Valorization of Chicken Slaughterhouse Byproducts to Obtain Antihypertensive Peptides.

Hypertension (HTN) is the leading cause of premature deaths worldwide and the main preventable risk factor for cardiovascular diseases. Therefore, there is a current need for new therapeutics to manage this condition. In this regard, protein hydrolysates containing antihypertensive bioactive peptides are of increasing interest. Thus, agri-food industry byproducts have emerged as a valuable source to obtain these hydrolysates as they are rich in proteins and inexpensive. Among these, byproducts from animal origin stand out as they are abundantly generated worldwide. Hence, this review is focused on evaluating the potential role of chicken slaughterhouse byproducts as a source of peptides for managing HTN. Several of these byproducts such as blood, bones, skins, and especially, chicken feet have been used to obtain protein hydrolysates with angiotensin-converting enzyme (ACE)-inhibitory activity and blood pressure-lowering effects. An increase in levels of endogenous antioxidant compounds, a reduction in ACE activity, and an improvement of HTN-associated endothelial dysfunction were the mechanisms underlying their effects. However, most of these studies were carried out in animal models, and further clinical studies are needed in order to confirm these antihypertensive properties. This would increase the value of these byproducts, contributing to the circular economy model of slaughterhouses.

Interplay of Walnut Consumption, Changes in Circulating miRNAs and Reduction in LDL-Cholesterol in Elders.

The mechanisms underlying the lipid-lowering effect of nuts remain elusive. This study explores whether one-year supplementation with walnuts decreases LDL-cholesterol (LDL-C) by affecting the expression of circulating microRNAs (c-miRNA). In this sub-study of the Walnuts and Healthy Aging (WAHA) trial, we obtained fasting serum at baseline and at 1 year from 330 free-living participants (63-79 year, 68% women), allocated into a control group (CG, abstinence from walnuts, n = 164) and a walnut group (WG, 15% of daily energy as walnuts, ~30-60 g/d, n = 166). Participants in the WG showed a 1 year decrease in LDL-C (-9.07, (95% confidence interval: -12.87; -5.73) mg/dL; p = 0.010 versus changes in the CG). We conducted a miRNA array in eight randomly selected participants in the WG who decreased in LDL-C. This yielded 53 c-miRNAs with statistically significant changes, 27 of which survived the correction for multiple testing. When validating them in the full population, statistical significance lasted for hsa-miR-551a, being upregulated in the WG. In mediation analysis, the change in hsa-miR-551a was unrelated to LDL-C decrease. Long-term supplementation with walnuts decreased LDL-C independently of the changes in c-miRNA. The hsa-miR-551a upregulation, which has been linked to a reduced cell migration and invasion in several carcinomas, suggests a novel mechanism of walnuts in cancer risk.

Dietary bovine milk miRNAs transported in extracellular vesicles are partially stable during GI digestion, are bioavailable and reach target tissues but need a minimum dose to impact on gene expression.

PURPOSE: Extracellular RNAs are unstable and rapidly degraded unless protected. Bovine-milk extracellular vesicles (EVs) confer protection to dietary miRNAs, although it remains unclear whether this importantly improves their chances of reaching host target cells to exert biological effects. METHODS: Caco-2, HT-29, Hep-G2 and FHs-74 cell lines were exposed to natural/labelled milk EVs to evaluate cellular uptake. Five frequently reported human milk miRNAs (miR-146b-5p, miR-148a-3p, miR-30a-5p, miR-26a-5p, and miR-22-3p) were loaded into EVs. The intracellular concentration of each miRNA in cells was determined. In addition, an animal study giving an oral dose of loaded EVs in C57BL6/ mice were performed. Gene expression regulation was assessed by microarray analysis. RESULTS: Digestive stability analysis showed high overall degradation of exogenous miRNAs, although EV-protected miRNAs better resisted gastrointestinal digestion compared to free miRNAs (tenfold higher levels). Importantly, orally delivered EV-loaded miRNAs reached host organs, including brain, in mice. However, no biological effect has been identified. CONCLUSION: Milk EVs protect miRNAs from degradation and facilitate cellular uptake. miRNA concentration in EVs from bovine milk might be insufficient to produce gene modulation. Nevertheless, sizable amounts of exogenous miRNAs may be loaded into EVs, and orally delivered EV-loaded miRNAs can reach tissues in vivo, increasing the possibility of exerting biological effects. Further investigation is justified as this could have an impact in the field of nutrition and health (i.e., infant formulas elaboration).

