Source: OHIO STATE UNIVERSITY, THE submitted to NRP
ACCELERATED INACTIVATION OF BACTERIAL SPORES BY INTERACTION OF ELECTRIC FIELDS WITH KEY SPORE COMPONENTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1027962
Grant No.
2022-67017-36290
Cumulative Award Amt.
$602,100.00
Proposal No.
2021-08186
Multistate No.
(N/A)
Project Start Date
Jan 1, 2022
Project End Date
Dec 31, 2025
Grant Year
2022
Program Code
[A1332]- Food Safety and Defense
Recipient Organization
OHIO STATE UNIVERSITY, THE
1960 KENNY ROAD
COLUMBUS,OH 43210
Performing Department
Food, Ag and Bio Engineering
Non Technical Summary
The quality of canned foods is often compromised because they must receive severe heat processing to kill bacterial spores that could cause botulism poisoning in humans. If it were possible to inactivate spores at lower temperatures, the same foods would still be as safe, but far better in flavor and nutritional value. We have found that applying electric fields in combination with heat can greatly accelerate the killing of bacteria, potentially resulting in improved product delivered to the consumer. We propose to investigate the reasons for the effectiveness of electricity by testing organisms that lack those components that protect bacterial DNA. If we can show that bacteria are inactivated because electric fields disrupt these components, we will be able to design a milder heat process that results in a safe product without excessive heating. We plan to test such a process in practice and evaluate the quality of the food using a sensory evaluation panel. If successful, many of tomorrow's canned food products could have greatly improved taste and nutritional value.
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
5015010110050%
7121411202050%
Knowledge Area
501 - New and Improved Food Processing Technologies; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1411 - Beans (fresh, fresh-processed); 5010 - Food;

