Search

The chemical composition, and antioxidant, antimicrobial and anti-proliferative activities of the essential oils of Ammoides pusilla (Apiaceae) and Thymus munbyanus (Lamiaceae), collected in Algeria, were studied. Cumin alcohol (44%), p-cymene (18%), limonene (14%), thymol (11%) and γ-terpinene (7%) predominated in A. pusilla essential oil, whereas carvacrol (71%) was dominant in T. munbyanus essential oil. The antibacterial activity of A. pusilla and T. munbyanus essential oils was studied by the agar diffusion method against Escherichia coli, Salmonella enterica serovar Thyphimurium, methicillin-resistant Staphylococcus aureus and Listeria monocytogenes. The tested bacteria were more susceptible to T. munbyanus essential oil. Ammoides pusilla essential oil was the most effective for scavenging the free radicals. Ammoides pusilla essential oil also had the best reducing power as well as the highest lipid peroxidation inhibition. Thymus munbyanus essential oil showed the best anti-proliferative activity of human acute monocytic leukemia cell line (THP-1). [ABSTRACT FROM AUTHOR]

Erophaca baetica is a legume endemic to the Mediterranean region. Although the fruits and seeds are large, the presence of the “locoism" which produces the alkaloid, swainsonine has prevented its use as animal feed or for human nutrition. Their protein content and chromatographic profile, amino acid composition, fatty acid composition, and polyphenol contents have been determined in order to explore the potential of the E. baetica seeds as a source of dietary protein with functional components. The protein content was found to be 36% (w/w), and an amino acid analysis revealed a deficiency in sulphur amino acids, tryptophane, and lysine. The low lysine content is probably due to the abundance of alkaloids metabolically derived from this amino acid. Oleic and linoleic acids are the major fatty acids in the seeds. The antioxidant activity of polyphenol extracts was higher than the activity of the polyphenols extracted from most edible legume seeds. Henee, E. baetica seeds represent a promising source of functional and nutritional components on the condition that the anti-nutritional alkaloids are previously removed. [ABSTRACT FROM AUTHOR]

The protein profile and the amino acid composition of eleven amaranth species have been studied. The following species were taken into account: A. viridis, A. powellii, A. muricatus, A. deflexus, A. graecizans, A. blitoides, A. retroflexus, A. blitum, A. albus, A. cruentus and A. hypochondriacus. Seed samples were obtained from wild populations located in the southwest of Spain. The protein profile was studied by gel filtration chromatography and denaturing electrophoresis. Profiles were similar in all taxa, with small variations in the molecular weights and amounts of the main seed proteins. Thus, after gel filtration chromatography six main fractions of around 300 kDa, 180 kDa, 120 kDa, between 40 and 50 kDa, 20 and 30 kDa and below 10 kDa were observed. On the other hand, the electrophoretic analysis showed peptides grouped into three main fractions, between 50 and 64 kDa, 33 and 37 kDa and 18 and 25 kda. The most balanced amino acid compositions were observed in the wild taxa A. muricatus, A. blitum and A. powellii showed the most equilibrated amino acid composition. A. hypochondriacus and A. graecizans showed the most deficient amino acid composition with limitations in five essential amino acids. These results show the potential of wild amaranthus taxa for their introduction as crops or their use in the improvement by hybridization mechanisms of other crops such as A. hypochondriacus. [ABSTRACT FROM AUTHOR]

Pharmaceuticals and pesticides can be considered hazardous compounds for Mediterranean coastal wetland ecosystems. Although many of these compounds co-occur in environmental samples, only a few studies have been dedicated to assessing the ecotoxicological risks of complex contaminant mixtures. We evaluated the occurrence of 133 pharmaceuticals and pesticides in 12 sites in a protected Mediterranean wetland, the Albufera Natural Park (ANP), based on conventional grab sampling and polar organic chemical integrative samplers (POCIS). We assessed acute and chronic ecological risks posed by these contaminant mixtures using the multi-substance Potentially Affected Fraction (msPAF) approach and investigated the capacity of a constructed wetland to reduce chemical exposure and risks. This study shows that pharmaceuticals and pesticides are widespread contaminants in the ANP, with samples containing up to 75 different compounds. POCIS samplers were found to be useful for the determination of less predictable exposure profiles of pesticides occurring at the end of the rice cultivation cycle, while POCIS and grab samples provide an accurate method to determine (semi-)continuous pharmaceutical exposure. Acute risks were identified in one sample, while chronic risks were determined in most of the collected samples, with 5-25% of aquatic species being potentially affected. The compounds that contributed to the chronic risks were azoxystrobin, ibuprofen, furosemide, caffeine, and some insecticides (diazinon, imidacloprid, and acetamiprid). The evaluated constructed wetland reduced contaminant loads by 45-73% and reduced the faction of species affected from 25 to 6%. Our study highlights the need of addressing contaminant mixture effects in Mediterranean wetlands and supports the use of constructed wetlands to reduce contaminant loads and risks in areas with high anthropogenic pressure., (© 2024. The Author(s).)

Bayesian network (BN) models are increasingly used as tools to support probabilistic environmental risk assessments (ERAs), because they can better account for uncertainty compared with the simpler approaches commonly used in traditional ERA. We used BNs as metamodels to link various sources of information in a probabilistic framework, to predict the risk of pesticides to aquatic communities under given scenarios. The research focused on rice fields surrounding the Albufera Natural Park (Valencia, Spain), and considered three selected pesticides: acetamiprid (an insecticide), 2-methyl-4-chlorophenoxyacetic acid (MCPA; a herbicide), and azoxystrobin (a fungicide). The developed BN linked the inputs and outputs of two pesticide models: a process-based exposure model (Rice Water Quality [RICEWQ]), and a probabilistic effects model (Predicts the Ecological Risk of Pesticides [PERPEST]) using case-based reasoning with data from microcosm and mesocosm experiments. The model characterized risk at three levels in a hierarchy: biological endpoints (e.g., molluscs, zooplankton, insects, etc.), endpoint groups (plants, invertebrates, vertebrates, and community processes), and community. The pesticide risk to a biological endpoint was characterized as the probability of an effect for a given pesticide concentration interval. The risk to an endpoint group was calculated as the joint probability of effect on any of the endpoints in the group. Likewise, community-level risk was calculated as the joint probability of any of the endpoint groups being affected. This approach enabled comparison of risk to endpoint groups across different pesticide types. For example, in a scenario for the year 2050, the predicted risk of the insecticide to the community (40% probability of effect) was dominated by the risk to invertebrates (36% risk). In contrast, herbicide-related risk to the community (63%) resulted from risk to both plants (35%) and invertebrates (38%); the latter might represent (in the present study) indirect effects of toxicity through the food chain. This novel approach combines the quantification of spatial variability of exposure with probabilistic risk prediction for different components of aquatic ecosystems. Environ Toxicol Chem 2024;43:182-196. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Pollution by agricultural pesticides is one of the most important pressures affecting Mediterranean coastal wetlands. Pesticide risks are expected to be influenced by climate change, which will result in an increase of temperatures and a decrease in annual precipitation. On the other hand, pesticide dosages are expected to change given the increase in pest resistance and the implementation of environmental policies like the European ´Farm-to-Fork` strategy, which aims for a 50 % reduction in pesticide usage by 2030. The influence of climate change and pesticide use practices on the ecological risks of pesticides needs to be evaluated making use of realistic environmental scenarios. This study investigates how different climate change and pesticide use practices affect the ecological risks of pesticides in the Albufera Natural Park (Valencia, Spain), a protected Mediterranean coastal wetland. We performed a probabilistic risk assessment for nine pesticides applied in rice production using three climatic scenarios (for the years 2008, 2050 and 2100), three pesticide dosage regimes (the recommended dose, and 50 % increase and 50 % decrease), and their combinations. The scenarios were used to simulate pesticide exposure concentrations in the water column of the rice paddies using the RICEWQ model. Pesticide effects were characterized using acute and chronic Species Sensitivity Distributions built with toxicity data for aquatic organisms. Risk quotients were calculated as probability distributions making use of Bayesian networks. Our results show that future climate projections will influence exposure concentrations for some of the studied pesticides, yielding higher dissipation and lower exposure in scenarios dominated by an increase of temperatures, and higher exposure peaks in scenarios where heavy precipitation events occur right after pesticide application. Our case study shows that pesticides such as azoxystrobin, difenoconazole and MCPA are posing unacceptable ecological risks for aquatic organisms, and that the implementation of the ´Farm-to-Fork` strategy is crucial to reduce them., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Mediterranean coastal wetlands are considered biodiversity hot-spots and contain a high number of endemic species. The biodiversity of these ecosystems is endangered by several pressures resulting from agricultural and urban expansion, climate change, and the alteration of their hydrological cycle. In this study we assess the state-of-the-art regarding the impact of several stressor groups on the biodiversity of Mediterranean coastal wetlands (i.e., lagoons, marshes, estuaries). Particularly, we describe the impacts of eutrophication, chemical pollution, invasive species, salinization, and temperature rise, and analyze the existing literature regarding the impact of multiple stressors on these ecosystems. Our study denotes a clear asymmetry both in terms of study areas and stressors evaluated. The majority of studies focus on lagoons and estuaries of the north-west parts of the Mediterranean basin, while the African and the Asian coast have been less represented. Eutrophication and chemical pollution were the most studied stressors compared to others like temperature rise or species invasions. Most studies evaluating these stressors individually show direct or indirect effects on the biodiversity of primary producers and invertebrate communities, and changes in species dominance patterns that contribute to a decline of endemic populations. The few available studies addressing stressor interactions have shown non-additive responses, which are important to define appropriate ecosystem management and restoration measures. Finally, we propose research needs to advance our understanding on the impacts of anthropogenic stressors on Mediterranean coastal wetlands and to guide future interventions to protect biodiversity., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

