Processed Food Samples Research Articles

Colorimetric loop‐mediated isothermal amplification (LAMP) for identification of crocodile meat in raw meat and commercial processed meat products

SummaryDeliberate and accidental adulteration or substitution of crocodile meat with other meats is possibly seen in meat products, which could be challenging to identify. This research aims to develop a new loop‐mediated isothermal amplification for accurately identifying crocodile meat in raw meat and processed food samples. Conditions for LAMP were initially optimised. The LAMP was proven for its high specificity to only crocodile DNA. The LAMP revealed a detection limit of 1 pg/μL crocodile DNA and 0.01% (w/w) crocodile meat in meat admixtures. From forty‐seven commercial processed meat samples, the LAMP detected crocodile content in ten products with the declaration of crocodile on their labels. Without the requirement of DNA purification, the direct LAMP showed its ability to detect crocodile content in both fresh meat and processed meat samples. This LAMP could become a promising alternative to support the food industry, especially for on‐site services and limited‐resource laboratories.

Sensitivity of commercial rapid test kit to pork contamination in processed foods

Guaranteeing halal products has now become a necessity, especially for food products. This is intended to ensure that the food produced is not contaminated with non-halal ingredients, including pork. Pork contamination in processed meat foods such as meatballs is still often found. Various tests can be done to detect the presence of pork in processed foods. One of them is a rapid test using the LFIA method. This test is widely used because it is more efficient, economical, and easy to prepare samples. A rapid pork contamination test kit (XEMA) has been circulating in Indonesia. In the research, the sensitivity of this rapid test kit was tested on processed meat foods with various concentrations of pork and variations in the main ingredients. The color test shows that the simulated samples of beef meatballs without added pork are dark greyish white, as are the simulated samples with concentrations of 1% and 10%. Meanwhile, samples with concentrations of 20% and 40% have a paler color. Meanwhile, there was no significant difference in the variation in pork concentration in meatballs with the main ingredients of chicken and fish. For smell and texture, there were no significant differences in the simulated samples, both the control and samples with varying concentrations. From testing, it is known that the test kit can detect the presence of pork up to a concentration of 10% in samples, with the main ingredients being beef, chicken, or fish. These results indicate that this rapid test kit can well detect pork contamination in processed food samples.

Development of a PCR Method to Detect a Mysid Species Causing False Positives in Tests for Crustacean Allergens

Mysids are small crustaceans that are closely related to shrimp/prawns and crabs but not subject to food allergen labeling requirements for raw materials. In the past, a processed food that contained Japanese smelt (wakasagi) was suspected of producing a false-positive result in shrimp/prawn and crab allergen test because of the presence of consumed mysids. However, there was no reported methods to confirm mysid presence. Therefore, we developed a PCR method to detect mysids. The developed PCR method had high specificity for a mysid species, with no amplification observed from samples of shrimp, crab, krill, mantis shrimp, or the meat of Japanese smelt. In addition, DNA extracted from the internal organs of Japanese smelt was amplified by this PCR method, and sequencing revealed mysid DNA. This confirmed that mysids remained in the internal organs of Japanese smelt following consumption. This PCR method for mysid detection even amplified Japanese smelt-containing processed food samples that were suspected to have produced a false-positive result in shrimp/prawn and crab ELISA. Thus, this PCR method would enable to detect such false positives are caused by mysid contamination.

Absolute quantification of targeted rabbit liver- and meat tissue-specific peptide markers in highly processed food products

A highly sensitive and selective method for the simultaneous absolute quantification of peptides unique to rabbit meat- and liver-specific tissue was developed using liquid chromatography - triple quadrupole mass spectrometry. Two rabbit skeletal muscle-specific peptides (SSVFVADPK and PHSHPALTPEQK), three rabbit liver tissue-specific peptides (FNLEALVTHTLPFEK, AILNYVANK, and TELAEPTSTR) and one peptide specific to both rabbit offal and skeletal muscle tissue (AFFGHYLYEVAR) were monitored. Analyses were performed using peptides labelled with stable isotopes (13C and 15N) as internal standards. Fifteen food samples containing rabbit meat and/or liver were analysed to verify compliance of the rabbit meat and liver composition with product labelling. One sample was adulterated with undeclared rabbit liver. The limit of detection and limit of quantification for the selected peptides of interest were in the range of 0.17 to 0.35 ng/mg and 0.57 to 1.17 ng/mg, respectively. The method may be useful for the determination of rabbit meat and liver tissue in highly processed food samples.

