Dynamics of physiological responses of potentially probiotic fruit-derived Limosilactobacillus fermentum in apple and orange juices during refrigeration storage and exposure to simulated gastrointestinal conditions.

This study evaluated the dynamics of the physiological responses of potentially probiotic fruit-derived Limosilactobacillus fermentum 139 and L. fermentum 263 in apple and orange juice during 28 days of refrigeration storage (4 °C) and when submitted to simulated gastrointestinal conditions. Physiological responses were measured with multiparametric flow cytometry using propidium iodide (PI), carboxyfluorescein diacetate (cFDA) and bis-1,3-dibutylbarbutiric acid (BOX). Viable counts were enumerated with plate count. L. fermentum strains had sizes of > 30% of cell subpopulations with non-permeabilized membrane and enzymatic activities (viable cells, PI-CFDA +) in apple and orange juices during storage and viable counts of > 6 log CFU ml-1. Sizes of cell subpopulations with permeabilized membrane without enzymatic activity (dead cells, PI + cFDA-) were low (< 15%) in apple and orange juices during storage. Sizes of cell subpopulations with non-permeabilized and depolarized membrane (PI-BOX +) were decreased (14%) on day 28 of storage. The sizes of permeabilized and depolarized membrane cell (PI + BOX-) subpopulations were variable among the examined strains in juices during storage. Both strains maintained high PI-cFDA + cell subpopulation sizes (> 35%) after exposure to ileum condition and viable counts of ≥ 5 log CFU/mL. PI-BOX + cell subpopulation sizes were low (< 13%) after exposure to ileum condition. L. fermentum 139 and L. fermentum 263 are capable of maintaining a high population of physiologically active and functional cells in apple and orange juice during 28 days of refrigeration storage and when exposed to gastrointestinal conditions.

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.

Mentha piperita L. essential oil inactivates spoilage yeasts in fruit juices through the perturbation of different physiological functions in yeast cells.

This study evaluated the efficacy of the essential oil from Mentha piperita L. (MPEO) to inactivate cells of the potentially spoilage yeasts Candida albicans, Candida tropicalis, Pichia anomala and Saccharomyces cerevisiae in cashew, guava, mango and pineapple juices during 72 h of refrigerated storage. Damage in different physiological functions caused by MPEO in S. cerevisiae in cashew and guava juices were investigated using flow cytometry (FC). The effects of the incorporation of an effective anti-yeast MPEO dose on sensory characteristics of juices were also evaluated. MPEO displayed minimum inhibitory concentration of 1.875 µL/mL against all tested yeasts. A >5 log reduction in counts of C. albicans, P. anomala and S. cerevisiae was observed in cashew and guava juices with 7.5 and 3.75 µL/mL MPEO. Tested MPEO concentrations (1.875, 3.75 and 7.5 µL/mL) were not effective to cause >5 log reduction in counts of target yeasts in mango and pineapple juices during 72 h of exposure. Incorporation of 1.875 µL/mL MPEO in cashew and guava juices strongly compromised membrane permeability, membrane potential, enzymatic activity and efflux pump activity in S. cerevisiae cells. This same MPEO concentration did not affect appearance, odor and viscosity in fruit juices, but negatively affected their taste and aftertaste. These results show the efficacy of MPEO to inactivate potentially spoilage yeasts in fruit juices through disturbance of different physiological functions in yeast cells. However, the combined use of MPEO with other technologies should be necessary to decrease its effective anti-yeast dose in fruit juices and, consequently, the possible negative impacts on specific sensory properties of these products.

Investigation of damage to Escherichia coli, Listeria monocytogenes and Salmonella Enteritidis exposed to Mentha arvensis L. and M. piperita L. essential oils in pineapple and mango juice by flow cytometry.

