Spread of Aspergillus flavus and aflatoxin accumulation in postharvested maize treated with biocontrol products

Maize is a major staple crop and calorie source for many people living in Sub-Saharan Africa. In this region, Aspergillus flavus causes ear rot in maize, contributing to food insecurity due to aflatoxin contamination. The biological control principle of competitive exclusion has been applied in both the United States and Africa to reduce aflatoxin levels in maize grain at harvest by introducing atoxigenic strains that out-compete toxigenic strains. The goal of this study was to determine if the efficacy of preharvest biocontrol treatments carry over into the postharvest drying period, the time between harvest and the point when grain moisture is safe for storage. In Sub-Sahara Africa, this period often is extended by weather and the complexities of postharvest drying practices. Maize grain was collected from fields in Texas and North Carolina that were treated with commercial biocontrol products and untreated control fields. To simulate moisture conditions similar to those experienced by farmers during drying in Sub-Sahara Africa, we adjusted the grain to 20% moisture content and incubated it at 28 °C for 6 days. Although the initial number of kernels infected by fungal species was high in most samples, less than 24% of kernels were infected with Aspergillus flavus and aflatoxin levels were low (<4 ppb). Both toxigenic and atoxigenic strains grew and spread through the grain over the incubation period, and aflatoxin levels increased, even in samples from biocontrol-treated fields. Our molecular analysis suggests that applied biocontrol strains from treated fields may have migrated to untreated fields. These results also indicate that the population of toxigenic A. flavus in the harvested grain will increase and produce aflatoxin during the drying period when moisture is high. Therefore, we conclude that preharvest biocontrol applications will not replace the need for better postharvest practices that reduce the drying time between harvest and storage.     

INFLUENCE OF MOISTURE CONTENT AND STORAGE TEMPERATURE ON THE PRODUCTION OF AFLATOXIN BY ASPERGILLUS FLAVUS EA-81 IN MAIZE AFTER EXPOSURE TO GAMMA RADIATION

The effect of gamma radiation on aflatoxin production by Aspergillus flavus EA-81 in maize with different initial moisture levels was determined over a 15-day period. The viability of A. flavus on maize decreased over time with increasing moisture contents and storage at 8C. After 45 days at 28C, levels of viable conidiospores of A. flavus increased from 4.5 × 10⁷ to about 3.0 × 10⁸ per gram of maize. Levels of aflatoxin B₁ produced by A. flavus were 10 μg kg^-1 in the maize stored at 8C after 45 days. Production of aflatoxin was highest at 40% moisture and 28C. Irradiation of 1.0 or 2.0 kGy greatly reduced the level of mold growth relative to unirradiated controls. A dose of 4.0 kGy eliminated all viable fungi. Aflatoxin B₁ production decreased with increased levels of irradiation and was negligible at 4.0 kGy. When maize was inoculated after irradiation and stored, the spore counts and aflatoxin levels were higher than in unirradiated and inoculated controls after 30 days. Apparently, the natural competitive microflora prevented growth and thus limited higher concentrations of aflatoxin in maize.     

Effect of γ‐irradiation on aflatoxin B1 production by Aspergillus flavus and chemical composition of three crop seeds

The effect of γ‐irradiation on aflatoxin B₁ production by Aspergillus flavus, and the chemical composition of some different crop seeds were investigated. A. flavus infected seeds behaved differently according to their principal constituents. A. flavus caused an increase in protein and decrease in lipids and carbohydrate contents of wheat, soyabean and fababean seeds. Growth of A. flavus and production of aflatoxin B₁ was inhibited at a dose level of 5 kGy. A. flavus utilizes carbohydrates of seeds for its growth and aflatoxin production. Crops were arranged, in descending order, according to aflatoxin produced in seeds as wheat > soyabean > fababean. There were no changes in chemical constituents of irradiated seeds, such as protein, lipids, and carbohydrates.     

