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.

Differences Due to Sex and Sweetener on the Bioavailability of (Poly)phenols in Urine Samples: A Machine Learning Approach.

Metabolic diseases have been related to the overdrinking of high-sugar content beverages. As a result, the demand for alternative formulations based on plant-based ingredients with health-promoting properties has increased during the last few years. Nonetheless, the design and production of effective formulations requires understanding the bioavailability of these compounds. For this purpose, a two-month longitudinal trial with 140 volunteers was conducted to measure the beneficial effects of a maqui-citrus beverage, rich in (poly)phenols. From data obtained by quantifying metabolites present in urine samples, biostatistical and machine learning (data imputation, feature selection, and clustering) methods were applied to assess whether a volunteer's sex and the sweetener added to the beverage (sucrose, sucralose, or stevia) affected the bioavailability of (poly)phenol metabolites. Several metabolites have been described as being differentially influenced: 3,4-dihydroxyphenylacetic acid and naringenin with its derivatives were positively influenced by stevia and men, while eriodictyol sulfate and homoeridictyol glucunoride concentrations were enhanced with stevia and women. By examining groups of volunteers created by clustering analysis, patterns in metabolites' bioavailability distribution as a function of sex and/or sweeteners (or even due to an uncontrolled factor) were also discovered. These results underline the potential of stevia as a (poly)phenol bioavailability enhancer. Furthermore, they also evidence sex affects the bioavailability of (poly)phenols, pointing at a sex-dependent metabolic pathway regulation.

Bioactive Compounds in Plasma as a Function of Sex and Sweetener Resulting from a Maqui-Lemon Beverage Consumption Using Statistical and Machine Learning Techniques.

The present study analyses the effect of a beverage composed of citrus and maqui (Aristotelia chilensis) with different sweeteners on male and female consumers. Beverages were designed and tested (140 volunteers) as a source of polyphenols, in a previous work. Plasma samples were taken before and after two months of daily intake. Samples were measured for bioactive-compound levels with metabolomics techniques, and the resulting data were analysed with advanced versions of ANOVA and clustering analysis, to describe the effects of sex and sweetener factors on bioactive compounds. To improve the results, machine learning techniques were applied to perform feature selection and data imputation. The results reflect a series of compounds which are more regulated for men, such as caffeic acid or 3,4-dihydroxyphenylacetic acid, and for women, trans ferulic acid (TFA) or naringenin glucuronide. Regulations are also observed with sweeteners, such as TFA with stevia in women, or vanillic acid with sucrose in men. These results show that there is a differential regulation of these two families of polyphenols by sex, and that this is influenced by sweeteners.

Influence of Sweeteners (Sucrose, Sucralose, and Stevia) on Bioactive Compounds in a Model System Study for Citrus-Maqui Beverages.

Recently, new formulations of beverages with low sugar and high bioactive compound contents are being demanded because of their association with metabolic health. However, the sweeteners' influences on the bioactive compounds remain underexplored. In this sense, this work aims to evaluate the interactions between different bioactive compounds such as flavonoids, vitamin C, and sweeteners (sucrose, stevia, and sucralose) in a functional citrus-maqui beverage. For this purpose, the phytochemical behavior was studied, in model system solutions, during one-month storage at 4 °C. The results obtained corroborated previous descriptions of the interactions between these compounds. However, when studying the bioactives in the model solutions, the loss of flavanones in the citrus solution increased up to 29%, while anthocyanin losses decreased to 27%. The vitamin C losses ranged from 100% (beverage) to 46% (ascorbic acid solution). Regardless, an influence of the sweeteners was observed. Sucrose reduced the anthocyanin and flavanone losses in both model solutions and the beverage, while sucralose increased flavanone loss. Finally, all sweeteners increased vitamin C degradation (up to 75%) when compared to the model solution. In conclusion, sweeteners added to beverages should be taken into account, depending on the bioactive compounds that should be preserved during storage.