In this paper, the surface area of coated polymer-based textiles, i.e., copper and nickel plated woven polyester fabric, copper and acrylic coated woven polyester fabric, and copper and acrylic coated non-woven polyamide fabric, is investigated. In order to evaluate the surface area of the woven fabrics, Peirce's geometrical model of the interlacing point and measurement using an electron microscope are used. Non-woven fabrics are evaluated using an optical method, handmade method, and MATLAB functions. An electrochemical method, based on the measurement of the resistance between two electrodes, is used for relative comparison of the effective surface area of the coated woven and non-woven fabrics. The experimental results show that the measured and calculated warp lengths do not differ within the standard deviation. The model for the surface area evaluation of the Pierce's geometrical model for monofilament (non-fibrous) yarns is extended to multifilament yarns and to a uniform sample size. The experimental results show the increasing trend of surface area evaluation using both modeling and electrochemical methods, i.e., the surface area of the copper and acrylic coated woven Polyester fabric (PES) is the smallest surface area of investigated samples, followed by the surface area of the copper and acrylic coated non-woven fabric, and by copper and nickel plated woven PES fabric. These methods can be used for surface area evaluation of coated polymer-based textiles in the development of supercapacitors, electrochemical cells, or electrochemical catalysts.

Background: Essential oils (EOs) have shown antimicrobial, antioxidant, and antiproliferative activity, which may, alone or in combination with other substances, potentially be used for the development of new drugs. However, their chemical variability, depending on the species, varieties, or geographical origin (among other factors) determines different bioactivities that need to be evaluated. Methods: The antioxidant activity of Corymbia citriodora and eight Eucalyptus species EOs was determined using two different methods: the scavenging ability of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS +• ) and peroxyl free radicals or oxygen radical absorbance capacity (ORAC). Antibacterial activity was evaluated using the microorganisms Streptococcus pneumoniae (strains D39 and TIGR4), and Haemophilus influenza (strain DSM 9999). The essential oils’ minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was assessed using a microdilution method. The antiproliferative activity was determined using the THP-1 cell line (human acute monocytic leukaemia) with methylthiazolyldiphenyl-tetrazolium bromide assay (MTT). Results: Corymbia citriodora and Eucalyptus viminalis EOs showed the highest ABTS and peroxyl free radical scavenging capacity. Eucalyptus globulus EO showed a high potential to treat Streptococcus pneumoniae infections. Haemophilus influenzae was the respiratory pathogen that showed the highest resistance to all EOs, including tea tree EO. After 96 h of incubation, at 25 μg/mL, Eucalyptus radiata and Eucalyptus viminalis EOs showed highest cytotoxic activity against the THP-1 cell line. Conclusions: Despite their specific bioactivities, no single EO showed simultaneously good antioxidant, antimicrobial, and antiproliferative activity.

