Production of milk-coagulating protease by fungus Pleurotus djamor through solid state fermentation using wheat bran as the low-cost substrate.

Proteases are enzymes that hydrolyze peptide bonds present in proteins and peptides. They are widely used for various industrial applications, such as in the detergent, food, and dairy industries. Cheese is one of the most important products of the dairy industry, and the coagulation stage is crucial during the cheese-making process. Enzymatic coagulation is the most common technique utilized for this purpose. Microbial enzymes are frequently used for coagulation due to their advantages in terms of availability, sustainability, quality control, product variety, and compliance with dietary and cultural/religious requirements. In the present study, we identified and subsequently characterized milk coagulant activity from the fungus Pleurotus djamor PLO13, obtained during a solid-state fermentation process, using the agro-industrial residue, wheat bran, as the fermentation medium. Maximum enzyme production and caseinolytic activity occurred 120 h after cultivation. When the enzyme activity against various protease-specific synthetic substrates and inhibitors was analyzed, the enzyme was found to be a serine protease, similar to elastase 2. This elastase-2-like serine protease was able to coagulate pasteurized whole and reconstituted skim milk highly efficiently in the presence and absence of calcium, even at room temperature. The coagulation process was influenced by factors such as temperature, time, and calcium concentration. We demonstrate here, for the first time, an elastase-2-like enzyme in a microorganism and its potential application in the food industry for cheese production.

Effectiveness of a natural coagulant based on common mallow (Malva sylvestris) in urban wastewater treatment.

This study evaluated the effectiveness of a natural coagulant based on common mallow (Malva sylvestris) to remove turbidity in urban wastewater. A 22 factorial design was selected to determine the optimal dose and the working pH of the natural coagulant. Its potential was studied in 50.0-450 mg/L and 4.00-10.0 ranges of doses and pH, respectively. A simplex lattice mixture evaluated its effectiveness as a coagulant aid combined with aluminum sulfate (conventional coagulant). Mixture proportions 0.000-1.00 were studied for each component, finding the proportion more effective. Results showed that the coagulation treatment could be feasible since a turbidity removal efficiency of 73.7% can be achieved under optimal conditions (50.0 mg/L and pH of 10.0). Likewise, a turbidity removal of 58.9% is obtained using 250 mg/L and maintaining wastewater pH (7.45). This efficiency can be increased using 31.0% natural coagulant mixed with 69.0% aluminum sulfate at 250 mg/L without modifying the wastewater pH. This improvement was associated with the natural coagulant's high molecular weight and long-chained structure since these properties enhance settling time, floc size and strength, and low sludge production. These results support using common mallow as a natural coagulant, making its use more feasible in alkaline water pH or as a coagulant aid combined with aluminum sulfate for urban wastewater treatment. A cost of USD 370/Kg of natural coagulant was estimated, which is higher than conventional coagulants. However, a cost-effectiveness analysis of its implementation should be performed since process scaling costs could significantly reduce its price.

Milk-Clotting Properties and Primary Proteolysis of a Lyophilised Extract from Artichoke Flowers (Cynara scolymus L.).

Research background: A few studies have investigated Cynara scolymus enzymes as a substitute for calf rennet in cheese making. They used aqueous extracts prepared by maceration of plant material and stored by freezing. However, it was indicated that lyophilisation is a better alternative to preserve the coagulant properties of plant extracts over a longer period of time, as it is a more controllable, stable and hygienic alternative with a better shelf life that is easier to transport, store and standardise. Experimental approach: We obtained a lyophilised extract of mature artichoke flowers, named CS, which was characterised for its milk-clotting properties at different pH and temperatures. In addition, the potential yield, whey composition and the primary hydrolysis profile of caseins by urea-polyacrylamide gel electrophoresis (PAGE) of mini curds prepared with different doses of coagulant were determined. Results and conclusions: The lyophilised extract was able to hydrolyse casein and showed stable proteolytic activity at pH=6.4 and 37 °C for 50 min, which decreased when the process temperature was increased to 41 and 45 °C and was lost at 70 °C. On the other hand, milk-clotting activity increased significantly (p<0.001) when the temperature increased from 37 to 45 °C and the pH of the milk decreased from 6.8 to 5.8. Potential yield ​​between 10 and 17 % was obtained for the mini curds prepared with the lyophilised artichoke extract, and the casein degradation pattern obtained by urea-PAGE was similar to that of the commercial coagulant. Novelty and scientific contribution: On a laboratory scale, our work has shown that the lyophilised artichoke extract has sufficient proteolytic and coagulant activity to be used as a milk coagulant, i.e. plant rennet, in cheese making as an alternative to animal rennet. As this extract is lyophilised, it has the advantage of being a better alternative in terms of preservation and shelf-life. It offers an innovative way to diversify cheese products and appeal to consumers with different dietary preferences and needs.

