Lectins are a class of proteins that bind to carbohydrates with a high degree of specificity. They are involved in various cellular processes such as, host - pathogen interactions, targeting of proteins within cells, cell - cell interaction, cellular segregation and development. They serve as important tools for probing the carbohydrate structures in biological systems such as cell membranes and also as model systems for elucidating protein - carbohydrate interactions. Lectins are distributed ubiquitously in nature ranging from microorganisms to the plants and animals. Plant lectins are a group of proteins that according to a recently updated definition comprise all plant proteins possessing at least one non-catalytic domain that binds reversibly to specific mono- or oligosaccharide. The majority of all currently known plant lectins may be classified into four major groups - (1) Legume lectins, (2) Chitin-binding lectins, (3) Type 2 Ribosome inactivating proteins and the (4) Monocot mannose binding lectins. The monocot mannose binding lectins are an extended superfamily of structurally and evolutionarily related proteins. Till now these proteins have been isolated from the following families, namely, Amaryllidaceae, Affiaceae, Araceae, Orchidaceae, Iridaceae and Li/iaceae. They exhibit marked sequence homology and a unique specificity for mannose. At present there is a wide interest in the monocot mannose-binding lectins because of: (1) their exclusive specificity towards mannose, (2) their anti - retroviral activity and (3) their potent entomotoxic properties. Of particular interest are lectins from the bulbs of garlic (Allium sativum) and ramson (A. ursinum), which contain more than one type of lectin. The first report of the presence of lectins in the bulbs of garlic {Allium sativum agglutinin, ASA) was made by Van Damme et al in 1991. Bulbs of garlic are known to accumulate two types of mannose binding lectins, the heterodimeric, ASAI and the hornodimeric, ASAII. Though these two lectins differ in the lengths of their polypeptide chains, they exhibit marked similarities with respect to their primary sequence, post translational modifications, serological properties, immunochemical attributes as well as carbohydrate binding properties. This thesis describes the successful cloning of the ASAI gene from the garlic genomic DNA and expression of the functional recombinant protein in insect cell lines. ASAI was subsequently characterized for its carbohydrate binding specificity by means of a sensitive enzyme based assay. Finer insights into this sugar binding topology of ASAI for its complementary ligands was obtained from the surface plasmon resonance studies. Lastly, the folding behaviour as well as an estimate of its conformational stability was investigated by differential scanning calorimetric and equilibrium solution denaturation studies. Chapter 1 provides a comprehensive review on lectins pertaining to their definition, historical background, occurrence in nature, three dimensional structure and architecture, modes of bonding, biological functions and implications as well as their applications in biomedical research. Chapter 2 describes the isolation and purification of the heterodimeric lectin, ASAI in two steps using affinity chromatography followed by gel filtration chromatography from the bulbs of garlic. The purified ASAI was then characterized for their serological, physico- and immuno-chemical properties by means of capillary electrophoresis, hemagglutination activity and generation of antisera against ASAI in rabbits. Chapter 3 revolves around the cloning of the gene encoding ASAI by PCR amplification from garlic genomic DNA. The authenticity of the ASA gene was established by means of gene sequencing, which in turn provided us with the primary sequence of this lectin. With the ASAI clone established innumerable attempts, as highlighted in the chapter, were made to express the functional protein in bacteria. All attempts yielded pure recombinant garlic lectin with no detectable activity. This prompted us to shift our efforts into expression of the recombinant protein in the baculovirus expression system using the Sf21 insect cell lines and the Autographa californica nuclear polyhedrosis virus (AcNPV). The choice of this system proved beneficial as we obtained functional recombinant garlic lectin with its hemagglutinating activity comparable to the native protein. Chapter 4 highlights the design of an elegant coupled enzyme-based colorimetric assay (Enzyme Linked Lectin Adsorbent Assay) for elucidation of the carbohydrate binding specificity of ASAI. This expansive and extensive study involved the assay of a wide range of mannooligosaccharides in order to gain an insight into the sugar binding details of ASAI. ASAI recognizes monosaccharides in the mannosyl configuration. The potencies of the ligands for ASAI is shown to increase in the following order: Mannobiose < Mannotriose Mannopentaose Man9 oligosaccharide. Mannononase glycopeptide (Man9GlcNAc2Asn), the highest oligomer studied exhibited the greatest binding affinity suggesting ASAI to possess a preference for cluster of terminal αl-2-linked mannosyl residues at the