Apart from the nutritional importance of proteins, their physicochemical, functional and bioactive properties are dependent on their structure as well as their resultant interactions with other proteins, water and lipids. We investigated the physicochemical and functional properties of hempseed protein isolate as well the bioactive properties of hemp seed protein hydrolysates (HPH) using antioxidant and antihypertensive assays. The results show that molecular mass decreased with increase in time of hydrolysis. Alcalase treated HPH had an increase in surface hydrophobicity. Increase in hydrolysis time increased protein solubility and water holding capacity of HPH, while foaming and fat absorption capacity decreased as time of hydrolysis increased. The <3 kDa permeates of thermolysin hydrolysate had the highest ACE-inhibition activity, while the <5 kDa permeates of alcalase hydrolysates had the highest renin inhibition value. The various fractions showed low scavenging activity of 1,1–diphenyl-2-picrylhydrazyl, while the superoxide radical scavenging activities were weak to nil. The three various fractions of thermolysin treated hydrolysates displayed the strongest chelating activity. For ferric reducing activities, <5 kDa thermolysin fraction was relatively high when compared to glutathione. The work concluded that HPH can be used not only as a source of nutrients but also as a functional ingredient in food systems as well as therapeutic agent against chronic diseases such as hypertension and oxidative stress-related disorders.

Apart from the nutritional importance of proteins, their physicochemical, functional and bioactive properties are dependent on their structure as well as their resultant interactions with other proteins, water and lipids. We investigated the physicochemical and functional properties of hempseed protein isolate as well the bioactive properties of hemp seed protein hydrolysates (HPH) using antioxidant and antihypertensive assays. The results show that molecular mass decreased with increase in time of hydrolysis. Alcalase treated HPH had an increase in surface hydrophobicity. Increase in hydrolysis time increased protein solubility and water holding capacity of HPH, while foaming and fat absorption capacity decreased as time of hydrolysis increased. The <3 kDa permeates of thermolysin hydrolysate had the highest ACE-inhibition activity, while the <5 kDa permeates of alcalase hydrolysates had the highest renin inhibition value. The various fractions showed low scavenging activity of 1,1–diphenyl-2-picrylhydrazyl, while the superoxide radical scavenging activities were weak to nil. The three various fractions of thermolysin treated hydrolysates displayed the strongest chelating activity. For ferric reducing activities, <5 kDa thermolysin fraction was relatively high when compared to glutathione. The work concluded that HPH can be used not only as a source of nutrients but also as a functional ingredient in food systems as well as therapeutic agent against chronic diseases such as hypertension and oxidative stress-related disorders.

The objective of this work was to produce bioactive peptides from the enzymatic hydrolysis of chicken skin proteins that could be used to treat hypertension, oxidative stress and associated health conditions using a metabolomics approach. Enzymatic hydrolysis of chicken thigh and breast muscle skin proteins was carried out using alcalase or a combination of pepsin/pancreatin (PP) at 1–4% enzyme concentrations. Chicken skin protein hydrolysates (CSPH) were each fractionated by membrane ultrafiltration into different molecular weight peptides (<1, 1–3, 3–5 and 5–10 kDa). Investigation of their in vitro antihypertensive and antioxidant activities showed that alcalase hydrolysates had significantly (p < 0.05) higher ACE-inhibitory activity compared to PP hydrolysates. ACE inhibition was inversely related to size of ultrafiltration membrane peptides. Renin-inhibitory activity varied from 15–36%, and was dependent on the type of protease; PP hydrolysates showed significantly (p < 0.05) higher inhibition than alcalase hydrolysates. CSPHs also significantly (p < 0.05) scavenged antioxidant radicals, increasing with enzyme concentration but decreased as peptide size increased. Kinetics studies revealed that peptide-dependent enzyme inhibition pattern was mostly of the mixed-type for both ACE and renin. Short-term (24 hr) oral administration of 100 mg peptides/kg body weight to spontaneously hypertensive rats (SHRs) led to maximum systolic blood pressure (SBP) reduction of –32.67 and –31.33 mmHg after 6 h for chicken thigh skin hydrolysate and chicken breast skin hydrolysate, respectively. During a 6-week feeding trial, CSPH at 1.0 and 0.5% feed substitutions had significant (p<0.05) antihypertensive effects in SHRs (-36 and -31 SBP reductions, respectively). SBP reduction was directly related to lower plasma ACE but not renin activity. Plasma total antioxidant capacity of the rats was also high. Metabolomics analysis revealed several metabolites with significant changes (≥ 2-fold changes, p < 0.05) in urine and plasma of SHRs fed CSPH, such as Symmetric Dimethylarginine (SDMA), N2-acetyl-L-ornithine, buthionine sulfoximine, uric acid, Vitamin E succinate, L-isoleucine and phospholipids which may be considered important biomarkers/pathways for hypertension and oxidative stress. We conclude that CSPHs may be used as ingredients to formulate functional foods and nutraceuticals for the management of oxidative stress and hypertension-related diseases. / October 2015

Thesis--PlanA (M.S.)--University of Wisconsin--Stout, 2003. / Includes bibliographical references.

