Modulation of gilthead sea bream gut microbiota by a bioactive egg white hydrolysate

Trabajo presentado en Aquaculture Europe 2020, celebrado en modalidad virtual del 12 al 15 de abril de 2021.[Introduction]: A bioactive egg white hydrolysate (EWH) treated with pepsin has demonstrated potent in vitro and in vivo antioxidant and anti-inflammatory properties, improving oxidative stress and inflammation biomarkers on genetically and diet induced obese rats (Requena et al., 2017). However, the effects of protein hydrolysates and bioactive food-derived peptides on gut microbiome remain relatively poorly studied in mammals and fish in particular. Thus, the aim of this study was to unravel the main effects on fish performance, histopathological scoring and mucosal adherent gut microbiota of EWH supplementation in a fish fed a formulation with a high replacement of marine feedstuffs by alternative plant ingredients, using gilthead sea bream as a farmed fish model. [Methods]: The feeding trial lasted 8 weeks (May-July) under natural photoperiod and temperature conditions. Juvenile fish (20-24 g initial body weight, 4.8-4.9 kg/m3) were fed near to visual satiety with control (CTRL) or low fish meal (FM)/fish oil (FO) diets with/without egg white hydrolysate (EWH) supplementation (7.5%). DNA from the adherent bacteria of the anterior intestine was collected and the V3-V4 region of the 16S rRNA of each sample was amplified and sequenced by Illumina MiSeq. Taxonomic assignment was performed with a custom-made pipeline using the RDP database. Alpha diversity was calculated using Phyloseq, and beta diversity using PERMANOVA and partial least-squares discriminant analysis (PLSDA) models. Metagenome prediction and pathway analysis were performed using Piphillin.[Methods]: The feeding trial lasted 8 weeks (May-July) under natural photoperiod and temperature conditions. Juvenile fish (20-24 g initial body weight, 4.8-4.9 kg/m3) were fed near to visual satiety with control (CTRL) or low fish meal (FM)/fish oil (FO) diets with/without egg white hydrolysate (EWH) supplementation (7.5%). DNA from the adherent bacteria of the anterior intestine was collected and the V3-V4 region of the 16S rRNA of each sample was amplified and sequenced by Illumina MiSeq. Taxonomic assignment was performed with a custom-made pipeline using the RDP database. Alpha diversity was calculated using Phyloseq, and beta diversity using PERMANOVA and partial least-squares discriminant analysis (PLSDA) models. Metagenome prediction and pathway analysis were performed using Piphillin.[Results]: Daily specific growth rates (SGR) varied significantly from 2.16 in CTRL fish to 1.88 in EWH fish as a result of a reduced feed intake. A slight impairment of feed conversion ratio, from 1.03 to 1.10, was also observed. Intermediate values on growth performance parameters were reported with the low FM/FO diet without EWH supplementation. No changes in total plasma antioxidant capacity, and faecal concentrations of lactic acid and short-chain fatty acids were found among dietary groups. The dietary replacement of FM/FO triggered a hyperplasic inflammation of the anterior intestine submucosa that was not alleviated by EWH supplementation. Conversely, alterations on the staining pattern and amount of goblet cells at the level of anterior intestine were reversed in EWH fish, together with a decreased accumulation of lipid vacuoles in the epithelium of posterior intestine, a high abundance of hepatic melanomacrophage centers, and depletion of hepatocyte lipid depots until the restoration of CTRL fish values. Illumina sequencing reads were assigned to 2,117 OTUs and a significantly lower richness was found in the EWH group. Indeed, at the phylum level, Proteobacteria reached the highest proportion in CTRL and EWH fish, whereas Firmicutes were decreased and Actinobacteria increased with the replacement of FM/FO. The proportion of Actinobacteria was restored to CTRL values with the dietary EWH supplementation. Additionally, EWH triggered the highest amount of Bacteroidetes and Spirochaetes phyla. Detailed differences in microbiota composition were analysed with a statistically validated PLS-DA which clearly separated CTRL fish from fish fed low FM/FO diets along x-axis (component 1, 37.4%), whereas component 2 (43.2%) separated the low FM/FO diets with/without EWH along y-axis (Fig. 1). This analysis disclosed 165 OTUs discriminating among diets (VIP ≥ 1), with 46 OTUs representing at least the 1% in one of the groups. For these abundant bacteria, a first type of response was mediated by 17 OTUs that were increasing with the FM/FO replacement and decreasing again in EWH fish. In this group, Neisseriaceae family and species of Ralstonia, Lactobacillus, Streptococcus, Corynebacterium and Nocardioides genera were included. A group of 14 OTUs were present in high proportion in the CTRL group, but decreased in fish fed the two low FM/FO diets. In this case, the dietary plant ingredients drove the decrease of the Comamonadaceae family and Mesorizhobium, Brochotrix, Bacillus, Clostridium sensu stricto and Exiguobacterium genera. The remaining 15 OTUs increased their proportion in fish fed the EWH diet, being in a very low proportion in the other two dietary groups. This response triggered the presence of Bacteroidetes phylum, Rhodospirilalles order and Granucatella, Bradyrizhobium, Propionibacterium and Streptophyta genera. Inferred metagenome results showed two pathways corresponding to primary bile acid biosynthesis and steroid degradation consistently underrepresented in the microbiota of EWH fish when compared to the other two groups[Conclusions]: These results reinforce the central role of gut microbiota in the regulation of host metabolism and lipid metabolism in particular (Hegyi et al., 2018), supporting a main role of the EWH as an anti-obesity and satiety factor in fish as suggested in rat models of obesity. The potential use of this functional food ingredient in finishing diets, and the role of gut microbiota in tuning fillet fatty acid composition of marketable fish merits further research.This work was funded by the TNA programme (AE150009) within H2020 AQUAEXCEL2020 project (652831) to GAWP for accessing to IATS-CSIC facilities

