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

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

Molecular identification of Brazilian Fusarium strains: sources of proteases with milk-clotting properties.

Fusarium is a genus of ubiquitous fungi that comprises mycotoxigenic animal and plant pathogens. These fungi have the ability to exploit a wide range of substrates and hosts, indicating their great potential for enzyme production; however, this aspect is understudied. Therefore, the present study aimed for revaluating the identity of twenty-three Fusarium strains maintained in the University Recife Mycology (URM) culture collection, Brazil, and to evaluate their potential for proteases production and the milk-clotting activity of these proteases. According to phylogenetic analysis of translation elongation factor 1-alpha (TEF1) gene partial sequences, these strains belonged to 12 species representing four species complexes: Fusarium concolor, F. fujikuroi, F. incarnatum-equiseti, and F. oxysporum. Four of these species are putatively novel to science. Notably, novel associations of Fusarium spp. with certain hosts/substrates were documented. The proteolytic activity ranged from 1.67 U ml-1 to 22.03 U ml-1 among the evaluated fungal isolates, with specific proteolytic activity reaching 205.86 U mg-1. The values for coagulant activity and specific activity were up to 157.14 U ml-1 and 1,424.11 U mg-1, respectively. These results indicate the potential of URM Fusarium strains as a source for the production of enzymes of industrial interest. Additionally, they reinforce the importance of applying DNA-based methods for reviewing the identification of fungal strains preserved in biodiversity repositories.

Thermostable trypsin-like protease by Penicillium roqueforti secreted in cocoa shell fermentation: Production optimization, characterization, and application in milk clotting.

The increased demand for cheese and the limited availability of calf rennet justifies the search for milk-clotting enzymes from alternative sources. Trypsin-like protease by Penicillium roqueforti was produced by solid-state fermentation using cocoa shell waste as substrate. The production of a crude enzyme extract that is rich in this enzyme was optimized using a Doehlert-type multivariate experimental design. The biochemical characterization showed that the enzyme has excellent activity and stability at alkaline pH (10-12) and an optimum temperature of 80°C, being stable at temperatures above 60°C. Enzymatic activity was maximized in the presence of Na+ (192%), Co2+ (187%), methanol (153%), ethanol (141%), and hexane (128%). Considering the biochemical characteristics obtained and the milk coagulation activity, trypsin-like protease can be applied in the food industry, such as in milk clotting and in the fabrication of cheeses.

Milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits on individual caseins.

In recent years, many attempts have been made to find new plant proteases to make artisan cheeses. The global increase in cheese consumption, together with a lower supply and increasing cost of calf rennet, religious factors (Islam and Judaism) and food choices (vegetarianism) have led to the search for suitable rennet substitutes for milk clotting. This study describes the milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits (SoAP) on individual caseins to explore its potential use as an alternative to animal rennet. The milk-clotting index obtained for SoAP was 8.4 times lower than that obtained for chymosin. SoAP showed a higher degree of hydrolysis on α-casein than on the other fractions under the proposed conditions. RP-HPLC, mass spectrometry analyses and sequencing of the hydrolysates allowed identifying five peptides from α-casein, one peptide from ß-casein, and three peptides from k-casein. In silico analysis showed that the peptides identified may display a wide variety of potential biological activities. These results demonstrate the possibility of using SoAP for the manufacture of new types or artisan cheeses, with the simultaneous added value of the potential health-promoting benefits of the bioactive peptides generated during the hydrolysis.

Exploring the Milk-Clotting and Proteolytic Activities in Different Tissues of Vallesia glabra: a New Source of Plant Proteolytic Enzymes.

Proteolytic enzymes are widely distributed in nature, playing essential roles in important biological functions. Recently, the use of plant proteases at the industrial level has mainly increased in the food industry (e.g., cheesemaking, meat tenderizing, and protein hydrolysate production). Current technological and scientific advances in the detection and characterization of proteolytic enzymes have encouraged the search for new natural sources. Thus, this work aimed to explore the milk-clotting and proteolytic properties of different tissues of Vallesia glabra. Aqueous extracts from the leaves, fruits, and seeds of V. glabra presented different protein profiles, proteolytic activity, and milk-clotting activity. The milk-clotting activity increased with temperature (30-65 °C), but this activity was higher in leaf (0.20 MCU/mL) compared with that in fruit and seed extracts (0.12 and 0.11 MCU/mL, respectively) at 50 °C. Proteolytic activity in the extracts assayed at different pH (2.5-12.0) suggested the presence of different types of active proteases, with maximum activity at acidic conditions (4.0-4.5). Inhibitory studies indicated that major activity in V. glabra extracts is related to cysteine proteases; however, the presence of serine, aspartic, and metalloproteases was also evident. The hydrolytic profile of caseins indicated that V. glabra leaves could be used as a rennet substitute in cheesemaking, representing a new and promising source of proteolytic enzymes.

