Continuous Preparation Of Casein Phosphopeptides Using Enzymatic Membrane Reactor And Anion-exchange Chromatography

The application of enzymatic membrane reactor (EMR) for preparation of bioactive peptides has steadily become an attractive method in the food industry. It is a specific mode for continuous reaction, simultaneous separation of products from the hydrolysates and separation of enzymes from end products with the help of a selective membrane. It brings inhanced production efficiency and economic benefits with continuous production. Therefore, the study of continuous isolation and purification of casein phosphopeptides (CPP) using combination of EMR and anion-exchange chromatography (AXC) was investigated.First, the continuous separation process of CPP using EMR and AXC was established. Alcalase(?) FG 2.4 L was used to continuously hydrolyse casein for 3 h at pH 8.5 and 50℃in a tangential flow filter (TFF) enzymatic membrane reactor to prepare casein bioactive peptides. The separation and purification of CPP from hydrolysates was achieved using AXC. The type of macroporous resin and the separation technique of AXC were investigated by static adsorption and desorption method. The parameters of dynamic adsorption and desorption were optimized by single factor design. The results showed that macroporous weakly basic anion exchanger D3O3 was the most suitable resin. With the optimum loading permeate flow rate 1 Bv/h, loading quantities 25 mg/mL, eluting temperature 45℃, eluting hydrochloric acid concentration 0.15 M, the recovery rate of P from the permeate was 93.11% and the molar ratio of nitrogen to phosphate was 7.21.The influence of proteases including Alcalase, Trypsin, Protease N, Papain on isolation of CPP was investigated. All the proteases were used to hydrolyse casein in EMR fitted with 10 kDa membrane with their optimum conditions of hydrolysis process. The degree of hydrolysis (DH), permeate flux (J), enzyme activity, yield and purity of CPP were detennined to select a suitable protease. It was shown that the DH and the yield of CPP were higher with Alcalase than with other proteases. Alcalase also reduced the Aieakage and membrane fouling, relatively.Enzyme leakage and membrane fouling are strong limitations of the application and development of this process. Therefore, the effect of molecular weight cut-off (MWCO) of the membrane and the operating conditions of ultrafiltration on both enzyme leakage and membrane fouling were studied. Alcalase was used to continuously hydrolyse an initial concentration of 6%(w/w) casein at pH 8.5 and 50℃in EMR fitted with 3 kDa,5 kDa and 10 kDa membrane, respectively. The DH, J, Sapparent, enzyme activity, yield and purity of CPP were determined to select a suitable membrane. Results show that with a high NMWCO membrane, high yield and purity of CPP was obtained and the value of Aresidual reached above 90%. However, serious membrane fouling, a lower Sapparent and a high increase in Aleakage were observed. The effect of operating conditions such as pressure and recirculation flow rate on isolation of casein bioactive peptides was studied by single factor experimental and the complex interrelationships of all the factors were correctly revealed. It was shown that with increasing operating pressure, a high increase in Ji and Sapparent as well as a serious enzyme leakage and membrane fouling were obtained. However, with a suitable recirculation flow rate of 140 mL/min, enzyme leakage and membrane fouling could be reduced together with a relatively high Sapparent.Preparation of CPP with high efficiency, continuous operation and large scale was achieved in this study. With the optimum operation conditions to continuously hydrolyse casein for 8 h in EMR, the results were as following:the Sapparent 70.93%, Aleakage 34.64%, the recovery rate of CPP from casein 20.03% and the molar ratio of nitrogen to phosphate 7.18.