Effects of sequential enzymatic hydrolysis on structural, bioactive and functional properties of Phaseolus lunatus protein isolate

Significant initiatives exist within the global food market to search for new, alternative protein sources with better technological, functional, and nutritional properties. Lima bean (Phaseolus lunatus L.) protein isolate was hydrolyzed using a sequential pepsin-pancreatin enzymatic system. Hydrolysis was performed to produce limited (LH) and extensive hydrolysate (EH), each with different degrees of hydrolysis (DH). The effects of hydrolysis were evaluated in vitro in both hydrolysates based on structural, functional and bioactive properties. Structural properties analyzed by electrophoretic profile indicated that LH showed residual structures very similar to protein isolate (PI), although composed of mixtures of polypeptides that increased hydrophobic surface and denaturation temperature. Functionality of LH was associated with amino acid composition and hydrophobic/hydrophilic balance, which increased solubility at values close to the isoelectric point. Foaming and emulsifying activity index values were also higher than those of PI. EH showed a structure composed of mixtures of polypeptides and peptides of low molecular weight, whose intrinsic hydrophobicity and amino acid profile values were associated with antioxidant capacity, as well as inhibiting angiotensin-converting enzyme. The results obtained indicated the potential of Phaseolus lunatus hydrolysates to be incorporated into foods to improve techno-functional properties and impart bioactive properties.

Evaluation of some residual bioactivities of microencapsulated Phaseolus lunatus protein fraction with carboxymethylated flamboyant (Delonix regia) gum/sodium alginate

Recent studies have shown the beneficial effect of peptides, an unexploited source could be Phaseolus lunatus being an important raw material for those functional products in order to improve their utilization. In addition to improve the beneficial effect of bioactive peptides the microencapsulation could be a way to protect the peptides against the environment to which they are exposed. P. lunatus protein fraction (<10 kDa of weight) was encapsulated using a blend of carboxymethylated flamboyant gum (CFG) and sodium alginate (SA) at different concentrations of CaCl2 and hardening times. After in vitro digestion of microcapsules the residual activity, in the intestinal system, both inhibition of agiotensin-converting enzyme (I-ACE) and antioxidant activity obtained were in a range of 0.019-0.136 mg/mL and 570.64-813.54 mM of TEAC respectively. The microencapsulation employed CFG/SA blends could be used controlled delivery of peptide fractions with potential use as a nutraceutical or therapeutic agents.

Antioxidant activity of black bean (Phaseolus vulgaris L.) protein hydrolysates

Abstract The objective of this work was to study the effect of enzymatic hydrolysis of black bean protein concentrate using different enzymes. Bean proteins were extracted and hydrolyzed over a period of 120 min using the enzymes pepsin or alcalase. The protein hydrolysates’ molecular weight was assayed by electrophoresis and the antioxidant activity was evaluated by the capturing methods of free radicals ABTS●+ and DPPH. Electrophoretic results showed that the bands above 50 kDa disappeared, when the beans protein was subjected to hydrolysis with pepsin. The bean protein hydrolysate obtained by hydrolysis with alcalase enzyme, showed higher antioxidant activity for inhibition of the radical ABTS●+. However, the hydrolysates obtained by hydrolysis with pepsin had higher antioxidant activity for inhibition of the radical DPPH. The use of pepsin and alcalase enzymes, under the same reaction time, produced black bean protein hydrolysates with different molecular weight profiles and superior antioxidant activity than the native bean protein.

Germination, initial growth and cotyledon protein content of bean cultivars under salinity stress

Excess salts in the root zone inhibit water uptake by plants, affect nutrient uptake and may result in toxicities due to individual salts in the soil solution. Excess exchangeable sodium in the soil may destroy the soil structure to a point where water penetration and root aeration become impossible. Sodium is also toxic to many plants. Beans (Phaseolus vulgaris L.) are consumed as protein source in northeastern Brazil, although little is known about common bean cultivar tolerance to salinity. The germination of bean cultivars under salt stress was studied. The cultivars 'Carioca' and 'Mulatinho' were submitted to germination test in a germinator at 25ºC, at the Seed Analysis Laboratory of the Brazilian Agricultural Research Corporation unit in the Semi- Arid region (Embrapa Semi Árido), Petrolina, Pernambuco State. These seeds were germinated on "germitest" papers imbibed in distilled water or in 10, 50, 100 e 200 mol.m-3sodium chloride (NaCl) solutions. At the first and second counts of the germination test, normal seedlings were counted, measured, weighed and dried, supplying data for vigor, total germination, fresh matter weight and dry matter weight and seedlings length. Total protein was quantified in cotyledons at 3, 6 and 9 days after sowing. The results indicated that the NaCl content influenced seed germination and concentrations above 50 mol.m-3 decreased germination and seedling growth.

