Título da Dissertação: Avaliação da atividade antibacteriana e esporicida dos óleos essenciais de Copaifera multijuga, Thymus vulgaris e nisina frente à Alicyclobacillus acidoterrestris.

Orientador: Prof. Dr. Benício Alves de Abreu Filho

Data da Defesa: 26/02/2015



INTRODUCTION. Alicyclobacillus acidoterrestris was first isolated in 1980 from apple juice. The name A. acidoterrestris was associated with the cyclic fatty acids that form its cell membrane and increase the resistance of its spores to thermal treatments, this behavior may also be related to the reduced membrane permeability in this species. A. acidoterrestris is a Gram-positive bacillus that is thermoacidophilic, aerobic, food spoiling, non-pathogenic and catalase positive with terminal or subterminal spores. Develops well in acid media (pH from 2.5 to 6.0) and at high temperatures (from 20 to 60 ºC). One of the important characteristics of A. acidoterrestris is its ability to spoil acidic fruit juices, such as orange juice. Spoilage caused by A. acidoterrestris occurs because of the production of 2-methoxyphenol (guaiacol), 2,6-dibromophenol and 2,6 dichlorophenol, which are substances associated with the unpleasant taste and odor of contaminated food. This spoilage process is characterized by a lack of gas production, low turbidity and sedimentation; thus, it is difficult to detect. The pasteurization process contributes to reducing pathogens and spoilage microorganisms, improving food safety and reducing chemical preservatives. However, spore-forming microorganisms can resist the highest temperatures used in pasteurization, and under appropriate conditions, these microorganisms develop inside packaged products, thus reducing the shelf life and profitability of affected products. Brazil is a major exporter of orange juice, and it accounts for 50% of the global production. To ensure orange juice quality, research is required to develop new antimicrobial agents that can contribute to food preservation by increasing the shelf life and preserving the sensory and nutritional characteristics of the juice, making it healthier. The demand for natural antimicrobial agents has increased because of the popularity of green consumerism and related concerns by consumers and regulatory agencies for the health and safety of food products. Several studies have indicated the potential of essential oils (EOs) and their isolates and nisin alone or in combination as antibacterial agents because of their bacteriostatic, bactericidal and/or sporicidal properties.
AIMS. Therefore, this study aimed to evaluate the antibacterial activity of the essential oils of Copaifera multijuga and Thymus vulgaris against the vegetative and sporulated forms of Alicyclobacillus acidoterrestris and assess the combined effects of the EOs and nisin.
MATERIAL AND METHODS. Bacterial strain and growth conditions: The A. acidoterrestris strains (CBMAI 0244T – source: soil). Bacillus acidoterrestris (BAT) culture medium with a final pH adjusted to 4.0. Inoculum preparation: The vegetative cells of A. acidoterrestris procedure was performed as recommended by the Clinical and Laboratory Standards Institute (CLSI) M7-A9. A. acidoterrestris spores was performed and stored in sterile deionized water at 4 ºC until used. Essential oils: The T. vulgaris (thyme) EO was provided by the Laboratory of Toxicology (Laboratório de Toxicologia) of UEM, state of Paraná, Brazil. The EO of C. multijuga (Amazonas) was provided by the Laboratory of Microbiology of Natural and Synthetic Products (Laboratório de Microbiologia Aplicada aos Produtos Naturais e Sintéticos) of the Department of Basic Health Sciences (Departamento de Ciências Básicas da Saúde) of UEM. Nisin: Nisin was commercially purchased, and a stock solution was prepared in 0.02 M hydrochloric acid (HCl) and sterilized in a 0.22 μm membrane. Determination of the antibacterial activity of the essential oils: The antibacterial activity of the EOs was determined as recommended by the CLSI M7-A9. It was possible to evaluate the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum sporicidal concentration (MSC). Checkerboard method: The checkerboard method is widely used for in vitro evaluations of the combined antibacterial activity of two drugs as antibacterial agent. The microdilution was performed as recommended by Schelz, Molnar and Hohmann (2006). Interpretation of the results was performed as Gutierrez, Barry-Ryan and Bourke (2008). Death time curve: This assay was performed as Ruiz et al. (2013) and shows the bacterial decay time in contact with antimicrobial agents. Dose-response effect: The determination of 50% inhibitory concentration (IC50) of vegetative cells of A. acidoterrestris has been accomplished through serial as microdiluições Tanaka et al. (2006). Cell viability assay: The cytotoxic activity of the EOs was evaluated by the MTT colorimetric method as described by Mosmann (1983). The EOs’ cytotoxicity to the Vero cells was compared to the selectivity index (SI), which was determined by dividing the 50% cytotoxic concentration to the Vero cells (CC50) by the 50% inhibitory concentration to the bacteria (IC50). Scanning electron microscopy: In this assay, it was possible to determine the morphological alterations of the vegetative cells and spores of A. acidoterrestris by comparison with a negative control (no treatment). This methodology was performed as Haddad et al. (2007). Flow cytometry: In this test was determined and the survival rates of cell membrane integrity as Anjos et al. (2013).
