Highly sensitive PCR-based detection method specific for Aspergillus flavus in wheat flour

Aspergillus flavus is frequently found in food, producing a wide variety of toxins, aflatoxins being the most relevant in food safety. A specific PCR-based protocol for this species is described which allowed discrimination from other closely related species having different profiles of secondary metabolites from the Aspergillus Section Flavi, particularly A. parasiticus. The specific primers were designed on the multi-copy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA) and were tested in a wide sample of related species and other fungal species commonly found in food. The PCR assay was coupled with a fungal enrichment and a DNA extraction method for wheat flour to enhance the sensitivity of the diagnostic protocol. The results indicated that the critical PCR amplification product was clearly observed for wheat flour contaminated by 10(2) spores after 16 h of incubation.     

Specific detection of Aspergillus parasiticus in wheat flour using a highly sensitive PCR assay

Aspergillus parasiticus is one of the most important aflatoxin-producing species that contaminates foodstuffs and beverages for human consumption. In this work, a specific and highly sensitive PCR protocol was developed to detect A. parasiticus using primers designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA). The assay proved to be highly specific for A. parasiticus when tested on a wide range of related and other fungal species commonly found in commodities, and allowing discrimination from the closely related A. flavus. Accuracy of detection and quantification by conventional PCR were tested with genomic DNA obtained from wheat flour artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10⁶ spore/g could be detected by the assay directly without prior incubation of the samples. The assay described is suitable for incorporation in routine analyses at critical points of the food chain within HACCP strategies.     

Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR

Aflatoxins are important mycotoxins that represent a serious risk for human and animal health. These mycotoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus, two closely related species with different array of aflatoxins. In this work, two specific quantitative PCR (qPCR) assays were developed to detect and quantify both species in wheat flour using primers based on the multicopy ITS2 rDNA target sequence. The species specificity of the assays was tested in a wide range of strains of these species and others colonizing the same commodities. The sensitivity of the assay was estimated in 2.5 pg/reaction in both species. Discrimination capacity for detection and relative quantification of A. flavus and A. parasiticus DNA were analyzed using samples with DNA mixtures containing also other fungal species at different ratios. Both qPCR assays could detect spore concentrations equal or higher than 10⁶ spores/g in flour samples without prior incubation. These assays are valuable tools to improve diagnosis at an early stage and in all critical control points of food chain integrated in HACCP strategies.     

Isolation and Identification of Bacillus spp. and Related Genera from Different Starchy Foods

ABSTRACT: Samples of butternut squash, potatoes, rice, and wheat flour were analyzed. Bacillus spp. and related species belonging to Paenibacillus and Brevibacillus genera were found in 96% of the samples. In butternut squash, predominant species were Bacillus pumilus and Paenibacillus polymyxa together with other Bacillus spp. species (B. cereus, B. licheniformis, B. sphaericus, and B. subtilis). In all the potato samples, Bacillus species were detected (B. cereus, B. mycoides, and B. licheniformis). Also, Bacillus spp. were detected in 100% of the unhusked rice samples, while incidence in white rice samples was 83%. In total rice samples, B. pumilus, Brevibacillus brevis, and Paenibacillus macerans were the main species and B. cereus, P. polymyxa, B. subtilis, and Brevibacillus laterosporus had the lower percentage. The most important species found in wheat flour was P. polymyxa with colony forming units per gram of about 10². As the identified species were potentially causatives of foodborne diseases, attention should be given to sanitary and temperature conditions as critical factors that influence the safety and shelf life of these products. Practical Application: Foodborne illness produce by B. cereus have been associated with a wide variety of food. In addition, some other Bacillus species have been related to foodborne disease in humans. Information about the virulence mechanisms of other Bacillus spp. is scanty and their risk is underestimated. Identifying the group of food and the food processes in which Bacillus cereus or other Bacillus spp. would be hazardous for human health is vital for the prevention of foodborne outbreak. In this study, we determined the incidence of Bacillus spp. and related genera in some food items of agriculture origin from Argentina. This research is relevant to identify the presence of potentially pathogen Bacillus species and related genera in this type of food.     

