Chemical Composition and Antioxidant Potential of Five Algae Cultivated in Fully Controlled Closed Systems.

In this study, the chemical composition and antioxidant profile of five edible macroalgae, Fucus vesiculosus, Palmaria palmata, Porphyra dioica, Ulva rigida, and Gracilaria gracilis, cultivated in fully controlled closed systems, were determined. Protein, carbohydrates, and fat contents ranged between 12.4% and 41.8%, 27.6% and 42.0%, and 0.1% and 3.4%, respectively. The tested seaweeds presented considerable amounts of Ca, Mg, K, Mn, and Fe, which reinforce their favorable nutritional profile. Regarding their polysaccharide composition, Gracilaria gracilis and Porphyra dioica were rich in sugars common to agar-producing red algae, and Fucus vesiculosus was composed mainly of uronic acids, mannose, and fucose, characteristic of alginate and fucoidans, whereas rhamnose and uronic acid, characteristic of ulvans, predominated in Ulva rigida. Comparatively, the brown F. vesiculosus clearly stood out, presenting a high polysaccharide content rich in fucoidans, and higher total phenolic content and antioxidant scavenging activity, determined by DPPH and ABTS. The remarkable potential of these marine macroalgae makes them excellent ingredients for a wide range of health, food, and industrial applications.

Extraction of Nannochloropsis Fatty Acids Using Different Green Technologies: The Current Path.

Nannochloropsis is a genus of microalgae widely recognized as potential sources of distinct lipids, particularly polyunsaturated fatty acids (PUFA). These may be obtained through extraction, which has conventionally been performed using hazardous organic solvents. To substitute such solvents with "greener" alternatives, several technologies have been studied to increase their extraction potential. Distinct technologies utilize different principles to achieve such objective; while some aim at disrupting the cell walls of the microalgae, others target the extraction per se. While some methods have been utilized independently, several technologies have also been combined, which has proven to be an effective strategy. The current review focuses on the technologies explored in the last five years to extract or increase extraction yields of fatty acids from Nannochloropsis microalgae. Depending on the extraction efficacy of the different technologies, distinct types of lipids and/or fatty acids are obtained accordingly. Moreover, the extraction efficiency may vary depending on the Nannochloropsis species. Hence, a case-by-case assessment must be conducted in order to ascertain the most suited technology, or tailor a specific one, to be applied to recover a particular fatty acid (or fatty acid class), namely PUFA, including eicosapentaenoic acid.

Design and Characterization of a Cheese Spread Incorporating Osmundea pinnatifida Extract.

Marine algae have been emerging as natural sources of bioactive compounds, such as soluble dietary fibers and peptides, presenting special interest as ingredients for functional foods. This study developed a cheese spread incorporating red seaweed Osmundea pinnatifida extract and subsequently characterized it in terms of nutritional, pH, and microbiological parameters and bioactivities including prebiotic, antidiabetic, antihypertensive, and antioxidant activities. This food was produced through incorporation of O. pinnatifida extract (3%), obtained via enzymatic extraction Viscozyme L in a matrix containing whey cheese (75%) and Greek-type yoghurt (22%). The product was then subjected to thermal processing and subsequently stored for 21 days at 4 °C. During storage, this food showed a high pH stability (variations lower than 0.2 units), the absence of microbial contamination and all tested bioactivities at the sampling timepoints 0 and 21 days. Indeed, it exerted prebiotic effects under Lactobacillus acidophilus LA-5® and Bifidobacterium animalis subsp. lactis BB-12®, increasing their viability to around 4 and 0.5 log CFU/g, respectively. In addition, it displayed antidiabetic (α-glucosidase inhibition: 5-9%), antihypertensive (ACE inhibition: 50-57%), and antioxidant (ABTS: 13-15%; DPPH: 3-5%; hydroxyl radical: 60-76%) activities. In summary, the cheese spread produced may be considered an innovative food with high potential to contribute toward healthier status and well-being of populations.

Inhibition of TRPM8 by the urinary tract analgesic drug phenazopyridine.

