Effect of Temperature of Milk Acidification on Rennet Gel Properties

Changes in mineral solubilization and rennet reaction rate were investigated after decreasing milk pH to 6.3 by lactic acid addition at a temperature of acidification (TAC) of 25 or 35 °C with a short period of equilibration. With increasing TAC, casein micelles retained higher amounts of Ca and P, and at a given temperature of coagulation, rennet clotting time was increased, and dG'/dt decreased. This effect was confirmed by the microstructure of casein micelles during the first stage of the enzymic coagulation indicating that the aggregation of para-k-casein was observed later at higher TAC. The effect of TAC on rennet milk gel formation could be attributed to the nature of the micellar mineral content and the conformational state of casein micelles before rennet action.     

Proteolytic and Milk Clotting Fractions in Milk Clotting Preparations

Five different milk clotting preparations were fractionated on Sephadex G-100 and then tested for milk clotting activity and for proteolysis of denatured hemoglobin. Two preparations were also tested for proteolytic activity on casein. Proteolytic activities on hemoglobin were correlated with clotting ability of bovine rennet, calf rennet, and rennin-pepsin mixture at pH 1.6 and with Mucor miehei protease at pH 5.2. Modified Mucor miehei protease activities on hemoglobin correlated equally well at pH 1.6 and 5.2. Gel filtration through Sephadex G-100 and elimination of nonclotting fractions reduced the proteolytic activities on hemoglobin at pH 5.2 of calf rennet, bovine rennet, Mucor miehei protease, modified Mucor miehei protease, and rennin-pepsin mixture by 68.6, 88.5, 3.7, 53.7, and 91.2%, respectively. At pH 1.6, proteolysis was reduced by 54.2, 41.2, 51.8, 59.5, and 60.8%. Proteolytic activities of bovine rennet and renninpepsin mixture on casein were reduced by 59.8 and 72%, respectively.     

Heat and Fermentation Effects on Total Nonprotein Nitrogen and Urea in Milk

Kinetic analysis was applied to the increase of NPN and the decrease in urea when milk was heated at 80, 100 and 120°C. NPN increase with heat followed zero-order kinetics with an energy of activation of 17 kcal/mole. Urea losses with heating followed pseudo first-order kinetics with an energy of activation of 20 kcal/mole. Commercial yogurt and leben fermentation processes were used to determine the effects of fermentation on NPN and urea. Yogurt and leben fermentations increased NPN by 150 mg/kg and 208 mg/L, respectively, but had no effect on urea.     

Milk Curdling by Rennet under High Pressure

The effect of pressure on milk curd formation initiated by rennet was studied under high pressures. The milk used was a 20% solution of skim milk powder in 0.02M CaCl₂. After holding the milk with rennet at 35°C for given periods under high pressure, up to 1300 kglcm², the extent of proteolytic digestion and the curd tension were measured under atmospheric pressure. Rennet and milk protein were not denatured by pressure under the experimental conditions used. The primary phase reaction of milk curdling by rennet (hydrolysis of casein) was not affected by compression. The secondary phase reaction (core formation of casein micelle aggregates) was delayed under high pressure and the tertiary phase reaction (milk curd formation) was accelerated.     

Effect of Processing on Yield, Color and Texture of Canned Mushrooms

Net yield of canned mushrooms during retorting was improved by vacuum hydration. Yield, color, and texture decreased with increased process time at all temperatures. Process-induced changes in all three parameters were modeled as first order reactions. For hydrated mush-rooms, activation energies for yield, color and texture were 16.0 Kcal/mole, 16.2 Kcal/mole, and 31.3 Kcal/mole, respectively. Corresponding values for nonhydrated mushrooms were 15.9 Kcal/mole, 38.4 Kcal/mole, and 38.5 Kcal/mole. Z values for yield, color and texture of hydrated samples were 44.0°C, 43.9°C, and 22.4°C, respectively, and for nonhydrated samples, 44.1°C, 18.7°C, and 18.3°C, respectively, Vacuum hydration caused color and texture to be relatively insensitive to process temperature.     

Effect of Heat-Stable Proteases on the Kinetic Parameters of Milk Clotting by Chymosin

The milk clotting per unit casein hydrolytic activities of proteases from 14 psychrotrophic pseudomonad isolates of raw milk ranged from 0.77 to 9.97 at 30°C. The milk clotting activities of chymodn and T16 protease were not completely additive, especially at high chymosin concentration when clotting time was relatively fast. The T16 protease was not effective in catalyzing the enzymic step of milk clbtting at O°C in the time expected on the basis of its milk clotting at 30°C. Milk incubated with the T25 motease for 8 days at 4°C and then clotted with chymosin at 30°C exhibited weak curd consistency.     

