Front-face Fluorescence Research Articles

Predicting lactulose concentration in heat-treated reconstituted skim milk powder using front-face fluorescence

Industrial processing of milk and dairy foods requires heat treatment, which induces undesirable effects in milk such as protein denaturation, organoleptic and nutritional properties alteration, browning, etc. The hypothesis of the present study is that it is possible to use front-face measurement of fluorescent markers such as tryptophan, dityrosine, Maillard intermediate compounds and riboflavin as an inline, cheap, easy and fast way to evaluate lactulose formation in milk. Thus, the specific objective of this study was modeling the kinetics of lactulose formation during heat treatment of milk as well as evaluating the correlation of lactulose concentration with fluorescent markers in order to obtain prediction models of lactulose concentration in heat-treated milk based on front-face fluorescence.Three replicates of the study were performed at three different temperatures (80, 90 and 100 °C) and six different times (0, 10, 15, 30, 45 and 60 min). Standard, low-heat, spray-dried skim milk powder, supplied by Chr. Hansen SL (Barcelona, Spain) was reconstituted at 12% and used in all trials to ensure consistency among the various experimental trials.Lactulose concentration was significantly correlated (P < 0.001) with fluorescence intensity of different markers such as maximum intensity and maximum emission wavelength of tryptophan as well as the fluorescence intensity of riboflavin. Fluorescence changes associated to thermal treatment of milk were characterized and a series of fluorescent markers were identified as predictors allowing prediction of lactulose concentration with an R2 of 0.91 at a temperature of 90 °C.

Front-face fluorescence spectroscopy combined with second-order multivariate algorithms for the quantification of polyphenols in red wine samples.

The potential of front-face fluorescence spectroscopy combined with second-order chemometric methods was investigated for the quantification of the main polyphenols present in wine samples. Parallel factor analysis (PARAFAC) and unfolded-partial least squares coupled to residual bilinearization (U-PLS/RBL) were assessed for the quantification of catechin, epicatechin, quercetin, resveratrol, caffeic acid, gallic acid, p-coumaric acid, and vanillic acid in red wines. Excitation-emission matrices of different red wine samples, without pretreatment, were obtained in front-face mode, recording emission between 290 and 450 nm, exciting between 240 and 290 nm, for the analysis of epicatechin, catechin, caffeic acid, gallic acid, and vanillic acid; and excitation and emission between 300-360 and 330-400nm, respectively, for the analysis of resveratrol. U-PLS/RBL algorithm provided the best results and this methodology was validated by an optimized liquid chromatographic coupled to diode array and fluorimetric detectors procedure, obtaining a very good correlation for vanillic acid, caffeic acid, epicatechin and resveratrol.

Synchronous front-face fluorescence spectroscopy for authentication of the adulteration of edible vegetable oil with refined used frying oil

Synchronous front-face fluorescence spectroscopy has been developed for the discrimination of used frying oil (UFO) from edible vegetable oil (EVO), the estimation of the using time of UFO, and the determination of the adulteration of EVO with UFO. Both the heating time of laboratory prepared UFO and the adulteration of EVO with UFO could be determined by partial least squares regression (PLSR). To simulate the EVO adulteration with UFO, for each kind of oil, fifty adulterated samples at the adulterant amounts range of 1–50% were prepared. PLSR was then adopted to build the model and both full (leave-one-out) cross-validation and external validation were performed to evaluate the predictive ability. Under the optimum condition, the plots of observed versus predicted values exhibited high linearity (R2>0.96). The root mean square error of cross-validation (RMSECV) and root mean square error of prediction (RMSEP) were both lower than 3%.

Partitioning of vanillic acid in oil-in-water emulsions: Impact of the Tween®40 emulsifier

Tween®40, a non-ionic emulsifier, was studied regarding its influence on the partitioning of vanillic acid, chosen as a model of phenolic antioxidants, in oil/water emulsions. Three oil-in-water (30:70) systems at pH3.5 were compared: an oil/water two-phase system, a system with oil/water/Tween just mixed, and an emulsified oil/water/Tween system. The partitioning of vanillic acid in the aqueous and oily phases was studied by 2D front-face fluorescence spectroscopy and UV spectroscopy. Tween®40 used at two concentrations 0.9% and 3.6% was only partitioned in the aqueous phase and at the interface between oil and water.Vanillic acid in the oil/water two-phase system reflected its amphiphilic nature: 3/4 was partitioned in the aqueous phase and 1/4 in the oily phase. In the presence of Tween®40, the major part of vanillic acid (90%) was found in the aqueous phase of the non-emulsified system, where a high proportion was associated with Tween®40 micelles. In the emulsion system, vanillic acid moved with Tween®40 to the o/w interface of the oil droplets. Fluorescence analyses demonstrated that the interactions between vanillic acid and Tween®40 were dependent on pH.

