Fluorescence spectroscopy: a rapid tool for analyzing dairy products

This paper gives a critical evaluation of the use of fluorescence spectroscopy for measuring chemical and physical changes in dairy products caused by processing and storage. Fluorescence spectroscopy is able to determine various properties of foods without use of chemicals and time-consuming sample preparation. This is shown by examples where the measurement of a given chemical parameter has been appropriately described and validated, as well as situations showing potential applications, but where further research and validation is required. The interpretation of fluorescence spectroscopic data is complex due to absorbance by other molecular groups, changes caused by variation in the sample matrix, etc. It is illustrated how advanced data analytical techniques are required to obtain optimal interpretation of the data. Even though the review focuses on examples from the dairy industry, the principles are broader and can be applied to other fields of food and agricultural research.     

Authentication of the botanical and geographical origin of honey by front-face fluorescence spectroscopy

Front-face fluorescence spectroscopy, directly applied on honey samples, was used for the authentication of 11 unifloral and polyfloral honey types (n = 371 samples) previously classified using traditional methods such as chemical, pollen, and sensory analysis. Excitation spectra (220-400 nm) were recorded with the emission measured at 420 nm. In addition, emission spectra were recorded between 290 and 500 nm (excitation at 270 nm) as well as between 330 and 550 nm (excitation at 310 nm). A total of four different spectral data sets were considered for data analysis. Chemometric evaluation of the spectra included principal component analysis and linear discriminant analysis; the error rates of the discriminant models were calculated by using Bayes' theorem. They ranged from <0.1% (polyfloral and chestnut honeys) to 9.9% (fir honeydew honey) by using single spectral data sets and from <0.1% (metcalfa honeydew, polyfloral, and chestnut honeys) to 7.5% (lime honey) by combining two data sets. This study indicates that front-face fluorescence spectroscopy is a promising technique for the authentication of the botanical origin of honey and may also be useful for the determination of the geographical origin within the same unifloral honey type.     

Effect of extended aging of parma dry-cured ham on the content of oligopeptides and free amino acids

The effect of the dry-curing processing time on the release of oligopeptides and amino acids was evaluated with 158 Parma hams subdivided into three groups: (1) traditional processing (450 days); (2) extended processing (570 days); and (3) extended aging (690 days). Most of the oligopeptides and free amino acids detected increased up to the last deadline (690 days); a sharp increase of peptides below 400 Da was the main change in most aged hams. In particular, gamma-glutamyl dipeptides showed a remarkable increase during ham extended aging, acting like permanent taste-active compounds, being unsuitable for further enzymatic breakdown. The pH of fresh hams showed negative relationships (P < 0.001) with most peptides. With regard to free amino acids, the pattern was modified by different processing lengths, together with their taste categories, so that the amino acids having monosodium glutamate-like and bitter tastes were enhanced in more aged hams.     

白洋淀沉积物中溶解有机质荧光特性及其与铜的相互作用

在实验条件下通过三维荧光光谱、荧光猝灭滴定和质子核磁共振（1HNMR）,分析了白洋淀污染水体沉积物中溶解有机质（DOM）与Cu（Ⅱ）的相互作用。结果表明：DOM的结构以碳水化合物及多糖为主且具有明显的类蛋白（峰B）和类富里酸（峰A和峰C）荧光峰,类蛋白的荧光强度较强于可见类富里酸;3类荧光基团荧光强度由于Cu（Ⅱ）猝灭,呈明显不同程度的降低;并且DOM与Cu（Ⅱ）络合常数及1HNMR分析表明DOM中荧光基因与Cu（Ⅱ）络合或螯合能力较大,配位的配位基较多。     

Authentication of the botanical origin of honey by front-face fluorescence spectroscopy. A preliminary study

The potential of front-face fluorescence spectroscopy for the authentication of unifloral and polyfloral honey types (n = 57 samples) previously classified using traditional methods such as chemical, pollen, and sensory analysis was evaluated. Emission spectra were recorded between 280 and 480 nm (excit: 250 nm), 305 and 500 nm (excit: 290 nm), and 380 and 600 nm (excit: 373 nm) directly on honey samples. In addition, excitation spectra (290-440 nm) were recorded with the emission measured at 450 nm. A total of four different spectral data sets were considered for data analysis. After normalization of the spectra, chemometric evaluation of the spectral data was carried out using principal component analysis (PCA) and linear discriminant analysis (LDA). The rate of correct classification ranged from 36% to 100% by using single spectral data sets (250, 290, 373, 450 nm) and from 73% to 100% by combining these four data sets. For alpine polyfloral honey and the unifloral varieties investigated (acacia, alpine rose, honeydew, chestnut, and rape), correct classification ranged from 96% to 100%. This preliminary study indicates that front-face fluorescence spectroscopy is a promising technique for the authentication of the botanical origin of honey. It is nondestructive, rapid, easy to use, and inexpensive. The use of additional excitation wavelengths between 320 and 440 nm could increase the correct classification of the less characteristic fluorescent varieties.     

