KILLING BEFORE MILLING: UTILIZATION OF VACUUM STEAM PASTEURIZATION FOR CONTROLLING ENTERIC PATHOGENS ON WHEAT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1021872
• Grant No.: 2020-67018-30782
• Cumulative Award Amt.: $498,615.00
• Proposal No.: 2019-06908
• Project Start Date: Jun 1, 2020
• Project End Date: May 31, 2025
• Grant Year: 2020
• Program Code: [A1332]- Food Safety and Defense

Recipient Organization
NORTH DAKOTA STATE UNIV
1310 BOLLEY DR
FARGO,ND 58105-5750

Performing Department
Plant Sciences

Non Technical Summary
Wheat flour is an agricultural product, which does not undergo a kill step that would effectively reduce microbial populations during the milling process. While flour is not intended to be consumed as a RTE food, some products such as raw cookie dough, frozen doughs, or cake batters are often tasted by consumers before baking. Additionally, failure to properly wash hands and surfaces after contact with raw dough can lead to cross-contamination of other foods. An increase in outbreaks and recalls associated with wheat flour and wheat flour products, as well as pressure from new legislative regulations have presented the milling industry with a new challenge. Traditional pasteurization methods utilize high temperatures to reduce microbial populations; however, high temperature treatment can greatly reduce the functional properties of wheat flour. Development of a process that can actively reduce microbial populations while maintaining wheat quality is of great interest to wheat processors (see letter of support from North Dakota Wheat Commission). Vacuum steam pasteurization (VSP) is the application of steam to food products under a vacuum, resulting in steam at temperatures below 100°C. Steam allows for improved heat transfer, and subsequent microbial inactivation, compared to application of dry heat. The Simsek and Bergholz labs have focused on the potential for vacuum steam pasteurization to inactivate pathogens on wheat grain.The data collected will provide needed information regarding the survival kinetics and duration of enteric pathogens on wheat kernels, whether pathogens can increase in numbers during tempering, and whether pathogens localize to the crease of the kernel. These data will be important for designing effective control measures for foodborne bacterial pathogens on wheat.Also, though it is important to have a treatment to reduce pathogen contamination in wheat, the flour's functionality must be maintained. Thus, it is of utmost importance to conduct flour and end-use quality evaluation for flours treated with VSP. The milling company must ensure that any treatment to the grain for the purpose of reducing microbial contamination will not negatively impact the functionality of the flour. These possible changes associated with the VSP treatment could also affect texture and flavor of the end products. It will be important to conduct sensory evaluation of products prepared from VSP treated flours to ensure that there are no changes to flavor or texture due to the VSP treatment. When considering a treatment to food products, consumer acceptance is one of the most important factors when determining that treatments viability as an addition to the processing of that food.Pilot-scale processing is an important step in the scale-up process from lab-scale experiments to commercial-scale processing. Pilot-scale vacuum steam pasteurization equipment with a capacity of ~250kg is available for experimental use from Napasol. Once we have determined the pasteurization parameters that result in maximal pathogen inactivation while maintaining flour functionality, and confirmed the appropriate surrogate strain, the vacuum steam pasteurization process for wheat grain will be validated on the pilot-scale equipment. Surrogate inactivation will be quantified and milling quality assessed, to demonstrate effective scale-up of this potential control measure for pathogens on wheat.The long-term goal of the project is to develop and validate the use of vacuum steam pasteurization to inactivate enteric pathogens on wheat grain.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	1549	1100	30%
712	1549	1100	30%
503	1549	1100	40%