Determination of Gluten Contamination in Foods Available on the Turkish Market via Enzyme-Linked Immunosorbent Assay (ELISA)

Wheat, along with maize and rice, is one of the “big 3” cereal crops, and although it is a major component of many food products, it is one of the top eight food allergens listed by the Codex Alimentarius. The wheat protein gluten is responsible for several diseases including wheat allergy (WA), gluten sensitivity (GS), and celiac disease (CD). The presence of a “gluten free” label on food products specially produced for these patients facilitates the selection of safe products. To enforce food labeling regulations and quantitatively assess the potential degree of gluten contamination in gluten-free foods, standardized analytical methodologies are required. In recent years, multiple techniques for detecting gluten utilizing enzyme-linked immunosorbent assays (ELISAs), lateral flow devices/dipsticks, polymerase chain reaction, and mass spectrometry have been developed. In this study, the R-Biopharm Ridascreen® Gliadin Kit, a commercial ELISA kit, was used to assess the degree of gluten contamination, if any, in naturally gluten-free foods and foods labeled “gluten free” obtained from Izmir Province and its surroundings. In total, 71 samples (45 naturally gluten-free food samples, and 26 processed food samples labeled “gluten free”) were investigated for gluten contamination. Gluten extraction was performed with cocktail solution (patented, R-Biopharm), and contamination was analyzed quantitatively using the ELISA method based on the antigen–antibody relationship with the R-Biopharm Ridascreen® Gliadin Kit. In total, 94.37% of the samples were found to be gluten-free, 4.23% were found to be low in gluten, and 1.40% were found to be beyond the limit. In conclusion, gluten-free product manufacturers should implement comprehensive measures in the production stage to eliminate contamination risks, and individuals with celiac disease or gluten sensitivity should be vigilant when reading labels and inquiring about food preparation to manage symptoms and maintain good health.

Quantification of Resistant starch, Non-Resistant Starch and Total Starch Content of Some Processed Foods Sold in Enugu Metropolis

Resistant starch, which is produced by the interaction between starch, lipids and proteins, is an important part of diet due to its ability to escape digestion in the gut, thus reducing the blood glucose concentration. Resistant starch contents of some processed food products sold in Enugu Metropolis were quantified using Megazyme resistant starch kit (Megazyme Bray, Ireland). The processed foods were grouped into cereals (Oatmeal flours, basmati rice flours), Legumes (beans flour), Fruits (Unripe plantain flours) and Tubers (yam flours and potato starch flours). Mean values were determined with MegaCalc and data were expressed as percentage weight per weight. Among the processed food samples analyzed, plantain flour (82.314% Ayoola foods plantain flour, 82.757 % for naked amala plantain flour and 65.648% for deluxe plantain flour) contains the highest concentration of resistant starch. In descending order of resistant starch concentration is as follows; rice samples (lamis basmati rice, 43.427%, TBR basmati rice (39.268%). The resistant starch content of yam samples was 38.769% (Ayo ola poundo yam flour) and 28.354% for Ola ola yam flour respectively. The least resistant starch content was detected in beans sample; deluxe bean flour (~6.788%), Ayo ola bean flour (~3.481 %) and tasty pot bean flour (~4.345 %) and oat flour; lecker white oat 1.503%), health-wise oat meal (3.497%) and elkaris swallow oat (1.325%) respectively. Plantain and rice flours should be considered most for consumption, due to the high resistant starch content that confer many of health benefits.

Bisphenol A contamination in processed food samples: an overview

Bisphenol A contamination in processed food samples: an overview

Optimization and evaluation of a multiplex PCR assay for detection of Staphylococcus aureus and its major virulence genes for assessing food safety.

