Organisms In Food Research Articles

Improved detection and quantification of cauliflower mosaic virus in food crops: assessing false positives in GMO screening based on the 35S promoter

The 35S promoter (P35S) of the cauliflower mosaic virus (CaMV) is frequently used in screening tests as a first step in detecting and quantifying genetically modified organisms (GMOs) in food and feed samples. However, PCR reactions targeting P35S do not distinguish between GMOs and naturally occurring CaMV introduced by infection or contamination, resulting in false positives. Initial attempts at specific CaMV detection were based on a limited number of CAMV sequences. To account for the broad variability in the viral genome, we designed a new real-time PCR assay based on the genomic sequences of 96 CaMV isolates collected worldwide. This system amplified a 112 bp fragment of ORFV in the double-stranded genome, with complete sequence homology to the primer and probe targets of the tested genomes. Tests on a CaMV isolate collection and GMOs revealed improved specificity compared to previously published methods as well as high sensitivity, with limits of detection (LOD) and quantification (LOQ) of three copies of CaMV.

The Road to Infection: Host-Microbe Interactions Defining the Pathogenicity of Streptococcus bovis/Streptococcus equinus Complex Members.

The Streptococcus bovis/Streptococcus equinus complex (SBSEC) comprises several species inhabiting the animal and human gastrointestinal tract (GIT). They match the pathobiont description, are potential zoonotic agents and technological organisms in fermented foods. SBSEC members are associated with multiple diseases in humans and animals including ruminal acidosis, infective endocarditis (IE) and colorectal cancer (CRC). Therefore, this review aims to re-evaluate adhesion and colonization abilities of SBSEC members of animal, human and food origin paired with genomic and functional host-microbe interaction data on their road from colonization to infection. SBSEC seem to be a marginal population during GIT symbiosis that can proliferate as opportunistic pathogens. Risk factors for human colonization are considered living in rural areas and animal-feces contact. Niche adaptation plays a pivotal role where Streptococcus gallolyticus subsp. gallolyticus (SGG) retained the ability to proliferate in various environments. Other SBSEC members have undergone genome reduction and niche-specific gene gain to yield important commensal, pathobiont and technological species. Selective colonization of CRC tissue is suggested for SGG, possibly related to increased adhesion to cancerous cell types featuring enhanced collagen IV accessibility. SGG can colonize, proliferate and may shape the tumor microenvironment to their benefit by tumor promotion upon initial neoplasia development. Bacteria cell surface structures including lipotheichoic acids, capsular polysaccharides and pilus loci (pil1, pil2, and pil3) govern adhesion. Only human blood-derived SGG contain complete pilus loci and other disease-associated surface proteins. Rumen or feces-derived SGG and other SBSEC members lack or harbor mutated pili. Pili also contribute to binding to fibrinogen upon invasion and translocation of cells from the GIT into the blood system, subsequent immune evasion, human contact system activation and collagen-I-binding on damaged heart valves. Only SGG carrying complete pilus loci seem to have highest IE potential in humans with significant links between SGG bacteremia/IE and underlying diseases including CRC. Other SBSEC host-microbe combinations might rely on currently unknown mechanisms. Comparative genome data of blood, commensal and food isolates are limited but required to elucidate the role of pili and other virulence factors, understand pathogenicity mechanisms, host specificity and estimate health risks for animals, humans and food alike.

Drosophila melanogaster as a Versatile Model Organism in Food and Nutrition Research.

Drosophila melanogaster has been widely used in the biological sciences as a model organism. Drosophila has a relatively short life span of 60-80 days, which makes it attractive for life span studies. Moreover, approximately 60% of the fruit fly genes are orthologs to mammals. Thus, metabolic and signal transduction pathways are highly conserved. Maintenance and reproduction of Drosophila do not require sophisticated equipment and are rather cheap. Furthermore, there are fewer ethical issues involved in experimental Drosophila research compared with studies in laboratory rodents, such as rats and mice. Drosophila is increasingly recognized as a model organism in food and nutrition research. Drosophila is often fed complex solid diets based on yeast, corn, and agar. There are also so-called holidic diets available that are defined in terms of their amino acid, fatty acid, carbohydrate, vitamin, mineral, and trace element compositions. Feed intake, body composition, locomotor activity, intestinal barrier function, microbiota, cognition, fertility, aging, and life span can be systematically determined in Drosophila in response to dietary factors. Furthermore, diet-induced pathophysiological mechanisms including inflammation and stress responses may be evaluated in the fly under defined experimental conditions. Here, we critically evaluate Drosophila melanogaster as a versatile model organism in experimental food and nutrition research, review the corresponding data in the literature, and make suggestions for future directions of research.

