A Robust and Poisson Validated Quantitative 5′ Nuclease TaqMan® Real-Time PCR Assay Targeting fimA for the Rapid Detection of Salmonella spp. in Food

A newly designed TaqMan® probe and an internal amplification control were implemented in a conventional PCR system targeting the major fimbrial subunit encoding gene fimA. This assay has an inclusivity and exclusivity of 100 % (n?=?126). The limit of detection (LOD) and the absolute quantification limit, both determined by advanced Poisson analyses, were three bacterial cell equivalents per quantitative real-time PCR reaction. The fimA assay achieved 100 % accuracy and performed very robustly, producing reproducible, reliable data for quantification of Salmonella spp., even at the LOD.     

Detection and Quantification of Enterobacter sakazakii by Real-time 5′-Nuclease Polymerase Chain Reaction Targeting the palE Gene

Enterobacter sakazakii is an emerging food-borne pathogen causing invasive infection with high mortality rates in neonates and infants. The aim of this study was to develop, optimize, and evaluate real-time 5′-nuclease polymerase chain reaction (PCR) for the specific detection and quantification of E. sakazakii. Original primers and TaqMan probe targeting a sequence of E. sakazakii palE gene were designed. The developed real-time PCR system was highly specific for E. sakazakii with 100% inclusivity determined using 54 E. sakazakii strains and 100% exclusivity determined using 99 other strains. Detection limits of 4 × 10² and 4 × 10¹ CFU ml⁻¹ were determined with 100% and 90% probability, respectively. The response of the 5′-nuclease PCR system was linear (correlation coefficient ≥ 0.997) in the range of 10¹ to 10⁸ CFU ml⁻¹. Five methods of DNA sample preparation were compared. The methods of DNA preparation using the InstaGene Matrix and the simple lysis by boiling with the Triton X-100 were the most sensitive with calibration lines applicable for quantification. The developed real-time PCR targeted to the palE gene provides an alternative possibility for the detection and quantification of E. sakazakii after the suitable sample preparation.     

An Overview on the Application of Chemometrics in Food Science and Technology—An Approach to Quantitative Data Analysis

During the last 30 years, food scientists and technologists all over the world are dealing with massive amounts of data derived from the use of different measuring devices (e.g. instrumental and sensory data), the integration of different analytical techniques and processes during the analysis and production of foods. Therefore, complementary disciplines and tools to the traditional ones used in food science such as statistics, experimental design and chemometrics have become essential in modern sciences and are an integral component in the day-to-day analysis of foods and derived products. The aim of this paper is to introduce as well as provide with an overview of different concepts, methods, techniques and general steps used in the quantitative analysis of foods when chemometrics or multivariate analytical methods are applied.     

Relative Accuracy,Specificity and Sensitivity of a 5′ Nuclease Real-Time PCR Assay for Listeria monocytogenes Detection in Naturally Contaminated Pork Cuts

In the present study, the relative sensitivity, specificity and accuracy of a real-time PCR assay for Listeria monocytogenes detection in naturally contaminated pork cuts were evaluated in comparison to the ISO 11290-1:2004 reference culture method. One hundred and sixty meat samples were collected from ten different lots over a year. The PCR method included a 24-h primary enrichment step, a DNA extraction step, and a final 5′ nuclease real-time PCR assay, including an internal amplification control (IAC) and targeting the hlyA gene. Based on the analysis of 480 sub-units (three sub-units for each sample), the relative sensitivity, specificity and accuracy of the real-time PCR assay were 77.3 %, 67.0 % and 73.1 %, respectively, corresponding to a Cohen’s kappa value of 0.69 (good agreement). Considering that real samples from the worse storage scenarios were included, these results suggest the PCR method as a rapid and accurate alternative method for the quick check of L. monocytogenes in meat lots before distribution.     

Transgenic and cloned animals in the food chain – are we prepared to tackle it?

Transgenic and cloned animal production for various purposes has been increasing rapidly in recent times. While the actual impact of these animals in the food chain is unknown, the significance of tracking and monitoring measures to curb accidental and or deliberate release has been discussed. Religious perspectives from different faiths and traditions have been presented. Although the concept of substantial equivalence satisfies the technical and nutritional requirements of these products when assessed against comparators, public opinion and religious concerns should also be considered by the regulators while developing policy regulations. In conclusion, measures to prevent real or perceived risks of transgenic and cloned animals in food production require global coordinated action. It is worthwhile to consider establishing effective tracking systems and analytical procedures as this will be a valuable tool if a global consensus is not reached on policy regulation. © 2015 Society of Chemical Industry     

Detection of 5-hydroxymethyl-2-methyl-3(2H)-furanone and of α-dicarbonyl compounds in reaction mixtures of hexoses and pentoses with different amines

Summary In reaction mixtures of hexoses with primary amines, heated at pH 5 and 7, distinctly higher amounts of fragmentation products are formed in the upper pH range. To estimate the amount of fragmentation, all-dicarbonyl compounds which react witho-phenylenediamine to quinoxaline derivatives were recorded. For the first time the-dicarbonyl intermediates in a pentose amine reaction mixture were determined and the formation of 2,3,4-pentantrione was detected. The 1,4-dideoxyosone was formed from hexoses and pentoses in higher yields in the presence of-amino acids, i.e. when Strecker degradation could take place. 5-Hydroxymethyl-2-methyl-3(2H)-furanone was identified as a new hexose product of Strecker degradation, and its structure established unequivocally by independent synthesis as well as by¹H and¹³C NMR.

---

Nachweis von 5-Hydroxymethyl-2-methyl-3(2H)-furanon und von -Dicarbonylverbindungen in Reaktionsgemischen von Hexosen und Pentosen mit verschiedenen Aminen

Zusammenfassung In Reaktionsgemischen von Hexosen mit priären Aminen, die bei pH 5 und pH 7 erhitzt wurden, findet man im höheren pH-Bereich wesentlich größere Mengen an Fragmentierungsprodukten. Zur Abschätzung des Fragmentierungsanteils werden alle-Di-carbonylverbindungen herangezogen, die mito-Pheny-lendiamin im Umsetzungsgemisch zu Chinoxalinen reagieren. Erstmals werden die-Dicarbonyl-Zwischenstu-fen in Pentose-Amin-Reaktionsans ätzen untersucht und die Bildung des 2,3,4-Pentantrions nachgewiesen. Interessant ist, daß bei Hexosen und Pentosen das 1,4-Didesoxyoson in wesentlich größeren Mengen entsteht, wenn -Aminosäuren an der Umsetzung beteiligt sind, d.h. wenn ein Strecker-Abbau stattfinden kann. 5-Hydroxymethyl-2-methyl-3(2H)-furanon wird als neues Hexoseprodukt des Strecker-Abbaus identifiziert. Die Absicherung der Struktur erfolgt durch Synthese und NMR-Un-tersuchungen.

     

Variants in the 3′ untranslated region of the ovine acetyl-coenzyme A acyltransferase 2 gene are associated with dairy traits and exhibit differential allelic expression

The acetyl-CoA acyltransferase 2 (ACAA2) gene encodes an enzyme of the thiolase family that is involved in mitochondrial fatty acid elongation and degradation by catalyzing the last step of the respective β-oxidation pathway. The increased energy needs for gluconeogenesis and triglyceride synthesis during lactation are met primarily by increased fatty acid oxidation. Therefore, the ACAA2 enzyme plays an important role in the supply of energy and carbon substrates for lactation and may thus affect milk production traits. This study investigated the association of the ACAA2 gene with important sheep traits and the putative functional involvement of this gene in dairy traits. A single nucleotide substitution, a T to C transition located in the 3′ untranslated region of the ACAA2 gene, was used in mixed model association analysis with milk yield, milk protein yield and percentage, milk fat yield and percentage, and litter size at birth. The single nucleotide polymorphism was significantly associated with total lactation production and milk protein percentage, with respective additive effects of 6.81 ± 2.95 kg and ?0.05 ± 0.02%. Additionally, a significant dominance effect of 0.46 ± 0.21 kg was detected for milk fat yield. Homozygous TT and heterozygous CT animals exhibited higher milk yield compared with homozygous CC animals, whereas the latter exhibited increased milk protein percentage. Expression analysis from age-, lactation-, and parity-matched female sheep showed that mRNA expression of the ACAA2 gene from TT animals was 2.8- and 11.8-fold higher in liver and mammary gland, respectively. In addition, by developing an allelic expression imbalance assay, it was estimated that the T allele was expressed at an average of 18% more compared with the C allele in the udder of randomly selected ewes. We demonstrated for the first time that the variants in the 3′ untranslated region of the ovine ACAA2 gene are differentially expressed in homozygous ewes of each allele and exhibit allelic expression imbalance within heterozygotes in a tissue-specific manner, supporting the existence of cis-regulatory DNA variation in the ovine ACAA2 gene. This is the first study reporting differential allelic imbalance expression of a candidate gene associated with milk production traits in dairy sheep.     

