Randomly amplified polymorphic DNA (RAPD) was used to analyzed 78 samples comprises of certified reference materials (soya and maize powder), raw seeds (soybean and maize), processed food and animal feed. Combination assay of two arbitrary primers in the RAPD analysis enable to distinguish genetically modified organism (GMO) reference materials from the samples tested. Dendrogram analysis revealed 13 clusters at 45% similarity from the RAPD. RAPD analysis showed that the maize and soybean samples were clustered differently besides the GMO and non-GMO products.
A vortex-assisted liquid-liquid-liquid microextraction method followed by high performance liquid chromatography-diode array detection for the determination of fourteen phenolic acids (cinnamic, m-coumaric, chlorogenic, syringic, ferulic, o-coumaric, p-coumaric, vanillic, p-hydroxybenzoic, caffeic, 2, 4-dihydroxybenzoic, sinapic, gentisic and gallic acids) in honey, iced tea and canned coffee drink samples has been developed. The separation was achieved using a Poroshell 120-EC-C18 column under a gradient elution at a flow rate of 0.6mLmin-1 and mobile phase composed of methanol and acetic acid (1%, v/v). Under the optimum chromatographic conditions, the fourteen phenolic acids were separated in less than 32min. The extraction was performed using a small volume (400µL) of ternary organic solvents (1-pentanol, propyl acetate and 1-hexanol) dispersed into the aqueous sample (10mL) and assisted by vortex agitation (2500rpm for 45s), the analytes were next back-extracted from the organic solvent using 0.02M KOH (40µL) with vortex speed and time of 2500rpm and 60s, respectively. Under these conditions, enrichment factors of 30-193-fold were achieved. The limits of detection (LODs) were 0.05-0.68µgL-1. Recoveries in honey, iced tea and canned coffee drinks were in the range 72.2-112%. The method was successfully applied for the determination of the phenolic acids in honey, iced tea and canned coffee drinks.
Surface enhanced Raman scattering (SERS) DNA biosensing is an ultrasensitive, selective, and rapid detection technique with the ability to produce molecule-specific distinct fingerprint spectra. It supersedes the long amplicon based PCR assays, the fluorescence and spectroscopic techniques with their quenching and narrow spectral bandwidth, and the electrochemical detection techniques using multiplexing. However, the performance of the SERS DNA biosensor relies on the DNA probe length, platform composition, both the presence and position of Raman tags and the chosen sensing strategy. In this context, we herein report a SERS biosensor based on dual nanoplatforms with a uniquely designed Raman tag (ATTO Rho6G) intercalated short-length DNA probe for the sensitive detection of the pig species Sus scrofa. In the design of the signal probe (SP), a Raman tag was incorporated adjacent to the spacer arm, followed by a terminal thiol modifier, which consequently had a strong influence on the SERS signal enhancement. The detection strategy involves the probe-target DNA hybridization mediated coupling of the two platforms, i.e., the graphene oxide-gold nanorod (GO-AuNR) functionalized capture probe (CP) and SP-conjugated gold nanoparticles (AuNPs), consequently enhancing the SERS intensity by both the electromagnetic hot spots generated at the junctions or interstices of the two platforms and the chemical enhancement between the AuNPs and the adsorbed intercalated Raman tag. This dual platform based SERS DNA biosensor exhibited outstanding sensitivity in detecting pork DNA with a limit of detection (LOD) of 100 aM validated with DNA extracted from a pork sample (LOD 1 fM). Moreover, the fabricated SERS biosensor showed outstanding selectivity and specificity for differentiating the DNA sequences of six closely related non-target species from the target DNA sequences with single and three nucleotide base-mismatches. Therefore, the developed short-length DNA linked dual platform based SERS biosensor could replace the less sensitive traditional methods of pork DNA detection and be adopted as a universal detection approach for the qualitative and quantitative detection of DNA from any source.