Contribution of the diverse experimental models to unravelling the biological scope of dietary (poly)phenols.

The health benefits associated with (poly)phenols need to be supported by robust and insightful information on their biological effects. The use of in vitro, ex vivo, and in vivo models is crucial to demonstrate functionalities in specific targets. In this regard, bioaccessibility, bioavailability, and tissue/organ distribution need to be fully understood and established. In addition, the structure-function relationships, concerning both descriptive and mechanistic information, between specific compounds and therapeutic objectives, need to be supported by results obtained from in vivo studies. Nevertheless, these studies are not always possible or have some limitations, particularly concerning the mechanistic information explaining the health benefits provided that should be covered with complementary experimental models. Based on these premises, this review aims to overview the contribution of the separate experimental approaches to gain insights into the bioaccessibility, bioavailability, and bioactivity of (poly)phenols. To achieve this objective, recent evidence available on the linkage of healthy/functional foods with the incidence of non-communicable pathologies is presented. The different experimental approaches provide complementary information that allows advances to be applied to the knowledge gained on the functional properties and mechanistic facts responsible for the health attributions of polyphenols. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

(Poly)phenolic compounds and gut microbiome: new opportunities for personalized nutrition.

For decades, (poly)phenols have been linked to cardiometabolic health, but population heterogeneity limits their apparent efficacy and the development of tailored, practical protocols in dietary interventions. This heterogeneity is likely determined by the existence of different metabotypes, sub-populations of individuals metabolizing some classes of (poly)phenols differently. The gut microbiota plays a major role in this process. The impact of microbiota-related phenolic metabotypes on cardiometabolic health is becoming evident, although the picture is still incomplete, and data are absent for some classes of (poly)phenols. The lack of a complete understanding of the main microbial actors involved in the process complicates the picture. Elucidation of the mechanisms behind phenolic metabotypes requires novel experimental designs that can dissect the inter-individual variability. This paper, in addition to providing an overview on the current state-of-the-art, proposes wider metabotyping approaches as a means of paving the way towards effective personalized nutrition with dietary (poly)phenols.

The use of alternative sweeteners (sucralose and stevia) in healthy soft-drink beverages, enhances the bioavailability of polyphenols relative to the classical caloric sucrose.

The comparison of non-caloric sweeteners (stevia and sucralose) and sucrose, on the plasma concentration and cumulative effects of phenolic compounds, was achieved. A long-term intervention, consisting of the daily intake of 330 mL of healthy citrus-maqui soft drinks, for 60 days, by 138 healthy overweight adults, was followed. A total of 24 bioavailable metabolites derived from caffeic acid, 3,4-di-hydroxyphenylacetic acid, eriodictyol, homoeriodictyol, hippuric acid, naringenin, 2,4,6-tri-hydroxybenzaldehyde, and vanillic acid were detected in peripheral blood plasma. A similar augment of bioactive compounds in plasma concentrations were found for the three beverages, in the range 12.3% (day 0)- 85.3% (day 60), depending on the analyte considered. Due to this, the present study highlights sucralose and stevia as valuable alternatives to sucrose, providing and non-significantly different plasma concentration and cumulative effect in the plasma, thus contributing to prevent a diversity of metabolic disorders and health constraints.

High Hydrostatic Pressure vs. Thermal Pasteurization: The Effect on the Bioactive Compound Profile of a Citrus Maqui Beverage.

