Enzymatic Activity and Its Relationships with the Total Phenolic Content and Color Change in the High Hydrostatic Pressure-Assisted Curing of Vanilla Bean (Vanilla planifolia).

Diverse enzymatic reactions taking place after the killing of green vanilla beans are involved in the flavor and color development of the cured beans. The effects of high hydrostatic pressure (HHP) at 50-400 MPa/5 min and blanching as vanilla killing methods were evaluated on the total phenolic content (TPC), polyphenoloxidase (PPO), and peroxidase (POD) activity and the color change at different curing cycles of sweating-drying (C0-C20) of vanilla beans. The rate constants describing the above parameters during the curing cycles were also obtained. The TPC increased from C1 to C6 compared with the untreated green beans after which it started to decrease. The 400 MPa samples showed the highest rate of phenolic increase. Immediately after the killing (C0), the highest increase in PPO activity was observed at 50 MPa (46%), whereas for POD it was at 400 MPa (25%). Both enzymes showed the maximum activity at C1, after which the activity started to decrease. As expected, the L* color parameter decreased during the entire curing for all treatments. An inverse relationship between the rate of TPC decrease and enzymatic activity loss was found, but the relationship with L* was unclear. HHP appears to be an alternative vanilla killing method; nevertheless, more studies are needed to establish its clear advantages over blanching.

Chloride Applied via Fertilizer Affects Plant Nutrition and Coffee Quality.

The present study had the objective to evaluate the effect of blends of KCl and K2SO4 fertilizers and their influence on the yield and the nutritional state of coffee plants, as well as on the chemical composition and quality of the coffee beverage. The experimental design was in randomized blocks with four repetitions and six treatments (T1: 100% KCl; T2: 75% KCl + 25% K2SO4; T3: 50% KCl + 50% K2SO4; T4: 25% KCl + 75% K2SO4; T5: 100% K2SO4; and a control, without application of K). The following analyses were performed: K and Cl content in the leaves and the soil, stocks of Cl in soil, yield, removal of K and Cl with the beans, cup quality of the beverage, polyphenol oxidase activity (PPO), electric conductivity (EC), potassium leaching (KL), the content of phenolic compounds, the content of total sugars (TS), and total titratable acidity (TTA). The stocks of Cl in the soil decreased as the proportion of KCl in the fertilizer was reduced. The fertilization with KCl reduces the cup quality and the activity of the polyphenol oxidase, probably due to the ion Cl. The increase in the application of Cl directly relates to the increase in potassium leaching, electric conductivity, and titratable acidity. Indirectly, these variables indicate damages to the cells by the use of Cl in the fertilizer. The activity of the polyphenol oxidase enzyme and the cup quality indicate that the ion Cl- reduces the quality of the coffee beverage. K content in the leaves was not influenced by the application of blends of K fertilizer while Cl content increased linearly with KCl applied. The application of KCl and K2SO4 blends influenced coffee yield and the optimum proportion was 25% of KCl and 75% of K2SO4. The highest score in the cup quality test was observed with 100% K2SO4.

Differential Responses of Antioxidative System during the Interaction of Soursop Fruits (Annona muricata L.) and Nectria haematococca at Postharvest Storage.

Soursop fruit (Annona muricata L.) production is diminished by the attack of pathogens such as Nectria haematococca. However, the fruit-pathogen interaction at the biochemical and molecular levels is still unknown. The objective of this study was to analyze the response of the soursop fruit to the presence of N. haematococca during postharvest storage. Soursop fruits were inoculated with the pathogen and total phenolic compounds, antioxidant capacity by Ferric reducing/antioxidant power (FRAP), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS•+), and 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH•), as well as enzymatic activity and transcript levels of polyphenol oxidase (PPO) and superoxide dismutase (SOD), were evaluated at 1, 3, and 5 days of storage. The noninoculated fruits were the controls of the experiment. The highest total phenol content was recorded on day one in the inoculated fruits. FRAP, ABTS, and DPPH activity presented the highest values on day three in the control fruits. Inoculated fruits recorded the highest PPO activity on day five and a five-fold induction in the PPO transcript on day three. SOD activity showed a decrease during the days of storage and 10-fold induction of SOD transcript on day three in the inoculated fruits. Principal component analysis showed that total phenols were the variable that contributed the most to the observed variations. Furthermore, a positive correlation between total phenols and SOD activity, PPO expression, and SOD expression, as well as between DPPH and FRAP, was recorded. The results showed a differential response in antioxidant capacity, enzymatic activity, and gene expression during the interaction of soursop fruits-N. haematococca at postharvest storage.

