Inhibition of Aspergillus carbonarius growth and Ochratoxin A production using lactic acid bacteria cultivated in an optimized medium.

The Gram-positive bacteria lactic acid bacteria (LAB) are used in the food industry but are also known for inhibiting certain food spoilage microorganisms, especially fungi. Sources of nitrogen (N) for culture media are generally organic and expensive. Many attempts have been made to formulate economical culture media with alternative N sources obtained from agricultural and industrial byproducts. This study describes the design and optimization of an inexpensive culture medium for Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) MZ809351 strain B31. The culture medium was optimized using statistical experimental designs to identify the factors with the most significant effects on biomass concentration to reduce the overall cost, aiming to obtain a biomass concentration similar to that obtained with the reference LAB culture medium (de Man, Rogosa and Sharpe; MRS). Sodium acetate and magnesium sulfate were the most significant factors (p < 0.005), and their contents were reduced by 22 % and 40 %, respectively, without affecting biomass concentration. Malt germ extract (MGE) was used as an alternative nitrogen source to replace meat extract (ME) and proteose peptone (PP). Through these experiments, the composition of a culture medium that is less expensive than MRS broth was defined, which produced a biomass concentration (3.8 g/L) similar to that obtained with MRS medium. The inhibitory effects of two LAB strains isolated from the Ivory Coast and Mexico on the growth and production of ochratoxin A (OTA) in an ochratoxigenic fungus was tested. The minimum cellular concentration of the LAB to prevent the development of Aspergillus carbonarius Ac 089 and the production of OTA was determined in a model assay in Petri dishes. The conditions to inhibit the germination of A. carbonarius Ac 089 and the production of OTA were found. Using the optimized medium and a ratio of 2 × 104 LAB/spore (1 × 108 CFU/mL) strain B7 (L. plantarum MZ809351) and 2 × 103 LAB/spore (1 × 107 CFU/mL) strain B31 (L. plantarum MN922335) completely inhibited the growth of the fungus. A ratio of 2 × 105 LAB/spore (1 × 109 CFU/mL) was required to inhibit OTA production with strains B7 and B31. This study indicates the potential of cultivating LAB in an optimized and inexpensive culture medium for use as a biological control agent against ochratoxigenic fungi in food.

Antifungal and Cytotoxic Activity of Diterpenes and Bisnorsesquiterpenoides from the Latex of Euphorbia resinifera Berg.

Euphorbia resinifera latex has been extensively utilized in traditional medicine due to its range of bioactivities. Chromatographic separations on silica gel of ethanol extract of E. resinifera latex led to the development of a new procedure for isolating resiniferatoxin (4) via dried E. resinifera latex and the identification of nine compounds. Among these, catechol (7), protocatechuic acid (8) and 3,4-dihydroxyphenylacetic acid (9), known phenolic compounds, were identified for the first time in E. resinifera latex. Herein we investigated the effects of major compounds of the latex of E. resinifera on the yeast Saccharomyces cerevisiae, on the growth of Aspergillus carbonarius, a widespread fungal contaminant, and on the breast cancer cell line MCF7 as well as on MCF10A normal breast cells. 12-deoxyphorbol-13-isobutyrate-20-acetate (2) had an inhibiting effect on the growth of A. carbonarius, and 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) showed a negative effect on yeast cell growth and also a cytotoxic effect on breast cancer cell line MCF7, but not on MCF10A cells. Deglucosyl euphorbioside A (5) and euphorbioside A (6) showed a discoloration effect that was possibly related to mitochondrial functionality in yeast, and also cytotoxicity only on the cancer cell line that was tested. Interestingly, treatment of MCF7 cells with 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) and deglucosyl euphorbioside A (5) not only led to a specific cytotoxic effect but also to the increase in the level of intracellular ROS.

Sodium alginate-cinnamon essential oil coated apples and pears: Variability of Aspergillus carbonarius growth and ochratoxin A production.

