MATERIAL AND METHODS:

1. Collection of samples:

The study regions were selected based on their importance as major peanut and peanut product producing areas, as well as for providing a contrasting environment for peanut cultivation, shelling, and processing. A total of (90) stakeholders were sampled, including farms, shelling stations, and processing factories.

Thirty soil samples, thirty raw peanut kernels, and thirty shelled peanuts were collected from farms and shelling stations along the Atlantic coast. Additionally, thirty peanut-based products were collected from processing factories across various Moroccan cities according to ¹². Table 1 provides a detailed breakdown of the samples collected from different regions in Morocco.

(30) peanut fields were selected for soil sampling, and the top 5 cm of the surface was collected from each field at 10-meter intervals as described by ^13,14. Raw peanut kernel samples were taken from the same farms during harvesting, taking peanuts from bottom, middle and top levels. Similarly, (30) raw peanut samples were obtained from sacks at shelling stations. Peanut-based product samples were collected from processing factories, with each sample weighing 500g. All samples were preserved at ambient conditions and analyzed within a 24-hour period following collection.

2. Isolation and morphological characterization of fungi:

Growth media (PDA, AFPA, CZ) were prepared according to the manufacturer’s instructions and were supplemented with 50mg/L of chloramphenicol to inhibit bacterial growth. The media were autoclaved, cooled, poured into Petri plates and stored at 4°C until inoculation.

For soil samples, 1 g of soil was mixed in 3 mL of distilled water and plated onto PDA plates as described by ¹⁵. For raw and shelled peanuts, seeds were directly plated onto PDA. For peanut-based products, the suspension-dilution technique was used to isolate fungi on agar medium. For all previous samples, three replicates were performed and Petri dishes were incubated at 28°C for 10 days.

Any visible Aspergillus section Flavi-like mycelial growth characterized by greenish-yellowish coloration was considered as the initial isolation criterion. The suspected Aspergillus section Flavi colonies were examined under electron microscope. Then, the representative isolates of Aspergillus section Flavi were purified through repeated sub-culturing on PDA multiple times. The isolates were identified by macro-morphological features as described by¹⁶ and ¹⁷.

3. Identification of mycotoxins production ability of isolates:

3.1 Detection of AFB1 production by Ultraviolet (UV) light test:

Isolates were cultivated on coconut cream agar (CCA) plates supplemented with chloramphenicol, as described by ¹⁸. Approximately 200 ml of commercial coconut milk was diluted in 1000 ml of water, homogenized, and added to 2% agar. The mixture was then autoclaved at 121 °C for 15 min. The plates were inoculated with spore suspensions from 7-day-old cultures, incubated at 25°C for 14 days and observed under UV light (365 nm) for fluorescence, which indicates aflatoxin production.

3.2 Detection of CPA production by TLC:

To analyze the isolates for (CPA), the cultures were grown in Erlenmeyer flasks for 10 days at 25-28°C. The cultures were extracted with chloroform, dried over sodium sulfate, and analyzed by thin-layer chromatography (TLC) using a mobile phase of toluene–ethyl acetate–formic acid (5:4:1, vol/vol/vol). This method is based on the procedure described by ¹⁹.

3.3 Quantitation of CPA and AFB1 by HPLC:

To quantify the levels of AFB1 and CPA, the chloroformic extracts of the culture broths were analyzed using a high-performance liquid chromatography (HPLC) system equipped with a fluorescence detector. The method involved injecting 50 µL of each extract into a C18 column with a particle size of 5 µm and a length of 25 cm. The column was eluted at a flow rate of 1 mL/min²⁰.For CPA quantification, the mobile phase was methanol/water (70:30) containing 300 mg ZnSO4·7H2O, as described by ²¹. For AFB1 quantification, the mobile phase was water/ acetonitrile/ methanol (60:25:15, v/v/v) as described by ²². The concentrations of CPA and AFB1 were quantified by measuring the peak absorbance at 284 nm and 365 nm, respectively. The concentrations were determined by comparing the sample peak areas to those of pure CPA and AFB1 standards.

4. Genetic identification of highly toxigenic isolate:

Genetic confirmation and aflatoxigenic production ability were determined through several steps. The internal transcribed spacer (ITS), partial beta-tubulin, and calmodulin genes were targeted for genetic confirmation, following the methodology described by^23–25 with slight modifications. DNA was extracted and its purity was measured using a spectrophotometer. Specific universal primers were used for amplification. The PCR product was purified after verifying its quality using gel electrophoresis. The sequencing reaction was performed using a commercial sequencing kit, and the purification was done according to the manufacturer's instructions. Sequencing was performed using a Sanger sequencer. The assembled results were compared to the NCBI nucleotide library collection. The toxigenic potential of the isolates was assessed using a multiplex PCR assay targeting several aflatoxin biosynthesis genes following the method described by ²⁶ with some modifications. This approach involved two separate multiplex PCR reactions. The first set targeted the omtA, glca, and pksA genes, while the second set targeted the aflR, ver‐1, and nor‐1 genes. The PCR cycling conditions were optimized to include an initial denaturation step, followed by multiple cycles of denaturation, annealing, and extension, and a final extension step.

