Orientador: Valdir Augusto / Banca: Valdir Augusto Neves / Banca: José Paschoal Batistuti / Banca: Jonas Contiero / Resumo: A família das anonáceas é composta por muitos gêneros e espécies; dentre essas, a atemóia (Annona cherimola Mill. X Annona squamosa L.), resultante do cruzamento entre a fruta-do-conde (Annona squamosa L.) e a cherimóia (Annona cherimola Mill.). No Brasil, a atemóia vem despertando grande interesse na produção/comercialização dos seus frutos; no entanto um dos obstáculos enfrentados é a facilidade de escurecimento enzimático que a fruta apresenta. Esse tem como responsável a polifenoloxidase (PPO, E.C. 1.14.18.1); que sob diferentes condições de armazenamento e processamento de vegetais na sua fase póscolheita pode atuar sob substratos naturais e resultar na formação de compostos escuros, acarretando diminuição do valor nutricional, modificação das propriedades organolépticas e sensoriais, com consequente rejeição. Os objetivos desse trabalho foram isolar, purificar e caracterizar algumas propriedades da polifenoloxidase de atemóia. Condições de extração para a PPO foram estabelecidas e a enzima foi isolada por precipitação com sulfato de amônio e eluição em Sephadex G-100. Somente um pico de atividade foi eluído no processo de purificação com um fator de purificação de 7,12. O peso molecular determinado foi 82 kDa com valores ótimos de pH 6,0 e 7,0 para 4-metil catecol e catecol, respectivamente; sendo estável por 12 e 24 horas à incubação na faixa de pH 4,0-8,0. A temperatura ótima de atividade foi 35°C e 28°C para 4-metil catecol e catecol, respectivamente. Os valores calculados de energia de ativação (Ea) para esses substratos foram 87,29 cal/mol-1 e 180,97 cal/mol-1. As constantes cinéticas Km e Vmax foram 5,52mmol/L e 1428,57 UA/mL, para 4-metil catecol, e 79,3 mmol/L e 5000 UA/mL, para catecol. A relação Km/Vmax determinou uma maior afinidade da enzima pelo primeiro substrato. A enzima mostrou desprezível atividade... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The family of annonaceae is composed of many genus and species, among these, the atemoya (Annona cherimola Mill. X Annona squamosa L.), resulting from a cross between custard apple (Annona squamosa L.) and cherimoya (Annona cherimola Mill.). In Brazil, the atemoya is attracting great interest in the production / marketing of its fruits, however one of the obstacles faced is the ease of enzymatic browning that gives the fruit. This is as responsible for polyphenoloxidase (PPO, EC 1.14.18.1), which under different conditions of storage and processing plant in its post-harvest can act on natural substrates and result in the formation of dark compounds, causing decrease in nutritional value, modification of the organoleptic and sensory properties, with consequent rejection. The objectives of this study were to isolate, purify and characterize some properties of polyphenoloxidase from atemoya. Conditions for the extraction of PPO were established and the enzyme was isolated by precipitation with ammonium sulfate and elution on Sephadex G-100. Only one peak of activity was eluted in the process of purification with a purification factor of 7.12. The molecular weight of 82 kDa was determined with the optimum values of pH 6.0 and 7.0 for 4-methylcatechol and catechol, respectively. The enzyme was stable for 12 and 24 hours incubation in the pH range 4.0-8.0. The optimum temperatures for activity were 35 °C and 28 °C for 4-methylcatechol and catechol, respectively. The values of activation energy (Ea) for these substrates cal/mol-1 were 87.29 and 180.97 cal/mol-1. The kinetic constants Km and Vmax were 5.52 mmol/L and 1428.57 UA/mL for 4-methylcatechol, and 79.3 mmol/L and 5000 AU / mL for catechol. The Km/Vmax determined a higher affinity of the enzyme by the first substrate. The enzyme showed negligible activity for caffeic acid and chlorogenic as any for L-DOPA, catechin... (Complete abstract click electronic access below) / Mestre