non-reducing end. This kind of exquisite specificity is unique in the lectins described so far. Among the glycoproteins assayed, invertase, soyabean agglutinin and ovalbumin displayed high binding affinity. Chapter 5 unravels the fine specificity of the mannose containing carbohydrate moieties for binding to ASAI with emphasis on their kinetics of binding. This has been achieved by invoking the principle of surface plasmon resonance allowing measurement of bimolecular interactions in real time. This investigation corroborates our earlier study about the special preference of garlic lectin for terminal a α1-2 linked mannose residues. Increase in binding propensity can be directly correlated to the addition of αl-2 linked mannose to the mannooligosaccharide at its non-reducing end. An analyses of these data reveals that the α1-2 linked terminal mannose on the α1-6 arm to be the critical determinant in the recognition of mannooligosaccharides by the lectin. While kI increases progressively from Man3 to Man7 derivatives, and more dramatically so for Man8 and Man9 derivatives, k-1 decreases relatively much less gradually from Man3 to Man9 structures. An unprecedented increase in the association rate constant for interaction with ASAI with the structure of the oligosaccharide ligand constitutes a significant finding in protein-sugar recognition. Chapter 6 deals with the thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism which shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers (A2 2U). Moreover, its conformational stability has been determined as a function of temperature; GdnCl concentration and pH using a combination of thermal and isothermal GdnCl induced unfolding monitored by DSC, far-UV CD and fluorescence, respectively. Analysis of these data yielded the heat capacity change upon unfolding (∆CP) as also the temperature dependence of the thermodynamic parameters, namely, ∆G, ∆H, ∆S. The protein appears to attain a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates very well with its X-ray structure. The final chapter summarizes the findings reported in the thesis.

Allium sativum has a long tradition in medicine. While much is known about its potential healthy effects, nearly nothing is known about wild garlic (allium sativum, ramson), which is very common in the area of Leipzig and has been used as a herbal remedy since centuries. The goal of the present study was to assess a potential anti-platelet activity of these two allium species and to try to identify the chemical active principle. For that purpose various extracts (hydrophilic and lipophilic) were prepared from Allium sativum and Allium ursinum, and analysed using thin layer chromatography and HPLC. After identifying an active, i.e. antiaggregatory extract (see below), this was fractionated and the active fraction was further sub-fractionated for subsequent chemical analysis by mass spectroscopy, ESI (Electrospray ionization), and COSY (Correlation effect spectroscopy), and functional testing. Anti-platelet activity was assessed in human platelets (platelet rich plasma) using a classical turbidimetric method. Platelets were stimulated with various agonists (arachidonic acid, ADP, epinephrine, collagen, A23187) with and without the addition of the extracts or the fractions /sub-fractions. Both Allium Ursinum and Allium sativum extracts exert antiaggregatory effects with EC50 values around 0.1 mg/ml. The garlic extracts are acting by inhibition of the ADP pathway comparable as known from the clinically used drug clopidegrol.The pharmacological active antiaggregatory component of the extracts appears to be lipophilic rather than hydrophilic. This is the first report on an antiplatelet activity of Allium Ursinum. One final structure determined by HPLC, MS, ESI and COSY which exerts the antiplatelets inhibitory effect is β-sitosterol-3-O-β-D-glucoside of the fraction 7-14 crystals. It is considered that about three up to five grams of dried leaves might be enough to exert antiaggregatory effects (comment: in pharmacy normally dried plant material is used in therapy). The second compound with antiaggregatory activity was identified as 1-β-D-galactopyranoside-2, 3-bis-linolenic glycerate. The problem of loosing the active volatile oily components by drying the leaves in future studies looking for the clinical use may be solved by looking for a raw or a refined extract which would be the form of a real phytomedical drug; for example capsules about 120 to 200 micrograms of an alcoholic or better an heptane / oily extract gained from wood garlic leaves would be an useful drug formulation to reach respective concentrations in blood. However, we have to admit that since our investigations were in-vitro, the in-vivo situation is somewhat different due to the metabolism, which is nearly unknown. Nevertheless, this study shows for the first time that allium ursinum does exert anti-platelet activity and that both allium species can unfold antiaggregatory effects which are worth to be investigated in subsequent in-vivo studies. β-sitosterol-3-O-β-D-glucoside and 1-β-D-galactopyranoside-2, 3-bis-linolenic glycerate could be identified as active antiaggregatory principals.

