Detection strategies for bioassays based on liquid chromatography, fluorescence spectroscopy and mass spectrometry

New detection strategies for bioassays based on liquid chromatography,\ud fluorescence spectroscopy and mass spectrometry were developed and are\ud presented within this thesis

The microbiological quality of raw and sour milk from local markets and smallholder dairy farms and associated potential risk for public health in The Gambia

Deckblatt-Impressum persönlicher Dank Inhaltsverzeichnis Tabellenverzeichnis Abkürzungsverzeichnis Einleitung Literaturübersicht Material und Methoden Ergebnisse Diskussion Schlussfolgerungen Zusammenfassung Summary Literaturverzeichnis Anhang DanksagungMilch wird als wesentlicher Lieferant von Energie, Proteinen und Kalzium angesehen, insbesondere für Kinder in Entwicklungsländern, die wenige Alternativen für diese Nährstoffe haben. Milch ist aber auch, vor allem in tropischen Ländern, ideal für das Wachstum von pathogenen Bakterien und Verderbniskeimen und kann für die Übertragungvon bakteriellen, viralen und parasitären Krankheiten verantwortlich sein. Pathogene Organismen in Milch können von der Kuh stammen aber auch vom Menschen und der Umgebung. In Gambia, wie in den anderen Ländern West-Afrikas, werden Kühe von Hand gemolken, nachdem das Kalb den Milchfluß durch kurzes Säugen stimuliert hat. Wasser ist an den Melkplätzen im allgemeinen nicht vorhanden und weder die Hände des Melkersnoch das Euter werden vor dem Melken gereinigt. Utensilien wie Melkeimer und Sammelbehälter werden nur unzureichend mit kaltem Wasser ausgespült. Mangelnde Kühlmöglichkeiten und lange Transportwege zu Milchhändlern, offenen Märkten und Konsumenten bieten Bakterien gute Bedingungen zur Vermehrung. Ziel dieser Arbeit war die Untersuchung des hygienischen Status der Milch, die in Gambia produziert und vermarktet wird und der Vergleich mit Grenzwerten, die für die Europäische Union oder Kenia als afrikanisches Vergleichsland gelten. Anhand der gewonnenen Information soll abgeschätzt werden, ob die auf Märkten angebotene Milch gesundheitsschädigend für Konsumenten sein kann. Dies ist besonders bedeutend in Ländern wie Gambia, in denen die öffentliche Gesundheitsversorgung unzureichend und ein Bewußtsein für lebensmittelbedingte Krankheiten kaum vorhanden ist. Für die Untersuchung wurden vier Märkte aus unterschiedlichen Verwaltungseinheiten Gambias ausgewählt. Die Milchverkäufer auf den jeweiligen Märkten wurden mit Hilfe von strukturierten Fragebögen interviewt; aus den Informationen wurde versucht, dieVermarktungskette von Milch zurückzuverfolgen und beliefernde Zwischenhändler und Produzenten zu identifizieren. Diese Vermarktungsketten bildeten die Grundlage für den Beprobungsplan. Dementsprechend wurden Milchproben auf unterschiedlichen Ebenen der Produktions- und Vermarktungskette genommen, angefangen mit Proben der Herdenmilchauf der Ebene der Produzenten über Proben der Sammelmilch auf der Ebene der Zwischenhändler bis zu den Endprodukten, die auf den Märkten den Konsumentenangeboten wurden. Roh- und Sauermilchproben wurden mikrobiologisch auf aerobe Gesamtkeimzahl, coliforme Bakterien, E. coli, Koagulase-positive Staphylokokken, Salmonella spp., Listeriaspp., H2S-reduzierende Clostridien und B. cereus untersucht. Die Ergebnisse wurden miteuropäischen und kenianischen Grenzwerten verglichen, um anhand der Standards für beide Regionen ein Gesundheitsrisiko für Verbraucher abzuschätzen. Es wurden 236 Rohmilch- und 142 Sauermilchproben untersucht. Die aerobe Gesamtkeimzahl lag bei 90,9% der Rohmilchproben mit über 2x10(hoch 6) KbE/ml außerhalb des Akzeptanzbereiches kenianischer Normen. Auch der Gehalt coliformer Bakterien lag bei 64% der Rohmilch- und 55% der Sauermilchproben über dem kenianischen Grenzwert von 5x10(hoch 4) KbE/ml. Keimzahlen über 1x10(hoch 5) KbE/ml E. coli wurden bei 22,6% der Rohmilch-und 23,7% der Sauermilchproben gefunden. 25% der Rohmilchproben enthielten mehr als 2x10(hoch 3) KbE/ml Koagulase-positive Staphylokokken und lagen damit über demeuropäischen Standard für Milch für die Herstellung von Milchprodukten. Listerien und Salmonellen konnten nur in Einzelfällen aus den Proben isoliert werden, Sporenbildner wie H2S- reduzierende Clostridien und Bacillus cereus wurden dagegen häufig nachgewiesen. 