The effects of high hydrostatic pressure (HHP) compared to thermal pasteurization (TP) were studied in healthy citrus-maqui beverages. The impact of the processing technologies on the microbiological and phytochemical profile was assessed by applying two HHP treatments at 450 and 600 MPa for 180 s and TP at 85 °C for 15 s. The shelf life under refrigeration (4 °C) and room temperature (20 °C) was monitored for 90 days. All treatments ensured microbiological stability at both storage temperatures. Aside from that, the physicochemical parameters were not significantly different after processing or throughout the storage period. Regarding color parameters, an increase in the reddish coloration was observed during storage for those beverages treated by HHP. In general, phenolic compounds were little affected by the processing technique, even when treatment under HHP was more stable than by TP during storage. On the other hand, vitamin C showed great degradation after processing under any condition. It can be concluded that HHP is an effective alternative to thermal treatments, achieving effective microbial inactivation and extending the shelf life of the juices by contributing to a better preservation of color and bioactive compounds.

Synergistic Interaction in the Analgesic-Like Effects of Maqui Berry and Citrus Is Antagonized by Sweeteners.

Although physiologically pain has a protective function, in many diseases, it is one of the most prominent symptoms. Today, new trends are focused on finding more natural alternatives to conventional treatments to alleviate it. Thereby, the purpose of this investigation was to obtain preclinical data of the antinociceptive properties of a lyophilized obtained from a newly designed maqui-citrus beverage alone and added with different sweeteners. To achieve this objective, maqui berry and citrus pharmacological activity were studied separately, as well as the interaction of both ingredients. In addition, due to the controversy generated regarding the intake of sugars, related to different metabolic diseases, the influence of different sweeteners (stevia, sucralose, or sucrose) was studied to determine their possible influence on the bioactive compounds of this product. For the attainment of our goals, a pharmacological evaluation, using the 1% formalin test, a nociceptive pain model in mice, was performed by using a sub-efficacious dosage of Maqui (25 mg/kg, i.p.) alone and combined with citrus, and then compared with the effects obtained in the presence of the different sweeteners. As a result, the antinociceptive response of the maqui was synergized in the presence of citrus in the neurogenic and inflammatory phases of the formalin test. However, this response was partially or totally reduced in the presence of the sweeteners. Our study gives preclinical evidence that a combination of maqui and citrus might exert beneficial actions to relieve pain, whereas the presence of sweeteners could reduce or avoid it.

Antinociceptive effects of maqui-berry (Aristotelia chilensis (Mol.) Stuntz).

Maqui-berry is characterised by presenting a high concentration of (poly)phenols, accounting anthocyanins (cyanidin and delphinidin) for over 85% of the total. These coloured flavonoids have demonstrated potential neurological activity, but the evidence of their antinociceptive properties is scarce. In order to cover this gap, different doses (suitable for human administration) of a maqui-berry powder (1.6% anthocyanin), using enteral and parenteral routes of administration, were compared at central and peripheral levels using a nociceptive pain model (formalin test) in mice. Gastric damage analysis as possible adverse effects of analgesic and anti-inflammatory drugs was also explored. Dose-antinociceptive response was confirmed using both routes of administration and in both neurogenic and inflammatory phases of the formalin test, without gastric damage. In conclusion, these preliminary data provide evidence of pharmacological properties of maqui-berry to alleviate nociceptive pain.

Sweetener influences plasma concentration of flavonoids in humans after an acute intake of a new (poly)phenol-rich beverage.

