Evaluating different levels of papain as texture modifying agent in bovine meat loaf containing transglutaminase.

In this study, bovine meat loaves were produced with different levels of papain (0.00125%, 0.0025%, 0.00375%, and 0.005%) combined with transglutaminase (1%). The effect of this reformulation on pH, instrumental color, water activity, proximate composition, texture, yield, and scanning electron microscopy (SEM) of meat loaves was investigated. In addition, the enzymatic activity of papain was also analyzed. The papain addition increased the pH and the yield of the samples. The hardness was progressively reduced with the increase of papain level. Such changes could be seen through the images recorded by SEM, where an extremely fragmented structure was observed in treatments with higher papain concentration. Papain showed an optimum temperature of 80 °C. This study allowed to observe an intense proteolytic effect in all treatments, despite the papain concentration. Therefore, lower levels should be applied so that the product does not alter its sensory characteristics, such as soft and crumbly texture.

Characterization and application of a crude bacterial protease to produce antioxidant hydrolysates from whey protein.

Bacillus sp. CL14 crude protease was partially characterized and applied to obtain antioxidant whey protein isolate (WPI) hydrolysates. Optimal activity occurred at pH 9.0 and 60 °C. Ca2+, Mg2+, and Mn2+ (5 mM) enhanced activity (12-26%), whereas Co2+, Cu2+, Fe2+, and Zn2+ inhibited it (50-94%). At 1% (v/v), Tween 20 and Triton X-100 enhanced activities (21-27%), ß-mercaptoethanol decreased it (15%), and dimethyl sulfoxide (DMSO) had no effect. Sodium dodecyl sulfate (SDS; 0.1%, w/v) increased activity by 36%. Complete inhibition by phenylmethylsulfonyl fluoride (PMSF), and 85% inhibition by ethylenediaminotetraacetic acid, indicates its serine protease character and the importance of cations for activity/stability. With 5 mM Ca2+, protease was optimally active at 65 °C and completely stable after 20 min at 40-55 °C. Crude protease preferentially hydrolyzed WPI and soy protein, followed by casein. WPI hydrolysis was then performed (55 °C, pH 9.0, 5 mM Ca2+) for 0-180 min. Contents of trichloroacetic acid (TCA)-soluble proteins in WPI hydrolysates (HWPI) increased from 29% (0 min) to 50-52% (60-180 min), accompanied by enhanced radical scavenging activity (14%, 0 min; ∼34%, 60-180 min) and Fe2+-chelating ability (56%, 0 min; ∼74%, 45-180 min). CL14 protease might represent an alternative biocatalyst to obtain antioxidant hydrolysates from WPI and, potentially, from other food proteins.

Asclepain cI, a proteolytic enzyme from Asclepias curassavica L., a south American plant, against Helicobacter pylori.

Helicobacter pylori is a Gram negative bacterium most frequently associated with human gastrointestinal infections worldwide. The increasing occurrence of antibiotic-resistant isolates of H. pylori constitutes a challenge. The eradication of the microorganism is currently being considered a "high priority" by the World Health Organization (WHO). In this context, bioactive compounds found in natural products seem to be an effective therapeutic option to develop new antibiotics against the pathogen. In this study, we investigated the effect of asclepain cI, the main purified proteolytic enzyme of the latex of petioles and stems from Asclepia curassavica L. (Asclepiadaceae), a South American native plant, against H. pylori; in order to obtain a natural therapeutic adjuvant and a safe nutraceutical product. Asclepain cI showed antibacterial activity against reference strains and drug-resistant clinical isolates of H. pylori in vitro. A range of minimal inhibitory concentration (MIC) from 1 to 2 µg/ml and minimal bactericidal concentration (MBC) from 2 to 4 µg/ml was obtained, respectively. The action of asclepain cI on the transcription of omp18, ureA, flaA genes showed a significantly decreased expression of the selected pathogenic factors. Furthermore, asclepain cI did not induce toxic effects at the concentrations assayed. Asclepain cI could be considered a highly feasible option to be used as a natural therapeutic adjuvant and a safe nutraceutical product against H. pylori.

