Antimicrobial activity of apitoxin from Apis mellifera in Salmonella enterica strains isolated from poultry and its effects on motility, biofilm formation and gene expression.

Salmonella is a major global food-borne pathogen. One of the main concerns related to Salmonella and other food-borne pathogens is their capacity to acquire antimicrobial resistance and produce biofilms. Due to the increased resistance to common antimicrobials used to treat livestock animals and human infections, the discovery of new antimicrobial substances is one of the main challenges in microbiological research. An additional challenge is the development of new methods and substances to inhibit and destruct biofilms. We determined the antimicrobial and antibiofilm activities of apitoxin in 16 Salmonella strains isolated from poultry. In addition, the effect of apitoxin on Salmonella motility and the expression of biofilm- and virulence-related genes was evaluated. The minimum inhibitory concentrations (MIC) of apitoxin ranged from 1,024-256 µg/mL, with 512 µg/mL being the most common. Sub-inhibitory concentrations of apitoxin significantly reduced biofilm formation in 14 of the 16 Salmonella strains tested, with significant increases in motility. MIC concentrations of apitoxin destroyed the pre-formed biofilm by 27.66-68.22% (47.00% ±â€¯10.91). The expression of biofilm- and virulence-related genes and small RNAs was differentially regulated according to the strain and the presence of apitoxin. The transcription of the small RNAs dsrA and csrB, related to antimicrobial resistance, was upregulated in the presence of apitoxin. We suggest that apitoxin is a potential antimicrobial substance that could be used in combination with other substances to develop new drugs and sanitizers against food-borne pathogens.

Tracing Recombinant Bovine Somatotropin Ab(Use) Through Gene Expression in Blood, Hair Follicles, and Milk Somatic Cells: A Matrix Comparison.

The use of recombinant bovine somatotropin (rbST) in dairy cattle is forbidden in the European Union. Due to the very low circulating concentration of rbST in treated animals, its direct detection is still a challenge. Therefore, the use of indirect methods to detect the ab(use) of rbST in dairy cattle appears as a good alternative. In the past few years, gene expression demonstrated its utility in screening the use of illicit substances in both humans and animals. In this study, a comparison of three types of matrices (milk somatic cells, blood, and hair follicles) was carried out to evaluate their potential use for routine control of rbST using 15 gene-expression profiles. A total of six rbST-treated cows and three control cows were included in the study. A subcutaneous injection containing 500 mg of rbST was administered to the treated group. Samples of the three matrices were collected before rbST administration, and at three and nine days after treatment. The quality of RNA extracted was higher in the blood and hair-follicle samples than in the milk somatic cells. In the three matrices, there were significant differences in the expression of some genes, with milk somatic cells and blood presenting the the best matrices. On this note, the cyclin D1 (CCND1), interleukin 1 beta (IL-1ß), tumor necrosis factor (TNF), and insulin-like growth factor 1 receptor (IGF-1R) genes showed potential as biomarkers of rbST treatment. Therefore, blood, somatic cells, and follicle hair should be considered as promising sources of RNA, and can be used in gene-expression assays to routinely control the illicit use of rbST.

Salmonella and Campylobacter biofilm formation: a comparative assessment from farm to fork.

It takes several steps to bring food from the farm to the fork (dining table), and contamination with food-borne pathogens can occur at any point in the process. Campylobacter spp. and Salmonella spp. are the main microorganisms responsible for foodborne disease in the EU. These two pathogens are able to persist throughout the food supply chain thanks to their ability to form biofilms. Owing to the high prevalence of Salmonella and especially of Campylobacter in the food supply chain and the huge efforts of food authorities to reduce these levels, it is of great importance to fully understand their mechanisms of persistence. Diverse studies have evaluated the biofilm-forming capacity of foodborne pathogens isolated at different steps of food production. Nonetheless, the principal obstacle of these studies is to reproduce the real conditions that microorganisms encounter in the food supply chain. While there are a wide number of Salmonella biofilm studies, information on Campylobacter biofilms is still limited. A comparison between the two microorganisms could help to develop new research in the field of Campylobacter biofilms. Therefore, this review evaluates relevant work in the field of Salmonella and Campylobacter biofilms and the applicability of the data obtained from these studies to real working conditions. © 2018 Society of Chemical Industry.

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.