Staphylococcus aureus is a natural commensal microflora of humans which causes opportunistic infections due to its large arsenal of exotoxins, invasion, immune evasion, and antibiotic resistance mechanisms. The primary goal of this study is to develop a multiplex PCR (mPCR) assay for simultaneous detection of Staphylococcus aureus (nuc) and its virulence genes coding for prominent exotoxins namely alpha hemolysin (hla), enterotoxins A (sea), enterotoxin B (seb), toxic shock syndrome toxin (tsst-1), and the gene coding for methicillin resistance (mecA). A competitive internal amplification control (IAC) was included in the assay to exclude the false negative outcomes. Highly specific primer pairs were designed for the target genes using in silico resources. At the outset, monoplex PCRs were standardized using reference S. aureus strains. Primer specificity to the target genes was authenticated through restriction digestion analysis of amplified PCR products. Multiplex PCR was optimized in increments of one gene starting with nuc and IAC amplified simultaneously using one pair of primers (nuc) in a competitive manner. The mPCR assay was found to be highly sensitive with a detection limit of ~10 CFUs per reaction for pure cultures. Multiplex PCR assay was further evaluated on the retail and processed food samples to test the prevalence of S. aureus and study their exotoxin profiles. Of the 57 samples examined, 13 samples (22.80%) were found to be contaminated with S. aureus whose DNA was extracted after a 6-h enrichment period. Among these, a high percentage of hemolytic and enterotoxin A positive strains were encountered. The mPCR assay developed in this study would be a useful tool for rapid and reliable monitoring of S. aureus for food quality testing and from clinical infections.

Determination and extraction of acrylamide in processed food samples using alkanol-based supramolecular solvent-assisted dispersive liquid-liquid microextraction coupled with spectrophotometer: Optimization using factorial design

In current study, simple, rapid, sensitive and selective alkanol-based supramolecular solvent-assisted dispersive liquid-liquid microextraction (SSA-DLLME) method was first time developed for the extraction and determination of acrylamide (AA) in coffee, chocolate, roasted nuts, French fries, cereals, biscuits, chips, bread and caramelized fruit using UV–visible spectrophotometer. Different parameters were investigated and optimized to know their effects on the extraction of AA. Studies have shown that effective extraction of AA was achieved at short vortex time (2 min) and acidic pHs. Extraction of AA was inversely proportional to sample volume. The linear range (LR) of the SSA-DLLME method was 0.6–350 ng mL−1. The limit of detection (LOD), and limit of quantification (LOQ) were found to be 0.2 ng mL−1 and 0.6 ng mL−1, respectively. In addition, enhancement factor and preconcentration factor was 114 and 200, respectively. Factorial design was utilized to better understand dual effluents of factors on extraction recovery of the AA. Optimized SSA-DLLME method was successfully applied to process food samples for the determination of AA.

Molecular Detection of Virulence Genes in Escherichia coli and Salmonella enterica Isolated from Minimally Processed Foods Sold within Kaduna Metropolis

Minimally processed foods are widely consumed in Kaduna, Nigeria and the world at large. However, poor quality control during their processing make them prone to contamination by enteropathogens known to possess multiple virulence genes, possibly causing morbidities and mortalities. Investigating the prevalence and virulence genes of food-borne bacteria is therefore paramount. This study was aimed at detecting virulence genes in enteropathogenic bacteria associated with minimally processed foods sold within Kaduna metropolis. Samples of sliced watermelons, peeled sugarcanes, peeled and sliced pineapples and unshelled coconuts (n=140) were collected from Kaduna North, Kaduna South and Igabi LGAs. Upon enrichment, samples were inoculated onto Eosin-Methylene Blue (EMB) and Cefixime-Tellurite-Sorbitol MacConkey (CT-SMAC) agar and incubated for 18 hours at 35oC. Isolates were identified using MicrobactTM 24E Gram Negative Bacteria Identification System for Enterobacteriaceae. Thereafter, E. coli and Salmonella enterica isolates’ DNA was extracted and purified using AccuPrep Genomic DNA Extraction Kit; used as a template for the PCR amplification of the bundle forming pilus (bfpA) and invasion (invA) genes; and then the PCR products were visualised using agarose gel electrophoresis documentation system. The results of the MicrobactTM analyses showed widespread contamination of the samples with E. coli (22 isolates) and Salmonella enterica (3 isolates). There was no statistically significant difference in the bacterial contaminants isolated from the various sampling areas (one-way ANOVA: p = 0.577); similarly, the type of the minimally processed food samples had no influence on the prevalence of E. coli and Salmonella enterica isolates (p = 0.345). PCR result revealed that the invA virulence gene (284bp) was present in one Salmonella enterica isolate. The presence of invA gene in the Salmonella enterica isolate indicated that the isolate is a virulent strain, which can cause food-borne infections. These results indicated the contamination of the minimally processed fruits with enteropathogens, hence, standards of quality control should be enshrined, towards safer foods and enhanced health of the consuming populace.