Characterization and protein fingerprinting of Botrytis cinerea isolates /

Botrytis cinerea isolates collected from Niagara region were treated with different concentrations of the fiingicide, iprodione to test their sensitivity to this fungicide. These Botrytis cinerea isolates were divided into two groups according to their sensitivity to iprodione. Those isolates whose growth was inhibited by iprodione at concentrations < 2|i,g/nil were classified as sensitive isolates. Isolates that were able to show considerable growth at 2|j,g/ml iprodione were classified as resistant isolates. Resistant and sensitive isolates were compared for their morphological and growth characteristics, conidial germination, virulence on grape berries and protein banding profiles. The fungicide iprodione at a concentration of 2|xg/nil inhibited mycelial growth, sporulation and conidial germination of sensitive isolates but not those of resistant isolates. The inhibitory effect of the fungicide was greater on mycelial growth than on conidia germination of the sensitive isolates. Sensitive isolates produced no sclerotia whereas resistant isolates produced large number of sclerotia. The fungicide iprodione affected sclerotial production in the resistant isolates. The number of sclerotia was decreased by the increase of iprodione in the medium. Sporulation of resistant isolates was improved significantly in the presence of iprodione. The resistant isolates were as virulent as the sensitive isolates on grape berries. The sensitive and resistant isolates showed similar protein banding profiles in the absence of iprodione in polyacrylamide gel electrophoresis studies. Similar protein profiles were also observed when these isolates were grown in the presence of low iprodione concentration (0.5|ig/nil). However, in the presence of concentration (0.5|ig/nil). However, in the presence of iprodione at concentration of 5|Xg/nil, one protein band with approximate molecular weight of 83 KDa was present in the growing resistant isolates (and the controls) but was missing in the inhibited sensitive isolates.

Regulation of the memory T cell development and islet beta-cell survival by DAPK-related apoptosis-inducing protein kinase 2 (Drak2)