The scope of the present study was to use selected fruits as model foods (wounded skin or slices of apples and pears), for the in situ assessment of the potential of natural antimicrobials to control fungal growth and OTA production and the investigation of alternative ways of their application, e.g., via edible coatings. Fresh fruits were cut: i) in halves or ii) across in round slices of ca. 1 cm thickness. Wounds were introduced into the skin and the center of the slice (5 mm deep; 4 mm diameter) and inoculated with a range of 2.0-7.5 × 103spores per wound of Aspergillus carbonarius. Following inoculation, samples were coated with Na - alginate supplemented with 0.3 (0.3% ECC) and 0.9% v/v (0.9% ECC) cinnamon ΕΟ. Inoculated samples without edible coating and EO (C) or with edible coating and without EO (EC) were used as negative or positive controls, respectively. All samples were stored under aerobic conditions at 15, 20, and 25 °C. Fungal growth was estimated by colony diameter measurements (n = 30), while OTA production was determined by HPLC (n = 4). Antimicrobial treatment with 0.9% EO was more effective on fungal growth when the inoculation took place on slices than in wounded skin (p < .05), regardless of storage temperature and fruit. The variability of µmax increased with EO concentration, except for the coated slices of apples with 0.9% v/v EO (at all temperatures), or pears with 0.3% and 0.9% v/v EO (at 15 and 20 °C), where no growth was observed. OTA was below the detection limit (1 ppb) on the majority of 0.9% ECC apples slices and in 0.3% ECC and 0.9% ECC pears slices, stored at 20 and 25 °C. However, the sample to sample variation in the produced amounts of OTA was remarkable. Thus, considering that inhibition of growth and toxin production do not always concur, the present study provided quantitative information on the variability in A. carbonarius growth and OTA production in real model foods in response to antimicrobial coating with natural active compounds. Such data could be of relevance to risk assessment and assist in designing effective control strategies for limiting OTA levels in foods and thus, protecting consumer health.

Essential Oils Modulate Gene Expression and Ochratoxin A Production in Aspergillus carbonarius.

Ochratoxin A (OTA) is a mycotoxin, mainly produced on grapes by Aspergillus carbonarius, that causes massive health problems for humans. This study aims to reduce the occurrence of OTA by using the ten following essential oils (E.Os): fennel, cardamom, anise, chamomile, celery, cinnamon, thyme, taramira, oregano and rosemary at 1 µL/mL and 5 µL/mL for each E.O.As a matter of fact, their effects on the OTA production and the growth of A. carbonarius S402 cultures were evaluated, after four days at 28 °C on a Synthetic Grape Medium (SGM). Results showed that A. carbonarius growth was reduced up to 100%, when cultured with the E.Os of cinnamon, taramira, and oregano at both concentrations and the thyme at 5 µL/mL. As for the other six E.Os, their effect on A. carbonarius growth was insignificant, but highly important on the OTA production. Interestingly, the fennel E.O at 5 µL/mL reduced the OTA production up to 88.9% compared to the control, with only 13.8% of fungal growth reduction. We further investigated the effect of these E.Os on the expression levels of the genes responsible for the OTA biosynthesis (acOTApks and acOTAnrps along with the acpks gene) as well as the two regulatory genes laeA and vea, using the quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) method. The results revealed that these six E.Os reduced the expression of the five studied genes, where the ackps was downregulated by 99.2% (the highest downregulation in this study) with 5 µL/mL of fennel E.O.As for the acOTApks, acOTAnrps, veA and laeA, their reduction levels ranged between 10% and 96% depending on the nature of the E.O and its concentration in the medium.

Effect of oxidant stressors and phenolic antioxidants on the ochratoxigenic fungus Aspergillus carbonarius.