BACKGROUND: and purpose: Phenazopyridine (PAP) is an over-the-counter drug widely used to provide symptomatic relief of bladder pain in conditions such as cystitis or bladder pain syndrome (BPS). Whereas the analgesic effect of PAP has been attributed to a local effect on the mucosa of the lower urinary tract (LUT), the molecular targets of PAP remain unknown. We investigated the effect of PAP on pain-related Transient Receptor Potential (TRP) channels expressed in sensory neurons that innervate the bladder wall. EXPERIMENTAL APPROACH: The effects of PAP on the relevant TRP channels (TRPV1, TRPA1, TRPM8, TRPM3) expressed in HEK293 or CHO cells was investigated using Fura-2-based calcium measurements and whole-cell patch-clamp recordings. Activity of PAP on TRPM8 was further analysed using Fura-2-based calcium imaging on sensory neurons isolated from lumbosacral dorsal root ganglia (DRG) of mice. KEY RESULTS: PAP rapidly and reversibly inhibits responses of TRPM8 expressed in HEK293 cells to cold and menthol, with IC50 values between 2 and 10 µM. It acts by shifting the voltage dependence of channel activation towards positive potentials, opposite to the effect of menthol. PAP also inhibits TRPM8-mediated, menthol-evoked calcium responses in lumbosacral DRG neurons. At a concentration of 10 µM, PAP did not significantly affect TRPA1, TRPV1, or TRPM3. CONCLUSION AND IMPLICATIONS: PAP inhibits TRPM8 in a concentration range consistent with PAP levels in the urine of treated patients. Since TRPM8 is expressed in bladder afferent neurons and upregulated in patients with painful bladder disorders, TRPM8 inhibition may underlie the analgesic activity of PAP.

Exploring Freeze-Drying as Strategy to Enhance Viability of Faecalibacterium duncaniae DSM 17677 upon Aerobic Storage and Gastrointestinal Conditions

Faecalibacterium duncaniae is an intestinal commensal bacterium proposed as a next-generation probiotic due to its promising outcomes in the treatment and prevention of several human diseases, which demonstrate its multiple contributions to the host's health. However, its strict anaerobic nature has created several hurdles in the development of functional foods, nutraceuticals, and biotherapeutic products. Herein, we explored freeze-dried formulations containing prebiotics, cryoprotectants, and antioxidant agents as a technological strategy to enhance the viability of F. duncaniae DSM 17677 upon aerobic storage and gastrointestinal tract conditions. Our results indicate that freeze-dried F. duncaniae in a matrix containing inulin, sucrose, cysteine, and riboflavin survived at levels higher than 106 CFU/g and around 105 CFU/g after 1 and 4 days of aerobic storage at room temperature, respectively. Thus, the freeze-dried formulation with inulin, sucrose, cysteine, and riboflavin presents as a protective strategy to improve F. duncaniae viability under aerobic environments. Nevertheless, incorporation of a suitable coating aimed at protecting F. duncaniae against the detrimental gastrointestinal passage effects is urgently required, given its high susceptibility to extreme acidic pH values and bile.

Development and Characterization of a Novel Sustainable Probiotic Goat Whey Cheese Containing Second Cheese Whey Powder and Stabilized with Thyme Essential Oil and Sodium Citrate.

Probiotic goat whey cheeses with added second cheese whey powder (SCWP) were developed, resulting in creamy and spreadable products. The products contained Lactobacillus rhamnosus and Bifidobacterium animalis, as well as thyme essential oil and sodium citrate. Matrices of probiotic whey cheeses, with and without additives, were produced and stored at 5 °C for 21 days. Microbial and chemical profiles were evaluated weekly. The composition of the optimum matrix, formulated with whey cheese, probiotic culture, SCWP, thyme essential oil and sodium citrate (WCPSTC) was, expressed in % (w/w): protein (10.78 ± 0.08), fat (7.59 ± 0.03), dry matter (25.64 ± 0.13), ash (2.81 ± 0.02) and lactose (3.16 ± 0.04). Viable cell numbers of both probiotic cultures in matrix WCPSTC remained above 107 CFU g-1. This finding is of the utmost importance since it proves that both probiotic bacteria, citrate and thyme essential oil can be combined in order to increase the shelf-life and functional value of dairy products. All matrices' pH values decreased during storage, yet only matrix WCPSTC remained above 5.0 pH units. The results indicated that the development of a probiotic whey cheese incorporating a dairy by-product, SCWP, is possible without compromising its chemical, microbiological or sensorial stability.

