Lifestyle, Eating Habits, and Health Behaviors Among Dietary Supplement Users in Three European Countries.

Dietary supplements (DS) are used by about 30-50% of adults in developed countries. However, only a few studies have compared the characteristics of DS users in different nations. This study aimed to identify and compare selected health-related behaviors of DS users from three European countries. A total of 3,588 adults (32.08 ± 8.04 years) from Poland (1,030 females, 287 males), Germany (994 females, 190 males), and the United Kingdom (911 females, 176 males) were included in the analysis. The study was based on a self-administered survey consisting of 70 questions regarding baseline characteristics, lifestyle, eating, and health habits. The associations of the obtained results were compared using the Kruskal-Wallis test, Pearson Chi-Square test, and Cramer's V value. The highest percentage of DS users (56.98%, n = 2,044) had a correct body weight, while higher body weight values were observed in 39.19% (n = 1,406). In terms of lifestyle, statistically significant differences (p < 0.05) were noted for alcohol consumption and the level of physical activity. Fruit and vegetables were most often consumed a few times a weeks (34.67%, n = 1,244). A similar result was observed for the consumption of whole grain (37.76%, n = 1,355), dairy (39.99%, n = 1,435), eggs (49.67%, n = 1,782), and meat (51.45%, n = 1,846). Most DS users did not have a chronic disease (66.72%, n = 2,394). Among the other conditions, a frequent occurrence (a few times a weeks) of gastrointestinal problems (28.29%, n = 1,015) and concentration disorders (29.15%, n = 1,046) was noted. Cramer's V values (<0.3) indicated a weak (but significant p < 0.05) relationship between the country of residence and most of the analyzed variables. In conclusion, DS users were characterized by a healthy lifestyle with appropriate behaviors but not healthy eating habits.

Novel Primer Sets for Rapid Detection of Zymoseptoria tritici in Wheat.

Zymoseptoria tritici is a fungal pathogen causing losses in wheat yields. Here, we present new primer sets for species-specific identification of this microorganism in wheat leaf samples using conventional PCR. Primer sets were validated in silico using tools available in genetic databases. Furthermore, in vitro tests were also carried out on 190 common wheat samples with visual symptoms of Septoria tritici blotch (STB) collected in Poland in three growing seasons (2015, 2016, 2017). The designed primer sets showed full hybridization to the available genetic resources deposited in the NCBI GenBank database, and their high specificity and sensitivity were demonstrated on wheat leaf samples and selected fungal strains.

Identification of stable reference genes for qPCR studies in common wheat (Triticum aestivum L.) seedlings under short-term drought stress.

BACKGROUND: Quantitative PCR (qPCR) is one of the most common and accurate methods of gene expression analysis. However, the biggest challenge for this kind of examinations is normalization of the results, which requires the application of dependable internal controls. The selection of appropriate reference genes (RGs) is one of the most crucial points in qPCR data analysis and for correct assessment of gene expression. Because of the fact that many reports indicate that the expression profiles of typically used RGs can be unstable in certain experimental conditions, species or tissues, reference genes with stable expression levels should be selected individually for each experiment. In this study, we analysed a set of ten candidate RGs for wheat seedlings under short-term drought stress. Our tests included five 'traditional' RGs (GAPDH, ACT, UBI, TUB, and TEF1) and five novel genes developed by the RefGenes tool from the Genevestigator database. RESULTS: Expression stability was assessed using five different algorithms: geNorm, NormFinder, BestKeeper, RefFinder and the delta Ct method. In the final ranking, we identified three genes: CJ705892, ACT, and UBI, as the best candidates for housekeeping genes. However, our data indicated a slight variation between the different algorithms that were used. We revealed that the novel gene CJ705892, obtained by means of in silico analysis, showed the most stable expression in the experimental tissue and condition. CONCLUSIONS: Our results support the statement, that novel genes selected for certain experimental conditions have a more stable level of expression in comparison to routinely applied RGs, like genes encoding actin, tubulin or GAPDH. Selected CJ705892 gene can be used as a housekeeping gene in the expression analysis in wheat seedlings under short-term drought. The results of our study will be useful for subsequent analyses of gene expression in wheat tissues subjected to drought.

Genome analysis of a wild rumen bacterium Enterobacter aerogenes LU2 - a novel bio-based succinic acid producer.

