Nutritional Ergogenic Aids in Cycling: A Systematic Review.

This systematic review aimed to evaluate the effectiveness of the independent or combined use of nutritional ergogenic aids belonging to Group A of the ABCD classification by the Australian Institute of Sport (AIS) in the context of cycling (caffeine, creatine, sodium bicarbonate, beta-alanine, nitrates, and glycerol). A comprehensive search was carried out using three databases: PubMed, Scopus, and Web of Science. All the databases were searched for Randomized Controlled Trials or crossover design studies assessing the effects of supplementation on cycling performance in comparison with placebos in healthy adults. The methodological quality of each study was evaluated using the Physiotherapy Evidence Database scale. Thirty-six articles involving 701 participants were included in this review, examining supplementation with caffeine (n = 5), creatine (n = 2), sodium bicarbonate (n = 6), beta-alanine (n = 3), and nitrates (n = 8). Additionally, supplemental combinations of caffeine and creatine (n = 3), caffeine and sodium bicarbonate (n = 3), caffeine and nitrates (n = 1), creatine and sodium bicarbonate (n = 1), and sodium bicarbonate and beta-alanine (n = 4) were analyzed. A benefit for cyclists' athletic performnce was found when consuming a caffeine supplement, and a potential positive effect was noted after the consumption of sodium bicarbonate, as well as after the combination of caffeine and creatine. However, no statistically significant effects were identified for the remaining supplements, whether administered individually or in combination.

Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens.

Loop-mediated isothermal amplification, LAMP, is nowadays the most popular isothermal nucleic acid amplification technique, and as such, several commercial, ready-to-use master mixes have flourished. Unfortunately, independent studies to determine their performance are limited. The current study performed an independent evaluation of the existing ready-to-use commercial LAMP master mixes WarmStart® LAMP Kit, LavaLAMP™ DNA Master Mix, Saphir Bst Turbo GreenMaster, OptiGene Fast Master Mix ISO-004, and SynLAMP Mix. To reduce bias, three different genes, namely ttr (Salmonella spp.), rfbE (E. coli O157), and hly (Listeria monocytogenes), were targeted. The comparison was based on amplification speed, performance with decreasing DNA concentrations, and the effect of five typical LAMP reaction additives (betaine, DMSO, pullulan, TMAC, and GuHCl). Significant differences were observed among the different master mixes. OptiGene provided the fastest amplification and showed less detrimental effects associated with the supplements evaluated. Out of the chemicals tested, pullulan provided the best results in terms of amplification speed. It is noteworthy that the different additives impacted the master mixes differently. Overall, the current study provides insights into the performance of commercial LAMP master mixes, which can be of value for the scientific community to better select appropriate reagents when developing new methods.

Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens.

Rapid identification of Shiga toxin-producing Escherichia coli, or STEC, is of utmost importance to assure the innocuousness of the foodstuffs. STEC have been implicated in outbreaks associated with different types of foods however, among them, ready-to-eat (RTE) vegetables are particularly problematic as they are consumed raw, and are rich in compounds that inhibit DNA-based detection methods such as qPCR. In the present study a novel method based on Loop-mediated isothermal amplification (LAMP) to overcome the limitations associated with current molecular methods for the detection of STEC in RTE vegetables targeting stx1 and stx2 genes. In this sense, LAMP demonstrated to be more robust against inhibitory substances in food. In this study, a comprehensive enrichment protocol was combined with four inexpensive DNA extraction protocols. The one based on silica purification enhanced the performance of the method, therefore it was selected for its implementation in the final method. Additionally, three different detection chemistries were compared, namely real-time fluorescence detection, and two end-point colorimetric strategies, one based on the addition of SYBR Green, and the other based on a commercial colorimetric master mix. After optimization, all three chemistries demonstrated suitable for the detection of STEC in spiked RTE salad samples, as it was possible to reach a LOD50 of 0.9, 1.4, and 7.0 CFU/25 g for the real-time, SYBR and CC LAMP assays respectively. All the performance parameters reached values higher than 90 %, when compared to a reference method based on multiplex qPCR. More specifically, the analytical sensitivity was 100, 90.0 and 100 % for real-time, SYBR and CC LAMP respectively, the specificity 100 % for all three assays, and accuracy 100, 96 and 100 %. Finally, a high degree of concordance was also obtained (1, 0.92 and 1 respectively). Considering the current technological advances, the method reported, using any of the three detection strategies, demonstrated suitable for their implementation in decentralized settings, with low equipment resources.

Whole genome sequencing in the palm of your hand: how to implement a MinION Galaxy-based workflow in a food safety laboratory for rapid Salmonella spp. serotyping, virulence, and antimicrobial resistance gene identification.