Insights into the Antimicrobial Resistance Profile of a Next Generation Probiotic Akkermansia muciniphila DSM 22959.

Akkermansia muciniphila is a Gram-negative intestinal anaerobic bacterium recently proposed as a novel probiotic candidate to be incorporated in food and pharmaceutical forms. Despite its multiple health benefits, the data addressing its antimicrobial susceptibility profile remain scarce. However, the absence of acquired resistance in probiotic strains is a compulsory criterion for its approval in the qualified presumption of safety list. This study aimed at characterizing the A. muciniphila DSM 22959 strain's antimicrobial susceptibility profile using phenotypic and in silico approaches. To establish the phenotypic antimicrobial susceptibility profile of this strain, minimum inhibitory concentrations of eight antimicrobials were determined using broth microdilution and E-test methods. Additionally, the A. muciniphila DSM 22959 genome was screened using available databases and bioinformatics tools to identify putative antimicrobial resistance genes (ARG), virulence factors (VF), genomic islands (GI), and mobile genetic elements (MGE). The same categorization was obtained for both phenotypic methods. Resistance phenotype was observed for gentamicin, kanamycin, streptomycin, and ciprofloxacin, which was supported by the genomic context. No evidence was found of horizontal acquisition or potential transferability of the identified ARG and VF. Thus, this study provides new insights regarding the phenotypic and genotypic antimicrobial susceptibility profiles of the probiotic candidate A. muciniphila DSM 22959.

Spray-Drying Encapsulation of the Live Biotherapeutic Candidate Akkermansia muciniphila DSM 22959 to Survive Aerobic Storage.

Akkermansia muciniphila is regarded as a promising next-generation probiotic or live biotherapeutic candidate. Effective delivery strategies must be developed to ensure high enough viability of the probiotic strain throughout its industrial formulation, distribution chain, shelf-life, and, ultimately, the host's gastrointestinal tract, where it should exert its beneficial effect(s). Among the possible methodologies, spray-drying is considered industrially attractive regarding its costs, efficiency, and scalability, with the due parameter customization. In this study, spray-drying was explored as a one-step process to encapsulate A. muciniphila DSM 22959, testing the drying settings and three different dairy-based matrices. Microcapsule morphology and size was assessed, and viability throughout storage at 4 or 22 °C and simulated gastrointestinal passage was determined. Akkermansia muciniphila microencapsulation by spray-drying, using 10% skim milk and inlet/outlet temperatures of 150/65 °C, is effective in terms of viability stabilization, both during prolonged aerobic storage and exposure to simulated gastrointestinal passage. Akkermansia muciniphila viability was maintained at around 107 CFU/g up to 28 days at 4 °C under aerobic conditions with viability losses inferior to 1 log reduction. This methodology provides the necessary conditions to efficiently deliver the recommended dose of live A. muciniphila in the human gut as a live biotherapeutic product.

Bradykinin induces peripheral antinociception in PGE2-induced hyperalgesia in mice.