A Meta-Analysis of Major Foodborne Pathogens in Chinese Food Commodities Between 2006 and 2016

Prevalence of pathogenic bacteria in food commodities in China have been reported in numerous publications over time. However, the results are scattered and varied. To calculate a robust point estimate with a higher statistical power, we applied meta-analytic approach for investigating the prevalence of common foodborne pathogens in major food items in China. Data, on prevalence of bacteria in various food commodities were extracted and analyzed from 361 (132 English and 229 Chinese) publications. Prevalence of eight most frequently reported pathogens on six broad food categories was used for pooled and subgroup meta-analysis by DerSimonian-Laird method in random-effects model. The estimated overall prevalence of pathogens in the foods was 8.5% (95% CI 8.2-8.7). The highest prevalence, irrespective of the pathogen type, was in the aquatic produce at 12.8% (12.0-13.5), while the least was in the vegetables at 3.0% (2.6-3.4). Among the pathogens, the most prevalent was Vibrio at 21.3% (19.6-23.1), whereas the least was pathogenic Escherichia coli at 4.3% (3.3-5.2). The major food pathogens in Chinese foods in decreasing order of prevalence were Vibrio parahaemolyticus, Campylobacter, Bacillus cereus, Staphylococcus aureus, Salmonella, Enterobacter, Listeria monocytogenes, and pathogenic E. coli. Presence of these organisms in foods equates the risk of microbiological food safety in China with other developed countries rather than the developing countries. This justifies the need of novel perspectives for formulating policies on microbiological food safety and risk mitigation.

Electrochemical genoassays on gold-coated magnetic nanoparticles to quantify genetically modified organisms (GMOs) in food and feed as GMO percentage

The integration of nanomaterials in the field of (bio)sensors has allowed developing strategies with improved analytical performance. In this work, ultrasmall core-shell Fe3O4@Au magnetic nanoparticles (MNPs) were used as the platform for the immobilization of event-specific Roundup Ready (RR) soybean and taxon-specific DNA sequences. Firstly, monodisperse Fe3O4 MNPs were synthesized by thermal decomposition and subsequently coated with a gold shell through reduction of Au(III) precursor on the surface of the MNPs in the presence of an organic capping agent. This nanosupport exhibited high colloidal stability, average particle size of 10.2 ± 1.3 nm, and spherical shape. The covalent immobilization of ssDNA probe onto the Au shell of the Fe3O4@Au MNPs was achieved through a self-assembled monolayer (SAM) created from mixtures of alkane thiols (6-mercapto-1-hexanol and mercaptohexanoic acid). The influence of the thiols ratio on the electrochemical performance of the resulting electrochemical genoassays was studied, and remarkably, the best analytical performance was achieved for a pure mercaptohexanoic acid SAM. Two quantification assays were designed; one targeting an RR sequence and a second targeting a reference soybean gene, both with a sandwich format for hybridization, signaling probes labelled with fluorescein isothiocyanate (FITC), enzymatic amplification and chronoamperometric detection at screen-printed carbon electrodes (SPCE). The magnetogenoassays exhibited linear ranges from 0.1 to 10.0 nM and from 0.1 to 5.0 nM with similar detection limits of 0.02 nM and 0.05 nM for the event-specific (RR) and the taxon-specific (lectin) targets, respectively. The usefulness of the approach was demonstrated by its application to detect genetically modified organisms (GMOs) in feed and food.

The Genus Macrococcus: An Insight Into Its Biology, Evolution, and Relationship With Staphylococcus.

