Processed Food Samples Research Articles

Impact of the preparation procedure on gliadin, glutenin and gluten contents of wheat starches determined by RP-HPLC and ELISA

Celiac disease patients rely on safe gluten-free products, and the use of purified wheat starch (WSt) as part of the diet is usually well tolerated. However, uncertainties about residual gluten amounts in WSt remain, because ELISAs target the alcohol-soluble prolamin (gliadin) fraction of gluten, but hardly detect the alcohol-insoluble glutelin (glutenin) fraction. Therefore, gliadin, glutenin and gluten contents of WSt prepared from doughs on a laboratory scale were monitored by RP-HPLC. WSt washed with water from optimally or overmixed doughs had the lowest gluten contents. Gliadins were removed more extensively than glutenins during consecutive washing steps, so that gluten contents analyzed by ELISA were lower than those by RP-HPLC in 17 out of 24 WSt samples. The inability to detect glutelins by ELISA may thus lead to an underestimation of gluten contents in WSt. These findings highlight the need for improved analytical methods capable of detecting both prolamins and glutelins in processed food samples.

Competitive immunosensor based on gliadin immobilization on disposable carbon-nanogold screen-printed electrodes for rapid determination of celiotoxic prolamins

The first competitive disposable amperometric immunosensor based on gliadin-functionalized carbon/nanogold screen-printed electrodes was developed for rapid determination of celiotoxic prolamins. To date, no competitive spectrophotometric or electrochemical immunoassays have yet been successfully applied to gluten detection in processed food samples, which require the use of complex prolamin extraction solutions containing additives with denaturing, reducing and disaggregating functions. Thus, in this work, great effort was put into the optimization and performance evaluation of the immunosensor in terms of suitability as a screening tool for analysis of cereal-based food samples. For this purpose, aqueous ethanol or complex extraction mixtures, as the patented Cocktail Solution®, were proved effective in the extraction of gliadin. Good sensitivity was achieved after optimization of the immunocompetitive assay, giving limit of detection and limit of quantitation of 8 and 22ng/ml of gliadin, respectively, for ethanol extracts. The immunosensor was proved to be suitable also for samples extracted with Cocktail Solution® after a proper dilution. Analysis of real samples of different flours proved the suitability of the immunosensing device as a powerful tool for safety assessment of raw materials used for the formulation of dietary products for celiac disease patients. This immunosensor combines good analytical performance using a very simplified set-up protocol with suitability for rapid screening analysis performed using inexpensive and portable instrumentation. Graphical abstract Depiction of the development and working principle of the competitive immunosensor.

Species detection using HyBeacon® probe technology: Working towards rapid onsite testing in non-human forensic and food authentication applications

Identifying individual species or determining species’ composition in an unknown sample is important for a variety of forensic applications. Food authentication, monitoring illegal trade in endangered species, forensic entomology, sexual assault case work and counter terrorism are just some of the fields that can require the detection of the biological species present. Traditional laboratory based approaches employ a wide variety of tools and technologies and exploit a number of different species specific traits including morphology, molecular differences and immuno-chemical analyses. A large number of these approaches require laboratory based apparatus and results can take a number of days to be returned to investigating authorities. Having a presumptive test for rapid identification could lead to savings in terms of cost and time and allow sample prioritisation if confirmatory testing in a laboratory is required later. This model study describes the development of an assay using a single HyBeacon® probe and melt curve analyses allowing rapid screening and authentication of food products labelled as Atlantic cod (Gadus morhua). Exploiting melt curve detection of species specific SNP sites on the COI gene the test allows detection of a target species (Atlantic cod) and closely related species which may be used as substitutes. The assay has been designed for use with the Field Portable ParaDNA system, a molecular detection platform for non-expert users. The entire process from sampling to result takes approximately 75min. Validation studies were performed on both single source genomic DNA, mixed genomic DNA and commercial samples. Data suggests the assay has a lower limit of detection of 31 pg DNA. The specificity of the assay to Atlantic cod was measured by testing highly processed food samples including frozen, defrosted and cooked fish fillets as well as fish fingers, battered fish fillet and fish pie. Ninety-six (92.7%) of all Atlantic cod food products, tested, provided a correct single species result with the remaining samples erroneously identified as containing non-target species. The data shows that the assay was quick to design and characterise and is also capable of yielding results that would be beneficial in a variety of fields, not least the authentication of food