Eating microRNAs: pharmacological opportunities for cross-kingdom regulation and implications in host gene and gut microbiota modulation.

Cross-kingdom communication via non-coding RNAs is a recent discovery. Exogenous microRNAs (exog-miRNAs) mainly enter the host via the diet. Generally considered unstable in the gastrointestinal tract, some exogenous RNAs may resist these conditions, especially if transported in extracellular vesicles. They could then reach the intestines and more probably exert a regulatory effect. We give an overview of recent discoveries concerning dietary miRNAs, possible ways of enhancing their resistance to food processing and gut conditions, their transport in extracellular vesicles (animal- and plant-origin) and possible biological effects on recipient cells after ingestion. We critically focus on what we believe are the most relevant data for future pharmacological development of dietary miRNAs as therapeutic agents. Finally, we discuss the miRNA-mediated cross-kingdom regulation between diet, host and the gut microbiota. We conclude that, despite many obstacles and challenges, extracellular miRNAs are serious candidates to be targeted pharmacologically for development of new therapeutic agents.

Bovine Milk-Derived Exosomes as a Drug Delivery Vehicle for miRNA-Based Therapy.

MicroRNAs (miRNAs) are small non-coding RNAs with a known role as mediators of gene expression in crucial biological processes, which converts them into high potential contenders in the ongoing search for effective therapeutic strategies. However, extracellular RNAs are unstable and rapidly degraded, reducing the possibility of successfully exerting a biological function in distant target cells. Strategies aimed at enhancing the therapeutic potential of miRNAs include the development of efficient, tissue-specific and nonimmunogenic delivery methods. Since miRNAs were discovered to be naturally transported within exosomes, a type of extracellular vesicle that confers protection against RNase degradation and increases miRNA stability have been proposed as ideal delivery vehicles for miRNA-based therapy. Although research in this field has grown rapidly in the last few years, a standard, reproducible and cost-effective protocol for exosome isolation and extracellular RNA delivery is lacking. We aimed to evaluate the use of milk-derived extracellular vesicles as vehicles for extracellular RNA drug delivery. With this purpose, exosomes were isolated from raw bovine milk, combining ultracentrifugation and size exclusion chromatography (SEC) methodology. Isolated exosomes were then loaded with exogenous hsa-miR148a-3p, a highly expressed miRNA in milk exosomes. The suitability of exosomes as delivery vehicles for extracellular RNAs was tested by evaluating the absorption of miR-148a-3p in hepatic (HepG2) and intestinal (Caco-2) cell lines. The potential exertion of a biological effect by miR-148a-3p was assessed by gene expression analysis, using microarrays. Results support that bovine milk is a cost-effective source of exosomes which can be used as nanocarriers of functional miRNAs with a potential use in RNA-based therapy. In addition, we show here that a combination of ultracentrifugation and SEC technics improve exosome enrichment, purity, and integrity for subsequent use.

One-year dietary supplementation with walnuts modifies exosomal miRNA in elderly subjects.

PURPOSE: Epidemiological studies and clinical trials support the association of nut consumption with a lower risk of prevalent non-communicable diseases, particularly cardiovascular disease. However, the molecular mechanisms underlying nut benefits remain to be fully described. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and play a pivotal role in health and disease. Exosomes are extracellular vesicles released from cells and mediate intercellular communication. Whether nut consumption modulates circulating miRNAs (c-miRNAs) transported in exosomes is poorly described. METHODS: Cognitively healthy elderly subjects were randomized to either control (n = 110, abstaining from walnuts) or daily supplementation with walnuts (15% of their total energy, ≈30-60 g/day, n = 101) for 1-year. C-miRNAs were screened in exosomes isolated from 10 samples, before and after supplementation, and identified c-miRNA candidates were validated in the whole cohort. In addition, nanoparticle tracking analysis and lipidomics were assessed in pooled exosomes from the whole cohort. RESULTS: Exosomal hsa-miR-32-5p and hsa-miR-29b-3p were consistently induced by walnut consumption. No major changes in exosomal lipids, nanoparticle concentration or size were found. CONCLUSION: Our results provide novel evidence that certain c-miRNAs transported in exosomes are modulated by walnut consumption. The extent to which this finding contributes to the benefits of walnuts deserves further research.

