Alfalfa has long been recognized as an outstanding hay plant in term. of both yield and feeding value. It is high in digestible protein, minerals, and vitamins. Because of its long life it can be economically produced. It is of exceptional value in maintaining soil fertility by helping to control erosion, by improving the physical condition of the soil, and by accumulating large amount of nitrogen.

Riboflavin transport is mediated, in part, by riboflavin transporter proteins 2 and 3, encoded by SLC52A2 and SLC52A3, respectively. Biallelic mutations in SLC52A2 and SLC52A3 impair riboflavin transporter protein function and riboflavin transport, causing disruptions to mitochondrial metabolism which result in sensory and motor neurodegeneration and give rise to riboflavin transporter deficiency (RTD) in humans. RTD is a rare neurodegenerative disease characterised by respiratory compromise, muscle and limb weakness, and vision and hearing impairments. RTD patients are treated with high-dose riboflavin supplementation which is effective in over 70% of cases but can be ineffective due to rapid excretion of riboflavin when its plasma concentration exceeds 0.5 μM. To address the need for alternative or supplemental RTD treatment, this study generated morpholino-mediated knockdown and CRISPR/Cas9 models of RTD in zebrafish. An RTD-like phenotype is observed in these RTD models including hearing loss, decreased motor axon length, and impaired locomotor activity. The slc52a3 morphant phenotype was found to be specific via coinjection of slc52a3 morpholino/human SLC52A3 mRNA, which achieved effective rescue of the morphant phenotype, as well as slc52a3 morpholino/p53 morpholino coinjection, which maintains the slc52a3 morphant phenotype. In line with clinical findings, riboflavin supplementation resulted in some improvement of the morphant phenotype. Probenecid was selected as a candidate drug due to its inhibitory effect on OAT-3, which mediates riboflavin excretion. However, supplementing riboflavin treatment with probenecid provided no additional benefit to the slc52a3 knockdown model. Further development of CRISPR/Cas9-knockout lines of slc52a2 and slc52a3, as well continued therapeutic screening of riboflavin and probenecid and consideration of alternative therapeutics will provide more opportunities to uncover novel therapeutic strategies to improve RTD treatment.

The effects of supplementation of riboflavin along with Bacillus subtilis was investigated on growth performance and intestinal health of broilers under coccidial challenge. Treatments are 3 × 2 × 2 factorial arrangement, which includes 3 levels of riboflavin (0.75, 6.6 (recommended), and 20 ppm), with or without Bacillus subtilis, and with or without coccidial challenge. Coccidial challenge impaired the intestinal morphology and reduced body weight (BW), body weight gain (BWG), and feed intake and increased FCR between d 14-28. However, the cocci impact on birds was reduced along with age, as the effects on internal organs was reduced, which allowed birds to recover. However, coccidiosis reduced overall BW and BWG and increased abdominal fat pad weight and slight woody breast incidence. In conclusion, riboflavin at tested level was unable to enhance growth performance and intestinal health, but its inclusion altered birds response to coccidial challege other than feeding Bacillus subtlis alone.

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The primary goal of this research was to evaluate the effects of light on soymilk, including the oil component. Soybean oil with added chlorophyll a (0, 1, and 2μg/ml), a photosensitizer, was exposed to light (no light [control], broad-spectrum light) and narrow-band wavelengths (430nm, 450nm, and 660nm] for 4h. Chlorophyll completely degraded under broad-spectrum light and 430nm treatments; 64% degradation occurred at 660nm. Oil with chlorophyll addition resulted in significantly higher peroxide values and malondialdehyde concentrations with light exposure to broad spectrum and 430nm wavelengths. Light at 430 and 660 nm degraded chlorophyll and increased risk of oxidation in soybean oil. Soymilk contains low concentrations of chlorophyll, the photosensitizer riboflavin, as well as highly susceptible oxidizable substrates from the soy oil. Soymilk (1% fat from soybean oil) was packaged under a positive flow hood into 5 high density polyethylene (HDPE) packages and stored for 36 days at 4°C under fluorescent lighting (1122 lux ± 439 lux). Control packaging had no light protective additive (LPA; positive (foil-wrapped) and negative control) and the experimental packaging treatments had three levels of LPA (low, medium, high). Chemical and sensory analyses to measure oxidation changes were completed on the product at days, 1, 4, 8, 15, 22, 29, and 36. HDPE packages with high LPA protected the sensory quality of the product as well as the positive control (foil-wrapped) packages for a minimum of 15 days. High-LPA HDPE protected soymilk for 29 days from degradation of riboflavin and limited development of aldehyde end-products associated with photooxidation. Soymilk was treated with food grade TiO2 at levels of 0, 0.5, and 1.0% by weight. TiO2 significantly whitened the product as demonstrated by L* values. TiO2-treated soymilks resulted in significantly improved hedonic scores for appearance, smell, taste, mouthfeel, and aftertaste compared to control soymilk. However, in a second experiment, overall acceptability of TiO2-treated soymilk, at additions of 0.1%, 0.3%, and 0.5% TiO2, was not higher than control soy milk. / Ph. D.

