Starches isolated from sixteen quinoa lines ranged in amylose content from 3 to 20%. With the exception of pasting temperature, large variations in pasting characteristics were found among starches and were correlated with amylose content. The gelatinization onset (44.7-53.7 ºC) and peak (50.5-61.7 ºC) temperatures and retrogradation tendencies (19.6-40.8%) were positively correlated with amylose content. No significant variation in gelatinization enthalpy was observed. Swelling, solubility, freeze-thaw stability and water-binding capacity also differed among starches and were correlated with amylose content. The wide variation in amylose content and physicochemical characteristics among quinoa starches suggests applications in a variety of food and non-food products. Two major polypeptides with apparent molecular masses of 56 and 62 kDa were present in quinoa starch and were identified as isoforms of Granule Bound Starch Synthase I (GBSSI). The content of the two isoforms was positively correlated with the concentration of amylose in starch. Starch synthase activity in developing seed was positively correlated with the amylose concentration in starch during seed development. An integrated process was developed for the fractionation of quinoa into starch, protein, oil and saponins. Seed was first roller milled, yielding a coarse bran fraction (48% of the seed weight) that was high in protein (22.9%, db), oil (8.8%, db), and saponins (7.4%, db), and a fine, starch-rich fraction [52% of the seed weight containing 77.2% (db) starch]. Protein, oil and saponins were extracted from the bran under optimized conditions. The protein extracts were concentrated and purified using isoelectric precipitation or ultrafiltration. The means of concentration as well as the presence of saponins strongly affected protein recovery and functionality. Starch was recovered using aqueous alkali (pH 9) to solubilize the protein followed by centrifugation, after which the starch-rich pellet was washed and the sediment which accumulated on top of the pellet was removed. The end-products of the integrated extraction process were a crude saponin extract, a crude oil product, and several protein and starch products. Forty-one percent of the protein present in the seed was recovered as a protein product that contained over 77% (db) protein. Sixty-eight percent of the starch was recovered as a starch product that contained 97% (db) starch and 1.2% (db) protein.

Starches isolated from sixteen quinoa lines ranged in amylose content from 3 to 20%. With the exception of pasting temperature, large variations in pasting characteristics were found among starches and were correlated with amylose content. The gelatinization onset (44.7-53.7 ºC) and peak (50.5-61.7 ºC) temperatures and retrogradation tendencies (19.6-40.8%) were positively correlated with amylose content. No significant variation in gelatinization enthalpy was observed. Swelling, solubility, freeze-thaw stability and water-binding capacity also differed among starches and were correlated with amylose content. The wide variation in amylose content and physicochemical characteristics among quinoa starches suggests applications in a variety of food and non-food products. Two major polypeptides with apparent molecular masses of 56 and 62 kDa were present in quinoa starch and were identified as isoforms of Granule Bound Starch Synthase I (GBSSI). The content of the two isoforms was positively correlated with the concentration of amylose in starch. Starch synthase activity in developing seed was positively correlated with the amylose concentration in starch during seed development. An integrated process was developed for the fractionation of quinoa into starch, protein, oil and saponins. Seed was first roller milled, yielding a coarse bran fraction (48% of the seed weight) that was high in protein (22.9%, db), oil (8.8%, db), and saponins (7.4%, db), and a fine, starch-rich fraction [52% of the seed weight containing 77.2% (db) starch]. Protein, oil and saponins were extracted from the bran under optimized conditions. The protein extracts were concentrated and purified using isoelectric precipitation or ultrafiltration. The means of concentration as well as the presence of saponins strongly affected protein recovery and functionality. Starch was recovered using aqueous alkali (pH 9) to solubilize the protein followed by centrifugation, after which the starch-rich pellet was washed and the sediment which accumulated on top of the pellet was removed. The end-products of the integrated extraction process were a crude saponin extract, a crude oil product, and several protein and starch products. Forty-one percent of the protein present in the seed was recovered as a protein product that contained over 77% (db) protein. Sixty-eight percent of the starch was recovered as a starch product that contained 97% (db) starch and 1.2% (db) protein.

