Milk-clotting Enzyme Research Articles

Cell immobilization for enhanced milk clotting enzyme production from Bacillus amyloliquefacien and cheese quality

BackgroundMilk clotting enzymes, essential for milk coagulation in cheese production, are obtained from the stomach of young ruminants, an expensive and limited source. This study was accomplished by finding a suitable alternative. Bacterial isolates recovered from honey were screened for milk clotting enzyme activity. and further, by immobilization of the microorganisms to enhance stability and facilitate their repeated use.ResultThe most effective enzyme was produced by a microbe identified as Bacillus amyloliquefaciens based on 16 S rRNA sequencing. The cells were encapsulated in Ca2+ alginate beads. These beads retained complete enzyme production after being used five times. Glucose and Soybean were selected as the most favorable carbon and nitrogen sources, respectively. The optimum temperature for activity was 35 ℃ for both free and immobilized cells but as the temperature was increased to 55 °C and above, the encapsulated form retained more activity than the free cells. The pH optimum shifted from 6.5 to 7 for the free cells to 7–7.5 for the immobilized cells. The immobilization process decreased the activation energy for enzyme production and activity, prolonged the enzyme half-life, and increased the deactivation energy. Enzyme produced by immobilized cells generated a more compact cheese.ConclusionsThe finding of this study was to identify a less expensive source of milk-clotting enzymes and confirm the success of cell immobilization in improving cell rigidity and stability. Also, immobilization of this B. amyloliquefaciens strain offers an enzyme source of value for industrial production of cheese.

Influence of physicochemical factors on the specific activity of proteases used in cheesemaking biotechnology

The formation of a clot under the influence of proteolytic enzymes is an essential stage in cheesemaking technology. Depending on their composition, milk-clotting enzyme preparations have a different effect on the quality characteristics of the clot, whey separation, and yield of the finished product. At present, the Russian market offers a wide range of domestic and imported milk-clotting enzyme preparations. In this connection, the choice of a milk coagulant by cheese makers should be carried out in accordance with the cheese group produced by the enterprise. The milk-clotting activity of enzyme preparations depends not only on the active acidity of the mixture, but also on the temperature of the solution used to obtain them. It is the solution temperature that has a determining effect on the duration of the coagulation process and, therefore, the quality of the formed clot. Research into the specific activity of milk-clotting enzyme preparations, obtained in a working solution with different active acidity (pH from 4.0 to 8.0), showed their milk-clotting activity to decrease significantly upon pH shifting to the alkaline region. Application of solutions with a pH of 4.0 led to an increase in the milk-clotting activity of bovine pepsin, VNIIMS SG-50 rennet-bovine preparation, and rennet enzyme by 94, 72, and 36%, respectively, relative to the control. Bovine pepsin demonstrated the highest sensitivity to fluctuations in active acidity. A study into the effect of substrate temperature in the range from 30 to 40 °C established rennet to be the most sensitive preparation to temperature changes. It is concluded that a properly selected enzyme composition is an effective means for affecting the quality of cheese products.

Fe3O4@silica-thermolysin: A robust, advantageous, and reusable microbial nanobiocatalyst for proteolysis and milk-clotting

Thermolysin (TLN) is a microbial highly-priced thermostable metallo-endoprotease with complementary substrate specificity to those of proteases widely used in science and industry for protein digestion and milk-clotting. This study is the first to immobilize TLN on aminated superparamagnetic nanoparticles (Fe3O4@silica-NH2) aiming for higher stability, recoverability, reusability, and applicability in proteolysis and as a microbial rennet-like milk-clotting enzyme. The nanobiocatalyst developed (Fe3O4@silica-TLN) displays hydrolytic activity on a synthetic TLN substrate and, apparently, was fully recovered from reaction media by magnetic decantation. More importantly, Fe3O4@silica-TLN retains TLN catalytic properties in the presence of calcium ions even after exposure to 60 °C for 48 h, storage at 4 °C for 80 days and room temperature for 42 days, use in proteolyses, and in milk-clotting for up to 11 cycles. Its proteolytic activity on bovine milk casein in 24 h furnished 84 peptides, of which 29 are potentially bioactive. Also, Fe3O4@silica-TLN catalyzed the digestion of bovine serum albumin. In conclusion, Fe3O4@silica-TLN showed to be a new, less autolytic, thermostable, non-toxic, magnetically-separable, and reusable nanobiocatalyst with highly attractive properties for both science (peptide/protein chemistry and structure, proteomic studies, and the search for new bioactive peptides) and food industry (cheese manufacture).