Characterization of Andean Blueberry in Bioactive Compounds, Evaluation of Biological Properties, and In Vitro Bioaccessibility.

The aim of this study was to evaluate Andean blueberries (Vaccinium floribundum Kunth) from Ecuador as a potential functional ingredient for the food and pharmaceutical industries. The analysis of bioactive compounds by HPLC-DAD-MSn determined a high content of (poly)phenols, mainly anthocyanins, and the presence of the carotenoid lutein. Regarding its biological properties, Andean blueberry did not show toxicity by the zebrafish embryogenesis test, showing also a lack of the antinutrients lectins. Moreover, the results of in vitro and in vivo antioxidant capacity evaluation suggested its possibility to be used as natural antioxidant. This fruit also exhibited antimicrobial activity toward Staphylococcus aureus and Escherichia coli in low doses. Finally, in vitro gastrointestinal (GI) digestion showed a partial bioaccessibility of (poly) phenols (~50% at the final step), showing high antioxidant capacity in the different GI phases. These results revealed Andean blueberry as an interesting candidate for being used as a functional ingredient and the development of further in vivo and clinical assays.

Postprandial Circulating miRNAs in Response to a Dietary Fat Challenge.

Postprandial lipemia has many physiopathological effects, some of which increase the risk of cardiovascular disease. MicroRNAs (miRNAs) can be found in almost all biological fluids, but their postprandial kinetics are poorly described. We aimed to profile circulating miRNAs in response to a fat challenge. In total, 641 circulating miRNAs were assessed by real-time PCR in plasmas from mice two hours after lipid gavage. Mice with intestine-specific loss of Dicer were screened to identify potential miRNAs released by the intestine. A total of 68 miRNAs were selected for further validation. Ten circulating miRNAs were finally validated as responsive to postprandial lipemia, including miR-206-3p, miR-543-3p, miR-466c-5p, miR-27b-5p, miR-409-3p, miR-340-3p, miR-1941-3p, miR-10a-3p, miR-125a-3p, and miR-468-3p. Analysis of their possible tissues of origin/target showed an enrichment of selected miRNAs in liver, intestine, brain, or skeletal muscle. miR-206, miR-27b-5p, and miR-409-3p were validated in healthy humans. Analysis of their predicted target genes revealed their potential involvement in insulin/insulin like growth factor (insulin/IGF), angiogenesis, cholecystokinin B receptor signaling pathway (CCKR), inflammation or Wnt pathways for mice, and in platelet derived growth factor (PDGF) and CCKR signaling pathways for humans. Therefore, the current study shows that certain miRNAs are released in the circulation in response to fatty meals, proposing them as potential novel therapeutic targets of lipid metabolism.

Biological Active Ecuadorian Mango 'Tommy Atkins' Ingredients-An Opportunity to Reduce Agrowaste.

Mango is a commercially important tropical fruit. During its processing, peel and seed kernel are discarded as waste but they could be recovered as an excellent and cost-effective source of health-promoting ingredients. This study aimed to characterize some of them, including carotenoids like the provitamin A ß-carotene and lutein, with an interest beyond its role in eye health. Other health-promoting compounds like tocopherols and polyphenols were also evaluated, as well as the in vitro antioxidant capacity of mango by-products. Regarding isoprenoids, α-tocopherol was mainly found in the peels and carotenoids concentration was higher in the pulps. ß-carotene was the most abundant carotene in pulp and seed kernel, whereas peel was the only source of lutein, with violaxanthin the most abundant xanthophyll in the different mango organs tested. With regard to polyphenols, peels exhibited greater variability in its phenolic composition, being the total content up to 85 and 10 times higher than the pulp and seed kernels, respectively. On the other hand, peels also stood out for being a very rich source of mangiferin. Seed kernels and peels showed higher antioxidant capacity values than the pulps. These results contribute to the valorization of mango by-products as new natural ingredients for the pharma and food industries.

