EVALUATING INACTIVATION OF SALMONELLA ON LOW-MOISTURE FOODS BY VACUUM STEAM PASTEURIZATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1008667
• Grant No.: 2016-69003-24851
• Project No.: ND05142
• Proposal No.: 2015-08058
• Program Code: A4131
• Project Start Date: Jan 15, 2016
• Project End Date: Jan 14, 2019
• Grant Year: 2016

Project Director
Bergholz, T.

Recipient Organization
NORTH DAKOTA STATE UNIV
(N/A)
FARGO,ND 58105

Performing Department
Vet and Micro Sciences

Non Technical Summary
Foodborne illness is a significant public health concern, with an estimated 48 million cases of foodborne illness each year in the U.S. In addition to health care costs, the presence of human pathogens in the food supply has a substantial economic effect on the food industry in the U.S. due to product recalls and loss of consumer confidence. Recently, a number of outbreaks of foodborne illness have been attributed to Salmonella on low-moisture foods. These foods were previously thought to be relatively low-risk for transmitting foodborne illness, as they do not support growth of microbes. Effective preventative controls are needed to reduce the risk of human illness associated with these foods. The use of vacuum steam pasteurization is expected to have greater efficacy against pathogens, as moist heat is more effective at inactivating microbes compared to dry heat. Characterizing the biological factors that impact thermal resistance of Salmonella on low-moisture foods will aid in development of effective strategies to inactivate this pathogen using vacuum steam pasteurization. This information should increase the options for food processors to effectively control Salmonella in low-moisture foods.

Animal Health Component

0%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

salmonella

low-moisture foods

pasteurization

Goals / Objectives
The goal of this project is to quantify the influence of biological variables on Salmonella thermal inactivation on low-moisture foods during vacuum steam pasteurization. This will be accomplished with the following objectives:1. quantify the effect of inoculum level on Salmonella inactivation by vacuum steam pasteurization on three different low-moisture foods2. quantify the amount of variation in inactivation parameters that are due to biological effects including strains, food matrix, and length of incubation time before processing

