Comprehensive proteomic analysis reveals omega-3 fatty acids to counteract endotoxin-stimulated metabolic dysregulation in porcine enterocytes.

Omega-3 polyunsaturated fatty acids (n-3 PUFA), such as the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are reported to beneficially affect the intestinal immunity. The biological pathways modulated by n-3 PUFA during an infection, at the level of intestinal epithelial barrier remain elusive. To address this gap, we investigated the proteomic changes induced by n-3 PUFA in porcine enterocyte cell line (IPEC-J2), in the presence and absence of lipopolysaccharide (LPS) stress conditions using shotgun proteomics analysis integrated with RNA-sequencing technology. A total of 33, 85, and 88 differentially abundant proteins (DAPs) were identified in cells exposed to n-3 PUFA (DHA:EPA), LPS, and n-3 PUFA treatment followed by LPS stimulation, respectively. Functional annotation and pathway analysis of DAPs revealed the modulation of central carbon metabolism, including the glycolysis/gluconeogenesis, pentose phosphate pathway, and oxidative phosphorylation processes. Specifically, LPS caused metabolic dysregulation in enterocytes, which was abated upon prior treatment with n-3 PUFA. Besides, n-3 PUFA supplementation facilitated enterocyte development and lipid homeostasis. Altogether, this work for the first time comprehensively described the biological pathways regulated by n-3 PUFA in enterocytes, particularly during endotoxin-stimulated metabolic dysregulation. Additionally, this study may provide nutritional biomarkers in monitoring the intestinal health of human and animals on n-3 PUFA-based diets.

Evaluation of Dietary Administration of Chestnut and Quebracho Tannins on Growth, Serum Metabolites and Fecal Parameters of Weaned Piglets.

In pig livestock, alternatives to in-feed antibiotics are needed to control enteric infections. Plant extracts such as tannins can represent an alternative as a natural source of functional compounds. The aim of this study was to evaluate the in vitro digestibility and in vivo effects of oral supplementation of combined chestnut (Ch) and quebracho (Qu) tannins in order to establish if they can induce a positive effect on weaned piglets' performance, metabolic status and fecal parameters. In vitro digestibility (dry matter, DM) of diets was calculated using a multi-step enzymatic technique. In vitro digested diet samples were further tested on an intestinal porcine enterocyte cell line (IPEC-J2). Weaned piglets (n = 120; 28 ± 2 day old) were randomly allotted to two groups (12 pens in total with 10 pigs per pen): control (Ctrl) and treatment (Ch/Qu). After one week of adaptation (day 0), 35-day-old piglets in the Ctrl group were fed a Ctrl diet and the Ch/Qu group were fed with 1.25% Ch/Qu for 40 days. Body weight and feed intake per pen were recorded weekly. At day 40, blood and fecal samples were collected. Principal metabolic parameters were evaluated from blood samples by enzymatic colorimetric analysis. Total phenolic compounds, urea, and ammonia in feces were analyzed (Megazyme International, Bray, Ireland). In vitro digestibility and cell viability assays showed that the inclusion of 1.25% Ch/Qu slightly reduced diet digestibility compared with the Ctrl diet, while intestinal cell viability was not altered with low concentrations of Ch/Qu digesta compared with Ctrl. In vivo results did not show any adverse effects of Ch/Qu on feed intake and growth performance, confirming that dietary inclusion of Ch/Qu at a concentration of 1.25% did not impair animal performance. The decreased diet DM digestibility in the Ch/Qu diet may cause increased serum concentration of albumin (Ctrl: 19.30 ± 0.88; Ch/Qu: 23.05 ± 0.88) and albumin/globulin ratio (Ctrl: 0.58 ± 0.04; Ch/Qu: 0.82 ± 0.04), but decreased creatinine (Ctrl: 78.92 ± 4.18; Ch/Qu: 54.82 ± 4.18) and urea (Ctrl: 2.18 ± 0.19; Ch/Qu: 0.95 ± 0.19) compared with Ctrl. Pigs in the Ch/Qu group contained higher (p < 0.05) concentrations of fecal phenolic compounds and nitrogen than the Ctrl group, while fecal ammonia and urea were not affected by tannins. In conclusion, Ch/Qu tannin supplementation did not influence growth performance. Although lower digestibility was observed in the diet supplemented with Ch/Qu tannins, Ch/Qu supplementation did not show any adverse effect on intestinal epithelial cell viability.