O aspartato é o precursor comum dos aminoácidos essenciais lisina, treonina, metionina e isoleucina. Devido à deficiência em lisina, a via metabólica do ácido aspártico tem sido estudada em cereais e os resultados obtidos indicaram a importância da aspartato quinase (AK), da homoserina desidrogenase (HSDH) e da dihidrodipicolinato sintase (DHDPS) como enzimas chave na regulação da síntese de lisina. Considerando-se a importância do milho e da quinoa como fontes de proteínas para humanos e animais, os objetivos deste trabalho foram: i) estudar as enzimas AK e HSDH nas sementes dos mutantes B77xB79o5; B37o7; W22o10, W22o11 e W22o13 e de seus respectivos tipos selvagens; ii) caracterizar a enzima DHDPS e analisar a composição de aminoácidos solúveis e incorporados nas proteínas das sementes dos mutantes Oh43o1, Oh43o2, Oh43fl1, Oh43fl2 e no respectivo tipo selvagem; iii) estudar nas sementes as enzimas AK, HSDH e DHDPS e alguns aspectos envolvidos na assimilação de nitrogênio e o perfil de aminoácidos de quinoa. Quando os mutantes B77xB79o5; B37o7; W22o10, W22o11 e W22o13 foram estudados, as análises demonstraram importantes variações nas atividades da AK e da HSDH entre os genótipos quando comparados com os respectivos tipos selvagens. A atividade da DHDPS foi fortemente inibida por lisina e não foram observadas variações significativas entre os genótipos Oh43o1, Oh43o2, Oh43fl1 e Oh43fl2, quando comparados com o genótipo Oh43+. A composição de aminoácidos revelou as maiores concentrações de lisina solúvel e incorporada em proteínas no mutante Oh43o2. Os estudos com quinoa demonstraram a presença de pelo menos duas isoenzimas da AK, uma sensível à inibição por lisina e a outra por treonina, duas isoenzimas da HSDH, uma resistente e a outra sensível à inibição por treonina e uma isoenzima da DHDPS sensível à inibição por lisina. As sementes de quinoa também apresentaram altas concentrações de lisina solúvel e incorporada em proteínas. / Aspartate is the common precursor of the essential amino acids lysine, threonine, methionine and isoleucine. Due to the deficiency in lysine, the aspartate metabolic pathway has been studied in cereal crops and the results obtained have indicated the importance of aspartate kinase (AK), homoserine dehydrogenase (HSDH) and dihydrodipicolinate synthase (DHDPS) as the key enzymes involved in the synthesis of lysine. Considering the importance of maize and quinoa as sources of protein for humans and animals, the objectives of this work were: i) to study the enzymes AK and HSDH in the seeds of the maize mutants B77xB79o5; B37o7; W22o10, W22o11 and W22o13 and their respective wild type; II) to characterize the enzyme DHDPS and to analyze the composition of soluble and amino acids incorporated into proteins in the seeds of the maize mutants Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2 and the wild type OH43+; iii) to study the enzymes AK, HSDH, DHDPS in quinoa seeds and some aspects involved in nitrogen assimilation and amino acids profile. When the mutants B77xB79o5; B37o7: W22o10, W22o11 and W22o13 were studied, the analysis revealed important variations on AK and HSDH activities among the genotypes when compared to their respective wild type. The results indicated that DHDPS was strongly inhibited by lysine, and that there was not significant variation among the mutants Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2 when compared to the wild type. The amino acids composition revealed high concentrations of lysine in the soluble form and incorporated into protein in the Oh43o2 mutant. The study of quinoa seeds allowed the identification of at least two AK isoenzymes, one sensitive to lysine inhibition and another sensitive to threonine, two HSDH isoenzymes, one resistant and another sensitive to threonine inhibition and one DHDPS isoenzyme sensitive to lysine inhibition. Quinoa also exhibited high concentration of soluble lysine and lysine incorporated into protein.

