A rapid PCR–RFLP method for the identification of Lophius species

The seven Anglerfish species, which belong to the genus Lophius, have a different value on the market, worldwide. If whole fishes can be identified by their morphological characteristics, they become indistinguishable when prepared or processed. In this study, a rapid method based on polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was developed for the authentication of the seven Lophius species, using a cytochrome b gene fragment of 566 bp. After a genus-specific PCR, a fast digestion with the restriction enzyme BfaI, followed by agarose gel electrophoresis, allowed a clear species identification by producing specific restriction patterns. The total time required was as low as 6 h, DNA extraction included. The method was then used to analyse 48 commercial samples, whose phylogenetic analysis confirmed the PCR–RFLP response at 100 %. Results showed that mislabelling occurs on the market regardless the kind of processing.     

Puffer fish-based commercial fraud identification in a segment of cytochrome b region by PCR–RFLP analysis

To identify the mislabeled or fraudulently substituted toxic puffer fish in thermally processed fish products, a polymerase chain reaction (PCR) method using restriction sites and sequence analysis has been developed in this study. A 376-bp fragment of the cytochrome b gene was produced after PCR amplification. Fish tissue samples were prepared under autoclaving conditions at 121 °C for 10–90 min at 10 min intervals. DNA fragments could not be detected after 90 min of autoclaving at 121 °C. For PCR product digestion, BsaJ I, Aci I, Hinf I, Taq I, and Sap I endonucleases were used to yield species-specific profiles for the identification of puffer fish species from 60 commercial market samples. Results from this study showed that the restriction fragment length polymorphism technique can be used to identify 17 puffer fish species from commercial products even after severe thermal processing.     

Determination of gellan gum by capillary electrophoresis and CE–MS

Intact gellan gum (0.1% m/v) was detectable by capillary electrophoresis (CE) with UV detection. Characteristic tetrasaccharide fragments, prepared with a newly characterised gellan-degrading enzyme, provided a clearer signal that was detectable in complex food products containing other polysaccharides. Food products spiked with gellan gum could be analysed reproducibly with high accuracy and specificity by CE–ESI–MS, which is recommended as the technique of choice. Gellan gum declared as a fruit flavour drink ingredient could not be identified by CE–ESI–MS. When added to the product at the start of sample preparation, before enzyme treatment, the gum was readily detectable, demonstrating that the method was compatible with this sample type. Possible explanations for the negative results are that gellan gum was used as a trace component, with other texturing agents; that its declaration was precautionary only; or that the product contained a chemically modified form. Further work will establish whether modified gellan gums can be similarly analysed.     

A procedure for olive oil traceability and authenticity: DNA extraction,multiplex PCR and LDR–universal array analysis

Traceability of olive oils is relevant not only in assessing their origin, but also in protecting against frauds. Here, we present an improvement of the assay previously developed for the genotyping of forty-nine frequently grown Mediterranean olive cultivars by ligation detection reaction (LDR)/universal array (UA), refining the entire procedure in order to address DNA extracted from monovarietal olive oils. Firstly, a simple and robust protocol to extract amplifiable DNA from olive oil was developed. Then, the SNP-containing DNA sequences were simultaneous amplified by multiplex PCR and used on a LDR-UA platform, which gave precise and accurate genotype results. Thirteen out of the seventeen investigated SNPs were amplifiable in multiplex PCR, and were sufficient to unequivocally discriminate the forty-nine cultivars. The availability of this semi-automated SNP genotyping assay should help food testing laboratories to verify the origin and authenticity of monovarietal extra-virgin olive oils. C. Consolandi and L. Palmieri equally contributed to this research paper.     

On-line analysis of intact olive fruits by vis–NIR spectroscopy: Optimisation of the acquisition parameters

In the last years, the potential of NIRS for quantitative and qualitative analysis of olives fruits and oils has been investigated. However, limited work has been published about the on-line implementation of NIR spectroscopy in this sector. NIRS application at factory level (olive mills) is desirable. However, prior its implementation, many parameters related to the on-line spectrum acquisition, must be studied and optimised. In this paper, the influence of parameters such as focal distance and integration time has been studied. On-line spectral measurements were performed on intact olives in the spectral range of 380–1690 nm using a diode array spectrometer located on the top of a conveyor belt. The statistical criteria used to evaluate the spectral repeatability for each level of parameters considered were the standard deviation (SD) of the log (1/R) values and the root mean square statistics (RMS). Results demonstrated that for on-line control of olives in movement, spectra acquisition process was more affected by the focal distance chosen than by the integration time used. A focal distance of 13 mm and an integration time of 5 s have been defined as the optimal operational conditions.     

