Título da Dissertação: Tabernaemontana catharinensis (A. DC): ATIVIDADE ANTIBACTERIANA EM Alicyclobacillus spp.

Orientador: Prof. Dr. Benício Alves de Abreu Filho

Data da Defesa: 27/03/2018



INTRODUCTION. Alicyclobacillus spp. genus is composed of bacteria acid-thermo resistant, aerobic, rod-shaped, Gram-positive, able to produce spores, nonpathogenic, involved in citrus fruit juice and tart food deterioration. Juice deterioration process was first described in 1981, over apple juice with taste and odor changes, due to the Alicyclobacillus acidoterrestris 0244T skill to produce 2-methoxyphenol (guaiacol) and 2,6 dibromophenol. Wall cell is composed by cyclic fatty acids that give resistance to temperature among 25 to 70 °C and low pH (2.0 to 6.5), with the capacity to create resistant spores that can cause some issues to the industry such as eventual lost of financial. In accordance with USDA (United States Department of Agriculture), considering the average of the last five orange crops, Brazil is responsible for 34% of the orange harvest and more than half of the orange juice production in the world, therefore, this country was responsible for 76% of orange juice global market in 2017. The export of concentrated orange juice (66 ºBrix) contributes significantly to the country 's trade balance and ensure the quality of concentrated and reconstituted juice, increase shelf life and find natural substances to avoid the development of bacterial species is essential for this sector. Tabernaemontana catharinensis (A. DC.) belongs in Apocynaceae family; it is a lactescent tree, found in Brazil, Argentina, Paraguay and Uruguay, used at popular medicine as an antidote for snake bites, toothache relief and vermifuges. It shows biological action as antinociceptive, anti-inflammatory, antimicrobial, trypanocidal, antiviral, antimycibacterial and antileishmaniasis. The plant T. catharinensis (A. DC) has laticifer living cells that secrete latex, an aqueous emulsion with various chemical substances and various bioactive compounds such as polyphenols and flavonoids, which are secreted in response to tissue damage in the plant.
OBJECTIVE. This research objective was evaluating crude latex antimicrobial skill from Tabernaemontana catharinensis (A. DC.), against microorganisms of Alicyclobacillus spp. genus.
MATERIAL AND METHODS. Microbial strains and growing conditions: A. acidoterrestris 0244T, A. hesperidum 0298T, A. acidiphilus 0247T, A. cycloheptanicus 0297T and A. acidocaldarius 0299 T. The growth medium used on this experiment was Bacillus acidoterrestris (BAT) Deinhard et al., (1987). Concentrated orange juice, 66 ºBrix, pH 4,0 was provided by a citrus juice industry located in Paraná northwest region, Brazil, and hydrated with ascetical distilled water over hygienic conditions, free from Alicyclobacillus spp. and spores, certified in our laboratory. Crude latex acquisition and preparation: Tabernaemontana catharinensis (A. DC) – Apocynaceae crude samples were collected in 2016 from plants grown at State University of Maringá campus, Maringá, Paraná, Brasil. Latex got by superficial incisions in T. catharinensis (A. DC.) stem, poured in a same volume water solution. The samples were spun (5.000 x g) in 10 °C for 25 minutes. The deposit was set aside; the supernatant was freeze-dried and used on the experiment. The part that embraces almost every soluble latex compound was named crude latex from T. catharinensis (A. DC.). Determination of inhibitory and minimum bactericidal concentrations: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for crude latex was determined in 96-well microdilution plates (TPP®, Switzerland), following the metodology of National Committee for Clinical Laboratory Standards (NCLSI M7-A9) (2012). Cytotoxicity analysis: T. catharinensis (A. DC.) crude latex cytotoxicity activity was made by colorimetric method, MTT (3-(4.5- dimethylthiazol-2-yl) 2.5-diphenyltetrazolium bromide), described by Mosmann (1983). Scanning electron microscopy: A. acidoterrestris 0244T cells were grown with T. catharinensis (A. DC.) crude latex by the following concentrations 1 x MIC, 4 x MIC and 8 x MIC in concentrated orange juice 66 °Brix, after adding distilled water, 11 °Brix, pH 4,0. The volume of the culture in each microtube was 1 mL and initial inoculum concentration was 104 CFU/mL. The microtubes were incubated at 45 °C for 24 hours. After incubation, A. acidoterrestris 0244T cells were washed with phosphate-buffered saline (PBS) at pH 7.2 and set with glutaraldehyde 2,5% (Sigma-Aldrich, St. Louis, MO) in sodium cacodylate buffer 0,1 M (SEM, Hatfield, PA) for 1 hour on room temperature. Then, the samples were taken from the setting solution, washed twice with cacodylate buffer 0,1M and put on a cover slip covered with Poli-L-lysine during 1 hour. The samples were washed three times with cacodylate buffer and later dehydrated using progressive ethanol concentrations (50, 70, 80, 90 and 100%). Afterwards, they were submitted to the CO2 critical point and covered by gold to be seeing through the scanning electron microscope QUANTA 250 (FEI Company).
