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Microbial milk-clotting enzymes present an economically viable alternative to traditional calf rennet, attributed to their expedited production cycles and facile fermentation processes. The objective of this study was to screen for novel microbial strains that produce milk-clotting enzymes with both high activity and minimal proteolytic effects. Molds isolated from traditional sufu served as the starting material for this selection process. Strains demonstrating high milk-clotting activity and low proteolytic activity were identified through ITS and mt SSU rDNA amplification sequencing for molecular characterization. Enzyme purification was conducted using a sequence of ammonium sulfate fractionation, DEAE-Sepharose Fast Flow anion exchange chromatography, and Sephadex G100 gel filtration chromatography. Subsequent analysis of the enzymatic properties of the purified milk-clotting enzyme was performed. Strain CQ3 emerged as a prominent candidate, showing substantial milk-clotting activity with reduced proteolytic effects, and was identified as Trametes versicolor. SDSPAGE analysis of the purified enzyme from strain CQ3 revealed a single band corresponding to a molecular weight of 61 kDa. The enzyme exhibited optimal activity at 45°C and a pH of 6.5, with Ca2 + markedly enhancing its activity. Stability tests demonstrated consistent enzyme performance within a pH range of 4.5 - 7.0 and at temperatures below 50°C. Protease inhibitor assays classified the enzyme as an aspartic protease. These results document a novel source of fungal strains for milk-clotting enzyme production, laying a theoretical groundwork for further assessment of Trametes versicolor enzymes in cheese manufacturing applications. [ABSTRACT FROM AUTHOR]

ABSTRACT: 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

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 m M CaCl 2. 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. [Display omitted] [ABSTRACT FROM AUTHOR]

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. 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. 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.

Abstract The milk-clotting enzyme (MCE) is a crucial ingredient in cheese manufacture. Due to the limits of traditional MCE, finding viable substitute is a pressing issue. This study aims to isolate and identify a wild strain with high milk-clotting activity (MCA) and low proteolytic activity (PA) and optimize the fermentation conditions for MCE production. A strain of Bacillus velezensis DB219 with high MCA/PA value (9.2) was isolated from dairy soil (Wuchang, Heilongjiang, China) and identified through 16S rRNA from 40 strains. The optimal wheat bran, carbon, nitrogen, inoculum size, volume and initial pH were 60 g/L, soluble starch 12.5 g/L, corn steep liquor 3 g/L, 5%, 40 mL and 6.15, respectively for improving DB219 MCE production through single factor experiment. The wheat bran concentration, corn steep liquor concentration and volume were the most critical factor and their changed range was determined through Plackett–Burman design and the steepest ascent/descent experiments. The response surface analysis experiment of three factors and three levels was conducted by Box–Behnken design. The theoretical optimal fermentation conditions for DB219 MCE were as follows: wheat bran concentration 60.14 g/L, soluble starch 12.5 g/L, corn steep liquor 3 g/L, inoculum size 5%, volume 40.08 mL and initial pH 6.15. DB219 MCE achieved the maximal MCA (3164.84 SU/mL) that was 101.9% of the predicted value (3104.49 SU/mL) and 4.3-fold higher than the control.

The milk-clotting enzyme (MCE) is a crucial ingredient in cheese manufacture. Due to the limits of traditional MCE, finding viable substitute is a pressing issue. This study aims to isolate and identify a wild strain with high milk-clotting activity (MCA) and low proteolytic activity (PA) and optimize the fermentation conditions for MCE production. A strain of Bacillus velezensis DB219 with high MCA/PA value (9.2) was isolated from dairy soil (Wuchang, Heilongjiang, China) and identified through 16S rRNA from 40 strains. The optimal wheat bran, carbon, nitrogen, inoculum size, volume and initial pH were 60 g/L, soluble starch 12.5 g/L, corn steep liquor 3 g/L, 5%, 40 mL and 6.15, respectively for improving DB219 MCE production through single factor experiment. The wheat bran concentration, corn steep liquor concentration and volume were the most critical factor and their changed range was determined through Plackett–Burman design and the steepest ascent/descent experiments. The response surface analysis experiment of three factors and three levels was conducted by Box–Behnken design. The theoretical optimal fermentation conditions for DB219 MCE were as follows: wheat bran concentration 60.14 g/L, soluble starch 12.5 g/L, corn steep liquor 3 g/L, inoculum size 5%, volume 40.08 mL and initial pH 6.15. DB219 MCE achieved the maximal MCA (3164.84 SU/mL) that was 101.9% of the predicted value (3104.49 SU/mL) and 4.3-fold higher than the control. [ABSTRACT FROM AUTHOR]

