Dietary aquaculture by-product hydrolysates: impact on the transcriptomic response of the intestinal mucosa of European seabass (Dicentrarchus labrax) fed low fish meal diets.

BACKGROUND: Aquaculture production is expected to double by 2030, and demands for aquafeeds and raw materials are expected to increase accordingly. Sustainable growth of aquaculture will require the development of highly nutritive and functional raw materials to efficiently replace fish meal. Enzymatic hydrolysis of marine and aquaculture raw materials could bring new functionalities to finished products. The aim of this study was to determine the zootechnical and transcriptomic performances of protein hydrolysates of different origins (tilapia, shrimp, and a combination of the two) in European seabass (Dicentrarchux labrax) fed a low fish meal diet (5%), for 65 days. RESULTS: Results were compared to a positive control fed with 20% of fish meal. Growth performances, anterior intestine histological organization and transcriptomic responses were monitored and analyzed. Dietary inclusion of protein hydrolysates in the low fish meal diet restored similar growth performances to those of the positive control. Inclusion of dietary shrimp hydrolysate resulted in larger villi and more goblet cells, even better than the positive control. Transcriptomic analysis of the anterior intestine showed that dietary hydrolysate inclusion restored a pattern of intestinal gene expression very close to the pattern of the positive control. However, as compared to the low fish meal diet and depending on their origin, the different hydrolysates did not modulate metabolic pathways in the same way. Dietary shrimp hydrolysate inclusion modulated more metabolic pathways related to immunity, while nutritional metabolism was more impacted by dietary tilapia hydrolysate. Interestingly, the combination of the two hydrolysates enhanced the benefits of hydrolysate inclusion in diets: more genes and metabolic pathways were regulated by the combined hydrolysates than by each hydrolysate tested independently. CONCLUSIONS: Protein hydrolysates manufactured from aquaculture by-products are promising candidates to help replace fish meal in aquaculture feeds without disrupting animal metabolism and performances.

Effects of protein hydrolysates supplementation in low fish meal diets on growth performance, innate immunity and disease resistance of red sea bream Pagrus major.

This study was conducted to evaluate the supplemental effects of three different types of protein hydrolysates in a low fish meal (FM) diet on growth performance, feed utilization, intestinal morphology, innate immunity and disease resistance of juvenile red sea bream. A FM-based diet was used as a high fish meal diet (HFM) and a low fish meal (LFM) diet was prepared by replacing 50% of FM by soy protein concentrate. Three other diets were prepared by supplementing shrimp, tilapia or krill hydrolysate to the LFM diet (designated as SH, TH and KH, respectively). Triplicate groups of fish (4.9 ± 0.1 g) were fed one of the test diets to apparent satiation twice daily for 13 weeks and then challenged by Edwardsiella tarda. At the end of the feeding trial, significantly (P < 0.05) higher growth performance was obtained in fish fed HFM and hydrolysate treated groups compared to those fed the LFM diet. Significant improvements in feed conversion and protein efficiency ratios were obtained in fish fed the hydrolysates compared to those fed the LFM diet. Significant enhancement in digestibility of protein was found in fish fed SH and KH diets and dry matter digestibility was increased in the group fed SH diet in comparison to LFM group. Fish fed the LFM diet showed significantly higher glucose level than all the other treatments. Whole-body and dorsal muscle compositions were not significantly influenced by dietary treatments. Histological analysis revealed significant reductions in goblet cell numbers and enterocyte length in the proximal intestine of fish fed the LFM diet. Superoxide dismutase activity and total immunoglobulin level were significantly increased in fish fed the diets containing protein hydrolysates compared to the LFM group. Also, significantly higher lysozyme and antiprotease activities were found in fish fed the hydrolysates and HFM diets compared to those offered LFM diet. Fish fed the LFM diet exhibited the lowest disease resistance against E. tarda and dietary inclusion of the hydrolysates resulted in significant enhancement of survival rate. The results of the current study indicated that the inclusion of the tested protein hydrolysates, particularly SH, in a LFM diet can improve growth performance, feed utilization, digestibility, innate immunity and disease resistance of juvenile red sea bream.

Effect of Early Peptide Diets on Zebrafish Skeletal Development.

Incorporation of dietary peptides has been correlated with decreased presence of skeletal abnormalities in marine larvae. In an attempt to clarify the effect of smaller protein fractions on fish larval and post-larval skeleton, we designed three isoenergetic diets with partial substitution of their protein content with 0% (C), 6% (P6) and 12% (P12) shrimp di- and tripeptides. Experimental diets were tested in zebrafish under two regimes, with inclusion (ADF-Artemia and dry feed) or lack (DF-dry feed only) of live food. Results at the end of metamorphosis highlight the beneficial effect of P12 on growth, survival and early skeletal quality when dry diets are provided from first feeding (DF). Exclusive feeding with P12 also increased the musculoskeletal resistance of the post-larval skeleton against the swimming challenge test (SCT). On the contrary, Artemia inclusion (ADF) overruled any peptide effect in total fish performance. Given the unknown species' larval nutrient requirements, a 12% dietary peptide incorporation is proposed for successful rearing without live food. A potential nutritional control of the larval and post-larval skeletal development even in aquaculture species is suggested. Limitations of the current molecular analysis are discussed to enable the future identification of the peptide-driven regulatory pathways.

A standardized, innovative method to characterize the structure of aquatic protein hydrolysates.

The performances of protein hydrolysates highly depend on their peptide composition (amount, size and diversity), which itself closely depends on raw material origin and the hydrolysis parameters of the manufacturing process. The current analyses that characterize protein hydrolysates provide information on the level of hydrolysis (degree of hydrolysis, DH). However, they need additional describers to better characterize peptide profiles and product standardization. To reach this objective, we developed a fast and standardized method to characterize the abundance and the diversity of low-molecular-weight peptides in protein hydrolysates. This method innovatively combines classical HPSEC and nLC-ESI-MS analytical tools to characterize any kind of hydrolysate, whether solid or liquid, in terms of peptide level and diversity, and then merge peptides into 2D diagrams to visualize comparisons between protein hydrolysates. The targeted applications of this new tool for characterizing complex protein hydrolysates are (i) verifying the standardization of the produced products across batches, and (ii) analyzing and understanding the consequences of the modifications of the hydrolysis process on the molecular profiles of the generated peptides. The sample standardization described in this study is therefore an essential prerequisite for the functional characterization of hydrolysates in vitro.

Transcriptomic and peptidomic analysis of protein hydrolysates from the white shrimp (L. vannamei).

An RNAseq approach associated to mass spectrometry was conducted to assess the composition, molecular mass distribution and primary sequence of hydrolytic peptides issued from hydrolysates of white shrimp (Litopenaeus vannamei) by-products. High performance size exclusion chromatography (HPSEC) analyses indicated that 69.2% of the 214-nm-absorbing components had apparent molecular masses below 1000 Da, and 88.3% below 2000 Da. OFFGEL-nLC-MALDI-TOF/TOF and nLC-ESI-MS/MS analyses led to the identification of 808 peptides based on the NCBI EST databank (161,397 entries) completed by the new L. vannamei databank (58,508 entries) that we created from the RNAs of tissues used for hydrolysate production. Whereas most of hydrolytic peptides have a MW below 2000 Da, preliminary investigations of antimicrobial properties revealed three antibacterial fractions that demonstrate functional activities. The abundance of small peptides as well as the biological activities detected could imply very interesting applications for shrimp hydrolysate in the field of aquaculture feeding.