Molecular Identification of Pectinolytic Bacteria Isolated from Rotten Fruit and Optimization of Pectinase Production Using Response Surface Methodology

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Résumé

Pectinases are enzymes of major industrial interest. This study aimed to isolate and characterize pectinolytic bacteria obtained from decomposing fruit and optimize the composition of the culture medium. Bacteria were isolated on a reconstituted, selective pectin-enriched medium. Active colonies were characterized by morphological observations, biochemical tests, and molecular identification of 16S rDNA. Of the 55 isolates selected, 12 (21.81%) were pectinolytic. All isolates were Gram-positive, predominantly bacillary, and positive for the citrate test. Five strains exhibited γ-haemolysis, indicating that they were non-haemolytic. Based on 16S rRNA gene sequencing, four of these bacteria that expressed high pectin degradation indices were related to Lactiplantibacillus plantarum, Acetobacter tropicalis, Bacillus amyloliquefaciens, and Bacillus subtilis. The B. amyloliquefaciens strain isolated from the decomposing cashew apple, which expressed the highest pectinolytic index (3.659) and had a non-haemolytic profile, was selected for optimizing its pectinase production. Unifactorial optimization of pectinase production by this strain showed that pectin was the most favourable carbon source, (NH4)2HPO4 was the best nitrogen source, and the optimum pH for activity was 7. Multifactorial optimization using four independent factors showed that the quadratic model optimized the yield by 160-fold, with a maximum activity of 19.3953 IU/ml at 32.50 °C, and pH of 7 in a buffered medium. Among the variables studied, pectin concentration appeared to be one of the most influential factors.

Mots-clés

Bacillus amyloliquefaciens, Cashew apple, Molecular identification, Optimization, Pectinolytic bacteria, Rotten fruit, Response surface methodology