Post-harvest handling practices and their influence on aflatoxin development in stored groundnut

Introduction

Groundnut is rich in protein, healthy fats, and micronutrients and at the same time, it serves as one of the most hospitable substrates for Aspergillus flavus and A. parasiticus, the fungal species responsible for producing aflatoxins [1] . This contrast between nutritional value and contamination risk defines the central challenge of groundnut post-harvest management. Aflatoxin B1, the most toxic and carcinogenic variant, has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer, with chronic exposure linked to liver cancer and immunosuppression [2,3,22] . Global aflatoxin contamination in groundnut causes estimated annual losses exceeding US$750 million in rejected exports from developing countries alone [4] . Regulatory limits vary widely: the European Union enforces 4 μg kg⁻¹ for total aflatoxin in ready-to-eat groundnuts, while the Codex Alimentarius sets 15 μg kg⁻¹ for total aflatoxin in processed nuts [5] . Japan follows its own Food Sanitation Act standard of 10 μg kg⁻¹ for total aflatoxin [6] . Aspergillus colonization and toxin production are strongly driven by moisture and temperature. Kernel moisture above 9-10% combined with temperatures of 25-35°C creates favorable conditions for rapid fungal growth [7,8] . Post-harvest handling including drying speed, storage container type, ventilation, and pest management determines whether field-safe groundnuts remain safe or become contaminated during storage [9,10] . Most published work on this topic originates from sub-Saharan Africa and South Asia, while data from East Asian smallholder contexts remain limited [11] . This research aimed to: (a) identify which post-harvest handling variables most strongly predict aflatoxin accumulation, (b) quantify the relationship between kernel moisture and AFB1 concentration, and (c) evaluate a combined intervention package for reducing contamination below regulatory thresholds [12] .

Aflatoxin Extraction Protocol

Aflatoxin extraction followed a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) approach adapted for groundnut matrices [13] . Subsamples of 25 g were homogenized in 100 mL of acetonitrile:water (84:16, v/v) for 3 minutes using a high-speed blender. Magnesium sulfate (6 g) and sodium chloride (1.5 g) were added, and the mixture was shaken for 1 minute and centrifuged at 4,000 rpm for 5 minutes. A 6 mL aliquot of the supernatant was cleaned through a dispersive solid-phase extraction tube containing 150 mg primarysecondary amine (PSA) and 900 mg MgSO₄. The cleaned extract was evaporated under nitrogen at 40°C and reconstituted in 1 mL of methanol:water (50:50, v/v) before injection into the HPLC-fluorescence detection system [14] .

Quality Control: Each analytical batch of 20 samples included one reagent blank, one spiked blank (at 2 and 10 μg kg⁻¹), and one certified reference material (FAPAS proficiency test material T04214). Recovery rates for the spiked samples ranged from 87.3% to 96.8% across all aflatoxin variants (B1, B2, G1, G2). The limit of detection (LOD) was 0.12 μg kg⁻¹ and the limit of quantification (LOQ) was 0.38 μg kg⁻¹ for AFB1. Intra-day and inter-day coefficients of variation were 4.7% and 7.2%, respectively, both within acceptable ranges for mycotoxin analysis [14,15] .

Material and Methods

Material:

The research was conducted at Kyoto University's Graduate School of Agriculture and in collaboration with agricultural cooperatives in Chiba and Ibaraki prefectures from June to December 2023. Institutional approval was granted under Kyoto University's research ethics framework (KU-AGR/2023-027, approved 22 May 2023). Ninety smallholder groundnut storage facilities were surveyed, representing three drying methods (sun drying, forced-air drying, and mechanical rotary drying) and three storage types (jute bags, polypropylene bags, and hermetic PICS bags). Three kernel samples of 500 g each were collected per facility at 0, 60, and 120 days postharvest [9,12] .

Methods

Kernel moisture was measured with a digital grain moisture meter (Kett PM-450, accuracy ±0.2%). Temperature and relative humidity inside storage containers were logged continuously using iButton sensors (DS1923). Insect damage was scored on a 0-4 scale based on the percentage of visibly bored kernels. Aspergillus colony counts were determined by dilution plating on Aspergillus flavus and parasiticus agar (AFPA). AFB1 was quantified by HPLC with post-column derivatization and fluorescence detection (λex 365 nm, λem 440 nm) on an Agilent 1260 Infinity II system [13,14] . Multiple regression analysis identified the relative contribution of each handling variable to AFB1 variance. Data were analyzed using JMP Pro 17 at p<0.05 [16] . 1). Both drying method (p<0.001) and storage type (p<0.001) had significant main effects, with a significant interaction (p = 0.014). Hermetic PICS bags reduced AFB1 by 58.4-70.4% compared to jute bags within the same drying category. Drying adequacy explained the largest share of AFB1 variance (28.4%), followed by storage ventilation (23.7%) and harvest timing (17.8%). All six variables were statistically significant predictors, and the full model explained 87.1% of total AFB1 variance (adjusted R² = 0.871, p<0.001) (Table 2).

Results

Discussion:

The exponential relationship between moisture content and AFB1 (R² = 0.871) confirms the well-established threshold effect reported globally: below 8-9% moisture, Aspergillus growth is effectively halted, but even a 2-3 percentage point increase above this threshold permits rapid colonization and toxin production [7,8] . The 1.73 vs. 14.87 μg kg⁻¹ contrast between mechanically dried and inadequately sundried lots demonstrates that the drying stage alone can determine whether a batch meets or fails regulatory limits [17] .

Hermetic PICS bags reduced AFB1 by 58-70% compared to jute bags, consistent with results from West African groundnut trials reporting 50-75% reductions [18] . The mechanism involves oxygen depletion within sealed containers, which suppresses both fungal metabolism and insect survival. The 91.2% compliance rate below 4 μg kg⁻¹ achieved by the combined intervention package (mechanical drying + hermetic bags + timely harvest) represents a practical target for smallholder extension programs. And while mechanical dryers involve upfront costs, cooperative ownership models could make them accessible at the village level [19] . The relatively low contribution of soil contact (7.3%) suggests that this factor, though often emphasized in extension messages, matters less than drying speed and storage airtightness in this temperate context [21] .

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