Effects of vitamin D3 on the expression of growth-related oncogene-α in THP-1 cells and human primary monocytes

Asthma and many autoimmune diseases, such as systemic lupus erythematosus, have been reported to associate with vitamin D deficiency recently. Growth-related oncogene-α (GRO-α)/CXCL1, a neutrophil-related chemokine, have an important influence on the chronic inflammation of these diseases. It is unknown whether vitamin D has regulatory effects on GRO-α expression in human monocytes. To this end, the human monocytic leukemia cell line, THP-1, and human primary monocytes were pretreated with 1α, 25-(OH)(2)D(3), and was stimulated with lipopolysaccharide (LPS). Supernatants were collected to determine GRO-α level by ELISA. The intracellular signaling was investigated by nuclear factor (NF)-κB inhibitor, the mitogen-activated protein kinase (MAPK) inhibitors, and Western blot. In our studies, LPS-induced GRO-α was significantly enhanced in THP-1 cells, but suppressed in human primary monocytes by 1α, 25-(OH)(2)D(3). Western blotting revealed that 1α, 25-(OH)(2)D(3) increased LPS-stimulated pp38 expression in THP-1 cells, but suppressed LPS-stimulated pMEK1/2-pERK and pJNK in human primary monocytes. In conclusion, the opposite effects of 1α, 25-(OH)(2)D(3) on GRO-α expression in THP-1 cells and human primary monocytes indicated that the data from THP-1 cells should be further confirmed by human primary monocytes. Moreover, vitamin D3 may have potentiality in treating GRO-α-related chronic inflammatory diseases, like asthma and autoimmune diseases.     

Water–water and water–macromolecule interactions in food dehydration and the effects of the pore structures of food on the energetics of the interactions

A molecular dynamics (MD) modeling and simulations approach has been rationally built and developed to study porous food systems constructed with amylose and dextran chains. The findings from our MD studies indicate that the presence of food macromolecules decreases the energetics of the water–water interactions for the nearby water molecules in the pore space, but provides additional water–macromolecule interactions that can significantly outweigh the partial loss of water–water interactions to make the adjacent water molecules strongly bound to the food macromolecules so that the water activity and water removal rate are decreased as dehydration proceeds and, thus, the dehydration energy requirement would be increased. The effects of pore structures are greater in systems with higher densities of food macromolecules, smaller in size pores, and stronger water–macromolecule interactions. Dehydration of food materials can thus be reasonably expected to start from the largest pores and from the middle of the pores, and to have non-uniform water removal rates and non-planar water–vapor interfaces inside individual pores as well as across sections of the food materials. The food porous structures are found to have good pore connectivity for water molecules. As dehydration proceeds, water content and the support from water–water and water–macromolecule interactions both decrease, causing the food porous structures to adopt more compact conformations and their main body to decrease in size. Dehydration in general also reduces pore sizes and the number of pore openings, increases the water–macromolecule interactions, and leads to the reduction of the overall thermal conductivity of the system, so that more energy (heat), longer times, and/or greater temperature gradients are needed in order to further dehydrate the porous materials. Our thermodynamic analysis also shows that the average minimum entropy requirement for food dehydration is greater when the water–macromolecule interactions are stronger and the food macromolecular density is higher. The importance of the physicochemical affinity of food molecules for water and of the compatibility of the resultant porous structures with water configurational structures in determining food properties and food processing through the water–macromolecule interactions, is clearly and fundamentally verified by the results and discussion presented in this work.     