Challenging genosensors in food samples: The case of gluten determination in highly processed samples

Electrochemical genosensors have undergone an enormous development in the last decades, but only very few have achieved a quantification of target content in highly processed food samples. The detection of allergens, and particularly gluten, is challenging because legislation establishes a threshold of 20ppm for labeling as gluten-free but most genosensors expresses the results in DNA concentration or DNA copies. This paper describes the first attempt to correlate the genosensor response and the wheat content in real samples, even in the case of highly processed food samples. A sandwich-based format, comprising a capture probe immobilized onto the screen-printed gold electrode, and a signaling probe functionalized with fluorescein isothiocyanate (FITC), both hybridizing with the target was used. The hybridization event was electrochemically monitored by adding an anti-FITC peroxidase (antiFITC-HRP) and its substrate, tetramethylbenzidine. Binary model mixtures, as a reference material, and real samples have been analyzed. DNA from food was extracted and a fragment encoding the immunodominant peptide of α2-gliadin amplified by a tailored PCR. The sensor was able to selectively detect toxic cereals for celiac patients, such as different varieties of wheat, barley, rye and oats, from non-toxic plants. As low as 0.001% (10mg/kg) of wheat flour in an inert matrix was reliably detected, which directly compete with the current method of choice for DNA detection, the real-time PCR. A good correlation with the official immunoassay was found in highly processed food samples.

Development of an Enzyme-Linked Immunosorbent Assay for the Detection of 2-Amino-3-Methylimidazo [4, 5-f] Quinoline (IQ) in Processed Foods

2-amino-3-methylimidazo [4, 5-f] quinoline (IQ), is a type of heterocyclic aromatic amines (HAAs) formed during the thermal treatment of food processing, which possesses potential carcinogenicity and mutagenicity for humans. In this work, a simple, selective and sensitive direct competitive enzyme-linked immunosorbent assay (dcELISA) for detecting IQ has been developed for the first time. 1H-pyrrolo [2, 3-f] quinoline (PQ), a structural analogue of IQ, was connected with two-carbon arm to obtain the hapten PQ acid (PQA), and then a specific polyclonal antibody was produced with PQA-bovine serum albumin (BSA) as immunogen. The half-maximum inhibition concentration (IC50) and limit of detection (LOD; calculated as IC15 value) of the assay were 50 ± 3 and 3 ± 0.3 μg L−1, respectively. The assay showed no cross-reactivity with other HAAs analogues except 2-amino-3,4-dimethylimidazo [4,5-f] quinoline (MeIQ) (12 %), which indicated that the assay had high specificity for the IQ. The proposed method was further applied successfully to determine IQ in fried beef, fried pork, and dried fish floss samples with satisfactory recoveries (84.96–116.23 %) and precision (CV%, 4.96–17.26 %). The LOD for IQ in processed foods was 15 μg kg−1. In addition, the results of the established method were in good agreement with the high-performance liquid chromatography (HPLC) (R 2 = 0.9917). All above shows the proposed immunoassay allows for a cost-effective, specific, accurate, and reliable quantitative detection of IQ in real processed food samples.

Direct-multiplex PCR assay for meat species identification in food products

This is the first time that direct PCR – DNA amplification without prior DNA extraction – was successfully developed and fully validated for rapid and economical simultaneous identification of six commonly consumed meat species. To achieve this, six species-specific primers were selected from previous reports and newly designed from the mitochondrial cytochrome b (cyt b), cytochrome oxidase I (COI), and 12s rRNA gene. The assay generated PCR products of 100, 119, 133, 155, 253, and 311bp for pork, lamb/mutton, chicken, ostrich meat, horsemeat and beef, respectively. Validation showed that the assay is robust, rapid, economical, reproducible, specific, and sensitive down to 12,500 mitochondrial copy (equating to seven fg). It could be used with a variety of raw meats and products, including highly degraded and processed food samples. This proposed method will be greatly beneficial to the consumers, food industry, and law enforcement.