RESULTS AND DISCUSSION. Minimum inhibitory and minimum bactericidal concentrations: T. catharinensis (A. DC.) crude latex MIC and MBC were determined against Alicyclobacillus spp. species. It can be observed MIC of 7.81 μg/mL for the five Alicyclobacillus species analysed. The MBC for the species A. acidoterrestris 0244T, A. hesperidum 0298T, A. acidiphilus 0247T and A. cycloheptanicus 0297T was 250 μg/mL. For the A. acidocaldarius 0299T strain, the MBC was 500 μg/mL. According to Holetz et al., (2002) studies, which evaluate the antimicrobial activity on several plants against many bacteria, it is possible to consider that a MIC under 100 μg/ml, presents great antimicrobial activity, from 100 to 500 μg/mL moderated antimicrobial activity, from 500 to 1000 μg/ml the activity is rated as weak and over 1000 μg/ml is considered inactive. Therefore we may consider that T. catharinensis (A. DC.) crude latex had shown great antimicrobial activity against Alicyclobacillus species studied in our research. Boligon et al., (2015) evaluated the antimicrobial activity of the crude extract of the bark of T. catharinensis (A. DC.) against Gram positive bacteria, Gram negative bacteria and fungi finding MIC values >1000 μg/mL. Studies performed by Gindri et al., 2011 with fração n-butanol from T. catharinensis (A. DC.) leaves had shown antibacterial effects against Micrococcus sp. (MIC 31.25 mg/mL), Aeromonas sp. (MIC 250 mg/mL), Proteus mirabilis and Enterococcus faecalis (MIC 62.50 mg/mL). Cytotoxicity: The experiment took part on Vero cells by MTT method. The cytotoxic effect of Tabernaemontana catharinensis (A. DC.) crude latex was evaluate by the number of survival cells considering that dead cells cannot metabolize MTT. CC50 values found for crude latex were 84.67 ± 8.14 μg/ml. Selectivity index was 10.84. A research made by Boligon et al. (2015) that evaluated T. catharinensis (A. DC) crude extract of shell cytotoxic activity of the bark showed results of CC50 = 59.53 ± 4.56 μg/mL, which allows us to realize that crude latex presents a highest value for CC50, that admits using 9 times the MIC and keeping within cytotoxicity limits, according to values determined in our analyses. Scanning electron microscopy: Morphological changes were seen on A. acidoterrestris 0244T vegetative cells treated with T. catharinensis (A. DC.) crude latex in concentrated orange juice, when compared with the untreated sample. Molva & Baysal (2015), on a study with pomegranate juice and grape seeds with apple juice and A. acidoterrestris DSM 3922, they reported morphological changes on bacterial cells structure and spores, similar to our results.
CONCLUSIONS. The latex extract of T. catharinensis (A. DC.) was effective against vegetative cells of Alicyclocacillus spp. and low concentrations are able to inhibit bacterial growth of five species of Alicyclobacillus spp. By applying the crude latex in the reconstituted orange juice and subjecting the scanning electron microscopy analysis, it was possible to find important morphological alterations, such as cell rupture and deformations in the cell wall of A. acidoterrestris. The selectivity index allowed to confirm the high ratio between toxicity and activity, demonstrating holding to the use of the extract. The crude latex cytotoxicity test showed results that allow the use of up to nine times MIC and although further in vivo toxicity studies are required for the application of this compound in foods. The application of the crude latex of T. catharinensis (A. DC.) In orange juice reconstituted as a preservative against Alicyclobacillus spp. emerges as a possibility for the future and requires more complementary studies since this study is unprecedented using this plant and these species of microorganisms.
Key-words: Antibacterial, Tabernaemontana catharinensis (A.DC.), latex, Alicyclobacillus acidoterrestris, Orange juice.

 Artigos Publicados Vinculados a Dissertação: