Antimicrobial and anti-aflatoxigenic activities of nanoemulsions based on Achillea millefolium and Crocus sativus flower extracts as green promising agents for food preservatives.

BACKGROUND: Although the mechanism of action of nanoemulsion is still unclear, the modern use of nanoemulsions made from natural extracts as antimicrobial and anti-aflatoxigenic agents represents a potential food preservation and a safety target. METHODS: Two natural nanoemulsion extracts of Crocus sativus (the saffron flower) and Achillea millefolium (the yarrow flower) were produced in the current study using a low-energy method that included carboxymethylcellulose and Arabic gum. The synthesized nanoemulsion was fully identified by different analytical methods. Detection of the volatile content was completed using GC-MS analysis. The antioxidant potential, and phenolic compounds content were analyzed in the extractions. The synthesized nanoemulsions were screened for their antimicrobial potential in addition to their anti-aflatoxigenic activity. RESULTS: The droplet size of Saffron flowers was finer (121.64 ± 2.18 nm) than yarrow flowers (151.21 ± 1.12 nm). The Zeta potential measurements of the yarrow flower (-16.31 ± 2.54 mV) and the saffron flower (-18.55 ± 2.31 mV) both showed high stability, along with low PDI values (0.34-0.41). The nanoemulsion of yarrow flower revealed 51 compounds using gas chromatography-mass spectrometry (GCMS), with hexanal (16.25%), ß-Pinene (7.41%), ß-Myrcene (5.24%), D-Limonene (5.58%) and Caryophyllene (4.38%) being the most prevalent. Additionally, 31 compounds were detected in the saffron nanoemulsion, with D-limonene (4.89%), isophorone (12.29%), 4-oxy isophorone (8.19%), and safranal (44.84%) being the most abundant. Compared to the nanoemulsion of the yarrow flower, the saffron nanoemulsion had good antibacterial and antifungal activity. Saffron nanoemulsion inhibited total fungal growth by 69.64-71.90% in a simulated liquid medium and demonstrated the most significant decrease in aflatoxin production. Infected strawberry fruits coated with nanoemulsion extracts exhibited high antimicrobial activity in the form of saffron flower and yarrow flower extract nanoemulsions, which inhibited and/or controlled the growth of Aspergillus fungi. Due to this inhibition, the lag phase was noticeably prolonged, the cell load decreased, and the stability time increased. CONCLUSION: This study will contribute to expanding the theoretical research and utilization of nanoemulsions as green protective agents in agricultural and food industries for a promising protection from the invasion of some pathogenic bacteria and fungi.

Insight into the substrate-dependent anti-aflatoxigenic effects of nanosized ZnO film: Electron transfer directed oxidative stress mechanisms.

Exploring new anti-aflatoxigenic materials and their mechanisms are critical to reduce the prevalence of drug-resistant fungi and the contamination of aflatoxins. Zinc oxide nanoparticles (ZnONPs) are promising antifungal candidates but supporting substrates generally affect their antifungal activities. In this study, ZnONPs with a three-dimensional flower-like hierarchical microstructure bound to different substrates as anti-aflatoxigenic composites were prepared using a facile deposition method. It was found that ZnO nanocomposites showed the substrate-dependent anti-aflatoxigenic activities. The antifungal activities of ZnO films toward A. flavus growth and aflatoxin B1 production decreased significantly in the order ZnO@Zn＞ZnO@Sn＞ZnO@Steel＞ZnO@Glass. The electrical conductivity of the substrate should play an important role for antifungal response. When compared with ZnO@Sn and ZnO@Steel, the conductivity value of ZnO@Zn was 2.07-fold and 14.84-fold of them, respectively. The higher the electrical conductivity of the substrate, the better the anti-aflatoxigenic efficiency of the composite. Such anti-aflatoxigenic activity was also due to ROS generation through electron transfer between fungi and the ZnO-substrate system, which could provoke the strength of intracellular oxidative stress. This mechanism was further confirmed using several assays such as hyphal morphology analysis, Zn2+ release, ROS evaluation, lipid peroxidation and antioxidant response. Collectively, improvement in knowledge regarding anti-aflatoxigenic performance of ZnONPs can help develop novel and effective strategies to reduce fungi growth and aflatoxin contamination in the food field.

Application and safety evaluation of an anti-aflatoxigenic chitosan pouch containing turmeric essential oil in the storage of traditional Chinese health food.