Основные технологические свойства заменителей сычужного фермента

The quality of the finished cheese depends on the process of obtaining a milk clot under the action of a milk-converting enzyme. Most types of cheeses can be produced using the full range of dairy coagulants available on the market. However, without taking into account their technological features, enterprises save pennies on the cost of enzymes and lose rubles on the quality of products. The main technological characteristics of rennet enzyme substitutes (proteolytic activity, sensitivity to Ca2+ ions, pH, temperature stability), important for regulating the coagulation process during cheese production, are investigated. Practical recommendations on the use of milk-clotting enzyme preparations are given.

Microbiological, chemical, and artificial sensory assessment of Sicilian cheeses made using different milk-clotting enzymes

In the present study, cheeses coagulated with different milk-clotting enzymes were investigated. In detail, calf rennet, microbial coagulant, and a kiwifruit enzymatic extract were used in three independent cheesemaking processes. Microbiological, chemical, and sensory profiles, by using E-Eye, E-Nose, and E-Tongue platforms, were evaluated. Based on microbiological data, mesophilic bacteria, Lactobacilli, and Lactococci showed similar viable count in cheeses clotted with animal rennet and with vegetable coagulant. Differently, higher value of mesophilic bacteria and lower count of both Lactobacilli and Lactococci were detected in samples obtained using microbial coagulant. Additionally, as revealed by the fatty acid composition, the kiwifruit enzymatic extract allowed to obtain a final product with improved health properties. Concerning the artificial senses, E-Eye, E-Nose, and E-Tongue, a similar sensory profile was revealed among cheeses clotted using the kiwifruit enzymatic extract and the calf rennet whereas a different sensory profile was observed in microbial clotted cheeses. This work demonstrated that the kiwifruit extract is a valid coagulant alternative to calf rennet and microbial coagulant.

Influence of different milk-clotting enzymes on the quality and shelf life of semihard cheeses

The study examined semi-hard cheeses made with milk-clotting enzymes (MCEs) of animal origin (Naturen Extra with a mass fraction of chymosin 95%, “Bovine Pepsin” with a mass fraction of chymosin 10%), microbial origin (Fromase 750 XLG) and recombinant origin (Chy-max Extra and Chy-max Supreme), at an introduction dose of MCE of 1,500 to 6,000 IMCU per 100 kg of milk. In cheeses at the age of 7, 60, and 150 days, pH, the degree of proteolysis, the content of peptides with a mass of 1–5 kDa, and the compressive stress at fracture were determined. Cheeses produced with a dose of MCE of 5,000–6,000 IMCU/100 kg of milk had a substantially (p<0.05) lower pH compared to cheeses made with doses of MCEs of 1,500–3,000 IMCU/100 kg of milk. At the same time points, there were no significant differences (p>0.05) in the degree of proteolysis between cheeses made using diverse types of MCEs at the same dose. The exceptions were cheeses produced with Chy-max Supreme, which had a substantially (p<0.05) lower level of proteolysis. Sensory assessment of the bitter taste intensity in cheeses is proportional to the content of peptides with a molecular weight of 1–5 kDa. With an increase in the MCE dose, the content of peptides with a molecular weight of 1 to 5 kDa increases in cheese. Chy-max Supreme forms the least number of bitter peptides in cheeses (p<0.05). There are no significant differences (p<0.05) in the magnitude of the compressive stress at fracture between the cheese variants produced with the same MCE doses of diverse types at the same time points.

