Salmon Bone Feasibility Study

Materials

Salmon bones from KCG Corporation Co., Ltd. (Bangkok, Thailand) were ground to a small size and dried at 60°C overnight. The bone was then filtered through a 150 micron sieve. Alcalase and Flavourzyme were purchased from Brentag (Mülheim, Germany). Neutrase was purchased from Novozymes (Bagsvaerd, Denmark). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), LPS from Escherichia coli, N-(1-Naphthyl) ethylenediamine (NED), penicillin-G, sodium nitroprusside (SNP), sulfanilamide and trifluoric acetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Determination of amino acid content

The amino acid content of the salmon bone was determined based on the standard AOAC 994.12 acid hydrolysis

The substrate was hydrolyzed separately using various concentrations (10, 25, and 50 mg/mL) of Alcalase, Flavourzyme, Neutrase and papain. The samples were activated with the individual enzyme conditions listed in Table 1. After the hydrolysis process, the SBP were immediately heated at 80°C for 20 min to inactivate the enzyme, followed by centrifugation at 5,000×g for 15 min and collection of the supernatants for further use.

DPPH radical scavenging assay

The antioxidant activity of SBP was measured with DPPH radicals according to the method of Chantaranothai et al. [8] Appropriate concentrations of SBP hydrolysate were mixed 1: 4 (v/v) with 200 M DPPH solution in anhydrous methanol for 30 min in the dark. The absorbance of the samples was recorded spectrophotometrically on a microplate reader (Multiskan Go, Thermo Fisher Scientific) at 517 nm. A control group containing DPPH solution without sample was also prepared. Ascorbic acid was used as a positive control.

ABTS radical scavenging