Milk-clotting Activity Research Articles

Milk-clotting activity of Withania coagulans hairy root extracts

This article presents data on the effect of fruit and hairy root extracts of Withania coagulans Dunal on the process of milk coagulation with a view to its potential application in cheese making. Cheese curd can be obtained by the action of milk-clotting enzymes on the protein fraction of milk. To that end, animal-derived enzymes are widely used. However, the as-produced cheeses may not suit some population groups. In this regard, studying the possibility of using plant proteases in cheese making, as well as their production in plant tissue cultures, appears to be a relevant research task. Hairy roots bioreactor-cultivated using the Agrobacterium rhizogenes soil bacterium contain, as a rule, not only root-specific metabolites but also compounds characteristic of other plant tissues. In this work, protein was extracted from Withania coagulans fruits and hairy roots using various buffers followed by assessing the milk-clotting activity of the extracts using fresh and powdered full cream milk. An animal milk-clotting enzyme was used as a control. The activity of Withania coagulans fruit extract was found to be 5 U/mg of protein. Coagulation carried out with hairy root extracts lasted for at least 6 hours, indicating the presence of a low concentration of milk-clotting enzyme. Since hairy roots of Withania coagulans have been studied in this respect for the first time, further optimization of their cultivation parameters, enzyme extraction, and its application is required.

From Beer to Cheese: Characterization of Caseinolytic and Milk-Clotting Activities of Proteases Derived from Brewer's Spent Grain (BSG).

This study explores the extraction and characterization of proteolytic enzymes from brewer's spent grain (BSG) and their potential as sustainable coagulants in the dairy industry. BSG samples from various beer types (Blonde Ale, IPA, Kölsch, Honey, and Porter) were obtained from two artisanal breweries in Mar del Plata, Argentina. Optimization of caseinolytic activity (CA) and protein extraction was conducted using a Plackett-Burman design, followed by a Box-Behnken design. Optimal protein concentration was achieved at intermediate pH and high temperature, while CA peaked at pH 8.0. The specific caseinolytic activity (SCA) varied among the extracts, with BSG3 showing the highest activity (99.6 U mg-1) and BSG1 the lowest (60.4 U mg-1). Protease inhibitor assays suggested the presence of aspartic, serine, metallo, and cysteine proteases. BSG3 and BSG4 showed the highest hydrolysis rates for α-casein (70% and 78%). For κ-casein, BSG1, BSG2, and BSG3 demonstrated moderate activity (56.5%, 49%, and 55.8), while BSG4 and BSG5 exhibited the lowest activity. Additionally, the milk-clotting activity (MCA) of BSG extracts was comparable to plant-based coagulants like Cynara cardunculus and Ficus carica. These findings highlight the potential of BSG-derived proteases as alternative coagulants for cheese production, offering a sustainable link between the brewing and dairy industries.

Proteomics-guided isolation of a novel serine protease with milk-clotting activity from tamarillo (Solanum betaceum Cav.)

Tamarillo is widely grown in Yunnan Province, China, and has been found that it can be used in cheese-making with a distinctive fruity flavour. However, this primary component responsible for curdling milk remains unclear. This study aimed to identify the main component in tamarillo responsible for curdling milk using proteomics and ammonium sulfate (AS) precipitation. Herein, 3199 proteins were identified in tamarillo, of which 546 exhibited hydrolase activity. In particular, a novel serine protease with milk-clotting activity (MCA) and a molecular weight of 79.1 kDa, named “MCP746”, was isolated from tamarillo. The milk-clotting proteases (MCPs) from tamarillo exhibited the highest MCA at 80 °C and stability under incubation temperatures below 70 °C, pH range of 5–8, and NaCl concentrations below 1 mol/L. This study revealed that serine protease is the primary MCPs of tamarillo along with a characterization of its milk-clotting characteristics, providing valuable insights into its potential application in cheese-making.

