Novel Sterilization Method for Food, Clinical and Pharmaceutical Applications

Non-Technical Summary:<br/>
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.
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Approach:<br/>
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.
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Progress:<br/>
2012/01 TO 2012/12<br/>
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.
<br/>PARTICIPANTS: Andrzej Paszczynski
<br/>TARGET AUDIENCES: Food, pharmaceutical and medical industries.
<br/>PROJECT MODIFICATIONS: Not relevant to this project.
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IMPACT: 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.