This paper reports the potential of synchronous front-face fluorescence spectroscopy in the characterization at the molecular level of milk changes during mild heating from 4 to 50 degrees C and acidification in the pH range of 6.8 to 5.1. Synchronous fluorescence spectra were collected in the 250-550 nm excitation wavelength range using offsets of 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, and 240 nm between excitation and emission monochromators. The potential of parallel factor (PARAFAC) analysis in the decomposition of the whole synchronous fluorescence data set into the contribution of each of the fluorescent compounds present in milk has been investigated for heating and acidification data sets. Models were fitted from 1 to 7 components. Considering the core consistency values, PARAFAC models with three components have been considered. The first three components explained 94.43% and 94.13% of the total variance for heating and acidification data sets, respectively. The loading profiles of the first and second components derived from PARAFAC analysis performed on heating and acidification data sets corresponded quite well with the characteristics of tryptophan and vitamin A fluorescence spectra, respectively. The third component corresponded to the riboflavin fluorescence spectrum. Considering the heating experiment, the profile of the concentration mode for the second component showed large variations according to the temperature, which were assigned to the melting of triglycerides between 4 and 50 degrees C. For the acidification experiment, drastic changes in the concentration modes of the three components were observed for pH below 5.6, in agreement with structural changes in casein micelles.
Fluorescence spectroscopy is an emerging tool for the analysis of biomolecules from complex matrices. We explored the potentialities of the method for the pseudomonad taxonomic purpose at the genus and species level. Emission spectra of three intrinsic fluorophores (namely, NADH, tryptophan, and the complex of aromatic amino acids and nucleic acid) were collected from whole bacterial cells. Their comparisons were performed through principal component analysis and factorial discriminant analysis. Reference strains from the Xanthomonas, Stenotrophomonas, Burkholderia, and Pseudomonas genera were well separated, with sensitivity and selectivity higher than 90%. At the species level, P. lundensis, P. taetrolens, P. fragi, P. chlororaphis, and P. stutzeri were also well separated, in a distant group, from P. putida, P. pseudoalcaligenes, and P. fluorescens. These results are in agreement with the generally admitted rRNA and DNA bacterial homology grouping but they also provide additional information about strain relatedness. In the case of environmental isolates, the method allows good discrimination, even for strains for which ambiguity still remained after PCR and API 20NE identification. Rapid, easy to perform, and low cost, fluorescence spectroscopy provides substantial information on cell components. Statistical analysis of collected data allows in-depth comparison of strains. Our results strongly support the view that fluorescence spectroscopy fingerprinting can be used as a powerful tool in a polyphasic approach to pseudomonad taxonomy.
The structure and rheology characteristics of Comté (hard cheese) and Raclette (semihard cheese) cheeses as a function of temperature were investigated using dynamic testing rheology and mid-infrared and synchronous front-face fluorescence spectroscopies. The storage modulus (G′), the loss modulus (G″), and the complex viscosity (η*) decreased while strain and phase angle (tan δ) increased as the temperature increased from 20 to 80°C. SF (250-500 nm with Δλ=80) and MIR (3,000-2,800 (fat region), 1,700-1,500 (protein region), and 1,500-900 cm −1 (fingerprint region)) spectra were recorded on cheese samples at 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80°C. The results showed that each spectroscopic technique provided relevant information related to the cheese protein and fat structures during melting, allowing the investigation of structural changes. In addition, the melting temperatures of cheese matrices and fats of the two cheeses were determined from the dynamic rheology data, SF spectra, and MIR spectra. Similar temperatures were obtained whatever the technique, since values of about 60 and 31°C were obtained for matrix and fat melting temperatures of Comté and Raclette cheeses, respectively. No significant difference was observed between the results obtained with the three methods (significance level of 5%).
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