In vitro versus in vivo protein digestibility techniques for calculating PDCAAS (protein digestibility-corrected amino acid score) applied to chickpea fractions.

Seven different in vitro methods to determine the protein digestibility for chickpea proteins were considered and also the application of these methodologies for calculating PDCAAS (protein digestibility-corrected amino acid score), seeking their correlations with the in vivo methodology. In vitro digestibility of raw and heated samples were determined using pepsin-pancreatin hydrolysis, considering soluble nitrogen via Kjeldahl (ppKJ) and hydrolysed peptide linkages using trinitrobenzenesulfonic acid and o-phthaldialdehyde. In vitro digestibility was also determined using trypsin, chymotrypsin and peptidase (3-Enz) or trypsin, chymotrypsin, peptidase and pronase solution (4-Enz). None of the correlations between in vitro and in vivo digestibilities were significant (at p<0.0500), but, strong correlations were observed between PDCAAS calculated by in vitro and in vivo results. PDCAAS-ppKJ, PDCAAS-3-Enz and PDCAAS-4-Enz presented the highest correlations with in vivo method, r=0.9316, 0.9442 and 0.9649 (p<0.0500), respectively. The use of in vitro methods for calculating PDCAAS may be promising and deserves more discussions.

Chemical characteristics and fractionation of proteins from Moringa oleifera Lam. leaves.

Moringa oleifera Lam. is a leguminous plant, originally from Asia, which is cultivated in Brazil because of its low production cost. Although some people have used this plant as food, there is little information about its chemical and nutritional characteristics. The objective of this study was to characterise the leaves of M. oleifera in terms of their chemical composition, protein fractions obtained by solubility in different systems and also to assess their nutritional quality and presence of bioactive substances. The whole leaf flour contained 28.7% crude protein, 7.1% fat, 10.9% ashes, 44.4% carbohydrate and 3.0mg 100g(-1) calcium and 103.1mg 100g(-1) iron. The protein profile revealed levels of 3.1% albumin, 0.3% globulins, 2.2% prolamin, 3.5% glutelin and 70.1% insoluble proteins. The hydrolysis of the protein from leaf flour employing sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (ME) resulted in 39.5% and 29.5%, respectively. The total protein showed low in vitro digestibility (31.8%). The antinutritional substances tested were tannins (20.7 mg g(-1)), trypsin inhibitor (1.45TIU mg g(-1)), nitrate (17 mg g(-1)) and oxalic acid (10.5 mg g(-1)), besides the absence of cyanogenic compounds. ß-Carotene and lutein stood out as major carotenoids, with concentrations of 161.0 and 47.0 µg g(-1) leaf, respectively. Although M. oleifera leaves contain considerable amount of crude protein, this is mostly insoluble and has low in vitro digestibility, even after heat treatment and chemical attack. In vivo studies are needed to better assess the use of this leaf as a protein source in human feed.

Caracterização de hambúrguer elaborado com farinha de folhas de Moringa (Moringa oleífera Lam. ) / Characterization of beef burger made with Moringa (Moringa oleífera Lam. ) leaves flour

The aim of this study was to determine the antioxidant activity of Moringa (Moringa oleifera Lam.) leaves flour in beef burger during storage for 120 days. Six hamburger formulations were processed: one control (without the use of additives), four with addition of Moringa leaves flour (0.10, 0.15, 0.20, and 0.25 g/100 g aggregate), and one with addition of synthetic antioxidant Propyl Gallate (0.01 g/100 g aggregate). The products were analyzed for their chemical composition with determinations of moisture, protein, dietary fiber, lipids, ash, carbohydrate, and caloric value after preparation. Microbiological and acceptance testing were performed at the beginning and after 120 days of storage. Determination of pH, instrumental color and lipid oxidation (TBARS) were performed at 1, 30, 60, 90 and 120 days of storage. All samples showed physical-chemical and microbiological tests in accordance with the Brazilian legislation. pH measurements were between 5.48 and 5.90; however, the intensity of red has changed according to the treatments and storage periods. The addition of Moringa leaves flour had no antioxidant effect on burgers, but its inclusion not only contributed to the improvement of nutritional quality, but also did not harm product acceptance.

