Research In Polypeptide Product Quality And Melanin Extracting And Content Determination From Black-bone Silky Fowl

Black-bone silky fowl (Gallus gellus demesticus brissen) is a kind of bantambreed which is native to South China, Taihe, Jiangxi Province. The originalblack-bone silky fowl (BSF) is well known in the orient and has been famous for itsmedicinal and health-promoting values for more than a thousand of years. As atraditional Chinese medicine, it has been used both as a source of edible chicken andas a source of traditional Chinese medicine for it can protect from emaciation andfeebleness, treat diabetes, reinforce body immunity and promote cell metabolization.It has benn used especially for curing women's disease, like weakness after givingbirth and so on. As an important bioactive component of BSF, melanin has beenstudied profoundly in many previous studies which revealed the pharmacologicaleffects of melanins include anti-mutagenic, enhance immunity, Anti-aging,elimination free radicals and anti-oxidation acitvities. By the way, certain degrees ofprotein hydrolysis can produce some bioactive peptides, which is an inevitable trendin developing the chicken processin industry.The effects of different extraction methods on melanin of BSF withcorresponding anaylsis methods, and major composition in bioactive polypeptides ofBSF had been studied in this paper for providing basic theoretic foundation for furtherstudies and the development of new products of BSF. The main results in the paperare mentioned as follows:1. The protein content and free amino acid content of three silkies biologicalpeptide were determined by Kjeldahl method, the amino acid analyzer automaticallyand 2,4-nitro-fluorophenyl pre-column derivatization HPLC method respectively. Thetotal protein content of three Silkies biological peptides were 94.30±0.36%,96.15±0.68% and 95.08±1.18% respectively. Total free amino acids were36.17±1.44%, 7.66±0.61% and 24.29±0.97% respectively. The polypeptide contentwere 53.80±0.87%, 75.59±0.44% and 46.90±1.13% respectively。2. The molecular weight distributions of three silkies biological polypeptidewere determination by gel chromatography. Gel chromatographic conditions were:column: Saphadex peptide column 10/300GL (300mm×7.8mm); mobile phase: PBSsolution (0.01 mol/L NaH₂PO₄; 0.01mol/L Na₂HPO₄; 0.01mol/L NaCl pH=6.86);temperature was set at 35℃; flow rate: 0.5 mL/min; injection sample was 20μl; detection wavelength was set at 220 nm. GPC software was used for data processing,the linear regression curve equation for the molecular weight was LogMW=-0.184 V+5.70 (V: elution volume, R=0.995). The results showed the method was appropriatefor determining the molecular weight of polypeptide.3. By scanning electron microscopy, ICP-AES, infrared scanning, anti-oxidationsystem the melanins extracted following hydrochloric acid extraction and enzymaticextraction were observed and compared. Enzymatic Hydrolysis Extraction method canbe considered as a more satisfactory way to extract melanin from Blackbone-silkyfowl for it can better maintain and retain its original structure. However the proteinresidue content was higher than that of acid extraction method. The elementexperiment showed that more minerals and trace elements were lost in acid treatment.Melanin extracted by enzymatic extraction showed a stronger antioxidant capacity.4. PTCA, SBF melanin oxidation product, was identified by HPLC-MS andquantitatived by HPLC method. The chromatographic conditions were: the KromasilC18 column (250 mm×4.6 mm, 5μm); detection wavelength was set at 270 nm;injection volume of 20μl; mobile phase A: 1% formic acid, mobile phase B: methanol;gradient elution flow rate of 0.5 mL/min. The melanin content in product extractedfollowing enzyme extraction was 63.19%.