BIOACTIVE PROPERTIES OF COLLAGEN PEPTIDES DERIVED FROM FISH SKIN USING ENZYMES OF MARINE ORIGIN
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Date
2018
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Fisheries College and Research Institute, Thoothukudi, Tamil Nadu Dr. J. Jayalalithaa Fisheries University
Abstract
In this study, unicorn leatherjacket (Aluterus monoceros) fish skin was used for the preparation of collagen peptides. Prior to this, crude collagenase was extracted from marine fish fins and the molecular mass was estimated as 29kDa in SDS-PAGE, and the average yield was 2.52±0.10%. On purification of crude collagenase enzyme in Sephadex G-100 column, two clear fractions were obtained and the average yields of fraction 1 (F1) was 0.52±0.02% and fraction 2 (F2) was 0.25±0.05%. The crude collagenase was used for the hydrolysis of unicorn leatherjacket skin at three different temperatures viz., 5 oC, 25 oC and 50 oC to obtain collagen hydrolysates viz., CH-5, CH-25 and CH-50, respectively.
Upon hydrolysis, the degree of hydrolysis (DH) increased linearly with the increase in reaction time reaching a maximum at 300 min. The maximum DH obtained was 7.6±0.01% for CH-50, 7.2±0.01% for CH-25 and 6.6±0.02% for CH-5. SDS-PAGE pattern of the collagen hydrolysates showed smear bands between 11kDa and 24kDa in CH-5, CH-25 and CH-50. From the collagen hydrolysates, collagen peptides of three different molecular masses viz., <3kDa, <10kDa and <30kDa were obtained by ultra-filtration using tangential flow filtration (TFF) system. The average yield of lyophilized collagen peptides obtained were 3.92±0.34%, 3.71±0.42% and 3.61±0.32% for CP-5, CP-25 and CP-50, respectively. FT-IR spectra of CP-5 showed major amide A peak at 3427.05 cm-1, amide II peak at 1574.17 cm-1 and amide III peak at 1079.52 cm-1, while these peaks were at 3425.53 cm-1, 1574.32 cm-1, and 1016.85 cm-1, respectively in CP-25. In CP-50, they were found at 3418.42 cm-1, 1558.96 cm-1, 1021.38 cm-1, respectively. Amide B and I peaks were not detected in collagen peptides. On the other hand, in fish skin collagen, the major peaks of amide A was at 3427.96 cm-1, amide B peak at 2923.87 cm-1, amide I peak at 1664.88 cm-1, amide II peak at 1550.95 cm-1, and amide III peak at 1239.72 cm-1.
Collagen peptides were examined for their in vitro antioxidative properties such as DPPH free radical scavenging activity, metal chelating ability, ferric reducing power, hydroxy radical scavenging activity, as well as anti-cancer, anti-diabetic, anti-inflammatory, and wound healing properties. The results indicated that CP-5 of <3kDa MWCO showed maximum DPPH and hydroxy radical scavenging activities of 70.26±0.02% (14 mg protein/mL) and 67.84±0.01% (14 mg protein/mL), respectively; while metal (Fe2+) chelating ability was 33.17±0.01% (12 mg protein/mL) and ferric reducing power activity was 0.3315 absorbance units (10 mg protein/mL). In MTT anti-cancer assay, CP-5 inhibited the viability of COLO320 cancer cells up to 49.78±0.01% at 1 mg/mL concentration, while the inhibition was only 29.92±0.01% by CP-50. Anti-diabetic activity as assessed by α-amylase inhibition was 80.45±0.02% by CP-5 at 1 mg/mL concentration, as against 75.81±0.01% and 71.17±0.02% by CP-25 and CP-50, respectively. Collagen peptides, in general, exhibited very low anti-inflammatory property, which ranged between 3.27±0.01% to 4.01±0.02%. In vitro wound healing property tested by scratch assay showed good migration of 3T3-L1 cells, when treated with CP-5, CP-25 and CP-50 at 0.2 mg/mL concentration. In vivo wound healing properties evaluated on BALB/c, an albino male mice by making subcutaneous circular incision wound showed that the percentage wound reduction was much faster in the collagen peptide treated groups (48.48±0.02%) than the standard (45.45±0.03%) and control (31.31±0.01%) groups.
In vitro gastro intestinal digestion study was conducted to examine the bioavailability of collagen peptides. The pepsin and pancreatin digests of <3kDa MWCO peptides contained more protein ie. 22.78% and 26.91% for CP-5; 22.01% and 26.61% for CP-25; 21.89% and 25.58% for CP-50, respectively than those of <10kDa and <30kDa. The rate of peptide absorption of <3kDa CP-5 was 32.56%, followed by CP-25 (32.43%) and CP-50 (32.35%) after gastric and pancreatin digestion. The collagen peptides after in vitro digestion were assessed for their in vitro antioxidant properties. The CP-5 of <3kDa MWCO had better antioxidative activities than CP-25 and CP-50. The DPPH and hydroxy radical scavenging activities of pepsin-digest of CP-5 were 36.72% and 54.68%, respectively; while metal (Fe2+) chelating ability was 28.14% and ferric reducing power activity was 0.2737 absorbance units. In the pepsin-pancreatin digest also,
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CP-5 showed the maximum DPPH and hydroxy radical scavenging activity of 48.32% and 66.29% respectively, while metal (Fe2+) chelating ability and ferric reducing power activity were 32.92% and 0.3100 absorbance units, respectively. The dialyzed content of CP-5, however had much lower DPPH radical scavenging, hydroxy radical scavenging, metal (Fe2+) chelating, and ferric reducing power activities of 30.74%, 47.74%, 22.72%, and 0.1672 absorbance units, respectively. Collagen peptides were also examined for any cytotoxic effect by mice experiment. The results showed that, there was no cytotoxic effects observed in the small intestinal epithelial cells when CP-5 of <3kDa MWCO were given as oral supplements for BALB/c, an albino mice for 15 days. Therefore, the skin collagen peptides derived from unicorn leatherjacket had good antioxidant, anti-cancer, anti-diabetic and wound healing properties when hydrolysed at 5 oC, rather than at 25 oC or 50 oC. In vitro digestion studies have also proven that the digestion of the collagen peptides by endogenous proteolytic enzymes yielded further smaller molecular weight peptides, which had enhanced their bioaccessibility to express more biofunctional properties.
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