Theses, Dissertations and Student Research Papers (Food Science and Technology)

Clostridium perfringens spores : inactivation, germination, and formation

Mon, 15 Apr 2013 00:00:00 GMT

Clostridium perfringens spores : inactivation, germination, and formation Udompijitkul, Pathima The enterotoxin-producing Clostridium perfringens type A isolates are responsible for the third most common foodborne illness in the United States and can also cause non-foodborne human gastrointestinal (GI) diseases such as antibiotic- associated and sporadic diarrheas. Three important factors contribute to the ability of C. perfringens to cause GI diseases, including its extremely rapid growth rate, its ubiquitous distribution in foods and environments, and its capability to form highly resistant endospores. In the first study, the antimicrobial peptide nisin was evaluated for its antimicrobial effect against enterotoxigenic C. perfringens food poisoning (FP) and non-foodborne (NFB) GI disease isolates. Nisin did not affect spore germination, whereas germinated spores were very susceptible to low concentration of nisin and thus spores outgrowth were arrested. Nisin also exerted its inhibitory effect against vegetative growth of C. perfringens FP and NFB isolates in rich medium; however, FP cells were less resistant to nisin than NFB cells. Nevertheless, nisin was not effective in controlling germination and outgrowth of C. perfringens spores in cooked meat products during storage at abusive temperature, even at ~ 4 times elevated concentration than the regulatory approved level. Strikingly, spores of NFB isolates also exhibited higher resistance to nisin than that of FP isolates in both laboratory medium as well as in meat systems. Collectively, despite its effectiveness in controlling spore outgrowth and vegetative cell growth in laboratory conditions, nisin showed no antimicrobial activity against C. perfringens spores inoculated into meat model systems. The main focus of the second study was to develop an effective spore inactivation strategy on food contact surfaces by inducing spore germination prior to inactivation of the more susceptible spores with commonly used surface disinfecting agents. The mixture of L-asparagine and KCl (AK) was the most effective germinant for spores of enterotoxigenic C. perfringens type A. Germination temperature had a significant influence on the germination extent and subsequent inactivation by variety of surface disinfectants. Implementation of germination step significantly increased the inhibitory effect of all tested disinfecting agents against spores of C. perfringens FP strain SM101 with lower efficacy against the spores of NFB strain NB16. Furthermore, spores of C. perfringens FP isolates could germinate with AK upon their adhesion onto stainless steel chips and were subsequently inactivated with disinfectant agents by i.e. 1.53 to 2.70 log reductions of colony forming units per chip. Overall, AK-induced germination followed by treatment with iodophore represents a promising strategy to inactivate spores of C. perfringens FP isolates on food contact surfaces. Spore germination is initiated upon sensing a variety of compounds, termed germinants, via the cognate germinant receptors. In the third study, we identified sodium ions and inorganic phosphate (NaPi) at pH ~ 6.0 as a novel germinant for spores of enterotoxin-producing C. perfringens FP isolates. The spores lacking germination proteins GerAA and GerKA-KC were severely impaired in their ability to germinate with NaPi, whereas GerKB-negative spores germinated to a similar extent as wild type spores with NaPi, but their initial rate of germination was lesser. Spores lacking GerO or GerO GerQ germinated to a lower extent and with a significantly slower rate than wild type spores. In contrast, gerQ spores exhibited only a slightly slower and lesser extent of germination with NaPi than its parent strains. Therefore, the germinant receptor proteins GerKA-KC, GerAA, and the putative antiporter GerO are essential for normal germination of C. perfringens spores with NaPi. In the fourth study, we demonstrated that polar, uncharged amino acids at pH 6.0 could efficiently trigger germination of spores of enterotoxigenic C. perfringens. While L-glutamine is a unique nutrient germinant for spores of C. perfringens FP isolates, L-asparagine, L-cysteine, L-serine, and L-threonine can induce germination of both FP and NFB spores. The germinant receptor GerKC is the major receptor involved in cysteine- and glutamine-induced germination and release of dipicolinic acid (DPA) from the spore’s core, whereas less pronounced germination defects were observed in gerAA and gerKB spores. GerKC also has a key role in L-asparagine germination. For serine and threonine (pH 6.0)-induced germination, GerKA is the dominant receptor and GerKC and GerKB are also required for efficient germination of FP spores. The objectives of the fifth study were to identify and characterize the putative sensor histidine kinases of C. perfringens. We identified six genes encoding putative sporulation-associated sensor histidine kinases in the genome of C. perfringens SM101. These putative kinase genes were highly expressed under sporulation- stimulating conditions. Two genes encoding putative orphan sensor histidine kinases, cpr1728 and cpr1055, were inactivated and roles of each putative kinase on various aspects in the life cycle of C. perfringens had been characterized. Inactivation of cpr1728 and cpr1055 significantly lowered C. perfringens sporulation capacity in two sporulation-inducing conditions. Moreover, sporulation delayed phenotype was also observed in strain lacking CPR1055. Inactivation of either cpr1728 or cpr1055 led to a marked defect in C. perfringens spore germination with all known germinants. Spores of two kinase mutants also exhibited slower outgrowth than their parental strain; however, no difference in colony forming efficiency was observed among tested strains. Additionally, mutations in cpr1728 and cpr1055 did not affect vegetative growth; however, both mutants grew at higher rate under sporulation-inducing conditions. In conclusion, this dissertation reports the experimental results that are relevant to various aspects of C. perfringens spores. These include the development of spore inactivation strategies in food products as well as on food contact surfaces, the identification of compounds triggering germination of spores of CPE-producing C. perfringens, and the insights into the roles of putative sensor histidine kinases in the process of spore formation and spore germination under a variety of conditions. Graduation date: 2013

