The Chemical Production of Hyaluronan from Chicken Combs

3 The Chemical Production of Hyaluronan from Chicken Combs 

Three main methods have been used to chemically produce hyaluronan from chicken 

The first method is a relatively simple and short process [5] that involves multiple water extractions of grounded rooster combs, precipitation from combined extracts of HA with acetic acid, treatment with base, enzymatic hydrolysis of the protein, ultrafiltration, ethanol precipitation and final dissolution of the precipitate hyaluronan in water. 

the rooster combs are kept under cold water that must be periodically changed to maintain temperature. Then the combs are removed from the water, squeezed to drain and carefully grounded. Water is added to the grounded tissue at a comb/water ratio of 1 2.5 and brought to a boil, after which the extract is separated. The extraction process is repeated six times. It is interesting that the fifth and sixth extracts are free of lipids, the protein content is quite minimal and the HA content is comparable to the first four extracts. The fifth and sixth extracts are then combined and the polysaccharide is precipitated with acetic acid at pH 5 for 18.5 h. The formed precipitate is separated, dissolved in distilled water at a ratio of 1 3, and the pH is adjusted to 8 with sodium hydroxide solution. 

Then a solution of papain (200ml of 0.1% solution of papain per 1kg of material) is added. Enzymatic hydrolysis of proteins is carried out for 20-40h. Every 6-8 h the pH must be adjusted to 6-7 (to optimize enzyme activity). The HA mixture is purified by ultrafiltration and precipitation with 96% ethanol. The final product, sodium hyaluronate, is dried by lyophilization and the precipitate is then dissolved in a water-alcohol mixture containing 40% ethanol. The final purification of hyaluronan is performed by membrane filtration of a solution containing 30-50% ethanol. 

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