Fructans production by cell-free sistems from Gluconacetobacter diazotrophicus culture supernatants

MARÍA LAURA MOLINARI; JOSÉ LUIS BOIARDI

San Miguel de Tucumán, Tucumán, Argentina

Congreso; 7mo Congreso Argentino de Microbiología General. SAMIGE del Bicentenario; 2011

Sociedad Argentina de Microbiología General (SAMIGE)

The main commercial production of fructans (levans and fructooligosaccharides (FOS)) comes from enzymatic transformation of sucrose by bacterial or fungal enzymes. The development of more efficient enzymes, with high activity and stability, is required and this has attracted the interest of biotechnologists and microbiologists. Frucatans have received particular interest because of their excellent biological and functional properties for use as prebiotic compounds. They are used as components of functional foods and generally recognized as safe by the FDA (Food and Drug Administration – U.S). They are calories free and non cariogenic sweeteners, stimulate the growth of bifidobacteria, and have been claimed to contribute towards the prevention of colon cancer and to reduce cholesterol, phospholipid and triglyceride levels in serum. Gluconacetobacter diazotrophicussecrets a constitutively levnasucrase (LsdA). This extracellular LsdA hidrolyzes sucrose to produce free glucose and fructans (which conteins β(2&→;6) linked fructosyl units) of low (FOS) and high (levans) molecular weight. In this work we approach and discuss the ability of G. diazotrophicusLsdA secreted to culture supernatants to produce fructans. G. diazotrophicus PAL5 was grown at 30 &º; C in LGI medium supplemented with 1,5 g/l yeast extract and 1,5 g/l triptone. 10 days-old cultures were centrifuged at 16000g 30 minutes. Supernatants were added to solutions of sucrose dissolved in a buffer sodium acetate 0,1 M, pH 5,2 and sterilized by filtration. These mixtures were incubated in order to follow fructans production. Different conditions have been tested: 1- enzyme-substrate ratios; 2- incubation temperature (30 &º; C and 40 &º; C); 3- sucrose concentration in the incubation mixture (100, 300 and 700 g/l). The fructan production was followed sampling the mixtures during 7 days. Levan production was assayed precipitating the polysaccharides by addition of 2 ethanol volumes to the mixture, recovering precipitates by centrifugation, drying and weighting. FOS production was tested by Thin Layer Chromatography (TLC). Glucose remanent in the mixture was assayed using an ezimatic kit test. Culture supernatants from sucrose containing media showed, although expressing a lower LsdA activity than supernatans from cultures performed with glycerol, gave a higher fructan production. Levan production was observed in all the conditions tested with concentrations increasing with time until about 96 hs of incubation. The highest levan production was obtained incubating at 40 &º; C in relation to mixtures incubated at 30 &º; C. FOS could be detected after 12 hs of incubation (mainly 1-kestose) in all the mixtures assayed. Higher concentrations of sucrose seems to favour production of low molecular weight fructans. Low sucrose concentration in the mixture showed a high rate of sucrose hydrolysis but most of it remained free in the mixture.