Detection of porcine species DNA on meat processed food samples (shredded meat) using Real-Time PCR

DNA detection of porcine species in processed meat samples (shredded meat) was carried out as an effort to enrich the literature and reference alternative testing methods in the control of halal products circulating in Indonesia. The purpose of this study was to detect the DNA of porcine species in samples using real-time PCR. The research method used is a qualitative technique. This technique analyzes the CT value of the sample compared to the positive control by looking at the formation of the sigmoid curve if amplification occurs. The controls used consisted of negative control, positive control, other DNA control, and LOD control. The extraction technique used is the column centrifuge technique. The extraction results were then measured using a nano photometer to see the purity and concentration of the extracted DNA. Based on the research, it is known that the extraction results read on a nanophotometer at a wavelength of A260 / A280 show a concentration of 24,350 with a purity value of 21,164. For sample testing, the results of real-time PCR analysis showed the sample was detected at CT 39.77, positive control at CT 31.66, and LOD at 28.32. for negative control and specificity using other DNA was not detected. The conclusion of this research is that the sample used can be detected by the presence of porcine DNA.

Novel powder-XRD method for detection of acrylamide in processed foods

Acrylamide (AA) is a toxin in food systems associated with neurotoxicity, carcinogenicity, genotoxicity, and cardiotoxicity. While several approaches exist, this work details the first-of-its-kind application of Powder X-Ray Diffraction (PXRD) for the detection of AA in food systems. A new methodology was validated as per ICH guidelines. Various x-ray tube combinations such as current (25–45 mA), voltage (25–45 kV), and scanning parameters such as step size (0.006–0.10°) and time-per-step (50–150 s) were experimented on 10–80° angle 2theta (2θ). In this investigation, more diffracted peaks were detected on time per step 151.210 [s], 0.0135[°] step size @ 35 mA/45 kV through phase quantification. The developed method was successfully applied to real processed food samples for qualitative analysis of AA. In addition, the influence of oil frying temperature–time conditions on AA content was studied. Overall, this work highlights the prospects of PXRD-based food quality and safety evaluation.

CRISPR-Cas system meets DNA barcoding: Development of a universal nucleic acid test for food authentication

Food authenticity is a critical issue owing economic or ethnic considerations. Herein, we introduce CRISPR-Cas system to food authentication via detecting DNA barcodes of livestock and poultry in both unprocessed and processed food samples, allowing for a specific molecular traceability of meat species. We integrated the specific sequence recognition of CRISPR/Cas12 system and the efficient amplification of PCR method to identify DNA barcodes to improve the accuracy of pork component identification. This method, termed CRISPR-Cas-based PCR DNA barcoding method (CAPCOD), can detect pork contamination diluted into 0.1% (w/w) in raw meat mixture, which ranks one of the most sensitive assays for food authenticity. And it has been applied to identify pork contents in complex processed halal food including beef luncheon meat, spiced beef and non-halal food including sausage, dried pork slices, pork balls and ham sausage. CRISPR-Cas-based DNA barcoding method is promising to serve as sensitive and reliable analytical tools for food authenticity.

Saltquanti – Development of a new portable device to analyse salt content in food

Excessive salt intake is a growing concern in society, impacting on a number of serious clinical conditions, such as the development of hypertension and increased mortality/morbidity caused by cardiovascular diseases. The urgent need to introduce in situ assessment of the salt content of processed foods led to the development of SALTQUANTI, a portable device developed and patented by a consortium promoted by the company EVOLEO Technologies and the University of Porto, which combines sample preparation with the analytical detection by means of a sodium selective electrode with potentiometric detection. The device includes the milling and stirring process of the sample, together with the analytical measurement and data interpretation via a touch display. Data for the analytical validation of the method were obtained from 20 different processed food samples. The linearity of the method was in the range from 0.06 to 6.00 g salt/100 g food. The precision was of 6% on average, considering repeatability of results for different types of processed food and the accuracy was of 9% on average. Interlaboratorial comparison showed deviation of results of less than 20 % for the different types of samples. The analytical procedure proved to be simple and reliable, and automation enabled the analysis in less than 5 min with an acceptable uncertainty.