原料肉的机械前处理工艺对猪肉切片火腿品质的影响

为了改善猪肉切片火腿出水、质构和口感问题,以精选猪后腿肉的股四头肌为主要原料,研究5种机械处理工艺对猪肉切片火腿系水性(蒸煮损失率、压榨失水率和杀菌失水率)、质构特性(硬度、弹性、内聚性和咀嚼度)、色泽和感官品质的影响。5种对原料肉的机械处理工艺包括只滚揉、注射腌制液后滚揉、嫩化后滚揉、注射后嫩化滚揉、腰刀绞制后滚揉。结果表明:注射和嫩化均可显著降低火腿的蒸煮损失率和压榨失水率,注射后嫩化滚揉的火腿的蒸煮损失率最低,为7.90%,而腰刀绞制后滚揉的火腿切片杀菌失水率最低,为2.66%,注射后嫩化滚揉与腰刀绞制后滚揉的火腿压榨失水率均最低,并且均显著低于其他处理组。注射和嫩化均可提高产品的硬度、内聚性和咀嚼度,注射嫩化滚揉组合处理与进行1种机械处理或2种机械处理相比,样品具有更好的质构特性,但是5种机械处理工艺中,腰刀绞制后滚揉的猪肉火腿硬度、内聚性和咀嚼度均最大。注射和嫩化有利于提高产品的亮度值(L*)和红度值(a*),降低产品的黄度值(b*)。通过感官评价可知,只经过滚揉的火腿具有最差的感官品质,注射后滚揉和嫩化后滚揉火腿的感官品质差异不大,注射后嫩化滚揉的火腿的质地和切片性低于腰刀绞制后滚揉的火腿,但是显肉性和色泽优于腰刀绞制后滚揉火腿。因此,工业化生产建议原料肉采用注射后嫩化滚揉处理,猪肉切片火腿具有最好的品质。     

Effect of cooking conditions on creatinine formation in cooked ham

The aim of this study was to evaluate the effect of different cooking procedures on the concentrations of creatine and creatinine and the ratio of creatinine/creatine in cooked ham. Two cooking methods (constant temperature and increasing temperature, constant T and DeltaT, respectively) were tested on different locations in porcine longissimus dorsi muscle and ham (semimembranosus, biceps femoris, and gluteus muscles). The results showed larger creatine conversion into creatinine in the surface layer than in the core as well as higher creatinine/creatine ratio values when applying the DeltaT in comparison to the constant T method. A correlation between the creatinine/creatine ratio and the heat treatment was established, and 15 samples of commercial cooked hams were analyzed to support these results. This creatinine/creatine ratio analyzed in the surface of the ham could be used as a rapid and nondestructive indicator to determine the effectiveness of the heat treatment in cooked ham processes.     

Native fluorescence from juvenile stages of common food storage insects

Insect infestation of stored foods has significant economic and health consequences; the development of novel methods of detection thus presents considerable opportunities. The fluorescence from nine species of storage insects (beetles and moths) was studied; the juvenile stages of all nine species exhibited fluorescence under long-wave (365 nm) UV light; none of the adult insects emit fluorescence, so the fluorophore(s) might be a compound(s) associated with the unsclerotized cuticle. The spectra of larval stages of Ephestia kuehniella, Oryzaephilus surinamensis, Corcyra cepahlonica, Tribolium castaneum, and Tribolium confusum exhibited excitation maxima in the range from 345 to 350 nm and emission maxima in the range from 421 to 427 nm, suggesting that fluorescence arises from a common chromophore; similarities in fluorescence properties implicate one of the many pteridine ring-containing compounds (pterins) commonly found in insects. Larvae and even eggs were readily imaged on foods using fluorescence under 365-nm excitation. Fluorescence thus appears to be ubiquitous in immature food storage insects, and fluorescence detection may be useful as a general method to detect insects in foods and agricultural commodities during storage or processing.     

