Título da Dissertação: CONTROLE DO CRESCIMENTO DE Alicyclobacillus acidoterrestris COM EXTRATOS DE Piperaceae EM SUCO DE LARANJA INDUSTRIALIZADO.

Orientador: Prof. Dr. Benício Alves de Abreu Filho

Data da Defesa: 03/03/2016



Alicyclobacillus acidoterrestris is a gram-positive, acidophilic, thermophilic, spore-forming, non-pathogenic bacillus that causes food to deteriorate drinks and industrialized food with acidic characteristics. Develops in environments with temperature 26 ºC - 60 ºC and pH 2.5 – 6.0. Its spore form is mainly found in soil and is easily transferred to industrial food production. The forms of contamination of A. acidoterrestris are associated with the processing, transport or handling of products, making strict control of food processing necessary. The presence of spores in juice concentrates deteriorates, the germination there of, after pasteurization and storage, because conventional survive the heat applied to process juices. Thus, the spores can germinate and develop into vegetative cells in the final product, resulting in sensory changes. The deterioration for A. acidoterrestris occurs due production of the 2,6-dibromofenol, 2,6-diclorofenol, 2-metoxifenol (guaiacol) components and causes off-flavors with antiseptic, astringent characteristics, but without changes in pH . Because of these effects, the microorganism is a major threat to the acidic beverage industry, causing production and economic losses. Brazil is a major exporter of concentrated orange juice, so it is important to invest in product quality, making sure that it can reach the final destination with quality. This way it is necessary to combine the heat treatment another process for control of deterioration caused by this microorganism. Nisin, the bacteriocin produced by Lactococcus lactis subsp lactis, has been approved as a food additive by the Food and Drug Administration – FDA. Studies show that nisin to associate with compounds of plant origin can promote synergistic associations, the effect of increasing and decreasing the concentration of antimicrobial agent applied in foods. Thus, the use of natural plant extracts as antimicrobial agents, has been the subject of research and is of growing importance to the food industry. The Piper genus used as a food flavoring, is used in popular medicine and exhibits a variety of biological and pharmacological activities.
The aim of the present study was to evaluate the antibacterial activity of crude extracts of P. marginatum, P. peltatum, its fractions and pure compound, and verify a synergistic action with nisin and its potential for application in fruit juice concentrates, such as natural biopreservative.
P. peltatum and P. marginatum leaves were crushed and subjected to the maceration process over a period of seven days. Then the extracts were filtered and rota-evaporated until complete removal of the solvent, leaving only the raw extracts. The extracts were subjected to antibacterial tests and extract showed better results against the microorganism under study was fractionated. Six fractions were obtained and used for the tests. The fraction with better antibacterial activity was subjected to chromatographic process the adsorption column to isolate the pure compound. The nisin used was purchased commercially and was prepared in 0.02 M hydrochloric acid (HCl) and sterilized in a 0.22 μm membrane. The microorganism Alicyclobacillus acidoterrestris (CBMAI 0244T) - Brazilian Collection of Environmental and Industrial Microorganisms - CBMAI and stored in the medium of BAT culture - Bacillus acidoterrestris, final pH adjusted at 4.0, with 30% glycerol at -20 ºC the Laboratório de Microbiologia da Água, Ambiente e Alimentos da Universidade Estadual de Maringá – UEM. To prepare the spore suspension of the microorganism was cultivated in tubes containing BAT broth, incubated at 45 °C to reach 80% sporulation, centrifuged, washed and stored at 4 °C until assayed. Determination of antibacterial activity for vegetative and sporulating cells of A. acidoterrestris was performed by microdilution in series, as CLSI M7-A9, using crude extracts, fractions, pure nisin and compound at varying concentrations. Thus, it was possible to evaluate the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Tests with the industrialized orange juice and reconstituted was performed using extracts, fraction and pure compound. The checkerboard method performed with the crude extract, fraction