Dissertations & Theses in Food Science and Technology

VALIDATION OF MICROWAVE HEATING INSTRUCTIONS FOR THE DESTRUCTION OF Salmonella spp. IN MICROWAVEABLE FOODS

Carol J. Valenzuela — Wed, 24 Apr 2013 13:25:29 PDT

Microwave heating instructions for three products (chicken nuggets, turkey pot-pies and mashed potato) were developed and validated based on end point temperatures using two microwave ovens (2,459 MHz; 700 W and 1,350 W). Heating instructions for chicken nuggets were validated using different configuration of product placement (edge or center of the carousel) and number of units (4, 6 and 8). Salmonella spp. reductions of 6.56 log CFU/g (700 W) were observed in chicken nuggets heated in groups of 4 and placed at the center of the carousel with 1 min 26 s of heating time with a target end point temperature of 73.8°C. Longer heating times (2 min 10 s) resulted in total Salmonella spp. reductions (7.22 log CFU/g) when chicken nuggets were placed in groups of 8. Similar Salmonella spp. reductions (p>0.05) were observed when chicken nuggets were placed at the center using shorter heating times (1350 W). Incorporation of standing time (2 min) eliminated Salmonella spp., regardless of the power of the microwave, location and the number of chicken nuggets. Heating instructions for turkey pot-pies were validated using inoculated product (at the geometric center or on the crust). Salmonella spp. reductions of 5.16 log CFU/g were observed following heating times of 9 min 31 s and 7 min 1 s for the low and high power microwave, respectively with a target end point temperature of 73.8°C. For the third product, different amounts of inoculated mashed potato (105 and 205 g) were used to validate the microwave heating instructions. Destruction of Salmonella spp. (8.73 log CFU/g) in mashed potato (105 g) can be achieved with a target end point temperature of 70°C at the geometric center, regardless of the power of the microwave oven. Salmonella spp. destruction (8.73 log CFU/g) was observed in mashed potato (205 g) using the high power microwave oven using an end point temperature of 72.2°C to calculate heating times. Salmonella spp. destruction in mashed potato is dependent on the amount of the product and the power of the microwave oven.

Advisor: Harshavardhan Thippareddi

"OCHRATOXIN A”: Evaluation of Methodologies for Determination of Ochratoxin A in Food Commodities, Contamination Levels in Different Products Available in the US Market and Evaluation of Fungal Microbiota Associated with Some of the Products

Lakshmi Gompa — Mon, 22 Apr 2013 10:30:39 PDT

Ochratoxin A (OTA) is one of the important mycotoxins that contaminate a wide range of food commodities available in the market. In this study, methodologies for analyzing OTA in commodities such as, roasted coffee, cocoa and meat were evaluated. The methods with the best recovery rates were used to analyze the incidence of OTA in these food products. Among different samples analyzed, predominantly 35% of the cocoa samples and 3% of meat samples were contaminated with OTA. Decaffeinated coffee samples showed the highest incidence of contamination (16.7%). Also as a part of this study, Veratox TM (Neogen, MI) ELISA test kit was validated for quantification of OTA in meat using a modified extraction method. Results for recovery, repeatability, cross-reactivity and robustness and linearity showed that the method was suitable for the analysis. Incidence of OTA levels in dried fruits was evaluated and among them raisins and dates showed high levels of incidence of OTA (100% and 70%, respectively). Fungal microbiota was also isolated from dried fruits was characterized and different strains of A. niger and A. tubingensis were identified. All the characterized isolates were tested for OTA production and 36.8% among them were OTA producers. Most of the OTA producers were identified as Aspergillus niger or Aspergillus tubingensis.

Adviser: Andreia Bianchini

Risk Assessment of Trace and Undeclared Allergens in Processed Foods

Benjamin C. Remington — Fri, 19 Apr 2013 10:14:18 PDT

Minimal eliciting doses for objective allergic reactions were found for 13 priority allergens and over 1800 individuals from published clinical literature or unpublished clinical data. Allergic populations did not vary when analyzed by age or geographic region. Results of this study show there are sufficient clinical data from food allergic individuals to use for risk assessment purposes for several allergenic foods.

Of 186 food products bearing advisory statements regarding peanut or 16 products that had peanut listed as a minor ingredient, 8.6% and 37.5% contained detectable levels of peanut (>2.5 ppm whole peanut). An additional market survey of 215 nutrition bars with peanuts as a minor ingredient and/or an advisory statement for peanuts found 24.6% tested positive for peanut compared to 4% of products with no mention of peanuts on the label. Probabilistic risk assessment showed the risk of reaction among peanut allergic consumers from advisory labeled nutrition bars was significant but brand-dependent. The probabilistic approach provides the food industry with a quantitative method to assist with determining when advisory labeling is most appropriate.

Agricultural commodity cross-contamination of soybean was detected in 62.8% of samples representing all forms of wheat flour. Conservative probabilistic risk assessments predict a risk of allergic reaction occurring in the most sensitive soy-allergic individuals. Experimental milling and stream separation with spiked soy in wheat samples did not produce a soy-free wheat flour stream. Additional cleaning measures will be needed to remove soy before wheat milling begins.