Enterobacter aerogenes LU2 was isolated from cow rumen and recognized as a potential succinic acid producer in our previous study. Here, we present the first complete genome sequence of this new, wild strain and report its basic genetic features from a biotechnological perspective. The MinION single-molecule nanopore sequencer supported by the Illumina MiSeq platform yielded a circular 5,062,651 bp chromosome with a GC content of 55% that lacked plasmids. A total of 4,986 genes, including 4,741 protein-coding genes, 22 rRNA-, 86 tRNA-, and 10 ncRNA-encoding genes and 127 pseudogenes, were predicted. The genome features of the studied strain and other Enterobacteriaceae strains were compared. Functional studies on the genome content, metabolic pathways, growth, and carbon transport and utilization were performed. The genomic analysis indicates that succinic acid can be produced by the LU2 strain through the reductive branch of the tricarboxylic acid cycle (TCA) and the glyoxylate pathway. Antibiotic resistance genes were determined, and the potential for bacteriocin production was verified. Furthermore, one intact prophage region of length ~31,9 kb, 47 genomic islands (GIs) and many insertion sequences (ISs) as well as tandem repeats (TRs) were identified. No clustered regularly interspaced short palindromic repeats (CRISPRs) were found. Finally, comparative genome analysis with well-known succinic acid producers was conducted. The genome sequence illustrates that the LU2 strain has several desirable traits, which confirm its potential to be a highly efficient platform for the production of bulk chemicals.

Development and Application of a New PCR Method for Detection of Blumeria graminis f. sp. tritici.

We developed new PCR assays that target beta-tubulin (TUB2) and 14 alpha-demethylase (CYP51) genes and used them for the species-specific detection of Blumeria graminis f. sp. tritici (Bgt). Based on fungi DNA sequences available in the NCBI (National Center for Biotechnology Information) GenBank database we developed simplex and duplex PCR assays. The specificities of the primer sets were evaluated using environmental samples of wheat leaves collected during the 2015/2016 growing season across Poland. Primer sets LidBg17/18 and LidBg21/22 strongly amplified fragments of the expected length for all 67 tested samples. Primer specificity was confirmed using field samples of Zymoseptoria tri-tici, Puccinia triticina (syn. P. recondita f. sp. tritici), P. striiformis f. sp. tritici, and Pyrenophora tritici-repentis.

A co-utilization strategy to consume glycerol and monosaccharides by Rhizopus strains for fumaric acid production.

The ability of Rhizopus oryzae to produce fumaric acid in the presence of glycerol and/or various monosaccharides as carbon sources was examined for seventeen different strains of this fungi. These strains were tested in shake-flask cultures on media containing glycerol and seven different carbohydrates, including glucose, fructose, galactose, mannose, xylose, arabinose, and rhamnose. An interesting and applicationally useful phenomenon was observed. This work presents a new approach to the conventional microbiological method of producing fumaric acid. In the presence of 40 g/l glycerol as the sole carbon source, fumaric acid production reached 0.16-6.1 g/l after 192 h. When monosaccharides were used as a single carbon source, the maximum fumaric acid concentration was much higher; for example, 19.8 g/l was achieved when 40 g/l xylose was used. In the co-fermentation of xylose (40 g/l) and glycerol (20 g/l), post-culture broth contained approx. 28.0 g/l of fumaric acid with a process yield of 0.90 g/g after 168 h. The production of fumaric acid by Rhizopus oryzae was also increased in the dual presence of glycerol and monosaccharides like fructose, galactose, and mannose. However, results obtained on glucose-glycerol-based medium did not follow this trend, showing instead complete utilization of glucose with significant glycerol consumption, but unexpectedly low final amounts of fumaric acid and process yields. Understanding how Rhizopus oryzae utilize various carbon sources may provide alternative avenues of fumaric acid fermentation.

Novel PCR Assays for the Detection of Biological Agents Responsible for Wheat Rust Diseases: Puccinia triticina and Puccinia striiformis f. sp. tritici.

