Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A incorporaÃÃo de compostos nutracÃuticos como vitaminas, probiÃticos, peptÃdeos bioativos e antioxidantes em sistemas alimentÃcios proporciona uma maneira simples de desenvolver novos alimentos funcionais que podem ter benefÃcios fisiolÃgicos ou reduzir os riscos de doenÃas. Pectinas tÃm sido investigadas por sua capacidade de produzir esferas de gÃis de pectinato de cÃlcio contendo bioativos. Quitosana tambÃm tem sido muito utilizada na preparaÃÃo de esferas por suas propriedades mucoadesivas. Neste estudo, trÃs tipos de esferas de pectina/cÃlcio/quitosana com diferentes graus de reacetilaÃÃo foram produzidas no intuito de encapsular um potente antioxidante natural extraÃdo da manga, a mangiferina. Inicialmente, duas amostras de pectina cÃtrica foram caracterizadas por FTIR, 1H RMN, Reologia e GPC. A pectina cÃtrica com menor grau de metoxilaÃÃo foi escolhida para a preparaÃÃo das esferas por apresentar melhores propriedades geleificantes. As esferas produzidas foram caracterizadas por FTIR, MEV e intumescimento. A formaÃÃo do complexo pectina/cÃlcio/quitosana reacetilada foi observada por FTIR e MEV. Foi realizado um estudo de liberaÃÃo controlada de mangiferina a partir de trÃs tipos de esferas obtidas por duas metodologias diferentes. Os valores de percentual de liberaÃÃo foram bastante significativos, chegando atà 75%. O comportamento dos mecanismos de liberaÃÃo de mangiferina das esferas de pectina/cÃlcio/quitosana mostrou que o bioativo foi liberado a partir da matriz principalmente por meio de relaxaÃÃo das cadeias polimÃricas. / Incorporation of nutraceuticals compounds such as vitamins, probiotics, bioactive peptides and antioxidants in food systems provides a simple way to develop new functional foods that can have physiological benefits or reduce risks of disease. Pectins have been investigated for their ability to produce spheres of calcium pectinate gels containing bioactive. Chitosan has also been widely used in the preparation of beads due to its mucoadhesive properties. In this study, three types of spheres of pectin/calcium/chitosan with different degrees of reacetylation were produced in order to encapsulate a powerful natural antioxidant extracted from mango, mangiferin. Initially, two samples of citrus pectin were characterized by FTIR, 1H NMR, Rheology and GPC. The citrus pectin with the lowest degree of methoxylation was chosen for the preparation of beads due to its better gelling properties. The beads produced were characterized by FTIR, SEM and swelling. The formation of the reacetylated complex pectin/calcium/chitosan was observed by FTIR and SEM. A study of controlled release of mangiferin was conducted from three types of beads obtained by two different methodologies. The values of percentage of release were quite significant, reaching up to 75%. The mechanisms behaviour for the release of mangiferin from spheres of pectin/calcium/chitosan showed that the bioactive was released from the matrix mainly through relaxation of the polymer chains.

