Dept. of Food Science and Human Nutrition Sat, 01 Oct 2016 05:00:02 GMT 2016-10-01T05:00:02Z Optimal yeast strains for the production of fuels and chemicals from lignocellulosic biomass Optimal yeast strains for the production of fuels and chemicals from lignocellulosic biomass Kim, Soo Rin The microbial production of fuels and chemicals has recently received much attention as an alternative to the limited fossil fuels. To become an economically viable option, the bioprocess must use non-food plants, called cellulosic biomass. Such renewable biomass, however, contains both hexose and pentose sugars, mainly glucose and xylose, which are not efficiently fermented by microbes. Despite numerous efforts for improving xylose fermentation during the last two decades, a method for engineering efficient xylose-fermenting strains has not been finalized yet. In this thesis study, by elucidating fundamental bottlenecks impeding the xylose metabolism, I identified and implemented powerful genetic perturbation targets for developing an efficient xylose-fermenting Saccharomyces cerevisiae strain. First, I used a genome library screening method to investigate the limiting steps in xylose metabolism by a S. cerevisiae strain expressing a heterologous xylose-assimilating pathway consisting of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XK), coded by XYL1, XYL2, and XYL3, respectively. Finding XYL2 as an overexpression target, I optimized the expression levels of XYL1/XYL2/XYL3 to maximize the ethanol yield with a minimum xylitol yield. Later, I confirmed that the optimized expression level of the xylose-assimilating pathway resulted efficient xylose fermentation regardless of the unbalanced cofactor requirements between XR and XDH: NADPH-specific XR (GRE3) was successfully substituted for NAD(P)H-specific XR (XYL1) in a S. cerevisiae strain expressing NAD+-specific XDH. Through evolutionary engineering of the engineered strain with the optimized xylose-assmilating pathway, deletion of PHO13 was found to be a key element in improving the growth rate on xylose. Lastly, after disruption of a major acetaldehyde dehydrogenase (ALD6) to prevent acetate accumulation, the final strain was able to ferment xylose efficiently under various conditions including dilute-acid hydrolyzates of cellulosic biomass. Using this engineered S. cerevisiae strain capable of fermenting xylose efficiently, we will be able to produce various fuels and chemicals from cellulosic biomass hydrolyzates. Saccharomyces cerevisiae; metabolic engineering; xylose fermentation; cellulosic biomass; ethanol; XYL1; GRE3; XYL2; XYL3; PHO13; ALD6; Genomic library; Evolutionary engineering Thu, 12 Jul 2012 00:00:00 GMT 2012-07-12T00:00:00Z Kim, Soo Rin Microencapsulation of tributyrin to improve sensory qualities and intestinal delivery Microencapsulation of tributyrin to improve sensory qualities and intestinal delivery Donovan, Joseph D Microencapsulation is commonly used in the food industry to provide functional and sensory benefits to a variety of compounds. Tributyrin (TB), a source of butyric acid, is characterized by a highly bitter taste and negative odor attributes. Its use in the maintenance of intestinal health and treatment of intestinal disorders shows promise. However, due to the negative sensory qualities and necessity to target the intestinal epithelium, TB has yet to be widely utilized in a food system for treatment. The overall objectives of this study were to: 1) determine the impact of protein type, inulin chain length, and gamma-cyclodextrin (GCD) on the stability and retention of microencapsulated TB, 2) measure which TB microcapsule formulation imparts the least overall sensory difference in an infant formula system, 3) determine the site of intestinal delivery and release of butyrate from microencapsulated TB, and 4) determine the sensory properties of food products containing microencapsulated TB. Microencapsulated TB in whey protein isolate (WPI)-based microcapsules resulted in higher (p<0.001) retention than soy protein isolate (SPI)-based microcapsules. The inclusion of inulin WPI-based wall materials improved (p<0.01) the retention of TB for all chain lengths over WPI-based microcapsules without inulin. The creation of altered surface morphology from inulin is due to the ability of inulin to interact with the WPI-based wall material. This interaction creates added wall flexibility during drying and may be responsible for increased TB retention. The use of GCD resulted in the highest TB retention (95%) when oven dried but the lowest when spray dried (62%). In infant formulas, GCD and TB oven dried (GCT OD) microcapsules were able to reduce the sensory perception of TB to a level indistinguishable from a control infant formula containing no TB. All other WPI, WPI-inulin, and