Progress 10/01/10 to 09/30/15
Outputs
Target Audience:Food Industry, academia and scientists in government laboratories Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two master of science students were trained for data collection and analysis for the project. First student developed a sensor for measuring the concentration of the chlorine dioxide sensor. The second student investigated the kinetics of bacterial inactivation using chlorine dioxide gas. Both students completed the masters degree and each wrote a master thesis. How have the results been disseminated to communities of interest?First student presented his results in the Conference in Food Engineering in 2012. The second student presented a poster at the 2015 Institute of Food Technologists Annual Meeting. A manuscript using the data from both students work was submitted which is currently going through the review process What do you plan to do during the next reporting period to accomplish the goals?We will continue on working to investigate the effectiveness of chlorine dioxide on preservation of fresh produce by expanding to other produce and other bacteria and also scaling up of the process and development of proper equipment for treatment of fresh produce with chlorine dioxide gas.
Impacts
What was accomplished under these goals?
The effect of chlorine dioxide on decontamination of fresh produce, specifically, spinach was investigated. Effect of chlorine dioxide gas on inactivating E.coli K12 attached to the spinach leaf surface was determined. Inactivation kinetics of E. coli was described by Weibull kinetics model more accurately than the first order kinetic model. The varying concentration of the gas was also incorporated into the model to describe the numbers of E.coli as a function of chlorine dioxide concentration and time of exposure. The effect of chlorine dioxide gas on color of spinach leaf was also investigated by calculating the Hue values for spinach leaves. Then, the time of treatment and the gas concentration for the treatment was optimized to obtain maximum bacterial inactivation and minimum color damage. It was found that low gas concentrations of 2mg/L with the relatively longer exposure times of 20 min resulted in the highest bacterial reductions and minimum or no color bleaching of spinach leaves.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2012
Citation:
Gehringer, R., 2012. Development of a chlorine dioxide gas concentration monitoring unit and kinetic analysis of the effect of chlorine dioxide treatment on color and microbial content change of spinach, Department of Food, Agricultural, and Biological Engineering. The Ohio State University
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2015
Citation:
Yang, W., 2015. Effect of Chlorine Dioxide Gas Treatment on Bacterial Inactivation Inoculated on Spinach Leaves and on Pigment Content, Department of Food, Agricultural, and Biological Engineering. The Ohio State University
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Graduate and undergraduate students, members of academia, food industry Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A doctoral student is continuing on the project for the analysis of stability of hydrogel particles and anthocyanin release by diffusion at low pH and by particle dissolution at higher pHs. 4 undergraduate students were trained by assigning shorter projects relevant to overall project 1 student has been working on effect of drying on particle stability 1 student has been working on effect of voltage and frequency on particle size 1 student has been working on creating smart biopolymer formulations by using other biopolymers 1 student has been working on mechanical properties of hydrogel particles How have the results been disseminated to communities of interest? One poster titled (Degradation kinetics of pH responsive alginate-pectin smart hydrogel for controlled nutrient delivery) was presented in ASABE meeting July 2014 One poster titled (Enhanced Stability And Targeted Delivery Of Microencapsulated Anthocyanins For Improved Food Quality And Human Health) was presented in Experimental Biology meeting April 2014 What do you plan to do during the next reporting period to accomplish the goals? We will continue to work on microencapsulation of anthocyanins and investigate the release kinetics of anthocyanins from hydrogel particles at low and high pH levels. We will investigate effect of encapsulation on anthocyanin stability and effect of drying on particle stability and anthocyanin release from the particles.
Impacts
What was accomplished under these goals?
