Milk-clotting Activity Research Articles

EXTRACTION, PARTIAL PURIFICATION AND SOME FACTORS AFFECTING MILK CLOTTING ACTIVITY OF A MILK COAGULANT PREPARED FROM SUNFLOWER (Helianthus annus) SEEDS

Crude extract of sunflower seeds was prepared using different extractants (distilledwater, 5% NaCl in distilled water, 5% NaCl in acetate buffer pH 5.0) and certain mixing ratios (1:2,1:5 and 1:10 seeds powder : extractant, respectively). Using of 5% NaCl in acetate buffer pH 5.0 atmixing ratio of 1:5 gave an extract with reasonable efficient clotting properties. Ammonium sulphateat 40-60% concentration was used in one single step for the partial purification of the extract.Ammonium sulphate, (40-60%) fraction showed milk clotting and specific activities of 440 sec and2.30 U/mg protein, respectively. Meanwhile a yield percentage and a fold of purification of 28.58 and2.02 were achieved. Clotting/proteolytic activity, water holding capacity and curd synersis of thepartially purified extract approved those of commercial calf rennet indicating the suitability of theextract as a rennet substitute. The optimum temperature, pH, Cacl2 and GDL concentration of theresultant extracts were found to be 65°C, 5.0, 0.04 % and 0.4%, respectively.

A comparison study on the cow and mare milk-clotting activity of Withania coagulans

The limitations of rennin application in cheese crafting usually urge the discovery of novel proteases. The Withania coagulans fruits are well-known for their caseinolytic activity in cheese production. The study aims to evaluate some of the factors affecting the milk-clotting activity (MCA) of W. coagulans fruit enzymatic extract in even and odd-toed hoofed-mammals milk. The extracts were prepared by distilled water and normal saline and their protein content were evaluated. The time necessary for the appearance of discernible and discrete particles in the mare and cow milk by the two concentrations of saline and hydro W. coagulans extracts (SE and HE, respectively) was assessed at 35 and 40°C while exposing different levels of calcium chloride. The interaction of these factors on MCA was evaluated using mixed-design ANOVA. Three significant interaction patterns considering the maximum number of factors were revealed (p < 0.05). The higher extract concentration and incubation temperature (40°C) was always effective in producing the utmost MCA in these interactions. The SE was faster than HE in milk clot formation. The cow milk was a more suitable substrate than mare milk for the enzyme activity.

STUDY OF THE BIOTECHNOLOGICAL POTENTIAL OF SELECTED LACTIC ACID BACTERIA CULTURES

Fermenting microflora has been selected by biotechnological activity markers, with various methodological approaches used, namely: directional selection, selection of bacteriophage-insensitive mutants, protoplast regeneration. The experimental data show a significant biotechnological potential of the selected lactic acid bacteria. They are characterized by high milk-clotting activity and yield, the ability to form aromatic compounds and/or viscous components, excellent organoleptic qualities of clots fermented by them, antago-nistic activity against pathogenic and opportunistic pathogenic microorganisms, and phage-resistance to species-specific virulent phages. The collection of industrial microorganisms of Institute of Food Resources of National Academy of Agrarian Sciences (IFR NAAS) has been supplemented with new bioactive strains, in particular, the species Streptococcus thermophilus, Lactobacillus acidophilus, Lactoba-cillus rhamnosus and Lactococcus lactis. Four bacterial compositions have been created. Three of them (Iprovit-LB-R; Iprovit-LB-A, and Iprovit-ST) are supposed to be used as functional enrichers for dry foodstuffs or as biologically active supplements. The bacterial prepara-tion Iprovit-Bifidolux is a universal composition. It can be introduced both as a fermenting culture for milk fermentation and as an enricher for dry and liquid foodstuffs. Biotechnologies for the production of dry bacterial preparations on the basis of selected strains have been field-proven at the State Research Enterprise of Starter Cultures that belongs to the IFR NAAS. The data on a wide range of clinical and therapeutic effects of the bacterial preparation Iprovit-Bifidolux allow recommending it for manufacturing functional foods.

Production, purification and characterization of a milk clotting enzyme from Bacillus methanolicus LB-1.