Assessment of Prickly Pear Fruit Peel Mucilage in Form of Gel as a Green Coagulant for the Tertiary Treatment of Domestic Wastewater.

The application of natural coagulants derived from food byproducts in domestic wastewater tertiary treatment, which contains a number of impurities as suspended colloidal particles, has a potential use as essential substitutes for traditional inorganic coagulants. These biomaterials are a sustainable and environmentally friendly alternative that can be used to improve water quality and human health. In this study, prickly pear (PP) fruit peel mucilage gel was evaluated as a novel coagulant for the tertiary stage of domestic wastewater treatment. Jar tests were performed on residual raw water at the inlet (influent) and outlet (effluent) of the tertiary wastewater treatment (constructed wetland) with a coagulant dose of 12 mg L-1 at a pH of 13. The efficiency of green (i.e., mucilage) and inorganic chemical (i.e., FeCl3) coagulants was compared on the basis of turbidity and color removal. The flocs produced by the coagulants were characterized structurally by FTIR spectroscopy and Zeta potential analysis and morphologically by scanning electron microscopy (SEM). The results showed that the turbidity and the color removal efficiency of the mucilage compared to the FeCl3 at the outlet of the treatment (effluent) were practically the same, reaching 94% turbidity and 85-87% color removal efficiency with both coagulants. The structure and morphology of the flocs generated by the coagulants showed a higher content of organic matter trapped in the flocs. The floc formation observed mechanisms were adsorption/bridging for mucilage and charge neutralization for FeCl3. The results of this study demonstrated that the PP mucilage green coagulant can be used to enhance the quality of treatment of domestic wastewater in an eco-friendly and biodegradable manner.

Evaluación antimicrobiana y coagulante de extractos de cinco vegetales de interés etnobotánico / Antimicrobial and coagulating evaluation of extracts of five vegetables of ethnobotanical interest

Resumen Objetivo: Evaluar la actividad antibacteriana de los extractos de Mimosa tenuiflora, Equisetum arvense, Syzygium aromaticum, Lippia graveolens y Aloe vera contra cepas bacterianas de S. mutans (ATCC700611) y S. sobrinus (ATCC33478) comparado con clorhexidina a 1200 µg/mL (0.12%) y la actividad coagulante en sangre humana. Materiales y métodos: Estudio comparativo, abierto, experimental, prospectivo y transversal in vitro. Se realizaron diluciones a 500 y 1000 µg/mL de cinco extractos y se probaron por triplicado contra microorganismos orales por medio de técnica de pozo en agar y en la evaluación de la actividad coagulante se probaron los cinco extractos por triplicado en sangre humana evaluando TP (tiempo de protrombina) y TTPa (tiempo de tromboplastina parcial activado) mediante coagulómetro. Resultados: El extracto de Lippia graveolens a 500 y 1000 µg/mL mostró un promedio de halos de inhibición sobre S. mutans de 26mm con respecto a clorhexidina a 1200 µg/mL que mostró un promedio de 15mm. Contra cepas de S. sobrinus mostraron un promedio de 19mm a 500 µg/mL y 23mm a 1000 µg/mL con respecto a 15mm de clorhexidina. El valor de TP (tiempo de protrombina) de la muestra de sangre fue 12.27 segundos, al aplicarle E. arvense y S. aromaticum ambos a 1000 µg/mL presentaron tiempos de 13.37 segundos. En cuanto al tiempo de tromboplastina parcial activada (TTPa) el valor de la muestra sin extracto fue 32.63 segundos, al aplicar M. tenuiflora a 500 µg/mL se aumentó el tiempo a 39.17 segundos. Conclusiones: Se concluye que Lippia graveolens tiene mejor efecto antibacteriano contra micrrorganismos orales y M. tenuiflora fue el extracto que aumentó por más tiempo el valor de TTPa.