The effective and efficient delivery of beneficial compounds require that they should be protected from environmental effects during processing and storage and after digestion to the point of absorption in the human body. The selection of encapsulant or shell material is critical for protection and functionality of the bioactive compounds. It is imperative for encapsulant material to be stable and intact under the conditions relevant to processing and storage of food and in the initial part of the GI tract but swell or degrade in the later part of the GI tract especially in intestines. Smart biopolymer mixture formulations were developed in our laboratory to encapsulate anthocyanins. The biopolymer mixtures investigated included alginate and pectin of two ratios by mass (43:57 Al:P and 82:18 Al:P). Spherical or disc shaped hydrogel particles were obtained by extrusion of biopolymer mixture through a needle and dripping into a pH 1 buffer solution. The stability of hydrogel particles were investigated by monitoring the dissolution behavior of particle at 3 temperature levels of 4, 24, and 37 degrees centigrade. Dissolution of particles were determined to follow zero order kinetics at all conditions investigated. Dissolution rate constants and the activation energies were calculated for spherical and disc shape particles, at two different pH levels of pH 5 and 7, and for two Al:P ratios to characterize the dissolution behavior of encapsulant material so that the release characteristics of anthocyanins can be predicted. The comparison of rate constants showed that at pH 5 and 7, the release of bioactive compounds is controlled by dissolution of Al:P hydrogel particles instead of diffusion. Therefore, kinetics of hydrogel particle dissolution has to be modeled and the parameters affecting the dissolution kinetics should be characterized. For fastest release of bioactive compounds in small intestine at pH7 and 37 ?C, hydrogel particles with 43:57 Al:P ratio, disc shaped particles should be used. Under these conditions hydrogel particles will release 70% of the bioactive compounds within 2.5 min following particle exposure to pH 7. For slowest release of bioactive compounds in small intestine at pH7 and 37 ?C, hydrogel particles with 82:18 Al:P ratio, sphere shaped particles should be used. Under these conditions hydrogel particles will release 70% of the bioactive compounds within 7 min following particles exposure to pH 7. Varying rate of bioactive compounds release can be obtained between 2.5 and 7 min by changing the particle shape and Al:P ratio of hydrogel. Hydrogel particles are expected to release 40% of the bioactive compounds within 4 - 5 min following particle exposure to pH 5 which is approximately pH of a full stomach. Given the higher residence times reported for stomach, particles within a beverage of pH 3 or lower should be consumed with an empty stomach so that they can be delivered intact to the intestines. Also, a system development is in progress to produce different size of particles by varying the voltage and frequency applied to the system during particle manufacturing. Following manufacturing, hydrogel particles were dried to various levels to reduce their water activities so as their storage stabilities were enhanced.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Huang, M., Kaletunc, M, St. Martin, S., Feller, M., McHale, L.K. 2014. Correlations of seed traits with tofu texture in 48 soybean cultivars and breeding lines, Plant Breeding, 133(1): 67-73.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Fresh produce industry, faculty and students from academia, government employees, extension educators Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? 1st project was collaboration among Kaletunc laboratory at Ohio State Universityand and US Army Natick Soldier Research, Development & Engineering Center, James Madison University, and Rutgers University. This project provided a training opportunity for a PhD student for the thermal characterization of enzyme and anzyme-tannin complexes. 2nd project was collaboration among Kaletunc laboratory and Horticulture and Crop Science at the Ohio State University. This project provided a training opportunity for a M.S student for the textural characterization of tofu prepared various food-grade soybean lines grown at the state of Ohio. How have the results been disseminated to communities of interest? 1st project results were presented in Conference in Food Engineering, NATAS Annual Conference, and Institute of Food Technologists Annual meeting. The results were also published as a journal article in Journal of Agricultural and Food Chemistry. 2nd project results were published as a journal article in Plant Breeding. What do you plan to do during the next reporting period to accomplish the goals? We will continue to work on microencapsulation of bioactive materials such as tannins for protection of sensitive bioactive materials during processing, storage, and passage through the initial section of gastrointestinal tract GIT and controlled and targeted delivery of bioactive materials in intestines. More specifically, we will be focusing on anthocyanins which are known to provide color and to have antioxidant properties.
Impacts
What was accomplished under these goals?
Effect of polyphenols (tannins) specifically from cranberry, grape, cocoa, and pomegrante on inhibiting starch digestion was studied. These compounds interact with proteins due to their numerous hydroxyl groups, which are suitable for hydrophobic associations. It was hypothesized that tannins could bind to the digestive enzymes α-amylase and glucoamylase, thereby inhibiting starch hydrolysis. Slowed starch digestion can theoretically increase satiety by modulating glucose “spiking” and depletion that occurs after carbohydrate-rich meals. Tannins were isolated from extracts of pomegranate, cranberry, grape, and cocoa and these isolates tested for effectiveness to inhibit the activity of α-amylase and glucoamylase in vitro. The compositions of the isolates were confirmed by NMR (nuclear magnetic resonance spectroscopy)and LC/MS (Liquid chromatography/mass spectrometry) analysis, and tannin−protein interactions were investigated using relevant enzyme assays and differential scanning calorimetry (DSC). The results demonstrated inhibition of each enzyme by each tannin, but with variation in magnitude. In general, larger and more complex tannins, such as those in pomegranate and cranberry, more effectively inhibited the enzymes than did less polymerized cocoa tannins. Interaction of the tannins with the enzymes was confirmed through calorimetric measurements of changes in enzyme thermal stability. Food-grade soybeans [Glycine max (L.) Merr] used to produce tofu have specific seed composition, shape, size and colour requirements. Seed qualities, such as protein content, have been correlated with tofu texture. The objective of this study was to determine the relationships between textural qualities of tofu and seed characters, including shape, size, density, weight, protein content and oil content. Tofu was produced from the seed of 48 high-protein or food-grade soybean lines grown at two locations in 2010. Four tofu textural traits were assessed including work to failure, deformability, stiffness and hardness. Seed protein and oil content were significantly correlated with stiffness and hardness, and protein was additionally correlated with work to failure. Pearson’s correlation coefficients ranged from 0.45 to 0.60 (adjusted P-values < 0.002) for protein and 0.49 to 0.35 for oil (adjusted P-values < 0.016). No significant correlations between tofu texture and seed volume or shape were detected. Seed protein content can be assessed for indirect selection for tofu firmness, whereas this study reveals no evidence that seed size affects tofu texture.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Huang, M., Kaletunc, M, St. Martin, S., Feller, M., McHale, L.K. 2013. Correlations of seed traits with tofu texture in 48 soybean cultivars and breeding lines, Plant Breeding, 132(6).
