Source: UNIV OF IDAHO submitted to
NOVEL STERILIZATION METHOD FOR FOOD, CLINICAL AND PHARMACEUTICAL APPLICATIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0231234
Grant No.
(N/A)
Project No.
IDA01478
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 1, 2012
Project End Date
Jun 30, 2017
Grant Year
(N/A)
Project Director
Paszczynski, AN.
Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
School of Food Science
Non Technical Summary
Foodborne microbial pathogens cause hundreds of thousands of cases of illness and hundreds of deaths in the United States each year. These pathogens cause illnesses ranging from diarrhea and vomiting to much more serious diseases including gastroenteritis, central nervous system disorders, severe bloodstream infections, and life-threatening illnesses such as botulism poisioning. Thus, there is a clear need for better processing and sterilization methods for foods and food packaging. These methods need to be affordable, robust enough to kill even the most resistant pathogens (e.g., bacterial endospores), scalable to industrial size, and have minimal deleterious effects on sterilized materials. Supercritical fluid technologies exhibit genuine promise as sterilization methods due to the fact that it achieves effective microbial lethality, exerts minimal impact on food quality, generates no waste or effluent, and is already commercialized on an industrial scale (e.g., for decaffeination or denicotinization applications). From work conducted in our laboratory, we have determined that inclusion of small amounts of modifier, such as hydrogen peroxide, has potential to further enhance the lethal effects of SCCD for food preservation and other sterilization applications. The use of modifier in conjunction with SCCD is expected to lessen treatment conditions required to achieve sterilization, thus preserving the quality of treated foods. However, to date, there are very limited studies in the literature regarding the use of modifiers in combination with SCCD for sterilization of food products, or how such combined treatments might affect sterilized microbes and their endospores as well as food quality. Thus, this research proposal is both novel and unique, and addresses a key issue affecting the state of Idaho and has national significance. The objectives of the proposed research program are to (1) demonstrate that SCCD containing a small amount of modifier (e.g., ~0.1% v/v peroxide) will sterilize raw foods such as meat, poultry, eggs, fish, shellfish, seed-derived foods such as fresh alfalfa sprouts, and fresh vegetables contaminated with bacteria (naturally or purposely), and also show that the technology will sterilize food packaging materials and pre-packaged foods. The second objective is to (2) develop a pilot-scale SCCD system design for testing in collaboration with the commercial food industry. Objective (3) is to develop an educational component of the program that trains graduate Food Science majors in the use of this new technology and the final objective (4) is to elucidate the killing mechanism imposed by the SCCD treatment on the vegetative cells and bacterial endospores. Knowledge generated from this work will be highly relevant for developing and understanding new sterilization methods as well as training future food science professionals in cutting-edge technologies related to food processing and food safety.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5015010110030%
5017299110030%
7125010110020%
7127299110020%
Goals / Objectives
Specific Objective 1: Demonstrate that supercritical carbon dioxide (SCCD) containing a small amount of modifier (e.g., ~0.1% v/v hydrogen peroxide, tert-butyl hydroperoxide, peracetate, tert-buty peracetate) will sterilize raw foods such as meat, poultry, eggs, fish, shellfish, seed-derived foods such as fresh alfalfa sprouts, and fresh and/or lightly processed vegetables contaminated with bacteria (naturally or purposely). We also will sterilize food packaging materials and pre-packaged foods. Specific Objective 2: Design and develop a pilot-scale (4-5 liter) SCCD food sterilization system for testing larger amounts of food for use in further quality evaluations. Specific Objective 3: Develop an educational component that trains undergraduate and graduate students in Food Science in the use and evaluation of this and other new technologies. PI will include supercritical fluid theory and practical use of supercritical equipment in his Instrumental Analysis class (SFS520 and MMBB520), offered every spring semester. Specific Objective 4: Elucidate killing mechanism imposed by SCCD treatment on bacterial endospores. We believe that the SCCD cannot be developed further without understanding details of biochemical mechanism of endospore protection. We will use proteomics and other analytical methods to compare