Bacillus methanolicus LB-1 isolated from traditional rice wine was found to produce a milk clotting enzyme (MCE), and its fermentation conditions were optimized using response surface methodology. Then the MCE was produced by ethanol precipitation, and further chromatography separation resulted in a 10.46-fold purification with a 59.28% recovery. The MCA (milk clotting activity) of the purified MCE reached 597,310 ± 0.13 SU/g. The optimal temperature of the MCE was determined to be 50°C and it was stable in the low temperature range of 40-45°C. The MCE had an optimum pH of 6.5, and it was stable under neutral conditions. Calcium chloride at the concentration of 25mM was found to be the most effective stimulus. The MCE was identified by LC-MS to be a putative protein (ID I3EB99) containing 759 amino acids with a molecular weight of 80.37kDa and a pI of 9.23.

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.

Drying kinetics and mass transfer properties in the drying of thistle flower

Abstract Thistle flowers, and particularly their stigmas, are used to coagulate milk in the production of a number of traditional Portuguese cheeses due to their high milk-clotting activity provided by the high content of aspartic proteases. The aim of the present work was to determine the mass transfer properties of thistle flower under different drying conditions: natural drying and convective drying. Convective drying took place in a convection chamber set at different temperatures (35 to 65 °C) and the process was terminated when the sample presented a moisture content of about 5% or less. The traditional drying method was also used, placing the thistle flowers in a dry place sheltered from the sun, and leaving them to dehydrate at the variable room temperature. The present work allowed for the conclusion that convective drying was much faster than natural drying, and that the drying rate increased with temperature. The drying curve revealed an initial constant rate period followed by a falling rate. All the five thin layer models tested to fit the experimental data were shown to adequately describe the drying of the thistle flowers, but the best one was the Page model. The drying constant increased with temperature as did the effective diffusivity and the mass transfer coefficient. The results allowed one to estimate the activation energy for moisture diffusion (57 kJ/mol) and for convective mass transfer (78 kJ/mol). Thus this study showed the possibilities for designing efficient drying processes for the thistle flower used for milk-clotting in the manufacture of traditional cheeses.

Characterization of Cynara cardunculus L. flower from Alentejo as a coagulant agent for cheesemaking

The cardoon (Cynara cardunculus L.) is a mandatory vegetable coagulant for certain Protected Designation of Origin Portuguese cheeses. It grows wild in Portugal and is used without any type of control regarding flower picking or extract preparation, representing some uncertainty in cheese manufacture. The variability in technological properties, in the context of traditional cheese manufacture, of cardoon flower ecotypes from the Alentejo region was evaluated, including milk clotting and proteolytic activities, coagulation properties and potential cheesemaking yield of flower extracts. Multivariate statistics highlighted the variability of flower properties for cheesemaking, but allowed the aggregation of the ecotypes into five groups under the major influence of milk clotting activity and effect on gel firmness and micellar aggregation rate, followed by proteolytic activity. These differences may have an impact on cheese properties and therefore can allow the selection of cardoon flower for the manufacture of different types of cheese.

A novel milk-clotting cysteine protease from Ficus johannis: Purification and characterization

Due to the need for calf rennet alternatives, many attempts have been made to find new proteases. A novel cysteine protease with milk-clotting activity was purified from Ficus johannis by cation exchange chromatography. The protease was stable in various pH (3.0–10.5) with the optimum at 6.5 and showed its maximum activity at 60 °C. The Km and Vmax values of the enzyme were obtained to be 0.604 mg/ml and 0.0273 μmol Tyr/min, respectively. The purified protease exhibited considerable activity towards κ-casein in comparison to α-casein and β-casein. The enzyme was almost completely active in the presence of high salt concentrations. Besides, it had high stability against autodigestion. The content of free amino acids was determined by HPLC, where leucine, lysine, valine, γ-aminobutyric acid and tyrosine were the most abundant amino acids. The cheese manufactured by using the purified protease showed similar textural properties and physico-chemical compositions to cheese produced using commercial rennet. Considering the special characteristics, including high milk-clotting activity, considerable stability over wide ranges of pH and temperature, resistance towards solvents, salts, and surfactants, the new protease might be the promising candidate for the dairy industry as well as other food and biotechnological industries.

