Diego García-Gonzalo

Chemical composition and antioxidant properties of Laurus nobilis L. and Myrtus communis L. essential oils from Morocco and evaluation of their antimicrobial activity acting alone or in combined processes for food preservation

BACKGROUND This study describes the antioxidant and antimicrobial activity of Laurus nobilis L. and Myrtus communis L. essential oils (EOs). This is the first report of the synergistic antimicrobial effect of these EOs in combination with physical food preservation treatments. RESULTS EOs obtained by steam distillation from aerial parts of Laurus nobilis and Myrtus communis were analysed by using gas chromatography-mass spectrometry. The main compounds were 1,8-cineole and 2-carene (L. nobilis EO); and myrtenyl acetate, 1,8-cineole and α-pinene (M. communis EO). L. nobilis EO showed higher antioxidant activity than M. communis EO in three complementary antioxidant tests. Although antimicrobial activity tests demonstrated the effectiveness of L. nobilis EO and the lack of bactericidal effect of M. communis EO, synergistic lethal effects were observed when combining each EO (0.2 µL mL(-1)) with mild heat (54°C for 10 min) or high hydrostatic pressure (175-400 MPa for 20 min). In contrast, combination of EOs with pulsed electric fields (30 kV cm(-1) for 25 pulses) showed no additional effects. CONCLUSION This study shows the great potential of these EOs in combined treatments with mild heat and high hydrostatic pressure to obtain a higher inactivation of foodborne pathogens, which might help in the design of safe processes applied at low intensity.

Inactivation of Escherichia coli O157:H7 in fruit juices by combined treatments of citrus fruit essential oils and heat

This work approaches the possibility of combining mild heat treatments with citrus fruit essential oils (EOs) to improve the effectiveness of heat treatments and thus to reduce treatment intensity. Concentrations between 10 and 200 μL/L of lemon, mandarin, or orange EO were tested at 54 °C for 10 min in laboratory media, determining that 200 μL/L of each EO was necessary to achieve a 5 log(10) reduction of the initial Escherichia coli O157:H7 concentration. A relationship could be established between sublethally injured cells after the heat treatment and inactivated cells after the combined process. In apple juice, the synergism in the inactivation of E. coli O157:H7 when adding 200 μL/L of lemon EO might suppose a reduction in the treatment temperature (of 4.5 °C) or in the treatment time (by 5.7 times) within the range of temperature assayed (54-60 °C). Addition of 75 μL/L of lemon EO was determined to achieve the same synergistic effect of the combined treatment when the initial inoculum was reduced from 3×10(7) to 3×10(4) CFU/mL. Since the addition of lemon EO did not decrease the hedonic acceptability of apple juice, the proposed combined treatment could be further studied and optimized for the production of new minimally processed juices.

Synergistic combinations of high hydrostatic pressure and essential oils or their constituents and their use in preservation of fruit juices.

This work addresses the inactivation achieved with Escherichia coli O157:H7 and Listeria monocytogenes EGD-e by combined processes of high hydrostatic pressure (HHP) and essential oils (EOs) or their chemical constituents (CCs). HHP treatments (175-400 MPa for 20 min) were combined with 200 μL/L of each EO (Citrus sinensis L., Citrus lemon L., Citrus reticulata L., Thymus algeriensis L., Eucalyptus globulus L., Rosmarinus officinalis L., Mentha pulegium L., Juniperus phoenicea L., and Cyperus longus L.) or each CC ((+)-limonene, α-pinene, β-pinene, p-cymene, thymol, carvacrol, borneol, linalool, terpinen-4-ol, 1,8-cineole, α-terpinyl acetate, camphor, and (+)-pulegone) in buffer of pH 4.0 or 7.0. The tested combinations achieved different degrees of inactivation, the most effective being (+)-limonene, carvacrol, C. reticulata L. EO, T. algeriensis L. EO and C. sinensis L. EO which were capable of inactivating about 4-5 log(10) cycles of the initial cell populations in combination with HHP, and therefore showed outstanding synergistic effects. (+)-Limonene was also capable of inactivating 5 log(10) cycles of the initial E. coli O157:H7 population in combination with HHP (300 MPa for 20 min) in orange and apple juices, and a direct relationship was established between the inactivation degree caused by the combined process with (+)-limonene and the occurrence of sublethal injury after the HHP treatment. This work shows the potential of EOs and CCs in the inactivation of foodborne pathogens in combined treatments with HHP, and proposes their possible use in liquid food such as fruit juices.

New insights in mechanisms of bacterial inactivation by carvacrol

To study the mechanism of bacterial inactivation by carvacrol and the influence of genetic and environmental factors in its antimicrobial activity.

Effect of citral on the thermal inactivation of Escherichia coli O157:H7 in citrate phosphate buffer and apple juice.

Inactivation and sublethal injury of Escherichia coli O157:H7 cells induced by heat in citrate phosphate buffer and apple juice (both at pH 3.8) were studied, and the effect of a combined preservation treatment using citral and heat treatments was determined. Heat resistance of E. coli O157:H7 was similar in both treatment media; after 27 min at 54°C, 3 log units of the initial cell population was inactivated in both treatment media. However, under less harsh conditions a protective effect of apple juice was found. Whereas inactivation followed linear kinetics in the citrate phosphate buffer, when cells were treated in apple juice the survival curves were concave downward. Heat treatment caused a great degree of sublethal injury; 4 min at 54°C inactivated less than 0.5 log CFU/ml but sublethally injured more than 3 log CFU/ml. The addition of 18 and 200 ppm of citral to the treatment medium acted synergistically with heat at 54°C to inactivate 3 × 10(4) and 3 × 10(7) CFU/ml, respectively. Addition of citral thus reduced the time needed to inactivate 1 log unit of the initial E. coli O157:H7 population from 8.9 to 1.7 min. These results indicate that a combined process of heat and citral can inactivate E. coli O157:H7 cells and reduce their potential negative effects.

Oxygenated monoterpenes citral and carvacrol cause oxidative damage in Escherichia coli without the involvement of tricarboxylic acid cycle and Fenton reaction.

Oxygenated monoterpenes citral and carvacrol are common constituents of many essential oils (EOs) that have been extensively studied as antimicrobial agents but whose mechanisms of microbial inactivation have not been totally elucidated. A recent study described a mechanism of Escherichia coli death for (+)-limonene, a hydrocarbon monoterpene also frequently present in EOs, similar to the common mechanism proposed for bactericidal antibiotics. This mechanism involves the formation of Fenton-mediated hydroxyl radical, a reactive oxygen species (ROS), via tricarboxylic acid (TCA) cycle, which would ultimately inactivate cells. Our objective was to determine whether E. coli MG1655 inactivation by citral and carvacrol follows a similar mechanism of cell death. Challenging experiments with 300μL/L citral and 100μL/L carvacrol inactivated at least 2.5log10cycles of exponentially growing cells in 3h under aerobic conditions. The presence of thiourea (an ROS scavenger) reduced cell inactivation in 2log10cycles, demonstrating the role of ROS in cell death. Decreased resistance of a ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) indicated that citral and carvacrol caused oxidative damage to DNA. Although the mechanism of E. coli inactivation by carvacrol and citral was similarly mediated by ROS, their formation did not follow the same pathways described for (+)-limonene and bactericidal drugs because neither Fenton reaction nor NADH production via the TCA cycle was involved in cell death. Moreover, further experiments demonstrated antimicrobial activity of citral and carvacrol in anaerobic environments without the involvement of ROS. As a consequence, cell death by carvacrol and citral in anaerobiosis follows a different mechanism than that observed under aerobic conditions. These results demonstrated a different mechanism of inactivation by citral and carvacrol with regard to (+)-limonene and bactericidal antibiotics, indicating the complexity of the mechanisms of bacterial inactivation among EO constituents. Advancements in the description of these mechanisms will help in extending and improving the use of these compounds as natural antimicrobials.

Role of general stress-response alternative sigma factors σS (RpoS) and σB (SigB) in bacterial heat resistance as a function of treatment medium pH

This investigation aimed to determine the role of general stress-response alternative sigma factors σ(S) (RpoS) and σ(B) (SigB) in heat resistance and the occurrence of sublethal injuries in cell envelopes of stationary-phase Escherichia coli BJ4 and Listeria monocytogenes EGD-e cells, respectively, as a function of treatment medium pH. Given that microbial death followed first-order inactivation kinetics (R(2)>0.95) the traditional D(T) and z values were used to describe the heat inactivation kinetics. Influence of rpoS deletion was constant at every treatment temperature and pH, making a ΔrpoS deletion mutant strain approximately 5.5 times more heat sensitive than its parental strain for every studied condition. Furthermore, the influence of the pH of the treatment medium on the reduction of the heat resistance of E. coli was also constant and independent of the treatment temperature (average z value=4.9°C) in both parental and mutant strains. L. monocytogenes EGD-e z values obtained at pH 7.0 and 5.5 were not significantly different (p>0.05) in either parental or the ∆sigB deletion mutant strains (average z value=4.8°C). Nevertheless, at pH 4.0 the z value was higher (z=8.4°C), indicating that heat resistance of both L. monocytogenes strains was less dependent on temperature at pH 4.0. At both pH 5.5 and 7.0 the influence of sigB deletion was constant and independent of the treatment temperature, decreasing L. monocytogenes heat resistance approximately 2.5 times. In contrast, the absence of sigB did not decrease the heat resistance of L. monocytogenes at pH 4.0. The role of RpoS in protecting cell envelopes was more important in E. coli (4 times) than SigB in L. monocytogenes (1.5 times). Moreover, the role of σ(S) in increasing heat resistance seems more relevant in enhancing the intrinsic resilience of the cytoplasmic membrane, and to a lesser extent, outer membrane resilience. Knowledge of environmental conditions related to the activation of alternative sigma factors σ(S) and σ(B) and their effects on heat resistance would help us to avoid and/or identify situations that increase bacterial stress resistance. Therefore, more efficient food preservation processes might be designed.

Individual Constituents from Essential Oils Inhibit Biofilm Mass Production by Multi-Drug Resistant Staphylococcus aureus

Biofilm formation by Staphylococcus aureus represents a problem in both the medical field and the food industry, because the biofilm structure provides protection to embedded cells and it strongly attaches to surfaces. This circumstance is leading to many research programs seeking new alternatives to control biofilm formation by this pathogen. In this study we show that a potent inhibition of biofilm mass production can be achieved in community-associated methicillin-resistant S. aureus (CA-MRSA) and methicillin-sensitive strains using plant compounds, such as individual constituents (ICs) of essential oils (carvacrol, citral, and (+)-limonene). The Crystal Violet staining technique was used to evaluate biofilm mass formation during 40 h of incubation. Carvacrol is the most effective IC, abrogating biofilm formation in all strains tested, while CA-MRSA was the most sensitive phenotype to any of the ICs tested. Inhibition of planktonic cells by ICs during initial growth stages could partially explain the inhibition of biofilm formation. Overall, our results show the potential of EOs to prevent biofilm formation, especially in strains that exhibit resistance to other antimicrobials. As these compounds are food additives generally recognized as safe, their anti-biofilm properties may lead to important new applications, such as sanitizers, in the food industry or in clinical settings.

Impact of Essential Oils on the Taste Acceptance of Tomato Juice, Vegetable Soup, or Poultry Burgers

Despite the vast body of available literature on the possibilities of essential oils (EOs) as food preservatives or functional ingredients, the sensory impact of their addition to foods has barely been approached. This work focuses on the hedonic taste acceptance of 3 food products (tomato juice, vegetable soup, and poultry burgers) when they are incorporated with potentially antimicrobial concentrations (20 to 200 μL/L) of 6 selected EOs (lemon, pennyroyal mint, thyme, and rosemary) and individual compounds (carvacrol, p-cymene). Although addition of 20 μL/L of pennyroyal mint or lemon EO did not change the taste acceptance of tomato juice, higher concentrations of these compounds or any concentration of the other 4 compounds did. In vegetable soup, the tolerance limit for rosemary EO, thyme EO, carvacrol, or p-cymene was 20 μL/L, while the addition of 200 μL/L of lemon EO was accepted. Tolerance limits in poultry burgers were established in 20 μL/L for carvacrol and thyme EOs, 100 μL/L for pennyroyal mint EO and p-cymene, and 200 μL/L for lemon and rosemary EOs. Moreover, incorporation of pennyroyal mint EO to tomato juice or poultry burgers, and enrichment of vegetable soup with lemon EO, could contribute to the development of food products with an improved sensory appeal.

Resistance of Staphylococcus aureus to UV-C light and combined UV-heat treatments at mild temperatures

In this investigation, the resistance of enterotoxigenic Staphylococcus aureus to short-wave ultraviolet light (UV-C) and to combined UV C-heat (UV-H) treatments in buffers and in liquid foods with different physicochemical characteristics was studied. Microbial resistance to UV-C varied slightly among the S. aureus strains tested. The UV-C resistance of S. aureus increased in the entry of stationary growth phase, which in part was due to the expression of the alternative sigma factor σ(B). The UV-C resistance of S. aureus was independent of the treatment mediums pH and water activity, but it decreased exponentially as the absorption coefficient increased. UV-C bactericidal efficacy in liquids of high absorption coefficients was improved synergistically when combined with a mild heat treatment at temperatures ranging from 50.0 to 57.5 °C. pH of the treatment medium modified the lethality of UV-H treatments and therefore the temperature of maximum synergy. The advantage of combined UV-H treatments was demonstrated in fruit juices and vegetable and chicken broths, inactivating 5 Log₁₀ cycles of S. aureus by applying UV-C treatments of 27.1 mJ/L for 3.6 min at 52.5 °C or 13.6 mJ/L for 1.8 min at 55.0 °C.