Chemical and organoleptic evaluation of fermented maize (Zea mays) gruel supplemented with fermented cowpea (Vigna unguiculata) flour and roasted melon seed (Citrullus vulgaris) paste

This work examined the chemical and organoleptic profile of fermented maize gruel enriched with roasted melon and fermented local species of cowpea ( oraludi ). The chemical composition of the processed food samples were assessed using standard methods. Blends were formulated in the ratio of 70: 30 (14 g protein basis) of maize gruel and cowpea flour or melon paste; and 70: 20: 10 (14 g protein basis) of maize gruel, cowpea flour and melon paste. Young students of the University community were selected for sensory evaluation. Sensory attributes were assessed with a nine-point Hedonic scale. Fermented maize gruel was the control. The proximate result reveals that melon paste had higher values than cowpea flour in protein, ash, crude fibre and fat (35.00: 29.75; 3.60: 2.40; 4.85: 2.33 and 39.95: 3.15%, respectively). Cowpea flour showed superior percentage value to melon paste in moisture and carbohydrate (8.76: 2.28; 57.26: 14.32%, respectively). Melon paste and cowpea flour had close values for iron (5.53: 5.48 mg/100 g, respectively) and zinc (0.019: 0.012 mg/100 g, respectively). There was not much difference in the phytate and tannins values of melon paste and cowpea flour (0.12: 0.18; 8.81: 10.48 mg/100 g, respectively). The blends were generally acceptable (5.46 to 6.88; p>0.05). Composite blends of locally available and under-utilized legumes should be used to add variety to infant diet. Keywords: Complementary food, gruel, chemical composition, organoleptic test. African Journal of Biotechnology Vol. 12(36), pp. 5549-5553

Evaluation of a multiplex selective enrichment broth SEL for simultaneous detection of injured Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes

Although many rapid and high throughput molecular methods have been developed in the recent years for the multiplex detection of foodborne pathogens, the simultaneous recovery and enrichment of sublethally injured cells is still a problem that needs to be considered. Combined with previous established multiplex real-time PCR assay, the capability of simultaneous recovery and enrichment of sublethally injured Salmonella, E. coli O157:H7 and L. monocytogenes cells was evaluated in a multiplex selective enrichment broth SEL. The injured cells were obtained by heat shock. After evaluation of different procedures, 1 h of recovery period prior to 20 h of enrichment was proved to be necessary for the detection of less than 10 CFU/5 mL broth of injured L. monocytogenes. When the detection method was applied to artificially contaminated ground beef, all the three injured pathogens could be simultaneously detected without discrimination by real-time PCR combined with SEL broth, the detection limit was < 5 CFU/10 g ground beef. Comparatively, when BPW was employed as the enrichment broth in the same detection procedure, injured L. monocytogenes could not be detected if the initially spiked level was below 10(2) CFU/10 g ground beef. Considering the capability of co-enrichment and high detection effectiveness, the real-time PCR assay combined with SEL broth herein appears to be a promising tool for high-throughput screening of a large number of processed food samples, which require either single or multiple pathogen detection. More important, the sublethally injured foodborne pathogen cells were also detectable.

A peach (Prunus persica)-specific gene, Lhcb2, used as an endogenous reference gene for qualitative and real-time quantitative PCR to detect fruit products

Among the methods used to detect food adulteration, the amplification of endogenous reference genes is particularly important. Endogenous reference genes for many different species, such as cotton, papaya, maize and others, have been reported, yet an endogenous reference gene for the peach is still lacking. In this paper, the chlorophyll a/b-binding protein (Lhcb2) gene was identified as a species-specific gene for the peach. Lhcb2 was assayed in 4 species of peaches and 8 non-peach species by both qualitative and quantitative PCR. No amplification was observed in other species. The detection limit of quantitative PCR was as low as 5 pg of DNA, equal to 9 copies, and Southern blot analysis confirmed that the Lhcb2 gene was present in a single copy in the peach genome. All of these experiments indicated that the Lhcb2 gene is a useful endogenous reference gene for the detection of peach material via both qualitative and quantitative PCR assays, even in the processed food samples such as juices containing peach.

