No description available.

Changes in histamine, putrescine, and cadaverine concentrations in fresh and stored bluefish (Pomatomus saltatrix) were determined using a new HPLC method. The HPLC method utilized a 5.0% (w/v) trichloroacetic acid (TCA) extraction, pre-column fluorescamine derivitization, and fluorescence detection. The derivatives were stable over 24 h. The 5% TCA extraction produced percent recoveries of 98.6%, 98.7, and 100.0% for histamine, cadaverine, and putrescine respectively. The HPLC process including extraction, derivatization, and HPLC analyses was conducted in less than 45 minutes. Fresh bluefish was found to contain between <1 ppm and 99 ppm histamine, and no cadaverine or putrescine. Fresh bluefish fillets were stored at 5, 10, and 15 degrees C until sensory rejection. Fresh bluefish fillets inoculated with Morganella morganii were also stored at the same conditions. Histamine levels as high as 2200 ppm were observed in the inoculated fish stored at 15 degrees C. Overall, histamine achieved higher levels in the bluefish pieces inoculated with Morganella morganii. Histamine was present in greater amounts than putrescine and cadaverine in the bluefish samples. Histamine levels at each temperature exceeded the 50 ppm advisory level established by the FDA before 100% sensory rejection. Putrescine levels increased at each temperature during storage. Cadaverine was present only in uninoculated bluefish stored at 15 degrees C. Consumer risk from histamine poisoning seems to be the greatest in those fish stored at 5 degrees C where acceptance levels were higher and histamine levels above 100 ppm were observed. The presence of histamine-forming bacteria in fish-processing facilities was studied. Environmental sampling techniques were conducted in the Hampton Roads area of Virginia in fish-processing facilities that regularly handle scombroid fish or other fish which are known to accumulate histamine levels greater than 50 ppm. Surfaces that come into contact with the fish were swabbed and the histamine-forming bacteria from these areas were identified. One isolate each of Klebsiella ozaenae and Vibrio alginolyticus, and two isolates of Aeromonas sp. were found in the processing facilities. The study concluded that histamine-forming bacteria do not make up a large part of the microflora associated with fish-processing facilities. Fishing vessels were also sampled and no histamine-forming bacteria were identified. / Master of Science

In comparison to other Pseudomonas, P. aeruginosa grows poorly in L-lysine as a sole source of nutrient while fast growth mutants can be obtained. The proposed catabolic pathway involves lysine decarboxylation to cadaverine and its subsequent degradation through g-glutamylation pathway to d-aminovalerate and glutarate. The lysine decarboxylase A (ldcA) gene, previously identified as a member of the ArgR regulon of L-arginine metabolism, was found essential for L-lysine catabolism. The ldcA gene encodes a decarboxylase which takes L-lysine but not L-arginine as substrate. Contrarily, the ldcA expression was inducible by L-arginine but not by L-lysine. This peculiar arginine control on lysine utilization was also noted from uptake experiments. The lack of lysine-responsive control on lysine catabolism and its tight connection to arginine regulatory network provided an explanation of lysine as poor nutrient for P. aeruginosa. Catabolism of cadaverine, a product from lysine decarboxylation, was investigated and compared to that of putrescine, another diamine of similar biochemical properties that is derived from arginine and ornithine. While the g-glutamylation pathway was first reported in E. coli for putrescine utilization, an expanded version of this pathway was found in P. aeruginosa with redundant enzymes for polyamine degradation. The PauR protein was identified as a transcriptional repressor of genes for the catabolism of putrescine and cadaverine, as well as their corresponding downstream metabolites, g-aminobutyrate (GABA) and d-aminovalerate (AMV). PauR shows distinct dimer configuration after glutaraldehyde crosslinkage, and possible conformational changes could be triggered by the presence of putrescine and cadaverine, but not GABA. A newly identified ABC transport system, encoded by the agtABCD operon, was found important for the uptake of GABA and AMV; and expression of which is controlled by the AgtSR two-component system. The CbrAB two-component system was proposed to regulate the catabolite repression control protein Crc through a small RNA CrcZ. A consensus CbrB recognition sequence was proposed based on the conserved palindromic nucleotide sequence in the upstream activating sequence of the crcZ promoter. Genetic studies indicated utilization of arginine, lysine and diamines (but not histidine, GABA and AMV) might be under CbrAB regulation through the CbrAB/CrcZ/Crc system in P. aeruginosa.

