The Preparation Of Lactoalbumin Hydrolysate And Its Application In BHK-21 Cell Culture

Protein hydrolysates are the products abtained after the hydrolysis of proteins as achieved by acid,alkali,and enzymes methods,mainly composed of peptides,Its also contain amino acids,carbohydrates,lipids,minerals and vitamins.According to the raw materials the products have various hydrolysates derived from animal,plant and microbial sources.Lactoalbumin hydrolysates was one of the vital protein hydrolysates,It was hydrolysised by protease or Peptidase,containing amino acids and peptides,can provide a variety of nutrients for cell growth,adherence factor and growth factor analogs,widely used in pharmaceuticals,vaccines,food and other industries.To investigate the manufacturing process in the present study,casein was used as the substrate,free amino acids content,hydrolysis degree,and molecular weight were used as the evaluation index,the trypsin,pepsin,papain,neutrase,alcalase and pancreatin six different enzymes were used to hydrolysis casein respectivly then screened the optimal enzymes under ultra-high pressure in this study.The optimal process of casein hydrolysis was optimized by single factor analysis and orthogonal experiments.The results showed that pancreatin was optimal enzyme to hydrolysis casein,the amino acids contents and hydrolysis degree were get 47.187%and 41.06%respectively and the molecular weight less than lOkD.The optimal process was as follows:15%substrate concentration,E/S = 1:5,150 MPa hydrolysis pressure,45? and pH 7.5 hydrolysised for 4 h.Under these conditions,52.18%free amino acids content of hydrolysate were gotten.Additionally,to investigate the physicochemical properties of casein hydrolysates(CH)by SDS polyacrylamide gel electrophoresis(SDS-PAGE),scanning electron microscopy(SEM),infrared spectroscopy(FTIR),X-ray diffraction(XRD),ion exchange chromatography(IEC)and size exclusion chromatography(SEC),The results showed:casein hydrolysates(CH)physicochemical properties is almost similar to the Hyclone lactoalbumin hydrolysate(HLH).They belong to matrix morphology,molecular weight less than 10KD.The secondary structure were mainly existed in a-helix and ?-turns,Contain protein(amid I),carboxyl,cellulose,nucleic acids,lipids and polysaccharide,consists of 19 kinds of same amino acid,and total content showed significant difference(P<0.05),24.04±0.768%and 46.202±2.263%,respectively.The results of Cellulose-60 results screen showed both CH and HLH contain F1,F2,F3,F4 and F5 five fractions respectively,but the contents was different.Finally,to investigate the effect of casein hydrolysate(CH)on growth and metabolism of BHK-21 adherent and suspension cells.At the same time,Hyclone lactoalbumin hydrolysate(HLH),Lactoalbumin hydrolysate-35(LH35)and Lactoalbuamin hydrolysate-80(LH-80)were used as the control group.he results showed:CH,LH35,LH80 and HLH showed supporting capability for BHK-21 adherent and suspension cells growth.CH showed better supporting capability than HLH for BHK-21 adherent cells growth.BHK-21 suspension cells supplemented with CH showed viable cell density almost equal to HLH,but lower than LH35 and LH80.BHK-21 suspension cells supplemented with Lactoalbuamin hydrolysates had different effection on the profiles of biochemical,enzymes and amino acids metabolites.In conclusion,the preparation process of the lactoalbumin hydrolysate were established,the products could suppor capability for BHK-21 cells growth as serum free medium additives.The study is benifit for the further exploitation and utilization of the casein and the application of protein hydrolysates in animal cell culture.