Introduction: Whole Genome Sequencing (WGS) implementation in food safety laboratories is a significant advancement in food pathogen control and outbreak tracking. However, the initial investment for acquiring next-generation sequencing platforms and the need for bioinformatic skills represented an obstacle for the widespread use of WGS. Long-reading technologies, such as the one developed by Oxford Nanopore Technologies, can be easily implemented with a minor initial investment and with simple protocols that can be performed with basic laboratory equipment. Methods: Herein, we report a simple MinION Galaxy-based workflow with analysis parameters that allow its implementation in food safety laboratories with limited computer resources and without previous knowledge in bioinformatics for rapid Salmonella serotyping, virulence, and identification of antimicrobial resistance genes. For that purpose, the single use Flongle flow cells, along with the MinION Mk1B for WGS, and the community-driven web-based analysis platform Galaxy for bioinformatic analysis was used. Three strains belonging to three different serotypes, monophasic S. Typhimurium, S. Grancanaria, and S. Senftenberg, were sequenced. Results: After 24 h of sequencing, enough coverage was achieved in order to perform de novo assembly in all three strains. After evaluating different tools, Flye de novo assemblies with medaka polishing were shown to be optimal for in silico Salmonella spp. serotyping with SISRT tool followed by antimicrobial and virulence gene identification with ABRicate. Discussion: The implementation of the present workflow in food safety laboratories with limited computer resources allows a rapid characterization of Salmonella spp. isolates.

Phage amplification coupled with loop-mediated isothermal amplification (PA-LAMP) for same-day detection of viable Salmonella Enteritidis in raw poultry meat.

Salmonella Enteritidis is the main serotype responsible for human salmonellosis in the European Union. One of the main sources of Salmonella spp. in the food chain are poultry products, such as eggs or chicken meat. In recent years, molecular methods have become an alternative to culture dependent methods for the rapid screening of Salmonella spp. In this work, the strain S. Enteritidis S1400, and previously isolated and characterized bacteriophage PVP-SE2, were used to develop and evaluate a same-day detection method combining Phage Amplification and Loop-mediated isothermal amplification (PA-LAMP) to specifically detect viable S. Enteritidis in chicken breast. This method is based on the detection of the phage DNA rather than bacterial DNA. The virus is added to the sample during pre-enrichment in buffered peptone water, where it replicates in the presence of viable S. Enteritidis. The detection of phage DNA allows, on the one hand to detect viable bacteria, since viruses only replicate in them, and on the other hand to increase the sensitivity of the method since for each infected S. Enteritidis cell, hundreds of new viruses are produced. Two different PA-LAMP detection strategies were evaluated, a real time fluorescence and a naked-eye detection. The present method could down to 0.2 fg/µL of pure phage DNA and a concentration of viral particles of 2.2 log PFU/mL. After a short Salmonella recovery step of 3 h and a co-culture of 4 h of the samples with phage particles, both real-time fluorescence and naked-eye method showed a LoD95 of 6.6 CFU/25 g and a LoD50 of 1.5/25 g in spiked chicken breast samples. The entire detection process, including DNA extraction and LAMP analysis, can be completed in around 8 h. In the current proof-of-concept, the novel PA-LAMP obtained comparable results to those of the reference method ISO 6579, to detect Salmonella Enteritidis in poultry meat.

Multiplex Real-Time PCR for the Detection of Shiga Toxin-Producing Escherichia coli in Foods.

Shiga toxin-producing Escherichia coli (STEC) is a group of human foodborne pathogens transmitted to humans through the consumption of different types of food. Their detection is mainly performed by targeting specific serogroups by classical microbiological methods and, later, by molecular typing with different techniques. The application of multiplex real-time PCR (qPCR) can significantly improve the turnaround time of the existing methodologies as in one single run it is possible to detect and characterize specific microorganisms. In the present chapter, a pentaplex qPCR assay is described for the identification of STEC which may also be applied for the rapid screening of these pathogens in different types of foods. The assay targets the most important virulence factors of these microorganisms, the genes stx1, stx2, and eae, along with the rfbE gene which encodes for the "O157" antigen as this is the most prevalent serogroup among all STEC, as well as an internal amplification control to rule out false-negative results due to qPCR inhibition.

Evaluation of the effect of outer primer structure, and inner primer linker sequences, in the performance of Loop-mediated isothermal amplification.