Abstract Objective: To evaluate the antimicrobial and coagulating activity from five vegetables of ethnobotanical interest extracts (Mimosa tenuiflora, Equisetum arvense, Syzygium aromaticum, Lippia graveolens and Aloe vera). Materials and methods: It was a Comparative, open, experimental, prospective and cross-sectional study through antimicrobial evaluation of the five extracts against bacterial strains of S. mutans (ATCC700611) and S. sobrinus (ATCC33478) by means of agar well technique and an evaluation of coagulating activity by measuring TP (prothrombin time) and APTT (activated partial thromboplastin time) using a coagulometer and comparing the results with those of a healthy patient. Results: It was found that the antimicrobial activity of the extracts on S. mutans at 500 and 1000ppm is statistically significant in the extracts of E. arvense and L. graveolens (p= 0.0057) and (p= 0.0000) respectively and on strains of S. sobrinus from the extracts of A. vera (p= 0.0011) and L. graveolens (p= 0.0089) in both concentrations, which show an antimicrobial effect superior to chlorhexidine. The PT patient's (prothrombin time) value was 12.27 seconds, no statistical difference was observed with a value of (p<0.05), however, E. arvense and S. aromaticum, both at 1000ppm, presented times of 13.37 seconds and at the activated partial thromboplastin time (PTPA) the value of the patient was 32.63 seconds, highlighting M. tenuiflora at 500 ppm, which presented times of 39.17 seconds. Conclusions: The extracts described above contain chemical compounds that are valuable alternatives against microorganisms and oral treatments, and it is also very important that research suggests materials and medications that are effective in the treatment of patients and that do not represent a health risk.

Use of tannin-based coagulant and chlorine dioxide in treating brewing water: reduction of trihalomethanes and impact on physicochemical and sensory quality.

This study aimed to evaluate an alternative to reduce trihalomethane (THM) formation in brewing water. THM affects the organoleptic properties of water and, consequently, the produced beer. Water treatment based on common chemicals such as alum and free chlorine could potentially form THM. Therefore, we studied the replacement of chemicals used in water treatment: aluminum sulfate by a tannin-based coagulant and sodium hypochlorite by chlorine dioxide. Experimentally, jar tests were conducted, and the role of coagulants and oxidizing agents was evaluated for: the removal of apparent color, turbidity, natural organic matter (NOM) and microorganisms; the formation of trihalomethanes (THM); and the sensory quality of the water. Using tannin-based coagulant with chlorine dioxide was associated with the lowest THM in treated water (1.7 µg/L) and higher satisfaction in the sensory analysis. However, using these chemicals make the water treatment more expensive than the current strategy. Overall, using the tannin-based coagulant and chlorine dioxide treatment is an alternative to produce water with a lower THM concentration, better physical-chemical, and sensory quality. These findings motivate further brewing experiments and a deeper economics evaluation considering the process's sustainability.

Use of Opuntia ficus-indica Fruit Peel as a Novel Source of Mucilage with Coagulant Physicochemical/Molecular Characteristics.