GCD-based microcapsules were significantly different from the control (R-index above 57% or at p<0.05) but not from free TB (p>0.05) as indicated by the rating method. During in vitro digestion, all microcapsules containing TB showed limited butyrate release (<5%) during oral and gastric stages. In then simulated small intestine, TB microcapsules released approximately 75% of their total butyrate content with no significant differences (p>0.05) across formulations. During fermentation, GCD-based microcapsules produced significantly more butyrate (p<0.001) on a molar basis than all WPI-based microcapsules. The microcapsule GCT OD was able to effectively deliver and produce butyrate in the small and large intestines. Results from the descriptive analysis (DA) test conducted using GCT OD in apple sauce, infant formula, and crackers, revealed that GCT OD behaved differently in each system due to the matricies physical properties. Combined common attribute principal correlation analysis (PCA) biplots show samples containing free TB were highly characterized by bitter taste and aftertaste. Cracker samples were not significantly different (p>0.05) for all measured attributes intensities as compared to free TB-containing samples, and were significantly higher in bitter taste and aftertaste. Apple sauce and infant formula containing GCT OD were significantly (p<0.05) reduced in bitter taste and aftertaste as compared to free TB samples. This bitter taste and aftertaste intensity for GCT OD was still, however, significantly higher (p<0.05) than the control for apple sauce and infant formula. The inclusion of GCT OD in infant formula had the least overall impact on the attributes that characterize control infant formula samples. Overall, findings from this research can be used to guide the production and application of microencapsulated TB for use in these and additional food products for the potential improvement of intestinal health or disease states. microencapsulation; spray drying; whey protein isolate; soy protein isolate; gamma-cyclodextrin; complexation; tributyrin; butyric acid; sensory evaluation; R-index; in vitro digestion; targeted release; descriptive analysis Wed, 17 Jun 2015 00:00:00 GMT 2015-06-17T00:00:00Z Donovan, Joseph D Autophagy: activation, function and regulation by a protein restricted diet during pregnancy and lactation Autophagy: activation, function and regulation by a protein restricted diet during pregnancy and lactation Wang, Huan Developmental protein restriction is associated with numerous diseases including obesity, diabetes and cardiovascular disease, both in the mother and offspring. Recent intensive research efforts have focused on the mechanisms behind the effects of a maternal low protein diet on diseases in offspring. However, thus far, the mechanisms behind the physiological and molecular adaptations in the mother in response to protein restriction during pregnancy and lactation are not well understood. Because skeletal muscle and liver have a critical role in energy metabolism, including pregnancy and lactation-associated metabolic adaptations, the present study will investigate the genetic and epigenetic mechanisms behind the physiological changes that occur in response to a protein restricted diet during pregnancy and lactation in the skeletal muscle and liver of rat dams. The amino acid response (AAR) pathway is important in responding to stress, such as amino acid limitation, and our previous data show that a protein restriction diet during pregnancy induces the AAR pathway in placenta and causes stunted growth in the offspring. The central regulator of the AAR pathway is the activating transcription factor 4, ATF4. It has been shown that ATF4 can regulate macroautophagy in response to amino acid limitation. The process of autophagy is essential for the maintenance of cellular homeostasis, and our results show that a gestational low protein (LP) diet induces the mRNA expression of autophagy-related genes in the skeletal muscle of rat dams and male offspring, providing strong evidence of maternal programming. Additionally, our study shows that a gestational and lactational LP diet stimulates autophagy and hepatic lipid accumulation, and decreases expression of histone deacetylase-3 (Hdac3) in the liver of rat dams. Histone acetylation is associated with increased gene transcription. Specifically, it has been reported that HDAC3 inhibition induces LC3B expression, a primary autophagy marker and lipid accumulation. More importantly, we show that amino acid limitation induces autophagy through HDAC3-dependent expression of LC3B and its increased association with lipid droplets in a hepatic cell line, HepG2. Investigating the role that HDAC3 may play in the activation of LC3B and lipid accumulation