Reliability of Listeria monocytogenes Identification by Specific PCR Assessed by Phenotypic and Genotypic Techniques

This study evaluates the possibility of using polymerase chain reaction (PCR) for rapid identification of food-borne Listeria monocytogenes as an alternative to API Listeria system and estimates the incidence of API Listeria misidentifications in food-borne Listeria species. A total of 198 strains, 11 L. monocytogenes, 28 other Listeria species, and 159 food isolates were phenotypically and genotypically characterized by API Listeria profiles and randomly amplified polymorphic DNA (RAPD) profiles, respectively. They were also tested for PCR amplification using genus- and species-specific primers. Clustering analysis of phenotypic and genotypic data showed discrepancies in species identification of some isolates by API Listeria profiles. Their identities were confirmed by 16S rDNA sequencing, and thus, it was revealed that 33% of Listeria innocua and 19% of Listeria welshimeri were misidentified as L. monocytogenes by API Listeria profiles. Reliable identification of L. monocytogenes was obtained by LM1–LM2 specific primers which allowed PCR amplification only in reference strains and isolates previously identified as L. monocytogenes by RAPD and 16S rDNA sequence analysis. These results corroborate the suitability of specific PCR as a rapid and accurate test for the identification of L. monocytogenes, avoiding misidentification with other Listeria species commonly found in food products.     

Impact of the fungal protease produced by Fusarium culmorum on the protein quality and breadmaking properties of winter wheat

Samples artificially infected by Fusarium culmorum were analyzed to assess the effects of Fusarium infection on the protein quality of winter wheat (Triticum aestivum L.). The Fusarium infection did not noticeably influence either the crude protein content or the water absorption ability of the wheat flour. The protease activity found in the wheat flour was inversely correlated to the sedimentation value. In contrast, it was positively correlated to both the free amino acid content and the degree of infection as expressed in Fusarium protein equivalents (FPE) quantified by an enzyme-linked immunosorbent assay. A distinct reduction in the content of both total glutenin and high molecular weight glutenin subunits was detected in the seriously infected samples (FPE>20 µg g^–1). The infection with Fusarium impaired the dough quality and led to a deformed loaf shape. The fungal protease was active over both a wide range of temperatures (from 10 to 100 °C) and a wide range of pH values (from 4.5 to 8.5). The maximum protease activity was displayed at 50 °C in the pH range 6.0–8.0. This property indicates that the protease produced by F. culmorum may impair storage proteins throughout all the processing procedures associated with wheat flour, thereby causing weak dough properties and, consequently, unsatisfactory bread quality.     

Molecular techniques for the detection of granary weevil (Sitophilus granarius L.) in wheat and flour

The granary weevil (Sitophilus granarius L.) is a stored grain pest that causes major economic losses. It reduces the quantity and quality of the grain by its feeding and excretion. Sequences of S. granarius mitochondrial cytochrome oxidase subunits genes mtCOI and mtCOII were analysed and compared with mtCOI/II sequences available in GenBank. The analysed genes displayed a high level of homology between corresponding subunits. Attempts were undertaken to develop detection methods for contamination by S. granarius in wheat and wheat flour based on the molecular biology techniques: standard and real-time polymerase chain reaction (PCR) with a TaqMan® molecular probe. (TaqMan probes are dual-labelled hydrolysis probes) Specific primers designed based on available sequences for mtCOI and mtCOII genes were applied and optimal reaction conditions established. The specificity of both methods was studied by using a species closely related to S. granarius: S. oryzae and S. zeamais. It is shown that the sensitivity threshold was very high?–?we were able to detect the equivalent of one beetle per 100 kg of flour when the real-time PCR with TaqMan probe method was applied to model samples. The primer sets used turned out to be species specific, and the technique was rapid, reliable and very sensitive.     

