Calorimetric Evaluation of Food and Biological Materials

<p>NON-TECHNICAL SUMMARY:<br/> Biological systems range from pure single phase to multicomponent and multiphase systems of solids, dilute and concentrated solutions of macromolecules, emulsions, foams, and bacteria. Characterization of these systems before and after processing will lead to rational design of processing conditions. Purpose: To addresses the thermal characterization of food and biological materials before and after exposure to physical and chemical treatments in the environment relevant to water contamination and food and pharmaceutical processing. Situation or Problem: Foods exhibit thermally-induced transitions over a temperature range between minus 50 degree C and 300 degree C. The thermal behavior of a food is mainly a reflection of its major component, however, with some change due to interactions with other components. For example, for cereal based
products, although the main component is starch, thermally-induced transitions are highly affected by the presence of other compounds in cereals such as proteins, non-starch carbohydrates, and lipids either due to competition for available water or direct interactions. The changes in phase transition information of major components due to minor component interactions can be utilized for efficient process design. Thermal analysis is particularly well suited for analysis of food materials mainly due to the relevance of the experimental protocols of calorimetry to the majority of processes employed in food preservation. Food processing methods that involve thermal treatment (heating, cooling, freezing) can be simulated by thermal analysis protocols. Determination of thermal properties of food materials such as specific heat as a function of temperature is essential for heat transfer and
energy balance calculations (Kaletunc, 2007). Generation of a reliable database to develop equations predicting thermal properties of food materials for optimization of food processes can be accomplished by use of calorimetry. Moreover, food materials and their components go through conformational and phase transitions during processing. Calorimetry data can be analyzed to evaluate the thermal and thermodynamic stability of various phases for a rational design of food product formulations and process conditions. The advantages of using calorimetry for study of biological materials can be outlined as follows: 1.)Direct measurement of the energetics of the transition is obtained. The experimental results are not model dependent, 2.)Calorimetry can be applied to a range of materials, pure or complex. Materials do not have to be optically transparent or have chromophores as required by
spectroscopic methods. 3.)Materials do not have to be uniform or have to be a homogeneous mixture. In fact, in addition to pure materials, the technique can be utilized to evaluate the interactions among the components in a complex system and how the interactions are altered by the processing. 4.)Calorimetry does not require elaborate or destructive sample preparation. Calorimetry has a high potential to use as a tool in process design and optimization as well as product development and improvement.
<p>APPROACH:<br/> Objective 1: To evaluate the influence of physical and chemical preservation methods on the thermal and thermodynamic stability of major food components using DSC. The effect of pressure treatment on development of new starch structure can be evaluated by DSC. The pressure treatment will use the High Pressure Processing Unit, ABB Quintus Food Processor QFP-6 Cold Isostatic Press (Columbus, OH). A computer controlled, pressure-variable, differential scanning calorimeter (DSC 111, Setaram, France) will be utilized for characterization of food componenets before and after treatment. All DSC measurements will be performed at 3C min-1 from 1C to 150C using fluid tight, stainless steel crucibles. After heating, samples will be cooled by liquid nitrogen and rescanned to observe the reversibility of thermal transitions. Alterations in the DSC profile after
exposure to the treatments will be evaluated by comparing starches of different botanical origin and to untreated starches. Objective 2: To evaluate the thermally-induced changes in major food and biological materials including proteins and starches using DSC: Starch gels produced by thermal methods or high pressure processing exhibit recrystallization during storage. The extent and the time of crystallization is an important factor determining the shelf life of the product. Recrystallization of starch gels will be characterized by using DSC. Objective 3: Develop predictive models for estimating crystallization kinetic parameters of starch gels. Crystallization kinetics of high pressure treated starch gels from different botanical origins will be compared using DSC. Avrami model of crystallization will be fitted to data to determine the crystallization kinetics parameters and to estimate
the shelf life of products.
<p>PROGRESS: 2013/01 TO 2013/09<br/>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.
<p>PROGRESS: 2012/01/01 TO 2012/12/31<br/>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.
<p>PROGRESS: 2011/01/01 TO 2011/12/31<br/>OUTPUTS: Calorimetry was utilized to evaluate effectiveness of tannins inhibition of ?-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
?-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.
<p>PROGRESS: 2010/01/01 TO 2010/12/31<br/>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 ?-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 ?-amylase through DSC analysis, and to correlate results with measured inhibition. Pressure variable DSC analysis of ?-amylase incubated with (10%) tannin was performed using a Setaram calorimeter. Inhibition of ?-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 DH5? 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.
<p>PROGRESS: 2009/01/01 TO 2009/12/31<br/>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.
<p>PROGRESS: 2008/01/01 TO 2008/12/31<br/>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 ?uorescent 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 ?uorescence microscopy. Among the DNA-binding ?uorescent dyes studied, Hoechst 33258 and DAPI bind in the minor groove of DNA with high sequence speci?cities 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 ?uorescence 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 ?uorescence microscopy. PARTICIPANTS: Kaletunc,G. TARGET AUDIENCES: Food industry, academia, and scientists in government laboratories. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
<p>PROGRESS: 2007/01/01 TO 2007/12/31<br/>
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.
<p>PROGRESS: 2006/01/01 TO 2006/12/31<br/>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.
<p>PROGRESS: 2005/01/01 TO 2005/12/31<br/>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.