Using Targeted Metabolomics to Unravel Phenolic Metabolites of Plant Origin in Animal Milk.

Milk holds a high nutritional value and is associated with diverse health benefits. The understanding of its composition of (poly)phenolic metabolites is limited, which necessitates a comprehensive evaluation of the subject. This study aimed at analyzing the (poly)phenolic profile of commercial milk samples from cows and goats and investigating their sterilization treatments, fat content, and lactose content. Fingerprinting of phenolic metabolites was achieved by using ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). Two hundred and three potential microbial and phase II metabolites of the main dietary (poly)phenols were targeted. Twenty-five metabolites were identified, revealing a diverse array of phenolic metabolites in milk, including isoflavones and their microbial catabolites equol and O-desmethylangolensin, phenyl-γ-valerolactones (flavan-3-ol microbial catabolites), enterolignans, urolithins (ellagitannin microbial catabolites), benzene diols, and hippuric acid derivates. Goat's milk contained higher concentrations of these metabolites than cow's milk, while the sterilization process and milk composition (fat and lactose content) had minimal impact on the metabolite profiles. Thus, the consumption of goat's milk might serve as a potential means to supplement bioactive phenolic metabolites, especially in individuals with limited production capacity. However, further research is needed to elucidate the potential health effects of milk-derived phenolics.

Nav1.8-expressing neurons control daily oscillations of food intake, body weight and gut microbiota in mice.

Recent evidence suggests a role of sensory neurons expressing the sodium channel Nav1.8 on the energy homeostasis control. Using a murine diphtheria toxin ablation strategy and ad libitum and time-restricted feeding regimens of control or high-fat high-sugar diets, here we further explore the function of these neurons on food intake and on the regulation of gastrointestinal elements transmitting immune and nutrient sensing.The Nav1.8+ neuron ablation increases food intake in ad libitum and time-restricted feeding, and exacerbates daily body weight variations. Mice lacking Nav1.8+ neurons show impaired prandial regulation of gut hormone secretion and gut microbiota composition, and altered intestinal immunity.Our study demonstrates that Nav1.8+ neurons are required to control food intake and daily body weight changes, as well as to maintain physiological enteroendocrine and immune responses and the rhythmicity of the gut microbiota, which highlights the potential of Nav1.8+ neurons to restore energy balance in metabolic disorders.

Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1ß production.

Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory condition resulting from monoallelic NLRP3 variants that facilitate IL-1ß production. Although these are gain-of-function variants characterized by hypersensitivity to cell priming, patients with CAPS and animal models of the disease may present inflammatory flares without identifiable external triggers. Here we find that CAPS-associated NLRP3 variants are forming constitutively active inflammasome, which induce increased basal cleavage of gasdermin D, IL-18 release and pyroptosis, with a concurrent basal pro-inflammatory gene expression signature, including the induction of nuclear receptors 4 A. The constitutively active NLRP3-inflammasome of CAPS is responsive to the selective NLRP3 inhibitor MCC950 and its activation is regulated by deubiquitination. Despite their preactivated state, the CAPS inflammasomes are responsive to activation of the NF-κB pathway. NLRP3-inflammasomes with CAPS-associated variants affect the immunometabolism of the myeloid compartment, leading to disruptions in lipids and amino acid pathways and impaired glycolysis, limiting IL-1ß production. In summary, NLRP3 variants causing CAPS form a constitutively active inflammasome inducing pyroptosis and IL-18 release without cell priming, which enables the host's innate defence against pathogens while also limiting IL-1ß-dependent inflammatory episodes through immunometabolism modulation.

Mitophagy curtails cytosolic mtDNA-dependent activation of cGAS/STING inflammation during aging.

Macroautophagy decreases with age, and this change is considered a hallmark of the aging process. It remains unknown whether mitophagy, the essential selective autophagic degradation of mitochondria, also decreases with age. In our analysis of mitophagy in multiple organs in the mito-QC reporter mouse, mitophagy is either increased or unchanged in old versus young mice. Transcriptomic analysis shows marked upregulation of the type I interferon response in the retina of old mice, which correlates with increased levels of cytosolic mtDNA and activation of the cGAS/STING pathway. Crucially, these same alterations are replicated in primary human fibroblasts from elderly donors. In old mice, pharmacological induction of mitophagy with urolithin A attenuates cGAS/STING activation and ameliorates deterioration of neurological function. These findings point to mitophagy induction as a strategy to decrease age-associated inflammation and increase healthspan.

