Assessment of aflatoxin presence in the Rwandan feed and milk value chains and mitigation potential using high voltage atmospheric cold plasma

Abstract

<p>Mycotoxins, particularly aflatoxins, are fungal metabolites, and under favorable growth conditions contaminate crops and animal feeds. They are associated with liver cancer, immunosuppression, and growth impairment. Human exposure to these mycotoxins is the result of ingestion of contaminated foods, or indirectly from consumption of animal source foods (e.g., dairy products and eggs) derived from animals previously exposed to aflatoxins in feeds. They also have an economic impact because contamination levels of aflatoxins and other mycotoxins in foods and feedstuffs can lead to rejection of these products in international markets.</p> <p>However, there has been a lack of data on the presence of aflatoxins in the food and feed supply of Rwanda. The absence of published data on the presence of aflatoxins in Rwandan feeds raises the possibility of underestimating the present risk, and missing opportunities to reduce the risk of mycotoxin contamination of animal source food (ASF), particularly milk, via consumption of contaminated animal feeds by livestock. The overall objective of this study was to assess the prevalence of aflatoxin and fumonisin contamination and associated risk factors in feed and feed ingredients, and aflatoxin M1 contamination in raw milk in Rwanda, and to explore the potential of high voltage atmospheric cold plasma (HVACP), a low-cost technology in its nascent form, to mitigate aflatoxin contamination of maize, a major ingredient of feeds in Rwanda.</p> <p>In total, 3328 feed and feed ingredient samples from the feed value chain in Rwanda were analyzed for aflatoxins and fumonisins using Enzyme-Linked Immunosorbent Assay (ELISA). Mean aflatoxin levels of 108.83 µg/kg (Median (MD): 43.65 µg/kg), 103.81µg/kg (MD: 48.4 µg/kg), 88.64 µg/kg (MD: 30.90 µg/kg) and 94.95 µg/kg (MD: 70.45 µg/kg) were determined for dairy farmers, poultry farmers, feed vendors and feed processors, respectively. Mean fumonisin levels were 1.52 mg/kg (MD: 0.71 mg/kg), 1.21 mg/kg (MD: 0.56 mg/kg), 1.48 mg/kg (MD: 0.76 mg/kg) and 1.03 mg/kg (MD: 0.47 mg/kg) for dairy farmers, poultry farmers, feed vendors and feed processors, respectively. Aflatoxin contamination was significantly affected by time of sampling and district from which feed samples originated (p<0.05). Fumonisins did not show any correlation trends. Ninety-two percent of survey participants were unaware of aflatoxins and fumonisins and their adverse effects.</p> <p>In total, 170 raw milk samples were collected during one sampling period; the mean AFM1 concentration in these samples was 0.89 ± 1.64 g/L (median: 0.33 g/L) with a maximum of 14.5 g/L. Ninety-one percent of milk samples exceeded 0.05 g/L (the European legal limit) and 38% of samples exceeded 0.5 g/L, the legal limit and maximum level established by the U.S. Food and Drug Administration (USFDA) and Codex Alimentarius, respectively.</p> <p>HVACP treatment significantly degraded pure AFs (each AF individually) powder on a glass slide exposed directly at 85 kV and 180 W (60 Hz) at laboratory conditions (25C, 80-90 % relative humidity) for 2, 5, 10, and 20 min using air. The electrode gap distance was maintained at 4.44 cm. After the treatment, AFs were extracted with methanol and the final concentration was 200 µM. AFB1 and AFG1 were more susceptible decreasing by 90% and 74% after 2 min, respectively comparing to the non-treated samples (0 min). After 10 and 20 min, AFG1 was not detected in samples. AFB2 and AFG2 were less susceptible decreasing by 38% and 79% after 20 min, respectively. Despite observed degradations, only one AFB1 DP (m/z: 331.08) was characterized presumably due to the limit of quantification of the technique used (< 10 mg/kg). AFB1 DP resulting in hydroxylation of the double bond in the position C8-C9 AFB1 was previously reported in published studies.</p> <p>HepG2 cells, a human hepatoma cell line, were exposed to AFB1treated at 85 kV with HVACP for 0, 2, 5, 10, and 20-minute periods for 72 hours (37C, 5% CO2). After HVACP treatment, AFB1 was extracted with DMSO (100 mL) and added to DMEM media (9.9 mL) for 100 µM AFB1 final concentration. AFB1 endpoint toxicities, i.e., cell viability (MTT assay using 1105 cells/mL), caspase-3 activity (2107 cells/mL), DNA fragmentation (1105 cells/mL), and protein carbonyl (80% confluent in 75-cm2 cell culture flask), were assessed for each treatment time. After 10 and 20 min treatment, cell viability in HepG2 cells exposed to AFB1 DP did not show a significant difference compared to non-exposed HepG2 cells (negative control) (p>0.05) but significantly differed from HepG2 cells exposed to AFB1(positive control) (p<0.05). Caspase-3 activity, DNA fragmentation values, and carbonyl contents in HepG2 cells exposed to AFB1 DP after 20 min treatment were similar to those in non-exposed HepG2 cells (p>0.05) but significantly differed from those in HepG2 cells exposed (p<0.05).</p> <p>Maize kernels (25 g) inoculated with A. flavus spores (106 spores/mL) were incubated for two weeks and treated with HVACP directly and indirectly at 85 kV and 180 W (60 Hz) at laboratory conditions (25C, 80-90 % relative humidity) for 7, 15, and 30 min. HVACP significantly reduced AFB1 in all treatment times. After 7 and 15 min, AFB1 decreased by 81.3 - 96.3 % and 78.1 – 85.0%, respectively. The 30 min treatment showed AFB1 decreasing from below the LOQ (< 5 µg/kg) to 88.9% ± 10.3. HVACP treatment inhibited A. flavus growth significantly reducing it by 2.4 and 1.9 log10 for direct and indirect exposure, respectively. A. flavus reduction was below the limit of detection for the 15 and 30 min treatments. HVACP treatment resulted in a slight change in yellow color but neither induced lipid oxidation nor changed flavor profile in treated maize samples.</p>