Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
Food Science & Technology
Non Technical Summary
Blueberry demand has increased significantly in the past decade mainly because of the health benefits associated with blueberry consumption. Despite the healthy outcomes of regular blueberry consumption, foodborne illnesses have been linked to fresh blueberries contaminated with pathogenic bacteria. Since fresh blueberries are not subjected to any antimicrobial treatment before being shipped to the market, the hygiene conditions in berry packing environment is extremely important for the production of microbiologically safe and wholesome products. In this project, we will evaluate the hygiene conditions of selected fresh blueberry packing lines and assess the microbial loads on blueberries collected from fresh fruit packing lines. We will also evaluate the efficacy of chemical sanitizers commonly used by blueberry packers in cleaning and decontaminating blueberry packing lines. The research will provide useful information to the blueberry industry.
Animal Health Component
100%
Research Effort Categories
Basic
0%
Applied
100%
Developmental
0%
Goals / Objectives
The demand for blueberries has exceeded its supply during the past decade, primarily as a result of the positive health benefits associated with blueberry consumption (Crozier et al. 2005). Blueberries are one of the most popular berries in the retail market, sold in numerous forms such as fresh, frozen, and processed fruits. The United States ranks first in the production of blueberries in the world, producing over 5.67 million pounds in 2014 (USDA-NASS, 2014). Georgia ranks first in the U.S. in total production and second in total cultivation acreage, yielding a farm gate value of $109 million. An estimated 51 percent of the harvested blueberries were sold as fresh fruit between 2009 and 2011 in the U.S. (Michalska, 2015). At present, fresh blueberries are available in the market all year long, sourced from different regions of the world based on growing seasons (Lin et al., 2003).Despite the healthy outcomes of blueberry consumption, foodborne illnesses have been linked to fresh blueberries contaminated with pathogens (Palumbo et.al 2013). A 2013 outbreak of 162 cases of hepatitis A infection was linked to berries mixed with pomegranate seeds (CDC, 2013a). A 2011 outbreak with 15 cases of E. coli O157:H7 infection in Oregon was linked to frozen strawberry (CDC, 2013b). A 2010 outbreak with 6 cases of S. Newport infection in Minnesota was linked to fresh blueberry consumption (Miller, Rigdon, Robinson, Hedberg, & Smith, 2013). A multistate outbreak of Salmonella Muenchen infection was associated with eating blueberries in 2009 (CDC, 2012). An outbreak of hepatitis A infection in New Zealand was linked to the consumption of fresh blueberries, which were likely contaminated by the berry handlers (Calder et.al, 2003). Fresh blueberries that are ready to be sent to the market do not undergo any treatment or washing before shipping due to the belief that washing will promote mold growth and shorten the shelf life of the product. Since fresh berries are consumed raw or minimally processed, they have the potential to serve as vectors for transmitting foodborne illnesses.The goal of the project is to generate knowledge needed to enhance the microbial safety of fresh blueberries. Specific objectives of the research include:Evaluate the hygenic conditions of fresh blueberry packing lines;Determine the microbial loads on blueberries collected direcly from the packing lines;Assess the biofilm-forming potentials of fecal coliforms isolated from blueberries or bleberry packing lines;Evaluate the clean-ability of blueberry contact surfaces by sanitizers commonly used by blueberry packers; andInvestigate whether pre-adaptation with sub-lethal concentrations of sanitizers will elevate the tolerance of fecal coliforms to antibiotics used in human medicine.Response to the comments made by National Program Leader at NIFA:Would have loved to see more recent numbers and linked threats. Information on berry-assoicated outbreaks has been updated in previous section.If no outbreaks since 2009 is this research really needed? Outbreaks associated with fresh and frozen berries continued to occur after 2009 (CDC, 2013a; 2013b; Miller et al., 2013). Although fresh blueberries are not considered as read-to-eat products, they are often consumed raw. Because of the way that blueberries are produced, it is possible for them to be contaminated with microorganisms, occasionally with human pathogens. Although the outbreaks of illness associated with blueberry do not occur as often as those associated with meat and poultry and leafy green vegetables and seed sprouts, a consumer group has considered berries as one of the ten most risky foods.The U.S. food safety regulation focuses on preventive measures. The current project will collect baseline data on the hygiene condition of blueberry packing lines, which will help blueberry industry setup self-imposed guidelines to produce blueberry with high microbial quality and comply with the new fresh produce safety rules.How will these objectives provide any new knowledge? The project will reveal the overall hygiene condition of blueberry packing lines, which segment(s) of the packing lines are most prone to microbial contamination, once blueberry contact surfaces are contaminated, whether the sanitizers and sanitation practice routinely used by blueberry packinghouses are sufficient to remove them.Some novelty and innovation is needed. There is currently no information on the hygiene conditions on blueberry packing lines. The growers had no knowledge on the problematic areas that they have in packing environment. The highbush blueberry association urgently needs the information to setup industry self-impose guidelines based on the results that will be gathered in the current study.