In vitro study of potential prebiotic properties of monovarietal extra virgin olive oils.

Olive oil, essential ingredient of the Mediterranean diet, is attracting a growing interest due to increasing evidence on its beneficial effects on human health. This study investigated whether extra virgin olive oil (EVOO) possess prebiotic properties. Twenty different monovarietal EVOO samples from 5 Marche region cultivars (Italy) were studied. The prebiotic activity of EVOOs was assessed monitoring the selective stimulation of gut bacterial species and the short chain fatty acids (SCFAs) production, using an in vitro fermentation system. All EVOOs selectively stimulated Lactobacillus spp., with a stronger activity than that observed in the inulin fermentation (positive control). Also, the bifidobacteria population increased; this bifidogenic stimulation was of EVOOs from Raggia cultivar. SCFAs appeared significantly higher after 24 h in all EVOO fermentations than in the control. Acetic and propionic acids production was particularly stimulated. Overall, most of the investigated EVOOs had a potential prebiotic activity, similar or stronger than inulin.

NMR Spectroscopic Identification of Urolithin G, a Novel Trihydroxy Urolithin Produced by Human Intestinal Enterocloster Species.

Urolithins are gut microbiota metabolites of ellagic acid. Here, we have identified and chemically characterized a novel urolithin produced from urolithin D (3,4,8,9-tetrahydroxy urolithin) by in vitro incubation with different human gut Enterocloster species under anaerobic conditions. Urolithin G (3,4,8-trihydroxy urolithin) was identified by 1H NMR, 13C NMR, UV, HRMS, and 2D NMR. For the identification, NMR spectra of other known urolithins were also recorded and compared. Urolithin G was present in the feces of 12% of volunteers in an overweight-obese group after consuming an ellagitannin-rich pomegranate extract. The production of urolithin G required a bacterial 9-dehydroxylase activity and was not specific to the known human urolithin metabotypes A and B. The ability to produce urolithin G could be considered an additional metabolic feature for volunteer stratification and bioactivity studies. This is the first urolithin with a catechol group in ring A while having only one hydroxyl in ring B, a unique feature not found in human and animal samples so far.

Ellagic acid and its metabolites urolithins A/B ameliorate most common disease phenotypes in cellular and mouse models for lysosomal storage disorders by enhancing extracellular vesicle secretion.

Niemann Pick diseases types A (NPDA) and C (NPDC) are lysosomal storage disorders (LSDs) leading to cognitive impairment, neurodegeneration, and early death. NPDA and NPDC have different genetic origins, being caused by mutations in the acid sphingomyelinase (ASM) or the cholesterol transport protein NPC1, respectively. However, they share a common pathological hallmark in the accumulation of lipids in the endolysosomal compartment. Here, we tested the hypothesis that polyphenols reduce lipid overload in NPD cells by enhancing the secretion of extracellular vesicles (ECVs). We show that among the polyphenols tested, the ellagic acid metabolites, urolithin A and B, were the safest and most efficient in increasing ECV secretion. They reduced levels of accumulating lipids and lysosomal size and permeabilization in cultured bone marrow-derived macrophages and neurons from ASMko and NPC1 mutant mice, which mimic NPDA and NPDC, respectively. Moreover, oral treatment with ellagic acid reduced lipid levels, ameliorated lysosomal alterations, and diminished microglia activation in the brain of NPD mice. These results support the therapeutic value of ECV secretion and polyphenols for NPDs, which may also help treat other LSDs characterized by intracellular lipid overload.

Gut Bacteria Involved in Ellagic Acid Metabolism To Yield Human Urolithin Metabotypes Revealed.

We aimed to elucidate the gut bacteria that characterize the human urolithin metabotypes A and B (UM-A and UM-B). We report here a new bacterium isolated from the feces of a healthy woman, capable of producing the final metabolites urolithins A and B and different intermediates. Besides, we describe two gut bacterial co-cultures that reproduced the urolithin formation pathways upon in vitro fermentation of both UM-A and UM-B. This is the first time that the capacity of pure strains to metabolize ellagic acid cooperatively to yield urolithin profiles associated with UM-A and UM-B has been demonstrated. The urolithin-producing bacteria described herein could have potential as novel probiotics and in the industrial manufacture of bioactive urolithins to develop new ingredients, beverages, nutraceuticals, pharmaceuticals, and (or) functional foods. This is especially relevant in UM-0 individuals since they cannot produce bioactive urolithins.

Separation of Isomeric Forms of Urolithin Glucuronides Using Supercritical Fluid Chromatography.