Optimization of media composition and growth conditions for production of milk-clotting protease (MCP) from Aspergillus oryzae DRDFS13 under solid-state fermentation.

This study reports the optimization of milk-clotting protease production from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) in both one-variable-at-a-time and response surface methodology (RSM). The production and optimization of milk-clotting protease obtained from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) using different agro-industrial wastes as solid substrates were studied. The agro-industrial wastes used included wheat bran, rice bran, pea bran, and grass pea bran. The chemical composition of the best solid substrate was tested using standard methods. Others cultivation parameters were studied, and the results showed that the optimum fermentation medium composed of wheat bran, casein (1% w/w), and glucose (0.5% w/w) and the conditions for maximum milk-clotting protease production were at the moisture content of 55.0%, inoculum of 0.5*106 spores/mL, incubation temperature of 30 °C, pH of 6.0, and fermentation time of 5 days. The highest milk-clotting activity was obtained from the crude enzyme extracted using 0.1 M NaCl and partial purification of the crude enzyme using chilled acetone, and 80% (NH4)2SO4 increased the ratio of MCA/PA from 0.56 to 1.30 and 0.65, respectively. Moreover, the highest MCA (137.58 U/mL) was obtained at a casein concentration of 0.5%, pH 4.0, and 25 °C, using RSM. Thus, results from the present study showed that the optimization of milk-clotting protease production from A. oryzae DRDFS 13 under SSF by both one-variable-at-a-time and RSM significantly increased the milk-clotting activity. This is the first report from a fungus in the Ethiopian setting and a modest contribution to highlight the potential of harnessing microbial protease enzymes for industrial applications.

Evaluation of the milk clotting properties of an aspartic peptidase secreted by Rhizopus microsporus.

Traditionally, chymosin has been used for milk-clotting, but this naturally occurring enzyme is in short supply and its use has raised religious and ethical concerns. Because milk-clotting peptidases are a promising substitute for chymosin in cheese preparation, there is a need to find and test the specificity of these enzymes. Here, we evaluated the milk-clotting properties of an aspartic peptidase secreted by Rhizopus microsporus. The molecular mass of this enzyme was estimated at 36 kDa and Pepstatin A was determined to be an inhibitor. Optimal activity occurred at a pH of 5.5 and a temperature range of 50-60 °C, but the peptidase was stable in the pH range of 4-7 and a temperature as low as 45 °C. Proteolytic activity was significantly reduced in the presence of Cu2+ and Al3+. When enzyme substrates based on FRET were used, this peptidase exhibited the highest catalytic efficiency for Abz-KNRSSKQ-EDDnp (4,644 ± 155 mM-1.s-1), Abz-KLRSSNQ-EDDnp (3,514 ± 130 mM-1.s-1), and Abz-KLRQSKQ-EDDnp (3,068 ± 386 mM-1.s-1). This study presents a promising peptidase for use in cheese making, due to its high stability in the presence of Ca2+ and broad pH range of 4-7, in addition to its ability to efficiently clot milk.

Biochemical characterization of a semi-purified aspartic protease from sea catfish Bagre panamensis with milk-clotting activity.

Pepsin from stomach of Bagre panamensis was semi-purified and biochemically characterized. The acid proteolytic activity and purification fold were 3875 U/mg protein and 91.85, respectively, after purification process. The optimum pH and temperature for semi-purified protease were 2-3 and 65 °C, respectively. The enzyme activity was stable after heating proteases at 50 °C for 120 min, but only 30% residual activity was detected after heating at 65 °C for 30 min. SDS-PAGE analysis showed two proteins bands after dialysis (26.1 and 38.6 kDa). Only the band of 38.6 kDa had proteolytic activity, which was inhibited using pepstatin A. Organic solvents, surfactants and reducing agents affect the proteolytic activity at different extent; however, metal ions or EDTA have no impact on protease activity. The semi-purified protease exhibited milk coagulant activity, with a maximum activity at 45 °C. The obtained results highlight the potential biotechnological use of B. panamensis pepsin.