BACKGROUND: Bradykinin (BK) is an endogenous peptide involved in vascular permeability and inflammation. It has opposite effects (inducing hyperalgesia or antinociception) when administered directly in the central nervous system. The aim of this study was to evaluate whether BK may also present this dual effect when injected peripherally in a PGE2-induced nociceptive pain model, as well as to investigate the possible mechanisms of action involved in this event in mice. METHODS: Male Swiss and C57BL/6 knockout mice for B1 or B2 bradykinin receptors were submitted to a mechanical paw pressure test and hyperalgesia was induced by intraplantar prostaglandin E2 (2 µg/paw) injection. RESULTS: Bradykinin (20, 40 and 80 ng/paw) produced dose-dependent peripheral antinociception against PGE2-induced hyperalgesia. This effect was antagonized by bradyzide (8, 16 and 32 µg/paw), naloxone (12.5, 25 and 50 µg/paw), nor-binaltorphimine (50, 100 and 200 µg/paw) and AM251 (20, 40 and 80 µg/paw). Bestatin (400 µg/paw), MAFP (0.5 µg/paw) and VDM11 (2.5 µg/paw) potentiated the antinociception of a lower 20 ng BK dose. The knockout of B1 or B2 bradykinin receptors partially abolished the antinociceptive action of BK (80 ng/paw), bremazocine (1 µg/paw) and anandamide (40 ng/paw) when compared with wild-type animals, which show complete antinociception with the same dose of each drug. CONCLUSION: The present study is the first to demonstrate BK-induced antinociception in peripheral tissues against PGE2-induced nociception in mice and the involvement of κ-opioid and CB1 cannabinoid receptors in this effect.

Pharmacological properties of TRPM3 isoforms are determined by the length of the pore loop.

BACKGROUND AND PURPOSE: Transient receptor potential melastatin 3 (TRPM3) is a non-selective cation channel that plays a pivotal role in the peripheral nervous system as a transducer of painful heat signals. Alternative splicing gives rise to several TRPM3 variants. The functional consequences of these splice isoforms are poorly understood. Here, the pharmacological properties of TRPM3 variants arising from alternative splicing in the pore-forming region were compared. EXPERIMENTAL APPROACH: Calcium microfluorimetry and patch clamp recordings were used to compare the properties of heterologously expressed TRPM3α1 (long pore variant) and TRPM3α2-α6 (short pore variants). Furthermore, site-directed mutagenesis was done to investigate the influence of the length of the pore loop on the channel function. KEY RESULTS: All short pore loop TRPM3α variants (TRPM3α2-α6) were activated by the neurosteroid pregnenolone sulphate (PS) and by nifedipine, whereas the long pore loop variant TRPM3α1 was insensitive to either compound. In contrast, TRPM3α1 was robustly activated by clotrimazole, a compound that does not directly activate the short pore variants but potentiates their responses to PS. Clotrimazole-activated TRPM3α1 currents were largely insensitive to established TRPM3α2 antagonists and were only partially inhibited upon activation of the µ opioid receptor. Finally, by creating a set of mutant channels with pore loops of intermediate length, we showed that the length of the pore loop dictates differential channel activation by PS and clotrimazole. CONCLUSION AND IMPLICATIONS: Alternative splicing in the pore-forming region of TRPM3 defines the channel's pharmacological properties, which depend critically on the length of the pore-forming loop. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

Revealing antimicrobial resistance profile of the novel probiotic candidate Faecalibacterium prausnitzii DSM 17677.

Faecalibacterium prausnitzii, a resident anaerobic bacterium commonly found in healthy gut microbiota, has been proposed as a next generation probiotic with high potential for application in food matrices and pharmaceutical formulations. Despite its recognized health benefits, detailed information regarding its antimicrobial susceptibility profile is still lacking. However, this information is crucial to determine its safety, since the absence of acquired antimicrobial resistance is required to qualify a probiotic candidate as safe for human and animal consumption. Herein, the antimicrobial susceptibility profile of F. prausnitzii DSM 17677 strain was evaluated by integrating both phenotypic and in silico data. Phenotypic antimicrobial susceptibility was evaluated by determining minimum inhibitory concentrations of 9 antimicrobials using broth microdilution and E-test® methods. Also, the whole genome of F. prausnitzii DSM 17677 was analysed, using several databases and bioinformatics tools, to identify possible antibiotic resistance genes (ARG), genomic islands (GI) and mobile genetic elements (MGE). With exception of erythromycin, the same classification (susceptible or resistant) was obtained in both broth microdilution and E-test® methods. Phenotypic resistance to ampicillin, gentamycin, kanamycin and streptomycin were detected, which was supported by the genomic context. Other ARG were also identified but they seem not to be expressed under the tested conditions. F. prausnitzii DSM 17677 genome contains 24 annotated genes putatively involved in resistance against the following classes of antimicrobials: aminoglycosides (such as gentamycin, kanamycin and streptomycin), macrolides (such as erythromycin), tetracyclines and lincosamides. The presence of putative ARG conferring resistance to ß-lactams could only be detected using a broader homology search. The majority of these genes are not encoded within GI or MGE and no plasmids were reported for this strain. Despite the fact that most genes are related with general resistance mechanisms, a streptomycin-specific ARG poses the only potential concern identified. This specific ARG is encoded within a GI and a MGE, meaning that it could have been laterally acquired and might be transferred to other bacteria present in the same environment. Thus, our findings provide relevant insights regarding the phenotypic and genotypic antimicrobial resistance profiles of the probiotic candidate F. prausnitzii DSM 17677.