Field Of Science
2020 - Engineering; 1100 - Bacteriology;
Goals / Objectives
1. Investigate the kinetics of combined electric field strength and temperature exposure on inactivation of a) wild-type and isogenic recA mutant spores of Bacillus subtilis in buffer in comparison to thermally processed controls subjected to identical temperature histories, and, b) under optimized conditions from the above, conduct the same comparisons for a pathogen within its relevant food matrix.2. Investigate the mechanism of inactivation of Bacillus cereus and Clostridium sporogenes spores in buffer subjected to combined electric fields and temperatures compared to purely thermal inactivation, and these spores in food matrices exposed to the optimal electric field treatments.3. Verify and demonstrate the above findings within products in realistic food processing scenarios.
Project Methods
Objective 1.We have previously developed a specialized ohmic kinetics test cell, which consists of a set of miniature sample heaters, made from capillary tubes, and containing samples inoculated with the spores. The miniature heaters are entirely within a larger ohmic heater, containing a fluid of the same electrical conductivity as the sample. The capillary sample cells may be made into miniature ohmic heaters using electrically conductive gel plugs at the ends, which allow passage of current when the capillaries are aligned parallel to the field. The sample cells may also be made into conventional heating cells by making the gel plugs nonconductive. In practice, the experiment is carried out by locating many sample cells (either in conventional or ohmic mode) within the large ohmic heater. The field is turned on to start heating. As the set point is achieved, a zero-time (Come-Up-Time, or CUT) sample is withdrawn (using a wire connected to the outside via a stuffing box) into the cooling chamber below (filled with ice water) to cool it rapidly. During the hold, the voltage is scaled back to maintain a constant temperature. Samples are withdrawn at each exposure time, and at the end of the experiment, all capillaries are recovered and plated to determine viable spores.We will test the following species and strains in buffer:Wild-type B. subtilis PS533, and resistant to kanamycinB. subtilis PS2318 that lacks the recA gene, as RecA is important in repair of a large amount of dormant spore DNA damage, when spores come back to life in germination and then outgrowth (strain resistant to chloramphenicol).Wild-type B. cereus T.Wild-type C. sporogenes ATCC15579 We will test the following species and strains in food matrices:Wild-type B. cereus T in a refried bean matrixWild-type C. sporogenes in pureed green bean baby food matrix. Samples will be treated under either electric field or no field to determine survivor counts, using analytical methods detailed below.Bacterial strains and culture conditions. Spores of strains of B. subtilis, B. cereus and C. sporogenes will be produced and purified at UConn Health in the Setlow lab: B. subtilis wild-type and recA (the latter defective in DNA repair) strain and wild-type B. cereus T; or the Mok lab, C. sporogenes. Spores of B. subtilis strains will be prepared at 37°C on 2xSG medium agar plates. C. sporogenes spores will be prepared in Robertson cooked meat broth at 37°C in an anaerobic chamber, and B. cereus spores will be prepared at 37°C in liquid.Food Matrix Inoculation: Canned (commercially sterile) refried beans will be inoculated in aseptic conditions by mixing with appropriate spore suspension. Refried beans will be inoculated with B.cereus spores. Baby food purees will be prepared from surface-sanitized vegetables (green beans, from an online recipe) ground using a sterilized blender under aseptic conditions, and aseptically mixed with C. sporogenes spores to receive the initial concentration of spores of 106 CFU/g. Both inoculated food matrices will be consistently kept refrigerated or on ice before and after the ohmic heating.Objective 2. Overall approach. We will use wild-type spores of B. subtilis and isogenic recA spores, and wild-type B. cereus and C. sporogenes spores. More rapid killing of B. subtilis recA spores than wild-type spores will show that the ohmic heating kills spores by DNA damage. This will be determined from the experiments described in Objective 1. In addition, we will check for mutants among survivors of wild-type B. cereus and C. sporogenes spore populations (killed ~95% by ohmic heating to ensure sufficient survivors for subsequent analyses) as we have done previously. A high level of mutants in these treated spores and more rapid killing of DNA repair-deficient than wild-type B. subtilis spores will show that ohmic heating kills spores by DNA damage. In contrast, this is not how thermal treatment alone kills spores, as determined for spores of a number of Bacillus species).Experimental approaches.Role of DNA repair (Sastry lab): We will compare inactivation rates of wild-type B. subtilis PS533 and PS2318 (which lacks the recA gene, thereby lacking DNA repair capacity). If our hypothesis of how an electric field increases thermal killing is correct, loss of DNA repair capacity will make PS2318 spores more sensitive to ohmic heat than wild-type spores, as thermal damage to naked DNA generated upon SASP-DNA dissociation in the electric field will be poorly and/or inaccurately repaired during outgrowth of the repair-deficient spores.Evidence of DNA Damage via Mutations (Setlow lab): For sporeformers B. cereus and C. sporogenes, for which recA mutants are not available, we will test for mutants. For this purpose, samples of these microorganisms, both within buffer and their test food matrices (refried beans and green bean puree) will be processed under conditions yielding optimum inactivation, as determined from kinetic data in Objective 1. Control treatments will involve conventional heat under an identical temperature history. (Details of replicates and number of experiments are as detailed above under Objective 1. Notably, however, we will process for short durations, to ensure ~ 95% survivors, so that a sufficient population exists to test for mutantsTest for mutants (Setlow lab). There is a simple method for simultaneous analysis of mutations generated in B. subtilis spores in >500 genes essential for either sporulation giving asporogenous (spo) mutants or small molecule biosynthesis giving auxotrophic (aux) mutants). Survivors of wild-type spore populations killed ~95% by thermal- or ohmic heat-treatment are obtained by spread-plating on rich medium plates. Then ~ 500 colonies are individually picked (~100 colonies/plate) and inoculated on both rich medium sporulation plates or minimal medium plates with only glucose, citrate, NH4+ and other salts, and plates are incubated for 2-4 d at 37°C. Mutants are then identified by inspection of the plates, as spo colonies (translucent) are easy to distinguish from wild type colonies (white crusty appearance), and aux colonies do not grow on minimal medium plates while wild-type cells do. In B. subtilis a-b- spore populations killed 90-99% by wet heat, 10-15% of survivors have acquired one or both of these types of mutations.Objective 3. The package in this case is made of MRE pouch stock, equipped with electrodes and an external heater to compensate for heating at cold edge zones; and modified to rectangular shape.The product (baby food based on green bean puree) will be prepared using recipes available online. We will treat and cool the product in a specialized ohmic retort setup described in that same publication. We can conduct three forms of verification. First, temperature verification, by comparing the temperature at three points within the pouch. Secondly, an electrical verification to ensure that the predicted current is close to the measured current, and finally, microbiological verification. Samples will be processed at various field strength-time combinations to allow for different inactivation levels. Control samples will be processed within retorts to temperatures consistent with no electric field (based on separately collected heat penetration data) and results will be similarly analyzed. Product inoculation and spore enumeration post-processing will be conducted as described above under Objective 1. A final demonstration of quality benefits deriving from a reduced ohmic process will be performed using sensory descriptive profiling combined with consumer acceptability testing. For this purpose, baby food puree made from green beans will be compared to a conventionally retorted sample. This work will be conducted by the sensory evaluation group of Dr. Chris Simons in the Department of Food Science and Technology at The Ohio State University.