Modulation of gilthead sea bream gut microbiota by a bioactive egg white hydrolysate: interactions between bacteria and host lipid metabolism

This study aimed to highlight the relationship between diet, animal performance and mucosal adherent gut microbiota (anterior intestine) in fish fed plant-based diets supplemented with an egg white hydrolysate (EWH) with antioxidant and anti-obesogenic activity in obese rats. The feeding trial with juveniles of gilthead sea bream (Sparus aurata) lasted 8 weeks. Fish were fed near to visual satiety with a fish meal (FM)/fish oil (FO) based diet (CTRL) or a plant-based diet with/without EWH supplementation. Specific growth rate decreased gradually from 2.16% in CTRL fish to 1.88% in EWH fish due to a reduced feed intake, and a slight impairment of feed conversion ratio. Plant-based diets feeding triggered a hyperplasic inflammation of the anterior intestine regardless of EWH supplementation. However, EWH ameliorated the goblet cell depletion, and the hepatic and intestinal lipid accumulation induced by FM/FO replacement. Illumina sequencing of gut mucosal microbiota yielded a mean of 136,252 reads per sample assigned to 2,117 OTUs at 97% identity threshold. The bacterial diversity was similar in all groups, but a significantly lower richness was found in EWH fish. At the phylum level, Proteobacteria reached the highest proportion in CTRL and EWH fish, whereas Firmicutes were decreased and Actinobacteria increased with the FM/FO replacement. The proportion of Actinobacteria was restored by dietary EWH supplementation, which also triggered a highest amount of Bacteroidetes and Spirochaetes. At a closer look, a widespread presence of Lactobacillales among groups was found. Otherwise, polysaccharide hydrolases secretors represented by Corynebacterium and Nocardioides were increased by the FM/FO replacement, whereas the mucin-degrading Streptococcus was only raised in fish fed the plant-based diet without EWH. In addition, in EWH fish, a higher abundance of Propionibacterium was related to an increased concentration of intestinal propionate. The antagonism of gut health-promoting propionate with cholesterol could explain the inferred underrepresentation of primary bile acid biosynthesis and steroid degradation pathways in the EWH fish microbiota. Altogether, these results reinforce the central role of gut microbiota in the regulation of host metabolism and lipid metabolism in particular, suggesting a role of the bioactive EWH peptides as an anti-obesity and/or satiety factor in fish.This work was supported by the EU H2020 Research Innovation Program under the TNA Program (project AE150009) at IATS-CSIC Research Infrastructure within AQUAEXCEL2020 Project (652831). This output reflects only the author’s view and the European Union cannot be held responsible for any use that may be made of the information contained herein. Additional funding was obtained by a Spanish MICINN project (Bream-AquaINTECH and RTI2018–094128-B-I00). MCP was funded by a Ramón y Cajal Postdoctoral Research Fellowship [RYC2018-024049-I/AEI/10.13039/501100011033 co-funded by the European Social Fund (ESF) and ACOND/2020 Generalitat Valenciana].Peer reviewe

Apocynin prevents vascular effects caused by chronic exposure to low concentrations of mercury.

