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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior This study aimed to evaluate the impact on digestibility and physical properties of enteral formulas by the addition of variable ingredients. Eight formulas were designed by a simplex-centroid evaluating different concentrations of inulin, medium-chain triglycerides (MCT) and whey protein isolate (WPI). Overall, the eight formulas developed presented mean values of 17.3% of protein, 62.3% of carbohydrates, 11.5% of lipids, a caloric value of 420 kcal/100 g, thus obtaining nutritionally adequate formulas. The emulsion stability of the suspended formulas was affected by all ingredients and the interactions between MCT – inulin, and MCT – WPI were effective in improving this parameter. Besides that, the use of inulin mostly affected the protein digestibility, according to the degree of hydrolysis and the peptide profile. The desirability function (d-value = 0.769) proposed a formulation containing 0.70% of inulin, 1.56% of MCT, and 1.73% of WPI. This proposed solution may improve enteral formulas because this has optimal emulsion stability and protein digestibility, which are essential characteristics for a product to be used by patients under special clinical conditions.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior The aim of this study was to evaluate the effect of inulin, medium-chain triglycerides (MCT) and whey protein isolate (WPI) on enteral nutrition formulas (ENF) and the influence of these ingredients on physicochemical parameters, protein in vitro digestion and functionality of bifidobacteria incorporated to the product. For this, a centroid simplex experimental design was used, including a central point and a control formula. Firstly, the formulas were characterized for chemical composition, emulsion stability and in vitro protein digestibility. In a second phase, the effect of the three ingredients on the viability of Bifidobacterium longum BL05 was evaluated during the product storage, for 120 days, at 4 oC, as well as the survival of the microorganisms under simulated gastrointestinal conditions. Overall, eight formulas were developed, presenting mean values of 17.3% of protein, 62.3% of carbohydrates, 11.5% of lipids, a caloric value of 420 kcal/100 g, all nutritionally adequate according to the Brazilian regulation. The emulsion stability of the suspended formulas was affected by all ingredients and the interactions between MCT – inulin, and MCT – WPI contributed positively to the improvement of this parameter. Regarding the protein digestibility, the presence of inulin promoted lower percentages of degree of hydrolysis and small peptides. B. longum BL 05 counts ranged from 9.05 to 9.79 log CFU g-1, and the WPI showed a positive effect on the viability at the end of the storage period. The presence of inulin exerted a protective effect on cells when subjected to simulated gastrointestinal conditions, with log reductions between 2.03 and 3.44 log CFU g-1 after 4 hours of simulated digestion. Thus, the developed formulas demonstrated adequacy regarding the composition and physicochemical parameters, as well as a potential food matrix for the incorporation of probiotic microorganisms. O objetivo deste trabalho foi avaliar o efeito da inulina, dos triglicerídeos de cadeia média (TCM) e da proteína isolada de soro de leite (WPI) em fórmulas para nutrição enteral (FNE) e a influência destes ingredientes nos parâmetros físico-químicos, na digestão in vitro das proteínas e na funcionalidade de bifidobactérias incorporadas ao produto. Para isso, foi utilizado um delineamento experimental centroid-simplex, incluindo um ponto central e uma fórmula controle. Em primeiro lugar, as fórmulas foram caracterizadas quanto à composição química, estabilidade da emulsão e digestibilidade in vitro das proteínas. Numa segunda etapa, avaliou-se o efeito dos três ingredientes na viabilidade de Bifidobacterium longum BL05 durante o armazenamento do produto, durante 120 dias, a 4 oC, bem como a sobrevivência dos micro-organismos sob condições gastrintestinais simuladas. Ao todo, oito fórmulas foram desenvolvidas, apresentando valores médios de 17,3% de proteína, 62,3% de carboidratos, 11,5% de lipídios, valor calórico de 420 kcal/100 g, todas nutricionalmente adequadas de acordo com a regulamentação brasileira. A estabilidade da emulsão das fórmulas foi afetada por todos os ingredientes e as interações entre TCM – inulina e TCM – WPI contribuiram positivamente para a melhoria deste parâmetro. Em relação à digestibilidade proteica, a presença de inulina resultou em menor liberação de pequenos peptídeos e aminoácidos livres. As contagens de B. longum BL 05 variaram entre 9,05 a 9,79 log UFC g-1, e o WPI apresentou um efeito positivo sobre a viabilidade no final do período de armazenamento. A presença de inulina exerceu um efeito protetor nas células quando submetidas a condições gastrointestinais simuladas, com reduções entre 2,03 e 3,44 log UFC g-1 após as 4 horas de digestão simulada. Assim, as fórmulas desenvolvidas demonstraram adequação quanto à composição e parâmetros físico-químicos, bem como uma potencial matriz alimentar para a incorporação de microrganismos probióticos.