Authentication of meat products is critical in the food industry. Meat adulteration may lead to religious apprehensions, financial gain and food-toxicities such as meat allergies. Thus, empirical validation of the quality and constituents of meat is paramount. Various analytical methods often based on protein or DNA measurements are utilized to identify meat species. Protein-based methods, including electrophoretic and immunological techniques, are at times unsuitable for discriminating closely related species. Most of these methods have been replaced by more accurate and sensitive detection methods, such as DNA-based techniques. Emerging technologies like DNA barcoding and mass spectrometry are still in their infancy when it comes to their utilization in meat detection. Gold nanobiosensors have shown some promise in this regard. However, its applicability in small scale industries is distant. This article comprehensively reviews the recent developments in the field of analytical methods used for porcine identification.
The bovine milk allergenic protein, 'β-lactoglobulin' is one of the leading causes of milk allergic reaction. In this research, a novel label-free non-faradaic capacitive aptasensor was designed to detect β-lactoglobulin using a Laser Scribed Graphene (LSG) electrode. The graphene was directly engraved into a microgapped (~ 95 µm) capacitor-electrode pattern on a flexible polyimide (PI) film via a simple one-step CO2 laser irradiation. The novel hybrid nanoflower (NF) was synthesized using 1,1'-carbonyldiimidazole (CDI) as the organic molecule and copper (Cu) as the inorganic molecule via one-pot biomineralization by tuning the reaction time and concentration. NF was fixed on the pre-modified PI film at the triangular junction of the LSG microgap specifically for bio-capturing β-lactoglobulin. The fine-tuned CDI-Cu NF revealed the flower-like structures was viewed through field emission scanning electron microscopy. Fourier-transform infrared spectroscopy showed the interactions with PI film, CDI-Cu NF, oligoaptamer and β-lactoglobulin. The non-faradaic sensing of milk allergen β-lactoglobulin corresponds to a higher loading of oligoaptamer on 3D-structured CDI-Cu NF, with a linear range detection from 1 ag/ml to 100 fg/ml and attomolar (1 ag/ml) detection limit (S/N = 3:1). This novel CDI-Cu NF/LSG microgap aptasensor has a great potential for the detection of milk allergen with high-specificity and sensitivity.
Food-derived bioactive proteins and peptides have gained acceptance among researchers, food manufacturers and consumers as health-enhancing functional food components that also serve as natural alternatives for disease prevention and/or management. Bioactivity in food proteins and peptides is determined by their conformations and binding characteristics, which in turn depend on their primary and secondary structures. To maintain their bioactivities, the molecular integrity of bioactive peptides must remain intact, and this warrants the study of peptide form and structure, ideally with robust, highly specific and sensitive techniques. Short single-stranded nucleic acids (i.e. aptamers) are known to have high affinity for cognate targets such as proteins and peptides. Aptamers can be produced cost-effectively and chemically derivatized to increase their stability and shelf life. Their improved binding characteristics and minimal modification of the target molecular signature suggests their suitability for real-time detection of conformational changes in both proteins and peptides. This review discusses the developmental progress of systematic evolution of ligands by exponential enrichment (SELEX), an iterative technology for generating cost-effective aptamers with low dissociation constants (Kd) for monitoring the form and structure of bioactive proteins and peptides. The review also presents case studies of this technique in monitoring the structural stability of bioactive peptide formulations to encourage applications in functional foods. The challenges and potential of aptamers in this research field are also discussed. Graphical abstract Advancing bioactive proteins and peptide functionality via aptameric ligands.
The performance of gas chromatography (GC) combined with a hybrid quadrupole time-of-flight (QTOF) mass spectrometry (MS) system for the determination of volatile and semi-volatile compounds in honey samples is evaluated. After headspace (HS) solid-phase microextraction (SPME) of samples, the accurate mass capabilities of the above system were evaluated for compounds identification. Accurate scan electron impact (EI) MS spectra allowed discriminating compounds displaying the same nominal masses, but having different empirical formulae. Moreover, the use of a mass window with a width of 0.005 Da provided highly specific chromatograms for selected ions, avoiding the contribution of interferences to their peak areas. Additional information derived from positive chemical ionization (PCI) MS spectra and ion product scan MS/MS spectra permitted confirming the identity of novel compounds. The above possibilities are illustrated with examples of honey aroma compounds, belonging to different chemical classes and containing different elements in their molecules. Examples of compounds whose structures could not be described are also provided. Overall, 84 compounds, from a total of 89 species, could be identified in 19 honey samples from 3 different geographic areas in the world. The suitability of responses measured for selected ions, corresponding to above species, for authentication purposes is assessed through principal components analysis.