Calf rennet has been traditionally used for cheese making all over the world since ancient times. It is primarily a type of aspartic protease. Calf rennet, also known as chymosin, is considered the best milk coagulant in cheese manufacturing. Its usage and demand are increasing day by day in the food industry; however, some ethical issues are also related since it is naturally present in the calf's stomach and obtained by the slaughtering of young animals. Therefore, researchers are trying to introduce some new and better alternatives for chymosin in the food industry. Mucor racemosus f. racemosus CBS 381, Mucor racemosus DSM 62760, and Aspergillus oryzae were cultivated by solid substrate fermentation using the design of experiment (DoE) (MODDE; Umetrics, Sweden) to optimize and analyze the various combinations of different factors and responses (milk‐clotting activity, proteolytic activity, specific activity). Based on the analysis of the screening and optimization results, Mucor racemosus CBS 381 was found to be the potential strain in terms of high production of aspartic protease, as well as had high milk‐clotting activity under a solid‐state fermentation system. However, molasses and casein were determined to be significant carbon and nitrogen sources, respectively, under conditions such as 70% moisture content and 25°C temperature. The molecular weight of the enzyme (Mucor CBS 381) is ∼30 KDa and it exhibits maximum activity at pH 4.8 at 45°C. The investigated enzyme was pronounced as thermal‐sensitive and lost activity completely after 10 min incubation at 55°C. The remarkable qualities of the studied enzyme, such as cost‐effective production, milk‐clotting and proteolytic activity make Mucor racemosus CBS 381 a promising alternate to calf chymosin in the cheese‐making industry. Practical Application: The milk‐clotting enzyme (aspartic protease) produced by the Mucor racemosus is the alternative to calf chymosin. It can be used to produce cheese on the industrial level with some desired properties such as good taste and texture that includes gumminess. Nowadays, consumers prefer products that do not involve any animal cruelty whereas a huge group of consumers oppose the use of genetically modified enzymes. Therefore, the enzyme by Mucor racemosus would produce the cheese that is going to meet the demands of various types of cheese consumers. [ABSTRACT FROM AUTHOR]

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, 2 enzymes were considered as vacuole enzymes according to a study on A. niger proteases. The 2 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. [ABSTRACT FROM AUTHOR]

In the cheese-making industry, commonly chymosin is used as the main milk-clotting enzyme. Bactrian camel (Camelus bactrianus) chymosin (BacChym) has a milk-clotting activity higher than that of calf chymosin for cow’s, goat’s, ewes’, mare’s and camel’s milk. A procedure for obtaining milk-clotting reagent based on recombinant camel chymosin is proposed here. Submerged fermentation by a recombinant yeast (Pichia pastoris GS115/pGAPZαA/ProchymCB) was implemented in a 50 L bioreactor, and the recombinant camel chymosin was prepared successfully. The activity of BacChym in yeast culture was 174.5 U/mL. The chymosin was concentrated 5.6-fold by cross-flow ultrafiltration and was purified by ion exchange chromatography. The activity of the purified BacChym was 4700 U/mL. By sublimation-drying with casein peptone, the BacChym powder was obtained with an activity of 36,000 U/g. By means of this chymosin, cheese was prepared from cow’s, goat’s, ewes’, camel’s and mare’s milk with a yield of 18%, 17.3%, 15.9%, 10.4% and 3%, respectively. Thus, the proposed procedure for obtaining a milk-clotting reagent based on BacChym via submerged fermentation by a recombinant yeast has some prospects for biotechnological applications. BacChym could be a prospective milk-clotting enzyme for different types of milk and their mixtures.

Bacillus subtilis is a generally recognized as safe probiotic, which is used as a starter for natto fermentation. Natto is a functional food with antithrombus function due to nattokinase. Compared with natto, fermented milk is a more popular fermented food, which is commonly fermented by Lactobacillus bulgaricus and Streptococcus. However, there is no report on B. subtilis –fermented milk. In this study, to produce a functional fermented milk with antithrombus function, a B. subtilis strain (B. subtilis JNFE0126) that produced both nattokinase and milk-clotting enzyme was isolated from traditionally fermented natto and used as the starter for the functional fermented milk. In liquid fermentation culture, the peak values of thrombolytic activity and milk-clotting activity were 3,511 U/mL at 96 h and 874.5 Soxhlet unit/mL at 60 h, respectively. The optimal pH and temperature were pH 7.0 at 40°C for nattokinase and pH 6.5 and 55°C for milk-clotting enzyme, respectively. The thrombolytic activity in the fermented milk reached 215.1 U/mL after 8 h of fermentation. Sensory evaluation showed that the acceptance of the milk fermented by B. subtilis JNFE0126 was similar to the traditional milk fermented by L. bulgaricus and S. thermophilus. More importantly, oral intake of the fermented milk by the thrombosis-model mice prevented the development of thrombosis. Our results suggest that B. subtilis JNFE0126–fermented milk has potential as a novel, functional food in the prevention of thrombosis-related cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Purpose: This study was conducted to find a microbial milk-clotting enzyme (MCE) with a high and stable milk-clotting activity (MCA) to proteolytic activity (PA) ratio suitable for the cheese industry. Methods: Microbial strains were isolated from soil suspensions cultured in solid casein medium. 16S rDNA of representative isolates were sequenced to identify the microbial species. Nutrition and fermentation conditions were systematically examined to optimize MCA of the selected MCE. Protease inhibitors were used to identify the type of MCE. The casein hydrolysis was analyzed through reversed-phase HPLC (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: The Bacillus licheniformis BL312 was identified from 50 bacterial strains. BL312 MCE achieved a maximal MCA (460 ± 15 SU/mL) at 48 h that was 2.7-fold higher than the control, and the MCA/PA ratio (9.0) and pH (6.6) remained stable throughout the fermentation process. Medium containing 30 g/L wheat bran shorts, 5 g/L glucose, and 3 g/L corn steep liquor was sufficient for optimal BL312 MCE production. Fermentation conditions of an inoculum size of 7.0% (v/v), fermentation temperature of 37 °C, agitation speed of 210 rpm, and initial pH 6.6 were required to achieve maximal MCA. BL312 MCE was inhibited by phenylmethanesulfonyl fluoride (PMSF) and high concentrations of ethylenediaminetetraacetic acid (EDTA) (5–25 mM). The αs-casein (αs-CN) and β-casein (β-CN) hydrolysates generated by BL312 MCE and calf rennet were different. Conclusions: BL312 MCE is a serine/alkaline protease that exhibits high MCA and various hydrolysis for caseins in comparison with calf rennet. [ABSTRACT FROM AUTHOR]