Changes in phytochemical composition, bioactivity and in vitro digestibility of guayusa leaves (Ilex guayusa Loes.) in different ripening stages.

BACKGROUND: Guayusa (Ilex guayusa Loes.) leaves, native of the Ecuadorian Amazon, are popularly used for preparing teas. This study aimed to assess the influence of leaf age on the phenolic compounds and carotenoids and the bioactivity and digestibility (in vitro) of aqueous and hydroalcoholic leaf extracts. RESULTS: In total, 14 phenolic compounds were identified and quantified. Chlorogenic acid and quercetin-3-O-hexose were the main representatives of the hydroxycinnamic acids and flavonols respectively. Seven carotenoids were quantified, lutein being the main compound. Ripening affected phenolic content significantly, but there was no significant difference in carotenoid content. Antioxidant capacity, measured by the DPPH• method, was also significantly affected by leaf age. The measurement of in vitro digestibility showed a decrease in phenolic content (59%) as well as antioxidant capacity, measured by the ABTS•+ method, in comparison with initial conditions of the guayusa infusion. Antibacterial and anti-inflammatory activities were assayed with young leaves owing to their higher phenolic contents. Guayusa did not show any antibacterial activity against Escherichia coli ATCC 25922 or Staphylococcus aureus ATCC 25923. Finally, the hydroalcoholic and aqueous extracts exhibited high in vitro anti-inflammatory activity (>65%). CONCLUSION: Young guayusa leaves have potential applications as a functional ingredient in food and pharmaceutical industries. © 2017 Society of Chemical Industry.

Guayusa (Ilex guayusa L.) new tea: phenolic and carotenoid composition and antioxidant capacity.

BACKGROUND: Guayusa (Ilex guayusa Loes) is an evergreen tree native of South America that grows particularly in the upper Amazon region of Ecuador. For its health benefits, it has been cultivated and consumed since ancient times by Amazon indigenous tribes. RESULTS: A total of 14 phenolic compounds were identified and quantified. Chlorogenic acid and quercetin-3-O-hexose were the main representatives of the hydroxycinnamic acids and flavonols, respectively. Five carotenoids were identified, showing lutein the highest concentration. Guayusa leaves revealed high antioxidant capacity determined by two analytical methods, DPPH and ORAC. The industrial processing applied to the leaves modified the composition of bioactive compounds and antioxidant capacity of guayusa. In general, blanched guayusa retained the concentration of phenolic compounds and some carotenoids and similar antioxidant capacity as untreated green leaves. In contrast, fermentation reduced the content of bioactive compounds and showed the lowest antioxidant capacity. CONCLUSION: Therefore, blanched guayusa has potential for product development as a functional ingredient in the food industry. © 2017 Society of Chemical Industry.

Banana Passion Fruit (Passiflora mollissima (Kunth) L.H. Bailey): Microencapsulation, Phytochemical Composition and Antioxidant Capacity.

Passiflora mollissima (Kunth) L.H. Bailey is an exotic fruit native to South America, known as taxo in Ecuador. This paper characterizes its flavonoid and carotenoid composition and antioxidant capacity and evaluates the effect of the spray-drying process on its phytochemical composition and antioxidant capacity. A total of 18 flavonoid compounds, nine proanthocyanidins and nine flavan-3-ol monomers, were identified and quantified. Glycosides of (epi)-afzelechin stood out as the most abundant flavonoid. Three carotenoids were identified, with ß-carotene having the highest concentration. The DPPH· and ORAC assay methods indicated a high antioxidant capacity. Furthermore, the bioactive content showed a positive and direct correlation with antioxidant capacity. On the other hand, the spray-drying process produced a stable phytochemical composition and antioxidant activity of taxo. These results demonstrate the potential applicability of microencapsulated taxo as a functional ingredient in the food industry.

Assessment of probiotic properties in lactic acid bacteria isolated from wine.