resistant and nonresistant Bacilli strains spores' components. Outcomes: This research and education program will result in (a) development of a novel method for sterilization of raw foods and food packaging that will be of practical value to the food industry as well as provide a new approach to decrease the incidence of foodborne disease in the United States and the world; and (b) will provide an interdisciplinary educational experience for undergraduate and graduate students in School of Food Science who will learn how to apply chemical, microbiological, and instrumental techniques to the areas of food safety and food quality. Students participating in this process/product development exercise will not only obtain expertise in SCCD processing, but will also be trained in steps of process development for use in their professional careers. Plans to communicate results to stakeholders and to the public: Results generated in the work described here will be published in graduate student theses and dissertations and in the peer-reviewed scientific literature within appropriate microbiology and food science journals. Summaries of progress will be posted on the University of Idaho web site (http://www.ebi.uidaho.edu/default.aspxpid=99130) which is accessible by the general public. Also, the findings from case study work conducted by FS 520 students will be posted on the course website (https://www.blackboard.uidaho.edu). The SCCD material will also be made available to instructors of food microbiology and food processing within the School of Food Science for use in training food science undergraduate students regarding the benefits and uses of SCCD technology. Appropriate patents will be filed through the University of Idaho Office of Technology Transfer, which will also conduct licensing activties with the food industry.
Project Methods
Procedures for Specific Objective 1. To perform the proposed research, we will use two SCCD systems that are housed in the University of Idaho School of Food Science, Dr. Paszczynski laboratory. One system, used extensively in our prior work consists of two ISCO SFX syringe pumps (ISCO model 260D, Lincoln NE) and pump controllers (series D and SFX 200) and a two-channel supercritical fluid extractor (SFX 220) equipped with a programmable restrictor temperature controller. The apparatus allows for effective delivery and maintenance of liquid CO2 and modifier pressure and flow rate to the sterilization chambers. Liquid CO2 is fed from a standard cylinder, equipped with a siphon tube, into one of the syringe pumps, all programmed and controlled by the ISCO SFX 200 pump controller. The second identical programmable pump is used to deliver modifier solution. Procedures for Specific Objective 2: To develop this innovative sterilization method for use by the commercial food industry, the collaborative efforts of academic scientists and private industry is needed. We have initiated these discussions with the private company Applied Separation, Allentown, PA, who has expressed an interest in developing a prototype SCCD sterilizer. To accomplish the goals of objective 2 we will produce via this collaboration an apparatus design proposal with which to approach food processors and packing firms. A prominent food processor (ConAgra Foods, Inc., Richland, WA) in the region has already been contacted, and has communicated their interest and support of the proposed research (see accompanying letters of support). ConAgra has further agreed to provide vegetable raw material and/or variably processed products as needed for use in the project. Analytical tests (microbial lethality and food quality assays described in Objective #1) will also be conducted to guide and confirm pilot-scale development of SCCD processes. Procedures for Specific Objective 3. Based on prior SCCD process development work, PI will include supercritical fluid theory and practical use of supercritical equipment in his Instrumental Analysis class (SFS520 and MMBB520), offered every spring semester. This effort involves direct training of students in supercritical process theory and practical operation of SF equipment, while at the same time provides valuable data regarding properties of SCCD-treated food products and other materials Procedures for Specific Objective 4. We believe that the SCCD cannot be developed further without understanding details of the biochemical mechanism of endospore protection. We will use proteomics and other analytical methods to compare biochemical composition of endospores of resistant and nonresistant Bacilli strains. Result from this objective will benefit further development of this and other sterilization techniques for food, medical, and pharmacological applications. To identify and quantify spore proteins we will use modern chromatographic and mass spectrometry techniques.