The species Puccinia triticina (Pt) and Puccinia striiformis f. sp. tritici (Pst) are devastating cereal pathogens that cause leaf and stripe rust diseases. We developed PCR assays for the species-specific detection of Pt and Pst, 2 biological agents that cause wheat rust disease. For each pathogen, we validated 3 primer sets that target the second largest subunits of the RNA polymerase II (rpb2) and ß-tubulin 1 (tub1) genes. The specificities of the primers were verified using naturally infected plant materials with visual symptoms of disease. All primer sets amplified a single DNA fragment of the expected length. The primer sets LidPr15/16, LidPr1/2, and LidPs13/14 were able to detect small amounts of pure fungal DNA with sensitivities of 0.1, 1, and 10 pg/µL, respectively. A sufficient detection limit (1 pg/µL to 5 ng/µL) was observed for all assays when the sensitivity test was performed with host plant DNA. The study also evaluated the simultaneous detection of both rust pathogens, and the multiplex PCR assay generated amplicons of 240 and 144 bp in length for Pts (LidPs9/10) and Pt (LidPr1/2), respectively.

A Review of Conventional PCR Assays for the Detection of Selected Phytopathogens of Wheat.

Infection of phyllosphere (stems, leaves, husks, and grains) by pathogenic fungi reduces the wheat yield and grain quality. Detection of the main wheat pathogenic fungi provides information about species composition and allows effective and targeted plant treatment. Since conventional procedures for the detection of these organisms are unreliable and time consuming, diagnostic DNA-based methods are required. Nucleic acid amplification technologies are independent of the morphological and biochemical characteristics of fungi. Microorganisms do not need to be cultured. Therefore, a number of PCR-based methodologies have been developed for the identification of key pathogenic fungi, such as Fusarium spp., Puccinia spp., Zymoseptoria tritici, Parastagonospora nodorum, Blumeria graminis f. sp. tritici, and Pyrenophora tritici-repentis. This article reviews frequently used DNA regions for fungus identification and discusses already known PCR assays for detection of the aforementioned wheat pathogens. We demonstrate that PCR-based wheat pathogen identification assays require further research. In particular, the number of diagnostic tests for Fusarium graminearum, Puccinia spp., and P. tritici-repentis are insufficient.

The Ability of a Novel Strain Scheffersomyces (Syn. Candida) shehatae Isolated from Rotten Wood to Produce Arabitol.

Arabitol is a polyalcohol which has about 70% of the sweetness of sucrose and an energy density of 0.2 kcal/g. Similarly to xylitol, it can be used in the food and pharmaceutical industries as a natural sweetener, a texturing agent, a dental caries reducer, and a humectant. Biotechnological production of arabitol from sugars represents an interesting alternative to chemical production. The yeast Scheffersomyces shehatae strain 20BM-3 isolated from rotten wood was screened for its ability to produce arabitol from L-arabinose, glucose, and xylose. This isolate, cultured at 28°C and 150 rpm, secreted 4.03 ± 0.00 to 7.97 ± 0.67 g/l of arabitol from 17-30 g/l of L-arabinose assimilated from a medium containing 20-80 g/l of this pentose with yields of 0.24 ± 0.00 to 0.36 ± 0.02 g/g. An optimization study demonstrated that pH 4.0, 32°C, and a shaking frequency of 150 rpm were the optimum conditions for arabitol production by the investigated strain. Under these conditions, strain 20BM-3 produced 6.2 ± 0.17 g/l of arabitol from 17.5 g/l of arabinose after 4 days with a yield of 0.35 ± 0.01 g/g. This strain also produced arabitol from glucose, giving much lower yields, but did not produce it from xylose. The new strain can be successfully used for arabitol production from abundantly available sugars found in plant biomass.

Variability of S-layer proteins in Lactobacillus helveticus strains.

The presence of S-layer proteins in the cell envelope of Lactobacillus helveticus may be technologically important. S-layer proteins are the adhesion site for cell envelope proteinase, which forms the proteolytic pathway in bacteria. Eleven strains of L. helveticus were examined for the presence of S-layer proteins and slpH genes. S-layer proteins from six strains were identified and sequenced. Multiple alignments of the deduced amino acid sequences demonstrated a strong sequence conservation of all Slp studied. Transmission Electron Microscopy analysis of the cells revealed the typical cell wall architecture of the S-layer. This is the first report on characterisation of glycosylated S-layer proteins from different strains of L. helveticus. The amino acid composition, the secondary structure, and the physical properties of these proteins were found to be quite similar to those of S-layer proteins from other lactobacilli. However, PCR analysis revealed that five of the examined strains of L. helveticus did not have slpH genes. This finding suggests that S-layer protein genes cannot be considered as housekeeping genes and cannot be used as molecular markers for L. helveticus.