Die Photophysik und Photochemie von Flavinen sind aufgrund ihrer biologischen Funktion, inbesondere von Flavoproteinen, von großen Interesse. Flavoproteine spielen eine große Rolle in einer Vielzahl von biologischen Prozessen, z.B. Biolumineszenz, Entfernung von Radikalen, die bei oxidativem Stress entstehen, Photosynthese und DNA-Reparatur. Die spektroskopischen Eigenschaften des Flavin-Cofaktors machen diesen zu einem natürlichen Reporter für Veränderungen innerhalb des aktiven Zentrums. Deshalb sind die Flavoproteine eine der am meisten untersuchten Enzymfamilien. Eine biologische Aktivität des Flavins führt über einen elektronisch angeregten Zustand, wo dann, abhängig von der Aminosäureumgebung, ein bestimmter Mechanismus zu einem biologischen Prozess führt (Photozyklus). Ein wichtiges Analysetool zum Verständnis des anfänglichen Photoanregungsschritts der Flavine sind die elektronische und die Schwingungsspektroskopie. In dieser Arbeit wurden die Prozesse von Riboflavin (RF) während und nach optischer Anregung mit theoretischen Mitteln beleuchtet. Dazu wurden quantenchemische Berechnungen für Schwingungsspektren (vibratorische) von Riboflavin, auch Laktoflavin oder Vitamin B2 genannt, dem Grundmolekül der Chromophore biologischer Blaulichtrezeptoren, in dessen elektronischem Grundzustand und dessen niedrigsten angeregten Zustand durchgeführt. Weiterhin wurden vibronische (vibratorische+elektronische) Absorptionsspektren und ein vibronisches Emissionsspektrum berechnet. Die so berechneten Schwingungs- und elektronischen Spektren sind in guter qualitativer wie quantitativer Übereinstimmung mit gemessenen Werten, und helfen so, die experimentellen Signale der Photoanregung von Flavinen zuzuweisen. Unmittelbar nach der Photoanregung wurde ein Verlust des Doppelbindungscharakters im polaren Bereich des Ringssystems beobachtet, was zu der vibronischen Feinstruktur im elektronischen Absorptions- und Emissionsspektrum führte. Hier zeigte sich zudem, dass neben den vibronischen Effekten auch die Lösungsmitteleffekte wichtig für das quantitative Verständnis der Photophysik der Flavine in Lösung sind. Um Details des optischen Anregungsprozesses als initialen, elementaren Schritt zur Signalweiterleitung zu entschlüsseln, wurden ultraschnelle (femtosekundenaufgelöste) Experimente durchgeführt, die die Photoaktivierung des Flavins untersuchen. Diese Arbeit soll zu einem weiteren Verständnis und der Interpretation dieser Experimente durch das Studium der Post-Anregungsschwingungsdynamik von Riboflavin und mikrosolvatisiertem Riboflavin beitragen. Dazu wurde eine 200 fs lange Molekulardynamik in angeregten Zuständen betrachtet. Durch die Analyse charakteristischer Atombewegungen und durch die Berechnungen zeitaufgelöster Emissionsspektren fand man heraus, dass nach der optischen Anregung Schwingungen im Ringssystem des Riboflavins einsetzen. Mit Hilfe dieser Berechnungen kann die Umverteilung der Energie im angeregten Zustand beobachtet werden. Neben den theoretischen Untersuchungen zu Riboflavin in der Gasphase und auch in Lösung wurde ein Modell für eine BLUF (Blue-Light Photoreceptor Using Flavin) Domäne, ein Flavin benutzender Photorezeptor, erstellt. Hierbei zeigt sich, dass man die in dieser Arbeit angewendeten Analysemethoden auch auf biologisch relevante Systeme anwenden kann. / The photophysics and photochemistry of flavins are due to their biological function, in particular of flavoproteins, of great interest. Flavoproteins play a major role in a variety of biological processes, eg Bioluminescence, removal of free radicals, resulting in oxidative stress, photosynthesis and DNA repair. The spectroscopic properties of the flavin cofactor make this a natural reporter for changes within the active site. Therefore flavoproteins are one of the most studied enzyme families. A biological activity of the flavin via an electronically excited state, which then performs a function of the amino acid environment, a specific mechanism to a biological process (photo cycle). An important analytical tool for the understanding of the initial step of the photoexcitation flavins are the electronic and vibrational spectroscopy. In this study, the processes of riboflavin (RF) during and after optical excitation illuminated by theoretical means. These quantum chemical calculations for the vibrational spectra (vibrational) of riboflavin, or vitamin B2 lactoflavin also been mentioned, the basic molecule of biological chromophores blue light receptors in the electronic ground state and the lowest excited state performed. Furthermore, vibronic (vibrational + electronic) absorption spectra and vibronic emission spectra were calculated. The calculated vibrational and electronic spectra are in good qualitative and quantitative agreement with measured values, and help to assign the experimental signals of the photo-excitation of flavins. Immediately after photoexcitation a loss of the double bond character was observed in the polar region of the ring system, leading to the vibronic fine structure in the electronic absorption and emission spectrum. It showed also that in addition to the vibronic effects, the solvent effects are important for a quantitative understanding of the photophysics of flavins in solution. To decipher details of the optical excitation process as initial, elementary step in signal transduction, ultrafast were performed (femtosecond-resolved) experiments that investigate the photoactivation of the flavin. This work will contribute to a further understanding and interpretation of these experiments by studying the post-excitation vibrational dynamics of riboflavin and riboflavin mikrosolvatisiertem. To a 200 fs long molecular dynamics in excited states was considered. By the analysis of characteristic atomic motions and by the calculations of time-resolved emission spectra, it was found that, after the optical excitation oscillations in the ring system of the riboflavin used. Using these calculations, the energy redistribution in the excited state can be observed. In addition to the theoretical studies of riboflavin in the gas phase and in solution, a model for a BLUF (Blue Light Photoreceptor Using flavin) domain, a flavin-use photoreceptor created. It is shown that one can apply the analytical methods employed in this work and to biologically relevant systems.

