A novel milk-clotting enzyme from Aspergillus oryzae and A. luchuensis is an aspartic endopeptidase PepE presumed to be a vacuolar enzyme

Aspergillus oryzae RIB40 has 11 aspartic endopeptidase genes. We searched for milk-clotting enzymes based on the homology of the deduced amino acid sequence with chymosins. As a result, we identified a milk-clotting enzyme in A. oryzae. We expected other Aspergillus species to have a homologous enzyme with milk-clotting activity, and we found the most homologous aspartic endopeptidase from A. luchuensis had milk-clotting activity. Surprisingly, two enzymes were considered as vacuole enzymes according to a study on A. niger proteases. The two enzymes from A. oryzae and A. luchuensis cleaved a peptide between the 105Phe-106Met bond in κ-casein, similar to chymosin. Although both enzymes showed proteolytic activity using casein as a substrate, the optimum pH values for milk-clotting and proteolytic activities were different. Furthermore, the substrate specificities were highly restricted. Therefore, we expected that the Japanese traditional fermentation agent, koji, could be used as an enzyme source for cheese production.

Influence of different milk-coating enzymes on the process of producing soft cheeses

The effect of the type of milk clotting enzyme (MCE) on the duration of milk coagulation, parameters of the composition of whey and cheeses, and the output of cheeses in the production of soft cheese such as “Lyubitel'skiy” were investigated. Three brands of MCE of different origins were investigated: Marzyme® (MCE of microbial origin based on Rhizomucor miehei protease), Naturen® (calf rennet) and Chy-max® M (recombinant camel chymosin). It was established that MCEs had different ratios of milk clotting activity (MCA) to total proteolytic activity (PA). It was determined that the MCA/PA ratio, which characterizes the degree of specificity of the MCE action with regard to kappa-casein, in Chy-max M 1000 is ~7 times higher than that of Naturen and ~50 times higher than that of Marzyme. Such differences did not lead to a negative effect when using the Marzyme preparation in the production of soft cheeses. There were no statistically significant differences in the amount of dry matter loss of the curd into the whey, physicochemical parameters and output between the variants of cheeses made with the studied brands of MCE. Shorter duration of milk clotting (16.5 min) was observed with Marzyme than with MCE of Naturen (20.5 min) and Chy-max M (22.5 min). The results of the coagulation duration were explained by the stimulation of the activity of MCE of microbial origin, by the pH level of milk before coagulation (below pH 6.4). It was shown that modern MCEs of microbial origin could be recommended as a cost-effective replacement for more expensive rennet and recombinant chymosins in the production of soft and fresh cheeses.

Purification and Characterization of Milk Clotting Enzyme from Edible Mushroom (Pleurotus florida)

Oyster mushroom (Pleurotus florida 14 MICC) is one of the most widely cultivated edible fungi in the world. Milk clotting enzyme (MCE) derived from the mold was developed as a calf rennet alternative; thus, it was purified and characterized, and its effect on different milk species was investigated. The highest MCE activity (75.49 SU/ml) was observed in mushroom fruit bodies dissolved in 0.2 M sodium acetate pH 5.0; as well as the highest total MCE activity (367.85 SU) was recorded at 20% of ammonium sulfate with a specific activity of 343.79 SU mg-1 protein while size exclusion column chromatography on Sephadex G-100 purified MCE 3.46-fold with 17.96% yield. Also, it could be capable of coagulating different milk species. Mushroom MCE exhibited their optimal activity at pH 5.0 for crude extract (CE) while at pH 6.0 for partial purified (PP) and purified (P) MCE fractions; as well as at 55 °C for CE and PP MCE fractions while 50 °C for P MCE. CaCl2 concentration (0.01%) recorded the maximal activity for CE while (0.04%) and (0.02%) for PP and P fractions, respectively. It could be concluded that MCE from Oyster mushroom may be a good candidate as a calf rennet substitute in cheese production.