Allium sativum has a long tradition in medicine. While much is known about its potential healthy effects, nearly nothing is known about wild garlic (allium sativum, ramson), which is very common in the area of Leipzig and has been used as a herbal remedy since centuries. The goal of the present study was to assess a potential anti-platelet activity of these two allium species and to try to identify the chemical active principle. For that purpose various extracts (hydrophilic and lipophilic) were prepared from Allium sativum and Allium ursinum, and analysed using thin layer chromatography and HPLC. After identifying an active, i.e. antiaggregatory extract (see below), this was fractionated and the active fraction was further sub-fractionated for subsequent chemical analysis by mass spectroscopy, ESI (Electrospray ionization), and COSY (Correlation effect spectroscopy), and functional testing. Anti-platelet activity was assessed in human platelets (platelet rich plasma) using a classical turbidimetric method. Platelets were stimulated with various agonists (arachidonic acid, ADP, epinephrine, collagen, A23187) with and without the addition of the extracts or the fractions /sub-fractions. Both Allium Ursinum and Allium sativum extracts exert antiaggregatory effects with EC50 values around 0.1 mg/ml. The garlic extracts are acting by inhibition of the ADP pathway comparable as known from the clinically used drug clopidegrol.The pharmacological active antiaggregatory component of the extracts appears to be lipophilic rather than hydrophilic. This is the first report on an antiplatelet activity of Allium Ursinum. One final structure determined by HPLC, MS, ESI and COSY which exerts the antiplatelets inhibitory effect is β-sitosterol-3-O-β-D-glucoside of the fraction 7-14 crystals. It is considered that about three up to five grams of dried leaves might be enough to exert antiaggregatory effects (comment: in pharmacy normally dried plant material is used in therapy). The second compound with antiaggregatory activity was identified as 1-β-D-galactopyranoside-2, 3-bis-linolenic glycerate. The problem of loosing the active volatile oily components by drying the leaves in future studies looking for the clinical use may be solved by looking for a raw or a refined extract which would be the form of a real phytomedical drug; for example capsules about 120 to 200 micrograms of an alcoholic or better an heptane / oily extract gained from wood garlic leaves would be an useful drug formulation to reach respective concentrations in blood. However, we have to admit that since our investigations were in-vitro, the in-vivo situation is somewhat different due to the metabolism, which is nearly unknown. Nevertheless, this study shows for the first time that allium ursinum does exert anti-platelet activity and that both allium species can unfold antiaggregatory effects which are worth to be investigated in subsequent in-vivo studies. β-sitosterol-3-O-β-D-glucoside and 1-β-D-galactopyranoside-2, 3-bis-linolenic glycerate could be identified as active antiaggregatory principals.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Apples are an important export commodity for the South African market, and postharvest losses that occur as a result of decay due to infection with pathogenic fungi such as Botrytis cinerea Pers., Penicillium expansum (Link) Thom. and Neofabraea alba (E.J. Guthrie) are of major concern for all parties concerned with fruit production and distribution. Decay control of these fungi is primarily managed through the use of synthetic fungicides; however, pathogen development of resistance to these fungicides and recent worldwide concern over healthier living and a greener environment has called for the discriminate use of synthetic chemicals. This has opened up an avenue for the development of safer and more environmentally friendly alternatives to control postharvest decays. The use of plant extracts and essential oils are favoured as natural sources of antimicrobials whilst still being safe for human consumption and having no negative impact on the environment. Allium sativum (garlic) is one such plant species that is well documented for its value in improving human health and is readily available for consumption not just as a flavour component of food but also to be taken as a daily herbal diet supplement. Given the antimicrobial effectiveness of garlic against human pathogens and ailments, its value as an antifungal agent against postharvest pathogens causing grey mould, blue mould and bull’s eye rot of apples was investigated in vitro and in vivo within this study. Furthermore, an attempt was made to elucidate the chemical components of garlic extracts by gas chromatography-mass spectrometry (GC-MS). All experiments in this study were carried out with garlic extracts prepared from fresh garlic bulbs. For