Age-related macular degeneration (AMD) is the leading cause of blindness in the United States of America. The pyridinium bis-retinoid A2-ethanolamine (A2E) has been implicated to play a role in AMD. We have observed novel pyridinium bis-retinoids through melanolipofuscin and human RPE extractions that may also play a role in the pathology of AMD. We have begun the construction of an amino-retinoid library in order to identify these ocular compounds. The compounds from the amino-retinoid library are also used in a targeted and triggered drug delivery system for treating cancer. Folic acid is coupled with the amino-retinoids to specifically target cancer cells. The first two amino-retinoids to be synthesized and characterized were A2-dopamine (A2D) and A2-cadaverine (A2C). Both pyridinium bis-retinoids were shown to generate cytotoxic oxidation products similar to A2E. Successful coupling of folic acid to A2C was achieved to form the folic acid-A2-cadaverine (FA-A2C) product. Preliminary irradiation results suggest that the FA-A2C product may be more photoreactive than initially anticipated. This could mean less drug and light exposure required to induce apoptosis and could eventually lead to a less invasive and toxic cancer treatment.

Biokatalys medför ett alternativt tillvägagångsätt för att kunna utforska och utveckla kemiskt syntetiserade vägar för produktionen av eftertraktade kemikalier, där hållbarhet och miljön tas till beaktan. I denna studie undersöktes potentialen av en enzymatisk kaskad för produktion av 5-hydroximetylfurfurylamin (HMFA). HMFA är en förening med tillämpningar inom flera industrier som till exempel jordbruks- och läkemedelsindustrin. Den enzymatiska kaskaden består av två reaktioner, varav den första involverar dekarboxylering av lysin med användning av lysindekarboxylas för att producera en så kallad ”smart amindonator” kadaverin. Den andra reaktionen i kaskaden består utav ett transaminas från Silicibacter pomeroyi (SpTA) som konverterarar 5-hydroximetylfurfuryl (HMF) till HMFA med hjälp av det framkallade kadaverinet från den första reaktionen i kaskaden. En enzymatisk kaskad tillåter mildare reaktionsbetingelser, mindre avfall och energisnål användning som därmed minskar miljöpåverkan samtidigt som det beaktar några av dem 12 principerna av grön kemi. Det uppstod utmaningar som hindrade slutförandet av den enzymatiska kaskaden, men trots detta erhölls värdefulla insikter. Denna studie belyser de invecklade reaktionsmekanismerna och några av de svårigheterna med immobilisering av enzym på EziG bärare. Trots att den avsedda kaskaden inte slutfördes, gav lärdomarna nya perspektiv samt potentiella områden att fortsätta undersöka för framtida framsteg inom biokatalys. / Biocatalysis is a promising alternative to chemical synthesis routes for high value chemicals which considers the sustainability and environmental aspect. In this study the feasibility of utilizing an enzymatic cascade for the production of 5-hydroxymethylfurfurylamine (HMFA) was explored. HMFA is a compound with diverse applications in industries such as agriculture and pharmaceuticals. The cascade consists of two main reactions, the first of which involves the decarboxylation of lysine using a lysine decarboxylase to produce cadaverine. The cadaverine produced will then be utilized as an amine donor in the second reaction, which involves the use of a transaminase derived from Silicibacter pomeroyi (SpTA) together with 5-hydroxymethylfurfural (HMF). This cascade considers the principals of green chemistry such as milder reaction conditions and less waste, hence aiming to reduce the environmental impact. Although there were challenges preventing the completion of the enzymatic cascade, valuable insights were gained. The contribution of this study sheds light on the intricate reaction mechanisms and some of the key difficulties with enzyme immobilisation. While the intended cascade was not finalized, the lessons learned will provide for new perspectives and potential future advancements in biocatalysis.

The aim of this thesis was to determine the content of biogenic amines (BA)and polyamines (PA)in selected types of cheese and the influence of storage time on the content of biogenic amines.

No description available.