Comparison of donor–acceptor and alumina columns for the clean-up of polycyclic aromatic hydrocarbons from edible oils

Two methods for clean-up and sample enrichment for the analysis of polycyclic aromatic hydrocarbons (PAHs) in edible oils are compared; a clean-up based on a donor–acceptor complex chromatography (DACC) column and a standardized method widely used in the food industry consisting in a low pressure column chromatography with alumina as stationary phase. Both methods are followed by a reversed-phase high-performance liquid chromatography with fluorescence detection for the separation and quantitation of each PAH. Certified materials were used in order to validate the methods. The limits of detection were lower than 1 ng/g and good selectivity was achieved in both cases. The DACC column clean-up is faster and better accuracies were obtained. The advantages and disadvantages of both methods are discussed.     

β-Cyclodextrin facilitates simultaneous analysis of six bioactive components in Rhizoma Smilacis Glabrae by capillary zone electrophoresis

A cyclodextrin-modified capillary zone electrophoretic method was developed for the separation and determination of trans-resveratrol, astilbin, taxifolin, shikimic acid, syringic acid and ferulic acid in Rhizoma Smilacis Glabrae. A running buffer comprising 20 mM borax and 2 mM β-cyclodextrin at pH 9.46 was used. These six components were well separated from each other within 8 min at a voltage of 25 kV, temperature of 25 °C and detection wavelength of 214 nm. The relative standard deviations of migration time ranged from 0.07% to 0.30% while those of the peak area ratios ranged from 2.66% to 4.12% for six determinations of analytes at the concentration of 5 and 25 μg mL⁻¹. The correlation coefficients of the calibration curves of analytes were all >0.9990, and the recoveries were from 97.5% to 108.3%. The method was successfully applied to determine the bioactive components in 12 samples of Rhizoma Smilacis Glabrae collected from China.     

A novel polymerase chain reaction (PCR) - denaturing gradient gel electrophoresis (DGGE) for the identification of Micrococcaceae strains involved in meat fermentations. Its application to naturally fermented Italian sausages

A new molecular method consisting of polymerase chain reaction (PCR) amplification and denaturing gradient gel electrophoresis (DGGE) of a small fragment from the 16S rRNA gene identified the Micrococcaceae strains isolated from natural fermented Italian sausages. Lactic acid bacteria, total aerobic mesophilic flora, Enterobacteriaceae and faecal enterococci were also monitored. Micrococcaceaea control strains from international collections were used to optimise the method and 90 strains, isolated from fermented sausages, were identified by biochemical tests and PCR-DGGE. No differences were observed between the methods used. The results reported in this paper prove that Staphylococcus xylosus is the main bacterium involved in fermented sausage production, representing, from the tenth day of ripening, the only Micrococcaceaea species isolated.     

The application of d-SPE in the QuEChERS method for the determination of PAHs in food of animal origin with GC–MS detection

This paper reports the evaluation of the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method for the determination of polycyclic aromatic hydrocarbons (PAHs) in food of animal origin with GC–MS detection. Although in the available literature, there is a lot of information about sample preparation method for PAHs determination in food samples, but the QuEChERS method application for PAHs determination in food of animal origin has not been reported as yet. The results showed that the best recovery ratios 72.4–110.8 % with relative standard deviation lower than 10 % for all determined compounds were received for the method with ethyl acetate as an extraction solvent, primary–secondary amine and C₁₈ sorbents and evaporation to dryness and dissolving the residues in the hexane. The limit of quantification ranged from 0.0003 to 0.0030 mg kg^?1 for pyrene and benzo[a]anthracene, respectively. This method was also used for the determination of PAHs in 15 samples of pork ham. In 8 of 15 samples selected, PAHs were identified. It was observed that in 6 cooked ham and one smoked and cooked samples, any PAHs were found. In other samples, which were smoked and roasted, some low concentration of PAHs was detected. In one sample benzo[a]pyrene (0.0015 mg kg^?1), in one sample benzo[b]fluoranthene (0.0015 mg kg^?1) and in one sample chrysene (0.0024 mg kg^?1) were detected. A number of other less harmful PAHs were also determined. There were no exceedances of maximum levels (according to Commission Regulation (EU) No 835/2011) for determined PAHs in any of the analysed samples.     

Optimised off-line SPE–GC–FID method for the determination of mineral oil saturated hydrocarbons (MOSH) in vegetable oils

An optimised off-line SPE–GC–FID method based on the use of silver-silica gel was developed for the determination of mineral oil saturated hydrocarbons (MOSH) in vegetable oils, including olive pomace oil. The method is specific in not including the aromatic hydrocarbons. The performance of different silica gels (untreated, activated and treated with silver nitrate) was compared in terms of capacity to retain fat and retention of interfering olefins present in particularly large amounts in refined olive oils. A coefficient of variation of 9% was obtained performing six replicate analyses of an extra virgin olive oil fortified with an amount of MOSH near the estimated LOQ (15 mg/kg). Recoveries were close to 100%. The use of activated aluminium oxide as an additional tool to eliminate interference by endogenous long-chain n-alkanes, is discussed.     