Barrett, A H, Ndou , T., Hughey, C., Straut, C, Howell, A., Dai, Z. and Kaletunc, G. 2013. Inhibition of ?-amylase and glucoamylase by tannins extracted from Cocoa, Pomegranates, Cranberries, and Grapes. J. Agric. Food Chem., 61, 1477?1486
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Worked on application of calorimetry to evaluate the extent that tannins inhibit the activity of a-amylase and glucoamylase (GA). Tannins from pomegranates, grape, cranberry, and cocoa were investigated by thermal analysis and enzyme kinetics studies. Enzyme and tannin mixtures were prepared at 1:1, 1:10, and 1:100 Enzyme:Tannin ratios. NMR studies were conducted to elucidate the structural differences among tannins from various botanical origins. DSC was used to determine the structural changes of GA as a function of pH and enzyme concentration and to investigate the relationships between the structural changes observed in thermograms and the activity of the enzyme. GA from Aspergillus niger was used for the studies. The optimum conditions for GA based on glucose production rate from maltose as substrate at a concentration of 10 mg/ml was investigated. The effect of enzyme concentration over 0.1 and 100 mg/ml and of pH over 4.5-5.5 on the thermal stability of GA was studied with DSC. PARTICIPANTS: The Ohio State University: Gonul Kaletunc, Zifei Dai; Natick Army Research Labs: Ann Barrett; James Madison University: Christine A. Hughey; Rutgers University: Amy Howell, Perla Relkin; AgroParis Tech, France TARGET AUDIENCES: Academia and food industry PROJECT MODIFICATIONS: Studies on to determine the structural changes of GA as a function of pH and enzyme concentration and to investigate the relationships between the structural changes observed in thermograms and the activity of the enzyme were added.
Impacts
Enzyme activity tests showed that pomegranate and cranberry had a similar inhibition effect on alpha-amylase (approximately 40%), and grape had a lower inhibition effect (approximately 20%) while cocoa tannin did not inhibit the alpha-amylase activity. Results suggest that larger and more complex tannins are better able to inhibit amylase, and that binding was also confirmed by DSC. Results of DSC studies showed the optimum pH and temperature of GA was found to be 5.5 and 55 C respectively based on the activity of the enzyme breaking maltose to glucose. The thermal stability of GA was highly depended on both the pH and enzyme concentration. While at the highest enzyme concentration and at pH 4.5, a single endothermic peak with a thermal stability of 78 C was observed, as the concentration decreased an overlapping endothermic and exothermic peaks were prominent. At 10 mg/ml enzyme concentration, the thermal stability of the major endothermic peak was not affected by pH. However, a lower temperature peak was highly affected by the pH of the environment even changing from an exothermic transition at pH 4.5 and 5 to an endothermic transition at pH 5.5. DSC studies provided valuable information to predict the unfolding behavior of enzymes which can be utilized in selection of optimum processing conditions for starch hydrolysis process. Impact: Close to 9% of US population have diabetes. Modulating blood glucose level is necessary for treating metabolic disorders such as diabetes and for maintaining optimal physical and mental performance. The findings of this research can be used for development of treatments for type II diabetes which affect 26 million children and adults in US.