Project Methods
Bacterial strains and low-moisture food products. We plan to use 36 strains of Salmonella - 8 each of S. Enteriditis, S. Tennessee, S. Agona, and S. Montevideo, all serotypes that have been linked to human outbreaks due to consumption of contaminated low-moisture foods. Each strain of Salmonella will be inoculated onto each of 3 different low-moisture foods: flaxseed, peppercorns, and sunflower kernels.Inoculation of low-moisture food products. Bacterial strains will be stored in BHI with 10% glycerol at -80°C. Strains will be transferred from freezer stocks to BHI agar plates and grown overnight at 37°C. A colony will be transferred to 5ml BHI broth and incubated at 37°C for 20 hours. 250 µl of the overnight broth culture will be plated uniformly onto BHI agar plate using spiral plater, and incubated at 37°C for 24 hours. The bacterial lawn will be collected and mixed well into a beaker containing a minimal amount of sterile water. Inoculation of these selected low moisture foods will be conducted in a bio-safety hood in the following manner. The culture will be added to food matrices of choice and mixed for 3-5 minutes to receive homogenous distribution of the bacteria. One such plate of bacterial lawn into 100g of food matrix is required to achieve 7-8 log CFU/g. For our preliminary trials on flaxseed, quinoa, and milled flaxseed we obtained 7-8 log CFU/g when 18 bacterial lawn plates were collected into 5ml of water and mixed into the 1800g of food. 8 samples (25g) of each inoculated food will be plated to determine the homogenous distribution of the bacteria before proceeding with pasteurization of the samples. Our preliminary data indicate fairly homogeneous distribution of Salmonella on flaxseeds using this inoculation method, with an average of 8.22 ± 0.12 and 8.18 ± 0.09 log CFU Salmonella per gram, where averages were calculated from 8 samples taken from each batch of inoculated seeds.Following inoculation, the product will be stored in an airtight chamber with saturated salt solutions to equilibrate the inoculated product to the original water activity of the product. The water activity equilibration would be achieved within 24-48 hours. After equilibration of water activity, the Salmonella strains will be tested for thermal resistance by subjecting 100g of inoculated foods in a dry oven set to 280°F/137°C for 15 minutes. A reduction of <2.5 log CFU from the initial count will be considered an acceptable heat resistance (1). Once equilibrated, inoculated product will be divided into 25g aliquots, placed into sterilized cotton sample bags, sealed and pasteurized. The packaging of samples, pasteurization runs and plating will be performed on the same day.Pilot-scale vacuum steam pasteurization. Pilot-scale lab equipment will be utilized for all vacuum steam pasteurization processing. This system uses vacuum and steam for pasteurization which works in three different stages a) pre-vacuum, b) sterilization, and c) post-vacuum. During pre-vacuum, a negative pressure of ~ 0.9 bar is applied inside the system which removes air from the food and the system. Then, steam is injected which is homogenously distributed inside the system to satisfactorily reach the target temperature at a pressure maintained between -0.5 and -0.6 bar to allow pasteurization for the designated amount of time. After pasteurization, vacuum is applied to extract all steam and moisture in the system leaving the product almost dry. One pasteurization cycle takes approximately 15-20 minutes to complete with 0-5 minutes set for pasteurization. Depending on the product, inoculated samples will be exposed to vacuum steam pasteurization in either a static or in a tumbler sterilizer system. However, both systems works on the same principal. We will use static processing which can process up to 300 lbs of a single product. Appropriate settings for vacuum and steam are required to obtain desired temperature in the sterilization system. Hence, three temperature data loggers will be used to monitor even distribution of temperature in the sterilization system, and these standardized settings will be used for future pasteurization of the inoculated samples for experimental purposes. The pasteurization settings may vary by product type and its volume. For the purpose of the experiment, approximately 100 lbs. of food (filler) will be run at each condition with the inoculated samples in sterilized cotton bags sandwiched between 100lbs of the product. Same product will be used as filler as the inoculated samples. Also, three temperature data loggers will be placed closer to the inoculated samples at each pasteurization cycles for careful observation of temperatures. Samples of multiple bacterial strains inoculated individually onto a single product type can be processed simultaneously. The pasteurization will be conducted at 75°C and 85°C, and Salmonella enumerated after 30, 60, 90, 120, 180, 240, and 300 seconds. Temperature profiles from the data loggers, time taken to achieve target temperature and time taken to reduce temperature following the pastuerization cycle will all be recorded for each run and included as variables in the assessment of inactivation.Bacterial enumeration. Following pasteurization, samples will be diluted with Butterfield buffer and homogenized in a stomacher for 90 seconds. Appropriate dilutions will be made and the homogenate will be plated in duplicate onto modified TSAYE agar with ammonium ferric citrate and sodium tetrathionate with a Spiral Plater. The plates will be incubated at 37°C for 48 hours, and black colonies indicative of Salmonella will be enumerated with a Q-Count automated colony counter. Three independent culture replicates will be assayed for each bacterial strain (biological replicates), and the experiment will be repeated three times to obtain 9 data points for each time point at each pasteurization temperature. For each technical replicate, three inoculated samples will be used as control, which will be transported to the pilot facility and handled in the same manner as the pasteurized samples, just without pasteurization.Data analysis. All colony count data will be deposited into the publically available database ComBase (http://www.combase.cc/). Colony count data for each strain, on each food, under each pasteurization temperature, will be fit to an appropriate primary survival model using the nlsMicrobio package in R (http://cran.r-project.org/web/packages/nlsMicrobio/nlsMicrobio.pdf). Distributions of inactivation parameters from all strains for each product will be determined. Significant differences among inactivation parameters by strain, serotype, pasteurization temperature, processing variables such as temperature variation and come-up and come-down time, and food matrix will be determined using ANOVA, or an appropriate non-parametric method, if needed.Assessment of Salmonella survival on each low-moisture food over time. Many low moisture foods may be stored at ambient temperature for lengthy periods of time prior to use. If Salmonella is present on the product during storage, there is the possibility the Salmonella may adapt to survive in this low moisture environment. Adaptations to this environment may impact subsequent thermal inactivation during pasteurization. Therefore, we want to determine if the length of time that Salmonella is associated with a low-moisture food product can impact subsequent thermal resistance. Each of the 3 food products will be inoculated, and once equilibrated, will be stored in Kraft poly-lined bags for three months. During the storage period, 4 25g samples will be removed every two weeks, and one sample used to enumerate Salmonella and the other three samples will be exposed to vacuum steam pasteurization.