Fourier transform infrared spectroscopy (FTIR) combined with multivariate calibration of partial least square (PLS) was developed and optimized for the analysis of Nigella seed oil (NSO) in binary and ternary mixtures with corn oil (CO) and soybean oil (SO). Based on PLS modeling performed, quantitative analysis of NSO in binary mixtures with CO carried out using the second derivative FTIR spectra at combined frequencies of 2977-3028, 1666-1739, and 740-1446 cm(-1) revealed the highest value of coefficient of determination (R (2), 0.9984) and the lowest value of root mean square error of calibration (RMSEC, 1.34% v/v). NSO in binary mixtures with SO is successfully determined at the combined frequencies of 2985-3024 and 752-1755 cm(-1) using the first derivative FTIR spectra with R (2) and RMSEC values of 0.9970 and 0.47% v/v, respectively. Meanwhile, the second derivative FTIR spectra at the combined frequencies of 2977-3028 cm(-1), 1666-1739 cm(-1), and 740-1446 cm(-1) were selected for quantitative analysis of NSO in ternary mixture with CO and SO with R (2) and RMSEC values of 0.9993 and 0.86% v/v, respectively. The results showed that FTIR spectrophotometry is an accurate technique for the quantitative analysis of NSO in binary and ternary mixtures with CO and SO.
The major compounds in honey are carbohydrates such as monosaccharides and disaccharides. The same compounds are found in cane-sugar concentrates. Unfortunately when sugar concentrate is added to honey, laboratory assessments are found to be ineffective in detecting this adulteration. Unlike tracing heavy metals in honey, sugar adulterated honey is much trickier and harder to detect, and traditionally it has been very challenging to come up with a suitable method to prove the presence of adulterants in honey products. This paper proposes a combination of array sensing and multi-modality sensor fusion that can effectively discriminate the samples not only based on the compounds present in the sample but also mimic the way humans perceive flavours and aromas. Conversely, analytical instruments are based on chemical separations which may alter the properties of the volatiles or flavours of a particular honey. The present work is focused on classifying 18 samples of different honeys, sugar syrups and adulterated samples using data fusion of electronic nose (e-nose) and electronic tongue (e-tongue) measurements. Each group of samples was evaluated separately by the e-nose and e-tongue. Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) were able to separately discriminate monofloral honey from sugar syrup, and polyfloral honey from sugar and adulterated samples using the e-nose and e-tongue. The e-nose was observed to give better separation compared to e-tongue assessment, particularly when LDA was applied. However, when all samples were combined in one classification analysis, neither PCA nor LDA were able to discriminate between honeys of different floral origins, sugar syrup and adulterated samples. By applying a sensor fusion technique, the classification for the 18 different samples was improved. Significant improvement was observed using PCA, while LDA not only improved the discrimination but also gave better classification. An improvement in performance was also observed using a Probabilistic Neural Network classifier when the e-nose and e-tongue data were fused.
Recent foodborne outbreaks in multiple locations necessitate the continuous development of highly sensitive and specific biosensors that offer rapid detection of foodborne biological hazards. This work focuses on the development of a reduced graphene oxide‑titanium dioxide (rGO-TiO2) nanocomposite based aptasensor to detect Salmonella enterica serovar Typhimurium. A label-free aptamer was immobilized on a rGO-TiO2 nanocomposite matrix through electrostatic interactions. The changes in electrical conductivity on the electrode surface were evaluated using electroanalytical methods. DNA aptamer adsorbed on the rGO-TiO2 surface bound to the bacterial cells at the electrode interface causing a physical barrier inhibiting the electron transfer. This interaction decreased the DPV signal of the electrode proportional to decreasing concentrations of the bacterial cells. The optimized aptasensor exhibited high sensitivity with a wide detection range (108 to 101 cfu mL-1), a low detection limit of 101 cfu mL-1 and good selectivity for Salmonella bacteria. This rGO-TiO2 aptasensor is an excellent biosensing platform that offers a reliable, rapid and sensitive alternative for foodborne pathogen detection.