Urolithins are gut microbiota metabolites produced in humans after consuming foods containing ellagitannins and ellagic acid. Three urolithin metabotypes have been reported for different individuals depending on the final urolithins produced. After absorption, they are conjugated with glucuronic acid (phase II metabolism), and these are the main circulating metabolites in plasma and reach different tissues. Different regioisomeric isomers of urolithin glucuronides have been described. Still, their identification and quantification in humans have not been properly reported due to resolution limitations in their analysis by reversed-phase high-performance liquid chromatography. In the present study, we report a novel method for separating these isomers using supercritical fluid chromatography. With this method, urolithin A 3- and 8-glucuronide, isourolithin A 3- and 9- glucuronide, and urolithin B 3-glucuronide (8-hydroxy urolithin 3-glucuronide; 3-hydroxy urolithin 8-glucuronide; 3-hydroxyurolithin 9-glucuronide; 9-hydroxyurolithin 3-glucuronide; and urolithin 3-glucuronide) were separated in less than 15 min. The proposed method was applied to successfully analyze these metabolites in urine samples from different volunteers belonging to different metabotypes.

Effect of Storage Conditions on the Stability of Polyphenols of Apple and Strawberry Purees Produced at Industrial Scale by Different Processing Techniques.

During a food product's life, storage conditions affect its composition of nutrients, bioactive compounds, and sensory attributes. In this research, strawberry and apple purees were selected as a model to examine how the storage of various purees industrially produced with different technologies affect the bioactive phenolic compounds, color, and sensory attributes. Specifically, fruit products processed on an industrial scale by different technologies including freezing, thermal treatment (mild and standard), and high-pressure processing were studied, as well as storage for up to 12 months at -20, 4, and 24 °C. In strawberry puree, storage conditions had a stronger impact on phenolic compound levels, particularly on anthocyanins, whereas in apple puree, the initial processing techniques exerted a greater influence than storage conditions, mainly caused by the hot or cold crushing processes. In general, proanthocyanidins were the major phenolic group and the most stable during storage, while anthocyanins were the group most affected by both processing and storage. Apple flavonols and dihydrochalcones were quite stable, while strawberry ellagitannins suffered higher degradations during storage. Through our analysis, it is found that during storage, the stability of polyphenols in each fruit is different, and processing and storage can be either detrimental or even beneficial. The selection of the ideal storage conditions (time and temperature) is a key factor to maintaining the polyphenol content in sensitive fruits such as strawberries. However, storage conditions are in some cases more important to minimizing the polyphenol losses than how the product is processed.

Pre-harvest Nitrogen Limitation and Continuous Lighting Improve the Quality and Flavor of Lettuce (Lactuca sativa L.) under Hydroponic Conditions in Greenhouse.

Short-term nitrogen limitation and continuous lighting (red/blue = 3:1) were applied individually and in combination to butterhead and red oak leaf lettuce for 1, 2, or 3 days before harvest to assess their effects on improving the nutritional value and sweet taste and reducing nitrate content and bitterness of lettuce. The results suggested that a 3-day nitrogen limitation combined with continuous lighting reduced the lettuce content of nitrate and sesquiterpene lactones and improved the quantities of soluble sugar, soluble protein, anthocyanins, and phenolic compounds without reducing the fresh weight of lettuce. In addition, in vitro simulated digestion results suggested that the 3-day nitrogen limitation combined with continuous lighting significantly improved the sweetness and reduced the bitterness of lettuce compared to the control. In conclusion, nitrogen limitation combined with continuous lighting for 3 days before harvest effectively enhanced the quality and taste of lettuce, showing great potential for its use in hydroponic lettuce production.

Ellagitannins, urolithins, and neuroprotection: Human evidence and the possible link to the gut microbiota.

Ellagitannins (ETs) and ellagic acid (EA) are dietary polyphenols poorly absorbed but extensively metabolized by the human gut microbiota to produce different urolithins (Uros). Depending on the individuals' microbial signatures, ETs metabolism can yield the Uro metabotypes A, B, or 0, potentially impacting human health after consuming ETs. Human evidence points to improved brain health after consuming ET-rich foods, mainly pomegranate juices and extracts containing punicalagin, punicalin, and different EA-derivatives. Although ETs and (or) EA are necessary to exert the effects, the precise mechanism, actual metabolites, or final drivers responsible for the observed effects have not been unraveled. The cause-and-effect evidence on Uro-A administration and the improvement of animal brain health is consistent but not addressed in humans. The Uro-A's in vivo anti-inflammatory, mitophagy, autophagy, and mitochondrial biogenesis activities suggest it as a possible final driver in neuroprotection. However, the precise Uro metabolic forms reaching the brain are unknown. In addition to the possible participation of direct effectors in brain tissues, the current evidence points out that improving blood flow, gut microbiota ecology, and gut barrier by ET-rich foods and (or) Uro-A could contribute to the neuroprotective effects. We show here the current human evidence on ETs and brain health, the possible link between the gut microbiota metabolism of ETs and their effects, including the preservation of the gut barrier integrity, and the possible role of Uros. Finally, we propose a roadmap to address what is missing on ETs, Uros, and neuroprotection.