Survival of Listeria monocytogenes, Salmonella spp., and Staphylococcus aureus in raw yellowfin tuna during refrigerated and frozen storage

Wed, 06 Mar 2013 00:00:00 GMT

Survival of Listeria monocytogenes, Salmonella spp., and Staphylococcus aureus in raw yellowfin tuna during refrigerated and frozen storage Mou, Jing The consumption of seafood in the United States has increased rapidly in recent years due to high quality protein and health benefits of seafood. Seafood can be a carrier for bacteria normally distributed in the marine environment and, in some cases, can be contaminated by human pathogens. Therefore, there is a potential health risk if seafood is consumed raw or undercooked. However, information regarding prevalence of foodborne pathogens in retail seafood products and the ability of pathogens to survive in the products during refrigerated and frozen storage is limited. The objective of this study was to generate such information for a better understanding of distribution of foodborne pathogens in seafood products and provide data which might be used for risk assessment of foodborne infection associated with seafood consumption. A total of 45 seafood products were collected from local retail stores and analyzed for aerobic plate counts (APC) and psychrotrophic bacterial counts (PBC) as well as presence of foodborne pathogens, including Escherichia coli O157:H7, Salmonella, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio vulnificus according to procedures described in the U.S. Food and Drug and Administration Bacteriological Analytical Manual (BAM). Presumptive isolates for each foodborne pathogen were further characterized by biochemical reactions using commercial identification kits and confirmed with polymerase chain reaction (PCR) assay. The samples had bacterial populations ranging from 1.90 to 6.11 CFU/g for APC and from 2.00 to 6.78 CFU/g for PBC. According to the microbiological criteria of International Commission on Microbiological Specifications for Foods (ICMSF), all 45 samples were considered acceptable quality (APC < 10⁷ CFU/g, E. coli < 3 MPN/g) with most samples (93.3%) being good quality (APC < 5 × 10⁵ CFU/g, E. coli < 3 MPN/g). No E. coli O157:H7, Salmonella, S. aureus, V. parahaemolyticus, and V. vulnificus was detected in any samples. Two previously frozen shrimp products (4.4%) were confirmed to carry L. monocytogenes. Studies of growth and survival of L. monocytogenes (3 strains), S. aureus (2 strains), and Salmonella (2 serovars) in raw yellowfin tuna meat stored at 5 - 7 °C for 14 days revealed that L. monocytogenes had the ability to multiply in the tuna meat during refrigerated storage while populations of S. aureus and Salmonella were reduced by 1 to 2 log CFU/g after 14 days at 5 - 7 °C. Studies of holding raw yellowfin tuna meat contaminated with L. monocytogenes, S. aureus, and Salmonella at -18 ± 2 °C for 12 weeks observed that all three pathogens, except Salmonella Newport, in tuna samples survived the frozen storage with less than 2- log of reductions in the populations over 12 weeks of storage. No viable cell of Salmonella Newport was detected in samples after 42 days storage at -18 °C. Raw seafood can be a carrier of foodborne pathogens, particularly L. monocytogenes, and many foodborne pathogens can survive in frozen products for several months. Consumption of raw or undercooked seafood products may lead to human infection if the products are contaminated with pathogens. Therefore, sanitation standard operating procedure (SSOP), good manufacturing practice (GMP) and hazards analysis and critical control points (HACCPs) programs shall all be implemented in the seafood industry to prevent seafood products from being contaminated with foodborne pathogens during handling and processing. Moreover, proper storage of raw seafood products and avoiding cross-contamination during handling at the retail levels also helps to minimize risk of human infection associated with ready-to-eat products. Graduation date: 2013