BACKGROUND AND AIM: The overconsumption of sucrose is closely related to sugar-sweetened beverages and one of the main factors associated with the increase of metabolic diseases, such as type 2 diabetes, obesity, and insulin resistance. So, the addition of alternative sweeteners to new fruit-based drinks could contribute to minimizing the incidence or severity of these pathologies. Nevertheless, current knowledge on the influence of these additives on the bioactive compounds present in these beverages is still scarce.new-onset hypertension, but few data were published in Asian. We aimed to investigate the association of lipid profiles with new-onset hypertension in a Chinese community-based non-hypertensive cohort without lipid-lowering treatment (n = 1802). METHODS AND RESULTS: Hence, to contribute to the understanding of this issue, the plasma concentration of phenolic compounds (anthocyanins and flavanones), after the ingestion of a new maqui-citrus-based beverage, supplemented with sucrose (natural high caloric), stevia (natural non-caloric), or sucralose (artificial non-caloric), was evaluated as evidence of their intestinal absorption and metabolism previous to renal excretion. The beverages were ingested by volunteers (n = 20) and the resulting phenolic metabolites in plasma were analyzed by UHPLC-ESI-MS/MS. A total of 13 metabolites were detected: caffeic acid sulfate, caffeic acid glucuronide, 3,4-dihydroxyfenylacetic, 3,4-dihydroxyfenylacetic sulfate. 3,4-dihydroxyfenylacetic acid di-sulfate, 3,4-dihydroxyfenylacetic di-glucuronide, 3,4-dihydroxyfenylacetic glucuronide-sulfate, trans-ferulic acid glucuronide, naringenin glucuronide, vanillic acid, vanillic acid sulfate, vanillic acid glucuronide-sulfate, and vanillic acid di-glucuronide, being recorded their maximum concentration after 30-60 min. CONCLUSION: In general, sucralose provided the greatest absorption value for most of these metabolites, followed by stevia. Due to this, the present study proposes sucralose and stevia (non-caloric sweeteners) as valuable alternatives to sucrose (high caloric sweetener), to avoid the augmented risk of several metabolic disorders.

Beverages Based on Second Quality Citrus Fruits and Maqui Berry, a Source of Bioactive (Poly)phenols: Sorting Out Urine Metabolites upon a Longitudinal Study.

The intake of sugar-sweetened beverages has been associated with an augmented prevalence of metabolic diseases, namely, obesity, type II diabetes, and metabolic syndrome. On the other hand, nowadays, it is broadly accepted that foods and beverages rich in (poly)phenols could contribute to reducing the incidence of these pathologies. In this sense, the objective of the work was to revalue second quality citrus fruits for the development of new beverages, rich in anthocyanins and flavanones (maqui berry and second qualities citrus-based), and evaluate the influence of alternative sweeteners (sucralose, sucrose, or stevia), regarding the bioaccessibility and bioavailability of these bioactive compounds in the frame of a chronic (longitudinal) intervention. To fulfill this objective, a longitudinal study of the urinary excretion of anthocyanins and flavanones, after 2-months of ingestion of the developed maqui-citrus beverage, by 138 volunteers (n = 46 per beverage) and the analysis of the resulting phenolic metabolites by ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-ESI-QqQ-MS/MS) was carried out. As major results, the bioavailable metabolites of caffeic acid (CA), catechol (CAT), 3,4-di-hydroxyphenylacetic acid (DHPAA), eriodictyol (E), homoeriodictyol (HE), hippuric acid (HA), naringenin (N), trans-ferulic acid (TFA), 2,4,6-tri-hydroxybenzaldehyde (THBA), trans-isoferulic acid (TIFA), and vanillic acid (VA) were detected. Accordingly, significantly different bioavailability was dependent on the sweetener used, allowing proposing stevia and, to a lower extent, sucralose, as valuable alternatives to sucrose.

Stevia vs. Sucrose: Influence on the Phytochemical Content of a Citrus-Maqui Beverage-A Shelf Life Study.

The consumption of sugar-sweetened beverages has been related with the risk of cardiovascular diseases and other pathophysiological situations, such as obesity or diabetes mellitus. Given the increasing awareness on this fact, food industries are developing new products to reduce the amount of added sugar in development of food products development. Accordingly, in the present work, new functional beverages, constituting a dietary source of bioactive phenolics and supplemented with stevia or sucrose, were designed in order to study the influence of the sweetener during processing and shelf-life. This study is of critical for the informed selection of the sweetener based on its effect on the final phytochemical profile of beverages, especially taking into consideration that there are no previous studies on Stevia rebaudiana. Physicochemical features and phytochemical composition, as well as stability of the different beverages concerning these parameters, were evaluated for 90 days during storage under different conditions (refrigeration (4 °C) and room temperature (25 °C) under light or darkness conditions). Physicochemical parameters (pH, titratable acidity, total soluble solids, and color) did not display statistically significant differences between beverages. Storage temperature was the greatest determinant affecting the stability of all the analyzed bioactive compounds (vitamin C, anthocyanins, and flavanones). The main difference between sweeteners was observed in flavanones, which exhibited a higher loss during storage under day light conditions when stevia was added instead of sucrose. In addition, the juices' colors were rather stable, keeping a reddish coloration and natural appearance throughout the shelf life. Hence, stevia could be considered as an alternative sweetener by the beverage industry.