the in vitro experiments, two extract preparations of garlic, one containing ethanol (Extract 1) and one where ethanol had been removed by evaporation (Extract 2), was tested for antifungal action within an amended media experimental design. Both extract preparations were each subjected to two dilution series (0-80% garlic extract) with water and ethanol as diluents. Both extract preparations were successful at retarding pathogen mycelial growth and spore germination; however, concentrations of Extract 2 (ethanol evaporated) and diluted with distilled water provided markedly better inhibition of B. cinerea and P. expansum than the ethanolic dilutions of extract 2. Both extract preparations yielded similar inhibitory results when tested against N. alba. Due to the results achieved in the amended media experiments, the use of a crude garlic extract without ethanol and diluted in water was considered to be the best option for further tests throughout the remainder of the study. In vitro volatile effects of crude garlic extracts at concentrations between 0 and 40% garlic extract were subsequently tested. Garlic volatiles were effective in inhibiting pathogen mycelial growth and spore germination of all three pathogens, at lower concentrations compared to the amended media experiments. In vitro volatile exposure with garlic extracts was more effective at inhibiting N. alba than direct application of the extracts. Curative and protective application of garlic extracts and clove oil for increased fungal inhibition through synergism was tested by direct and volatile exposure to the pathogens in vivo on three economically important apple cultivars; ‘Granny Smith’, ‘Golden Delicious’, and ‘Pink Lady’. Direct exposure of artificially wounded and inoculated fruit to the garlic extract and clove oil revealed that garlic extracts applied curatively but not protectively effectively controlled decay caused by B. cinerea and P. expansum on all apple cultivars. Both curative and protective applications were ineffective in controlling N. alba. In vivo volatile exposure to the garlic extracts and clove oil did not inhibit decay on any of the cultivars and was not effective against any of the three pathogens investigated. A full chemical profile analysis was done by GC-MS analysis of garlic extract samples. The compounds diallyl disulphide, allyl methyl trisulphide, allyl methyl disulphide and dimethyl trisulphide were detected in relatively high amounts. This result suggests that the abundance of sulphur and sulphur related compounds detected may be responsible for the antifungal action noted in the experimental studies. In conclusion, garlic was shown to have antifungal activity against B. cinerea, P. expansum and N. alba. The pathogens used in this study were not compared with each other, but undoubtedly each pathogens reacts differently to exposure to the garlic extracts. It would therefore be advisable to investigate the effects of the extracts on each of the pathogens in a more in-depth study. More investigations into the application of the garlic extracts is required before it may be recommended for use; however, results for the use of garlic extracts against these postharvest pathogens and the postharvest decay they cause are promising. / AFRIKAANSE OPSOMMING: Appels is ‘n belangrike uitvoerproduk vir die Suid-Afrikaanse vrugtebedryf, maar noemenswaardige na-oes verliese word weens bederf deur patogeniese swamme soos Botrytis cinerea Pers., Penicillium expansum (Link) Thom. en Neofabraea alba (E.J. Guthrie) ervaar. Dit raak alle partye betrokke met die produksie en verspreiding van hierdie vrugsoort. Hierdie swamme word hoofsaaklik met behulp van kunsmatige swamdoders beheer, alhoewel weerstand-ontwikkeling en wêreldwye bewusmaking van ‘n gesonder leefstyl en omgewing die gebruik van kunsmatige middels streng aanspreek en die ontwikkeling van veiliger en meer omgewingsvriendelike alternatiewe middels verlang. Plant-ekstrakte en essensiële olies kan dien as sulke middels en is natuurlike bronne van anti-mikrobiese aktiwiteit, is veilig vir menslike verbruik en het ook geen negatiewe invloed op die omgewing nie. Allium sativum (knoffel) is so ‘n plantspesie wat as alternatiewe middel gebruik kan word. Dit is bekend vir sy waarde in die verbetering van menslike gesondheid, is maklik bekombaar en word nie net as ‘n geurmiddel vir voedsel gebruik nie, maar ook as ‘n daaglikse krui-aanvulling. Gegewe die anti-mikrobiese doeltreffendheid van knoffel teenoor menslike patogene en kwale, is die werking (in vitro en in vivo) teen na-oes patogene wat grys skimmel, blou skimmel en teikenvrot in appels veroorsaak, in hierdie studie ondersoek. Bepaling van die chemiese samestelling van die knoffel-ekstrak is ook met behulp van gaschromatografie massa spektrometrie (GK-MS) onderneem.Vars knoffelbolle is vir elke eksperiment