Probiotic properties are highly strain-dependent but rarely studied in enological lactic acid bacteria (LAB). In this study, the probiotic features of 11 strains of Lactobacillus spp., Pediococcus spp., and Oenococcus oeni, including saliva and acid resistance, bile tolerance and exopolysaccharides' production, were investigated. The assays included two probiotic reference strains (Lactobacillus plantarum CLC 17 and Lactobacillus fermentum CECT5716). The Lactobacillus and Pediococcus strains showed high resistance to lysozyme (>80% resistance to 100 mg/L of lysozyme under conditions simulating the in vivo dilution by saliva) and were capable of surviving at low pH values (pH 1.8) and bile salts, suggesting good adaptation of the wine strains to gastrointestinal conditions. The ability of the strains to adhere to the intestinal mucosa and the inhibition of the adhesion of Escherichia coli to human intestinal cells were also evaluated. Adhesion levels of enological LAB to Caco-2 cells varied from 0.37% to 12.2%, depending on the strain. In particular, Pediococcus pentosaceus CIAL-86 showed a high percentage of adhesion to intestinal cells (>12%), even higher than that shown by the probiotic reference strains, and a high anti-adhesion activity against E. coli CIAL-153 (>30%), all of which support this wine LAB strain as a potential probiotic.

Genetic diversity of Oenoccoccus oeni isolated from wines treated with phenolic extracts as antimicrobial agents.

Molecular techniques have been applied to study the evolution of wine-associated lactic acid bacteria from red wines produced in the absence and presence of antimicrobial phenolic extracts, eucalyptus leaves and almond skins, and to genetically characterize representative Oenococcus oeni strains. Monitoring microbial populations by PCR-DGGE targeting the rpoB gene revealed that O. oeni was, as expected, the species responsible for malolactic fermentation (MLF). Representative strains from both extract-treated and not-treated wines were isolated and all were identified as O. oeni species, by 16S rRNA sequencing. Typing of isolated O. oeni strains based on the mutation of the rpoB gene suggested a more favorable adaptation of L strains (n = 63) than H strains (n = 3) to MLF. Moreover, PFGE analysis of the isolated O. oeni strains revealed 27 different genetic profiles, which indicates a rich biodiversity of indigenous O. oeni species in the winery. Finally, a higher number of genetic markers were shown in the genome of strains from control wines than strains from wines elaborated with phenolic extracts. These results provide a basis for further investigation of the molecular and evolutionary mechanisms leading to the prevalence of O. oeni in wines treated with polyphenols as inhibitor compounds.

Assessment of the impact of the addition of antimicrobial plant extracts to wine: volatile and phenolic composition.

BACKGROUND: Antimicrobial plant phenolic-rich extracts have been proposed as alternative to sulfites in the control of the malolactic fermentation (MLF) during winemaking. This addition may affect wine organoleptic properties. In this paper, we have investigated the changes in wine volatile and phenolic composition, after MLF, of a red wine treated with antimicrobial extracts from eucalyptus leaves and almond skins. RESULTS: Although addition of both extracts led to statistically significant changes (P < 0.05) in the concentration of several esters, alcohols, C13 nor-isoprenoids and volatile phenols, only few of these volatile compounds showed values of odour activity > 1 aroma unit; that is to say, whose concentrations were higher than their corresponding odour thresholds. With regard to phenolic compounds, the addition of both extracts did not significantly modify the content of anthocyanins, which predicts minor changes in wine colour. However, the content of non-anthocyanin phenolics was significantly higher in the wines treated with antimicrobial extracts, especially for flavonols, being the dose-over-taste factor for these wines significantly higher. Finally, principal component analysis showed that wines were mainly differentiated on the basis of whether MLF was conducted or not, and its method of performance (inoculated/spontaneous). CONCLUSION: Addition of antimicrobial extracts leads to some compositional changes in the wine, whose relevance needs to be addressed in future experiments, including wine sensorial analysis.

Potential of wine-associated lactic acid bacteria to degrade biogenic amines.

