This indicated that after exposing to the drying stress during sand inoculum preparation, this pre-storage stress response enhanced the survivability of Salmonella during storage. This also indicated the impact of inoculation carriers on the survival of pathogens. The differences observed between selective and non-selective agar indicate the formation of stressed or injured cells during storage. For example, in the wet-inoculated peaches made with sulfur treatment, after 5 days of storage at 5 °C, there were 7.21 log CFU/g of L. monocytogenes recovered from TSAR while only 5.32 log CFU/g of L. monocytogenes recovered from MOXR, indicating the formation of approximately 1.89 log CFU/g of stressed or injured cells. The difference between selective and non-selective agar could show up as early as Day 0. For example, when looking into Table 2.4, when E. coli O157:H7 was wet-inoculated onto dried peaches made without sulfur treatment, the differences between TSAR and MACR were up to 1.47 log CFU/g and up to 1.76 log CFU/g . Sometimes, differences between the selective agar counts and the non-selective agar counts increased as the storage length increased. For example, in the same table , on dry-inoculated peaches made without sulfur treatment, blueberry packaging containers when the storage temperature was at 20 °C, the differences between selective and non-selective agar were 1.26 log CFU/g on Day 0 and 2.65 on Day 60 . The difference between selective and non-selective agar counts were seen in the studies conducted by Blessington et al. .
Microorganisms exist in different metabolic states and growth phases under various environmental conditions. Active replication of cells is not always included in all phases . The formation of the viable but non-culturable state and the sub-lethally injured cells pose distinct threat to food safety as the conventional culturing methods might under-estimate the pathogen levels and injured cells might regain their ability to be cultured and cause infections in humans . Although the use of both selective agar and non-selective agar help us minimize the chance of underestimating surviving cells in this study, other detection methods, such as molecular-based detection protocols, may also needed in future studies to better evaluate the surviving levels of various pathogens.The global production of dried fruits increased from 2,246,739 metric tons in 2009/2010 to 3,222,767 metric tons in 2019/2020 . In California, dried fruits are economically valuable specialty crops and 1,174,000 tons of raisins, 325,500 tons of dried prunes, 11,000 tons of dried apricots, and 6,900 tons of dried freestone peaches were produced in 2017 . Among different dried fruits, table dates, which accounted for 35% of world dried fruit production, showed the most significant increase over the last decade . The consumption of dates also has the most significant increase among all dried fruits in the past 10 years . Medjool dates are one of the major varieties in the United States . Medjool dates have also been involved in a recent recall due to possible contamination with hepatitis A . Two types of Medjool dates prepared with different drying methods were used in this study.
Low-moisture dates are dates allowed to continue drying in nets after falling off the tree; high-moisture dates are harvested directly from the tree and have softer texture compared with the low-moisture ones. In addition to dates, the other two dried fruits, dried pluots made with sulfur dioxide treatment and sundried tomatoes were also used in this study. California is the major domestic supplier of sundried tomatoes in the United States . Unfortunately, sundried tomatoes were involved in a foodborne outbreak of hepatitis A .Sulfur dioxide treated dried pluots were also chosen in this study due to its lower pH compared with dates and sundried tomatoes. Dried fruits can get contaminated throughout the production and supply chain from various sources, such as raw materials, processing equipment, and personnel handling . Pathogens such as S. Typhimurium and Listeria monocytogenes have been detected on freshly harvested pears . In a 2014-2015 listeriosis outbreak associated with caramel apples, outbreak isolates of L. monocytogenes were recovered from whole apples . Listeria spp. including L. monocytogenes have been repeatedly detected from fruits or other produce processing facilities . Cross contamination from fruit surfaces to the edible portions can occur during peeling, cutting, and slicing .Inoculation and samples collection of dried fruits with wet and dry carriers. Sundried tomatoes, dried pluots processed with sulfur, high- and low-moisture Medjool dates were inoculated and sampled in the same manner as the dried peaches in Chapter 2 . Dried pluots processed with sulfur and sundried tomatoes were inoculated using the wet carrier. High-moisture Medjool dates and low-moisture Medjool dates were inoculated using the dry carrier. Triplicate samples for microbial enumeration were collected on the day of inoculation, after 48 h of drying , Day 5, Day 15, Day 30, and then every 30 days after that for a total of 6 months. Two trials of the survival study were performed . Water activity and pH measurement. pH and aw were measured following protocols described in Liu et al. .
