Functional Properties of Protein Hydrolysates on Growth, Digestive Enzyme Activities, Protein Metabolism, and Intestinal Health of Larval Largemouth Bass (Micropterus salmoides)

Abstract

The present study was conducted to investigate the effects of dietary inclusion of protein hydrolysates on growth performance, digestive enzyme activities, protein metabolism, and intestinal health in larval largemouth bass (Micropterus salmoides). The experimental feeding trial presented in this study was based on five isonitrogenous and isolipidic diets formulated with graded inclusion levels of protein hydrolysates, and it showed that protein hydrolysates improved growth performance, reduced larval deformity rate, and increased the activity of digestive enzymes, including pepsin and trypsin. Gene expression results revealed that the supplementation of protein hydrolysates upregulated the expression of intestinal amino acid transporters LAT2 and peptide transporter 2 (PepT2), as well as the amino acid transporters LAT1 in muscle. Dietary provision of protein hydrolysates activated the target of rapamycin (TOR) pathway including the up-regulation of TOR and AKT1, and down-regulation of 4EBP1. Additionally, the expression of genes involved in the amino acids response (AAR) pathway, ATF4 and REDD1, were inhibited. Protein hydrolysates inhibited the transcription of some pro-inflammatory cytokines, including IL-8 and 5-LOX, but promoted the expression of anti-inflammatory cytokines TGF-β and IL-10. The 16S rRNA analysis, using V3-V4 region, indicated that dietary protein hydrolysates supplementation reduced the diversity of the intestine microbial community, increased the enrichment of Plesiomonas and reduced the enrichment of Staphylococcus at the genus level. In summary, protein hydrolysates have been shown to be an active and useful supplement to positively complement other protein sources in the diets for largemouth bass larvae, and this study provided novel insights on the beneficial roles and possible mechanisms of action of dietary protein hydrolysates in improving the overall performance of fish larvae.

Keywords: intestinal development; intestinal microbiota; larval fish; protein hydrolysates; protein metabolism.

Figure 1

Relative expression of Peptide and AA transporter, L-type amino acid transporter 1 (LAT1) (A), L-type amino acid transporter 2 (LAT2) (B), oligopeptide transporter 2 (PepT2) (C), in intestine of largemouth bass fed the experimental diets for 26 days. Values (means ± standard error of the mean, SEM) in bars that have the same letter are not significantly different (P > 0.05; Duncan’s test) among treatments (N = 3). LAT1: P _linear = 0.001, ${\text{R}}_{\text{linear}}^2=0.605$ ; P _quadratic = 0.003, ${\text{R}}_{\text{quadratic}}^2=0.612$ ; LAT2: P _linear = 0.005, ${\text{R}}_{\text{linear}}^2=0.462$ ; P _quadratic = 0.023, ${\text{R}}_{\text{quadratic}}^2=0.467$ ; PepT2: P _linear < 0.001, ${\text{R}}_{\text{linear}}^2=0.706$ ; P _quadratic < 0.001, ${\text{R}}_{\text{quadratic}}^2=0.798$ .

Figure 2

Relative expression of AA transporter, L-type amino acid transporter 1 (LAT1) (A), L-type amino acid transporter 2 (LAT2) (B), in muscle of largemouth bass fed the experimental diets for 26 days. Values (means ± standard error of the mean, SEM) in bars that have the same letter are not significantly different (P > 0.05; Duncan’s test) among treatments (N = 3). LAT1: P _linear = 0.011, ${\text{R}}_{\text{linear}}^2=0.400$ ; P _quadratic = 0.005, ${\text{R}}_{\text{quadratic}}^2=0.582$ ; LAT2: P _linear = 0.235, ${\text{R}}_{\text{linear}}^2=0.107$ ; P _quadratic = 0.175, ${\text{R}}_{\text{quadratic}}^2=0.252$ .

Figure 3

Relative expression of TOR signaling pathway, target of rapamycin (TOR) (A), protein kinase B α (AKT1) (B), ribosomal protein S6 (S6) (C), and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4EBP1) (D), in muscle of largemouth bass fed the experimental diets for 26 days. Values (means ± standard error of the mean, SEM) in bars that have the same letter are not significantly different (P > 0.05; Duncan’s test) among treatments (N = 3). TOR: P _linear = 0.040, ${\text{R}}_{\text{linear}}^2=0.285$ ; P _quadratic = 0.002, ${\text{R}}_{\text{quadratic}}^2=0.664$ ; AKT1: P _linear = 0.007, ${\text{R}}_{\text{linear}}^2=0.438$ ; P _quadratic = 0.003, ${\text{R}}_{\text{quadratic}}^2=0.619$ ; S6: P _linear = 0.673, ${\text{R}}_{\text{linear}}^2=0.014$ ; P _quadratic = 0.796, ${\text{R}}_{\text{quadratic}}^2=0.037$ ; 4EBP1: P _linear = 0.005, ${\text{R}}_{\text{linear}}^2=0.467$ ; P _quadratic = 0.010, ${\text{R}}_{\text{quadratic}}^2=0.536$ .