The Gram-positive genus Macrococcus is composed of eight species that are evolutionarily closely related to species of the Staphylococcus genus. In contrast to Staphylococcus species, species of Macrococcus are generally regarded to be avirulent in their animal hosts. Recent reports on Macrococcus have focused on the presence of novel methicillin resistance genes in Macrococcus caseolyticus and Macrococcus canis, with the discovery of the first plasmid-encoded methicillin resistance gene in clinical Staphylococcus aureus of probable macrococcal origin generating further interest in these organisms. Furthermore, M. caseolyticus has been associated with flavor development in certain fermented foods and its potential as a food bio-preservative has been documented. The potential application of these organisms in food seems at odds with the emerging information regarding antibiotic resistance and is prompting further examination of the potential safety issues associated with such strains, given the European Food Safety Authority framework for the safety evaluation of microorganisms in the food chain. A comprehensive understanding of the genus would also contribute to understanding the evolution of staphylococci in terms of its acquisition of antibiotic resistance and pathogenic potential. In this review, we discuss the current knowledge on Macrococcus with regard to their phenotypic capabilities, genetic diversity, and evolutionary history with Staphylococcus. Comparative genomics of the sequenced Macrococcus species will be discussed, providing insight into their unique metabolic features and the genetic structures carrying methicillin resistance. An in-depth understanding of these antibiotic resistance determinants can open the possibilities for devising better preventative strategies for an unpredictable future.

Genetically modified foods in the opinion of the second-year students of biology, biotechnology and tourism and recreation of the Jan Kochanowski University in Kielce – a preliminary study

Abstract The aim of this work was to assess knowledge of and to identify awareness in second-year students of biology, biotechnology and tourism and recreation, regarding the use of genetically modified organisms (GMO) in food. The analysis of obtained results shows that about 98% of respondents know the concept of GMO and highly appreciate their knowledge of this topic. The main source of knowledge about GMO for the students is the Internet and the University. It is worth noting that 59% of respondents are aware of the use of GMO in food, while more than half do not know how the GMO in food should be labeled. In particular, students of biotechnology showed a distinctive knowledge about GMO. Over half of students of the Jan Kochanowski University in the fields of biology, biotechnology, and tourism and recreation (55%) recognized that the use of GMO poses a threat to human health.

Optimization and Verification of Droplet Digital PCR Even-Specific Methods for the Quantification of GM Maize DAS1507 and NK603.

In recent years, digital polymerase chain reaction (dPCR), a new molecular biology technique, has been gaining in popularity. Among many other applications, this technique can also be used for the detection and quantification of genetically modified organisms (GMOs) in food and feed. It might replace the currently widely used real-time PCR method (qPCR), by overcoming problems related to the PCR inhibition and the requirement of certified reference materials to be used as a calibrant. In theory, validated qPCR methods can be easily transferred to the dPCR platform. However, optimization of the PCR conditions might be necessary. In this study, we report the transfer of two validated qPCR methods for quantification of maize DAS1507 and NK603 events to the droplet dPCR (ddPCR) platform. After some optimization, both methods have been verified according to the guidance of the European Network of GMO Laboratories (ENGL) on analytical method verification (ENGL working group on "Method Verification." (2011) Verification of Analytical Methods for GMO Testing When Implementing Interlaboratory Validated Methods). Digital PCR methods performed equally or better than the qPCR methods. Optimized ddPCR methods confirm their suitability for GMO determination in food and feed.

Epidemiology of Helicobacter pylori infection.

The study of Helicobacter pylori genetic variability brought us interesting data on the history of mankind. Based on multilocus sequence typing and more recently on whole-genome sequencing, paleomicrobiology still attracts the attention of global researchers in relation to its ancestor roots and coexistence with humans. Three studies determining the prevalence of virulence factors illustrates the controversial results obtained since 30years by studies trying to associate prevalence of different virulence markers and clinical outcomes of H.pylori infection. Three articles analyzed the prevalence and risk of multiple (genetically distinct isolates) and mixed (susceptible and resistant isolates) infections. A number of studies confirm that H.pylori prevalence is falling worldwide especially in the developed world and in children but that the level of infection is higher in certain ethnic minorities and in Migrants. There is little new in identifying the mode of H.pylori transmission though intrafamilial spread appears to be important. There have, however, been some interesting papers on the presence of the organism in food, water, and the oral cavity.

