Enzymatic Preparation Of High Fischer Ratio Oligo-peptides From Casein

In the mixture of amino acids,the molar ratio of branched chain amino acids to aromatic amino acids is named F ratio.The High Fischer Ratio 0ligo-peptides are low molecular biologically active peptides which are formed by the hydrolysis of food protein.The High Fisher Ratio Oligo-peptides have special amino acid composition and peptide structural pattern, with functional properties of relieving hepatic encephalopathy symptom、adjuvant the cure of phenylketonuria、improving the protein nutritional status of the patients after operation and lying in bed for a long time、inhibitting cancer cell、lowering blood pressure and the concentration of serum cholesterol and so on.Now, a huge number of studies on the preparation of the high F oligopeptides have been done in the world, which have already formed a fairly developed prodution technology from the selection of materials to the purification procedure. In recent years, studies on the production of the high F oligo-peptide from the plant protein, such as the soy protein and corn protein, are very well done, but study on the animal protein, such as casein, is very rare in China.Our studies focused on the preparation of high F oligo-peptides from milk casein via two step hydrolysis, working out the key processing procedures and parametres. Our research will help the milk industry to produce value added products.Our reserch includes optimiztion of hydrolysis conditions by Alcalase and Flavourzyme, purification by membrane and adsorption by active carbon as well as desalinization and dibitterness of purified peptides. The relative molecular mass and F ratio were also studied. The main results indicated:1. The optimum hydrolysis condition of Alcalase by L9(34)the orthogonal test is 2% CGM、pH8.5、temperature55℃、enzyme dosage 20μL。The degree of hydrolysis is over 30.56%.Among all the parameters studied, pH was the most important one for the efficiency of hydrolysis and followed by CGM、temperature、enzyme dosage.2. The optimum hydrolysis conditions by Flavourzyme was pH6.5、enzyme dosage 20μL、temperature 50℃and hydrolysis for 3 hours. The analysis of free amino acids showed that AAA was greatly released by the hydrolysis of Flavourzyme. 3. The peptide mixtures obtained from complex enzyme digestion contained casein protein、poly-peptides、oligo-peptides、free amino acids as well as a great deal of salts. In order to obtain active high F oligo-peptide, we used membrane purification to purify the zymolyte. Thus the components of protein、poly-peptides were removed with molecular weight above 5 KDa. The best activated carbon was selected from five varieties which had the best adsorption result and the optimum conditions were identified as: the ratio of the weight of activated carbon to the volume of hydrolysate 1∶9 0、pH8.0、temperature 50℃for 1.5 hour.4. During the hydrolysis process, the pH should be stable and maintained at the optimum value for obtaining optimum enzyme activity. Thus hydrochloride and NaOH alkaline solution were continually added to the hydrolysate, which led to large amounts of salts left in the solution. So it is necessary to desalinization the solution. This research used ion exchange resin to desalinization. During desalinization, we found the flow rate was very important both to the casein recycling and desalinization rate. The faster the sample flow, the lower the desalinization rate is and the higher the protein recovery rate is. Vise the versa. The performance of the treatment was the best when the flow rate of the hydrolysate was six times of column volume per hour. The nitrogen retention of the hydrolysate was 85.34% and the ratio of desalinization was 82.11% after passing the ion exchange collumn.5. The casein hydrolysate tasted bitter and the bitterness will affect the quality of protein hydrolysates as food ingredients. So the process of debittering was needed. Our study usedβ-cyclodextrin, which has specific molecular structure, to embed and mask bitterness. When the dosage rose to 0.9%, the bitter value became zero. Therefore the usage level ofβ-cyclodextrin was 0.9%.6. The obtained product was characterized as white color、no-bitterness, with molecular weight below 1000. The result of UV scanning indicated that active carbon was efficient to absorb aromatic amino acids. The amino acid analysis showed that the F value of obtained oligo-peptides was 21.06, higher than 20, falling in the definition of high F oligo-peptides.