RESULTS AND DISCUSSION. Determination of the antibacterial activity of the essential oils: The MIC and MBC of the EOs of C. multijuga and T. vulgaris were determined for the vegetative cells and spores of A. acidoterrestris. For both EOs, the best effectiveness was obtained against the vegetative cells, resulting in moderate antibacterial activity (concentrations ranging from 100 to 500 μg mL-1). The EOs had a weak antibacterial activity against A. acidoterrestris spores (concentrations ranged from 500 to 1,000 μg mL-1). The resistance of spores to treatment with EOs may be related to the presence of dipicolinic acid found in the endospores. This characteristic confers high resistance to the spores to thermal and chemical treatments (Paredes-Sabja, Setlow and Sarker, 2011). The MIC and MBC of the EO of C. multijuga against the vegetative forms of A. acidoterrestris were 300 μg mL-1 and > 1,000 μg mL-1, respectively. For the spores, a reduction of 3.07 log CFU mL-1 was observed when they were treated with 500 μg mL-1. The MIC and MBC of the EO of T. vulgaris against the vegetative forms of A. acidoterrestris were 500 μg mL-1 and > 1,000 mL-1, respectively. For treatments using spores, reductions of 0.64 log CFU mL-1 and 2.05 log CFU mL-1 were observed when 500 μg mL-1 and 1,000 μg mL-1 EO were used, respectively. Nisin exhibited good bacteriostatic, bactericidal and sporicidal activity against A. acidoterrestris. The MIC and MBC for the vegetative forms were 15.60 μg mL-1 and 31.25 μg mL-1, respectively. The MSC with total elimination of the spores was reached at 62.50 μg mL-1. The results observed in our assays were similar to the results of Ruiz et al. (2013). Checkerboard method: The EO evaluations (C. multijuga and T. vulgaris) in combination with nisin were performed through the checkerboard method. For both treatments (C. multijuga + nisin or T. vulgaris + nisin), the FIC index was 0.75, and the combination of EOs + nisin produced an additive effect. Nisin and EOs have great potential for use as antibacterial agents of natural origin, and their combined effect provides a promising alternative for controlling several microorganisms and for use in applications in food matrices as preservatives, can be used as food preservatives. Death time curve: The death time curve method was conducted to assess the antibacterial activity of the EOs. At a concentration of 8x MIC C. multijuga EO, there was complete reduction of the bacterial load of A. acidoterrestris in the first three hours after treatment. With concentrations of 4x MIC and 2x MIC, a longer treatment was necessary, with 48 h required to eliminate the vegetative forms of A. acidoterrestris. With 1x MIC for 24 h, a reduction of approximately 4.65 log CFU mL-1 was obtained. The treatment with T. vulgaris EO exhibited a smaller reduction compared with the treatment using the C. multijuga EO. Total bacterial elimination was reached after 48 h of treatment using the concentration of 8x MIC. In the treatment using 1x MIC for 24 h and 48 h, there was a reduction of 3.81 log CFU mL-1 and approximately 4.11 Log CFU mL-1, respectively. Dose-response effect and cell viability: The results obtained in this assay revealed that the EOs of C. multijuga and T. vulgaris had CC50 values of 54.2 μg mL-1 and 142.3 μg mL-1, respectively, causing a reduction of 50% of the viable Vero cells at these concentrations. The IC50 results of A. acidoterrestris using the EOs of C. multijuga and T. vulgaris were 500 μg mL-1 and 1,000 μg mL-1, respectively, and there was 50% inhibition of bacterial growth with these concentrations. A comparison of the CC50 with IC50 showed that the EOs were less selective to A. acidoterrestris and more toxic to Vero cells. The selectivity index (SI) of the EO of C. multijuga and T. vulgaris were 0.11 and 0.14, respectively. Scanning electron microscopy: In this trial we observed morphological changes in both external and cellular forms treatments compared to the control. Flow cytometry: In this assay, the vegetative cells of A. acidoterrestris treated with the EOs of C. multijuga and T. vulgaris at their respective MICs had a higher percentage of cells with alterations in the cell membrane integrity compared with cells of the negative control. Therefore, we cannot conclude that the main mechanism of action of the EOs is changes in the cell membrane because the percentage of cells with membrane alterations was low in all treatments, with values ranging from 29.94% to 4.24%.
CONCLUSIONS. Our studies showed that the tested substances had good antibacterial properties because they reduced the viability of A. acidoterrestris. Thus, it is evident that the EOs of C. multijuga and T. vulgaris have potential for use as antibacterial agents against A. acidoterrestris.
Key words: Antibacterial, Copaifera multijuga, Thymus vulgaris, Alicyclobacillus acidoterrestris, orange juice.

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