will bring valuable insight into the epigenetic control of hepatic lipid accumulation by a LP diet during pregnancy and lactation in rat dams. In summary, our results demonstrate that protein restriction during gestation and lactation induces autophagy in skeletal muscle and liver, respectively; investigating the epigenetic regulation of autophagy will shed light on potential therapies that could diagnose and treat imbalanced maternal nutrition-related metabolic disorders in mother and offspring. Low protein; Autophagy Mon, 31 Aug 2015 00:00:00 GMT 2015-08-31T00:00:00Z Wang, Huan The coaggregative abilities of lactobacilli and Campylobacter jejuni The coaggregative abilities of lactobacilli and Campylobacter jejuni Wooten, Jessica Michelle Campylobacter jejuni is a pathogen commonly found in poultry that is one of the main bacterial causes of foodborne diarrheal disease in humans. The aim of this study was to analyze the coaggregative abilities of lactic acid bacteria and C. jejuni as a means to reduce the colonization of C. jejuni in poultry. In this study, various lactic acid bacteria were screened to analyze their coaggregative abilities with C. jejuni. The coaggregative abilities of LAB strains within the same species were compared. L. crispatus’ ability to coaggregate with C. jejuni mutant strains was tested. The generation of non-coaggregating L.crispatus variants was performed. DNA sequencing was performed on the wildtype strains of L.crispatus (MJM 207 and 207) along with three non-coaggregating variants. All of these measures were used to help pinpoint a genetic determinant for the coaggregative ability of the LAB strains tested. L.plantarum, L. acidophilus, and L. fermentum did not display significant variation of coaggregative abilities with C.jejuni. L. crispatus was not able to coaggregate with C.jejuni mutant strains (MJM 376, 378, 379, 381 and 382). Findings from this study can provide knowledge to help better understanding the coaggregative relationship between lactobacilli and C. jejuni. Lactobacillus; Campylobacter jejuni; coaggregation Thu, 10 Dec 2015 00:00:00 GMT 2015-12-10T00:00:00Z Wooten, Jessica Michelle Engineering yeast strains for producing fuels and value-added chemicals from cellulosic biomass Engineering yeast strains for producing fuels and value-added chemicals from cellulosic biomass Oh, Eun Joong The biotechnological production of fuels and value-added chemicals from cellulosic biomass is a sustainable and ecofriendly process. Pretreatment and hydrolysis of the biomass produce six (glucose) and five-carbon (xylose) sugars, and toxic fermentation inhibitors, such as acetic acid. The efficient conversion of the cellulosic sugars in hydrolyzates by fermentation microorganisms is one of the key steps for the economically feasible production of biofuels and chemicals. Most xylose-fermenting engineered yeast cannot utilize xylose until glucose is completely consumed. In addition, acetic acid derived from hemicellulose during the pretreatment negatively influences the fermentation performance by yeast. The overall goal of my thesis study is to develop metabolically engineered yeast strains to overcome the abovementioned challenges. Simultaneous co-utilization of cellobiose (a dimer of glucose) and xylose could bypass the inhibition of xylose uptake by glucose and improve ethanol production. To this end, heterologous genes coding for cellodextrin transporter (cdt-1) and β-glucosidase (gh1-1) were integrated into the genome of the yeast, and the expression cassettes were massively amplified through laboratory evolution, resulting in significantly improved cellobiose fermentation. Besides ethanol production, the simultaneous co-conversion of cellobiose and xylose strategy was successfully applied to improve the production of value-added products, such as xylitol. Furthermore, integration of cellobiose, xylose, and acetic acid utilizing pathways into one microbial platform enabled efficient bioconversion of the mixed substrates in a synergistic way. Next, cellodextrin transporter 2 (CDT-2), which has the potential to surpass the performance of CDT-1 because of its energetic benefits under stress conditions, was engineered to improve cellobiose uptake under acidic conditions. The mutant CDT-2 (I96N/T487A) was isolated through the directed evolution of CDT-2 followed by high-throughput screening using flow cytometry, and its cellobiose consumption rate under low pH conditions was much higher than that of the wild type CDT-2. Lastly, a genetic perturbation for enhancing the acetic acid tolerance has been identified via genomic library screening. Overexpression of RCK1 coding for protein kinase involved in the oxidative stress response in yeast significantly improved both glucose and xylose fermentations under acidic