Harvest maturity of Cascade and Willamette hops

Fri, 18 Jan 2013 00:00:00 GMT

Harvest maturity of Cascade and Willamette hops Sharp, Daniel C. (Daniel Collier) Hops (Humulus lupulus L.) are primarily used to provide specific characteristics to beer, such as bitterness, aroma, flavor, and microbial stability. The chemical composition of hops, relative to how they are used during the brewing process, dictates the expression of these characteristics. Of the raw ingredients that go into making beer, hops are perhaps the most costly. Considerable resources are required to grow quality hops, and therefore, brewers and hop growers alike have a common goal of obtaining the highest quality hops possible. However, quality can be a relative term. While it is commonly agreed upon that high brewing values, such as α-acids and essential oil content, and robust structural integrity are indicators of quality hops, there are many opinions of the ideal aroma. Changes in the chemical composition of hops during plant maturation are a dynamic process requiring a comprehensive, in-depth chemical and sensory analysis in order to maximize the characteristics of interest to brewers. The complex aroma chemistry associated with hops in beer has been a confounding variable for the practical brewer, and a deeper understanding of hop aroma development during cultivation is needed. The effect of harvest date, location, and cultivar on key chemical components of Willamette and Cascade hops was investigated for the 2010 and 2011 growing seasons. Hops were harvested at 3 time points (Early, Typical, and Late), within a 3-week interval from 2 different farms in the Willamette Valley, Oregon. A split-plot experimental design for each cultivar was used; each farm represented a main plot and harvest years were designated as subplots. American Society of Brewing Chemist standard methods of analysis were used to measure moisture content, hop acids and their homologs, Hop Storage Index, total essential oil content and volatile profile by GC-FID. Additionally, difference testing, descriptive analysis, and consumer acceptance testing was conducted using beers brewed with either Typical or Late harvested Cascade hops from the 2010 harvest year. The response of analytes was dependent on the cultivar being examined, its location within the Willamette Valley, as well as days until harvest. Hop acids did not change appreciably during plant maturation for the period examined, while hop oil content increased hyperbolically to a plateau as the hops aged on the bine. Increases in oil quantity were strongly correlated (r > 0.90) with increases in α-pinene, β-pinene, myrcene, limonene, methyl heptanoate, and linalool concentrations. For Cascade, α-pinene, β-pinene, myrcene, limonene, ρ-cymene, caryophyllene, E, β-farnesene, and humulene all increased from Early to Typical points but no increase was observed between the Typical and Late time point. Linalool and methyl heptanoate increased between each time point while citral and humulene epoxide differed between Early harvest and Late harvest, but not between Early and Typical or Late and Typical harvests. For Willamette hops, α-pinene, β-pinene, myrcene, limonene, ρ-cymene, and linalool all increased between each time point. Caryophyllene, E β-farnesene, humulene, farnesol and citral all increased from Early harvest to Typical harvest but no difference was observed between Typical and Late. Clear sensory differences were found between beers brewed with Typical harvest Cascade hops and Late harvest Cascade hops, in terms of difference testing, descriptive analysis and consumer preference tests. Graduation date: 2013

Development of antioxidant dietary fibers from wine grape pomace and their applications as functional food ingredients

