Mapping the functional and strain diversity of the main microbiota involved in cocoa fermentation from Cote d'Ivoire.

The variable quality of cocoa produced by farmers is still a problem in the value chain, strongly depending on microbial activities. We analyzed the variability of cocoa microbiota from all twelve producing regions in Cote d'Ivoire, and described the geographical distribution of isolated microbiota, using a mapping. Microbial species were identified by ribosomal genes sequencing, strains were typed by RFLP and their techno-functional capacities were further investigated. Results showed a restricted diversity of lactic acid bacteria (LAB) and acetic acid bacteria (AAB) with respectively 10 and 5 strains. The dominant LAB and AAB strains, notably Lactobacillus plantarum 1 A, Acetobacter pasteurianus 1 A, Acetobacter okinawensis 2 A, and Acetobacter tropicalis 3 A, were found in all regions assuming that the acid microbiota was weakly variable. In contrast, the distribution of their functional performance such as acidification capability was variable, stronger in strains from Nawa and Haut-Sassandra regions and weaker in Indenie-Djuablin and San Pedro; this distribution seemed to be random. Moreover, the study also revealed a complex yeasts population showing a wide genetic diversity with 22 species and 45 strains indicating an intraspecific heterogeneity. Strains were generally different from a region to another and the resulting yeasts microbiota was globally variable in the regions. Likewise, the functional capacities such as pectinolytic was weak in P. kudriazevii strain 2 K from Gboklè and strong in P. kudriazevii strain 2 A from Loh-Djiboua. Additionally, the quality of fermented beans was also variable in the regions. The great variation of yeasts strains in the different regions may be the main microbial factors responsible for variation of the fermented cocoa quality observed.

Acetic acid bacteria (AAB) involved in cocoa fermentation from Ivory Coast: species diversity and performance in acetic acid production.

In this study, we investigated the diversity of AAB from fermenting cocoa and the production of acetic acid in response to various environmental conditions. Ribosomal 16S gene sequence analysis and PCR-RFLP showed a restricted microbiota mainly composed of Acetobacter pasteurianus, Acetobacter tropicalis and Acetobacter okinawensis sp., consistently found in all six regions studied. Meanwhile Acetobacter malorum, Acetobacter ghanensis and Gluconobacter oxydans were isolated as minor species in specific regions. The dominant species were mainly isolated in the first 72 h period of natural cocoa fermentation while the minor species were present toward the later stages. Acetobacter okinawensis, a newly isolated species, was able to yield an unusually high quantity, up to 62 g/L of acetic acid at 30 °C. However, a shift of temperature to 35 °C severely impaired acid production in most strains of this species. While acetic acid production increases for up to 6 days in Acetobacter okinawensis and Acetobacter pasteurianus, it decreases beyond 4 days in Acetobacter tropicalis strains. The production of acetic acid was strongly dependent on environmental conditions, with optimal production between pH 4 and 5, under ethanol concentration below 8% and temperatures above 35-40 °C, corresponding to conditions prevailing in the first half of fermentation process. Acetobacter tropicalis was more productive at higher ethanol concentration and Acetobacter okinawensis at low pH. Species diversity and different behavior of strains highlight the importance of valuable starter selection for well-controlled cocoa fermentation.

Antimicrobial resistance and virulence associated genes in Campylobacter jejuni isolated from chicken in Côte d'Ivoire.

INTRODUCTION: Campylobacter jejuni is one of the major causes of gastroenteritis worldwide of the last century. The aim of this study was to investigate the antibiotics profiles and the virulence gene in C. jejuni strains isolated from chicken in Côte d'Ivoire. METHODOLOGY: A total of 336 chicken ceaca samples recovered from market of two municipality of Abidjan were examined by conventional microbiological methods and molecular test using PCR. The antibiotic susceptibility tests of the isolates were determined by disk diffusion method. The presence of virulence genes was examined using simple PCR method. RESULTS: Among of 336 samples, 168 (50%) were positives for C. jejuni. Among the C. jejuni isolates, 159 strains (94.64%) were resistant to one or more antimicrobial agents. The highest percentage of antimicrobial resistance was found for Nalidixic acid (85.33%), Tétracyclin (71.76%) and Ciprofloxacin (55.65%). Moreover, MDR including 3, 4, 5 and 6 antibiotics families was detected in 16.66% of isolates. On the other hand, detection of virulence putative gene shows presence of cadF in 100% of tested strains. In addition, cdtA, cdtB and cdtC genes were detected in 100%; 89.51% and 90.32% respectively of C. jejuniisolates. CONCLUSION: Because of the key role of broiler chicken in human campylobacteriosis infection, it will important in first time to monitoring using of antibiotics in chicken farms and in second time to verify presence of campylobactériosis in country.

Lactic acid bacteria involved in cocoa beans fermentation from Ivory Coast: Species diversity and citrate lyase production.