TRPM3 Is Expressed in Afferent Bladder Neurons and Is Upregulated during Bladder Inflammation.

The cation channel TRPM3 is activated by heat and the neurosteroid pregnenolone sulfate. TRPM3 is expressed on sensory neurons innervating the skin, where together with TRPV1 and TRPA1, it functions as one of three redundant sensors of acute heat. Moreover, functional upregulation of TRPM3 during inflammation contributes to heat hyperalgesia. The role of TRPM3 in sensory neurons innervating internal organs such as the bladder is currently unclear. Here, using retrograde labeling and single-molecule fluorescent RNA in situ hybridization, we demonstrate expression of mRNA encoding TRPM3 in a large subset of dorsal root ganglion (DRG) neurons innervating the mouse bladder, and confirm TRPM3 channel functionality in these neurons using Fura-2-based calcium imaging. After induction of cystitis by injection of cyclophosphamide, we observed a robust increase of the functional responses to agonists of TRPM3, TRPV1, and TRPA1 in bladder-innervating DRG neurons. Cystometry and voided spot analysis in control and cyclophosphamide-treated animals did not reveal differences between wild type and TRPM3-deficient mice, indicating that TRPM3 is not critical for normal voiding. We conclude that TRPM3 is functionally expressed in a large proportion of sensory bladder afferent, but its role in bladder sensation remains to be established.

Bioconversion of Fish Discards through the Production of Lactic Acid Bacteria and Metabolites: Sustainable Application of Fish Peptones in Nutritive Fermentation Media.

In the current work, we study the capacity of 30 peptones obtained by enzyme proteolysis of ten discarded fish species (hake, megrim, red scorpionfish, pouting, mackerel, gurnard, blue whiting, Atlantic horse mackerel, grenadier, and boarfish) to support the growth and metabolite production of four lactic acid bacteria (LAB) of probiotic and technological importance. Batch fermentations of Lactobacillus plantarum, L. brevis, L. casei, and Leuconostoc mesenteroides in most of the media formulated with fish peptones (87% of the cases) led to similar growths (quantified as dry-weight biomass and viable cells) and metabolites (mainly lactic acid) than in commercial control broth (MRS). Comparisons among cultures were performed by means of the parameters obtained from the mathematical fittings of experimental kinetics to the logistic equation. Modelling among experimental and predicted data from each bioproduction was generally accurate. A simple economic assessment demonstrated the profitability achieved when MRS is substituted by media formulated with fish discards: a 3-4-fold reduction of costs for LAB biomass, viable cells formation, and lactic and acetic acid production. Thus, these fish peptones are promising alternatives to the expensive commercial peptones as well as a possible solution to valorize discarded fish biomasses and by-products.

Why the emperor penguin reigns where elephants shiver.

Accurate sensing of the environmental temperature is crucial for the survival and wellbeing of organisms. In vertebrates, the cold- and menthol-activated ion channel TRPM8 acts as the prime molecular sensor of cool temperatures. By comparing TRPM8 in vertebrates with different habitat temperatures, from elephants to penguins, Yang et al. identify key residues within the pore domain that determine the channel's cold sensitivity. Strikingly, mice engineered to express penguin TRPM8 show a remarkable tolerance to cold.