Progress 09/01/12 to 06/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported 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? Nothing Reported

Impacts
What was accomplished under these goals? PI for this project has retired and the annual report filed on 2/22/2017 will serve as the final report.

Publications


    Progress 10/01/15 to 09/30/16

    Outputs
    Target Audience:Food, pharmaceutical and medical industries Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Specific Objective 3 Develop an educational component that trains undergraduate and graduate students in Food Science Based on prior SCCD process development work, PI has included supercritical fluid theory and practical use of supercritical equipment in his Instrumental Analysis class (SFS520 and MMBB520), offered every spring semester from 2012 to 2016. This effort involved direct training of students in supercritical process theory and practical operation of SF equipment, while at the same time provided valuable data regarding properties of SCCD-treated food products and other materials. Educational classroom component: An existing course, SFS 520 (instrumental Analysis - taught annually in the spring semester), was the vehicle through which this objective was implemented. This course is specifically designed as a 'capstone' experience for food and other biological sciences students, and requires integration of scientific principles in use and understanding of various analytical instruments in a modern life science laboratory. The class covered the theory and practical application of modern analytical methods used to analyze biological and chemical samples, including hands-on practice with equipment used in modern life and food science laboratories. Dr. Paszczynski has taught this course for the past20 years and during that time about 300 hundred students attended, about 60 students during the reporting period. A primary benefit of this arrangement is that students are directly introduced to a novel food processing (supercritical fluid technology) with true commercial potential. 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? Nothing Reported

    Impacts
    What was accomplished under these goals? In general, problematic microorganisms of interest to the food industry are human pathogens (relevant to human health) and spore-formers (relevant to food sterilization performance) groups. Our and others researchsuggest that the spore formers often have a combination of the following phenotypes: thermophiles, psychrophiles, desiccation tolerant, organisms that can survive exposure to electromagnetic radiation (UV, γ radiation), sterilizing chemicals, and low oxygen conditions encountered during food processing, sterilization, storage and transport. Hence, the identification of tolerance factors and the potential to assess for tolerance factors bears specific relevance to addressing stringency requirements as these factors can (A) be further validated as an assay to bolster an understanding of hardy organisms to assist the food industry in predictive risk assessments of food contamination, and (B) rapidly screen samples containing endospore formers and focus growth studies to isolate a larger and comprehensive breadth of organisms for the development and confirmation of specification values for the food industry. A bacterial endospore is a metabolically dormant form of life that is much more resistant to environmental challenges than their parental vegetative bacterial cell. These include heat, desiccation, lack of nutrients, and exposure to UV and gamma radiation, as well as organic chemicals and oxidizing agents. This exceptional resistance is attributed to the spore's structure and the biochemical properties of its components, many of which are specific to bacterial endospores. As a proof of concept we focused on candidate organisms that have one or more of the following phenotypes: food contaminant organisms that can survive exposure to heat, oxidizing chemicals such as hydrogen peroxide or chorine based cleaners, and that can survive exposure to electromagnetic radiation (UV and γ radiation). Organisms for study was obtained from various sources, including American Type Culture Collection (ATCC), ESA Microbial Collection at the DSMZ, Bacillus Genetic Stock Center, and other appropriate sources.

    Publications


      Progress 10/01/14 to 09/30/15

      Outputs
      Target Audience: Nothing Reported Changes/Problems: Nothing Reported 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?We will work on Specific Objective 4: Elucidate killing mechanism imposed by SCCD treatment on bacterial endospores.