The peels obtained as a byproduct from the processing of fruits (prickly pears) of the Cactaceae family are a rich source of mucilage, a hydrocolloid biopolymer that may have potential application in water/wastewater treatment as a natural coagulant. In this study, the structural (UPLC-QTOF-MS, FTIR, Raman, NMR, XRD, and zeta potential), morphological (SEM), and thermal (DSC/TGA) characterizations of the mucilage extracted from the peels of Opuntia ficus-indica (OFI) fruits were carried out. UPLC-QTOF-MS results revealed the presence of a branched polymer with an average molecular weight of 0.44 KDa for this mucilage in aqua media. The NMR spectra of mucilage in DMSO-d6 indicated that it seemed well-suited as a coagulant with its typical oligosaccharide structure. FTIR studies confirmed the presence of hydroxyl and carboxyl functional groups in the mucilage, indicating its polyelectrolyte nature that could provide coagulating properties through binding and adsorption mechanisms. Likewise, the zeta potential of -23.63 ± 0.55 mV showed an anionic nature of the mucilage. Power XRD technique evidenced the presence of crystalline poly(glycine-ß-alanine), glutamic acid, and syn-whewellite. SEM images revealed an irregular and amorphous morphology with cracks, which are suitable characteristics for adsorption mechanisms. The mucilage exhibited two endothermic transitions, with a decomposition temperature in uronic acid of 423.10 °C. These findings revealed that mucilage obtained from OFI fruit peels has molecular and physicochemical characteristics that are suited to its possible application as a natural coagulant in water/wastewater treatments.

Chemically enhanced primary treatment of dairy wastewater using chitosan obtained from shrimp wastes: optimization using a Doehlert matrix design.

Dairy operations generate large volumes of polluted wastewater that require treatment prior to discharge. Chemically enhanced primary treatment (CEPT) is a widely utilized wastewater treatment strategy; but it requires the use of non-biodegradable coagulants that can lead to toxic-byproducts. In this study, chitin from shrimp shell waste is extracted and converted into chitosan. Chitosan was demonstrated to be a natural, low-cost alternative coagulant compatible with the CEPT. Following treatment, dissolved air flotation allowed for the removal of turbidity, COD, and UV254 from the synthetic dairy effluent (SDE). Doehlert matrix was used to optimize the chitosan dosage and pH of the CEPT; as well as to model the process. The mechanisms behind the coagulation-flocculation were revealed using zeta potential analysis. FTIR spectroscopy was utilized to confirm the functional groups present on the chitosan. Chitosan with a degree of deacetylation equal to 81% was obtained. A chitosan dose of 73.34 mg/L at pH 5.00 was found to be optimal for the removal of pollutants. Removals of COD, turbidity and UV254 were 77.5%, 97.6%, and 88.8%, respectively. The amount of dry sludge generated to treat 1 m³ of SDE was 0.041 kg. Coagulation-flocculation mechanisms involved in chitosan-mediated treatment of SDE involve the neutralization of electrostatic charges carried on the amine groups present in cationic chitosan at pH 5.00. Doehlert matrix proved to be a useful tool in optimizing parameters throughout the coagulation-flocculation process. Chitosan from shrimp waste is a low-cost, eco-friendly coagulant alternative for the removal pollutants from dairy effluent using the CEPT.

Combined system for wastewater treatment: ozonization and coagulation via tannin-based agent for harvesting microalgae by dissolved air flotation.

This study evaluated the performance of urban wastewater treatment in pilot-plant by an integrated system consisting of anaerobic reactor, microalgae cultivation, Venturi tube ozone recirculation, coagulation/flocculation with tannin-based agent natural coagulant, and dissolved air flotation (DAF). Ozone concentrations (without ozone, 0.13 and 0.25 mg O3/mg of biomass) and tannin dosages (65, 85 and 105 mg/L) were evaluated regarding microalgae separation and their influences on wastewater treatment performance. During the experiments, it was verified that the treatment efficiency increased when ozone was applied and with higher tannin dosages. The best results were found with 0.13 mg O3/mg of biomass and 105 mg/L of tannin, obtaining excellent removal of turbidity removal (99.4%), apparent colour at 420 nm (94.5%), TN (83.2%), N-NH3 (100%), TOC (86.8%), BOD5 (86.5%) and COD (100%), 47.6% reduction in electric conductivity, 46.1% in TDS, 66.4% TP removal for the integrated system and 84% microalgae biomass recovery were obtained. Our results showed that the system proved to be a viable alternative for the treatment of urban wastewater and the recovery of microalgae through the insertion of ozone via Venturi tube combined with tannin-based agent.

