Occurrence of mycotoxins and microbial communities in artisanal infant flours marketed in Côte d'Ivoire.

The aim of this study was to determine the microbial diversity and mycotoxin profile of artisanal infant flours commonly vended in public healthcare centres and retail markets in Côte d'Ivoire. Thus, maize, millet, sorghum, soya and multigrain (mix of different cereals) flour samples collected from different localities were first, analysed for nutritional composition, then for microbial communities using high-throughput sequencing and for mycotoxins through UHPLC-MS/MS method. Firmicutes was the most abundant bacterial phylum and the dominant genera were Weissella, Staphylococcus, Pediococcus. Potential pathogenic genera such as Bacillus, Enterobacter, Acinetobacter and Burkholderia were also found. The fungal community was composed of two dominant phyla (Ascomycota and Basidiomycota) and 31 genera with > 0.1% relative abundance. In samples from public healthcare centres, Candida, Hyphopichia, Trichosporon, and Cyberlindnera were the most dominant genera according to the flour type while in samples from retail markets, they were Cyberlindnera, Clavispora, Nakaseomyces, Aureobasidium and Candida. Possible toxigenic genera Fusarium and Aspergillus were also detected. Aflatoxin B1 (AFB1), Ochractoxin (OTA), Fumonisin B1 (FB1) and B2 (FB2) were the mycotoxins found in the analysed flours. AFB1 was detected in 100% of maize (range 1.2-120.5 µg/kg; mean: 44.2 µg/kg) and 50-83.3% of millet flours (range 0.2-31.5 µg/kg; mean: 31.5 µg/kg). Its level in all maize and rice flour samples exceeded EU standard (0.1 µg/kg). For OTA and fumonisins, millet and maize flours showed the highest levels of sample exceeding the EU standard. Thus, artisanal infant flours marketed in Côte d'Ivoire, mainly maize and rice flours, although containing potentially beneficial bacteria, represent potential health risks for children.

Phenotypic characterization of indigenous Saccharomyces cerevisiae strains associated with sorghum beer and palm wines.

In order to phenotypically characterized Saccharomyces cerevisiae strains isolated from sorghum beer and palm wines for a possible selection of a starter culture, 30 strains were tested for killer activity, temperature resistance, ethanol tolerance, carbohydrate fermentation, enzyme profile and sorghum wort fermentation. Of the tested strains, three showed a killer profile, while four showed a neutral profile and 23 were found to be sensitive to K2 toxin. Temperatures of 40 °C and 44 °C allowed to distinguish strains into four thermal groups with only three strains may grow at 44 °C. Almost tested strains were tolerant to 5% ethanol with viability rates up to 73%. But at 10% and 15% ethanol, respectively 18 and 7 strains were tolerant. Carbohydrate fermentation revealed 13 fermentation profiles, including one typical and 12 atypical profiles. The typical profile strains (16.13% of the strains) fermented glucose, galactose, fructose, sucrose, maltose, trehalose and raffinose. Most of the strains secreted lipases (mainly esterase and esterase-lipase), proteases (mainly valine and cysteine arylamidase, chrymotrypsin) and phosphatases (mainly acid phosphatase and naphthol phosphohydrolase). On contrary, only five strains isolated from sorghum beer exhibited glucosidase activity, mainly α-glucosidase. The analyse of fermented sorghum wort revealed that fermentative performance is strain dependent. Furthermore, the Hierarchical Cluster Analysis showed that the strains were separated in three distinct clusters with the strains from sorghum beer clustered separately.

Genetic diversity and population structure of Saccharomyces cerevisiae strains isolated from traditional alcoholic beverages of Côte d'Ivoire.

In order to assess the genetic diversity and population structure of indigenous S. cerevisiae from Côte d'Ivoire, a total of 170 strains were isolated from four traditional alcoholic beverages through nine regions. Microsatellite analysis performed at 12 loci revealed that strains of palm oil and raffia wine were genetically related, unlike those of tchapalo and ron wine which formed two s from palm oil wine and raffia wine were clearly inbred. In comparison with the European, North American, Asian and others West African populations, Ivorian population was well defined, although most of these strains were admixed. Among these strains, only isolates from raffia wine appeared to have alleles in common to all populations.

Molecular identification of yeast, lactic and acetic acid bacteria species during spoilage of tchapalo, a traditional sorghum beer from Côte d'Ivoire.

Yeasts, lactic and acetic acid bacteria are responsible of microbial spoilage of alcoholic beverages. However species involved in deterioration of sorghum beer produced in Côte d'Ivoire has not been investigated. This study was carried out to identify species of yeast, LAB and AAB during spoilage of tchapalo in order to define the best strategy for beer preservative. Thus, a total of 210 yeasts, LAB and AAB were isolated from samples of tchapalo stored at ambient temperature and at 4 °C for 3 days. Based on PCR-RFLP of the ITS region and the sequencing of D1/D2 domain, yeast isolates were assigned to seven species (Saccharomyces cerevisiae, Candida tropicalis, Rhodotorula mucilaginosa, Trichosporon asahii, Kluyveromyces marxianus, Meyerozyma guilliermondii and Trichosporon coremiiforme). During the storage at ambient temperature and at 4 °C, S. cerevisiae was the predominant species (> 76%). Excepted R. mucilaginosa, occurrence of non-Saccharomyces species was sporadic. LAB species detected in fresh samples using molecular methods were Pediococcus acidilactici, Lactobacillus paracasei, Lb. curvatus, Lb. fermentum and Weisssella paramesenteroides. P. acidilactici was the dominant species (47.8%) followed by Lb. paracasei (17.5%). W. paramesenteroides and Lb. fermentum were not detected during the spoilage at ambient temperature while at 4 °C W. paramesenteroides and Lb. paracasei have not been detected. For AAB, the species found were Acetobacter pasteurianus sub paradoxus and Acetobacter cerevisiae. These species were common to all samples during spoilage and A. pasteurianus sub paradoxus was the most frequently detected.