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Thesis (MScEng) -- University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The grape juice concentrate (GJC) plant of the KWV at Robertson spent significant amounts of money on the decolourisation of grape juice concentrate. A chemically activated powdered activated carbon (PAC) purchased from Norit, namely CA1, was used as decolourisation product. Apart from the expenses involved, it contributed largely to the solid waste produced at this plant. A way was sought to minimise or prevent GJC discolourisation (and possibly solid waste) without increasing operating expenses. Browning reactions in GJC are as old as the product itself. Numerous researchers have studied the origins of these reactions, the reactants and products involved, as well as the reaction kinetics of these reactions. From the work of these researchers four possible browning reaction pathways were identified, namely: • enzymatic oxidative browning, • non-enzymatic oxidative browning, • non-enzymatic browning (the Maillard reaction), and • caramelisation. It was also identified that 5-hydroxymethylfurfural (HMF) are indicative of the browning potential of GJC. A method to analyse for HMF (quantitative and qualitative) was develop for the purposes of this study, namely positive electron-spray ionisation preceded by high-pressure liquid chromatography (HPLC) and followed by dual mass spectrometry. This method showed good repeatability and was used to analyse all samples generated during this study. It was confirmed that the manufacturing process at this plant favours nonenzymatic browning reactions, since mild heat treatment deactivates enzymes. Further investigation indicated that the overruling browning reaction on this plant was non-enzymatic oxidative browning. It was shown that neither the presence, nor the absence of protein had any effect on the rate of formation of HMF. It was, however, confirmed that HMF formation could be attributed to high temperatures and prolonged exposure to these temperatures. Other adsorption products were evaluated against the then current PAC (CA1), namely a steam activated PAC supplied by Norit, SA4, and a polymeric adsorbent, Polyclar V (polyvinylpolypyrrolidone/PVPP). Both SA4 and PVPP indicated superior HMF adsorption capacities. Replacing CA1 with SA4 could result in operating expenses savings and possible solid waste reduction. However, PVPP were too expensive to be considered an economically viable replacement for CA1. Improved concentration technologies such as reverse osmosis (RO) membrane concentration followed by centrifugal evaporation (CE) or twostage CE should be considered as possible replacement for the existing concentration technology (multi-stage falling film evaporator). This should decrease heat treatment/exposure by more than 90% and thus reduce browning significantly. An added advantage could be the reduction of solid waste, since less (if not no) decolourisation will be required. Alternatively, juice should be stored with added sulphur dioxide (SO2), since it was shown that this juice contained