O aspartato é o precursor comum dos aminoácidos essenciais lisina, treonina, metionina e isoleucina. Devido à deficiência em lisina, a via metabólica do ácido aspártico tem sido estudada em cereais e os resultados obtidos indicaram a importância da aspartato quinase (AK), da homoserina desidrogenase (HSDH) e da dihidrodipicolinato sintase (DHDPS) como enzimas chave na regulação da síntese de lisina. Considerando-se a importância do milho e da quinoa como fontes de proteínas para humanos e animais, os objetivos deste trabalho foram: i) estudar as enzimas AK e HSDH nas sementes dos mutantes B77xB79o5; B37o7; W22o10, W22o11 e W22o13 e de seus respectivos tipos selvagens; ii) caracterizar a enzima DHDPS e analisar a composição de aminoácidos solúveis e incorporados nas proteínas das sementes dos mutantes Oh43o1, Oh43o2, Oh43fl1, Oh43fl2 e no respectivo tipo selvagem; iii) estudar nas sementes as enzimas AK, HSDH e DHDPS e alguns aspectos envolvidos na assimilação de nitrogênio e o perfil de aminoácidos de quinoa. Quando os mutantes B77xB79o5; B37o7; W22o10, W22o11 e W22o13 foram estudados, as análises demonstraram importantes variações nas atividades da AK e da HSDH entre os genótipos quando comparados com os respectivos tipos selvagens. A atividade da DHDPS foi fortemente inibida por lisina e não foram observadas variações significativas entre os genótipos Oh43o1, Oh43o2, Oh43fl1 e Oh43fl2, quando comparados com o genótipo Oh43+. A composição de aminoácidos revelou as maiores concentrações de lisina solúvel e incorporada em proteínas no mutante Oh43o2. Os estudos com quinoa demonstraram a presença de pelo menos duas isoenzimas da AK, uma sensível à inibição por lisina e a outra por treonina, duas isoenzimas da HSDH, uma resistente e a outra sensível à inibição por treonina e uma isoenzima da DHDPS sensível à inibição por lisina. As sementes de quinoa também apresentaram altas concentrações de lisina solúvel e incorporada em proteínas. / Aspartate is the common precursor of the essential amino acids lysine, threonine, methionine and isoleucine. Due to the deficiency in lysine, the aspartate metabolic pathway has been studied in cereal crops and the results obtained have indicated the importance of aspartate kinase (AK), homoserine dehydrogenase (HSDH) and dihydrodipicolinate synthase (DHDPS) as the key enzymes involved in the synthesis of lysine. Considering the importance of maize and quinoa as sources of protein for humans and animals, the objectives of this work were: i) to study the enzymes AK and HSDH in the seeds of the maize mutants B77xB79o5; B37o7; W22o10, W22o11 and W22o13 and their respective wild type; II) to characterize the enzyme DHDPS and to analyze the composition of soluble and amino acids incorporated into proteins in the seeds of the maize mutants Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2 and the wild type OH43+; iii) to study the enzymes AK, HSDH, DHDPS in quinoa seeds and some aspects involved in nitrogen assimilation and amino acids profile. When the mutants B77xB79o5; B37o7: W22o10, W22o11 and W22o13 were studied, the analysis revealed important variations on AK and HSDH activities among the genotypes when compared to their respective wild type. The results indicated that DHDPS was strongly inhibited by lysine, and that there was not significant variation among the mutants Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2 when compared to the wild type. The amino acids composition revealed high concentrations of lysine in the soluble form and incorporated into protein in the Oh43o2 mutant. The study of quinoa seeds allowed the identification of at least two AK isoenzymes, one sensitive to lysine inhibition and another sensitive to threonine, two HSDH isoenzymes, one resistant and another sensitive to threonine inhibition and one DHDPS isoenzyme sensitive to lysine inhibition. Quinoa also exhibited high concentration of soluble lysine and lysine incorporated into protein.

