Fate of hydroxyapatite nano particles during in vitro gastrointestinal digestion : a thesis presented in partial fulfilment of the requirements for the degree of Master in Food Technology at Massey University, Riddet Institute and Massey Institute of Food Science and Technology, Palmerston North, New Zealan

The following figures have been removed for copyright reasons, but may be accessed through their sources listed in the References: Figures 2.2 (Yada et al., 2014, Fig.1), 2.3 (Rivera-Munoz, 2011), 2.4 (Sakae et al., 2015, Fig. 2 & 3), 2.5 (Ehrlich et al., 2009, Fig. 3), 2.6 (Lynn & Bonfield, 2005, Fig. 1), 2.7 (Ehrlich et al., 2009, Fig. 6), 2.8 (Mafé et al., 1992, Fig. 1), 2.9 (Gaucheron et al., 2005, Fig. 2). Composite Figure 2.10 remains for clarity's sake.There is an increasing change in population demographics towards an aging population in the world, which had led to the availability of various commercial nutritionally supplemented products. Hydroxyapatite (HA), with chemical formula Ca10(PO4)6(OH)2, is an insoluble calcium salt used for calcium supplementation because of its similarity to the minerals found in human bone and teeth. The insoluble calcium salts are preferred over the soluble ones because of their high heat stability during milk processing under high heat treatment. However, the drawback of insoluble calcium salts is the tendency to sediment during storage resulting in unfavourable gritty texture. Thus, reduction in particle sizes into micron to nano-size improves the dispersion of these insoluble salts. However, the application of nano-sized particles in food products have raised concerns from both the regulatory organizations and consumers on the implications related to both the environmental and health safety aspects. Thus, the objective of the study is to determine the digestion behaviour of nano-sized needle/rod shaped HA (nHA) when added into skim milk during in vitro gastrointestinal digestion. Determination of calcium such as soluble and ionic calcium was conducted to determine the dissolution of nHA. The structural changes and the crystallographic changes of nHA were determined using electron microscopy and x-ray diffraction techniques. The results of in vitro gastric digestion showed presence of undissolved nHA particles even after 240 min of gastric and 120 min of intestinal digestion when examined under TEM, while the XRD analysis detected the presence of crystalline nHA in the first 120 min of gastric digestion. Thus, the possible mechanisms leading to the incomplete dissolution of nHA under acidic conditions of the stomach are discussed subsequently

Expert Report of Kinley Larntz, PH.D.

While working in this matter, the author undertook the task of analyzing the statistical relationship between law school acceptance and ethnicity. In particular, focusing on the strength of the relationship between law school acceptance and being a member of certain ethnic groups, controlling for qualifications for admission such as undergraduate grade point average, Law School Admission Test score, and selection index, and for other factors such as residency in the State of Michigan, gender, and a measure of economic disadvantage, waiver of the fee for application

Comparative aquatic toxicity of a commercial naphthenic acid and processes for mitigating risks

Comparative toxicity data can inform predictions of relative risk, and can be used to implement strategies for altering exposures to mitigate risk. Naphthenic acids (NAs) are a complex group of carboxylic acids that naturally occur in petroleum sources and energy-derived process waters (e.g. refinery effluents and oil sands process affected waters). These compounds are relatively persistent in water and can be a source of toxicity to aquatic organisms. In the first experiment of this thesis, responses of sentinel aquatic organisms to 7-d exposures of commercial (Fluka) NAs were measured (in terms of acute toxicity) to discern relative sensitivities. In terms of sensitivities, fish\u3einvertebrates\u3eplant for exposures to Fluka NAs. Once toxicity was determined, two potential processes for altering exposures were investigated. In the second experiment, photocatalytic degradation of Fluka NAs was measured using fixed-film titanium dioxide (TiO2) irradiated with sunlight for 8 hours. Confirmation of changes in NA concentrations by photocatalytic degradation was accomplished analytically and with toxicity tests using sentinel vertebrate and invertebrate species. The half-life for Fluka NAs achieved by photocatalytic degradation was approximately 1 hour, with toxicity eliminated to both test species (Pimephales promelas and Daphnia magna) by the 5th hour of the sunlight exposure. In the third experiment, environmental conditions that can influence aerobic degradation for altering exposures of NAs were evaluated. Effects of nitrogen and phosphorus concentrations, dissolved oxygen concentrations, pH, and temperature on rates and extents of aerobic degradation of Fluka NAs were measured. Environmental conditions that positively influenced aerobic degradation rates of Fluka NAs included nutrients (C:N 10:1-500:1, C:P 100:1-5000:1), DO (4.76-8.43 mg/L), pH (6-8), and temperature (5-25ºC). At an initial Fluka NA concentration of 61 mg/L (±8), a removal rate of 11.7 mg/L day-1 was achieved (half-life approximately 2.5 days) in treatments with C:N and C:P molar ratios of 10:1 and 100:1, respectively (with other macro- and micronutrients supplied), DO \u3e8 mg/L, pH ≈8, and temperatures \u3e23ºC. Commercial NAs differ structurally from energy-derived NAs (e.g oil sands process affected waters), but environmental conditions systematically evaluated in this study are also expected to affect rates and extents of aerobic degradation of compositionally complex NAs. Ultimately, experiments conducted in this thesis can serve as a model approach for evaluating comparative toxicity of NAs, in terms of relative sensitivities of a taxonomic range of sentinel species, and using that information to implement effective strategies for mitigating risks in aquatic systems