conditions, reducing intracellular reactive oxygen species (ROS) accumulation. We demonstrated that the rational and combinatorial metabolic engineering strategies enabled engineered yeast strains to overcome the challenges in fermentation for the conversion of cellulosic biomass. Moreover, this study provided valuable findings that can be applied to industrial fermentation processes. Metabolic engineering; Saccharomyces cerevisiae; Cellulosic biomass Thu, 03 Dec 2015 00:00:00 GMT 2015-12-03T00:00:00Z Oh, Eun Joong Sensory discrimination testing methodology selection based on beverage complexity Sensory discrimination testing methodology selection based on beverage complexity Bloom, David J Sensory discrimination testing is a vital tool used by sensory professionals within the food industry. While many testing methods are available for selection, various methods have proven to differ in power. By utilizing a more powerful method, sensory professionals can limit the resources needed for testing and increase their ability to find significant differences between products. The inherent variability of food and beverage systems in addition to multidimensional changes often made during reformulation of food products can make the task of method selection challenging. The goal of this study was to determine the optimal sensory discrimination methodologies for use with multidimensional beverage systems with confusable difference comparison. Complex beverage systems with product variations relevant to actual testing within the food industry were utilized to compare experimental results to those expected from theoretical modeling predictions. The findings from this study led to the exploration of Researcher-Designated and Panelist-Articulated specific discrimination methods to determine which procedure resulted in greater power for use in comparison of complex beverage systems. Once a more powerful specific testing procedure was established, it was then used in product comparisons with non-specific methods to determine how the degree of difference between samples influenced complex beverage systems in one-dimensional or multidimensional formulation changes. A model beverage system was created which could be altered in formulation to create multidimensional changes between samples and alter d’. The model beverage was utilized to study the impact of sample dimensionality and d’ for both specified and non-specified methods. Differences in the proportion of correct response, method power, and overdispersion were observed between methods when tested with the model beverage system. The findings from these studies emphasize the need for sensory professional to look beyond the categorization of methods by specified versus non-specified. Findings suggest methods should instead be fit to the range of d’ and multidimensional makeup relevant to the samples used in testing. Discrimination Testing; Statistical Power; Beverages; Complexity; Dimensionality Thu, 15 Oct 2015 00:00:00 GMT 2015-10-15T00:00:00Z Bloom, David J Dietary broccoli impedes Western diet-enhanced fatty liver and hepatocellular carcinoma development Dietary broccoli impedes Western diet-enhanced fatty liver and hepatocellular carcinoma development Chen, Yung-Ju Liver is the metabolic center for energy homeostasis in our body, maintaining a balance between carbohydrate and fat metabolism. The “Westernized” dietary pattern, which is known for high saturated fat and refined sugar and rooted in the lifestyle of a large proportion of the world’s population, may greatly disrupt energy balance, resulting in obesity and obesity-related diseases. Non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma, which is also a possible endpoint of NAFLD, are both enhanced by adiposity and inflammation, and almost symptomless until serious damage is caused in liver. However, these diseases can be preventable, by changing lifestyle and diet. Broccoli, a well-accepted brassica vegetable in the United States, has the potential to reduce cancer risk and ameliorate inflammation. Therefore, in this study, we aimed to understand the impact of dietary broccoli on the development of NAFLD and liver cancer in mice fed a Western diet. Accordingly, we proposed a whole dietary broccoli intervention. A combined Western diet-fed and diethylnitrosamine (DEN)-treated mouse liver cancer models was used in order to evaluate the changes in hepatic lipidosis, macrophage activation, and tumorigenesis after long-term consumption of broccoli. Our results show that dietary broccoli decreased hepatic lipidosis as early as 3 months after initiation, and effectively down-regulated liver damage. The enlarged hepatic triglyceride pool due to the Western diet was narrowed by dietary broccoli, lowering the influx of non-esterified fatty acids but