El objetivo de este trabajo fue verificar la acción antioxidante de una harina de hojas de Moringa (Moringa oleífera Lam.) en carne de hamburguesa bovina durante su almacenamiento por 120 días. Se procesaron seis formulaciones de hamburguesa: una de control, sin uso de aditivos, cuatro adicionadas con harina de hojas de Moringa (0,10; 0,15; 0,20 y 0,25 g/100 g de masa) y una adicionada con antioxidante sintético Propil Galato (0,01 g/100 g de masa). Los productos se analizaron, en cuanto a su composición química, con determinaciones de humedad, proteína, fibra alimentaria, lípidos, cenizas, carbohidratos y valor calórico, y, tras la preparación, análisis microbiológicos y pruebas de aceptación, realizados tanto al inicio como al cabo de 120 días de almacenamiento. También se analizaron, con determinación de pH, Color instrumental y oxidación lipídica (TBARS), a los 1, 30, 60, 90 y 120 días de almacenamiento, respectivamente. Todas las muestras presentaron características físico-químicas y microbiológicas de acuerdo con la legislación nacional vigente. Los valores de pH oscilaron entre 5,48 y 5,90 y solamente la intensidad del rojo sufrió modificaciones debido a los tratamientos y periodos de almacenamiento. La adición de harina de hojas de Moringa no ejerció efecto antioxidante en las hamburguesas, sin embargo su inclusión contribuyó para mejorar la calidad nutricional y no perjudicó la aceptación del producto.

O objetivo deste trabalho foi verificar a ação antioxidante de uma farinha de folhas de Moringa (Moringa oleífera Lam.) em produto hambúrguer bovino durante estocagem por 120 dias. Foram processadas seis formulações de hambúrguer, sendo uma controle, sem o uso de aditivos, quatro adicionadas de farinha de folhas de Moringa (0,10; 0,15; 0,20 e 0,25 g/100 g de massa) e uma com adição de antioxidante sintético Propil Galato (0,01 g/100 g de massa). Os produtos foram analisados quanto à composição química, com determinações de umidade, pro¬teína, fibra alimentar, lipídeos, cinzas, carboidratos e valor calórico após o preparo, além de análises microbiológicas e teste de aceitação realizados no início e aos 120 dias de armazenamento, e determinação de pH, cor instrumental e oxidação lipídica (TBARS), realizadas com: 1, 30, 60, 90 e 120 dias de armazenamento. Todas as amostras apresentaram características físico-químicas e microbiológicas de acordo com a legislação nacional vigente. As medidas de pH ficaram compreendidas entre 5,48 e 5,90, e somente a intensidade de vermelho sofreu alterações de acordo com os tratamentos e períodos de estocagem. A adição de farinha das folhas de Moringa não exerceu efeito antioxidante nos hambúrgueres, porém sua inclusão contribuiu para a melhoria da qualidade nutricional e não prejudicou a aceitação do produto.

Partial characterization of proteins from baru (Dipteryx alata Vog) seeds.

BACKGROUND: Baru (Dipteryx alata Vog.) is a fruit distributed throughout the Brazilian savanna and contains a seed with a high protein content, whose properties have been rarely explored. The purpose of this study was to characterize this protein, especially by isolation and quantifying its fractions and measuring some of its molecular properties. RESULTS: Baru seeds contain 244 g kg(-1) protein on a dry weight basis. Solubility profiles showed a preponderance of globulins. This fraction dominated the seed composition, with 61.7 wt% of the total soluble proteins. Albumins and glutelins accounted for 14 and 3.3 wt%, respectively. SDS-PAGE resolution of albumin and globulin showed main bands with molecular weights of 84 kDa and 64, 66 and 73 kDa, respectively. The total protein of the flour and the globulin showed values of in vitro digestibility of 85.59% and 90.54%, relative to casein. Total globulin produced only one chromatographic peak, both on Sepharose CL-6B gel filtration and on DEAE-cellulose ion-exchange columns, eluted at a concentration of 0.12 mol L(-1) NaCl. CONCLUSION: The baru seed had high protein content with large quantities of storage proteins. The chromatographic and solubility profiles indicate the predominance of a fraction with characteristics of a legumin-type protein.

Soy ß-conglycinin (7S globulin) reduces plasma and liver cholesterol in rats fed hypercholesterolemic diet.