LC-MS/MS identified fourteen known allergens in industry representative soy product samples, including all subunits of Gly m 5 (β-conglycinin) and Gly m 6 (glycinin), the Kunitz trypsin inhibitor, Gly m Bd 28K, and Gly m Bd 30K. Method refinements or different techniques might be necessary to detect low abundance proteins such as Gly m 3 and 4. The relative amount of an allergen in a sample correlated positively to the intensity of IgE binding at the expected molecular weight using sera from soy-allergic individuals.

Advisers: Stephen L. Taylor and Joseph L. Baumert

New Technologies for Whole Wheat Processing: Addressing Milling and Storage Issues

Andres F. Doblado-Maldonado — Mon, 03 Dec 2012 12:37:25 PST

Whole wheat flour production and demand has increased dramatically during the last decade due to evidence supporting the benefits of whole grains in the diet. Hence, the food industry has provided a wide variety of new whole grain products. There are unique challenges that accompany whole wheat flour production, especially related to milling and storage. The present thesis provides new strategies on the adaptation of new technologies to overcome whole wheat processing issues. These issues are first discussed in a literature review and then followed by three research studies. In the first study, retail whole wheat flours were evaluated for particle size distribution to determine variations in currently available products. Significant differences were found for particle size distribution among and within brands. Compositional data elucidated differences in the degree to which the bran fraction of the kernel was milled. In the second study we aimed to produce whole wheat flour in the laboratory that could be used for end-use quality testing. We varied the moisture content during milling to produce flours with different particle size distributions and evaluated the functional properties of the flours. Mean particle size of the coarse fraction (>230 μm) decreased as moisture content decreased. Wheat milled at lower moisture contents (i.e., 6.89-7.98%) provided flours with better functionality and mixing properties. In the third study, salts were added to wheat during tempering to reduce lipolytic activity in an effort to extend shelf life of whole wheat flour. This strategy was effective at inhibiting lipase, and provided flour with better baking properties than the control after 6 months of storage. The outcomes of these studies serve as new strategies for the production and evaluation of whole wheat flour.

Adviser: Devin J. Rose

The Evolution of Host Specificity in the Vertebrate Gut Symbiont Lactobacillus reuteri

Steven Frese — Fri, 30 Nov 2012 13:55:32 PST

The vertebrate gut is home to one of the densest populations of life on Earth. This microbial community has a profound effect on host health, nutrition, development, behavior, and evolution. However, very little is known about how these microbes have evolved with their vertebrate hosts, how and whether they select hosts or how they remain associated with their hosts. Recent work identified Lactobacillus reuteri as an organism that is composed of host-specific sub-populations, each population associated with a different host animal. Representatives from each host-associated population were tested for their ability to colonize gnotobiotic mice, which only rodent strains could accomplish. To determine the genetic source of these differences, genome sequences from representative strains (from humans, pigs, chickens, and rodents) and genomic microarrays were used to identify host-specific genes tied to ecological success in vivo. Many of the rodents pecific genes were essential to the ecological success of this strain. Overall, the types of genes which were essential suggested that they were related to biofilm formation. Using a novel method of biofilm quantification and a germ-free mouse model, the ability of L. reuteri strains to form biofilms on the forestomach of germ-free mice was examined. In vivo biofilm formation was exclusive to the rodent associated strains. Using genome comparisons, in vitro biofilm formation, and related models of biofilm formation, genes essential to biofilm formation were identified. Genes responsible for adherence, aggregation, and gene regulation were all critical to biofilm formation. To determine whether the observed host specificity of rodent associated L. reuteri in mice extended to other host-associated lineages, a crossover human experimental trial was conducted. Three species of Lactobacillus were tested for their ability to persist in the human gastrointestinal tract. A human-associated strain of L. reuteri, as well as a related species isolated from humans reached higher populations than a non-associated strain, suggesting that host-specificity of L. reuteri extends to other lineages as well. Together, these experiments demonstrated the host-specificity of the species and identified the molecular mechanisms by which rodent-associated L. reuteri colonize their host.

Advisor: Dr. Jens Walter

EVIDENCE OF TRANSMISSION OF ESCHERICHIA COLI O157:H7 TO THE TISSUES OR PHYLLO-PLANE OF WHEAT, FROM CONTAMINATED SOIL, SEEDS OR WATER

Bismarck Antonio Martinez — Thu, 29 Nov 2012 11:40:42 PST

Escherichia coli O157:H7 is a human pathogen that can cause a wide spectrum of disease symptoms, such as bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Escherichia coli O157:H7 illness are mainly associated with undercooked beef; however, in recent years outbreaks have been linked to fresh produce such as spinach, lettuce, and sprouts. In 2009, flour was implicated as a contamination source in the consumption of raw cookie dough resulting in 77 illness-cases. The objective of this research was to determine the possible route of transmission of E. coli O157:H7 into the phyllo-plane of wheat using contaminated seed, soil or irrigation water. Levels of contamination were 6.88 log CFU/g, 6.60 log CFU/g and 6.76 log CFU/ml of Kanamycin resistant E. coli O157:H7. One hundred plants per treatment were sown in pots trays with 50 g of sterile soil, watered every day with 5 ml of dilution water and harvested after 9 days post-inoculation. In a fourth experiment, flowering wheat heads were spray-inoculated with water containing 4.19 log CFU/ml of E. coli O157:H7 and analyzed for survival after 15 days, close to the harvest period. To detect low levels of internalization, BioTecon q-PCR detections assays were used to determine the presence of E. coli O157:H7 in the wheat plants using a Roche Applied Science LightCycler 2.0.