Rennet Coagulability of High-Heat Treated Milk Influenced By Time of pH Adjustment

The coagulability of high-heat treated (91°C for 16 or 60 set) milk was measured with a Formagraph. Coagulation (hysteresis) and curd formation by such milk was increased by direct acidification to pH<6.4 before rennet addition. Coagulation properties were increased greatly when such milk was pH-cycled (i.e., acidified to pH 5.5, held overnight at 4°C, then neutralized to pH 6.2–6.4) before rennet addition.     

Influence of fat, heat treatments and species on milk rennet clotting properties and glycomacropeptide formation

The influence of species, fat and heat treatment (70 °C for 30 min) of milk on rennet clotting time, k₂₀ value (time as the curd is firm enough to be cut) and glycomacropeptide formation was studied. Rennet clotting properties were affected by the three studied factors. The results obtained could indicate that the fat and the heat treatment have a different influence in the rennet clotting properties of cow's, ewe's and goat's milks; however, those differences, although significant, are not high and its influence in cheese manufacture perhaps have not a great importance. The slope of the glycomacropeptide formation as a function of the incubation time of milk with rennet was calculated applying a linear regression analysis. The slope decreased significantly when whole or skim cow's milk was heated indicating a slower glycomacropeptide formation. Fat and heat treatments had no significant influence on glycomacropeptide formation of ewe's and goat's milk. The results obtained indicate that the fat concentration and the fat globule membrane could influence the initial aggregation of the destabilised casein micelles.     

Effect of Modified Manufacturing Parameters on the Quality of Cheddar Cheese made from Ultrafiltered (UF) Milk

Cheddar cheese was made from milk concentrated twofold by ultrafiltration (UF). Lowering the cooking and cheddaring temperature from 39°C to 35°C resulted in faster acid development, promoted more proteolysis, caused faster degradation of lactose, and contributed smoother body and texture to the cheese. Starter culture at 2% by weight of unconcentrated milk in combination with low cooking and cheddaring temperature reduced pH at faster rate and shortened the cheese making time by approximately 45 min, compared to cheese made using the traditional temperature (39°C). For the traditional temperature (39°C) of cooking and cheddaring, the addition of 0.2 mL/ kg rennet of unconcentrated milk produced the same rate of proteolysis in both control and cheese made from UF retentate. Composition (fat, protein, salt and moisture) and yield of the UF cheeses with modified temperature treatments were not significantly different from control.     

Enhanced Thermostability of Lactase (Escherichia coli) in Milk

Denaturation of lactase in phosphate buffer was a first-order process with a half-life of 1.12 min at 60°C. Denaturation in milk was a biphasic first-order process with a half-life of 115 min at 60°C. Electrophoresis, isoelectric focusing and kinetic analysis all indicated the presence of two isozymes. The minor isozyme exhibited 9% of the total activity and was less thermostable in milk than the major isozyme. In the temperature range 53.5–60°C, the rate of denaturation of the major isozyme in milk was more than 100 times less than the rate in buffer. Arrhenius plots of denaturation in milk and buffer were biphasic. Above 56°C, E_a denaturation in milk and buffer was 187 Kcals/mole and 19.8 Kcals/mole, respectively; below 56°C E_a denaturation in milk and buffer was 36.8 Kcals/mole and 158 Kcals/mole, respectively.     

MILK CLOTTING AND CHEESE MAKING PROPERTIES OF A CHYMOSIN-LIKE ENZYME FROM HARP SEAL MUCOSA

Pepsin A was isolated from the gastric mucosa of two year old harp seal (Pagophilus groenlandicus). Seal pepsin A has a relatively high CU/PU ratio of 0.074, although it is lower than that of calf chymosin (0.170). Equivalent milk clotting units of chymosin and seal pepsin A catalyzed formation of the same amount of nonprotein nitrogen (NPN) when incubated with 2% casein and for both there was no appreciable increase in NPN formation in the second hour of incubation. Seal pepsin A was similar to chymosin in clotting milk substrate with respect to the influence of pH, dilution, calcium chloride and temperature. Cheddar cheeses prepared with either calf rennet or pepsin A as coagulating agent were similar in yield, chemical composition and taste as judged by sensory preference tests. Estimation of protein degradation in the aged cheeses by ultraviolet absorption of citrate-HC1 extracts, gel filtration chromatography of protein soluble in 6 N urea, and determination of amino acids indicated there was slightly more protein degradation in cheese prepared with calf rennet after 30 weeks aging. Sal pepsin A appears to make a major contribution to the excellent cheese-making characteristic of crude seal pepsin(s). However, other components of the seal mucosa extract are probably responsible for the accelerated aging of Cheddar cheese noted in previous reports.     