Screening of the fungal strains, Aspergillus niger, Trichoderma harzianum, and Rhizopus microsporus var. chinensis for protease production was realised by monitoring the presence of clear zones on skimmed milk agar plates. Additionally, screening of the radial growth on vanilla waste, cane bagasse, and pineapple crown plates was performed. Radial growth data of the fungal strains on skimmed milk agar and agro-industrial waste plates were adjusted to the Gompertz model. The results indicate that R. microsporus var. chinensis and pineapple crown were adequate for the production of milk-clotting enzymes by solid-state fermentation. Crude enzyme extract was produced via solid-state fermentation using two particle sizes [2–2.6 mm (SSF1) and a mixture of less than 1 mm with 2–2.6 mm, at a ratio of 1:1(SSF2)]. The highest milk-clotting and proteolytic activities were shown within 12 h and 48 h of fermentation for SSF1 and SSF2, respectively. The optimum milk-clotting activity presented by the crude enzyme extract was at pH 6.5, 40 °C, and 0.04 M CaCl2. The ratio of milk-clotting activity to proteolytic activity (9.7) presented by the crude enzyme extract indicates potential for use as a calf rennet substitute. [ABSTRACT FROM AUTHOR]

[Display omitted] • High MCE-producing Bacillus megaterium strain LY114 was isolated from soil. • LY114 MCE was metalloprotease and characterized with remarkable specific activity. • LY114 MCE showed suitable characteristics and unique hydrolysis for cheese making. • LY114 MCE was successfully used in cheese making and had great sensory. • LY114 MCE had the potential to be a new traditional MCE substitute. 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 CaCl 2. The Michaelis constant (K m) and maximal velocity (V m) 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. [ABSTRACT FROM AUTHOR]

World nutritional science recognizes cheese as a nutritious, biologically wholesome, an easily digestible product. It is an indispensable and essential component of the human diet. In recent years the market of cheeses has changed. Instead of planned deliveries to the industrial centers and the regions of the North and the Far East, shipments of cheese for export and laying it for long-term storage the industry turned to the sales of products under direct contracts with consumers. The changes occurred in the relationship between milk producers, dairy industry and trade, as well as the need for a sharp increase in production of cheeses put forward the task of finding sound technologies, the range of schemes and the organization of cheese production. The main ones are smoothing the seasonality in the production of cheese and the creation of technologies to reduce production cycles. The purpose of this paper is to examine the characteristics and prospects for the production of soft cheese. Depending on the features of production and technological parameters soft cheeses can be divided into several independent groups with different types of milk clotting, applicable bacterial preparations, ripening conditions, temperature and time regimes generation, use of raw materials of non-dairy origin and fungi of the genus of and some other factors. The basis of the production of soft rennet cheeses is the clotting of milk. It comes under the influence of two agents (milk-clotting enzyme and bacterial starter). The work presents characteristics of individual groups of soft cheeses. The influence of the quantity of bacterial starter on the duration of milk coagulation, using different doses of milk coagulating enzymes at different temperature has been investigated. The comparative evaluation of the organoleptic and physico-chemical indices in individual groups of soft cheeses is given. The dynamics of microflora in the development of mild acid-rennet cheese has been studied.

Enzymes obtained from different sources and made ready for further use, which also coagulate caseins, are called milk coagulant enzyme, cheese rennet or rennet. For thousands of years, rennet (in general rennin/chymosin) from the abomasus section in stomachs of young ruminants animals has been obtained, and it has been used as milk coagulant in cheese making. As cheese production and consumption in the world have been increasing gradually, the steady declining supply of calf rennet has accelerated the acquisition of enzymes that can be used in place of alternative sources of calf rennet, and the search for highly efficient production methods has become compulsory. Studies on alternative sources for calf rennet are categorized as (i) the coagulants obtained from herbal sources (herbal rennet), (ii) the rennet source, the microbial cheese rennets consisting of proteolytic enzymes and obtained directly from microorganisms (microbial rennet), (iii) the transfer of genetic material responsible for rennin enzyme production to certain microorganisms, (recombinant chymosin), which is composed of enzymes identical to the renin enzyme that it produces. Recombinant chymosin is rapidly becoming a subject of research together with developing gene technology. In this review, the effects of animal, plant and microbial origin enzymes/cheese rennets on milk clotting and cheese ripening stages are discussed. [ABSTRACT FROM AUTHOR]

Cucumisin [EC 3.4.21.25] was first purified from Cucumis melo var. reticulatus juice by three-phase partitioning (TPP). Optimum purification parameters of the TPP system were determined as 60% ammonium sulfate saturation with 1.0:1.25 ratio of crude extract: t -butanol at pH and temperature of 8.0 and 20 °C, respectively. Cucumisin was purified with 4.61 purification fold and 156% activity recovery. The molecular weight of the recovered cucumisin was determined as 68.4 kDa and its isoelectric point is 8.7. Optimum pH and temperature of cucumisin were pH 9.0 and 60–70 °C, respectively. The protease was very stable at 20–70 °C and a pH range of 2.0–12.0. Km and V max constants were 2.24 ± 0.22 mgmL −1 and 1048 ± 25 μ Mmin −1 , respectively. The enzyme was stable against numerous metal ions and its activity was highly enhanced by Ca 2+ , Mg 2+ , and Mn +2 . Cucumisin activity was 2.35-folds increased in the presence of 5 mM of CaCl 2 . It was inactivated by Co 2+ , Cd 2+ , Zn 2+ and Fe 2+ and dramatically by PMSF. Cucumisin milk-clotting activity was highly stable when stored under freezing (−20 °C) compared at 4 °C and 25 °C. Finally, TPP revealed to be a useful strategy to concentrate and purify cucumisin for its use as a milk-clotting enzyme for cheese-making. [ABSTRACT FROM AUTHOR]

