RESUMEN
The presence of the various protein crosslinks GOLD 2, MOLD 3, GODIC 4, MODIC 5, DODIC 6, and glucosepan 7 in foods has been established for the first time by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). In compounds 2 and 3 two lysine moieties, in 4-7 a lysine and an arginine side chain are joined by the crosslink. Unequivocal identification of 2-7 was achieved with independently synthesized reference material. The quantitative results for the investigated foodstuffs show MODIC 5 to be the most important Maillard crosslink. The concentrations of 5 and GODIC 4 are 5-10 fold higher than those of the corresponding imidazolium compounds 3 and 2, establishing 5 and 4 as the major food protein crosslinks derived from methylglyoxal and glyoxal, respectively. The maximum value of 151 mg MODIC 5/kg protein (equivalent to 0.42 mmol/kg protein) was found in a butter biscuit sample which also shows the highest overall Maillard crosslink content with 0.71 mmol 47/kg protein. These first quantitative results suggest that compounds 4-7 can be jointly responsible for protein polymerization in the course of food processing.
Asunto(s)
Arginina/química , Reactivos de Enlaces Cruzados/química , Lisina/análogos & derivados , Lisina/química , Reacción de Maillard , Arginina/análisis , Cromatografía Líquida de Alta Presión , Análisis de los Alimentos , Manipulación de Alimentos , Cromatografía de Gases y Espectrometría de Masas , Glioxal/química , Imidazoles/química , Lisina/análisisRESUMEN
Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates is largely unknown. Investigations are reported on how the cross-linking unit 2-ammonio-6-[2-[(4-ammonio-5-oxido-5-oxopentyl)amino]-6,7-dihydrox y-4,5,6,7,8,8a-hexahydroimidazo[4,5-b]azepin-4-yl] hexanoate (7), designated as glucosepan, can be identified and quantified from D-glucose-bovine serum albumin (BSA) incubations. Independent synthesis and unequivocal structural characterization are given for glucosepan 7. A protocol was established for its determination by LC-MS with electrospray ionization (ESI). BSA and D-glucose were incubated at 37 degrees C, pH 7.4 for eight weeks and the time-dependent formation of 7 was observed. Since glucosepan 7 is unstable under acid proteolytic conditions, BSA was cleaved enzymatically. The maximum value obtained from a solution containing 50 g/L BSA and 100 mM D-glucose after eight weeks incubation time corresponds to an arginine derivatization rate of 1.38 +/- 0.07 mmol 7/mol Arg (equivalent to 31.7 +/- 1.6 mmol 7/mol BSA). From these results, it seems justified to expect 7 to play an important role in the cross-linking of proteins in vivo as well as in foodstuffs. The structural similarity of glucosepan 7 and pentosidine 1 made it obvious to also look for an eventual parallelism in the respective formation pathways.
Asunto(s)
Arginina/química , Azepinas/química , Reactivos de Enlaces Cruzados/química , Glucosa/química , Lisina/química , Ornitina/análogos & derivados , Proteínas/química , Animales , Arginina/análogos & derivados , Bovinos , Lisina/análogos & derivados , Reacción de Maillard , Ornitina/química , Albúmina Sérica Bovina/metabolismoRESUMEN
We have developed a simple, fast, and efficient procedure to detect enteroviruses in water samples. Aliquots of water are subjected to two-step filtration, with the second filter containing a positively charged nylon membrane that holds back virus particles. Viruses thus adsorbed are directly lysed, and RNA is isolated by hybridization to specific oligonucleotides bound to magnetic beads. The solution used contains guanidine thiocyanate, which lyses virus particles, inactivates enzymes, e.g., RNases, allows mild hybridization conditions, and does not influence biotin-streptavidin interaction on magnetic beads. Detection and specific identification are accomplished by reverse transcription PCR of the highly conserved noncoding region at the 5' end of virus RNA combined with Southern hybridization. The system was tested with tap water artificially spiked with poliovirus vaccine and yielded a detection limit of 20 50% tissue culture infective doses per liter. We used the same procedure to investigate the water quality of surface water at public beaches by rivers and lakes. Of 40 samples tested, 7 were positive for enteroviruses. A comparison with enterobacterial contamination determined by PCR and classical microbiological methods in parallel showed that enteroviruses were found only in samples also positive for Escherichia coli. In conclusion, this procedure can easily be adapted to test large water samples and is simple enough to be used for routine determinations of water quality in terms of virus contamination.
Asunto(s)
Enterovirus/genética , Enterovirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Microbiología del Agua , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , ADN Viral/genética , ADN Viral/aislamiento & purificación , Infecciones por Enterovirus/prevención & control , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Estudios de Evaluación como Asunto , Agua Dulce , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Sensibilidad y Especificidad , Transcripción Genética , Abastecimiento de Agua/normasRESUMEN
A comparison was made of three approaches for the detection of Listeria monocytogenes in naturally contaminated soft cheese and semi-soft cheese after enrichment. Enrichment broths were tested by plating them onto different selective agars, by "Gen Probe" DNA hybridization and by the polymerase chain reaction (PCR). Based on two-step enrichment, all three approaches showed high specificities (90% or more) in detecting L. monocytogenes. In contrast, the sensitivity of the Gen-Probe test was low (33% or less), whereas high sensitivities were obtained with selective plating and PCR (83% or more). Based on one-step enrichment, specificities again were high for selective plating and PCR assay (100%), whereas for the Gen-Probe assay the specificity was lower (88% or more). The best sensitivities were observed with selective plating (67%) and PCR (75%). In terms of sensitivity, specificity and analysis time, PCR applied to a two-step enrichment was the most powerful assay for detecting L. monocytogenes in soft and semi-soft cheese.
Asunto(s)
Queso , Sondas de ADN/genética , Microbiología de Alimentos , Listeria monocytogenes/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Medios de Cultivo , Hibridación in Situ , Técnicas In Vitro , Listeria monocytogenes/genéticaRESUMEN
A previously described polymerase chain reaction (PCR) assay (B. Furrer, U. Candrian, C. Höfelein, and J. Lüthy, J. Appl. Bacteriol. 70:372-379, 1991) was used to analyze food for the presence of Listeria monocytogenes. Food samples were artificially contaminated to develop two procedures to detect the organism following enrichment steps. Procedure A was based on dilution of the enrichment broth followed by lysis of the bacteria and direct analysis of the lysate with PCR. With procedure A and artificially contaminated food samples, it was possible to detect fewer than 10 bacteria per 10 g of food. In procedure B, centrifugation was used to concentrate bacteria before lysis and PCR. With procedure A, 330 naturally contaminated food samples of several types were analyzed. Twenty samples were found to be positive for L. monocytogenes, which was in agreement with the classical culture technique. By using procedure B on a subset of 100 food samples, 14 were found to be positive by PCR whereas the classical culture method detected only 13. Analysis times, including enrichment steps, were 56 and 32 h with procedures A and B, respectively.