The antioxidant activity of carnosine and phytic acid was investigated using several model systems. Carnosine and phytic acid alone inhibited metal ion-catalyzed deoxyribose degradation. Carnosine strongly inhibited metal ion-catalyzed lipid peroxidation in liposomes and in ground beef homogenates. Phytic acid facilitated oxidation of Fe (II) to Fe (III), and it inhibited hemeprotein + H202-catalyzed lipid peroxidation in linoleic acid micelles. Antioxidant and color stabilizing effects of carnosine and phytate were investigated in a beef model system. Both compounds increased the rate of pH decline in pre-rigor beef muscle and stabilized fresh meat color by inhibiting metmyoglobin formation and lipid peroxidation in raw samples during storage at 4°C. Both compounds inhibited heme degradation and lipid peroxidation in cooked beef during storage at 4°C. Iron released from heme was strongly related to lipid peroxidation in cooked beef. Ascorbic acid inhibited metmyoglobin formation on the surface of ground beef patties but not in the bulk of the product. In contrast, camosine inhibited metmyoglobin formation and brown color development throughout the product. Carnosine increased cook yield and salt-soluble protein, but ascorbic acid had no effect on cook yield and decreased salt-soluble protein. Carnosine was more effective on inhibition of lipid peroxidation than was ascorbic acid. Phytate greatly enhanced water-holding capacity of raw and cooked meat in a dilute beef model system. Effects of 0.5% sodium phytate, sodium pyrophosphate, and sodium tripolyphosphate, along with 1% NaCl, on physicochemical properties of restructured raw and cooked beef were compared. In raw beef, the treatments with sodium phytate, sodium pyrophosphate, and sodium tripolyphosphate increased meat pH and salt-soluble protein level, and inhibited metmyoglobin formation and lipid peroxidation, compared to the control. In cooked beef, the treatments with sodium phytate, sodium pyrophosphate, and sodium tripolyphosphate increased bind strength, cooked yield, moisture level, and meat pH, and inhibited lipid peroxidation. The treatments with sodium pyrophosphate and sodium tripolyphosphate increased inorganic orthophosphate level in both raw and cooked beef, compared to sodium phytate and the control. These results indicate that carnosine and phytate can be used as meat preservatives for extending shelf-life and enhancing water-holding capacity of meat and meat products.

The purpose of this study was to assess factors that can influence the colour stability of fresh sausage products using a pork patty model system over a typical distribution and display period. Fresh sausage is usually sold in raw; and it should have minimum 7.5% meat protein and 9% total protein. Losses of meat quality were evidenced through the discolouration of meat, depletion of endogenous antioxidant activities, proliferation of spoilage microorganisms, and reduction in the meat redox potential. <p>Both ground pork and fresh pork patties were made from pork picnic shoulder. In the first study, the quality of both ground pork patties and fresh pork sausage patties decreased over time during storage at 4°C. The fresh sausages contained ingredients that could prolong their shelf life. The activities of these antioxidant enzymes in both ground pork and fresh sausage were depleted by day 5 of the display period. Ground pork, however, had significantly (p<0.05) higher activity of catalase, glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and total antioxidant activity (TEAC) than fresh sausage due to the effect of the salt. Moreover, there was no significant treatment effect on microbial numbers but there was a significant (p<0.05) elevation of microbial colony forming units by day 5 of the display period. The elevation of microbial numbers by the end of the display period was consistent with the drop in redox potential that was measured near the surface of the patties at the end of the incubation period. <p>In the second study, there was no synergistic effect (p<0.05) between sodium erythorbate and lemon juice powder that were used to enhance colour stability during storage and display in terms of antioxidant activity, colour and microbiological profile. The addition of sodium erythorbate alone, however, had a significant effect (p<0.05) on catalase activity and a* values. In other words, this catalase activity was more effective in protecting against oxidation with the addition of sodium erythorbate so that the redness of the fresh sausages (a*values) was preserved. Furthermore, the combined addition of sodium erythorbate and lemon juice powder did not have any antimicrobial activity because there was no significant difference in total microbial counts (Brochothrix thermosphacta count and lactic acid bacteria) following the addition of those ingredients. The measurement of redox potential near the surface of fresh pork patties could not be conclusively correlated with the addition of non-meat ingredients or microbiological activity. However, the measurement of redox potential in the middle of fresh pork patties showed that the addition of sodium erythorbate lowered the redox of the fresh sausage B (0.05% sodium erythorbate) and D (0.25% lemon juice powder and 0.05% sodium erythorbate) compared to fresh sausage A (0.00% lemon juice powder and 0.00% sodium erythorbate).