Biophysical evaluation of milk-clotting enzymes processed by high pressure.

High pressure processing (HPP) is able to promote changes in enzymes structure. This study evaluated the effect of HP on the structural changes in milk-clotting enzymes processed under activation conditions for recombinant camel chymosin (212MPa/5min/10°C), calf rennet (280MPa/20min/25°C), bovine rennet (222MPa/5min/23°C), and porcine pepsin (50MPa/5min/20°C) and under inactivation conditions for all enzymes (600MPa/10min/25°C) including the protease from Rhizomucor miehei. In general, it was found that the HPP at activation conditions was able to increase the intrinsic fluorescence of samples with high pepsin concentration (porcine pepsin and bovine rennet), increase significantly the surface hydrophobicity and induce changes in secondary structure of all enzymes. Under inactivation conditions, increases in surface hydrophobicity and a reduction of intrinsic fluorescence were observed, suggesting a higher exposure of hydrophobic sites followed by water quenching of Trp residues. Moreover, changes in secondary structure were observed (with minor changes seen in Rhizomucor miehei protease). In conclusion, HPP was able to unfold milk-clotting enzymes even under activation conditions, and the porcine pepsin and bovine rennet were more sensitive to HPP.

Biochemical and milk-clotting properties and mapping of catalytic subsites of an extracellular aspartic peptidase from basidiomycete fungus Phanerochaete chrysosporium.

For a long time, proteolytic enzymes have been employed as key tools of industrial processes, especially in the dairy industry. In the present work, we used Phanerochaete chrysosporium for biochemical characterization and analysis of catalytic specificity of an aspartic peptidase. Our results revealed an aspartic peptidase with molecular mass ∼38kDa, maximal activity at pH 4.5 and 50°C, and stability above 80% in the pH range of 3-8 and temperature up to 55°C for 1h. In a milk-clotting assay, this peptidase showed maximal milk clotting activity at 60-65°C and maintenance of enzymatic activity above 80% in the presence of 20mM CaCl2. In a specificity assay, we observed stronger restriction of catalysis at the S1 subsite, with a preference for lysine, arginine, leucine, tyrosine, and phenylalanine residues. The restricted proteolysis and milk-clotting potential are attractive properties for the use in cheese production.

Potencial do latex da fruta pão (Artocarpus altilis) como agente coagulante do leite

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.

A fruta-pão é uma árvore exótica no Brasil, onde se aclimatou muito bem. Embora seja uma planta lactífera, o conhecimento sobre seu látex é escasso. No entanto, sabe-se que enzimas proteolíticas correspondem a mais de 50% da composição do látex em plantas lactíferas. O objetivo deste estudo foi avaliar o potencial do látex da fruta-pão (Artocarpus altilis var. Apyrena) como fonte de protease coagulante do leite e caracterizar parcialmente a enzima. A atividade proteolítica da fração de extrato bruto foi avaliada utilizando azocaseína e, para a quantificação das proteínas totais, o ácido bicinconínico (BCA). A atividade de coagulação do leite foi testada utilizando leite desnatado a 12%. A protease foi testada em diferentes condições de temperatura (35 a 80°C) e pH (5,8 a 10,7) e apresentou atividade ótima a 50°C e pH alcalino (8,5) sendo estável a estas variáveis durante 120 minutos. A atividade coagulante no leite foi diretamente proporcional à temperatura na melhor concentração de CaCl2 a 10µmol L-1. Os resultados indicam que a enzima analisada é uma possível alternativa à quimosina.

Potencial do latex da fruta pão (Artocarpus altilis) como agente coagulante do leite / Potential of breadfruit (Artocarpus altilis) latex as a milk-clotting agent

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.(AU)

