Detecção e quantificação de células viáveis de Bacillus sporothermodurans e de Bacillus cereus em leite através de PCR convencional e de PCR em tempo real associadas ao propídio monoazida

摘要

The presence of Bacillus spp. in milk is an important problem for the dairy industry due to their capability of sporulation and the possibility of spore resistance to heat treatment by ultra high temperature (UHT). Bacillus sporothermodurans survive to the UHT system, germinating and growing in stored milk and, if not correctly identified and quantified, can exceed the criterion established for mesophilic aerobic, besides altering the quality of dairy products when in high concentrations. On the other hand, contamination of milk by Bacillus cereus is not only an important cause of deterioration, but is also associated with the occurrence of diarrhea and emetic syndromes. Traditionally, these microorganisms are identified and quantified in food using conventional microbiological techniques, but the Polymerase Chain Reaction (PCR) based methods have been widely used for the same purpose. However, PCR cannot distinguish between viable and dead cells, which can be overcame with the use of DNA intercalating, such as propidium monoazide (PMA). PMA binds to DNA derived from cells with damaged membranes, preventing their amplification by PCR, allowing, thus, the selective detection of viable cells. Therefore, this thesis aimed to characterize the thermal resistance of B. sporothermodurans and to develop methods of detection and quantificatification of viable cells of B. sporothermodurans and B. cereus in milk samples by qPCR associated with PMA. Isothermal and non-isothermal treatments allowed the determination of the profile of heat resistance of B. sporothermodurans spores to heat UHT process, predicting that to 121°C was found a D value between 2 a 4 min. The selective detection and quantification of B. sporothermodurans and B. cereus by PMA-qPCR were developed targeting 16S rRNA gene and hemolysin gene, respectively. The treatment with PMA from pure culture and artificially contaminated UHT milk were standardized by end-point PCR for the detection of viable cells of these microorganisms. The inhibition of amplification of DNA from dead cells was obtained at a concentration of 30μg/mL PMA. The standardization of qPCR assays were performed using hydrolysis probes (TaqMan® system) specific to each target gene. The quantification limit from UHT milk artificially contaminated was 2. 5 x 102 CFU/mL for B. sporothermodurans and 7. 5 x 102 CFU/mL for B. cereus. The assays were applied to 135 samples of UHT milk of different commercial brands, comparing with the conventional method of cultivation for each microorganism. B. sporothermodurans and B. cereus were respectively detected in 14 (10. 4%) and 44 (32. 6%) of the samples by molecular methods developed, and in 11 (8. 1%) and 15 (11. 1%) by conventional culturing methods. The PMA-qPCR methods developed in this study were specific and sensitive for the detection and quantification of viable B. sporothermodurans and B. cereus cells, being applicable for the evaluation of milk samples, reducing the time for the analysis of this product. Furthermore, the results showed that B. cereus can be found in UHT milk.