The Water Is On Fire: Current Circuit Approaches to Fee-Shifting in Citizen-Suits Under the Clean Water Act and the Need for Clearer and More Uniform Standards

This Note will start by providing a short explanation of the origins of and congressional goals for the fee-shifting provision in the CWA [Clean Water Act]. It will then offer a brief summary of how Supreme Court precedent has both clarified and confused this issue. Then, it will dive into an examination of how the different circuits and their district courts have interpreted the CWA’s fee-shifting provision and how these interpretations have struggled with past Supreme Court decisions. Finally, this Note will explore the costs and benefits associated with these fee-shifting standards and offer a potential solution to this problem. Ultimately, this Note will argue that, in the absence of Supreme Court guidance, the circuits must adopt clearer, more uniform standards in order to fulfill congressional objectives, bolster the viability of CWA citizen-suits, and ensure that citizens do not pay the price for their public service. This abstract has been adapted from the author\u27s introduction

Imagining a Nation: Society, Regionalism, and National Identity in the Greek War of Independence

ABSTRACT: Modern Greece has held a marginal existence in the study of nationalism, and yet there is a wealth of information that it provides, which can broaden our understanding of nationalism and state-building, especially in the Balkans. The purpose of this article is to examine the various facets of Greek identity during the outbreak of the independence movement, and how identity shaped and affected the movement itself. This article argues that Greek identity was too multifarious to create a strongly defined national identity. Furthermore, this lack of national identity led to several years of civil war during the independence movement and the political strife that characterised the newly formed state. This article examines the weak sense of national identity and the complexities of the Greek War of Independence through identity within the various socioeconomic groups of the Greek-speaking lands, which have rarely been addressed

Exploring effective factors for improving instructors’ job satisfaction in vocational and traditional training institutes in Bhutan

This article aims to study the effective factors for improving instructors\u27 job satisfaction in vocational and traditional training institutes in Bhutan. The study was conducted using quantitative and qualitative methods. The population consisted of 75 instructors working in six technical training institutes and one traditional training institute in Bhutan. A descriptive survey was carried out with 75 instructors and interviews were conducted with 33 instructors. The career ladder, conducive learning environment, adequate tools and equipment were the main reasons leading to job satisfaction of the instructors. Institute management also leads to their job satisfaction. Their general opinions indicated that a clear and appropriate career ladder for instructors must be put in place, timely (four years) promotion should be awarded, the conducive working environment should be created, opportunities for training and retraining must be provided and acknowledging their contributions for achieving the common goals of the institute would enhance the instructors’ job satisfaction. Based on the findings, it is recommended to improve and develop the management practices and increase the level of job satisfaction of instructors by extending support from the Ministry of Labour and Human Resources. This should prevent job dissatisfaction and lead to a better-quality training system in Bhutan

PREVALENCE and BIOLOGICAL CONTROL of SALMONELLA CONTAMINATION in RENDERING PLANT ENVIRONMENTS and the FINISHED RENDERED MEALS