Project Methods
Year 1: Hygienic conditions of blueberry packing linesFresh blueberry packing lines in six packing establishments in Georgia will be selected for this research project. A delimited area on each of the ten selected sites on the bluberry packing lines will be swabbed with sterile sponges in the morning, at lunchtime, and in the evening of a packing day, and each of the 6 packing lines will be swabbed twice in two separate packing days. Following swabbing, the sponges will be sampled for total aerobic bacteria, yeasts and molds, total coliforms, fecal coliforms, and enterococci. Swab samples will be taken in a sequential manner, from the dumping site to the packaging area along a packing line. Before sampling, the sterile sponges will be hydrated with 25 ml of Dey-Engley (DE) neutralization broth in a sample bag. A surface area (100 cm2) on each site along blueberry packing lines will be swabbed with ten horizontal and ten vertical passes, with a force similar to what is used to remove dry blood from a surface. After swabbing, the sponges will be returned to the sample bags and transported in a car cooler to the laboratory for analysis.The sponges will be hand massaged for 1 min to release collected microbial cells to DE buffer. The buffer will be diluted in 0.1% phosphate buffered saline. Appropriate dilutions will be surface plated onto tryptic soy agar for total aerobic bacteria, potato dextrose agar for yeasts and molds, MacConkey agar for total and presimptive fecal coliforms, and enterococcus agar for presumptive enterococci. The agar plates for total aerobic bacteria, total coliforms, and enterococci will be incubated for 24 h at 37°C. Fecal coliforms will be incubated at 45°C for 24 h. The yeasts and molds will be incubated for 48-72 h at 25°C. All the samples will be plated in duplicates. To determine the differences in microbial loads at selected sampling sites, on different packing lines, and at different sampling times, Fisher's least significant difference (LSD) test in general linear model will be conducted using Statistical Analysis Software (SAS) (version 9.4; SAS Institute Inc., Carey, N.C.).Year 2: Microbial quality of fresh blueberries collected from the packing linesBlueberries will be collected in duplicate in the morning, at lunchtime, and in the evening from the six packing lines in two separate packing days. Collected fruits will be rinsed or homogenized followed by microbial enumeration.Fresh blueberry samples will be collected at the dumping, and packing, areas of the six packing lines. The samples, in duplicate, will be obtained in the morning, at lunchtime, and in the evening of two separate packing days. The collected fruits will be transported in a car cooler for analysis.Microorganisms on the berry samples will be collected using either surface rinsing or homogenization of the fruits. For rinsing, 25 g of fresh blueberries will be aseptically placed in a sterile Whirl-Pak sample bag with 100 ml of 0.1% phosphate buffered saline. The blueberries in the sample bags will be rinsed for 20 min on a platform shaker. For homogenization, 25 g of blueberries in a sterile sample bag containing 100 ml of DE broth will be pummeled for 2 min at normal speed. The rinsates and homogenates will be serially diluted in 0.1% PBS and appropriate dilutions will be plated for total aerobic bacteria, yeasts and molds, total and fecal coliforms, and enterococci as described above.The influence of packing (unpacked vs. packed), sample source (packing lines), sampling time (morning, lunchtime, and evening), and berry sampling method (rinsing vs. homogenization) on berry-borne microbial counts will be determined by Fisher's LSD test in the general linear model using SAS (version 9.4; SAS Institute Inc., Carey, N.C.). Year 3: Ease of microbial accumulation on blueberry contact surfacesTwelve fecal coliforms, two isolated from each of the 6 blueberry