Aflatoxin contamination is one of the most important factors jeopardizing the quality of traditional Chinese health food (TCHF) during storage. Based on our previous work, we investigated the stability of chitosan (CH) films containing turmeric essential oil (TEO) and employed CH-TEO films as inner pouches, then stored them with inoculated Coix seed, nutmeg, and Ziziphi Spinosae Semen (ZSS). We found that the stability of CH-TEO was most affected by high temperature, and these pouches dramatically decreased aflatoxin accumulation and maintained levels of marker components of each TCHF. We found that glycerol tristearat in Coix seed and jujuboside A and spinosin in ZSS were negatively correlated with aflatoxin accumulation. After three months of storage with a CH-TEO pouch, we found little change in marker components contents, but observed that Coix seed had the relative lower sensory characteristics score. In addition, acute and 90-day subchronic toxicity test in Coix seed stored with the largest amount of TEO showed no significant signs of toxicity or treatment-related changes in animals. The present study is the first report on the study of a green, efficient, and low toxicity solution for aflatoxic contamination in TCHF, and provides strong support for its future use.

GC-EIMS analysis, antifungal and anti-aflatoxigenic activity of Capsicum chinense and Piper nigrum fruits and their bioactive compounds capsaicin and piperine upon Aspergillus parasiticus.

GC-EIMS analysis, antifungal- and anti-aflatoxigenic activities of the ethanolic extract of Capsicum chinense and Piper nigrum fruits and their main bioactive compounds were evaluated upon Aspergillus parasiticus. The GC-EIMS analysis showed capsaicin (50.49%) and piperine (95.94%) as the major constituents in C. chinense and P. nigrum, respectively. MIC50 values revealed that capsaicin (39 µg/mL) and piperine (67 µg/mL) were lower than those from fruit extracts of C. chinense (381 µg/mL) and P. nigrum (68 µg/mL). Extracts and bioactive compounds showed anti-aflatoxigenic activity. Maximum aflatoxin inhibition occurred at 150 µg/mL of extracts and compounds. The present study showed satisfactory results concerning the effects of ethanolic extract of C. chinense and P. nigrum fruits upon A. parasiticus, showing the capabilities of inhibiting fungal growth development and altering aflatoxins production.

Inhibition of aflatoxin B1 biosynthesis and down regulation of aflR and aflB genes in presence of benzimidazole derivatives without impairing the growth of Aspergillus flavus.

Aflatoxins are mutagenic secondary metabolites produced by certain ubiquitous saprophytic fungi. These contaminate agricultural crops and pose a serious health threat to humans and livestock all over the world. Benzimidazole and its derivatives are biologically active heterocyclic compounds known for their fungicidal activity. In the present study, second and sixth position substituted benzimidazole derivatives are tested for their antifungal and anti-aflatoxigenic activity. Aflatoxigenic strain of Aspergillus flavus cultured in Yeast extract sucrose (YES) medium as well as in rice in the presence and absence of test compounds. 2-(2-Furyl) benzimidazole (FBD) showed complete inhibition of fungal growth at 50 µg/mL. However, the polar derivatives of FBD viz. 6-NFBD, 6-AFBD, 6-CAFBD, and 6-CFBD did not impair the fungal growth but effectively inhibited aflatoxin B1 biosynthesis. Significant down-regulation of aflR gene involved in regulation and aflB structural gene for aflatoxin B1 biosynthesis was observed in presence of 6-NFBD. These benzimidazole derivatives also showed good anti-aflatoxigenic activity in rice, though the IC50 concentrations in rice were comparatively higher than those in YES medium. This study summarizes the most notable structure-activity relationship (SAR) of 2-(2-Furyl) benzimidazoles for anti-aflatoxigenic and anti-fungal activities. These molecules can be further studied for their applications in industrial fermentation processes vulnerable to mold growth and subsequent aflatoxin B1 synthesis like koji fermentation, cheese production, etc.

Isolation of a novel deep-sea Bacillus circulus strain and uniform design for optimization of its anti-aflatoxigenic bioactive metabolites production.

The deep-sea bacterium strain FA13 was isolated from the sediment of the South Atlantic Ocean and identified as Bacillus circulans based on 16S ribosomal DNA sequence. Through liquid fermentation with five media, the cell-free supernatant fermented with ISP2 showed the highest inhibition activities against mycelial growth of Aspergillus parasiticus mutant strain NFRI-95 and accumulation of norsolorinic acid, a precursor for aflatoxin production. Based on ISP2, uniform design was used to optimize medium formula and fermentation conditions. After optimization, the inhibition efficacy of the 20-time diluted supernatant against A. parasiticus NFRI-95 mycelial growth and aflatoxin production was increased from 0-23.1% to 100%. Moreover, compared to the original protocol, medium cost and fermentation temperature were significantly reduced, and dependence on seawater was completely relieved, thus preventing the fermentor from corrosion. This is the first report of a deep-sea microorganism which can inhibit A. parasiticus NFRI-95 mycelial growth and aflatoxin production.

