposures to carvacrol was observed • Resistant variants to carvacrol showed cross-resistance to heat and to antibiotics.

Minimal Processing Technologies for Production and Preservation of Tailor-Made Foods§.

Tailor-made foods, also known as foods with programmable properties, are specialised systems with unique composition prepared by different methods, using the known mechanisms of action of their bioactive ingredients. The development of tailor-made foods involves the evaluation of individual components, including bioactive substances derived from waste products of other productions, such as essential oils. These components are evaluated both individually and in combination within food compositions to achieve specific functionalities. This review focuses on the application of minimal processing technologies for the production and preservation of tailor-made foods. It examines a range of approaches, including traditional and emerging technologies, as well as novel ingredients such as biomolecules from various sources and microorganisms. These approaches are combined according to the principles of hurdle technology to achieve effective synergistic effects that enhance food safety and extend the shelf life of tailor-made foods, while maintaining their functional properties.

Phenotypic and Genotypic Comparison of Antimicrobial-Resistant Variants of Escherichia coli and Salmonella Typhimurium Isolated from Evolution Assays with Antibiotics or Commercial Products Based on Essential Oils.

On account of the widespread development and propagation of antimicrobial-resistant (AMR) bacteria, essential oils (EOs) have emerged as potential alternatives to antibiotics. However, as already observed for antibiotics, recent studies have raised concerns regarding the potential emergence of resistant variants (RVs) to EOs. In this study, we assessed the emergence of RVs in Escherichia coli and Salmonella enterica Typhimurium after evolution assays under extended exposure to subinhibitory doses of two commercial EOs (AEN and COLIFIT) as well as to two antibiotics (amoxicillin and colistin). Phenotypic characterization of RVs from evolution assays with commercial EOs yielded no relevant increases in the minimum inhibitory concentration (MIC) of E. coli and did not even modify MIC values in S. Typhimurium. Conversely, RVs of E. coli and S. Typhimurium isolated from evolution assays with antibiotics showed increased resistance. Genotypic analysis demonstrated that resistance to commercial EOs was associated with enhanced protection against oxidative stress and redirection of cell energy toward efflux activity, while resistance to antibiotics was primarily linked to modifications in the cell binding sites of antibiotics. These findings suggest that AEN and COLIFIT could serve as safe alternatives to antibiotics in combating the emergence and dissemination of antimicrobial resistance within the agrifood system.

Combination of SPE and fluorescent detection of AQC-derivatives for the determination at sub-mg/L levels of biogenic amines in dairy products.

Biogenic amines (BAs) are compounds generated by decarboxylation of their amino acid precursors. Their intake, even at low concentrations, can lead to several types of health problems in sensitive individuals. As they can be easily formed in fermented dairy products, their quantitative determination is very relevant. In the present paper, a method for the quantitative determination of four biogenic amines in different dairy products has been developed, validated and applied to 37 samples of milk, 23 of yogurt, and 14 of kefir. Amines were selectively extracted using solid phase extraction, subsequently derivatizatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and further determined by High Performance Liquid Chromatography with fluorescence detection. The method's sensitivity was highly satisfactory, with limits of detection lower than 0.2 mg/L. Optimal linearity and repeatability were also achieved. BAs were not detected in most of the milk samples, but they were found frequently at high levels in yogurt and kefir samples, reaching values of up to 79 mg/kg total BAs in kefir samples. Levels measured should not be a cause for concern for the population at large, but should be known by BAs-sensitive individuals.

Potential of histamine-degrading microorganisms and diamine oxidase (DAO) for the reduction of histamine accumulation along the cheese ripening process.