A fruta-pão é uma árvore exótica no Brasil, onde se aclimatou muito bem. Embora seja uma planta lactífera, o conhecimento sobre seu látex é escasso. No entanto, sabe-se que enzimas proteolíticas correspondem a mais de 50% da composição do látex em plantas lactíferas. O objetivo deste estudo foi avaliar o potencial do látex da fruta-pão (Artocarpus altilis var. Apyrena) como fonte de protease coagulante do leite e caracterizar parcialmente a enzima. A atividade proteolítica da fração de extrato bruto foi avaliada utilizando azocaseína e, para a quantificação das proteínas totais, o ácido bicinconínico (BCA). A atividade de coagulação do leite foi testada utilizando leite desnatado a 12%. A protease foi testada em diferentes condições de temperatura (35 a 80°C) e pH (5,8 a 10,7) e apresentou atividade ótima a 50°C e pH alcalino (8,5) sendo estável a estas variáveis durante 120 minutos. A atividade coagulante no leite foi diretamente proporcional à temperatura na melhor concentração de CaCl2 a 10µmol L-1. Os resultados indicam que a enzima analisada é uma possível alternativa à quimosina.(AU)

Potencial do latex da fruta pão (Artocarpus altilis) como agente coagulante do leite

A fruta-pão é uma árvore exótica no Brasil, onde se aclimatou muito bem. Embora seja uma planta lactífera, o conhecimento sobre seu látex é escasso. No entanto, sabe-se que enzimas proteolíticas correspondem a mais de 50% da composição do látex em plantas lactíferas. O objetivo deste estudo foi avaliar o potencial do látex da fruta-pão (Artocarpus altilis var. Apyrena) como fonte de protease coagulante do leite e caracterizar parcialmente a enzima. A atividade proteolítica da fração de extrato bruto foi avaliada utilizando azocaseína e, para a quantificação das proteínas totais, o ácido bicinconínico (BCA). A atividade de coagulação do leite foi testada utilizando leite desnatado a 12%. A protease foi testada em diferentes condições de temperatura (35 a 80°C) e pH (5,8 a 10,7) e apresentou atividade ótima a 50°C e pH alcalino (8,5) sendo estável a estas variáveis durante 120 minutos. A atividade coagulante no leite foi diretamente proporcional à temperatura na melhor concentração de CaCl2 a 10µmol L-1. Os resultados indicam que a enzima analisada é uma possível alternativa à quimosina

Efecto del recunnto de células somáticas sobre a aptitud quesera de la leche yy la calidad fisicoquímica y sensorial del queso campesino / Somaticcell counnt effect on cheese yield and quality of fresh cheese

Antecedentes: en Colombia, el alto recuento de células somáticas (RCS) en la leche es un problema para la industria lechera. Altos recuentos pueden afectar de manera considerable los rendimientos y calidad final del queso. Varios países han establecido límites máximos estandarizados para el RCS. Colombia no lo ha hecho de manera oficial y tan solo unas pocas industrias manejan sus propios límites. Objetivos: Determinar el efecto del RCS sobre parámetros de aptitud quesera de la leche y la calidad sensorial del queso campesino. Métodos: Se tomaron muestras de leche de seis tanques con altos y bajos RCS y se realizaron mezclas para obtener 30 baches con diferentes RCS (desde 150.000 hasta 1.200.000 cel/ml). Con estas mezclas se elaboraron 30 quesos tipo campesino a los cuales se les determinaron variables de aptitud quesera (tiempo de coagulación, rendimientos y pérdidas en suero) y la calidad organoléptica a través de una prueba sensorial descriptiva de puntajes con panel de seis jueces con experiencia previa y entrenados en queso campesino. Las variables de aptitud quesera fueron analizadas por regresión múltiple y los resultados de la evaluación sensorial con la prueba no paramétrica de Friedman. Resultados: La aptitud quesera disminuyó con RCS superiores a 200.000 cel/ml. El tiempo de coagulación (R² = 0.74; P< 0.001) y las pérdidas de proteína en el lactosuero (R² = 0.55; P<0,001) aumentaron, mientras que los rendimientos (R²=0.31; P<0.01) disminuyeron a medida que aumentó el RCS. La calificación de los panelistas respecto de la textura y la apariencia disminuyó con RCS mayores a 600.000 cel/ml (P<0.01) y el sabor y el aroma, a partir de 800.000 cel/ml (P<0,01). Conclusiones: Aumentos en el RCS en leche afectan negativamente parámetros de aptitud quesera y la calidad sensorial del queso campesino. Se sugiere que los impactos serán menores sobre el rendimiento cuanto menor sea el RCS, mientras que la calidad organoléptica mejorará cuando la leche tenga RCS por debajo de 600.000 cel/ml.