Event-specific detection of genetically modified wheat B73-6-1 based on the 3′-flanking sequence

In this study, 3′-flanking sequence between the host plant DNA and the integrated gene construct of pHMW1Dx5 vector in transgenic wheat B73-6-1 was revealed by means of adaptor PCR; thus, the fragment with the length of 3.1?kb was obtained, including a 190-bp wheat genomic DNA, which demonstrates that this HMW-GS gene was located on the wheat chromosome 3B. And the event-specific PCR primers were designed based upon the revealed 3′-flanking sequence; the conventional qualitative PCR and quantitative SYBR real-time PCR detection methods employing these primers were successfully developed. In conventional qualitative PCR assay, the limit of detection was 0.1?% for B73-6-1 wheat genomic DNA for one reaction. In the quantitative SYBR real-time PCR assay, the limit of detection and limit of quantification were 10 and 100 haploid genome copies, respectively. In addition, three mixed blind wheat samples with known B73-6-1 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event-specific real-time PCR detection systems were reliable, sensitive and accurate.     

Interaction of phosphatidylcholine and α-tocopherol on the oxidation of sunflower oil and content changes of phosphatidylcholine and tocopherol in the emulsion under singlet oxygen

Abstract: Interaction of phosphatidylcholine (PC) and α‐tocopherol (α‐Toc) on the oxidation of oil in the emulsion consisting of sunflower oil and water under singlet oxygen at 25 °C was studied by determining peroxide value (PV) and conjugated dienoic acid (CDA) contents. Singlet oxygen was produced by chlorophyll b under 1700 lux. Single addition of PC or α‐Toc decreased the values of peroxides and CDAs of oil in the emulsion via singlet oxygen quenching. PC and α‐Toc showed simply additive interaction in decreasing the singlet oxygen oxidation of oil in the emulsion. α‐Toc was a physical quencher of singlet oxygen in the emulsion, but PC involved chemical quenching in the antioxidant action. Chlorophyll and PC contents were decreased in the emulsion under singlet oxygen, while α‐Toc was not. α‐Toc protected chlorophyll and PC from degradation, and was a more important component than PC in the oil oxidation under singlet oxygen in the emulsion.     

Effect of various calcium concentrations on the interactions between β-lactoglobulin and epigallocatechin-3-gallate

β-Lactoglobulin (β-lg) is a globular protein accounting for 11% of all milk proteins. Recently, the use of β-lg as vehicles for the delivery of bioactive compounds such as green tea catechins has been explored. The objective of this study was to determine the impact of various calcium concentrations on the interactions between β-lg and epigallocatechin-3-gallate (EGCG), the most abundant green tea catechins. Five different calcium concentrations were tested for their effect on complex formation and characteristics. Our results showed that when EGCG was present, the increase in calcium concentration influenced ζ-potential and more importantly, drastically increased particle size. The presence of EGCG was essential to the formation of larger complexes, stabilized mainly by electrostatic interactions via calcium bridging when calcium is present.     

Changes on the levels of serotonin precursors – tryptophan and 5-hydroxytryptophan – during roasting of Arabica and Robusta coffee

The levels of free and total tryptophan and of 5-hydroxytryptophan (5-HTP) were investigated in green and roasted grains and beverages of Coffea arabica L. (Arabica) and Coffea canephora Pierre var. robusta (Robusta). Grains were light, medium and dark roasted. Free and protein tryptophan were extracted before and after hydrolysis. The levels of tryptophan and 5-HTP were quantified simultaneously by ion-pair HPLC and fluorimetric detection after derivatisation with o-phthalaldehyde. Robusta green coffee had higher total and protein tryptophan, whereas Arabica had higher free tryptophan levels. 5-HTP was not detected in the samples before and after roasting. Free tryptophan was completely degraded during roasting. Roasting significantly affected protein tryptophan. The rate of loss was smaller in Arabica compared to Robusta at every roasting degree. A beverage prepared the Brazilian way with a medium-roasted coffee provided 1.4–2.5 mg tryptophan/50 ml cup.     

