Mechanism of Bacterial Inactivation by (+)-Limonene and Its Potential Use in Food Preservation Combined Processes (original) (raw)
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Strategic approach of multifaceted antibacterial mechanism of limonene traced in Escherichia coli
Scientific Reports, 2021
Antibacterial potential of Limonene against Multi Drug Resistant (MDR) pathogens was studied and mechanism explored. Microscopic techniques viz. Fluorescent Microscopy (FM), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) indicated membrane disruption, cellular leakage and cell death of Escherichia coli (E. coli) cells when treated with limonene. Leakage of intracellular proteins, lipids and nucleic acid confirmed membrane damage and disruption of cell permeability barrier. Further, release of intracellular ATP, also suggested disruption of membrane barrier. Interaction of limonene with DNA revealed its capability in unwinding of plasmid, which could eventually inhibit DNA transcription and translation. Differential expression of various proteins and enzymes involved in transport, respiration, metabolism, chemotaxis, protein synthesis confirmed the mechanistic role of limonene on their functions. Limonene thus can be a potential candidate in drug devel...
PLoS ONE, 2014
+)-limonene is a lipophilic antimicrobial compound, extracted from citrus fruits' essential oils, that is used as a flavouring agent and organic solvent by the food industry. A recent study has proposed a common and controversial mechanism of cell death for bactericidal antibiotics, in which hydroxyl radicals ultimately inactivated cells. Our objective was to determine whether the mechanism of Escherichia coli MG1655 inactivation by (+)-limonene follows that of bactericidal antibiotics. A treatment with 2,000 mL/L (+)-limonene inactivated 4 log 10 cycles of exponentially growing E. coli cells in 3 hours. On one hand, an increase of cell survival in the DacnB mutant (deficient in a TCA cycle enzyme), or in the presence of 2,29-dipyridyl (inhibitor of Fenton reaction by iron chelation), thiourea, or cysteamine (hydroxyl radical scavengers) was observed. Moreover, the DrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) was more sensitive to (+)limonene. Thus, this indirect evidence indicates that the mechanism of exponentially growing E. coli cells inactivation by 2,000 mL/L (+)-limonene is due to the TCA cycle and Fenton-mediated hydroxyl radical formation that caused oxidative DNA damage, as observed for bactericidal drugs. However, several differences have been observed between the proposed mechanism for bactericidal drugs and for (+)-limonene. In this regard, our results demonstrated that E. coli inactivation was influenced by its physiological state and the drug's concentration: experiments with stationary-phase cells or 4,000 mL/L (+)limonene uncovered a different mechanism of cell death, likely unrelated to hydroxyl radicals. Our research has also shown that drug's concentration is an important factor influencing the mechanism of bacterial inactivation by antibiotics, such as kanamycin. These results might help in improving and spreading the use of (+)-limonene as an antimicrobial compound, and in clarifying the controversy about the mechanism of inactivation by bactericidal antibiotics. Citation: Chueca B, Pagán R, García-Gonzalo D (2014) Differential Mechanism of Escherichia coli Inactivation by (+)-Limonene as a Function of Cell Physiological State and Drug's Concentration. PLoS ONE 9(4): e94072.
Isolation and screening of d-limonene-resistant microorganisms
Brazilian Journal of Microbiology, 2007
This study reports the isolation of microorganisms that are resistant to environment containing limonene, the most important residue in the citrus industry. For the isolation, samples collected from strategic places of a citrus processing plant (yellow water, entrance and exit of the bagasse tank, effluent and deteriorated fruits found in bins, machine straps, fruit washers and plant floor), some citrus fruit and mint from local market were used. The samples were incubated in rotary shaker at 30ºC/150rpm for 48h or 7 days in YM medium containing 0.1% limonene. Great part of the 112 strains recovered after 48h and the 126 strains recovered after 7 days were identified as Gram positive bacilli, followed by Gram negative bacilli, yeasts and Gram positive cocci, besides five fungi. About half of the Gram positive and Gram negative bacilli and yeasts and some Gram positive Cocci were resistant to limonene concentrations up to 2% in the medium broth. Amongst them seventy were able to grow in mineral medium containing limonene as sole carbon source. The research described in this paper is the initial step for the exploration of flavor compounds production via biotransformation of limonene, a nonexpensive by-product of citrus industry.
Limonene, a major component of citrus peel oil, has a number of applications related to microbiology. The antimicrobial properties of limonene make it a popular disinfectant and food preservative, while its potential as a biofuel component has made it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity remains enigmatic. In this study, we characterized a limonene-tolerant strain of Escherichia coli and found a mutation in ahpC, encoding alkyl hydroperoxidase, which alleviated limonene toxicity. We show that the acute toxicity previously attributed to limonene is largely due to the common oxidation product limonene hydroperoxide, which forms spontaneously in aerobic environments. The mutant AhpC protein with an L-to-Q change at position 177 (AhpC L177Q ) was able to alleviate this toxicity by reducing the hydroperoxide to a more benign compound. We show that the degree of limonene toxicity is a function of its oxidation level and that nonoxidized limonene has relatively little toxicity to wild-type E. coli cells. Our results have implications for both the renewable production of limonene and the applications of limonene as an antimicrobial. . 2015. Acute limonene toxicity in Escherichia coli is caused by limonene hydroperoxide and alleviated by a point mutation in alkyl hydroperoxidase AhpC. Appl Environ Microbiol 81:4690 -4696.