Background: Present study aims to isolate and optimize fermentation conditions of milk-clotting enzyme producing rhizobacteria Bacillus amyloliquefaciens SP1. Results: A bacterium producing an extracellular milk-clotting enzyme (MCE) was isolated from the rhizosphere of the planted population of apple trees growing at Distt. Chamba of Himachal Pradesh, India. According to morphological, physiological, biochemical, and molecular characterization, isolate was identified as B. amyloliquefaciens. Single-factor testing was used to study the optimum physical conditions and nutritional parameters for production of the MCE per proteolysis activity which insures its usefulness as new source of milk coagulant for cheese making. The optimum conditions for production of the milk-clotting enzyme were: temperature, 30 °C; inoculum size, 1 %; and initial pH of the medium, 6.0. The maximum milk-clotting activity and milk-clotting activity per proteolysis activity were found using soyabean as nitrogen source and sucrose as carbon source. Conclusion: These optimized conditions resulted in a 1.9-fold increase in production of the milk-clotting enzyme. This study reported a plant growth promoting rhizobacteria, i.e., B. amyloliquefaciens as source of milk-clotting enzyme which has potential as a calf rennet substitute. [ABSTRACT FROM AUTHOR]

This study investigated the influence of high pressure processing (HPP) on the proteolytic and milk-clotting activities of calf rennet and milk coagulation using processed enzyme by rheological assay and confocal microscopy. The process was carried out at 25 °C, using pressure range from 50 to 300 MPa and time between 5 and 30 min. It was found that HPP (175–285 MPa for 14–23 min) increased the enzyme proteolytic activity by up to 23% and the milk-clotting activity by up to 17%. Furthermore, the G’ values obtained during milk coagulation were higher for calf rennet processed at 280 MPa for 20 min than for the non-processed enzyme, forming more consistent gels (25.8% higher G′ value after 90 min). The evaluation of the milk coagulation by confocal microscopy confirms the results obtained on the rheology. Pretreatment of calf rennet using HPP accelerates the coagulation of milk and produces firmer and more consistent gels. [ABSTRACT FROM AUTHOR]

Amylomyces rouxii is one of the main fungi usually coexisting with yeasts in Chinese yeast ball, the starter of chiu-niang, a traditional Chinese fermented product from rice. In the present study, growth and production of milk-clotting enzyme (MCE) in gelatinous rice liquid culture of A. rouxii as influenced by waxy (gelatinous) rice content in the medium (5–20 %), temperature (25–40 °C), cultivation time (1–6 days), shaking speeds (0–150 rpm) and metal ions (Na+, K+, Zn2+, Mg2+, Mn2+, Cu2+, Ca2+, Fe3+ and Al3+) were investigated. Results revealed that rice content in the medium, shaking speed, temperature and cultivation time all affected the mycelial propagation and the production of milk-clotting enzyme by A. rouxii in the rice liquid culture. The maximum milk-clotting enzyme activity of ca. 1.22 unit/mL of medium was observed in the 3-day static culture of test organism grown at 30 °C in the medium containing 20 % of gelatinous rice, while mycelial propagation increased with the increase of cultivation time and shaking speed. Furthermore, a significant increase (p

The need to surmount the limitation of obtaining rennin, has been actively pushed researches to find new substitutes that present high milk-clotting activity which enables the production of high yields of cheese. In this study, the production of extracellular milk-clotting protease by locally isolated fungal specie, Aspergillus niger FFB1 under solid-state fermentation (SSF) using cheep agro-industrial byproduct (wheat bran) was optimized. The effects of several physicochemical and environmental factors were investigated to select the optimal conditions that ensure the best milk-clotting activity by application of "One-factor-at-a-time" method. A trial of cheese production using the crude extract was also carried out. The maximum enzyme activity (830 SU/g bran with a ratio MCA/PA of 4.25) was obtained under the optimum conditions of temperature (30?C), spores concentration (106 spores/mL), incubation time (72 hours), and moisture content of solid substrate (39.2%) adjusted suitably with mineral solution (Czapek-Dox) of pH 4. [ABSTRACT FROM AUTHOR]

Breadfruit is an exotic tree in Brazil, very well acclimatized. Despite of being a laticifer plant, the knowledge about its latex is scarce. However, it is known that proteolytic enzymes represents over 50% of the latex composition in laticifers plants. The aim of this study was to evaluate the potential of breadfruit (Artocarpus altilis var. Apyrena) latex as a source of milk clotting proteases and partially characterize it. The proteolytic activity of the fraction of crude extract was assessed using azocasein and quantitation of total protein, the bicinchoninic acid (BCA). Milk-clotting activity was analyzed using skim milk at 12%. The enzyme activity was analyzed under different temperature conditions (35 to 80°C) and pH (5.8 to 10.7) presenting optimum activity in alkaline pH (8.5) and 50°C; being stable to the two variables at 120 minutes during the test. The clotting activity was directly proportional to the temperature at the better concentration of CaCl2 (10mmol L-1). The results indicate that enzyme is a possible replacement for calf rennet. [ABSTRACT FROM AUTHOR]