increasing the excretion of very-low-density lipoprotein. Activation of hepatic macrophages, was lowered by continues consumption of broccoli. Furthermore, DEN-induced liver tumor size and hepatic neoplasm-related lesion formation were both decreased by dietary broccoli. In addition, as an incidental finding, intraperitoneal DEN-induced nasal epithelial neoplasm-related lesions in B6C3F1 mice are first reported in this study. Overall, whole broccoli dietary intervention has the potential to impede the progression of NAFLD, from hepatic steatosis, through steatohepatitis, to hepatocellular carcinoma. This study fills gaps of knowledge about the impact of broccoli on hepatic lipid metabolism, supports the cancer preventive effect of brassicas revealed by epidemiologic studies, and further encourages the whole food dietary intervention. Translation to clinical studies is needed. Broccoli; Western diet; Non-Alcoholic Fatty Liver Disease (NAFLD); Liver cancer Fri, 28 Aug 2015 00:00:00 GMT 2015-08-28T00:00:00Z Chen, Yung-Ju Engineering Saccharomyces cerevisiae for cellulosic ethanol production Engineering Saccharomyces cerevisiae for cellulosic ethanol production Xu, Haiqing There are two long-existing obstacles for cellulosic ethanol production in Saccharomyces cerevisiae. The first one is inefficient xylose utilization and the second one is sequential fermentation of glucose and xylose in engineered Saccharomyces cerevisiae. This study mainly focused on solving these two problems by using cutting edge synthetic biology tools. cellulosic ethanol production; xylose; CRISPR/Cas9; co-fermentation; co-culture system Mon, 07 Dec 2015 00:00:00 GMT 2015-12-07T00:00:00Z Xu, Haiqing Production of a functional human milk oligosaccharide, 2'-fucosyllactose, using microbial cell factories Production of a functional human milk oligosaccharide, 2'-fucosyllactose, using microbial cell factories Pathanibul, Panchalee Human breast milk is the gold standard for infant nutrition. In human milk, the oligosaccharides may protect babies by acting as decoy receptors for pathogens. Also, human milk oligosaccharides (HMOs) enhance the proliferation of probiotics to strengthen the host immune system. Among HMOs, 2’-fucosyllactose (2-FL), composed of L-fucose and D-lactose, is known to possess an anti-infection capability against many harmful organisms such as Campylobacter jejuni, enteropathogenic Escherichia coli, and even Norovirus. Consequently, great quantities of 2-FL are being demanded for food applications and thorough investigation of its biological properties. The current synthetic methods of 2-FL including chemical production and enzymatic catalysis are complicated, expensive, and thus impractical for a large-scale synthesis. In my research study, an alternative route to produce 2-FL using a microbial cell factory was devised. First, E. coli, as a host strain to produce 2-FL, was engineered to overexpress genes in the metabolic pathway of GDP-L-fucose, a donor of L-fucose, and harbor the fucosyltransferase enzyme (FucT2) which catalyzes the transfer of L-fucose onto a lactose molecule enabling 2-FL production. Second, the production conditions of 2-FL in the engineered E. coli were optimized in order to pinpoint the appropriate conditions promoting the efficient and consistent synthesis of 2-FL. Third, the key enzyme in 2-FL production, FucT2, from different sources was compared in search for the FucT2 leading to an enhanced synthesis of 2-FL. Fourth, the possibility to produce 2-FL was demonstrated in a different host, Saccharomyces cerevisiae, which is a generally recognized as safe (GRAS) organism. Through metabolic engineering, the minimum three components required to construct a 2-FL-producing S. cerevisiae consisting of GDP-L-fucose production, lactose internalization, and functional expression of FucT2 were confirmed. Additionally, the gene target perturbation, deletion of GDA1, which resulted in an improved production of GDP-L-fucose was identified. GDP-L-fucose is a beneficial sugar nucleotide which serves as a precursor to innumerable compounds aside from 2-FL. From this study, the feasibility and scalability of employing the microbial cell factory approach for 2-FL and GDP-L-fucose production were manifested. 