Extraction And Application Of Eggshell Membrane Polysaccharide

The eggshell and eggshell membrane was separated by hydrochloric acid immersion method assisted by ultrasonic. The eggshell membrane polysaccharide was extracted from separated eggshell membrane by double enzyme method, alkali-salt method and ultrasonic assisted alkali-salt method and purified by macroporous resin. The purity, molecular weight and antioxidant of purified eggshell membrane polysaccharide were studied while a formula of the eggshell membrane polysaccharide functional beverage was given. The major findings were presented as follows:The effect of dosage of hydrochloric acid, ultrasonic time, temperature and power on membrane yield was studied through single factor and orthogonal designed experiment. The results indicated the optimal separation conditions:dosage of hydrochloric acid was 20mL, ultrasonic time was 45min, ultrasonic temperature was 55℃, and ultrasonic power was 500W, which resulted in a membrane yield of 4.206%, eggshell residue rate was 0.23%.The effect of enzyme dosage, enzymatic hydrolysis time, pH and temperature on polysaccharide yield of both papain and trypsin extraction method was studied through single factor and orthogonal designed experiment. The results indicated the optimal reaction conditions of eggshell membrane polysaccharide extraction:papain enzyme dosage was 4200U/g, enzymatic hydrolysis time was 2.3h, pH was 8.0 and temperature was 50℃; trypsin enzyme dosage was 3800U/g, enzymatic hydrolysis time was 2.2h, pH was 8.7 and temperature was 47℃, which resulted in a polysaccharide yield of 77.50%.The effect of liquid-solid ratio, alkali-salt ratio, ultrasonic time, temperature and power on polysaccharide yield of ultrasonic assisted alkali-salt method was studied through single factor and orthogonal designed experiment. The results indicated the optimal reaction conditions of eggshell membrane polysaccharide extraction:liquid-solid ratio was 44:1 mL/g, alkali-salt ratio was 12:1 mL/mL, ultrasonic time was 55min. temperature was 60℃ and power was 500W, which resulted in a polysaccharide yield of 65.23%.The effect of liquid-solid ratio, alkali-salt ratio, extraction time, and temperature on polysaccharide yield of alkali-salt method was studied through single factor and response surface methodology. The results indicated the optimal reaction conditions of eggshell membrane polysaccharide extraction:extraction time was 8h, extraction temperature was 65℃, alkali-salt ratio was 6:1 mL/mL and liquid-solid ratio was 15:1 mL/g, which resulted in an extraction yield of 74.00%. Compared with the other methods, the double enzyme method was better.The eggshell membrane crude polysaccharide was purified by S-8 macroporous resin. The effect of sample pH, volume, flow rate and the pH of elution solution on the adsorption of eggshell membrane polysaccharide was studied. The results indicated the optimal purified conditions of eggshell membrane polysaccharide purification:sample pH was 7, volume was 10mL, flow rate was 1mL/min, which resulted in an adsorption rate of 89.86%; the pH of elution solution was 9 which resulted in a desorption rate of 30.31%.The maximum absorption wavelength of eggshell membrane crude polysaccharide was determined by means of ultraviolet spectroscopy with a result of 250 nm. The molecular weight of purified eggshell membrane polysaccharide and standard chondroitine sulfate was determined by means of GFC (Gel Filtration Chromatography) with a result of 41006 and 3137982; HPLC (High Performance Liquid Chromatography) were used to confirm the eggshell membrane polysaccharide is chondroitin sulfate and the purity of purified product was 89.51%.The clearance effect of O2-·,·OH and DPPH radical were determined and compared with that of vitamin C. The results exhibited that the O2- ·,· OH scavenging ability was strong, while the DPPH scavenging ability was weaker. However, both three scavenging ability of eggshell membrane polysaccharide were less than the same concentration of vitamin C.Based on the addition amount of eggshell membrane polysaccharide was 5mg/mL, the effect of the addition amount of xylitol, sodium citrate and concentrated juice on sensory test score of eggshell membrane polysaccharide functional beverage was studied through single factor and orthogonal designed experiment. The results indicated the optimal formula of the eggshell membrane polysaccharide functional beverage:the addition amount of xylitol was 6%, sodium citrate was 0.15% and concentrated juice was 10mL.