There is increasing interest in the commericalization of native fruits for utilization as foods and medicinal extracts. This study was undertaken to determine the physicochemical properties and phenolic composition of selected Saskatchewan native fruits, including buffaloberry (<i>Shepherdia argentea</i> Nutt.), chokecherry (<i>Prunus virginiana</i> L.) and sea buckthorn (<i>Hippophae rhamnoides</i> L.). The physicochemical analyses included carbohydrate content, CIELAB colour values, organic acid composition, phenolic content, % seed, soluble solids, pH, total solids, total titratable acidity and proximate composition. Fruit samples were collected and analyzed over four crop years. The proanthocyanidin content was also determined photometrically after acid depolymerization in acid-butanol. Buffaloberry contained a proanthocyanidin concentration of 505 ± 32 mg/100 g fresh fruit and this level was 10 fold higher than that of chokecherry and sea buckthorn. Chokecherry was found to contain an anthocyanin concentration of 255 ± 35 mg/100 g fresh fruit, as determined using the pH differential method. Two high performance liquid chromatography (HPLC) methods were developed for simultaneous determination of seven phenolic classes, including anthocyanins, hydroxybenzoic acids, hydroxycinnamic acids, flavanols, flavanones, flavones and flavonols in aqueous methanol extracts. Based on the semi-quantitative analysis of the total phenolic chromatographic index (TPCI), chokecherry contained the highest levels of phenolic compounds with a concentration of 3,327 ± 469 µg/g fresh fruit followed by buffaloberry (578 ± 73 µg/g fresh fruit) and sea buckthorn (477 ± µg/g fresh fruit). The antioxdant activity of the fruit extracts was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2´-azinobis-3-ethylbenzthiazoline-sulphonic acid (ABTS) radical scavenging assays. Buffaloberry and chokecherry produced the highest radical scavenging activity and were at least five fold greater than that of sea buckthorn. The major radical scavenging compounds in buffaloberry were ascorbic acid and proanthocyanidins. Radical scavenging activity of chokecherry fruit was largely attributable to its anthocyanins, flavonols and hydroxycinnamic acids. Prominent antioxidants in sea buckthorn included ascorbic acid, proanthocyanidins and flavonols. Certain individual compounds in the phenolic extracts were identified by HPLC-photodiode array and HPLC-mass spectrometry. Rutin was found in all of the extracts. Other phenolic compounds identified included catechin in sea buckthorn, and chlorogenic acid and quercetin in chokecherry. The chokecherry fruit pigments were comprised of two major anthocyanins and these were identified as cyanidin 3-glucoside and cyanidin 3-rutinoside. A preparative scale purification method for these anthocyanins using centrifugal partition chromatography (CPC) was determined. Under the CPC conditions employed, cyanidin 3-glucoside and cyanidin 3-rutinoside were purified to concentrations of 84 and 90%, respectively.

There is increasing interest in the commericalization of native fruits for utilization as foods and medicinal extracts. This study was undertaken to determine the physicochemical properties and phenolic composition of selected Saskatchewan native fruits, including buffaloberry (<i>Shepherdia argentea</i> Nutt.), chokecherry (<i>Prunus virginiana</i> L.) and sea buckthorn (<i>Hippophae rhamnoides</i> L.). The physicochemical analyses included carbohydrate content, CIELAB colour values, organic acid composition, phenolic content, % seed, soluble solids, pH, total solids, total titratable acidity and proximate composition. Fruit samples were collected and analyzed over four crop years. The proanthocyanidin content was also determined photometrically after acid depolymerization in acid-butanol. Buffaloberry contained a proanthocyanidin concentration of 505 ± 32 mg/100 g fresh fruit and this level was 10 fold higher than that of chokecherry and sea buckthorn. Chokecherry was found to contain an anthocyanin concentration of 255 ± 35 mg/100 g fresh fruit, as determined using the pH differential method. Two high performance liquid chromatography (HPLC) methods were developed for simultaneous determination of seven phenolic classes, including anthocyanins, hydroxybenzoic acids, hydroxycinnamic acids, flavanols, flavanones, flavones and flavonols in aqueous methanol extracts. Based on the semi-quantitative analysis of the total phenolic chromatographic index (TPCI), chokecherry contained the highest levels of phenolic compounds with a concentration of 3,327 ± 469 µg/g fresh fruit followed by buffaloberry (578 ± 73 µg/g fresh fruit) and sea buckthorn (477 ± µg/g fresh fruit). The antioxdant activity of the fruit extracts was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2´-azinobis-3-ethylbenzthiazoline-sulphonic acid (ABTS) radical scavenging assays. Buffaloberry and chokecherry produced the highest radical scavenging activity and were at least five fold greater than that of sea buckthorn. The major radical scavenging compounds in buffaloberry were ascorbic acid and proanthocyanidins. Radical scavenging activity of chokecherry fruit was largely attributable to its anthocyanins, flavonols and hydroxycinnamic acids. Prominent antioxidants in sea buckthorn included ascorbic acid, proanthocyanidins and flavonols. Certain individual compounds in the phenolic extracts were identified by HPLC-photodiode array and HPLC-mass spectrometry. Rutin was found in all of the extracts. Other phenolic compounds identified included catechin in sea buckthorn, and chlorogenic acid and quercetin in chokecherry. The chokecherry fruit pigments were comprised of two major anthocyanins and these were identified as cyanidin 3-glucoside and cyanidin 3-rutinoside. A preparative scale purification method for these anthocyanins using centrifugal partition chromatography (CPC) was determined. Under the CPC conditions employed, cyanidin 3-glucoside and cyanidin 3-rutinoside were purified to concentrations of 84 and 90%, respectively.