Characterization and Cheese-Making Properties of Rennet-Like Enzyme Produced by a Local Algerian Isolate of Aspergillus niger

An ochratoxin free extracellular acid protease was produced by solid state cultivation of Aspergillus niger FFB1. The purified enzyme (48.7 kDa) showed an optimal milk clotting activity at pH 5.5 and 45°C in the presence of 0.01 M CaCl₂. The enzyme was stable at least 24 h at 35°C in the pH range of 5.5–7.0. Thermal denaturation started above 45°C. Fresh cheese manufactured with reconstituted cow milk and the purified enzyme showed similar basic characteristics (pH 4.5, acid taste, white color) as marketed cheeses obtained with calf rennet. This emphasizes the value of exploiting local biological resources for value added food processing in developing countries.     

Effects of Enzyme Choice and Fractionation of Commercial Enzyme Preparations on Protein Recovery in Curd

Five different commercial milk clotting preparations (bovine rennet, calf rennet, calf rennet-porcine pepsin mixture, Mucor miebei protease, and modified Mucor miehei protease) were adjusted to equivalent milk clotting activities and then used to clot milk. Percentages of protein in the resulting wheys were compared. Calf rennet, bovine rennet, or modified Mucor miehei protease caused less loss of protein to the whey than Mucor miehei protease or calf rennet-porcine pepsin mixture. The five enzyme preparations were then fractionated by gel filtration. Fractions with milk clotting activity were pooled. Original enzyme preparations and the pooled fractions made from them were standardized to the same clotting activity, then used to coagulated milk to compare their effect on protein loss to the whey. Fractionation significantly improved protein recovery when bovine rennet and calf rennet-porcine pepsin mixture were used as coagulants but not when calf rennet, Mucor miehei protease, or modified Mucor miehei protease were used.     

Effects of Somatic Cell Counts and Milk Composition on the Coagulating Properties of Milk

Forty-two Holstein cows were selected to provide monthly milk samples with varying SCC for 1 yr. Coagulating properties of samples measured as rennet clotting time, rate of curd firming, and curd firmness at cutting were determined by a formagraph. Milk samples were analyzed for fat, protein, lactose, total solids, casein, individual caseins, urea, SCC, and pH. Least squares analyses of data, after adjustments were made for the effect of milk composition, indicated that elevated SCC were associated with a significant increase in rennet clotting time and slower rate of curd firming. An increase of SCC from 100,000 to 500,000 SCC/ml resulted in an increase of approximately 2.1 and 2.2% in RCT and K20, respectively. A further increase of SCC to above 1,000,000/ml resulted in an overall increase of 20.7 and 13.84% in RCT and K20, respectively. Regression analyses indicated that K20 was decreased by 5.42 min and curd firmness at cutting was increased by 12.92 mm for every percentage in milk casein. Rennet clotting time, rate of curd firming, and curd firmness at cutting were increased by 3.52, 3.41, min and decreased by 9.45 mm, respectively, for every unit increase in milk pH.     

Serum Protein Aggregates in the High-Heated Milk and Their Gelation Properties in Rennet-Induced Milk Gel

The influence of different heat treatments on the protein aggregates and changes in gelation properties of rennet-induced milk gels were investigated. In the heated milk, a visible difference in milk serum proteins was found resulting from the formation of protein aggregates. Meanwhile, the size of protein aggregates increased from 25 to 170 nm with increasing the intensity of heat treatment. Furthermore, the differences in textural variables of rennet gels were found among the heat treatments using the principal component analysis. The water holding capacity and cheese curd yield of rennet gels obtained from the heated milk were significantly greater than those of raw milk (p < 0.05). It was also found heat treatments above 80°C could endow rennet-induced milk gels with novel textural properties.     

Evaluation of Harp Seal Gastric Protease as a Rennet Substitute for Cheddar Cheese

A crude preparation of gastric proteases from Harp Seal (Pagophilus groenlandicus) was found to coagulate milk over a wider pH range than porcine pepsin and had a higher ratio of milk clotting to proteolytic activity with hemoglobin at pH 1.8. Cheddar cheese prepared with seal gastric protease (SGP) gave significantly higher sensory scores than cheese made with calf rennet. Chemical analysis of the cheeses revealed a lower concentration of citrate-HCl soluble nitrogen and less free and peptide-bound amino acids in SGP cheese than in the cheeses made with calf rennet and Mucor miehei protease.     