Using pH variations to improve the discrimination of wines by 3D front face fluorescence spectroscopy associated to Independent Components Analysis

Wine composition in polyphenols is related to the variety of grape that it contains. These polyphenols play an essential role in its quality as well as a possible protective effect on human health. Their conjugated aromatic structure renders them fluorescent, which means that 3D front-face fluorescence spectroscopy could be a useful tool to differentiate among the grape varieties that characterize each wine. However, fluorescence spectra acquired simply at the natural pH of wine are not always sufficient to discriminate the wines. The structural changes in the polyphenols resulting from modifications in the pH induce significant changes in their fluorescence spectra, making it possible to more clearly separate different wines.9 wines belonging to three different grape varieties (Shiraz, Cabernet Sauvignon and Pinot Noir) and from 9 different producers, were analyzed over a range of pHs. Independent Components Analysis (ICA) was used to extract characteristic signals from the matrix of unfolded 3D front-face fluorescence spectra and showed that the introduction of pH as an additional parameter in the study of wine fluorescence improved the discrimination of wines.

Monitoring changes in whiting (Merlangius merlangus) fillets stored under modified atmosphere packaging by front face fluorescence spectroscopy and instrumental techniques

Quality assessment of whiting (Merlangius merlangus) fillets stored in normal air (control group) and modified atmosphere packaging (MAP1: 50% N2/50% CO2 and MAP2: 80% N2/20% CO2) for up to 15days at 4°C was performed. The physico-chemical [pH, drip loss, moisture content, total volatile basic nitrogen (TVB-N), thiobarbituric acid reactive substances (TBARS) and peroxide value (PV)], textural (i.e., hardness, fragility, gumminess, chewiness, springiness, cohesiveness), and color (i.e., L∗, a∗, b∗) parameters were determined. Front face fluorescence spectroscopy (FFFS) emission spectra were also scanned on the same samples with excitation set at 290 and 360nm. The results indicated that MAP treatment, particularly MAP1 had an obvious preservative effect on fish quality by reducing pH value, TBARS and TVB-N contents, and retarding the softening of fish texture compared to control samples. Principal component analysis (PCA) applied to physico-chemical and instrumental data sets showed a clear discrimination of fish samples according to both their storage time and condition. A complete (100%) of correct classification was obtained by the concatenation of spectral, physico-chemical, and instrumental data sets. The results demonstrated that storage under MAP can be recommended to improve quality of whiting fillets, which in turn, can be evaluated by FFFS as a rapid and non-destructive technique.

Investigation of Heat‐Induced Degradation of Virgin Olive Oil Using Front Face Fluorescence Spectroscopy and Chemometric Analysis

AbstractA simple setup using a 365‐nm light‐emitting diode coupled to a USB spectrometer through an optical fiber, in a front‐face fluorescence configuration, was used to investigate the heat‐induced deterioration of virgin olive oil at different heating temperatures and times. The samples were heated for 30, 60, 120 and 180 min for every temperature setting of 140, 160 and 180 °C, respectively. Two important results are reported in this article. First, a neo‐formed compound around 665 nm due to the degradation of chlorophyll was observed. This new peak was attributed to pyropheophytins. The second result showed an important rise of the peak around 489 nm, which corresponded to the oxidation products. The correlation obtained between the peroxide value and the 489 nm peak using principal component analysis revealed the mechanism of the oxidation process. It further showed that the peak around 489 nm is a direct consequence of the degradation of hydroperoxide.