Some lactic acid bacteria (LAB) isolated from fermented foods have been proven to degrade biogenic amines through the production of amine oxidase enzymes. Since little is known about this in relation to wine micro-organisms, this work examined the ability of LAB strains (n=85) isolated from wines and other related enological sources, as well as commercial malolactic starter cultures (n=3) and type strains (n=2), to degrade histamine, tyramine and putrescine. The biogenic amine-degrading ability of the strains was evaluated by RP-HPLC in culture media and wine malolactic fermentation laboratory experiments. Although at different extent, 25% of the LAB isolates were able to degrade histamine, 18% tyramine and 18% putrescine, whereas none of the commercial malolactic starter cultures or type strains were able to degrade any of the tested amines. The greatest biogenic amine-degrading ability was exhibited by 9 strains belonging to the Lactobacillus and Pediococcus groups, and most of them were able to simultaneously degrade at least two of the three studied biogenic amines. Further experiments with one of the strains that showed high biogenic amine-degrading ability (L. casei IFI-CA 52) revealed that cell-free extracts maintained this ability in comparison to the cell suspensions at pH 4.6, indicating that amine-degrading enzymes were effectively extracted from the cells and their action was optimal in the degradation of biogenic amines. In addition, it was confirmed that wine components such as ethanol (12%) and polyphenols (75 mg/L), and wine additives such as SO(2) (30 mg/L), reduced the histamine-degrading ability of L. casei IFI-CA 52 at pH 4.6 by 80%, 85% and 11%, respectively, in cell suspensions, whereas the reduction was 91%, 67% and 50%, respectively, in cell-free extracts. In spite of this adverse influence of the wine matrix, our results proved the potential of wine-associated LAB as a promising strategy to reduce biogenic amines in wine.

Comparative study of the inhibitory effects of wine polyphenols on the growth of enological lactic acid bacteria.

This paper reports a comparative study of the inhibitory potential of 18 phenolic compounds, including hydroxybenzoic acids and their derivatives, hydroxycinnamic acids, phenolic alcohols and other related compounds, stilbenes, flavan-3-ols and flavonols, on different lactic acid bacteria (LAB) strains of the species Oenococcus oeni, Lactobacillus hilgardii and Pediococcus pentosaceus isolated from wine. In general, flavonols and stilbenes showed the greatest inhibitory effects (lowest IC50 values) on the growth of the strains tested (0.160-0.854 for flavonols and 0.307-0.855 g/L for stilbenes). Hydroxycinnamic acids (IC50 > 0.470 g/L) and hydroxybenzoic acids and esters (IC50 >1 g/L) exhibited medium inhibitory effect, and phenolic alcohols (IC50 > 2 g/L) and flavanol-3-ols (negligible effect) showed the lowest effect on the growth of the LAB strains studied. In comparison to the antimicrobial additives used in winemaking, IC50 values of most phenolic compounds were higher than those of potassium metabisulphite for O. oeni strains (e.g., around 4-fold higher for quercetin than for potassium metabisulphite), but lower for L. hilgardii and P. pentosaceus strains (e.g., around 2-fold lower for quercetin). Lysozyme IC50 values were negligible for L. hilgardii and P. pentosaceus, and were higher than those corresponding to most of the phenolic compounds tested for O. oeni strains, indicating that lysozyme was less toxic for LAB than the phenolic compounds in wine. Scanning electron microscopy confirmed damage of the cell membrane integrity as a consequence of the incubation with antimicrobial agents. These results contribute to the understanding of the inhibitory action of wine phenolics on the progress of malolactic fermentation, and also to the development of new alternatives to the use of sulphites in enology.

Role of specific components from commercial inactive dry yeast winemaking preparations on the growth of wine lactic acid bacteria.

The role of specific components from inactive dry yeast preparations widely used in winemaking on the growth of three representative wine lactic acid bacteria (Oenococcus oeni, Lactobacillus hilgardii and Pediococcus pentosaceus) has been studied. A pressure liquid extraction technique using solvents of different polarity was employed to obtain extracts with different chemical composition from the inactive dry yeast preparations. Each of the extracts was assayed against the three lactic acid bacteria. Important differences in the effect of the extracts on the growth of the bacteria were observed, which depended on the solvent employed during the extraction, on the type of commercial preparations and on the lactic acid bacteria species. The extracts that exhibited the most different activity were chemically characterized in amino acids, free monosaccharides, monosaccharides from polysaccharides, fatty acids and volatile compounds. In general, specific amino acids and monosaccharides were related to a stimulating effect whereas fatty acid composition and likely some volatile compounds seemed to show an inhibitory effect on the growth of the lactic acid bacteria. These results may provide novel and useful information in trying to obtain better and more specific formulations of winemaking inactive dry yeast preparations.