Production of Marine Probiotic Bacteria in a Cost-Effective Marine Media Based on Peptones Obtained from Discarded Fish By-Products.

The industrial production of marine bacteria with probiotic properties is limited by the excessive cost of the culture media adequate for their growth. The present work aimed to study the suitability of 30 marine media formulated with nitrogen sources (fish peptones) from different fish discards and seawater, for the growth of two marine probiotic bacteria (MPB), namely Phaeobacter sp. and Pseudomonas fluorescens. These fish peptones were produced from several discarded fish and by-products (heads, skins, and whole individuals of megrim, mackerel, gurnard, hake, etc.). In all cultivations, no significant differences were found on cell viability when the microorganism grew on commercial or alternative media. In relation to the biomass production, the growth of Phaeobacter sp. on waste media was commonly similar or a 20% lower than observed in the control cultures. For P. fluorescens, various peptones (skin peptones of pouting and blue whiting) showed even higher productive ability than commercial peptones. An initial economical evaluation revealed that low-cost media reduced until 120 times the cost of production of MPB.

Valorization of Mushroom By-Products as a Source of Value-Added Compounds and Potential Applications.

Nowadays, the food sector is highly concerned with environmental issues and foreseen to develop strategies to reduce waste and losses resulting from activities developed in the food system. An approach is to increment added value to the agro-industrial wastes, which might provide economic growth and environmental protection, contributing to a circular economy. Mushroom by-products represent a disposal problem, but they are also promising sources of important compounds, which may be used due to their functional and nutritional properties. Research has been developed in different fields to obtain value added solutions for the by-products generated during mushroom production and processing. Bioactive compounds have been obtained and applied in the development of nutraceutical and pharmaceutical formulations. Additionally, other applications have been explored and include animal feed, fertilizer, bioremediation, energy production, bio-based materials, cosmetics and cosmeceuticals. The main purpose of this review is to highlight the relevant composition of mushroom by-products and discuss their potential as a source of functional compounds and other applications. Future research needs to explore pilot and industrial scale extraction methods to understand the technological feasibility and the economic sustainability of the bioactive compounds extraction and valorization towards different applications.

Commensal Obligate Anaerobic Bacteria and Health: Production, Storage, and Delivery Strategies.

In the last years several human commensals have emerged from the gut microbiota studies as potential probiotics or therapeutic agents. Strains of human gut inhabitants such as Akkermansia, Bacteroides, or Faecalibacterium have shown several interesting bioactivities and are thus currently being considered as food supplements or as live biotherapeutics, as is already the case with other human commensals such as bifidobacteria. The large-scale use of these bacteria will pose many challenges and drawbacks mainly because they are quite sensitive to oxygen and/or very difficult to cultivate. This review highlights the properties of some of the most promising human commensals bacteria and summarizes the most up-to-date knowledge on their potential health effects. A comprehensive outlook on the potential strategies currently employed and/or available to produce, stabilize, and deliver these microorganisms is also presented.

Gain of channel function and modified gating properties in TRPM3 mutants causing intellectual disability and epilepsy.

Developmental and epileptic encephalopathies (DEE) are a heterogeneous group of disorders characterized by epilepsy with comorbid intellectual disability. Recently, two de novo heterozygous mutations in the gene encoding TRPM3, a calcium permeable ion channel, were identified as the cause of DEE in eight probands, but the functional consequences of the mutations remained elusive. Here we demonstrate that both mutations (V990M and P1090Q) have distinct effects on TRPM3 gating, including increased basal activity, higher sensitivity to stimulation by the endogenous neurosteroid pregnenolone sulfate (PS) and heat, and altered response to ligand modulation. Most strikingly, the V990M mutation affected the gating of the non-canonical pore of TRPM3, resulting in large inward cation currents via the voltage sensor domain in response to PS stimulation. Taken together, these data indicate that the two DEE mutations in TRPM3 result in a profound gain of channel function, which may lie at the basis of epileptic activity and neurodevelopmental symptoms in the patients.