To investigate mechanisms responsible for the turnover of endogenous muscle protein, lysosomotropic proteinase inhibitors have been employed to elucidate the relative contributions of lysosomal and non-lysosomal degradation pathways functioning under varying nutritional states and for different classes of intracellular proteins. Proteolysis in cultured bovine aortic smooth muscle cells was measured as the percentage of ³H-phenylalanine released per hour from pre-labelled cellular proteins. To reduce background radioactivity, the intracellular ³H-phenylalanine pool was depleted by serial extraction at 37°C, effecting equilibration between the intracellular pool and the phenylalanine-free medium. Reutilization of labelled amino acids during subsequent incubation periods was minimized by the presence of excess non-labelled phenylalanine in the medium. ³H-phenylalanine was released at a constant rate of 1,5 % per hour for at least 4 h, from cells pre-labelled for 16 h ('long-lived' proteins). Leupeptin, an inhibitor of thiol proteinases including cathepsin 8, inhibited degradation by 12 %, whereas the general lysosomal inhibitors chloroquine and NH₄Cl inhibited degradation by 30 %, presumably the contribution by the lysosomal pathway. In the case of 'short-lived' proteins (pre-labelled for 1 hour), the initial degradation rate was 6,5% per hour, which rapidly declined, reaching the basal rate of 1,5 % after 4 h. Chloroquine and NH₄Cl reduced proteolysis by only 12-15% and leupeptin had no significant inhibition, consistent with the view that the majority of short-lived proteins a degraded by non-lysosomal pathways. Proteolysis rates of 'abnormal' proteins containing the arginine-analogue, canavanine, were found to be significantly elevated (80 %) over controls. Leupeptin had no significant inhibition, and chloroquine and NH₄Cl only reduced degradation by 12-16 %, showing that the rapid removal of 'abnormal' intracellular proteins proceeds mainly via extra-lysosomal mechanisms. Incubation of the cells under nutritional step-down conditions, increased the average degradation rate of long-lived proteins to 3% per hour, and chloroquine and NH₄Cl inhibited degradation by 55-60 %, indicating that the accelerated proteolytic condition is due to increased activity of the lysosomes. Nutritional deprivation did not increase the rate of degradation of short-lived proteins. The results were clarified by the parallel use of the well-characterized LDL degradation system in this cell type, known to occur almost exclusively via lysosomes. This allowed the effectiveness of lysosomotropic inhibitors to be tested. Chloroquine inhibited LDL degradation by over 90 % and NH₄Cl inhibited by 80-95 % in all cases. Other proteinase inhibitors such as chymostatin, pepstatin and the chloromethyl ketones were also tested, and of these chymostatin seemed to be the most valuable because of its additivity to the effect of chloroquine, indicating its selective inhibition of non-lysosomal degradative mechanisms. Incubations of smooth muscle cells under anoxic conditions or with metabolic inhibitors such as fluoride, azide and cyanide, resulted in an inhibition of protein degradation which was greater than, and partially additive to, the effect of chloroquine, i.e. both lysosomal and non-lysosomal degradation pathways have some energy-dependence. The degradation of long-lived proteins appeared to be more sensitive to temperature than that of short-lived proteins, further indicating the activity of distinct proteolytic mechanisms for these two classes of intracellular proteins. Preliminary studies have indicated a role for Ca⁺⁺ in the regulation of proteolysis, since degradation rates were increased by elevated levels of Ca⁺⁺ in the extracellular medium. Inhibition of this increased proteolysis by leupeptin has indicated a