Biotechnology factors influence on formation of curd cheese quality indicators

Considering the growing interest of the Russian society in fermented dairy products, in particular, cream cheese enriched with probiotic cultures and biologically active substances (BAS), it is relevant to conduct research aimed at scientifically justifying the choice of biotechnology of curd production as a raw material for cream cheese by studying the influence of biotechnological and technological factors on the formation of properties of the used curd. The generally accepted methods of qualitative prediction were used. The study of consumer opinion revealed the demand of Russian buyers for curd products enriched with BAS and probiotic cultures. The analysis of the regulatory documentation allowed us to form a set of mandatory requirements for the quality of curd cheese and the curd used, as well as a list of raw materials, biotechnological and technological factors required in the technology of curd cheese production. The study of the influence of each type of biotechnological and technological factors on the properties of the finished product has shown that in the biotechnology of enriched cream cheese it is necessary to use starter from pure cultures of lactococci or a mixture of lactococci and thermophilic lactic acid streptococci, acid method of coagulation of milk protein, whey separation by separation or ultrafiltration, introduction of BAS and flavoring fillers with cream in a separate method of normalization, as well as to exclude the use of milkclotting enzyme, and also to eliminate the use of milk-clotting enzymes. The results obtained are the basis for the development of new biotechnologies of curd cheese enriched with BAS and/or probiotic cultures.

Технологические свойства препаратов сычужного фермента

The quality of the finished cheese is determined by all stages of its production, but the basis of cheese making is the production of a milk clot under the action of a milk-converting enzyme. The duration of coagulation and the structural and mechanical properties of the cheese clot are critical indicators that affect the yield of cheese, consistency, pattern, taste. In practice, it is necessary to take into account that the coagulating ability of the enzyme may vary depending on the properties of the milk mixture prepared for coagulation (protein content, mineral salts, acidity, temperature). The main technological characteristics of rennet enzyme preparations (proteolytic activity, qualitative composition, sensitivity to Caions, pH, thermal stability), important for the regulation of the coagulation process, have been studied. Practical recommendations on the use of milk-clotting enzyme preparations are given.

Анализ и перспективы развития отрасли сыроделия: региональный аспект

Cheese is a unique product of animal origin. The history of cheese-making dates back millennia, and every nation has its own traditional cheese formulations. Recent decades have seen a growing public interest in craft cheese-making and national cheese-making. As a multinational country, Russia boasts a wide range of traditional cheeses. However, the domestic cheese market belongs mainly to such popular foreign cheese sorts as Mozzarella, Camembert, Tilsit, Brie, etc. The steady increase in consumer demand for cheese products encourages cheese-makers to expand their product range, and, in their search for new formulations, they often appeal to traditional cheeses, e.g., those produced in the Altai Region. Despite this trend, the average annual per capita consumption of cheese in Russia remains at 6.5 kg while in some other developed countries this criterion is as high as 16 kg. The present development assessment of the Russian cheese-making industry showed a severe shortage in domestic direct-application starter cultures. Russian cheese-makers prefer imported recombinant milk-clotting enzymes, which significantly hampers industrial development. A new domestic production could provide enough raw materials to satisfy the growing demand for rennet products. At present, the raw milk production capabilities cannot meet the ever-increasing needs. The domestic market of dairy raw materials is dominated by cow milk while goat milk and sheep milk are associated mainly with soft cheeses, and buffalo milk occupies a very narrow sector of craft cheese-making.