BACKGROUND: There are few studies dealing with the relationship between oxidative stress and ochratoxin A (OTA) biosynthesis. In this work, we analyzed the effect of the oxidant stressor menadione and the antioxidants 3,5-di-tert-butyl-4-hydroxytoluene (BHT), catechin, resveratrol and a polyphenolic extract on growth, generation of reactive oxygen species (ROS), OTA production and gene expression of antioxidant enzymes of Aspergillus carbonarius. RESULTS: Exposure to menadione concentrations higher than 20 µmol L(-1) led to increases in ROS and OTA levels and a decrease in growth rate. Exposure to 2.5-10 mmol L(-1) BHT also led to higher ROS and OTA levels, although growth rate was only affected above 5 mmol L(-1). Naturally occurring concentrations of catechin, resveratrol and polyphenolic extract barely affected growth rate, but they produced widely different effects on OTA production level depending on the antioxidant concentration used. In general, gene expression of antioxidant enzymes superoxide dismutase (SOD) and peroxiredoxin (PRX) was downregulated after exposure to oxidant and antioxidant concentrations that enhanced OTA production level. CONCLUSION: Aspergillus carbonarius responds to oxidative stress, increasing OTA production. Nevertheless, the use of naturally occurring concentrations of antioxidant phenolic compounds to reduce oxidative stress is not a valid approach by itself for OTA contamination control in grapes.

Hydrolytic enzymes production by Aspergillus section Nigri in presence of butylated hydroxyanisole and propyl paraben on peanut meal extract agar.

BACKGROUND: In the last years, food grade antioxidants are used safely as an alternative to traditional fungicides to control fungal growth in several food and agricultural products. AIMS: In this work, the effect of butylated hydroxyanisole (BHA) and propyl paraben (PP) on two hydrolytic enzyme activity (ß-d-glucosidase and α-d-galactosidase) by Aspergillus section Nigri species under different water activity conditions (aW; 0.98, 0.95 and 0.93) and incubation time intervals (24, 48, 72 and 96h) was evaluated on peanut-based medium. METHODS: The activity of two glycosidases, ß-d-glucosidase and α-d-galactosidase, was assayed using as substrates 4-nitrophenyl-ß-d-glucopyranosido and 4-nitrophenyl-α-d-galactopyranosido, respectively. The enzyme activity was determined by the increase in optical density at 405nm caused by the liberation of p-nitrophenol by enzymatic hydrolysis of the substrate. Enzyme activity was expressed as micromoles of p-nitrophenol released per minute. RESULTS: The major inhibition in ß-d-glucosidase activity of A. carbonarius and A. niger was found with 20mmoll(-1) of BHA or PP at 0.98 and 0.95 aW, respectively, whereas for α-d-galactosidase activity a significant decrease in enzyme activity with respect to control was observed in A. carbonarius among 5 to 20mmoll(-1) of BHA or PP in all conditions assayed. Regarding A. niger, the highest percentages of enzyme inhibition activity were found with 20mmoll(-1) of BHA or PP at 0.95 aW and 96h. CONCLUSIONS: The results of this work provide information about the capacity of BHA and PP to inhibit in vitro conditions two of the most important hydrolytic enzymes produced by A. carbonarius and A. niger species.

Effect of antioxidant mixtures on growth and ochratoxin a production of Aspergillus section Nigri species under different water activity conditions on peanut meal extract agar.

The effect of mixtures of antioxidants butylated hydroxyanisol (BHA) and propyl paraben (PP) on lag phase, growth rate and ochratoxin A (OTA) production by four Aspergillus section Nigri strains was evaluated on peanut meal extract agar (PMEA) under different water activities (a(w)). The antioxidant mixtures used were: BHA + PP (mM), M1 (0.5 + 0.5), M2 (1.0 + 0.5), M3 (2.5 + 0.5), M4 (0.5 + 1.0), M5 (1.0 + 1.0), M6 (2.5 + 1.0), M7 (5.0 + 2.5) and M8 (10 + 2.5). The mixture M8 completely suppressed mycelial growth for all strains. A significant stimulation in OTA production was observed with mixtures M1 to M5 mainly at the highest a(w); whereas M6, M7 and M8 completely inhibited OTA production in all strains assayed; except M6 in A. carbonarius strain (RCP G). These results could enable a future intervention strategy to minimize OTA contamination.