Application of fluorescence spectroscopy in the evaluation of light-induced oxidation in cheese

Light-induced oxidation of semihard cheese has been evaluated by fluorescence spectroscopy. The cheese was packaged in two packaging materials and exposed to different storage conditions, which included light/dark storage, oxygen availability, and storage time (0, 4, 7, 14, 21, 42, 70, or 84 days). Fluorescence excitation-emission matrices (EEM) were analyzed by PARAFAC, which gave an estimation of the pure excitation and emission spectra of the fluorophores and the concentrations of these. This analysis showed the presence of components such as tryptophan, tyrosine, vitamin A, fluorescent oxidation products, and riboflavin. Effects of packaging material, light or dark storage, and storage time were seen. However, there was no effect of the oxygen availability on the fluorescence measurements. The score values obtained by the PARAFAC models and chemical and physical measurements were analyzed together by principal component analysis (PCA). The loadings showed a separation of the variables into three groups; the first group was related to oxidation, the second group was related to the degradation of both riboflavin and vitamin A, and the third group was linked to the protein structure.     

Analysis of the early stages of lipid oxidation in freeze-stored pork back fat and mechanically recovered poultry meat

An analytical method that can detect low levels of oxidation in food earlier than a sensory panel would be a valuable tool for food manufacturers as well as research institutes. Two model matrixes, pork back fat and mechanically recovered poultry meat (MRPM), were freeze-stored in air at -20 degrees C for 26 weeks. Peroxide value, thiobarbituric acid reactive substances, volatiles analyzed with dynamic headspace gas chromatography-mass spectrometry (GC-MS) and a gas-sensor array technique (electronic nose), chemiluminescence, and front-face fluorescence were evaluated against sensory analysis with regard to detection of early oxidation and correlation with sensory data. Fluorescence and GC-MS could detect oxidative changes in pork back fat earlier than the sensory panel and the electronic nose at the same time. The three methods were highly correlated with sensory attributes (r = 0.8-0.9). GC-MS gave the best results with regard to detection of small oxidative changes in MRPM.     

Thermal and Spectroscopic Characterization Of Composts From Municipal Solid Wastes

Six composts from municipal solid wastes (MSW) were sampled after composting times of 3, 7, 15, 45, 60 and 350 days and analyzed by chemical analysis, fluorescence spectroscopy in emission, excitation and synchronous scan modes, Fourier transform infrared spectroscopy and differential scanning calorimetry, without carrying out any organic matter extraction. Thermal and spectroscopic techniques appeared to be powerful tools to investigate the evolution of MSW organic matter during composting, and provided evidence that the chemical transformations occurring in these heterogeneous materials yield products with molecular complexity and stability, similar to humic substances.     

Spectroscopic and Thermal Investigation Of Hydrophobic and Hydrophilic Fractions Of Dissolved Organic Matter

Hydrophobic (HO) and hydrophilic (HI) fractions of dissolved organic matter (DOM) isolated from several substrates including a source separated urban waste compost, a mechanically separated urban waste compost, an anaerobically digested municipal sewage sludge, a composted sewage sludge and a cow slurry were characterised by Fourier transform infrared spectroscopy (FT-IR), monodimensional fluorescence spectroscopy in the emission, excitation and synchronous scan modes, tridimensional excitation emission matrix (EEM) fluorescence spectroscopy, ¹H nuclear magnetic resonance (¹H-NMR) spectroscopy, and differential scanning calorimetry (DSC). The FT-IR spectra indicated that the HI fractions were rich in both aromatic and aliphatic structures, whereas the HO fractions were rich in carboxylic groups and polysaccharides. The synchronous scan spectra of HO fractions showed the main peak at a wavelength much longer than that of the HI fractions, which indicated a greater molecular complexity of the HO fractions with respect to HI fractions. The ¹H-NMR data indicated a great percentage of aliphatic structures in all samples, whereas the HO samples were also characterised by the presence of aromatic structures. The DSC curves confirmed the more distinct aromatic nature of HO fractions, as compared to the corresponding HI fractions.     

Limited effects of land use on soil dissolved organic matter chemistry as assessed by excitation–emission fluorescence spectroscopy and molecular weight fractionation

Dissolved organic matter (DOM) in soil solution represents a complex mixture of organic molecules and plays a central role in carbon and nitrogen cycling in plant–microbial–soil systems. We tested whether excitation–emission matrix (EEM) fluorescence spectroscopy can be used to characterize DOM and support previous findings that the majority of DOM is of high molecular weight (MW). EEM fluorescence spectroscopy was used in conjunction with MW fractionation to characterize DOM in soil solution from a grassland soil land management gradient in North Wales, UK. Data analysis suggested that three distinct fluorescence components could be separated and identified from the EEM data. These components were identified as being of humic‐like or fulvic‐like origin. Contrary to expectations, the majority of the fluorescence signal occurred in the small MW (<1 kDa) fraction, although differences between soils from the differently managed grasslands were more apparent in larger MW fractions. We conclude that following further characterization of the chemical composition of the fluorophores, EEM has potential as a sensitive technique for characterizing the small MW phenolic fraction of DOM in soils.     