Anthocyanin Metabolites in Human Urine after the Intake of New Functional Beverages.

Sugar intake abuse is directly related with the increase of metabolic diseases such as type 2 diabetes, obesity, and insulin resistance. Along this line, the development of new beverages using alternative sweeteners could help with combatting the pathophysiological disorders associated to the consumption of sugar. To provide evidence on this issue, in the present work, the bioavailability of anthocyanins was evaluated after the acute ingestion of a new maqui-citrus-based functional beverage rich in polyphenols, and supplemented with a range of sweeteners including sucrose (natural high caloric), stevia (natural non-caloric), and sucralose (artificial non-caloric), as an approach that would allow reducing the intake of sugars while providing bioactive phenolic compounds (anthocyanins). This approach allowed the evaluation of the maximum absorption and the diversity of metabolites excreted through urine. The beverages created were ingested by volunteers (n = 20) and the resulting anthocyanin metabolites in their urine were analyzed by UHPLC-ESI-MS/MS. A total of 29 degradation metabolites were detected: Caffeic acid, catechol, 3,4-dihidroxifenilacetic acid, hippuric acid, trans-ferulic acid, 2,4,6-trihydroxybenzaldehyde, trans-isoferulic acid, and vanillic acid derivatives, where peak concentrations were attained at 3.5 h after beverage intake. Sucralose was the sweetener that provided a higher bioavailability for most compounds, followed by stevia. Sucrose did not provide a remarkably higher bioavailability of any compounds in comparison with sucralose or stevia. The results propose two sweetener alternatives (sucralose and stevia) to sucrose, an overused high calorie sweetener that promotes some metabolic diseases.

Alternative Sweeteners Modify the Urinary Excretion of Flavanones Metabolites Ingested through a New Maqui-Berry Beverage.

Dietary sugar has been largely related to the onset of metabolic diseases such as type 2 diabetes and obesity, among others. The growing awareness on the close relationship between the dietary habits and this health disturbance has encouraged the development of new beverages using alternative sweeteners that could contribute to combat the above referred pathophysiological disorders. To gain further insight into this issue, the present work, upon an acute dietary intervention, evaluated the urinary excretion of flavanones ingested through polyphenols-rich beverages composed of maqui berry and citrus, with the aim of establishing the highest urinary excretion rate and metabolite profiles. The functional beverages evaluated were supplemented with a range of sweeteners including sucrose (natural and high caloric), stevia (natural and non-caloric), and sucralose (artificial and non-caloric) as an approach that would allow reducing the intake of sugars and provide bioactive phenolics (flavanones). The juices developed were ingested by volunteers (n = 20) and the resulting flavanones and their phase II metabolites in urine were analyzed by Ultra-High Performance Liquid Chromatography ElectroSpray Ionization Mass Spectrometry (UHPLC-ESI-MS/MS). A total of 16 metabolites were detected: eriodyctiol, naringenin, and homoeriodyctiol derivatives, where peak concentrations were attained 3.5 h after beverage intake. Sucralose and stevia were the sweeteners that provided the highest urinary excretion for most compounds. Sucrose did not provide a remarkable higher elimination through urine of any compounds in comparison with sucralose or stevia. These results propose two alternative sweeteners to sucrose (sucralose and stevia), an overused, high caloric sweetener that promotes some metabolic diseases.