Effectiveness of potassium lactate and sodium diacetate in combination with irradiation to control Listeria monocytogenes on frankfurters

ABSTRACT:  The use of antimicrobial ingredients in combination with irradiation is an effective antilisterial intervention strategy for ready-to-eat meat products. Microbial safety was evaluated for frankfurters formulated with 0% or 3% added potassium lactate/sodium diacetate solution and inoculated with Listeria monocytogenes before or after treatment with irradiation (0, 1.8, or 2.6 kGy). Frankfurters were stored aerobically or vacuum packaged and L. mo nocytogenes counts and APCs were determined while refrigerated. The incorporation of lactate/diacetate with or without irradiation had a strong listeriostatic effect for aerobically stored frankfurters. Outgrowth was suppressed and counts were not different from initial counts (5.2 log CFU/frank compared with 5.0 log CFU/frank); however, those without the additive increased steadily (5.4 to 9.3 log CFU/frank). Irradiation treatments alone had higher L. monocytogenes counts after 3 wk. For vacuum-packaged frankfurters, both the addition of lactate/diacetate and irradiation were effective at controlling growth after 8 wk. Large and incremental reductions in total counts were seen for irradiation treatments. Initial counts were reduced by 3 log CFU with the application of 1.8 kGy while 2.6 kGy decreased counts over 5 log CFU. These reductions were maintained throughout storage for lactate/diacetate-treated frankfurters. By 8 wk, L. monocytogenes counts on 1.8 and 2.6 kGy irradiated frankfurters without lactate/diacetate increased to 7.43 and 6.13 log CFU, respectively. Overall, lactate/diacetate retarded the outgrowth of L. monocytogenes on frankfurters throughout aerobic storage and the combination of irradiation and 3% lactate/diacetate reduced and retarded growth of L. monocytogenes , especially during the last 2 wk of vacuum-packaged storage.     

Use of PCR for Detecting Aspergillus flavus in Maize Treated by Gamma Radiation Process

The aim of this study was to verify the effects of gamma radiation process on the fungal DNA and the application of PCR in the detection of Aspergillus flavus in irradiated maize grains. The samples were inoculated with a toxigenic strain and incubated under controlled conditions of relative humidity, water activity, and temperature for 15 days. After incubation, the samples were treated with gamma radiation with doses of 5 and 10 kGy and individually analyzed. The use of PCR technique showed the presence of DNA bands of Aspergillus flavus in all irradiated samples that showed no fungal growth in agar medium.     

Effects of gamma irradiation on the viability and phenotypic characteristics of Salmonella Enteritidis inoculated into specific-pathogen-free eggs

The goal of this study was to determine the effects of various levels of gamma irradiation on the phenotypic characteristics of 20 strains of Salmonella Enteritidis inoculated separately into specific-pathogen-free shell eggs. Bacterial strains were inoculated into egg yolks and exposed to (60)Co radiation at doses of 0.49 to 5.0 kGy. The eggs were maintained at 25°C and analyzed for the presence of Salmonella on days 1, 2, 4, and 7, and the recovered Salmonella isolates were characterized biochemically. All strains were resistant to doses of 0.49, 0.54, 0.59, 0.8, and 1 kGy; colony counts were ≥10(5) CFU/ml of egg yolk except for one strain, which was detected at 96 h and at 7 days after irradiation at 1 kGy, with a population reduction of 2 log CFU/ml. For the other evaluated doses, 12 strains (60.0%) were resistant at 1.5 kGy and 7 strains (35.0%) were resistant at 3.0 kGy. Among all analyzed strains, 5.0 kGy was more effective for reducing and/or eliminating the inoculated bacteria; only two (10%) strains were resistant to this level of irradiation. Salmonella colony counts were significantly reduced (P < 0.01) with increasing doses from the day 1 to 7 of observation, when microbial growth peaked. Loss of mobility, lactose fermentation, citrate utilization, and hydrogen sulfide production occurred in some strains after irradiation independent of dose and postirradiation storage time. Increases in antibiotic susceptibility also occurred: seven strains became sensitive to β-lactams, two strains became sensitive to antifolates, and one strain each became sensitive to fluoroquinolone, phenicol, nitrofurans, tetracyclines, and aminoglycosides. The results indicate that up to 5.0 kGy of radiation applied to shell eggs inoculated with Salmonella Enteritidis at 4 log CFU per egg is not sufficient for complete elimination of this pathogen from this food matrix.     