Monitoring of thermal changes in meat by synchronous fluorescence spectroscopy

Here we report the potential of synchronous front-face fluorescence spectroscopy in combination with chemometric tools to detect thermal changes in cooked meat. Samples of bovine meat (Longissimus dorsi muscle) were cooked at 66, 90 & 237°C for 0, 1, 2, 5, 7, and 10min. Synchronous front-face fluorescence spectra were collected on meat samples in the excitation wavelength range of 250–550nm using offsets (Δλ) of 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, and 160nm between excitation and emission wavelengths. The data from the synchronous fluorescence landscape containing 1080 spectra were analyzed using PCA & PARAFAC models. PCAs were applied to determine the potential of synchronous spectra to differentiate these samples as a function of cooking time and to retrieve additional information from different offset values. These results confirmed that the synchronous fluorescence spectra provide information related to the molecular structure of meat, allowing classification of samples as a function of cooking time & temperature. The best PARAFAC model was obtained with 3 components, having 55% core consistency values and 98% of the explained variance. The loading profiles of 1st, 2nd and 3rd components had an optimal Δλ of 60, 30 and 80nm, respectively, allowing the determination of excitation (exc.) and emission (em.) maxima wavelengths of 1st (fluorescence band at about exc.: 291 & em.: 351), 2nd (exc.: 297nm & em.: 327nm) and 3rd (exc.: 468nm & em.: 548nm) components. Loadings and scores of the PARAFAC model developed from the synchronous front-face fluorescence spectra enabled us to get information regarding the changes occurring in meat fluorophores during cooking of meat at 66, 90 & 237°C from 0 to 10min.

Phytochemical composition and antioxidant activity of Tuscan bee pollen of different botanic origins

Within the apicultural products, the honey bee-pollen is growing in commercial interest due to its high nutritional properties. For the first time, bee-pollen samples from Tuscany (Italy) were studied to evaluate botanical origin, phytochemical composition and antioxidant activity. The investigated pollen loads were composed of three botanical families: Castanea, Rubus and Cistus. The highest levels of proteins and lipids were detected in Rubus pollen. Castanea pollen contained greater polyphenols, flavonoids and anthocyanins content, while the highest flavonols level was detected in Cistus pollen. These results were also confirmed by front-face fluorescence spectroscopy, used here, for the first time, as a fast tool to characterize bee-pollens.

Front-face fluorescence spectroscopy coupled with chemometric tools for monitoring fish freshness stored under different refrigerated conditions

Front-face fluorescence spectroscopy was used as a rapid tool for assessing freshness of whiting fish (Merlangius merlangus) fillets stored at 4 ± 1 °C for up to 12 days under different conditions. Some physicochemical analysis [i.e., water content, pH, peroxide value, thiobarbituric acid reactive substances (TBARS)] were performed. With increasing storage time, the TBARS and pH values increased progressively in all whiting fish fillets, in particular for those exposed to light. Principal component analysis and factorial discriminant analysis applied separately to the tryptophan (excitation: 290 nm, emission: 305–450 nm) and nicotinamide adenine dinucleotide (NADH) (excitation: 340 nm, emission: 360–600 nm) spectra gave only a slight discrimination between the investigated whiting fish fillets. These results were improved when data from all the fluorescence spectra were analyzed jointly, since nearly three-quarters of samples were correctly classified. The best results were obtained by concatenating the fluorescence and physicochemical data sets, giving more than 90% of correct classification.

Assessment of the action spectrum for photooxidation in full fat bovine milk

The action spectrum for photooxidation in full fat bovine milk was measured. Samples of milk with air or argon in headspace were exposed to narrow wavelength bands of light in the range 400–700nm. Photooxidation in terms of off-flavors was measured by a sensory panel, volatile compounds by headspace solid phase micro extraction (SPME–GC–MS), and photobleaching of photosensitizers in milk (riboflavin, protoporphyrin IX and a chlorophyllic compound) by front face fluorescence spectroscopy. The action spectrum deviated significantly from the absorption spectrum of milk. Significant oxidation was induced by wavelengths around 400nm and 500–650nm in milk with air in headspace. Argon in headspace gave significant oxidation also at 700nm. It is suggested that protoporphyrin IX and chlorophyll are responsible for oxidation induced by wavelengths >500nm, and that also riboflavin is contributing from 400 to 500nm.

A method for determining supernatant-free spectra generated from fluorescently stained cells in suspension

Here we present a fluorometric method for direct determination of supernatant-free fluorescence spectra generated from fluorescently stained cells in suspension. The key element in the new technique is the design of an adapter to a standard cuvette holder that makes it possible to measure front-face fluorescence spectra from thin layers of cells spun down to the bottom of a spectrofluorometric cuvette. We have demonstrated the applicability of this approach and its analytical potential using the suspensions of yeast cells stained with the potentiometric dye of 3,3’-dipropylthiadicarbocyanine, diS-C3(3), and with the specific cell-wall marker calcofluor.