Purification and characteristics of a novel milk-clotting metalloprotease from Bacillus velezensis DB219

Milk-clotting enzyme (MCE) is the essential active agents in dairy processing. The traditional MCE is mainly obtained from animal sources, in which calf rennet is the most widely used in cheese industry. Traditional MCE substitute is becoming necessary due to its limited production and increased cheese consumption. A novel traditional MCE substitute was produced from Bacillus velezensis DB219 in this study. The DB219 MCE exhibited a notable specific activity of 6,110 Soxhlet units/mg and 3.16-fold purification yield with 28.87% recovery through ammonium sulfate fractionation and DEAE-Sepharose Fast Flow. The purified DB219 MCE was a metalloprotease with a molecular weight of 36 kDa. The DB219 MCE was weak acid resistance and stable at pH 6.0 to 10.0 and temperature <45°C. The highest milk-clotting activity was observed in substrate at pH 5.5 added with 20 to 30 mM CaCl2. The Michaelis constant and maximal velocity for casein were 0.31 g/L and 14.22 μmol/min. The DB219 MCE preferred to hydrolyze β-casein instead of α-casein. The DB219 MCE hydrolyzed α-casein, β-casein, and κ-casein to generate significantly different peptides in comparison with calf rennet and ES6023 MCE (fungal MCE) through SDS-PAGE and reversed-phase HPLC analysis. The DB219 MCE mainly cleaved Thr124-Ile125 and Ser104-Phe105 bonds in κ-casein and had unique casein cleavage sites and peptide composition through LC-MS/MS analysis. The DB219 MCE was potential to be a new milk coagulant and enriched kinds of traditional MCE substitute.

The effect of thioredoxin and prochymosin coexpression on the refolding of recombinant alpaca chymosin.

The milk-clotting enzyme chymosin is a member of the group of aspartate proteinases. Chymosin is the main component of rennet traditionally obtained from the stomachs of dairy calves and widely used to coagulate milk in the production of various types of cheese. Another source of chymosin, which does not require the killing of animals, is based on recombinant DNA technology. Recombinant alpaca chymosin has a number of valuable technological properties that make it attractive for use in cheese-making as an alternative to recombinant bovine chymosin. The purpose of this work is to study the effect of coexpression of thioredoxin and prochymosin on the refolding of the recombinant zymogen and the activity of alpaca chymosin. To achieve this goal, on the basis of the pET32a plasmid, an expression vector was constructed containing the thioredoxin A gene fused to the N-terminal sequence of the marker enzyme zymogen, alpaca prochymosin. Using the constructed vector, pET-TrxProChn, a strain-producer of the recombinant chimeric protein thioredoxin-prochymosin was obtained. The choice of prochymosin as a model protein is due to the ability of autocatalytic activation of this zymogen, in which the pro-fragment is removed, together with the thioredoxin sequence attached to it, with the formation of active chymosin. It is shown that Escherichia coli strain BL21 transformed with the pET-TrxProChn plasmid provides an efficient synthesis of the thioredoxin-prochymosin chimeric molecule. However, the chimeric protein accumulates in inclusion bodies in an insoluble form. Therefore, a renaturation procedure was used to obtain the active target enzyme. Fusion of thioredoxin capable of disulfide-reductase activity to the N-terminal sequence of prochymosin provides optimal conditions for zymogen refolding and increases the yield of recombinant alpaca chymosin immediately after activation and during long-term storage by 13 and 15 %, respectively. The inclusion of thioredoxin in the composition of the chimeric protein, apparently, contributes to the process of correct reduction of disulfide bonds in the prochymosin molecule, which is reflected in the dynamics of the increase in the milk-clotting activity of alpaca chymosin during long-term storage.

Production, purification, and characterization of a novel milk-clotting metalloproteinase produced by Bacillus velezensis YH-1 isolated from traditional Jiuqu