in hierdie studie gebruik met die voorbereiding van die knoffel-ekstrak. Vir die in vitro eksperiment is twee knoffel-ekstrakte voorberei, naamlik: ‘n ekstrak wat etanol bevat (Ekstrak 1) en een waarvan die etanol verwyder is met verdamping (Ekstrak 2). Die ekstrakte is getoets vir werking teen fungi in kultuur-medium.. Albei ekstrakte is verdun tot twee konsentrasie reekse (0-80%) met water en etanol as verdunningsmiddels. Albei ekstrakte het suksesvolle werking getoon teenoor die patogene ten opsigte van vertraging van miseliumgroei en spoor-ontkieming, alhoewel konsentrasies van Ekstrak 2, verdun met gesuiwerede water, patogene B. cinerea en P. expansum beter onderdruk het as Ekstrak 2 verdunnings met etanol.. Beide ekstrakte en hul afsonderlike verdunnings met etanol en water het soortgelyke resultate gelewer met onderdrukking van N. alba. Volgens resultate wat verkry is van die kultuur-medium eksperimente, is Ekstrak 2 verdun met gesuiwerde water beskou as die geskikste vir verdere toetse in hierdie studie. Die vlugtige effek van Ekstrak 2 is in vitro getoets by konsentrasies tussen 0 tot 40%. Die vlugtige stowwe van knoffel het al drie patogene se groei en spoor-ontkieming effektief onderdrukby laer konsentrasies as wat gebruik is in die kultuur-medium eksperiment. Dus is in vitro blootstelling van N. alba aan die vlugtige stowwe meer effektief as direkte toediening van die ekstrakte. Die voorkomende en beskermende effek van die knoffel-ekstrak, asook naeltjie-olie, is in vivo ondersoek om te bepaal of die stowwe saam sterker onderdrukking van die patogene kon bewerkstellig. Direkte en vlugtige blootstelling is op drie ekonomies-belangrike appel-kultivars getoets, naamlik: ‘Granny Smith’, ‘Golden Delicious’ en ‘Pink Lady’. Direkte blootstelling met die knoffel-ekstrak en naeltjie-olie aan gewonde en ge-inokuleerde vrugte het aangedui dat B. cinerea- en P. Expansum-bederf net beheer kon word indien knoffel voorkomend toegedien is vir al die ondersoekte appel-variëteite. Voorkomende en beskermende toediening was onsuksesvolle om N. alba te beheer. In vivo blootstelling van die drie patogene aan die knoffel-ekstrak en naeltjie-olie se vlugtige stowwe kon nie enige van die patogene effektief onderdruk nie en was onsuksesvol in bederf-beheer. ‘n Volledige chemiese profiel is saamgestel deur GK-MS ontleding van die knoffelekstrakte. Hoë vlakke van verbindings dialliel disulfied, alliel-metiel-tri-sulfied, alliel-metieldisulfied en dimetiel-trisulfied is bespeur. Die aantal vrye sulfied en sulfied-verwante verbindings in die ekstrak kan moontlik ‘n verduideliking bied vir die anti-swam werking waargeneem gedurende hierdie studie. Ten slotte: knoffel toon ‘n anti-swam werking teenoor B. cinerea, P. expansum en N. alba. Die patogene in hierdie studie is nie met mekaar vergelyk nie, omdat elkeen uniek en uiteenlopend op knoffel reageer het. Alhoewel die huidige studie alreeds belowende resultate gelewer het, moet die ekstrak se effek op elke patogeen onderskeidelik nog in diepte ondersoek word, asook die wyse van die toediening in die na-oes praktyk voordat hierdie middel aanbeveel kan word vir gebruik.

Causes and patterns of invasive plant species establishment and success depend broadly upon their ecology, including habitat suitability and interactions with other plants and animals. Here I examine the traits and distribution of invasive plants in Vermont, using spatial analysis, laboratory and field studies. I used GIS to investigate environmental factors correlated with presence of 19 invasive plant species in Vermont campgrounds. My results support the assumption that human dispersal of invasive plant seed and stock may be more important than natural dispersal of these plant species to new sites. I also investigate in-depth the relationships of invasive herbaceous garlic mustard (Alliaria petiolata) with native tree seedlings and co-occurring herbaceous plants in the greenhouse and Vermont forests, respectively. Shade from > 1 m tall A. petiolata plants may effect root:shoot ratios of neighboring tree seedlings and interact with nutrition quality of sites to affect their growth patterns. Invasive plants' integration into novel environments is also mediated by their interactions with native invertebrate species. A. petiolata is associated with a unique assemblage of aboveground invertebrates compared with neighboring native plants. Observations indicate A. petiolata may also serve as an attractant for ants, bees, and wasps who feed from water and nectar at the base of the flower or silique during its flowering and seeding period. These results collectively inform our understanding of plant invasion patterns and management strategies of A. petiolata in Vermont. Community interactions are probably more important than allelopathy in determining the influence of Alliaria petiolata on native ecosystems.

