Reduction In Aflatoxin Levels Research Articles

Impact of using cinnamon (Cinnamomum verum) essential oil and its pectin-chitosan nano-emulsion on survival of Aspergillus flavus and total aflatoxin inhibition in beef burger patties

Fungal growth is one of the most common and serious problems faced during food storage. Aspergillus flavus is one of the major storage fungi producing aflatoxins, which are highly toxic and carcinogenic and pose a marked hazard to human health. Cinnamon oil is a natural essential oil with no obvious hazard effect on human and animal health. So, the goal of this research was to assess the efficacy of cinnamon essential oil (CEO) and cinnamon nanoparticles (CEO-NPs) on Aspergillus flavus growth and aflatoxin inhibition. In this research, CEO components were determined using GC/MS. Moreover, the characterization of nanoparticles by transmission electron microscopy (TEM) has also been studied. Initially, the antifungal activity and Minimal inhibitory concentration (MIC) has been determined by employing agar well-diffusion test and macro-dilution method, respectively. Then, depending on MIC results, the antifungal effects of CEO (3.125 μl/ml) and CEO-NPs (1.56 μl/ml) against A. flavus were studied. Furthermore, the inhibitory effect of CEO and CEO-NPs on aflatoxin content was evaluated. Additionally, the sensory evaluation of cinnamon and its nanoparticles in beef burger patties was assessed. The obtained results showed that the A. flavus count in experimentally contaminated beef burger samples was elevated from 4.06 ± 0.06 to 5.65 ± 0.04 (log10 CFU/g) in control samples while decreasing to 2.49 ± 0.15 in the presence of the CEO; however, it was not detected in the presence of CEO-NPs by the 9th day of the chilled storage. Regarding the aflatoxins, the CEO and its nanoparticles exhibited a reduction in aflatoxin levels to 40.9% and 68.2%, respectively, as determined by the ELISA technique. In conclusion, this study confirms the inhibitory role of CEOs and CEO-NPs in controlling A. flavus growth with a potential reduction in aflatoxin levels. The results open a promising avenue for their use as a safe, bioactive natural preservative in the food industry.

Evaluation of the Level of Aflatoxins in Raw Peanuts and Reduction Levels of Aflatoxins in Peanuts through Nixtamalization (Lime Treatment) and Calcium Enrichment

Nixtamalization has been widely used as a method of reducing aflatoxin contamination in peanuts as well as in calcium enrichment. The aim of this study is to investigate the effects of nixtamalization on aflatoxin and nutrient content in peanuts among wholesalers and retailers at Nyamakima Market in Nairobi, Kenya. A representative sample was drawn from a population of 70 market vendors using a systematic random sampling technique, resulting in 35 raw peanut samples for analysis via ELISA to determine initial aflatoxin levels. These samples were then subjected to nixtamalization treatments of 4, 8, and 16 minutes. The findings revealed that the average concentration of total aflatoxins in peanuts initially exceeded the accepted safety limit, with 71% of the samples failing to meet the standards. However, nixtamalization led to a substantial reduction in aflatoxin levels by 49%, 48%, and 50% after treatment durations of 4, 8, and 16 minutes, respectively. Statistical analysis, including ANOVA and Two-sample T-tests, confirmed significant distinctions between initial and final mean aflatoxin levels post-treatment, as well as variations between treatments. The study also observed a significant variation in calcium levels between the different time treatments, supported by linear regression analysis, which demonstrated a robust correlation between time treatments and calcium levels (R2=0.83, P=0.011). Furthermore, the analysis revealed significant differences in mean moisture contents across various treatments (P=0.000), and a linear relationship between mean moisture content and exposure time (P=0.02). The results emphasize the positive impact of nixtamalization in reducing aflatoxin levels and enhancing calcium content in peanuts. However, it is crucial to note the negative effect on moisture content, which exceeded stipulated limits, potentially raising concerns about product safety and quality.