Publications
- Kaletunc,G. and Relkin, P. "Comparison of thermal stability and activity of glucoamylase for starch saccharification process optimization." [Abstract]. NATAS Proceedings. Vol. 41. Orlando: NATAS. (Aug 2012) www.natasinfo.org
- Barrett, A.H.; Kaletunc, G.; Hughey, C.A.; Straut, C.; Howell, A. and Ndou, T. "Dietary incorporation of proanthocyanidins/tannins that inhibit digestive enzymes offers an approach to engineering foods for blood sugar control." [Abstract]. Conference of Food Engineering 2012. Leesburg: Conference of Food Engineering. (Apr 2012)
- Ann Barrett, Christine A. Hughey, Christine Straut, Amy Howell, Tshina Ndou, Zifei Dai and Gonul Kaletunc. Inhibition of a-amylase and glucoamylase by tannins extracted from cocoa, pomegranates, cranberries and grapes. Journal of Agricultural and Food Chemistry, Accepted for publication, Nov 15, 2012.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Calorimetry was utilized to evaluate effectiveness of tannins inhibition of α-amylase and glucoamylase, starch degrading enzymes. Tannins are known to form complexes with proteins thereby inhibiting their enzymatic activity. Apple juice, grape juices and berry juices are all high in tannins. Pomegranates contain a diverse array of tannins, particularly hydrolyzable tannins. Most berries, such as cranberries, strawberries and blueberries, contain both hydrolyzable and condensed tannins. The binding of the tannins to starch hydrolysis enzymes was evaluated using differential scanning calorimeter (DSC) analysis and results correlated with changes in enzyme activity measured by starch hydrolysis assay. Starch hydrolysis involves two major enzymes at several major steps. Starch is first broken into oligosaccharides by α-amylase and then oligosaccharides are broken into glucose by glucoamylase. Enzyme and tannin mixtures were prepared at 1:1, 1:10, and 1:100 Enzyme:Tannin ratios. Then thermal analysis of alpha-amylase and glucoamylase incubated with tannin. DSC thermograms of enzymes and tannin alone and in combination were recorded between 10 to 100 degree C at 5 degree C/min heating rate. Inhibitions of alpha-amylase and glucoamylase activities were monitored by measuring the absorbance at 540 nm based on maltose calibration curve by using a spectrophotometer. Analysis of DSC thermograms enzymes with or without tannins showed the denaturation of enzyme depended on both origin of tannin and the type of enzyme. Tannins were heat stable. NMR studies showed that grape tannins are mostly low molecular weight, pomegranate tannins are large molecular weight hydrolysable tannins, cranberry and cocoa tannins are proanthocyanidins with different linkages. The higher thermal stability of enzyme with protein binding may be attributed to binding of tannins to native protein while the lower thermal stability may be binding of tannin to denatured protein. Enzyme activity tests showed that pomegranate and cranberry had a similar inhibition effect on alpha-amylase (approximately 40%), and grape had a lower inhibition effect (approximately 20%) while cocoa tannin did not inhibit the alpha-amylase activity. Results suggest that larger and more complex tannins are better able to inhibit amylase, and that binding was also confirmed by DSC. PARTICIPANTS: The Ohio State University:Gonul Kaletunc, Zifei Dai, Natick Army Research Labs: Ann Barrett TARGET AUDIENCES: Academia and food industry PROJECT MODIFICATIONS: Studies for controlled release of beneficial compounds in food materials for improving food quality and enhancing targeted delivery in human body were added.
Impacts
Close to 9% of US population have diabetes. Slowed starch digestion by use of tannins in the diet can modulate glucose "spiking" and depletion that occurs after ingestion of a carbohydrate-rich meal. These studies confirmed amylase activity can be inhibited by tannin derivatives naturally occurring in plant-based foods. Such inhibition of starch hydrolysis with plant based tannins is potentially a healthy way of controlling blood sugar through consumption of fruits. Fruits rich in tannins may provide nutraceutical treatment for type II diabetes and obesity. The findings of this research can be used for nutraceutical treatment for type II diabetes which affect 26 million children and adults in US.