Influence of γ‐irradiation and maize lipids on the production of aflatoxin B1 by Aspergillus flavus

The effect of γ‐irradiation and maize lipids on aflatoxin B₁ production by Aspergillus flavus artificially inoculated into sterilized maize at reduced water activity (a_w 0.84) was investigated. By increasing the irradiation doses the total viable population of A. flavus decreased and the fungus was completely inhibited at 3.0 kGy. The amounts of aflatoxin B₁ were enhanced at irradiation dose levels 1.0 and 1.5 kGy in both full‐fat maize (FM) and defatted maize (DM) media and no aflatoxin B₁ production at 3.0 kGy γ‐irradiation over 45 days of storage was observed. The level in free lipids of FM decreased gradually, whereas free fatty acid values and fungal lipase activity increased markedly by increasing the storage periods. The free fatty acid values decreased by increasing the irradiation dose levels and there was a significant enhancement of fungal lipase activity at doses of 1.0 and 1.50 kGy. The ability of A. flavus to grow at a_w 0.84 and produce aflatoxin B₁ is related to the lipid composition of maize. The enhancement of aflatoxin B₁ at low doses was correlated to the enhancement of fungal lipase activity.     

Radiation processing for elimination of Salmonella typhimurium from inoculated seeds used for sprout making in India and effect of irradiation on germination of seeds

The effect of radiation processing on the germination of the sprout seeds mung (Phaseolus aureus), matki (Phaseolus aconitifolius), chana (Cicer arietinum), and vatana (Pisum sativum) in terms of percent germination, germination yield, sprout length, vitamin C content, and texture was investigated. Gradual decreases in the percent germination, germination yield, and sprout length with increases in radiation dose (0.5 to 2.0 kGy) were observed. Vitamin C content and texture remained unaffected for the seeds treated with doses of up to 2 kGy. To determine the efficacy of radiation treatment in elimination of foodborne pathogens, seeds inoculated with 4 log CFU/g of Salmonella Typhimurium were treated with radiation doses of 1 and 2 kGy. A reduction in counts of Salmonella Typhimurium in inoculated seeds after radiation treatment was observed. A radiation dose of 2 kGy resulted in the complete elimination of 4 log CFU/g of Salmonella Typhimurium from the inoculated seeds. However, on sprouting for 48 h, the count of Salmonella Typhimurium reached 8 log CFU/g for the control seeds and the seeds treated with a 1-kGy radiation dose. The aerobic plate counts for seeds were 2.0 to 2.6 log CFU/g, which were reduced to 0.9 to 1.2 log CFU/g on treatment with a 2-kGy radiation dose. On sprouting for 48 h, the aerobic plate count reached 8 log CFU/g for both the control and radiation-treated seeds. The study demonstrates that irradiation can control bacterial levels on seeds but not contamination introduced during posttreatment handling. Therefore, radiation processing of the final product (sprouts) is recommended, rather than of the seeds.     

Assessing Purdue Improved Crop Storage (PICS) bags to mitigate fungal growth and aflatoxin contamination

Storing maize in regions of the world without sufficient drying and storage capacity is challenging due to the potential risk of aflatoxin contamination produced by Aspergillus flavus. This study sought to determine if storage of maize in Purdue Improved Crop Storage (PICS) bags prevents mold growth and aflatoxin accumulation. PICS bags are a three-layer, hermitic bag-system that forms a barrier against the influx of oxygen and the escape of carbon dioxide. Maize conditioned at 12, 15, 18, and 21% grain moisture was inoculated with 50 g of maize kernels infected with fluorescent-marked strain of A. flavus. The grain was stored in either PICS or woven bags at 26 °C, and percent oxygen/carbon dioxide levels, fungal growth, aflatoxin, moisture content, and kernel germination were assessed after 1 and 2 months incubation. Maize stored in woven bags was found to equilibrate with the ambient moisture environment over both storage periods, while PICS bags retained their original moisture levels. Aspergillus flavus growth and aflatoxin accumulation were not observed in maize stored in any PICS bags. No aflatoxin B1 was detected in woven bags containing low-moisture maize (12 and 15%), but detectable levels of aflatoxin were observed in high moisture maize (18 and 21%). The percentage of oxygen and carbon dioxide within PICS bags were dependent on initial grain moisture. Higher carbon dioxide levels were observed in the bags stored for 1 month than for 2 months. High initial moisture and carbon dioxide levels correlated with low kernel germination, with the 18 and 21% treatment groups having no seeds germinate. The results of the study demonstrate that storage of maize in PICS bags is a viable management tool for preventing aflatoxin accumulation in storage.     