Although the rendering process serves as invaluable means for the disposal of inedible animal by-products, the finished products often harbor pathogenic and opportunistic microorganisms such as Salmonella and enterococci, respectively. The temperatures used during the rendering process far exceed the heat tolerance threshold of most bacterial species, so cross-contamination from the environment and/or from the incoming raw material is the proposed source of the contamination. Research has demonstrated that the raw material coming into the rendering facility is highly contaminated with pathogenic bacteria including Salmonella. While not in a rendering facility, studies have also demonstrated that bacteria such as Salmonella can persist on food processing equipment and be transferred into the product upon contact. The objectives of this study were to: 1) isolate and characterize Salmonella and enterococci from finished animal by-products, 2) produce and optimize a bacteriophage cocktail against Salmonella, 3) apply the bacteriophages to reduce Salmonella levels on environmental surfaces found in a rendering facility and in raw offal, and 4) use the bacteriophage as a feed additive to reduce or prevent Salmonella infection in mice. To determine the prevalence of Salmonella and enterococci, two hundred finished meals provided by various rendering facilities across the U.S. were analyzed. While the animal meals were shown to not be a suitable environment for bacterial growth (moisture content 1.9 to 11.5%), these products did contain enterococci and Salmonella. Enterococci were detected in 83% of the samples and accounted for up to 54% of the total bacterial count, which ranged from 1.7 to 6.8 log CFU/g. Characterization of the enterococci isolates revealed that only 3 isolates were resistant to vancomycin (32 µg/ml). PCR analysis revealed that none of these 3 VRE isolates were E. faecalis or E. faecium. In addition, no VRE isolates were of the VanA or VanB-type, which confer the highest levels of resistance to vancomycin. Salmonella (n = ? ) was isolated from 8.7% of the finished meal samples. There were 13 Salmonella serotypes identified among the isolates with 16 pulse field gel electrophoresis (PFGE) patterns. Thermal tolerance studies revealed that these Salmonella isolates had D-values of 9.27-9.99, 2.07-2.28, and 0.35-0.40 min at 55°C, 60°C, and 65°C, respectively. As a means to prevent or reduce Salmonella contamination, bacteriophages were isolated from raw chicken offal that would be used for rendering. Bacteriophages were isolated by using the Salmonella spp. isolated previously from the finished meals as the host bacteria. For further study, five of the isolated bacteriophages (n = ? ) were selected to produce a cocktail for bacteriophage treatment studies. The selection was based on which bacteriophage had the highest lytic activity against 5 pre-determined Salmonella serotypes, i.e., Enteritidis, Idikan, Johannesburg, Mbandaka, and Typhimurium. Prior to bacteriophage treatment studies, the optimal bacteriophage cocktail concentration was determined by multiplicity of infection (MOI) optimization. Initial results indicated that an actively growing culture is needed for lytic activity of bacteriophages. When using an actively growing Salmonella cocktail, the effectiveness of the bacteriophage cocktail was shown to increase by raising the MOI from 1 to 10, whereas MOI of 50 didn\u27t enhance the lytic activity further. MOI optimization also revealed that resistance strains of the Salmonella spp. are selected for quickly (12 h), but that the lytic activity of the bacteriophage treatment is easily extended through the addition of a different bacteriophage cocktail at the 12 h mark. The optimized bacteriophage cocktail was able to successfully reduce Salmonella levels on all tested environmental surfaces (HDPE plastic, cement, rubber, stainless steel). Treatment of Salmonella cells attached to the environmental surfaces resulted in a 2 log CFU/cm2 reduction at 40° and 30°C, and ca. 1 log CFU/cm2 reduction at 20°C on all surface materials. The presence of an organic layer on the surface had identical levels of reduction, indicating the organic material does not interfere with the bacteriophage\u27s lytic activity. Treatment of the single species biofilm resulted in ca. 1-2, 2-3, and 1 log CFU/cm2 reduction in S. Enteritidis H4717 populations on all surface materials at 20, 30, and 40°C, respectively, as compared with ca. 0.5, 1.5-2, and 0.5 log CFU/cm2 reduction in S. Enteritidis populations of the double species biofilm under the same experimental condition. In addition to the surface materials, the bacteriophage cocktail was shown to reduce Salmonella levels in raw chicken offal. When treating irradiated raw offal that was artificially contaminated with the Salmonella cocktail (105 CFU/g), the bacteriophage cocktail reduced Salmonella levels by ca. 2.0, 2.7, and 2.5 log CFU/g at 20°, 30°, and 40°C, respectively. The bacteriophage was also capable of reducing Salmonella levels to the same degree, i.e., ca. 2.0, 2.2, and 2.2 log CFU/g at 20°, 30°, and 40°C, respectively, in the non-sterile raw chicken offal at the presence of background microorganisms. The bacteriophage cocktail was also evaluated as a feed additive. The bacteriophage cocktail was lyophilized and added into the animal meals (blood, feather, and poultry), which were artificially contaminated with a five-strain Salmonella cocktail at initial level of ca. 105 CFU/g. A series of dehydration studies revealed that the addition of dehydrated bacteriophages to finished meals does not reduce the level of Salmonella present upon rehydration; however, there was an observable difference between those samples containing the bacteriophages and those that did not after 12 h of rehydration, with those containing the bacteriophages having lower levels of Salmonella (ca. 2 log CFU/g difference ??). Our results also revealed that the bacteriophage cocktail\u27s stability was reduced quickly when applied in a dehydrated form. The concentration of the dehydrated bacteriophage decreased by 1.5 log PFU/g within the rendered meal over a 4 week period at 30°C. However, the stability of the bacteriophage was maintained well when the bacteriophage was added to the animal feed in liquid form and stored under refrigeration conditions (4°C). Under these conditions, the bacteriophage cocktail\u27s concentration decreased by 0.23 log PFU/g over a 4 week period. The liquid bacteriophage was then supplemented into animal feed and given to mice during an animal trial. The mice that had been given a diet containing the bacteriophage for a period of a week prior to Salmonella inoculation were not infected as evidenced through fecal sampling. These mice shed no Salmonella in their feces over a 4 week period. Mice that had not been given the diet supplemented with the bacteriophage shed Salmonella in their feces for a period of 2.5 weeks. Histological analysis of the liver and intestine also indicated no observable signs of inflammation in those mice given the bacteriophages. In mice not receiving the bacteriophage treatment, venous dilation, cholangiohepatitis, and monocytes in the portal areas were observed in the liver. Our results indicate that the Salmonella contamination of finished rendered meals is likely the source of cross-contamination between the environment or the incoming raw material and the finished products. The risk for cross-contamination may be reduced through the use of bacteriophage treatment which was found to reduce ca. 99 to 99.9% of Salmonella levels on both the environmental surfaces and raw material. The bacteriophage cocktail was also found to have the potential to be used as a feed additive to reduce pathogen levels within an animal host when added to the feed in liquid form just prior to consumption