Browsing by Author "MaryAnne Drake, Committee Member"

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Comparison of RNA and DNA-Based Amplification Methods for the Discrimination of Viable from Non-Viable Salmonella Typhimurium
(2008-11-26) Bingham, James Edmund; MaryAnne Drake, Committee Member; Trevor Phister, Committee Member; Lee-Ann Jaykus, Committee Chair
Although nucleic acid-based foodborne pathogen detection strategies offer promise, a consistent concern has been their poor correlation to bacterial cell viability, largely due to long-term persistence of DNA even after cell death. Evidence has suggested that RNA may be a better target than DNA for detection of viable cells. The purpose of this study was to compare a real-time RNA amplification method (nucleic acid sequence-based amplification or NASBA) to a similar real-time DNA-based PCR method with respect to the ability to detect viable bacterial cells. Salmonella enterica serovar Typhimurium was grown in both pure culture and a chicken carcass rinse and subjected to different time and temperature conditions (no heat, 2.5-5 min at 60ï‚°C, 10-20 min at 60ï‚°C, and 15 min at 121ï‚°C) followed by isolation and purification of both DNA and RNA and cultural enumeration by direct plating. Isolated RNA and DNA were amplified by real-time NASBA or molecular Beacon, SYBR Green, and/or TaqManÂ® real-time PCR, respectively. Detection limits for each assay were determined using a modified MPN approach. The SYBR Green and the TaqManÂ®-based PCR assays had a detection limit of 102-103 CFU/ml for each replicate, with a similar log linear quantification range (approximately 2.0-9.0 log10 CFU/ml); in comparison, the real-time NASBA assay had a detection limit of 104 CFU/ml indicating a less sensitive assay. Exposure of S. Typhimurium cells to 60ï‚°C for 2.5-5 min resulted in a 4 log10 reduction in viable count when compared to the control (unheated) samples, while an >8 log10 reduction in viable count was observed after exposure to heat for 10-20 min at 60ï‚°C or 15 min at 121ï‚°C, regardless of the matrix. Based on the MPN equivalents estimate, there was no statistically significant difference between detection of S. Typhimurium when comparing treatments for any one real-time PCR method in either matrix. There was a gradual reduction in real-time NASBA signal as the heat treatments increased in severity for each matrix. The RNA-based amplification assay was more indicative of bacterial cell viability than was a parallel DNA-based amplification assay, suggesting that RNA may be a more reliable target in this regard. While the real-time NASBA assay was a better indicator of cell viability, it appears that mRNA can still be detected up to one hour after cell death. Consequently, the detection of either DNA or RNA using nucleic acid amplification methods cannot be relied upon to completely indicate the presence of live target bacterial cells.
Comparison of Sweetened Condensed Skim Milk and Whey Protein Ingredients in Caramels
(2003-12-08) Flint, Mandy Pauline; Frank Smith, Committee Member; MaryAnne Drake, Committee Member; E. Allen Foegeding, Committee Chair
Caramels may be described as 'soft glasses' that are viscous in nature and contain a dispersion of milk protein and an emulsion of fat. Milk proteins have traditionally been used in the confectionary industry for contributing distinct flavor, color, and texture, with sweetened condensed milk and milk powders being among the most popular. There are two main types of proteins in milk: caseins and whey proteins. Two popular ingredients made from whey proteins are whey protein isolates (WPI) and concentrates (WPC). Whey protein concentrates (WPC) contain between 25 and 80% protein and whey protein isolates (WPI) contain approximately 90% protein, with the remaining constituents being water, ash, lipid, and lactose. The goal of this research was to evaluate the acceptability and functionality of using whey protein ingredients in caramel confections by replacing the sweetened condensed skim milk with an imitation sweetened skim milk made with whey protein ingredients. A control formula containing sweetened condensed skim milk (SCSM) and one with an imitiation sweetened condensed skim milk made with whey protein isolate (I-SCSM) were evaluated. Formulations were cooked to 113°C, 116°C, and 119&$176;C. Properties of both treatments were highly influenced by cook temperature. Creep recovery testing was used to evaluate viscoelastic properties of caramels. All caramels showed minimal recovery, indicating they were mainly viscous (fluid) in nature. Cold flow, the flow of caramels at room temperature over time under the force of gravity, was evaluated by measuring sample area over time. Minimal cold flow was seen in caramels cooked to 116 and 119°C. However, caramels cooked to 113°C showed cold flow in both formulations, with caramels made with WPI exhibiting more cold flow than the control caramel. There were perceptible color differences between control and those made with WPI processed to 119°C; however, few differences were seen at 113&3176;C and none were seen at 116°C. The relationship between glass transition temperature and maximum compliance was similar between both caramel treatments, suggesting no change in the mechanism responsible for rheological properties. Based on all of the properties measured, whey proteins can be substituted for SCSM in caramels with an endpoint temperature of 116°C. However, color and textural differences were seen