Virulence factors and determination of antifungal susceptibilities of Candida species isolated from palm wine and sorghum beer.

Since it has been considered that Candida species in food or drinks may, following ingestion, enter the bloodstream and cause fungaemia, the presence of these yeast species in traditional alcoholic beverages may be of some clinical significance. Thus we attempted to assess virulence factors and antifungal susceptibility profile of Candida strains and other potential pathogenic yeasts isolated from palm wine and sorghum beer (tchapalo). Of the 23 yeast isolates from palm wine, phospholipase, esterase and haemolysin production was revealed amongst 69.6%, 65.2% and 100% isolates respectively with high activity belonging to Candida tropicalis strains. All the isolates were biofilm producers at variable degree but none showed proteinase activity. When the isolates were tested for their susceptibility to five antifungal agents, we found that ketoconazole (91.3%) followed by fluconazole (78.3%) and amphotericin B (73.9%) were the most potent agents. Of the 14 isolates from tchapalo, 57.1%, 87.5% and 57.1% exhibited phospholipase, haemolysin and esterase activity respectively. They did not also show proteinase activity while 87.5% produced biofilm. The majority of the isolates were susceptible to azoles (92.7%) and amphotericin B (85.3%) but they were 5-flucytosine resistant like palm wine strains.

Candida species in tchapalo and bangui, two traditional alcoholic beverages from Côte d'Ivoire.

The increase of infections due to non-Candida albicans species made it very necessary to conduct adequate characterization to be able to identify the species of Candida isolated from traditional fermented foods. In this study, based on their hue on Candida Chromogenic Agar medium, a total of 136 yeast strains were isolated from tchapalo and bangui. Molecular identification based on PCR-RFLP of internal transcribed spacers of rDNA (ITS) and sequencing of the ITS and the D1/D2 regions allowed us to assign these isolates to seven species: Candida tropicalis, Candida inconspicua, Candida rugosa, Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Trichosporon asahii. With the respect to each beverage, six species were found among with four species are regarded as opportunistic pathogens. From these, C. tropicalis, C. inconspicua and K. marxianus were the most commonly encountered. The enzyme activities of the potential pathogens assessed using API ZYM system showed that almost strains had esterase, esterase lipase, valine and cystine arylamidase, alpha chymotrypsin, alkaline phosphatase and naphthol phosphohydrolase activities. The activity of α-glucosidase was found only in C. tropicalis and C. inconspicua strains isolated from tchapalo while ß-glucosidase activity was found in all strains from tchapalo and only in C. inconspicua isolated from bangui.

Identification of yeasts isolated from raffia wine (Raphia hookeri) produced in Côte d'Ivoire and genotyping of Saccharomyces cerevisiae strains by PCR inter-delta.

Raffia wine is a traditional alcoholic beverage produced in several African countries where it plays a significant role in traditional customs and population diet. Alcoholic fermentation of this beverage is ensured by a complex natural yeast flora which plays a decisive role in the quality of the final product. This present study aims to evaluate the distribution and the diversity of the yeast strains isolated in raffia wine from four sampling areas (Abengourou, Alépé, Grand-Lahou and Adzopé) in Côte d'Ivoire. Based on the D1/D2 domain of the LSU rDNA sequence analysis, nine species belonging to six genera were distinguished. With a percentage of 69.5 % out of 171 yeast isolates, Saccharomyces cerevisiae was the predominant species in the raffia wine, followed by Kodamaea ohmeri (20.4 %). The other species isolated were Candida haemulonii (4.1 %), Candida phangngensis (1.8 %), Pichia kudriavzevii (1.2 %), Hanseniaspora jakobsenii (1.2 %), Candida silvae (0.6 %), Hanseniaspora guilliermondii (0.6 %) and Meyerozyma caribbica (0.6 %). The molecular characterization of S. cerevisiae isolates at the strain level using the PCR-interdelta method revealed the presence of 21 profiles (named I to XXI) within 115 isolates. Only four profiles (I, III, V and XI) were shared by the four areas under study. Phenotypic characterization of K. ohmeri strains showed two subgroups for sugar fermentation and no diversity for the nitrogen compound assimilations and the growth at different temperatures.

Production of freeze-dried yeast culture for the brewing of traditional sorghum beer, tchapalo.

Freeze-drying is a well-known dehydration method widely used to preserve microorganisms. In order to produce freeze-dried yeast starter culture for the brewing purpose of African sorghum beer, we tested protective agents (sucrose, glucose, glycerol) in combination with support materials (millet, maize, sorghum, and cassava flours) at 1:1 ratio (v/v). The yeast strains Saccharomyces cerevisiae F 12-7 and Candida tropicalis C 0-7 previously isolated from sorghum beer were used in a mixed culture at a ratio of 2:1 (C. tropicalis/S. cerevisiae). After the freeze-drying, the residual water contents were between 0.78 -2.27%, 0.55 -4.09%, and 0.40-2.61%, respectively, with sucrose, glucose and glycerol. The dried yeasts viabilities were between 4.0% and 10.6%. Among the protective agents used, sucrose was found to be the best protectant giving cell viabilities of 8.4-10.6%. Considering the support materials, millet flour was the best support after drying. When the freeze-dried yeast powders were stored at 4°C and room temperature (25-28°C) for up to 3 months, the survival rates were the highest with cassava flour as the support material.