Moringa oleifera Plant as potent alternate to Chemical Coagulant in Water Purification

Abstract Quality of groundwater is threatened due to pollution by industrial, domestic and agricultural waste. A large number of populations are residing in rural areas which are unable to afford high cost water purifiers due to their low income as well as limited awareness. However, limited availability of fresh water has become a critical issue in developing countries. Around 1.2 billion population is deprived of affordable and safe water for their domestic need. Additionally, chemical coagulants which are nowadays being used for water purification pose severe and numerous health hazards to human. Thus utilization of easily accessible natural coagulant for water purification might offer a sustainable, practical and cost effective solution to the current alarming situation in developing countries. Several experimental findings have shown strong efficiency of Moringa oleifera plant extracts obtained from different solvents in the improvement of water quality parameters including physicochemical (such as pH, hardness, turbidity, metallic impurities, total dissolved solid) and biological (E.coli count) parameter. We have also highlighted the limitations and advantages of chemical coagulation in water purification. Altogether, this review summarizes one such miracle tree which has shown significant potential as a natural coagulant and its associated underlying mechanism in water purification process.

Uso do coagulante/floculante emergente à base de moringa no tratamento de água com verificação da composição e toxicidade do lodo produzido: tratamento de água com Moringa e toxicidade do lodo / Use of emergent moringa-based coagulant/flocculant for water treatment with verification of composition and toxicity of the produced sludge: water treatment with Moringa and toxicity of the sludge

RESUMO O presente estudo consistiu em avaliar a eficiência dos processos de tratamento convencional de água, por meio do coagulante natural Moringa oleifera Lam, com e sem casca, para o tratamento de água bruta captada do Rio Poxim, Aracaju, SE, Brasil. Após essa avaliação, realizou-se a análise da composição química e da toxicidade do lodo. As etapas de coagulação/floculação e decantação (30 e 60 minutos) foram realizadas em jar test com o coagulante, seguidas de filtração rápida descendente. A eficácia dos coagulantes foi analisada por meio da caracterização da água nas dosagens de 100, 200, 300 e 400 mg.L-1, por meio de análises físico-químicas, englobando os parâmetros pH, cor aparente e turbidez. O lodo proveniente do tratamento, em ambas as situações em estudo, foi caracterizado em relação à composição de proteína, amido e lipídio, além de ter sido verificada a toxicidade do resíduo por intermédio do crescimento e da germinação da Lactuca sativa. Como resultado, a moringa demonstrou potencial no tratamento de água pra consumo humano, principalmente o extrato de moringa com casca, tendo como dosagem ótima 300 mg.L-1 tanto para os parâmetros de cor quanto para a turbidez. Em relação ao pH, constatou-se que os tratamentos não promoveram variação significativa em relação aos valores da água bruta. Quanto à caracterização do lodo, foi possível verificar que em sua constituição há presença tanto de lipídio quanto de proteínas e ausência de amido; além disso, seu resíduo apresentou-se como atóxico para o meio ambiente.

ABSTRACT The present study aimed at evaluating the efficiency of conventional water treatment processes using the natural coagulant Moringa oleifera Lam, with and without shell, for the treatment of raw water captured in the Poxim River, Aracaju, state of Sergipe, Brazil. After this evaluation, the chemical composition of the sludge and its toxicity was analyzed. The stages of coagulation/flocculation and decanting (30 and 60 min) were performed in jar test with the coagulant, followed by rapid descending filtration. The effectiveness of the coagulants was analyzed by the characterization of the water in dosages of 100, 200, 300, and 400 mg.L-1, according to physicochemical analyses, including the parameters pH, apparent color, and turbidity. The treatment sludge, in both situations under study, was characterized in relation to the composition of protein, starch and lipid, in addition to verifying the toxicity of the residue by the growth and germination of Lactuca sativa. As a result, moringa showed potential in the treatment of water for human consumption, mainly the extract of moringa in shell, with an optimal dosage of 300 mg.L-1 for both color and turbidity parameters. Regarding pH, the treatments did not promote significant variation in the raw water values. In terms of sludge characterization, the presence of lipids and proteins was verified, but not that of starch. In addition, the sludge residue was considered nontoxic to the environment.