The hydrolysis of milk proteins by the recombinant AprX-SK37 protease and the changes in the rheological properties of the milk gel generated with Apr X- SK37 and glucono-δ-lactone ( GDL) were investigated. The Apr X- SK37 and rennet selectively hydrolysed κ-casein to yield a 16-kDa band, while subtilisin hydrolysed all of the casein components. Milk treated only with Apr X- SK37 formed softer gel. Storage modulus (G′) values of the combined gels increased with GDL concentrations up to 7 g/L. High tan δ was observed in the combined gel at 8.75 g/L GDL alongside syneresis. AprX-SK37 is a promising milk-clotting enzyme when combined with an optimal GDL concentration. [ABSTRACT FROM AUTHOR]

Abstract: Submerged cultured higher basidiomycetes were screened for milk-clotting activity. The native liquid of Coprinus lagopides demonstrated high milk-clotting activity, while having relatively low general proteolytic activity. Optimization of growth media composition and ultrafiltration of the native liquid allows increasing the ratio of milk-clotting and proteolytic activities. The Enzyme was purified and characterized. [Copyright &y& Elsevier]

In order to improve the production of the milk-clotting enzyme under submerged fermentation, two statistical methods were applied to optimize the culture conditions of Bacillus amyloliquefaciens D4 using wheat bran as nutrient source. First, initial pH, agitation speed, and fermentation time were shown to have significant effects on D4 enzyme production using the Plackett-Burman experimental design. Subsequently, optimal conditions were obtained using the Box-Behnken method, which were as follows: initial pH 7.57, agitation speed 241 rpm, fermentation time 53.3 h. Under these conditions, the milk-clotting enzyme production was remarkably enhanced. The milk-clotting enzyme activity reached 1996.9 SU/mL, which was 2.92-fold higher than that of the initial culture conditions, showing that the Plackett-Burman design and Box-Behnken response surface method are effective to optimize culture conditions. The research can provide a reference for full utilization of wheat bran and the production of milk-clotting enzyme by B. amyloliquefaciens D4 under submerged fermentation. [ABSTRACT FROM AUTHOR]

Aspartic proteinases are an important class of proteinases which are widely used as milk-coagulating agents in industrial cheese production. They are available from a wide range of sources including mammals, plants, and microorganisms. Various attempts have been made in order to get insights into enzyme structure/function relationships for designing improved biocatalysts. This review provides an overview of historical background and recent achievements on the classification and structural characteristics of such enzymes as related to their functional properties, mechanism of catalysis, pH, and temperature dependence, substrate specificities, mechanism of inhibition, enzyme engineering, and technological applications with the focus on cheese manufacturing. [ABSTRACT FROM AUTHOR]

To enhance the yield and productivity of milk-clotting enzyme (MCE) by Bacillus amyloliquefaciens, a two-stage oxygen supply control strategy was proposed and successfully applied in the MCE fermentation. During the first 16 h, Ka was controlled at 72.2 h to obtain high cell growth rate ( v) and MCE activity (MCA) productivity ( r). Subsequently, Ka was controlled at 33.9 h to maintain high specific MCA productivity ( q). Using this strategy, MCA peaked at 36 h with the MCA of 6,590.41 SU ml, which was 18 h earlier than other investigated processes. The concept and results described represent the basis of an industrial scale-up process to achieve high MCE yield, MCA productivity and MCA/proteolytic activity. [ABSTRACT FROM AUTHOR]

Abstract: A novel milk-clotting serine protease, named religiosin B, is purified from Ficus religiosa. The molecular mass of the protein is 63,000 with pI value of pH 7.6. The proteolytic activity of the enzyme is strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and chymostatin. Religiosin B acts optimally at pH 8.0–8.5 and temperature 55°C. The molar absorption coefficient of the enzyme is 149,725M−1cm−1 with 23 tryptophan, 15 tyrosine and 7cysteine residues per molecule of the enzyme. The enzyme shows broad substrate specificity with natural as well as synthetic substrates. Religiosin B is highly stable against denaturants and metal ions as well as over a wide range of pH and temperature. The de novo sequencing confirms the novelty of the enzyme. In addition to its high milk-clotting ability, it could be used in the cheese industry, as well as other food and biotechnological industries. [Copyright &y& Elsevier]

A milk-clotting enzyme named YS-1 was purified from a Bacillus subtilis ( B. subtilis) YB-3, which we have isolated from Tibetan Plateau, Gansu, China. The enzyme YS-1 was identified as a metalloproteinase. SDS-PAGE and MALDI-TOF-MS analysis of the purified enzyme gave a molecular weight of 42 kDa. YS-1 was stable over a wide range of temperature from 20 to 60 °C. Purified YS-1 was also active over a wide range of pH from 5.0 to 9.0. It can be activated by Ca and Al but inhibited by Zn, Fe and Cu. The milk-clotting enzyme YS-1 exhibited high specificity to substrate β-casein and yak milk casein and led to a 75% more rapid coagulation of yak milk than cow milk, due to high β-casein content in yak milk. Together, our findings confirmed that the enzyme YS-1 has a potential to be used in yak cheese industry. [ABSTRACT FROM AUTHOR]