Antimicrobial Effect of a Proteolytic Enzyme From the Fruits of Solanum granuloso-leprosum (Dunal) Against Helicobacter pylori.

Helicobacter pylori is a gram-negative, helix-shaped, and microaerophilic bacteria that colonizes the human gastric mucosa, causing chronic infections, gastritis, peptic ulcer, lymphomas associated with lymphoid mucosa tissue, and gastric cancer. H. pylori is considered a Type 1 human carcinogen by WHO. The prevalence of the infection is estimated in more than half of the world population. Treatment of H. pylori infection includes antibiotics and proton pump inhibitors, but the increasing antibiotic resistance promotes the research of novel, more effective, and natural antibacterial compounds. The aim of this work was to study the effect of the partially purified proteolytic extract (RAP) of the fruits from Solanum granuloso-leprosum (Dunal), a South American native plant, and a purified fraction named granulosain I, against H. pylori, to obtain natural food additives for the production of anti-H. pylori functional foods. Furthermore, granulosain I and RAP could be used as natural adjuncts to conventional therapies. Granulosain I and RAP antibacterial activity was evaluated as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against H. pylori NCTC 11638 (reference strain) and twelve H. pylori wild strains, using a microdilution plating technique (Clinical and Laboratory Standards Institute). All the strains tested were susceptible to granulosain I with MIC from 156.25 to 312.5 µg/mL and MBC from 312.5 to 625 µg/mL, respectively. Besides, all the strains tested were susceptible to the RAP with MIC from 312.5 to 625 µg/mL and MBC from 625 to 1,250 µg/mL, respectively. The effect of granulosain I and RAP on the transcription of H. pylori genes encoding pathogenic factors, omp18, ureA, and flaA, with respect to a housekeeping gene (16S rRNA), was evaluated by RT-PCR technique. The band intensity between pathogenic factors and control gene was correlated under treated or untreated conditions, using the ImageJ program. Granulosain I and RAP significantly decreased the expression of pathogenic factors: omp18, ureA, and flaA. The combined inhibitory effect of granulosain I or RAP and an antibiotic such as, amoxicillin (AML, 10 µg), clarithromycin (CLA, 15 µg), levofloxacin (LEV, 5 µg), and metronidazole (MTZ, 5 µg) was evaluated, using the agar diffusion technique. Granulosain I and RAP showed significant synergistic effect on AML, CLA, and LEV, but no significant effect on MTZ was observed. Besides, granulosain I and RAP did not show toxicological effects at the concentrations studied. Finally, granulosain I and RAP could be used as safe natural food additives and as adjuvants for conventional therapies against H. pylori.

A Collagenolytic Aspartic Protease from Thermomucor indicae-seudaticae Expressed in Escherichia coli and Pichia pastoris.

Proteases are produced by the most diverse microorganisms and have a wide spectrum of applications. However, the use of wild microorganisms, mainly fungi, for enzyme production has some drawbacks. They are subject to physiological instability due to metabolic adaptations, causing complications and impairments in the production process. Thus, the objective of this work was to promote the heterologous expression of a collagenolytic aspartic protease (ProTiN31) from Thermomucor indicae seudaticae in Escherichia coli and Pichia pastoris. The pET_28a (+) and pPICZαA vectors were synthesized containing the gene of the enzyme and transformed into E. coli and P. pastoris, respectively. The recombinant enzymes produced by E. coli and P. pastoris showed maximum activity at pH 5.0 and 50 °C, and pH 5.0 and 60 °C, respectively. The enzyme produced by P. pastoris showed better thermostability when compared to that produced by E. coli. Both enzymes were stable at pH 6.0 and 6.5 for 24 h at 4 °C, and sensitive to pepstatin A, ß-mercaptoethanol, and Hg2+. Comparing the commercial collagen hydrolysate (Artrogen duo/Brazil) and gelatin degradation using protease from P. pastoris, they showed similar peptide profiles. There are its potential applications in a wide array of industrial sectors that use collagenolytic enzymes.