Whey protein concentrate hydrolysates, capable of inhibiting the activity of angiotensin-converting enzyme, were prepared in the present study. Several parameters, such as type of enzyme, enzyme:substrate ratio, and use of ultrafiltration were evaluated. The angiotensin-converting enzyme inhibitory activity was observed only after the enzymatic treatment and high rates of inhibition were obtained, ranging from 17.29 to 91.88%. Most hydrolysates obtained with pancreatin showed higher angiotensin-converting enzyme inhibitory activity than those obtained with papain both in the absence and presence of ultrafiltration. The smallest enzyme:substrate ratio for pancreatin resulted in better angiotensin-converting enzyme inhibitory activity values. The use of ultrafiltration proved to be disadvantageous for inhibitory activity of hydrolysates obtained with papain.

In this work, the influence of some reactional parameters in the hydrolysis of whey protein concentrate (WPC) was evaluated, in terms of the nutritional quality of peptide profiles of the hydrolysates as well as the reduction of costs for scaling-up the process. Two enzymes (subtilisin and pancreatin) were used for preparing 18 hydrolysates, using different E:S ratios and reaction times, and the distribution of peptides according to chain length was analyzed by size-exclusion chromatography. The studied parameters affected the peptide profiles of WPC hydrolysates and the best result was similar for subtilisin and pancreatin, both using an E:S ratio of 4:100, after 5 h and 10 h, respectively. In these conditions, these enzymes gave rise to the smallest large peptide contents (12.28% and 12.34%, respectively) and one of the highest amount of di- and tripeptides (13.34% and 13.00%, respectively) as well as of free amino acids (45.56% and 47.26%, respectively). However, in terms of number of samples the action of pancreatin was more advantageous than subtilisin, since among the nine hydrolysates, four showed appropriate peptide profiles (P1, P2, P5, and P6), from the nutritional point of view, while the same happened only with one hydrolysate prepared by using subtilisin (S3). Also, the economical advantage of using smaller E:S ratio and reaction time was observed in several cases for both enzymes.

The aim of this study was to prepare enzymatic hydrolysates from whey protein concentrate with a nutritionally adequate peptide profile and the ability to inhibit angiotensin-converting enzyme (ACE) activity. The effects of the type of enzyme used (pancreatin or papain), the enzyme:substrate ratio (E:S ratio=0.5:100, 1:100, 2:100 and 3:100) and the use of ultrafiltration (UF) were investigated. The fractionation of peptides was performed by size-exclusion-HPLC, and the quantification of the components of the chromatographic fractions was carried out by a rapid Corrected Fraction Area method. The ACE inhibitory activity (ACE-IA) was determined by Reverse Phase-HPLC. All parameters tested affected both the peptide profile and the ACE-IA. The best peptide profile was achieved for the hydrolysates obtained with papain, whereas pancreatin was more advantageous in terms of ACE-IA. The beneficial effect of using a lower E:S ratio on the peptide profile and ACE-IA was observed for both enzymes depending on the conditions used to prepare the hydrolysates. The beneficial effect of not using UF on the peptide profile was observed in some cases for pancreatin and papain. However, the absence of UF yielded greater ACE-IA only when using papain.O objetivo deste estudo foi preparar hidrolisados enzimáticos do concentrado proteico do soro de leite com perfil peptídico nutricionalmente adequado e com capacidade para inibir a atividade da enzima conversora da angiotensina (ECA). Os efeitos do tipo de enzima usado (pancreatina ou papaína), da relação enzima:substrato (E:S=0,5:100, 1:100, 2:100 e 3:100) e do uso da ultrafiltração (UF) foram investigados. O fracionamento dos peptídeos foi feito por CLAE de exclusão molecular e a quantificação dos componentes das frações cromatográficas foi realizada pelo método da Área Corrigida da Fração. A atividade inibitória da ECA (AI-ECA) foi determinada por CLAE de fase reversa. Todos os parâmetros testados afetaram tanto o perfil peptídico quanto a AI-ECA. O melhor perfil peptídico foi atingido para os hidrolisados obtidos com papaína, enquanto a pancreatina foi mais vantajosa em termos da AI-ECA. O efeito benéfico do uso de menor relação E:S sobre o perfil peptídico e a AI-ECA foi observado para ambas as enzimas dependendo das condições usadas para o preparo dos hidrolisados. O efeito benéfico da ausência da UF sobre o perfil peptídico foi observado em alguns casos para pancreatina e papaína. No entanto, a ausência da UF produziu maior AI-ECA somente quando a papaína foi usada.