O alho é uma hortaliça importante não só pelo atrativo culinário, como também pelo grande número de propriedades medicinais. A utilização efetiva de recursos genéticos, conservados em bancos de germoplasma, em programas de melhoramento depende de criteriosa caracterização, utilizando em combinação caracteres agromorfológicos e marcadores moleculares. Neste estudo, 16 novos locos microssatélites específicos para a espécie foram desenvolvidos, 10 polimórficos. Os bancos de germoplasma de alho, das instituições IAC, ESALQ e Embrapa, foram caracterizados utilizando 17 locos microssatélites polimórficos, sete deles disponíveis na literatura. A maioria dos locos foi considerada moderado a altamente informativo, com PIC médio de 0,545 e máximo de 0,851. Foram encontrados 90 alelos, com riqueza alélica média de 2,258 alelos por loco e índice de Shannon de 1,176. A coleção da Embrapa apresentou destaque, com maior número de alelos privados e genótipos multi locos (MLGs). Os 151 acessos avaliados foram representados por 65 MLGs. A estratégia M foi utilizada para definição de uma coleção nuclear, que foi constituída por 16 acessos, com cobertura de 100% dos alelos e mínima redundância. O GST geral foi de 0,200 e para MLGs de 0,068, indicando alta diferenciação genética dentro dos bancos de germoplasma, e GIS de 0,195, indicando excesso de heterozigotos, comum em espécies de propagação vegetativa como o alho. A estruturação genética dos MLGs resultou na distinção de dois subgrupos pelo método Bayesiano, consistente com os agrupamentos observados no dendrograma e PCA. Os grupos formados apresentaram coerência com a classificação de acessos de acordo com o ciclo fenológico, em nobres e seminobres. Os marcadores microssatélites desenvolvidos neste trabalho são ferramentas valiosas para estudos de diversidade genética, conservação de germoplasma, mapeamento genético e associativo e espera-se que sejam úteis em futuros programas de melhoramento da espécie. / Garlic is an important crop not only for its culinary attractiveness, but also because of a huge number of medicinal properties. The genetic resources in germplasm require characterization by morphological traits and molecular markers to be effectively used in breeding programs. A novel set of 16 SSR loci specific to garlic were developed. Ten loci were polymorphic, moderate to highly informative, with an average PIC of 0.545 and maximum of 0.851. A total of 151 accessions of garlic from three Brazilian germplam, IAC, ESALQ, and Embrapa, were evaluated for genetic diversity using 10 novel polymorphic SSR loci and other 7 available in the literature. A total of 90 alleles were detected, the average allelic richness was 2.258 alleles per locus and the Shannon index 1.176. The Embrapa collection had the vast majority of private alleles and multi locus genotypes (MLGs). The whole collection was reduced to 65 MLGs. The M strategy were used to define a core collection, 16 accessions were selected with 100% coverage of alleles and minimum redundancy. Overall GST was 0.200 and for MLGs, 0.068, indicating a high genetic differentiation within collections, and GIS was 0.195, indicating an excess of heterozygosity, which was expected as garlic is vegetatively propagated. MLGs were structured in two subgroups by Bayesian approach, consistent with the clustering based on genetic distances and PCA. The groups were coherent with the classification of accessions according to phenology (noble and semi-noble). The new SSR loci are tools for future studies of genetic diversity, germplasm conservation, mapping, and associative genetics, and hopefully will shed light on garlic breeding programs.

Tez (Yüksek Lisans) - Süleyman Demirel Üniversitesi, Fen Bilimleri Enstitüsü, Gıda Mühendisliği Anabilim Dalı, 2008. / Kaynakça var.

Thesis (M.S.)--University of Toledo, 2009. / Typescript. "Submitted as partial fulfillment of the requirements for The Master of Science Degree in Biology (Ecology-track)." "A thesis entitled"--at head of title. Bibliography: leaves 44-55.

Thesis (M.S.)--Miami University, Dept. of Botany, 2006. / Title from first page of PDF document. Includes bibliographical references.