Classification and characterization of beef muscles using front-face fluorescence spectroscopy

The objective of this study was to evaluate the potential of fluorescence spectroscopy to identify different muscles and to predict some physicochemical and rheological parameters. Samples were taken from three muscles (Semitendinosus, Rectus abdominis and Infraspinatus) of Charolais breed. Dry matter content, fat content, protein content, texture and collagen content were determined. Moreover emission spectra were recorded in the range of 305–400nm, 340–540nm and 410–700nm by fixing the excitation wavelength at 290, 322 and 382nm, respectively. The results obtained were evaluated by partial least square discriminant analysis and partial least square regression. Results of our research work show that front-face fluorescence spectroscopy and chemometrics offer significant potential for the development of rapid and non-destructive methods for the identification and characterization of muscles.

3D front face solid-phase fluorescence spectroscopy combined with Independent Components Analysis to characterize organic matter in model soils

Soil organic matter (SOM) is a very complex and heterogeneous system which complicates its characterization. In fact, the methods classically used to characterize SOM are time- and solvent-consuming and insufficiently informative. The aim of this work is to study the potential of 3D solid-phase front face fluorescence (3D-SPFFF) spectroscopy to quickly provide a relevant and objective characterization of SOM as an alternative to the existing methods.Different soil models were prepared to simulate natural soil composition and were analyzed by 3D front-face fluorescence spectroscopy without prior preparation. The spectra were then treated using Independent Components Analysis. In this way, different organic molecules such as cellulose, proteins and amino acids used in the soil models were identified.The results of this study clearly indicate that 3D-SPFFF spectroscopy could be an easy, reliable and practical analytical method that could be an alternative to the classical methods in order to study SOM.The use of solid samples revealed some interactions that may occur in natural soils (self-quenching in the case of cellulose) and gave more accurate fluorescence signals for different components of the analyzed soil models.Independent Components Analysis (ICA) has demonstrated its power to extract the most informative signals and thus facilitate the interpretation of the complex 3D fluorescence data.

EEM fluorescence spectroscopy as a fast method to assess the brine composition of salted herring

Ripening of barrel-salted herring (Clupea harengus) is evaluated by the use of fluorescence spectroscopy and protein determinations. During ripening, protein degradation takes place in the herring and protein is extracted into the brine. The present study aims at identifying parameters which are correlated to the ripening characteristics of barrel-salted herring and which can provide a better understanding of the ripening process. Front face fluorescence landscapes were obtained by measuring directly on the brine from barrel-salted herring. These data were analyzed by parallel factor analysis (PARAFAC), which revealed four fluorophores, tryptophan (two states), vitamin B6 and riboflavin. All four parameters showed an increase in concentration during the storage period corresponding to an increase in protein content that varied from 3g/100g at day 60 to 5g/100g after 277 days of storage. It was not possible to see a difference in the development of the four fluorophores during the ripening period. The protein content was predicted from the fluorescence landscapes by partial least squares (PLS). The use of unfolded fluorescence spectra gave an RMSECV of 0.26 g/100g and a correlation between the measured protein content and the predicted values of 0.86.

Characterization of fatty acid profile by FFFS

The purpose of the study was to assess potential application of front face fluorescence spectroscopy as a rapid and non-destructive technique to discriminate between fats of animal and plant origin based on their fatty acid profiles, and to predict concentration of fatty acids from fluorescence spectra. Vitamin E emission spectra (300–500 nm) of butterfat and vegetable oil samples were recorded with excitation wavelength set at 295 nm. Fatty acid composition of the samples was determined by gas chromatography. Principal component analysis and partial least squares regression analysis were applied to the gas chromatography and fluorescence spectroscopy data. The butter-fats and vegetable oils were discriminated based on the total saturated and unsaturated fatty acids respectively. Tocopherols and tocotrienols accounted for the variability among various oils. A good prediction model was established with R2 = 0.745–0.992 for saturated fatty acids. The unsaturated fatty acids were characterized by low coefficients of determination (R2 < 0.339). The fatty acid profiles predicted from fluorescence spectra did not show significant difference to those determined by gas chromatography used as references. A good association was established between the two data tables. The study demonstrated great potential of front face fluorescence spectroscopy to rapidly discriminate between fats of animal and plant origin, and predict their saturated fatty acids composition, which could in turn be used for detection of milk fat adulteration with vegetable oil.