much lower HMF concentrations than diluted concentrate (stored for the same time). This should reduce heat exposure by up to 50% and thus minimise browning reactions. / AFRIKAANSE OPSOMMING: Die druiwesapkonsentraat (DSK) aanleg van die KWV in Robertson het jaarliks aansienlike bedrae geld spandeer tydens die ontkleuringsproses van DSK. ‘n Chemies geaktiveerde verpoeierde koolstof (GVK) verkrygbaar van Norit, naamlik CA1, is gebruik as ontkleuringsproduk. Buiten die kostes verbonde aan hierdie produk het dit ook grootliks bygedra tot soliede afval by hierdie aanleg. Oplossings is gesoek om die verbruining/ontkleuring van DSK (en dalk ook soliede afval) te verminder (of selfs te voorkom) sonder om bedryfskostes te verhoog. Verbruiningsreaksies in DSK bestaan al so lank soos DSK self. Verskeie navorsers het die oorsake, reaktante, produkte en reaksiekinetika van hierdie reaksies oor die jare heen bestudeer. Uit die werk van sommige van hierdie navorsers kon vier moontlike verbruiningsreaksieroetes geïdentifiseer word, naamlik: • ensiematiese oksidatiewe verbruining, • nie-ensiematiese oksidatiewe verbruining, • nie-ensiematiese verbruining (die Maillard-reaksie), en • karamelisering. Daar was verder geïdentifiseer dat 5-hidroksiemetielfurfuraal (HMF) aanduidend is van die verbruiningspotensiaal van DSK. ‘n Analitiese metode (kwalitatief en kwantitatief) om vir HMF te analiseer is vir die doel van hierdie studie ontwikkel, naamlik positiewe elektronsproei ionisasie, voorafgegaan deur hoëdruk vloeistof chromatografie en gevolg deur dubbele massa spektrometrie. Hierdie analitiese metode het goeie herhaalbaarheid getoon en was deurgaans gebruik om monsters te analiseer gedurende hierdie studie. Dit was bevestig dat die vervaardigingsproses by hierdie aanleg nieensiematiese verbruiningsreaksies begunstig, aangesien geredelike hittebehandeling ensieme deaktiveer. Verdere navorsing het getoon dat die oorheersende verbruiningsreaksies by hierdie aanleg nie-ensiematiese oksidatief van aard is. Resultate het getoon dat proteinstabiliteit geen invloed op die vormingstempo van HMF het nie. Dit was egter bevestig dat vorming van HMF direk verband hou met hoë temperature en lang blootstellingsperiodes aan hierdie temperature. Ander adsorpsieprodukte was vergelyk met die huidige GVK (CA1), naamlik ‘n stoom geaktiveerde verpoeierde koolstof (Norit se SA4) en ‘n polimeriese adsorbant, Polyclar V (polivinielpolipirrolidoon/PVPP). Beide SA4 en PVPP het CA1 oortref wat betref HMF adsorpsie. Moontlike bedryfskostebesparings (en soliede afval verminderings) potensiaal bestaan indien CA1 vervang word met SA4. Die teenoorgestelde is egter waar vir PVPP wat bedryfskoste aangaan. Instede van die huidige verdampinstegnologie, naamlik vallendefilmverdamping, hoort verbeterde konsentrasietegnologieë soos tru-osmose membraankonsentrasie gevolg deur sentrifugale verdamping, of, alternatiewelik, twee-stadium sentrifugale verdamping, orrweeg te word. Op hierdie wyse behoort hittebehandeling (en dus verbruining) met sowat 90% verminder te word. ‘n Moonlike addisionele voordeel is die vermindering van soliede afval aangesien minder ontkleuring nodig sal wees. Indien die verbeterde tegnologieë te duur is moet daar gekyk word daarna om die ongekonsentreerde sap met addisionele swaweldioksied (SO2) te stoor, aangesien veel laer HMF konsentrasies in sulke sap waargeneem is as in verdunde direkte konsentraat wat vir dieselfde typerk gestoor is. Hittebehandeling sal op hierdie wyse met tot 50% verminder word (en dus verbruining ook).