Progress 01/01/24 to 12/31/24

Outputs
Target Audience:Food industry, regulators, process authorities and academic researchers. Changes/Problems:Because of our extensive activities relating to mutants, we were able to identify specific mechanisms for spore kill by ohmic heating. In this area, we accomplished far more than expected. We requested a no-cost extension to complete studies on Bacillus cereus in refried beans, and final sensory trials. These will be completed in the upcoming year. What opportunities for training and professional development has the project provided?We have presented the results to various interested professional groups, and are exploring further collaboration, possibly with the US Army Combat Feeding Division. Our team has learned much about spores by the interaction during this project with Dr. Peter Setlow and his research group at UConn Health. This has included the learning of a graduate student working on the project and our research scientist (microbiologist). How have the results been disseminated to communities of interest?Various presentations have been made at professional meetings, as reported earlier. We will continue dissemination over the coming year and into the future. What do you plan to do during the next reporting period to accomplish the goals?In the final year, we will complete studies on inactivation of Bacillus cereus spores within refried beans (previously a product of concern); and will complete consumer panel evaluation of green bean puree processed in pouches by ohmic and conventional heating.

Impacts
What was accomplished under these goals? Objective 1: We found those properties of spores that make them highly heat-resistant are associated with mutants with enhanced membrane stability. Most other core components (e.g. RecA proteins) were all found to interact with electric fields. In all cases, increased electric field strength resulted in greatly accelerated spore killing, even with short exposure times. We found that the same acceleration of spore kill observed in buffer suspensions was also observed in food matrices. Thus, we expect to be able to kill sporeformers far faster than conventional heat treatments. We tested a variety of mutant types with different missing core components, and also conducted molecular dynamics simulations and flow cytometry to identify the pathways to spore killing. Objective 2: Clostridium sporogenes inactivation was also greatly accelerated by electric fields, both in buffer and food media. Work on Bacillus cereus will be completed in the upcoming year. Objective 3: We processed green bean puree using ohmic heating in pouches equipped with electrodes, and compared sensory attributes (by a trained panel) with those of conventionally heated green bean puree while achieving the same kill of Clostridium sporogenes in both cases. In all cases, the ohmically treated puree retained better color and aroma quality than the conventionally processed product. In summary, we have exciting possibilities for greatly improved shelf-stable products in the future.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Singh, S.K., Ali, M., Mok, J.H., and Sastry, S.K. 2024. Effects of field strength and frequency on inactivation of Clostridium sporogenes during ohmic heating. J. Food Engineering 375:110280 https://doi.org/10.1016/j.jfoodeng.2024.112080
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Singh, S.K., Ali, M.M., Mok, J.H., Korza, G., Setlow, P., and Sastry, S.K. 2024. Mechanistic insight into roles of alpha/beta-type small acid soluble proteins, RecA and inner membrane proteins during bacterial spore inactivation by ohmic heating. Journal of Applied Microbiology, 2024, 135, lxae151 https://doi.org/10.1093/jambio/lxae151
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Singh, S.K, Ali, M., Setlow, P. and Sastry, SK. Accelerated inactivation of Clostridium sporogenes spore in Green Bean Puree using Ohmic heating and mechanistic insights for this rapid killing of spores using genetically modified strains of Bacillus subtills. Presented at the annual meeting of the Institute for Thermal Processing Specialists, Nashville, TN, March 12-14.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Singh, S.K, Ali, M., Setlow, P. and Sastry, SK. Mechanistic insights into role of alpha/beta-type small acid soluble protein and inner membrane proteins during bacterial spore inactivation by ohmic heating. Presented at the International Association for Food Protection annual meeting, Long beach, CA, July 14-17, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Singh, S.K, Ali, M., Korza, G., Setlow, P. and Sastry, SK. Bacterial spore proteins and their role in influencing spore inactivation during ohmic heating. Presented at the annual meeting of Conference of Food Engineering, Seattle, WA, August 25-28, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Sastry, S.K., Singh, S.K., Ali, M.M., Setlow, P., Korza, G., Liu, H. 2024. Mechanisms of accelerated inactivation of bacterial spores by ohmic heating. Presentation at the 2024 EFFoST/IFT-NPD Workshop on Nonthermal Processing of Foods, Potsdam, Germany, October 7-9, 2024.