Production of a fungal aspartic protease via solid‐state fermentation using a rotating drum bioreactor

AbstractBACKGROUNDSolid‐state fermentation (SSF) has gained attention recently as a promising approach for producing a range of high‐value industrial products. SSF has several benefits, including a high product yield, a lesser chance of contamination, a resemblance to some fungi and bacteria's native environment, a lower energy need, and cost‐effectiveness. Additionally, the enzyme extraction from concentrated solutions facilitates the recovery procedure. Mucor racemosus, a filamentous fungus, has the potential to produce a milk‐clotting enzyme (aspartic protease) cultivated on various combinations of the solid media to investigate the effect of media on the production and activity of the milk‐clotting enzyme.RESULTSThe fermentation was scaled up by utilizing Terrafors‐IS Infors HT in‐situ sterilizable rotating solid‐state bioreactor to cultivate the fungus. The effect of agitation (rotation on the day of inoculation and day 1), aeration (air flow rates of 1 L and 2 L/min), and moisture content (60% and 90%), during SSF were investigated. The profiles of various operational parameters (that is, temperature, pressure, flow rate, exit O2, and exit CO2) were monitored using the provided software, and data were collected at 10‐min intervals during the whole course of fermentation. The results suggest that aeration has a remarkable effect on the quantity of biomass produced, but it doesn't assure high enzyme production. The highest milk clotting activity of ~570 U/mL was recorded with a moisture content of 60% and a flow rate of 1 L/min.CONCLUSIONA high yield of ~570 U/mL of the enzyme with desired characteristics was obtained with minimum moisture availability at low temperatures. The results narrated in this study validate the SSF process for producing aspartic protease from wheat bran using a rotating solid‐state bioreactor to be used as a potential milk coagulant in the cheese industry. © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

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.

Investigation of miniature goat cheese produced from Cynara scolymus L. pistil cell suspension culture extracts elicited with melatonin and salicylic acid.

The present study aimed to evaluate the potential of enzymes produced by artichoke (Cynara scolymus L.) flower cell suspension cultures elicited by melatonin (5 μm) and salicylic acid (50 μm) on the production and characteristics of miniature goat cheeses. In this study, five types of fresh miniature goat cheese were produced using whole artichoke flower extract, salicylic acid (50 μm) or melatonin (5 μm) treated artichoke suspension cell culture extracts, a culture extract without elicitor treatment (control) and rennin enzyme. The milk clotting activity values of the enzymes were measured in the range 0.52-0.74. The effect of the enzymes on the titratable acidity, water-soluble nitrogen, ripening index, αs-caseins and β-caseins of goat cheese was significant (P < 0.05). The highest level of casein degradation was observed in the cheese produced with the enzyme containing melatonin, followed by the cheese produced with the enzyme containing salicylic acid. For the enzymes produced by the suspension cell culture method, the addition of melatonin and salicylic acid had a slightly positive effect on the proteolytic activity of the extracts. It was also found that the enzymes obtained from artichokes by the suspension cell culture method did not achieve successful cheese production in terms of chemical, textural and biochemical aspects compared to those obtained from animal enzymes. © 2024 Society of Chemical Industry.

Comparison of milk-clotting activity measurement using the Berridge's operator-based approach and a proposed digital method

The milk clotting activity (MCA) analysis of coagulants used in the production of cheese is an important parameter used in the modern dairy industry. The MCA is calculated using the estimated milk-clotting time (MCT). The standard method used for estimation of MCT is presented in the ISO 23058/IDF 199 Standard using the Berridge's operator-based approach. In this paper a new method is presented to estimate the MCT using digital image acquisition and signal processing. The estimate is obtained by processing a sequence of images of the wall of a round bottom flask containing milk and the coagulant. Digital signal processing and filter techniques were used to remove interferences and artifacts and improve the clotting time estimation. A set of 28 samples of milk plus coagulant were processed. The method's accuracy is about 1/30 s and the average relative error, when compared to an experienced human operator, was με = 2.82%, according to the experimental results, with an associated standard deviation of σε = 3.07% and a median of mε = 1.92%. The method may be used to replace the human operator during the MCT estimation procedure.

Microsyringe-assisted visual volume detection based on phase separation: the case of chymosin milk-clotting activity study.