Loop-mediated isothermal amplification, or LAMP, is nowadays the most popular isothermal nucleic acid amplification technique. This technique implements a minimum of four primers, named outer (F3/B3) and inner primers (FIP/BIP). The inner primers hybridize in two distinct regions, and some studies have reported that the usage of a linker, typically composed of four thymines, in the middle of these primers can improve assay performance. In addition to this, dual-priming oligonucleotides, DPO, have been reported to provide highly specific reducing non-specific amplifications. Considering the large number of primers implemented in LAMP assays, in the current study the suitability of DPO primers replacing regular outer primers; and their combination with different linker sequences in the inner primers were explored. The results demonstrated that replacing standard F3/B3 by DPO primers does not significantly affect that overall performance of the assay, and provides additional stability to temperature changes. This observations were consistent regardless the type of linker implemented in the inner primers, out of which in the current study a linker composed of thymines significantly outperformed the other options tested, most likely due to a combination of sequence and physical structure.

Evaluation of Covalent Organic Frameworks for the low-cost, rapid detection of Shiga Toxin-producing Escherichia coli in ready-to-eat salads.

BACKGROUND: Ready-to-eat products, such as leafy greens, must be carefully controlled as they are directly consumed without any treatment to reduce the presence of potential pathogens. Food industries, especially those that process products with short shelf-life, demand rapid detection of foodborne pathogens such as Shiga Toxin-producing Escherichia coli (STEC). In this sense, molecular methods can fulfill both requirements of turnaround time and consumer safety. The most popular rapid methods are those based on real-time PCR (qPCR) however, vegetables contain inhibitory compounds that may inhibit the amplification reaction thus, there is a need for novel sample preparation protocols. RESULTS: In the current study, a low-cost sample treatment based on sequential filtration steps was developed. This protocol was combined with covalent organic frameworks (COFs), and compared against a chelating resin, to evaluate their performance by multiplex qPCR targeting the major virulence genes of STEC, namely stx1, stx2, and eae, along with the rfbE for the specific identification of serogroup O157 due to its particularly high incidence, and an Internal Amplification Control to assess reaction inhibition. The optimized sample treatment effectively removed vegetable qPCR inhibitory compounds, and it was possible to detect STEC in spiked ready-to-eat salad samples in one working day, roughly 5 h, with an LOD50 of 8.7 CFU/25 g with high diagnostic sensitivity and specificity. The method was also assessed in samples with cold-stressed bacteria with good results, further demonstrating its applicability. SIGNIFICANCE: It was demonstrated for the first time that COFs are suitable for DNA extraction and purification. In addition to this, due to the tunable nature of these materials, it is envisioned that future modifications in terms of pore size or combination with magnetic materials, will allow to further improve their performance. In addition to this, the rapid and low-cost sample treatment protocol developed demonstrated suitable for the rapid screening of STEC vegetable samples.

The Human Gut Virome and Its Relationship with Nontransmissible Chronic Diseases.

The human gastrointestinal tract contains large communities of microorganisms that are in constant interaction with the host, playing an essential role in the regulation of several metabolic processes. Among the gut microbial communities, the gut bacteriome has been most widely studied in recent decades. However, in recent years, there has been increasing interest in studying the influences that other microbial groups can exert on the host. Among them, the gut virome is attracting great interest because viruses can interact with the host immune system and metabolic functions; this is also the case for phages, which interact with the bacterial microbiota. The antecedents of virome-rectification-based therapies among various diseases were also investigated. In the near future, stool metagenomic investigation should include the identification of bacteria and phages, as well as their correlation networks, to better understand gut microbiota activity in metabolic disease progression.

Evaluation of the Novel mTA10 Selective Broth, MSB, for the Co-Enrichment and Detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes in Ready-to-Eat Salad Samples.

Multiplex assays implementing DNA-based methods have been demonstrated as suitable alternatives to culture-based microbiological methods; however, in most cases, they still require a suitable enrichment step. Finding suitable enrichment conditions for different bacteria may result in challenges. In the present study, a novel selective broth named MSB (mTA10 selective broth) was formulated for the simultaneous recovery of Salmonella spp., E. coli O157:H7 and L. monocytogenes. Attention was paid to ensure the optimal enrichment of L. monocytogenes as its enrichment is more challenging. To this end, cellobiose was added to increase the growth of L. monocytogenes, and sodium pyruvate was also added to improve the recovery of stressed bacteria. Four selective agents were added, namely nalidixic acid, sodium cholate, lithium chloride and potassium tellurite, to control the growth of interfering microorganisms. It was concluded that the novel broth was suitable for the simultaneous enrichment of the target pathogens, allowing them to reach concentrations higher than 7 log CFU/mL for each bacterium in pure culture. Furthermore, all heavily contaminated ready-to-eat salad samples reached concentrations higher than 5 log CFU/g. Finally, after 24 h of enrichment of spiked salad, it was possible to detect concentrations below 10 CFU/25 g.