UNLABELLED: Mercury increases the risk of cardiovascular disease and oxidative stress and alters vascular reactivity. This metal elicits endothelial dysfunction causing decreased NO bioavailability via increased oxidative stress and contractile prostanoid production. NADPH oxidase is the major source of reactive oxygen species (ROS) in the vasculature. Our aim was to investigate whether treatment with apocynin, an NADPH oxidase inhibitor, prevents the vascular effects caused by chronic intoxication with low concentrations of mercury. Three-month-old male Wistar rats were treated for 30 days with a) intramuscular injections (i.m.) of saline; b) HgCl(2) (i.m. 1(st) dose: 4.6 µg/kg, subsequent doses: 0.07 µg/kg/day); c) Apocynin (1.5 mM in drinking water plus saline i.m.); and d) Apocynin plus HgCl(2). The mercury treatment resulted in 1) an increased aortic vasoconstrictor response to phenylephrine and reduced endothelium-dependent responses to acetylcholine; 2) the increased involvement of ROS and vasoconstrictor prostanoids in response to phenylephrine, whereas the endothelial NO modulation of such responses was reduced; and 3) the reduced activity of aortic superoxide dismutase (SOD) and glutathione peroxidase (GPx) and increased plasma malondialdehyde (MDA) levels. Treatment with apocynin partially prevented the increased phenylephrine responses and reduced the endothelial dysfunction elicited by mercury treatment. In addition, apocynin treatment increased the NO modulation of vasoconstrictor responses and aortic SOD activity and reduced plasma MDA levels without affecting the increased participation of vasoconstrictor prostanoids observed in aortic segments from mercury-treated rats. CONCLUSIONS: Mercury increases the vasoconstrictor response to phenylephrine by reducing NO bioavailability and increasing the involvement of ROS and constrictor prostanoids. Apocynin protects the vessel from the deleterious effects caused by NADPH oxidase, but not from those caused by prostanoids, thus demonstrating a two-way action

Effect of apocynin treatment on endothelial modulation of the vasoconstrictor response to phenylephrine.

<p>Concentration-response curve to phenylephrine (Phe) in intact (Control) and endothelium removal (E-) aortic segments of rats (A) untreated, (B) treated with apocynin (Apo), (C) mercury (HgCl₂), and (D) apocynin plus mercury (ApoHg). The results (mean±SEM) are expressed as a percentage of the response to 75 mmol/l KCl. The number of animals is indicated in parentheses. *P<0.001 by Two-Way ANOVA. (E) Differences in the area under the concentration-response curves (dAUC) in endothelium denuded and intact segments of the four experimental groups. * P<0.05 <i>vs</i>. Untreated and ^#<i>vs</i>. HgCl₂-treated by <i>t</i>-test.</p

Effect of apocynin treatment on systolic and diastolic blood pressure.

<p>Values of (A) systolic (SBP) and (B) diastolic blood pressure (DBP, mmHg) in the aorta of rats untreated (n = 9) or treated with mercury (HgCl₂, n = 8), apocynin (Apo, n = 9) or apocynin plus mercury (ApoHg, n = 9). The results are expressed as the mean±SEM, <i>t</i>-test *P<0.05 <i>vs</i>. Untreated.</p

Effect of apocynin treatment on ROS modulation of the vasoconstrictor response to phenylephrine.

<p>Concentration-response curve to phenylephrine (Phe) in aortic segments of rats (A) untreated, (B) treated with apocynin (Apo), (C) mercury (HgCl₂), and (D) apocynin plus mercury (ApoHg) in the absence (Control) and the presence of the NADPH oxidase inhibitor Apocynin (0.3 mM). The results (mean±SEM) are expressed as a percentage of the response to 75 mmol/l KCl. The number of rats is indicated in parentheses. *P<0.001 by Two-Way ANOVA. (E) Differences in the area under the concentration-response curve to phenylephrine (dAUC) in aortic segments incubated in the absence and the presence of apocynin of the four experimental groups. * P<0.05 <i>vs</i>. Untreated and ^#<i>vs</i>. HgCl₂-treated by <i>t</i>-test.</p

Effects of apocynin treatment of rats in the absence and in the presence of HgCl₂ on maximum response (R_max) and sensitivity (pD2) to acetylcholine and sodium nitroprusside.

<p>Parameters of maximal response (Rmax) and sensitivity (pD2) of the concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP) in aortas from rats untreated, treated with mercury (HgCl₂), apocynin and apocynin plus mercury (ApoHg) in intact segments (Control). Results are expressed as mean±SEM. R_max, maximal effect (expressed as a percentage of the previous contraction to phenylephrine) and pD2 expressed as -log one-half R_max; <i>t</i>-test: *P<0.05 vs. Untreated; ^#P<0.05 <i>vs</i>. HgCl₂-treated.</p

Effect of apocynin treatment on SOD and GPx activity.

<p>Values of (A) SOD and (B) GPx activities in the aortas of rats untreated (n = 6) and treated with mercury (HgCl₂, n = 6), apocynin (Apo, n = 6) and apocynin plus mercury (ApoHg, n = 6). Data are expressed as the mean±SEM. <i>t</i>-test *P<0.05 <i>vs</i>. Untreated and ^#P<0.05 <i>vs</i>. HgCl₂-treated.</p

Effect of apocynin treatment on the vascular relaxation response to acetylcholine and sodium nitroprusside.

<p>Concentration-response curves to (A) acetylcholine (ACh) and (B) sodium nitroprusside (SNP) in the aortas of rats untreated, treated with mercury (HgCl₂) or apocynin plus mercury (ApoHg) pre-contracted with phenylephrine. The results (mean±SEM) are expressed as a percentage of the response to phenylephrine. The number of animals used is indicated in parentheses. Two-Way ANOVA *P<0.05.</p