In Vitro Digestion of Chestnut and Quebracho Tannin Extracts: Antimicrobial Effect, Antioxidant Capacity and Cytomodulatory Activity in Swine Intestinal IPEC-J2 Cells.

Quebracho (Qu) and chestnut (Ch) are natural sources of tannins and they are currently used in animal nutrition as feed ingredients. However, to date the bio-accessibility, antimicrobial, antioxidant, and intestinal epithelial cell stimulatory doses of Qu and Ch have not been determined. Our study investigates the antioxidant and E. coli F4+ and F18+ growth inhibitory activity of Qu, Ch, and their combinations after solubilization in water (to evaluate the already bio-accessible molecules) and after simulated gastro-intestinal digestion in vitro. The effect of an in vitro digested Ch and Qu combination was also tested on intestinal epithelial IPEC-J2 cells experimentally stressed with hydrogen peroxide (H2O2) and Dextran Sodium Sulfate (DSS). The results showed that undigested Qu and Ch alone, and in combination, exerted a valuable antioxidant capacity and E. coli F4+ and F18+ growth inhibitory activity. The concentration of 1200 µg/mL exhibited the highest E. coli growth inhibitory activity for all the samples tested. In addition, after in vitro digestion, Qu and Qu50%-Ch50% maintained E. coli growth inhibitory activity and a modest antioxidant capacity. Three hours pre-treatment with in vitro digested Qu50%-Ch50% counteracted the H2O2 and DSS experimentally-induced stress in the intestinal IPEC-J2 cells. Ch and Qu tannin extracts, particularly when combined, may exert E. coli F4+ and F18+ growth inhibitory activity and valuable antioxidant and cell viability modulation activities.

Whey protein lowers systolic blood pressure and Ca-caseinate reduces serum TAG after a high-fat meal in mildly hypertensive adults.

Epidemiological studies show an inverse association between dairy consumption and blood pressure (BP) but there are few data on the postprandial effects of milk proteins. This study examined their effects, compared to maltodextrin, on postprandial BP and other CVD risk markers in volunteers with mild and pre-hypertension over an 8 h period. In this double-blinded, randomised, cross-over, controlled study 27 adults ingested a high-fat, isoenergetic breakfast and lunch with 28 g whey protein, 28 g Ca-caseinate or 27 g maltodextrin. Whey protein reduced systolic BP compared with Ca-caseinate (-15.2 ± 13.6 mmHg) and maltodextrin (-23.4 ± 10.5 mmHg) up to 5 h post-ingestion. There was an improvement in arterial stiffness after whey protein compared with maltodextrin (incremental Area Under the Curve- iAUC0-8h: +14.4 ± 6.2%). Despite similar glucose levels after both whey protein and Ca-caseinate, whey protein induced a higher insulin response than Ca-caseinate (iAUC0-8h: +219.5 ± 54.6 pmol/L). Ca-caseinate induced less suppression of non-esterified fatty acids than whey protein (iAUC0-5h: -58.9 ± 135.5 µmol/L) and maltodextrin (iAUC0-5h: -106.9 ± 89.4 µmol/L) and induced a smaller postprandial triacylglycerol response than whey protein (iAUC0-8h: -1.68 ± 0.6 mmol/L). Milk proteins co-ingestion with high-fat meals may have the potential to maintain or improve CVD risk factors.

Short-Communication: A Comparison of the In Vitro Angiotensin-1-Converting Enzyme Inhibitory Capacity of Dairy and Plant Protein Supplements.