Development and inter-laboratory assessment of droplet digital PCR assays for multiplex quantification of 15 genetically modified soybean lines

Quantification of genetically modified organisms (GMOs) in food and feed products is often required for their labelling or for tolerance thresholds. Standard-curve-based simplex quantitative polymerase chain reaction (qPCR) is the prevailing technology, which is often combined with screening analysis. With the rapidly growing number of GMOs on the world market, qPCR analysis becomes laborious and expensive. Innovative cost-effective approaches are therefore urgently needed. Here, we report the development and inter-laboratory assessment of multiplex assays to quantify GMO soybean using droplet digital PCR (ddPCR). The assays were developed to facilitate testing of foods and feed for compliance with current GMO regulations in the European Union (EU). Within the EU, the threshold for labelling is 0.9% for authorised GMOs per ingredient. Furthermore, the EU has set a technical zero tolerance limit of 0.1% for certain unauthorised GMOs. The novel multiplex ddPCR assays developed target 11 GMO soybean lines that are currently authorised, and four that are tolerated, pending authorisation in the EU. Potential significant improvements in cost efficiency are demonstrated. Performance was assessed for the critical parameters, including limits of detection and quantification, and trueness, repeatability, and robustness. Inter-laboratory performance was also determined on a number of proficiency programme and real-life samples.

Análise microscópica do amido extraído de milho (Zea mays) convencional e transgênico

Os programas de melhoramento genético têm desenvolvido uma grande variedade de sementes de milho, com características que conferem maior resistência às adversidades do plantio, e permitem a extensão do seu cultivo nas mais diversas condições climáticas. A análise microscópica de grãos é uma ferramenta de grande relevância na caracterização das matérias primas e modificações do amido. Levando-se em conta o fato do uso de transgênicos em alimentos ser ainda controverso, e principalmente para os consumidores, o considerável aumento de sua utilização como ingrediente em produtos alimentícios, o presente trabalho investigou a possibilidade de detectar, por meio de microscopia, a variação das características dos grãos de amido de milho transgênico em comparação com os grãos de amido de milho convencional. Os resultados indicaram diferenças entre os grãos de amido extraídos de milho convencional e naqueles extraídos de milho transgênico. O amido extraído de milho convencional, em sua maioria, caracterizou-se pela presença de grânulos de formato poliédrico, alguns ligeiramente abaulados, com hilo em formato estrelado. No amido extraído dos grãos de milho transgênico, o hilo apresentou formato linear, pontual ou ausente. Estas propriedades serão úteis para caracterizá-los nos trabalhos de pesquisa e de identificação em produtos alimentícios.

Rapid Screening and Identification of Living Pathogenic Organisms via Optimized Bioorthogonal Non-canonical Amino Acid Tagging

Pathogenic bacteria can be a major cause of illness from environmental sources as well as the consumption of contaminated products, giving rise to public health concerns globally. The surveillance of such living organisms in food and water supplies remains an important challenge in mitigating their deleterious societal effects. Here, we have developed an optimized bioorthogonal non-canonical amino acid tagging approach to the imaging, capture, and interrogation of shigatoxigenic/verotoxigenic Escherichia coli (VTEC) and Listeria that enables the distinction between living wild-type pathogenic bacteria. The approaches utilize homopropargylglycine (HPG), as well as optimized growth media, that restricts endogenous methionine biosynthesis in a variety of species of public health concern. Endogenous methionine residues are then replaced with HPG, which can then be modified using a myriad of compatible bioorthogonal reactions for tagging of exclusively live bacteria. The methods reported allow for the very rapid screening and identification of living pathogenic organisms.