Comparison of a sodium-based and a chloride-based approach for the determination of sodium chloride content of processed foods in the Netherlands

This study reports and discusses the sodium content of a set of 1016 processed food samples collected in the Netherlands, which covered 10 food groups (cakes/pastries, chips/nuts, sauces, processed meat, conserves, snacks, ready-to-eat products, cheese, bread, and soups) and 100 food commodities. The food groups that showed highest sodium contents were processed meat (1030mg Na 100g−1), cheese (820mg Na 100g−1), and sauces (752mg Na 100g−1). Lowest sodium concentrations were determined in conserves (286mg Na 100g−1), cakes/pastries (322mg Na 100g−1), and soups (355mg Na 100g−1). In addition, two different approaches for the assessment of sodium chloride content in the same sample set have been compared for all 1016 samples: determination of sodium by flame emission spectroscopy and determination of chloride by potentiometric titration. The sodium chloride content was then calculated converting the sodium and chloride content into the corresponding salt (NaCl) content. For the NaCl contents determined by the two approaches, significant differences for seven out of the ten food groups were observed, and the sodium contents of nearly half of the commodities showed significantly different NaCl levels. At food group level, the NaCl contents calculated from sodium were significantly higher (p<0.05) than the NaCl content calculated from chloride for cakes/pastries, processed meat, snacks, cheese and soups, whereas it was significantly lower for the chips/nuts and the bread group. These differences can be explained by additional sources of sodium and/or chloride, e.g. certain food additives and the natural sodium and chloride content of the ingredients. Although most legal recommendations specify NaCl levels, the present study shows that sodium and chloride concentrations do not go necessarily hand in hand since they may originate from different sources.

Single-Tube Nested Real-Time PCR as a New Highly Sensitive Approach to Trace Hazelnut

Hazelnut is one of the most commonly consumed tree nuts, being largely used by the food industry in a wide variety of processed foods. However, it is a source of allergens capable of inducing mild to severe allergic reactions in sensitized individuals. Hence, the development of highly sensitive methodologies for hazelnut traceability is essential. In this work, we developed a novel technique for hazelnut detection based on a single-tube nested real-time PCR system. The system presents high specificity and sensitivity, enabling a relative limit of detection of 50 mg/kg of hazelnut in wheat material and an absolute limit of detection of 0.5 pg of hazelnut DNA (1 DNA copy). Its application to processed food samples was successfully achieved, detecting trace amounts of hazelnut in chocolate down to 60 mg/kg. These results highlight the adequacy of the technique for the specific detection and semiquantitation of hazelnut as potential hidden allergens in foods.

Multiplex Real-Time PCR Assay for Simultaneous Detection ofOmphalotus guepiniformisandLentinula edodes

A rapid multiplex real-time PCR assay was developed to achieve highly specific, simultaneous detection of two kinds of mushrooms, Omphalotus guepiniformis and Lentinula edodes. Primers and TaqMan minor groove binder probes were designed according to the internal transcribed spacers 1-5.8S region of rDNA and evaluated by the specificity for fruiting bodies of 17 O. guepiniformis, 16 L. edodes and samples from 57 other species. DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.00025 ng of DNA. Threshold cycle (Ct) values for raw and processed fruiting bodies and for fruiting bodies (1% (w/w)) mixed with foodstuffs or artificial gastric juice contents ranged from 17.16 to 26.60 for both examined species. This new assay proved specific to the target species, highly sensitive, and applicable to processed food samples and gastric juice contents, making it useful for rapidly identifying O. guepiniformis and L. edodes.

Occurrence of Aflatoxins in Selected Processed Foods from Pakistan

A total of 125 (ready to eat) processed food samples (70 intended for infant and 55 for adult intake) belonging to 20 different food categories were analyzed for aflatoxins contamination using Reverse Phase High Performance Liquid Chromatography (RP-HPLC) with fluorescent detection. A solvent mixture of acetonitrile-water was used for the extraction followed by immunoaffinity clean-up to enhance sensitivity of the method. The limit of detection (LOD) (0.01–0.02 ng·g−1) and limit of quantification (LOQ) (0.02 ng·g−1) was established for aflatoxins based on signal to noise ratio of 3:1 and 10:1, respectively. Of the processed food samples tested, 38% were contaminated with four types of aflatoxins, i.e., AFB1 (0.02–1.24 μg·kg−1), AFB2 (0.02–0.37 μg·kg−1), AFG1 (0.25–2.7 μg·kg−1) and AFG2 (0.21–1.3 μg·kg−1). In addition, the results showed that 21% of the processed foods intended for infants contained AFB1 levels higher than the European Union permissible limits (0.1 μg·kg−1), while all of those intended for adult consumption had aflatoxin contamination levels within the permitted limits.

