Screening for oligosaccharides producing microrganisms isolated from Brazilian biomes

Author(s)

Saartje Hernalsteens

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia de Alimentos
• Defense date: 2006-12-12

Examining board members

Francisco Maugeri Filho; Maria Isabel Rodrigues; Gabriela Alves Macedo; Jonas Contiero; Telma Teixeira Franco; Eloizio Julio Ribeiro

• Advisor: Francisco Maugeri Filho

Abstract

In response to the increasing demand for healthier foods and as a result of the expanding applications of oligosaccharides in the cosmetic, agrochemical, pharmaceutical and food industries, the search for ¿new¿ enzymes concerning the oligosaccharides production, became necessary. The present study reports on the screening for high transfructosylating enzymes in yeasts strains isolated from fruits and flowers obtained from tropical Brazilian biomass. The efforts made to screen for high extra-cellular transfructosylating enzyme producing yeasts provided very promising results. Although the enzymes from the strains Candida sp. LEB-I3, Rhodotorula sp. LEB-U5 and Cryptococcus sp. LEB-V2 showed high hydrolytic activity, the production of fructooligosaccharides (FOS) by the Rhodotorula sp. LEB-V10 enzyme was successful, showing a continuous increase in FOS concentration up to the end of the synthesis reaction. The best operational conditions for these enzymes, considering the transfructosylating activities, were determined to be in the pH range from 4.0 to 5.0 and temperatures from 65 to 70°C. While the fructofuranosidase activities had shown the optimum activity on pH values from 3.0 to 4.0 and temperatures between 55 and 75°C. The enzymatic kinetic (fructosyl transferase activity) of the Candida sp. LEB-I3 showed a Michaellis-Mentem behavior, while the Rhodotorula sp. LEB-U5 and Cryptococcus sp. LEB-V2 showed a substrate inhibitory kinetic and the Rhodotorula sp. LEB-V10 showed a sigmoid shape, similar to that of allosteric enzymes. Considering that the Rhodotorula sp. LEB-V10 process was the only one that may be regarded as economically possible, the response surface methodology was employed to study the fermentation and the synthesis condition aiming the process optimization. On basis of the experimental results, the optimum conditions to obtain high fructosyl transferase activity were: 250 rpm, 30-35°C, 9% ± 1% (w/v) corn steep liquor and 7.5% ± 0.7% (w/v) of total reducing sugar from sugar cane molasses. The synthesis of FOS was also optimized (55 to 65% of yield), being the optimum conditions: 50% sucrose (P.A., commercial or from sugar cane molasses), 50°C (± 1°C), pH 5.0 (± 0.5) and 6.5 FTA.ml-1 (± 0.5). This data is very similar to those from the commercial process, and the use of commercial sucrose and sugar cane molasses led to a reduction on the production cost, consequently, further studies on the enzyme and fructooligosaccharides production conditions may show its potential for commercial application (AU)