The constantly diverse demand scenarios for rapid on-site analysis have put forward high requirements for developing low-cost and user-friendly visual detection methods. Therefore, developing a visual detection method with simple operation and intuitive results has important practical value in the field of analysis and detection, but it is also challenging. In this work, we propose a microsyringe-assisted visual volume detection method based on phase separation, and apply it to analyze the milk-clotting activity of chymosin. Chymosin can cause phase separation of milk with whey in the mobile phase and curd in the gel state. The network structures of casein in curd can trap water molecules, resulting in separation of whey from curd gradually. Therefore, the analysis of chymosin milk-clotting activity can be realized according to the volume of whey measured using a portable microsyringe. This method shows a good linear correlation when the concentration of chymosin ranges from 1.02 U L-1 to 1020 U L-1 and the limit of detection of this method for chymosin is calculated to be 0.03 U mL-1. This work successfully realizes the visual analysis of chymosin milk-clotting activity based on the enzyme-triggered phase separation. It also shows great promise to be applied in other phase separation-based detection systems with the advantages of high accuracy, great portability and user-friendliness.

Effect of chymosin/pepsin ratio on milk coagulation and physical properties of model cheese

Chymosin and pepsin are the two major enzymes in coagulants for the cheese industry. They affect milk coagulation properties, curd composition, casein hydrolysis and the consequent development of cheese texture. Limited knowledge is available on the effect of the chymosin/pepsin ratio on the mentioned factors. This study aimed (1) to better understand the effect of chymosin/pepsin ratio on milk coagulation properties, casein hydrolysis, rheological and textural properties of model cheese during storage, and (2) to link casein hydrolysis to the rheological and textural properties. For this purpose, coagulants were prepared in which the ratio between the two enzymes was systematically varied. Our results showed that even when milk clotting activity was standardized, coagulants with different chymosin/pepsin ratios had a significant effect on curd firming rate and final firmness, which could be attributed to the different specificity of chymosin and pepsin for hydrolyzing κ-CN. During 4 weeks of storage, the chymosin/pepsin ratio influenced the hydrolysis of αs1-CN, and the subsequent properties of the protein network and the cheese texture. For coagulants with a higher proportion of pepsin, less intact αs1-CN and a higher degree of casein hydrolysis (expressed as protein fraction soluble at pH 4.6) were found. Correspondingly, the cheese had a weaker and less brittle network, and was softer, less elastic and more sticky. Commercial calf rennet showed more extensive hydrolysis of αs1-CN than other coagulants, which led to the weakest protein network and the softest cheese. The present study's findings can be used to control and design cheese with desired textures. For example, coagulants with varying chymosin/pepsin ratio or from a specific source can be used to modulate the casein hydrolysis.

Chemical Composition, Antioxidant Activity, and Milk-Clotting Activity of Aqueous Extracts from Leaves, Stems, and Flowers of Three Tunisian Ecotypes of Spontaneous and Cultivated Onopordum nervosum ssp. platylepis Murb.: A Potential Novel Vegetable Rennet Option

This study aimed to compare the chemical composition of aqueous extracts from different aerial plant parts (leaves, stems, and flowers) of Onopordum nervosum ssp. platylepis growing in different regions (Sousse, Kairouan, and Nabeul) in Tunisia, as well as their antioxidant and milk-clotting properties for both spontaneously grown and cultivated plants. Results showed that phenolic composition varies significantly among ecotypes and plant organs (p &lt; 0.05), with flowers containing the highest amounts of total phenols and flavonoids in both plant types. The flowers from Nabeul (NA) region showed the highest amounts of total phenols and higher phenolic contents compared to leaves and stems, with 44.75 mg GAE/g and 39.79 mg GAE/g in spontaneous and cultivated plants, respectively. However, flowers of spontaneously plants grown in Sousse (SO) showed the highest total flavonoid contents (11.42 mg QE/g). Additionally, the findings indicated that flowers contained higher concentrations of mono- and disaccharides than leaves and stems. The antioxidant activity showed that the radical scavenging activity of O. platylepis aqueous extracts is significantly affected by the organ and genotype (p &lt; 0.05). NA genotype revealed the highest potency in inhibiting free radicals, with flowers having the lowest IC50s values in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) tests, registering 0.13 ± 0.02 and 0.14 ± 0.01 mg/mL, respectively. Milk-clotting activity was limited to flowers of this thistle. Moreover, SO and NA genotypes were identified as the most potent populations to coagulate milk in spontaneous and cultivated plants. The principal component analysis confirms the organ and genotype variability in this thistle, with the first two axes explaining 64.15% of the variance and highlighting a distinct flower group. The obtained results suggest that the domestication of this thistle could be useful for the conservation of biodiversity and the promotion of genotypes with potential technological properties.