Lentilactobacillus parabuchneri is the main bacteria responsible for the accumulation of histamine in cheese. The goal of this study was to assess the efficiency of potential histamine-degrading microbial strains or, alternatively, the action of the diamine oxidase (DAO) enzyme in the reduction of histamine accumulation along the ripening process in cheese. A total of 8 cheese variants of cow milk cheese were manufactured, all of them containing L. parabuchneri Deutsche Sammlung von Mikroorganismen 5987 (except for the negative control cheese variant) along with histamine-degrading strains (Lacticaseibacillus casei 4a and 18b; Lactobacillus delbrueckiisubsp. bulgaricus Colección Española de Cultivos Tipo (CECT) 4005 and Streptococcus salivariussubsp.thermophilus CECT 7207; two commercial yogurt starter cultures; or Debaryomyces hansenii), or DAO enzyme, tested in each cheese variant. Histamine was quantified along 100 days of cheese ripening. All the degrading measures tested significantly reduced the concentration of histamine. The highest degree of degradation was observed in the cheese variant containing D. hansenii, where the histamine content decreased up to 45.32 %. Cheese variants with L. casei, or L. bulgaricus and S. thermophilus strains, also decreased in terms of histamine content by 43.05 % and 42.31 %, respectively. No significant physicochemical changes (weight, pH, water activity, color, or texture) were observed as a consequence of the addition of potential histamine-degrading adjunct cultures or DAO in cheeses. However, the addition of histamine-degrading microorganisms was associated with a particular, not unpleasant aroma. Altogether, these results suggest that the use of certain histamine-degrading microorganisms could be proposed as a suitable measure in order to decrease the amount of histamine accumulated in cheeses.

Biopreservation of Chocolate Mousse with Lactobacillus helveticus 2/20: Microbial Challenge Test

Probiotic bacteria are used for food biopreservation because their metabolic products might contribute to ensuring food microbiological safety and/or increase its shelf life without the addition of chemical preservatives. Moreover, biopreserved foods are excellent vehicles for the delivery of probiotic bacteria. The aim of the study was to investigate the potential of chocolate mousse food matrix for the delivery of the probiotic strain Lactobacillus helveticus 2/20 (Lb. helveticus 2/20) and to investigate its capacity to inhibit the growth of two foodborne pathogenic bacteria (Staphylococcus aureus and Escherichia coli). Therefore, the populations of free or encapsulated in calcium alginate Lb. helveticus 2/20 cells and/or of each pathogen (used to voluntarily contaminate each sample) were monitored both in complex nutrient medium (MRS broth) and in chocolate mousse under refrigeration conditions and at room temperature. Lb. helveticus 2/20 alone in free or encapsulated state effectively inhibited the growth of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 in chocolate mousse when stored at 20 ± 2 °C. Practically no viable unwanted bacteria were identified on the 7th day from the beginning of the process. High viable Lb. helveticus 2/20 cell populations were maintained during storage under refrigerated conditions (4 ± 2 °C) and at room temperature. Chocolate mousse is thus a promising food matrix to deliver probiotic Lb. helveticus 2/20 cells, which could also protect it from contamination by unwanted bacteria.

Analytical strategies for the determination of biogenic amines in dairy products.

Biogenic amines (BA) are mainly produced by the decarboxylation of amino acids by enzymes from microorganisms that emerge during food fermentation or due to incorrectly applied preservation processes. The presence of these compounds in food can lead to a series of negative effects on human health. To prevent the ingestion of high amounts of BA, their concentration in certain foods needs to be controlled. Although maximum legal levels have not yet been established for dairy products, potential adverse effects have given rise to a substantial number of analytical and microbiological studies: they report concentrations ranging from a few mg/kg to several g/kg. This article provides an overview of the analytical methods for the determination of biogenic amines in dairy products, with particular focus on the most recent and/or most promising advances in this field. We not only provide a summary of analytical techniques but also list the required sample pretreatments. Since high performance liquid chromatography with derivatization is the most widely used method, we describe it in greater detail, including a comparison of derivatizing agents. Further alternative techniques for the determination of BA are likewise described. The use of biosensors for BA in dairy products is emerging, and current results are promising; this paper thus also features a section on the subject. This review can serve as a helpful guideline for choosing the best option to determine BA in dairy products, especially for beginners in the field.

Effect of Intramuscularly Administered Oxytetracycline or Enrofloxacin on Vancomycin-Resistant Enterococci, Extended Spectrum Beta-Lactamase- and Carbapenemase-Producing Enterobacteriaceae in Pigs.