The purpose of this study was to assess factors that can influence the colour stability of fresh sausage products using a pork patty model system over a typical distribution and display period. Fresh sausage is usually sold in raw; and it should have minimum 7.5% meat protein and 9% total protein. Losses of meat quality were evidenced through the discolouration of meat, depletion of endogenous antioxidant activities, proliferation of spoilage microorganisms, and reduction in the meat redox potential. <p>Both ground pork and fresh pork patties were made from pork picnic shoulder. In the first study, the quality of both ground pork patties and fresh pork sausage patties decreased over time during storage at 4°C. The fresh sausages contained ingredients that could prolong their shelf life. The activities of these antioxidant enzymes in both ground pork and fresh sausage were depleted by day 5 of the display period. Ground pork, however, had significantly (p<0.05) higher activity of catalase, glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and total antioxidant activity (TEAC) than fresh sausage due to the effect of the salt. Moreover, there was no significant treatment effect on microbial numbers but there was a significant (p<0.05) elevation of microbial colony forming units by day 5 of the display period. The elevation of microbial numbers by the end of the display period was consistent with the drop in redox potential that was measured near the surface of the patties at the end of the incubation period. <p>In the second study, there was no synergistic effect (p<0.05) between sodium erythorbate and lemon juice powder that were used to enhance colour stability during storage and display in terms of antioxidant activity, colour and microbiological profile. The addition of sodium erythorbate alone, however, had a significant effect (p<0.05) on catalase activity and a* values. In other words, this catalase activity was more effective in protecting against oxidation with the addition of sodium erythorbate so that the redness of the fresh sausages (a*values) was preserved. Furthermore, the combined addition of sodium erythorbate and lemon juice powder did not have any antimicrobial activity because there was no significant difference in total microbial counts (Brochothrix thermosphacta count and lactic acid bacteria) following the addition of those ingredients. The measurement of redox potential near the surface of fresh pork patties could not be conclusively correlated with the addition of non-meat ingredients or microbiological activity. However, the measurement of redox potential in the middle of fresh pork patties showed that the addition of sodium erythorbate lowered the redox of the fresh sausage B (0.05% sodium erythorbate) and D (0.25% lemon juice powder and 0.05% sodium erythorbate) compared to fresh sausage A (0.00% lemon juice powder and 0.00% sodium erythorbate).