Drak2 est un membre de la famille des protéines associées à la mort et c’est une sérine/thréonine kinase. Chez les souris mutantes nulles Drak2, les cellules T ne présentent aucune défectuosité apparente en apoptose induite par activation, après stimulation avec anti-CD3 et anti-CD28, mais ont un seuil de stimulation réduit, comparées aux cellules T de type sauvage (TS). Dans notre étude, l’analyse d’hybridation in situ a révélé que l’expression de Drak2 est ubiquiste au stade de la mi-gestation chez les embryons, suivie d’une expression plus focale dans les divers organes pendant la période périnatale et l’âge adulte, notamment dans le thymus, la rate, les ganglions lymphatiques, le cervelet, les noyaux suprachiasmatiques, la glande pituitaire, les lobes olfactifs, la médullaire surrénale, l’estomac, la peau et les testicules. Nous avons créé des souris transgéniques (Tg) Drak2 en utilisant le promoteur humain beta-actine. Ces souris Tg montraient des ratios normaux entre cellules T versus B et entre cellules CD4 versus CD8, mais leur cellularité et leur poids spléniques étaient inférieurs comparé aux souris de type sauvage. Après activation TCR, la réponse proliférative des cellules T Tg Drak2 était normale, même si leur production d’interleukine (IL)-2 et IL-4 mais non d’interféron-r était augmentée. Les cellules T Tg Drak2 activées ont démontré une apoptose significativement accrue en présence d’IL-2 exogène. Au niveau moléculaire, les cellules T Tg Drak2 ont manifesté une augmentation moins élevée des facteurs anti-apoptotiques durant l’activation; un tel changement a probablement rendu les cellules vulnérables aux attaques subséquentes d’IL-2. L’apoptose compromise dans les cellulesT Tg Drak2 a été associée à un nombre réduit de cellules T ayant le phénotype des cellules mémoires (CD62Llo) et avec des réactions secondaires réprimées des cellules T dans l’hypersensibilité de type différé. Ces résultats démontrent que Drak2 s’exprime dans le compartiment des cellules T mais n’est pas spécifique aux cellules T; et aussi qu’il joue des rôles déterminants dans l’apoptose des cellules T et dans le développement des cellules mémoires T. En outre, nous avons recherché le rôle de Drak2 dans la survie des cellules beta et le diabète. L’ARNm et la protéine Drak2 ont été rapidement induits dans les cellules beta de l’îlot après stimulation exogène par les cytokines inflammatoires ou les acides gras libres et qui est présente de façon endogène dans le diabète, qu’il soit de type 1 ou de type 2. La régulation positive de Drak2 a été accompagnée d’une apoptose accrue des cellules beta. L’apoptose des cellules beta provoquée par les stimuli en question a été inhibée par la chute de Drak2 en utilisant petit ARNi. Inversement, la surexpression de Drak2 Tg a mené à l’apoptose aggravée des cellules beta déclenchée par les stimuli. La surexpression de Drak2 dans les îlots a compromis l’augmentation des facteurs anti-apoptotiques, tels que Bcl-2, Bcl-xL et Flip, sur stimulation par la cytokine et les acides gras libres. De plus, les expériences in vivo ont démontré que les souris Tg Drak2 étaient sujettes au diabète de type 1 dans un modèle de diabète provoqué par de petites doses multiples de streptozotocine et qu’elles étaient aussi sujettes au diabète de type 2 dans un modèle d’obésité induite par la diète. Nos données montrent que Drak2 est défavorable à la survie des cellules beta. Nous avons aussi étudié la voie de transmission de Drak2. Nous avons trouvé que Drak2 purifiée pouvait phosphoryler p70S6 kinase dans une analyse kinase in vitro. Lasurexpression de Drak2 dans les cellules NIT-1 a entraîné l’augmentation de la phosphorylasation p70S6 kinase tandis que l’abaissement de Drak2 dans ces cellules a réduit la phosphorylation. Ces recherches mécanistes ont prouvé que p70S6 kinase était véritablement un substrat de Drak2 in vitro et in vivo. Cette étude a découvert les fonctions importantes de Drak2 dans l’homéostasie des cellules T et le diabète. Nous avons prouvé que p70S6 kinase était un substrat de Drak2. Nos résultats ont approfondi nos connaissances de Drak2 à l’intérieur des systèmes immunitaire et endocrinien. Certaines de nos conclusions, comme les rôles de Drak2 dans le développement des cellules mémoires T et la survie des cellules beta pourraient être explorées pour des applications cliniques dans les domaines de la transplantation et du diabète.

Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine.

Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 ± 9.6%) compared to the unvaccinated and challenged animals (45.8 ± 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was slightly improved in the rAdP97c vaccinated pigs (0.672 ± 0.068 kg/day) compared to the unvaccinated and challenged animals (0.568 ± 0.104 kg/day). A bacterin-based commercial vaccine (Suvaxyn® MH-one) was more efficient to induce a protective immune response than rAdP97c even if it did not evoke a P97 specific immune response. These results suggest that immunodominant antigens other than P97 adhesin are also important in the induction of a protective immune response and should be taken into account in the future development of M. hyopneumoniae subunit vaccines.

Preparation of Low-Protein Natural Rubber Latex: Effect of Polyethylene Glycol

Low-protein content natural rubber latex was produced by using a nonionic surfactant-polyethylene glycol (PEG). Extractable protein content of natural rubber latex was found to decrease with PEG treatment and reduction increased with increase in the molecular weight of PEG. The low-protein latex samples were characterized by tensile testing, Fourier transform infrared and thermogravimetric analysis. The results have shown 35% reduction in the extractable protein content, without any compromise on the mechanical properties of the latex; however, thermal stability of low-protein latex was found to be reduced marginally with PEG treatment.