Single step purification via magnetic nanoparticles of new broad pH active protease from Penicillium aurantiogriseum.

A new set of applications can be achieved when using high stability proteases. Industrially, high costs can be related to production medium and purification process. Magnetic nanoparticles have been successfully used for rapid and scalable purification. In this work, azocasein were immobilized on magnetite nanoparticles and applied in a single step purification of protease produced by Penicillium aurantiogriseum using soybean flour medium, and the new purified enzyme was characterized. Glutaraldehyde activated nanoparticles were used in azocasein immobilization and then incubated with dialyzed 60-80% saline precipitation fraction of crude extract for purification. Adsorbents were washed 7 times (0.1 M NaCl solution) and eluted 3 times (1 M NaCl solution), these final elutions contained the purified protease. This protease was purified 55.68-fold, retaining 46% of its original activity. Presented approximately 40 kDa on SDS-PAGE and optimum activity at 45 °C and pH 9.0. Maintained over 60% of activity from pH 6.0 to 11.0. Kept more than 50% activity from 15 to 55 °C, did not lose any activity over 48 h at 25 °C. Inhibitors assay suggested a serine protease with aspartic residues on its active site. Results report a successful application of an alternative purification method and novel broad pH tolerant protease.

A stimuli-responsive and bioactive film based on blended polyvinyl alcohol and cashew gum polysaccharide.

In this study, a stimuli-responsive, biodegradable and bioactive film was produced by blending cashew gum polysaccharide (CGP) and polyvinyl alcohol (PVA). The film presented malleability and mechanical properties enabling an easy handling. Wetting the film changed the optical property from opacity to levels of transparency higher than 70% and resulted in up to 2-fold increase in its superficial area. Different swelling indexes were obtained varying the pH of solvent, which allows classifying the CGP/PVA film as pH sensitive stimuli-responsive material. The bioactivity was achieved through covalent immobilization of papain, which remained active after storage of CGP/PVA-papain film for 24h in the presence of buffer or in a dry form. These results evidenced that CGP/PVA-papain film is a very promising material for biomedical applications.

Pseudomonas spp. and Serratia liquefaciens as Predominant Spoilers in Cold Raw Milk.

The storage of fresh raw milk at low temperature does not prevent proliferation of psychrotrophic bacteria that can produce heat-resistant proteolytic enzymes contributing to the reduced shelf life of dairy products. This study aimed to identify the dominant psychrotrophic proteolytic enzyme-producing population of raw milk from Brazil. Raw milk samples collected in 3 different cooling tanks in Brazil were stored at optimal (45 h at 4 °C followed by 3 h at 7 °C) and suboptimal (45 h at 7 °C followed by 3 h at 10 °C) conditions to simulate farm storage and transportation allowed by Brazilian laws. The highly proteolytic enzyme-producing strains isolated from stored cold raw milk were characterized by repetitive sequence-based Polymerase Chain Reaction (PCR) analysis. This clustering resulted in 8 different clusters and 4 solitary fingerprints. The most proteolytic isolates from each rep-cluster were selected for identification using miniaturized kit, 16S rDNA and rpoB gene sequencing. Serratia liquefaciens (73.9%) and Pseudomonas spp. (26.1%) were identified as the dominant psychrotrophic microorganisms with high spoilage potential. The knowledge of milk spoilage microbiota will contribute to improved quality of milk and dairy products.

