Antimicrobial Effect of Lippia citriodora Extract in Combination with Gallic Acid or Octyl Gallate on Bacteria from Meat.

Chicken meat and its derivatives are easily alterable. They are a nutritionally healthy food, and their consumption has seen a remarkable increase worldwide in recent years. At the same time, consumer demand for the use of natural products to control microbial growth is increasing. In this context, the antimicrobial capacity of a commercial extract of the lemon verbena (Lippia citriodora) plant, (LCE) was tested in binary combination with gallic acid or octyl gallate against two strains of lactic acid bacteria (LAB) of meat origin: Carnobacterium divergens ATCC 35677 and Leuconostoc carnosum ATCC 49367. First, the antimicrobial potential was evaluated by the checkerboard microdilution method at the optimal growth temperature of each and at 4 °C, pH 5.7 and 6.7, in culture medium. Octyl gallate was the most effective antimicrobial against the two bacteria under all study conditions. At 4 °C, the combination of LCE with octyl gallate had a similar antimicrobial effect on the two LAB, being bactericidal at pH 6.7. In chicken breast, this effective combination was tested in normal or modified atmosphere and refrigerated (4-8 °C) for 9 days. LCE + OG in modified atmosphere reduced the different microbial groups studied, including the lactic acid bacteria as the main microorganisms responsible for the spoilage of fresh meat. Further research could pave the way for the development of novel strategies contributing to the technological stability, security, and functional properties of chicken meat.

Assessment of (-) epicatechin as natural additive for improving safety and functionality in fresh "Piel de Sapo" melon juice.

Epicatechin (EC) is a very abundant flavonoid in vegetable tissues that presents high antioxidant activity in living systems. The minimum inhibitory concentration (MIC) of (-)EC was determined in three species of bacteria commonly associated with foodborne illness of plant origin: Listeria (L.) monocytogenes, Escherichia (E.) coli -serogroups O157: H7 and O111- and Bacillus (B.) cereus; two strains of probiotic-type lactic acid bacteria (PT-LAB) and two control strains. All 10 strains were assayed under three temperature conditions (30º, 10º, and 4ºC) and at each temperature under two pH conditions (6.7 and 5.5). Mean EC MIC values were generally lower at refrigeration (4º and 10ºC) temperatures and at standard pH (6.7). By inoculating with each of the strains separately, both melon juice (MJ) and MJ supplemented with EC (ECSMJ), at the accepted maximum sensorial limit, and storing them at 4ºC for 10 days; the final counts (CFU/mL) were lower for ECSMJ than for plain MJ both for pathogenic bacteria and for PT-LAB. The presence of EC during refrigerated storage counteracted the ability of MJ as a growth medium for all the pathogenic bacteria. ECSMJ increased the antioxidant activity of MJ significantly to levels similar to those of EC alone. (-) Epicathechin would be a promising ingredient for increasing the functional properties of "Piel de Sapo" MJ (phenolic compounds and antioxidant ability) while contributing to improving the safety of this type of juice during prolonged refrigerated storage at 4ºC.

Combination of Carvacrol and Thymol: Antimicrobial Activity Against Staphylococcus aureus and Antioxidant Activity.

Plant and essential oil extracts have been used for some time as antimicrobials and antioxidants, although little is known about the interactions between the main components of these plant materials. This knowledge could help to design more potent antimicrobial and antioxidant mixtures. Carvacrol and thymol, the main components of the essential oils of the Lamiaceae family of plants, were assessed in combination to evaluate their antioxidant activity and antimicrobial effect against 19 strains of Staphylococcus aureus (S. aureus) of different origins (clinical, meat, milk, and other) and mostly (12) enterotoxin producers. The microdilution test assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the two phenolics alone and in combination. Based on the fractional inhibitory concentration index (FICI), no antimicrobial interaction (0.5 < FICI <4) between carvacrol and thymol was observed against 42% of the S. aureus strains and an antagonistic interaction (FICI >4) was observed in the rest, which indicates different behavior among strains in relation to this antimicrobial combination. Particularly, an antagonistic effect was observed in 29% of the meat origin strains and 57% of the dairy origin strains. Combinations of carvacrol and thymol were bactericidal (differences in MIC and MBC values not more than twofold) for 60% of the tested strains. At low concentrations of both components, the antioxidant effect is additive. However, at high concentrations (2.50 or 2.66 mM) of at least one of the components of the combination, it is antagonistic. The different types of interactions of the components in the combination can depend on many factors (ratio, structural characteristics, and the establishment of intermolecular complexes). The results could be used as reference to apply this combination in foods to control S. aureus, to maintain the organoleptic properties and to extend the shelf-life of them.