2'-fucosyllactose; metabolic engineering Fri, 04 Dec 2015 00:00:00 GMT 2015-12-04T00:00:00Z Pathanibul, Panchalee Manufacture and utilization of a biodegradable sensor platform from gold coated zein nanophotonic films to detect acrylamide and Ara-h1 using SERS Manufacture and utilization of a biodegradable sensor platform from gold coated zein nanophotonic films to detect acrylamide and Ara-h1 using SERS Gezer, Pervin Gizem With the current developments in biosensor and nanotechnology, detection of analyses that are important to food industry are becoming more commonplace. One of the strong tools that nanotechnology enabled is Surface Enhanced Raman Spectroscopy (SERS). SERS is a highly sensitive and specific technique which provides molecular fingerprinting, with the enhancement effect as a result of roughened noble metal surfaces. The platform for these surfaces are generally made out of non-biodegradable, plastic materials. As the one-time use, large scale applications are needed for many fields such as medical, forensic and food industry, disposability of these sensors will pose a problem in the future. In the scope of this dissertation, we investigated the feasibility of a biodegradable sensor platform that is made of zein, a corn protein, utilized in SERS measurements of food analytes. First, the effect of parent substrate (the surface, which zein was cast on) and plasticizer, oleic acid content, on the surface hydrophilicity of resulting zein films was analyzed. It was found that the surface chemistry of the parent substrate was more important than the topography of the parent substrate. Oxygen plasma was used to make the polydimethylsilohexane (PDMS) surfaces more hydrophilic and it was found that zein film surfaces that were in contact with PDMS also had more hydrophilic surfaces, compared to regular PDMS, which is a hydrophobic material. Water contact angle (WCA) method was used to quantify the hydrophilicity of zein films. WCA reached values as low as 20 degrees with a high oleic acid content. Increase in oleic acid content in the formulation of zein films as well as the parent substrate chemistry was found to influence the water affnity of zein films. In the development of the fabrication method of nanopatterned and gold coated zein surfaces, a simultaneous three-dimensional transfer was used. Four different nanopatterns, namely positive pyramids, inverted pyramids, nano pillars and nano pores were transferred onto zein films along with either 80 or 200 nm gold coating by using solvent casting technique. Scanning microscopy images showed that the patterns were transferred onto zein films with high fidelity and success. The enhancement effect of these SERS substrates were tested by using a model molecule, Rhodamine 6G. It was found that the best enhancement effect was provided by inverse-pyramid structures coated with 200 nm gold. For the rest of the study, these structures were used. Zein-SERS substrates were utilized in two different food analyte detection purpose, acrylamide and peanut allergen protein Ara h1. Acrylamide is a potential carcinogenic compound that is formed during high temperature food processing. French fries, potato chips, bread and coffee are some of the food products that may contain high amounts of acrylamide. Since Food and Drug Administration released a draft advisory for mitigation strategies for acrylamide content in foods, there is a need for routine testing technique of acrylamide in food products. In this research, acrylamide was detected by using zein-SERS substrates as a proof of concept. Limit of detection was found to be 10 micrograms/milliliter. Calibration curve was obtained with an R2 value of 0.93 and 0.97 for log-log version. Peanut allergies are among the most common food allergies, and they can result in life-threatening reactions in allergic patients. For this reason, it is extremely important to monitor the presence or cross-contamination of peanuts into food products. There are 8 identified peanut allergen proteins and Ara h1, consists of the largest percentage of protein content, in addition to causing reactions in almost 100 % of the patients. Zein-SERS substrates were utilized in detection of Ara h1. With the use of statistical clustering technique called principal component analysis (PCA), it was possible detect and quantify Ara h1 protein. Limit of detection was found to be 0.14 mg/ml. The surface of the zein-SERS substrates were functionalized with monoclonal antibody and tested for capturing Ara h1 as a proof-of-concept. With this research, utilizing zein as a biodegradable sensor platform for SERS measurements were investigated for the first time. It was shown that detection of both acrylamide and Ara h1, peanut protein, was possible. The methods developed in this study for controlling the surface hydrophilicity of zein films and direct transfer of both micro and nano-scale patterns onto zein along with noble metals can be employed in other biosensor and biopolymer applications as well in the future. This kind of biodegradable platforms might be an alternative solution for environmentally friendly and large scale sensor applications. Zein; biosensor; Surface Enhanced Raman Spectroscopy (SERS); Raman; acrylamide; peanut allergen; detection; surface Tue, 01 Dec 2015 00:00:00 GMT 2015-12-01T00:00:00Z Gezer, Pervin Gizem