Steering Innovation For Treatment Of Liquid Foods To Eliminate Pathogenic Microbes And Toxins Using Low Wave-Length Uv Irradiation

<p>Optical Property Measurements and SimulationOptical property measurements procedure will be carried out as described by Jacques (2013).UV Dose DeterminationUV dose determination will carried out by developing a MATLAB based Monte Carlo program to deduce the photon fluence in a 5 ml volume in a 10ml beaker. The simulations will automatically assume irradiance to be 1W/m2. This will be adjusted to get the fluence for the correct irradiance values. The program has been described by Wang (1995). Collimated Beam Set-UpAFC will assist in building and designing a collimated beam unit for irradiating samples (Figure 1). This unit will consist of a low-pressure lamp operating at 254 nm wave-length within built cooling device and an automatic timer. The CB beam will be designed and machined so as to deliver a uniform irradiance over a 5ml sample volume. A black plastic tube will be attached from top of the lamp to increase the degree of collimation. Bioassay & UV Sensitivity Testing in the context of this document, an empirical assessment of the inactivation response of a specific microorganism to a controlled dose of UV light, usually in UV reactors/systems is termed as biodosimetry. In order to determine the sensitivity of the challenge organisms, irradiations will be performed in buffered water or test fluid using a collimated beam irradiation device. The test fluid will be normally placed on the horizontal surface below the bottom of the collimating tube. The intensity of the light will be controlled by the distance between the end of the collimating tube and the irradiation vessel. For clean water and microbial surrogates (target micro-organisms). The organism sensitivity is the inverse of the slope and expressed as mJcm-2. This parameter is very important in designing target doses for various microbial surrogates. The same procedure will be used for monitoring UV dose delivery in liquid foods. The dose delivery testing and UV sensitivity are based on the methods described in the UVDGM (USEPA, 2006).Chemical Profiling Analytical methods from the literature based on reverse- phase high performance liquid chromatography (diode array detector or evaporative light scattering detection) and gas chromatography-mass spectrometry for the determination of polyphenols, vitamins, sugars and amino acids will be adapted (Tsao & Yang, 2003; Ma et al. 2014; Neves & Morais, 1997). If possible the method will be streamlined in order to facilitate a high throughput of samples. The above listed compounds will be measured by reverse-phase high performance liquid chromatography (Agilent 1100 series and Shimadzu CLASS-VP System) equipped with a UV or ELSD detector. Separations will be performed on a classical C18 column using a binary solvent system. Limit of detection, limit of quantification and compounds recovery will be carried out to assess the validity of the methods. HMF will be quantified as per the method described by Arif?na et al. 2014), if present. Microbial Analysis (Bacterial strains and growth conditions) Salmonella enterica serovar Typhimurium ATCC 14028, Salmonella enterica serovar Montevideo ATCC 8387, and E. coli O157:H7 strains ATCC 700728, ATCC 35150, and Listeria monocytogenes strains ScottA and V7 (4b) will be used in this study. Strains of Salmonella and E. coli O157:H7 will be stored with 20% glycerol at −80 °C in tryptic soy broth, (Difco Co.) and L. monocytogenes in Listeria enrichment broth (Difco Co.).Growth and inactivation studies. A loop inocula obtained from frozen bacterial cultures will be respectively introduced to tubes containing 10 ml of tryptic soy broth or Listeria enrichment broth propagating medium and placed at respective incubating temperatures. The E. coli O157:H7 and Salmonella strains will be inoculated in tryptic soy broth tubes prior to incubation at 35 °C for 24 h. Listeria monocytogenes strains will be inoculated in Listeria enrichment broth (LEB) and incubated for 24 h at 37 °C. The E. coli O157:H7 and Salmonella cultures will be streaked on tryptic soy agar (TSA) plates and Listeria monocytogenes cultures will be streaked on Listeria enrichment agar plates.A single colony of each strain of E. coli O157:H7, Salmonella L. monocytogenes will be picked and then added to 50 mL tubes containing 25 mL of each selective medium and incubated overnight while shaken at 37 °C. 5 mL of the overnight of each strain of E. coli O157:H7, Salmonella L. monocytogenes will be transferred to 500 mL of fresh corresponding medium and incubated at 37 °C for 18 h to obtain early-stationary phase cells. Incubation for 24 h allowed the respective bacteria to approach the stationary phase of growth. For E. coli O157:H7, Salmonella, and L. monocytogenes strain inactivation trials, the overnight culture of each strain will be homogenized and distributed in ten 1 mL Eppendorf tubes that will be centrifuged at 1000 rpm for 2 min at an ambient temperature. 0.8 mL of the supernatant phase will be discarded and the other 0.2 mL of all ten tubes will be spread on 50 mL of fresh samples (liquid products), to obtain a concentration of cells around 8 log10 CFU/ml. Afterwards, 5 mL of the homogenized mixture will be transferred to a 10ml beaker for irradiation experiments. For enumeration, decimal dilutions will be made with 0.1% peptone water and samples will be surface plated in triplicate on respective medium. For Salmonella onto Xylose-Lysine-Desoxycholate (XLD) selective Agar (BD, Franklin Lakes, NJ) plates; E. coli O157:H7 onto sorbitol-MacConkey agar, supplemented with potassium tellurite and cefixime; and L monocytogenes will be plated on Listeria enrichment agar plates. The plates will be incubated at 37 °C for 24 h and counted. To determine the D10 value (mJcm-2) for different targets, log inactivation data will be plotted against selected doses.MS2 (single stranded RNA virus) and T1 (double-stranded DNA virus) enumeration and testing will be carried out at GAP ENVIROMICROBIAL SERVICES INC, Canada. The host used for T1 enumeration and propagation is E. coli CN13 ATCC 700609 and MS2 uses an E. coli host strain, ATCC 23631. GAP uses these two strains of E.Coli for virus culturing and platting. Cytotoxicity AnalysisThe complete cell culture media will be treated with a range of low, medium and high doses of UV. After irradiation, the media will be filtered through 0.2µm filters. Effects of irradiated culture media on human cells will be established using cell viability assay and colony formation assay. Normal human intestinal myofibroblasts cells (Lonza) will be maintained in non-irradiated culture media. Cells will be seeded in a 96-well plate. After 24 hours, cell culture media will be replaced with irradiated cell culture media. After another 24, 48, 72 or 96 hours of treatment, cells will be subject to MTT assay to determine cell viability as we previously established (Qiu et al., 2011). In colony formation assay, cells will be cultured in 6-well plates for 24 hours, and then treated with irradiated media for 10 days. The numbers and sizes of colonies formed will be quantified after staining with crystal violet (Nutakul et al., 2011). We will utilize flow-cytometry analysis for cell cycle and apoptosis to further determine the effects of irradiated media on the cell cycle progression and cell death of normal human intestinal myofibroblasts cells. As we previously described, cells will be cultured in 6-well plates for 24 hours, and then treated with irradiated media for 24, 48, 72 or 96 hours. Cells will be harvested, stained, and analyzed by flow-cytometry (Qiu et al., 2011).</p>