Table 1.2 shows the pH values of dried fruits before and after using PBS or MilliQ water as the wet carrier and then homogenizing with PBS or MilliQ water. As shown in Table 1.3, no difference was observed between pH measurements taken from samples inoculated with PBS or MilliQ water and homogenizing with PBS or MilliQ water for most of the samples. The only significant difference in pH measurement was observed from dried peaches that were inoculated with PBS and dried for 48 h. When these samples were homogenized with PBS or MilliQ water for pH measurement, the pH taken from samples homogenized with MilliQ water were higher than the pH taken from samples homogenized with PBS . Evaluation of homogenization methods for recovering pathogenic cells from inoculated sand. The efficacy of recovering Salmonella from inoculated sand by stomaching or manually shaking was evaluated. Inoculated sand samples were taken right after inoculation and after 48 h of drying in the oven. The concentration of the Salmonella cocktail was 11.07 ± 0.04 log CFU/mL. Since 20 g of sand was mixed with 1 mL of liquid culture, the theoretical inoculation level in sand is 9.77 log CFU/g. As shown in Table 1.4, 9.51 ± 0.04 log CFU/g of Salmonella was recovered from freshly inoculated sand by stomaching. After 48 hours of drying, 6.93 ± 0.09 Log CFU/g of Salmonella was recovered from the inoculated sand by stomaching. Drying at 40 °C for 48 h caused an approximately 2.5 log reduction of Salmonella. The differences between counts obtained from TSAR and XLT-4R indicated the formation of injured cells during drying.
Comparing the cell numbers recovered by stomaching with the cell numbers recovered by shaking methods, blueberry box no difference in TSAR counts was observed from inoculated sand that has been dried for 48 h . When looking at the XLT-4R counts, stomaching method had higher counts on XLT-4R than shaking method . Similar trend was observed from freshly inoculated sand. Based on this result, stomaching was used in the following studies for recovering pathogens from inoculated sand or sand-inoculated dried fruits.The objective of this chapter was to determine the inoculation carriers for different dried fruits, the buffer system for pH measurement, as well as the pathogen recovery method for sand-inoculated dried fruits. Based on the results, both wet and dry inoculation changed the pH of low-moisture dates, while generating no significant impact on the aw of the samples . A similar observation of aw before and after inoculation was found in high-moisture dates. Dry inoculation reduced the pH of high-moisture dates more than the wet-inoculation. However, when looking at the physical properties of the inoculated dates, wet inoculation caused the skin to start to peel from the dates. Based on these observations, dry inoculation was chosen to inoculate dates. In addition, since dates are typically grown in sandy regions where sand storms are common and some dates are cleaned by air pressure without any contact with water before packaging . For dried peaches, the wet inoculation reduced the aw slightly compared to the noninoculated ones . This reduction in aw might be caused due to the additional 48 h drying after inoculation. 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 in the original food.
Dry inoculation did not impact the aw of either dried peaches. Neither dry nor wet inoculation altered the pH of dried peaches without sulfur treatment significantly. They both reduced the pH of dried peaches made with sulfur treatment. Based on the measurement, there is no strong preference between two inoculation methods. In this case, it was decided to use both wet and dry carriers to inoculate dried peaches.For dried pluots and sundried tomatoes, wet inoculation generated no impact on their pH or aw. In addition, since pluots and tomatoes are also often processed in large quantities and washed before drying like the peaches . Using a liquid carrier could help represent possible contamination from water during washing. The impact of different inoculation methods on the behavior of inoculated pathogens can be determined by the types of products. As discussed earlier, both Blessington et al. and Beuchat and Mann did not find any significant differences between wet- and dry inoculated products. Bowman et al. monitored the survival of Salmonella on black peppercorns and cumin seeds. The tested methods included wet-inoculation with Salmonella cells grown in TSB wet-inoculation with Salmonella grown on TSA biofilm inoculation , and dry inoculation with TSAgrown-Salmonella-inoculated sand. Their results showed that the biofilm inoculation method had the least Salmonella reduction. When comparing the dry inoculation method vs. the wet inoculation methods , the dry inoculation method had no difference with wet inoculation method on black peppercorn but had less reduction of Salmonella on cumin seed. One potential explanation for these differences might be the content and release of different amounts of antimicrobials from the food during wet inoculation . Deng et al. investigated the impact of aw, pH and temperature on the survival of E. coli O157:H7 in a commercial dry infant rice cereal. Results showed that much better survival was seen when the pH was at 6.8 than 4.0. In the current study, if PBS is chosen to carry out the wet inoculation, how it impacts the pH and pH measurement needs to be understood before the experiment. PBS is more often used as a carrier than MilliQ water because of its ability to prevent cells bursting due to osmosis .
