Título da Dissertação: AVALIAÇÃO DA ATIVIDADE ANTIBACTERIANA DOS EXTRATOS DE Piperaceae E NISINA EM Alicyclobacillus acidoterrestris

Orientador: Prof. Dr. Benício Alves de Abreu Filho

Data da Defesa: 02/03/2012



 INTRODUCTION. Alicyclobacillus acidoterrestris is a gram-positive aerobic terminal or sub-terminal endospore-forming bacterium. This bacterium resists pasteurization temperature and low pH, and is usually related to the spoiling of reconstituted orange juice. Juice spoilage by A. acidoterrestris may be observed by its disagreeable smell and taste, without any gas formation and pH alterations, albeit with a slight increase in turbidity, and the presence or absence of sediments within the packing. Pasteurization is the thermal treatment that may trigger spore germination. Another process should be added to the thermal treatment for the spoilage produced by the micro-organism since high temperature may cause the loss of the product’s sensorial and nutritional characteristics. The nisin is the only bacteriocine acknowledged by the World Health Organization as food preservative and an alternative for the decrease of hardness of thermal treatment. The genus Piper (Piperaceae) has a wide antibacterial and antifungal range of biological and pharmacological activities as an alternative for its use as biopreservative in food.
AIMS. Current research evaluates the antibacterial activity of the species Piper (Piperaceae) and nisin on Alicyclobacillus acidoterrestris. Subject the active species to the isolation of pure compound and to evaluate the synergism in combination with nisin.
MATERIALS AND METHODS. The plant species (Piper amalago, Piper aduncum, Piper arboreum, Piper hispidum, Piper xylosteoides and Piper crassinervium) were re-ground with hydroalcoholic solvent 90%. Crude extracts (CE) were filtered and rota-evaporated and an aqueous and chloroform extract obtained. A microbiological activity of extract and nisin was conducted on Alicyclobacillus acidoterrestris. Minimum Inhibitory Concentration (MIC) and Minimum Bactericide Concentration (MBC) were determined by broth microdilution. MBC was determined by the sub-culture of each well and positive control. The species P. aduncum (chloroformic phase) had a higher biological activity and was selected for the subsequent assays and fractioned by chromatography in a vacuum adsorption column in silica gel. The six fractions obtained were evaluated and dichloromethane fraction (F.3) was found to have the lowest MIC/MBC. F.3 was fractioned in an adsorption column for the isolation of the pure compound. The isolated compound was identified by spectrometric methods and biological assays conducted. Antibacterial activity of the isolated compound was confirmed by time-kill curve. The minimum concentration esporocidal was determined by treatment of spores with extract  together and temperature heat shock. The interaction of nisin combination with
the crude extract, dichloromethane fraction and the isolated compound of P.  aduncum were evaluated by checkerboard method. Cytotoxicity assay of the crude extract, fraction and isolated compound of P. aduncum was undertaken by Sulforhodamine B technique. Morphological changes of A. acidoterrestris cells were verified by scanning electronic microscopy.
RESULTS AND DISCUSSION. The CE (chloroformic phase) of species P. aduncum, P. amalago, P. crassinervium and P. hispidum had a good inhibitory activity with MIC lower than 100 μg/ml. Species P. arboreum and P. xylosteoides showed a moderate activity with MIC higher than 100 μg/ml. Piper aduncum (chloroformic phase) had the lowest MIC and MBC, with 15.63 μg/ml, and thus selected for fractioning. After fractioning, six fractions were obtained in which the dichloromethane fraction had the lowest MIC/MBC (7.81μg/ml). The dichloromethane fraction underwent fractioning and the pure compound was obtained. Based on the spectra analyses, the compound was identified as prenylated chromene. The rate of bactericide and sporocide concentration of prenylated chromene was equal to 7.81μg/ml. The compound did not diminish activity by temperature in spore treatment. Nisin showed bactericide and sporocide activities respectively at 31.25μg/ml and 7.81μg/ml. The combination  of crude extract of P. aduncum and nisin showed a synergic interaction with FIC (Fractional Inhibitory Concentration) = 0.24 index, whereas in the case of the dichloromethane fraction and isolated compound there was an addition effect when combined to nisin. The diversity of compounds present in the crude extract that may contribute towards synergism. Pure compound’s activity was
confirmed by the time-kill curve with complete inhibition of bacterial growth and bactericide effects after 12 hours of incubation. Crude extract, fraction and prenylated chromene of P. aduncum had a selective activity on bacteria. The effect of prenylated chromene on the morphology of A. acidoterrestris was examined in cells treated with the compound by scanning electronic microscopy, in which changes in cell morphology and intumescence occurred.
CONCLUSION. Crude extract, fractions and isolated compound of P. aduncum showed good antibacterial activity against spores and vegetatives cells of A. acidoterrestris. Prenylated chromene was isolated and identified as the main antibacterial compound of the plant species. Nisin and P. aduncum together inhibited the bacterial growth and may be a alternative to use in combined concentrations as biopreservatives. However, further studies should be undertaken to explain the behavior of extracts’ activity on in vivo microorganism and cytotoxicity.
Keywords. Alicyclobacillus acidoterrestris, Piperaceae, nisin, prenylated chromene, synergism.

Artigos Publicados Vinculados a Dissertação:

Mestrado: https://doi.org/10.1111/1750-3841.12283