REDUCTION OF FUNGI AND MYCOTOXINS FORMATION IN SEEDS BY GAMMA-RADIATION

Ninety samples of maize, chick-peas and groundnut seeds collected from the Egyptian market were found to be heavily contaminated by molds. Alternaria, Aspergillus, Cladosporium, Eurotium, Fusarium, Mucor, Penicillium and Rhizopus were the most common fungal genera isolated from nondisinfected seeds . Aspergillus alutaceus, A. flavus, Fusarium verticillioides and F. oxysporum were isolated from all surface-disinfected seeds and were reported to produce ochratoxin A, aflatoxin B₁ and zearalenone, respectively. Irradiation at a dose 4.0 kGy reduced the mold growth greatly relative to unirradiated controls. There was no growth at dose 5.0 kGy. On the basis of the radiation survival data, the decimal reduction values D₁₀ for A. alutaceus, A. flavus and F. verticilliodies were 0.70. 2.10 and 0.93 kGy in maize. A dose of 5 kGy inhibited the toxigenic molds and mycotoxin formation in seeds. Aflatoxin B₁ and ochratoxin A were detected in maize and chick-peas, whereas zearalenone was detected in maize samples. Application of radiation at a dose of 6.0 kGy detoxified aflatoxin B₁ by 74.3–76.7%, ochratoxin A by 51.3–96.2% and zearalenone by about 78%.     

Potential of aflatoxin B1 production by Aspergillus flavus strains on commercially important food grains

In this study, we investigated the potential of aflatoxin B₁ (AFB1) production by five Aspergillus flavus strains previously isolated from sorghum grains on cereals (barley, maize, rice, wheat and sorghum), oilseeds (peanuts and sesame) and pulses (greengram and horsegram). Five strains of A. flavus were inoculated on all food grains and incubated at 25 °C for 7 days; AFB1 was extracted and estimated by enzyme‐linked immunosorbent assay. All A. flavus strains produced AFB1 on all food grains ranging from 245.4 to 15 645.2 μg kg^?1. Of the five strains tested, strain Af 003 produced the highest amount of AFB1 on all commodities ranging from 2245.2 to 15 645.2 μg kg^?1. Comparatively, the AFB1 accumulation was high on rice grains ranging from 3125.2 to 15 645.2 μg kg^?1, followed by peanuts ranging from 2206.2 to 12 466.5 μg kg^?1. Less AFB1 accumulation was observed in greengram and sesame seeds ranging from 645.8 to 2245.2 and 245.4 to 2890.6 μg kg^?1, respectively. Our results showed that all food grains tested are susceptible to A. flavus growth and subsequent AFB1 production.     

Use of low dose e-beam irradiation to reduce E. coli O157:H7, non-O157 (VTEC) E. coli and Salmonella viability on meat surfaces

This study determined the extent that irradiation of fresh beef surfaces with an absorbed dose of 1 kGy electron (e-) beam irradiation might reduce the viability of mixtures of O157 and non-O157 verotoxigenic Escherichia coli (VTEC) and Salmonella. These were grouped together based on similar resistances to irradiation and inoculated on beef surfaces (outside flat and inside round, top and bottom muscle cuts), and then e-beam irradiated. Salmonella serovars were most resistant to 1 kGy treatment, showing a reduction of ≤ 1.9 log CFU/g. This treatment reduced the viability of two groups of non-O157 E. coli mixtures by ≤ 4.5 and ≤ 3.9 log CFU/g. Log reductions of ≤ 4.0 log CFU/g were observed for E. coli O157:H7 cocktails. Since under normal processing conditions the levels of these pathogens on beef carcasses would be lower than the lethality caused by the treatment used, irradiation at 1 kGy would be expected to eliminate the hazard represented by VTEC E. coli.     