La détection et l'identification de bactéries pathogènes revêt une grande importance dans de nombreux domaines tels que la santé et l’industrie agroalimentaire. Dans ce contexte, les travaux de thèse s’intéressent à détection non invasive de Salmonella via la fraction volatile de son métabolome dont les métabolites volatils caractéristiques sont le sulfure d’hydrogène et la cadavérine. Ils illustrent également le concept de substrats osmogènes libérant des mCOV exogènes sous l’action d’enzyme spécifique d’Escherichia coli. Un premier capteur colorimétrique capable de distinguer le sulfure d’hydrogène du méthanethiol a été préparé. Il s’agit d’une matrice de silicate nanoporeuse dopée avec les réactifs N,N-diméthyl-p-phénylènediamine et ions Fe3+. Une bonne stabilité de l’intermédiaire réactionnel issu de ces réactifs, la quinonediimine (QD), est obtenue pour une forte concentration d’acide chlorhydrique. La réaction entre QD et 1000 ppm de sulfure d’hydrogène et de méthanethiol entraîne l’apparition respective d’une coloration verte et rouge-marron du capteur. Le capteur fluorimétrique de cadavérine, basé sur la formation d’un complexe fluorescent entre le Naphthol AS-BI déméthylé (ArOH) et la cadavérine, permet de détecter 250 ppb de cadavérine. La preuve de concept de substrats osmogènes a été illustrée avec la détection de p-nitrophénol (pNP) et de β-naphthylamine (β-NA) libérés en présence d’enzymes de E. coli, β-D-glucuronidase et L-alanine- β-naphthylamidase. Les capteurs nanoporeux produits, de taille de pores contrôlée, peuvent détecter 100 ppm de pNP, composé coloré (jaune) et 100 ppm de β-NA, composé fluorescent, ou encore 100 ppm de β-NA par dérivation chimique de ce dernier avec le diméthyl-p-aminocinnamaldéhyde (formation d’un produit rouge). En milieu biologique, l’eau est un interférent majeur. / Microbial contamination of food and biological samples is a big issue in the industry as much as in the medical field. In that context, the present thesis brings innovative solutions. A first explored way is the identification of Salmonella by detecting and measuring the specific metabolomics volatile organic compounds (mVOC) released, H2S and cadaverine. Another new concept is the use of osmogenic substrates able to release mVOC under the action of specific enzyme of Escherichia coli.A first colorimetric sensor able to discriminate H2S from CH3SH was produced, using a nanoporous silicate matrix doped with N,N-dimethyl-p-phenylenediamine and Fe3+ ions. A very acidic medium is needed to stabilize the “key” intermediate of the reaction, the quinonediimine species (QD), which gives with H2S and CH3SH a green and red-brown product, respectively. The fluorimetric sensor of cadaverine is based on the formation of a fluorescent complex between AS-BI demethylated Naphthol and cadaverine and can detect 250 ppb of cadaverine. A proof of concept of osmogenic substrates is given with the detection of p-nitrophenol (pNP) et de β-naphthylamine (β-NA) released under the action of Escherichia coli enzymes, β-D-glucuronidase et L-alanine- β-naphthylamidase. Various nanoporous sensors are produced with tailored pore size, which can detect 100 ppm of the yellow pNP, 100 ppm of the fluorescent β-NA and 100 ppm of the red product issued from the derivation of β-NA with dimethyl-p-aminocinnamaldehyde. In biological media, water remains the major interfering agent.