Bacillus velezensis YH-1 isolated and identified from conventional rice wine starter (Jiuqu) was shown to produce a milk-clotting enzyme (MCE) with remarkable milk-clotting activity (MCA) (642.96 SU/mL). The MCE was produced and purified with a specific 5762.7 SU/mg activity. LC–MS/MS combined with 3D structural modeling was used for the identification of MCE and this analysis indicated that it was a Zn2+ metalloproteinase. The MCE was shown to be relatively stable between pH 6–7 at temperatures below 50 °C. Certain parameters (Km and Vmax are 8.58 mg/mL and 15.65 U/min) were calculated for MCE that could cleave casein at the Lys21-Ile22 and Tyr30-Val31 links. The purpose is to destabilize casein structure and allow coagulation. Whole-genome sequencing and Liquid Chromatograph Mass Spectrometer/Mass Spectrometer (LC-MS/MS) analysis determined the MCE zymogen-producing gene from B.velezensis YH-1 to be 1566 bp long, yielding a protein that is 56.8 kDa in mass and containing 521 amino acid residues. According to an additional structural simulation study, the MCE was hydrophilic and included more-helices, contributing to its structural stability. The signal peptide was in the first 27 amino acids of the MCE. When compared to commercial rennet, MCE was superior in its ability to increase the milk curd's flexibility and viscosity and speed up the coagulation process. This novel study document the presence of a metalloproteinase milk-clotting factor from B. velezensis, the MCE has the potential to replace chymosin in cheese production.

Characteristics and application in cheese making of newly isolated milk-clotting enzyme from Bacillus megaterium LY114

Milk-clotting enzyme (MCE) is a crucial active agent in cheese making. It is necessary to find traditional MCE substitutes due to the limited production of traditional MCE (e.g., calf rennet) and increased cheese consumption. Bacillus megaterium strain LY114 with good milk-clotting activity (MCA) (448 SU/mL) and a high MCA/proteolytic activity (PA) ratio (6.0) was isolated and identified from agricultural soil in Laiyang (Shandong, China) through 16S rRNA sequencing of 45 strains. The Bacillus megaterium LY114 MCE had a remarkable specific activity (7532 SU/mg) and displayed a 4.83-fold purification yield with 34.17% recovery through ammonium sulfate fractionation and DEAE-Sepharose Fast Flow. The purified LY114 MCE was a metalloprotease with a molecular weight of 30 kDa. LY114 MCE was stable at pH 5.0–7.0 and temperature <40 °C. The highest MCA appeared at a substrate pH of 5.5 with 30 mM CaCl2. The Michaelis constant (Km) and maximal velocity (Vm) for casein were 0.31 g/L and 14.16 μmol/min, respectively. LY114 MCE preferred to hydrolyze α-casein (α-CN) rather than β-casein (β-CN) and had unique α-CN, β-CN and κ-casein (κ-CN) cleavage sites. LY114 MCE hydrolyzed casein to generate significantly different peptides compared with calf rennet and fungal MCE as determined by SDS-PAGE analysis. Chemical index analysis and sensory evaluation confirmed the usefulness of LY114 MCE in cheese making. LY114 MCE had the potential to be used in dairy processing and enriched traditional MCE substitutes.

Plant extracts and essential oils in the dairy industry: A review

Plants have been used as food additives worldwide to enhance the sensory qualities of foods and extend their shelf life by reducing or eliminating foodborne pathogens. They also serve as therapeutic agents due to their beneficial effects on human health through their anti-cancerous, anti-inflammatory, antioxidant, and immune-modulatory properties. &#x0D; Plants can be added to food as a dry powder, grated material, paste, juice, or as an extract that can be produced by a variety of methods. Plant extracts and essential oils are concentrated sources of bioactive phytochemicals that can be added to food in small amounts in a variety of forms. These forms include liquid, semi-solid, or dry powder for easy and uniform diffusion. Encapsulation can protect bioactive compounds from temperature, moisture, oxidation, and light, as well as allow for controlling the release of the encapsulated ingredients. Nanoemulsions can enhance the bioactivity of active components. &#x0D; This review explains how plant extracts and essential oils are used in the dairy industry as antimicrobial materials, analyzing their impact on starter bacteria; as natural antioxidants to prevent the development of off-flavors and increase shelf life; and as technological auxiliaries, like milk-clotting enzymes, stabilizers, and flavoring agents. Therefore, plant extracts and essential oils are a better choice for the dairy industry than plants or their parts due to a wide range of applications, homogeneous dispersion, and ability to control the concentration of the bioactive ingredients and enhance their efficiency.