The aim of this study was to examine the comparative hypocholesterolemic effect of soybean 7S fraction in rats fed a high-cholesterol diet. Soybean 7S globulin (ß-conglycinin) was administered orally once a day to rats, and the effects were measured after 28 days. Wistar rats were divided into four groups: standard diet (STD) (casein alone), hypercholesterolemic (HC) diet (STD plus 1 g/100 g cholesterol and 0.5 g/100 g cholic acid), HC+7S(1) diet (HC diet plus 200 mg of 7S/kg of body weight/day), and HC+7S(2) diet (HC diet plus 300 mg of 7S/kg of body weight/day). Food intake, weight gain, animals' growth, and feeding efficiency ratio were similar among the STD and three HC groups, indicating that these parameters were not affected by treatments. Animals that had received different doses of soybean 7S globulin had lower total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL)/high-density lipoprotein (HDL) ratio in serum and lower levels of hepatic TC and TG than those fed only the HC diet. The atherogenic indexes of HC+7S(1) and HC+7S(2) groups were 40% and 55% lower than that of the HC group, respectively. The results showed that the oral daily administration of ß-conglycinin in the diet to HC rats, at between 1.85% and 2.75% of total ingested protein, promotes the reduction of TC, LDL-cholesterol, and TG and an increase in HDL-cholesterol in the plasma, besides a small but significant reduction in cholesterol and TG levels in the liver of the animals as well as a reduced atherogenic index.

Some Biochemical properties of polyphenoloxidase from spearmint (Mentha arvensis)

Polyphenoloxidase (PPO; EC 1.14.18.1) extracted from Mentha arvensis leaves was isolated by (NH4)2SO4 precipitation and extensive dialysis. Its optimum pH and temperature varied with the substrate. The PPO showed activity with various diphenols. Km values were found 0.825, 0.928 and 7.41mM for caffeic acid, 4-methylcatechol and catechol, respectively. On heat-inactivation, half of the activity was lost after 60 and 15 sec at 70 and 75ºC, respectively. Measuring of residual activity showed a stabilizing effect of sucrose at various temperatures with activation energy (Ea) for inactivation increasing with sucrose concentration from 0 to 40 percent (w/w). Ea values of 78.13; 80.37; 82.79 and 81.00 kJ/Mol were found for 0, 15; 30 and 40 percent sucrose, respectively. PPO was inhibited by ascorbic, benzoic, cinnamic, ferulic, p-coumaric, protocatechuich acids, sodium metabisulfite, pyrogallol and resorcinol. The Ki values showed that ascorbic acid was the most effective inhibitor. The type inhibition was determined for each inhibitor.

Polifenoloxidase (PPO, EC 1.14.18.1) extraída de folhas de Mentha arvensis foi isolada por precipitação com (NH4)2SO4 e diálise extensiva. Seu pH e temperatura ótimos variaram com o substrato. A PPO apresentou atividade com vários difenóis. Valores de Km foram 0,825; 0,928 e 7,41 mM para ácido caféico, 4-metilcatecol e catecol, respectivamente. Na inativação térmica, 50 por cento da enzima foi inativada após 60 e 15 segundos a 70 e 75ºC, respectivamente. A medida de atividade residual mostrou um efeito estabilizante de sacarose a várias temperaturas e uma energia de ativação (Ea) para inativação aumentando com a concentração de sacarose de 0 a 40 por cento (p/p). Valores de energias de ativação de 78,13; 80,37; 82,79 and 81,00 kJ/Mol foram encontradas para 0, 15, 30 e 40 por cento de sacarose, respectivamente. A PPO foi inibida pelos ácidos ascórbico, benzóico, cinamico, ferulico, p-cumárico, protocatéquico, além de metabisulfito de sódio, resorcinol e pirogalol. Os valores de Ki mostram que o ácido ascórbico foi o mais efetivo inibidor. O tipo de inibição foi determinado para cada inibidor.

Enzymatic hydrolysis of sweet lupin, chickpea, and lentil 11S globulins decreases their antigenic activity.

We investigated the effects of treatments with the enzymes pepsin and trypsin on the in vitro immunological reactivity of the major globulins found in the seeds of sweet lupin, chickpea, and lentil. Polyclonal major globulin-specific antiserum was obtained by immunization of rabbits with a solution of the 11S globulin of each legume. The globulins were hydrolyzed with pepsin and trypsin for 1, 5, 15, and 30 min. The native globulins and their hydrolysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to identify the polypeptide bands with antigenic activity, and the hypoantigenicity of the hydrolysates was analyzed by enzyme-linked immunosorbent assay. Our results show that enzymatic treatment of the major storage protein (11S globulin) of sweet lupin, chickpea, and lentil with pepsin or trypsin lead to the formation of large amounts of short peptides and free amino acids that do not allow antibody binding, resulting in a weakened immunoreactivity.