      Impacts
      What was accomplished under these goals? In this reporting period PI addressed the educational component of the proposal which include training of undergraduate and graduate students in use of supercritical fluid technologies. PI included information related to the supercritical fluid theory and practical use of supercritical sterilization equipment in his Instrumental Analysis Class - FS520, offered during the 2015 spring semester. Briefly, during the SF520 class students were familiarized with principle of operation of ISCO supercritical fluid sterilization/extraction system. Carbon dioxide to reach supercritical stage require 72 atm pressure and 32 degree C of temperature. To handle CO2 in supercritical stage a specialized equipment is required. The ISCO supercritical sterilization equipment consisted of two ISCO SFX syringe pumps (ISCO model 260D, Lincoln NE) and pump controllers (series D and SFX 200) and a two-channel supercritical fluid extractor (SFX 220) equipped with a programmable temperature controlled venting tubes. The apparatus allowed for effective delivery and maintenance of liquid carbon dioxide and modifier flowrate to the two 30 ml temperature controlled compartments. The sterilization chamber was able to accommodate different type of solid samples. Liquid carbon dioxide was delivered from a standard pressurized cylinder equipped with siphon, into one of the syringe pumps, all programmed and controlled by the ISCO SFX 200 controller. The second similar programmable pump (series D and SFX 200) was used to deliver modifier solution. The dynamically mixed SF-CO2 and modifier solutions were delivered to the SFX-220 apparatus equipped with two independently controlled 30 mL extraction/sterilization chambers. After completion of each sterilization cycle, CO2 and modifier were released through a restricted vent heated to 50 °C at a flow rate 1.5mL/min into a collecting flask. The restricted vent was used only during dynamic sterilization process. In spring semester 2015 the Instrumental Analysis class - FS520was attended by seventeen graduate and undergraduate students from University of Idaho and Washington State University. After completing all class requirement, I can gladly report that all students received passing grades. The class also covered the theory and practical application of various methods used to analyze biological, chemical and food samples, including hands-on practice with equipment used in modern life science analyticallaboratories. The course emphasized spectroscopic and chromatographic instruments and covered theory of chromatographic separation methods. Topics covered included: pre-experiment planning, preparative methods, analytical methods, statistical evaluation of data, and examination and presentation of results. The course also introduces students to basics of computer modeling and visualization of biological molecules.

      Publications


        Progress 10/01/13 to 09/30/14

        Outputs
        Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? We will publishreportedresults in near future. What do you plan to do during the next reporting period to accomplish the goals? PI willaddressthe educational component of this proposal which include training of undergraduate and graduate studentsin use of supercritical fluid technologies. PI will include supercritical fluid theory and practical use of supercritical equipment in his Instrumental Analysis class - SFS520, offeredduring the 2015 spring semester. In this semesterFS520 is attended by 14 graduate students from University of Idaho and Washington State University. We will also work on Specific Objective 4: Elucidate killing mechanism imposed by SCCD treatment on bacterial endospores.

