ISOLATION AND CHARACTERIZATION OF BACTERIAL STRAIN CAPABLE OF LIMONENE BIOTRANSFORMATION.

PILATTI, L*, TAKITA, M. A.2

1*

Centro de Ciências Agrárias, Universidade Federal de São Carlos, Rodovia Anhanguera, Km 174, Araras– SP. *lipilatti@gmail.com

2* _{Centro APTA de Citros Sylvio Moreira/IAC, Cordeirópolis – SP, Rodovia Anhanguera, Km 158,} Cordeirópolis – SP.

Keywords: bacteria, biotransformation, characterization, isolation, limonene.

The citrus industry has a key role in the Brazilian agribusiness, with the production of frozen concentrated orange juice as the main activity and having the State of Sao Paulo as the main producer, processor, and exporter. The orange essential oil is a byproduct of the juice production, which makes Brazil the world’s largest producer of this kind of essential oil. Limonene is the main component of citrus essential oil, accounting for nearly 95% of its composition. Because of the high production of essential oil from sweet orange in Brazil, limonene has a very low commercial value. One way of obtaining other products from limonene with higher commercial value is through the use of microorganisms capable of biotransforming this terpene. Thus this work aimed to isolate and identify bacterial strains from sweet orange fruits that can grow in presence of limonene and therefore metabolize it. Using a fruit at the beginning of decomposition stage we isolated some bacteria from the peel, the main source for essential oil in citrus. This was done by adding the extract to a medium containing (NH4)2SO4 37.83mM; (NH4)2HPO4 12.34mM; NaCl

8.55mM; MgSO4.7H2O 1.62mM; CaCl2.2H2O 4.08mM; KCl 28.83mM; FeSO4.7H2O

0.03mM; ZnSO4.7H20 0.03mM; and CuSO4.5H2O 0.04mM. The suspension was incubated

for one day at 37ºC with rotation at 250rpm and from that we isolated bacteria on plates. The isolated bacteria were grown in LB medium and had their 16S-23S intergenic region amplified for identification using universal primers. The amplicons were purified using the Geneclean kit (BIO 101) and cloned into pJet1.2 (Fermentas). The plasmidial DNA was extracted through alkaline mini preparations using 100µL of Solution I (glucose 50mM, Tris-HCl pH 8.0 25mM, EDTA pH 8.0 10mM), 150µL of Solution II (NaOH 0.2 N, SDS 1%), and 200µL of Solution III (60mL of potassium acetate 5M, 11.5mL of glacial acetic acid, in a total of 100mL). The cell debris was removed by centrifugation, and the DNA was purified with Geneclean kit (BIO 101). The sequencing was done with BigDye 3.1 (Applied Biosystems) using primers specific for pJET1.2. The sequences were obtained in an ABI 3730 automatic sequencer (Applied Biosystems) and processed in the LASERGENE 99 (DNASTAR), resulting in contigs that were then used for comparison with sequences from the public database Genbank, using the blastn tool. We observed similarity with sequences from citrus chloroplast as well as uncultured bacteria with the different sides of the cloned insert.

Financial support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; 2010)

ISOLATION LACTIC-ACID BACTERIA OF FRESH MILK FROM PELOTAS,RS FREIRE, V. A. P.1, DANNEMBERG, G.1, OLIVEIRA, L. C.1, SOUZA, J. M. 1, SILVA, W. P.1, FIORENTINI, M. A.1

1*

Depto. de Ciência e Tecnologia agroindustrial, ,Faculdade de Agronomia Elizeu Maciel, Campus Capão do Leão, Universidade Federal de Pelotas, s/n, CEP 96010-900, caixa postal 354, Pelotas, RS. vagnafreire@gmail.com

Keywords: Lactic-Acid Bacteria, fresh milk.

The Lactic Acid Bacteria (LAB) are a group of microorganisms naturally found in foods, including milk and derivates. The microorganims in this group have several morphological, physiological and metabolic characteristics in commom: They are Gram positive, non-spore forming, catalase negative, fastidious, anaerobic, aero-tolerants and acid-tolerants. Several studies describe the participation of LAB as a constituent of the microflora of milk and its derivates, especially artisan cheeses. However, in Brazil the studies for the identification and characterization of lactic microflora in these products are scarce.The isolation and identification of microorganisms from natural sources has been a way widely used for obtaining useful and genetically stable strains (ADNNA and TAN, 2007). The objective this study was to isolate LAB of samples of fresh milk. The LAB were isolated of 03 samples of Milk fresh from different farms localizated in Pelotas, Rio Grande do Sul. For the isolation was used ágar and broth MRS (Man, Rugosa and Sharpe). From the pure cultures held tests of Gram, catalase, morphology as well as tests of their growth in MRS broth under the following conditions: 3.0 and 4.0% NaCl, 0.3 and 1.0 % bile salts and pH 3.0 and 4.0. In this study were obtained 12 isolates of lactic acid bacteria, all were characterized as Gram-positive and catalase negative. All isolates showed resistance to pH 3 and 4. This information is extremely important to predict the resistence of culture at pH values that the bacteria will be submitted after being ingested with the food that contains. In the same way, the isolated microorganisms showed resistant in media added 3-4% NaCl. This information is important to evaluate the possible applications of bacteria isolated. However, was possible to identify that there was no growth of any microorganism isolated in medium containing 1% bile salts, while 100% of them were resistant microorganisms in the medium containing 3% of salt. The LAB acid-tolerants and bile tolerants can be isolated in conditions of stress. The LAB are used for conservation of foods through fermentation promoting beneficial effects to health, preventing contamination caused by pathogenic microorganisms. These isolates can be tested in dairy products for checks its beneficial properties replacing the use of commercial cultures. The importance to use LAB tolerants to several concentrations of NaCl, bile salts and several pH ranges is in to diversification of foods products with the same benefits offered by these microorganisms.

Financial support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

ISOLATION OF BLACK YEASTS (CHAETOTHYRIALES) FROM LEAF-