EFFECT OF TRANSGLUTAMINASE TREATMENTON TRANSITION OF SOLIDS FROM MILK TO CHEESE

Transglutaminase is an enzyme that forms cross-links between protein molecules and affects such protein properties as the ability to gel, thermal stability, water retention, etc. One of the important tasks of the food industry is to increase the yield of products, in particular soft cheeses, in the production of which a significant part of the whey with dissolved substances is separated from milk. Therefore, the aim of the work was to study the effect of transglutaminase on the transition of milk solids to cheese, depending on the stage of application of the enzyme. Transglutaminase in an amount of 0.05% by weight of milk (1.6 units in terms of 1 g of protein) was introduced in two versions: simultaneously with the milk-clotting enzyme and after cutting the clot. It was found that the use of transglutaminase does not affect the duration of acid-rennet coagulation of milk. The data obtained indicate that when transglutaminase is introduced into milk simultaneously with the milk-converting enzyme, the transition of milk solids to cheese increases by 5.15%, including fat by 3.07%, compared to samples without transglutaminase. When transglutaminase is added at the stage of processing the clot, a denser consistency of cheese is formed.

Kinetic, Catalytic and Thermodynamic Properties of Immobilized Milk Clotting Enzyme on Activated Chitosan Polymer and its Application in Cheese Making

Milk clotting enzyme (MCE) from Bacillus circulans 25 was immobilized by covalent binding, ionic binding and entrapment methods using various carriers. MCE covalently immobilized on activated chitosan polymer with the bifunctional agent glutaraldehyde (Ch-MCE) exhibited highest immobilization yield (74.6 %). Comparing to the native MCE, Ch-MCE exhibited higher optimum pH, higher optimum reaction temperature, lower activation energy, higher half-life time, lower deactivation rate constant and higher energy for denaturation. After immobilization, maximum reaction rate, Michaelis-Menten constant, specificity constant, turnover number, and catalytic efficiency of the enzyme were significantly changed. Calculated thermodynamic parameters for denaturation (enthalpy, entropy and Gibbs free energy) confirmed that the catalytic properties of MCE were significantly improved after immobilization. Reusability tests showed that after 7 catalytic cycles, the Ch-MCE retained about 71 % of its activity confirming its suitability for industrial applications.

Extraction, Partial Purification And Characterization Of Milk- Clotting Enzyme From Local Coagulants On The Yield Of Cheese Using Cow Milk

Milk-clotting activity is the most important property of enzymes used in cheese making. The study conducts at the University of Gondar, Institute of biotechnology microbial and cellular biology laboratory. Latex extracted from each plant using an appropriate procedure. Five hundred ml of milk added to each beaker in all extract labels with its replication, heated, each coagulant was added and heated till a separate layer of cheese and whey happened. Then after clotting time, whey product, cheese- fresh weight, cheese dry weight, and the percentage of cheese yield were recorded using appropriate measurement and calculation. All data analyzed using SAS Studio, and analysis of variance showed that highly significant differences have existed among plant extract used for all studied parameters. The highest (59, 74.67, 91.33 gm) amount of cheese- dry weight observes in lemon juice. The highest (11.34, 14.36, and 17.56)% of cheese- yield observe in lemon juice for all extract labels, respectively. The lowest amount (32.67, 25.67, and 17.00) of cheese formation starting time observed from lemon juice, respectively. Therefore, lemon juice, calotropis procera, and Solanum incanum could use in the dairy industry and local community as an alternative source of rennet.

Express method for assessing proteolysis in cheese and aromatic addi- tives with cheese flavor

The method based on the determination of the amount of active amino groups using o-Phthaldialdehyde (OPA method) can be applied in practice to assess accurately the degree of proteolysis in cheeses. The work establishes that the OPA method gives results that strictly correlate (R2 > 0.80, p < 0.01) with the results of assessing the degree of proteolysis by the Kjeldahl method. The results of the OPA method, expressed in the absorption intensity of the colored sample at a wavelength of 340 nm (OD340), can be converted to the content of soluble nitrogenous substances in cheese (WSN), using the calibration relationship between these indicators.The accuracy of the calibration relationship between WSN and OD340 can be increased (R2 > 0.91, p< 0.01) when using the OPA method in relation to a homogeneous group of cheeses produced by the same technology using the same type of milk clotting enzyme and lactic acid starter culture and having a similar shape of the molecular mass distribution of proteolysis products.The OPA method can be used to assess the content of proteolysis products, which form cheese flavor, in EMС. The results of assessing the degree of proteolysis by the OPA method (OD340) are proportional to both the total content of soluble nitrogen and the proportion of nitrogenous substances in it with a mass of less than 0.5 kDa, which make the greatest contribution to the formation of cheese flavor.The advantage of using the OPA method for assessing proteolysis in cheeses and EMC instead of the Kjeldahl method is a simpler measurement procedure and the possibility of studying more samples in less time.