Milk-clotting enzymes produced by microorganisms have been developed to replace calf rennet, yet the enzymatic level and the ratio of milk-clotting activity to proteolytic activity still need further improvement. This work described a strain Bacillus amyloliquefaciens JNU002 that screened from wheat bran that has a promising characterization. After optimization, B. amyloliquefaciens JNU002 showed a high milk-clotting activity (4969 SU/mL) and low proteolytic activity (4.02 U/mL) at 48 h with an inoculum size of 0.2% (v/v) at initial pH 6.0 in 15-L bioreactor. After purification, the purified enzyme gave a single protein band on SDS-PAGE, corresponding to 28 kDa. The purified enzyme showed a high ratio (2,575) of milk-clotting activity to proteolytic activity at 35 °C, and the ratio would be even higher (22,992) at 70 °C. The milk-clotting reaction and proteolytic reaction were prevented at 75 °C. This enzyme was stable at pH 4-6 and below 40 °C, and this was convenient for storage and transportation. [ABSTRACT FROM AUTHOR]

A bacterium producing an extracellular milk-clotting enzyme was isolated from yak grazing soil in the Tianzhu Tibetan autonomous county on the Tibetan Plateau and identified as Bacillus amyloliquefaciens. We used single-factor testing to study the optimum physical conditions and nutritional parameters for production of the milk-clotting enzyme, while the temperature and pH stability of the enzyme were also studied. The optimum conditions for production of the milk-clotting enzyme were: temperature, 37°C; inoculum size, 4% (1.8 × 10 CFU/mL); agitation speed, 170 rpm; and initial pH of the medium, 6.2. The maximum milk-clotting activity and milk-clotting activity per proteolysis activity were found using wheat bran juice at a concentration 18 g/100 mL, 4% (w/v) sucrose and 2% (w/v) skim milk powder. These optimized conditions resulted in a 2.68-fold increase in production of the milk-clotting enzyme. The crude enzyme exhibited good temperature and pH stability; 80% of the milk-clotting activity was retained after incubation at 40°C for 30 min and more than 70% of the activity was retained between pH 5.5 and 7.5. This B. amyloliquefaciens milk-clotting enzyme has potential as a calf rennet substitute. [ABSTRACT FROM AUTHOR]

Abstract: Prato cheeses were manufactured using coagulant from Thermomucor indicae-seudaticae N31 or a commercial coagulant. Cheeses were characterised using the following analysis: yield; fat; acidity; moisture; ash; salt; pH; total nitrogen; total protein; NS-pH 4.6/NT*100; NS-TCA 12%/NT*100; casein electrophoresis; and RP-HPLC. The results were statistically analysed and revealed that the proteolytic indices were not significantly different throughout the 60days of ripening of cheeses made with either coagulant. Even though there were some quantitative differences in the peptide profile of cheeses, the enzyme from T. indicae-seudaticae N31 was used in the production of good quality Prato cheese without having to change the established technological parameters of the process. [Copyright &y& Elsevier]

The milk-clotting enzyme from Bacillus amyloliquefaciens D4 was purified to 17.2-fold with 20% recovery by precipitation in ammonium sulfate and ion-exchange chromatography. The molecular mass of the enzyme was 58.2 kDa as determined by SDS-PAGE, and it was proved to be a metalloprotease by EDTA inhibition. The enzyme was active in the pH range 5.5-7.0 and was inactivated completely by heating at 55 °C for 20 min. The highest level of enzyme activity was obtained at 65 °C, pH 5.5, in the presence of 25mM CaCl. The milk-clotting activity was inhibited only slightly by Na and K and significantly by Cu, Zn and Sn. The K value of this enzyme was 0.471 mg/mL. The high level of milk-clotting activity coupled with a low level of thermal stability suggested that the milk-clotting enzyme from B. amyloliquefaciens D4 should be considered as a potential substitute for calf rennet. [ABSTRACT FROM AUTHOR]

Glutinous rice wine mash liquor is a traditional food of south of China and its ability to coagulate the milk has been proved. The aim of this work was to extract milk-clotting enzyme from glutinous rice wine mash liquor. A partial purified extract of enzyme was obtained by fractional precipitation with (NH4)2SO4. The fractions obtained by precipitation, 40–90% possessed the milk-clotting activity (MCA) (145.72 U/mg). The 40–90% (NH4)2SO4 fraction was further purified by sephadex G-100 and DEAE-sephadex A-50 with MCA (4,360±50 U/mg), which was confirmed by SDS-PAGE that showed only one band with a molecular mass of 36.0 kDa. Highest MCA was attained at 36 °C. The enzyme was completely inactivated by heating for 20 min at 60 °C. The MCA increased with the decreasing of milk pH from 8.0 to 5.5, and it was active at the wide range of pH 1 to 7. The metal ions Mg2+, Ca2+, Ba2+, Mn2+, Al3+, Fe2+ had a very clear function to accelerate milk coagulation whereas Na+ and K+ decelerated the activity slightly. The curd effect of the milk-clotting enzyme has primarily been studied. [ABSTRACT FROM AUTHOR]

Abstract: Factors affecting the production of milk-clotting enzyme (MCE) by Bacillus subtilis (natto) Takahashi, a ready available commercial natto starter, were studied. Remarkable milk-clotting activity (MCA), 685.7SU/ml or 12,000SU/g, was obtained when the bacteria were cultivated in the medium containing sucrose (50g/L) and basal salts at pH 6, 37°C with shaking at 175rpm for 1 day. The MCA and MCA/PA ratio of the crude enzyme obtained are comparable with those of Pfizer microbial rennin and Mucor rennin. The crude enzyme showed excellent pH and thermal stability; it retained 96% of MCA after incubation for 40min at 40°C and retained more than 80% of its activity between pH 4 and pH 7 for more than 30min at 30°C. The MCE of B. subtilis (natto) Takahashi has potential as calf rennet substitutes. [Copyright &y& Elsevier]

