Preparation And Application Of Gelatin Peptides With Whitening Activity

Gelatin peptide is a general term that refers to functional peptides with 2-20 amino acids,which can be obtained by hydrolysis of gelatin or collagen.The different functional properties mainly depend on the amino acid composition,sequence and molecular weight size.The whitening characteristics of gelatin peptides was assessed via enzymolysis of gelatin and verification of the activity by B16 cells.The puried gelatin peptides for the assessment was obtained by a simple purification combined technique by comparing ultrafiltration and adsorption.The tyrosinase inhibition mechanism was investigated and then the gelatin peptides after purification were incorporated into a cream and their whitening characteristics in cosmetic usage assessed which provided a good scientific basis on the application of gelatin peptides in the whitening cosmetic.Specific contents were as follows:To achieve this objective,the basic composition,molecular weight distribution,and amino acid composition of different rich sources?fish-skin,bovini-skin,and porcine-skin gelatins?were measured.The results showed that there was no significant difference in the basic composition among the three kinds of gelatin.Though the molecular weight distribution of the three samples was averagely around 10⁵ g/mol,the fish-skin gelatin was relatively concentrated.The total amino acid content relative to the tyrosinase inhibition activity reached 42.56%?38.84%and 40.98%,respectively for fish-skin,bovini-skin,and porcine-skin gelatins.Five protease were chosen to hydrolyze three kinds of gelatin to prepare the gelatin peptides with whitening activity.The measurement of the tyrosinase inhibition activity as the main index and the degree of hydrolysis as the secondary index during enzymatic hydrolysis were used to determine the gelatin peptides with whitening activity on the raw and enzyme.The fish-skin gelatin was hydrolyzed with neutrase with enzyme dosage of 1200 U/g and enzymatic time of 180 min.The tyrosinase inhibition activity and the degree of hydrolysis were respectively,31.02%and 19.02%on the optimal preparation process.The safety and whitening activity of gelatin peptides were verified by B16 cells.The results of methyl thiazolyl tetrazolium?MTT?demonstrated that gelatin peptides had no cytotoxicity in0.01-100 logarithmic gradient range.The results of the effects of gelatin peptides on tyrosinase inhibition activity and melanin production showed that tyrosinase inhibition activity and melanin production inhibition rate were 41.2%and 27.52%respectively,when the gelatin peptides were 50 mg/m L.The ultrafiltration-cut-off and macroporous resin adsorption-desorption were used to separate gelatin peptides.The results of ultrafiltration separation showed that the peptides yield of F₁?>3 KDa??F₂?1-3 KDa?and F₃?<1 KDa?were 12.13%?56.35%and 21.62%respectively,while and the tyrosinase inhibition specific activity were 1.64?4.44 and 5.25,in that order.When gelatin peptides were adsorbed by DA201-C in the period of 5 h to reach adsorption equilibrium,adsorption capacity and adsorption rate were 74.95%and 54.3 mg/g,respectively.Tyrosinase inhibition specific activity reached 5.47 under 75%ethanol desorption.The correlation analysis between the hydrophobic and tyrosinase inhibition activity revealed that the hydrophobic fractions were better and produced higher tyrosinase inhibition activity.The final purification process was determined to be 3 KDa ultrafiltration membrane as the cut-off,while>3 KDa fractions were discarded.Fractions under 3 KDa components were adsorbed by DA201-C and desorbed by 75%ethanol.The results of the purified gelatin peptides showed that the peptides yield and tyrosinase inhibition specific activity of purified peptides were respectively 77.31%and 6.08.The tyrosinase inhibition mechanism of purified peptides was explored from tyrosinase monophenolase,tyrosinase diphenolase and free radical scavenging capacity.Results showed gelatin peptides had certain lag effects on the monophenolase.In the meantime,the steady state speed decreased and hysteresis time extended gradually with the increase of gelatin peptides.The hysteresis time was extended from 2 min to 7 min and the steady state speed decreased from 0.0087 to 0.0051 when the gelatin peptides reached 40 mg/m L.The inhibition type of diphenolase was reversible noncompetitive inhibition and the inhibition constant K_m was 4.88 mg/m L and K_i was 0.22 mg/m L.Tyrosinase fluorescent structure displayed obvious blue shift in characteristic adsorption peak of tyrosinase,which may be due that gelatin peptides combined with tyrosinase to a certain degree.The ability of removing radicals of gelatin peptides displayed the capacity of DPPH and OH free radicals of gelatin peptide were55%and 3.6%respectively,when the gelatin peptides reached 50 mg/m L.Gelatin peptides had no effects on p H,rheological properties and skin irritation due to cream usage when gelatin peptides were added into the cream.The effects of gelatin peptides on the cream whitening efficacy was also investigated by the human skin test including skin melanin content MI value and skin bright white L value.There were 15 volunteers who showed positive results among the 20 volunteers that participated in the study.The skin?L increased and?MI decreased according to the mean of positive results of the gelatin peptides cream compared the blank cream.The skin?MI decreased from 5.27 to-22.27 and the?L increased from-0.19 to 1.19 with gelatin peptides cream was applied from 5d to 30 days.