and pure compound in combination with nisin as recommended by Schelz, Molnar and Hohmann (2006). Interpretation of the results was done according to Gutierrez, Barry-Ryan and Bourke (2008). The determination of 50% inhibitory concentration (IC50) of vegetative cells of A. acidoterrestris has been accomplished through serial microdilution according to Tanaka et al. (2006). The cytotoxicity assay was performed by the colorimetric MTT assay as described by Mosmann (1983), using LLCM-K2 cells (epithelial cells from the kidney of Macaca mulatta). The cytotoxicity of the extracts against LLCM-K2 cells was compared using the selectivity index (SI) which was determined by dividing the IC50 and CC50 interpreted according to Santos et al. (2008). Scanning electron microscopy was used in order to verify the morphological alterations of the vegetative cells and spores of A. acidoterrestris by comparison with the negative control (no treatment). This methodology was performed as Haddad et al. (2007).
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the microdilution method. The extracts show good antibacterial activity. P. marginatum presented MIC/MBC of 62.50 while P. peltatum showed lower MIC and MBC of 15.62 μg/mL and was subjected to fractionation. Six fractions were obtained and evaluated and the fraction hexane: dichloromethane showed lower MIC/MBC of 31.25 μg/mL. The fraction hexane: dichloromethane and dichloromethane: ethyl acetate were further fractionated and pure compound 4-Nerolidylcatechol (4-NC) was isolated. 4-NC substance showed MIC and MBC of 62.50 μg/mL. Other research with Piperaceae extracts also show great antibacterial properties. The nisin presented good bactericidal activity, where as the MIC and MBC for vegetative cells was 15.62 μg/mL and 31.25 μg/mL, respectively. In relation to the spores, the extracts were also effective, reducing the initial microbial load. However noticed a greater resistance in relation spores, vegetative cells, probably due to the fact that the presence of dipicolinic acid found in spores. Sporicidal effect was observed when using P. peltatum extract at a concentration of 250 μg/ml, while P. marginatum this same concentration was able to reduce 2.76 Log. The fraction hexane: dichloromethane at a concentration of 1.000 μg/mL completely reduced the existing bacterial load and pure compound 4-NC was down 2.49 Log in concentration of 250 μg/mL. The application of extracts and isolated fraction of reconstituted orange juice contaminated with vegetative cells A. acidoterrestris in the concentration of 1 x MIC, we noticed lower reduction between the antimicrobials tested. In concentration of 4 x MIC was observed greater decrease in the crude extract of P. marginatum, 4.96 Log reducing substance isolated and 4-NC reduced microbial load of 3.93 Log. A concentration of 8 x MIC, the crude extract of P. peltatum reduced 1.5 Log and for the fraction, there was a reduction of 5 Log, conditions similar to our studies have found Molva and Baysal (2015a). The checkerboard method showed a synergistic effect with nisin for the extracts and the fraction, where FICTotal index was less than 0.5 and an additive effect on the pure compound 4-NC with FICTotal = 0.98. The dose-response effect and cell viability through the selectivity index showed values above 1.0 revealing that the extracts tested were more selective to the microorganism and less toxic to the LLCM K2-cells. By scanning electron microscopy, it observed changes in cell membrane integrity of vegetative and sporulated cells of A. acidoterrestris when subjected to treatment with antimicrobials under test.
The Piperaceae extracts, fractions and the pure compound 4-NC were effective against vegetative and sporulated cells of A. acidoterrestris, whereas low concentrations were effective at eliminating the microbial load. When applied to the reconstituted orange juice, they proved effective as antibacterial agents applied in foods. A synergistic effect with nisin demonstrates the possible use of these extracts as antimicrobial agents. The addition of P. peltatum and P. marginatum extracts to orange juice acted as a natural preservative against the microorganism studied, indicating a promising future, although requires further studies of the sensory characteristics of the orange juice.
Key words: Piperaceae, Alicyclobacillus acidoterrestris, antimicrobial, orange juice

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