Binding of the wheat germ lectin to Cryptococcus neoformans chitooligomers affects multiple mechanisms required for fungal pathogenesis

The principal capsular component of Cryptococcus neoformans, glucuronoxylomannan (GXM), interacts with surface glycans, including chitin-like oligomers. Although the role of GXM in cryptococcal infection has been well explored, there is no information on how chitooligomers affect fungal pathogenesis. In this study, surface chitooligomers of C. neoformans were blocked through the use of the wheat germ lectin (WGA) and the effects on animal pathogenesis, interaction with host cells, fungal growth and capsule formation were analyzed. Treatment of C. neoformans cells with WGA followed by infection of mice delayed mortality relative to animals infected with untreated fungal cells. This observation was associated with reduced brain colonization by lectin-treated cryptococci. Blocking chitooligomers also rendered yeast cells less efficient in their ability to associate with phagocytes. WGA did not affect fungal viability, but inhibited GXM release to the extracellular space and capsule formation. In WGA-treated yeast cells, genes that are involved in capsule formation and GXM traffic had their transcription levels decreased in comparison with untreated cells. Our results suggest that cellular pathways required for capsule formation and pathogenic mechanisms are affected by blocking chitin-derived structures at the cell surface of C. neoformans. Targeting chitooligomers with specific ligands may reveal new therapeutic alternatives to control cryptococcosis.

Empirical and fundamental mechanical tests in the evaluation of dough and bread rheological properties

Bread dough and particularly wheat dough, due to its viscoelastic behaviour, is probably the most dynamic and complicated rheological system and its characteristics are very important since they highly affect final products’ textural and sensorial properties. The study of dough rheology has been a very challenging task for many researchers since it can provide numerous information about dough formulation, structure and processing. This explains why dough rheology has been a matter of investigation for several decades. In this research rheological assessment of doughs and breads was performed by using empirical and fundamental methods at both small and large deformation, in order to characterize different types of doughs and final products such as bread. In order to study the structural aspects of food products, image analysis techniques was used for the integration of the information coming from empirical and fundamental rheological measurements. Evaluation of dough properties was carried out by texture profile analysis (TPA), dough stickiness (Chen and Hoseney cell) and uniaxial extensibility determination (Kieffer test) by using a Texture Analyser; small deformation rheological measurements, were performed on a controlled stress–strain rheometer; moreover the structure of different doughs was observed by using the image analysis; while bread characteristics were studied by using texture profile analysis (TPA) and image analysis. The objective of this research was to understand if the different rheological measurements were able to characterize and differentiate the different samples analysed. This in order to investigate the effect of different formulation and processing conditions on dough and final product from a structural point of view. For this aim the following different materials were performed and analysed: - frozen dough realized without yeast; - frozen dough and bread made with frozen dough; - doughs obtained by using different fermentation method; - doughs made by Kamut® flour; - dough and bread realized with the addition of ginger powder; - final products coming from different bakeries. The influence of sub-zero storage time on non-fermented and fermented dough viscoelastic performance and on final product (bread) was evaluated by using small deformation and large deformation methods. In general, the longer the sub-zero storage time the lower the positive viscoelastic attributes. The effect of fermentation time and of different type of fermentation (straight-dough method; sponge-and-dough procedure and poolish method) on rheological properties of doughs were investigated using empirical and fundamental analysis and image analysis was used to integrate this information throughout the evaluation of the dough’s structure. The results of fundamental rheological test showed that the incorporation of sourdough (poolish method) provoked changes that were different from those seen in the others type of fermentation. The affirmative action of some ingredients (extra-virgin olive oil and a liposomic lecithin emulsifier) to improve rheological characteristics of Kamut® dough has been confirmed also when subjected to low temperatures (24 hours and 48 hours at 4°C). Small deformation oscillatory measurements and large deformation mechanical tests performed provided useful information on the rheological properties of samples realized by using different amounts of ginger powder, showing that the sample with the highest amount of ginger powder (6%) had worse rheological characteristics compared to the other samples. Moisture content, specific volume, texture and crumb grain characteristics are the major quality attributes of bread products. The different sample analyzed, “Coppia Ferrarese”, “Pane Comune Romagnolo” and “Filone Terra di San Marino”, showed a decrease of crumb moisture and an increase in hardness over the storage time. Parameters such as cohesiveness and springiness, evaluated by TPA that are indicator of quality of fresh bread, decreased during the storage. By using empirical rheological tests we found several differences among the samples, due to the different ingredients used in formulation and the different process adopted to prepare the sample, but since these products are handmade, the differences could be account as a surplus value. In conclusion small deformation (in fundamental units) and large deformation methods showed a significant role in monitoring the influence of different ingredients used in formulation, different processing and storage conditions on dough viscoelastic performance and on final product. Finally the knowledge of formulation, processing and storage conditions together with the evaluation of structural and rheological characteristics is fundamental for the study of complex matrices like bakery products, where numerous variable can influence their final quality (e.g. raw material, bread-making procedure, time and temperature of the fermentation and baking).