Publications
- Kaletunc, G. Ohio State Univ., Columbus, OH; Barrett, A. H. Ph.D. , U.S. Army Natick Soldier RD&E Ctr., Natick, MA; Tshina Ndou, M.S., R.D., U.S. Army Natick Soldier RD&E Ctr., Natick, MA, Inhibition of starch hydrolysis by binding of tannins to alpha-amylase 39th Annual Conference of North American Thermal Analysis Society, Demoines, Iowa, Abstract. August 7-10, 2011 http://www.natasinfo.org/publications/conference-proceedings/
- Ann H. Barrett, Ph.D. , U.S. Army Natick Soldier RD&E Ctr., Natick, MA; Christine Straut, Ph.D., Battelle, Natick, MA; Gonul Kaletunc, Ohio State Univ., Columbus, OH; Tshina Ndou, M.S., R.D., U.S. Army Natick Soldier RD&E Ctr., Natick, MA, Differentiation of condensed tannins from fruit extracts by nuclear magnetic resonance (NMR) and relationship of structure toalpha-amylase binding 2011 IFT Annual Meeting, June 11 - 14, New Orleans LA, http://www.ift.org/Meetings-and-Events/Past-Meeting-Resources/Technic al%20Abstract%20Search%20Details.aspx?id=52103
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Researched using calorimetry to evaluate inactivation of bacteria in food preservation and to characterize effectiveness of tannins in slowing down starch hydrolysis. (a) In food preservation high hydrostatic pressure (HHP) is used as alternative to thermal processing, but processing above 600 MPa can adversely alter texture and color of foods and increase operating costs. Thus multiple preservation techniques with milder conditions are sought. Research evaluated effects of HHP and nisin treatment alone and in combination on cellular components and viability of two Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis) strains using differential scanning calorimetry (DSC) and plate counting. Using HHP (up to 200 MPa) or the nisin alone did not affect the viability and cellular components of either strain, but in combination achieved a reduction of bacterial load. The decrease in apparent enthalpy (DSC) was used to monitor the bacterial reduction and was compared to plate count results. (b) Inhibition of α-amylase, a digestive enzyme, may help control blood sugar by modulating release of glucose, thus preventing glucose and insulin spikes. Natural, plant-derived catechin constituents (tannins) are known to bind proteins. Study was to elucidate effectiveness of four tannin sources, pomegranate, cranberry, grape, and cocoa, on binding of the tannins to α-amylase through DSC analysis, and to correlate results with measured inhibition. Pressure variable DSC analysis of α-amylase incubated with (10%) tannin was performed using a Setaram calorimeter. Inhibition of α-amylase was determined through starch digestion assays and spectrophotometric determination of released maltose. DSC thermograms of amylase solutions showed interaction with pomegranate: specifically, pomegranate raised onset and peak temperatures for denaturation endotherms, indicating greater thermal stability of the protein due to binding. Pomegranate tannins effectively inhibited amylase (>80 %), with a more moderate effect (~30%) shown by cranberry, and negligible or no effect produced by treatment with grape or cocoa. Results suggest that more complex tannins are better able to inhibit amylase and that such interaction is confirmable by DSC. (c) Starch hydrolysis reaction can be improved by using a thermostable glucoamylase from a hyperthermophilic microorganism. However, thermophilic organisms grow slowly and produce low yield. High temperature stable protein production can be increased through recombinant DNA technology. The encoding gene (SSO0990) from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was cloned and expressed in Escherichia coli DH5α cells to obtain a thermostable recombinant glucoamylase. The GluA then was purified and thermal stability was characterized by using spectroscopy and differential scanning calorimetry in addition to enzyme activity test. The DSC thermogram for the purified enzyme shows an exothermic peak at 77.6C and an endothermic peak at 90C. This may indicate that the other proteins are present in purified enzyme and they aggregate first, followed by thermostable GluA denaturation at 90C. PARTICIPANTS: The Ohio State University:Gonul Kaletunc, Jaesung Lee, Zifei Dai, Natick Army Research Labs: Ann Barrett TARGET AUDIENCES: Academia and food industry PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
a) Developed new strategies for optimizing food processing to eliminate bacterial contamination and to produce safe food. b) Inhibit digestion of starch as a potentially healthful means of controlling blood sugar and hunger. c) Developed methods to improve the efficiency of industrial starch processing to reduce cost of material and energy.
Publications