Alternaria toxins in Argentinean wheat,bran, and flour

Alternaria species have been reported to infect a wide variety of vegetables, fruits, and cereal crops. Wheat is one of the most consumed cereal worldwide. A sensitive HPLC-DAD methodology was applied to quantify alternariol (AOH), alternariol methyl ether (AME) and tenuazonic acid (TeA) in 65 samples of whole wheat, bran, and flour. The extraction methodology allowed extracting the three toxins simultaneously. Limits of detection in wheat were 3.4, 4.5, and 0.5 µg kg^?1 for AOH, AME and TeA, respectively. For bran, these data were 3.1, 4.5, and 12 µg kg^?1 and for flour 50, 70, and 14 µg kg^?1, respectively. The studied recoveries were higher than 70% and RSD was below 10%. Wheat and bran samples showed low AOH and AME contamination compared to TeA. The averages levels found for TeA in wheat, bran and flour were 19,190, 16,760, and 7360 µg kg^?1, respectively.     

Multiplex real-time PCR for detection and quantification of mycotoxigenic Aspergillus, Penicillium and Fusarium

The most agriculturally and economically important classes of mycotoxins are produced by species of Aspergillus, Penicillium, and Fusarium. Rapid methods to detect mycotoxigenic fungi could help prevent mycotoxins from entering the food chain. The purpose of this research was to develop a multiplex real-time PCR assay to detect and quantify multiple species of mycotoxigenic fungi. A pair of broad-spectrum PCR primers was designed for amplification of the internal transcribed spacer (ITS) regions of rDNA from the mycotoxigenic species. An in silico analysis of the primers revealed the presence of amplification in more than 40 Aspergillus species, 23 Fusarium species, and 32 Penicillium species as well as 64 other fungal genera. Genus-specific Taqman probes were designed from the ITS sequences of the most important mycotoxigenic species of Fusarium, Penicillium, and Aspergillus. The specificity of the probes was established against a wide range of fungal species. As a multiplex assay, the linear range of detection was 1 pg to 10 ng of DNA. The assay was validated by analyzing fungal growth in distiller's grain (DG), an animal feedstock that is a by-product when ethanol is produced from corn. This assay could be used as an initial step to evaluate the mycotoxigenic potential of DG and various other agricultural commodities.     

Development of a polyclonal antibody-based indirect competitive ELISA for determination of sterigmatocystin in wheat and corn flours

Sterigmatocystin (STC) is a toxic secondary metabolite produced by more than 50 fungal species, including Aspergillus flavus, A. parasiticus, A. nidulans, and A. versicolor. The Joint FAO/WHO Expert Committee on Food Additives concluded that sterigmatocystin is genotoxic and carcinogenic with the critical effect determined to be carcinogenicity. The present study describes a simple method to prepare hapten and immunogens in order to generate polyclonal antibodies against this metabolite. We developed a sensitive and specific polyclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ciELISA) for monitoring STC in wheat and corn flours without the need for derivatisation of STC or clean-up of samples by immunoaffinity chromatography for quantification. The half inhibitory concentration (IC₅₀) of the established method was 4.52 ± 0.81 ng mL^?1, with the limit of detection (IC₁₀) being 0.19 ± 0.04 ng mL^?1 in wheat and corn flour matrices with the coefficient of variation of less than 22%.The assay was very specific to STC and showed no cross-reactivity with its analogue structures. The ELISA allowed for up to 5% methanol without significant influence on the IC₅₀ value. Validation of the assay was performed by spiking STC into a blank flour matrix and the recoveries were in the range of 75.3 % to 104.5 % with a coefficient of variation less than 15%. A small retail survey was conducted by purchasing wheat (n = 8) and corn flours (n = 2) from local grocery stores. All of these retail samples were negative for STC using the developed ELISA method and were confirmed by LC-MS/MS. We demonstrated a rapid, simple, and reliable method for screening STC in wheat and corn flours.     