Spectrofluorometric characterization of beta-lactoglobulin B covalently labeled with 2-(4'-maleimidylanilino)naphthalene-6-sulfonate.

Bovine beta-lactoglobulin, genetic variant B, has been labeled with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid through covalent attachment through the Cys-121 thiol group for the study of stepwise pressure denaturation of this whey protein by fluorescence spectroscopy. The labeling was performed under nondenaturing conditions with a factor of 5 excess of the fluorophore in dimethylformamide/water (1:10) to yield the whey protein highly labeled after chromatographic separation. MALDI-TOF mass spectroscopy confirmed labeling. The emission from the fluorophore, which is sensitive to the microenvironment, has been characterized for the labeled protein (aqueous pH 7.4 solution, 25 degrees C) and has a lambda(em,max) = 410 nm (lambda(ex,max) = 318 nm) with a fluorescence lifetime of 6.1 +/- 0.2 ns. Fluorescence anisotropy increases and fluorescence quantum yield (Phi(f) = 0.103 at 320 nm) decreases with increasing excitation wavelength. For increasing hydrostatic pressure, fluorescence quantum yield showed a minimum at approximately 50 MPa, corresponding to the pre-denatured "pressure-melted" state in which thiol reactivity previously was found to increase prior to reversible protein unfolding.     

基于前表面荧光光谱鉴别新鲜与冻融大黄鱼

为研究反复冻融对水产品品质的影响,通过理化方法检测了不同冻融次数处理对大黄鱼解冻损失、pH 值、色泽、硫代巴比妥酸值、羰基含量等指标的影响,并采用前表面荧光光谱结合主成分分析（principal component analysis,PCA）和 Fisher 线性判别分析法（Fisher linear discriminant analysis,FLDA）对不同冻融次数的大黄鱼进行区分。结果显示随着冻融次数增加,大黄鱼的解冻损失显著增加（P<0.05）;pH 值呈先上升后下降的趋势;L*（亮度）值、b*（黄度）值均有不同程度的增加（P<0.05）,a*（红度）值下降（P<0.05）;羰基含量和硫代巴比妥酸反应物值（thiobarbituric acid reactive substances,TBARS）增加（P<0.05）,反复冻融导致大黄鱼的品质下降。色氨酸和烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide,NADH）的荧光光谱分别结合 PCA 和 FLDA 对不同冻融处理组进行分析,结果表明 FLDA 识别效果优于 PCA。通过 FLDA 建立了新鲜大黄鱼与冻融大黄鱼荧光光谱判别模型,发现色氨酸原始判别的准确率和交叉验证的准确率分别为68.3%和66.7%,NADH 原始判别的准确率和交叉验证的准确率均达到100%。由此可见,利用 NADH荧光光谱结合化学计量分析可以鉴别不同冻融处理的大黄鱼。研究结果为水产品新鲜度的快速评价提供参考。     

Fluorescence spectroscopy of sedimentary pore-water humic substances: a simple tool for retrospective analysis of lake ecosystems

Purpose

The feasibility of applying the method of single-scan fluorescence emission spectra of humic substances (HSs) without chemical pretreatments of sediment pore-water samples was tested to ascertain the past productivity and sources of organic matter of lakes.

Materials and methods

Sediment samples were collected from ten Estonian lakes (located between 57°36′ and 59°25′N and 22°12′ and 26°59′E) covering all levels of the trophic scale. The height (fluorescence intensity), location (fluorescence maximum) and shape (fluorescence index, the ratio of intensities at the emission wavelengths 450 and 500 nm) of the fluorescence emission spectrum at an excitation of 340 nm were under consideration.

Results and discussion

Pore-water humic substances (pwHSs) from sediments of eutrophic lakes had generally a high fluorescence intensity and fluorescence index and their fluorescence maximum was located at shorter wavelengths. Characteristic features of pwHSs from oligotrophic lakes were low fluorescence intensity, emission of maximum fluorescence at shorter wavelengths and high fluorescence index values. Pore-water humic substances from sediments of dystrophic lakes were characterized by a low fluorescence intensity and fluorescence index and their spectral peak was shifted to longer wavelengths. The study also demonstrated that a shift in the peak location of pwHSs fluorescence was accompanied with a change in the C/N ratios of sedimentary organic matter, and the alteration in the fluorescence index of pwHSs was synchronous with the changes in their molecular weight.