The catalytic and kinetic characterization of Bacillus subtilis MK775302 milk clotting enzyme: comparison with calf rennet as a coagulant in white soft cheese manufacture

BackgroundCalf rennet is considered the traditional source of milk clotting enzyme (MCE). However, increasing cheese consumption with decreasing the calf rennet supply had encouraged the quest for new rennet alternatives. The purpose of this study is to acquire more information about the catalytic and kinetic properties of partially purified Bacillus subtilis MK775302 MCE and to assess the role of enzyme in cheese manufacture. ResultsB. subtilis MK775302 MCE was partially purified by 50% acetone precipitation with 5.6-fold purification. The optimum temperature and pH of the partially purified MCE were 70 °C and 5.0, respectively. The activation energy was calculated as 47.7 kJ/mol. The calculated Km and Vmax values were 36 mg/ml and 833 U/ml, respectively. The enzyme retained full activity at NaCl concentration of 2%. Compared to the commercial calf rennet, the ultra-filtrated white soft cheese produced from the partially purified B. subtilis MK775302 MCE exhibited higher total acidity, higher volatile fatty acids, and improved sensorial properties. ConclusionsThe partially purified MCE obtained in this study is a promising milk coagulant that can replace calf rennet at a commercial scale to produce better-quality cheese with improved texture and flavor.

Screening and isolation of milk-clotting enzymes from seaweed

Cheese-making involves milk coagulation as a crucial step where calf rennet has been used traditionally as the milk-clotting enzyme in the cheese industry. This study investigated milk-clotting enzymes from seven species of seaweed and evaluated the parameters for their isolation and partial purification including post-harvest processing, extraction and purification methods. The saturation degree of ammonium sulphate and the precipitation stages were evaluated to obtain optimal purification conditions, and three desalting methods, namely dialysis, desalting column and the combination of dialysis and desalting column, were investigated to determine the most suitable method for ammonium sulphate removal. Protein extracts of all seven species of seaweeds exhibited some caseinolytic activity, but the extract of unprocessed (whole) dried samples had higher protein yields and stronger caseinolytic activities. The extract from one species, Gracilaria edulis, demonstrated the ability to clot milk. The evaluation of the purification method for G. edulis extract revealed an optimum single step of 50% saturation and ammonium sulphate precipitation with dialysis as the desalting method.

THE OBTAINING OF THE RECOMBINANT CAMEL CHYMOSIN BY SUBMERGE FERMENTATION IN THE PILOT BIOREACTOR

In the cheese making industry, chymosin is used as a milk-clotting enzyme. With its high specific activity against κ-casein, chymosin better than other proteolytic enzymes. Bactrian camel chymosin has a milk-clotting activity higher than calf chymosin. A scheme for obtaining a milk-clotting preparation based on recombinant camel chymosin is proposed. Submerge fermentation of recombinant yeast Pichia pastoris was carried out in a 50-liter bioreactor and recombinant camel chymosin was obtained. The activity of chymosin in the yeast culture was 174.5 U/mL. Chymosin was concentrated 5.6-fold by cross-flow ultrafiltration with 10 kDa cut-off membrane, and chymosin was purified by ion exchange chromatography. The activity of purified chymosin was 4700 U/mL. By sublimation drying with casein peptone, the powder chymosin was obtained with an activity of 36,000 U/g. The proposed scheme for obtaining a milk-clotting drug based on recombinant camel chymosin using submerge fermentation of recombinant yeast has the prospect of being used at biotechnological enterprises.