<p>U ovoj doktorskoj disertaciji analizirano je bra&scaron;no strnih žita (p&scaron;enice, spelte, ječma,<br />raži, tritikalea, ovsa), prosolikog žita (kukuruza) i pseudocerealija (heljde i<br />amarantusa) primenom gasne hromatografije sa masenom spektrometrijom (GC-MS) u<br />kombinaciji sa multivarijantnom analizom, u cilju ispitivanja mogućnosti za<br />određivanje autentičnosti navedenih biljnih vrsta. U tu svrhu, iz bra&scaron;na navedenih<br />cerealija i pseudocerealija ekstrahovani su mali molekuli (lipidi i &scaron;ećeri). Lipidne<br />komponente bra&scaron;na ekstrahovane su heksanom. Nakon vi&scaron;estrukog obezma&scaron;ćivanja<br />istih uzoraka bra&scaron;na heksanom i su&scaron;enja, usledila je ekstrakcija &scaron;ećernih komponenata<br />96%-tnim etanolom. Dobijeni heksanski i etanolni ekstrakti derivatizovani su<br />odgovarajućim reagensima. Za derivatizacaju lipidnih komponenata kori&scaron;ćen je<br />rastvor TMSH (trimetilsulfonijum hidroksida, 0,2 M u metanolu). Time se masne<br />kiseline prevode u odgovarajuće metil-estre. Za derivatizaciju &scaron;ećernih komponenata<br />kori&scaron;ćen je etanolni rastvor natrijum-hidroksida i hidroksilamin-hidrohlorida u<br />kombinaciji sa BSTFA (bis-(trimetilsilil-trifluoroacetamidom). Na taj način su prosti<br />&scaron;ećeri prevedeni u odgovarujuće trimetilsilil-oksime. Ovako pripremljeni ekstrakti<br />uzoraka bra&scaron;na su analizirani na GC&ndash;MS uređaju.<br />Dobijeni hromatogrami međusobno pokazuju veliku sličnost, a naročito u okviru<br />uzoraka iste botaničke vrste. U obradi hromatograma i masenih spektara, kori&scaron;ćen je<br />MSD Productivity ChemStation program uz primenu Wiley 275 biblioteke masenih<br />spektara. Dobijeni podaci analizirani su na tri načina. Prvi način podrazumeva<br />kreiranje numeričkih matrica u modu ukupne jonske struje (TIC). Drugi postupak<br />podrazumeva kreiranje numeričkih matrica izolovanjem karakterističnih fragmentnih jona sa hromatograma (kod lipida 74 m/z, a kod &scaron;ećera 73 m/z u kombinaciji sa bar<br />jednim od sledećih jona: 204 m/z, 217 m/z, i 361 m/z). Kod trećeg načina obrade<br />podataka, analiza je urađena kreiranjem matrica primenom binarnog sistema (1/0), gde<br />&bdquo;1&ldquo; označava prisustvo određene komponente, a &bdquo;0&ldquo; označava njeno odsustvo u<br />posmatranom uzorku. Tako dobijeni podaci podvrgnuti su multivarijantnoj analizi<br />primenom statističkih programa &ndash; PAST i STATISTICA. U sva tri slučaja ispitivani<br />uzorci grupisani su u međusobno jasno odvojene grupe, prema odgovarajućoj biljnoj<br />vrsti. Uočeno je jasno razlikovanje pseudocerealija (heljde i amarantusa) i prosolikog<br />žita (kukuruza) od grupe strnih žita (p&scaron;enice, spelte, ječma, raži, tritikalea i ovsa).<br />Primenjenim metodama postiže se manje selektivna klasifikacija biljnih vrsta u okviru<br />strnih žita, sa izuzetkom uzoraka ovsa i spelte. Ovi rezultati pokazuju da je novim<br />navedenim postupcima moguće utvrditi autentičnost bra&scaron;na različitih botaničkih vrsta<br />cerealija i pseudocerealija.<br />Na isti način tretirani su i uzorci proizvedenog hleba (kore i sredine) u kojima je<br />p&scaron;enično bra&scaron;no supstituisano sa 0, 20, 40, 50, 60, 80 i 100% heljdinog bra&scaron;na.<br />Multivarijantnom analizom GC-MS podataka dobijaju se jasno odvojeni klasteri u<br />skladu sa porastom udela bra&scaron;na heljde u kori i sredini hleba. Najbolji rezultati<br />postignuti su koreliranjem sastava i sadržaja ugljenohidratnih komponenata u kori<br />hleba.<br />Krajnji ishod razvoja ovih novih originalnih metoda je mogućnost njihove praktične<br />primene u cilju rutinskih kontrola kvaliteta bra&scaron;na i gotovih pekarskih proizvoda u<br />laboratorijama, proizvodnim pogonima i inspekcijskim službama. Posebna pogodnost<br />primene predloženih metoda je to &scaron;to su one semi-kvalitativnog i semi-kvantitativnog<br />karaktera, pa se mogu izvoditi bez tačne identifikacije eluirajućih malih molekula</p> / <p>In this doctoral thesis various flour samples of small grains (wheat, spelt, barley, rye,<br />triticale, oats), corn and pseudocereals (buckwheat and amaranth) were analyzed using<br />gas chromatography - mass spectrometry (GC-MS) in combination with multivariate<br />analysis, in order to examine the possibilities for determining the authenticity of