Based on our results, wet inoculation with PBS did not have a significant impact on the pH of the dried fruit and using PBS for pH measurement also did not impact the result . Thus, PBS was appropriate to use as the liquid carrier for the dried fruits as changes in pH can influence pathogen survival in dried foods. In the Blessington et al. study, in which both sand and chalk were tested as dry carriers, bigger impact on the weight of the inoculated nuts was observed from the chalk inoculated nuts, especially almonds . Based on our own lab’s data, chalk and talc are lighter and tend to fly around when conducting the inoculation. When dealing with pathogens, it is not safe. Thus, sand is a better option compared with other dry carriers. Unfortunately, sand is an abrasive substance and could potential damage cells during inoculation. Sahin used sand in their study to disrupt bacterial cell walls, indicating that sand can lead to cell abrasion if used in a particular way. When comparing stomaching and shaking by hand in this study for homogenization of samples, Salmonella was recovered at significantly higher levels from stomached samples than shaken samples . When looking at the cell counts obtained from the selective agar, stomaching recovered a higher number of Salmonella from sand samples that had been dried for 48 h. The major difference observed in this study was the injured cells formed during drying as indicated by the differences between counts on TSA and XLT-4 agars. Based on these results, stomaching was chosen for recovering pathogens from sand-inoculated samples.Sulfur treatments are often used in dried fruits to preserve color . In other food applications, blueberry package sulfur is often used as an antimicrobial but it has not been evaluated in this capacity for dried fruits. Based on a recent report , dried fruits produced in the U.S. contain free SO2 ranging from 20 to 635 ppm. Among all the products tested, dried pineapple contains the least free SO2 while dried apples contain the highest levels of SO2. Dried peaches contain 355 ppm SO2 when measured in slurry . Witthuhn et al. evaluated the sulfur dioxide content in various commercial South African dried fruits and the microbial quality associated with these products. Results showed that raisins, Clingstone peaches, apricots, nectarines, and prunes contained approximately 1,302, 866, 1,318, and 806 mg/kg of SO2 respectively. However, no correlation between the SO2 content and the microbial counts was identified. On the other hand, Karabulut et al. showed that the total number of microbes present on sulphurated dried apricots were 3 logs less compared to the non-sulfured dried apricots. Türkyılmaz et al. reported a significantly lower total microbial population on dried apricots made with the sulfur dioxide treatment compared to non-sulfured dried apricots. In the Liu et al. study , the authors have shown that pathogens survived at higher levels for a longer period on dried apricots made without sulfur treatment.
Sulfur dioxide is considered by the FDA as generally recognized as safe . The FDA does not set limits on the amount of sulfur dioxide permitted in foods. However, proper labeling is required for foods containing levels of sulfur dioxide that exceeds 10 ppm. In dried fruits, less than 100 ppm is typically found . However, when inhaled or ingested by sensitive groups, it can induce asthma, even in low concentrations . The California Office of Environmental Health Hazard Assessment has developed a proposed MADL for SO2 of 220 µg/day . Taking dried peaches as an example and assuming 26 grams of dried peaches are consumed each day, the estimated exposure to SO2 is 191 µg/day .Bacterial strains and preparation. The strains of bacteria used for this survival study were provided courtesy of Dr. Linda J. Harris at the University of California, Davis. Five strains each of rifampicin-resistant Salmonella spp., Listeria, and E. coli O157:H7 were used. The strains are summarized in Table 2.1. Preparation of inocula. Individual frozen stock cultures were streaked onto TSAR and incubated at 37 °C overnight. Each isolated colony was transferred into 10 ml of TSBR, 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, 6 plates 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 2 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 and a total of 15 mL cell suspension was recovered for each strain. Once all plates were scraped, 15 mL of the recovered culture from each strain were combined to make the 5-strain cocktail. A 5-strain cocktail was made for each of the pathogens and was then used to inoculate dried fruits. Each cocktail was diluted and plated onto TSAR for calculating the inoculum levels. Inoculation of dried peaches with wet or dry carriers. Before inoculation, 3 samples of uninoculated dried peaches were homogenized with 100 mL of PBS. One hundred microliters of each of the homogenates were plated on to TSAR to check for the presence of rifampicin-resistant bacteria. This was done to ensure that any bacteria present on plates during the study were from the rifampicin-resistant inoculum used and not from background microbes. Once the 5-strain cocktails were prepared, they were used to inoculate the dried peaches or sand. For sand inoculation, 1 mL of each 5-strain cocktail was added for every 20 g of fine white sand and mixed thoroughly. The inoculated sand was then spread as thinly as possible across a sheet of filter paper in a metal tray. The tray was placed in a gravity oven at 40 °C to dry for 48 h. Once dry, 160 g of the inoculated sand was used to inoculate 800 g of dried peaches and massaged into the fruit for 1 min by hand. The dry inoculated peaches were transferred into Ziploc bags and placed in a plastic container for storage at ambient or refrigerated temperature for 6 months. For wet inoculation, the remaining 5-strain cocktail was added to enough PBS to make a 1:10 dilution.Dry-inoculation, due the cell loss during the drying of inoculated sand, had lower initial inoculation levels. On Day 0 , there were 7.15 ± 0.11 log CFU/g of Salmonella on dried peaches. Storage temperatures directly impacted the die-off pattern of Salmonella.