role for a thiol proteinase, possibly Ca⁺⁺-activated neutral proteinase. In similar studies with cultured L8 skeletal muscle cells, an average proteolysis rate of 1,2 % per hour was found, which was increased by 50 % under nutritional step-down conditions. Once again, the lysosomal pathway was found to account for only about one-third of basal protein degradation but fully accounted for the increased proteolysis under nutrient deprivation. The degradation characteristics of intracellular smooth and skeletal muscle cell proteins was examined using double isotope labelling. It was found that large molecular weight proteins and glycoproteins tended to be degraded more rapidly than small proteins and non-glycoproteins. In smooth muscle cells, these correlations were markedly reduced or absent under the accelerated proteolysis associated with nutrient deprivation, possibly confirming the increased activity of the non-selective autophagic lysosomal pathway under these conditions. A similar loss of correlations was not so clearly seen for skeletal muscle cell proteins.

Introdução - Devido ao perfil de mortalidade e de danos patológicos associados, as doenças cardiovasculares são consideradas um sério problema de saúde pública. Níveis de colesterol plasmático elevados fazem parte dos fatores de risco mais importantes para o desenvolvimento dessas doenças. Pesquisas recentes demostraram que a proteína do feijão caupi promove a redução dos níveis de colesterol em hamsters e em seres humanos, possivelmente pela ação de peptídeos bioativos advindos da dieta. Entretanto, a via pela qual o colesterol é inibido por esses peptídeos, assim como os efeitos do processamento na ação biológica ainda são desconhecidos. Objetivo - Verificar a via de ação hipocolesterolêmica dos hidrolisados do feijão caupi e o efeito do processamento térmico nesta propriedade. Métodos - Parte da farinha integral foi submetida ao isolamento de proteína e o restante dos grãos foi submetido à cocção em autoclave e à extrusão. Após ser cozido em autoclave, o feijão cozido também teve sua proteína isolada. Posteriormente, a proteína isolada do feijão integral e do feijão cozido foi submetida à hidrólise in vitro. O processo de extrusão foi modelado em função da expansão dos extrusados segundo a metodologia de superfície de resposta. A farinha do feijão extrusado foi submetida à hidrólise enzimática in vitro sem isolamento prévio da proteína. Os três hidrolisados foram submetidos à ultrafiltração e a fração menor que 3 kDa foi utilizada nos ensaios de inibição da enzima 3-hidroxi-3-metilglutaril coenzima A redutase (HMGR) e no ensaio de inibição da solubilização micelar do colesterol para avaliar a ação dos hidrolisados na via hepática e na via entérica do metabolismo do colesterol respectivamente. Resultados - Os hidrolisados provenientes dos isolados proteicos apresentaram comportamentos semelhantes. Em doses mais elevadas de proteína (acima de 70 µg/mL), a inibição apresentou-se estável, por volta dos 75 por cento. Em relação ao hidrolisado da farinha de feijão extrusado, à medida que se aumenta a quantidade de proteína a capacidade inibitória diminui. Os hidrolisados foram capazes de inibir a solubilização micelar do colesterol de 5 a 39 por cento. O processamento térmico foi fator determinante para diminuir a solubilização do colesterol in vitro. Conclusão Os hidrolisados do feijão caupi são capazes de inibir a enzima HMGR e reduzir a solubilização micelar do colesterol in vitro, mesmo após o feijão ser processado termicamente. A capacidade dos hidrolisados de insolubilizar o colesterol foi melhorada pelo cozimento em autoclave e pela extrusão / Introduction- The cardiovascular diseases, due to mortality and associated pathological damage, are considered a serious public health problem. Elevated plasma cholesterol levels are