A Box–Behnken Design for Determining the Optimum Experimental Condition of Cake Batter Mixing

The application of Box-Behnken design to establish the optimum experimental condition of cake batter mixing is important because optimal mixing can ensure the dispersion of ingredients, and the incorporation and retention of air efficiently. A Box-Behnken with three factors such as mixing time, mixing speed, and cake loading was selected to observe the effects on batter density, cake density, hardness, springiness, cohesiveness, gumminess, chewiness, and resilience. The mixing times ranged from 6 to 20 min, mixing speeds from 90 to 120 rpm, and cake loadings from three to five cakes. A total of 15 runs of experiments where three levels attributed to each factor at high, central and low, and with additional three replicated center points were conducted. Using goal settings for each response based on quality parameters attained from the best definition of each response to achieve high cake quality, an optimum and feasible experimental condition of batter mixing was obtained at 9 min of mixing time, 90 rpm of mixing speed, and three cakes loading.     

Processes for the Production of Xylitol—A Review

Xylitol is a sugar alcohol that is used as a sweetener for diabetics and also for other purposes (e.g., in chewing gum). Industrially, xylitol is manufactured through a chemical process that has some disadvantages, including a high energy requirement, extensive purification steps, and a high cost of product. The microbial production of xylitol has been examined as an alternative to the chemical process, which on an industrial scale is time-consuming mainly due to sterilization and inoculum development. The enzymatic production of xylitol from lignocellulosics is an attractive and promising alternative to the chemical process, potentially eliminating the major disadvantages of conventional processes. This article reviews the literature on xylitol production processes and identifies ways to further improve xylitol synthesis to compete with the current chemical process.     

Monitoring the oxidation of almond oils by HS-SPME–GC–MS and ATR-FTIR: Application of volatile compounds determination to cultivar authenticity

In this study, the oxidative process of Spanish and American almond oils was monitored by using headspace solid-phase microextraction/gas chromatography–mass spectrometry (HS-SPME/GC–MS) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Oxidative stability of samples was evaluated after thermal treatment at 100 °C for 1, 3, 5, 7, 10, 15 and 20 days, in order to accelerate the lipid oxidation process. Variations observed in bands of infrared spectra were used to monitor the progress of the oxidation process of almond oils. HS-SPME–GC–MS was used for isolation and determination of volatile compounds in oxidised almond oils. The following aldehydes were identified in all samples: hexanal, (E)-2-heptenal, (E)-2-octenal, nonanal, (E)-2-nonenal, (E,E)-2,4-nonadienal and (E,E)-2,4-decadienal. Determination of volatile compounds resulting from lipid oxidation from three different almond cultivars (Spanish Guara and Marcona and Butte from USA) was performed to obtain a set of parameters for discrimination between Spanish and American cultivars. Successful discrimination was obtained in samples kept under thermal treatment for 7 days, by using HS-SPME/GC–MS combined with multivariate stepwise linear discriminant analysis (LDA).     

Levels of dioxins and dioxin-like PCBs in food of animal origin in the Netherlands during the period 2001–2011

ABSTRACT

The aim of this study was to assess levels of dioxins (polychlorinated dibenzo-p-dioxins and dibenzofurans, PCDD/Fs) and dioxin-like polychlorinated biphenyls (PCBs) (DL-PCBs) in food of animal origin produced in the Netherlands, including potential trends in time. Test results from about 2500 samples of animal derived food products (beef, veal, lamb, chicken, pork, deer, milk and eggs), sampled for the National Residue Monitoring Plan from 2001–2011, were evaluated. Most samples were screened with a bioassay and, if suspected, analysed by GC-HRMS. The fraction of samples which were non-compliant with European maximum levels was rather low, being below 1% for most food products, except for lamb. Exceedance of action levels was particularly observed for lamb and beef. To obtain an insight into background levels, a randomly taken part of the samples was directly analysed by GC-HRMS. In general, only minor decreases in mean PCDD/F and DL-PCB concentrations could be observed for the period 2001–2011. This may be due to a plateauing of current background levels but also to factors like the sensitivity of the analytical method.     

