Survival of Porcine Pepsin During Cheddar Cheese Making and Its Effect on Casein During Cheese Ripening

A modification of a linear diffusion test for measuring milk clotting enzymes at concentrations of 1 x 10^-4 to 1 x 10^-1 chymosin units/ ml was developed to permit quantitative assay of porcine pepsin in Cheddar cheese with a standard deviation of 6%. The amount of porcine pepsin retained in Cheddar cheese curd was dependent on pH of milk at setting. Milk at pH 6.6, 6.4, 6.2, and 6.0 was set with porcine pepsin, acidified with lactic acid and glucono-~-lactone and made into Cheddar cheese. After pressing the corresponding curd contained 0, 3.64+0 .12 %, 4.79+0.52%, and 5.94+0.30% of the pepsin activity added to the milk. Polyacrylamide gel electrophoresis of cheese revealed increasing degradation of the ~s-casein band with increasing residual pepsin in the curd. However, some degradation of the ~s-casein band was evident in curd set at pH 6.6 which showed no residual pepsin. Curd was made by ultrafiltration from whole milk followed by acidification to pH 5.2 with hydrochloric acid and glucono-6- lactone and vacuum evaporated to 39% moisture at 40 C (no clotting enzymes and no starter) . Degradation of the ~s- casein in this curd was similar to that observed in pepsin cheese set at pH 6.6. All degradation in ~s-casein cannot be attributed to milk clotting enzymes or starter bacteria. Porcine pepsin does not contribute to protein digestion in cheese curd during ripening unless the milk is below pH 6.6 at setting

Evaluation of novel protease enzymes on growth performance and apparent ileal digestibility of amino acids in poultry: enzyme screening

Three experiments were conducted to evaluate eight neutral and six acid proteases on growth performance and apparent ileal amino acid digestibility (AID) of poults (Experiment 1) or chicks (Experiments 2 and 3). Two basal diets were formulated: a nutrient adequate positive control (PC), which met or exceeded the nutrient requirements for poults (Experiment 1) or chicks (Experiments 2 and 3) and a negative control (NC) formulated to achieve 85% (Experiments 1 and 2) or 80% (Experiments 3) of the requirement for protein and amino acids. Phytase was included in all diets to provide 500 phytase units (FTU)/kg and xylanase was included in all diets to provide 10,000 (Experiments 1 and 2) or 16,000 (Experiments 3) xylanase units (BXU)/kg. Proteases were supplemented in the NC diet at an equivalent amount of enzyme protein to create 16 experimental diets. There were five birds/pen and 10 replicate pens per treatment in each experiment. In experiment 1, birds fed the PC diet gained more (P < 0.05) than birds fed the NC. There were no differences in growth performance in birds fed the PC or NC in experiments 2 or 3. In all three experiments, birds fed the NC supplemented with neutral protease 1 had reduced (P < 0.05) feed intake (FI) or body weight gain (BWG) and increased (P < 0.05) feed conversion ratio (FCR) compared with birds fed the NC. Birds fed the NC diet supplemented with neutral protease 3, 7 (Experiment 1), or acid protease 4 (Experiment 3) had increased (P < 0.05) FCR and birds fed neutral protease 6 (Experiment 2) had reduced (P < 0.05) BWG compared with birds fed the NC. Apparent ileal amino acid digestibility was improved (P < 0.05) with protease supplementation to the NC diets (Experiment 1 or 3), but this was dependent on the protease and the amino acid. In conclusion, novel protease supplementation improved AID of amino acids but this was not reflected in improvements in growth performance of poults or chicks

Protein chemical characterization of Mucor pusillus aspartic proteinase Amino acid sequence homology with the other aspartic proteinases, disulfide bond arrangement and site of carbohydrate attachment

AbstractThe amino acid sequence of Mucor pusillus aspartic protenaise was determined by analysis of fragments obtained from cleavage of the enzyme by CNBr and limited tryptic digestion. The protenaise is a single polypeptide chain protein containing 361 amino acid residues, cross-linked by two disulfide bonds. A sugar moiety composed of two GlcNAc residues and four neutral sugar residues is asparagine-linked to the chain. The sequence of M. pusillus proteinase is highly homologous with the M. miehei protonaise (83% identity). The homology with other aspartic proteinases is low (22–24%) and indicates that the Mucor proteinases diverged at an early evolutionary phase. The most conservative regions of the molecule are those involved in catalysis and forming the binding cleft and the core region of the molecule