Utilisation of Giant African snail (Achatina fulica) meal as protein source by laying hens

A 12-week experiment was carried out to investigate the effects of substituting Giant African snail meal for fish meal in laying hens diet. Four diets were formulated to contain snail meal as replacement for fish meal at 0 (control), 33, 67 and 100%. A total of 120 Shaver Brown pullets aged 18 weeks were allocated to the dietary treatments in a randomised design. Each treatment consisted of three replicates and ten birds per replicate. Feed intake increased only for the 33% treatment as compared to the 67% replacement diet but did not differ from the other treatments. There were no significant treatment effects on egg performance parameters observed (egg production, egg weight, total egg mass, feed conversion ratio and percent shell). The overall feed cost of egg production reduced on the snail meal-based diets. The organoleptic evaluation of boiled eggs revealed no difference between the treatments. Based on these results it was concluded that total replacement of fish meal with cooked snail meat meal does not compromise laying performance or egg quality. The substitution is beneficial in terms of production cost reduction and the reduction of snails will have a beneficial impact especially where these snails are a serious agricultural pest. The manual collection and processing of snails can also become a source of rural income.

Intake, live-weight gain and carcass characteristics of beef cattle given diets based on forage maize silage harvested at different stages of maturity

Advancing maturity of forage maize is associated with increases in the proportion of dry matter (DM) and starch, and decreases in the proportions of structural carbohydrates in the ensiled crop. This experiment investigated the effects of three maize silages of 291 (low), 339 (medium) and 393 (high) g DM per kg fresh weight on the performance of 48 Simmental. Holstein-Friesian cattle. Equal numbers of steers (mean start weight = 503 (s.d. 31.3) kg) and heifers (mean start weight = 378 (s.d. 11.2) kg) were offered individually isonitrogenous diets composed of the three silages plus a protein supplement with minerals once daily until slaughter at the target live weight of 575 and 475 kg for steers and heifers, respectively. Intake was reduced on the low diet (P < 0.01) compared with the other two treatments. Dietary starch intake increased by a total of 1 kg/day between low and medium diets but by only 0.2 kg/day between medium and high diets. Unlike starch intake, total neutral-detergent fibre intake showed no significant difference (P > 0.05) between diets. There were no differences in live-weight gain between treatments but differences (P < 0.05) in food conversion efficiency indicated relative gains of 115, 100 and 102 g gain per kg DM intake for diets low, medium and high, respectively. There were no differences between diets in carcass weights, fat score and overall conformation.

Effect of breed, gender, housing system and dietary crude protein content on performance of finishing beef cattle fed maize-silage-based diets

Maize silage-based diets with three dietary crude protein (CP) supplements were offered to 96 finishing cattle of contrasting breed (Holstein Friesian (HF) v. Simmental x HF (SHF)) and gender (bull v. steer) housed in two types of feeding system (group fed v. individually fed). The three protein supplements differed either in CP or protein degradability (degradable (LUDP) v. rumen undegradable (HUDP)) and provided CP concentrations of 142 (Con), 175 (LUDP) and 179 (HUDP) g/kg dry matter (DM) respectively, with ratios of degradable to undegradable of 3.0, 1.4 and 0.9:1 for diets Con, LOP and HUDP respectively. DM intakes were marginally higher (P = 0. 102) for LOP when compared with Con and HOP Rates of daily live-weight gain (DLWG) were higher (P = 0.005) in LUDP and HOP when compared with Con. HF had higher DM intakes than SHF although this did not result in any improvement in HF DLWG. Bulls had significantly better DM intakes, DLWG and feed conversion efficiency than steers. Conformation scores were better in SHF than HF (P < 0.001) and fat scores lower in bulls than steers (p < 0.001). There was a number of first order interactions established between dietary treatment, breed, gender and housing system with respect to rates of gain and carcass fat scores.

Retention time prediction and protein identification

Proteins are commonly identified through enzymatic digestion and generation of short sequence tags or fingerprints of peptide masses by mass spectrometry. Separation methods, such as liquid chromatography and electrophoresis, are often used to fractionate complex protein or peptide mixtures and these separations also provide information on the different species, such as molecular weight and isoelectric point from electrophoresis and hydrophobicity in reversed-phase chromatography. These are also properties that can be predicted from amino acid sequences derived from genomic sequences and used in protein identification. This chapter reviews recently introduced methods based on retention time prediction to extract information from chromatographic separations and the applications to protein identification in organisms with small and large genomes. Novel data on retention time prediction of posttranslationally modified peptides is also presented.

