Maximum Milk-clotting Activity Research Articles

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<0.05) higher moisture, ash and yield, but lower fat content than the rennet cheese. No significant difference (p>0.05) was found in the calcium and protein content of both cheeses. The prepared cheese using ginger protease had significantly (p<0.05) higher spreadability, but lower aftertaste than the rennet cheese. However, non-significant difference (p>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<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.

Partial purification and characterization of Rhizomucor miehei protease from wild-type and mutated strains

Characteristics of the partially purified Rhizomucor miehei proteases from the wild-type isolate (Rm4) and novel microwaves-induced mutant (MW150) were studied. Using SDS-PAGE technique, the molecular weights of Rm4 and MW150 proteases were estimated at 39.77 kDa, and 40.62 kDa, respectively. Maximum milk-clotting activity (MCA) was achieved at 57.5 °C, pH 5, and 9 g/L CaCl2. Maximum proteolytic activity (PA) was achieved at 57.5 °C, and pH 4. Both enzymes were stable at pH values ranging from 3 to 7 for 48 h and temperatures of 50 °C and 60 °C for 24 h and 15 min, respectively. The effect of NaCl by stimulating MCA and inhibiting PA. Maximum PA of Rm4 and MW150 proteases was achieved using 4 % and 2 % casein, respectively. For reconstituted skimmed and whole milk, maximum MCA of Rm4 and MW150 proteases were achieved at the concentrations of 10 % and 15 %, and 4 % and 3.36 %, respectively.

Factors affecting acid protease production by Mucor circinelloides MG603064.1 through SmF process: characterization and fromage frais making.

The exploitation of food industry wastes and their conversion into value-added products present a promising and continuously growing field, given the diversity of elaborated wastes. The current work aimed to utilize sweet cheese whey as a growth medium for acid protease production by a local fungus strain. The biochemical and physicochemical properties of the cheese whey, such as pH, conductivity, chemical oxygen demand, biological oxygen demand (BOD5), total nitrogen and protein contents, and mineral salts, were assessed using various analytical methods. The effect of certain parameters on acid protease production by Mucor circinelloides MG603064.1 through the SmF process was investigated using the conventional design method "One factor at a time". Subsequent to characterization, the crude extract was used in a trial to create fromage frais, compared to the commercial rennin CHY-MAX® Powder Extra. Cheese whey characterization revealed its richness in total nitrogen (1.044 ± 0.044 g/l), protein content (6.52 ± 0.04 g/l), and principal mineral salts: calcium (1.637 ± 0.037 g/l), phosphorus (1.173 ± 0.023 g/l), and chloride (1.66 ± 0.09 g/l). The optimal values of the SmF process for acid protease production, such as the inoculum size, beef extract, and KH2PO4 supplements, the initial pH of cheese whey, and incubation temperature were, respectively, 11% (v/v), 0.4% (w/v), 0.5% (w/v), 5.5, and 30°C. Under these conditions, the lowest milk-clotting time of 290 s was achieved, representing an 18.41-fold increase compared to the initial step using the unoptimized medium. The enzyme showed maximum milk-clotting activity at pH 5, a temperature of 60°C, and in the presence of 0.025 M of CaCl2. The enzyme activity also significantly improved with sonication (35 kHz) for 10 min. The crude extract of M. circinelloides ensured the production of fresh cheese samples with characteristics roughly similar to those obtained by the control (CHY-MAX® rennin). The acid protease of M. circinelloides could successfully substitute the conventional rennin in the manufacture of fresh cheese.

Extraction, Partial Purification and Utilization of Milk Coagulating Enzyme from Kiwifruit (Actinidia Deliciosa) in Fresh Cheesemaking