        Impacts
        What was accomplished under these goals? Materials: Bacillus pumilus 107 strips from Raven Labs (Omaha, NE), frozen vegetables from J.R. Simplot Company (Caldwell, ID): frozen edamame, parfried diced potatoes, RoastWorks Vegetables & Fruit: flame-roasted corn & black bean fiesta, flame-roasted peppers & onions. Sterilization method: The machine used for sterilization is an extractor (Spe-ed SFE) by Applied Separations (Allentown, PA) with a dual piston pump (Model 1500) by Scientific Systems, Inc., (State College, PA) for application of modifier. The extractor uses a pump system that provides dynamic flow of SFCO2 at a set flow rate, pressure, temperature and duration. The SFCO2 flow rate was set to approximately 2 L/m for all sterilization treatments. The metering valve temperature was set to 100°C. The machine was primed prior to the first sterilization treatment of the day, by running it for 45 minutes at 50°C, 100 atms, and 0.3 ml/min of modifier. A 3% H2O2 modifier was used in all sterilizations. Sample preparation with miracloth: Small "pockets" of Miracloth were made by folding the Miracloth and stapling one side shut. Sterile tweezers were used to transfer one piece of vegetable into sterile Miracloth. Miracloth-wrapped vegetables were transferred into a sterile container with lid. Thesterilizationj chamber was filled to approximately 75% capacity with glass beads. Three Miracloth-wrapped vegetables were transferred into the chamber on top of the glass beads. Chamber was sealed and sterilization process was started. After sterilization was complete, sterile scissors were used to cut the Miracloth and the vegetables were removed with sterile tweezers. Sterilized samples were place on a TSA plate and incubated at 37°C for up to one week. Glass bead and cotton method: Chamber was filled approximately 75% with glass beads. Three bare vegetable samples were added on top of the glass beads using sterile tweezers. Remaining empty space above vegetables was filled with sterile cotton. Chamber was sealed and sterilization process started. After sterilization, sterile tweezers were used to remove the cotton plug. Vegetables were place on separate TSA plates using sterile tweezers. TSA plates were incubated at 37°C for up to one week. Homogenization method: Frozen vegetables were placed in 50 mL of 0.1% Tween 80. They were homogenized for 30 seconds (used homogenizer by Brinkmann Instruments Co., Westbury, NY). Performed serial dilutions from original suspension until 10-10. Plated 0.1 mL from each dilution onto a TSA or PDA plate. Incubated at 37 or 30°C, respectively. Counted the number of colonies on each plate after 24 hours. Homogenization and filter method: Frozen vegetables were placed in 50 mL of 0.1% Tween 80. They were homogenized for 30 seconds. Filtered homogenization liquid through gauze stretched over a funnel. Performed serial dilutions from original suspension until 10-10. Plated 0.1 mL from each dilution onto a TSA plate. Incubated at 37°C. Counted the number of colonies on each plate after 24 hours. Shaken method: Placed frozen vegetable in 10 mL of 0.1% Tween 80. Shook for 15 minutes at 25°C and 250 rpm's. Performed serial dilutions from original suspension until 10-10. Plated 0.1 mL from each dilution onto a TSA plate. Incubated at 37°C. Counted the number of colonies on each plate after 24 hours. Results and Discussion A different extraction machine (Spe-ed SFE by Applied Separations) was used for this research than had been previously used to perform sterilizations. Bacillus pumilus 107 strips were used to validate the sterilization capabilities of this machine, and sterilization was achieved at 35°C, 80 atmospheres, 0.15 ml/min of a 3% hydrogen peroxide modifier, and 45 minutes. These conditions were then used to test frozen corn. However, the corn was not sterilized at these conditions and the plates developed outgrowth. Temperature, pressure, and modifier amount were increased in an attempt to find sterilizing conditions. Sterilization of the frozen corn was achieved at 45°C, 100 atm, 0.3 ml/min of modifier, and 45 minutes. These conditions were then used on other frozen vegetables, such as red pepper, green pepper, onion, black bean, edamame, and potato. Sterilization was only achieved with corn, black bean, and edamame. Sterilization of a larger quantity (3-4 pieces per miracloth) of mixed frozen vegetables was then attempted. The vegetables were wrapped together in a layer of miracloth and sterilized at the previously mentioned conditions. Sterilization was only achieved with one of the mixes of corn, onion, black bean and red pepper. The method was then changed slightly by removing the glass beads from the chamber, plugging one end of the chamber with cotton, adding the vegetable mix (not wrapped in Miracloth, 8-12 pieces of vegetable), then plugging the other side with cotton. However, sterilization was still not achieved, even after raising the temperature to 50°C. The method was further modified by filling the extraction chamber with glass beads, adding the vegetable, and plugging the rest of the empty space with cotton. Sterilization of potato was achieved at 50°C, 100 atm, 0.3 ml/min of modifier, and 45 minutes, as well as at a reduced modifier amount of 0.15 ml/min. Black bean, green pepper and onion were all sterilized at these conditions as well. We then tested to see if lyophilization of the vegetables would affect sterilization. The vegetables were added to 50 mL plastic containers, with small holes in the cap, and lyophilized for three days. They were then sterilized using the glass beads and cotton method at 50°C, 100 atms, 0.3 ml/min of modifier, and 45 minutes. Using this method, all of the lyophilized vegetables were successfully sterilized, with no growth even up to a week, with the exception of the green pepper. The success of sterilizing the lyophilized vegetables compared to the frozen vegetables could be due to water acting as a barrier to the SFCO2 and H2O2 modifier. With the freeze-dried vegetables this excess water was removed, perhaps allowing better access to the vegetables. Attempts were made to estimate the amount of microorganisms on all of the frozen vegetables using serial dilutions, but they were unsuccessful. We tried homogenizing the vegetables in 10 mL of a 0.1% Tween 80 solution and serially diluting out to 10-10, but the growth on the plates was inconsistent and did not follow the normal pattern of serial dilutions. We also tried shaking the whole vegetable in 0.1% Tween 80 for 15 minutes at 25°C, 250 rpm and doing serial dilutions from the liquid but there was no resulting growth on the plates. We tried filtering the homogenization of vegetable through gauze so that the pieces of vegetable wouldn't be present in the serial dilutions but there was no growth as a result of this method. It is probable that the microorganisms need specific nutrients from the vegetables to grow, and with the homogenization method inconsistent pieces of vegetable in the Tween 80 may have interfered with growth, while with the filter and shaken methods there were insufficient nutrients for growth. We plated serial dilutions on TSA and potato dextrose agar (PDA) plates and incubated at 37 and 30°C respectively, for up to one week. Different modifiers: We considered using different modifiers than 3% hydrogen peroxide, such as peracetic acid, lactic acid and tert-butyl hydroperoxide. However, using a 3% peracetic acid solution caused our machine to malfunction after running two sterilizations with it. The corn was successfully sterilized, but we abandoned our efforts in this area because of the apparent damage to the sample. Future Directions The lyophilization method gave promising results; so further research should be conducted in this area. Different modifiers could also be explored, although they would require more research to avoid any equipment damage.