Proteases from Calotropis gigantea stem, leaf and calli as milk coagulant source

Abstract Background Universal demand for cheese keeps the search for appropriate enzymes from plants mimicking animal rennet action in scientific focus. Objective To associate distribution of milk clotting potential and profile of whole/κ-casein hydrolysis by Calotropis gigantea stem, leaf and respective calli crude enzymes (CE). Materials and methods Milk clotting activity and index were assayed for CE. Caseinolytic activity (CA) was evaluated spectrophotometrically. 0.5 CA units of CE and Enzeco® were used for studying whole/κ-casein hydrolysis pattern by Tricine SDS-PAGE. Inhibition studies were performed for enzyme characterisation. Results Traditionally propagated (TP) stem and its callus CE exhibited high specific milk clotting activity (1297.30±0.2 U/mg of protein and 926.74±44.13 U/mg of protein, respectively) and milk clotting index (103.562±1.162 and 79.365±4.93, respectively). Comparison of whole casein hydrolytic pattern by 0.5 CA units of CE revealed closer resemblance between leaf callus and Enzeco®. However, κ-casein specificity analysis revealed TP leaf to be closely mimicking the performance by Enzeco®. Conclusion Study suggests CE from TP leaf to be a potential vegetable coagulant to work as an effective and low-cost milk clotting mediator in cheese industry.

Milk-clotting activity of high pressure processed coagulants: Evaluation at different pH and temperatures and pH influence on the stability

Abstract This study evaluated the milk-clotting activity (MCA) at different pH values and temperatures of milk and the influence of pH on the stability of commercial coagulants subjected to high pressure processing (HPP). The pressurized enzymes showed higher MCA for almost all conditions studied, as compared to the non-processed samples, and the differences were more pronounced at higher temperatures and lower pH values. For the conditions used in industrial cheese making (pH 6.6 at 35 °C), the HPP led to an increase in MCA of 27, 9, 8, and 5% for the recombinant camel chymosin, bovine rennet, calf rennet, and porcine pepsin, respectively. In addition, HPP increased the pH stability for most of the enzymes. Therefore, HPP emerges as an effective technology to improve the hydrolytic profile of these enzymes as a function of pH and temperature. Industrial relevance HPP was able to promote increased in milk-clotting activity and stability of coagulants at different temperatures and pH values. For some enzymes the pressurization increased the activity at a natural pH of the milk (pH 6.6), resulting in activity similar to that observed for native enzyme at lower pH and higher temperatures. Thus, such conditions lead the use of less enzyme concentration in coagulation process, reducing costs. Additionally, it was also shown that lyophilization preserved the changes induced by the HPP in enzymes solution. Therefore, HPP technology is a unit operation can be used to increase activity and stability of milk-clotting enzymes.

Calf rennet production and its performance optimization

Milk clotting enzymes are one of the most significant cheese making raw materials impacting and regulating milk coagulation activity. Calf rennet is one of the enzymes which extracted from suckling calf abomasum. The rennet extracted was optimized for its clotting time t (s) by varying three levels of temperature (30, 35 and 40oC), pH (4, 5 and 6) and CaCl2 concentration (0.1, 0.2 and 0.3 gm/500 ml of milk) using response surface method (RSM) and also its milk clotting activity (MCA) was determined. Based on the optimized result, temperature 39.13oC, pH 4 and CaCl2 concentration of 0.21 gm/500ml had resulted minimum clotting time of 91.27 s. Using this minimum clotting time, the strength was found to be 1: 13148, establishing the fact that the calf rennet is the best natural coagulant of milk.

Characterization and milk coagulating properties of Cynanchum otophyllum Schneid. proteases

The herbaceous plant Cynanchum otophyllum Schneid. is widely used as a milk coagulant to make a Chinese traditional milk product, milk cake. However, the milk-clotting compounds and their mechanism remain unclear. In this study, crude proteases were extracted from the dried leaves of Cynanchum otophyllum Schneid. using citric acid-phosphate buffer and then partially purified by weak anion exchange chromatography. Two proteases, QA and QC, with molecular weights of 14 and 27 kDa, respectively, were shown to exhibit milk-clotting activity. A study of the effects of pH and temperature on the milk-clotting activity and proteolytic activity of the proteases showed that they exhibited good pH stability from pH 5.5 to 7.5 and good thermal stability at temperatures from 50 to 70°C. The QA and QC were the cysteine proteases, able to hydrolyze β-casein and κ-casein completely, and α-casein partially. The cleavage site on κ-casein determined by Orbitrap (Thermo Fisher Scientific, San Jose, CA) analysis showed that QA and QC could cleave κ-casein at Ser132-Thr133. Overall, the results suggest that the Cynanchum otophyllum Schneid. proteases are a promising milk-clotting enzyme that could be used for manufacturing milk cake and cheese.