Characterization of polyphenol oxidase from purple sweet potato (Ipomoea batatas L. Lam) and its affinity towards acylated anthocyanins and caffeoylquinic acid derivatives.

Biochemical characterization of polyphenol oxidase (PPO) present in purple sweet potato (PSP) is a key step in developing efficient methodologies to control oxidative damage caused by this enzyme to the valuable components of PSP, such as caffeoylquinic acid derivatives and acylated anthocyanins. Thus, this work focused on the assessment of the effects of pH, temperature, and chemical agents on the PPO activity as well as characterization of the PPO substrate specificity towards major phenolic compounds found in PSP. The optimum conditions of enzyme activity were pH 7 and a temperature range of 20-30 °C at which phenolic substrates were oxidized with 72.5-99.8% yield. Zn2+ ions remarkably reduced PPO activity while Cu2+ ions improved enzyme performance. The highest substrate preference was shown for 3,4,5-tri-caffeoylquinic and 3,5-di-caffeoylquinic acid, followed by 5-caffeoylquinic and caffeic acid, 3,4- and 4,5-di-caffeoylquinic acids, peonidin-3-caffeoyl-p-hydroxybenzoyl-sophoroside-5-glucoside. The highest Km values were found for 4,5-feruloyl-caffeoylquinic acid and catechol.

Insight of Polyphenol Oxidase Enzyme Inhibition and Total Polyphenol Recovery from Cocoa Beans.

A full factorial design (ascorbic acid/l-cysteine inhibitors, temperature, and time as factors) study was conducted to enhance inhibition of polyphenol oxidase (PPO) activity without decreasing cocoa polyphenol concentrations. The data obtained were modelled through a new equation, represented by Γ, which correlates both high polyphenol content with reduced specific PPO activity. At optimized values (70 mM inhibitory solution at 96 °C for 6.4 min, Γ = 11.6), 93.3% PPO inhibition and total polyphenol of 94.9 mg GAE/g were obtained. In addition, microscopy images confirmed the cell morphological changes measured as the fractal dimension and explained the possible cell lysis and denaturation as a result of heat treatment and chemical inhibitors. Results also showed that PPO enzyme was most suitable (higher vmax/Km ratio) for catechol, with a reduction in its affinity of 13.7-fold after the inhibition heat treatment. Overall, this work proposed a suitable and food-safe procedure for obtaining enriched polyphenol extract with low enzyme activity.

Effects of processing on the chemical, physicochemical, enzymatic, and volatile metabolic composition of pitaya (Hylocereus polyrhizus (F.A.C. Weber) Britton & Rose).

The effects of processing on the chemical, physicochemical, enzymatic, and volatile metabolic composition of pitaya pulp were assessed for the first time. To this end, the following treatments to obtain pitaya pulp were evaluated: Treatment A (TA, pulp processing without ascorbic acid), Treatment B (TB, whole fruit processing with ascorbic acid), and Control (whole fruit processing without ascorbic acid). The treatment employed in TB resulted in low polyphenol oxidase and peroxidase activity, and no significant chemical or physicochemical alterations in most parameters evaluated. In addition, TB presents high yields and fiber content compared to the TA or Control. For metabolic analysis, Gas Chromatography-Mass Spectrometry (GC-MS) was effective for the simultaneous determination of 80 volatile metabolites in pitaya. Chemometric analyses was used to efficiently distinguish the volatile compounds of each treatment, and demonstrated that TB presents an interesting volatile profile due the conservation or agregation of compounds.

Purification and biochemical characterization of polyphenol oxidase from soursop (Annona muricata L.) and its inactivation by microwave and ultrasound treatments.