at 113&$176;C and 119°C. Based on the similarity seen in caramels made with WPI and SCSM, three brands of 34% whey protein concentrates (WPC) were explored as a more complex system containing higher levels of lipid, lactose, and minerals. There were no significant effects (p>0.05) due to brand of 34% whey protein concentrate (WPC) in compliance from the creep and recovery test, viscosity, percent recovery, glass transition temperature, moisture content or water activity. Differences due to WPC brand were seen in retardation time and color. A consumer acceptance test (n = 106) revealed that a caramel formulation made with one brand of WPC was similar to the control caramel with SCSM with the exception of stickiness. Three brands of commercial caramels were evaluated in order to validate that the data from analytical testing was similar to that found in experimental caramels. Only slight differences were seen in rheological properties amongst commercial caramels and between commercial caramels and experimental caramels. Differences were mostly seen in color, which may be attributed to by final cook temperature (unknown) or ingredient formulations. Color values did fall within the range of experimental caramel formulations using SCSM, WPI, and WPC.
Identification of dairy beverage and marketing opportunities to address the needs of the growing united states hispanic population
(2008-06-25) Thompson, Jacob Lorin; Tim Sanders, Committee Member; MaryAnne Drake, Committee Member; Allen Foegeding, Committee Member; Jon Bartley, Committee Member
The role of molecular methods in the detection of pathogens in food
(2004-03-18) Stevens, Kelly A.; Lee-Ann Jaykus, Committee Chair; Wondwossen Gebreyes, Committee Member; Craig Altier, Committee Member; MaryAnne Drake, Committee Member
The research described here addresses several issues associated with testing food samples for the presence of pathogens. Based on three different perspectives, the following major objectives are noted: (i) the use of molecular amplification methods to detect Listeria monocytogenes and Salmonella enterica serovar enteritidis directly from food samples, bypassing the need for cultural enrichment; (ii) evaluation of automated ribotyping for the differentiation of Salmonella enterica serovar Typhimurium strains; and (iii) development of a decision model to evaluate a pathogen testing decision in industry. In the first study, we developed and evaluated a method for the direct detection of foodborne pathogens without prior cultural enrichment. Eleven-gram samples of plain nonfat yogurt or mild cheddar cheese were seeded with L. monocytogenes or S. enterica Enteritidis at levels of 10, ², - 10, ⁶, CFU per sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9,700 x g) followed by detection using both cultural and molecular methods. Molecular detection limits of 10, ³,and 10, ¹, CFU per 11 g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. In a second and related study, alternative nucleic acid preparation methods were evaluated to improve the direct detection of Listeria monocytogenes from a frankfurter matrix. Using a combined concentration / extraction sample preparation, 11-g samples were concentrated 100-fold to 100 μl with recovery of target nucleic acids which were further purified by column chromatography and specific bacterial rRNA isolation using two magnetic bead-based technologies, i.e., MICROBEnrich® and MICROBExpress®. PCR detection limits were 10, ⁵, CFU/11g sample and RT-PCR detection limits were 10, ³, CFU/11g. Detection limits were improved an additional 10-fold (to 102 CFU/11g) when extracted RNA was further purified using MICROBExpress®. The third study evaluated the RiboPrinter®l microbial characterization unit for its ability to differentiate thirty-nine isolates of multi-drug resistant Salmonella enterica serovar. Typhimurium. Phenotypically, the isolates varied by phage type and marginally by antibiotic resistance pattern. However, the strains could not be meaningfully differentiated from one another using ribotyping, suggesting that in this case, phenotypic methods may be more discriminatory than this molecular typing method. In the final study, decision analysis tools were used to develop a model addressing the issues encountered when making a testing decision. From a food processors perspective, three potential consequences of foodborne pathogen contamination were considered as elements of the decision, e.g., no consequences, regulatory recall without disease, and disease outbreak. Accordingly, the inputs of the model were (i) costs associated with food-borne contamination (business and health related costs); (ii) reliability of testing; and (iii) prevalence of contamination. In general, the model indicated that testing for highly prevalent pathogens may provide an improvement in food safety but end product testing for pathogens of low prevalence should be carefully considered and may not be justified due to limited return on investment. These efforts represent continued progress in harnessing the power and diversity of molecular methods for the identification and characterization of foodborne pathogens. The development of systematic approaches to making testing decisions is also justified and needed by the industry. Taken together, these studies add to our understanding of the impediments to application of rapid methods for the detection of foodborne pathogens, and provide some solutions to facilitate the practical use of these methods in the future.