Chickpea is a grain legume widely consumed in the Mediterranean region and other parts of the world. Chickpea seeds are rich in proteins but they also contain a substantial amount of free amino acids, especially arginine. Hence chickpea may represent a useful source of free amino acids for nutritional or pharmaceutical purposes. Arginine is receiving great attention in recent years because it is the substrate for the synthesis of nitric oxide, an important signaling molecule involved in numerous physiological and pathological processes in mammals. In this work we describe a simple procedure for the purification of arginine from chickpea seeds, using nanofiltration technology and an ion-exchange resin, Amberlite IR-120. Arginine was finally purified by precipitation or crystallization, yielding preparations with purities of 91% and 100%, respectively. Chickpea may represent an affordable green source of arginine, and a useful alternative to production by fermentation or protein hydrolysis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The role of the peptides-phenolic compounds (PC) interaction on the antioxidant capacity profile (ACP) of protein hydrolysates from rapeseed (Brassica napus) was studied in 36 hydrolysates obtained from a PC-rich and PC-reduced protein substrate. The latent profile analysis (LPA), with data of seven in vitro methods and one assay for cellular antioxidant activity (CAA), allowed identifying five distinctive groups of hydrolysates, each one with distinctive ACP. The interaction of peptides with naturally present PC diminished in vitro antioxidant activity in comparison with their PC-reduced counterparts. However, CAA increased when peptides-PC interaction occurred. The profile with the highest average CAA (62.41 ± 1.48%), shown by hydrolysates obtained by using alcalase, shared typical values of Cu(2+)-catalysed β-carotene oxidation (62.41 ± 0.43%), β-carotene bleaching inhibition (91.75 ± 0.22%) and Cu(2+)-chelating activity (74.53 ± 0.58%). The possibilities for a sample to exhibit ACP with higher CAA increased with each unit of positively charged amino acids, according to multinomial logistic regression analysis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A method for determination of the non-protein amino acid l-α-amino-γ-(guanidinooxy)-n-butyric acid (L-canavanine) and other free amino acids in Vicia disperma is presented. Seed extracts were derivatized by reaction with diethyl ethoxymethylenemalonate and analyzed by reverse-phase high-performance liquid chromatography. Calibration curves showed very good linearity of the response. The limit of detection and quantification were 0.15 and 0.50 μM, respectively. The method has a high intra- (RSD=0.35%) and inter-repeatability (RSD=2.86%), and a remarkable accuracy with a 99% recovery in spiked samples. The method is very easy to carry out and allows for ready analysis of large number of samples using very basic HPLC equipment because the derivatized samples are very stable and have very good chromatographic properties., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The antioxidant and antiproliferative activities of the essential oils from Thymbra capitata and Thymus species grown in Portugal were evaluated. Thymbra and Thymus essential oils were grouped into two clusters: Cluster I in which carvacrol, thymol, p-cymene, α-terpineol, and γ-terpinene dominated and Cluster II in which thymol and carvacrol were absent and the main constituent was linalool. The ability for scavenging ABTS(•+) and peroxyl free radicals as well as for preventing the growth of THP-1 leukemia cells was better in essential oils with the highest contents of thymol and carvacrol. These results show the importance of these two terpene-phenolic compounds as antioxidants and cytotoxic agents against THP-1 cells.

Essential oils (EO) possess antimicrobial, anti-inflammatory, insect repellent, anti-cancer, and antioxidant properties, among others. In the present work, the antioxidant, anti-inflammatory and anti-proliferative activities of Moroccan commercial EOs (Citrus aurantium, C. limon, Cupressus sempervirens, Eucalyptus globulus, Foeniculum vulgare and Thymus vulgaris) were evaluated and compared with their main constituents. T. vulgaris EO showed the best free radicals scavenging capacity. This EO was also the most effective against lipid peroxidation along with C. limon and F. vulgare EOs. C. sempervirens EO was the most effective in scavenging NO free radicals, whereas C. limon EO showed the best chelating power. Not all of the major compounds of the EO were responsible for the whole activity of the EOs. T. vulgaris EO showed the best anti-proliferative activity against THP-1 cells in contrast to that of F. vulgare. The antioxidant and anti-inflammatory activities of the EOs were plant species dependent and not always attributable to the EOs main components. Nevertheless, the EOs anti-proliferative activities were more related to their main components, as with T. vulgaris, C. limon, E. globulus and C. sempervirens.

A method for determination of β-cyano-L-alanine, γ-glutamyl-β-cyano-L-alanine and other free amino acids in Vicia sativa is presented. Seed extracts were derivatized by reaction with diethyl ethoxymethylenemalonate and analyzed by reverse-phase high-performance liquid chromatography. Calibration curves showed very good linearity of the response. The limit of detection and quantification was 0.15 and 0.50 μM, respectively. The method has high intra- (RSD = 0.28-0.31%) and interrepeatability (RSD = 2.76-3.08%) and remarkable accuracy with a 99% recovery in spiked samples. The method is very easy to carry out and allows for ready analysis of large number of samples using very basic HPLC equipment because the derivatized samples are very stable and have very good chromatographic properties. The method has been applied to the determination of γ-glutamyl-β-cyano-L-alanine, β-cyano-L-alanine, and common free amino acids in eight wild populations of V. sativa from southwestern Spain.