Queijo prato com teor reduzido de gordura adicionado de enzima proteolítica: características físicas e sensoriais / Reduced fat prato cheese added of proteolytic enzyme: physical and sensorial

O queijo Prato, segundo queijo mais consumido no Brasil, obtido por coagulação enzimática do leite e maturado por pelo menos 25 dias, é classificado como gordo e de média umidade. Devido à preocupação com a saúde, os consumidores de queijos têm procurado produtos em suas versões com menor teor de gordura. Contudo, a gordura confere as características sensoriais desejáveis, como sabor, cremosidade, maciez e textura aos queijos. Alterações têm sido introduzidas no processo tecnológico de fabricação dos queijos com teor reduzido de gordura, com o intuito de efetuar melhoria nesses produtos; e o uso de enzimas proteolíticas é uma importante estratégia a ser considerada. A capacidade de derretimento, cor e avaliação sensorial são fundamentais indicadores da qualidade dos produtos obtidos. O presente trabalho analisou as características físicas e sensoriais de queijo Prato com teor reduzido de gordura adicionado da enzima proteolítica fastuosaína, extraída do fruto verde do gravatá. A adição da fastuosaína não interferiu na capacidade de derretimento, tampouco promoveu o desenvolvimento de amargor, que é característica comum não apreciada em queijos com teor reduzido de gordura.

The Prato cheese is the second most consumed cheese in Brazil. It is produced by milk enzymatic coagulation,and maturated for at least 25 days; it is classified as fatty cheese and of medium moisture. Due to the concern to health, the cheeses consumers have been seeking for products with low fat contents; however fat is essential for providing desirable sensory and physiologic characteristics, such as flavor, softness and texture to cheeses. Alterations on the technological processing of low fat cheeses have been made seeking for improved products, and the use of proteolytic enzymes has been a significant strategy. The meltability, color and sensory characteristics are fundamental quality indicators of the final products. This study reports the findings from the analyses on the physical and sensory characteristics of low fat Prato cheese with addition of proteolytic enzyme – fastuosain, that is extracted from unripe gravata fruit. The addition of fastuosainimproved the quality of the product, as this additive neither affected the meltability, nor produced bitterness,which is a common unpleasant taste in low fat cheeses.

Reduced fat Prato cheese added of proteolytic enzyme: physical and sensorial characteristics / Queijo Prato com teor reduzido de gordura adicionado de enzima proteolítica: características físicas e sensoriais

The Prato cheese is the second most consumed cheese in Brazil. It is produced by milk enzymatic coagulation,and maturated for at least 25 days; it is classified as fatty cheese and of medium moisture. Due to theconcern to health, the cheeses consumers have been seeking for products with low fat contents; howeverfat is essential for providing desirable sensory and physiologic characteristics, such as flavor, softness andtexture to cheeses. Alterations on the technological processing of low fat cheeses have been made seeking forimproved products, and the use of proteolytic enzymes has been a significant strategy. The meltability, colorand sensory characteristics are fundamental quality indicators of the final products. This study reports thefindings from the analyses on the physical and sensory characteristics of low fat Prato cheese with additionof proteolytic enzyme fastuosain, that is extracted from unripe gravata fruit. The addition of fastuosainimproved the quality of the product, as this additive neither affected the meltability, nor produced bitterness,which is a common unpleasant taste in low fat cheeses.

O queijo Prato, segundo queijo mais consumido no Brasil, obtido por coagulação enzimática do leite ematurado por pelo menos 25 dias, é classificado como gordo e de média umidade. Devido à preocupaçãocom a saúde, os consumidores de queijos têm procurado produtos em suas versões com menor teor degordura. Contudo, a gordura confere as características sensoriais desejáveis, como sabor, cremosidade,maciez e textura aos queijos. Alterações têm sido introduzidas no processo tecnológico de fabricaçãodos queijos com teor reduzido de gordura, com o intuito de efetuar melhoria nesses produtos; e o uso deenzimas proteolíticas é uma importante estratégia a ser considerada. A capacidade de derretimento, cor eavaliação sensorial são fundamentais indicadores da qualidade dos produtos obtidos. O presente trabalhoanalisou as características físicas e sensoriais de queijo Prato com teor reduzido de gordura adicionadodaenzima proteolítica fastuosaína, extraída do fruto verde do gravatá. A adição da fastuosaína não interferiuna capacidade de derretimento, tampouco promoveu o desenvolvimento de amargor, que é característicacomum não apreciada em queijos com teor reduzido de gordura.