Efficacy of short-synthetic antifungal peptides on pathogenic Aspergillus flavus

The efficacies of three short synthetic antifungal peptides were tested for their inhibitory action on pathogenic fungi, Aspergillus flavus. The sequences of the short synthetic peptides are PPD1- FRLHF, 66-10-FRLKFH, 77–3- FRLKFHF, respectively. These test peptides inhibited fungal growth and showed a membranolytic activity. The fungal biomass and ergosterol levels were significantly low in peptides treated samples. Further, the fungal cell wall component chitin was also found to be lower in peptides treated samples. Scanning electron microscopic images also showed highly wrinkled fungal mycelia. Significant membrane permeabilisation as well as potassium ion leakage was also observed in fungal samples treated with peptides. To assess the membrane damage, the uptake of Sytox green dye was employed. At tested concentration, peptides induced fungal membrane damage as evidenced by the green fluorescence. Further, at tested concentration, these peptides induced an oxidative stress in A.flavus as evidenced by an increase in the ROS production, malondialdehyde levels, increase in the antioxidant enzymes - superoxide dismutase, catalase with concomitant decrease in the reduced glutathione content. Additionally, a growth dependent reduction in aflatoxin levels were also observed in peptides treated samples. Docking studies on the interaction of the peptides with a trans-membrane protein calcium ATPase of A. flavus showed that all the peptides were able to bind to the protein with high z rank score. The activity of the calcium ATPase was significantly decreased in peptides treated fungal samples, thereby validating the docking results. Among all the tested peptides, 77–3 peptide exhibited the maximal membrane damage property.

Trial Summary on the Comparison of Various Non‐Aflatoxigenic Strains of Aspergillus flavus on Mycotoxin Levels and Yield in Maize

Core Ideas Biocontrol strains are effective at reducing AF levels in maize. Native and commercially available biocontrol strains are equally effective in reducing AF levels. Deploying strains of opposite mating types in combination can lead to the greatest reduction in AF contamination. The fungus Aspergillus flavus can contaminate maize (Zea mays L.) by producing aflatoxins (AFs), secondary metabolites that have been shown to have adverse health impacts for humans and animals when ingested in large quantities or over extended lengths of time. The FDA strictly regulates that corn contaminated with more than 20 parts per billion (ppb) AFs cannot be marketed for human consumption; therefore, AFs cost US corn growers billions of dollars every year. Current methods to curb aflatoxin contamination in fields involve dense applications of non‐aflatoxigenic biological control (biocontrol) strains, either Afla‐Guard or AF36, that outcompete native strains and reduce toxicity levels throughout the field. This fungus is heterothallic and sexual reproduction occurs between isolates of opposite mating types, either MAT1−1 or MAT1−2. Both biocontrol strains are of a single mating type MAT1−2. The implications of adding a strain of opposite mating type (MAT1−1) to this formulation are unknown. Here we examine the ability of native non‐aflatoxigenic strains applied singly and in combination to reduce AF concentrations in a cornfield in Rocky Mount, NC. We show that native, non‐aflatoxigenic A. flavus strains reduced aflatoxin levels and increased yield when compared with untreated controls. Moreover, the strain formulations that included sexually compatible MAT1−1 and MAT1−2 strains showed the greatest reduction in aflatoxin levels. We propose that using a combination of native isolates of opposite mating types reduces AF levels further than current biocontrol agents of a single mating‐type strain and could potentially provide a more long‐term form of control.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the α-amylase Inhibitor from Lablab purpureus L.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of α-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. α-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus α-amylase enzyme and belongs to the lectin-arcelin-α-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus α-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values. The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%-72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%-88% reduction in aflatoxin levels.

Control of Aspergillus flavus growth and aflatoxin production in transgenic maize kernels expressing a tachyplesin-derived synthetic peptide, AGM182

Aspergillus flavus is an opportunistic, saprophytic fungus that infects maize and other fatty acid-rich food and feed crops and produces toxic and carcinogenic secondary metabolites known as aflatoxins. Contamination of maize with aflatoxin poses a serious threat to human health in addition to reducing the crop value leading to a substantial economic loss. Here we report designing a tachyplesin1-derived synthetic peptide AGM182 and testing its antifungal activity both in vitro and in planta. In vitro studies showed a five-fold increase in antifungal activity of AGM182 (vs. tachyplesin1) against A. flavus. Transgenic maize plants expressing AGM182 under maize Ubiquitin-1 promoter were produced through Agrobacterium-mediated transformation. PCR products confirmed integration of the AGM182 gene, while RT-PCR of maize RNA confirmed the presence of AGM182 transcripts. Maize kernel screening assay using a highly aflatoxigenic A. flavus strain (AF70) showed up to 72% reduction in fungal growth in the transgenic AGM182 seeds compared to isogenic negative control seeds. Reduced fungal growth in the AGM182 transgenic seeds resulted in a significant reduction in aflatoxin levels (76–98%). The results presented here show the power of computational and synthetic biology to rationally design and synthesize an antimicrobial peptide against A. flavus that is effective in reducing fungal growth and aflatoxin contamination in an economically important food and feed crop such as maize.

