The extracted phytochemicals created inhibition zones against Bacillus subtilis, Staphylococcus aureus, E. coli, and Salmonella spp. of 13, 15, 9, and 11 mm respectively . These results suggest that the phytochemicals found in dried fruits could play a role in the survival of pathogens. Dry fruit related outbreaks. While not many, there have been several foodborne outbreaks associated with low moisture foods . In 2020, an outbreak of the hepatitis A virus associated with dates occurred in the United Kingdom . Twenty eight people were infected and the dates, which were imported from Jordan, were subsequently voluntarily recalled. Another outbreak of hepatitis A occurred in England from semi-dried tomatoes, which infected two people . An outbreak of salmonellosis in Norway was associated with consumption of a Salmonella Agbenicontaminated dried fruit and nut mix . In this outbreak, 39 people were infected. Another outbreak was linked to Salmonella Phage type 13a in a dried vegetable spice mix, in which 108 people were infected in Sweden . Objectives. As discussed above, dried fruits are of great economic importance to California and different processors follow distinct protocols to prepare their dried fruits. In addition to the various drying methods used, pre- and post-drying treatments can also be applied. Unfortunately, there has not been a systematic evaluation of the antimicrobial efficacy of different drying methods or pre- and post-drying treatments or a combination of them.Pathogen contamination of the final products can happen at any processing point. Once happened, it is critical to better understand the behavior of these pathogens in dried fruits. The goal of this study is to fill in the current knowledge gaps associated with microbial safety risks of dried fruit by conducting a challenge study. The three pathogens selected include Salmonella, E. coli O157:H7, and L. monocytogenes. Dried fruits, including peaches, pluots, tomatoes, blueberry grow bag and dates were purchased from local farmers markets for this study.When conducting challenge studies and preparing artificially contaminated food items, there are different carriers that can be used.
The currently available inoculation methods can be grouped into three major categories: a liquid-carrier method, a dry-carrier method, and a no-carrier method. Examples of liquid carriers include peptone water, saline buffers, and ultrapure water. Cocoa butter oil has also been used to carry out inoculation . Dry carriers that have been tested include sand, chalk, and talc . The no-carrier method utilizes freeze- or vacuum-dried cultures or cell pellets and directly applies them to products . Choosing the correct inoculation carrier is critical. Although liquid carriers, such as buffered peptone water or ultrapure water, have been more widely used for delivering pathogens onto product surfaces, the addition of liquid or the introduction of additional moisture into the products changes the moisture content and aw of a dry substrate and may requires additional or extended drying steps . As indicated by Palipane and Driscoll , moisture adsorption/desorption isotherms are inherently non-equivalent, the aw of the product after an additional drying step may not be the same as the original food. Beuchat and Mann used two different methods for inoculating dried cranberries, raisins, and strawberries and date paste. No difference in Salmonella behavior was observed between two inoculation methods . Similar observation was made by Blessington et al. , in which no difference in Salmonella decline was observed between dry-inoculated and wet-inoculated nut kernels. Both studies indicated that when choosing the proper carriers fordried products, the key features or changes that need to be monitored are the physical or chemical properties. In addition, inoculation methods should try to mimic real life contamination scenarios to give the most accurate representation of survival after a contamination event. It is a water-based salt solution containing sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate. It helps maintain the osmolarity balance of bacteria when being used as the carrier for inoculation. However, since another key function if this buffer is to help maintain a constant pH, there is a concern over the use of PBS for inoculation and sample homogenization when measuring the pH of inoculated dried fruits. In this case, before the long-term survival study, the impact of PBS on the pH measurement needs to be studied. An efficient recovery method that can release and recover target bacteria from food surfaces, is the foundation for accurate detection and enumeration.
Stomaching, shaking, rubbing, sonication, pummeling, pulsing, as well as blending have been tested and used for releasing and recovering bacteria from food or environmental samples . Based on the various physical and chemical properties of the samples, the efficacy of these cell recovery method change. As shown by Kim et al. , bacterial populations recovered from pummeled and pulsed iceberg lettuce, perilla leaves, cucumber, and green pepper samples were higher than those recovered from sonicated and hand-shaken samples. However, this trend was not observed on cherry tomatoes. Thus, which bacteria recovery method is more appropriate for sand-inoculated dried fruit needs to be determined.Dried fruits. Dried fruits used for this survival study were purchased from local farmers markets. Fruits used included sundried tomatoes, peaches, peaches processed with sulfur, pluots processed with sulfur, low-moisture Medjool dates, and high-moisture Medjool dates . High-moisture dates are harvested directly from the tree; low-moisture dates are dates allowed to continue drying in nets after falling off the tree and have a harder texture compared to the high-moisture dates. Tomatoes and the sulfured peaches and pluots were sun dried. The peaches that were not processed with sulfur were dried using a dehydrator. Once purchased, the dried fruits were stored at room temperature for up to 1 week prior to use in experiments. Inoculation with wet and dry carriers. Dried fruits were combined with either water or sand by the following methods. Briefly, ultrapure water was added to every 100 g of each of dried fruits and massaged by hand for 1 min. The dried fruits were then transferred to plastic containers with drying racks lined with filter paper . The lids of the containers were taped down slightly ajar with a piece of mesh to cover the opening . The containers of fruit were set out to dry at room temperature for 48 h.