Impact of storage environment on the efficacy of hermetic storage bags

Small hermetic bags (50 and 100 kg capacities) used by smallholder farmers in several African countries have proven to be a low-cost solution for preventing storage losses due to insects. The complexity of postharvest practices and the need for ideal drying conditions, especially in the Sub-Sahara, has led to questions about the efficacy of the hermetic bags for controlling spoilage by fungi and the potential for mycotoxin accumulation. This study compared the effects of environmental temperature and relative humidity at two locations (Indiana and Arkansas) on dry maize (14% moisture content) in woven polypropylene bags and Purdue Improved Crop Storage (PICS) hermetic bags. Temperature and relative humidity data loggers placed in the middle of each bag provided profiles of environmental influences on stored grain at the two locations. The results indicated that the PICS bags prevented moisture penetration over the three-month storage period. In contrast, maize in the woven bags increased in moisture content. For both bag types, no evidence was obtained indicating the spread of Aspergillus flavus from colonized maize to adjacent non-colonized maize. However, other storage fungi did increase during storage. The number of infected kernels did not increase in the PICS bags, but the numbers in the woven bags increased significantly. The warmer environment in Arkansas resulted in significantly higher insect populations in the woven bags than in Indiana. Insects in the PICS bags remained low at both locations. This study demonstrates that the PICS hermetic bags are effective at blocking the effects of external humidity fluctuations as well as the spread of fungi to non-infected kernels.     

Preservation treatment of fresh raspberries by e-beam irradiation

E-beam irradiation was studied as a post-harvest treatment for red raspberries (Rubus idaeus L.). Microbial inactivation (natural microbiota and potential pathogenic bacteria) and bioactive properties (phenolic content, vitamin C content and antioxidant activity and cytotoxicity) of these fruits were evaluated before and after irradiation and during storage of 14 days at 4 °C. A reduction of 2 log CFU/g of mesophilic bacteria and 3 log CFU/g on filamentous fungi, and no detection of foodborne inoculated pathogens (3 log CFU/g) was achieved with an e-beam treatment at 3 kGy and during 7 days of refrigerated storage. Regarding bioactive properties, the results suggested that irradiation could preserve the phenolic content and antioxidant activity of raspberries through 7 days of cold storage, even though a decrease of 80% on ascorbic acid concentration was observed. Furthermore, no in vitro inhibitory effect on human cells lines was observed for the extracts from e-beam-treated raspberries. The overall results suggested that use of e-beam irradiation as post-harvest treatment of raspberries as an emergent, clean and environmental friendly process to extend the shelf-life of this fruit with safety and preservation of bioactivity.Industrial relevanceRed raspberries are known to demonstrate high bioactivity that could be beneficial to human health, but are highly perishable and often associated with foodborne outbreaks, which makes its safety and commercialization a challenge. The use of a terminal control such as irradiation might reduce the burden of disease transmission and extend the quality of fresh red raspberries. The present research indicated that e-beam irradiation can be used as post-harvest treatment of raspberries, guarantying its safety and quality with the add-value of shelf-life extension.     

Use of mild irradiation doses to control pathogenic bacteria on meat trimmings for production of patties aiming at provoking minimal changes in quality attributes

The objectives of the present work were to assess the use of moderate doses of gamma irradiation (2 to 5 kGy) and to reduce the risk of pathogen presence without altering the quality attributes of bovine trimmings and of patties made of irradiated trimmings. Microbiological indicators (coliforms, Pseudomonas spp and mesophilic aerobic counts), physicochemical indicators (pH, color and tiobarbituric acid) and sensory changes were evaluated during storage. 5 kGy irradiation doses slightly increased off flavors in patties. Two pathogenic markers (Listeria monocytogenes and Escherichia coli O157:H7) were inoculated at high or low loads to trimming samples which were subsequently irradiated and lethality curves were obtained. Provided that using irradiation doses ≤ 2.5 kGy are used, reductions of 2 log CFU/g of L. monocytogenes and 5 log CFU/g of E. coli O157:H7 are expected. It seems reasonable to suppose that irradiation can be successfully employed to improve the safety of frozen trimmings when initial pathogenic bacteria burdens are not extremely high.     