Detection by real-time PCR and pyrosequencing of the cry1Ab and cry1Ac genes introduced in genetically modified (GM) constructs

ABSTRACTThe presence of genetically modified organisms (GMOs) in food and feed is mainly detected by the use of targets focusing on promoters and terminators. As some genes are frequently used in genetically modified (GM) construction, they also constitute excellent screening elements and their use is increasing. In this paper we propose a new target for the detection of cry1Ab and cry1Ac genes by real-time polymerase chain reaction (PCR) and pyrosequencing. The specificity, sensitivity and robustness of the real-time PCR method were tested following the recommendations of international guidelines and the method met the expected performance criteria. This paper also shows how the robustness testing was assessed. This new cry1Ab/Ac method can provide a positive signal with a larger number of GM events than do the other existing methods using double dye-probes. The method permits the analysis of results with less ambiguity than the SYBRGreen method recommended by the European Reference Laboratory (EURL) GM Food and Feed (GMFF). A pyrosequencing method was also developed to gain additional information thanks to the sequence of the amplicon. This method of sequencing-by-synthesis can determine the sequence between the primers used for PCR. Pyrosequencing showed that the sequences internal to the primers present differences following the GM events considered and three different sequences were observed. The sensitivity of the pyrosequencing was tested on reference flours with a low percentage GM content and different copy numbers. Improvements in the pyrosequencing protocol provided correct sequences with 50 copies of the target. Below this copy number, the quality of the sequence was more random.

A Quantitative PCR-Electrochemical Genosensor Test for the Screening of Biotech Crops.

The design of screening methods for the detection of genetically modified organisms (GMOs) in food would improve the efficiency in their control. We report here a PCR amplification method combined with a sequence-specific electrochemical genosensor for the quantification of a DNA sequence characteristic of the 35S promoter derived from the cauliflower mosaic virus (CaMV). Specifically, we employ a genosensor constructed by chemisorption of a thiolated capture probe and p-aminothiophenol gold surfaces to entrap on the sensing layer the unpurified PCR amplicons, together with a signaling probe labeled with fluorescein. The proposed test allows for the determination of a transgene copy number in both hemizygous (maize MON810 trait) and homozygous (soybean GTS40-3-2) transformed plants, and exhibits a limit of quantification of at least 0.25% for both kinds of GMO lines.

Food Analysis Using Organelle DNA and the Effects of Processing on Assays.

Extrachromosomal DNA such as organelle DNA are increasingly targeted in molecular detection assays where samples have been degraded by physical or chemical means. Owing to multiple organelles per cell and greater copy numbers than nuclear genes, organelle gene targets provide a more robust signal in polymerase chain reaction (PCR), quantitative PCR (qPCR), and other emerging molecular technologies. Because of these advantages, direct analysis of organelle DNA in food matrices is used for detection of contaminants and identification and authentication of food ingredients and allergens. Non-nuclear DNA is also used as an assay normalizer for detection of genetically modified organisms (GMOs) in foods. This review describes these protocols plus the effects of processing on efficacy, with special emphasis on thermally produced DNA fragmentation. Future research may incorporate molecular techniques beyond detection, used instead as time-temperature indicators in thermal food processing or quality indicators in food fermentation or acidification.

Shielding calculations for industrial 5/7.5MeV electron accelerators using the MCNP Monte Carlo Code

High energy X-rays from accelerators are used to irradiate food ingredients to prevent growth and development of unwanted biological organisms in food, and by that extend the shelf life of the products. The production of X-rays is done by accelerating 5 MeV electrons and bombarding them into a heavy target (high Z). Since 2004, the FDA has approved using 7.5 MeV energy, providing higher production rates with lower treatments costs. In this study we calculated all the essential data needed for a straightforward concrete shielding design of typical food accelerator rooms. The following evaluation is done using the MCNP Monte Carlo code system: (1) Angular dependence (0-180°) of photon dose rate for 5 MeV and 7.5 MeV electron beams bombarding iron, aluminum, gold, tantalum, and tungsten targets. (2) Angular dependence (0-180°) spectral distribution simulations of bremsstrahlung for gold, tantalum, and tungsten bombarded by 5 MeV and 7.5 MeV electron beams. (3) Concrete attenuation calculations in several photon emission angles for the 5 MeV and 7.5 MeV electron beams bombarding a tantalum target. Based on the simulation, we calculated the expected increase in dose rate for facilities intending to increase the energy from 5 MeV to 7.5 MeV, and the concrete width needed to be added in order to keep the existing dose rate unchanged.