The study aimed to determine the potential of kiwifruit milk clotting enzyme in cheesemaking. The kiwifruit crude enzyme, extracted with sodium phosphate buffer (pH 7.0), was partially purified by 30-80% ammonium sulfate precipitation. 50% ammonium sulfate saturation exhibited maximum milk clotting activity (MCA), along with 1.56 purification fold, and 78.84% activity recovery. From SDS-PAGE analysis, the partially purified protease showed two bands with a molecular mass of 24 kDa and 23 kDa respectively. The optimum conditions (temperature and pH of milk) for a minimum time of coagulation (TOC) and maximum MCA were determined by response surface methodology (RSM). From the numerical optimization study, the optimum conditions for cheesemaking were pH 6.5 and temperature 55oC, having 0.94 desirability. The cheese prepared by kiwifruit protease had significantly (p<0.05) higher moisture, ash, calcium content, and yield than rennet cheese, while significantly (p<0.05) lower fat content and acidity were observed in kiwifruit protease coagulated cheese. However, a non-significant (p>0.05) difference in protein content was obtained between both cheeses. This study highlighted that kiwifruit protease has the ability to be used as an efficient milk clotting enzyme in fresh cheesemaking.

Identification, structure, and caseinolytic properties of milk-clotting proteases from Moringa oleifera flowers

The protein extract of Moringa oleifera flowers is reported to have milk-clotting activity (MCA), but information regarding its protease is unclear. In this study, two milk-clotting proteases (MoFP 12 and MoFP 13) with molecular weights of 42.304 kDa and 31.741 kDa, respectively, were isolated and identified from M. oleifera flowers using liquid chromatography-mass spectrometry (LC-MS/MS). Bioinformatics analysis showed that these two milk-clotting proteases were primarily involved in hydrolase activity and catabolic process, and exhibited hydrophilicity. The secondary structure of MoFP 12 consisted of 43.65% helix, 13.23% strand, and 43.12% coil, and MoFP 13 consisted of 26.51% helix, 20.14% strand, and 53.35% coil. The proteases were stable in the pH range of 5.0 to 8.0 and showed their maximum MCA at 70℃. Additionally, by investigating the effect of proteases on caseins, κ-casein (CN) was observed to preferentially be hydrolyzed by the two proteases, followed by α-CN, and to a lesser extent β-CN. These findings revealed the main milk-clotting proteases in M. oleifera flowers and its milk-clotting properties, indicating its potential for application in the dairy and food sectors, especially in the cheese-making industry.

Characterization of nettle leaves (Urtica dioica) as a novel source of protease for clotting dromedary milk by non-destructive methods

This study investigates the valorization of the nettle leaves (Urtica dioica) as a novel source of a protease for clotting dromedary milk. The aim of this work is to study the effect of extracting pH on the enzymatic activity of nettle leaves extracts. The extraction was achieved in phosphate citrate buffer at different pH values (from 3 to 6.5) and the obtained extracts were used to coagulate dromedary milk. The characterization of the obtained extracts was carried out using non-destructive methods namely FT-MIR, fluorescence spectroscopy and turbiscan instrument. The extract prepared at pH = 4 had the highest proteolytic activity. The fluorescence and turbiscan measurements revealed a substantial effect of the pH value on chlorophyll residues extraction and stability, respectively. At an acidic environment (pH range of 3 – 4), the enzymatic extracts were unstable (with turbiscan stability index (TSI) values ~ 20), while at a nearly neutral pH value (pH range of 5 – 6.5), they were found to be more stable as indicated by the low TSI values ~ 1. The maximum milk-clotting activity (MCA) (0.021 U/mL) was obtained for the extracts prepared at pH = 4.

Optimization of Crude Papaya (Carica papaya) Protease in Soft-Unripened Cheese Preparation

The use of plant protease instead of chymosin for producing cheese has become a trend which is aimed at lacto-vegetarian consumers and religion based ecological markets. In this context, the present investigation was carried out in order to utilize milk clotting enzyme from Papaya (Carica papaya). Numerical optimization study revealed that maximum milk clotting activity was achieved at pH 6.5, temperature 70℃ and enzyme concentration 1 g/1000 ml milk using papaya protease as coagulant. Protein, ash and calcium showed no significant (p>0.05) difference among the cheeses made using different coagulants. However, significantly (p<0.05) higher levels of moisture and ash, and lower levels of fat were observed in the cheese produced by papaya protease compared to that made using rennet. Papaya protease significantly enhanced the spreadability of cheese while the other sensory properties were similar to the control except aftertaste. The results revealed that the papaya latex as crude papaya protease may have potential application for the manufacture of soft-unripened cheese and further could be utilized as a milk coagulant in cheese making.

PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF ASPARTIC PEPTIDASE PRODUCED BY NOVEL ISOLATE MUCOR CIRCINELLOIDES (VON TIEGHEM) USING SSF PROCESS

Microbial peptidases are among the most important hydrolytic enzymes which have a potential application in a wide number of industrial processes. In this study, the milk-clotting enzyme of a newly isolated strain Mucor circinelloides (von Tieghem) MG603064 was produced by solid-state fermentation using wheat bran as the substrate. The crude extract exhibited a maximum milk-clotting activity of 1500 ± 50.943 SU/mL after 72 h of incubation at 25 °C. Purification of the enzyme using fractionation at 20‒70% (NH4)2SO4 followed by size exclusion chromatography on Sephadex G-100 allowed us to obtain a 20-fold purified peptidase with a recovery of 18.41%. The highest activity of the purified enzyme (30 kDa) was obtained in 25 mM CaCl2, at pH 5.0 and temperature of 60 °C. The enzyme was stable between pH 3.0‒4.0 for 24 h at 4 °C in 0.1 M citrate buffer and retained more than 80% of its maximum activity at 45 °C for 1 h with complete inactivation at 75 °C. The enzyme inhibition of 94.5 and 98.6% by 0.02 and 0.1 mM Pepstatin A, respectively, confirmed that the enzyme is an aspartyl peptidase. A partial inhibition of 78.17% was noted for EDTA at 14 mM. The enzyme activity was improved significantly by Mg2+, Fe2+, Mn2+ and Zn2+ by 40.5, 59.5, 75.6 and 85%, respectively, and strongly inhibited by Al3+ (93.1%) and Hg2+(94.4%), at a concentration of 10 mM. Activity stimulation of 120% was maximum in the presence of Ba2+ using the same concentration. The crude and pre-purified extracts were applied in a trial of semi-hard cheese making (type Edam) as possible rennet substitutes.

Plumeria Alba Latex as a New Plant Protease for Waragashi Cheese Production: A Comparative Assessment of Yield and Physicochemical and Textural Characteristics

The Peuhl cheese (Waragashi, Warangachi, Wagashi, or Wara in local language) is a soft cheese with a high nutritional value, prepared by coagulation of whole milk under the action of calotropaïne, a plant enzyme from Calotropis procera. In this study, <i>Plumeria alba</i> latex has been used as an alternative source of enzymes for the production of Waragashi cheese. The effect of temperature on the milk-clotting activity (MCA), as well as the physicochemical properties of the Waragashi cheeses were investigated and compared with the ones prepared with <i>C. procera</i> and <i>C. papaya</i> latices. <i>C. procera</i> and <i>C. papaya</i> latices exhibited a maximum MCA at temperatures close to 55°C, while <i>P. alba</i> latex exhibited a maximum MCA at temperatures close to 80°C. Waragashi cheese produced using <i>P. alba</i> showed textural properties comparable with those prepared using <i>C. procera</i>, while the yield of production varied as a function of the quantity of the different coagulants and milk temperature. The results of these studies, it is clear that the <i>P. alba</i> latex can be used to produce Waragashi cheese, similar to the latices of C. procera and <i>C. papaya</i>. the next challenge of this study is to verify the toxicity of <i>P. Alba</i> latex.

The non-edible parts of fennel (Fœniculum vulgare) as a new milk-clotting protease source

Although fennel stems and leaves represent an abundant biomass, they remain poorly exploited in terms of extraction of biomolecules. This work aimed at the valorization of the non-edible parts of fennel by the extraction and the characterization of a new milk-clotting protease. Both, enzymatic extraction and milk-clotting conditions were optimized in the context of developing new aspects of cheese. Fennel stems and leaves were sorted and coarsely grounded. The extraction consists in an aqueous maceration and extracts were used to coagulate raw cow's milk. Maximum milk-clotting activity was obtained under a temperature of 20°C, extraction time of 12h and solid-to-solvent ratio of 150:100gmL−1. Besides, the impact of extract concentration, milk pH, calcium content and temperature on clotting-time were studied. The statistical analysis of the results confirmed significant effects of the four studied parameters. The use of specific inhibitors suggested the presence of a serine protease in the crude enzymatic extracts. The findings of this research confirmed that the use of fennel protease could be a new alternative for the commercial proteases in dairy products.