Mother's milk provides multiple benefits to the offspring. However, xenobiotics transferred into breast milk may pose a risk to the nursing infant. The breast cancer resistance protein (BCRP) actively transports xenobiotics into breast milk. BCRP also transports nutrients, like riboflavin, and together with recently identified riboflavin transporters (RFT), may provide a mechanism for riboflavin secretion into breast milk. Expression of RFT in the mammary gland remained unknown. Our objective was to characterize Bcrp and Rft mRNA expression in the mammary gland of FVB/N mice, and investigate a strategy to decrease excretion of BCRP-transported xenobiotics into the milk using riboflavin intervention. Rft and Bcrp mRNA were upregulated in the mammary gland of lactating mice. An intravenous riboflavin administration significantly reduced the levels of BCRP-transported cimetidine in milk. This study demonstrates the use of riboflavin to exploit the function of mammary BCRP in order to reduce xenobiotic secretion into breast milk.

Mother's milk provides multiple benefits to the offspring. However, xenobiotics transferred into breast milk may pose a risk to the nursing infant. The breast cancer resistance protein (BCRP) actively transports xenobiotics into breast milk. BCRP also transports nutrients, like riboflavin, and together with recently identified riboflavin transporters (RFT), may provide a mechanism for riboflavin secretion into breast milk. Expression of RFT in the mammary gland remained unknown. Our objective was to characterize Bcrp and Rft mRNA expression in the mammary gland of FVB/N mice, and investigate a strategy to decrease excretion of BCRP-transported xenobiotics into the milk using riboflavin intervention. Rft and Bcrp mRNA were upregulated in the mammary gland of lactating mice. An intravenous riboflavin administration significantly reduced the levels of BCRP-transported cimetidine in milk. This study demonstrates the use of riboflavin to exploit the function of mammary BCRP in order to reduce xenobiotic secretion into breast milk.