O alho (Allium sativum L.) originário das zonas temperadas da Ásia Central é uma planta herbácea da família Alliaceae; possui como principais constituintes funcionais a alicina e a inulina. A alicina é um componente ativo e responsável pelo cheiro característico e atividade antimicrobiana, enquanto que a inulina é classificada como prebiótico e como fibra alimentar solúvel por ser resistente à digestão na parte superior do trato intestinal. Neste trabalho foram estudadas as propriedades físico-químicas do alho in natura, além do processo de branqueamento em água e vapor e a desidratação por ar quente e liofilização no alho. Os bulbos de alho foram limpos e selecionados considerando a ausência de injúrias visuais e infecções, bem como a uniformidade de tamanho e cor. O alho in natura apresentou umidade de 64,15±0,09 %, atividade de água de 0,986±0,001, sólidos solúveis de 36,00±0,85 °Brix e pH de 6,41±0,008. As concentrações de inulina, glicose e frutose foram de 56,62±0,89, 2,37±0,03 e 2,23±0,05 g/100g de matéria seca respectivamente. Os bulbilhos do alho foram descascados e cortados em rodelas com diâmetros de 15±2,40 mm e espessuras de 1±0,35 mm. A seguir as amostras passaram pelo processo de branqueamento em água previamente aquecido a 80 e 90 °C e em vapor a 100 °C à pressão atmosférica. Foram empregados tempos de 1, 2, 4, 6, 8 e 10 minutos em ambos os casos. Nesta etapa verificou-se o efeito do tempo e temperatura de branqueamento sobre a atividade das enzimas peroxidase, polifenoloxidase e inulinase e, mediram-se os parâmetros de cor L*, a* e b*, avaliando-se a cinética de inativação dessas enzimas e das mudanças de cor. A análise das concentrações de inulina, glicose e frutose foram realizadas para as melhores condições de branqueamento utilizando Cromatografia Líquida de Alta Eficiência. A melhor condição de branqueamento foi obtida no vapor por 4 minutos, onde não se observaram mudanças na textura, com redução da atividade enzimática de 93,53 %, 92,15 % e 81,96 % para a peroxidase, polifenoloxidase e inulinase respectivamente. Nesta condição a concentração da inulina diminuiu 3,72 %, enquanto que a glicose aumentou 0,67 % e a frutose 0,55 %, quando comparadas ao alho in natura, devido à atividade residual da inulinase. Durante o branqueamento em água e vapor o parâmetro de cor L* aumentou com o tempo, tornando as amostras mais claras, enquanto que a* e b* diminuíram, obtendo-se rodelas mais esverdeadas e azuladas. Na secagem, as rodelas de alho sem ou com branqueamento em vapor por 4 minutos, foram levadas a um secador de ar forçado às temperaturas de 50, 60 e 70 °C durante 6 horas. Para a liofilização as rodelas foram previamente congeladas a -80 °C por 24 horas e dispostas em bandejas dentro de um liofilizador empregando pressão de 64 μmHg, onde permaneceram por tempo médio de 48 horas. Ao estudar a cinética de secagem em ar forçado usando os modelos propostos por Henderson-Pabis, Page e Newton constatou-se o aumento da constante da taxa de secagem com o aumento da temperatura e com o uso do branqueamento. Os valores de umidade e de atividade de água no equilíbrio, obtidos a partir da porção assintótica das curvas de secagem em condições dinâmicas, estiveram na faixa de 0,086 a 0,102 g/g de matéria seca e de 0,376 a 0,521 aw respectivamente, sendo estes menores com o aumento da temperatura e com emprego do branqueamento. Posteriormente as amostras desidratadas por ar quente e liofilização, foram moídas para medição dos parâmetros de cor, tamanho de partículas e determinação dos teores de inulina, glicose e frutose, temperatura de transição vítrea e observação da microestrutura por microscopia eletrônica de varredura. As amostras liofilizadas tiveram valores de L* significativamente maiores, e parâmetros de a* e b* menores que as amostras desidratadas em ar forçado, sendo as amostras liofilizadas mais claras, esverdeadas e azuladas, próxima à cor do alho in natura. Também foi encontrado a diminuição da concentração de inulina e aumento dos teores de glicose e frutose nas amostras desidratadas, indicando a possível hidrólise da inulina, podendo estar relacionada à atividade residual da enzima inulinase. Também foi observado nas amostras branqueadas menores concentração de inulina, glicose e frutose, que quando não branqueadas, devido à possível lixiviação na água. Nas amostras liofilizadas os diâmetros médios das partículas foram de 79,35 μm e 104,79 μm no alho sem e com branqueamento respectivamente, sendo menores que às desidratadas em ar quente que variaram de 119,28 μm à 302,04 μm, observando-se, por microscopia eletrônica, menores danos na superfície das amostras liofilizadas, pois este processo origina menor contração e conseqüentemente menor rugosidade nas amostras quando comparado com a secagem em ar. As temperaturas de transição vítrea do alho desidratado em pó variaram de 99,39±1,98 °C à 120,15±3,80 °C, sendo maior quanto menor o valor de atividade de água, confirmando o efeito plasticizante da água. / Garlic (Allium sativum L.) originated