Results showed that internalization was possible using contaminated seed, soil, and irrigation water in wheat seedlings with an internalization rate of 2%, 5% and 10% respectively. Even though this rate is low, this is the first study to demonstrate the ability of this strain to reach the phyllo-plane in wheat. In the head contamination experiment, all samples tested positive, showing the ability of E. coli O157:H7 to survive on the wheat head phyllo-plane. Although possible this research does not provide evidence for efficient uptake of E. coli O157:H7 into the internal tissue of wheat plants from a contaminated environment. However, surface contamination and the ability of E. coli O157:H7 to survive long-term on the wheat plants is an issue to be considered when addressing food safety issues in products derived from wheat.

Adviser: Jayne Stratton

Soybean allergy: Effect of genetic modification (GM), heat and enzymatic treatment on overall allergenicity

Rakhi Panda — Mon, 19 Nov 2012 11:00:52 PST

Soybean allergy affects approximately 0.4% of children worldwide. At least 16 proteins in soybean bind IgE from some soybean allergic subjects. Although the relative allergenic importance and abundance of individual proteins in soybean varieties is not understood, the allergenicity assessment guideline for the safety of genetically modified (GM) food crops (Codex, 2003) includes assessing potential increases in expression of endogenous allergens in an allergenic crop like soybeans that might be due to insertion of the new DNA. The studies described in this dissertation included comparison of binding of IgE from individual soybean allergic subjects to proteins in three transgenic soybean lines, their respective near-isogenic and other commercial lines. The results indicated no evidence that the transgenic soybean lines present an increased risk for soybean allergic subjects especially since those with soybean allergy should avoid all soybeans. Furthermore, based on the observed variation among commercial lines, it is not clear that similar tests are useful to evaluate food safety for typical GM varieties.

Soybean products are widely used in food because of their functionality, nutritional properties and low cost. Some soybean ingredients are processed either by heat treatment or enzymatic hydrolysis to attain desirable functional properties or in some cases to reduce the allergenicity. However, few studies have investigated the effect various processing conditions have on allergenicity of soybean products and their efficacy in reducing allergenicity of soybean. Additional studies described in this dissertation evaluated potential changes in IgE binding to soybean proteins that are heat-treated under conditions that mimic some commercial processing or undergo enzyme hydrolysis. Results indicated that majority of thermal treatment conditions utilized in making soybean products will not affect their allergenicity and hydrolysis of soybean proteins by different enzymes does not make them less allergenic compared to the untreated proteins and may increase their allergenicity.

Advisor: Richard E. Goodman

Characterization of Starch by Vibrational Spectroscopy

Brandon H. Holder — Tue, 23 Oct 2012 14:15:29 PDT

To develop a dispersive Raman spectroscopic method for measuring amylose-amylopectin ratios of corn starch mixtures, 67 mixtures were prepared by randomly mixing waxy and normal corn starches. Amylose contents were measured using a dual wavelength iodine binding colorimetric method. Raman data were collected from 250 to 3200 cm^-1 using optimized instrument parameters. Partial least-squares (PLS) and principal components regression (PCR) were used to prepare multivariate calibration models; however, PLS commonly outperformed PCR. Truncating the spectra to 250 to 2000 cm^-1 improved the results (r² of validation = 0.831, SEP = 2.90%). Removal of a cold water swelling starch from the data also offered a slight improvement in results (r² of validation = 0.860, SEP = 2.70%). Dispersive Raman spectroscopy may not be well suited for quantifying amylose content of starch mixtures; however, the method was easily capable of discriminating between waxy and normal starches. This may allow the method to be used for confirming the identity of starch shipments.

A dispersive Raman spectroscopic method for measuring retrogradation in corn starch gels was investigated. Thirty-six gels were prepared, stored at 4° C and measured at regular time intervals (0 h, 24 h, 48 h, 72 h, 120 h, 168 h after preparation). After each measurement, the gels were freeze-dried, then each resultant dried gel was ground into a powder and measured using X-ray diffraction. Relative crystallinity was determined, and intensity changes in the Raman band at 480 cm^-1 were measured. No correlation was found between changes in the 480 cm^-1 band and the relative crystallinity of the gels (r² < .1). The low starch concentration used may have caused the poor Raman signal strength and the unpredictable changes in the X-ray diffraction data. The experiment found that measuring retrogradation in very dilute starch gels could be problematic, and that more development is needed in order to apply Raman spectroscopy to in a food system like white pan bread.

