Título da Dissertação: Formação de biofilmes mono e multi-espécie de Staphylococcus aureus  e  Escherichia coli e bioatividade do óleo essencial de orégano frente a Alicyclobacillus acidoterrestris.

Orientadora: Prof. Dr. Benício Alves de Abreu Filho

Data da Defesa: 25/02/2019




The biofilm consists of a community of sessile cells, mono or multi species, that produce an exopolysaccharide (EPS) responsible for its adhesion on surfaces such as stainless steel AISI 304, used in food industries. Among biofilm-forming bacteria, can be highlight Staphylococcus aureus, Gram-positive microorganism, mesophilic, normal constituent of the human microbiota and cause food spoilage; and Escherichia coli, a Gram-negative microorganism, present in the gastrointestinal tract of humans and animals, characteristic of poor hygiene and fecal contamination, resistant to environmental stresses, disinfectants and antibiotics.

Alicycobacillus spp. is a deteriorating citrus food, it is a Gram-positive bacillus, acidophilus, and spore-forming. There are more than 21 species identified, among them the most important is A. acidoterrestris because of its ability to form the coproduct,as guaiacol, responsible for the astringent taste in acidic juices. Studies report the use of plant essential oils as an antimicrobial agent in the fight against this microorganism, as well as the user associated with chemical agents such as nisin; in the case of oregano essential oil, its antioxidant and antimicrobial activity may be related to the presence of the compounds carvacrol and thymol in its composition.


As goals in the first article, we have the training evaluation and quantification of the biofilm of E. coli and S. aureus mono and a multi-species surface of stainless steel AISI 304 with food matrices such as exudate of meat and milk, in addition to the Muller Hinton broth (MH).

While in the second article the objectives were to characterize the chemical composition of oregano essential oil (Origanum vulgare), to evaluate its antimicrobial antioxidant activities, as well as to test its association with nisin in order to combat the deteriorating Alicyclobacillus spp.


The strains of E. coli ATCC 25922 and S. aureus ATCC 25923, obtained from the microbiology laboratory of the Federal Technological University of Paraná, Campo Mourão campus, were used for the formation of the biofilm, as well as the culture media to be used. The surface used was an AISI 304 stainless steel coupon; the milk was commercially acquired, and meat exudate was obtained from the methodology of Midelet (2002). Biofilm formation was continued at 25 °C and 35 °C for 24h and 48h; after counting the formation of the biofilms by plating serial dilutions, with results expressed in CFU/cm2. To verify the biofilm formation, a scanning electron microscopy was performed using the Shimadzu SS-550.

In the second article, the strain of Alicyclobacillus spp. was obtained from the Brazilian Collection of Microorganisms (CBMAI), located at Unicamp's Research Center in Chemistry, Biology, and Agriculture (CPQBA). Oregano essential oil was obtained commercially from the Ferquima brand, as well as nisin (Sigma-Aldrich). The chemical characterization of the essential oil was performed by Gas Chromatography coupled to the Mass Spectrum (GC-MS). The antioxidant activity was determined by the free radical methods DPPH and ABTS, with statistical evaluation ANOVA and Tukey's test; already the antibacterial activity was obtained from the analysis of minimum inhibitory concentration (MIC) and minimum bactericidal (CBM) in 96-well microplate. The agar diffusion method was also used to evaluate the antimicrobial activity. To evaluate the effect of the combination of the essential oil with nisin, the Checkerboard method was performed, also in a 96-well microplate, with a result expressed in fractional inhibitory concentration (FIC). Seeking to observe the damage caused in the cells of Alicyclobacillus spp. the scanning electron microscopy was performed.

 RESULTS AND DISCUSSIONcoli formed a stainless-steel biofilm in both the MH broth and the meat and milk matrices evaluated at both temperatures and contact times in the MH and Eosin Methylene Blue (EMB) media; the exudate of meat presented favoring the formation of the biofilm at 25 °C. In milk the adhesion and formation of the biofilm occurred more slowly, after 48h of contact the biofilm was in exponential phase of growth. For the S. aureus, it was observed that in meat exudate at 25 ºC growth occurred in up to 48 hours; already in the temperature of 35 °C, after 24h there was decline in the counting of the adhered cells in both Baird Parker (BP) and MH medium. For the biofilm formed in the milk, the S. aureus followed with its exponential development observed through the quantification carried out with 48 hours in both incubation temperatures. When placed together for multi-species growth there was a greater adaptation of Gram-negative to Gram-positive bacteria, suggesting that the presence of E. coli inhibits the growth of S. aureus, as can be observed by electron microscopy scanning (SEM).

In the second article, the result of the characterization of oregano essential oil has shown that the major compound present is carvacrol acetate, with 65.51% of the 93.1% of the compounds identified. As for the antioxidant activity, there was a difference between the methods tested, being 363 μmol trolox/mg by the DPPH method; and 1142 μmol trolox/mg by ABTS, suggesting a good antioxidant activity of the essential oil. The antibacterial activity was evaluated against A. acidiphilus, A. cycloheptanicus, A. herbarius and A. acidoterrestris, with results of 125 μg/mL, 125 μg/mL, 62.5 μg/mL and 62.5 μg/mL respectively; with a minimum bactericidal concentration of 1000 μg/mL and a reduction of 2 logs against the four strains tested. The combined use of oregano essential oil with nisin showed indifferent activity. The disc diffusion method confirmed the inhibition caused by the essential oil through the formation of halo, at concentrations of 2000, 1000, 500 and 250 μg/mL. Finally, scanning electron microscopy allowed the visualization of the action of the essential oil deforming the structure of the A. acidoterrestris bacillus.


In the first article concluded that Escherichia coli and Staphylococcus aureus can form biofilm separately and together on stainless steel surface, with favored formation in meat matrix and at the highest temperature evaluated, with E. coli prevalence. In the second article, the essential oil of oregano showed antimicrobial activity against the genus Alicyclobacillus spp. and a significant antioxidant. Possibly caused by its majority compound characterized as ethyl acetate; leading to the possibility of using this essential oil as a natural preservative in food. Thus, regardless of the pathogenic or deteriorating characteristics of bacteria, they can form biofilms and contaminate the entire food production line, making it important to study and develop new alternatives for its control and elimination.

Key words: stainless steel AISI 304, meat exudate, milk, antimicrobial activity, antioxidant activity, chemical composition.

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