Applied and Environmental Microbiology, 2015
Limonene, a major component of citrus peel oil, has a number of applications related to microbiology. The antimicrobial properties of limonene make it a popular disinfectant and food preservative, while its potential as a biofuel component has made it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity remains enigmatic. In this study, we characterized a limonene-tolerant strain of Escherichia coli and found a mutation in ahpC , encoding alkyl hydroperoxidase, which alleviated limonene toxicity. We show that the acute toxicity previously attributed to limonene is largely due to the common oxidation product limonene hydroperoxide, which forms spontaneously in aerobic environments. The mutant AhpC protein with an L-to-Q change at position 177 (AhpC L177Q ) was able to alleviate this toxicity by reducing the hydrope...
Nanoemulsified D-Limonene Reduces the Heat Resistance of Salmonella Senftenberg over 50 Times
Nanomaterials, 2017
Salmonella Senftenberg is a pathogen agent causative of foodborne disease and it is considered the most heat-resistant serovar within this genus. Food industries use heat treatment and chemical antimicrobials in order to eliminate this microorganism in food, but consumers prefer natural antimicrobials as essential oils and their components. This study evaluates the combined effect of thermal treatments and different concentrations of D-limonene nanoemulsion on the inactivation of Salmonella (S.) Senftenberg. The results showed an important effect of the nanoemulsified D-limonene on the heat resistance of S. Senftenberg. The δ50 °C value was reduced by 85%, 96% and 98% when 0.1, 0.5 and 1 mM of nanoemulsified D-limonene was added to the heating medium. The effect was kept along all the heating temperatures researched and the shape of the survival curves did not change with the addition of the antimicrobial. The results obtained in this research could be very useful for food industries for optimizing or improving heat treatments applied to food.
Journey of Limonene as an Antimicrobial Agent
Journal of Pure and Applied Microbiology, 2021
Injudicious consumption of antibiotics in the past few decades has arisen the problem of resistance in pathogenic organisms against most antibiotics and antimicrobial agents. Scenarios of treatment failure are becoming more common in hospitals. This situation demands the frequent need for new antimicrobial compounds which may have other mechanisms of action from those which are in current use. Limonene can be utilized as one of the solutions to the problem of antimicrobial resistance. Limonene is a naturally occurring monoterpene with a lemon-like odor, which mainly present in the peels of citrus plants like lemon, orange, grapefruit, etc. The study aimed to enlighten the antimicrobial properties of limonene as per previous literature. Advantageous contributions have been made by various research groups in the study of the antimicrobial properties of limonene. Previous studies have shown that limonene not only inhibits disease-causing pathogenic microbes, however, it also protects v...
Food Microbiology, 2017
The application of D-limonene in form of nanoemulsion has been proved to reduce dramatically the thermal resistance of Listeria monocytogenes in culture media. The present research shows very promising results on the application in food products. The thermal resistance of L. monocytogenes was reduced 90 times when 0.5 mM nanoemulsified D-limonene was added to apple juice. This is the biggest reduction in the heat resistance of a microorganism caused by an antimicrobial described ever. However, no effect was found in carrot juice. A carrot juice system was prepared in an attempt to unravel which juice constituents were responsible for the lack of effect. When fat and fibre were not included in the carrot juice system formulation, the thermal resistance of L. monocytogenes was, again, dramatically reduced in presence of nanoemulsified D-limonene, so these components were shown to interfere with the effect. Once this interaction with food constituents becomes solved, the addition of nanoemulsified antimicrobials would allow to reduce greatly the intensity of the thermal treatments currently applied in the food processing industry.
Grapefruit bioactive limonoids modulate E. coli O157: H7 TTSS and biofilm
International journal of food microbiology, 2010
Limonoids are important constituents of the grapefruit and other citrus fruits. Research on health benefits suggests that citrus limonoids may act as anti-cancer, cholesterol lowering, anti-HIV and anti-feedant compounds. However, antimicrobial activities of citrus limonoids are not reported. In the present investigation, limonoids were purified from grapefruit seed and evaluated for their potential to antagonize cell-to-cell communication, biofilm formation and expression of Enterohemorrhagic Escherichia coli (EHEC) type three secretion ...
Scientific Reports, 2020
Consumers’ demands for ready-to-eat, fresh-like products are on the rise during the last years. This type of products have minimal processing conditions that can enable the survival and replication of pathogenic microorganisms. Among them, Listeria monocytogenes is of special concern, due to its relatively high mortality rate and its ability to replicate under refrigeration conditions. Previous research works have shown that nanoemulsified essential oils in combination with thermal treatments are effective for inactivating L. monocytogenes. However, previous research works were limited to isothermal conditions, whereas actual processing conditions in industry are dynamic. Under dynamic conditions, microorganism can respond unexpectedly to the thermal stress (e.g. adaptation, acclimation or increased sensitivity). In this work, we assess the combination of nanoemulsified D-limonene with thermal treatments under isothermal and dynamic conditions. The nanoemulsion was prepared following an innovative methodology using soya lecithin, a natural compound as well as the essential oil. Under isothermal heating conditions, the addition of the antimicrobial enables a reduction of the treatment time by a factor of 25. For time-varying treatments, dynamic effects were relevant. Treatments with a high heating rate (20 °C/min) are more effective than those with a slow heating rate (1 °C/min). This investigation demonstrates that the addition of nanoemulsified D-limonene can greatly reduce the intensity of the thermal treatments currently applied in the food industry. Hence, it can improve the product quality without impacting its safety.