Figure 4

Relative expression of AAR signaling pathway, eukaryotic initiation factor 2α (eIF2α) (A), ATF4, activating transcription factor 4 (ATF4) (B), and regulated in development and DNA damage responses 1 (REDD1) (C), in muscle of largemouth bass fed the experimental diets for 26 days. Values (means ± standard error of the mean, SEM) in bars that have the same letter are not significantly different (P > 0.05; Duncan’s test) among treatments (N = 3). eIF2α: P _linear = 0.408, ${\text{R}}_{\text{linear}}^2=0.053$ ; P _quadratic = 0.720, ${\text{R}}_{\text{quadratic}}^2=0.053$ ; ATF4: P _linear = 0.007, ${\text{R}}_{\text{linear}}^2=0.438$ ; P _quadratic = 0.013, ${\text{R}}_{\text{quadratic}}^2=0.514$ ; REDD1: P _linear < 0.001, ${\text{R}}_{\text{linear}}^2=0.661$ ; P _quadratic = 0.001, ${\text{R}}_{\text{quadratic}}^2=0.671$ .

Figure 5

Relative expression of inflammation, tumor necrosis factor α (TNF-α) (A), interleukin-8 (IL-8) (B), and 5-lipoxygenase (5-LOX) (C), and transforming growth factor β (TGF-β) (D), interleukin-10 (IL-10) (E), in intestine of larval largemouth bass fed the experimental diets for 26 days. Values (means ± standard error of the mean, SEM) in bars that have the same letter are not significantly different (P > 0.05; Duncan’s test) among treatments (N = 3). TNF-α: P _linear = 0.704, ${\text{R}}_{\text{linear}}^2=0.011$ ; P _quadratic = 0.406, ${\text{R}}_{\text{quadratic}}^2=0.140$ ; IL-8: P _linear = 0.004, ${\text{R}}_{\text{linear}}^2=0.478$ ; P _quadratic = 0.008, ${\text{R}}_{\text{quadratic}}^2=0.555$ ; 5-LOX: P _linear = 0.002, ${\text{R}}_{\text{linear}}^2=0.533$ , P _quadratic = 0.001, ${\text{R}}_{\text{quadratic}}^2=0.693$ ; TGF-β: P _linear < 0.001, ${\text{R}}_{\text{quadratic}}^2=0.620$ ; P _quadratic = 0.003, ${\text{R}}_{\text{quadratic}}^2=0.621$ . IL-10: P _linear < 0.001, ${\text{R}}_{\text{linear}}^2=0.606$ ; P _quadratic < 0.001, ${\text{R}}_{\text{quadratic}}^2=0.914$ .

Figure 6

The alpha diversity comparisons analysis, including Shannon diversity index (A), Simpson diversity index (B), Ace species richness index (C) and Chao species richness index (D) of microbial communities in the intestine of larval largemouth bass between the PH0 and PH100 group. Values (mean ± standard error of the mean, SEM) in bars that have the same letter are not significantly different (P > 0.05; Welch’s t-test) between treatments (N = 3).

Figure 7

The beta diversity comparisons analysis, including non-metric multidimensional scaling (NMDS) (A), principal component analysis (PCA) (B), and unweighted uniFrac distance matrix (C) of microbial communities at genus level in the intestine of larval largemouth bass between the PH0 and PH100 group.

Figure 8

Relative abundances (%) of dominant phyla (A) and comparison of five high abundance phyla (B) in the intestine of largemouth bass between the PH0 and PH100 group at the phylum level, the phyla with relative abundances lower than 1% were assigned as “others” in Bar map. Heatmap showing the relative abundance of the top 50 most abundant genera in bacterial communities between the PH0 and PH100 group (C). Relative richness of five high abundance genera selected for comparisons in largemouth bass intestine for multiple comparisons among PH0 and PH100 group (D), respectively. * 0.01 < P ≤ 0.05, ** 0.001 < P ≤ 0.01 (Welch’s t-test, N = 3).

Figure 9

Venn diagram analysis of microbial communities in the intestine of larval largemouth bass between the PH0 and PH100 group. The number (A) and community bacterial phylum (B) and unique (C, D) of overlapping in the intestine of largemouth bass were identified ( Figure 9.1 ). The number (A) and community bacterial genera (B) and unique (C, D) of overlapping in the intestine of largemouth bass were identified ( Figure 9.2 ).

Figure 10

Cladogram showing the phylogenetic distribution of the bacterial lineages associated with dietary protein hydrolysates inclusion. Taxonomic representation of statistically and biologically consistent differences between intestinal microbiota of largemouth bass between the PH0 and PH100 group (A). Differences were represented by the color of the most abundant class (red indicates control group; blue indicates PH100 group). Histogram of linear discriminant analysis (LDA) scores for differentially abundant taxon (B). For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.