Production, purification and characterisation of fish scale collagen and microbial collagenase and their application in the formation of bioactive collagen peptides

Abstract

This study describes a biorefinery approach for the valorisation of fish waste to value-added products such as collagen, collagenase and collagen peptides. Collagen from fish waste has been recently explored as a substitute source for mammalian collagen, with fish scales and skin being utilised the most. In the present study, acid soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted from scales of cape salmon (Atractoscion aequidens) and northern red snapper (Lutjanus campechanus). Northern red snapper scales resulted in a collagen yield of 2.17 ± 0.23% and 2.92 ± 0.31% for acid and pepsin treatments, respectively, whereas cape salmon scales yielded 1.60 ± 0.12% ASC and 4.65 ± 0.39% PSC on weight-byweight basis. Various physicochemical techniques were used to characterise the extracted collagen, including UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), Differential Scanning Calorimetry (DSC), Zeta potential, and Scanning Electron Microscopy (SEM). Maximum absorbance peaks at 232-233 nm for ASC and PSC of both northern red snapper and cape salmon were observed on UV–vis spectroscopy profiles, which are mainly associated with the peptide bond absorptions of the C=O, –COOH, and CONH2 groups in polypeptide chains of collagen. FTIR spectra of cape salmon and northern red snapper collagens displayed the type I collagen characteristic peaks, Amide A and B, as well as Amide I, II, and III, corresponding to the main functional groups in the collagen protein. The presence of distinct α1 and α2 chains and a β-chain observed on SDS-PAGE confirmed the type I nature of the extracted collagens. The amino acid analysis showed the dominance of three amino acids: glycine, proline, and alanine which are characteristic features of type I collagen. The differential scanning calorimetry displayed the high denaturation temperatures of collagen from cape salmon scales as 112.27°C (ASC) and 113.41°C (PSC), and for northern red snapper it was 110.09°C (ASC) and 112.84°C (PSC). Zeta potential analysis demonstrated isoelectric point (pI) for collagens at pH 5.6 (ASC) and 4.8 (PSC) for cape salmon and pH 6.7 (ASC) and 6.4 (PSC) for northern red snapper. The extracted collagen protein also showed the ability to form fibrils in the presence of low concentrations of NaCl, which is a promising characteristic for its biomedical application. Subsequently, protease producers were isolated from environmental samples on casein agar plates, which resulted in 56 bacterial cultures. Among them, seven isolates displayed potent collagenolytic properties during further screening on gelatin agar plates. The best collagenolytic protease-producing isolate was identified as Bacillus sp. strain SC2 by 16S rRNA gene sequencing and was subjected to enzyme production in media containing fish scales as the main carbon source. The initial enzyme production by Bacillus sp. strain SC2 was 13.3 ± 0.34 U/mL, which was improved to 21.89 ± 0.90 U/mL (~1.65 fold) by supplementing the fermentation media with starch and peptone as additional carbon and nitrogen sources, respectively. Subsequently, the collagenolytic protease was purified to homogeneity using ammonium sulphate precipitation and diethylaminoethyl anion exchange (DEAE)-cellulose chromatography. The purified collagenase (~53.62 kDa) exhibited optimal activity at pH 8.0 and 40°C and showed stability over a pH range of 7.0 to 9.0 and temperatures ranging from 4 to 50°C. However, the collagenolytic protease activity was substantially inhibited in the presence of ethylenediaminetetraacetic acid (EDTA), proposing that this collagenase belongs to the metalloprotease family. In contrast, the enzyme activity of collagenase was enhanced in the presence of Ca2+, Zn2+, Mg2+, and Ba2+ ions, while it was reduced in the presence of Hg2+, Ag, Pb, and Fe3+ ions, SDS and Tween 80 surfactants, and phenylmethylsulfonyl fluoride (PMSF). The Km and Vmax of the purified collagenase were shown to be 2.24 ± 0.15 mg/mL and 1.28 ± 0.08 × 10-3 mg/mL/min, respectively. Subsequently, the purified collagenase was used to produce collagen peptides from cape salmon and red snapper collagen. The collagen peptides thus formed exhibited bioactivities such as antioxidant, antihypertensive and antidiabetic potential.