Expression, activation and processing of a novel plant milk-clotting aspartic protease in Pichia pastoris

Galium verum, also known as Lady’s Bedstraw or Cheese Rennet, is an herbaceous perennial plant traditionally used in cheese-making. We used RACE PCR to isolate novel enzymes from Galium verum with the ability to clot milk. This approach generated two cDNA sequences (named preprogaline A and B) encoding proteins displaying the typical plant aspartic protease primary structure. Preprogaline B was expressed in the yeast Pichia pastoris, after deleting and replacing its original signal peptide with the yeast α-factor signal peptide from Saccharomyces cerevisiae. The secreted recombinant protein was obtained by growing P. pastoris in YPD medium and had the ability to clot milk. The mature form of progaline B is a heterodimeric glycosylated enzyme, with a molecular weight of approximately 48 kDa, that contains a heavy (30.7 kDa) and a light (13.5 kDa) polypeptide chains linked by disulfide bonds. Western blot analysis revealed that progaline B is activated by the acidification of the yeast culture medium and that enzymatic activation requires two steps. First the precursor protein is cleaved into two polypeptide chains by partial removal of the plant-specific insert (PSI) present in plant aspartic proteases; this is later followed by propeptide removal. By altering the pH of the P. pastoris culture medium, we were able to obtain either active or inactive forms of the enzyme. Recombinant progaline B displayed a κ-casein hydrolysis pattern analogous to those produced by the animal and microbial coagulants currently used in the dairy industry, but it exhibited a different digestion profile on α- and β-caseins. The plant protease progaline B displays milk-clotting activities suitable for the production of novel dairy products.

PROTEOLYTIC ENZYMES IN CHEESE MAKING

There has been an increasing consumer demand for cheese along with a search for products with new organoleptic parameters, which has resulted in extensive research on alternative milk coagulants. The ratio of proteolytic activity to milk-clotting activity determines the requirements for proteases used in the cheese making process. To date, plant enzymes have largely been used for this purpose, along with traditional enzymes of animal origin, chymosin and pepsin. The most popular types of proteases used in the food industry, especially in cheese making, are plant proteases belonging to the cysteine (papain, bromelain, ficin), aspartate (cinarase, cardosin), and serine (kukumizin, leucine) group of proteases. The aspartate proteases of microbial origin mucorpepsin and endotyapepsin have found wide application in cheese production due to low production costs and high organoleptic characteristics of the final product. The use of plant and microbial milk-clotting enzymes as an alternative to animal-derived enzymes allows not only to diversify the assortment of cheeses on the market but also to solve ethical and economic issues. In addition, vegetable and microbial preparations meet the requirements of vegetarianism, halal, and kosher food, thus further opening the market.

Crude Enzymes from a &lt;i&gt;Hericium&lt;/i&gt; Edible Mushroom Isolated in Japan: Variability in Milk-clotting Activity and the Ability to Coagulate Ultra-high-temperature Pasteurized Milk

Crude Enzymes from a &lt;i&gt;Hericium&lt;/i&gt; Edible Mushroom Isolated in Japan: Variability in Milk-clotting Activity and the Ability to Coagulate Ultra-high-temperature Pasteurized Milk

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.