- Lee, J,, Kaletunc, G. 2010. Inactivation of Salmonella Enteritidis strains by combination of high hydrostatic pressure and nisin Int J Food Microbiol. 140(1):49-56.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Research focus on application of calorimetry to evaluate effectiveness of food preservation for inactivation of bacteria and to characterize hyperthermophiles. Preservation of food products by using high hydrostatic pressure (HHP) is gaining interest in the food industry as an alternative to thermal processing. However, as reported in 2007, preservation by HHP processing requires pressure levels above 600 MPa which can adversely alter texture and color of many foods as well as increase initial and maintenance costs, promote wear, and shorten the life of the equipment. Therefore, applying a combination of moderate levels of food preservation methods might achieve desired food safety levels. In our research, a combination of HHP and bacteriocin was used. The effects of high hydrostatic pressure (HHP) and nisin treatment alone and in combination on cellular components and viability of two Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis) strains were evaluated by differential scanning calorimetry (DSC) and plate counting in order to evaluate the relative resistance and optimize the treatment conditions. Salmonella Enteritidis FDA and OSU 799 strains were subjected to HHP (0.1- 550 MPa for 10 min at 25oC) alone and in combination with nisin (200 IU/ml nisin) in culture broth. HHP (up to 200 MPa) or the nisin alone did not affect the viability and cellular components of either strain. An 8-log cfu/ml reduction was observed after a pressure treatment at 500 MPa for the FDA strain and 450 MPa for the OSU 799 strain. When nisin was added, a similar reduction was obtained at 400 MPa for FDA strain and 350 MPa for the OSU 799 strain. The decrease in apparent enthalpy appeared to be mainly due to reduction in the ribosome denaturation peak for both the pressure alone and the pressure-nisin combination treatments. HHP facilitates penetration of nisin into the cell above 100 MPa pressure. Monitoring through DNA binding probes the effect of pressure and nisin treatments on DNA in vivo showed nisin does not affect DNA at 200 IU/ml. Development of an understanding for inactivation of Gram-negative bacteria by pressure-nisin treatment allows optimizing HHP-nisin combinations that yield desired reduction of Gram-negative bacteria in food products. In this study, we also used bisbenzimide as a probe to identify the impact of HHP-nisin treatment on the thermal stability of the cellular DNA transition in vivo. Publication by Lee, J, Kaletunc, G. "Inactivation of Salmonella Enteritidis strains by combination of high hydrostatic pressure and nisin" will be pubshied in International Journal of Food Microbiology" in 2010. PARTICIPANTS: Kaletunc,G., Lee, J. TARGET AUDIENCES: Food industry, academia, and scientists in government laboratories. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Calorimetry data is used to evaluate the effectiveness and mechanism of each hurdle on overall inactivation of bacterial cell as well as their effect on cellular components so that hurdle levels and intensities can be optimized to achieve necessary inactivation levels. This information can be used to minimize large scale trial-and-error approach by reducing the time, labor, and material cost for optimization of process variables
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Research investigated use of calorimetry to evaluate effectiveness of food preservation techniques for inactivation of bacteria and to characterize hyperthermophiles. In 2008 focus was hyperthermophiles. Aeropyrum pernix(hAp) is a thermophile isolated from a coastal solfataric thermal vent with an optimum growth temperature between 90 and 95 degrees centigrade and optimum growth pH around 7. Hyperthermophilic organisms are mostly investigated for isolation of thermostable enzymes for use in biotechnological applications. Although their adaptation to heat is apparent, it is not entirely understood which cellular components are responsible for their thermal tolerance. Results showed that the DNA, which is one of the most stable macromolecules in bacteria, was a relatively thermally less stable macromolecule in the hAp. To further investigate DNA stability inside the cell, a commercially available DNA-binding fluorescent probes, Hoechst 33258, DAPI and acridine orange, were used to identify the DNA peak in DSC thermograms and to visualize the DNA molecules inside cells using fluorescence microscopy. Among the DNA-binding fluorescent dyes studied, Hoechst 33258 and DAPI bind in the minor groove of DNA with high sequence specificities for adenine and thymine. Since the DNA-Hoechst 33258 complex has a higher thermal stability than DNA alone in vitro(Lee and Kaletunc, 2005) it can be used to identify DNA transition in a complex whole-cell thermogram where the transitions of the individual cellular components are difficult to identify without comparisons with DSC transitions of the isolated components. Protocols to study microbial cell structures have been designed mainly for mesophilic organisms. Therefore, these protocols have to be adapted in order to be applicable to organisms such as Aeropyrum pernix that has optimum growth above 80 degrees C. Based on the DSC and fluorescence studies, Hoechst 33258 would be a good DNA marker in vivo for DSC studies. However, DAPI, Hoechst 33258 and acridine orange are not suitable probes for fluorescence microscopy, as they cannot clearly distinguish between living and dead A. pernix cells. The commercially available Live/Dead BacLightTM kit was the most suitable probe for detection of cell viability of A. pernix cells using fluorescence microscopy. PARTICIPANTS: Kaletunc,G. TARGET AUDIENCES: Food industry, academia, and scientists in government laboratories. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This project provides a new method for distinguishing which cellular components are responsible for thermal inactivation of bacteria, including foodborne pathogens. This knowledge is important for design of better and safer methods for processing food which enhance storage stability and safety.