Knowledge Area
501 - New and Improved Food Processing Technologies; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 503 - Quality Maintenance in Storing and Marketing Food Products;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

salmonella and enterohemorraghic e. coli

flour

flour recall

vacuum steam pasteurization

wheat

Goals / Objectives
The three primary objectives to achieve our long-term goal are:1. Determine the fate of enteric pathogens on wheat grains during storage and tempering2. Quantify pathogen inactivation, microbial shelf-life, functional and sensory impacts of vacuum steam pasteurized soft wheat3. Validation of microbial inactivation and milling quality of wheat processed with large scale vacuum steam pasteurization equipmenta) Project objectives and their relationship to program area priorities: Based on the above rationale, this research will address the AFRI Foundational and Applied Science Program priority area for Food Safety and Defense (A1332) involving the i) development and validation of innovative technologies for food processing and ii) development of effective interventions for reducing contaminants in foods.b) Potential for long range improvement and sustainability of U.S. food systems: In recent years there have been several multi-state outbreaks of pathogen contamination of wheat flour in the U.S. These outbreaks are resulting in major loss and waste of food products in the U.S. These recalls result in losses of millions of dollars for milling companies. In the last 10 years there have been four outbreaks directly related to wheat flour. Of particular note are the two recent outbreaks in the United States in 2016 and 2019. These outbreaks caused 84 illnesses, which resulted in 20 hospitalizations. Results from the proposed research will provide long range improvements and sustainability to the U.S. food system by reducing costs associated with product recalls and outbreak management.c) Novel ideas and contributions: The proposed study provides data on the use of vacuum steam pasteurization from lab scale to large scale processing, and will provide not only the microbial safety data, but essential product quality and end-user sensory data. This holistic set of data and analyses are necessary for adoption of an effective control measure by the food industry.