Nutritional responses of rats to diets based on chickpea ( Cicer arietinum L.) seed meal or its protein fractions.

The aim of this study was to isolate the protein fractions from chickpea, var. IAC-Marrocos, as well as to evaluate its in vivo nutritional protein quality. Among the proteins, albumins showed better nutritional value in the in vivo assays and amino acid contents, despite their higher trypsin inhibitor contents. Trypsin inhibitors were found to be heat labile in all samples, but the digestibility results for unheated and heated flour and albumins suggest that their contents are not very decisive. The PER values for casein (not supplemented) were very similar to those of heated flour and unheated or heated albumin and total globulins. The albumin and glutelin fractions showed the best results for PDCAAS, however, lower than those of casein. Despite the high digestibility of the globulin the very low essential amino acid content lowered its PDCAAS, and it had the lowest values.

Polyphenol oxidase from yacon roots (Smallanthus sonchifolius).

Polyphenol oxidase (E.C. 1.14.18.1) (PPO) extracted from yacon roots (Smallanthus sonchifolius) was partially purified by ammonium sulfate fractionation and separation on Sephadex G-100. The enzyme had a molecular weight of 45 490+/-3500 Da and Km values of 0.23, 1.14, 1.34, and 5.0 mM for the substrates caffeic acid, chlorogenic acid, 4-methylcatechol, and catechol, respectively. When assayed with resorcinol, DL-DOPA, pyrogallol, protocatechuic, p-coumaric, ferulic, and cinnamic acids, catechin, and quercetin, the PPO showed no activity. The optimum pH varied from 5.0 to 6.6, depending on substrate. PPO activity was inhibited by various phenolic and nonphenolic compounds. p-Coumaric and cinnamic acids showed competitive inhibition, with Ki values of 0.017 and 0.011 mM, respectively, using chlorogenic acid as substrate. Heat inactivation from 60 to 90 degrees C showed the enzyme to be relatively stable at 60-70 degrees C, with progressive inactivation when incubated at 80 and 90 degrees C. The Ea (apparent activation energy) for inactivation was 93.69 kJ mol-1. Sucrose, maltose, glucose, fructose, and trehalose at high concentrations appeared to protect yacon PPO against thermal inactivation at 75 and 80 degrees C.

Changes in protein fractions, trypsin inhibitor and proteolytic activity in the cotyledons of germinating chickpea

The chickpea seed germination was carried out in 6 days. During the period it was observed a little variation on total nitrogen contents, however the non protein nitrogen was double. A decrease of 19.1 and 20.6 in relation to total nitrogen was observed to the total globulin and albumin fractions, respectively. The gel filtration chromatography on Sepharose CL-6B and SDS-PAGE demonstrated alterations on the distribution patterns of the albumin and total globulin fractions between the initial and the sixth day of germination suggesting the occurrence of protein degradation in the germination process. The assay for acid protease only appeared in the albumin fraction with casein and chickpea total globulin as substrates, whereas the former was more degraded than the latter, however the transformations detected in the protein fractions appear indicated that others enzymes could be acting during the process. The trypsin inhibitor activity had a little drop after six day of germination indicating a possible increase on the digestibility of the proteins.

Peroxidase from peach fruit: thermal stability

Peroxidase from peach fruit was purified 28.9 fold by DEAE-cellulose, Sephadex G-100 and hydroxylapatite chromatography. The purified enzyme showed only one peak of activity with an optimum pH of 5.0 and temperature of 40§C. The calculated activation energy (Ea) for the reaction was 7.97 kcal/mol. The enzyme was heat-labile in the temperature range of 60 to 80§C with a fast inactivation at 80§C. PAGE of the inactivation course at 70§C showed only one band of activity. Different sugars increased the heat stability of the activity in the following order: sucrose>glucose>fructose. Measurement of residual activity showed a stabilizing effect of sucrose at various temperature/sugar concentrations (10 to 40 per cent, w/w) with the Ea for inactivation with sucrose concentration from 0 to 20 per cent (w/w). After inactivation at 70§C the enzyme was able to be reactivated by up to 40 per cent of the initial activity when stored at 30§C