Electrochemical detection of magnetically-entrapped DNA sequences from complex samples by multiplexed enzymatic labelling: Application to a transgenic food/feed quantitative survey

Monitoring of genetically modified organisms in food and feed demands molecular techniques that deliver accurate quantitative results. Electrochemical DNA detection has been widely described in this field, yet most reports convey qualitative data and application in processed food and feed samples is limited. Herein, the applicability of an electrochemical multiplex assay for DNA quantification in complex samples is assessed. The method consists of the simultaneous magnetic entrapment via sandwich hybridisation of two DNA sequences (event-specific and taxon-specific) onto the surface of magnetic microparticles, followed by bienzymatic labelling. As proof-of-concept, we report its application in a transgenic food/feed survey where relative quantification (two-target approach) of Roundup Ready Soybean® (RRS) was performed in food and feed. Quantitative coupling to end-point PCR was performed and calibration was achieved from 22 and 243 DNA copies spanning two orders of magnitude for the event and taxon-specific sequences, respectively. We collected a total of 33 soybean-containing samples acquired in local supermarkets, four out of which were found to contain undeclared presence of genetically modified soybean. A real-time PCR method was used to verify these findings. High correlation was found between results, indicating the suitability of the proposed multiplex method for food and feed monitoring.

Statistical optimization of exopolysaccharide production by Lactobacillus plantarum NTMI05 and NTMI20

In this study, 27 strains of Lactic acid bacteria (LAB) were isolated and identified from different milk sources. All the isolates were biochemically characterized and screened for their ability to produce exopolysaccharides (EPS), among which two isolates namely Lactobacillus plantarum NTMI05 (197mg/L) and Lactobacillus plantarum NTMI20 (187mg/L) showed higher EPS production. Both the isolates were molecular characterized and tested for their probiotic properties. The chemical composition of EPS from L. plantarum NTMI05 and NTMI20 revealed the presence of 95.45% and 92.35% carbohydrates, 14±0.1and 11±0.15mg/L lactic acid, 10.5±0.2 and 9±0.1mg/mL of reducing sugar, respectively. HPLC analysis showed galactose at the retention time of 2.29.The maximum EPS yield was optimized for the media components like glucose (20g/L), yeast extract (25g/L) and ammonium sulphate (2g/L) using Central Composite Design and Response Surface Methodology (RSM). Under optimum conditions the predicted maximum EPS production was 0.891g/L, 0.797g/L, while the actual experimental value was 0.956g/L and 0.827g/L for L. plantarum NTMI05 and NTMI20, respectively. The antioxidant capacity was also evaluated by DPPH and reducing power assay proving the potentiality of these organisms in food and dairy industries.

Inter-laboratory studies for the validation of two singleplex (tE9 and pea lectin) and one duplex (pat/bar) real-time PCR methods for GMO detection

This paper presents the inter-laboratory studies for the successful validation of qualitative real-time Polymerase Chain Reaction methods for the detection of Genetically Modified Organisms in food and feed. Two singleplex TaqMan methods were developed for the detection of the tE9 terminator present in several genetically modified events and the detection of the endogenous lectin gene from pea; the tE9 terminator donor organism. In addition, a TaqMan duplex method was validated, which allows the simultaneous detection of the pat and bar elements.The paper explains the different acceptance parameters used during the in-house validation, the method transferability study and finally the inter-laboratory validation, how this validation was conducted and the problems encountered. Statistical methods for the validation of the qualitative methods are also presented. This information can be used by laboratories for the verification of these methods during their implementation and for the validation of new methods.

Highly sensitive detection of dipicolinic acid with a water-dispersible terbium-metal organic framework

The sensitive detection of dipicolinic acid (DPA) is strongly associated with the sensing of bacterial organisms in food and many types of environmental samples. To date, the demand for a sensitive detection method for bacterial toxicity has increased remarkably. Herein, we investigated the DPA detection potential of a water-dispersible terbium-metal organic framework (Tb-MOF) based on the fluorescence quenching mechanism. The Tb-MOF showed a highly sensitive ability to detect DPA at a limit of detection of 0.04nM (linear range of detection: 1nM to 5µM) and also offered enhanced selectivity from other commonly associated organic molecules. The present study provides a basis for the application of Tb-MOF for direct, convenient, highly sensitive, and specific detection of DPA in the actual samples.