INTRODUCTION: There is a lack of information on the trans fatty acid (TFA) content in Malaysian foods. The objective of this study is to determine the TFA content of bakery products, snacks, dairy products, fast foods, cooking oils and semisolid fats, and breakfast cereals and Malaysian fast foods. This study also estimated the quantity of each isomer in the foods assayed.
METHODS: The trans fatty acid content of each food sample was assessed in duplicate by separating the fatty acid methyl esters (FAME) in a gas chromatography system equipped with HP-88 column (USA: split ratio 10: 1) for cis/trans separation. Five major TFA isomers, palmitoelaidic acid (16: 1t9), petroselaidic acid (18:1t6), elaidic acid (18:1t9), vaccenic acid (18: 1t11) and linoelaidic acid (18:2t9, 12), were measured using gas chromatography (GC) and the data were expressed in unit values of g/100 g lipid or g/100 g food.
RESULTS: The total TFA contents in the studied foods were < 0.001 g-8.77 g/100 g lipid or < 0.001 g-5.79 g/100 g foods. This value falls within the standard and international recommendation level for TFA. The measured range of specific TFA isomers were as follows: palmitoelaidic acid (< 0.001 g-0.26 g/100 g lipid), petroselaidic acid (< 0.001 g - 3.09 g/100 g lipid), elaidic acid (< 0.001 g-0.87 g/100 g lipid), vaccenic acid (< 0.001 g-0.41 g/100 g lipid) and linoelaidic acid (< 0.001 g-6.60 g/100 g lipid).
CONCLUSION: These data indicate that most of the tested foods have low TFA contents (< 1 g/100 g lipid).
A new extraction and cleanup procedure with gas chromatography was developed for the sensitive determination of acephate, dimethoate, malathion, diazinon, quinalphos, chlorpyrifos, profenofos, alpha-endosulfan, beta-endosulfan, chlorothalonil and carbaryl using 1-chloro-4-fluorobenzene as an internal standard in fruits and vegetables. Several extracting and eluting solvents for solid-phase extraction were investigated. The overall extracting solvent with a mixture of acetone:ethyl acetate:hexane (10:80:10, v/v/v) and a eluting solvent of 5% acetone in hexane used with the RPC18 cartridge gave the best recovery for all of the investigated pesticides, and minimized the interference from co-extractants. Under the optimal extraction and clean-up conditions, recoveries of 85 - 99% with RSD < 5.0% (n = 3) for most of the pesticides at the 0.02 - 0.5 mg/kg level were obtained. The limit of detection was between 0.005 - 0.01 mg/kg and the limit of quantification was 0.01 mg/kg. This analytical procedure was characterized with high accuracy and acceptable sensitivity to meet requirements for monitoring pesticides in crops.
Gelatin is a highly purified animal protein of pig, cow, and fish origins and is extensively used in food, pharmaceuticals, and personal care products. However, the acceptability of gelatin products greatly depends on the animal sources of the gelatin. Porcine and bovine gelatins have attractive features but limited acceptance because of religious prohibitions and potential zoonotic threats, whereas fish gelatin is welcomed in all religions and cultures. Thus, source authentication is a must for gelatin products but it is greatly challenging due to the breakdown of both protein and DNA biomarkers in processed gelatins. Therefore, several methods have been proposed for gelatin identification, but a comprehensive and systematic document that includes all of the techniques does not exist. This up-to-date review addresses this research gap and presents, in an accessible format, the major gelatin source authentication techniques, which are primarily nucleic acid and protein based. Instead of presenting these methods in paragraph form which needs much attention in reading, the major methods are schematically depicted, and their comparative features are tabulated. Future technologies are forecasted, and challenges are outlined. Overall, this review paper has the merit to serve as a reference guide for the production and application of gelatin in academia and industry and will act as a platform for the development of improved methods for gelatin authentication.