the<br />plant species listed. Small molecules (lipids and sugars) were extracted from flour<br />samples of the above-mentioned cereals and pseudocereals. The lipid components of<br />the flour were extracted with hexane. The extraction of sugar components from the<br />same flour samples was performed with 96% ethanol solution, after multiple defatting<br />with hexane and drying of the samples. The obtained hexane and ethanol extracts were<br />derivatized with appropriate reagents. For the derivatization of the lipid components, a<br />solution of TMSH (trimethylsulfonium hydroxide, 0.2 M in methanol) was used. Thus,<br />the fatty acids were converted into the corresponding methyl esters. For the<br />derivatization of the sugar components, an ethanol solution of sodium hydroxide and<br />hydroxylamine hydrochloride combined with BSTFA (bis- (trimethylsilyl<br />trifluoroacetamide) was used. Thus, the free sugars were converted into the<br />corresponding trimethylsilyl oximes. The derivatized extracts prepared in this manner<br />were analyzed on a GC -MS device.<br />The obtained chromatograms were very similar, and especially within the samples of<br />the same botanical species. In the processing of the chromatograms and mass spectra,<br />MSD Productivity ChemStation program was used with the Wiley 275 Mass Spectra<br />Library. The obtained data were processed in three ways. The first method involves the<br />creation of numerical matrices in the total ion current mode (TIC). The second method<br />involves the creation of numerical matrices by isolating characteristic ion fragments from the TIC chromatograms (74 m/z for the lipid components, and 73 m/z in<br />combination with at least one of the following ions: 204 m/z, 217 m/z, and 361 m/z, for<br />the sugar components). In the third way of data processing, the analysis was done by<br />creating a matrix using a binary system (1/0), where &ldquo;1&rdquo; denotes the presence of a<br />particular component, and &ldquo;0&rdquo; denotes its absence in the observed sample. The data<br />thus obtained were subjected to multivariate analysis using statistical programs - PAST<br />and STATISTICA. In all three cases, the analyzed samples were grouped in clearly<br />separated groups according to the appropriate plant species. A clear distinction was<br />observed between pseudocereals (buckwheat and amaranth), corn and the group of<br />small grains (wheat, spelt, barley, rye, triticale, oats). The applied methods achieved a<br />less selective classification of plant species within the group of small grains, with the<br />exception of oats and spelt samples. These results show that it is possible to determine<br />the authenticity of flour of various botanical species of cereals and pseudocereals,<br />applying new methods described in this doctoral thesis.<br />The samples of produced bread (crust and crumbs) in which wheat flour was<br />substituted with 0, 20, 40, 50, 60, 80 and 100% of buckwheat flour were treated in the<br />same way. Multivariate analysis of GC-MS data provided clearly separated clusters in<br />accordance with the increase in the share of buckwheat flour in bread crusts and<br />crumbs. The best results were achieved by correlating the composition and content of<br />the carbohydrate components in bread crusts.<br />The ultimate outcome of the development of these new original methods is the<br />possibility of their practical application for the purpose of routine quality control of<br />flour and bakery products in laboratories, production facilities and inspection services.<br />A particular advantage of applying the proposed methods is that they are semiqualitative<br />and semi-quantitative. Therefore, they can be performed without the exact<br />identification of eluting small molecules (lipids and free sugars), nor their<br />quantification using analytical standards.</p>