Impact of Recombinant Bovine Somatotropin on Bovine Milk Composition and Fatty Acidome: A Multidose Longitudinal Study.

Somatotropin is a species-specific polypeptide hormone produced in the pituitary gland of vertebrates. When administered exogenously to cattle, it can increase milk yield. However, the trade and administration of recombinant bovine somatotropin (rbST) to farm animals have been banned in the European Union (EU). Aside from food safety issues, very little is known about the effects of this hormone on milk composition and quality. In this work, a wide profile of fatty acids (the so-called fatty acidome) was determined by GC-FID in raw milk collected from control and rbST-treated lactating cows in a multidose longitudinal study. Milk composition (lactose, protein, fat, dry matter), including minerals (Ca, K, Mg, Na, P), was also determined, and milk yield was recorded. A tendency toward a less saturated profile was observed in the milk collected from animals treated with rbST, with higher concentrations of monounsaturated fatty acids. In addition, less calcium and potassium and more lactose and protein content were observed in milk from treated animals than in regular milk. As a result of this multicomponent profiling of milk, a clear impact of somatotropin treatment on milk quality was observed. The obtained results should be particularly interesting for those countries that permit the use of this hormone in dairy production.

Nutritional composition, heavy metal content and in vitro effect on the human gut microbiota of Talitrus saltator, an underutilized crustacean from the Atlantic coast.

In this study, an undervalued marine crustacean (Talitrus saltator) was characterized in terms of nutritional and heavy metal composition and its potential to affect human gut microbiota. Nutritional analysis of this crustacean revealed that it complies with the criteria established in European legislation to include nutritional claims in their labeling, such as "source of fiber," "low in fat," "low in sugars" and "high in protein." The analysis of the heavy metal content did not reveal any risk derived from the presence of Cd, Hg, or Pb, whereas essential metals contained in 100 g exceeded the minimum daily requirements recommended in Europe for Zn (19.78 mg/kg), Cu (2.28 mg/kg), and Fe (32.96 mg/kg). Using an in vitro system, the effect of T. saltator on the human colonic microbiota shows some beneficial effects, such as fermentation-maintained populations of Bifidobacterium or Lactobacillus, did not increase Firmicutes phylum counts, decreased the Firmicutes/Bacteroidetes ratio, and stimulated 11 metabolic pathways with respect to baseline. These results are unusual in a high protein content-food. However, negative effects were also found in gut microbiota relative proportions, such as an increase in the Proteobacteria phylum and especially some opportunistic bacteria from this phylum, probably due to the antimicrobial effect of chitin on other groups more sensitive to its effect. This work shows for the first time the effect of T. saltator on human colonic microbiota using and in vitro system. The presence of chitin in its composition could provide some beneficial effects by modulating the microbiota, but as T. saltator is a high-protein food, more studies should be carried out showing these benefits.

A2 Milk: New Perspectives for Food Technology and Human Health.

Although milk consumption is increasing worldwide, in some geographical regions, its consumption has persistently declined in recent decades. This fact, together with the increase in milk production prices, has caused both milk producers and the dairy industry to be immersed in a major crisis. Some possible solutions to this problem are to get people who do not currently consume milk to start drinking it again, or to market milk and dairy products with a higher added value. In this context, a type of milk called A2 has recently received attention from the industry. This type of milk, characterized by a difference in an amino acid at position 67 of the ß-casein polypeptide chain, releases much smaller amounts of bioactive opioid peptide ß-casomorphin 7 upon digestion, which has been linked to harmful effects on human health. Additionally, A2 milk has been attributed worse technological properties in the production of some dairy products. Thus, doubts exist about the convenience for the dairy industry to bet on this product. The aim of this review is to provide an update on the effects on human health of A2 milk, as well as its different technological properties to produce dairy products.

Cow's Milk Antigens Content in Human Milk: A Scoping Review.

The functionality of breast milk in terms of immunity is well-known. Despite this, a significant proportion of breastfed infants exhibit sensitization to different potentially allergenic proteins and clinical reactivity (including anaphylaxis) early in life and before the introduction of complementary feeding for the first time. The potential induction of early oral tolerance to overcome early allergic sensitization through exposure to allergens in breast milk also remains controversial and not yet well-established. The objective of this scoping review is to provide a critical appraisal of knowledge about the content of cow's milk antigens in human milk. The amount of dietary derived milk antigens found in human milk and the analytical methodologies used to detect and quantify these antigens, the allergic status of the mother, the stage of lactation, the time of sampling (before or after ingestion of food), and the impact of human milk allergen on the infant were the outcomes that were assessed. Allergy risk was explored in all reviewed studies and could help to better elucidate its role in the context of allergic disease development. According to the included literature, we can conclude that there are mainly fragments derived from bovine proteins in human milk, and the presence of potentially allergenic molecules is greater in the milk of mothers with an allergic tendency. A clear relationship between maternal diet and allergen content in breast milk could not be firmly concluded though. Also, infants receiving milk from human milk banks, where donor milk is pasteurized for preservation, may be subject to greater risk of allergy development, especially for ß-lactoglobulin.