Laboratory tests for assessing efficacy of atoxigenic Aspergillus flavus strains as biocontrol agents

Biocontrol by competitive inhibition using atoxigenic Aspergillus flavus strains has been shown to be an effective method for controlling aflatoxin production in peanuts, maize and cottonseed. Selecting biocontrol strains is not straightforward, as it is difficult to assess fitness for the task without expensive field trials. Reconstruction experiments have been generally performed under laboratory conditions to investigate the biological mechanisms underlying the efficacy of atoxigenic strains in preventing aflatoxin production and/or to give a preliminary indication of strain performance when released in the field. The study here described was conducted in order to evaluate the potential of the different atoxigenic A. flavus strains, colonizing the corn fields of the Po Valley, in reducing aflatoxin accumulation when grown in mixed cultures together with atoxigenic strains; additionally, we developed a simple and inexpensive procedure that may be used to scale-up the screening process and to increase knowledge on the mechanisms interfering with mycotoxin production during co-infection.     

Comparative study of toxigenic potential of Aspergillus flavus and Aspergillus niger isolated from Barley as affected by temperature,water activity and carbon source

The objective of the present work was to evaluate the ability of two mycotoxigenic species Aspergillus flavus and Aspergillus niger, isolated from barley, to produce aflatoxin B1 (AFB1) and ochratoxin A (OTA), respectively, as affected by nutritional and environmental factors. Six carbon sources (D-fructose, D-glucose, D-galactose, lactose, sucrose and starch) and different water activities (0.90, 0.95 and 0.98), temperature (20 and 28 °C) and incubation time (5 and 10 days) were tested. The results showed that optimal conditions for growth on Barley Meal Extract Agar (BMEA) medium were 28 °C and 0.95 a_w for A. niger strain and 28 °C and 0.98 a_w for A. flavus strain. Optimal conditions for OTA and AFB1 production were largely different for the two tested strains. A. niger had an optimal OTA production at 0.98 a_w and 20 °C after 10 days of incubation while A. flavus had an optimal AFB1 production at 0.95–0.98 a_w and 28 °C after 5 days of incubation. These results indicates that A. flavus has a higher optimum temperature for mycotoxin synthesis than A. niger and takes greater advantage of drier conditions for maximum AFB1 production. In the current study, both OTA and AFB1 production of A. niger and A. flavus were highly influenced by carbon sources. The sugar that provided the highest toxin levels in the cultures of the two species was sucrose with the lowest levels given by starch. OTA production by A. niger was also highly induced by fructose as carbon sources, while, AFB1 production by A. flavus was favored by glucose. Globally, our results showed, significantly different optimal conditions for production of AFB1 and OTA, respectively, by A. flavus and A. niger.     

Effect of sublethal concentrations of propionic or butyric acid on growth and aflatoxin production by Aspergillus flavus

Propionic or butyric acid was added at sublethal doses (0.1–2 mg/ml) to a growth medium supporting growth of Aspergillus flavus and subsequent aflatoxin production. A reduction in growth and aflatoxin production occurred when the acids were added at the time of inoculation. Addition of the acids to cultures at different times resulted in little effect on growth but production of aflatoxin after 12 days was reduced with earlier time of application for both propionic and butyric acid. When the acids were added to rough rice with a moisture content of 21% and inoculated with A. flavus fungal growth and aflatoxin production were reduced relative to non-inoculated controls. Early application of acids resulted in lower yields of aflatoxin.     

Effects of the repair treatment on improving the heat resistance of Lactiplantibacillus plantarum LIP-1

To improve the heat resistance of lactobacilli during spray-drying, we first investigated the effect of heat shock and repair treatment on the heat resistance of Lactiplantibacillus plantarum LIP-1, and the specific cell repair mechanism. Compared with control group, the reduction of the strain after the heat treatment (75 °C for 40 s) decreased from 1.05 to 0.36 log CFU/mL (initial cell counts 9.30 log CFU/mL) by heat shock (44 °C for 10 min). The residuals of the strain after heat-treated increased by 0.90 log CFU/mL by heat shock firstly, and then the repair treatment (30 °C for 10 min).During recovery period, the relative content of unsaturated long-chain fatty acids (C18:1n9 & C18:2n6) induced by heat shock proteins increased by 7.0%, and the amount of DnaK protein anchored on the cell membrane increased by 0.17 pg/mL. Cell membrane damage caused by heat shock was reduced and strain heat resistance was improved.Industrial relevanceOur research found that the repair treatment after heat shock reduced cell membrane damage caused by heat shock, which is a very promising technology for improving the heat resistance of L. plantarum LIP-1. This technology is also expected to be widely used in the preparation of LAB powders by spray-drying technology.