Publications
- Cirnigoj, M., Kostanjsek, R., Kaletunc, G., Poklar Ulrih, N. 2008. Effect of different fluorescent dyes on thermal stability of DNA and cell viability of the hyperthermophilic archaeon Aeropyrum pernix. World J Microbiol Biotechnol, 24(10):2115-2123
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Research focus on application of calorimetry to evaluate effectiveness of food preservation for inactivation of bacteria and to characterize hyperthermophiles. Topic 1:High hydrostatic pressure (HHP)inactivates bacteria while maintaining the quality attributes of food. Preservation by HHP processing requires pressure levels above 600 MPa which may adversely alter food quality as well as increase equipment costs and shorten the life of the equipment. Hurdle technology has been applied to inactivate pathogenic bacteria by combining HHP with low pH or antimicrobial peptides. Nisin, an antimicrobial peptide, was shown to be effective against Gram-positive bacteria but not against Gram-negative bacteria due to impermeable outer membrane. Our goal was to inactivate Gram-negative bacteria by moderately high pressure-nisin treatment and optimize the HHP-nisin combinations using differential scanning calorimeter (DSC). Salmonella strains were subjected to HHP up to 550 MPa alone and
in combination with nisin. An 8-log cfu/ml reduction was observed for pressure treatment alone at 500 MPa for the FDA strain and 450 MPa for the OSU strain. When nisin was added, a similar reduction was obtained at 400 MPa for FDA strain and 350 MPa for the latter. A linear relationship between the logarithm of fractional viability based on apparent enthalpy data and plate count data was obtained for each organism. The decrease in apparent enthalpy appeared to be mainly due to reduction in the ribosome denaturation peak for both the pressure alone and the pressure-nisin combination treatments. HHP facilitates penetration of nisin into the cell most likely by alteration in the outer membrane. Topic 2:Archaea have cell structures and biocomponents different from those found in bacteria. Aeropyrum pernix is a thermophile isolated from a coastal solfataric thermal vent. It has an optimum growth temperature between 90 and 95 degrees centigrade and optimum growth pH around 7. A. pernix
grows in the presence of NaCl with an optimum of 3.5%. Hyperthermophilic organisms are mostly investigated for isolation of thermostable enzymes for use in biotechnological applications. Although their adaptation to heat is apparent, it is not entirely understood which cellular components are responsible for their thermal tolerance. Our goal was to characterize the thermally-induced transitions in whole cells of the A. pernix and to assign the transitions to cellular components based on calorimetric data of isolated cell components in order to identify cellular components responsible for the thermal stability of the organism. Studies of A. pernix show that the viability of cells after heat treatment and cell recovery depends on the temperature of treatment. DSC thermograms show five visible endothermic transitions with two major transitions. DSC analysis of isolated crude ribosomes shows that the two major peaks observed in whole cell thermograms are due to denaturation of ribosomal
structures. A comparison of partial and immediate full rescan thermograms of whole cells indicates that both major peaks are irreversible.
TARGET AUDIENCES: Food industry, academia, and scientists in government laboratories.
Impacts
Topic 1 Impact: The apparent enthalpy data obtained from DSC can be used to evaluate pressure levels necessary to reduce a microbial population in the presence of nisin and provide information about viability in shorter time as compared to plate counts. Topic 2 Impact: We demonstrated that in contrast to mesophilic organisms, hyperthermophilic A. pernix ribosomes are more thermally stable than DNA, but in both organisms irreversible changes leading to cell death occur due to ribosomal denaturation.
Publications
- Milek, I., Crnigoj, M., Poklar Ulrih, N., Kaletunc, G. 2007. In vivo characterization of thermal stabilities of Aeropyrum pernix cellular components by DSC, Can J Microbiol 53:1-8.
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Progress 01/01/06 to 12/31/06
Outputs
The application of antimicrobial agents during food processing allows the use of mild heating in order to preserve nutritional and textural qualities while maintaining an extended shelf-life. This approach is known as hurdle technology. The most commonly employed hurdles to reduce the intensity of heat treatment include controlling water activity, increasing acidity, and use of preservatives. The effectiveness of hurdle technology can be enhanced if hurdles target different cellular components of bacteria such as membrane, nucleic acids, and proteins. The optimal conditions for hurdle technology requires understanding of the effect of chemical agents on major cellular components leading to cell injury and death. Differential Scanning Calorimetry(DSC) was used to monitor changes in cellular components induced by chemical agents in vivo by comparing the thermograms of bacteria before and after treatment. Acetic acid, hydrochloric acid, ethanol or NaCl induced changes in
the major cellular components of E. coli and DSC were evaluated. The plate count method was performed to evaluate the viability of the chemically treated E. coli cells. E. coli cells were grown to a final concentration of 1.0 plus minus 0.1 x 109 cfu ml minus 1. Then, ethanol, sodium chloride (NaCl), hydrochloric acid (HCl), or acetic acid. were added to the growth media. Ethanol (95%) was added to the broth to achieve a final concentration of 6, 10, 12 or 15% (vol/vol). NaCl was added to attain 1.1 M or 1.9 M sodium chloride concentration in the growth media. HCl (36%, wt/vol) was titrated into the growth media to reduce the pH of the medium to 3.0 or 4.0. Similarly, glacial acetic acid was added to the growth media to reach 0.04 N, 0.1 N, 0.2 N, or 0.4 N acetic acid concentration in the medium. Results showed the thermal stability for ribosomal subunits denaturation and the total apparent enthalpy decreased with increasing ethanol, salt, and acid concentration. The reduction of
ribosomal subunit denaturation peak was the primary contributor to the decrease in the total apparent enthalpy. DSC thermograms showed that even at concentrations at which less than 0.4 log reduction of cell viability with a concomitant minimal reduction of total apparent enthalpy occurred, a decrease in onset temperature of ribosomal transition was evident. Acid treatments at pH 3 induced by HCl and by the 0.4 N acetic acid caused the DNA denaturation temperature in vivo to decrease. Application of chemical treatment prior to heat treatment noticeably reduced the viability of E. coli cells at all the heat treatment temperatures (60, 62.5, and 65C) compared to that of heat treatment alone thereby suggesting an increased sensitivity of bacteria to heat treatment. Results showed DSC studies in vivo can be used to assess the effectiveness of hurdles when thermal processing with hurdles are designed.