The soursop (Annona muricata L.) is a climacteric fruit that may undergo enzymatic browning during ripening, mainly by the activity of polyphenol oxidase (PPO). Soursop PPO was purified 160-fold by hydrophobic interaction and ion-exchange chromatography. The native structure has a molecular weight of 112 kDa corresponding to a dimeric structure. The protein has an optimum pH and temperature of 6.5 and 25°C, respectively; and activation energy of 40.97 kJ·mol-1 . The lowest Km value was observed for caffeic acid (0.47 mM); the best substrate was 4-methyl-catechol (1,067 U·mM-1  min-1 ). Inactivation assays showed that PPO was completely inactivated by tropolone, Na2 S2 O5 and ascorbic acid, and thermally at 55°C for <5 min, microwave exposure reduced activity to 57% at 70 W in 30 s and ultrasound treatment diminished activity to 43% at 120 W in 220 s. This study allows a better understanding of soursop PPO behavior and provides inactivation information. PRACTICAL APPLICATIONS: The conservation of fresh fruits is complicated due to the enzymatic reactions that are present in fruits, such as enzymatic browning. The enzymes responsible for these reactions can be inactivated by, different chemical compounds as well as by the use of emerging technologies, such as microwaves and sonication, which seek to satisfy the consumer needs to obtain fresh products with good nutritional characteristics and adequate safety.

Efecto de la procedencia y el almacenamiento en la calidad del café / Effect of provenance and storage on coffee quality

Resumen Antecedentes. La calidad del grano de café ha sido relacionada con su procedencia, su manejo agronómico y sus condiciones de almacenamiento. Objetivo. Determinar la actividad de la polifenil oxidasa, el contenido de lípidos, el color y las características organolépticas de cafés provenientes de 3 subestaciones experimentales. Materiales y métodos. Se siguió un diseño completamente aleatorio en arreglo factorial factorial 3x6 (lugares de procedencia del café y tiempo de almacenamiento respectivamente). Resultados. La actividad de la polifenil oxidasa es mayor en el café fresco-para las tres procedencias. El café procedente de Naranjal presentó actividades enzimáticas más altas que los cafés provenientes de las subestaciones Supía y la Catalina. El análisis de varianza mostró el efecto de la procedencia sobre la variable actividad enzimática. La actividad de la polifenil oxidasa en los cafés estudiados decrece con el tiempo de almacenamiento. El contenido de lípidos es menor a menor en la subestación de la Catalina. Todos los cafés fueron caracterizados de buena calidad en el tiempo cero de almacenamiento. Las características de aroma, intensidad del aroma y cuerpo presentaron altibajos en los diferentes meses de almacenamiento. El café de Naranjal, obtuvo en promedio una calificación aceptable a lo largo de los seis meses de almacenamiento. Conclusiones. Se encontraron diferencias significativas para las variables estudiadas por efecto de la procedencia y el almacenamiento. La actividad enzimática de la PFO presentó etapas de activación/inhibición, durante los seis meses de almacenamiento.

Abstract Background. The quality of the coffee bean has been related to its origin, the agronomic management and the storage conditions. Objective. To determine the activity of the polyphenyl oxidase, the lipid content, the color and the organoleptic characteristics of coffees from 3 experimental substations. Materials and methods. A completely randomized design was followed in a 3x6 factorial arrangement (places of coffee origin and storage time respectively). Results. The activity of polyphenyl oxidase is greater in fresh coffee-for the three provenances. The coffee from Naranjal presented higher enzymatic activities than the coffees from the Supía and the Catalina substations. The analysis of variance showed the effect of provenance on the enzyme activity variable. The activity of the polyphenyl oxidase in the coffees studied decreases with storage time. The lipid content is lower at a lower height in the Catalina. All coffees were characterized as good quality at zero storage time; but the characteristics of aroma, intensity of aroma and body presented ups and downs in the different months of storage. Naranjal coffee, on average, obtained an acceptable rating throughout the six months of storage. Conclusions. Significant differences were found for the variables studied due to the effect of provenance and storage. The enzymatic activity of the PFO showed activation / inhibition stages, during the six months of storage.

Combined effect of pseudomonas sp. and trichoderma aureoviride on lettuce growth promotion / Efeito combinado de Pseudomonas sp. e Trichoderma aureoviride na promoção do crescimento de alface