Identification of 11 sea cucumber species by species-specific PCR method

A species-specific PCR (polymerase chain reaction) method was developed to identify the 11 sea cucumber species, even in highly processed food samples such as frozen or dried sea cucumbers (beche-de-mer). Universal primers were used for the amplification of 16S rRNA gene in each species. The alignment of the obtained sequences was the basis for the design of 11 species-specific reverse primers located in the 16S rRNA gene region. The developed species-specific PCR method was successfully applied to authenticate species of commercial sea cucumber products. Therefore, this method really provides a simple, fast and reliable protocol for the accurate identification of sea cucumber species, and also can be very useful for correct labeling of commercial products, traceability, and protection of the consumer's rights.

Development of a multiplex real-time PCR method for simultaneous detection of Salmonella enterica, Shigella flexneri and Listeria monocytogenes in processed food samples

The present work is focused on the development of a TaqMan multiplex real-time PCR method for the detection of Salmonella, Shigella and L. monocytogenes in seafood, meat and ready-to-eat products. The aim of this study is to detect the three pathogens in one single test including an enrichment medium for the simultaneous growth of the bacteria of interest and an Internal Amplification Control (IAC) to monitor PCR inhibitors. For this purpose, three genes were selected, invA for Salmonella, ipaH for Shigella and hlyA for L. monocytogenes. Also, no. 17 broth without dextrose and further modified by adding Tween 80 was used for the enrichment step. Specificity of the method was checked against a panel of 24 non-target bacterial strains. RT-PCR efficiency obtained for the simultaneous amplification of all three pathogens was 102.5% for Salmonella, 108.9% for Shigella and 106.4% for L. monocytogenes. The limit of detection (LOD) was evaluated in seafood, meat and ready-to-eat products, being established within 3 and 22 cfu in 25 g of sample for the three bacteria analyzed. Seventy-eight samples were analyzed with multiplex RT-PCR including spiked and natural samples collected from different laboratories. Even though several RT-PCR methods have been developed for the detection of Salmonella, Shigella and L. monocytogenes, as far as we know this is the first method developed for the simultaneous detection of these three pathogens, coupling RT-PCR with an enrichment in the same broth and being tested in a wide range of different processed food samples with a low LOD. The application of this method can significantly reduce costs and time of analysis in laboratories, what would be reflected in a faster response in those risk situations when they are detected.

Aluminium content of some processed foods, raw materials and food additives in China by inductively coupled plasma–mass spectrometry

The level of aluminium in 178 processed food samples from Shenzhen city in China was evaluated using inductively coupled plasma–mass spectrometry. Some processed foods contained a concentration of up to 1226 mg/kg, which is about 12 times the Chinese food standard. To establish the main source in these foods, Al levels in the raw materials were determined. However, aluminium concentrations in raw materials were low (0.10–451.5 mg/kg). Therefore, aluminium levels in food additives used in these foods was determined and it was found that some food additives contained a high concentration of aluminium (0.005–57.4 g/kg). The results suggested that, in the interest of public health, food additives containing high concentrations of aluminium should be replaced by those containing less. This study has provided new information on aluminium levels in Chinese processed foods, raw materials and a selection of food additives.

A highly sensitive real-time PCR system for quantification of wheat contamination in gluten-free food for celiac patients

A quantitative real-time PCR system (Q-PCR) for the detection and quantification of wheat DNA in food for celiacs has been developed and optimised. The DNA was extracted with a new procedure, based on the SDS/Guanidine–HCl/Proteinase K method, but faster, with a higher yield and enough purity to avoid an additional purification step. This is the first report that compares the information provided by a Q-PCR technique with the prolamin levels determined with the most frequently used commercially available ELISA, based on the R5 monoclonal antibody. With the exception of some hydrolysed and highly processed food samples (such as beers, syrups, malt extracts, breakfast cereals…), the rest of the food with prolamin levels above the R5 ELISA quantification limit (1.5 mg/kg) have given positive signals with the Q-PCR system. Moreover, some food samples with prolamin levels below 1.5 mg/kg have also given positive signals with the Q-PCR system, indicating a better sensitivity of the DNA technique. Therefore, the developed Q-PCR system can be used as a non-immunological tool in order to confirm, by the “DNA pathway”, the presence of wheat in food not only for celiacs but also for individuals with wheat allergy.

