| The purpose of limited proteolysis of defatted soy flour (DSF) is to obtain productions which are accordant with animal nutritional mode, but the existing evaluating system can't meet the request of study in this field. Four analytical methods were established in this study: degree of hydrolysis (DH) of DSF, average molecular weight, molybdenum's modality distributions and release ratio of phosphorus, all of which were applied to the study of limited hydrolysis to obtain productions with both nutritional and functional properties. There are mainly three methods in the determination of proteins: Coomassie brilliant blue G-250 (CBB G-250), UV absorption (UV) and micro-Kjeldahl. Studies indicated that the exposed degree of alkaline radical or aromaticring amino acid residual were significantly different because the source and proteolysis procedure of DSF were different, so it was difficult for CBB G-250 and UV to determine the concentration of proteins in the hydrolysates correctly. Micro-Kjeldahl was immune to the described factors and had good exactitude and veracity. The average molecular weight of amides can reflect the real DH of DSF better. The objects of ninhydrine areα-amino acids and using single amino acid as the reference substance is not suitable to determine the number of amino in the hydrolysate. The result of formaldehyde titration was the summation of amino in peptides and amino acids, pH meter was used to indicate the end of titration and the colors of samples were overcame, RSD<2.5%. Molybdenum is one of the necessary microelements and its absorption by animals is different according to its concentration and modality. A new photometry for molybdenum modality distribution was established based on the reaction of MBASF with Mo(VI), the apparent molar absorptivity of complex is 1.54×105 L. mol-1.cm-1, the color reaction system were sensitive and selective. Hydrolysate (A) was precipitated at its isoelectric point to obtain the filtrate (B), subsequently, B was transferred to the chromatogram column with D301R resin, the column were purged by 20ml HAc-NaAc(pH3.6) and 80mL NH4Cl-NH3H2O(pH9) buffer solution orderly and obtained the effluent solution C and D. B, C and D solutions were used to spectrophotometric determination of hydrolyzed Mo, chelated Mo by peptide or amino acid and dissociative Mo, study on molybdenum's modality distributions was realized. Phytase hydrolyzes phytate which can't be absorbed by animals to form inorganic phosphorus, in the medium of H2SO4 and the presence of polyvinyl alcohol, malachit Green-molybdic acid ammonium react with phosphate to form a green complex which gives maximum absorption at 635nm with an apparent molar absorptivity is 9.30×104 L. mol-1.cm-1, the method was selective. It was determined that the content of phosphorus in DSF is 0.87% and about 62.13% of which were phytate, higher release ratio of phosphorus can be obtained after the addition of 0.67% phytase and incubation of 45min at pH4.5, 45℃.The whole proteolysis was broken down into three parts: pretreatment of DSF, enzyme hydrolysis and the synergies of proteases and phytase. Incubation at pH3, 40℃for about 2h and then heated on the boiling water bath for 15min, following 30min hydrolysis ofα-amylase at pH6,40℃were used as pretreatment and the degree of hydrolysis can be improved by 35% mostly. Among the schemes of single protease, double proteases and triple protease mulriples, triple protease mulriple gave the best performance. Phytase shows no inhibiting effect to protease when added during the acid lixiviate process, so the optimal scheme is acid lixiviate, phytase→heat treatment→α-amylase→acid protease→neutral protease→alkaline protease. The main technical parameters are: total DH is 61.56%, protein lixiviate ratio is 66.13%, degree of soluble protein hydrolysis is 93.09%, average molecular weight of the amide is 640 and the hydrolysates was bitterless, molybdenum and phosphorus release ratio reached 88.15% and 47.17% separately. HPLC and amino acid analysis indicate that about 87.47% of the hydrolysates is lower than 1000.
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