Influence of different milk-clotting enzymes on the process of producing semihard cheeses

The effect of milk-clotting enzymes (MCEs) of animal origin (Naturen Extra with a mass fraction of chymosins of 95%, “Bovine Pepsin” with a mass fraction of chymosin of 10%), as well as MCEs of microbial origin (Fromase 750 XLG) and recombinant origin (Chy-max Extra and Chy-max Supreme) on the duration of milk coagulation and processing of cheese curd, as well as on the composition of whey and fresh cheeses in the manufacture of semihard cheeses, at an introduction dose of MCE of 1,500 to 6,000 IMCU per 100 kg of milk. Increasing the MCE dose from 1,500 to 2,000–3,000 IMCU/100 kg of milk leads to a decrease in the coagulation duration by an average of 20 min (p&lt;0.05). A further increase in the MCE dose to 5,000–6,000 IMCU/100 kg of milk does not lead to a reduction in the coagulation duration. With equal introduction doses, MCE brands of Fromase and “Pepsin” show greater losses of fat (by 0.15–0.60%) and dry matter (by 0.30–0.50%) in whey than MCEs of Naturen brands, Chymax Extra and Chy-max Supreme. Cheese variants made using different types and doses of MCEs did not have statistically accurate differences in the content of dry matter, fat, protein, salt and pH (p&gt;0.05). The use of MCEs of Naturen, Chy-max Extra and Chy-max Supreme brands at a dosage of 5,000–6,000 IMCU/100 kg of milk gives an average of 1.5% higher actual cheese yield compared to MCEs of the Fromase and “Pepsin” brands at an introduction dose of 1500 IMCU/100 kg of milk.

Enzymatic proteolysis during the conversion of milk into cheese

The transformation of milk into cheese occurs under the influence of many physicochemical, biochemical and microbiological processes, among which proteolysis plays a very important role. Proteolysis belongs to the most complex type of irreversible post-translational modification of proteins. Enzymatic proteolysis catalysts at different stages of cheese production are native milk enzymes, exo- and endopeptidases of starter and non-starter microorganisms, and milk-clotting enzymes. The article presents a brief overview of modern ideas about the properties, mechanism of action and specificity of the main representatives of enzymes that hydrolyze milk proteins at the stages of preparing milk for coagulation, during rennet coagulation and subsequent maturation of cheeses. These include the plasmin system of milk, enzymes of psychrotrophic bacteria and lactic acid microorganisms that enter milk both accidentally (non-starter microflora) and planned in the form of starter cultures from specially selected strains. Milk-clotting enzymes, having fulfilled their main function — milk coagulation — partially pass into cheese and, along with enzymes of starter microorganisms and plasmin, participate in proteolytic processes during cheese ripening. It is generally accepted that proteolysis in ripening cheeses is the most significant biochemical process that affects the formation of taste, aroma and texture along with lipolysis and glycolysis. The combination of proteolysis products (peptides, amino acids, amines, etc.) is individual for different types of cheese and varies depending on the technological parameters of production, including the duration of maturation. Proteolysis in cheeses has been studied by many scientists in various aspects. This review supplements the known information with new information, without claiming to be comprehensive.

Characterization of a Novel Milk-Clotting Aspartic Protease from Penicillium sp. and Structural Explanation for its High Milk-Clotting Index.

A novel milk-clotting enzyme isolated from Penicillium sp. ACCC 39790 (PsMCE) was prepared by heterologous expression. The recombinant PsMCE had an apparent molecular mass of 45 kDa and exhibited maximum casein hydrolysis activity at pH 4.0 and 50 °C. The PsMCE activity was enhanced by calcium ions and strongly inhibited by pepstatin A. Through hydrolysis pattern and cleavage site analyses, the milk-clotting activity of PsMCE was related to its specific hydrolysis between Phe105 and Met106 in the κ-casein proteins. The structural basis of PsMCE was characterized using homology modeling, molecular docking, and interactional analysis. The P1' region of PsMCE is critical for its selective binding to the hydrolytic site in κ-casein, and the hydrophobic forces play a decisive role in the specific cleavage of Phe105 and Met106. These interactional analyses between PsMCE and the ligand peptide clarified the fundamentals of its high milk-clotting index (MCI). PsMCE could be applied in cheese making due to its thermolability and high MCI value as a potential milk-clotting enzyme.