Nowadays, there is a great concern about the prevalence of multidrug resistant Enterococcus spp. and Enterobacteriaceae in food-producing animals. The aim of this work was to evaluate the effect of oxytetracycline or enrofloxacin treatment on vancomycin-resistant enterococci (VRE), extended spectrum ß-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae in pigs. A total of 26 piglets were received and distributed in three groups. Group 1 was treated with enrofloxacin (N = 12), group 2 with oxytetracycline (N = 10) and group 3 did not receive any treatment (control group) (N = 4). A higher number of vancomycin-resistant E. faecium were recovered compared to E. faecalis. In the pigs treated with enrofloxacin, vancomycin resistant E. faecium was found in a higher percentage of animals than in the control group. ESBL-producing E. coli was not detected in rectal samples from control animals. However, it was detected in 17-20% of animals treated with oxytetracycline on days 6 to 17 and in 17-50% of the animals treated with enrofloxacin. Carbapenemase-producing E. coli was isolated in animals treated with oxytetracycline, but not in animals treated with enrofloxacin or in the control group. This study highlights that the use of oxytetracycline or enrofloxacin in food-producing animals could select ESBL and carbapenemase-producing E. coli. Further studies shall be needed to validate the results obtained, considering a more robust and extended experimental design.

Carvacrol Selective Pressure Allows the Occurrence of Genetic Resistant Variants of Listeria monocytogenes EGD-e.

Essential oils and their constituents, such as carvacrol, are potential food preservatives because of their great antimicrobial properties. However, the long-term effects of these compounds are unknown and raise the question of whether resistance to these antimicrobials could emerge. This work aims to evaluate the occurrence of genetic resistant variants (RVs) in Listeria monocytogenes EGD-e by exposure to carvacrol. Two protocols were performed for the RVs selection: (a) by continuous exposure to sublethal doses, where LmSCar was isolated, and (b) by reiterative exposure to short lethal treatments of carvacrol, where LmLCar was isolated. Both RVs showed an increase in carvacrol resistance. Moreover, LmLCar revealed an increased cross-resistance to heat treatments at acid conditions and to ampicillin. Whole-genome sequencing identified two single nucleotide variations in LmSCar and three non-silent mutations in LmLCar. Among them, those located in the genes encoding the transcriptional regulators RsbT (in LmSCar) and ManR (in LmLCar) could contribute to their increased carvacrol resistance. These results provide information regarding the mode of action of this antimicrobial and support the importance of knowing how RVs appear. Further studies are required to determine the emergence of RVs in food matrices and their impact on food safety.

Development of a new dual electrochemical immunosensor for a rapid and sensitive detection of enrofloxacin in meat samples.

A novel dual electrochemical immunosensor was fabricated for the rapid and sensitive detection of enrofloxacin (EF) antibiotic in meat. Anti-quinolone antibody was immobilized onto screen-printed dual carbon electrodes via carbodiimide coupling. A new electrochemical probe was synthesized by conjugating difloxacin and aminoferrocene, whose oxidation was measured at + 0.2 V vs. Ag/AgCl by differential pulse voltammetry. The detection principle was based on the competitive binding of this conjugate and free EF on immobilized antibodies. The proposed immunosensor allowed detection of EF at concentrations ranging from 0.005 µg.mL-1 to 0.01 µg.mL-1 with a detection limit of 0.003 µg.mL-1. The immunosensor was stable for at least 1 month at 4 °C and displayed a good specificity for other fluoroquinolones. The new dual electrode design offered an improved accuracy as one electrode was used as negative control. The efficiency of the sensor and the adequacy of the extraction process were finally validated by detecting EF in different meat samples.

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.

Optimization and Validation of a New Microbial Inhibition Test for the Detection of Antimicrobial Residues in Living Animals Intended for Human Consumption.

Even though antibiotics are necessary in livestock production, they can be harmful not only due to their toxicity, but also in view of their contribution to the emergence of antimicrobial resistance. Screening tests based on microbial growth inhibition appeared to be useful tools to prevent its entry into the food chain. They have nevertheless been traditionally carried out post mortem, leading to great economical loss and harm to the environment in case a positive sample is found. Hence, the objective was to evaluate the use of a screening test as an ante mortem alternative for the detection of antibiotic residues in meat: thus, Explorer®-Blood test was optimized and validated. After adapting the procedure for matrix preparation, the assay parameters were assessed from 344 antibiotic-free blood serum samples. Limits of Detection (LoDs) were defined by spiking blood serum with several of the most common antimicrobials used in veterinary practice. LoDs were similar to those obtained for meat and were at or below the maximum residue limits set by EU legislation for muscle. Analyses of in vivo injected samples, previously characterized by LC-MS/MS, demonstrated the method's accuracy and proved that Explorer®-Blood can be considered a suitable alternative to conventional post mortem screening methods.