Plant growth promotion by microorganisms may be a viable alternative to increase lettuce production through pathogens control and nutrients absorption increase. Trichoderma and Pseudomonasgenus are examples of widely studied microorganisms with the capacity to promote plant growth. However, there are still gaps regarding the action of the combined effect of these two microorganisms. Therefore, the objective of this study was to evaluate the combined effect of Pseudomonas sp. UAGF14 and Trichoderma aureoviride URM5158 on the development of lettuce plants. The experimental design was completely randomized, with five treatments: CONT (control), CM (soil with organic fertilization), CMB (soil withorganic fertilization and Pseudomonas sp.), CMF (soil with organic fertilization and T. aureoviride), and CMFB (soil with organic fertilization, Pseudomonas sp. and T. aureoviride), with ten repetitions. At 30, 40 and 60 days after sowing, the following parameters were analyzed: plant and canopy height and number of leaves. At 60 days after emergence, shoot dry matter, leaf area, root dry matter, root length and chlorophyll were analyzed. Catalase, peroxidase and polyphenol oxidase enzymatic activity were determined. The CMFB treatment had the highest means of lettuce growth promotion, confirming the synergistic effect of the combination of the two microorganism types, as it increased height, canopy, shoot and root dry matter, and chlorophyll levels compared to CONT, although did not differ from CM in some variables. Enzymatic activity was also influenced by the action of these microorganisms combined, evidencing by polyphenol oxidase increase. The CMFB or CM were efficient in promoting lettuce growth, showing positive response to the plant morphological and physiological characteristics. However, few responses were observed in lettuce plant growth in the first cycle evaluated after 60 days, compared CM and CMFB treatments, but both treatments showed superiority in lettuce plant growth submitted to CONT treatment. Therefore, further studies are needed to estimate the long-term effects of combined effect of Pseudomonas sp. UAGF14 and T. aureoviride URM5158 on crop productivity in field conditions.

A promoção do crescimento das plantas por micro-organismos pode ser uma alternativa viável para aumentar a produção de alface através de controle de patógenos e aumento da absorção de nutrientes. O gênero fúngico Trichoderma e o gênero bacteriano Pseudomonas são exemplos de micro-organismos amplamente estudados com capacidade para promover o crescimento da planta. No entanto, ainda existem lacunas quanto à ação do efeito combinado desses dois micro-organismos. Portanto, o objetivo deste estudo foi avaliar o efeito combinado de Pseudomonas UAGF14 e Trichoderma aureoviride URM5158 sobre o desenvolvimento de plantas de alface. O delineamento experimental foi completamente casualizados, com cinco tratamentos: CONT (controle, sem fertilização orgânica), CM (solo com fertilização orgânica), CMB (solo com fertilização orgânicae Pseudomonas sp.), CMF (solo com fertilização orgânica e T. aureoviride) e CMFB (solo com fertilização orgânica, Pseudomonas sp. e T. aureoviride), com dez repetições. Aos 30, 40 e 60 dias após a semeadura, foram analisados os seguintes parâmetros: altura da planta e dossel e número de folhas. Aos 60 dias após a emergência, a matéria seca da parte aérea, a área foliar, a massa seca das raízes, o comprimento radicular e a clorofila foram analisados. Catalase, peroxidase e atividade enzimática da polifenol oxidase foram determinadas. O CMFB apresentou o maior crescimento de alface, confirmando o efeito benéfico da combinação dos dois tipos de micro-organismos com a planta, na medida em que aumentou a altura, o dossel, a matéria seca da parte aérea e da raiz, e os níveis de clorofila em relação ao CONT, embora não tenha diferido do CM em algumas variáveis. As atividades enzimáticas também foram influenciadas pela ação desses micro-organismos combinados, evidenciada pelo aumento de polifenol oxidase. O CMFB ou CM foram eficientes na promoção do crescimento da alface, mostrando respostas positivas às características morfológicas e fisiológicas. Entretanto, poucas respostas foram observadas no crescimento da alface noprimeiro ciclo da planta avaliado depois de 60 dias, comparando os tratamentos CM e CMFB, mas ambos tratamentos mostraram superioridade em relação ao crescimento das plantas de alface submetidas ao tratamento controle. Por isso, são necessários futuros estudos para estimar à longo prazo o efeito combinado de Pseudomonas sp. UAGF14 e Trichoderma aureoviride URM5158 na produção de cultura em condições de campo.

Reduced Enzymatic Browning in Potato Tubers by Specific Editing of a Polyphenol Oxidase Gene via Ribonucleoprotein Complexes Delivery of the CRISPR/Cas9 System.

