Diversity in Secondary Metabolites Including Mycotoxins from Strains of <i>Aspergillus </i>Section <i>Nigri </i>Isolated from Raw Cashew Nuts from Benin, West Africa

<p>In a previous study, raw cashew kernels were assayed for the fungal contamination focusin on strains belonging to the genus Aspergillus and on aflatoxins producers. These sample showed high contamination with Aspergillus section Nigri species and absence o aflatoxins. To investigate the diversity of secondary metabolites, including mycotoxins, th species of A. section Nigri may produce and thus threaten to contaminate the raw cashe kernels, 150 strains were isolated from cashew samples and assayed for their productio of secondary metabolites using liquid chromatography high resolution mass spectrometr (LC-HRMS). Seven species of black Aspergilli were isolated based on morphological an chemical identification: A.Tubingensis (44%), A. niger (32%), A. brasiliensis (10%), A. carbonariu (8.7%), A. luchuensis (2.7%), A. aculeatus (2%) and A. aculeatinus (0.7%). Fro these, 45 metabolites and their isomers were identified. Aurasperone and pyranonigrin A produced by all species excluding A. aculeatus and A. aculeatinus, were most prevalen and were encountered in 146 (97.3%) and 145 (95.7%) isolates, respectively. Three mycotoxin groups were detected: fumonisins (B2 and B4) (2.7%) ochratoxin A (13.3%), an secalonic acids (2%), indicating that these mycotoxins could occur in raw cashew nuts Thirty strains of black Aspergilli were randomly sampled for verification of species identit based on sequences of β-Tubulin and calmodulin genes. Among them, 27 isolates wer positive to the primers used and 11 were identified as A. niger, 7 as A.Tubingensis, 6 as A carbonarius, 2 as A. luchuensis and 1 as A. welwitschiae confirming the species names a based on morphology and chemical features. These strains clustered in 5 clades in A. sectio Nigri. Chemical profile clustering also showed also 5 groups confirming the speciespecific metabolites production.</p

Raw cashew nut quality as function of contamination by mycotoxins and other secondary metabolites of Aspergillus spp. and farmer practices

Cashew nut is a cash crop in tropical regions of Africa including Benin, providing substantial revenues to small-scale farmers and contributing significantly to the national gross domestic product. In Beninese context, about 95% of raw cashew nuts are exported. Therefore, the production of raw cashew nuts of good quality is mandatory to enter the international market. Moreover, health issues related to the consumption of nuts contaminated with mycotoxins are of high concern for human being. The objective of this research was to investigate the occurrence of mycotoxins and other secondary metabolites of Aspergillus sp. on raw cashew nuts and to evaluate farmers’ practices that affect the quality of raw nuts.In surface disinfected samples 90.2% of raw kernels were contaminated by strains of A. section Nigri in the northern guinea (NG) zone compared to 87.2% in the southern guinea (SS) zone. The level of contamination of raw cashew kernels by strains of A. section Flavi was 6.7% in the NG zone whereas it was 4.6% in the SS zone. When non disinfected kernels were plated, A. section Nigri was predominant in both NG and SS zones, with percentages of 89.7% and 93.4%, respectively. Ten and 15.7% of raw cashew kernels were contaminated by strains of A. section Flavi in NG and SS zones, respectively. A. flavus, A. tamari, A. costaricaensis, A. minisclerotigenes and A. nomius were identified as strains in A. section Flavi. The average water content and the cashew nut count were respectively 8.6% and 172 nuts/kg in the NG zone and 8.7% and 174 nuts/kg in the SS zone.The occurrence of aflatoxins, analysed by ultra-high performance liquid chromatography tandem mass spectrometry, showed that none of the analysed samples was positive for natural occurrence of aflatoxins with a detection limit of 0.05 - 0.2 µg/kg. All the above results showed that, at present, cashew nuts from Benin were in the range of good quality for export.Seven species of black aspergilli were isolated based on morphological and chemical identification namely A. tubingensis (44% of total strains), A. niger (with a chemical profile similar to A. welwitschiae) (32%), A. brasiliensis (10%), A. carbonarius (8.7%), A. luchuensis (synonym to A. kawachi or A. acidus) (2.7%), A. aculeatus (2%) and A. aculeatinus (0.7%). Forty five different metabolites and their isomers were identified and classified in 2 groups. Group A of known mycotoxins: fumonisin B2, fumonisin B4, ochartoxin B, ochratoxin A and secalonic acid A, secalonic acid D and secalonic acid F. Group B included other secondary metabolites like nigragillin, pyranonigrin A, nigerazine A, nigerazine B, nigerapyrone E, tensyuic acid A, tensyuic acid F, pyranonigrin B, pyranonigrin C, pyranonigrin D, fonsecin, tensidol A, pyrophen, atromentin, tensyuic acid B, funalenone, rubrofusarin, orlandin, asperazine, tensyuic acid C, tensyuic acid D, nigerasperone A, tensidol B (pestalamide A), fonsecin B, malformin A2, tubingensin A, tubingensin B, malformin C, kotanin, nominine, antafumicin A, antafumicin B, aurasperone C, aurasperone F, aurasperone E, aurasperone B, flavasperone and nafuredin. DNA analysis resulted in a phylogenetic trees based on combined sequences data of β-tubulin and calmodulin of 27 strains of A. section Nigri that clustered in 5 clades. Also, the chemical profile clustering showed also 5 groups confirming the species specific metabolites production.Sorting and grading resulted in two grades. First (good) grade (Grade 1) consisted of nuts with less than 50% of their shell surface affected by fungi and/or insect injuries, whereas second (bad) grade (Grade 2) were nuts that had 50% or more of their shell surface covered by fungi and/or insect symptoms. Nut counts in Grade 1 were from 151 to 174 nuts/kg and from 142 to 182 nuts/kg in NG and SS, respectively. In Grade 2, nut counts were from 168 to 202 nuts/kg in NG and from 171 to 197 nuts/kg in SS. These results indicated that sorting and grading resulted in Grade 1 of better quality with high nut count, and low fungal contamination.Interviews revealed the dominance of men (95%) in cashew nut cultivation indicating that it as a gendered activity. Cashew nut farming is labour intensive with 55% of farmers in the range of 35 to 55 years old. Unfortunately, most cashew farmers were less educated with up to 76% not exceeding primary educational level, making the educational level of farmers to be the main constrain for the production of cashew nut of good quality. This resulted of 80% of cashew farmers to select their seedlings from uncertified sources and 59% to never ask or received advice from extension services about cashew farming practices. Cashew nut farmers reported that cashew revenue contributed significantly to the household income for 44% and 54% of farmers in the NG and the SS zone, respectively. Other constrains for good cashew nut production were lack of sorting, drying and farm management. The farmer’s education level, limited access to extension services, lack of sorting, drying and farm management are major parameters that affect the quality of raw cashew nuts. Improvement of these parameters can positively affect raw nut quality and therefore contribute to better revenue from raw cashew production.Together with good farming and post-harvest management practices, the implementation of specific extension service for cashew growers and an incentive for sorting and grading as low cost technology, will impact the quality parameters of raw cashew nuts intended for export from Benin