part of the most important risk factors for the development of these diseases. Recent research indicated that Cowpea protein promotes the reduction of cholesterol levels in hamsters and humans, possibly by the action of bioactive peptides from the diet. However, the route by which cholesterol is inhibited by peptides, as well as the processing effects on biological action are still unknown. Objective - To verify the hypocholesterolemic pathway of hydrolyzed cowpea and the effect of thermal processing on this property. Methods - Part of wholemeal flour was subjected to isolation of protein, and the remaining of the grains was subjected to retort and extrusion cooking processes. After autoclaving the grain, protein was also isolated from the flour. Subsequently, the isolated protein from wholemeal and cooked flour was subjected to in vitro hydrolysis. The extrusion process was optimized according to response surface methodology using the expansion ratio of extrudates as the dependent variable. The extruded bean flour was subjected to in vitro enzymatic hydrolysis without the isolation of the protein. The three hydrolysates were subjected to ultrafiltration and and fractions smaller than 3 kDa was used for inhibition assays of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and for inhibition assay of micellar solubilization of cholesterol to evaluate their effect on the liver and enteral cholesterol metabolism respectively. Results - The hydrolysates from the protein isolates showed similar. At higher doses of protein (above 70 mg/mL), the inhibition was stable at around 75 per cent. Regarding the hydrolyzate of bean flour extruded as it increases the amount of protein inhibitory capacity decreases. The hydrolysates were able to inhibit the micellar solubilization of cholesterol between 5 and 39 per cent. The thermal processing was the determining factor to decrease the solubility of cholesterol in vitro. Conclusion The hydrolysates of cowpea are able to inhibit the enzyme HMGR micellar solubilization and reducing cholesterol in vitro, even after being thermally processed beans. The ability of hydrolysates insolubilize cholesterol was improved by cooking in an autoclave and by extrusion

Whey Protein Concentrate (WPC) is used as a functional ingredient in many food products. To increase the applicability of WPC as well as other food proteins, it is often necessary to enhance the functional properties of the protein. Various protein modification techniques can be used for this purpose; this includes chemical, physical and enzymatic modification. In present study acid hydrolysis, a chemical modification, was investigated as a means to improve functionality of WPC, emulsifying, foaming and gelatin. Most of the previous work on WPC has been directed at enzymatic hydrolysis. / Dispersions of WPC (8%) in organic acids (0.5 N, 1 N and 1.5 N acetic acid, citric acid phosphoric acid and mixture of these acids) were subjected to acid hydrolysis (6, 18 and 48 h) and the effects of this modification on functional properties was assessed. The degrees of hydrolysis were measured and freeze-dried hydrolysates were evaluated for their foam capacity and stability, emulsifying activity and stability index and toughness. Highest foam capacity was found in the hydrolysate obtained using 0.5 N acetic acid (6 h hydrolysis, foaming capacity of 140%); acid hydrolysis increased foam stability, in general. In addition, acid hydrolysis did not affect emulsifying activity index but gave higher emulsifying stability index and toughness of prepared gels. / Results of PAGE indicated that acidic modification led to progressive decrease in the $ alpha$-lactalbumin and BSA. $ alpha$-lactalbumin was found to be the most sensitive protein with significant degradation after 6 h hydrolysis. (Abstract shortened by UMI.)