Numerous attempts have been made to replace calf rennet with other milk clotting proteases because of limited supply and increasingly high prices. The aim of this work was to investigate the characteristic of the milk-clotting enzyme from Nocardiopsis sp. The partial purification extract was obtained by fractional precipitation with ammonium sulphate. Of the fractions obtained by precipitation, 40–60% possessed the milk-clotting activity (156.25 U/mg). The chromatography of 40–100% ammonium sulphate fraction in DEAE-cellulose yielded four fractions (F4, F5, F6, F7) with milk-clotting activity. The F5 yielded the best milk-clotting activity (20 U/ml). Both crude and partially purified extract were active at the range pH 4.5–11.0, however, optimum activity was displayed at pH 11.0 and pH 7.5, respectively. The milk-clotting activity was highest at 55 °C for both crude and partially purified extract. The crude and partial purification extract were inactivated at 65 and 75 °C after 30 min. [Copyright &y& Elsevier]

Milk-clotting enzyme is considered largely denatured after the cooking step in hard cheeses. Nevertheless, typical hydrolysis products derived from rennet action on α1-casein have been detected during the ripening of hard cheeses. The aim of the present work was to investigate the influence of residual milk-clotting enzyme on αs1-casein hydrolysis in Reggianito cheeses. For that purpose, we studied the influence of cooking temperature (45, 52, and 60°C) on milk-clotting enzyme residual activity and αs1-casein hydrolysis during ripening. Milk-clotting enzyme residual activity in cheeses was assessed using a chromatographic method, and the hydrolysis of αs1-casein was determined by electrophoresis and high performance liquid chromatography. Milk-clotting enzyme activity was very low or undetectable in 60°C- and 52°C-cooked cheeses at the beginning of the ripening, but it increased afterwards, particularly in 52°C-cooked cheeses. Cheese curds that were cooked at 45°C had higher initial milk clotting activity, but also in this case, there was a later increase. Hydrolysis of αs1-casein was detected early in cheeses made at 45°C, and later in those made at higher temperatures. The peptide αs1-I was not detected in 60°C-cooked cheeses. The results suggest that residual milk-clotting enzyme can contribute to proteolysis during ripening of hard cheeses, because it probably renatures partially after the cooking step. Consequently, the production of peptides derived from αs1-casein in hard cheeses may be at least, partially due to this proteolytic agent. [ABSTRACT FROM AUTHOR]

Site-directed mutagenesis was carried out to investigate the functional roles of amino acid residues of Rhizomucor pusillus pepsin (RMPP) in substrate-binding and catalysis. Mutations of two amino acid residues, E13 in the S3 subsite and N219 in the S3/S4 sub-sites, caused marked changes in kinetic parameters for two substrate peptides with different sequences. Further site-directed mutagenesis at E13 suggested that E13 plays a critical role in forming the correct hydrogen bond network around the active center. In the crystal structure of Rhizomucor miehei pepsin (RMMP), which is an aspartic proteinase produced by Rhizomucor miehei and shows 81% amino acid identity to RMPP, the Oe atom of N219 forms a hydrogen bond with the N-H of isovaline in pepstatin A, a statine-type inhibitor, at the P3 position, suggesting that the loss of the hydrogen bond causes an unfavorable arrangement of the P3 residue. Among the mutants constructed, the E13A mutant showed a 5-fold increase in the ratio of clotting versus proteolytic activity without significant loss of clotting activity. This mutant may present a promising candidate for a useful milk coagulant. [ABSTRACT FROM AUTHOR]

[Display omitted] • The sources and types of calf rennet substitutes were comprehensively summarized. • The advantages and disadvantages of different calf rennet substitutes were discussed. • Characteristics and modification of microbial milk-clotting enzymes were elaborated. • The influence of calf rennet substitutes on cheese quality was summarized. • Future perspective and challenges of calf rennet substitutes were proposed. Milk coagulation is an important step in cheese production, and milk-clotting enzymes (MCEs) play a major role in this process. Calf rennet is the most widely used MCE in the cheese industry. The use of calf rennet substitutes is becoming necessary due to the limited availability of calf rennet and the increase in cheese consumption. The objective of this review is to summarize the latest findings on calf rennet substitutes (animal MCEs, plant-derived MCEs, recombinant MCEs and microbial MCEs) and their application in cheese production. Special emphasis has been placed on aspects of the effects of these substitutes on hydrolysis, functional peptides, cheese variety and cheese yield. The advantages and disadvantages of different calf rennet substitutes are discussed, in which microbial MCEs have the advantages of less expensive production, greater biochemical diversity, easier genetic modification, etc. In particular, some of these MCEs have suitable characteristics for cheese production and are considered to be the most potential calf rennet substitutes. Moreover, challenges and future perspectives are presented to provide inspiration for the development of excellent calf rennet substitutes. [ABSTRACT FROM AUTHOR]

Plant aspartic proteinases contain a plant-specific insert (PSI) of about 100 amino acids of unknown function with no similarity with the other aspartic proteinases but with significant similarity with saposins, animal sphingolipid activator proteins. PSI has remained elusive at the protein level, suggesting that it may be removed during processing. To understand the molecular relevance of PSI, the proteolytic processing of cardosin A, the major aspartic proteinase from the flowers of cardoon (Cynara cardunculus L.) was studied. Procardosin A, a 64-kDa cardosin A precursor containing PSI and the prosegment was identified by immunoblotting using monospecific antibodies against PSI and the prosegment. Procardosin A undergoes proteolytic processing as the flower matures. PSI was found to be removed before the prosegment, indicating that during processing the enzyme acquires a structure typical of mammalian or microbial aspartic proteinase proforms. In vitro studies showed that processing of PSI occurs at pH 3.0 and is inhibited by pepstatin A and at pH 7.0. Sequence analysis allowed the identification of the cleavage sites, revealing that PSI is removed entirely, probably by an aspartic proteinase. Cleavage of the PSI scissile bonds requires, however, a conformation specific to the precursor since isolated cardosins and pistil extracts were unable to hydrolyse synthetic peptides corresponding to the cleavage sites. In view of these results, a model for the proteolytic processing of cardosin A is proposed and the molecular and physiological relevance of PSI in plant aspartic proteinase is discussed. [ABSTRACT FROM AUTHOR]