Partial Purification and Characterization of an Acid Phosphatase from Papaya

A phosphatase in papaya was extracted, partially purified, and characterized. With p-nitrophenyl phosphate as substrate, the enzyme had a pH optimum of 6.0, which categorized it as an acid phosphatase, a temperature optimum of 37°C, and a Km of 1.0 mM. Heat inactivation of papaya acid phosphatase was biphasic, and the kinetics of both phases were first order reactions. D values at 60°, 65°, 70°C for the heat resistant phase were 21.0, 11.7, and 4.0 min, respectively. For the heat labile and heat resistant isozymes of papaya acid phosphatase, the activation energies, E_a, for thermal inactivation were 60.0 Kcal/mole and 37.8 Kcal/mole, respectively. The apparent molecular weight of the enzyme as determined by gel filtration was 120,000 daltons.     

Extraction of Milk Clotting Enzyme from Sodom Apple (Calotropis procera)

A milk clotting enzyme with low proteolytic activity was extracted with ammonium sulfate, at 0.40-0.65 saturation, from sodom apple leaves. The enzyme with apparently a basic isoelectric point was activated by cysteine and was more active at 65°C than at 35°C. Milk clotting activity increased with pH at 65°C, with the enzyme being almost twice as active at pH 6.4 as at pH 5.4-5.7. Storage at 4°C for 15 days resulted in a 30-50% loss in enzyme activity.     

Influence of protein concentration and coagulation temperature on rennet-induced gelation characteristics and curd microstructure

This study characterized the coagulation properties and defined the cutting window (CW; time between storage modulus values of 35 and 70 Pa) using rheometry for milk standardized to 4, 5, or 6% protein and set at 28, 32, or 36°C. Milks were standardized to a protein-to-fat ratio of approximately 1 by blending ultrafiltration retentate, skim milk, and whole milk. The internal curd microstructure for selected curd samples was analyzed with transmission electron microscopy and scanning electron microscopy. Lowering the coagulation temperature caused longer rennet coagulation time and time to reach storage modulus of 35 Pa, translating into a wider CW. It also led to a lower maximum curd-firming rate (MCFR) with lower firmness at 40 min at a given protein level. Increasing protein levels resulted in the opposite effect, although without an effect on rennet coagulation time at a given temperature. On coagulation at 28°C, milk with 5% protein resulted in a similar MCFR (～4 Pa/min) and CW (～8.25 min) compared with milk with 4% protein at 32°C, which reflects more standard conditions, whereas increasing milk to 6% protein resulted in more than doubling of the curd-firming rate (MCFR = 9.20 Pa/min) and a shorter CW (4.60 min). Gels set at 28°C had lower levels of rearrangement of protein network after 40 min compared with those set at 36°C. Protein levels, on the other hand, had no influence on the levels of protein network rearrangement, as indicated by loss tangent values. The internal structure of curd particles, as investigated by both scanning electron microscopy and transmission electron microscopy, appeared to have less cross-linking and smaller casein aggregates when coagulated at 28°C compared with 36°C, whereas varying protein levels did not show a marked effect on aggregate formation. Overall, this study showed a marked interactive effect between coagulation temperature and protein standardization of milk on coagulation properties, which subsequently requires adjustment of the CW during cheesemaking. Lowering of the coagulation temperature greatly altered the curd microstructure, with a tendency for less syneresis during cutting. Further research is required to quantify the changes in syneresis and in fat and protein losses to whey due to changes in the microstructure of curd particles arising from the different coagulation conditions applied to the protein-fortified milk.     

Evaluation of Thiol Activated Proteases from Clam Viscera as a Rennet Substitute for Cheese-Making

Clam rennet, which is a crude enzyme preparation of cathepsin B-like protease from clam viscera was characterized and compared to porcine pepsin and calf rennet for its suitability as a milk coagulant in cheesemaking. Clam rennet was more proteolytic and produced a softer curd than the other two coagulants. However, influences of the pH and temperature on milk clotting with clam rennet were very similar to those of calf rennet. The cheddar cheese made from clam rennet was not inferior to the Cheddar cheese made from calf rennet. Quality enhancement occurred despite the view that high ratio of proteolytic to clotting activity is generally considered to be unfavorable for cheese-making. The higher proteolytic activity appeared to accelerate the ripening process. A small yield loss occurred as a result of excessive proteolysis during cheese-making.