Philosophiae Doctor - PhD / The role of phenolic acids in mitigating salt stress tolerance have been well documented. However, there are contradicting reports on the effect of exogenously applied phenolic acids on the growth and development of various plants species. A general trend was observed where phenolic acids were shown to inhibit plant growth and development, with the exception of a few documented cases. One of these such cases is presented in this thesis. This study investigates the role of exogenously applied p-coumaric acid (p-CA) on physio-biochemical and molecular responses of chia seedlings under salt stress. This study is divided into three parts. Part one (Chapter 3) focuses on the impact of exogenous p-coumaric acid on the growth and development of chia seedlings. In this section, chia seedlings were supplemented with exogenous p-CA and the various biochemical and plant growth parameters were measured. The results showed that exogenous p-CA enhanced the growth of chia seedlings. An increase in chlorophyll, proline and superoxide oxide contents were also observed in the p-CA treatment relative to the control. We suggested that the increase in chia seedling growth could possibly be via the activation of reactive oxygen species-signalling pathway involving O2− under the control of proline accumulation (Chapter 3). Given the allopathy, nature of p-coumaric acid it is noteworthy that the response observed in this study may be species dependent, as contrasting responses have been reported in other plant species. Part two (Chapter 4) of this study investigates the influence of piperonylic acid (an inhibitor of endogenous p-coumaric acid) on the growth and development of chia seedlings. In trying to illustrate whether p-CA does play a regulatory role in enhancing pseudocereal plant growth, we treated chia seedlings with the irreversible inhibitor of C4H enzyme, to inhibit the biosynthesis of endogenous p-CA. In this section, chia seedlings were treated with piperonylic acid and changes in plant growth, ROS-induced oxidative damage, p-CA content and antioxidant capacity was monitored. Inhibition of endogenous p-CA restricted chia seedling growth by enhancing ROS-induced oxidative damage as seen for increased levels of superoxide, hydrogen peroxide and the extent of lipid peroxidation. Although an increase in antioxidant activity was observed in response to piperonylic acid, this increase was not sufficient to scavenge the ROS molecules to prevent oxidative damage and ultimate cellular death manifested as reduced plant growth. The results presented in this section support our hypothesis that p-CA play an important regulatory role in enhancing chia seedling growth and development as shown in Chapter 3. Part three (Chapter 5) seeks to identify and functionally characterise p-coumaric acid induced putative protein biomarkers under salt stress conditions in chia seedlings. Previous studies have shown that p-CA reversing the negative effect caused by NaCl-induced salt stress. While these studies were able to demonstrate the involvement of p-CA in promoting plant growth under salt stress conditions, they focussed primarily on the physiological aspect, which lacks in-depth biochemical and molecular analysis (ionomic and proteomic data) which could help in detecting the genes/proteins involved in salt stress tolerance mechanisms. A comparative ionomics and proteomic study was conducted, with the aim of elucidating the pivotal roles of essential macro elements and/or key protein markers involved in p-CA induced salt stress tolerance in chia seedlings. With the exception of Na, all the other macro elements were decreased in the salt treatment. Contrary to what was observed for the salt treatment most of the macro elements were increased in the p-CA treatment. However, the addition of exogenous p-CA to salt stressed seedlings showed an increase in essential macro elements such as Mg and Ca which have been shown to play a key role in plant growth and development. In the proteomic analysis we identified 907 proteins associated with shoots across all treatments. Interestingly, only eight proteins were conserved amongst all treatments. A total of 79 proteins were unique to the p-CA, 26 to the combination treatment (NaCl + p-CA) and only two proteins were unique to the salt stress treatment. The unique proteins identified in each of the treatments were functionally characterised to various subcellular compartments and biological processes. Most of the positively identified proteins were localised to the chloroplast and plays key roles in photosynthesis, transportation, stress responses and signal transduction pathways. Moreover, the protein biomarkers identified in this study (especially in the p-CA treatment) are putative candidates for genetic improvement of salt stress tolerance in plants.