Impacts
DSC studies can reveal the effect of chemical treatment on a cellular component without the need for isolating the cellular component. Therefore, it provides not only information about the effectiveness of the treatment but the target of the chemical treatment by evaluating the heat sensitivity of the cellular component as a function of the treatments. The DSC studies in vivo can be used to assess the effectiveness of hurdles so that use of hurdles can be optimized when current thermal processing technologies are modified.
Publications
- Lee J. and Kaletunc G. 2005. Evaluation by differential scanning calorimetry of the effect of acid, ethanol, and NaCl on Escherichia coli. J of Food Protection 68 (3): 487-493
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Progress 01/01/05 to 12/31/05
Outputs
Bacterial cells exposed to different physical and chemical treatments suffer injury that could be reversible in food materials during storage. Injury has been observed for many bacterial cells. The injured cells can repair in a medium containing the necessary nutrients under the conditions of optimum pH and temperature leading to outbreaks of foodborne disease and food spoilage. The structural and functional components known to be damaged by sublethal stresses are the cell wall, cytoplasmic membrane, ribosomal RNA and DNA, as well as some enzymes. High hydrostatic pressure (HHP) is one of the emerging technologies proposed as an alternative to thermal processing and has been investigated to enhance safety and shelf life of many perishable foods. The ability to inactivate foodborne pathogens at pressures between 300 to 600 MPa without detrimental effects on important quality characteristics of foods has increased the interest in HHP applications on milk and dairy
products. The primary pressure damage occurs at pressures of 400 MPa or higher but damage of ribosomal units were observed at pressures lower than 400 MPa. The damage caused by HHP may be repairable and the cells can potentially grow after repairing the site of injury during storage. Our objective was to evaluate the effect of storage temperature on repair of injured foodborne pathogens in HHP treated milk. Two Gram-positive (Listeria monocytogenes CA and Staphylococcus aureus 485) and two Gram-negative (Escherichia coli O157:H7 933 and Salmonella enteritidis FDA) relatively pressure resistant strains of foodborne pathogens were pressurized at 350, 450 and 550 MPa in milk (pH 6.65) and stored at 4, 22 and 30 degrees C. The three states of cells just after pressure treatment were defined as (i) active cells (AC): can form visible colonies on both selective and non-selective agar; (ii) primary injury (I1) : can form visible colonies on non-selective agar but not on selective agar,
however colonies were formed on selective agar during prolonged storage; and (iii) secondary injury (I2): can not form visible colonies on either non-selective or selective agar, however colonies were first formed on non-selective agar and later on selective agar during prolonged storage. The results of shelf life studies indicated two types of injury, I1 and I2, for all of the pathogens studied. I2 type injury is a major injury and after its repair (I2 to I1), the cells can form colonies on non-selective but not on selective agar. The formation of colonies on both selective and non-selective agar occurs only after full recovery of injury (I1 to AC). Even if injured cells are not detected immediately after HHP treatment, I2 type injury could be potentially present in the food system. Therefore, it is imperative that shelf life studies must be conducted over a period of time for potential repair of I2 type injury either to detectable injury (I1) or to active cells (AC) to ascertain
microbiological safety of low acid food products.
Impacts
The presence of injured bacteria, especially in low acid food products, can be an important safety consideration during prolonged storage conditions where injury may be repaired leading to presence of active pathogens. If injury and repair are not considered, safety of products during storage is underestimated. On the other hand, injury can be advantageous in high acid foods, where lower pressure can be used to produce injured cells that could not repair in acidic medium.
Publications
- Bozoglu, F., Alpas , H., and Kaletunc, G. 2004. Effect of storage temperature on injury recovery of foodborne pathogens in High Hydrostatic Pressure (HHP) treated milk FEMS, 40: 231-235.
- Kaletunc, G., Lee, J., Alpas , H., and Bozoglu, F. 2004 . Evaluation of structural changes induced by high hydrostatic pressure in Leuconostoc mesenteroides Appl. Environ. Microbiol , 70: 1116-1122.
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