From multispectral imaging of autofluorescence to chemical and sensory images of lipid oxidation in cod caviar paste

The potential of multispectral imaging of autofluorescence to map sensory flavour properties and fluorophore concentrations in cod caviar paste has been investigated. Cod caviar paste was used as a case product and it was stored over time, under different headspace gas composition and light exposure conditions, to obtain a relevant span in lipid oxidation and sensory properties. Samples were divided in two sets, calibration and test sets, with 16 and 7 samples, respectively. A third set of samples was prepared with induced gradients in lipid oxidation and sensory properties by light exposure of certain parts of the sample surface. Front-face fluorescence emission images were obtained for excitation wavelength 382 nm at 11 different channels ranging from 400 to 700 nm. The analysis of the obtained sets of images was divided in two parts: First, in an effort to compress and extract relevant information, multivariate curve resolution was applied on the calibration set and three spectral components and their relative concentrations in each sample were obtained. The obtained profiles were employed to estimate the concentrations of each component in the images of the heterogeneous samples, giving chemical images of the distribution of fluorescent oxidation products, protoporphyrin IX and photoprotoporphyrin. Second, regression models for sensory attributes related to lipid oxidation were constructed based on the spectra of homogeneous samples from the calibration set. These models were successfully validated with the test set. The models were then applied for pixel-wise estimation of sensory flavours in the heterogeneous images, giving rise to sensory images. As far as we know this is the first time that sensory images of odour and flavour are obtained based on multispectral imaging.

Predicting rheological behavior of wheat dough based on machine learning and front-face fluorescence spectroscopy on wheat flour.

Predicting rheological behavior of wheat dough based on machine learning and front-face fluorescence spectroscopy on wheat flour.

Use of Front-Face Fluorescence Spectroscopy to Differentiate Sheep Milks from Different Genotypes and Feeding Systems

The objective of the present study was to assess the potential of front-face fluorescence spectroscopy coupled with chemometric tools for the evaluation of the quality of milk samples according to the feeding system and genotype. Fifty (n = 50) ewe's milk samples were scanned after excitation set at 250, 290, 322, and 380 nm and emission set at 410 nm. Thirty out of the 50 samples composed the first trial and were obtained from two different genotypes (i.e., Comisana versus Sicilo-Sarde); the second trial was composed of 20 samples obtained from the Sicilo-Sarde genotype with two different feeding systems in pen (soybean versus scotch bean). Milk samples were divided into four groups named Sicilo-Sarde with pasture feeding (Spas), Comisana with pasture feeding (Cpas), Sicilo-Sarde feeding on scotch bean (Ssco), and Sicilo-Sarde feeding on soybean (Ssoy). The factorial discriminant analysis was applied to the: (i) four groups (i.e., Spas, Ssco, Ssoy, and Cpas) and (ii) three groups composed only of Sicilo-Sarde genotype (i.e., Spas, Ssco, and Ssoy). Considering the four groups, the best result was obtained with the excitation vitamin A spectra since correct classification amounting to 76% was observed. When the factorial discriminant analysis was performed with the three groups belonging to the Sicilo-Sarde genotype, the best result was obtained again with vitamin A spectra (i.e., emission and excitation spectra) since 88.6% of correct classification was observed. Concatenation technique applied to the five fluorescence spectra improved the rate of classification between the four groups since 44 out of 50 samples were correctly classified. No misclassification was observed between milk samples collected from ewes with pasture feeding from the pen feeding. It was concluded from the obtained results that fluorescence spectroscopy could be considered as a powerful tool for differentiating between raw milks according to both genotype and feeding system.

Prediction of Aerobic Plate Count on Beef Surface Using Fluorescence Fingerprint

The potential of fluorescence fingerprint (FF) spectroscopy was investigated to develop a nondestructive prediction method of aerobic plate count on a beef surface. Sixty samples (e.g., 30 lean meat slices each of Australian cattle and Japanese cattle) stored aerobically at 15 °C were analyzed by front-face fluorescence spectrophotometry. FF and aerobic plate count (APC) were measured after 0, 12, 24, 36, and 48 h of storage. FFs were collected in both excitation and emission wavelength ranges of 200–900 nm. Partial least-squares regression (PLSR) performed on an FF dataset predicted an APC in the bacterial contamination load range from 1.7 to 7.8 log colony-forming units (cfu)/cm2 with a prediction error of 0.752 log cfu/cm2. The regions where the regression coefficient of the PLSR model was relatively high were consistent with those of the FF peaks of five intrinsic fluorophores: tryptophan, NAD(P)H, vitamin A, porphyrins, and flavins. This suggests that changes in the autofluorescence of these intrinsic fluorophores due to the metabolism of bacterial flora on meat are reflected in the PLSR model for predicting APC from the FF dataset. FF spectroscopy coupled with multivariate analysis appeared to be applicable to the nondestructive determination of APC on the surface of lean beef.