Polyphenol Oxidases (PPOs) catalyze the conversion of phenolic substrates to quinones, leading to the formation of dark-colored precipitates in fruits and vegetables. This process, known as enzymatic browning, is the cause of undesirable changes in organoleptic properties and the loss of nutritional quality in plant-derived products. In potato (Solanum tubersoum L.), PPOs are encoded by a multi-gene family with different expression patterns. Here, we have studied the application of the CRISPR/Cas9 system to induce mutations in the StPPO2 gene in the tetraploid cultivar Desiree. We hypothesized that the specific editing of this target gene would result in a lower PPO activity in the tuber with the consequent reduction of the enzymatic browning. Ribonucleoprotein complexes (RNPs), formed by two sgRNAs and Cas9 nuclease, were transfected to potato protoplasts. Up to 68% of regenerated plants contained mutations in at least one allele of the target gene, while 24% of edited lines carried mutations in all four alleles. No off-target mutations were identified in other analyzed StPPO genes. Mutations induced in the four alleles of StPPO2 gene, led to lines with a reduction of up to 69% in tuber PPO activity and a reduction of 73% in enzymatic browning, compared to the control. Our results demonstrate that the CRISPR/Cas9 system can be applied to develop potato varieties with reduced enzymatic browning in tubers, by the specific editing of a single member of the StPPO gene family.

In vitro and in vivo inhibition of Hass avocado polyphenol oxidase enzymatic browning by paeonol, ß-cyclodextrin, and paeonol:ß-cyclodextrin inclusion complex.

Polyphenol oxidase (PPO) was extracted from Hass avocados and its physicochemical properties were analyzed. The optimum pH and temperature of the enzyme were pH 7.5 and 20°C. This PPO showed a high thermal stability, since 26% of the initial activity was retained by the enzyme after heating at 60°C for 40 min. Inhibition studies were performed using different chemical reagents, and the order in the inhibition efficiency was paeonol > 4-hydroxybenzaldehyde > ß-cyclodextrin (ß-CD). The first two inhibitors presented a non-competitive mechanism while the inhibition by ß-CD results from a mixed type mechanism. Since the aqueous solubility of paeonol (a natural compound) is very low, the inclusion complex between this drug and ß-CD was obtained in solution and solid state. The stoichiometry of the paeonol:ß-CD complex was 1:1 and its ΔG° of formation was -26 kJ/mol. The complexation of paeonol by ß-CD not only enhances the aqueous solubility and thermal stability of the drug, but also improves the in vitro inhibition efficiency against PPO. Colorimetric analysis on avocados pulp (in vivo) showed that the inclusion complex does not increase the inhibitory effect of paeonol, remaining practically unchanged. However, the formulation of paeonol:ß-CD inclusion complex allows employing this compound as PPO inhibitor in aqueous solutions.

Red clover silage: an alternative for mitigating the impact of nitrogen excretion in ovine production systems

The objective was to quantify the flow of intestinal nutrients and nitrogen excretion and retention in sheep receiving isoproteic diets. Eight Texel x Lacaune wethers (average body weight = 25±2.5 kg) were fitted with duodenal cannula and housed in metabolic cages. Wethers were assigned to the treatments in a crossover design with two periods of 20 days each, and all feces and urine produced by the wethers were collected. The treatments consisted of two isoproteic (160 g kg−1 of crude protein on dry matter basis) diets composed of red clover (RC) or lucerne (LU; Medicago sativa) silages plus corn silage and concentrate feed. The digestible organic matter and metabolizable energy intake did not differ between treatments. The intestinal non-ammonia N (NAN) flow was 5.9 g day−1 (37%) higher in RC wethers than in those of the LU treatment. This result was a consequence of both an increase in the efficiency of microbial protein synthesis (12.7% higher) and a decrease in ruminal degradable protein (RDP) content (20% lower) of the diet. However, the increase in the intestinal NAN flow was accompanied by a reduction in intestinal digestibility of N, resulting in similar daily N retention between treatments. The reduction of RDP content was probably the main reason for reductions in N urinary excretion in RC wethers compared with those in the LU treatment, showing that RC silage may be a tool for mitigating the impact of N excretion in ovine production systems, without changes in N retention.(AU)

Phenotypic Characterization and Synthesis of Extracellular Catecholase from a Newly Isolated Bacterium Pseudomonas sp. BSC-6

Abstract Catecholase (EC 1.10.3.1), an oxidoreductase enzyme is a key member of polyphenol oxidase family which catalyze the degradation of catechol. This enzyme possesses vast applications in diverse areas and is found in bacteria, fungi, mushrooms, higher plants, arthropods, amphibians and mammals. Catechol, a phenolic compound, is used as a starting material in the synthesis of various industrial compounds such as inhibitors, antioxidants, pesticides etc. The release of this phenolic compound in the environment causes toxicity to both flora and fauna. In the present studies, emphasis has been laid on isolation, screening and characterization of catechol degrading bacterium coupled with synthesis of catecholase enzyme. Further, the selected isolated strain was phenotypically characterized and was found to be member of genus Pseudomonas. Among all the isolates, BSC-6 was found as best isolate with maximum extracellular catecholase activity of 152.32 IU/L obtained after scale up studies. The herein synthesized bacterial catecholase may be employed for wide applications particularly in bioremediation of phenol enriched polluted sites.