22,3% der Rohmilch- und 14,4% der Sauermilchproben enthielten H2S-reduzierendeClostridien und in 17% respektive 12,7% waren B. cereus auffindbar. Die mangelhafte hygienische Qualität der Milch ist zum einen auf die schlechten hygienischen Verhältnisse beim Melken zurückzuführen und zum anderen auf die ungenügende Reinigung der Melkutensilien und Milchgefäße. In die Milch gelangte Keime finden gute Bedingungen zur Vermehrung, da die Milch nicht gekühlt wird und die Wege zum Verbraucher meist lang sind. Die Ergebnisse dieser Arbeit geben Gründe zu der Annahme, dass der Verzehr von sowohl roher als auch von fermentierter Milch in Gambia Krankheitssymptome auslösen kann. Rückschlüsse auf die tatsächlichen Gesundheitsrisiken durch den Verzehr von lokaler Milch zu ziehen bleibt dennoch schwierig. Fehlende Labordiagnosen bei menschlichen Erkrankungen mit gastrointestinalen Symptomen, das Fehlen von Systemen zur Rückverfolgung von kontaminierten Lebensmitteln und das fehlende Bewußtsein der Bevökerung für Gesundheitsrisiken durch Lebensmittel im Allgemeinen lassen nur Vermutungen über das tatsächliche Risiko zu.Milk is considered as being an attractive source of energy, proteins and calcium especiallyfor children in developing countries, who have only few alternatives for these nutrients.But milk is equally suitable for the growth of spoilage and pathogenic bacteria and can bethe vehicle for the transmission of bacterial, viral and parasitic diseases. Pathogens in milk can derive from the cow but also from the milker or the environment. In The Gambia, as in other West-african countries, cows are milked by hand, after the stimulation of milk let-down by a suckling calf. Water is usually not available at the milking place and neither themilker s hands nor the udder is cleaned before milking. Milking equipment and containers are inadequately rinsed with cold water. The lack of cooling facilities and long distances between producers, traders and consumers provide optimal conditions for the growth of bacteria. The objective of this study was the identification and quantification of bacterial contaminants and pathogenic agents in milk at producer s, trader s and vendor s level and the comparison with hygiene standards of the European Union and Kenya. Results of this investigation were used to evaluate the potential risk for consumers in The Gambia. This is particularly important in countries such as The Gambia, where the public healthinfrastructure is insufficient and the public awareness for health risks related to food is more or less inexistant. Four local markets in different Divisions were selected and milk vendors were interviewed using structured questionnaires. With the information acquired through the interviews, existing milk marketing chains were identified, which served as a basis for the sampling of milk along the marketing ways. Raw and fermented milk samples were tested for total bacterial count, coliform bacteria, E.coli, coagulase-positive Staphylococci, Salmonella spp., Bacillus cereus, Listeria spp.and H2S- reducing Clostridia. The results were compared with European and Kenyanhygiene standards to estimate the potential risk for consumers of milk in The Gambia. 236 raw and 142 fermented milk samples were investigated. The total bacteria count of 90.9%of raw milk samples was above Kenyan standard with a highest acceptable count of 2x10(hoch6) cfu/ml. 64% of raw milk and 55% of fermented milk samples contained more than 5x104cfu/ml, which is the acceptance limit in Kenya. E.coli counts above 1x104 cfu/ml were found in 22.6% of raw milk and 23.7% of fermented milk. 25% of raw milk samplesexceeded the European acceptance limit of 2x103 cfu/ml coagulase-positive Staphylococci.Listeria spp. and Salmonella spp. were only isolated in a few samples. Sporeforming bacteria such as Bacillus cereus and H2S- reducing Clostridia were more frequentlyisolated. 22.3% of raw milk and 14.4% of fermented milk contained H2S- reducing Clostridia. Bacillus cereus could be found in 17% of raw milk and in 12.7% of fermented milk. The poor hygienic quality of milk is mainly caused by the poor hygienic conditions around milking and to the inadequate cleaning of milking equipment and containers. Bacterial contaminants in milk find good conditions for growth as milk is not cooled and usually transported over long distances to traders and consumers. Results of this study lead to the assumption that the consumption of both raw and fermented milk in The Gambia poses a public health hazard. However, poor public health infrastructure, lack of laboratory confirmed cases and the low public awareness for food-borne diseases in general make it difficult to establish a direct relationship between the high contamination of milk and the health risk for consumers

A preferred vision for administering secondary schools : a reflective essay

The chief purpose of this paper is to discuss the components of six leadership styles: autocratic, transactional, transformational, situational, expert thinking, and value-added. Throughout this paper, my objective is to identify the six styles and compare how each can be used in school administration. Finally, I will discuss my growth in school leadership along with plans for future improvements