PRODUCTION OF RECOMBINANT MILK-CONVERTING ENZYME IN YEAST PICHIA PASTORIS

Chymosin is the main enzyme with a high degree of specificity towards k-casein and is therefore used in the cheese industry. This work presents the results on the expression of the goat prochymosin gene in the yeast Pichia pastoris and the study of the milk-clotting activity of recombinant goat chymosin. The goat prochymosin gene was integrated into the chromosomal DNA of yeast and the secretion of prochymosin into yeast culture was carried out by methanol induction. After activation, chymosin was purified by sequential anion and cation exchange chromatography. A biochemical study showed that a decrease in the milk-clotting activity of the enzyme was observed with an increase in pH. The highest activity of recombinant goat chymosin was manifested at 60 °C and pH 4.5-5.0 and amounted to 7680 ± 0.32 and 8727 ± 0.39 for cow's and goat's milk, respectively. The total proteolytic activity of the enzyme was 7769.2±0.38 U/mg. The results obtained suggest that recombinant goat chymosin can be used in the cheese industry and has good prospects for practical application in the production of cheese from goat's milk.

Isolation and identification of milk-clotting proteases from Prinsepia utilis Royle and its application in cheese processing

The aim of this study was to isolate and identify the main milk-clotting proteases from Prinsepia utilis Royle. Protein isolates obtained using precipitation with 20 %–50 % ammonium sulfate (AS) showed higher milk-clotting activity (MCA) at 154.34 + 0.35 SU. Two milk-clotting proteases, namely P191 and P1831, with molecular weight of 49.665 kDa and 68.737 kDa, respectively, were isolated and identified using liquid chromatography-mass spectrometry (LC-MS/MS). Bioinformatic analysis showed that the two identified milk-clotting proteases were primarily involved in hydrolase activity and catabolic processes. Moreover, secondary structure analysis showed that P191 structurally consisted of 40.85 % of alpha-helices, 15.96 % of beta-strands, and 43.19 % of coiled coil motifs, whereas P1831 consisted of 70 % of alpha-helices, 7.5 % of beta-strands, and 22.5 % of coiled coil motifs. P191 and P1831 were shown to belong to the aspartic protease and metalloproteinase types, and exhibited stability within the pH range of 4–6 and good thermal stability at 30–80 °C. The addition of CaCl2 (<200 mg/L) increased the MCA of P191 and P1831, while the addition of NaCl (>3 mg/mL) inhibited their MCA. Moreover, P191 and P1831 preferably hydrolyzed kappa-casein, followed by alpha-casein, and to a lesser extent beta-casein. Additionally, cheese processed with the simultaneous use of the two proteases isolated in the present study exhibited good sensory properties, higher protein content, and denser microstructure compared with cheese processed using papaya rennet or calf rennet. These findings unveil the characteristics of two proteases isolated from P. utilis, their milk-clotting properties, and potential application in the cheese-making industry.

Exploring New Fruit- and Vegetable-Derived Rennet for Cheese Making

Cheese production is an ancient practice to preserve a perishable food, such as milk, for a long time. The first step of cheese processing involves the addition of rennet, which contains the enzymes necessary for the hydrolysis and coagulation of the caseins present in milk. Typically, animal-derived rennet, such as calf rennet containing chymosin, are used as source of enzymes for cheese processing. Alternatively, microbial chymosin or recombinant chymosin is used. However, recently, plant-derived rennet such as the ones derived from thistle and bitter orange flowers and from artichoke (Cynara cardunculus var. scolymus) have also been demonstrated to be valid sources of enzymes for cheese processing. Therefore, herein, different plant and fruit extracts were tested and compared for their ability to coagulate milk caseins. In particular, beyond artichoke and cardoon (Cynara cardunculus) extracts, those from pineapple (Ananas comosus (L.) Merr.), papaya (Carica papaya L.), common fig (Ficus carica L.) milky sap, and oyster mushroom (Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm.) were investigated for their proteolytic, esterase, and milk-clotting activities. The extracts were then exploited as vegetable and fruit rennet for the experimental production of cheeses, which were examined, after 30 days of maturation, for their moisture, fat, protein, and free fatty acid (FFA) content. Interestingly, the artichoke, cardoon, and thistle mushroom extracts showed high proteolytic activity compared to calf rennet, while the level of esterase activity appeared to be similar for all the extracts. The papaya extract showed the lowest proteolytic and esterase activity. Although the pH, moisture, fat, and protein contents were very similar to those of cheese made with calf rennet, the medium- and long-chain FFAs broadly differed among produced cheeses, with variations in the lipid quality indices.