in the temperate zones of Central Asia and is a herbaceous plant of the Alliaceae family; its main functional components are allicin and inulin. Allicin is the active component responsible for the characteristic odor and anti-microbial activity, whereas inulin is classified as a prebiotic substance and as a soluble dietary fiber since it is resistant to digestion in the small intestine. The physicochemical properties of garlic in natura were studied in the present work, and also the processes of water and steam blanching and dehydration in hot air and by freeze-drying. The garlic cloves were cleaned and selected considering the absence of visual injuries and infections and also uniformity of size and color. The in natura garlic presented a moisture content of 64.15±0.09 %, water activity of 0.986±0.001, soluble solids of 36.00±0.85 °Brix and pH of 6.41±0.008. The inulin, glucose and fructose concentrations were 56.62±0.89, 2.37±0.03 and 2.23±0.05 g/100g of dry matter, respectively. The garlic cloves were peeled and cut into slices with diameters of 15±2.40 mm and thicknesses of 1±0.35 mm. The samples were submitted to blanching in water baths previously heated to 80 and 90 ºC and, steam at a temperature of 100 ºC at atmospheric pressure. Times of 1, 2, 4, 6, 8 and 10 minutes were employed for both water and steam blanching. At this stage the effects of blanching time and temperature on the activities of the enzymes peroxidase, polyphenoloxidase and inulinase were determined, and the color parameters of L*, a* and b* were measured, evaluating the kinetics of inactivation of these enzymes and the color changes. The concentrations of inulin, glucose and fructose were determined by high performance liquid chromatography in the samples treated with the best of the blanching conditions. The best blanching conditions were obtained using steam for 4 minutes. Under these conditions no changes in texture were observed, and the enzymatic activities were reduced by 93.53 %, 92.15 % and 81.96 % for peroxidase, polyphenoloxidase and inulinase, respectively. Under these conditions the inulin concentration decreased by 3.72 % and the glucose and fructose concentrations increased by 0.67 % and 0.55 %, respectively, when compared to the in natura garlic, due to the residual inulinase activity. The color parameter L* increased with time during both steam and water blanching, whereas a* and b* decreased, obtaining slices which were greener and bluer. For drying, the garlic slices, with or without steam blanching for 4 minutes, were placed in forced air dryers at 50, 60 and 70 ºC for 6 hours. For freeze drying, the slices were previously frozen at -80 ºC for 24 hours and then placed in trays in the freeze drier at a pressure of 64 μmHg, where they remained for a mean time of 48 hours. The models proposed by Henderson-Pabis, Page & Newton were used to study the drying kinetics, and it was shown that the drying rate constant increased with increase in temperature and with the use of blanching. The equilibrium values for moisture content and water activity (aw), obtained from the asymptotic part of the drying curves under dynamic conditions, were in the range from 0.086 to 0.102 g/g of dry matter and from 0.376 to 0.521 respectively, these values decreasing with increase in temperature and with the use of blanching. After drying the samples using both hot air and freeze-drying, they were ground for subsequent determination of the color parameters, particle size, inulin, glucose and fructose contents and glass transition temperatures, and for observation of the microstructure by scanning electronic microscopy. The freeze-dried samples showed significantly higher values for L* and lower values for a* and b* than the samples dried by forced air, and thus the freeze-dried samples were lighter in color and more greenish or bluish, closer to the color of the in natura garlic. All the dehydrated samples showed a decrease in the inulin contents and increase in the glucose and fructose contents, indicating a possible hydrolysis of the inulin which could be related to the residual activity of the enzyme inulinase. There were also lower inulin, glucose and fructose concentrations in the blanched samples as compared to the non-blanched samples, possibly due to leaching into the water. In the freeze-dried samples the mean particle diameters were 79.35 μm and 104.79 μm for the non-blanched and blanched samples, respectively, smaller than the values obtained in the forced air-dried samples, whose diameters varied from 119.28 μm to 302.04 μm. By way of scanning electronic microscopy, it was observed that the surface of the freeze-dried samples was less damaged than that of the forced air-dried samples, since freeze-drying results in less contraction and consequently less wrinkling than air drying. The glass transition temperatures of the dehydrated garlic powders varied from 99.39±1.98 °C to 120.15±3.80 °C, increasing with decrease in the water activity, confirming the plasticizing effect of water.