Functional components and lipid quality assessment in vegetable foods: analytical and technological approaches

Lipids are important components that contribute very significantly to nutritional and technological quality of foods because they are the least stable macro-components in foods, due to high susceptibility to oxidation. When rancidity take place, it makes food unhealthy and unacceptable for consumers. Thus, the presence of antioxidants, naturally present of added to foods, is required to enhance shelf life of foods. Moreover, antioxidant like phenolic compounds play an important role in human health enhancing the functionality of foods. The aim of this PhD project was the study of lipid quality and lipid oxidation in different vegetable foods focusing on analytical and technological aspects in order to figure out the effects of lipid composition and bioactive compounds (phenolic compounds, omega-3 fatty acids and dietary fiber) addition on their shelf life. In addition, bioavailability and antioxidant effects of phenolic compounds in human and animals, respectively, were evaluated after consumption of vegetable foods. The first section of the work was focused on the evaluation of lipid quality impact on technological behaviour of vegetable foods. Because of that, cocoa butter with different melting point were evaluated by chromatographic techniques (GC, TLC) and the sample with the higher melting point showed the presence of fatty acids, triglycerides, 2-monoglycerides and FT-IR profile different from genuine cocoa butter, meaning an adding of foreign fat (lauric-fat) not allowed by the law. Looking at lipid quality of other vegetable foods, an accelerated shelf life test (OXITEST®), was used to evaluate of lipid stability to oxidation in tarallini snacks made up using different lipid matrices (sunflower oil, extravirgin olive oil and a blend of extravirgin olive oil and lard). The results showed a good ability of OXITEST® to discriminate between lipid unsaturation and different cooking times, without any samples fat extraction. In the second section, the role of bioactive compounds on cereal based food shelf life was studied in different bakeries by GC, spectrophotometric methods and capillary electrophoresis. It was examined the relationships between phenolic compounds, added with flour, and lipid oxidation of tarallini and frollini. Both products showed an increase in lipid oxidation during storage and antioxidant effects on lipid oxidation were not as expected. Furthermore, the influence of enrichment in polyunsaturated fatty acids on lipid oxidation of pasta was evaluated. The results proved that LC n-3 PUFA were not significantly implicated in the onset of oxidation in spaghetti stored under daylight and accelerated oxidation in a laboratory heater. The importance of phenolic compounds as antioxidant in humans and rats was also studied, by HPLC/MS in the latter section. For this purpose, apigenin and apigenin glycosides excretion was investigated in six women’s urine in a 24 hours study. After a single dose of steamed artichokes, both aglicone and glucuronide metabolites were recovered in 24 h urine. Moreover, the effect of whole grain durum wheat bread and whole grain Kamut® khorasan bread in rats were evaluated. Both cereals were good sources of antioxidants but Kamut® bread fed animals had a better response to stress than wheat durum fed, especially when a sourdough bread was supplied.