Advisor: Randy Wehling

HOST-MICROBE-DIET INTERPLAY: DIETARY MODULATION OF THE GUT MICROBIOTA IN RELATION TO HEALTH

Inés Martínez — Mon, 23 Jul 2012 10:17:19 PDT

Vertebrates are associated with trillions of bacteria, with the densest populations residing in the large intestine. The symbiosis between vertebrates and their gut microbiota has resulted in important implications of the gut microbiome on host health. Diet is an important factor that shapes gut microbiota composition, and because of the interplay between host-microbiome-diet, dietary strategies that modulate gut microbiome structure are deemed a relevant tool to improve host health. However, gaps in knowledge exist with respect to these interactions, and it is essential to obtain a mechanistic understanding of how these relations take place to develop successful therapeutic strategies that target the gut microbiome.

In order to address these gaps, human trials were performed to assess the impact of primary components of the human diet, resistant starches and whole grains, on the gut microbiota. Overall, the impact of diet was temporal and varied across subjects. Resistant starches substantially modulated the gut bacterial community of the subject population, especially increasing the abundance of Bifidobacterium adolescentis. Ruminococcus bromii, Eubacterium rectale, and Parabacteroides distasonis were also significantly enriched. Dietary incorporation of whole grains increased the proportions of Eubacterium rectale and acetogens such as Blautia wexlerae. Of note, whole grains significantly improved in inflammation and glycemic parameters. The shifts in Eubacterium rectale correlated with glycemic improvements. Moreover, distinct abundances of Dialister were determined among subjects that differed in terms of their in inflammatory improvement. To gain mechanistic insight on the host-microbe-diet interplay, animal experiments were conducted to evaluate the effects of grain sorghum lipids and plant sterol esters in the context of dyslipidemia. Significant and consistent alterations in gut microbiota composition were detected in both experiments, especially involving shifts in Coriobacteriaceae and Erysipelotrichaceae abundance, which displayed remarkable correlations to host cholesterol markers. Mathematical modeling of these associations revealed them to be inhibitory interactions, suggesting that changes in host metabolism affected gut microbiome structure through an antimicrobial effect of cholesterol, which was conformed in vitro against selected gut microbes.

In conclusion, the studies presented in this dissertation allowed new insights on the impact of diet on the gut microbiota and its consequences for health.

Adviser: Jens Walter

ANALYSIS OF MICROBIAL DIVERSITY BY AMPLICON PYROSEQUENCING

Ryan Legge — Thu, 19 Jul 2012 12:18:30 PDT

Microorganisms numerically dominate terrestrial biodiversity, and play important biochemical and geochemical roles in the environments they inhabit. To understand structure and function of complex ecosystems, it is essential to identify primary drivers of microbial diversity and community structure. Historically, the study of microbial ecology was reductive, limited to microbes able to be cultured and enumerated. Microbes meeting this criterion were thought to comprise the dominating members of the environments they were isolated from, however, estimates suggesting up to 99% of the endogenous species are uncultivable with existing methodologies; a concept that reflects experimental failure, rather than a verifiable conclusion. Therefore surveys of microbial community members relying solely on culture-based techniques will severely underestimate the extent of microbial diversity. Analytical methods for DNA sequencing have progressed over the last 30 years allowing for increasingly detailed analysis of microbial communities. Microbes can be recognized and their function can be understood at the DNA/RNA level without cultivation bias through molecular techniques which analyze content based on microbial DNA isolated from environmental samples. Using high-capacity sequencing, environmental samples can be characterized at resolution, ultimately allowing communities to be compared on the basis of their taxonomic or phylogenetic content as well as on functions the microbes carry out. In this dissertation research, two unique studies were explored. Studies focus on differences in composition of microbial communities as a phenotype in the GI tract of animals in a genetic selection experiment and as a measure of contamination risk in food production with 454-based pyrosequencing of the 16s rRNA amplicon. These two studies give credence to the applications of “-omics” techniques in addressing questions relevant to fundamental and applied biological disciplines. Ultimately, studies like these are creating paradigm shifts in how we view food production and human health as they begin to uncover the entire microbial community to unparalleled levels. Continued advancements in the technology itself and the associated bioinformatics tools will influence a broad cross-section of problems in food production, heath care, and water and land management.