Phytochemical composition, antioxidant activity, pigment stability, bioactive properties, and in-vitro absorption of polyphenolics in acai fruit (Euterpe oleracea Mart.) were investigated. Detailed characterization of phenolic compounds present in acai fruit, acai fruit pulp, and a polyphenolic-enriched acai oil were conducted by HPLCESI- MSn analyses and their stability and influence on antioxidant capacity determined. Anthocyanins were predominant in acai fruits, which also contained several flavone and flavonol glycosides, flavanol derivatives, and phenolic acids. In-vitro absorption and antiproliferative effects of phytochemical extracts from acai pulp and acai oil were determined as a function of chemical composition. Polyphenolic mixtures from both acai pulp and acai oil extracts significantly inhibited HT-29 colon cancer cell proliferation, also inducing the generation of reactive oxygen species. In-vitro intestinal absorption using Caco-2 cell models demonstrated that phenolic acids and monomeric flavanol derivatives are readily transported through cell monolayers in-vitro. The influence of polyphenolic cofactors on the stability of anthocyanins in acai fruit under varying conditions of temperature and pH was evaluated. Significant time, temperature, and pH-dependent anthocyanin losses were observed in all models, yet the presence of phenolic acids, procyanidins, and flavone-C-glycosides had a positive influence on anthocyanin stability. External addition of flavone-C-glycosides significantly enhanced visual color, increased anthocyanin stability during exposures to high pH or storage temperatures, and had comparable effects to those of a commercial anthocyanin enhancer. Anthocyanin polymerization reactions occurring during storage of acai fruit juice models were investigated and potential mechanisms and reaction products identified. Polymeric anthocyanin fractions contained several anthocyanin-flavanol adducts based on cyanidin or pelargonidin aglycones and their presence was related to increased anthocyanin sulfite bleaching resistance and to the appearance of large, unresolved peaks in HPLC chromatograms. A reaction mechanism involving the nucleophilic addition of anthocyanins in their hydrated form to flavanol carbocations resulting from cleavage of interflavanic bonds was proposed for the formation of flavanol-anthocyanin adducts in acai fruit juices. Antiproliferative activity and in-vitro absorption of monomeric and polymeric anthocyanin fractions were also evaluated. Both fractions inhibited HT-29 colon cancer cell growth in a similar, concentration-dependent manner, yet in-vitro absorption trials using Caco-2 intestinal cell monolayers indicated the presence of anthocyanin polymers may influence anthocyanin absorption in acai fruit products.

Evidence shows that consumption of a Mediterranean diet can lower the risk of all-cause and cause-specific mortality suggesting that this diet has an overall effect on health. Antioxidants found within olive oil, the primary source of fat in the Mediterranean diet, may be leading contributors to the decreased disease risk. More specifically, hydroxytyrosol (HT), one of the most active and powerful antioxidants found in olive oil, has the ability to increase total antioxidant status and lower levels of lipid peroxidation. In addition to a healthy diet, physical activity decreases the risk of cardiovascular morbidity and mortality; however, aerobic exercise of sufficient intensity or duration can induce oxidative stress. Therefore, the purpose of this study was to investigate the effects of 6 weeks of HT supplementation on antioxidant status and markers of inflammation in healthy, recreationally active males before and throughout acute aerobic exercise bouts. Using a randomized, double-blind, repeated-measures, placebo-controlled design, sixty-one (n=61) participants were randomly assigned to consume a placebo (PLA), low dose of HT (LHT, 50 mg/day), or high dose of HT (HHT, 150 mg/day). Throughout the course of the study, the participants performed four time trial rides (TT1-TT4) on cycle ergometers. TT1 occurred before supplementation, TT2 halfway through the supplementation period, and TT3 and TT4 occurred in the sixth week and final two days of supplementation. Blood was drawn prior to (pre) and just before termination (end) of each time trial to measure markers of antioxidant status and inflammation during exercise. We did not observe significant main effects for treatment on any of the markers for antioxidant status (TEAC) or for markers of inflammation (oxLDL, CRP, 8IP, TNFα, IL-6, IL-10, IL-1β, or IL-1ra). Significant treatment-by-time interactions occurred for CRP, 8IP, and IL-6 although significant treatment differences in these measures were not detected. We conclude that chronic and acute HT supplementation does not improve antioxidant status nor decrease markers of inflammation in this population at rest, during, or following exercise. / text

Mitochondria serve as the major source of reactive oxygen species (ROS) production in cells resulting in antioxidant systems and cell signaling pathways that are unique to mitochondria. Thioredoxin-2 (Trx-2) is the mitochondrial member of the thioredoxin superfamily, and acts specifically to reduce the mitochondrial peroxidase, peroxiredoxin-3. It has been proposed that Trx-2 associates with cytochrome c, which functions in mitochondrial respiration and apoptosis. Homozygous Trx-2 deletion in mice is embryonic lethal and it is hypothesized here that Trx-2 lethality is caused by loss of mitochondrial function and oxidative stress. Results of experiments investigating mitochondrial integrity, cell viability, and ROS levels in Trx-2(-/-) mouse embryonic fibroblasts (MEFs), and results from Trx-2 siRNA MEFs, are similar to findings of knockouts in previously reported proteins that function in mitochondrial respiration and support the involvement of Trx-2 in this process. Mitochondrial ROS have also been implicated as major secondary messengers in cell signaling. Results reported here using cancer cells and cancer cells depleted of mitochondrial DNA, which consequently produce few ROS, have indicated that mitochondrial ROS produced in hypoxia are necessary for HRE and ARE activation, and are fundamental in the activation of SP-1 during reoxygenation. However, mitochondrial ROS are not required for HIF-1&amp;#945; protein expression in hypoxia, indicating a unique relationship between HIF-1&amp;#945;, hypoxia, and mitochondrial ROS.