Tue, 18 Dec 2012 00:00:00 GMT

Development of antioxidant dietary fibers from wine grape pomace and their applications as functional food ingredients Tseng, Angela Y. Wine grape pomace (WGP), the byproduct from winemaking, is a good source of polyphenols and dietary fibers, and may be utilized as antioxidant dietary fibers (ADF) for food applications. The objectives of this thesis research were to first determine the phenolic compounds, antioxidant and antimicrobial activities in red WGP under different drying processes for long-term storage, and to further evaluate the feasibility of using WGP as a functional food ingredient in yogurt and salad dressing for enhancing the nutritional value and improving storability of the products. Two types of WGP samples, pomace containing seeds and skins (P) and pomace with skins only (S) from Pinot Noir (PN) and Merlot (M) were studied. Samples were subjected to four different drying conditions: 40 °C conventional and vacuum oven, 25 °C ambient air and freeze dry. Total phenolic content (TPC, by Folin-Ciocalteu assay), anthocyanins (ACY, by pH differential method) and flavanols content (TFC, by vanillin assay) of the samples along with their antioxidant activity (DPPH radical scavenge method, RSA) and antibacterial activity (minimum inhibition concentration, MIC) were determined during 16 weeks of storage under vacuum condition at 15±2 °C. Meanwhile, dietary fiber profile was evaluated by using gravimetric-enzyme method. Results showed that dietary fiber contents of PN-P, PN-S, M-P and M-S were 57-63% d.m. with the majority of insoluble fraction. Freeze dried WGP retained the highest bioactive compounds with TPC 21.19-67.74 mg GAE/g d.m., ACY of 0.35-0.76 mg Mal-3-glu/g d.m., TFC of 30.16-106.61 mg CE/g d.m. and RSA of 22.01-37.46 mg AAE/g d.m., followed with ambient air dried samples. Overall, TPC, TFC and RSA were higher in PN than in M, and higher in pomace than in skins, while reverse results were observed in ACY. All samples lost significant amount of bioactive compounds during storage, in which ambient air and freeze dried samples had TPC reduction of 32-56% and 35-58%, respectively at the end of 16 weeks of storage. RSA in PN-P and M-P remained more than 50 mg TE/g d.m., meaning WGP still met the criteria of ADF definition after 16 weeks of storage. WGP extracts showed higher antibacterial efficiency against L. innocua than that of E. coli with MIC of 2, 7, 3 and 8% against L. innocua, and 3, 6, 4 and 9% against E. coli for PN-P, PN-S, M-P and M-S samples, respectively. This study demonstrated that Pinot Noir and Merlot pomace are good sources of ADF even after 16 weeks of storage at 15 °C and vacuum condition. Due to the highest antioxidant activity (RSA 37.46 mg AAE/g) and dietary fiber content (61%), PN-P was selected as ADF to be fortified in yogurt and salad dressing. Three types of WGP: whole powder (WP), liquid extract (LE) and freeze dried extract (FDE) with different concentrations were incorporated into yogurt (Y), Italian (I) and Thousand Island (T) salad dressings. TPC, RSA and dietary fiber content, major quality attributes including pH and peroxide value (PV) during the shelf life and consumer acceptance of fortified products were evaluated. The highest ADF were obtained in 3% WP-Y, 1% WP-I and 2% WP-T samples with the dietary fiber contents of 1.98%, 2.12% and 1.83% and RSA of 935.78, 585.60 and 706.67 mg AAE/kg, respectively. WP fortified products had more dietary fiber content than that of LE and FDE fortified ones because of the insoluble fractions. The pH dropped from 4.52 to 4.32 for 3% WP-Y during three weeks of storage at 4 °C, but remained stable in WGP-I and WGP-T samples after four weeks of storage at 4 °C. Adding WGP resulted in 35-65% reduction of PV in all samples compared to the control. In WGP-Y, the viscosity increased, but syneresis and lactic acid percentage were stable during storage. The 1%WP-Y, 0.5%WP-I and 1%WP-T samples were mostly liked by consumers. Study demonstrated that WGP can be used as a functional food ingredient for enhancing nutraceutical content and extending shelf-life of the food products. This study provided important information about the economically feasible drying methods for retaining the bioactive compounds in WGP during processing and storage and also suggested that WGP can be utilized as antioxidant dietary fiber to be fortified in consumer products to promote nutritional benefit and extend product shelf-life. Graduation date: 2013