Advisor: Andrew Benson

INFLUENCE OF DIETARY FIBERS AND WHOLE GRAINS ON FECAL MICROBIOTA DURING IN VITRO FERMENTATION

Junyi Yang — Thu, 24 May 2012 12:25:10 PDT

There has been a dramatic increase in obesity, which presents a risk of many chronic health problems. Recent studies have shown that obese individuals harbor an abnormal gut microbiota (dysbiosis), which has sparked interest in the gut microbiota as a target for weight management. Dietary fiber and whole grains that can be metabolized by gut microbiota have been shown to promote the growth of beneficial bacteria in the gut. Increased consumption of dietary fiber and whole grains may improve host / gut microbiota interactions in obesity and other metabolic diseases by normalizing gut dysbiosis. The present thesis describes two research projects to assess the impact of dietary fibers and whole grain on gut microbiota. In the first study, six dietary fibers [pectin, guar gum, inulin, arabinoxylan, β-glucan, and resistant starch type 2 (RS-2)] were subjected to in vitro digestion and fermentation using fecal samples from obese and normal weight individuals. Pyrosequencing was used to assess the impact of each dietary fiber on the gut microbiota community. Short/branched chain fatty acids (SCFA/BCFA) and carbohydrate utilization were correlated with proportions of bacterial taxa. The data showed that RS-2 caused the most dynamic change of the whole microbiota community and Bifidobacterium increased almost 10-fold on pectin substrate compared with the control. Certain taxa may be targeted to increase SCFA production or increase dietary fiber utilization. For instance, Ruminococcaceae and Faecalibacterium displayed positive correlations with butyrate production and while a strong positive relationship was shown between β-glucan utilization and Firmicutes. In the second study, since SCFA can influence hormones involved in energy absorption, utilization, and storage, the SCFA profile from five whole grains (wheat, rye, corn, rice, and oats) using an in vitro method was evaluated. There were large differences in fermentation profiles among individuals, even when supplied with the same dietary fiber. The obese type microbiota was less efficient at butyrate production and less metabolically active than the normal weight type microbiota initially, but given sufficient whole grain substrates the bacteria quickly became metabolically active.

Characterization of IgG and IgE Binding to Parvalbumin Derived from Commercially Important Fish Species

Poi-Wah Lee — Wed, 18 Apr 2012 13:06:41 PDT

Parvalbumin is a calcium-binding muscle protein that is present in all vertebrates. Despite being a pan-allergen in fish and frog, fish-specific IgE and antiparvalbumin IgG antibodies displayed varying cross-reactivity among fish species. In this research, the parvalbumin-binding characteristics of several antibodies were investigated, including 3 IgG antibodies against frog, carp, and cod parvalbumins, and human IgE.

By immunoassay and IgG-immunoblotting, 3 antiparvalbumin antibodies revealed inconsistent specificity among 29 raw fish muscle extracts, which may be partially attributed to the decreased levels of fish muscle parvalbumin from anterior to posterior positions. Parvalbumin-binding by antibodies was unaffected in 112 days of frozen-stored fish muscles. Anticod parvalbumin polyclonal antibody (anticod PoAb) was the most suitable for detecting parvalbumins as it reacted to the widest range, but not all fish species.

IgE-immunoblotting demonstrated intra- and inter-individual diversity in IgE-binding to fish and frog proteins. Of 39 fish-allergic individuals, >50% subjects bound to purified cod and carp parvalbumins, and proteins corresponding to parvalbumins in 21 fish extracts, whereas

Heating, calcium-depletion and Maillard reactions affected the 3 antiparvalbumin IgG antibodies binding to fish muscle extracts and cod parvalbumin, although anticod PoAb was less affected. Both Maillard and heat treatments reduced IgE binding to cod parvalbumin and these effects were more pronounced without calcium.

Parvalbumins among fish revealed higher sequence identity than non-fish species. Both calcium-binding loops representing Gad c 1 epitopes were conserved among fish and non-fish, whereas low homology in AB domain and AB/CD inter-domain junction of fish parvalbumins may contribute to variable cross-reactivity among fish. Phylogenetic analysis showed that all fish parvalbumins but zebrafish and pike were closely related. Teleost beta-parvalbumins were closely related to beta-parvalbumins of amphibians and reptiles, but divergent from alpha-parvalbumin in mammals and non-mammals.

Adviser: Steve L. Taylor

VALIDATION OF THERMAL PROCESSING TO CONTROL SALMONELLA SPP. AND CLOSTRIDIUM PERFRINGENS DURING PRIME RIB PREPARATION FROM INTACT AND NON-INTACT MEAT

Maria A. Calle — Wed, 07 Mar 2012 19:14:34 PST

Beef prime rib is a delicacy cooked at low temperatures for long period and extended hot holding prior to consumption. Due to the potential survival and outgrowth of foodborne pathogens, this process may pose a public health risk. Considering an increase in the availability of non-intact beef, the microbial safety of the preparation of prime rib from intact and mechanically tenderized beef was evaluated.