The loss of efficiency and performance of bioprocesses on scale-up is well known, but not fully understood. This work addresses this problem, by studying the effect of some fermentation gradients (pH, glucose and oxygen) at a larger scale in a bench-scale two compartment reactor (PFR + STR) using the cadaverine-producing recombinant bacterium, Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT. The initial scale down strategy increased the magnitude of these gradients by only increasing the mean cell residence time in the plug flow reactor (τ_PFR). The cell growth and product related rate constants were compared as the τ_PFR was increased; differences were significant in some cases, but only up to 2 min residence time. For example, losses in cadaverine productivity when compared to the control fed-batch fermentation on average for the τ_PFR of 1 min, 2 min and 5 min were 25 %, 42 % and 46 % respectively. This indicated that the increasing the τ_PFR alone does not necessarily increase the magnitude of fermentation gradients. The new scale-down strategy developed here, increased the magnitude of fermentation gradients by not only increasing the τ_PFR, but also considering the mean frequency at which the bacterial cells entered the PFR section (f_m). The f_m was kept constant by reducing the broth volume in the STR. Hence, the bacterial cells also spent shorter times in the well mixed STR, as the τ_PFR was increased (hypothesised as giving the bacterial cells less time to recover the non-ideal PFR section of the SDR). On adoption of this strategy cadaverine productivity decreases for the τ_PFR of 1 min, 2 min and 5 min were 25 %, 32 % and 53 % respectively. Thus, highlighting that loss in performance is most likely to occur as the magnitude of heterogeneity within the fermentation environment increases. However, Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT did show some resilience in its biomass productivity. It was only marginally affected in the harshest of conditions simulated here.

The thesis deals with the use and effectiveness of some less common methods of conservation of fish meat. The formation of biogenic amines in meat is observed in connection with the non-traditional preservative methods. Amines can serve as indicators of protein degradation. The quality of fish was considered in connection with the increasing content of selected biogenic amines (putrescine, cadaverine, spermidine, spermine, 2-fenylathylamine, histamine, tyramine and tryptamine). Ultra performance liquid chromatography (UPLC) was used as the method for determination of biogenic amines. Amines were derivatized by dansylchloride before their UPLC separation. The fish flesh was vacuum-packed. Samples were stored for several weeks in a thermostat at the selected storage temperature after the application of selected preservative technique. Beta-irradiation and high hydrostatic pressure were used for the preservation of fish flesh. Control samples were not exposed to the â-irradiation and high pressure. The organoleptic properties were studied for all samples (smell/odor, insight and shape). Beta-irradiation was applied to fish meat of common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). Both these species of freshwater fish are economically significant. Carp and trout are the species being mostly consumed in the Czech Republic. Fish meat is considered to be provided the flesh is fresh. A testing series of samples was created with common carp to determine the appropriate dose of â-irradiation. The maximum permissible dose of irradiation for fish meat is 3 kGy. Fish samples were exposed this dose in the first experiment. The dose of irradiation was reduced in following experiments based on the experience from the initial experiment. The doses of 0.25, 0.5, 0.75, 1.0 and 2.0 kGy were applied to rainbow trout. The value of 0.75 kGy of â-irradiation or higher (1.0, 2.0 and 3.0 kGy) prolonged the shelf life of fish meat, which was stored for three months (98 days). Prolonging of the shelf life of fish meat to approximately 98 days at 3.5 °C is redundant from technical point of view. For that reason lower doses 0.25, 0.5 and 0.75 kGy were tested in more detail in the repeated experiment with carp meat. Lower doses of â-irradiation are considered to be more acceptable and-at the same time-sufficiently effective for delaying the beginning of degradation processes. 6 High hydrostatic pressure was applied to meat of common carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss) and pike (Exos lucius). Pike is another very popular kind of freshwater fish. Pike flesh is very tasty, but in spite of this, pike is not so much popular among consumers compared to carp and trout. The cause is its high price. Samples of pike were stored at standard temperature 3.5 °C and also at higher temperature 12 °C (unlike experiments with â-irradiation). Lower temperature of storage (3.5 °C) followed the conditions of storing of fish meat in industrial refrigeration facilities and households. The high pressure might not be sufficient for preservation at higher storage temperatures. This assumption was based on available information. Samples were treated by high pressure and stored at both 3.5 °C and 12 °C to verify this assumption. Higher temperature simulated either failure of refrigeration equipment or unsuitable store temperature of meat. In all species selected freshwater fish two levels of high pressure were applied ? 300 and 500 MPa. Both levels had significantly reduced the formation of biogenic amines, especially in samples stored at 3.5 °C. At this temperature, the effect of 300 and 500 MPa delayed start of degradation processes in fish meat by 3?4 weeks. At 12 °C and 500 MPa, high pressure extended the sustainability of meat by no more than one week. 500 MPa is effective treatment at the lower temperature of 3.5 °C. High pressure is not reliable preservative techniques at higher temperature.