Destructive Water-Borne Pressure Waves

Many energetic sources (blasting, pile-driving, seismic exploration, …) near or within a water body may produce destructive, water-borne pressure waves. Pressure waves of sufficient amplitude can impact water-side structures and aquatic fauna. Such energetic sources produce pressure waves and cavitation that can cause concrete to spall, deform metal sheets that are near to the source, kill aquatic organisms, and/or damage hearing of marine mammals at large distances. Any program (energetic systems) that causes pressure waves within the water layer may be conducted in a manner that reduces the pressure waves’ severity. The methodology to reduce pressure waves does not need to reduce the efficiency of the energetic source. Basic physical laws govern the transmission and attenuation of the pressure waves. Exploration of the site’s geology and assessment of potential adverse impacts should be conducted before the specifications for the project are written. Reduction of the pressure waves without other added mitigation differs for each class of sources. Additional mitigation varies by source class, the site’s geology, the water depth at the source and at the protected zone, potential adverse impacts from the pressure waves, needed pressure-wave reduction, and the range of azimuths from the source region requiring protection

A dual fluorophore system for simultaneous bioassays

A detection scheme for the simultaneous evaluation of two bioassays based on fluorescence spectroscopy is presented. For the determination of hydrogen peroxide-generating enzymes or peroxidases, the non-fluorescent 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH) is converted to the strongly fluorescent 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole (MNBDA). Phosphatases are detected based on the cleavage of the non-fluorescent 5-fluorosalicyl phosphate (5-FSAP) under formation of the fluorescent 5-fluorosalicylic acid (5-FSA). While excitation of the fluorophores may be carried out at the same wavelength, their emission spectra differ significantly. This allows the read-out of both assays using commercially available microplate readers without additional chemometric tools. Compared with individual assays, limits of detection are similar, and linearity of the calibration functions for both enzymes is observed over 2–3 concentration decades starting at the limit of quantification. The simultaneous determination of glucose oxidase and acid phosphatase in honey is presented as example for the application of the detection scheme

Fluorescence and Mass Spectrometric Detection Schemes for Simultaneous Enzymatic Conversions: Method Development and Comparison

Fluorescence and mass spectrometric detection schemes are developed and compared for the simultaneous activity determination of two enzymes in solution. As model system, the following reactions are used: The alkaline phosphatase catalyzed reaction with 5-fluorosalicyl phosphate yields the fluorescent 5-fluorosalicylic acid, whereas microperoxidase 11 reacts with 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole and H2O2 to the strongly fluorescent 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole. As the emission spectra of the fluorescent products as well as the molecular masses of substrates and products do not interfere with each other, is it possible to determine both reactions in parallel with both detection schemes. The measurements resulted in the same limits of detection, limits of quantification and linear ranges of the single/simultaneous enzyme determination for fluorescence and MS detection. While the relative standard deviations were significantly lower in case of fluorescence detection (1.4–3.2%) than in mass spectrometry (5.7–10.1%), the latter proved to be the more versatile approach for multianalyte determination

Back to Nature: Native Flora for Wildlife and People. The Lincoln University Native Plants Program

Presentation made at Latinos in the Heartland (12th : 2014 : St. Louis, Mo.) and published in the annual conference proceedingThe overall objective of the Lincoln University Cooperative Extension (LUCE) Sprouts and Roots Program (SRP) is to improve the well-being of minorities and other underserved communities through gardening and to promote intergenerational activities in Jefferson City and elsewhere in Missouri. To meet this objective, training is offered on nutrition, wellness, and gardening. The effects of gardening and healthy habits on the physical and psychological health of seniors and youth are being evaluated in an ongoing research study. Recruitment was done in schools, senior centers, Boys and Girls clubs, churches, and other locations with the assistance of the LUCE Paula Carter Center on Minority Health and Aging. Flyers, emails, calls, and personal visits were used to contact potential participants. Children and seniors attended indoor and outdoor classes in spring and fall, and pretests and posttests were provided to determine their change in knowledge of particular topics. Participants practiced their new knowledge to grow their own food at the community garden located on campus. Sixteen adult seniors and 16 children participated in 2012. The farmers market, also located on campus and adjacent to the garden, is offering the opportunity to SRP participants and area farmers to sell produce, baked goods, and other value-added products. An average of 14 vendors, and a total of 43, attended the market in 2012 where fresh or value-added products were sold on Thursdays and Saturdays during the growing season. Some vendors continued selling their products during the winter months. More than 4,000 people of different ethnicities, ages, and genders attended the market on Saturdays in 2012, compared to 1,305 in 2011. The SRP is providing communities the opportunity to develop their own value-added products by facilitating their access to a commercial kitchen recently opened to the public, located next to the market and community garden