Progress 01/15/16 to 01/14/19

Outputs
Target Audience:The target audience reached includes food safety researchers and professionals. Changes/Problems:A major change made in the middle of year 1 was the switch from pilot scale vacuum steam pasteurization to a lab scale vacuum steam pasteurization system. This switch occured because the company that was providing access to the pilot scale system closed. The switch led to a delay as we had to obtain the lab scale system and run initial experiments to validate the set up. Because of this delay, we did not obtain data for Salmonella inoculated onto three different low moisture foods. The complete study design was conducted for all 32 strains of Salmonella on flaxseeds, and assessed vacuum steam pasteurization for one strain on whole wheat kernels. What opportunities for training and professional development has the project provided?Two graduate students, Sahar Malekmohammadi and Manoj Shah have participated in this project. Sahar presented her work at the North Central Branch ASM meeting in 2018. How have the results been disseminated to communities of interest?Through presentations at regional and national meetings. A manuscript has been submitted for peer-review. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Throughout the project, 32 strains of Salmonella were used for survival and thermal resistance assays on flaxseed. This strain set included 8 strains from each of 4 serovars: Agona, Enteriditis, Montevideo, and Tennessee. The commonly used S. Enteriditis PT30 strain was included in this set. All strains were inoculated onto individual aliquots of flaxseed, allowed to equilibrate to pre-inoculation water activity, and then stored in sealed mylar bags over a period of 6 months. Survival on the flaxseed over time was quantified, as well as thermal resistance to vacuum steam pasteurization at 4 time points. Over the 6 month storage period, Salmonella survival on flaxseed was non-linear. The average maximum rate of reduction was significantly different among the serovars, with strains of serovar Agona having the lowest rate of reduction, 0.37 ± 0.11 log CFU/g/week. Serovars Enteriditis and Montevideo had average rates of reduction that were significantly higher than Agona, 0.60 ± 0.14, and 0.50± 0.14 log CFU/g/week, respectively. The average rate of reduction for serovar Tennessee was 0.48 ± 0.10 log CFU/g/week, which was not statistically different from the other serovars. As Salmonella cell density changed over the storage time of the flaxseeds, we wanted to determine if differences in cell density would impact inactivation rates when inoculated flaxseeds were treated with vacuum steam pasteurization. a subset of 8 strains, 2 randomly selected from each serovar, were inoculated onto flaxseeds at a level of 8 log CFU/g and at 6 log CFU/g. After the 2 day water activity equilibration process, samples were treated with vacuum steam pasteurization at 71ºC. Inactivation rates were similar between the different levels of inoculum, with average maximum inactivation rates of 3.4 ± 0.6 log CFU/g/min for the 8 log/g inoculum and 3.7 ± 0.7 log/CFU/g/min for the 6 log/g inoculum. Comparison of average inactivation rates between the different inoculum levels for each strain also found no significant differences in inactivation rate due to inoculum level. Thermal resistance of Salmonella was assessed after water activity equilibration,and after 8, 16, and 24 weeks of storage. The average kmax,T0 of Enteritidis, Montevideo, Tennessee, and Agona were 3.9±0.9 , 4.1±0.8, 4.1±0.8, and 3.3±0.9 log10 CFU/g/min respectively. Agona has the lower kmax,T0than Montevideo and Tennessee (adj. p<0.05). The average kmax,T8week of Enteritidis, Montevideo, Tennessee, and Agona were 3.6±1.2 , 3.6±1.3, 4.2±1.3, and 4.1±1.1 log10 CFU/g/min respectively. There were no significant differences in inactivation rates among serovars at 8 weeks post inoculation. The average kmax,T16week of Enteritidis, Montevideo, Tennessee, and Agona were 2.6±0.9 , 4.4±1.8, 3.8±1.5, and 2.8±0.9 log10 CFU/g/min respectively. 16-week post inoculation, Montevideo exhibited lower thermal resistance compared to Agona and Enteritidis (adj. p<0.05). The average kmax,T24week of Enteritidis, Montevideo, Tennessee, and Agona were 2.7±0.8, 2.8±1.2, 2.9±1.7, and 2.6±1.7 log10 CFU/g/min respectively. 24-week post inoculation, there were no significant differences observed among serovars. We also compared inactivation rates within serovars over storage time. The average kmax,T0 of Enteritidis, Montevideo, Tennessee, and Agona were 3.9±0.9 , 4.1±0.8, 4.1±0.8, and 3.3±0.9 log10 CFU/g/min respectively and kmax,T24week was significantly decreased to 2.7±0.8, 2.8±1.2, 2.9±1.7, and 2.6±1.7 log10 CFU/g/min respectively. These data demonstrate that over storage time on flaxseeds, average thermal resistance increased. As our data demonstrated no significant differences in thermal resistance due to inoculum level, these results suggest that Salmonella may be adapting to survive under low water activity conditions, which could impact thermal resistance. Salmonella Enteriditis strain PT30, commonly used in low moisture food studies, was inoculated onto whole wheat kernels to determine a pasteurization temperature and time that would be effective at reducing cell numbers on this low moisture food. Pasteurization at 85ºC for 10 minutes led to 6 log reduction of Salmonella. Additional work would be required to determine if treatment at this temperature had any negative impacts on wheat or flour quality and function.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Malekmohammadi, S, and T. M. Bergholz. Evaluation of long term survival of four Salmonella serovars on flaxseed. North Central Branch American Society for Microbiology Annual Meeting, Mankato, Minnesota. September 28, 2018
• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Malekmohammadi, S., M. K. Shah, M. K. Townsend Ramsett, and T. M. Bergholz. Variation in survival and thermal resistance phenotypes among Salmonella serovars on flaxseed.