packing lines will be used in this study. Biofilm-forming ability of the selected fecal coliforms will be assessed on coupons made of blueberry-contact materials. Two fecal coliforms isolated from each packing line will be streaked onto Luria-Bertini no salt (LBNS) agar and incubated at 37oC for 24 h. One colony of each fecal coliform culture will be transferred into 9 ml of LBNS broth to make 6 bi-strain mixtures. The resulting cultures will be diluted (1:40) in LBNS broth. The diluted cultures will be used for biofilm formation on the surfaces described above. The LBNS not inoculated with fecal coliforms will be used as controls.De-contaminated surface coupons will be placed in a sterile glass container with 200 ml of LBNS broth inoculated with each bi-strain mixture of fecal coliforms. After incubation at 10°C for 7 days, developed biofilms will be quantified using the crystal violet binding assay as previously described by Stepanovic et al. (2014).Two replications of each experiment with duplicate samples will be performed. Data collected will be analyzed using the general linear model of SAS (SAS Institute Inc.). Fisher's LSD at 95% confidence interval will be used to identify the differences in biofilm accumulation on various blueberry contact surfaces.Year 4: Clean-ability of blueberry-contact surfacesThe efficacy of sanitizers that are commonly used by blueberry packers in removing the biofilms formed on coupons made of blueberry contact surface materials will be assessed.Four commercial sanitizers that are commonly used by blueberry packers, including aqueous chlorine dioxide (ClO2), quanternary ammonium compound (QAC), ozonated water (OW), and sodium hypochlorite (NaOCl), will be used in the study. The working solutions of ozonated water and ClO2 will be 3 ppm and 5 ppm, respectively whereas that of QAC and NaOCl will be 200 ppm.The surface coupons with developed biofilms will be rinsed with sterile water three times. Rinsed coupons will be placed in 200 mL of each sanitizing solutions for 1 min at room temperature (ca. 22oC). After the treatments, coupons will be immediately immersed in double strength DE neutralizing broth for 10 min followed by air drying at 60?C for 2 h. The residual biofilm mass on various coupons will be quantified using the crystal violet binding assay described above. The differences in biofilm mass on treated vs. untreated coupons will be compared and the clean-ability of each blueberry contact surfaces by selected sanitizers will be reported.Two replications of each experiment with duplicate samples will be performed. Data collected will be analyzed using the general linear model of SAS (SAS Institute Inc.). Fisher's LSD at 95% confidence interval will be used to determine the significance of differences among the efficacies of sanitizing treatments.Chapter 5: Effect of preadaptation by sub-lethal concentrations of sanitizers on the susceptibility of fecal coliforms to antibioticsThe susceptilibity of fecal coliforms isolated from blueberry packing lines to 15 selected antibiotics will be tested using the disc diffusion assay. Twelve fecal coliform isolates, two from each of the six packing lines will be pre-adapted in sublethal concentrations of sanitizers. The minimal inhibitory concentrations (MICs) of selected antibiotics against sanitizer-adapted and nonadpated cells of fecal coliforms will be compared.Sanitizer adaptation will be done according to the protocol described above, but using pre-determined sub-lethal concentrations. The antibiotics to be tested for their MICs against selected fecal coliforms will be diluted in two-fold series in Mueller Hinton broth, after being dissolved in appropriate solvents. The lowest concentration that did not have visible bacterial growth after overnight incubation at 37oC will be regarded as the MIC. The MICs for sanitizer-adapted and non-adapted cells of fecal coliforms will be compared.