Differences in Kernel Shape, Size, and Density between Healthy Kernels and Mold Discolored Kernels and their Relationship to Reduction in Aflatoxin Levels in a Sample of Shelled Corn

Abstract. Unacceptably high levels of aflatoxin in shelled corn drastically reduce its market value and result in substantial economic losses. This study looked at the reduction in aflatoxin levels that could be achieved by removal of kernels discolored by mold and presumed to contain aflatoxin. The study related kernel physical characteristics to the reduction achieved using screening and density separation techniques which are currently in use in the grain industry. Physical properties of corn kernels discolored by mold and healthy corn kernels were determined in a sample taken from a 737 kg corn lot known to have relatively high levels of aflatoxin. Properties measured included kernel diameters, sphericity, and kernel density. Differences between discolored and healthy kernels in major diameter, minor diameter, sphericity, and density were statistically significant. Tests were also conducted at a commercial inbred seed corn processing facility using a screen cleaner and a gravity table. Removal of fine material (approximately 10% by weight) reduced aflatoxin levels by 84%. In two successive passes through a screen cleaner, removal of smaller kernels and kernel pieces with a round-hole sieve further reduced aflatoxin levels by 1.8% and 9.4%, respectively. After the smaller kernels and kernel pieces were added back to the lot, two successive passes through a gravity table reduced aflatoxin levels by 12.6% and 16.4%, respectively. Kernel density distributions of a sample taken before gravity table density sorting, and of samples of the higher and lower density fractions after sorting, were also determined. The effect of screening with a slotted sieve was studied by screening a 3 kg sample from the corn lot using a table top Clipper cleaner. This 3 kg sample had been previously screened to remove fines. The results indicate that, at least for some corn lots, aflatoxin levels can be reduced using a screen cleaner and a gravity table. The authors also concluded that insight into the effectiveness of aflatoxin reduction in corn by size and density sorting could be gained by conducting a size and density analysis on a representative sample of kernels taken from the corn being sorted. Keywords: Aflatoxin, Corn, Density, Gravity table, Physical properties, Screening, Sieves.

The Fate of Aflatoxin in Corn Fermentation

A lab-scale ethanol fermentation was investigated to determine where aflatoxin concentrated during each phase of production. Four corn samples with high levels of aflatoxin (ranging from 7750 – 17,208 parts per billion) and their replicates were compared with a replicated negative control. Fractions were taken from the fermented mash, distilled ethanol, stillage, and dried corn solids (DCS). These fractions were analyzed using two different immunoassay methods and liquid chromatography tandem mass spectrometry (LC-MS/MS). Results indicated no aflatoxin was found in the distilled ethanol. Some aflatoxin (13%) was detected in the stillage, but most of the toxin was recovered in the DCSs ranging from 31% to 58%. A second series of experiments were conducted to investigate the effect of binders on dried distillers grains (DDGs). A brewers dried yeast anti-caking binder that contains glucomannon (MTB-100?), was mixed with contaminated DDGs. Addition of the binder showed a significant reduction in aflatoxin levels in comparison to a positive control. Aflatoxin binding at 2% binder w/w reached 72.5% and showed a minimal binding percentage increase of 80% at 6% binder w/w. Testing was also conducted to determine if environmental variables such as pH and temperature had any effect on the binding capabilities. Temperature near 0?C resulted in binding at 19.7% at a pH range of 6 to 8. Additionally, at a temperature of 40?C resulted in binding of 36%, 47%, and 45% at pHs 6, 7, and 8, respectively. These findings suggest that the addition of sorbents may be an effective way of salvaging contaminated DDGs.