In the first study, lethality of the prime rib (from intact and non-intact, blade tenderized beef) cooking process to Salmonella spp. was evaluated. Boneless beef rib eye was surface inoculated with a five-strain cocktail of Salmonella spp. to obtain ca. 5.9 log CFU/g. The inoculated blade tenderized subprimals were passed with the fat side facing the blades to prepare non-intact beef rib eye. The subprimals were seared for 15 min at 260°C, cooked at 121°C to internal temperatures of either 37.8 or 48.9°C and held at 60°C for up to 8 h. The searing of blade tenderized rib eye resulted in 0.63 log CFU/g reduction in Salmonella spp. and cooking to internal temperatures of 37.8 and 48.9°C resulted in 2.86 and 3.58 log CFU/g reduction in Salmonella spp., respectively. Subsequent holding of the blade tenderized prime rib at an oven temperature of 60°C for 8 h resulted in an increase of 1.99 log CFU/g in Salmonella spp. population. Thermal processing of intact rib eye resulted in 5.22 and 5.54 log CFU/g reduction of Salmonella spp. population after cooking to 37.8 and 48.9°C respectively. Subsequent holding at 60°C during 8 h resulted in an increase in Salmonella spp. population of 1.07 and 0.44 log CFU/g, respectively.

In the second study, the growth of C. perfringens during hot holding and cooling was evaluated. Ground rib eye was dispensed in 5-g pouches, inoculated to obtain 2.5 spores/g of C. perfringens, and vacuum-packaged. C. perfringens spores were heat activated and the pouches with the inoculated meat were held at 43 or 49°C. Hot holding of inoculated rib eye resulted in germination and outgrowth of C. perfringens spores by 6.5 and 6.05 log CFU/g. Abusive cooling of cooked prime rib from 57.2 to 5°C within 6, 9, 12, and 15 h resulted in C. perfringens spore germination and outgrowth by 0.2, 0.4, 0.8, and 2.2 log CFU/g respectively.

Use of non-intact rib eye can pose an enhanced risk of foodborne illness when prime rib is cooked to low end point temperatures, held for extended periods of times at these low temperatures, and cooled at slow rates.

Advisor: Harshavardhan Thippareddi

Adaptation and Validation of Food Product Specific Analytical Methods for Monitoring Prebiotics Present in Different Types of Processed Food Matrices

Rebbeca M. Duar — Tue, 14 Feb 2012 14:49:47 PST

Prebiotic carbohydrates are now added to a variety of processed foods to beneficially affect the gut microbial composition and activities. However, published data remain limited on the stability of prebiotics during food processes, such as baking, extrusion, pasteurization, high temperature heating, low pH condition, etc. As the complexity of the food matrix may also affect the ability to test for prebiotics, product specific analytical methods, including UV-vis spectroscopy, GC and HPLC, were developed and validated to monitor the stability of FOS, inulin and GOS in different types of processed food (breakfast cereal, cookie, muffin, sports drink and a nutritional bar). The results showed satisfactory linearity (r>0.9) for all the validated methods, however differences in the complexity of the ingredients were reflected in method accuracy and precision. Method precision was determined by calculating the percent relative standard deviation (% RSD) of spiked samples (n=5) below (0.5%) and above (2%) the target amount to be supplemented in the food products (1%) with results ranging from 1-39%. Methods for monitoring FOS and GOS resulted in low detection and quantitation limits allowing analysis of prebiotics of less than 1% in the presence of complex matrices. On the other hand the inulin method presented high detection and quantitation limits. Additionally, accuracy was affected by compounds present in the food matrix, which was accessed by determining the percent recoveries of spiked prebiotic in the control samples and applying corresponding correction factors to the supplemented processed foods when the accuracy was below 90% or above 110%. The chemical fate of the prebiotics was determined by applying the optimized and validated extraction and analytical method to prototype food products with 1% supplemented prebiotic. Recoveries ranged from 25-300% depending on method’s performance, complexity of the matrix and the severity of the processing effects. Finally the fate of the prebiotics in a cereal and sports drink that were prepared under various processing conditions indicated a high stability of GOS, while FOS and inulin were affected by low pH and high temperatures.

Adviser: Vicki L. Schlegel.

Identification and Characterization of Putative Allergens in Pecan Species

Jelena Spiric — Wed, 30 Nov 2011 10:08:50 PST

The identification and characterization of soluble pecan proteins was found to be qualitatively dependent on the chemical characteristics of the extraction buffer that also had a significant influence on the total protein content of an extract. The ionic strength of the extraction buffer had a greater impact on protein extractability than pH. A high salt phosphate buffer (0.01 M PBS, 1M NaCl, pH 7.2) was used as an optimal extraction solution that allowed remarkable detection of clinically relevant allergens.

The 2S albumin family of seed storage proteins has been shown to be one of the most stable allergens, thus potentially making these proteins clinically relevant for allergic sensitization. In this study, we sought to purify native 2S albumin from pecan to further characterize this putative allergen. The protocol for purification and isolation of 2S albumin protein from pecan, Car i 1, was developed employing two size-exclusion chromatography steps. Sequence identification of pecan proteins was done by means of LC-MS/MS ion trap mass spectrometry.

The soluble high molecular weight pecan proteins were rapidly digested by pepsin in simulated gastric fluid (SGF) whereas the low molecular weight proteins were more stable or were reduced to digestion resistant proteins and polypeptides. The purified 2S albumin, Car i 1, from pecan was found to be resistant to digestion in SGF and comparatively stable to proteolysis by trypsin and pancreatin in simulated intestinal fluid (SIF).

