Studies on the Extraction, Isolation, Identification and Functional Properties of Bioactive Peptides from Lantern Fish and Tilapia’s Fish Scale

• Main Authors: Huey-Jine Chai, 蔡慧君
• Other Authors: Chyuan-Yuan Shiau
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/49350687490884802940

博士 === 國立臺灣海洋大學 === 食品科學系 === 100 === The purposes of this thesis were to prepare testing protein hydrolysates from lantern fish (Benthosema (B.) pterotum) and tilapia (Oreochromis sp.) scales by using proteases-hydrolyzed methods. First the bioactive peptides were purified and identified from protein hydrolysate of B. pterotum (BPH), and then to evaluate the food safety, stability and functional properties including antioxidant, ACE inhibitory effect, anti-aging and protected effect of neuroblastoma cells (SHSY5Y) for BPH. Regarding the effects of molecular mass and conformations of fish scale collagen peptide (FSCP) on facial skin qualities and transdermal penetration efficiency were also studied. This research provided potential methods to utilize low-valued marine wastes including lantern fish and fish scales to the high-added-valued protein sources. B. pterotum and Diaphus splendidus are the large genera stock of lantern fishes (Myctophidae) with 6 genera, and 17 species around Taiwan Ocean. As compared to D. splendidus, B. pterotum contained high levels of proteins, free amino acids and functional components such as phospholipids, unsaturated fatty acids and minerals. B. pterotum is also free of wax esters and hazard trace elements such as Cd, Hg, Pb, and As. The protein hydrolysate of B. pterotum (BPH) using Protease N possessed 77.5% of peptides with Mw less than 1500 Da in the hydrolysate. BPH had good antioxidative activities. The in vitro effective peptide concentrations for eliminating 50% free radicals (EC50) on DPPH scavenging, Fe2+-chelating, reducing power and superoxide oxide dismutase (SOD)-like activity were 1.06 ± 0.07, 0.59 ± 0.01, 2.31 ± 0.38 and 10.6 ± 0.70 mg peptide/mL, respectively. Subsequently the amino acid sequences of the purified antioxidant peptides from BPH have been identified as Phe-Tyr-Tyr (Mw = 529 Da) and Asp-Trp (Mw = 337 Da) by consecutive chromategraphic methods and automated Edman degradation with N-terminal sequencer. For food safety, BPH was evaluated by both Ames test (using Salmonella Typhimurium serious stain) and acute toxicity in a rat model. No mutagenicity arose on the levels of BPH ranging from 625-5000 μg/plate with/without liver microsomal activation (S9). No treatment- related changes were found in rats. For the no-observed-adverse-effect- level (NOAEL), BPH was estimated to be no less than 15 g/kg in both male and female rats, thus BPH was concluded to be safe. Furthermore, BPH was discovered to possess antioxidative activities and ACE (angiotensin I converting enzyme) inhibitory effect in simulated gastrointestinal (GI) digestion assays. As an antioxidant, the EC50 on DPPH scavenging, reducing power and SOD-like activity were determined no significantly changes during digestion processing. However, the activity of Fe2+-chelating was 2.42 ± 0.41 mg peptide/mL significantly decreased to be 0.53 ± 0.01 mg peptide/mL after pepsin-pancreatin digestion. In addition, the in vitro effective concentration for inhibiting 50% ACE ability (IC50) was 0.31 mg peptide/mL significantly decreased to be 0.14 mg peptide/mL. Moreover BPH with bioactive potential not only resisted physiological digestion in gastrointestinal (GI) tract by determinate in vitro pepsin- pancreatin simulated GI digestion, but also provided a protective barrier against H2O2-induced oxidative damage, and dose-dependently increased neuroblastoma cell (SHSY5Y) proliferation at the concentration of 0.01-5 mg/mL by MTT assay. Meanwhile, the BPH with 1.2-2.5 mg/mL also significantly increased the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxide (GPx), respectively, and consequently scavenge reactive oxygen species (ROS), thus protected against the H2O2 induced the DNA fragments as both the neuroblastoma cell (SHSY5Y) survival rate and synaps outgrowth significantly increased in a dose-dependent manner as opposed to the H2O2 injury cells. In a H2O2- and beta-amyloid (Aβ)-toxicity test, the proliferation and antioxidative enzyme activities of neuroblastoma cell (SHSY5Y) were increased at the concentration of 1.2-2.5 mg/mL when compared to those added no BPH, revealing BPH is able to prevent neuronal cell against from H2O2- and Aβ-induced oxidative damage. BPH has been proved to ameliorate D-galactose-induced memory and learn deficit by three individual animal model tests including passive avoidance task, active avoidance task and water-maze task. Meanwhile, BPH could prevent against D-galactose induced raise of thiobarbiturivc acid reactive substances (TBARS) and activity of endothelial nitric oxide synthase (eNOS) as well as increased the activities of superoxide dismutase (SOD) and glucose-6-phosphate dehydrogenase (G6PDH) and brain- derived neurotrophy factor (BDNF) contents. Based on above results demonstrated BPH is a promising biomaterial for antioxidative healthy products, and expected to develop as physiologically functional foods for treating oxidative damage-derived neurodegenerative disorders. Fish scale collagen peptides (FSCP) were isolated from tilapia (Oreochromis sp.) scales by enzymatic hydrolysis. The FSCP-based skin essences were first formulated into 5%, 7%, and 10% of total FSCP as the common basal ingredients. Sixty-two voluntary women (within 23 to 60 years of age) were subjected to the FSCP-based skin essence treatment on facial skin twice a day for 30 days. FSCP-based skin essences did improve facial skin qualities (moisture and elasticity) in a time- and dose-dependent manner in these tested subjects. FSCP (0.4-200 μg/mL) can dose dependently stimulate the cell proliferation in both fibroblast cells, Detroit 551 cells (human embryonic skin fibroblast line) and STO cells (mouse embryonic fibroblast). Likewise, the procollagen type I synthesis was found mass produced in a concentration-dependent manner in the STO cells with FSCP treatments. The procollagen synthesis in the concentration of 200 μg/mL FSCP was found most prominent and peaked as high as 250%. The transdermal penetration capabilities of the fractionationed FSCP were evaluated using the Franz-type diffusion cell model. Results showed that the highest cumulative penetration amounts for heavier FSCP, 3,500 and 4,500 Da, at 24 hours were 1,825 and 1,986 μg/cm2, respectively. In contrast, the FSCP mixture (total FSCP hydrolates without ultrafiltration membrane refining) showed the lowest transdermal penetration capability. The penetration depths for each FSCP group were also measured by confocal spectral microscopy. The distances for 1,300, 2,000, 3,500, 4,500 Da and the mixture were 21.21, 21.44, 28.72, 27.73, and 14.58 μm, respectively. The heavier FSCP exhibited a coil-like structure in epidermis as opposed to lighter groups, which showed less folded or in a linear form. As a result, the transdermal penetration efficiency of given FSCPs are positively correlated to FSCPs mass and/or conformations thereof.