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Collagen is a major component of skin, bones, tendons, cartilage and other connective tissues of the body. One of the potential applications of collagen hydrolysates is in the field of skin health. Traditionally, the source for collagen, gelatin and their hydrolysates has been the connective tissue from farm animals including pigs and cows; however, there is increasing interest in utilizing the collagen obtained from fishes as well as poultry as alternative sources due to fear of bovine spongiform encephalopathy and religious reasons. However, there is limited research in preparing collagen from chicken and there is no published report of using collagen from spent hens. Given this background, the purpose of this study was to determine the factors (both enzymatic preparation methods and profiles of constituent peptides) underlying the ability of different collagen hydrolysates to exert beneficial effects on inflammatory molecule expression, oxidative stress, type I collagen synthesis and cell proliferation, using cultured human dermal fibroblasts (HDF) as a model system.
Chicken meat was excised manually from spent hens and trimmed of external fat for collagen isolation. Protein hydrolysates were prepared by digestion with 2% w/w enzyme for 2 h. Protein content of the samples was estimated by a LECO TruSpec machine according to the manufacturer’s instructions using 5.55 as the protein factor for gelatin, the denatured form of collagen. Each preparation of collagen hydrolysate is composed of a number of different peptides. To determine the relative hydrophilic and hydrophobic nature of the constituent peptides, 5 μL of each hydrolysate (10 mg/mL) was injected onto BEH C18 (1.7 μm 2.1 × 100 mm) column attached to Waters Acquity UPLC system. Column temperature was 35 °C and the peptides were eluted by a gradient of 100% water containing 0.1% TFA to 30% acetonitrile with 0.1% TFA over 45 min. The peptides were detected by PDA detector at 254 nm. A later time point of elution indicated a greater hydrophobic nature. Commercially available adult HDFs were obtained for cell culture. All experiments were performed on 70-80% confluent cells grown in tissue culture grade plastic 48-well plates. All collagen hydrolysates were de-salted, freeze-dried and reconstituted in sterile distilled water prior to use in cell culture studies. To measure Type I procollagen levels, confluent monolayers of HDF were incubated for 24 h with quiescing medium containing 2.5 mg/mL of different collagen hydrolysate samples. At the end of this incubation period, cell free supernatants were collected and analyzed for their type I procollagen content by a commercially available ELISA kit.
In general, no major difference was noted in moisture level, dry matter or protein content among the different hydrolysate preparations. An analysis of peptide molecular weights by gel filtration chromatography suggested a diversity of peptide size among the different preparations. In general, use of a single enzyme produced hydrolysates with a greater frequency of larger peptides. Digestion with Protease M in particular yielded more small peptides compared to others. Our results showed that use of a second digestion with another enzyme significantly reduced peptide size and yielded hydrolysates containing shorter peptides (
In conclusion, these findings demonstrate that differences in enzymatic treatments generate collagen hydrolysates with distinct chemical and biological profiles. Use of two enzymes in sequential digestion improves the production of bioactive peptides that may be better suited to wound healing and maintenance of skin health.