Rosa roxburghii, also known as "Burr Rose" or "Chestnut Rose", originated in southwest China and was introduced to the botanic garden in Calcutta around 1824. It was named after William Roxburgh who was the superintendent. The extract of fruit of the Rosa roxburghii plant is the base ingredient of a range of products that is commercially sold under the Cili Bao label. The extract is composed of a wide range of substances of nutritional value, in particular a relatively high amount of antioxidants such as ascorbate and plant phenols. It has been reported before that supplementation with the fruit extract resulted in increased red blood cell superoxide dismutase, catalase and the reduced form of glutathione. An enhancement of the antioxidant status could contribute to the protection against several diseases where oxidative stress is a major factor in the pathology, such as atherosclerosis, cancer and immunity stress. Several anecdotal reports with little (published) scientific support claim that human supplementation of the Rosa roxburghii extract to the diet has a protective effect against several diseases, including the above mentioned. Medicinal and herbal plants are used in large sections in developing countries for primary care and there is now also an increase in the use of natural therapies in developed countries. However, plant extracts can also consist of anti-nutritional and possible toxic components, such as oxalic acid and nitrates, which could express cytotoxic and genotoxic activities. Therefore, understanding the health benefits but also the potential toxicity of these plants is important. The objective of this study was to investigate the beneficial properties of Rosa roxburghii extract from an antioxidant potential perspective and in particular to investigate the safety of the product for human consumption. For this purpose in vitro evaluation of the cellular toxicity, mutagenicity and genotoxicity was performed. In addition, specific biochemical parameters relating to the antioxidant status of the product, i.e. antioxidant capacity, oxidative stress prevention and glutathione redox state profiles were investigated in vitro as well as in vivo. The results indicated that Rosa Roxburghii fruit extract was not mutagenic when tested with Salmonella typhimurium strains TA 98, TA 100 and TA 102 in the Ames test. The results, however, pointed towards an antimutagenic effect of the extract in these strains against metabolic activated mutagens 2- acetylaminoflurorene (2-AAF) and aflatoxin B1, and the direct-acting mutagen, methanesulfonate (MMS). In primary rat hepatocyte, Rosa roxbughii extract did not elicit double or single strand DNA damage and cell viability loss using the single cell gel electrophoresis (Comet assay), lactate dehydrogenase leakage test or the mitochondria1 conversion test of MTT to formazan (MTT test). Again the opposite effect was observed: pre-treatment of hepatocytes with Rosa roxbughii extract significantly reduced the effect of oxidative stress-induced cellular- and genotoxicity. These results point to a protective effect against oxidative stress which is reflected in an increase of the antioxidant capacity and glutathione redox state (GSH/GSSG) in vitro (lymphoblasts) and in vivo (humans) reported in this study. This study underlines the previously suggested potential of this plant extract as a natural and safe antioxidant supplement. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2004.