Introdução - Devido ao perfil de mortalidade e de danos patológicos associados, as doenças cardiovasculares são consideradas um sério problema de saúde pública. Níveis de colesterol plasmático elevados fazem parte dos fatores de risco mais importantes para o desenvolvimento dessas doenças. Pesquisas recentes demostraram que a proteína do feijão caupi promove a redução dos níveis de colesterol em hamsters e em seres humanos, possivelmente pela ação de peptídeos bioativos advindos da dieta. Entretanto, a via pela qual o colesterol é inibido por esses peptídeos, assim como os efeitos do processamento na ação biológica ainda são desconhecidos. Objetivo - Verificar a via de ação hipocolesterolêmica dos hidrolisados do feijão caupi e o efeito do processamento térmico nesta propriedade. Métodos - Parte da farinha integral foi submetida ao isolamento de proteína e o restante dos grãos foi submetido à cocção em autoclave e à extrusão. Após ser cozido em autoclave, o feijão cozido também teve sua proteína isolada. Posteriormente, a proteína isolada do feijão integral e do feijão cozido foi submetida à hidrólise in vitro. O processo de extrusão foi modelado em função da expansão dos extrusados segundo a metodologia de superfície de resposta. A farinha do feijão extrusado foi submetida à hidrólise enzimática in vitro sem isolamento prévio da proteína. Os três hidrolisados foram submetidos à ultrafiltração e a fração menor que 3 kDa foi utilizada nos ensaios de inibição da enzima 3-hidroxi-3-metilglutaril coenzima A redutase (HMGR) e no ensaio de inibição da solubilização micelar do colesterol para avaliar a ação dos hidrolisados na via hepática e na via entérica do metabolismo do colesterol respectivamente. Resultados - Os hidrolisados provenientes dos isolados proteicos apresentaram comportamentos semelhantes. Em doses mais elevadas de proteína (acima de 70 µg/mL), a inibição apresentou-se estável, por volta dos 75 por cento. Em relação ao hidrolisado da farinha de feijão extrusado, à medida que se aumenta a quantidade de proteína a capacidade inibitória diminui. Os hidrolisados foram capazes de inibir a solubilização micelar do colesterol de 5 a 39 por cento. O processamento térmico foi fator determinante para diminuir a solubilização do colesterol in vitro. Conclusão Os hidrolisados do feijão caupi são capazes de inibir a enzima HMGR e reduzir a solubilização micelar do colesterol in vitro, mesmo após o feijão ser processado termicamente. A capacidade dos hidrolisados de insolubilizar o colesterol foi melhorada pelo cozimento em autoclave e pela extrusão / Introduction- The cardiovascular diseases, due to mortality and associated pathological damage, are considered a serious public health problem. Elevated plasma cholesterol levels are part of the most important risk factors for the development of these diseases. Recent research indicated that Cowpea protein promotes the reduction of cholesterol levels in hamsters and humans, possibly by the action of bioactive peptides from the diet. However, the route by which cholesterol is inhibited by peptides, as well as the processing effects on biological action are still unknown. Objective - To verify the hypocholesterolemic pathway of hydrolyzed cowpea and the effect of thermal processing on this property. Methods - Part of wholemeal flour was subjected to isolation of protein, and the remaining of the grains was subjected to retort and extrusion cooking processes. After autoclaving the grain, protein was also isolated from the flour. Subsequently, the isolated protein from wholemeal and cooked flour was subjected to in vitro hydrolysis. The extrusion process was optimized according to response surface methodology using the expansion ratio of extrudates as the dependent variable. The extruded bean flour was subjected to in vitro enzymatic hydrolysis without the isolation of the protein. The three hydrolysates were subjected to ultrafiltration and and fractions smaller than 3 kDa was used for inhibition assays of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and for inhibition assay of micellar solubilization of cholesterol to evaluate their effect on the liver and enteral cholesterol metabolism respectively. Results - The hydrolysates from the protein isolates showed similar. At higher doses of protein (above 70 mg/mL), the inhibition was stable at around 75 per cent. Regarding the hydrolyzate of bean flour extruded as it increases the amount of protein inhibitory capacity decreases. The hydrolysates were able to inhibit the micellar solubilization of cholesterol between 5 and 39 per cent. The thermal processing was the determining factor to decrease the solubility of cholesterol in vitro. Conclusion The hydrolysates of cowpea are able to inhibit the enzyme HMGR micellar solubilization and reducing cholesterol in vitro, even after being thermally processed beans. The ability of hydrolysates insolubilize cholesterol was improved by cooking in an autoclave and by extrusion

No description available.