Antimicrobial efficacy of Lippia citriodora natural extract against Escherichia coli and Enterococcus faecalis in "Piel de Sapo" melon juice.

BACKGROUND: The minimal inhibitory concentration (MIC) of an aqueous extract of Lippia citriodora with reported functional properties (PLX®) was determined on two strains of Escherichia coli (E. coli) belonging to serogroups commonly associated with foodborne illnesses (E. coli O157:H7 ATCC 700728 and E. coli O111 isolate 172) in vegetable products and two control strains for antimicrobial tests assays (E. coli ATCC 25922 and Enterococcus-En. faecalis ATCC 29212). RESULTS: Mean MIC values at standard pH (7.4) in broth for the E. coli strains tested ranged from 4,444 µg/ml (35ºC) to 1,250 µg/ml (10ºC) and to 182 µg/ml (4ºC). At pH 5.5, conditions resembling those of melon juice, MIC was about 2 times higher at 35 and 10ºC compared with 4ºC. The MIC of En. faecalis was similar or slightly lower than those of E. coli at the conditions tested. In melon juice fortified with PLX® (2,500 µg/ml, maximum sensorial acceptable limit), the three strains of E. coli maintained their viability although none showed growth potential after 4 days at 4ºC. CONCLUSIONS: PLX® could be added to melon juice to control E. coli O157:H7 and E. coli O111 during refrigerated storage, reducing the risk of microbiological contamination in this food.

Stress in Phycomyces blakesleeanus by glucose starvation and acetate growth: response of the antioxidant system and reserve carbohydrates.

The objective of this study was to analyze the response of Phycomyces blakesleeanus to glucose starvation and acetate growth stress. At the onset of the exponential growth phase, the fungus shows a high tolerance to both stresses, being higher for the glucose starvation. In both stresses we have found higher activities of catalase and glutathione peroxidase, and a decrease of the pools of D-erythroascorbate (D-erythroascorbate+D-erythroascorbate monoglucoside) and glutathione (GSH+GSSG), while the intracellular GSH/GSSG redox balance becomes more reducing. Gallic acid was not detected under both stresses. Glycogen breakdown and the high levels of trehalose seem to be part of the stress response. Both stress, under the conditions of this study, seem to lead to a qualitatively similar response in P. blakesleeanus, with regard to the behavior of antioxidant system, the content of secondary metabolites and the role of the reserve carbohydrates.

Antioxidant defence system during exponential and stationary growth phases of Phycomyces blakesleeanus: response to oxidative stress by hydrogen peroxide.

An analysis of the components of the antioxidant defence system in exponential and stationary growth phases of filamentous fungus Phycomyces blakesleeanus and the response to the oxidative stress hydrogen peroxide were performed. There is a strong positive correlation between mycelial antioxidant capacity and the contents of gallic acid, d-erythroascorbate (d-EAA) or d-erythroascorbate monoglucoside (d-EAAG). These secondary metabolites are specifically synthesized by this fungus and reach maximal values in the stationary growth phase, suggesting that they can play some role in the antioxidant defence system of this fungus. There is a differential expression of the two more notable antioxidant activities, catalase (CAT) and superoxide dismutase (SOD), depending of the growth stage of P. blakesleeanus, CAT being expressed in the exponential and SOD in the stationary phase. Phycomyces blakesleeanus showed a high resistance to the oxidative stress caused by H2O2 (50 and 200 mM) which was higher in exponential phase. This higher resistance can be explained by the presence of CAT, glutathione peroxidase (GPx), and the probable contribution of glutathione-S-transferase (GST) and high levels of reduced form of glutathione (GSH). The transition to stationary phase was accompanied with a higher physiological oxidative damage illustrated by the higher protein carbonylation. In this growth stage the resistance of the fungus to the oxidative stress caused by H2O2 could be explained by the presence of SOD, GPx, and the probable contribution of GST as well as of secondary metabolites, mainly d-EAA and d-EAAG. These results highlight a specific response to oxidative stress by H2O2 depending on the growth phase of P. blakesleeanus.