A Practical Approach to Identifying Processed White Meat of Guinea Fowl, Rabbit, and Selected Fish Species Using End-Point PCR.

Among the foodstuff, most often adulterated are white meat and meat products as well as fish and fish products. For this reason, we evaluated in practice the possibilities of identifying selected species of white meat, i.e., guinea fowl and rabbit as well as four fish species, namely, pollock, hake, sole, and panga, in thermally treated samples. The aim was to check whether the previously published in the scientific literature species-specific primers allows for the identification of processed meat using the end-point PCR technique. To identify the six species, the short sequence fragments (from 130 to 255 bp) of 12S rRNA, COX3, mitochondrial ATP synthase Fo subunit 6 (ATP6) gene, pantophysin (Pan I) gene, 5S rRNA gene, and microsatellite markers (locus: Phy01-KUL) were selected. Stability and specificity of the six pair primers were evaluated on cooked and autoclaved meat, and commercially processed food samples such as rabbit and guinea pâtés, ready-made baby food, and breaded, fried, and deep-frozen fish products. The method proved to be useful for the authentication of severely processed food products against fraudulent species substitution and mislabelling and this approach may be an alternative to more advanced and more expensive PCR techniques.

A broad-spectrum antibody based bio-barcode fluorescence immunosensor for simultaneous detection of eight heterocyclic aromatic amines (HAAs) in heat processed meat

• A broad-spectrum specific antibody prepared can equivalently recognize 8 HAAs. • This immunosensor has achieved detecting total amount of 8 HAAs in one test process. • This immunosensor is simple, convenient, and effective for detection of 8 HAAs. • The bio-barcode assay has been introduced to achieve signal amplification detection. • This immunosensor has good reliability and practicability for detection of 8 HAAs. Heterocyclic aromatic amines (HAAs) as carcinogenic and mutagenic substance are naturally produced in heat processed meat. In this work, we have proposed a novel and sensitive fluorescence immunosensor for simultaneous detecting eight HAAs in heat processed meat samples. Herein, a broad-spectrum antibody simultaneously recognizing eight HAAs, amino modified ssDNA and Alexa Flour 488 labeled ssDNA are connected to silica nanoparticles for preparing the bio-barcode immune signal probe. Coating antigens are linked to AuNPs to form the coupling complex as fluorescent quencher. Based on the competition between quencher and analyte for binding to antibody of signal probe, as the analyte increases, the amount of quenchers combined to the signal probe decreases, and the fluorescence intensity increases. With MeIQx as the analyte, the immunosensor has a wide linear range from 0.1–200 μg L −1 with low limit of detection of 0.01 μg L −1 . The applicability of the fluorescence immunosensor is evaluated by measuring total amount of eight HAAs in real heat processed meat. The measured results of this fluorescence immunosensor are in good agreement with that of UPLC-MS/MS and ELISA. This fluorescence immunosensor provides an effective detection tool for the simultaneous detection of multiple HAAs in heat processed food samples.

Heavy Metal Tolerance Trend in Extended-Spectrum β-Lactamase Encoding Strains Recovered from Food Samples.