Effect of Blanching on Enzyme Activity and Bioactive Compounds of Blackberry

ABSTRACT The inactivation of peroxidase and polyphenol oxidase and the degradation of phenolic compounds, anthocyanins, and antioxidant activity in blackberry were investigated during blanching in water at 80-90 °C, and in steam, from 0 to 10 minutes. The enzyme kinetics indicated the presence of two isoenzymes with different thermal stability, following two-phase model, in which the reaction rate constant increased with increasing temperature for both the heat labile and heat resistant component. The phenolic compounds also followed a biphasic pattern, indicating a more heat resistant fraction, and the rate constants increased with increasing temperature. For the antioxidant activity and anthocyanins, the first order kinetic model (R2>0.982) indicated an increase in degradation rate constants with temperature, from 0.050 to 0.137min-1 for DPPH, 0.085 to 0.285min-1 for ABTS, and 0.017 to 0.052 min-1 for anthocyanins.

Heat stability and effect of pH on enzyme activity of polyphenol oxidase in buriti (Mauritia flexuosa Linnaeus f.) fruit extract.

Polyphenol oxidase (PPO) was extracted and characterized from ripe fruit of Mauritia flexuosa. Buriti PPO showed optimum activity at pH 7.0 and 35°C, with complete inactivation in between 2.0≤pH>10, using catechol as substrate. The enzyme had optimum temperaturet 35°C and was relatively stable at 77°C, with 59.93% loss of activity. These results demonstrate that the enzyme has heat stability at higher temperatures and the possibility of being used to construct biosensors and other analytical methods in various fields of science.

Induction of defense-related enzymes in patchouli inoculated with virulent Ralstonia solanacearum

Background: Defense-related anti-oxidative response is a vital defense mechanism of plants against pathogen invasion. Ralstonia solanacearum is an important phytopathogen. Bacterial wilt caused by R. solanacearum is the most destructive disease and causes severe losses in patchouli, an important aromatic and medicinal plant in Southeast Asia. The present study evaluated the defense response of patchouli inoculated with virulent R. solanacearum. Results: Results showed that the basic enzymatic activities differed not only between the leaves and stems but also between the upper and lower parts of the same organ of patchouli. POD, SOD, PPO, and PAL enzymatic activities were significantly elevated in leaves and stems from patchouli inoculated with R. solanacearum compared to those in control. The variation magnitude and rate of POD, PPO, and PAL activities were more obvious than those of SOD in patchouli inoculated with R. solanacearum. PAGE isoenzymatic analysis showed that there were one new POD band and two new SOD bands elicited, and at least two isoformic POD bands and two SOD bands were observably intensified compared to the corresponding control. Conclusion: Our results suggest that not only defense-related enzymatic activities were elevated but also the new isoenzymatic isoforms were induced in patchouli inoculated with R. solanacearum.

Enzymatic browning in avocado (Persea americana) revisited: History, advances, and future perspectives.

Considering nearly 80 years of research regarding one of the enzymes responsible for catalyzing the formation of pigments in higher animals, plants, fungi and bacteria, this review will focus on collecting and categorizing the existing information about polyphenol oxidase (PPO) in fruits, with particular emphasis on the information in relation to avocado, which is one of the hardiest species in terms of inactivation, has documented dual activity (EC 1.14.18.1/EC 1.10.3.1), and represents one of the oldest challenges for food science research and fruit processors. It is expected that this review will contribute to the further development of the field by highlighting the questions that have arisen during the characterization of PPO, the progress that has been made and the questions that remain today, in addition to new methodologies that are being applied to study this system. Holistic methodologies offer unexplored potential for advancing our understanding of the complex phenomena that govern PPO activity in fruits, because these methodologies will enable the characterization of this family of enzymes in all of its complexity. Subsequently, it will be possible to develop better techniques for controlling enzymatic browning in this valuable fruit.