Whole wheat and white wheat flour— the mycobiota and potential mycotoxins

Mold growth has detrimental effects on the quality of flour and may result in mycotoxin contamination. The search for potential mycotoxins—almost 400 are known—is time-consuming and expensive. However, detailed knowledge about the mycobiota and especially the toxin producing fungi enables the effective search for these toxic fungal metabolites. Therefore, a whole wheat flour and a white wheat flour (type 405) were investigated for their total qualitative as well as quantitative mycobiota. Overall, 51 species belonging to 14 different genera could be isolated. Total fungal counts of the whole wheat flour amounted to 1833 molds while the white wheat flour contained 1730 cfu 2 g⁻¹. The mycobiota of both flours was dominated by Aspergillus spp. accounting for 84% and 77·3% of the isolations, respectively. Fungi of the genus Penicillium spp. occurred only to a minor degree: 8% of the isolations in whole wheat flour and 15% in white wheat flour. Aspergillus candidus was the most frequently encountered mold.Penicillium aurantiogriseum , Cladosporium cladosporioides, A. flavus, Eurotium herbariorum,P. griseofulvum , P. brevicompactum and P. viridicatum were isolated to a lesser degree. From the 3563 identified fungi 93·3% (32 species) belong to the group of toxigenic molds.     

The Use of Molecular Methods for Detecting and Discriminating Salmonella Associated with Foods — A Review

The genus Salmonella is composed of two species, Salmonella enterica and Salmonella bongori. Only S. enterica subsp. enterica is considered of human clinical significance and consists of 1478 serotypes. A large number of virulence genes and virulence-enhancing genes have been described for Salmonella. There are more than 30 Salmonella specific genes that have been used for the polymerase chain reaction to detect and characterize Salmonella. The sensitivity of detection of Salmonella from complex matrices such as food and feces by PCR is invariably enhanced using nonselective or selective enrichment, particularly if followed by immuno-magnetic separation in addition to coupling the PCR with ELISA formats. R-plasmids are considered to be the main factors responsible for the horizontal transfer of antibiotic resistance genes in Salmonella. A sizeable number of primer pairs are available for determining by the PCR the presence of many antibiotic resistance genes in Salmonella isolates that are not necessarily specific for Salmonella. The collective PCR detection of members of the genus Salmonella in foods and environmental samples has been achieved by amplification of invA gene sequences that are highly conserved among all Salmonella serotypes in addition to the amplification of his gene sequences also present throughout the genus Salmonella. Amplification of 16S rDNA sequences have also been found useful for genus specific detection of Salmonella. d-Tartrate (dT⁺) fermenting strains have been found to result in less severe gastrointestinal infections than d-tartrate-nonfermenting (dT^-) strains. Primers have therefore been developed for distinguishing between (dT⁺) and (dT^-) strains. Among the molecular techniques available for strain discrimination of Salmonella isolates, pulsed field gel electrophoresis, random amplified polymporphic DNA analysis, ribotyping, multilocus sequence typing, subtracted finger printing, and enterobacterial repetitive intergeneric consensus typing have been found useful. Multiplex PCR has been found effective for simultaneously detecting Salmonella and other pathogens in foods, particularly with real-time PCR.     

Potentially toxigenic fungi from selected grains and grain products

A total of 85 grain and grain product samples (including corn meal, corn muffin mix, popcorn, various types of rice, and self‐rising, all‐purpose unbleached and whole wheat flour) from U.S. retail were tested for fungal contamination levels and profiles using conventional plating as well as molecular methods. The results of this study showed that over 90% of wheat flour and corn product samples and 73% of rice samples tested carried live fungi. Popcorn carried the highest fungal levels reaching 5.45 log₁₀ colony forming units (cfu) per gram followed by corn meal (reaching 5.38 log₁₀ cfu/g). Mold and yeast counts in rice and wheat flour reached 3.30 log₁₀ and 3.28 log₁₀ cfu/g, respectively. The predominant molds in wheat flour were aspergilli and fusaria found in 50 and 46% of samples, respectively; Fusarium spp. were the most frequent contaminants of corn‐based products found in 74% of the samples followed by penicillia (present in 44% of tested samples). Rice, conversely, contained mainly Aspergillus, Fusarium, and yeasts (each found in 21% of the samples).

Practical applications

Toxigenic molds are often contaminating stored grains and grain products and under improper storage conditions could cause spoilage of these commodities accompanied with production of toxic secondary metabolites, mycotoxins. Mycotoxins are known to cause illnesses in humans and animals. Therefore, monitoring the presence and inhibiting the growth of these organisms is critical for achieving and maintaining high quality products, suitable for human and animal consumption, and free of health hazards. Establishing toxigenic mold profiles in stored grains and their derivatives can point to correct storage management and thus reduction/elimination of spoilage and mycotoxin production in these products. In this study we tested several corn, rice, and wheat flour commodities for live potentially toxigenic fungal species. Our findings can help select proper storage management techniques for these commodities.     