Comparison of ELISA and PCR vis-à-vis cultural methods for detecting Aeromonas spp. in foods of animal origin

The present study was conducted to assess the best method of the most commonly used methods for detection of aeromonads in foods of animal origin. With this objective an OMP based indirect plate ELISA and a duplex-PCR using primers targeting aerolysin gene and 16S rRNA gene and yielding amplicons of 252 bp and 599 bp, respectively, were standardized. The standardized protocols and the conventional cultural method were then compared for their respective sensitivities and specificities for detecting aeromonads from chicken and milk samples. Both the standardized assays were found to be highly specific for Aeromonas. The efficiency of the standardized indirect-ELISA and duplex-PCR protocols was assessed by artificial inoculation studies with varying concentrations of Aeromonas cells inoculated in chicken and milk samples followed by enrichment in Alkaline Peptone Water supplemented with 10 mg/ml cephalothin (APW-C) for 12 h. The results revealed that indirect-ELISA was able to detect a minimum of 10(3) cells/ml or g of Aeromonas cells in spiked milk and chicken samples, respectively. Whereas, duplex-PCR and cultural method were able to detect as low as 1 cell/ml or g of Aeromonas cells in spiked milk and chicken samples. The developed assays were also tested for their efficiency to detect Aeromonas spp. in naturally contaminated milk and chicken samples. Out of a total 50 milk samples screened for presence of Aeromonas by the three methods viz., indirect-ELISA, duplex-PCR and cultural method only 1 (2%) turned out to be positive showing positive results by all three methods. Similarly, 50 samples of chicken were tested by all three methods. Three samples (6%) turned out to be positive and here again by all the three methods.     

Classification of vegetable oils according to their botanical origin using amino acid profiles established by High Performance Liquid Chromatography with UV–vis detection: A first approach

A preliminary study using amino acid profiles to classify oils according to their botanical origin has been performed. Amino acid profiles were obtained from hydrolysis of proteins present in vegetable oils, and established by High Performance Liquid Chromatography (HPLC) with UV–vis detection. Proteins present in hazelnut, corn, soybean, olive, avocado, peanut and grapeseed oils were precipitated with acetone, and the residue was hydrolysed in acid medium. The amino acids obtained were derivatized with o-phthaldialdehyde and separated by HPLC. Peaks corresponding to 18 amino acids were observed using a C18 column and a gradient of acetonitrile–water in the presence of a 5 mM citric/citrate buffer at pH 6.5. The 16 peaks observed in each sample (arginine–serine and phenylalanine–leucine peaks appeared overlapped) were used to construct linear discriminant analysis (LDA) models. Ratios of the peak areas selected by pairs were used as predictors. With a LDA model, the oils were correctly classified with assignment probabilities higher than 99%.     

A multiplex polymerase chain reaction for the identification of cows’, goats’ and sheep's milk in dairy products

A multiplex PCR able to identify cows’, goats’ and sheep's milk in dairy products was developed. Specific primers were designed in the mitochondrial 12s and 16s rRNA genes so as to generate fragments of different length.The assay was applied to 19 cheeses from the retail trade in order to verify the label statements. The multiplex PCR results were confirmed by PCR-restriction fragment length polymorphism.The proposed multiplex PCR represents a rapid and sensitive method applicable on a routine basis. In fact it enables to detect, in a single step, goats’, sheep's and cows’ milk in dairy products with a good sensitivity threshold (0.5%).     

Analytical techniques for the study of polyphenol–protein interactions

This mini review focuses on advances in biophysical techniques to study polyphenol interactions with proteins. Polyphenols have many beneficial pharmacological properties, as a result of which they have been the subject of intensive studies. The most conventional techniques described here can be divided into three groups: (i) methods used for screening (in-situ methods); (ii) methods used to gain insight into the mechanisms of polyphenol–protein interactions; and (iii) methods used to study protein aggregation and precipitation. All of these methods used to study polyphenol–protein interactions are based on modifications to the physicochemical properties of the polyphenols or proteins after binding/complex formation in solution. To date, numerous review articles have been published in the field of polyphenols. This review will give a brief insight in computational methods and biosensors and cell-based methods, spectroscopic methods including fluorescence emission, UV-vis adsorption, circular dichroism, Fourier transform infrared and mass spectrometry, nuclear magnetic resonance, X-ray diffraction, and light scattering techniques including small-angle X-ray scattering and small-angle neutron scattering, and calorimetric techniques (isothermal titration calorimetry and differential scanning calorimetry), microscopy, the techniques which have been successfully used for polyphenol–protein interactions. At the end the new methods based on single molecule detection with high potential to study polyphenol–protein interactions will be presented. The advantages and disadvantages of each technique will be discussed as well as the thermodynamic, kinetic or structural parameters, which can be obtained. The other relevant biophysical experimental techniques that have proven to be valuable, such electrochemical methods, hydrodynamic techniques and chromatographic techniques will not be described here.