Project Methods
Objective 1:Salmonella and EHEC strains will be used for all of the experiments in objective 1. The stains include S. Enteritidis PT30 (ATCC BAA-1045), S. Agona FSL S9-0322, S. Tennessee FSL R6-0494, and S. Montevideo FSL R8-3881.The bacterial strains will be streaked onto LB agar and incubated at 37°C for 20 hours. Single colonies will be transferred to LB broth and incubated at 37°C for 16 hrs. Cultures will be used to spread inoculate LB agar plates. Following inoculation, wheat will be conditioned with saturated LiCl to reduce the aw. Inoculated wheat will be subdivided into 50g portions in Whirl-Pak bags, which will then be placed into Mylar bags and sealed for long term storage at 21-22°C. Duplicate 25g samples will be opened, diluted in Butterfield's buffer, homogenized in the Stomacher, and plated on the appropriate selective agar over a period of 9 months.Wheat will be inoculated with each strain as described in Task 1. Inoculated wheat will be tempered according to AACCI Method 26-95.01. A control sample of inoculated wheat will also be monitored over the tempering time, but no water will be added to allow for direct comparison of changes in pathogen numbers due to the addition of water during tempering. Uninoculated wheat will also be tempered and monitored over time. The tempering process takes 16 hours, and eight 100g aliquots of inoculated wheat for each strain will be placed into tempering jars and tempered. Aliquots will be diluted with Butterfield's buffer, homogenized in the Stomacher, and plated on the appropriate selective agar.Plasmid pFCcGi, containing constitutively expressed mCherry will be obtained from AddGene and transformed into each bacterial strain. This plasmid was selected as it has been designed for use in pathogenic enteric bacteria (Salmonella), and has constitutive expression of the fluorescent protein. Transformation will be verified by detection of fluorescent colonies. mCherry tagged strains will be inoculated onto wheat as described in Task 1, and used for microscopic evaluation of where the pathogen localizes on the wheat kernel.Fluorescent tagged strains will be inoculated onto wheat and prepared for long term storage as described above. For each strain, 10 kernels will be sampled and prepared for microscopic imaging after aw equilibration and every week for 4 weeks. Kernels will be imaged in 3-D using the Zeiss Axio Observer Z1 LSM700 at the Advanced Imaging and Microscopy Core laboratory at NDSU.Fluorescent tagged strains will be inoculated onto wheat and prepared for tempering as described above. The tempering process will also be carried out as described above, and 10 kernels will be sampled at each of the 8 tempering time points. These data over the duration of tempering will be compared to determine if pathogens are collecting in the crease of the kernel.Objective 2: The lab scale VSP system to be used in this set of experiments has been described previously. HOBO Thermocouple Data Loggers are used to record processing temperatures in 15 sec intervals. Processing temperatures of 65 and 75°C required the vacuum to be maintained within a range of 230-275 mbar and 356-421 mbar, respectively.Eleven pathogens will be inoculated onto soft wheat kernels at a target density of 8 log CFU/g. Following equilibration of water activity after inoculation, 25g portions of wheat will be placed into sterile cotton bags. 1kg batches of soft wheat kernels will be pre-heated to 40°C for 60 minutes. Three of the 25g portions for each strain will be placed within the 1kg batch of wheat for pasteurization. Pathogen numbers will be enumerated after 0, 2, 4, 6, 8, and 10 minutes at the target temperature. Three independent replicates of each strain will be inoculated onto wheat grain and treated at both temperatures.To verify E. faecium can also serve as an effective surrogate on wheat, E. faecium NRRL B-2354 (ATCC 8459) will be inoculated onto