Characteristics and oil absorption in deep‐fat fried batter prepared from ball‐milled wheat flour

BACKGROUND: The porous structure generated during frying influences oil absorption and textural qualities. The alteration in physical properties of wheat flour is suspected to affect the structure formation. The present study investigated the effect of physicochemical changes in wheat flour by the ball‐milling process on structure formation and consequently oil absorption of a fried wheat flour batter model. RESULTS: Batter models containing 600 g kg^?1 moisture were made of 0–10 h ball‐milled wheat flour and then fried in frying oil at 150 °C for 1–7 min. The samples made of milled flour possess larger pores and exhibit lower oil absorption than sample made of 0 h milled flour. The fracture force of a fried sample prepared from 5 and 10 h milled flour is lower than that of a sample prepared from 0 h milled flour. CONCLUSION: The decrease in glass transition temperature (T_g) and melting temperature (T_m) of milled flour affect the microstructure formation in the fried wheat flour batter. The microstructure is responsible for oil absorption and fracturability in fried food. The samples made of flour of longer ball‐milling time have lower oil absorption and higher crispness. Ball‐milling may be a tool to produce mechanically modified wheat flour which can reduce oil absorption for fried batter. Copyright © 2009 Society of Chemical Industry     

Stability of the mycotoxin deoxynivalenol (DON) during the production of flour-based foods and wheat flake cereal

Deoxynivalenol (DON) is a mycotoxin found in cereal grains and cereal-based foods. DON concentrations in finished products are reduced under some processing conditions, but not others. DON concentrations in flour, wheat and selected foods made from them under commercially relevant conditions were compared by GC with electron capture detection. Average concentrations (n?=?9/item) in cookies, crackers and pretzels ranged from 61% (cookies) to 111% (pretzels) compared with flour (100%?=?0.46?µg?g^?1). Lesser amounts were found in donuts and bread: their respective DON concentrations were 44% and 30% that of flour. Mass balance estimates for DON (µg?g^?1 flour equivalents) ranged from 50% (bread?=?0.23?µg?g^?1 flour equivalents) to 120% (donuts), indicating that dilution by recipe ingredients contributed to DON reductions in bread and accounted for all of the apparent reduction in donuts. Mass balance estimates averaged 76% (crackers) to 107% (pretzels) for the other flour products. DON concentrations were higher in cereal flakes (0.55?µg?g^?1 in the finished product and 0.58?µg?g^?1 on a mass balance basis) than in wheat (0.40?µg?g^?1), suggesting that DON concentrations might increase during processing of wheat cereals under some conditions. In summary, DON concentrations of finished food products were reduced?≥50% only in bread and donuts. Reduction in bread resulted from a combination of DON ‘loss’ and dilution by recipe ingredients whereas the reduction in donuts was due entirely to dilution. These results are further evidence of DON stability during the preparation of popular flour or wheat-based products.     

Application of Different Molecular Techniques for Characterization of Catalase‐Positive Cocci Isolated from Sucuk

This study was carried out for the characterization and discrimination of the indigenous Gram positive, catalase‐positive cocci (GCC) population in sucuk, a traditional Turkish dry‐fermented sausage. Sucuk samples, produced by the traditional method without starter culture were collected from 8 local producers in Kayseri/Turkey and a total of 116 GCC isolates were identified by using different molecular techniques. Two different molecular fingerprinting methods; namely, randomly amplified polymorphic DNA‐PCR (RAPD‐PCR) and repetitive extragenic palindrome‐PCR (rep‐PCR), were used for the clustering of isolates and identification at species level was carried out by full length sequencing of 16S rDNA. Combining the results obtained from molecular fingerprinting and 16S rDNA sequencing showed that the dominant GCC species isolated from the sucuk samples was Staphylococcus saprophyticus followed by Staphylococcus succinus and Staphylococcus equorum belonging to the Staphylococcus genus. Real‐time PCR DNA melting curve analysis and high‐resolution melting (HRM) analysis targeting the V1 + V3 regions of 16S rDNA were also applied for the discrimination of isolates belonging to different species. It was observed statistically different Tm values and species‐specific HRM profiles for all except 2 species (S. saprophyticus and Staphylococcus xylosus) that have high 16S rDNA sequence similarity. The combination of rep‐PCR and/or PCR‐RAPD with 16S rRNA gene sequencing was an efficient approach for the characterization and identification of the GCC population in spontaneously fermented sucuk. On the other hand, intercalating dye assays were found to be a simple and very promising technique for the differentiation of the GCC population at species level.     