        Publications


          Progress 01/01/13 to 09/30/13

          Outputs
          Target Audience: Food Science and Food Industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? My graduate student Miss Aleksandra Chencinska successfully defendedher Ph.D. Dissertation in December 2013. Dissertation title: DEVELOPMENT OF SUPERCRITICAL FLUID CO2-H2O2 STERILIZATION METHOD AND ELUCIDATION OF BACILLI ENDOSPORES’ TOLERANCE TO HYDROGEN PEROXIDE AND HEAT. How have the results been disseminated to communities of interest? Results have been submitted for publication in peer review journal, BMC Microbiology. What do you plan to do during the next reporting period to accomplish the goals? We will continue research related to the objectives 1 and 4 of this proposal.Specific objective 1: Demonstrate that supercritical carbon dioxide (SCCD) containing a small amount of modifier (e.g., ~0.1% v/v hydrogen peroxide, tert-butyl hydroperoxide, peracetate, tert-buty peracetate) will sterilize raw foods such as meat, poultry, eggs, fish, shellfish, seed-derived foods such as fresh alfalfa sprouts, and fresh and/or lightly processed vegetables contaminated with bacteria (naturally or purposely). We also will sterilize food packaging materials and pre-packaged foods.Specific objective 4: Elucidate killing mechanism imposed by SCCD treatment on bacterial endospores. We believe that the SCCD cannot be developed further without understanding details of biochemical mechanism of endospore protection. We will use proteomics and other analytical methods to compare resistant and nonresistant Bacilli strains spores' components.

          Impacts
          What was accomplished under these goals? In order to understand the mechanism(s) of tolerance to extreme conditions and further develop our SF-CO2-hydrogen peroxidesterilization process, the biochemical makeup and functions of individual bacterial endospore components have to be elucidated (Objective 4 of the proposal). We hypothesize that components of spore structure contribute to the resistance. In the presented research we targeted the following questions:What are these components? What is their function? How do they contribute to the spore’s high tolerance? Which components should be targeted by novel/improved sterilization methods? In the research for this reporting period, we compared chaperones (heat shock proteins) in endospores of four Bacilli species and quantified several protein families in Bacillus pumillusSAFR-032 endospores obtained at 26°C and 42°C. Among all proteins identified in spores of four Bacilli species, chaperones constituted 3% of total amino acids sequences identified. In the spores of B. pumilus SAFR-032 produced in elevated temperatures GroEL, DnaK, and Tig protein level significantly increased, but the level of CspB, CspC, CspD did not change. We also observed a significant increase in the concentration of all small acid soluble proteins, except for SspA. Interestingly, the protein concentrations of both ribosomal subunits did not change except for a few that showed increased induction in the spores of SAFR-032 obtained at 42oC. The enzymes related to DNA and RNA metabolism were much less affected by elevated sporulation temperatures, although, concentration of DNA methylase, DNA directed RNA polymerase subunits alpha and beta and spore photoproduct lyase increased in endospores produced in 42oC. There is seemingly no consensus about the role of chaperones in spores of Bacilli species in the scientific community, however the results from our studies show the presence of chaperones in dormant spores of four investigated species and a significant increase of their levels in the dormant mature spores produced at elevated temperature, confirming earlier heat shock studies. Our observation that increased temperature resistance of endospores obtained at 42°C suggests that chaperones may play a critical role in helping germinating endospores protein to recover from any heat or other stress encountered during the dormancy period. The heat resistance assay confirmed the higher survivability of spores obtained at 42°C

