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 milk-clotting enzymes of animal and microbial origin on the quality and shelf life of soft cheeses

A comparative test was carried out for milk-clotting enzymes (MCE) of animal origin (Naturen® Extra), microbial origin (Marzyme®) and MCE based on recombinant camel chymosin (Chy-max® M) in the production of soft cheese “Lyubitelskiy”. By the end of the shelf life of the cheeses (12 days at a temperature of 3 ± 1 °C), differences were noted in the degree of proteolysis (DP) and the value of the complex modulus G*, which were the following ones for cheeses produced with MCE of the brands: Naturen® — DP = 17.86 ± 0.24%; G* = 4164 ± 587 Pa; Marzyme® — DP = 17.98 ± 0.49%; G* = 4581±786 Pa; Chy-max® M — DP = 9.85 ± 0.63%; G* = 7949 ± 1157 Pa. Cheeses made with Chy-max® M MCE had a denser texture than cheeses made with MCE of Naturen or Marzyme, which did not differ significantly in consistency. In the studied cheeses, the severity of the bitter taste was proportional to the content of water-soluble peptides with a mass of 0.5–3 kDa. Cheeses with Marzyme® MCE had a more intense bitterness than cheeses with Naturen® MCE. There was no bitter taste in cheeses produced with MCE of Chy-max® M. It was concluded that in the production of soft cheeses, recombinant camel chymosin can be used to increase the shelf life, and MCE of microbial origin can be recommended to replace more expensive MCE of animal origin.

Characterization of a Novel Aspartic Protease from Rhizomucor miehei Expressed in Aspergillus niger and Its Application in Production of ACE-Inhibitory Peptides

Rhizomucor miehei is an important fungus that produces aspartic proteases suitable for cheese processing. In this study, a novel aspartic protease gene (RmproB) was cloned from R. miehei CAU432 and expressed in Aspergillus niger. The amino acid sequence of RmproB shared the highest identity of 58.2% with the saccharopepsin PEP4 from Saccharomyces cerevisiae. High protease activity of 1242.2 U/mL was obtained through high density fermentation in 5 L fermentor. RmproB showed the optimal activity at pH 2.5 and 40 °C, respectively. It was stable within pH 1.5–6.5 and up to 45 °C. RmproB exhibited broad substrate specificity and had Km values of 3.16, 5.88, 5.43, and 1.56 mg/mL for casein, hemoglobin, myoglobin, and bovine serum albumin, respectively. RmproB also showed remarkable milk-clotting activity of 3894.1 SU/mg and identified the cleavage of Lys21-Ile22, Leu32-Ser33, Lys63-Pro64, Leu79-Ser80, Phe105-Met106, and Asp148-Ser149 bonds in κ-casein. Moreover, duck hemoglobin was hydrolyzed by RmproB to prepare angiotensin-I-converting enzyme (ACE) inhibitory peptides with high ACE-inhibitory activity (IC50 of 0.195 mg/mL). The duck hemoglobin peptides were further produced at kilo-scale with a yield of 62.5%. High-level expression and favorable biochemical characterization of RmproB make it a promising candidate for cheese processing and production of ACE-inhibitory peptides.

Milk-clotting proteases from Pleurotus albidus: an innovative alternative for the production of Minas frescal cheese

Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.

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.

Development of a Novel Ultrasonic Spectroscopy Method for Estimation of Viscosity Change during Milk Clotting

Ultrasonic testing is an emerging non-destructive testing technology with high repeatability and precision. Milk is a very complex liquid and the change of its viscosity is a highly relevant property throughout conversion into other dairy products. In the following paper, we propose a novel method for the monitoring of viscosity during enzymatic milk clotting by ultrasonic spectroscopy. An ultrasonic transducer–receiver couple with a 250 kHz nominal frequency was submerged in the samples and an enveloped sweep (“chirp”) signal was applied in a through-transmission mode. Simultaneously, the change in viscosity was measured with a rotational viscometer at a constant shearing speed. The data were analyzed with an algorithm developed by the authors for spectral ultrasonic testing. Estimations yielded a high adjusted R2 (0.963–0.998) and low cross-validated estimation error (RPD: 4.38–14.22), suggesting that the method is suitable for industrial use given the right instrumentation.

Methods of Assessing Milk Proteins Coagulation as a Part of the Forecasting System of Technological Properties

Introduction. An integrated monitoring of raw milk’s contents, quality, and security is a key factor that guarantees the high-quality dairy production. As a result, new research methods of rennet clotting are a topical and urgent area of study. The research objective was to systemize and analyze the basic criteria and assessment methods of rennet-induced protein gels in milk systems. Study objects and methods. The authors reviewed the official Russian criteria and methods of raw milk assessment by its rennet coagulation properties. The research also featured the most widespread instrumental approaches used in best practices from around the world. Results and discussion. In Russian and foreign cheese production, milk is always tested for its physicochemical and microbiological indicators, with a mandatory check of its coagulation characteristics. Russian cheese-makers use different modifications of rennet and rennet-fermenting tests based on subjective sensory evaluation, while international scientific trends in this area aim at developing and improving instrumental methods by using a complex indicator MCP. This indicator characterizes the parameters of rennet coagulation with the help of such devices as Formagraph, Lattodinamografo, Optigraph, etc. Formagraph is a simple instrument for measuring rheological properties of milk clotting. It was popular in the late XX century. The resent years saw the development of other methods of instrumental control, including those based on optical measurements. For instance, Ortigraph is one such instrument that receives unified data on coagulation properties of raw milk by using near-infrared waves. Sensory and instrumental methods have their advantages and disadvantages. However, the most significant criteria for a quick test of cheeseability, yield, and quality are the speed of analysis and the comparability and reproducibility of results. Conclusion. To improve cheeseability testing, Russian cheese-makers need to use the international experience, which offers standardized evaluation criteria in combination with various instrumental approaches. However, the integration of foreign regulations and methods into Russian studies is not the only option. The improvement and development of national instrumental methods of evaluating milk protein coagulation is also crucial.

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.