5. Determination of levels of AFB1 in samples:

Contamination of collected samples was evaluated. Raw and shelled peanuts were finely ground before use, while soil and peanut-based products were directly utilized. 25 grams of each sample were dissolved in 125 milliliters of 80% methanol and volumes of 50µl were injected into an HPLC system equipped with a fluorescence detector using the same protocol described in section (3.3Quantitation of CPA and AFB1 by HPLC). Kruskal-Wallis statistical analysis and pairwise comparisons tests at 5 % significance level were used to compare the medians of mycotoxins production in each group of samples. P < 0.05 was considered significant. The results were analyzed by Minitab version 18.

RESULTS AND DISCUSSIONS:

1. Isolates Identification:

The color of the colonies on PDA medium was used for first identification of the isolates. Table 2 shows fungal Contamination in peanut products; the fungi isolated with their overall contamination percentage are as follow: Aspergillus section flavi (96.7%), Aspergillus section nigri (69.2%), Penicillium spp (48.3%), Rhizopus (45%), Fusarium sp. ( 43.3%), Alternaria sp. ( 13.3%). Results revealed a clear dominance of Aspergillus section flavi species. The Aspergillus section Flavi isolates exhibited colonies with a greenish-yellow hue.

The conidia produced were smooth to slightly rough in texture and had a globose shape with thin walls. The predominant morphotype was biseriate. The identity of the isolates as A.flavus was confirmed by the distinctive orange coloration observed on the underside of the colonies on AFPA medium. This coloration is a result of a chemical reaction involving ferric citrate aspergellic acid produced by the fungus ²⁷.

The micro and macro morphology of A. flavus colonies on PDA is shown in Figure 1 a-b-c-d-e. Initially, the mycelial growth appeared white. After 3 days of incubation, the colony developed yellow-green conidia with rough texture. The colonies typically had a flat border and a raised center, surrounded by a white circle. The reverse side of the colonies appeared pale in color. At the microscopic level, the conidiophores are relatively long, colorless, more or less rough and bearing vesicles which are generally globose. Small columnar heads are possible, especially in aerial mycelium. The, the phialides are uni or bi-seriate, the conidia are globose, more or less ornamented.

a b

c d

Figure 1: Morphological characteristics of A.flavus colonies, showing the yellow-green conidia with the white circle (a), the reverse side of the colony (b), the rough texture of the colony (c), the initial white color of the colony (d) and the microscopic observation showing the long colorless conidiophores bearing the vesicles and the globose conidia (e).

1.2 Mycotoxins production and chemotypes identification

Table 3 presents the classification of A. flavus isolates from peanut samples according to their ability to produce AFB1and CPA. The results show that the majority of the isolates (92.9%, 145 out of 156) produced mycotoxins, with 7.1% (11 isolates) being non-toxigenic. The levels of CPA and AFB1 generated by the isolates ranged from 20.2-405.9 μg/g and 46.3-2175 μg/g, respectively.

The isolates were categorized into four groups based on their mycotoxin production profiles (AFB1 and CPA), following ¹⁰ with slight modifications. Table 4 shows the percentages of A. flavus chemotypes based on AFB1 and CPA production; Group I: 55.7% of the isolates produced both CPA and AFB1. Group II: 27.5% of the isolates produced AFB1 but not CPA. Group III: 9.6% of the isolates produced CPA but not AFB1. Group IV: 7% of the isolates were non-toxigenic, producing neither CPA nor AFB1.

A. flavus (RP-6) isolated from a raw peanut sample was found to be the most mycotoxigenic with AFB1 production of (2175 μg/ml) and CPA production of (405.9µg/ml), and was therefore selected as test fungus for genetic confirmation. The identity of the highly mycotoxigenic isolate was confirmed through genetic analysis. Sequencing of three genomic regions - the internal transcribed spacer (ITS), beta-tubulin, and calmodulin genes - was performed. Comparison of the obtained sequences to public databases using sequence alignment tools showed over 97% similarity to A. flavus species reported in the literature.

A reliable identification of A. flavus species was achieved through the polyphasic approach, integrating macro and micro morphological, chemical, and genetic characterization. This approach is significant because it highlights the co-occurrence of both AFB1 and CPA in some strains, which can increase their combined toxic effect in peanuts. Several studies have employed similar methods to identify the chemotype diversity and mycotoxin production abilities in Aspergillus section Flavi strains. These studies have similarly demonstrated the significant diversity in mycotoxin production profiles exhibited by A. flavus isolates across different agricultural commodities. ^28–31.

The presence of highly toxin-producing A. flavus strains is a significant concern in the peanut supply chain. These toxigenic fungal isolates can contaminate peanuts starting from the soil, and then persist through the various processing stages, ultimately leading to aflatoxin contamination in the final peanut-based products. This highlights the need for robust control measures to mitigate the risk of aflatoxin exposure from peanut commodities³².

Table 4 : A. flavus chemotypes according to their AFB1 and CPA production.