          Publications


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

            Outputs
            OUTPUTS: Sterilization with supercritical fluid carbon dioxide (SF-CO2) is an alternative to commonly-used methods of sterilization for the removal of microorganisms from food. The traditional, commonly used methods are: steam autoclaving, dry heat sterilization, ethylene oxide vapor treatment, and gamma-irradiation. Carbon dioxide is in its supercritical fluid state when both the temperature and pressure equal or exceed the critical point of 32 C and 73 atm. In its supercritical state, SF-CO2 has both gas-like and liquid-like properties, and it is this dual characteristic of supercritical fluids that provides the ideal conditions for fast delivery of sterilizing compounds to the bacterial cells/viruses and spores with a high degree of efficiency in a short period of time. Hydrogen peroxide as a microbe sterilization agent has been used for many years in the medical and food packaging fields with no negative impact on instrument materials or subsequent instrument performance. The task is how to employ H2O2 as a sterilant that is low in concentration and compatible with the sensitive materials and equipment. A solution of H2O2 in water was thus considered a prime candidate as an additive to SF-CO2 to create a universal sterilant. fluid and unique sterilization process. Recent research demonstrated that H2O2 added as a modifier to SF-CO2 indeed created a more effective sterilization process, requiring lower temperature, lower pressure and shorter cycle times. In this project the SF-CO2 enriched with small amount of hydrogen peroxide was investigated to determine the combinations of pressures, temperatures and time that effect the fastest and most complete inactivation of bacterial and fungal spores and microorganisms in biofilm structures. PARTICIPANTS: Andrzej Paszczynski TARGET AUDIENCES: Food, pharmaceutical and medical industries PROJECT MODIFICATIONS: Not relevant to this project.

            Impacts
            Foodborne microbial pathogens cause hundreds of thousands of cases of illness and hundreds of deaths in the United States each year. These bacteria are typically transmitted to humans from contaminated raw meat, vegetables, cheese, eggs, and milk. These pathogens cause illnesses ranging from diarrhea and vomiting to much more serious diseases including gastroenteritis, central nervous system disorders, severe bloodstream infections, and life-threatening illnesses such as listeriosis. Rapid molecular-based methods currently exist for the detection of foodborne Salmonella, E. coli O157:H7, Vibrio and for Listeria. However, there are no rapid molecular-based methods to detect spore formers which are responsible for an increasing number of foodborne illnesses. Spore forming species, such as Geobacillus spp, Bacillus cereus, Clostridium botulinum, C. perfingens, Paenibacillus spp., Ailcyclobacillus spp. and others, are often implicated in food poisoning and/or spoilage resulting in huge losses each year to the US economy. Spore formers are the bacteria most frequently responsible for food poisoning and spoilage of heat-treated foods because endospores are resistant to desiccation and often can survive food processes and sterilization methods (e.g. Pasteurization). It has been suggested that the pasteurization process for milk selects for endospore formers by killing competing vegetative bacteria and that heat from pasteurization can "shock" endospores to germinate. Thus, there is a clear need for better food sterilization method that will be as robust as heat sterilization (e.g. autoclaving) but, as gentle as Pasteurization.

            Publications

            • No publications reported this period