Submitted in complete fulfillment for the degree of Doctor of Philosophy (Food Science and Technology), Durban University of Technology, Durban, South Africa, 2016. / Bambara groundnut (Vigna substerranea) is an indigenous legume of African origin which is currently experiencing a low level utilisation. It is tolerant to drought and can grow under poor soil conditions in which other lucrative crops such as groundnut cannot grow. Bambara is a good source of protein comparable to that of cowpea and slightly lower than soya bean. In order to assess the potential use of bambara protein as a functional ingredient in food systems and as an important ingredient for the formulation of therapeutic product, the knowledge of its protein composition, structure and functionality becomes important. The main goal of this thesis was to determine the composition and bioactive properties of bambara protein and its hydrolysates. Specifically, a comparative study was carried out on the protein content, yield and functional properties of protein concentrates prepared from three different bambara landraces using different extraction methods (Salt solubilisation and Acid precipitation). There was no significant difference in protein content, yield and functional properties of the landraces. However, the method of extraction had an influence on their physicochemical and functional properties. Acid precipitation produced bambara protein concentrates with high protein content and yield (79% and 52% respectively) when compared to salt solubilisation (protein content - 57% and yield - 25%). Protein concentrates prepared through salt solubilisation method exhibited better functional properties in terms of water absorption capacity, oil absorption capacity, foaming capacity, foaming stability and emulsion activities when compared to concentrates obtained through acid precipitation. Furthermore, the composition of bambara proteins produced through isoelectric precipitation was determined. SDS PAGE revealed four major bands; a broad band at 55 kDa which was analysed to be vicilin, two medium bands at 62 kDa and 80 kDa and a high molecular weight (HMW) protein at 141 kDa. Further investigation of bambara protein revealed vicilin (55 kDa) with two sub units as the major protein in bambara and this was also confirmed by the proteomic map. The proteomic map revealed acidic amino acids as the major protein of bambara which is characteristic of vicilin, the map also showed that there were differences in the number of spots across the landraces with 77 spots matching each other. Circular dichroism spectroscopy exhibited reductions in α-helix, and β-pleated sheet conformations as pH varies. In addition, the tertiary structures as observed from the near-UV CD spectra were also influenced by shifts in pH conditions. Differential scanning calorimetry thermograms showed two endothermic peaks at around 67 and 81oC respectively. These can be attributed to thermal denaturation of vicilin and the HMW protein. Subsequent studies used isolates from red bambara since the composition of the landraces were similar. Bambara protein isolate was subjected to enzymatic hydrolysis using three proteases (alcalase, pepsin and trypsin) to produce various bambara protein hydrolysates (BPHs). BPHs were investigated for antioxidant and antihypertensive activities. The in vitro structural and functional characteristics of bambara protein and its enzymatic protein hydrolysate revealed that bambara groundnut possessed antioxidant properties against a variety of physiologically relevant free radicals. High surface hydrophobicity and the molecular size of the peptide seem to be important for scavenging of hydroxyl radicals, ferric reducing power and metal chelation. BPHs and peptide fractions were able to scavenge DPPH radicals with greater affinity for smaller size. Less than 1 and 1-3 kDa pepsin fraction was able to scavenge DPPH radical more than glutathione, BPHs and its fractions scavenge ABTS•+ three folds than the isolate. Scavenging of superoxide radicals was generally weak except for 5-10 kDa peptide fractions. All BPHs inhibited linolenic acid oxidation with greater affinity for the lower molecular size peptide. BPHs showed potential antihypertensive properties because of the in vitro inhibition of activities of angiotensin converting enzyme (ACE) and renin inhibition. The molecular size had significant effect on the ACE inhibitory properties with low molecular weight peptide (<1 kDa) fractions exhibiting significantly higher (p<0.05) inhibitory activities. However, enzyme type had synergistic effects on renin inhibition with alcalase hydrolysate showing highest inhibition at 59% when compared to other hydrolysates and their membrane fractions. The fractions with <1 and 1-3 kDa peptides showed a higher potential as antihypertensive and antioxidant peptides. Based on this study, incorporation of bambara protein isolate as an ingredient may be useful for the manufacture of high quality food products. Likewise, the bambara protein hydrolysates, especially the <1 kDa and 1-3 kDa fraction represent a potential source of bioactive peptides in formulating functional foods and nutraceuticals. / D