EXPLORING THE MILK-CLOTTING PROPERTIES OF EXTRACTS FROM Bromelia pinguin FRUIT

Plants represent an attractive source of milk-clotting proteases with potential use for chymosin substitution in cheesemaking process. A crude enzymatic extract from B. pinguin fruit showed capacity to clot milk efficiently in a short time. It showed a maximum milk-clotting activity around 4 U/mL in the range of 70-80 ºC. This value indicated that one mL of the extract was capable to clot around 400 mL of milk in 40 min at the specified condition. B. pinguin extract presented only 25% of its maximum activity under standard temperature (30-35 ºC) for cheese-making. Proteases inhibitors indicated that cysteine and serine proteases were present in the extract and could be responsible for the milk-clotting activity found. SDS-PAGE analysis of casein hydrolysis indicated that proteases in B. pinguin extract were more proteolytic than chymosin. The high presence of milk-clotting proteases in B. pinguin fruit offers an alternative new source of proteases for biotechnological processes.

Technological properties of milk gels produced by chymosin and wild cardoon rennet optimized by response surface methodology

The aim of this paper was to optimize extraction conditions of C. cardunculus rennet by response surface methodology, in order to maximize its milk-clotting activity (MCA). The second objective was to examine the effect of different amounts of the optimized extract and the type of milk, on technological characteristics of the obtained gels. Results of the central composite design showed that the optimum extraction conditions, corresponding to maximum MCA (9.550CAU/mL), were selected as follows: Grinding time 30s, pH 3, extraction time 50min and solid to liquid ratio 15g/100mL. According to technological properties, it can be concluded that C. cardunculus extract exhibited an excellent efficiency on raw milk, in term of dynamic moduli and gel firmness, as compared to chymosin. A good gelation time and WHC were also obtained. Consequently, the optimized C. cardunculus rennet has the potential to be employed as an efficient milk-clotting agent.

Effect of oxygen supply on milk-clotting activity expressed by Paenibacillus spp. strain BD3526

Paenibacillus spp. BD3526, a novel bacteria species isolated from raw yak (Bos grunniens) milk collected in Tibet, is able to produce metalloproteinase with milk-clotting activity (MCA) in wheat bran broth. To determine the critical factor favoring enzyme productivity, response surface methodology and the Box-Behnken design were adopted to preliminarily optimize BD3526 cultivation conditions for maximum MCA in shaking flasks. The highest degree of MCA (7072 ± 519 SU/mL) was obtained at a liquid volume of 30 mL and 300 r/min, which implies that oxygen supply is crucial for enzyme synthesis. MCA levels were enhanced as agitation speeds and aeration rates were increased in a 7-L scale fermentor. MCA in the supernatant was further boosted by adding hydrogen peroxide (H2O2) to the broth during cultivation. Maximum MCA (14778 ± 1100 SU/mL) and MCA/proteolytic activity (PA) (33000 ± 918) were achieved when cultivation was carried out at an agitation speed of 400 r/min, aeration rate of 5.5 L/min and H2O2 flow of 500 mmol/L at a feeding rate of 2.5 mL/h after 24 h of cultivation.

Strain variability of Irpex Lacteus basidiomycetes in the synthesis of specific milk-clotting proteinases

The ability to synthesize milk-clotting (rennet) proteinases was studied in eight strains of Irpex Lacteus basidiomycetes. It was found that the studied I. lacteus strains are characterized by different enzyme activities in their culture liquid. For I. lacteus strains 2425, 2426, and 2427, the maximum milk-clotting activity was detected during the exponential growth phase on the 15th day of cultivation on glucose–peptone medium. These I. lacteus strains are the most prospective milk-clotting enzyme producers for further research and practical application. I. lacteus strains 2421, 2422, 2423, 2424, and 2428 showed lower values of the enzyme activity and require additional research to determine the optimal culture conditions.

Optimization of milk-clotting enzyme production by Bacillus amyloliquefaciens SP1 isolated from apple rhizosphere

Present study aims to isolate and optimize fermentation conditions of milk-clotting enzyme producing rhizobacteria Bacillus amyloliquefaciens SP1. 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. 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.

