Exploring the Potential of Olive Flounder Processing By-Products as a Source of Functional Ingredients for Muscle Enhancement

Abstract

Olive flounder (OF) is a widely aqua-cultivated and recognized socioeconomic resource in Korea. However, more than 50% of by-products are generated when processing one OF, and there is no proper way to utilize them. With rising awareness and interest in eco-friendly bio-materialization recycling, this research investigates the potential of enzymatic hydrolysis of OF by-products (OFB) to produce functional ingredients. Various enzymatic hydrolysates of OFB (OFBEs) were generated using 11 commercial enzymes. Among them, Prozyme 2000P-assisted OFBE (OFBP) exhibited the highest protein content and yield, as well as low molecularization. The muscle regenerative potential of OFBEs was evaluated using C2C12 myoblasts, revealing that OFBP positively regulated myoblast differentiation. In an in vitro Dex-induced myotube atrophy model, OFBP protected against muscle atrophy and restored myotube differentiation and Dex-induced reactive oxygen species (ROS) production. Furthermore, zebrafish treated with OFBEs showed improved locomotor activity and body weight, with OFBP exhibiting outstanding restoration in the Dex-induced muscle atrophy zebrafish in vivo model. In conclusion, OFBEs, particularly OFBP, produce hydrolysates with enhanced physiological usability and muscle regenerative potential. Further research on its industrial application and mechanistic insights is needed to realize its potential as a high-quality protein food ingredient derived from OF processing by-products.

Keywords: dexamethasone-induced ROS production; dexamethasone-induced muscle atrophy; enzymatic hydrolysis; fish by-product; myogenesis; zebrafish.

Figure 1

The enzymatic hydrolysis procedure using OFB. During the hydrolysis procedure, steps ①–② involve preparing the raw OFB ingredient for extraction. Step ③ involves calibrating the dry mass in OFB. Step ④ involves setting the enzymatic hydrolysis conditions and inactivating the enzyme. Step ⑤ involves filtering the hydrolysates and confirming the extract performance. Steps ⑥–⑧ involve post-extraction manipulations for storage.

Figure 2

The extract performances of OFBEs. The list of extract yield percentage of OFBEs (A) and a graphical figure of hydrolysate yield percentage (B). The graphical figure of hydrolysis efficiency percentage (C). The comparative images of OFBEs’ turbidity (D). Data are expressed as mean ± SD. **** p < 0.0001 vs. OFB_Control (n = 3).

Figure 3

The molecular weight distribution of OFBEs. The SDS-PAGE results indicate the molecular weight distribution of OFBEs stained by Coomassie blue staining (A). The percentage of molecular weight distribution of OFBEs was determined by way of the major mass list analysis using HPLC-MS (B).

Figure 4

The comparative profiling associating myoblast functional enhancement by OFBEs. The OFBEs treatment induced an extent of cytotoxicity rate (A), glucose uptake (B), and the regulation of cell proliferation (C). The OFBEs treatment-mediated myogenesis-related factors (MRFs) were investigated via immunoblotting (D) and the quantified value of an immunoblot was represented graphically (E). All the data are shown as mean ± SD (n = 3). Control (Con) is a vehicle-treated group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. OFB_Control (n = 3).

Figure 5

The recovery of myotube formation, which is inhaled by Dex treatment by OFBEs treatment. The cellular Myosin heavy chain (MyH) was targeted for immunofluorescence staining to determine the myotube formation in the Dex-induced C2C12 myotube atrophy model (A). The graphically quantified value of MyH immunofluorescent images applied OFBEs (B). Entire data are presented as mean ± SD (n = 3). (1) DW, (2) Protamex, (3) Flavourzyme, (4) Protana prime, (5) Neutrase, (6) Alcalase, (7) Prozyme EXP 5000, (8) Prozyme 2000P, (9) Prozyme 1000L, (10) Foodpro Alkaline, (11) Foodpro PNL, and (12) Sumizyme. * p < 0.05, ** p < 0.01, and **** p < 0.0001 vs. Blank. ^#### p < 0.0001 vs. Control. (Sacle bar = 1000 μm).

Figure 6

The restoration of imbalance on swimming in Dex-induced muscle atrophy zebrafish model by OFBEs supplementation. The experimental schedule of in vivo model (A); 1% of OFBEs contained zebrafish diet was supplemented to zebrafish prior to 0.01% Dex-induced muscle atrophy stimulation. The locomotion tracking of OFBEs supplemented the Dex-induced zebrafish muscle atrophy model (B). The graphically quantified values of velocity (C), acceleration (D), and distance moved (E). The change in body weight of the zebrafish experimental group supplemented with OFBEs was measured at the initial and final time points (F). All data were indicated to mean ± SD (n = 6). * p < 0.05 and ** p < 0.01 vs. Blank. ^# p < 0.05 and ^### p < 0.001 vs. Control. Blue and red arrows are indicating Dex and OFBP group respectively. ns (Not significant).

Figure 7

The potential OFBP effect on muscular regeneration in C2C12 myoblast in vitro. The MyH immunofluorescent staining evaluating myotube formation efficiency by way of a series of OFBP concentrations (A) and the myotube diameter changes represented by quantitative graphical values of diameter changes in 40 myofibers (B), the percentage of myotube coverage change (C), and the promotion of MRFs protein expression change through OFBP supplementation in a dose-dependent manner (D). The MyH immunofluorescent staining estimating a Dex-induced myotube atrophy restoration by OFBP in dose-dependent treatment in vitro (E) and the myotube diameter changes represented by quantitative graphical values of diameter changes in 40 myofibers (F), the percentage of myotube coverage change (G), and the OFBP-mediated recoveries of MRFs protein expressions, which were inhibited by Dex stimulation (H). OFBP reduced the ROS production in Dex-induced C2C12 myotube (I) and in AAPH alkyl radical stimulated C2C12 myotube (J). All data are expressed as mean ± SD (n = 6). * p < 0.05 and ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. Blank. ^#### p < 0.0001 vs. Control. (Sacle bar = 1000 μm).