This study evaluates bacteriological profiles in ready-to-eat (RTE) foods and assesses antibiotic resistance, extended-spectrum β-lactamase (ESBL) production by gram-negative bacteria, and heavy metal tolerance. In total, 436 retail food samples were collected and cultured. The isolates were screened for ESBL production and molecular detection of ESBL-encoding genes. Furthermore, all isolates were evaluated for heavy metal tolerance. From 352 culture-positive samples, 406 g-negative bacteria were identified. Raw food samples were more often contaminated than refined food (84.71% vs. 76.32%). The predominant isolates were Klebsiella pneumoniae (n = 76), Enterobacter cloacae (n = 58), and Escherichia coli (n = 56). Overall, the percentage of ESBL producers was higher in raw food samples, although higher occurrences of ESBL-producing E. coli (p = 0.01) and Pseudomonas aeruginosa (p = 0.02) were observed in processed food samples. However, the prevalence of ESBL-producing Citrobacter freundii in raw food samples was high (p = 0.03). Among the isolates, 55% were blaCTX-M, 26% were blaSHV, and 19% were blaTEM. Notably, heavy metal resistance was highly prevalent in ESBL producers. These findings demonstrate that retail food samples are exposed to contaminants including antibiotics and heavy metals, endangering consumers.

Development of a new and sensitive method for the detection of pork adulteration in gelatin and other highly processed food products

ABSTRACT Porcine adulteration has become a major concern amongst communities including Muslims and Jews. Reliable detection of the presence of porcine DNA in gelatin and highly processed food samples is essential for Halal and kosher food control. In this study, a forensic approach involving DNA isolation and real-time polymerase chain reaction (PCR) was performed to detect the presence of porcine DNA in commercial gelatin and processed foods containing porcine by-products. The method was compared with commercially sensitive porcine DNA detection kits (Biotecon and R-Biopharm) which are commonly used in food control laboratories for Halal and kosher authentication. The results indicated that a newly developed method called TübiGel was at least 10 times more sensitive for porcine DNA detection. The TübiGel method was found to have a detection limit of 0.01% porcine gelatin, whilst the Biotecon method had 0.1% and R-Biopharm method detected >5% porcine gelatin. The forensic DNA isolation approach of the TübiGel method was found to be a critical step. In addition, real-time PCR of TübiGel method was also found to detect porcine DNA better than real-time PCR of commercial kits.

Salting-Out Induced Liquid-Liquid Microextraction: an Environmentally Friendly Approach to Preservative Determination in Food Samples

The salting-out induced liquid-liquid microextraction technique was properly evaluated for preservative determination in processed food samples. In the best conditions, 0.5 g sample and 4.5 mL acetate buffer, at pH 4.2, were extracted with 1 mL acetonitrile, followed by agitation, addition of 2 g MgSO4, a new agitation step, and centrifugation. Afterwards, the extract was diluted with ultrapure water and analyzed by high-performance liquid chromatography with ultraviolet detection. Correlation coefficients above 0.99, LOQs between 0.03 and 0.07 mg kg−1, and recoveries between 80 and 103%, with RSD ≤ 11%, were achieved. When applied to preservative determination in food samples, the method under evaluation provided similar results to those obtained by a previously validated one. Finally, this method is a good alternative to determining preservatives in food samples, with the advantages of being greener, cheaper, and faster than the official ones.

Establishment and validation of a loop-mediated isothermal amplification (LAMP) assay targeting the ttrRSBCA locus for rapid detection of Salmonella spp. in food

Salmonella infection represents a common foodborne zoonosis of public concern worldwide. Rapid and user-friendly nucleic acid-based detection methods like loop-mediated isothermal amplification (LAMP) can contribute to the improvement of food safety and prevention of foodborne illness. In the present study, a LAMP assay was established and validated as a specific and rapid tool for detecting Salmonella spp. in various food matrices. Primers targeted a region within the ttrRSBCA locus and enabled 100% inclusivity of the 88 tested Salmonella strains, while no false positive signal occurred in any of the 92 tested non-Salmonella strains. After 20-h enrichment of artificially contaminated minced meat and ready-to-eat salad, initial contamination levels of 1 CFU/25 g were detectable via LAMP. The establishment of a simplified DNA-isolation method, which was adapted for field applications, enabled similar detection probability after 20-h enrichment of artificially contaminated food samples. During validation, 82 variously processed food samples underwent comparative analysis by LAMP and the standard culture method. The LAMP assay was further validated by the investigation of reference material of unknown Salmonella contamination status. Results showed that the ttrRSBCA-based LAMP detection of Salmonella spp. is a suitable tool for rapid sample screening within 24 h including first enrichment. Additionally, all Salmonella isolates obtained during cultural examination of naturally contaminated samples were correctly identified by LAMP, thus making it a suitable confirmation method for official applications.