Antibacterial Residue Excretion via Urine as an Indicator for Therapeutical Treatment Choice and Farm Waste Treatment.

Many of the infectious diseases that affect livestock have bacteria as etiological agents. Thus, therapy is based on antimicrobials that leave the animal's tissues mainly via urine, reaching the environment through slurry and waste water. Once there, antimicrobial residues may lead to antibacterial resistance as well as toxicity for plants, animals, or humans. Hence, the objective was to describe the rate of antimicrobial excretion in urine in order to select the most appropriate molecule while reducing harmful effects. Thus, 62 pigs were treated with sulfamethoxypyridazine, oxytetracycline, and enrofloxacin. Urine was collected through the withdrawal period and analysed via LC-MS/MS. Oxytetracycline had the slowest rate of degradation (a half-life time of 4.18 days) and the most extended elimination period in urine (over 2 months), followed by enrofloxacin (a half-life time of 1.48 days, total urine elimination in ca. 3 weeks) and sulfamethoxypyridazine (a half-life time of 0.49 days, total urine elimination in ca. 1 week). Bacterial sensitivity and recommendations for responsible use are limiting when selecting the treatment. Nevertheless, with similar effectiveness, sulfamethoxypyridazine would be the choice, as waste treatment would only need to be implemented for 1 week after treatment. Thus, more in-depth knowledge regarding antibacterial elimination would improve resource management, while protecting animals and consumers' health.

Emerging mutant populations of Listeria monocytogenes EGD-e under selective pressure of Thymbra capitata essential oil question its use in food preservation.

Due to their excellent antimicrobial properties, essential oils (EO) have been proposed as potential preservatives for certain kinds of foods, such as dairy products. However, the occurrence of pathogenic populations that are resistant to EOs could pose a health risk. This report seeks to assess the emergence of resistant populations in Listeria monocytogenes EGD-e growth at 37 °C under selective pressure of Thymbra capitata EO (TCO), to characterise their resistance in laboratory media, and to identify their genotypic changes, as well as to evaluate the resistance in skimmed milk. TCO cyclic treatment allowed the isolation of two L. monocytogenes EGD-e resistant strains against the EO: LmSTCO by sublethal doses (75 µL/L TCO) and LmLTCO by lethal doses (300 µL/L TCO) after 20 and 30 cycles, respectively. Both strains displayed an increase of the minimum inhibitory and bactericidal concentration against TCO and a higher survival rate after lethal treatments than the wild-type strain (LmWT). Growth kinetics revealed a better adaptation of LmSTCO in presence of TCO, while LmLTCO grew more slowly compared to LmWT, even in the absence of the antimicrobial. Moreover, a slight increase in cross-resistance to antibiotics was observed: LmSTCO to ß-lactams and LmLTCO to a series of broad-spectrum antibiotics. The genomic study revealed one sole nucleotide change in LmSTCO located in plsC gene codifying an enzyme involved in the production of phosphatidic acid, a precursor in cell membrane synthesis. Five genetic variations were found in LmLTCO: among them, the deletion of an ATP-synthesis system involved in slowing bacterial growth. Inhibition and inactivation assays in skimmed milk confirmed the increased resistance of both strains, thereby indicating a safety risk in case these strains emerge in the food chain. These results strongly suggest that the occurrence of such resistances should be taken into account in order to ensure the efficacy of natural antimicrobials in the design of food preservation strategies.

Histamine accumulation in dairy products: Microbial causes, techniques for the detection of histamine-producing microbiota, and potential solutions.