Summary The action of various proteases was tested for preparing whey protein concentrate (WPC) hydrolysates with high degree of hydrolysis (DH), appropriate peptide profiles and reduced phenylalanine (Phe) content. The peptide profile analysis included the fractionation of hydrolysates by size-exclusion HPLC. The rapid correct fraction area method was used to quantify the components of the chromatographic fractions. Activated carbon (AC) was used to remove Phe, and its efficiency was evaluated by measuring the amount of Phe by second-derivative spectrophotometry. The results showed that the DH of WPC hydrolysates increased and that the protease from Aspergillus oryzae yielded the highest DH value. This protease also produced the best peptide profile, that is, the highest di- and tripeptide content (16.14%), the highest amounts of free amino acids (18.43%) and the lowest amount of large peptides (18.76%). The proteases from both A. oryzae and Bacillus subtilis produced the highest Phe removals (79.0 and 77.8%, respectively).

Summary The action of proteases from Bacillus licheniformis and Aspergillus oryzae was studied with the aim of preparing hydrolysates from whey protein concentrate with nutritionally appropriate peptide profile. Various enzyme/substrate ratios were used, and the peptides were fractionated by size-exclusion HPLC followed by their quantification using the rapid method of correct fraction area (CFA). The protease from B. licheniformis (E:S of 8:100) produced the best peptide with a much lower amount of large peptides (44.61%), greater of di- and tripeptides (8.79%) and higher sum of the di- and tripeptides with free amino acids (9.99%) than the other hydrolysates. The advantage of using a lower E:S ratio to obtain a nutritionally adequate peptide profile was observed for the protease of A. oryzae when it passed from 3:100 to 2:100.

Optimization of enzymatic protein extraction from beans. Considering the importance of beans in the Brazilian diet and the fact that it is forbidden in phenylketonuric feeding, it is relevant to promote the extraction of its proteins, for phenylalanine removal. Aiming for the optimization of protein extraction from beans, two enzymatic methods were initially employed. For the one that produced the highest protein extract yield (PEY), a study was conducted to assess de effect of several parameters, such as centrifugation velocity, reaction time, initial pH and type of enzyme (one of Bacillus licheniformis, two of Bacillus subtilis, one of Aspergillus sojae and one of Papaya carica). The results showed that the best method was the one that employed a concentration of raw material of 1:10 (w v -1 ), initial pH of 10.5, temperature of 50°C, in the absence of ultra- turrax treatment. With regard to the parameters tested, the largest PEY (93.1%) was obtained by using the enzyme from Bacillus licheniformis, the centrifugation velocity of 10,640 x g, the reaction time of 3h and initial pH of 10.5.

Considerando a importância do leite na dieta do brasileiro e a sua restrição para pacientes fenilcetonúricos, este trabalho teve como objetivo reduzir o teor de fenilalanina (Phe) deste alimento. Para isso, vários hidrolisados enzimáticos foram preparados, empregando uma protease do Bacillus subtilis e testando diversos parâmetros de reação, como o efeito do tempo, da temperatura e da relação E:S. A remoção da Phe foi realizada empregando o carvão ativado (CA) como meio adsorvente, e avaliou-se as relações proteína:CA de 1:22, 1:44 e 1:88. A efi ciência da remoção foi avaliada por espectrofotometria derivada segunda, determinando-se o teor de Phe livre no leite e em seus hidrolisados, após tratamento com CA. A maior remoção de Phe (75,9%) foi obtida com o tempo de 5h, a temperatura de 50ºC, a relação E:S de 4:100 e a relação proteína:CA de 1:22. Este ensaio originou um leite com teor fi nal de 36,9mg de Phe/100ml, o que permitiria a utilização do mesmo por pacientes fenilcetonúricos.