Digestion of purified Car i 1 in SGF and SIF resulted in formation of different digestion-resistant peptides, which included important epitopes that were retained despite extensive in vitro digestion. These peptides were capable of binding IgE antibodies from allergic individuals. Digestion stability of Car i 1and formation of stable, digestionresistant antigenic polypeptides may contribute to allergic sensitization to pecans in susceptible individuals.

Advisor: Joseph L. Baumert

Studies on the Adherence Properties of Plant Lectins and Bacterial Adhesins and their Inhibition by Prebiotic Oligosaccharides and Bovine Colostrum Fractions

Maria X. Maldonado-Gomez — Wed, 30 Nov 2011 06:38:23 PST

To initiate colonization and infection, most microbial pathogens must first recognize and adhere to cells in the host tissues. Adherence inhibition, therefore, can be used as a strategy for preventing infectious disease. In this research, we developed a simplified model using plant lectins and HEp-2 tissue culture cells to simulate bacterial adhesins-receptors interactions. The effect of lectin ligands and two prebiotic carbohydrates, galactooligosaccharide (GOS) and polydextrose (PDX) was measured with this approach. Lectins that were able to bind to the target cells were inhibited in the presence of the cognate ligands and at least one of the prebiotic carbohydrates. The inhibition was generally proportional to the structural similarity between the prebiotic and the cognate ligands. In particular, GOS significantly inhibited attachment of most of the lectins, consistent with the established role of GOS as an anti-adherence agent. The results support the hypothesis that cell binding inhibition is caused by competition of the GOS for the lectin. Experiments were completed to assess the anti-adherence properties of several bovine colostrum fractions against Escherichia coli, Cronobacter sakazakii and Salmonella enterica serovar Typhimurium. An ultra-filtration permeate and an oligosaccharide fraction significantly inhibited binding of all four pathogens to HEp-2 cells. In contrast, a nano-filtration retentate inhibited Salmonella adherence, but not other pathogens, while a colostrum whey fraction had no effect for any of the test organisms. The effectiveness of the inhibitory fractions was mainly attributed to the presence of oligosaccharides and/or peptides. Several methods for extracting oligosaccharides and removing lactose from bovine colostrum were evaluated. Enzymatic hydrolysis and microbial fermentation were effective methods for degrading lactose. In contrast, the physical-chemical separation methods were limited in their ability to separate and enrich oligosaccharides. The results obtained from this study provide evidence to support the concept that bovine milk components can promote consumer health. These constituents could be used as antimicrobial agents in foods and/or in pharmacological applications. In particular, bovine colostrum whey appears to be a suitable source for these bioactive compounds.

Advisor: Robert W. Hutkins

Effect of Sodium Nitrite, Sodium Erythorbate and Organic Acid Salts on Germination and Outgrowth of Clostridium perfringens Spores in Ham during Abusive Cooling

Mauricio A. Redondo — Wed, 23 Nov 2011 12:46:44 PST

The effect of sodium nitrite (NaNO₂), sodium erythorbate, sodium chloride (NaCl) and organic acid salts (potassium lactate and sodium diacetate) on C. perfringens spore germination and outgrowth in ham was evaluated. The research was divided in two parts. The first experiment consisted of potential C. perfringens spore germination and outgrowth in ham containing combinations of NaNO₂ (0, 50, 100, 150 or 200 ppm) and sodium erythorbate (0 or 557 ppm) during cooling. This experiment included the evaluation of residual nitrite levels as affected by temperature (heat shock at 75°C for 20 min and subsequent cooling from 54.4°C to 4.4°C within 15 h), storage time (3 and 24 h at 5°C) and sodium erythorbate concentration. Inhibition of C. perfringens spore germination and outgrowth was observed after 3 h of ham preparation in all the treatments evaluated. Greater inhibition of C. perfringens was observed with higher concentrations of NaNO₂. Addition of sodium erythorbate resulted in greater C. perfringens populations subsequent to 15 h of abusive cooling. Residual nitrite concentrations were similar under the experimental conditions used in this study. Residual oxygen in the ham could explain the inhibition observed during cooling of the ham within 3 h of preparation. During the second experiment, the effect of NaNO₂ (0 or 100 ppm), NaCl (1 or 2%) and organic acid salts (Opti.Form PD4®; 0, 1.5 or 2.5%) on C. perfringens spore germination and outgrowth during abusive cooling was evaluated. Incorporation of Opti.Form PD4® (1.5%) inhibited C. perfringens spore germination and outgrowth regardless of the cooling rate (9, 15 or 21 h). Addition of NaNO₂ enhanced the antimicrobial activity of organic acid salts. Shorter time period between ham preparation and heat treatment/cooling resulted in greater inhibition of C. perfringens spore germination and outgrowth. Antimicrobial agents should be incorporated into ham formulations in case reductions in NaCl and NaNO₂ concentrations are considered in view of the recommendation to reduce sodium content in the diet.