Proteolytic, milk-clotting, and stability properties of proteases from Solanum elaeagnifolium fruit

Solanum elaeagnifolium (trompillo) has been considered a new alternative source of proteolytic enzymes for cheese-making. However, properties for this proteolytic source still need to be described. This study evaluated the proteolytic (PA) and milk-clotting activity (MCA) as well the stability properties for the proteases found in S. elaeagnifolium fruit extract (FE). Proteolytic enzymes were active in a broad pH range with a maximum at pH 10.0 and 80 °C and showed high stability in a wide pH (5.0–12.0) and temperature ranges (30–75 °C). MCA increased with the temperature in a range of 30–85 °C. Proteases from S. elaeagnifolium FE presented high thermostability, retaining 40% of the activity after 15 min of incubation at 80 °C. Temperatures higher than 85 °C caused the total PA loss. Phenylmethylsulphonyl fluoride (PMSF) inhibited more than 95% of the PA, indicating that the activity is mainly related to serine proteases. In addition, a casein hydrolysis profile, by SDS-PAGE, showed that proteases degrade α, β, and κ-casein with high affinity. S. elaeagnifolium fruit contains abundant proteolytic enzymes with high catalytic properties and activity over different protein substrates. Therefore, S. elaeagnifolium fruit represents a potential source of serine proteases for use in different biotechnological processes, offering attractive properties such as high activity in a broad pH and temperature range, high thermal stability, and preference over different natural substrates.

Development of Probiotic Indian Cheese using Rennet by Potential Microbes Isolated from Natural Sources

Rennet is an exogenous enzymes used in dairy processing industries and food sectors.Rennin acts on the milk protein in two stages, through enzymatic and non-enzymatic action,resulting in the milk coagulation.Traditionally, the enzyme is extracted from whole abomasum of ruminants.Microbial rennet contains enzymes having a high ratio of milk clotting activity and proteolytic activity This review aims to explore techniques of microbial production, characterization and cost benefits over current technologies used in dairy industries. Among the dairy product, cheese is an major choice for consumption with high nutritional values and probiotic activity. Cheese is a nutrient dense dairy product providing essential biomolecules such as proteins,minerals,fats. The viability of probiotic microorganism in cheese is high as found in other dairy product and shelf life is extended as compared to with other alternatives of production.The manufactured probiotic cheese have certain benefits over traditional cheese production processes.

Evaluation of milk-clotting activity of kiwifruit juice by rheological method

Evaluation of milk-clotting activity of kiwifruit juice by rheological method

Utilization of Partially Purified Milk Clotting Protease from Ginger Rhizome in the Manufacturing of Fresh Cheese

This study was carried out to utilize the partially purified ginger rhizome protease in fresh cheesemaking. The crude ginger rhizome extract was partially purified by employing 30-80% (NH4)2SO4 saturations (w/v). The maximum milk clotting activity was achieved at 50% saturation, exhibiting the purification fold of 1.64 and activity recovery of 85.76%. The milk pH and temperature for optimum time of coagulation and milk clotting activity were optimized by using response surface methodology (RSM). A numerical optimization study revealed that the optimum milk pH and temperature for producing cheese were 6.5 and 50 C respectively. The cheese produced by ginger protease had significantly (p&lt;0.05) higher moisture, ash and yield, but lower fat content than the rennet cheese. No significant difference (p&gt;0.05) was found in the calcium and protein content of both cheeses. The prepared cheese using ginger protease had significantly (p&lt;0.05) higher spreadability, but lower aftertaste than the rennet cheese. However, non-significant difference (p&gt;0.05) was observed in the flavor and overall acceptance between the two cheeses. Hence, this study demonstrated that the ginger rhizome protease has the potential to be utilized as an effective milk coagulating enzyme in the manufacturing of fresh cheese.