Histamine poisoning is a significant public health and safety concern. Intoxication from ingestion of food containing high amounts of histamine may cause mild or severe symptoms that can even culminate in cardiac arrest. Nonetheless, although histamine levels in dairy products are not subject to any regulation, important outbreaks and severe adverse health effects have been reported due to intake of dairy products with a high histamine content, especially ripened cheeses. Histamine, a biogenic amine, can accumulate in dairy products as a result of the metabolism of starter and nonstarter lactic acid bacteria, as well as yeasts that contribute to the ripening or flavoring of the final product, or even as a result of spoilage bacteria. The aim of this review is to describe the microbiological causes of the presence of histamine in fermented milk products, and to propose control measures and potential methods for obtaining histamine-free dairy products. Thus, this manuscript focuses on histamine-producing microbiota in dairy products, highlighting the detection of histamine-producing bacteria through traditional and novel techniques. In addition, this review aims to explore control measures to prevent the access of histamine-producing microbiota to raw materials, as well as the formation of histamine in dairy products, such as a careful selection of starter cultures lacking the ability to produce histamine, or even the implementation of effective food processing technologies to reduce histamine-producing microbiota. Finally, the removal of histamine already formed in dairy products through histamine-degrading microorganisms or by enzymatic degradation will also be explored.

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-ß- 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-ß-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-ß-CD, normally used in the encapsulation of carvacrol and thymol. HP-ß-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. © 2020 Society of Chemical Industry.

Improvement of the Shelf-Life Status of Modified Atmosphere Packaged Camel Meat Using Nisin and Olea europaea Subsp. laperrinei Leaf Extract.

The impact of combined biopreservation treatment with Olea europaea subsp. laperrinei leave extracts (laper.OLE) and nisin on the quality attributes of camel steaks packaged under high O2 (80%) and CO2 (20%) atmosphere was investigated during refrigerated (1 ± 1 °C) long-term storage. As measured by reversed phase HPLC/DAD analysis, oleuropein is the phenolic compound most present in the chemical composition of laper.OLE (63.03%). Camel steaks treated with laper.OLE had a lower concentration of thiobarbituric acid-reactive substances (TBA-RSs) in the course of 30 days of storage. Surface metmyoglobin (MetMb) increased at a reduced rate in laper.OLE-treated samples compared to control samples. Neither modified atmosphere packaging (MAP) nor biopreservation treatments significantly altered the tenderness of camel steaks, expressed in terms of Warner-Bratzler shear force (WBSF), as compared to control samples. After 30 days of storage, psychrotrophic bacteria and Pseudomonas spp. counts were significantly lower in camel steaks treated with a combination of laper.OLE and nisin than in untreated steaks. Moreover, samples treated with laper.OLE received higher scores on bitterness acceptability. In sum, the use of combined biopreservation methods could be a sustainable solution for the preservation and promotion of the quality characteristics of camel meat in arid regions.

Physiological alterations involved in inactivation of autochthonous spoilage bacteria in orange juice caused by Citrus essential oils and mild heat.

This study investigated physiological alterations involved in the inactivation of Levilactobacillus (L.) brevis and Leuconostoc (Lc.) mesenteroides in orange juice caused by Citrus lemon essential oil (CLEO) and C. reticulata essential oil (CREO) alone and combined with mild heat treatment (MHT). Damage in DNA, membrane integrity, membrane potential, metabolic and efflux activity of bacterial cells were measured after exposure (6 and 12 min) to CLEO or CREO (0.5 µL/mL) and/or MHT (54 °C) using flow cytometry. Limonene was the major constituent in CLEO (66.4%) and CREO (89.4%). The size of the damaged cell subpopulations increased (p < 0.05) after longer exposure time and varied with the tested essential oil and/or bacterial isolate. After exposure to CLEO and CREO alone, the cell subpopulations with damage in measured physiological functions were in a range of 19.6-66.8% and 23.8-75.9%, respectively. Exposure to CREO resulted in larger Lc. mesenteroides cell subpopulations (35.4-68.7%) with damaged DNA, permeabilized and depolarized membrane and compromised metabolic or efflux activity compared to L. brevis (23.8-58.0%). In contrast, exposure to CLEO led to higher damaged L. brevis cell subpopulations (35.1-77%) compared to Lc. mesenteroides (25.3-36.6%). Exposure to combined treatments (CLEO or CREO and MHT) affected the measured physiological functions in almost the entire L. brevis and Lc. mesenteroides cell population (up to 99%), although the damage extension on each isolate varied with tested essential oil. Results show that inactivation of L. brevis and Lc. mesenteroides cells caused by CLEO and CREO alone and combined with MHT in orange juice involves a multi-target action, which causes DNA damage, altered permeability and depolarization of membrane and compromised metabolic and efflux activities.

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.