Made available in DSpace on 2015-04-22T22:17:25Z (GMT). No. of bitstreams: 1 SALOMAO ROCHA.pdf: 785933 bytes, checksum: 2a406f595cf4495ac889e48923ddd00c (MD5) Previous issue date: 2012-08-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / During the freezing of fruits pulps, the enzyme activity is not finished completely. Sensory, nutritional and coloring changes may occur on fruits due to the action of oxidative enzymes such as peroxidase and polyphenoloxidase. The frozen cupuaçu pulps, sold in Brazil, have a shelf life of one year and become browned during this period. The aim of this study was to evaluate the effect of frozen storage on the physicochemical characteristics, polyphenoloxidase activity and soluble and ionically bound peroxidases presented in the pulps of four new cupuaçu genotypes over twelve months. The cupuaçu genotypes developed by the West Amazonian Agroforestry Research Center (EMBRAPA) were pulped, frozen and stored at 30 °C. The polyphenoloxidase of the four cupuaçu genotypes showed an increase in activity according to the storage time with peaks in the sixth, ninth and tenth months, but the peroxidases exhibited oscillations in the enzyme activity. The physicochemical properties of the pulps showed variations during the twelve months of storage under freezing. The vitamin C content of D 28-10 and P 3-10 genotypes decreased from the fourth and tenth months, respectively. Moreover P 9-8 e B 28-7 genotypes remained stable. In relation the acidity of citric acid, the B-28-7, D 28-10 and P 9-8 samples were not different, but P 3-10 genotype presented a reduction. The pH and total soluble solids of all genotypes decreased over the study period. There was an increase in sugar concentration of B 28-7, P 3-10 and P 9-8 genotypes, except for D 28-10 sample which remained unchanged. All genotypes were in accordance with physical-chemicals standards required by legislation, except for P 3-10 genotype that showed a lower acidity. In respect of the enzymatic parameters, there were variations in the activity of peroxidase and polyphenoloxidases of all genotypes. / No congelamento de polpas de frutas a atividade enzimática não é completamente cessada. Podem ocorrer mudanças sensoriais, nutricionais e de coloração devido à ação de enzimas oxidativas, como a peroxidase e a polifenoloxidase. Considerando que as polpas de cupuaçu congeladas, comercializadas no Brasil, têm um prazo de validade de um ano e tornam-se escurecidas ao longo deste período, objetivou-se neste estudo avaliar o efeito do tempo de congelamento nas características físico-químicas e nas atividades da polifenoloxidase e peroxidases solúvel e insolúvel presentes nas polpas de quatro novos genótipos de cupuaçu, durante doze meses. Os frutos dos genótipos de cupuaçu, desenvolvidos pela Embrapa Amazônia Ocidental, foram despolpados, congelados e armazenados à temperatura de -30 ºC. A polifenoloxidase das polpas dos quatro genótipos apresentou aumento na sua atividade com picos no sexto, nono e décimo mês e as peroxidases apresentaram oscilações na atividade enzimática. As propriedades físico-químicas das polpas apresentaram variações durante os doze meses de armazenamento sob congelamento. O teor de vitamina C dos genótipos D 28-10 e P 3-10 diminuiu a partir do 4º e 10º mês, respectivamente. Por outro lado os genótipos B 28-7 e P 9-8 permaneceram estáveis. Em relação à acidez em ácido cítrico, as amostras B-28-7, D 28-10 e P 9-8 não diferiram, havendo redução no genótipo P 3-10. O valores de pH e sólidos solúveis totais de todos os genótipos diminuíram ao longo do período avaliado. Houve aumento na concentração de açúcares das polpas dos genótipos B 28-7, P 3-10 e P 9-8, com exceção da amostra D 28-10 que permaneceu inalterada. Todos os genótipos apresentaram-se dentro dos padrões físico-químicos exigidos pela legislação, com exceção do genótipo P 3-10 que apresentou acidez inferior. Em relação aos parâmetros enzimáticos, houve variações na atividade das peroxidases e polifenoloxidases de todos os genótipos avaliados.