Development of a low-alcoholic fermented beverage employing cashew apple juice and non-conventional yeasts

Cashew apples are by‐products in the production of cashew nuts, which are mostly left to rot in the fields. Cashew apple juice (CAJ), a highly nutritious beverage, can be produced from them. It is rich in sugars and ascorbic acid, but its high polyphenol content makes it bitter and astringent, and therefore difficult to commercialize. The kingdom of fungi contains more than 2000 yeast species, of which only a few species have been studied in relation to their potential to produce aroma compounds. The aim of this research was to develop a new low‐alcoholic fermented beverage to valorize cashew apples. For this purpose, a screening was carried out employing non‐conventional yeast species and some species of the genus Saccharomyces for comparison, followed by a more detailed study with four selected strains cultured at different conditions. The production of volatile aroma compounds as a function of the presence of oxygen, temperature, and yeast species was investigated. The results showed that the more diverse aroma profiles appeared at 25 °C under anaerobic cultivation conditions, where Saccharomyces cerevisiae WUR 102 and Hanseniaspora guilliermondii CBS 2567 excelled in the synthesis of certain aroma compounds, such as β-phenylethanol and its acetate ester (rose aroma). Further studies are needed to test consumer acceptance of these new products.</p

Mycotoxins and other secondary metabolites of <i>Aspergillus</i> section <i>Nigri</i> isolated from cashew nuts.

<p>Mycotoxins and other secondary metabolites of <i>Aspergillus</i> section <i>Nigri</i> isolated from cashew nuts.</p

<i>Aspergillus</i> section <i>Nigri</i> strains isolated from cashew nuts characterized by sequence analysis.

<p><i>Aspergillus</i> section <i>Nigri</i> strains isolated from cashew nuts characterized by sequence analysis.</p

Base Peak Chromatogram (BPC) of <i>Aspergillus niger</i> extract.

<p>The Analysis was done by reversed phase ultra-high-performance liquid chromatography quadrupole time of flight mass spectrometry. Positive electrospray ionization (<i>ESI</i>⁺, <i>m/z</i> 100–1000). <i>A</i>. <i>niger</i> was cultured on yeast extract sucrose agar for 5 days in dark. The BPC showed the production of fumonisin B₂ and fumonisin B₄ and other secondary metabolites.</p

Mycotoxins and other secondary metabolites of <i>Aspergillus</i> section <i>Nigri</i> isolated from cashew nuts.

<p>Mycotoxins and other secondary metabolites of <i>Aspergillus</i> section <i>Nigri</i> isolated from cashew nuts.</p