The effects of Mentha arvensis L. (MAEO; 0.625 µL/mL) and M. piperita L. (MPEO; 1.25 µL/mL) essential oils on viable cell counts and physiological functions in Escherichia coli, Listeria monocytogenes and Salmonella enterica Serovar Enteritidis in pineapple and mango juice after a 15 min-exposure under refrigeration were evaluated in this study. The physiological functions of the bacterial cells were assessed by flow cytometry using the fluorochromes thiazole orange, propidium iodide, bis-1,3-dibutylbarbutiric acid, ethidium bromide, and 5-cyano-2,3-ditolyl tetrazolium chloride to investigate membrane integrity, membrane potential, efflux activity, and respiratory activity. MAEO and MPEO sharply reduced (>5 log10 CFU/mL cycles) the counts of E. coli, L. monocytogenes and Salmonella Enteritidis in pineapple juice, and caused smaller reductions (0.61-1.58 log10 CFU/mL cycles) in mango juice. Bacterial cells exposed to MAEO and MPEO in pineapple and mango juice showed increased membrane permeability, membrane depolarization and changes in efflux pump and respiratory activity. More physiological damage occurred in bacterial cell populations exposed to MAEO or MPEO in pineapple juice than in mango juice. These results indicate that MAEO and MPEO inactivate E. coli, L. monocytogenes and Salmonella Enteritidis cells in pineapple and mango juice through a multi-target action mode that disrupts cytoplasmic membranes, increases permeability and potential depolarization, as well as inhibits efflux pump and respiratory activity.

The efficacy of Mentha arvensis L. and M. piperita L. essential oils in reducing pathogenic bacteria and maintaining quality characteristics in cashew, guava, mango, and pineapple juices.

This study evaluated the ability of the essential oil from Mentha arvensis L. (MAEO) and M. piperita L. (MPEO) to induce ≥5-log reductions in counts (CFU/mL) of E. coli, L. monocytogenes, and Salmonella enterica serovar Enteritidis in Brain-Heart Infusion broth (BHIB) and cashew, guava, mango, and pineapple juices during refrigerated storage (4±0.5°C). The effects of the incorporation of these essential oils on some physicochemical and sensory parameters of juices were also evaluated. The incorporation of 5, 2.5, 1.25, or 0.625µL/mL of MAEO in BHIB caused a ≥5-log reduction in counts of E. coli and Salmonella Enteritidis after 24h of storage; but only 5µL/mL was able to cause the same reduction in counts of L.monocytogenes. The incorporation of 10µL/mL of MPEO in BHIB caused a ≥5-log reduction in counts of E. coli, Salmonella Enteritidis, and L. monocytogenes after 24h of storage; smaller reductions were observed in BHIB containing 5, 2.5, and 1.25µL/mL of MPEO. Similar reductions were observed when the MAEO or MPEO was incorporated at the same concentrations in mango juice. The incorporation of MAEO or MPEO at all tested concentrations in cashew, guava, and pineapple juices resulted in a ≥5-log reduction in pathogen counts within 1h. The incorporation of MAEO and MPEO (0.625 and 1.25µL/mL, respectively) in fruit juices did not induce alterations in °Brix, pH, and acidity, but negatively affected the taste, aftertaste, and overall acceptance. The use of MAEO or MPEO at low concentrations could constitute an interesting tool to achieve the required 5-log reduction of pathogenic bacteria in cashew, guava, mango, and pineapple fruit juices. However, new methods combining the use of MAEO or MPEO with other technologies are necessary to reduce their negative impacts on specific sensory properties of these juices.

The Potential of the Incorporation of Essential Oils and Their Individual Constituents to Improve Microbial Safety in Juices: A Review.

The juice sector is one of the fastest growing sectors in the food industry. Although juices are important because of their nutritional value and convenience, their composition and physicochemical properties affect their microbiological safety and overall quality during their shelf-life. Furthermore, the thermal process classically applied in juices partially reduces the occurring microflora, and the use of chemical additives is perceived negatively by consumers. For these reasons, researchers have proposed the use of nonthermal technologies as antimicrobial preservatives in juices. This paper covers the recent literature on the use of essential oils (EOs) and the individual constituents (ICs) found therein, used alone or in combination with other emerging technologies, for the preservation of juices. From this perspective, this paper discusses the growing importance of the use of EOs and their ICs, either alone or in association with other emerging technologies, in juices and their effects on the safety and physicochemical and sensory quality attributes of these products. The results of papers currently available in the literature reveal that EOs and their ICs are promising alternatives to achieve microbial safety and stability in juices. However, extensive studies considering the effects of each EO or IC on sensory characteristics, primarily taste and aroma, are still needed to establish each of these substances/compounds as feasible preservatives for use in juices. Finally, further studies could focus on the combination of low amounts of EOs or ICs with other nonthermal technologies to achieve a balance between the microbial safety and sensory acceptability of juices.