wheat using the procedure described for pathogens. Survival during vacuum steam pasteurization will be quantified in triplicate at the same time as the pathogens. Survival parameters for E. faecium will be compared to those of the Salmonella and EHEC strains.Three 1kg batches of soft wheat will be pasteurized for 5 and 10 minutes at 65°C and 75°C with the lab-scale VSP unit. 3 batches of unpasteurized grain will be included as the control. Control and pasteurized wheat will be tempered to 14-15% moisture content and milled on a Buhler laboratory mill to produce whole wheat flour. Flour will be packaged in air tight plastic packaging and stored at room temperature (22-25°C) for 3 months. Total aerobic microbes, coliforms, yeasts and molds will be quantified immediately after milling, and after 1, 2, and 3 months of storage. At each sampling time, 2 x 25g samples will be collected from whole wheat flour from each time/temperature combination, homogenized in a Stomacher with Butterfield's dilution buffer, serially diluted, and plated onto plate count agar (PCA), violet red bile agar (VRBA), and DG18 agar. Colonies will be enumerated after incubation at 37°C for 24 h (PCA and VRBA) or 25°C for 7 days (DG18).The flour will be tested for lipid oxidation according to AACCI approved method 58-16.01. Moisture content and aw will also be monitored. Enzyme activities will be measured using AACCI approved methods and Megazyme test kits.Flour quality and functionality will be following AACCI approved methods. To evaluate end-product quality pan bread, sponge cake and cookies will be made and evaluated using AACCI standard methods. Changes in protein quality will be determined by measuring wet gluten and protein composition will be analyzed with HPLC.Sensory evaluation will be done on bread, cake and cookie samples. A trained panel will evaluate the samples for rancidity and staleness using AACCI approved methods. A triangle test will also be performed according to AACCI approved method to determine if changes in stored flours can be detected.Objective 3: Wheat will be shipped to the Napasol pilot-scale facility in France for processing in 180kg batches. Wheat inoculated with the surrogate will also be shipped to Napasol to be included in 4 processing runs. Prior to processing, wheat will be pre-heated to 40°C using forced air in a closed bin. Come-up time, temperature, and processing time will be recorded for all processing runs and included in the analysis. 10 inoculated samples will be included in each processing run, distributed throughout the processing chamber. 10 inoculated samples will be shipped but not processed, and will serve as the 'transport controls' to monitor for any changes in bacterial numbers that may occur during the shipping and handling of samples. A total of 50 wheat samples inoculated with the surrogate will be enumerated by Napasol's independent contract laboratory in France.Four batches of 180kg wheat will be processed in runs separate from the surrogate validation. Processed wheat will be shipped to NDSU for milling quality evaluation, using AACCI standard methods. The wheat will be tempered and milled as described in objective 2 task 3. Flour quality (moisture, protein, ash, color, total starch and starch damage) will be determined according to AACCI approved methods. Flour functionality will be evaluated during by measuring starch pasting profile according to AACCI approved method 76-21.02, dough quality will be evaluated using a farinograph following method 54-21.02. To evaluate end-product quality pan bread, sponge cake and cookies will be made and evaluated using AACCI standard methods. Changes in protein quality will be determined by measuring wet gluten 51 and protein composition will be analyzed with HPLC. Enzyme activities will be measured to evaluated changes in amylase, polyphenol oxidase and xylanase using AACCI approved methods and Megazyme test kits.