Microbial fermentation is an indispensable process for high quality chocolate from cocoa bean raw material. lactic acid bacteria (LAB) are among the major microorganisms responsible for cocoa fermentation but their exact role remains to be elucidated. In this study, we analyzed the diversity of LAB in six cocoa producing regions of Ivory Coast. Ribosomal 16S gene sequence analysis showed that Lactobacillus plantarum and Leuconostoc mesenteroides are the dominant LAB species in these six regions. In addition, other species were identified as the minor microbial population, namely Lactobacillus curieae, Enterococcus faecium, Fructobacillus pseudoficulneus, Lactobacillus casei, Weissella paramesenteroides and Weissella cibaria. However, in each region, the LAB microbial population was composed of a restricted number of species (maximum 5 species), which varied between the different regions. LAB implication in the breakdown of citric acid was investigated as a fundamental property for a successful cocoa fermentation process. High citrate lyase producer strains were characterized by rapid citric acid consumption, as revealed by a 4-fold decrease in citric acid concentration in the growth medium within 12h, concomitant with an increase in acetic acid and lactic acid concentration. The production of citrate lyase was strongly dependent on environmental conditions, with optimum production at acidic pH (pH<5), and moderate temperature (30-40°C), which corresponds to conditions prevailing in the early stage of natural cocoa fermentation. This study reveals that one of the major roles of LAB in the cocoa fermentation process involves the breakdown of citric acid during the early stage of cocoa fermentation through the activity of citrate lyase.

Regulation of the synthesis of pulp degrading enzymes in Bacillus isolated from cocoa fermentation.

Pectin degrading enzymes are essential for quality of product from cocoa fermentation. Previously, we studied purified pectate lyases (Pel) produced by Bacillus strains from fermenting cocoa and characterized the cloned pel genes. This study aims to search for biological signals that modulates Pel production and regulators that influence pel gene expression. Strains were grown to the end of exponential phase in media containing various carbon sources. Pel enzymes production in Bacillus was unaffected by simple sugar content variation up to 2%. Additionally, it appeared that pel gene is not under the control of the most common carbon and pectin catabolism regulators ccpA and kdgR, which could explain the insensitivity of Pel production to carbon source variation. However, a 6-fold decrease in Pel production was observed when bacteria were grown in LB rich medium as opposed to basal mineral medium. Subsequently, bioinformatics analysis of cloned pel gene promoter region revealed the presence of DegU binding site. Furthermore, the deletion of degU gene dramatically reduces the pel gene expression, as revealed by real time quantitative PCR, showing an activation effect of DegU on Pel synthesis in Bacillus strains studied. We assumed that, during the latter stage of cocoa fermentation when simple sugars are depleted, production of Pel in Bacillus is stimulated by DegU to supply microbial cells with carbon source from polymeric pectic compounds.

Characterisation of a highly saturated Irvingia gabonensis seed kernel oil with unusual linolenic acid content.

The search for new sources of oil with improved characteristics has focused our attention on the characterisation of Irvingia gabonensis seed kernel oil. Physicochemical analysis have revealed the following assets: refractive index (1.42 ± 0.00), free fatty acids (2.3 ± 0.8%), peroxide value (3.33 ± 0.57 meq O(2)/kg), iodine value (32.43 ± 1.22 g I(2)/100 g), saponification value (233.75 ± 2.60 mg KOH/g), unsaponifiable matter (1.5 ± 0.02%), carotenoids (63 ± 0.01 mg ß-carotene/100 g) and phospholipids (2.1 ± 0.01%). Absorbance of this oil decreased abruptly in the range of UV-B and UV-A wavelengths. Gas chromatography analysis showed that the major fatty acids were saturated, being mainly composed of lauric (C12:0, 39.35 ± 0.01%) and myristic acids (C14:0, 20.54 ± 0.01%). Nevertheless, an unusually high amount (6.44 ± 0.02%) of linolenic acid was also noted. Mass spectrometer analysis of volatile compounds highlighted the presence of various aromatic and aliphatic organic compounds. I. gabonensis seed kernel oil also showed oxidative stability at 60 °C after 12 days of storage with maximum peroxide value of 34.66 meq O(2)/kg. In view of these interesting characteristics, I. gabonensis seed kernel could be used as an alternative source of oil for lipid industries.

Chemical composition and insecticidal activity of essential oils of two aromatic plants from Ivory Coast against Bemisia tabaci G. (Hemiptera: Aleyrodidae).

Essential oils of aromatic plants with insecticidal properties are nowadays considered as alternative insecticides to protect cultures from attack by insect pest. The aims of the present work were to evaluate the toxicity of the essential oils vapors of two aromatic plants (Lippia multiflora Mold. and Aframomum latifolium K. Schum) against Bemisia tabaci and to characterize their chemical composition. The highest fumigant toxicity against B. tabaci adults was observed with the L. multiflora oil: by exposure to 0.4 microL/L air, the lethal time inducing 90% mortality (LT90) was below 2 hours for this essential oil whereas it reached 15 h in the case of the A. latifolium oil. Both oils were analyzed by GC-FID and GC-MS on two capillary columns. The oil of L. multiflora contained a majority of oxygenated terpenoids mainly represented by the two acyclic components linalool (46.6%) and (E)-nerolidol (16.5%); the oil of A. latifolium was dominated by hydrocarbonated terpenoids among them beta-pinene (51.6%) and beta-caryophyllene (12.3%) were the two major components.