Effect of addition of protein fractions extracted from flours of different baking quality on gluten rheology

The effect of change of the rheological properties of gluten with the addition of fractions with specific molecular weight was investigated. Fractions extracted from Hereward, Riband and Soissons flours were added to the dough prior to gluten extraction. Once extracted, the glutens were subjected to temperature sweeps and creep recovery rheological tests. In the temperature sweeps, Hereward fractions containing the larger polypeptides had a strengthening effect on the gluten, indicated by a decrease in tan delta and an increase in elastic creep recovery, while those fractions that comprised monomeric gliadins had a weakening effect. Adding total gluten also had a strengthening effect. For the biscuit-making flour Riband, the results were quite the reverse: all fractions appeared to strengthen the gluten network, while the addition of total gluten did not have a strengthening effect. For Soissons gluten, the addition of total gluten had a strengthening effect while adding any individual fraction weakened the gluten. The results were confirmed with creep-recovery tests.

Comparison of heat-treated rapeseed expeller and solvent-extracted soya-bean meal as protein supplements for dairy cows given grass silage-based diets

Sixteen early to mid lactation Finnish Ayrshire dairy cows were used in a cyclic change-over experiment with four 21-day experimental periods and a 4 5 2 factorial arrangement of treatments to evaluate the effects of heat-treated rapeseed expeller and solvent-extracted soya-bean meal protein supplements on animal performance. Dietary treatments consisted of grass silage offered ad libitum supplemented with a fixed amount of a cereal based concentrate (10 kg/day on a fresh weight basis) containing 120, 150, 180 or 210 g crude protein (CP) per kg dry matter (DM). Concentrate CP content was manipulated by replacement of basal ingredients (g/kg) with either rapeseed expeller (R; 120, 240 and 360) or soya-bean meal (S; 80, 160 and 240). Increases in concentrate CP stimulated linear increases (P < 0.05) in silage intake (mean 22.5 and 23.8 g DM per g/kg increase in dietary CP content, for R and S, respectively) and milk production. Concentrate inclusion of rapeseed expeller elicited higher (P < 0.01) milk yield and milk protein output responses (mean 108 and 3.71 g/day per g/kg DM increase in dietary CP content) than soya-bean meal (corresponding values 62 and 2.57). Improvements in the apparent utilization of dietary nitrogen for milk protein synthesis (mean 0.282 and 0.274, for R and S, respectively) were associated with higher (P < 0.05) plasma concentrations of histidine, branched-chain, essential and total amino acids (35, 482, 902 and 2240 and 26, 410, 800 and 2119 mu mol/l, respectively) and lower (P < 0.01) concentrations of urea (corresponding values 4.11 and 4.52 mmol/l). Heat-treated rapeseed expeller proved to be a more effective protein supplement than solvent-extracted soya-bean meal for cows offered grass silage-based diets.

Glutathione and related thiol compounds II: the importance of protein bound glutathione and related protein-bound compounds in gluten proteins

Protein-bound glutathione (PSSG) and protein-bound related thiol compounds, i.e. cysteine (PSSCys), glutamyl-cysteine (PSSGlu-Cys) and cysteinyl-glycine (PSSCys-Gly), were analysed in proteins of Osborne fractions, i.e. gliadin, glutenin and gliadin-, glutenin-subfractions separated by gel filtration chromatography, gel protein and the total gluten proteins separated from wheat varieties with varying breadmaking performances. The results showed that PSSG and some protein-bound related thiol compounds were found in monomeric gliadins, indicating that glutathione and some related thiol compounds are able to form disulphide bonds (SS) with sulphydryl group (SH) of those proteins and the formation of those disulphide bonds may prevent those monomeric proteins from binding to other proteins. It was also observed that a larger amount of PSSG in glutenin proteins was negatively correlated with the molecular weight (M-w) distribution of glutenin polymers, suggesting that PSSG and protein-bound related thiol compounds may play an important role in controlling polymerisation of glutenin. Furthermore, it was found that the level of PSSG in gel protein from flours with poor breadmaking performances was constantly higher and significantly different (p < 0.05) from that of flours with good breadmaking performance. The same trend was observed with gluten samples from breadmaking and biscuitmaking flours. (C) 2003 Elsevier Ltd. All rights reserved.