Progress 06/01/23 to 05/31/24

Outputs
Target Audience:The target audience is food safety, cereal science and food science researchers and industry professionals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided opportunities for training and professional development of an undergraduate student, two graduate students, and a postdoctoral scientist. How have the results been disseminated to communities of interest?Results have been disseminated to cereal science, food safety researchers and milling industry professionals through presentations at the annual IAFP, IFT meetings,publication of thesis and peer-reviewed research articles. What do you plan to do during the next reporting period to accomplish the goals?We will build upon our current findings regarding the impact of inoculum growth method on pathogen survival on wheat grain and focus on assessing the impact of inoculum growth method on pathogen reduction during tempering. We will also utilize fluorescence scanning microscopy to determine how pathogens may transfer from contaminated to uncontaminated wheat kernels during the tempering process.

Impacts
What was accomplished under these goals? In the past year, we have focused on determining the fate of enteric pathogens on wheat grain during storage and tempering, especially as tempering solution additives are seen as a low cost solution by the milling industry. Previous studies have shown inoculum prepared with the surface growth impacted the population stability and inactivation kinetics of Salmonella, and we aimed to quantify and model the survival kinetics of Salmonella and STEC on wheat grain using four different inoculum growth methods. Three strains of Salmonella (serovars Agona, Enteritidis, and Mbandaka) and three strains of STEC (serotypes O157:NM, O26:H11, and O121:H19) were inoculated onto wheat at 8 log CFU/g using different inoculum growth methods: aerobic liquid growth in LB (L+), aerobic and anaerobic surface growth on LB agar (S+/S-), and aerobic surface growth on tryptic soy +1% glucose agar for acid-adaptation (SA+). After inoculation, aw was adjusted to 0.45. Bacterial populations were quantified by plate count after inoculation, after water activity equilibration (Day 2), and during storage at 20°C, 65% RH for up to six months. Weibull-Mafart's model was used to fit the inactivation of Salmonella and STEC populations after water activity equilibration. After equilibration, aw of wheat grain was 0.47±0.02 and remained stable during storage. Differences in inactivation rates were found to vary by inoculum growth method. The first decimal reduction ranges from 14.2 to 34.5 days for L+, 26.0 to 73.6 days for S+, 20.1 to 60.7 days for S-, and 17.4 to 57.2 days for SA+. Pathogens grown using the L+ method had significantly lower survival rates on wheat compared to those prepared by S+ and S-. S. Enteritidis survived significantly better than S. Agona and STEC on wheat when the inoculum was prepared with the S+ and S- methods. Survival kinetics of Salmonella and STEC on wheat grains are impacted by the inoculum growth method; differences between species and within species among isolates were also observed. During the past year, within Vacuum Steam Pasteurized (VSP) (75oC for 8 min) treated and untreated soft red wheat samples which were single and double milled using a stone mill, microbial activity measurements were done. These stone-milled whole wheat flour samples (both treated and untreated) obtained were then stored for 84 days at room temperature. At specific time intervals (Day 0, 14, 28, 42, 56, and 84) the stored flour samples were taken out and flour quality was measured in terms of microbial activity. For microbial activity, 10g flour samples were suspended in 90 mL Butterfield's dilution buffer, mixed, and serial dilutions were made. For each sample, 10µL of each dilution was plated in duplicates onto plate count agar (PCA), violet red bile agar (VRBA), and DG18 agar plates. The plates were incubated at 37°C for 24 h for PCA and VRBA and 25°C for 7 days for DG18 agar plates and then the colonies were enumerated. The DG 18 agar was used to enumerate yeast and mold from the whole wheat flour samples. There were only mold colonies observed in the samples. Over the period of storage, VSP treated flour had or tended to have less mold than did the untreated flour. The VRB agar was used for detection and enumeration of coliform bacteria in the flour samples and there was a (p<0.05) reduction in log CFU/g of these microbes in VSP treated flour as compared to untreated flours, and there were no significant (p<0.05) changes in the coliforms over the period of storage. PCA is used for enumeration of viable bacteria growth in the whole wheat flour samples. Overall, VSP resulted in significant (p<0.05) reduction in log CFU/g plate count as compared to untreated flour samples. Over the period of storage, no (p<0.05) changes in log CFU/g of VSP single milled and untreated single milled flour samples, but there were some changes in VSP double milled and untreated double milled samples, but they did not follow any particular pattern. Thus, this study highlights the use of VSP in reducing the microbial activity of whole soft red wheat flours.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Lin, Y., Q. Suehr, K. Dolan, S. Simsek, T. M. Bergholz. Inactivation of Salmonella and Shiga-toxin producing Escherichia coli on soft wheat kernels using vacuum steam pasteurization. 2023. International Journal of Food Microbiology. DOI: 10.1016/j.ijfoodmicro.2023.110375
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lin, Y., C. Peterson, and T. M. Bergholz. Inoculum growth method impacts the survival kinetics of Salmonella and Shiga-Toxin Producing Escherichia coli inoculated onto wheat grain. International Association for Food Protection Annual Meeting, Toronto, Canada. 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: H. Kaur, D. Olsen, S. Simsek, S. Islam, and A. Vegi. Effect of milling, storage and vacuum steam pasteurization on soft red wheat flour quality, functionality and end-product quality. IFT First, Chicago, IL, USA. 2023.
• Type: Theses/Dissertations Status: Published Year Published: 2023 Citation: H. Kaur. 2023. Impact of Vacuum Steam Pasteurization on Whole Wheat Stone Milling and Flour Functionality of Soft Red Wheat. M.S. Thesis. North Dakota State University, Fargo, North Dakota.