Investigating the Dietary Impact on Trans-Vaccenic Acid (Trans-C18:1 n-7) and Other Beneficial Fatty Acids in Breast Milk and Infant Formulas.

Maternal diet plays a significant role in the fatty acid composition of breast milk. Dietary products such as milk and meat are the primary sources of natural TFAs for humans. These peculiar fatty acids hold nutritional significance as they not only lack the detrimental effects of industrially produced trans fats on the endothelium characteristic, but they also exhibit anti-inflammatory properties. The relationship between the presence of eight fatty acids in breast milk (including natural TFAs trans-vaccenic and conjugated linoleic acid) and the maternal diet has been explored, and their abundance has been compared to that of infant formulas. Two cohorts of lactating women, originating from a Spanish region, participated in this study; they adhered to the Southern European Atlantic diet or the Atlantic diet. While the consumption of conventional meat or dairy products does not seem to increase the abundance of TFAs in breast milk, trans-vaccenic and oleic acid are among the most distinctive features of breast milk fat in mothers consuming naturally improved dairy products with an improved fatty acid profile. The most significant differences between natural breastfeeding and formula feeding lie in natural TFAs, since formulas are notably deficient in natural TFAs while being overfortified in alpha-linolenic acid in comparison to breast milk. We suggest an improvement in the formulation of these products through using cow's milk with an optimal fatty acid profile that better mimics the fatty acid composition found in human milk.

Structure elucidation and HPLC-MS/MS determination of a potential biomarker for estradiol administration in cattle.

Administration of hormonal compounds as growth promoters in livestock farming was banned by Council Directive 96/22/EC. However, this kind of substances is sometimes reported within the framework of European monitoring residue plans. Various analytical methods have been previously developed to screen for their misuse, and they are now especially efficient for monitoring the illegal administration of synthetic and semisynthetic hormones. Nevertheless, proving an exogenous administration of hormones from natural origin (i.e., estradiol-17ß or progesterone) still remains a challenge for European authorities. These target compounds are indeed always present in the animal matrix, and the establishment of reference thresholds appears very difficult because of the extreme variability existing among animals. In 2011, a metabolomics study was performed on serum samples obtained from cows treated with estradiol-17ß (or its ester estradiol benzoate) and from control animals using a high-performance liquid chromatography (HPLC)-LTQ-Orbitrap system. After appropriate data processing and multivariate statistical analysis (orthogonal partial least squares discriminant analysis), it was possible to highlight one potential biomarker candidate of estradiol treatments in bovine animals. Now, this biomarker has been structurally elucidated as a dipeptide, and its usefulness has been tested through a targeted HPLC-MS/MS method. Its presence in the previous samples has been confirmed and also in additional samples from estradiol-treated animals.

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.

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.

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.

Quick, Easy, Cheap, and Effective Method for the Synthesis of Rugged Magnetic Molecularly Imprinted Stir-Bars.

This contribution describes a fast and facile method for fabrication of robust magnetic stir-bars composed of molecularly imprinted polymers (MIPs) combined with magnetite particles. This was achieved through a prior optimization of the protocol presented here, in particular, the selection of cross-linker and porogen suited for obtaining a durable monolithic magnetic stir-bar. In-house prepared magnetite nanoparticles (Fe3O4) are used as magnetic core, coating them with molecularly imprinted polymers through a simple process of bulk polymerization. Procedures for magnetite synthesis, preparation of polymerization mixture, stir-bar synthesis, and analytical application are described.

Human Milk Oligosaccharides (HMOs) and Infant Microbiota: A Scoping Review.

Human milk oligosaccharides (HMOs) are the third most abundant solid component of breast milk. However, the newborn cannot assimilate them as nutrients. They are recognized prebiotic agents (the first in the newborn diet) that stimulate the growth of beneficial microorganisms, mainly the genus Bifidobacterium, dominant in the gut of breastfed infants. The structures of the oligosaccharides vary mainly according to maternal genetics, but also other maternal factors such as parity and mode of delivery, age, diet, and nutritional status or even geographic location and seasonality cause different breast milk oligosaccharides profiles. Differences in the profiles of HMO have been linked to breast milk microbiota and gut microbial colonization of babies. Here, we provide a review of the scope of reports on associations between HMOs and the infant gut microbiota to assess the impact of HMO composition.

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.