Antibacterial activity against foodborne Staphylococcus aureus and antioxidant capacity of various pure phenolic compounds.

Six pure phenolic compounds (hydroquinone, thymol, carvacrol, butylated hydroxyanisole, gallic acid, and octyl gallate) were tested for their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against several strains of Staphylococcus aureus isolated from dairy and meat products. In addition, S. aureus reference strains (American Type Culture Collection) for antimicrobial studies and/or isolated from human infections and outbreaks of food poisoning were included in the study. Of the compounds tested, octyl gallate and hydroquinone were the most effective against S. aureus (mean MIC values of 20.89 and 103.05 µg/mL, respectively) and carvacrol and thymol the least (mean MIC values of about 413 µg/mL). The mean MBC values were 40.84, 194.37, 417.46, and 581.90 µg/mL for octyl gallate, hydroquinone, carvacrol, and thymol, respectively. Meat isolates were more resistant than those of dairy origin to hydroquinone, gallic acid, and octyl gallate, as well as to penicillin G (used as a control of the methodology used); gallic acid and penicillin G showed the highest differences in MIC values between the groups of strains (about 10 and 200 times, respectively). On the other hand, when we tested the isolates included in each group of strains (dairy, meat, and other/mixed sources) we only detected significant differences (p < 0.05) among dairy and isolates from other/mixed sources for hydroquinone and thymol, respectively. However, strains of meat origin exhibited significant differences among each other (p < 0.05) to most of the phenolic compounds tested (hydroquinone, carvacrol, gallic acid, and octyl gallate). The relationship between MICs and MBCs for each of the phenolic compounds tested suggested a bactericidal mechanism of action against S. aureus. Gallic acid and octyl gallate exhibited the highest antioxidant capacity and thymol and carvacrol the lowest. So, octyl gallate is an agent with both antimicrobial and antioxidant properties, which would be of interest to use in the food industry.

Production, stability, and antioxidative and antimicrobial activities of two L-ascorbate analogues from phycomyces blakesleeanus: D-erythroascorbate and D-erythroascorbate glucoside.

D-erythroascorbate (D-EAA), a five-carbon analogue of L-ascorbate (L-AA), and D-erythroascorbate monoglucoside (D-EAAG) are accumulated in Phycomyces blakesleeanus grown on glucose (99.5 and 1084 µg/g mycelial dry weight, respectively) and also excreted into the culture medium. Both compounds showed UV spectral properties and ionization constants similar to those of L-AA. D-EAAG was much more stable to aerobic oxidation than D-EAA and L-AA at acidic pH. D-EAAG is synthesized from D-erythroascorbate by a mycelial glucosyltransferase activity that uses UDP-glucose as glucose substrate donor with K(m) = 2.5 mM and 41.3 µM for D-EAA. This glucosyltransferase activity was maximal in the stationary growth phase in parallel with maximal production of D-EAAG. The presence of D-arabinose or D-arabinono-1,4-lactone in the culture medium produces the maximal accumulation of D-EAA and D-EAAG (about 30- and 4-fold with respect to that obtained in glucose culture). Both compounds showed greater antioxidant activity than L-AA and other standard antioxidants, with a capacity similar to that of L-AA to inhibit the growth of Escherichia coli.

Screening of phenolic antioxidants for their inhibitory activity against foodborne Staphylococcus aureus strains.

Seventeen phenolic compounds that are allowed to be used in the European food industry as aromatizants or antioxidants or that are naturally present in plants were tested for their ability to inhibit 19 strains of Staphylococcus aureus by using a standardized paper disc assay. Most of the strains assayed were foodborne (dairy and meat products). Human isolates and/or strains recommended for testing antimicrobial agents were also included in the study, and some of the test strains were enterotoxin producers. When the content was 200 microg/disc, various phenolic compounds had shown antimicrobial activity against all (hydroquinone, thymol, carvacrol, butylated hydroxyanisole, octyl gallate, and tannic acid) or most (gallic acid, propyl gallate, and ellagic acid) of the S. aureus strains tested. Significant differences in the inhibition zones (p < 0.05) among strains of the same, or similar, origin and among the different origins were observed for most of the phenolic compounds that showed antimicrobial activity for all or most of the strains tested.