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:The target audience is food safety, cereal science and food science researchers and industry professionals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided opportunities for training and professional development of an undergraduate student, two graduate students, and a postdoctoral scientist. How have the results been disseminated to communities of interest?Through presentations at professional meetings such as the Cereals & Grains Association, International Association for Food Protection and in peer-reviewed journal articles. What do you plan to do during the next reporting period to accomplish the goals?For objective 1: evaluate the impact of varying storage temperatures on survival of Salmonella and STEC on wheat grain. We plan to continue working on the remaining project experiments to accomplish the project goals.

Impacts
What was accomplished under these goals? Objective 2: In previous lab-scale studies, vacuum steam treatment was able to reduce Salmonella Enteritidis PT30 and Shiga-toxin producing Escherichia coli (STEC) O121 levels on soft wheat kernels while maintaining flour quality and gluten functionality. We used a newly designed lab-scale vacuum steam pasteurizer (VSP) to evaluate its efficacy to inactivate multiple strains of Salmonella and STEC on soft wheat by modeling the non-isothermal time-temperature history during treatment and reduction of the microbial populations. The results demonstrated that vacuum steam treatment could effectively disinfect wheat grains inoculated with enteric pathogens. In this study, Salmonella strains were less thermally resistant than STEC strains. The D75°C of Salmonella strains were 2.8 and 3.2 min, and the D75°C of STEC ranged from 3.1 to 4.6 min. E. faecium had a D75°C of 3.3 min, which indicates that it could be used as surrogate for larger scale evaluation of vacuum steam pasteurization in the future but was not conservative compared to some of the STEC strains. Also, VSP (75oC for 8 min) treated and untreated soft red wheat samples were single and double milled using a stone mill. Whole wheat flour samples obtained were then stored for 84 days at room temperature in individual kraft-paper bags. At specific time intervals (Day 0, 14, 28, 42, 56, and 84) the stored flour bags were taken out and flour and final product quality analyses were done; specifically, solvent retention capacity (SRC), enzymatic activity (alpha-amylase, polyphenol oxidase (PPO), xylanase), color, protein content, starch pasting profile, cookie and cake quality changes were measured. VSP treated and stored flour samples had better flour functionality than the untreated samples. The cake quality of VSP treated samples was lower in terms of volume index only and the cookie quality was not affected by treatment, milling or storage. Thus, this study can help understand changes in soft wheat flour quality after VSP treatment during storage.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Lin, Y, S. Simsek, and T. M. Bergholz. Impact of chlorinated water on pathogen inactivation during wheat tempering and resulting flour quality. 2022. Journal of Food Protection, 85(8): 1210-1220. DOI: 10.4315/JFP-22-076
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Lin, Y., S. Simsek, and T. M. Bergholz. Fate of Salmonella and Shiga toxin-producing Escherichia coli on wheat grain during tempering. 2023. Food Microbiology 104194.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Lin, Y., S. Simsek, and T. M. Bergholz. Impact of chlorinated water on pathogen inactivation during wheat tempering and resulting flour quality. International Association for Food Protection Annual Meeting, Pittsburgh, Pennsylvania. 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Lin, Y., K. Dolan, S. Simsek, and T. M. Bergholz. Inactivation of Salmonella and Shiga-toxin producing Escherichia coli on soft wheat kernels using vacuum steam pasteurization. International Association for Food Protection Annual Meeting, Pittsburgh, Pennsylvania. 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: H. Kaur, D. Olsen, and A. Vegi. Effect of kernel moisture content and milling sequence on the quality of stone-milled soft white whole wheat flour and its products. Cereals & Grains 22 Conference, Bloomington, Minnesota. 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: H. Kaur, and A. Vegi. Effect of vacuum steam pasteurization, milling, and storage on soft red wheat flour functionality, and end product quality. 2023 Milling and Baking Division, Cereals & Grains Spring Technical Conference, Atlanta, Georgia. 2023.