Phytoestrogen content of fruits and vegetables commonly consumed in the UK based on LC–MS and C-labelled standards

Phytoestrogens are a group of non-steroidal secondary plant metabolites with structural and functional similarity to 17β-oestradiol. Urinary and plasma phytoestrogens have been used as biomarkers for dietary intake, however, this is often not possible in large epidemiological studies or to assess general exposure in free-living individuals. Accurate information about dietary phytoestrogens is therefore important but there is very limited data concerning food contents. In this study, we analysed the phytoestrogen (isoflavone, lignan and coumestrol) content in more than 240 different foods based on fresh and processed fruits and vegetables using a newly developed sensitive method based on LC–MS incorporating ¹³C₃-labelled standards. Phytoestrogens were detected in all foods analysed with a median content of 20 μg/100 g wet weight (isoflavones: 2 μg/100 g; lignans 12 μg/100 g). Most foods contained less than 100 μg/100 g, however, 5% of foods analysed contained more than 400 μg/100 g, in particular soya-based foods and other legumes. The results published here will contribute to databases of dietary phytoestrogen content and allow the more accurate determination of phytoestrogen exposure in free-living individuals.     

Detection and identification of the cross-linking amino acidsNτ-andNπ-(2′-amino-2′-carboxy-ethyl)-l-histidine (“histidinoalanine”, HAL) in heated milk products

Summary An unknown ninhydrin positive compound, X, was detected in acid hydrolysates of heated skim milk samples by amino acid analysis, eluting between phenylalanine and pyridosine in the chromatogram. The formation of X correlated with heating time and temperature. preparative ion-exchange chromatography enabled the isolation of X and a second minor compound from a milk protein hydrolysate and from a model mixture consisting of N-acetylhistidine and methyl-2-acetamidoacrylate (acetyldehydroalaninmethylester), in a relative abundance of 8 to 1. By¹H-NMR spectroscopy, the two compounds could be identified as the N- and N-isomers of N-(2-amino-2-carboxy-ethyl)-l-histidine (histidinoalanine), a cross-link amino acid that has not been described in food proteins up to now. In a number of foods containing milk protein, the N-histidinoalanine contents were between 50 and 1800 mg/kg protein, which is in a concentration range comparable to the potential nephrotoxic cross-link lysinoalanine, which was determined simultaneously.

---

Nachweis und Identifizierung der Crosslink-AminosäurenN -undN -(2-Amino-2-carboxy-ethyl)-l-histidin (Histidinoalanin, HAL) in erhitzten Milchprodukten

Zusammenfassung In Säurehydrolysaten erhitzter Magermilch konnte am Aminosäureanalysator im Elutionsbereich zwischen Phenylalanin und Pyridosin eine unbekannte Verbindung, X, nachgewiesen werden, deren Bildung mit der Erhitzungszeit und -temperatur korrelierte. Durch präparative Ionenaustauschchromatographie gelang die Isolierung von X und einer zweiten Minorkomponente aus einem Milchproteinhydrolysat sowie einem Modellansatz bestehend aus N-Acetylhistidin undx` 2-Acetamidoacrylsäuremethylester (Acetyldehydroalaninmethylester) im Mengenverhältnis von 8 zu 1.¹H-NMR-spektroskopische Untersuchungen ermöglichten die eindeutige Identifizierung der beiden Verbindungen als N- und N-Isomer vonN-(2-Amino-2-carboxyethyl)-l-histidin (Histidinoalanin). Diese Crosslink-Aminosäuren waren bisher noch nicht in Lebensmitteln nachgewiesen worden. Die Gehalte an N-Histidinoalanin in einer Anzahl milchproteinhaltiger Lebensmittel lagen mit 50 bis 1800 mg/kg Protein in einer mit der simultan quantifizierten, potentiell nierentoxischen Crosslink-Aminosäure Lysinoalanin vergleichbaren Größenordnung.

---

---

Dedicated to Prof. Dr. F. Kiermeier on the occasion of his 85th birthday