Progress 01/01/23 to 12/31/23

Outputs
Target Audience:Food industry, regulators, process authorities and academic researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have made various presentations at meetings, and expect to incorporate findings into coursework. How have the results been disseminated to communities of interest?Presentations at professional meetings, as detailed in Products. What do you plan to do during the next reporting period to accomplish the goals?Under objective 3, we are planning to conduct studies on spore inactivation in a pouch filled with a baby food formulation (green-bean puree) together with sensory evaluation of non-inoculated pouches to assess quality effects in a food processing scenario.

Impacts
What was accomplished under these goals? Under objective 1, we have determined how electric fields interact with specific spore components by comparing their effects on wild-type spores and mutants lacking specific components. Results show that electric fields interact with Small Acid Soluble Proteins (SASPs) in the spore core, as well as the inner membranes of spores. Under objective 2, we have found greatly accelerated inactivation of bacterial spores under ohmic heating (combined electric fields and temperatures). The same accelerated spore inactivation effects have been found to occur within a food matrix (green bean puree). Activity under objective 3 is planned for this year (final year of project).

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Singh, S.K., Ali, M., Mok, J.H., and Sastry, S.K. 2023. Effects of field strength and frequency on inactivation of Clostridium sporogenes during ohmic heating. J. Food Engineering (revised version in review).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Singh,S.K., Ali, M., Mok,J.H., Korza, G., Liu,H., Setlow, P, and Sastry, S.K. 2023. Accelerated Inactivation of Clostridium sporogenes and Bacillus subtilis by Ohmic heating. Presentation at the Annual Meeting of the International Association for Food Protection (IAFP), Toronto, Canada, July 16-19, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Singh, S.K, Ali, M., Mok, J.H., Liu, H., Korza, G., Setlow, P. and Sastry, SK. Accelerated Inactivation of Bacterial Spores by Interaction of Electric Fields with Key Spore Components. Presented at the 14th edition of the International Congress on Engineering and Food, Nantes, France, June 20-23, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Singh, S.K, Ali, M., Mok, J.H., Liu, H., Korza, G., Setlow, P. and Sastry, SK. Accelerated Inactivation of Bacterial Spores by Interaction of Electric Fields with Key Spore Components. Presented a poster at the annual meeting of institute of Food Technologists, Chicago, IL, July 16-19th 2023.


Progress 01/01/22 to 12/31/22

Outputs
Target Audience:Food industry, students and faculty at academic institutions. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We plan to share these results widely with industry, government agencies and academic institutions. The data will also form part of lectures for graduate students in food engineering and food science and technology. 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?We will complete analysis of data and begin by presentations at meetings during 2023. These will then be published. We will also make industry presentations as and when appropriate.

Impacts
What was accomplished under these goals? 1. We have found that when exposed to an electric field, wild-type spores are inactivated much faster than with heat alone. However, spores of Bacillus subtilis PS 578 that lack small acid-soluble proteins (SASPs) do not show such marked differences at low field strengths, suggesting that SASPs play a role in susceptibility to electric fields. However, the SASP-lacking PS578 spores show remarkable sensitivity to electric fields at field strengths of 50 V/cm, suggesting that there are additional components that affect inactivation by electric fields. 2. In studying C. sporogenes mutants inactivated to about 95%, the survivors have not shown evidence of mutants. We are still analyzing these data and will attempt to draw inferences shortly. We have still to study the effects of food matrices. 3. We have not yet begun studies on this objective, and will do so once Objectives 1 and 2 are complete.

Publications