Synergistic effect of serine protease inhibitors and a bothropic antivenom in reducing local hemorrhage and coagulopathy caused by Bothrops jararaca venom.

Snakebite accidents are a public health problem that affects the whole world, causing thousands of deaths and amputations each year. In Brazil, snakebite envenomations are caused mostly by snakes from the Bothrops genus. The local symptoms are characterized by pain, swelling, ecchymosis, and hemorrhages. Systemic disturbances can lead to necrosis and amputations. The present treatment consists of intravenous administration of bothropic antivenom, which is capable of reversing most of the systemic symptoms, while presenting limitations to treat the local effects, such as hemorrhage and to neutralize the snake venom serine protease (SVSP). In this context, we aimed to evaluate the activity of selective serine protease inhibitors (pepC and pepB) in combination with the bothropic antivenom in vivo. Further, we assessed their possible synergistic effect in the treatment of coagulopathy and hemorrhage induced by Bothrops jararaca venom. For this, we evaluated the in vivo activity in mouse models of local hemorrhage and a series of in vitro hemostasis assays. Our results showed that pepC and pepB, when combinated with the antivenom, increase its protective activity in vivo and decrease the hemostatic disturbances in vitro with high selectivity, possibly by inhibiting botropic proteases. These data suggest that the addition of serine protease inhibitor to the antivenom can improve its overall potential.

Functionalized magnetite nanoparticles with Moringa oleifera with potent antibacterial action in wastewater.

Contaminations by Staphylococcus aureus in food industry environments have been extended to industrial Effluent Treatment Plant (ETP). The methodologies used in ETP for bacterial removals and quality parameters adjustment commonly use products toxic to the environment, being mostly inefficient against virulent bacteria such as S. aureus. Seeds of Moringa oleifera Lam. (MO) have potential to be used in ETP as an alternative to harmful products, as it has both the ability to regulate the physicochemical parameters of water and has antibacterial action. Functionalization of MO with magnetite magnetic nano particles (Fe3O4) at nano scale focusing on coagulation and flocculation of wastewater has been gaining prominence. Therefore, the present study evaluated the potential use of the magnetic coagulant MO-Fe3O4 in the elimination of S. aureus in synthetic dairy effluent; concomitantly sought to adjust the quality levels of physicochemical parameters. MO-Fe3O4 added to synthetic dairy effluent at different concentrations amounted to 16 treatments, which were evaluated for removal of color, turbidity, UV254nm and S. aureus on the effluent surface and sludge after 30 min of sedimentation. The results confirmed the efficient elimination of S. aureus simultaneously with a significant reduction of the physicochemical values, with constant efficiency up to 30 min. Scanning electron microscopy images confirm the removal of S. aureus on the effluent surface and sludge. Thus, this study was able to present a natural coagulant capable of remove bacteria and adjust the quality levels of the effluent after 10 min of sedimentation, making this biotechnological innovation highly applicable to ETP.

Synergistic effect of serine protease inhibitors and a bothropic antivenom in reducing local hemorrhage and coagulopathy caused by Bothrops jararaca venom

Snakebite accidents are a public health problem that affects the whole world, causing thousands of deaths and amputations each year. In Brazil, snakebite envenomations are caused mostly by snakes from the Bothrops genus. The local symptoms are characterized by pain, swelling, ecchymosis, and hemorrhages. Systemic disturbances can lead to necrosis and amputations. The present treatment consists of intravenous administration of bothropic antivenom, which is capable of reversing most of the systemic symptoms, while presenting limitations to treat the local effects, such as hemorrhage and to neutralize the snake venom serine protease (SVSP). In this context, we aimed to evaluate the activity of selective serine protease inhibitors (pepC and pepB) in combination with the bothropic antivenom in vivo. Further, we assessed their possible synergistic effect in the treatment of coagulopathy and hemorrhage induced by Bothrops jararaca venom. For this, we evaluated the in vivo activity in mouse models of local hemorrhage and a series of in vitro hemostasis assays. Our results showed that pepC and pepB, when combinated with the antivenom, increase its protective activity in vivo and decrease the hemostatic disturbances in vitro with high selectivity, possibly by inhibiting botropic proteases. These data suggest that the addition of serine protease inhibitor to the antivenom can improve its overall potential.

