Physicochemical, technofunctional, in vitro antioxidant, and in situ muscle protein synthesis properties of a sprat (Sprattus sprattus) protein hydrolysate

Abstract

Introduction: Sprat (Sprattus sprattus) is an underutilized fish species that may act as an economic and sustainable alternative source of protein due to its good amino acid (AA) profile along with its potential to act as a source of multiple bioactive peptide sequences.

Method and results: This study characterized the physicochemical, technofunctional, and in vitro antioxidant properties along with the AA profile and score of a sprat protein enzymatic hydrolysate (SPH). Furthermore, the impact of the SPH on the growth, proliferation, and muscle protein synthesis (MPS) in skeletal muscle (C2C12) myotubes was examined. The SPH displayed good solubility and emulsion stabilization properties containing all essential and non-essential AAs. Limited additional hydrolysis was observed following in vitro-simulated gastrointestinal digestion (SGID) of the SPH. The SGID-treated SPH (SPH-SGID) displayed in vitro oxygen radical antioxidant capacity (ORAC) activity (549.42 μmol TE/g sample) and the ability to reduce (68%) reactive oxygen species (ROS) production in C2C12 myotubes. Muscle growth and myotube thickness were analyzed using an xCELLigence™ platform in C2C12 myotubes treated with 1 mg protein equivalent.mL^-1 of SPH-SGID for 4 h. Anabolic signaling (phosphorylation of mTOR, rpS6, and 4E-BP1) and MPS (measured by puromycin incorporation) were assessed using immunoblotting. SPH-SGID significantly increased myotube thickness (p < 0.0001) compared to the negative control (cells grown in AA and serum-free medium). MPS was also significantly higher after incubation with SPH-SGID compared with the negative control (p < 0.05).

Conclusions: These preliminary in situ results indicate that SPH may have the ability to promote muscle enhancement. In vivo human studies are required to verify these findings.

Keywords: C2C12 cells; antioxidant; muscle growth; muscle protein synthesis; sprat protein hydrolysates; technofunctional.

Figure 1

Aqueous protein solubility (%) of the sprat (Sprattus sprattus) protein hydrolysate as a function of pH. Values represent mean ± SD (n = 3).

Figure 2

Effect of pH on the emulsion activity index (EAI -•) and emulsion stability (ES % -Δ) of the sprat (Sprattus sprattus) protein hydrolysate. Values represent mean ± SD (n = 3). Different lowercase letters show significant differences for EAI at different pHs (p < 0.05). Different uppercase letters show significant differences for ES at different pHs (p < 0.05).

Figure 3

Viability of muscle myotube (C2C12) cells treated with 0.1, 0.5, and 1.0 mg protein equivalent.mL⁻¹ sprat (Sprattus sprattus) protein hydrolysate subjected to simulated gastrointestinal digestion (SPH-SGID). The negative control was amino acid and serum-free media without SPH. Treatment of cells was for 4 h after 1 h of nutrient deprivation. The results represent mean ± SD (n = 3) and are expressed relative to the negative control.

Figure 4

Effect of sprat (Sprattus sprattus) protein hydrolysate subjected to simulated gastrointestinal digestion (SPH-SGID) treatment on cell index (area under the curve) and myotube diameter in skeletal muscle cells. C2C12 myotubes were nutrient deprived for 1 h followed by 4 h treatment with 1 mg protein equivalent.mL⁻¹ of SPH-SGID. Myotube growth was monitored every 2 min over 4 h. (A) Representative graph comparing myotube growth (cell index and area under the curve) in the presence of sample relative to the negative control. (B) Representative graph comparing myotube diameter in the presence of sample relative to the negative control. (C) Quantification of myotube diameter 4 h post-treatment as measured by microscopy. Images of myotubes treated with sample were taken at 4X magnification following 4 h treatment. All values are expressed as mean ± SD (n = 6). A p-value of < 0.05* compared to the negative control (amino acid and serum-free media). Ctl-: negative control (amino acid and serum-free media), Ctl+: positive control (100 ng.ml⁻¹ IGF-1), SPH-SGID: sprat protein hydrolysate subjected to simulated gastrointestinal digestion.

Figure 5

Phosphorylation of mTOR, 4E-BP1, and ribosomal S6 incubated with 1 mg protein equivalent.mL-1 sprat (Sprattus sprattus) protein hydrolysate (SPH) subjected to simulated gastrointestinal digestion (SPH-SGID) (n = 4). C2C12 myotubes were nutrient deprived for 1 h followed by treatment with SPH-SGID plus 1 uM puromycin for 4 h. Data reported as the ratio of phosphoproteins relative to the total protein. All values were expressed as a percent of the negative control within each assay. Phosphorylation of mTOR (A), rpS6 (B), and 4-EBP1 (C) following SPH-SGID treatment, and their corresponding representative immunoblot. (D) Muscle protein synthesis (MPS) after treatment with SPH-SGID and a representative immunoblot of MPS (measured by puromycin incorporation) relative to total protein (loading control). Data reported as mean ± SEM, *compared to negative control, p < 0.01. Ctl-: negative control (amino acid and serum-free media), Ctl+: positive control (100 ng. mL-1 IGF-1).