Inhibitory Effect of Flower-Shaped Zinc Oxide Nanostructures on the Growth and Aflatoxin Production of a Highly Toxigenic Strain of Aspergillus flavus Link.

Flower-shaped zinc oxide (ZnO) nanostructures were prepared via a simple aqueous precipitation strategy at room temperature. The as-grown nanostructures were characterized by UV⁻vis spectroscopy, UV⁻vis diffuse reflectance spectroscopy (DRS), spectrofluorometry, Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection (ATR), X-ray diffraction (XRD), and field emission scanning electron microscopy (FESEM). The antifungal and anti-aflatoxigenic activities of the ZnO nanostructures were further investigated using a highly toxigenic strain of Aspergillus flavus Link under in vitro and in situ conditions. The results showed that the A. flavus isolate was inhibited to various extents by different concentrations of ZnO nanostructures, but the best inhibitions occurred at 1.25, 2.5, and 5 mM in the culture media. At these concentrations, suppression of aflatoxin biosynthesis (99.7%) was also observed. Moreover, a reasonable reduction in the aflatoxin content (69%) was observed in maize grains treated with the lowest ZnO concentration that exhibited the strongest inhibitory activity in the liquid media. SEM micrographs clearly indicate multiple degenerative alterations in fungal morphology after treatment with ZnO such as damage of the tubular filaments, loss of hyphae shape, as well as hyphae rupture. These results suggest that flower-shaped ZnO nanostructures exhibit strong antifungal and anti-aflatoxigenic activity with potential applications in the agro-food system.

Rice Phyllosphere Bacillus Species and Their Secreted Metabolites Suppress Aspergillus flavus Growth and Aflatoxin Production In Vitro and In Maize Seeds.

The emergence of super-toxigenic strains by recombination is a risk from an intensive use of intraspecific aflatoxin (AF) biocontrol agents (BCAs). Periodical alternation with interspecific-BCAs will be safer since they preclude recombination. We are developing an AF-biocontrol system using rice-associated Bacilli reported previously (RABs). More than 50% of RABs inhibited the growth of multiple A. flavus strains, with RAB4R being the most inhibitory and RAB1 among the least. The fungistatic activity of RAB4R is associated with the lysis of A. flavus hyphal tips. In field trails with the top five fungistatic RABs, RAB4R consistently inhibited AF contamination of maize by Tox4, a highly toxigenic A. flavus strain from Louisiana corn fields. RAB1 did not suppress A. flavus growth, but strongly inhibited AF production. Total and HPLC-fractionated lipopeptides (LPs) isolated from culture filtrates of RAB1 and RAB4R also inhibited AF accumulation. LPs were stable in vitro with little loss of activity even after autoclaving, indicating their potential field efficacy as a tank-mix application. A. flavus colonization and AF were suppressed in RAB1- or RAB4R-coated maize seeds. Since RAB4R provided both fungistatic and strong anti-mycotoxigenic activities in the laboratory and field, it can be a potent alternative to atoxigenic A. flavus strains. On the other hand, RAB1 may serve as an environmentally safe helper BCA with atoxigenic A. flavus strains, due its lack of strong fungistatic and hemolytic activities.

A review on the inhibitory potential of Nigella sativa against pathogenic and toxigenic fungi.

Nigella sativa (N. sativa) grows in various parts of the world, particularly in Iran. It has been traditionally used as a folk remedy to treat a number of diseases. The seeds of this plant contain moisture, proteins, carbohydrates, crude fiber, alkaloids, saponins, ash, fixed oils and essential oil. The major components of the essential oil are thymoquinone, p-cymene, trans-anethole, 2-methyl-5(1-methyl ethyl)-Bicyclo[3.1.0]hex-2-en and γ-terpinene. So far, several pharmacological effects such as anti-oxidant, anti-inflammatory, anti-cancer and anti-microbial have been reported for N. sativa or its active compounds. Thymoquinone, thymohydroquinone and thymol are the most active constituents which have different beneficial properties. The oil, extracts and some of N. sativa active components possessed moderate in vitro and in vivo inhibitory activity against pathogenic yeasts, dermatophytes, non-dermatophytic filamentous fungi and aflatoxin-producing fungi. The main morphological changes of pathogenic and toxigenic fungi treated with N. sativa oil were observed in the cell wall, plasma membrane and membranous organelles, particularly in the nuclei and mitochondria. Although this review represents first step in the search for a new anti-fungal drug, the full potential of N. sativa as a fungitoxic agent has not been exploited and necessitates further investigations.