Conclusions

The obtained results suggest that fluorescence spectroscopy of pwHSs without using chemical pretreatments has a great potential in the reconstruction of past lake conditions.     

Simple Measurement of 4,4’-bis(2-sulfostyryl)-biphenyl in River Water by Fluorescence Analysis and Its Application as an Indicator of Domestic Wastewater Contamination

A characteristic peak of fluorescent whitening agents (FWAs) was detected by fluorescence excitation spectrum (FES) measurement of river water samples. The main causative chemical was 4,4’-bis(2-sulfostyryl)-biphenyl (DSBP), which is commonly added to household detergents in Japan. As the fluorescence of DSBP overlaps with that of fulvic-like organic matter in the spectral fluorescent signatures, DSBP concentration was determined by the newly proposed calculation method, which uses fluorescence intensity at three excitation wavelengths of 320, 345 and 360 nm at emission wavelength of 430 nm for baseline correction. The concentration of DSBP calculated using this method showed strong correlation (correlation coefficient: r = 0.992) with that obtained by high-performance liquid chromatography analysis. The concentrations of DSBP detected in river water samples were 0.28 to 1.84 μg l^?1, with high concentrations observed at the stations with relatively high flow rates of upstream sources of treated domestic wastewater and untreated gray water (domestic wastewater excluding flush toilet wastewater). It was proved that the concentration of DSBP in river water is useful for giving rough estimation of the magnitude of domestic wastewater contamination in river water.     

Fluorescence Tracing of Diffuse Landfill Leachate Contamination in Rivers

Landfill leachates are composed of a complex mixture of degradation products which include a wide range of potentially fluorescent organic molecules and compounds. Here we investigate the use of fluorescence excitation–emission matrix (EEM) analysis in detecting diffuse landfill leachate contamination in rivers. Landfill leachates from three unlined landfill sites adjacent to our study river are characterised by intense fluorescence at excitation wavelength 220–230 nm, and emission wavelength 340–370 nm, which derives from fluorescent components of the xenobiotic organic matter fraction. Seven surface water sample sites on an adjacent polluted river system were analysed for fluorescence and water quality properties. The 220–230 nm excitation wavelength, 340–370 nm emission wavelength fluorescent centre was also detected in this river system at the sample locations downstream of the landfills, but not at upstream control sites, demonstrating its use as a tracer of landfill leachate contamination. Negative correlations are observed between this fluorescence centre and dissolved oxygen in the river water samples, demonstrating the water quality implications of leachate contamination at this study site. The fluorescence intensity at the 220–230 nm excitation wavelength, 340–370 nm emission wavelength fluorescent centre in landfill leachates is such that it remains detectable at dilutions of 10²–10³, and the fluorescence EEM technique is rapid and cost-effective for use by river managers and water quality regulators.     

畜禽粪便堆肥溶解态有机质三维荧光光谱特征及Cu络合

畜禽粪便中溶解态有机质（DOM）易与Cu发生络合,从而促进其向土壤溶液和地表水体中迁移。该文对比分析堆肥前后猪粪和牛粪DOM三维荧光光谱特征变化,并通过荧光猝灭滴定法研究了堆肥对猪粪和牛粪中DOM与Cu络合的影响。研究发现,未经堆腐的猪粪和牛粪DOM中均存在较强的类蛋白荧光峰,包括类酪氨酸峰和类色氨酸峰;除了类蛋白峰,牛粪DOM中还出现了类腐殖质荧光峰。经过堆肥后,猪粪DOM中类酪氨酸峰和类色氨酸峰强度显著减弱,并在可见光激发区域出现类腐殖质峰;与猪粪堆肥类似,牛粪堆肥后类酪氨酸峰和类色氨酸峰也隐没不现,在紫外激发区域出现类腐殖质峰,同时可见光激发区的类腐殖质峰荧光强度减弱,位置发生红移。荧光猝灭试验结果显示,猪粪和牛粪堆肥后与Cu络合容量显著降低。因此,堆肥后改变了畜禽粪便DOM组成,生成大量胡敏酸和富里酸物质,从而降低畜禽粪便中DOM-Cu络合物的迁移性和生物可利用性。