EVALUATION OF RENNET SUBSTITUTE FROM ARTICHOKE (Cynara scolymus L.) FLOWERS EXTRACTS: STUDY THE FACTORS AFFECTING THE ACTIVITY OF MILK CLOTTING

Factors affecting milk clotting activity of rennet substitute extracted from green bracts of artichoke (Cynara scolymus) flowers in different buffer solutions were evaluated. These factors include extraction pH, clotting temperature, extract concentrations, addition of CaCl2, NaCl and glucono delta-lactone (GDL) at different concentrations. The proteolytic activity of different extract solutions and rheological properties of produced curd was studied as well. The obtained results indicated that the optimum clotting activity of artichoke crude extracts improved water holding capacity and susceptibility to syneresis obtained at pH value of 5.0-6.0, temperature of 65-70°C and 3%, 0.04-0.05, 3%, 0.5% of crude extract, CaCl2, NaCl and GDL concentrations, respectively. The optimum clotting activity of artichoke crude extracts indicated that the sodium acetate buffer solution T4 (5% NaCl in sodium acetate buffer, pH 5.0) and sodium phosphate buffer, T5 (5% NaCl in sodium phosphate buffer, pH 5.5), T6 (5% NaCl in sodium phosphate buffer, pH 6.5), and T7 (5% NaCl in sodium phosphate buffer, pH 7). Results also indicated that artichoke crude extracted in sodium phosphate buffer solutions and sodium acetate buffer (T4) had higher proteolytic activity than other buffers. The best rheological properties of resultant curds were noticed in sodium phosphate buffers (T5, T6, T7) and sodium acetate buffer (T4).

Study the influence of culture conditions on rennin production by Rhizomucor miehei using solid-state fermentations.

Investigations were conducted on the production of Rennin enzyme from the fungi Rhizomucor miehei 3420 NRRL using Solid-State fermentation. Wheat bran was used as a substrate. The influence of moisture content, incubation temperature, and the initial pH of fermentation medium were studied. The protein content, milk clotting activity (MCA), specific activity, proteolytic activity (PA), and (MCA/PA) ratio of the extracted enzyme were calculated after 4 days of incubation to evaluate the quality of the enzyme. The results showed that the optimal conditions for production were as follows: incubation temperature of 40 °C, moisture content of 60%, and pH of (3). Under these conditions, a production process of Rennin enzyme was established, and the values of protein content, milk clotting activity, specific activity, proteolytic activity, and (MCA/PA) ratio reached to 4 mg/mL, 600 SU/mL, 150 SU/mg, 45 PU/mL, 13.3 respectively.

Utility of Milk Coagulant Enzyme of Moringa oleifera Seed in Cheese Production from Soy and Skim Milks.

In this study, the potential use of Moringa oleifera as a clotting agent of different types of milk (whole, skim, and soy milk) was investigated. M. oleifera seed extract showed high milk-clotting activity followed by flower extract. Specific clotting activity of seed extract was 200 times higher than that of flower extract. Seed extract is composed by four main protein bands (43.6, 32.2, 19.4, and 16.3 kDa). Caseinolytic activity assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and tyrosine quantification, showed a high extent of casein degradation using M. oleifera seed extract. Milk soy cheese was soft and creamy, while skim milk cheese was hard and crumbly. According to these results, it is concluded that seed extract of M. oleifera generates suitable milk clotting activity for cheesemaking. To our knowledge, this study is the first to report comparative data of M. oleifera milk clotting activity between different types of soy milk.

Functional and structural characterization of synthetic cardosin B-derived rennet.

The potential of using a synthetic cardosin-based rennet in cheese manufacturing was recently demonstrated with the development and optimization of production of a recombinant form of cardosin B in Kluyveromyces lactis. With the goal of providing a more detailed characterization of this rennet, we herein evaluate the impact of the plant-specific insert (PSI) on cardosin B secretion in this yeast, and provide a thorough analysis of the specificity requirements as well as the biochemical and structural properties of the isolated recombinant protease. We demonstrate that the PSI domain can be substituted by different linker sequences without substantially affecting protein secretion and milk clotting activity. However, the presence of small portions of the PSI results in dramatic reductions of secretion yields in this heterologous system. Kinetic characterization and specificity profiling results clearly suggest that synthetic cardosin B displays lower catalytic efficiency and is more sequence selective than native cardosin B. Elucidation of the structure of synthetic cardosin B confirms the canonical fold of an aspartic protease with the presence of two high mannose-type, N-linked glycan structures; however, there are some differences in the conformation of the flap region when compared to cardosin A. These subtle variations in catalytic properties and the more stringent substrate specificity of synthetic cardosin B help to explain the observed suitability of this rennet for cheese production.