An Evaluation of the Pathogenic Potential, and the Antimicrobial Resistance, of Salmonella Strains Isolated from Mussels.

Salmonella spp. and antimicrobial resistant microorganisms are two of the most important health issues worldwide. In the present study, strains naturally isolated from mussels harvested in Galicia (one of the main production areas in the world), were genetically characterized attending to the presence of virulence and antimicrobial resistance genes. Additionally, the antimicrobial profile was also determined phenotypically. Strains presenting several virulence genes were isolated but lacked all the antimicrobial resistance genes analyzed. The fact that some of these strains presented multidrug resistance, highlighted the possibility of bearing different genes than those analyzed, or resistance based on completely different mechanisms. The current study highlights the importance of constant surveillance in order to improve the safety of foods.

Complete Genome Sequences of Six Salmonella enterica Strains (S. enterica subsp. arizonae, S. enterica subsp. diarizonae, and S. enterica subsp. salamae) Isolated from Poultry Houses.

Non-enterica subspecies of Salmonella enterica are associated with cold-blood animals. We report the complete genomes of six S. enterica strains (one S. enterica subsp. arizonae strain, four S. enterica subsp. salamae strains, and one S. enterica subsp. diarizonae strain) isolated from Spanish poultry houses. This will increase our knowledge of these non-enterica subspecies.

Bacterial Diversity of Breast Milk in Healthy Spanish Women: Evolution from Birth to Five Years Postpartum.

The objective of this work was to characterize the microbiota of breast milk in healthy Spanish mothers and to investigate the effects of lactation time on its diversity. A total of ninety-nine human milk samples were collected from healthy Spanish women and were assessed by means of next-generation sequencing of 16S rRNA amplicons and by qPCR. Firmicutes was the most abundant phylum, followed by Bacteroidetes, Actinobacteria, and Proteobacteria. Accordingly, Streptococcus was the most abundant genus. Lactation time showed a strong influence in milk microbiota, positively correlating with Actinobacteria and Bacteroidetes, while Firmicutes was relatively constant over lactation. 16S rRNA amplicon sequencing showed that the highest alpha-diversity was found in samples of prolonged lactation, along with wider differences between individuals. As for milk nutrients, calcium, magnesium, and selenium levels were potentially associated with Streptococcus and Staphylococcus abundance. Additionally, Proteobacteria was positively correlated with docosahexaenoic acid (DHA) levels in breast milk, and Staphylococcus with conjugated linoleic acid. Conversely, Streptococcus and trans-palmitoleic acid showed a negative association. Other factors such as maternal body mass index or diet also showed an influence on the structure of these microbial communities. Overall, human milk in Spanish mothers appeared to be a complex niche shaped by host factors and by its own nutrients, increasing in diversity over time.

Salmonella spp. quorum sensing: an overview from environmental persistence to host cell invasion.

Salmonella spp. is one of the main foodborne pathogens around the world. It has a cyclic lifestyle that combines host colonization with survival outside the host, implying that Salmonella has to adapt to different conditions rapidly in order to survive. One of these environments outside the host is the food production chain. In this environment, this foodborne pathogen has to adapt to different stress conditions such as acidic environments, nutrient limitation, desiccation, or biocides. One of the mechanisms used by Salmonella to survive under such conditions is biofilm formation. Quorum sensing plays an important role in the production of biofilms composed of cells from the same microorganism or from different species. It is also important in terms of food spoilage and regulates the pathogenicity and invasiveness of Salmonella by regulating Salmonella pathogenicity islands and flagella. Therefore, in this review, we will discuss the genetic mechanism involved in Salmonella quorum sensing, paying special attention to small RNAs and their post-regulatory activity in quorum sensing. We will further discuss the importance of this cell-to-cell communication mechanism in the persistence and spoilage of Salmonella in the food chain environment and the importance in the communication with microorganisms from different species. Subsequently, we will focus on the role of quorum sensing to regulate the virulence and invasion of host cells by Salmonella and on the interaction between Salmonella and other microbial species. This review offers an overview of the importance of quorum sensing in the Salmonella lifestyle.