The consumption of supplements based on dairy or plant proteins may be associated with bioactive potential, including angiotensin-1-converting enzyme inhibitory (ACE-1i) activity, which is linked with blood pressure reduction in vivo. To gain insight into this proposed mechanism, the ACE-1i potential of protein-based supplements, including a selection of dairy (n = 10) and plant (n = 5) proteins were in vitro digested. The total digest was filtered and permeate and retentate were obtained. ACE-1i activity was measured as the ability of proteins (pre-digestion, 'gastric', permeate, and retentate) to decrease the hydrolysis of furanacroloyl-Phe-Glu-Glu (FAPGG) substrate for the ACE-1 enzyme. Permeate and retentate of dairy proteins exerted a significantly higher ACE-1i activity (mean of 10 proteins: 27.05 ± 0.2% and 20.7 ± 0.2%, respectively) compared with pre-digestion dairy proteins (16.7 ± 0.3%). Plant protein exhibited high ACE-1i in 'gastric' and retentate fractions (mean of five proteins: 54.9 ± 0.6% and 35.7 ± 0.6%, respectively). The comparison of the in vitro ACE-1i activity of dairy and plant proteins could provide valuable knowledge regarding their specific bioactivities, which could inform their use in the formulation of specific functional supplements that would require testing for blood pressure control in human randomly-controlled studies.

Whey protein lowers blood pressure and improves endothelial function and lipid biomarkers in adults with prehypertension and mild hypertension: results from the chronic Whey2Go randomized controlled trial.

BACKGROUND: Cardiovascular diseases (CVDs) are the greatest cause of death globally, and their reduction is a key public-health target. High blood pressure (BP) affects 1 in 3 people in the United Kingdom, and previous studies have shown that milk consumption is associated with lower BP. OBJECTIVE: We investigated whether intact milk proteins lower 24-h ambulatory blood pressure (AMBP) and other risk markers of CVD. DESIGN: The trial was a double-blinded, randomized, 3-way-crossover, controlled intervention study. Forty-two participants were randomly assigned to consume 2 × 28 g whey protein/d, 2 × 28 g Ca caseinate/d, or 2 × 27 g maltodextrin (control)/d for 8 wk separated by a 4-wk washout. The effects of these interventions were examined with the use of a linear mixed-model ANOVA. RESULTS: Thirty-eight participants completed the study. Significant reductions in 24-h BP [for systolic blood pressure (SBP): -3.9 mm Hg; for diastolic blood pressure (DBP): -2.5 mm Hg; P = 0.050 for both)] were observed after whey-protein consumption compared with control intake. After whey-protein supplementation compared with control intake, peripheral and central systolic pressures [-5.7 mm Hg (P = 0.007) and -5.4 mm Hg (P = 0.012), respectively] and mean pressures [-3.7 mm Hg (P = 0.025) and -4.0 mm Hg (P = 0.019), respectively] were also lowered. Flow-mediated dilation (FMD) increased significantly after both whey-protein and calcium-caseinate intakes compared with control intake [1.31% (P < 0.001) and 0.83% (P = 0.003), respectively]. Although both whey protein and calcium caseinate significantly lowered total cholesterol [-0.26 mmol/L (P = 0.013) and -0.20 mmol/L (P = 0.042), respectively], only whey protein decreased triacylglycerol (-0.23 mmol/L; P = 0.025) compared with the effect of the control. Soluble intercellular adhesion molecule 1 and soluble vascular cell adhesion molecule 1 were reduced after whey protein consumption (P = 0.011) and after calcium-caseinate consumption (P = 0.039), respectively, compared with after control intake. CONCLUSIONS: The consumption of unhydrolyzed milk proteins (56 g/d) for 8 wk improved vascular reactivity, biomarkers of endothelial function, and lipid risk factors. Whey-protein supplementation also lowered 24-h ambulatory SBP and DBP. These results may have important implications for public health. This trial was registered at clinicaltrials.gov as NCT02090842.