Breast Milk: A Source of Functional Compounds with Potential Application in Nutrition and Therapy.

Breast milk is an unbeatable food that covers all the nutritional requirements of an infant in its different stages of growth up to six months after birth. In addition, breastfeeding benefits both maternal and child health. Increasing knowledge has been acquired regarding the composition of breast milk. Epidemiological studies and epigenetics allow us to understand the possible lifelong effects of breastfeeding. In this review we have compiled some of the components with clear functional activity that are present in human milk and the processes through which they promote infant development and maturation as well as modulate immunity. Milk fat globule membrane, proteins, oligosaccharides, growth factors, milk exosomes, or microorganisms are functional components to use in infant formulas, any other food products, nutritional supplements, nutraceuticals, or even for the development of new clinical therapies. The clinical evaluation of these compounds and their commercial exploitation are limited by the difficulty of isolating and producing them on an adequate scale. In this work we focus on the compounds produced using milk components from other species such as bovine, transgenic cattle capable of expressing components of human breast milk or microbial culture engineering.

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.

Probiotics as a Possible Strategy for the Prevention and Treatment of Allergies. A Narrative Review.

Allergies are an increasing global public health concern, especially for children and people living in urban environments. Allergies impair the quality of life of those who suffer from them, and for this reason, alternatives for the treatment of allergic diseases or reduction in their symptoms are being sought. The main objective of this study was to compile the studies carried out on probiotics as a possible therapy for allergies. The most studied allergies on which probiotics have been shown to have a beneficial effect are rhinitis, asthma, and atopic dermatitis. Most studies have studied the administration of Lactobacillus and Bifidobacterium spp. in children and have shown beneficial effects, such as a reduction in hyperreactivity and inflammation caused by allergens and a decrease in cytokine release, among other beneficial effects. In the case of children, no clear beneficial effects were found in several studies, and the potential risk from the use of some opportunistic bacteria, such as probiotics, seems controversial. In the studies that reported beneficial results, these effects were found to make allergy symptoms less aggressive, thus reducing morbidity in allergy sufferers. The different effects of the same probiotic bacteria on different patients seem to reinforce the idea that the efficacy of probiotics is dependent on the microbial species or strain, its derived metabolites and byproducts, and the gut microbiota eubiosis of the patient. This study is relevant in the context of allergic diseases, as it provides a broader understanding of new alternatives for the treatment of allergies, both in children, who are the main sufferers, and adults, showing that probiotics, in some cases, reduce the symptoms and severity of such diseases.

Probiotic Effects against Virus Infections: New Weapons for an Old War.

This review aimed to gather the available literature investigating the effects of probiotics against the most common viral infections using in vitro trials in cell lines and in vivo clinical trials in both experimental animals and humans. Probiotics were employed to prevent and reduce symptoms of infections caused by common viruses, especially respiratory tract viruses, but also for viral digestive infections (such as rotavirus, coronavirus, or norovirus) and other viral infections (such as viruses that cause hepatitis, human papillomavirus, human immunodeficiency virus, and herpes simplex virus). Different probiotics have been studied to see their possible effect against the abovementioned viruses, among which different Lactobacillus species, Bifidobacterium, Clostridium, Enterococcus, and Streptococcus can be highlighted. In many cases, mixtures of various probiotic strains were used. Although the results obtained did not show similar results, in most cases, probiotic supplementation improved both barrier and biochemical immune responses, decreased susceptibility to viral infections, and enhanced the effects of concomitant vaccines. Works collected in this review show a beneficial effect of probiotics in the prevention and treatment of different viral infections. We found interesting results related to the prevention of viral infections, reduction of the duration of diseases, and decrease of symptoms.

Determination of naturally occurring progestogens in bovine milk as their oxime derivatives using high performance liquid chromatography-electrospray ionization-tandem mass spectrometry.