Advisor: Harshavardhan Thippareddi

BIOLOGICAL ANALYSIS OF PREBIOTICS IN VARIOUS PROCESSED FOOD MATRICES

Kristina Elise Moore — Tue, 01 Nov 2011 13:35:24 PDT

The addition of prebiotic to a variety of food products has become a more common occurrence in recent years. Although research on the stability of prebiotics has been conducted, knowledge of the stability of prebiotics in processed foods is limited. The purpose of this research was to determine the biological stability of five prebiotics in a variety of food matrices when processed under various conditions. A biological test, the prebiotic activity assay, was used to test for stability of six food products (muffin, cookie, granola bar, breakfast cereal, sports drink, and bread) containing five different prebiotics (fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), resistant starch, and polydextrose). The prebiotic activity assay reflects the capability of a prebiotic to support the growth of a probiotic strain relative to an enteric strain and relative to growth of both on a non-prebiotic substrate. Due to the complex matrices of the food products as well as low concentrations of prebiotics, the prebiotic activity assay was not sufficiently sensitive to assess biological stability in these food products. Additional food products (cracker, granola, and sports drink) were produced without background sugars and included a higher concentration of prebiotic, 10%. The prebiotic activity assay was used to assess the biological stability of prebiotics within these food matrices. Overall, FOSand inulin were stable when exposed to mild to moderate heating, but were biologically degraded when exposed to an acidic environment and moderate heat. GOS was stable when exposed to mild to moderate heat as well as when exposed to an acidic environment that was processed with moderate heat. Resistant starch posed problems with being accurately tested for prebiotic stability, and no strong conclusions were able to be made based on the results obtained through this method. The prebiotic activity assay was able to assess the biological stability of prebiotics in food matrices when exposed to several processing conditions, although the assay was better suited for certain prebiotics as well as certain food matrices.

Advisor: Robert W. Hutkins

PULSED ELECTRIC FIELD (P.E.F) AND PECTINASE FOR THE EXTRACTION OF POLYPHENOLS FROM GRAPE POMACE AND PEEL

Julien Khalil — Wed, 27 Jul 2011 06:37:14 PDT

Grapes are an important source of bioactive compounds such as gallic acid, resveratrol, and catechin. The consumption of grapes is associated with a lower risk of diseases such as cardiovascular diseases and some types of cancer. Grape pomace and skins are good sources of many phytochemicals known for their antioxidant potential.

In this research, the peel of the Chilean “Flame” grape cultivar and the pomace of St. Croix, Frontenac and St. Pepin grape cultivars were subjected to a pulsed electric field (5 KV, 1 μFarad, 20 Pulses) and to an enzymatic treatment (Pectinase, 5KU). The total phenolic content, determined in gallic acid equivalents using the Folin-Ciocalteau assay was analyzed. In addition to that, some of the individual phenolics present in the extracts were identified and quantified using HPLC. Finally, the antioxidant potential of the extracts was calculated using the FRAP assay.

This research explored the possibility of establishing if by-products generated by wineries could become a potential source of polyphenols. Pulsed electric field and pectinase treatments were both effective in enhancing the extraction of polyphenols from grape pomace and peel. The extracts showed a strong antioxidant power.

Adviser: Durward A. Smith

Adaptation and Validation of Existing Analytical Methods for Monitoring Prebiotics Present in Different Types of Processed Food Matrices

Pei Tze Ang — Fri, 10 Jun 2011 13:48:27 PDT

Interest in resistant starch (RS) as a prebiotic is increasing due to its putative impacts on the gastrointestinal microbiome and thus colon health. Application of RS as a food ingredient is also increasing because of its unique functionality. However, a major obstacle when correlating RS containing food systems to the potential health benefits is the discrepancy in the analytical tests applied and the limitations of those methods used throughout the literature. Therefore, the Megazyme resistant starch assay was adapted and developed to monitor RS present in different types of products commonly consumed in the United States, including an extruded ready-to-eat breakfast cereal, a cookie, a muffin, a sport drink, and a nutritional bar. The linearity of the calibration curve based on glucose standards (0.1-1.0 mg/mL) from the validation of the Megazyme RS assay was Y= 0.9918x + 0.0057 with a correlation coefficient of R_² = 0.9995. The low detection (0.0013%) and quantitation limits (0.0048%) of blank samples were needed to assay the final food products. Each processed food required minimal optimization, with the exception of the sports drink, that included increasing the centrifuge time (cookie), to changing the entire formulation (extruded cereal), to removing the moisture and fat prior to analysis (granola, muffin). As a result, % recoveries of 1% spiked samples ranged from 66-100% and the detection limit was lower than 1% for all the processed foods except for muffin and cookie. Application of the validated method to 1% supplemented processed foods showed processing effects, especially for the extruded cereal. RS was also significantly affected when the sports drink was reformulated (sweetener composition and pH) but only non-significant trends resulted when changing the extrusion parameters (barrel temperatures and screw speeds) for the cereal. In summary, application of a more robust and reliable RS assay is important in determining the chemical fate of RS under different processing treatment in processed food supplemented with RS.