Stability of phenolic compounds in apple and strawberry: Effect of different processing techniques in industrial set up.

Different technologies commonly employed by the fruit processing industry affect the phenolic content. This study aimed to establish the extent to which different processing techniques and conditions, tested at an industrial scale, affect phenolics, color, and sensory attributes of fresh strawberries and apples. The effects of freezing, thermal treatments, and high-pressure processing were investigated. In strawberries, mild and standard thermal treatments showed similar patterns for most phenolic groups; an increase in proanthocyanidins, no change in ellagic acid conjugates, and a major decrease in flavonols and anthocyanins. In apples, mild treatments and high-pressure processing had similar effects in all phenolic groups, with increases in dihydrochalcones, hydroxycinnamics, and proanthocyanidins and decreases in flavonols. However, the standard thermal treatment increased flavonols and dihydrochalcones concentrations. This study shows that each fruit behaves differently, and both technology and processing conditions should be customized accordingly to preserve or even increase the phenolic content.

Biodegradation of Punicalagin into Ellagic Acid by Selected Probiotic Bacteria: A Study of the Underlying Mechanisms by MS-Based Proteomics.

Pomegranate (Punica granatum L.) is a well-known source of bioactive phenolic compounds such as ellagitannins, anthocyanins, and flavanols. Punicalagin, one of the main constituents of pomegranate, needs to be biodegraded by bacteria to yield metabolites of medicinal interest. In this work, we tested 30 lactic acid bacteria (LAB) and their capacity to transform punicalagin from a punicalagin-rich pomegranate extract into smaller bioactive molecules, namely, ellagic acid and urolithins. These were identified and quantified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS2). Further, we evaluated the molecular mechanism governing this transformation through label-free comparative MS-based proteomics. All tested LAB strains were capable of transforming punicalagin into ellagic acid, while the biosynthesis of urolithins was not observed. Proteomic analysis revealed an increase of generic transglycosylases that might have a hydrolytic role in the target phenolic molecule, coupled with an increase in the quantity of ATP-binding cassette (ABC) transporters, which might play a relevant role in transporting the resulting byproducts in and out of the cell.

Untargeted Metabolomics Reveals New Markers of Food Processing for Strawberry and Apple Purees.

In general, food processing and its conditions affect nutrients, bioactive compounds, and sensory characteristics of food products. This research aims to use a non-targeted metabolomics approach based on UPLC-ESI-QTOF-MS to determine how fruit processing can affect the metabolic profile of fruits and, through a comprehensive metabolic analysis, identify possible markers to assess their degree of processing. The present study uses a real case from the food industry to evaluate markers of the processing of strawberry and apple purees industrially elaborated with different processing techniques and conditions. The results from the multivariate analysis revealed that samples were grouped according to the type of processing, evidencing changes in their metabolic profiles and an apparent temperature-dependent effect. These metabolic profiles showed changes according to the relevance of thermal conditions but also according to the exclusively cold treatment, in the case of strawberry puree, and the pressure treatment, in the case of apple puree. After data analysis, seven metabolites were identified and proposed as processing markers: pyroglutamic acid, pteroyl-D-glutamic acid, 2-hydroxy-5-methoxy benzoic acid, and 2-hydroxybenzoic acid ß-d-glucoside in strawberry and di-hydroxycinnamic acid glucuronide, caffeic acid and lysoPE(18:3(9Z,12Z,15Z)/0:0) in apple purees. The use of these markers may potentially help to objectively measure the degree of food processing and help to clarify the controversial narrative on ultra-processed foods.

A novel combined analytical UV and MS approach for the quantification of oleuropein metabolites in human biological samples when authentic standards are not available.