Chemotype	Number of strains	CPA	AFB1
I	87 (55.7%)	+	+
II	15 ( 9.6%)	+	-
III	43 ( 27.5%)	-	+
IV	11 (7%)	-	-

2. AFB1 levels in samples

Table 5 shows the Statistical analysis of AFB1 levels in peanuts samples. The "shelled peanuts" group has the highest mean rank (79.8), indicating that it tends to have the largest contamination values. The "peanut based products" group has the lowest mean rank (46.7), indicating that it tends to have the smallest contamination values. The "raw peanuts" and "soil" groups have intermediate mean ranks (59.1 and 56.4 respectively). P-value is less than 0.05, the null hypothesis of equality of medians is rejected. This means that there is at least one significant difference between the medians of the 4 groups. Pairwise comparisons show that the "peanut based products" and "shelled peanuts" groups as well as the "raw peanuts" and "shelled peanuts" groups have significant median differences. The other pairs of groups do not differ significantly.

The notably higher contamination values of “shelled peanuts” compared to the other groups are consistent with the results reported by²² which demonstrated that the upstream stages of the peanut manufacturing chain have the greatest impact on aflatoxin levels. In particular, the shelling stage appears to be a critical point, which could be explained by the traditional methods used by many shelling stations. These traditional practices often involve soaking the peanut pods in water without proper subsequent drying, which can contribute to increased aflatoxin contamination.

The mean ranks also indicate that certain sample groups surpassed the maximum permissible limits for AFB1 contamination as per the Moroccan regulations for peanuts and peanut-based products. These findings are consistent with the results reported in the studies by ^33–35, which also found elevated levels of aflatoxin contamination exceeding the regulatory limits.

The results underscore the need for more quality control measures and improvements in peanut processing practices, especially during the shelling stage, to effectively mitigate aflatoxin contamination and ensure the safety of peanut-based products for consumers.

Table 5: Statistical analysis of AFB1 levels in peanuts samples

Group	N	Median	Mean Rank	Z-value
Peanut based products	30	4.08	46.7	-2.51
Raw peanuts	30	4.05	59.1	-0.25
Shelled peanuts	30	50.00	79.8	3.51
Soil	30	2.05	56.4	-0.74
Overall	120	-	60.5	-

(a): descriptive statistics

Method	Df	H-value	P-value
Unadjusted for tied ranks	3	14.43	0.002
Adjusted for tied ranks	3	14.67	0.002

(b): Kruskal-Wallis test; Null Hypothesis (H₀): All medians are equal. Alternative Hypothesis (H₁): At least one median is different

Compared Groups	Difference in Mean Ranks	Z-value	Adjusted P-value
Peanut based products vs. Raw peanuts	-12.4	-2.51	0.791
Peanut based products vs. Shelled peanuts	-33.1	-3.51	0.002
Peanut based products vs. Soil	-9.7	-2.51	0.273
Raw peanuts vs. Shelled peanuts	20.7	2.81	0.015
Raw peanuts vs. Soil	2.7	-0.25	1.000
Shelled peanuts vs. Soil	-23.4	-0.74	1.000

(c): Pairwise Comparisons.

CONCLUSION:

This in-depth study of A. flavus strains present in the peanut production chain has provided valuable insights into the risks associated with aflatoxin contamination. A particularly concerning finding is the co-occurrence of AFB1 and CPA in certain identified strains, which can potentially amplify their adverse health effects on consumers. These discoveries highlight the urgent need for effective monitoring and control measures to prevent peanut contamination, starting from the field and throughout the production chain, given the high levels of contamination observed. Building on the information gained from this study, it will be possible to guide mycotoxin risk management strategies and ensure the safety of peanuts for human consumption.

CONFLICT OF INTEREST:

The authors do not have any conflict of interest.

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Received on 10.07.2024 Revised on 04.11.2024

Accepted on 07.01.2025 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):751-756.

DOI: 10.52711/0974-360X.2025.00111

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Fungi flora	Samples				Overall contamination percentage
Fungi flora	Soil	Raw peanuts	Shelled peanuts	Peanut based products	Overall contamination percentage
Aspergillus section Flavi	29/30	29/30	30/30	28/30	96.7%
Aspergillus section Nigri	20/30	23/30	25/30	15/30	69.2%
Penicillium sp.	1/30	23/30	25/30	9/30	48.3%
Alternaria sp.	1/30	6/30	9/30	0/30	13.3%
Fusarium sp.	19/30	18/30	15/30	0/30	43.3%
Rhizopus sp.	1/30	25/30	28/30	0/30	45%

CPA Production (μg/g)	0	Inf a 100	100-400	Sup a 400	Minimum value	Maximum value
Number of isolates	54	90	09	3	20.2	405.9
AFB1 Production (μg/g)	0	Inf a 100	100-1000	Sup a 1000	Minimum value	Maximum value
Number of isolates	26	119	7	4	46.3	2175

Samples	Sampling zone
Samples	Kenitra (W)*	Larache (N)*	Tangier (N)*	Agadir (S)*	Oujda (E)*
Soil	15	15	0	0	0
Raw peanuts	15	15	0	0	0
Shelled peanuts	15	15	0	0	0
Peanut based products	5	3	3	16	3