Optimization of Three Phase Partitioning Purification of Papain from Carica papaya Latex by Response Surface Methodology

Three phase partitioning (TPP), a novel and non-chromatographic method was effectively applied for the extraction and recovery of papain from Carica papaya latex. The effect of change in ammonium sulfate concentration, crude extract to solvent ratio and pH on extraction efficiency was studied, and these parameters were optimized by response surface methodology (RSM) using Box-Behnken Design. The protease was found to be concentrated more at the aqueous phase (AP) with 51.08% ammonium sulfate precipitation, crude extract to tert-butanol ratio of 1:0.78 (v/v), and pH 6.05 resulting 9.242 purification fold and 159.806% activity recovery. Compared to the intermediate phase (IP), the AP demonstrated higher recovery and purification fold. Purified papain showed significantly (p&lt;0.05) greater activity at a temperature of 60°C and pH of 6.0. It also demonstrated stability within the temperature range of 40 to 70°C and pH range of 6.0 to 8.0. Enzyme retained maximum milk clotting activity when stored under freezing (-20°C) for 3 weeks. This study showed that the TPP can be used to extract and purify papain from papaya latex economically and it can also be employed as a vegetable coagulant for cheesemaking.

Analysis of some biochemical properties of recombinant siberian roe deer (Capreolus pygargus) chymosin obtained in the mammalian cell culture (CHO-K1)

The structure of the Siberian roe deer (Capreolus pygargus) chymosin gene has been established for the first time and its exon/intron organization has been determined. The coding part of the C. pygargus chymosin gene was reconstructed and obtained as a DNA clone using the Golden Gate method. Comparative analysis of the sequences of prochymosins of roe deer, cow and single-humped camel revealed a number of amino acid substitutions in the sites forming the substrate-binding cavity of the enzyme and affecting the specificity subsites S4 and S1′ + S3′. The recombinant plasmid pIP1-Cap was constructed using the integration vector pIP1 for the expression of the roe deer prochymosin gene in CHO-K1 cells. A polyclone of CHO-K1-CYM-Cap cells was obtained, providing synthesis and secretion of recombinant prochymosin into the culture fluid of the producer. As a result of zymogen activation, a recombinant roe deer chymosin preparation with a total milk-clotting activity of 468.4 ± 11.1 IMCU/ml was obtained. The yield of recombinant roe deer chymosin was 500 mg/liter or ≈ 468,000 IMCU/liter, which exceeds the yield of genetically engineered chymosins in most of the expression systems used. The main biochemical properties of the obtained enzyme were compared with commercial preparations of recombinant chymosins of single-humped camel (Camelus dromedarius) and cow (Bos taurus). The specific milk-clotting activity of recombinant C. pygargus chymosin was 938 ± 22 IMCU/mg of protein and was comparable with the indicators of comparison enzymes. The nonspecific proteolytic activity of recombinant roe deer chymosin was 1.4-4.5 times higher than that of cow and camel enzymes. In terms of coagulation specificity, the recombinant C. pygargus chymosin occupied an intermediate position between the genetically engineered analogues of B. taurus and C. dromedarius chymosins. The threshold of thermal stability of recombinant roe deer chymosin was equal to 55°C. At 60°C, the enzyme retained

Improvement of camel milk-clotting: Usefulness of crude extract from green pods of carob (Ceratonia siliqua. L) as a substitute for commercial rennet.

Camel milk transformation into cheese remains an objective to be improved today. This study aimed to improve camel milk clotting using a crude extract from green pods of carob as a substitute for commercial rennet. The composition of the crude carob extract was determined for dry matter and protein content. Milk clotting conditions were studied at different temperatures, pH and CaCl2 concentrations. Milk clotting properties were assessed by milk clotting activity, specific activity and proteolytic activity. Enzymatic hydrolysis of camel milk caseins by crude carob extract and its inhibition were demonstrated by SDS-polyacrylamide gel electrophoresis. Crude carob extract analysis showed a protein and dry matter content of 23.26±0.5 mg/ml and 30.66±0.5 g/l, respectively. Optimal milk clotting activity was observed at 53.6 °C, pH 4.5, and 0.09 M CaCl2. The crude carob extract showed a high milk clotting activity (4.97 U/ml) and a low proteolytic activity (0.04U/ml) with camel milk. The cheese yield of curd produced from camel milk using crude carob extract was the highest (23.95%) compared with that of Camel chymosin (20.5%). The high ratio of milk-clotting to proteolytic activity shows the potential of this extract as a substitute for commercial rennet in the dairy industry.