Replacement of Trp39 of Rhizomucor pusillus pepsin (RMPP) by Asn or Cys resulted in a marked decrease in the milk-clotting and proteolytic activities. Kinetic analysis with chromogenic synthetic oligopeptides as substrates revealed that the mutations caused marked changes in the kcat value, but only slight changes in the Km value. Similar enzymatic properties were observed in mutants of Tyr75, which was shown to have a role in enhancing the catalytic activity. Both Tyr75Asn and Trp39Asn mutants rapidly lost the activity at high temperatures due to autocatalytic digestion at two sites. The structures of several aspartic proteinases including RMPP, as revealed by X-ray crystallographic studies, showed that Trp39 occupies a position close to Tyr75 and the Nδ atom of Trp39 within hydrogen-bonding distance of the hydroxyl side chain of Tyr75. These observations suggest that Trp39 plays a role in maintaining Tyr75 in the correct orientation in aspartic proteinases, including RMPP. [ABSTRACT FROM AUTHOR]

Further characterization of an aspartyl protease from Mucor bacilliformis with milk-clotting activity was performed. An extinction coefficient, ε278 cm = 1.61 mL/mg/cm, a molecular mass of 35,400 Da and a pI of 5.2 were determined. Proteolytic activity and kinetic parameters were evaluated by using the hexapeptide Leu-Ser-pNO2-Phe-Nle-Ala-Leu-OMe as the substrate. The effect of pH and temperature on peptide cleavage, as well as protease heat stability, was determined. Such properties, taken as a whole, indicate that the M. bacilliformis protease can be considered a potential substitute for bovine chymosin in cheese manufacture. [ABSTRACT FROM AUTHOR]

[Display omitted] • Proteomics was used to explore the bio-functions of D. sinensis stems proteins. • Proteomics can provide insights in the purification of milk-clotting enzymes. • A cysteine protease with MW of 25.8 kDa and stable properties was identified. Dregea sinensis (D. sinensis) stems have traditionally been used as milk coagulant in Dali of Yunnan Province, China. In this study, proteomics was used to investigate the bio-functions of D. sinensis stem proteins, leading to the purification and identification of the milk-clotting enzyme. A total of 205 proteins mainly involved in the catalytic and metabolic processes were identified, of which 28 proteins exhibited hydrolase activity. Among the 28 proteins, we focused on two enzymes (M9QMC9 and B7VF65). Based on proteomics, a cysteine protease (M9QMC9) with a molecular weight of 25.8 kDa and milk-clotting activity was purified from D. sinensis stems using double ammonium sulfate precipitation and was confirmed using liquid chromatography-mass spectrometry (LC-MS/MS). The milk-clotting temperature using the purified enzyme was around 80 °C (specific activity at 314.38 U/mg), and it was found to be stable in the pH range of 6–9 in NaCl concentration of <0.8 mol/L. These findings indicated that the enzyme isolated from D. sinensis stems has potential in the dairy and food sectors, especially in the cheese-making industry. [ABSTRACT FROM AUTHOR]

A new milk-clotting enzyme (MCE) was produced from Bacillus licheniformis BL312. The characteristics of BL312 MCE were compared with those of Calf rennet, R. miehei MCE, and Chymopapain. BL312 MCE showed a remarkable activity of 5291 SU/mg after purification by ammonium sulfate fractionation and DEAE-Sepharose Fast Flow, which yielded 5.09-fold purification with a recovery of 39.24%. The purified BL312 MCE gave a single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, corresponding to 27 kDa. The α s -casein (α s -CN) and β-casein (β-CN) hydrolysates generated by BL312 MCE were different from those by Calf rennet, R. miehei MCE, and Chymopapain through SDS-PAGE and reversed-phase HPLC (RP-HPLC) analyses. The main cleavage sites in κ-casein (κ-CN) were the Met106-Ala107 and Asn123-Thr124 bonds for BL312 MCE, as determined by mass spectrometry analysis. BL312 MCE hydrolyzed α s -CN to a greater level than β-CN. It was stable at a wide pH range of 5.5–11.0 and temperature below 45 °C. The milk-clotting activity (MCA) reached the maximum when the substrate contained 50 mM CaCl 2 at pH 5.5. The optimal temperature of MCE was 55 °C. The successful application in Monascus -fermented cheese production confirmed the usefulness of BL312 MCE in food and dairy processing. • Isolated and purified a novel milk-clotting enzyme (MCE) from a soil-borne Bacillus licheniformis BL312 strain. • BL312 MCE was characterized with remarkable milk-clotting activity, weak pH sensitivity, and unique hydrolysis properties. • The milk-clotting mechanism for BL312 MCE was the primary hydrolysis of the Met106-Ala107 and Asn123-Thr124 bonds in κ-CN. • The application in Monascus -fermented cheese production showed the BL312 MCE was potential as a Calf rennet substitute. [ABSTRACT FROM AUTHOR]