For sand inoculation, 20 g of sand was added to 100 g of each dried fruit and massaged and shaken for 1 min. The dried fruits were then transferred to gallon storage zipper bags and stored at room temperature. For the water-inoculated samples, the pH and aw was measured before and right after the inoculation. After 48 h, the pH and aw of both the wet and dry inoculated dried fruits were both measured. A pH meter and water activity meter were used to take the measurements. Effect of phosphate buffered saline on pH measurement of dried fruits. Phosphate buffered saline was chosen as the wet carrier with which to inoculate the dried fruits. Since PBS is a buffer solution, the impact of PBS on the pH measurement of dried fruits was tested. To do so, dried fruits were combined with either ultrapure water or PBS and massaged and shaken by hand for 1 min. The pH of the sample was measured before the addition of the liquid, immediately after massaging, and after 48 h of drying. To measure the pH, blueberry grow bag size each fruit sample was combined with water or PBS equal to 40% of the sample mass and then stomached for 1 min at the fast setting to homogenize. The pH meter was used to take the measurements. Bacterial cultures and inoculum preparation. The strains of bacteria used for this study were provided courtesy of Dr. Linda J. Harris at the University of California, Davis. Five strains of rifampicin-resistant Salmonella were used. The strains are summarized in Table 1.1.Individual frozen stock cultures were streaked onto tryptic soy agar , Sparks, MD, supplemented with 50 µg/mL of rifampicin , and incubated at 37 °C overnight. Each isolated colony was transferred into 10 mL of tryptic soy agar supplemented with rifampin at 50 µg/mL , and then incubated at 37 °C overnight. One 10-µL loopful of the overnight culture was transferred to 10 ml of fresh TSBR and incubated at 37 °C for another 24 h. The newly inoculated broth was spread onto TSAR plates, 250 µL per plate, one plate per strain, and incubated for 24 h at 37 °C. To recover bacterial lawns from plates, 1 mL of phosphate-buffered saline was pipetted onto each plate, and an L-shaped plastic cell spreader was used to loosen and scrape the lawn. The re-suspended cells were then pipetted into a 15- mL Falcon™ tube . The addition of PBS and lawn scraping was repeated two more times for each plate, for a total of 3 mL of PBS used per plate. Approximately 2.5 mL of culture was recovered from each plate. Once all plates were scraped, 15 mL of the recovered culture from each strain were combined to make the 5-strain cocktail of Salmonella. The cocktail was diluted and plated onto TSAR for calculating the inoculum level. Evaluation of homogenization methods for recovering pathogenic cells from inoculated sand. Salmonella-inoculated sand was used to test the recovery method used for dried fruit . Twenty grams of sand was inoculated with 1 mL of the 5- strain Salmonella cocktail and they were mixed together by hand for 1 min. Samples of the inoculated sand were sampled immediately after mixing and after 48 h of drying. The drying process was done at 40 °C for 48 h in a gravity oven . At each sampling point, three 10-g sub-samples were taken for the analysis of Salmonellacounts.
Each 10-g inoculated sand sample was divided into two portions . These two portions were added to two 24-oz filter bags together with 95 mL of PBS in each bag. One bag was stomached for 1 min using a smasher , while the other bag was shaken by hand for 1 min. The contents were then serially diluted appropriately with PBS and two 100 µL suspensions from each dilution were spirally plated onto TSA with rifampicin and Xylose Lysine Tergitol 4 agar with 50 µg/mL rifampicin . After 24 h of incubation at 37 °C, colonies were counted and the populations determined. Statistical Analysis. One trial was conducted for every test performed in this section. At each sampling point, three samples were analyzed . Means comparison were performed using Excel to determine whether carrier type had a significant impact on pH and water activity of dried fruits as well as if homogenization methods for inoculated sand had a significant impact on recovery of pathogenic cells. Differences between mean values were considered significant at P < 0.05.Inoculation with a wet or dry carrier. Table 1.2 shows the pH and water activity of dried fruits before and after the addition of either water as a wet-carrier or sand as a dry carrier. In low-moisture dates, the initial pH was 5.83 ± 0.06. With both wet and dry-inoculation, the change in pH was statically significant, dropping to 5.24 ± 0.05 and 5.59 ± 0.04 respectively. The initial aw of the low-moisture dates was 0.62 ± 0.03 and showed no significant change after either wet or dry-inoculation, with their values being 0.64 ± 0.00 and 0.61 ± 0.00, respectively. In high-moisture dates the initial pH was 5.59 ± 0.04. The pH did not change significantly afterwet-inoculation. The pH dropped after the dry-inoculation to 5.39 ± 0.04. The initial aw of the high-moisture dates was 0.55 ± 0.02. No significant change was observed after either wet or dry inoculation. The initial pH of the dried peaches was 3.94 ± 0.07, and neither the wet nor the dry inoculation generated significant change on the pH. Additional loss of moisture might have occurred during the 48-h of drying after wet inoculation, which might be the reason why there was differences between the aw before and after wet-inoculation. A similar observation was made in dried peaches made with sulfur treatment, as the aw decreased after wet-inoculation. Both the wet- and dry-inoculation slightly reduced the pH value of the products, changing from 3.59 to 3.51 and 3.48 respectively. In dried pluots, although neither wet- nor dry-inoculation generated any impact on aw, dry-inoculation reduced the pH of the products . For sundried tomatoes, inoculation had no impact on pH but the dry inoculation significantly reduced the aw of the products .