Milk-clotting proteases from Pleurotus albidus: an innovative alternative for the production of Minas frescal cheese

Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.(AU)

Milk-clotting proteases from Pleurotus albidus: an innovative alternative for the production of Minas frescal cheese

Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.

Effectiveness of the mixture of nopal and cassava starch as clarifying substances in water purification: A case study in Colombia.

Aluminum sulfate is one of the most used chemical coagulants in the world, but research has shown that high concentrations of aluminum in the body are associated with neuropathological conditions. Because of this, different alternatives have been evaluated such as natural coagulants, which are considered safe for human health and contain fewer contaminants than chemicals due to their biodegradation properties. The main objective of this study was to evaluate the efficiency of mixing nopal mucilage and cassava starch for turbidity removal in water purification. In this paper, test jars and the treatment equipment (TA-scale FQ-005/PE manufactured by Generatoris SA de CV of Mexico) was applied in order to measure turbidity and pH parameters before and after the process of coagulation-flocculation, which was applied to water from the Magdalena River in Colombia. Samples from two sampling periods were assessed. One was evaluated during the rainy season and the other was evaluated without precipitation (drought) with initial turbidities of 316 NTU and 80 NTU, respectively. It was found that aluminum sulfate as a coagulant reference obtained better turbidity removal results (up to 99%) as compared to nopal (up to 60.4%), and nopal-starch combination of cassava (up to 67%), indicating that this mixture increases the effectiveness of natural coagulants used individually. Our results indicate that this should be considered as an alternative in the water purification process.

Toxinological characterization of venom from Leptodeira annulata (Banded cat-eyed snake; Dipsadidae, Imantodini).

We investigated the histology of Duvernoy's venom gland and the biochemical and biological activities of Leptodeira annulata snake venom. The venom gland had a lobular organization, with secretory tubules formed by serous epithelial cells surrounding each lobular duct. The latter drained into a common lobular duct and subsequently into a central cistern. In contrast, the supralabial gland was mucous in nature. SDS-PAGE revealed a profile of venom components that differed from pitviper (Bothrops spp.) venoms. RP-HPLC also revealed greater complexity of this venom compared to Bothrops venoms. The venom had no esterase, l-amino acid oxidase or thrombin-like activity, but was proteolytic towards elastin-Congo red, fibrin, fibrinogen, gelatin and hide powder azure. The venom showed strong α-fibrinogenase and fibrinolytic activities and reduced the rate and extent of plasma recalcification. The proteolytic activity was inhibited by EDTA and 1,10-phenanthroline (metalloproteinase inhibitors) but not by AEBSF and PMSF (serine proteinase inhibitors). The venom had phospholipase A2 (PLA2) activity that was inhibited by varespladib. The venom cross-reacted with antivenoms to lancehead (Bothrops spp.), coralsnake (Micrurus spp.) and rattlesnake (Crotalus durissus terrificus) venoms. The venom did not aggregate rat platelets or inhibit collagen-induced aggregation, but partially inhibited thrombin-induced aggregation. The venom was hemorrhagic (inhibited by EDTA) and increased the vascular permeability (inhibited by varespladib) in rat dorsal skin. In gastrocnemius muscle, the venom caused myonecrosis and increased serum creatine kinase concentrations. In conclusion, L. annulata venom has various enzymatic and biological activities, with the local effects being mediated primarily by metalloproteinases and PLA2.

Noni (Morinda citrifolia L.) fruit as a new source of milk-clotting cysteine proteases.