High Milk-Clotting Activity Expressed by the Newly Isolated Paenibacillus spp. Strain BD3526.

Paenibacillus spp. BD3526, a bacterium exhibiting a protein hydrolysis circle surrounded with an obvious precipitation zone on skim milk agar, was isolated from raw yak (Bos grunniens) milk collected in Tibet, China. Phylogenetic analysis based on 16S rRNA and whole genome sequence comparison indicated the isolate belong to the genus Paenibacillus. The strain BD3526 demonstrated strong ability to produce protease with milk clotting activity (MCA) in wheat bran broth. The protease with MCA was predominantly accumulated during the late-exponential phase of growth. The proteolytic activity (PA) of the BD3526 protease was 1.33-fold higher than that of the commercial R. miehei coagulant. A maximum MCA (6470 ± 281 SU mL−1) of the strain BD3526 was reached under optimal cultivation conditions. The protease with MCA was precipitated from the cultivated supernatant of wheat bran broth with ammonium sulfate and purified by anion-exchange chromatography. The molecular weight of the protease with MCA was determined as 35 kDa by sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and gelatin zymography. The cleavage site of the BD3526 protease with MCA in κ-casein was located at the Met106–Ala107 bond, as determined by mass spectrometry analysis.

Expression and characterization of camel chymosin in Pichia pastoris

Chymosin efficiently coagulates milk and so is widely used in commercial cheese production. Traditional chymosin production requires the slaughter of a large numbers of unweaned calves. In the present study, a full-length camel prochymosin gene was synthesized and cloned into the pPIC9K vector, which was then inserted into the yeast strain, Pichia pastoris GS115. Expression of the chymosin gene in yeast was under the control of an AOX1 inducible promoter. The yeast system produced approximately 37mg/L of recombinant enzyme under lab conditions. SDS–PAGE of the raw supernatant revealed two molecular bands, which were approximately 42kDa and 45kDa in size. The 45kDa band disappeared after treatment of the supernatant with N-glycosidase F (PNGase F), indicating that the recombinant protein was partially glycosylated. When subjected to a low pH, recombinant prochymosin was converted into mature and active chymosin. The active chymosin was capable of specifically hydrolyzing κ-casein. A pH of 5.04, and temperature range of 45–50°C, was optimum for milk clotting activity. Maximum milk clotting activity was detected with the inclusion of 20–40mM CaCl2. The recombinant enzyme was highly active and stable over a wide pH range (from 2.5 to 6.5) at 20°C for 8h. Thermostability of the recombinant enzyme was also analyzed. Pilot-scale production (300mg/L) was attained using a 5L fermenter. We demonstrated that expression of the camel chymosin gene in P. pastoris could represent an excellent system for producing active camel chymosin for potential use in the commercial production of cheese.

PsoP1, a Milk-Clotting Aspartic Peptidase from the Basidiomycete Fungus Piptoporus soloniensis

The first enzyme of the basidiomycete Piptoporus soloniensis, a peptidase (PsoP1), was characterized after isolation from submerged cultures, purification by fractional precipitation, and preparative native-polyarylamide gel electrophoresis (PAGE). The native molecular mass of PsoP1 was 38 kDa with an isoelectric point of 3.9. Similar to chymosin from milk calves, PsoP1 showed a maximum milk-clotting activity (MCA) at 35-40 °C and was most stable at pH 6 and below 40 °C. The complete inhibition by pepstatin A identified this enzyme as an aspartic peptidase. Electrospray ionization-tandem MS showed an amino acid partial sequence that was more homologous to mammalian milk clotting peptidases than to the chymosin substitute from a fungal species, such as the Zygomycete Mucor miehei. According to sodium dodecyl sulfate-PAGE patterns, the peptidase cleaved κ-casein in a way similar to chymosin and hydrolyzed β-casein slowly, as it would be expected from an efficient chymosin substitute.

Screening fermentation parameters of the milk-clotting enzyme produced by newly isolated Bacillus amyloliquefaciens D4 from the Tibetan Plateau in China

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 × 107 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.