Visando o preparo de leite com baixo teor de fenilalanina (Phe), para ser introduzido na dieta de fenilcetonúricos, inicialmente as proteínas foram hidrolisadas pela ação de uma protease do "Aspergillus sojae", tendo sido testados o efeito da relação E:S, da temperatura e do tempo de reação. Posteriormente, os hidrolisados protéicos foram tratados com carvão ativado (CA) para remoção de Phe, empregando-se as relações proteína:CA de 1:22, 1:44 e 1:88. A eficiência da remoção foi avaliada por espectrofotometria derivada segunda, determinando-se o teor de Phe livre no leite e em seus hidrolisados, após tratamento com CA. Dentre todos os parâmetros estudados, o melhor resultado foi obtido ao se empregar o tempo de 3h, a temperatura de 50ºC, a relação E:S de 4:100 e a relação proteína:CA de 1:22, tendo atingido 64,6% de remoção e o teor final de Phe de 54,2mg/100 mL de leite, permitindo assim a utilização deste alimento por pacientes fenilcetonúricos.

A fenilcetonúria (PKU) é uma desordem metabólica congênita na qual os indivíduosnão tratados ainda nos primeiros meses de vida desenvolvem retardo mental irreversível, sendo o leite proibido na alimentação destes pacientes. Visando o desenvolvimento de leite com teor reduzido de Phe, para ser utilizado por pacientes fenilcetonúricos, foram testadas 32 condições de hidrólise enzimática das proteínas,empregando-se, separadamente, uma protease do Aspergillus sojae e uma subtilisina, como também, alguns parâmetros de remoção de Phe, usando o carvão ativado (CA) como meio adsorvente. Foram avaliados os efeitos do tempo e da temperatura de reação, assim como da relação enzima:substrato (E:S) e da relação proteína:CA, visando a redução dos custos e a adaptação do processo em larga escala. Após passagem do leite contendo proteínas hidrolisadas por coluna de CA, a Phe foi dosadapor espectrofotometria derivada segunda (EDS). Desta forma, empregando-se a protease do A. sojae, o melhor resultado foi obtido ao se utilizar o tempo de reação de 3h, a temperatura de 50 ºC, a relação E:S de 4:100 e a relação proteína:CA de 1:22, tendo atingido 64,6% de remoção e o teor final de Phe de 54,2mg de Phe/100 ml de leite. Já para a subtilisina, os parâmetros estudados que levaram ao maior teor deremoção de Phe foram quase todos os mesmos observados para a protease do A. sojae, exceto o tempo de reação, que foi de 5h, obtendo-se 75,9% de remoção, o que equivale a um teor final de 36,9mg de Phe/100 ml. Conclui-se, portanto, que as duas enzimas testadas, assim como as condições avaliadas, foram eficientes na obtençãode leite com teor reduzido de Phe, situando-se abaixo do limite máximo permitido pela legislação brasileira. Phenylketonuria (PKU) is a metabolic disease in which the untreated patients can show irreversible mental retardation, and the use of milk by phenylketonurics patients is forbidden . Aiming the development of milk with reduced Phe content to be used by phenylketonurics patients with no restriction, 32 conditions of enzymatic hydrolysis were tested employing, separately, a protease from Aspergillus sojae and a subtilisin, as well as some parameters of Phe removal were also evaluated, employing activated carbon (AC) as adsorbent support. The effects of reaction time and temperature, as well as of the enzyme:substrate ratio (E:S) and protein:activated carbon ratio were evaluated, aiming cost reduction and adjustment of the process for the scaling-up. After passing the milk containing hydrolyzed proteins through an AC column, the Phe content was evaluated by second derivative spectrophotometry (SDS). Thus, using the protease from A. sojae,the best result was obtained by using a reaction time of 3h, a temperature of 50 oC, an E:S ratio of 4:100 and a protein:AC ratio of 1:22, reaching 64.6% of Phe removal and a final content of 54.2mg/100 ml of milk. For subtilisin, the studied parameters that led to the highest Phe removal were almost the same as those observed for the A. sojae protease, except for the reaction time, which was 5h, obtaining 75.9% of removal which corresponds to a final Phe content of 36.9mg /100 ml. It can be inferred that both tested enzymes and the evaluated conditions showed to be efficient in obtaining milk with reduced Phe content, which is bellow the maximum limit stated by the Brazilian legislation.