BACKGROUND: Hormones and hormone-like substances which are present in the environment have been repeatedly accused of being the cause of most endocrine disruption. However, the possible role of endogenous hormones in food of animal origin deserves to be discussed as well. The relation between steroid hormones and several human health problems has been previously reported, such as prostate and breast cancer, perturbation of human reproduction and endocrine disruption on humans and wildlife. This research is particularly concerned with cow's milk, which contains a considerable amount of sex hormones. RESULTS: A liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous detection and quantification of four naturally occurring steroid hormones in commercial bovine milk (pregnenolone (P5), progesterone (P4), 17-hydroxypregnenolone (17-OHP5), 17-hydroxyprogesterone (P4)). Oxime derivatives of steroids were analyzed in positive ionization and multiple reaction monitoring mode. Methodology has been validated according to Decision 2002/657/EC criteria. CONCLUSION: This method has been successfully used in real samples. It is fast and easy-handling and provides a useful tool for the assessment of progestogens in bovine milk.

Association between Breast Milk Mineral Content and Maternal Adherence to Healthy Dietary Patterns in Spain: A Transversal Study.

The composition of breast milk is influenced by many factors, some of which dependent on the mother and others on the child. Changes in lactation and other factors depending on the mother's physiology and anthropometric characteristics, as well as her nutritional status and diet, are of key importance. Breast milk minerals have been extensively studied with highly uneven results. In this work, a comparison will made with data across the world. To understand the factors that might explain the disparity, several minerals (Na, K, Ca, P, Mg, Fe, Se and I) have been analyzed using ICP-MS in a set of human milk samples (n = 75). The samples had an identical geographical origin (Galicia, in northwestern Spain) but different lactation circumstances, including maternal anthropometric data, lactating time, newborn sex and maternal adherence to healthy dietary patterns (Mediterranean Diet, MD, or Atlantic Diet, AD). The required concentrations of essential elements reported in the literature are similar to those found in these Spanish women. A univariate approach revealed that factors such as lactating time, body mass index (BMI) and newborn sex have a significant influence in breastmilk mineral content. According to multivariate linear regression analysis, minerals in milk are particularly associated with lactating time, but also with newborn sex, maternal BMI, age and diet pattern in some cases. More precisely, these results suggest that the iron and selenium concentrations in the milk of Galician donors may be positively influenced by maternal adherence to AD and MD, respectively.

Antimicrobial Activity of Five Apitoxins from Apis mellifera on Two Common Foodborne Pathogens.

Antimicrobial resistance is one of today's major public health challenges. Infections caused by multidrug-resistant bacteria have been responsible for an increasing number of deaths in recent decades. These resistant bacteria are also a concern in the food chain, as bacteria can resist common biocides used in the food industry and reach consumers. As a consequence, the search for alternatives to common antimicrobials by the scientific community has intensified. Substances obtained from nature have shown great potential as new sources of antimicrobial activity. The aim of this study was to evaluate the antimicrobial activity of five bee venoms, also called apitoxins, against two common foodborne pathogens. A total of 50 strains of the Gram-negative pathogen Salmonella enterica and 8 strains of the Gram-positive pathogen Listeria monocytogenes were tested. The results show that the minimum inhibitory concentration (MIC) values were highly influenced by the bacterial genus. The MIC values ranged from 256 to 1024 µg/mL in S. enterica and from 16 to 32 µg/mL in L. monocytogenes. The results of this study demonstrate that apitoxin is a potential alternative agent against common foodborne pathogens, and it can be included in the development of new models to inhibit the growth of pathogenic bacteria in the food chain.

Application of dummy molecularly imprinted solid-phase extraction in the analysis of cyproheptadine in bovine urine.

Due to the difficulty of monitoring trace levels of cyproheptadine (CYP) residues in complicated biological matrices, specific adsorption materials for the preconcentration and clean-up of CYP are indispensable. In this work, CYP was extracted from urine using dummy molecularly imprinted SPE (DMISPE) to avoid leakage of the imprinting molecules during the desorption phase. For synthesis of DMISPE, azatadine (AZA) was employed as the dummy template, methacrylic acid (MAA) as the monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, 2,2'-azobis(2-methylpropionitrile) (AIBN) as the initiator, and dichloromethane as the porogen solvent. An LC-MS/MS method was used to analyze CYP. Two MRM (multiple reaction monitoring) transitions for each analyte were monitored using diphenylpyraline hydrochloride (DPP.HCl), which was used as an internal standard. The advantages of DMISPE include obtaining less complex chromatograms and reducing ion suppression in ESI. The process efficiencies for DMISPE and SPE were 80% and 12%, respectively. In addition, the demonstrated reusability of the DMISPE cartridges is an advantage compared with single-use SPE cartridges or immunoaffinity materials.