Sensory Texture and Fundamental Rheology of Agar and Agarose Gels
(2006-01-18) Barrangou, Lisa; MaryAnne Drake, Committee Member; Christopher R. Daubert, Committee Co-Chair; Den Truong, Committee Member; E. Allen Foegeding, Committee Chair
Texture properties of foods are an important component of food quality perception and acceptability. In order to design specific textures with predictable sensory attributes, a molecular understanding of food structures and their corresponding texture is necessary. Fundamental rheological methods are valuable tools for investigating structural mechanisms because they are based on physical and chemical theory, and when combined with descriptive sensory analysis, structure-function relationships can be established. The overall objective of this research was to utilize model food systems to further elucidate how physical properties of foods relate with the dynamic sensory perception of texture. Agar and agarose gels were used as model food gel systems. Initially, rheological profiles of agarose gels were developed, including linear, non-linear and fracture properties. Gel properties were examined under conditions of varying agarose concentration, solvent conditions, and strain rate. Increasing concentrations of agarose produced an increasingly stronger, more brittle network, while increasing concentrations of glycerol produced an increasingly stronger, more deformable network. All fracture properties and non-linear behaviors increased with increasing strain rate in a similar manner, suggesting a general mechanism responsible for strain rate effects that is similar for non-linear and fracture behavior. Additionally, a new model was proposed to reliably describe and quantify non-linear behavior. Descriptive analysis was used to quantify the perceived hand texture characteristics of agarose gels, and results were compared with fundamental rheological profiles to determine if structure-function relationships could be established. Sensory small-strain and fracture causing forces were capable of differentiating the gels equally as well, indicating that relative gel strength was perceived similarly with non-destructive and fracture causing deformations. Surprisingly, hand force terms correlated more highly with fracture modulus (fractures stress / fracture strain) values (r ≥ 0.98, p ≤ 0.001) than fracture stress values (r = 0.76-0.82, p ≤ 0.05), suggesting sensory perception of force includes a coupling of stress and strain. Additionally, sensory deformation perceived at fracture correlated highly with fracture strain values (r = 0.98, p ≤ 0.001). Small-strain rheological tests could not distinguish gels as sensitively as fracture properties, indicating that fracture properties relate to sensory texture better than small-strain rheology. Descriptive sensory analysis and fundamental large-strain rheological methods were also used to characterize texture characteristics of agar gels. Gels were differentiated in the same manner by sensory texture analysis in the mouth and rheological properties (p ≤ 0.05), and significant correlations between sensory and rheological properties were reported. First bite and chew-down sensory terms highly correlated with each other and with fracture properties. Specifically, the first bite sensory term of force required to cause fracture correlated well with the chew-down sensory term chewiness (r > 0.99, p ≤ 0.0001), and both of these sensory terms highly correlated with the fundamental rheological property, fracture modulus (r > 0.94, p ≤ 0.05). The first bite sensory term deformability perceived at fracture highly correlated with fracture strain values (r = 0.88, p ≤ 0.05), while the chew down property of particle breakdown negatively correlated with fracture stress values (r = -0.97, p ≤ 0.05). Additionally, the sensory properties that contribute to perception of strain-hardening were determined, and were found to correlate with non-linear rheological behavior, which is an important first step in understanding how non-linearity influences sensory perception of texture. These findings clearly demonstrate that fundamental large-strain rheological properties give valuable information toward the understanding of sensory perception of physical properties of foods.