The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely used in exotic foods and traditional medicines. Currently available polymerase chain reaction (PCR) assays to identify MBT lack automation and involve long targets which break down in processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this research gap for the first time through the combination of 120- and 141-bp targets from MBT and eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal preparations. This authentication ensures better security through automation, internal control and short targets that were stable under the processing treatments of foods and medicines. A melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25-153.00%, PCR efficiency of 99-100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50-7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32-28.90 ± 0.42) and melting curves (74.63-78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth.
The influence of temperature-time combinations on non-volatile compound and taste traits of beef semitendinosus muscles tested by the electronic tongue was studied. Single-stage sous-vide at 60 and 70 °C (6 and 12 h), and two-stage sous-vide that sequentially cooked at 45 °C (3 h) and 60 °C (either 3 or 9 h) were compared with traditional cooking at 70 °C (30 min). Umami was better explained in the given model of partial least squares regression than astringency, sourness, saltiness, bitterness, and richness. Sous-vide at 70 °C for 12 h characterized the most umami, likely adenosine-5'-monophosphate (AMP) and guanosine-5'-monophosphate (GMP) as significant contributors. Two-stage sous-vide projected higher histidine, leucine, inosine, and hypoxanthine with the astringent and sour taste significant after 6 and 12 h cooking, respectively. Equivalent umami concentration (EUC) between umami amino acids and umami nucleotides showed a strong relationship to umami taste assessed by the electronic tongue.
Clenbuterol (CLB) is an antibiotic and illegal growth promoter drug that has a long half-life and easily remains as residue and contaminates the animal-based food product that leads to various health problems. In this work, electrochemical immunosensor based on poly(3,4-ethylenedioxythiophene)/graphene oxide (PEDOT/GO) modified screen-printed carbon electrode (SPCE) for CLB detection was developed for antibiotic monitoring in a food product. The modification of SPCE with PEDOT/GO as a sensor platform was performed through electropolymerization, while the electrochemical assay was accomplished while using direct competitive format in which the free CLB and clenbuterol-horseradish peroxidase (CLB-HRP) in the solution will compete to form binding with the polyclonal anti-clenbuterol antibody (Ab) immobilized onto the modified electrode surface. A linear standard CLB calibration curve with R² = 0.9619 and low limit of detection (0.196 ng mL-1) was reported. Analysis of milk samples indicated that this immunosensor was able to detect CLB in real samples and the results that were obtained were comparable with enzyme-linked immunosorbent assays (ELISA).
An efficient and rapid method for the analysis of pesticide residues in cocoa beans using gas and liquid chromatography-tandem mass spectrometry was developed, validated and applied to imported and domestic cocoa beans samples collected over 2 years from smallholders and Malaysian ports. The method was based on solvent extraction method and covers 26 pesticides (insecticides, fungicides, and herbicides) of different chemical classes. The recoveries for all pesticides at 10 and 50 μg/kg were in the range of 70-120% with relative standard deviations of less than 20%. Good selectivity and sensitivity were obtained with method limit of quantification of 10 μg/kg. The expanded uncertainty measurements were in the range of 4-25%. Finally, the proposed method was successfully applied for the routine analysis of pesticide residues in cocoa beans via a monitoring study where 10% of them was found positive for chlorpyrifos, ametryn and metalaxyl.
Graphene-magnetite composite (G-Fe3O4) was successfully synthesized and applied as adsorbent for magnetic solid phase extraction (MSPE) of two phenolic acids namely 4-hydroxybenzoic acid (4-HB) and 3,4-dihydroxybenzoic acid (3,4-DHB) from stingless bee honey prior to analysis using high performance liquid chromatography with ultraviolet-visible detection (HPLC-UV/Vis). Several MSPE parameters affecting extraction of these two acids were optimized. Optimum MSPE conditions were 50 mg of G-Fe3O4 adsorbent, 5 min extraction time at 1600 rpm, 30 mL sample volume, sample solution pH 0.5, 200 µL methanol as desorption solvent (5 min sonication assisted) and 5% w/v NaCl. The LODs (3 S/N) calculated for 4-HB and 3,4-DHB were 0.08 and 0.14 µg/g, respectively. Good relative recoveries (72.6-110.6%) and reproducibility values (RSD