Briefly, for aw measurement, dried fruits were first cut into slices and the aw was measured with the water activity meter. To measure the pH of the dried fruits, 10 g of each dried fruit were homogenized with 4 mL of MilliQ water in a 24-oz filter bag using the Smasher at fast speed for 1 min. The pH was then measured using the pH meter. All measurements were repeated three times at each sampling or testing point. Microbiological analysis. At each sampling point, three 25-g subsamples were taken from each pathogen × dried fruit combination. Each subsample was combined with 100 mL of PBS in a 24-oz filter bag. The mixture was then homogenized in the Smasher for 1 min, serially diluted with PBS, and spirally plated onto TSAR and selective agar. The same sampling, homogenizing and plating methods were followed as described in Chapter 2. Xylose Lysine Tergitol 4 agar with 50 µg/mL of rifampicin , MacConkey agar with 50 µg/mL of rifampicin , and Modified Oxford agar with 50 µg/ml of rifampicin for Salmonella spp., E. coli O157:H7, and L. monocytogenes, respectively. Statistical analysis. Triplicate samples were analyzed in each of the two trials of the survival study . Means comparisons were performed using Excel to determine whether sampling day and type of media had significant impact on pathogen survival, pH, and aw. Analysis of variance , and Tukey multiple comparison test were performed using R studio to determine whether sampling day, storage temperature, or media significantly impactpathogen survival, as well as sampling day, type of dried fruit, and storage temperature significantly impact pH and aw. Differences between mean values were considered significant at P < 0.05.Survival of Salmonella on various dried fruits. Figure 3.1 and Table 3.1 summarize the survival of Salmonella on two types of dates, dried pluots, and sundried tomatoes. Salmonella was dry inoculated onto dates. The initial inoculation levels were 6.92 ± 0.03 and 6.43 ± 0.07 log CFU/g for low- and high-moisture dates respectively. Salmonella gradually decreased on both low- and high-moisture dates at 5 °C. At the end of storage , 5.31 ± 0.16 log CFU/g of Salmonella were still recovered from low-moisture dates. When the storage temperature increased to 20 °C, blueberry packing boxes a greater than 2.5 log reduction was seen from Day 60 to Day 90. By Day 150, no Salmonella could be recovered from inoculated high- or low-moisture dates by enrichment. Salmonella decreased much more rapidly when inoculated onto dried pluots and sundried tomatoes. The Salmonella population on the wet-inoculated dried pluots and sundried tomatoes immediately after inoculation were 9.39 ± 0.32 and 9.73 ± 0.14 log CFU/g respectively . The initial wet-inoculation levels on dried pluots and sundried tomatoes were 8.09 ± 0.07 and 7.95 ± 0.10 log CFU/g respectively . When looking at the dried pluots, a greater than 3 log reduction was observed during the first 5-day of storage. That reduction was even higher if we compare the XLT-4R data . From Day 5 to Day 15, the population of Salmonella further decreased and another sharp decrease in TSAR count was seen on Day 30. On Day 30, Salmonella could only bedetected by enrichment.
Starting from Day 60, no Salmonella could be detected by neither direct plating nor enrichment from dried pluots. The decreasing Salmonella was more rapidly when the storage temperature was at 20 °C. Starting from Day 30, no Salmonella could be recovered from inoculated dried pluots stored at 20 °C. The survival of Salmonella on sundried tomatoes was better than dried pluots when the storage temperature was at 20 °C. Salmonella gradually decreased from Day 0 to Day 30. After that, a greater 4.92 log reduction of Salmonella was seen between Day 30 and Day 60. Starting from Day 60, Salmonella could no longer be detected even by enrichment from inoculated sundried tomatoes. Another interesting observation made from inoculated sundried tomatoes was the more rapid die-off rate of Salmonella when the storage temperature was at 5 °C than at 20 °C. At 5 °C, Salmonella can only be counted by using TSAR plate on Day 5. Greater than 4 log of injured cells were formed from Day 0 to Day 5 by showing no colony on XLT-4R and 6.13 log CFU/g from TSAR. Starting from Day 15, Salmonella could not be detected by neither direct plating nor enrichment from inoculated sundried tomatoes stored at 5 °C. Survival of E. coli O157:H7. Figure 3.2 and Table 3.2 summarize the survival of E. coli O157:H7 on the dried fruits. The initial dry-inoculation level of E. coli O157:H7 were 5.46 ± 0.75 and 6.04 ± 0.24 log CFU/g on low- and high-moisture dates respectively . The number of surviving E. coli O157:H7 on low-moisture dates gradually decreased when the storage temperature was at 5 °C. A 2.61 Log reduction was seen between Day 150 and Day 180. When looking at the selective agar, surviving cells could not be counted on MACR since Day 120, indicating the formation of injured cells during storage. When the storage temperature was at 20 °C, the grater than 2.61 log reduction happened between Day 60 and Day 90, earlier than samples stored at 5 °C. On Day 180, E. coli O157:H7 on low-moisture dates could no longer be recovered by even enrichment. When comparing the survival of E. coli O157:H7 on low-moisture dates than that on high-moisture dates, a great reduction of cell counts obtained from MACR was seen on Day 60 on high-moisture dates while the similar reduction was observed on Day 120 on low-moisture dates . At 20 °C, that reduction was seen on Day 30 and Day 90 respectively for high- and low-moisture dates. The E. coli O157:H7 population on the wet-inoculated pluots and sundried tomatoes immediately after inoculation were 9.18 ± 0.37 and 9.40 ± 0.20 log CFU/g respectively . On Day 0, a higher initial wet-inoculation level was seen from sundried tomatoes than dried pluots. Based on TSAR, 8.70 ± 0.71 log CFU/g of E. coli O157:H7 was inoculated onto sundried tomatoes while 6.28 ± 0.95 log CFU/g of E. coli O157:H7 was inoculated onto dried pluots. During storage, E. coli O157:H7 decreased rapidly on dried pluots. E. coli O157:H7 could no longer be detected from dried pluots starting from Day 30 and Day 15 respectively for storage temperatures of 5 and 20 °C. For sundried tomatoes, a greater than 4.92 log CFU/g reduction was seen on Day 60 at 5 °C. A similar reduction was seen on Day 30 when the storage temperature was at 20 °C. No E. coli O157:H7 could be detected from sundried tomatoes after 60 days of storage at 5 °C and 30 days of storage at 20 °C. Survival of L. monocytogenes. Figure 3.3 and Table 3.3 summarize the survival of L. monocytogenes on the dried fruits. The initial dry inoculation levels on low- and high-moisture dates were 6.19 ± 0.15 and 6.57 ± 0.46 log CFU/g respectively. L. monocytogenes decreased gradually on both types of dates. On Day 150, L. monocytogenes could only be detected byenrichment from high-moisture dates stored at 5 °C. On Day 180, L. monocytogenes could no longer be detected from either high- or low-moisture dates. L. monocytogenes decreased faster at 20 °C. Starting from Day 120, L. monocytogenes could not be detected from either type of date at 20 °C.