A microbial survey of food served to Oncohematology patients at a University Hospital

Although everyone is susceptible to foodborne diseases, the immunocompromised patients are particularly at risk of contracting foodborne illnesses and suffer more serious sequelae as a result of infection. The aim of the present work was to measure the degree of microbial contamination in different food stuffs, served to immunocompromised patients admitted at the Oncohematology unit in our hospital. Several types of foods were selected from those served to the oncohematology patients in our hospital. Samples of each food were taken at random from the central kitchen, the wards’ pantry as well as from the patients. Food included processed food stuffs as well as non-processed food stuffs. Samples were processed according to the protocol obtained from the Centre Hospitalier Universitaire Bicetre (2001). The total bacterial count, the total coliform count, the presence of Staphylococcus aureus, as well as the presence of fungal food contamination were determined and values obtained were compared to the accepted values cited in the protocol provided by the CHU Bicetre (2001). Twenty one out of 28 examined processed food samples (75%) were free from any contamination. The remaining 7 samples (25%) yielded positive results on culture, yet the counts were considered acceptable. On the other hand, all of the non-processed food samples examined were contaminated (100%). Identified organisms were Pseudomonas, Aeromonas, Enterocci, Diphtheroid and Anthracoid. All examined samples were positive for fungal contamination with Aspergillus niger being the most commonly isolated mold. We concluded that non-processed food represents a potential risk of microbial exposure to the immunocompromised patients.

Carotene location in processed food samples measured by Cryo In-SEM Raman

Cryo In-SEM Raman has been used for the first time to localise carotene compounds in a food matrix. Raman spectra of lycopene and β-carotene have been obtained from sampling oil droplets and plant cell structures visualised with cryo-SEM in tomato and carrot based emulsions containing 5% oil. It was possible to identify the carotenoids in both the oil droplets and the cell walls. Furthermore our results gave some indication that the carotenoids were in the non-crystalline state. It has been suggested that a higher amount of carotenes solubilised into the oil phase of the food matrix would lead to a higher bioaccessibility, thus understanding the effect of processing conditions on micronutrients distribution in a food matrix might help the design of plant based food products with a better nutritional quality. This shows improved structural characterisation of the cryo-SEM with the molecular sensitivity of Raman spectroscopy as a promising approach for complex biological problems.

Antibiotic Sensitivity and Sequence Amplification Patterns of Genes in Multidrug Resistant Enterobacteria Isolates from Processed Foods in Some West African Countries

Diarrhoea, dysentery and other diseases due to other enteric bacteria have reportedly been found to resist chemotherapeutic treatment in some West African communities with fatal consequences in some cases. This study was carried out to determine multidrug resistance patterns of Enterobacteria isolates from processed ready-to-eat foods. Indigenously processed food samples of different types were collected from two Francophone and two Anglophone countries in the West African sub-region during the wet and dry seasons of a sampling period of two years. Enterobacteria were isolated from the samples using standard techniques. Amplification of chromosomal DNA of the isolates using the Polymerase Chain Reaction was carried out. The results obtained were subjected to statistical analyses. All isolates showed resistance to cefuroxime (90.7%), nitrofurantoin (90.6%), augmentin (86.1%) and ampicillin (51.2%) while all were sensitive to gentamycin and ciprofloxacin. There was amplification indicating the presence of invA gene at a position of 240 bp. There was no amplification at all for the spvC gene in any of the isolates tested. Multidrug resistant enteric bacteria in these foods containing the invA gene could lead to infections with uncontrolled antibiotic use. The presence of enteric bacteria in the foods analyzed which provide undeniable evidence of the poor microbiological quality of these foods could form the basis of a useful databank in formulation of food-borne disease control and prevention strategies.