Background: A high milk somatic cell count (SCC) is a problem for milk industry in Colombia. These high counts can affect considerably yield and final quality of cheese. Several countries have established maximum limits for SCC. In Colombia these limits have not been established officially, only a few industries have their own limits. Objectives: To determine the effect of somatic cell count (SCC) on milk potential for cheese making and sensorial quality of fresh cheese. Methods: Six milk samples with high and low SCC, were taken from bulk tanks and mixed to obtain 30 samples with SCC of 150.000 to 1.200.000 somatic cells/mL. Thirty fresh cheeses were prepared and clotting time, yield (protein, fat, dry matter) and whey losses were determined. Additionally, score descriptive sensorial quality test was performed by 6 trained judges. Protein, fat, dry matter in milk and cheese yields were analyzed by multiple regressions and information of sensorial test by Friedman method. Results: When milk SCC (somatic cells/ml) increased from 150.000 to 1.200.000, clotting time (R² = 0,74; P<0.001), and whey protein losses increased (R² = 0.55; P<0.001) and cheese yield decreased (R²=0.31, P<0.01). According to panelists, texture and appearance were affected negatively when SCC was higher than 600.000 cells per ml (P<0.01), flavor and aroma when they were higher than 800000 cells/ml. Conclusions: Increases in SCC have a negative effect on milk potential for cheese making and quality sensorial parameters on fresh cheese. It is suggested that minor impacts in fresh cheese yield would be obtained with a lower SCC and for good sensorial quality when the milk has SCC, lower than 600.000 cells per ml.

Comparison of the milk-clotting properties of three plant extracts.

Several proteases from plant sources have been proposed as milk coagulants, however, limited research has been done on their milk-clotting properties. The effect of temperature on the milk-clotting activity of kiwi fruit, melon and ginger extracts was evaluated, as well as the effects of the different extracts on curd properties. Melon extracts showed high milk-clotting activity over a broad temperature range (45-75 °C) while kiwi fruit and ginger extracts showed high activity over a narrower temperature range, with a maximum at 40 and 63 °C, respectively. Curds produced using kiwi extracts had textural properties comparable with those obtained using commercial rennet, while melon extracts produced a fragile gel and low curd yield. The milk-clotting behavior of the three plant extracts was related to the protease specificity present in these extracts. The kiwi proteases displayed chymosin-like properties and thus hold the best potential for use as a milk coagulant in cheese production.

Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

The partial characterization and purification of milk clotting enzyme obtained from the (root latex) of Jacaratia corumbensis O. kuntze was studied, by fractional precipitation with ammonium sulphate and ion exchange chromatography. The ammonium sulphate precipitate showed five fractions (AS1- 0-20 percent; AS2 - 20-40 percent; AS3 - 40-60 percent; AS4 - 60-80 percent; AS5 - 80-100 percent) and among the fractions obtained, the 40-60 percent fraction (AS3) showed the highest milk clotting activity with a purification factor of 1.2 fold in relation to the crude extract. This fraction when applied on Mono Q column yielded two protein peaks (p1 and p2), but p1 pool showed the best milk-clotting activity. The optimal pH for the crude and partially purified extract was 6.5 and 7.0, respectively. The maximum milk-clotting activity was at 55ºC for the both crude and partially purified extracts. The enzyme was inhibited by iodoacetic acid which suggested that this enzyme was a cysteine protease, with molecular weight of 33 kDa.

A enzima coagulante de leite obtida de látex de raiz de Jacaratia corumbensis O. kuntze foi caracterizada parcialmente e purificada, por precipitação fracionária com sulfato de amônio e cromatografia de troca de íon. Foram utilizadas cinco frações de sulfato de amônio (AS1 - 0-20 por cento; AS2 - 20-40 por cento; AS3 - 40-60 por cento; AS4 - 60-80 por cento; AS5 - 80-100 por cento), a fração 40-60 por cento (AS3) mostrou alta atividade coagulante com um fator de purificação de 1,2 vezes em relação ao extrato bruto. Esta fração foi aplicada em coluna Mono Q obtendo dois picos de proteína (p1 e p2), o p1 mostrou melhor atividade coagulante. O pH ótimo para o extrato bruto e parcialmente purificado foi 6,5 e 7,0, respectivamente. A atividade coagulante foi atingida a 55ºC para ambos os extratos, bruto e parcialmente purificado. A enzima foi inibida por ácido iodoacético que sugere que esta enzima é uma cisteína protease, com peso molecular de 33 kDa.