DULCE AIME MOREIRA DA SILVA

Título da Dissertação: PREVENÇÃO DO CRESCIMENTO DE ALICYCLOBACILLUS ACIDOTERRESTRIS EM SUCO DE LARANJA INDUSTRIALIZADO USANDO ÓLEO ESSENCIAL DE ALECRIM E NISINA.

Orientador: Prof. Dr. Benício Alves de Abreu Filho 

Data da Defesa: 21/02/2017

RESUMO GERAL

INTRODUCTION. Alicyclobacillus spp. are Gram-positive, non-pathogenic, aerobic and deteriorating bacteria, which have the ability to form spores, grow at temperatures ranging from 26 to 60 °C, and at pH 2.0 to 6.0. The most important specie is Alicyclobacillus acidoterrestris. Its appearance has been reported in juices such as orange, apple, tomato, among others. They can still be found after the heat treatment, due to its characteristics, which help in its resistance. A. acidoterrestris can produce odor-causing substances in the juice, such as guaiacol (2-methoxyphenol), 2,6-dibromophenol and 2,6-dichlorophenol. Brazil is the third largest producer of the orange fruit, and a large part of this production goes to juice production and then exportation. Thus, it is necessary study and ensures the quality of the product until its final destination. Essential oils have been increasingly used by the food and pharmaceutical industry due to its antimicrobial and antioxidants compounds. Rosemary essential oil has been applied against different bacteria (Escherichia coli, Staphylococcus aureus and Listeria monocytogenes). Another natural antimicrobial that has also been used is nisin which is produced by Lactococcus lactis subsp. Lactis as it is approved by the Food and Drug Administration (FDA) and has good stability in acidic media.
OBJECTIVES. The objectives of this study were evaluate the antimicrobial activity of rosemary essential oil (RO) and its combination with nisin (NR) against the vegetative cells and spores of A. acidoterrestris in industrialized and reconstituted orange juice. Evaluate if the addition of these compounds might increase the total phenolic compounds content and the antioxidant activity of orange juice, and also if the microorganism might deteriorates the antioxidant compounds present in orange juice, in two different temperatures (28 and 45 ºC) during 96 h.
MATERIALS AND METHODS. The microorganism used was A. acidoterrestris obtained from the Brazilian Collection of Microorganisms in the Environment and Industry (CBMAI – 0244T), located at the Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas — CPQBA/UNICAMP — São Paulo — Brazil. The strain was maintained using the BAT culture medium (Bacillus acidoterrestris) with 20% glycerol and maintained at -20 °C. Rosemary essential oil (dissolved in 1% Tween-80), and nisin (2 mg of nisin in 1 ml of 0.02 N HCl), was used for the experiment. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of rosemary oil and nisin in BAT medium at 45 °C were determined. After that, the antimicrobial combination of the oil and nisin was made by the Checkerboard method and the fractional inhibitory concentration index (FIC) was calculated, verifying if the compounds had a synergism effect. The growth inhibition test was then performed in reconstituted orange juice, by adding concentrations of 1, 4 and 8 x MIC of RO (125, 500 and 1000 μg/mL, respectively) or 1, 4 and 8 x MNR (MIC of combination) (1.95/15.62 μg/mL, 7.8/62.5 μg/mL, 15.62/125 μg/mL, nisin/RO) at temperatures of 28 to 45 °C during 96 h. The total phenolic content and the antioxidant activity (DPPH and ABTS) of orange juice and orange juice with NR (1x MNR) (with or without microorganism) were determined. In order to verify the changes in treated cells, the scanning electron microscopy and flow cytometry tests were performed. All analyzes were performed in triplicate. The data were assessed by analyzes of variance using the general linear model (GLM) with SPSS (v.15.0) and when differences were statistically significant, a Tukey test was performed with statistical significance set at p < 0.05.
RESULTS AND DISCUSSION. The result for MIC and MBC of RO was 125 μg/mL for both analyzes. Several studies have proposed the use of natural compounds, eucalyptus leaves, and major compounds of essential oils (eugenol and limonene), to inhibit A. acidoterrestris. After, the MIC and MBC of nisin were performed, which resulted in 15.62 and 31.25 μg/mL, respectively. Then, the antimicrobial combination of RO and nisin was evaluated, obtaining a FIC = 0.15, which corresponds to a synergism effect. Based on this result, the concentrations of 15.62 and 1.95 μg/mL for oil and nisin, respectively, were used for the subsequent application in orange juice.
Analyzing the use of rosemary oil individually, only with 8 x MIC at 28 °C it was possible to obtain a sporostatic effect during the 96 h, this may be associated to the optimum temperature growth of the microorganism (45 °C), which makes it more difficult to prevent the microorganism from multiplying. In relation to NR, the lowest concentration tested (1 x MNR) had a sporicidal effect at the first hours, for both the vegetative cells and spores. In this work, the growth of A. acidoterrestris against nisin (alone) during 96 h (data not shown) was also analyzed, and the concentration of 1.95 μg/mL showed the lowest population count, but, still had microorganism growth. After, the total phenolic compounds and antioxidant activity of rosemary essential oil was evaluated, and the results showed a low antioxidant activity. After NR application in orange juice no significant difference between treatments was observed, which was already expected due to the results obtained from RO. Scanning electron microscopy was performed and showed differences in the structure of treated cells. In flow cytometry, it was observed that the stained cells were in Q2, which corresponds to the injured cells, and although there was no great cell death, the treatment was efficient and the cells did not develop even in an appropriate environment, as shown in Table 2.
CONCLUSIONS. In this study was evaluated the effect of rosemary oil and its combination with nisin on growth inhibition of A. acidoterrestris in industrialized and reconstituted orange juice. At the concentration of 1000 μg/mL, rosemary oil had a sporostatic effect, while the association of nisin + rosemary oil, (1.95 μg/mL and 15.62 μg/mL, respectively), resulted in a sporicidal effect. Thus, results indicated that the application of rosemary essential oil and nisin as an alternative in the prevention of the Alicyclobacillus development in juices.
Keywords: Alicyclobacillus, essential oil, nisin, orange juice, bioconservation, synergism

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