The beneficial health effects of phytochemicals depend on their bioavailability and the form under which they reach systemic circulation, usually as phase II metabolites. The lack of authentic standards for these metabolites makes their quantification in biological samples challenging. A new analytical approach to get a more accurate quantification of oleuropein metabolites in biological samples after ingestion of olive leaf extract was proposed. This approach was based on the calculation of a response factor in QTOF MS for each metabolite, comparing their quantification in UV and MS using urine samples concentrated in the metabolites of interest. Glucuronide and sulfate conjugates of hydroxytyrosol and homovanillyl alcohol were more accurately quantified in plasma and urine and for the first time, oleuropein aglycone conjugates and their hydroxylated and hydrogenated derivatives were quantified after consumption of olive products. This approach could be extensible to the analysis of other phenolic metabolites when authentic standards are not available, opening a valuable method for bioavailability studies.

Introduction to Novel Approaches in the Valorization of Agricultural Wastes and Their Applications.

Sustainable food production, waste minimization, and valorization are critical concerns for the agricultural and food industry considering their environmental, social, and economic impacts. Foods are processed to prolong shelf life, make products available out of season, produce new products, and improve nutritional value. During processing, a significant amount of waste is generated, which attracts the attention of scientists and industrialists to valorize the value-added components, including dietary fibers, proteins, and bioactive components. This special issue focuses on the latest developments in waste valorization, covering different industries and methodologies. Within the special issue, waste valorization of some specific food groups, including fruit juice wastes, honeybee byproducts, soybean processing waste, grape pomace materials, and olive mill solid waste, are evaluated. On the other hand, novel approaches to valorizing agricultural wastes and their applications, also covering the recovery of natural pigments with potential health effects, are considered. Besides, nanotechnological and microbial bioconversion methods are used to improve the bioavailability and bioactivity of phytochemicals derived from wastes. It can be concluded that there are still some challenges in waste management and valorization concerning economic, safety, sensory, consumer acceptance, and regulative aspects, and further research is required to overcome these challenges and improve the quality and quantity of value-added products obtained from agricultural wastes and byproducts.

Novel Regioselective Synthesis of Urolithin Glucuronides─Human Gut Microbiota Cometabolites of Ellagitannins and Ellagic Acid.

Urolithins (dibenzo-pyran-[b,d]-6 one derivatives) are human gut microbiota metabolites produced from the natural food antioxidant ellagic acid. Urolithins are better absorbed than ellagic acid and demonstrate biological activities that suggest that they are responsible for the health effects observed after consuming ellagitannin- and ellagic acid-containing foods. Urolithins occur in the systemic circulation as glucuronide conjugates following phase II metabolism. These phase II conjugates are essential for testing the urolithin mechanisms of action in human cell line bioassays. Urolithin glucuronides are not commercially available, and their biosynthesis leads to mixtures of regional isomers. This study describes a novel and regioselective synthesis of urolithin A (3,8-dihydroxy urolithin) 3- and 8-glucuronides and isourolithin A (3,9-dihydroxy urolithin) 3- and 9-glucuronides. The metabolites were characterized using 1H and 13C NMR spectroscopy and UV spectrophotometry. The presence of these metabolites in human subjects belonging to different urolithin metabotypes was also investigated.

Production of New Microbially Conjugated Bile Acids by Human Gut Microbiota.

Gut microbes have been recognized to convert human bile acids by deconjugation, dehydroxylation, dehydrogenation, and epimerization of the cholesterol core, but the ability to re-conjugate them with amino acids as an additional conversion has been recently described. These new bile acids are known as microbially conjugated bile acids (MCBAs). The aim of this study was to evaluate the MCBAs diversity produced by the gut microbiota through a metabolomics approach. In this study, fresh fecal samples from healthy donors were evaluated to explore the re-conjugation of chenodeoxycholic and 3-oxo-chenodeoxycholic acids by the human gut microbiota. No significant differences were found between the conversion trend of both BAs incubations. The in vitro results showed a clear trend to first accumulate the epimer isoursochenodeoxycholic acid and the dehydroxylated lithocholic acid derivatives in samples incubated with chenodeoxycholic and 3-oxo-chenodeoxycholic acid. They also showed a strong trend for the production of microbially conjugated dehydroxylated bile acids instead of chenodeoxycholic backbone conjugates. Different molecules and isomers of MCBAs were identified, and the new ones, valolithocholate ester and leucolithocholate ester, were identified and confirmed by MS/MS. These results document the gut microbiota's capability to produce esters of MCBAs on hydroxyls of the sterol backbone in addition to amides at the C24 acyl site. This study opens a new perspective to study the BAs diversity produced by the human gut microbiota.