Effect of flaxseed and wheat flour blends on dough rheology and bread quality

The rheological and baking properties of flaxseed/wheat composite flours were studied. Flaxseed flour was used to replace 50, 100, 150 and 200 g kg^?1 of wheat flour in bread. Farinographic studies showed that water absorption, dough development time and mixing tolerance index increased as the amount of flaxseed flour increased, while dough stability decreased at 100, 150 and 200 g kg^?1 of flaxseed flour substitution. The extensographic energy of dough also decreased at 150 and 200 g kg^?1 flaxseed levels. The addition of increasing amounts of flaxseed flour caused a decrease in extensibility. Doughs containing 100, 150 and 200 g kg^?1 flaxseed flour showed resistance to extension comparable to that of control dough. The specific volume of flaxseed flour breads was similar to that of control bread. Crust L, a, b values of breads with flaxseed flour were lower than those of control bread. Breads with flaxseed flour gave lower crumb L and b values and higher a values than control bread. The sensory properties showed that an acceptable bread could be produced using flaxseed flour up to a level of 200 g kg^?1. Copyright © 2007 Society of Chemical Industry     

Energy use in flour production

Energy use at three UK flour mills was studied. The primary energy required to provide the fuel and power used in milling averaged 1.16 GJ t^?1 flour. Electricity accounted for approximately 80% of this value and was the sole energy source used directly in the milling process. Electricity consumption per tonne of flour showed no significant variation between a soft grist and a hard grist. The total primary energy requirement for the milling and transport of flour delivered in bulk was 1.43 GJ t^?1 compared with 1.88 GJ t^?1 for flour packed in 32-kg bags, where transport and the provision of packaging material require additional energy use. Primary energy use in wheat growing was evaluated for Canada, USA, France and the UK but did not show marked differences, at about 4.4 GJ t^?1 wheat. The primary energy requirement for fuel used in transporting wheat to UK flour mills was estimated to range from 1.46 GJ t^?1 of Canadian wheat to 0.08 GJ t^?1 of home grown wheat. In order to make available at a UK bakery flour milled from a grist of 30% Canadian, 40% French and 30% home grown wheat, the total primary energy requirement (including wheat growing, milling, transport of wheat and flour) was 6.41 GJ t^?1 flour. However, flour production results in a nett production of food energy.     

Detection of wheat adulteration of spelt flour and products by PCR

 To date, no general method to detect wheat [Triticum aestivum vulgare (Vill.) Mackey] adulteration of spelt [T. aestivum spelta (L.) Thell.] exists. Exploiting a published allelic difference in γ-gliadin gene GAG56D, we developed and evaluated three PCR-based methods to determine the proportion of wheat in spelt flour and products. A sensitive wheat-specific PCR system was designed. Moreover, a heterologous internal standard was constructed for quantitative competitive (QC-)PCR. The standard was arbitrarily calibrated to 5% wheat DNA in spelt DNA. A PCR-RFLP system consisting of a GAG56D-specific PCR system with subsequent restriction was used to estimate the relative proportions of wheat and spelt in DNA mixtures. The three methods were successfully applied to seven commercial spelt samples and one self-produced bread. Five and three of the seven samples were found to contain more than 5% and 10% wheat, respectively. One sample appeared to contain no spelt at all. Analyzing the bread crumb with PCR-RFLP, we were able to estimate the wheat content of the flour which was used to produce the bread and thus demonstrated the applicability of PCR-RFLP to quantify processed products. Received: 24 March 2000