This work reports the characterisation of caseinolytic and milk-clotting activities of proteases extracted from ripe fruits of Morinda citrifolia L., as a potential of their use in cheese production. Noni puree extract (NPE) was obtained by homogenising the fresh puree in 150 mM NaCl/50 mM sodium phosphate buffer (pH 7.0). The resulting protein concentration was of 0.367 ±â€¯0.006 mg/mL, and an electrophoretic profile of the extract revealed protein bands ranging from 14 to 55 kDa. The proteolytic activity of NPE was higher when the extract had been previously incubated at pH 6.0 (8.859 ±â€¯0.216 U/mg), whereas the optimum caseinolytic activity was observed at 50 °C. Noni puree proteases were strongly (98%) inhibited by iodoacetamide and E-64, suggesting the presence of only cysteine proteases in the crude extract. NPE proteases showed a milk-clotting activity (MCA) of 238.80 ±â€¯5.29 U/mL, a specific milk-clotting activity (SMCA) of 9950.17 ±â€¯220.74 U/mg, and an SMCA/PA ratio of 1124.31 ±â€¯24.94, this last being comparable to those of commercial calf rennet. The cheese manufactured using NPE presented brittle and soft texture, high humidity, and showed sanitary conditions compatible with current Brazilian regulations. The product showed a slightly bitter taste, but still good acceptability, rating between 6 and 7 in the hedonic scale for flavour, texture, and overall acceptance. Lastly, there was 60% of positive purchase intent, demonstrating that noni fruit is a promising source of milk-clotting enzymes for the dairy industry.

Edematic and coagulant effects caused by the venom of Bothrops rhombeatus neutralized by the ethanolic extract of Piper auritum.

ETHNOPHARMACOLOGICAL RELEVANCE: In Colombia, the only authorized treatment to cure snakebite envenomation is with the use of antivenom. The antivenom neutralizes the systemic effects properly, but is not very effective at neutralizing local effects, thus several cases have lead to complications. On the other hand, rural communities turn to the use of plants that are easily accessible and available for basic health care. One of these plants is named Piper auritum (PA), which is traditionally highlighted in some indigenous communities of Antioquia and Chocó. AIM OF THE STUDY: The main objective of this work was to characterize the venom's toxicity by determining the Minimum Edema Dose (MED), the Minimum Coagulant Dose-Plasma (MCD-P), the Minimum Hemorrhagic Dose (MHD) and to determine the neutralizing power of the Total Ethanolic Extract (TEE) from leaves of PA on the localized and systemic effects caused by the Bothrops rhombeatus venom. MATERIALS AND METHODS: To begin, the minimum dose that causes edema-forming, coagulant and hemorrhagic activities was determined. The protocols investigated include coagulant and edematic activities caused by the venom of Bothrops rhombeatus which were neutralized by the TEE of PA. RESULTS: The MCD-P was found to be 0.206 ±â€¯0.026 µg, the MED is the same at 0.768 ±â€¯0.065 µg, and the MHD is 3.553 ±â€¯0.292 µg, which are different from the reports for Bothrops asper and Bothrops ayerbei. Next, a phytochemical screening was done to the TEE where mainly triterpenes, steroids, coumarins, saponins, and lignans were identified. Also present were 43,733 ±â€¯2106 mg AG/g ES of phenols, which are secondary metabolites that are probably responsible for the neutralization of coagulant and edematic activities at rates of 2363.870 µL and 1787.708 µL of extract/mg of venom, respectively. As a comparative parameter, the National Institute Health's (NHI) effective dose of the antivenom was used as a comparative parameter. In addition, we determined the toxicity of the TEE of PA on to Artemia salina, being moderately toxic at 6 and 24 h, while the essential oil of PA at the same observation hours is in the extremely toxic range. CONCLUSIONS: The results reflect that the extract of P. auritum has an anti-inflammatory effect similar to that of the NIH serum. It could be used as a complement of NIH antivenom, using them together so it contributes to effectively reduce inflammation and the socio-economic impact generated by the permanence of a patient victim of snakebite in health centers. CLASSIFICATIONS: Immunological products and vaccines.