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:The target audience is food safety and food science researchers and industry professionals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided opportunities for training and professional development of two graduate students. How have the results been disseminated to communities of interest?Results have been disseminated through 1 peer reviewed publication and 1 presentation at a national meeting. What do you plan to do during the next reporting period to accomplish the goals?For objective 1, we will complete the proposed microscopy experiments to determine if pathogens localize to the crease of the wheat kernel. For objective 2, we will finish quantifying pathogen inactivation on soft wheat kernels during vacuum steam pasteurization. We will finish the shelf life study, flour functionality and sensory analysis of the whole wheat soft wheat flour/other end products from vacuum steam pasteurized wheat. We plan to continue working on the remaining project experiments accomplish the project goals.

Impacts
What was accomplished under these goals? Objective 1: evaluating pathogen behavior on wheat kernels during tempering. Soft wheat kernels were inoculated with four Salmonella strains and seven STEC strains at 6 log CFU/g. The lab-scale tempering process was conducted using 50g aliquots following AACC method 26-95 to achieve the final desired moisture content of 15% after 16 hours. Water activity was measured, and pathogens were enumerated by plating at eight time points over 16 hours. Uninoculated wheat samples were also tempered, and total plate count and coliform counts were measured at each time point. The water activity increased from 0.51±0.01 to 0.81±0.04 during the first half-hour after the addition of water. Changes in Salmonella cell density ranged from -0.72 to +0.28 log CFU/g while changes in STEC cell density ranged from -0.44 to +0.37log CFU/g over the first half hour. Over the 16 hours of tempering, the aw increased by 0.25±0.13, and the cell density changes for Salmonella ranged from -0.93 to -0.11 log CFU/g, while changes in STEC cell density ranged from -0.91 to -0.01 log CFU/g. The data show minimal changes in Salmonella or STEC numbers during tempering. Chlorinated water at 400-700ppm has been used in the flour milling industry as a tempering aid to control growth of yeast and mold in tempering bins. However, the effectiveness of chlorinated water for inactivating enteric pathogens on wheat kernels remained unknown. Five strains of STEC and two strains of Salmonella were inoculated onto hard red spring wheat at 7 log CFU/g and stored at room temperature for 1-month. Inoculated wheat was tempered with four concentrations (0, 400, 800, 1200ppm) of chlorinated water (pH 6.5). The reduction due to chlorine was determined by calculating change in cell density at each chlorine level using the response at 0ppm as a reference. Uninoculated wheat tempered with chlorinated water was used to measure flour quality parameters. Changes in pathogen density over 18 hours ranged from -2.35 to -0.30 log CFU/g with 800ppm chlorinated water and were not significantly different from changes at 400ppm and 1200ppm. Significant (p< 0.05) differences in the extent of reduction were observed among strains. However, the effect of chlorinated water at reducing native microbes on wheat kernels was minimal, with an average reduction of 0.39 log CFU/g for all concentrations. No significant (p>0.05) changes occurred in flour quality and gluten functionality, or during breadmaking for grains tempered at 400 and 800ppm chlorinated water. There were small but significant (p<0.05) changes in flour protein content, final viscosity, and water absorption when tempered with 1200ppm chlorinated water. The data showed that the level of chlorinated water currently used in industry for tempering could reduce enteric pathogen numbers by 1.22 log CFU/g for STEC and 2.29 log CFU/g for Salmonella, with no significant effects on flour quality and gluten functionality. Objective 2: In previous lab-scale studies, vacuum steam treatment was capable of reducing microbial loads on soft wheat kernels while maintaining the flour quality and gluten functionality. While demonstrating the potential for pathogen reduction, the previous system was limited in capacity and fine temperature control. This study used a redesigned lab-scale vacuum steam pasteurizer to assess how effective the system could inactivate the pathogens on wheat kernels. The improvements include greater capacity, an improved insulated treatment chamber, a computer-controlled steam and vacuum system, and continuous sample temperature recording. STEC O26:H11 and Salmonella enterica Agona were inoculated onto soft red wheat at 8 log CFU/g and water activity adjusted to 0.51 ± 0.02 after inoculation. The treatment chamber was filled with 1.9 kg of wheat kernels and was preheated to 45C with steam. Three technical replicates of 25g inoculated samples were placed in gauze bags individually and buried in the pre-heated wheat before sealing the chamber and activating the vacuum to 272mbar. A thermocouple placed in the middle of the filler wheat continuously recorded the temperature. Samples were enumerated at 0, 2, 4, 6 mins at the target temperature of 75C to quantify changes in cell density over time. A log reduction of 4.47±0.14log CFU/g was observed for Salmonella Agona, and reduction of 4.64±0.43 log CFU/g was observed for E. coli O26:H11 after 6 minutes at 77 ± 1C. Vacuum steam treatment of soft wheat for 6 minutes effectively leads to a 4.5 log reduction of Salmonella and E. coli O26:H11.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Lauer, J.R., S. Simsek, and T. M. Bergholz. Fate of Salmonella and Enterohemorrhagic E. coli on wheat grain. Journal of Food Protection, 2021 Dec 1; 84(12):2109-2115. doi: 10.4315/JFP-21-076
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Lin, Y., S. Simsek, and T. M. Bergholz. Fate of Salmonella and STEC on soft wheat kernels during tempering. International Association for Food Protection Annual Meeting, Phoenix, Arizona. 2021.

Progress 06/01/20 to 05/31/21

Outputs
Target Audience:The target audience is food science and food safety researchers, and wheat milling industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided opportunites for training and professional development of a graduate student. How have the results been disseminated to communities of interest?The results have been disseminatedthrough a thesis. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue working on the remaining project experiments to work towards completeing the work to accomplish these goals.