Progress 01/15/17 to 01/14/18

Outputs
Target Audience:Food safety researchers and professionals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students, Sahar Malekmohommadi and Manoj Shah have participated in this project. Sahar presented her work at IAFP in 2017. How have the results been disseminated to communities of interest?Through presentations at national conferences. What do you plan to do during the next reporting period to accomplish the goals?We have completed one replicate of the entire experiment, and are in progress with the second replicate. We will also be completing the inoculum level experiment, to determine how inoculum level may or may not influence inactivation rates. We also have access to genome information for all of the strains used in this study, and we will explore associations between thermal resistance levels and genome content.

Impacts
What was accomplished under these goals? 8 strains of each of the following Salmonella serovars (Montevideo, Tennessee, Agona, and Enteriditis) were inoculated onto flaxseed and stored over a 6 month period. Survival of each strain was measured over time by enumerating Salmonella from the flaxseeds every 2 weeks. Survival on flaxseeds was non-linear, and we calculated the kmax values for the rate of inactivation over the entire 6 month period. Inactivation rates are presented as the maximum rate of log reduction per week, and were slowest for strains of serotype Agona (0.32 +/- 0.03) compared to Enteriditis (0.63 +/- 0.14), Montevideo (0.52 +/- 0.13), and Tennessee (0.44 +/- 0.10). 2 months post-inoculation, strains of serotypes Enteriditis and Montevideo had greater reductions in cell density (2.6 to 2.7 log reduction) compared to serotypes Agona and Tennessee (1.8 to 1.9 log reduction). Overall log reductions were similar at 4 months post-inoculation, while overall log reductions at 6 months post-inoculation were fairly similar across the serotypes. Overall log reduction at 6 months post-inoculation was 3.6 +/- 0.6 for serotype Tennessee, 3.9 +/- 0.4 for Agona, 4.0 +/- 0.8 for Montevideo, and 4.3 +/- 0.9 for Enteriditis. Post-inoculation, samples were subjected to vacuum steam pasteurization at 72°C, and Salmonella survival over a 3 minute period was measured. Survival under vacuum steam pasteurization was also non-linear, and kmax values were determined. 2 days post-inoculation, kmax values ranged from 4 to 5.3 log reduction per minute across all strains. Survival under vacuum steam pasteurization was also assessed for samples 2 and 4 months post-inoculation. Strains of serotype Enteriditis became more sensitive to the heat treatment over time, as average kmax values for those strains increased over storage time, from 4.2 +/- 0.9 at 2 days post-inoculation, to 4.6 +/- 1.4 at 2 months post-inoculation, and 6.3 +/- 1.5 at 4 months post-inoculation.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Malekmohammadi, S. and T. M. Bergholz. Evaluation of Survival on Flaxseeds and Subsequent Heat Resistance among Four Salmonella Serovars. International Association for Food Protection Annual Meeting, Tampa, Florida. 2017

Progress 01/15/16 to 01/14/17

Outputs
Target Audience: Nothing Reported Changes/Problems:Our initial plan was to utilize pilot scale pasteurization equipment at a local business. During the first year of the project, that business closed. We have obtained a lab scale vacuum pasteurization system from the Grimsby Institute in the UK, that has been previously described in a number of food safety publications (for example, DOI:10.1111/j.1365-2672.2004.02511.x). The pasteurization data we have collected so far are all from use of this lab scale device. Acquistion of this device took a few months, and as a result, we are slightly behind our originally planned time frame. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Over the next year, we will complete the second replicate of inoculation onto flaxseed, long term storage, and evaluation of pasteurization efficacy for the 32 strains. We will also complete the assessment of the impact of different inoculation levels on survival during pasteurization. We will also start a similar set of experiments with a different low moisture food, sunflower kernels.

Impacts
What was accomplished under these goals? 8 strains of each of the following Salmonella serovars (Montevideo, Tennessee, Agona, and Enteriditis) were inoculated onto flaxseed and stored over a 6 month period. Survival of each strain was measured over time by enumerating Salmonella from the flaxseeds every 2 weeks. After 2 and 4 months, samples of the inoculated seed was subjected to vacuum steam pasteurization at 75°C, and Salmonella survival over a 3 minute period was measured. Considerable variation in survival on flaxseeds among the different strains has been observed, with ~ 1 log difference in surviving Salmonella across the strains. The pasteurization data so far also indicate variation in survival during pasteurization among the different strains, though this data set is not complete yet.

Publications