Efficacy of ozone as a fungicidal and detoxifying agent of aflatoxins in peanuts

Peanut contamination by fungi is a concern of processors and consumers owing to the association of these micro-organisms with quality deterioration and aflatoxin production. In this study the fungicidal and detoxifying effects of ozone on aflatoxins in peanuts was investigated. Peanut kernels were ozonated at concentrations of 13 and 21 mg L⁻¹ for periods of 0, 24, 48, 72 and 96 h. Ozone was effective in controlling total fungi and potentially aflatoxigenic species in peanuts, with a reduction in colony-forming units per gram greater than 3 log cycles at the concentration of 21 mg L⁻¹ after 96 h of exposure. A reduction in the percentage of peanuts with internal fungal populations was also observed, particularly after exposure to ozone at 21 mg L⁻¹. A reduction in the concentrations of total aflatoxins and aflatoxin B1 of approximately 30 and 25% respectively was observed for kernels exposed to ozone at 21 mg L⁻¹ for 96 h. It was concluded that ozone is an important alternative for peanut detoxification because it is effective in controlling potentially aflatoxigenic fungi and also acts in the reduction of aflatoxin levels in kernels.

Carry-Over of Aflatoxins to Fig Molasses from Contaminated Dried Figs

The carry-over of aflatoxins to fig molasses produced by using two different processing techniques from contaminated dried figs (> 1000 ppb of total aflatoxins) were examined by using a HPLC technique. The effects of extraction, bleaching and concentration steps on the reduction of aflatoxin levels were also investigated. The reductions in total aflatoxin levels in fig molasses produced by using the two techniques were detected to be 62 and 38%, respectively. Extraction, bleaching and concentration steps were observed to cause 22% reduction in total aflatoxin levels.

Effect of parboiling and bran removal on aflatoxin levels in Sri Lankan rice

Commercial parboiling of rice in Sri Lanka and many south Asian countries provides ideal conditions for the occurrence of aflatoxins because the rice is steeped (allowing fermentation) thus providing ideal conditions for growth of toxigenic Aspergillus species. However the traditional 'cottage' method of parboiling rice, which does not involve steeping, appears to reduce Aspergillus growth even after long storage periods. Preferential infection of parboiled rice by Aspergillus flavus was observed. Aflatoxin contents in inoculated rice produced by commercial parboiling (AFB1 60-92 mg/kg) were significantly higher than that in inoculated 'cottage' processed rice (AFB1 12-29 micrograms/kg). The steeping (precooking/soaking) process in commercial parboiling appears to increase the susceptibility of rice grains to fungal infection. Aflatoxin content in grains increased considerably with the increase in duration of soaking. However, the addition of 10 ppm calcium hypochlorite (bleach) to soaking water appreciably reduced A. flavus contamination and subsequent aflatoxin content in parboiled rice. No significant reduction in aflatoxin levels were observed after bran removal of contaminated rice.

Decontamination of aflatoxin‐contaminated peanut meal using monomethylamine:Ca(0H)2

AbstractRadiolabeled aflatoxin B1 was added to naturally contaminated peanut meal, and the fate of aflatoxin‐related by‐products after decontamination by monomethylamine:Ca(OH)2 was monitored. The decontamination process resulted in a 94‐100% reduction in aflatoxin levels, depending on the level of contamination and the chemical structure of the aflatoxin. Following air drying and extractions with chloroform/water, methanol, acetic acid and water, the majority (69‐86%) of the, decontamination by‐products remained with the residue. Significant amounts of aflatoxin B1, (12%) and aflatoxin‐related compounds (27%) were liberated from the residue after enzymic digestion. Isolation and identification studies revealed a wide variety of aflatoxin‐related decontamination by‐products. High performance liquid and thin layer Chromatographie separation of chloroform‐soluble compounds revealed compounds that exhibited some degree of toxicity. Comparative toxicity tests showed that, although some decontamination by‐products exhibited elevated responses to specific toxicity tests, the relative toxicity was inferior to aflatoxin B1. The moisture, protein and nitrogen levels did not change significantly. There was a significant change in the mold flora following the decontamination process, and the product was susceptible to recontamination.

Reducation of aflatoxin levels in cottonseed and peanut meals by ozonization.

AbstractCottonseed and peanut meals were treated with ozone to destroy or eliminate aflatoxins. High meal moistures (cottonseed 22%, peanut 30%), high temperature (100C), and longer treatment times favored inactivation as measured by thin‐layer chromatography. Aflatoxins B1 and G1 were readily destroyed by the ozone processes whereas aflatoxin B2 appeared relatively resistant. In cottonseed meal, 91% of the total aflatoxins was destroyed in 2 hr, a decrease from 214 to 20 ppb; in peanut meal, 78% was destroyed in 1 hr, a decrease from 82 to 18 ppb. In both meals, aflatoxin B1 was totally inactivated within the times specified.