project presentation modified
- 1. Food processing methods such as pasteurisation, concentration by evaporation, spray drying and the addition of chemical and sugar additives are becoming less favored by consumers due to the desire of more nutritious, chemical free and higher quality foods. New technologies are now required to inactivate deleterious or pathogenic micro organisms without leaving chemical residues in the food or destroying healthy compounds within the food. UV light irradiation holds considerable promise in food processing as an alternative to traditional thermal processing. It is widely accepted that the disinfection efficiency of Pulsed UV (PUV) technology takes a much shorter time than continuous UV light technology due to higher intensities. Treatment : During phase logarithm, each bacteria was subjected to PUV treatment (Control = no treatment, 20P, 40P, 60P, 80P, 120P and 160P). 100 µl of the treated bacteria was plated following serial dilutions at 37oC for 24hours and Total Viable Count (TVC) performed. Protein analysis : Immediately post treatment, 1 ml of the each treated bacteria was also transferred to a sterile Eppendorf tube which was centrifuged at 8,000rpm for 10min for protein and DNA analysis. The BCA Protein Assay was used to determine the loss of internal microbial proteins against PUV treatment. Standards created from known concentrations of BSA (Bovine Serum Albumin) (Fig. 1.2.) were used to determine the protein content of the supernatant with the aid of the colorimetric assay. Figure 1.2. Standard curve of BSA at 562nm. DNA analysis : DNA extraction was performed using a DNA purification kit – High Pure PCR Template Preparation Kit. The kit is composed of a cell lysis buffer, proteinase K enzyme, binding buffer, elution wash buffers, a pre warmed final elution buffer and filter tubes. Quantitative PCR (q PCR) was then performed using the Fast Start Essential DNA Probes Master. A primer specific for a region – 16S rRNA of all Bacillus spp. was used to amplify genomic DNA sequences of Bacillus megaterium and Bacillus cereus in this study . Cycle threshold (Ct) values of known bacteria numbers were used to determine the level of DNA damage in the samples. Table 1.1. DNA quantification standards. qPCR was also performed in conjunction with Propidium Monazide (PMA) dye prior to DNA extraction and purification. The PMA works in conjunction with PCR methods in identification of viable only bacterial cells. Black, J. G., 2008. Microbiology. 7th ed. Asia: John Wiley and Sons Inc. Farrell, H.P., Garvey, M. Cormican, M., Laffey, J.G., Rowan, N.J., 2009. Investigation of critical inter related factor affecting the efficacy of pulsed light for inactivation of clinically relevant bacterial pathogens. Journal Applied Microbiology, 91 (2009) pp 1494 – 1508. Fox, C., 2011. Studies on the variation in strain susceptibility of Staphylococcus aureus to high intensity pulsed UV light irradiation. Garvey, M. (2009) Pulsed UV light inactivation of Cryptosporidium Parvum and other microbial species in drinking water supplies in Ireland. Garvey, M., Farrell, H., Cormican, M., Rowan, N., 2014. Investigations of the relationship between use of in vitro cell culture quantitative PCR and a mouse based bioassay for evaluating critical factors affecting the disinfection performance of pulsed UV light for treating Cryptosporidium parvum oocysts in saline. Journal of Microbiology Methods. 80 (3) pp 267 – 273. Patrasa, A. Bruntona, N.P O’Donnell, C. Tiwari, B.K. 2010. Effect of thermal processing on anthocyanin stability in foods; mechanisms and kinetics of degradation, trends in food science and technology. 21 (2010) pp. 3 – 11. Awuah, G.B. Ramaswamy, H.S. Economides A. 2006. Thermal processing and quality principles and overview, chemical engineering and food processing. 46 (2007) pp. 584 – 602. Goosen, N., Moolenaar, G.E., 2008. Repair of UV damage in bacteria. DNA repair. 7 (2008) pp 353 – 379. Special acknowledgment to Prof. Neil Rowan, AIT, Dr. Alessia Stocca, AIT, Dr. Damien Brady, AIT, Dr. Sile O Flaherty, AIT and class colleagues and friends. The effectiveness of PUV in this study is demonstrated in Fig. 1.2. PUV inflicts 4 types of cellular damage – photo hydration, photo splitting, photo crossing and photo dimerization, all of which prevent DNA replication. 100 % reduction in both Bacillus spp. was seen at 120P and 160P. Bacillus megaterium was deemed the more sensitive of the two bacteria as total reduction of the bacteria was also seen at 80P (Fig. 1.4.). Increase in protein concentration as a result of internal cellular protein damage (Fig 1.5.) also demonstrates the effectiveness of PUV. Less protein was released by Bacillus megaterium as a result of PUV. The measurement of increase in protein as a result of intracellular damage does not correlate the results of TVC. Therefore the increase in protein demonstrates protein leakage of the cell rather than bacterial inactivation. As the DNA analysis performed in this study was not successful due to a non functioning primer, replication of the study is required to determine the relationship between protein leakage and DNA damage following PUV treatment. Further research may also be warranted which determines the effect of PUV on the pathogenic toxins released by both Bacillus spp. Project By: Lorraine Hannon. BSc. (Hons) in Applied Biosciences Supervisor: Prof. Neil Rowan, Athlone Institute of Technology. Bacillus spp. are often involved in issues related to food poisoning and food spoilage characteristics. Widely found in soil and water, Bacillus spp., are rod shaped, endospore forming, gram positive bacteria. They can be either facultative anaerobic or obligate aerobic and are also a highly resistant genera due to the production of endospores from vegetative cells enabling the bacterium to resist unfavourable conditions. Bacillus cereus (Fig. 1.1.) has been recognized as an agent of food poisoning since 1955 and causes two types of food borne illness – emetic and diarrheal based on the toxins released. Bacillus megaterium is involved in increasing numbers of studies examining its pathogenic potential due to the discovery of a heat stable toxin along with several toxin encoding genes present. Figure 1.1. Bacillus cereus viewed under fluorescent microscope (Black, 2008). . Factors Governing Disinfection Performance Of Pulsed Light Technology For Food Applications. Figure 1.3. Bacterial Inactivation of Bacillus cereus and Bacillus megaterium following PUV. Figure 1.4. Total inactivation of Bacillus megaterium at 80P (Author, 2015). Figure 1.5. Protein concentration increase as a result of internal microbial protein loss following PUV. To determine the relationship between PUV light irradiation and the occurrence of molecular and cellular damage in bacteria of food pathogenic relevance – Bacillus cereus and Bacillus megaterium. Table 1.1. Ct Values for Standards Bacteria Numbers Bacillus megaterium Bacillus cereus 1 x 109 13.543 16.929 1 x 108 14.465 20.728 1 x 107 16.987 24.461 1 x 106 18.486 27.732 1 x 105 21.432 30.399 1 x 104 22.543 32.105
Editor's Notes
- #2: Copyright Colin Purrington (http://colinpurrington.com/tips/academic/posterdesign).