Made available in DSpace on 2014-06-11T19:23:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-05-29Bitstream added on 2014-06-13T20:30:22Z : No. of bitstreams: 1 santos_irc_me_arafcf.pdf: 1670522 bytes, checksum: 94cf024bcd75478eee1c840ef55cc77f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A família das anonáceas é composta por muitos gêneros e espécies; dentre essas, a atemóia (Annona cherimola Mill. X Annona squamosa L.), resultante do cruzamento entre a fruta-do-conde (Annona squamosa L.) e a cherimóia (Annona cherimola Mill.). No Brasil, a atemóia vem despertando grande interesse na produção/comercialização dos seus frutos; no entanto um dos obstáculos enfrentados é a facilidade de escurecimento enzimático que a fruta apresenta. Esse tem como responsável a polifenoloxidase (PPO, E.C. 1.14.18.1); que sob diferentes condições de armazenamento e processamento de vegetais na sua fase póscolheita pode atuar sob substratos naturais e resultar na formação de compostos escuros, acarretando diminuição do valor nutricional, modificação das propriedades organolépticas e sensoriais, com consequente rejeição. Os objetivos desse trabalho foram isolar, purificar e caracterizar algumas propriedades da polifenoloxidase de atemóia. Condições de extração para a PPO foram estabelecidas e a enzima foi isolada por precipitação com sulfato de amônio e eluição em Sephadex G-100. Somente um pico de atividade foi eluído no processo de purificação com um fator de purificação de 7,12. O peso molecular determinado foi 82 kDa com valores ótimos de pH 6,0 e 7,0 para 4-metil catecol e catecol, respectivamente; sendo estável por 12 e 24 horas à incubação na faixa de pH 4,0-8,0. A temperatura ótima de atividade foi 35°C e 28°C para 4-metil catecol e catecol, respectivamente. Os valores calculados de energia de ativação (Ea) para esses substratos foram 87,29 cal/mol-1 e 180,97 cal/mol-1. As constantes cinéticas Km e Vmax foram 5,52mmol/L e 1428,57 UA/mL, para 4-metil catecol, e 79,3 mmol/L e 5000 UA/mL, para catecol. A relação Km/Vmax determinou uma maior afinidade da enzima pelo primeiro substrato. A enzima mostrou desprezível atividade... / The family of annonaceae is composed of many genus and species, among these, the atemoya (Annona cherimola Mill. X Annona squamosa L.), resulting from a cross between custard apple (Annona squamosa L.) and cherimoya (Annona cherimola Mill.). In Brazil, the atemoya is attracting great interest in the production / marketing of its fruits, however one of the obstacles faced is the ease of enzymatic browning that gives the fruit. This is as responsible for polyphenoloxidase (PPO, EC 1.14.18.1), which under different conditions of storage and processing plant in its post-harvest can act on natural substrates and result in the formation of dark compounds, causing decrease in nutritional value, modification of the organoleptic and sensory properties, with consequent rejection. The objectives of this study were to isolate, purify and characterize some properties of polyphenoloxidase from atemoya. Conditions for the extraction of PPO were established and the enzyme was isolated by precipitation with ammonium sulfate and elution on Sephadex G-100. Only one peak of activity was eluted in the process of purification with a purification factor of 7.12. The molecular weight of 82 kDa was determined with the optimum values of pH 6.0 and 7.0 for 4-methylcatechol and catechol, respectively. The enzyme was stable for 12 and 24 hours incubation in the pH range 4.0-8.0. The optimum temperatures for activity were 35 °C and 28 °C for 4-methylcatechol and catechol, respectively. The values of activation energy (Ea) for these substrates cal/mol-1 were 87.29 and 180.97 cal/mol-1. The kinetic constants Km and Vmax were 5.52 mmol/L and 1428.57 UA/mL for 4-methylcatechol, and 79.3 mmol/L and 5000 AU / mL for catechol. The Km/Vmax determined a higher affinity of the enzyme by the first substrate. The enzyme showed negligible activity for caffeic acid and chlorogenic as any for L-DOPA, catechin... (Complete abstract click electronic access below)