Molecular identification and pectate lyase production by Bacillus strains involved in cocoa fermentation.

We have previously reported the implication of Bacillus in the production of pectinolytic enzymes during cocoa fermentation. The objective of this work was to identify the Bacillus strains isolated from cocoa fermentation and study their ability to produce pectate lyase (PL) in various growth conditions. Ninety-eight strains were analyzed by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Four different banding patterns were obtained leading to the clustering of the bacterial isolates into 4 distinct ARDRA groups. A subset of representative isolates for each group was identified by 16S rRNA gene partial sequencing. Six species were identified: Bacillus subtilis, Bacillus pumilus, Bacillus sphaericus, Bacillus cereus, Bacillus thuringiensis, together with Bacillus fusiformis which was isolated for the first time from cocoa fermentation. The best PL producers, yielding at least 9 U/mg of bacterial dry weight, belonged to B. fusiformis, B. subtilis, and B. pumilus species while those belonging to B. sphaericus, B. cereus and B. thuringiensis generally showed a low level of activity. Two kinds of PL were produced, as revealed by isoelectrofocusing: one with a pI of 9.8 produced by B. subtilis and B. fusiformis, the other with a pI of 10.5 was produced by B. pumilus. Strains yielded about 2 fold more PL in a pectic compound medium than in glucose medium and maximum enzyme production occurred in the late stationary bacterial growth phase. Together all these results indicate that PL production in the bacilli studied is modulated by the growth phase and by the carbon source present in the medium.

Biochemical properties of pectate lyases produced by three different Bacillus strains isolated from fermenting cocoa beans and characterization of their cloned genes.

Pectinolytic enzymes play an important role in cocoa fermentation. In this study, we characterized three extracellular pectate lyases (Pels) produced by bacilli isolated from fermenting cocoa beans. These enzymes, named Pel-22, Pel-66, and Pel-90, were synthesized by Bacillus pumilus BS22, Bacillus subtilis BS66, and Bacillus fusiformis BS90, respectively. The three Pels were produced under their natural conditions and purified from the supernatants using a one-step chromatography method. The purified enzymes exhibited optimum activity at 60 degrees C, and the half-time of thermoinactivation at this temperature was approximately 30 min. Pel-22 had a low specific activity compared with the other two enzymes. However, it displayed high affinity for the substrate, about 2.5-fold higher than those of Pel-66 and Pel-90. The optimum pHs were 7.5 for Pel-22 and 8.0 for Pel-66 and Pel-90. The three enzymes trans-eliminated polygalacturonate in a random manner to generate two long oligogalacturonides, as well as trimers and dimers. A synergistic effect was observed between Pel-22 and Pel-66 and between Pel-22 and Pel-90, but not between Pel-90 and Pel-66. The Pels were also strongly active on highly methylated pectins (up to 60% for Pel-66 and Pel-90 and up to 75% for Pel-22). Fe(2+) was found to be a better cofactor than Ca(2+) for Pel-22 activity, while Ca(2+) was the best cofactor for Pel-66 and Pel-90. The amino acid sequences deduced from the cloned genes showed the characteristics of Pels belonging to Family 1. The pel-66 and pel-90 genes appear to be very similar, but they are different from the pel-22 gene. The characterized enzymes form two groups, Pel-66/Pel-90 and Pel-22; members of the different groups might cooperate to depolymerize pectin during the fermentation of cocoa beans.

Alpha-mannosidases from the digestive fluid of Rhynchophorus Palmarum larvae as novel biocatalysts for transmannosylation reactions.

Two biological fluids, namely hemolymph and digestive fluid from the larval stage of Rhynchophorus palmarum Linneaus, a serious pest in agroecosystem exploiting oil palm, were screened for hydrolytic activities, by the use of synthetic and natural glycoside substrates. Several exo and endoglycosidase activities were observed but, the interesting alpha-mannosidase activity (0.41 +/- 0.04 UI) had attracted our attention. So, we have previously demonstrated that this activity harbours four distinctive alpha-mannosidase isoforms named RpltM, RplM1, RplM2 and RplM3. We have extended this work to determine the ability of these enzymes to catalyze synthesis reactions. Finally, we have revealed that, alpha-mannosidases from the digestive fluid of R. palmarum larvae catalyze transmannosylation reactions. The stability of the enzymes and the optimization of the transfer product yield were studied as functions of pH, enzyme unit, starting concentration of donor or acceptor and time. It was shown that, in experimental optimum conditions, average yields of 12.34 +/- 0.75, 12.15 +/- 0.79, 5.59 +/- 0.35 and 8.43 +/- 0.50% were obtained for the alpha-mannosidases RpltM, RplM1, RplM2 and RplM3, respectively. On the basis of this work, alpha-mannosidases from the digestive fluid of Rhynchophorus palmarum larvae appear to be a valuable tool for the preparation of neoglycoconjugates.