The erythrocyte agglutinating activity of polyphenol extracts from six grain legumes was investigated. Polyphenols are amphipathic molecules that can bind to proteins and lipids through hydrophobic and polar interactions, leading to agglutination of liposomes and bacteria. The extracts from four of the six legumes that were studied caused erythrocyte agglutination at concentrations in the μM range. Soybean extracts had the highest activity, followed by the extracts from lentils, broad bean, and chickpea. As a good representative of these legumes, binding of the polyphenols extracted from lentils to erythrocytes was investigated in more detail, showing that agglutination was mediated by binding of 84% of the polyphenols present in the incubation, which corresponds to 2.42 μg bound polyphenols/mg erythrocytes, and a maximum polyphenol binding of 96% according to Lineweaver-Burk plots. The relatively high concentrations that are required for agglutination justify that polyphenols more probably do not agglutinate erythrocytes in vivo, but the possibility still exists that in vivo binding without agglutination could occur, which could have some effects on the metabolism and health-promoting properties of polyphenols., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

One of the main difficulties in the study of emotion is the induction of a real emotional response by means of artificial techniques. The aim of the current study is to validate the Spanish version of a set of films with the capacity to induce emotions (PIE) under laboratory conditions and to analyze its capacity to provoke differentiated basic emotions. A sample of 127 subjects took part in the study; 57 excerpts of Spanish-dubbed films with capacity in previous studies to induce 7 emotions: anger, fear, sadness, disgust, amusement, tenderness, and neutral emotion were used. Subjective emotional response was measured using the Self-Assessment Manikins and the Discrete Emotions Questionnaire. Films included showed a good capacity to induce positive and negative affects, high levels of emotional activation and variations in the perception of emotional control. They induced basic emotions of amusement and fear in a differentiated way. However, sadness and disgust could not be significantly differentiated from anger; or anger and tenderness from sadness. The PIE could be a useful tool for the experimental research of emotions in Spanish populations.

Plant protein hydrolysates are a source of bioactive peptides. There are peptides that decrease the micellar cholesterol solubility from bile acids and therefore may reduce in vivo cholesterol absorption. The presence of these peptides in sunflower protein hydrolysates has been studied. Sunflower protein hydrolysates produced with alcalase plus flavourzyme or with pepsin plus pancreatin inhibited in some degree the cholesterol incorporation to micelles. Protein hydrolysates generated after 30 min of hydrolysis with alcalase, and after 30 min of hydrolysis with pepsin, were the inhibitoriest of the cholesterol incorporation to micelles. The average amino acid hydrophobicity of inhibitory peptides in cholesterol micelles was higher than the observed in the corresponding protein hydrolysates. This high hydrophobicity probably favours their inclusion in the lipid micelles. In vivo, this inhibition may translate in a decrease of cholesterol absorption. Reported results show that a combination of different characteristics such as peptide size or hydrophobicity may be responsible of the inhibitory activity of generated peptides.

The growth of mammalian cells in vitro requires the use of rich culture media that are prepared by combining serum with specific nutrient formulations. Serum, the most expensive component of culture media, provides a complex mixture of growth factors and nutrients. Protein hydrolysates that can support in vitro cell growth and eliminate or reduce the need to use serum have been obtained from different sources. Here we describe the use of two food grade proteases to produce a chickpea protein hydrolysate that has been added to cell culture medium in order to determine whether it can be used as a substitute for serum. Medium containing the hydrolysate has been tested using two human cells lines: the monocytic THP-1 cell line which grows in suspension, and the epithelial Caco-2 cell line which grows as a monolayer. The chickpea protein hydrolysate was a good substitute for serum in the first case, but did not allow growth of Caco-2 cells. Supplementation of culture media with this inexpensive and safe hydrolysate would greatly reduce the cost of cell culture.