Solanum melongena polyphenol oxidase biosensor for the electrochemical analysis of paracetamol.

A new strategy for the construction of a polyphenol oxidase carbon paste biosensor for paracetamol detection is reported. The eggplant (Solanum melongena) was processed to collect the polyphenol oxidase as an enzyme that was incorporated in the carbon paste sensor construction. The constructed sensor displayed high sensitivity and good selection for paracetamol detection and recognition. Optimized conditions included pH 6.0 (highest activity), pH 7.0 (highest stability), pulse amplitude of 50 mV, and 15% of vegetable extract per carbon paste. The sensor displayed a linear range from 20 to 200 µM, with a detection limit of 5 µM. Application of the sensor to paracetamol determination in tablet and oral solutions have shown satisfactory results. The efficiency of the method showed very good repeatability ranging between 1.26 and 1.72% relative standard deviation for interday analysis, while recoveries for paracetamol varied between 97.5 and 99.8% for the voltammetric determination. The strategy for a simple, low cost, and efficient eggplant polyphenol oxidase sensor showcased in this work provides an opportunity for the detection of other phenolic compounds in various matrices.

Characterization of polyphenol oxidase from Cape gooseberry (Physalis peruviana L.) fruit.

Cape gooseberry (Physalis peruviana) is an exotic fruit highly valued, however it is a very rich source of polyphenol oxidase (PPO). In this study, Cape gooseberry PPO was isolated and biochemically characterized. The enzyme was extracted and purified using acetone and aqueous two-phase systems. The data indicated that PPO had the highest substrate affinity for chlorogenic acid, 4-methylcatechol and catechol. Chlorogenic acid was the most suitable substrate (Km=0.56±0.07 mM and Vmax=53.15±2.03 UPPO mL(-1) min(-1)). The optimal pH values were 5.5 for catechol and 4-methylcatechol and 5.0 for chlorogenic acid. Optimal temperatures were 40°C for catechol, 25°C for 4-methylcatechol and 20°C for chlorogenic acid. In inhibition tests, the most potent inhibitor was found to be ascorbic acid followed by L-cysteine and quercetin. This study shows possible treatments that can be implemented during the processing of Cape gooseberry fruits to prevent browning.

Influência da temperatura e do tempo de armazenamento sobre a atividade de enzimas oxidativas em Hancornia speciosa Gomes

A espécie Hancornia speciosa Gomes é uma frutífera nativa do Brasil sobre a qual tem crescido o interesse por parte da indústria de polpas, sucos e sorvetes. A exploração do fruto da mangabeira é dificultada pela sua reduzida vida útil pós-colheita. A perda rápida das características apreciáveis desse fruto está associada à atividade de enzimas oxidativas como a peroxidase (POD) e a polifenoloxidase (PPO). Neste trabalho foi quantificada a atividade dessas duas enzimas na polpa de frutos de mangaba submetida a três temperaturas de refrigeração (6, 10 e 18°C) em função de diferentes tempos de armazenamento (0, 3, 6, 9, 12 e 15 dias). Os níveis de PDO na polpa dos frutos de mangaba foram sempre superiores aos de PPO para as três temperaturas testadas, para todos os tempos de armazenamento analisados. Analisando-se cada enzima em particular, não foi detectada diferença na atividade de POD ou de PPO entre as temperaturas testadas e ao desdobrar-se o tempo dentro de cada temperatura testada, foi encontrada uma correlação de 0,035, para a atividade de PPO.(AU)

The species Hancornia speciosa is a native fruit of Brazil over which it has been growing interest by industry of the pulps, juices and ice cream. The exploration of the fruit of mangabeira is hampered by their limited shelf-life. The rapid loss of the appreciable features of this fruit is associated with the activity of oxidative enzymes such as peroxidase and polyphenol oxidase. In this study we quantified activity of these two enzymes in the fruits pulp of the mangaba subjected to three refrigeration temperatures (6, 10 and 18 º C) for different storage times (0 ,3, 6, 9, 12 and 15 days). Levels of PDO in the pulp mangaba were always higher than the PPO for the three temperatures tested, for ali storage periods analyzed. Analyzing each particular enzyme, no difference was detected in the activity of POD and PPO between the temperatures tested and unfold the time within each temperature tested, found a correlation of 0.035 for the PPO activity.(AU)