La conservation des mangues 4ème gamme est limitée par le brunissement enzymatique et la perte de fermeté dus aux différentes opérations de transformation. Cette thèse propose un nouvelle approche pour améliorer la conservation de mangues 4ème gamme : l’application de traitements thermiques par immersion et/ou la conservation sous atmosphère modifiée : passive ou active (5% O2 – 5 % CO2) ou des enrobages (chitosane). Ce travail a permis tout d’abord de déterminer un couple température / temps optimal: 50 °C / 30 min qui permet de maintenir la qualité de plusieurs variétés de mangue (Keitt, Kent, Tommy Atkins). Les effets du traitement thermique sur l’intensité respiratoire, la couleur, la fermeté, les teneurs en antioxydants : vitamine C, caroténoïdes et phénols totaux, et sur l’activité de plusieurs enzymes (pour la fermeté : PME, PG, b-GAL ; et pour la couleur : PPO et PAL) ont été étudiés. Ces travaux ont montré que durant 9 jours à 6 °C, le traitement thermique maintient principalement la couleur par réduction de l’activité de la PPO et réduit la perte en fermeté par réduction de l’activité des enzymes pectolytiques (PME et b-GAL). Par ailleurs, les atmosphères modifiées semblent moins efficaces pour le maintien de la couleur et de la fermeté des mangues 4ème gamme. L’association du traitement 50°C/ 30 min avec une conservation sous atmosphère modifiée apporte peu d’effets additionnels et a même parfois un effet inhibiteur / The storage of fresh-cut mangoes is limited by the enzymatic browning and the loss of firmness due to the various operations of process. This work proposes a new approache to improve the storage of fresh-cut mangoes by: application of heat treatments by dipping with, or not, a storage under modified atmosphere: passive or active (5% O2 - 5% CO2) or using coatings (chitosane). The first step of this work was to determine the traitement 50 °C/30 min as an optimal treatment which maintian the quality of several varieties of mango (Keitt, Kent, Tommy Atkins). Effects of the heat treatment on the respiratory intensity, the color, firmness, the contents antioxydants: vitamin C, carotenoids and phenols total, and on the activity of several enzymes (PME, PG, b-GAL: for firmness; and PPO and PAL for colour changes) were studied. This work showed that during 9 days at 6 °C, the heat treatment maintains mainly the color by reduction of the activity of PPO and reduces the loss in firmness by reduction of the activity of the pectolytic enzymes (PME et b-GAL). In addition, the modified atmospheres seem less effective for the maintenance of the color and the firmness of freshcut mangoes. Combination of heat treatment with a modified atmosphere storage and have less additional effects and has even sometimes an inhibiting effect

Non-enzymatic browning (NEB) due to ascorbic acid degradation is one of the most common reasons the shelf life of many processed foods is reduced. Different methods to minimize or retard the formation of browning pigments have been studied; however, to date, refrigeration is still the most preferable. Unfortunately, the use of low temperatures to preserve food is not always available in many parts of the world. Indeed, an area of concern due to NEB has been identified in meal-ready-to-eat (MRE) individual military rations, specifically diced peaches with syrup. This product was once part of soldiers' menus; however, it was removed due to browning and textural deterioration that occurred when stored under field conditions. We examined two general approaches to reduce NEB: the replacement of ascorbic acid by a more stable form and the use of flavonoids as antibrowning antioxidants in peach systems. These approaches were studied in three objectives. In our first objective, ascorbyl-2-phosphate showed better stability than ascorbic acid at 40°C in peach puree model systems, but not at 50 or 60°C. In the second objective, after the evaluation of the effect of two forms of vitamin C (ascorbic acid and ascorbyl-2-phosphate) and Pycnogenol (0%, 0.01% and 1%) on the quality of diced peaches in retortable pouches, we concluded that neither ascorbyl-2-phosphate nor pycnogenol resulted in improved color or ascorbic acid stability. Finally, in our third objective, after the evaluation of the effect of peach source (fresh, individually quick frozen and canned), addition of calcium chloride, and the addition of a water soluble flavonoid (°-glucosylrutin, °-GR) in diced peaches packaged in retortable pouches stored at 24, 40 and 51°C, there were no significant effects of °-GR on any of the peach sources at 51°C. However, at 40°C, °-GR improved the quality of diced peaches in pouches made of individually quick frozen and canned peaches, but not for fresh peaches. Quality was assessed by color (CIELAB system), which was measured using a handheld colorimeter, and ascorbic acid levels of peaches, which was determined using high performance liquid chromatography. / Master of Science