Copper-chelating peptides were purified from sunflower protein hydrolysates by affinity chromatography using immobilized copper. A variety of protein hydrolysates were obtained by incubation with the proteases Alcalase and Flavourzyme for different periods of time. Chelating activity was indirectly determined by measuring the inhibitory effect of hydrolysates on the oxidation of beta-carotene by copper. Copper-binding peptides purified from the two hydrolysates that inhibited oxidation by copper the most contained 25.4 and 42.0% histidine and inhibited beta-carotene oxidation 8 and 3 times more than the original hydrolysates, which had 2.4 and 2.6% histidine, respectively. Thus, histidine content is not the only factor involved in antioxidant activity, and probably other factors such as peptide size and amino acid sequence are also important. This work shows that affinity chromatography can be used for the purification of copper-chelating peptides and probably other metals of nutritional interest such as calcium, iron, and zinc. In addition to their antioxidant potential, chelating peptides are of nutritional interest because they increase bioavailability of minerals.

Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.

Brassica carinata protein isolates were hydrolyzed using the digestive enzymes trypsin, chymotrypsin, and carboxypeptidase A in order to obtain hydrolyzates with a high Fischer's ratio. The proteases were immobilized using two glyoxyl-agarose supports of different porosity, 4 and 10% agarose gels, in order to evaluate the effect of substrate diffusion into the support containing the enzyme on the hydrolytic process. Reaction time, substrate concentration, and the enzyme to substrate ratio were optimized in an attempt to increase the Fischer's ratio in the resulting hydrolyzates. Gel filtration chromatography of a hydrolyzate with a degree of hydrolysis of 36% yielded a fraction that represented 31% of the total hydrolyzed proteins and had a Fischer's ratio of 28.3 with a phenylalanine + tyrosine content below 1.5%. This material could be used for preparing special diets when there is a need to increase the supply of branched amino acids and/or reduce the intake of aromatic amino acids.

A lung extract rich in angiotensin converting enzyme (ACE) and pure ACE were immobilized by reaction with the activated support 4 BCL glyoxyl-agarose. These immobilized ACE derivatives were used for purification of ACE inhibitory peptides by affinity chromatography. The immobilized lung extract was used to purify inhibitory peptides from sunflower and rapeseed protein hydrolysates that had been obtained by treatment of protein isolates with alcalase. The ACE binding peptides that were retained by the derivatives were specifically released by treatment with the ACE inhibitor captopril and further purified by reverse-phase C18 HPLC chromatography. Inhibitory peptides with IC50 50 and 150 times lower than those of the original sunflower and rapeseed hydrolysates, respectively, were obtained. The derivative prepared using pure ACE was used for purification of ACE inhibitory peptides from the same type of sunflower protein hydrolysate. ACE binding peptides were released from the ACE-agarose derivatives by treatment with 1 M NaCl and had an IC50 a little higher than those obtained using immobilized extract and elution with captopril. Affinity chromatography facilitated the purification of ACE inhibitory peptides and potentially other bioactive peptides present in food proteins.

The assay of angiotensin-converting enzyme (ACE) inhibition by food-derived peptides is usually carried out by using soluble ACE in a batch process. The purification of this enzyme from tissues is not an easy task, and the resulting preparation loses activity very fast. In addition, ACE commercial preparations are very expensive. In this work the immobilization of ACE, through lysine amino groups, to 4% beads cross-linked (4 BCL) glyoxyl-agarose is described. The amount of immobilized enzyme increased with increasing concentrations of enzyme and with incubation time until a saturation point was reached at 50 mg protein/mL gel and 3.5 hours, respectively. The IC50 values for a noncompetitive sunflower peptide inhibitor were similar for the soluble (30.56 microM) and immobilized (32.7 microM) enzymes. An immobilized derivative was obtained that was 60 times more stable than the soluble enzyme at 60 degrees C. This procedure yields a derivative that can be reused and has increased thermal stability compared to that of the soluble enzyme. Thus, ACE immobilization is a good alternative to using soluble freshly prepared or commercial preparations because of economical and practical reasons.

Sunflower protein isolates and the proteases pepsin and pancreatin were used for the production of protein hydrolysates that inhibit angiotensin-I converting enzyme (ACE). Hydrolysates obtained after 3 h of incubation with pepsin and 3 h with pancreatin were studied. An ACE inhibitory peptide with the sequence Phe-Val-Asn-Pro-Gln-Ala-Gly-Ser was obtained by G-50 gel filtration chromatography and high-performance liquid chromatography C18 reverse phase chromatography. This peptide corresponds to a fragment of helianthinin, the 11S globulin from sunflower seeds, which is the main storage protein in sunflower. These results show that sunflower seed proteins are a potential source of ACE inhibitory peptides when hydrolyzed with pepsin and pancreatin.