Impacts
What was accomplished under these goals? Objective 1: Hard red spring wheat was inoculated with strains of four serovars of Salmonella enterica (Enteritidis, Agona, Tennessee, and Montevideo) and six serotypes of EHEC (O157:H7, O26:H11, O121:H19, O45:NM, O111:H8, and O103:H2) in triplicate, sealed in Mylar bags to maintain the water activity, and stored at room temperature (22 ± 1°C). The survival of each pathogen was evaluated by plating onto differential media. Viable counts of strains from all four serovars of Salmonella (Enteritidis, Agona, Tennessee, and Montevideo) were detected on wheat grain stored at room temperature (22 ± 1°C) for the duration of the study (52 weeks). Viable counts of strains from EHEC serotypes O45:NM, O111:H8, and O26:H11 were only detected for 44 weeks and strains from serotypes O157:H7, O121:H19, and O103:H2 were only detected for 40 weeks until they passed below the limit of detection (2.0 log CFU/g). D-values were found to be significantly different between Salmonella and EHEC (adj. p ≤ 0.05) with Salmonella D-values ranging from 22.9 ± 2.2 to 25.2 ± 1.0 weeks and EHEC D-values ranging from 11.4 ± 0.6 to 13.1 ± 1.8 weeks. There were no significant differences amongst the four Salmonella strains or amongst the six EHEC strains (adj. p > 0.05). These observations highlight the wide range of survival capabilities of enteric pathogens in a low moisture environment and confirm these pathogens are a food safety concern when considering the long shelf life of wheat grain and its products. Objective 2:Soft red winter wheat samples underwent VST for 4 and 8min at 65, 75, and 85°C prior to milling. SRW flour was evaluated for end product quality. Increasing VST temperatures negatively impacted bread quality due to protein denaturation. Cake and cookie quality parameters resulted in little to no significant (p<.05) difference with increasing VST temperatures. SRW wheat was also inoculated withEscherichia coliO121 andSalmonellaEnteritidis PT30. The treatments resulted in a maximum average microbial reduction of 5.09±0.50log CFU/g forSalmonellaand 7.34±0.11log CFU/g forE.coli.Vacuum steam treatment could be used by the milling industry to effectively inactive pathogens without compromising soft wheat flour quality. Objective 3: Salmonella enterica and Enterohemorrhagic Escherichia coli are tolerant to desiccation stress and can survive for extended periods of time in a low-moisture environment. On wheat grain, strains of four different serovars of Salmonella enterica were able to survive for 52 weeks with an average decimal reduction time of 24 weeks. Viable counts of strains from EHEC serotypes O45:NM, O111:H8, and O26:H11 were detected for 44 weeks and strains from serotypes O157:H7, O121:H19, and O103:H2 were detected for 40 weeks until they passed below the limit of detection (2.0 log CFU/g). The average decimal reduction time for the EHEC serotypes was 12 weeks. A significant difference in the survival of Salmonella and EHEC was noted, but both were able to persist on wheat grain for an extended period of time at a high enough level to cause infection. We also determined that there is no uniform response to desiccation stress between Salmonella enterica and EHEC and that the growth method used to prepare the bacteria can also affect desiccation tolerance. A similar trend among Salmonella strains of higher desiccation tolerance from the liquid culture was noted. When six strains of the serovar S. Agona were observed, a consistent response within the serovar was seen with total log reductions ranging from 0.26 ± 0.10 log CFU/mL to 0.37 ± 0.18 log CFU/mL under the broth growth condition and ranged from 0.53 ± 0.34 log CFU/mL to 0.89 ± 0.22 log CFU/mL under the lawn growth condition. When six strains of EHEC O157:H7 were observed, the desiccation tolerance varied both among strains and between growth methods. RpoS functionality was determined to be a crucial factor in desiccation tolerance with strains that were determined to be RpoS negative having a significantly lower desiccation tolerance than the strains that were determined to be RpoS positive. Knowledge of the desiccation tolerance and long-term survival capabilities of these pathogens is key to performing accurate risk assessments in the food industry. It was determined that there are many influential factors that can affect these responses. These factors include, growth method, the composition of the low-moisture surface, environmental conditions, and more. It was also shown here that strain selection is vitally important when evaluating desiccation tolerance and long-term survival in a low-moisture environment as it cannot always be assumed that a single strain will be representative of an entire serotype. The differing results from different bacterial growth methods must also be taken into consideration when performing studies like this in order to ensure the results obtained are an accurate representation of what would occur outside of a lab environment. In conclusion, the information provided by this research enhances the knowledge on the desiccation tolerance and long-term survival of Salmonella enterica and EHEC and is useful for risk assessments and determining appropriate preventative measures in the food industry.

Publications

• Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2021 Citation: Lauer, J. 2021. THE RESPONSE OF SALMONELLA ENTERICA AND ENTEROHEMORRHAGIC ESCHERICHIA COLI IN LOW-MOISTURE ENVIRONMENTS. M.S. Thesis. North Dakota State University, Fargo, North Dakota.