Characterization of novel probiotics and prebiotics

The role of the human gut microbiota in impacting host’s health has been widely studied in the last decade. Notably, it has been recently demonstrated that diet and nutritional status are among the most important modifiable determinants of human health, through a plethora of presumptive mechanisms among which microbiota-mediated processes are thought to have a relevant role. At present, probiotics and prebiotics represent a useful dietary approach for influencing the composition and activity of the human gut microbial community. The present study is composed of two main sections, aimed at elucidating the probiotic potential of the yeast strain K. marxianus B0399, as well as the promising putative prebiotic activity ascribable to four different flours, naturally enriched in dietary fibres content. Here, by in vitro studies we demonstrated that K. marxianus B0399 possesses a number of beneficial and strain-specific properties desirable for a microorganism considered for application as a probiotics. Successively, we investigated the impact of a novel probiotic yoghurt containing B. animalis subsp. lactis Bb12 and K. marxianus B0399 on the gut microbiota of a cohort of subjects suffering from IBS and enrolled in a in vivo clinical study. We demonstrated that beneficial effects described for the probiotic yoghurt were not associated to significant modifications of the human intestinal microbiota. Additionally, using a colonic model system we investigated the impact of different flours (wholegrain rye and wheat, chickpeas and lentils 50:50, and barley milled grains) on the intestinal microbiota composition and metabolomic output, combining molecular and cellular analysis with a NMR metabolomics approach. We demonstrated that each tested flour showed peculiar and positive modulations of the intestinal microbiota composition and its small molecule metabolome, thus supporting the utilisation of these ingredients in the development of a variety of potentially prebiotic food products aimed at improving human health.

Wheat and low-input agriculture: agronomic, nutritional and nutraceutical implications

Recently, the increasing interest in organic food products and environmental friendly practices has emphasized the importance of selecting crop varieties suitable for the low-input systems. Additionally, in recent years the relationship between diet and human health has gained much attention among consumers, favoring the investigations on food nutraceutical properties. Among cereals, wheat plays an important role in human nutrition around the world and contributes to the daily intake of essential nutrients such as starch and protein. Moreover, whole grain contains several bioactive compounds that confer to wheat-derived products unique nutraceutical properties (dietary fibre, antioxidants). The present research provided interesting insights for the selection of wheat genotypes suitable for low-input systems and the development of specific breeding programs dedicated to organic farming. The investigation involved 5 old not dwarf genotypes (Andriolo, Frassineto, Gentil rosso, Inallettabile, Verna) and 1 modern dwarf variety (Palesio), grown under biodynamic management, over two consecutive growing seasons (2009/2010, 2010/2011). Results evidenced that under low-input farming some investigated old wheat genotypes (Frassineto, Inallettabile) were comparable to the modern cultivar in terms of whole agronomic performance. As regards the nutritional and nutraceutical properties, some old genotypes (Andriolo, Gentil rosso, Verna) emerged for their relevant content of several investigated phytochemicals (such as insoluble dietary fibre, polyphenols, flavonoids, in vitro antioxidant activity) and nutrients (protein, lipid, minerals). Despite of the low technological features, the six wheat varieties grown under low-input management may efficiently provide raw material for the preparation of traditionally processed bread with valuable sensory and nutritional properties. Results highlighted that old wheat varieties have peculiar phytochemical composition and may be a valuable source of nutraceutical compounds. Some of the genetic material involved in the present study may be used in breeding programs aimed at selecting varieties suitable for low-input farming and rich in health-promoting compounds.

Fusarium species responsible for mycotoxin production in wheat crop: involvement in food safety

Fusarium Head Blight (FHB) is a worldwide cereal disease responsible of significant yield reduction, inferior grain quality, and mycotoxin accumulation. Fusarium graminearum and F. culmorum are the prevalent causal agents. FHB has been endemic in Italy since 1995, while there are no records about its presence in Syria. Forty-eight and forty-six wheat kernel samples were collected from different localities and analyzed for fungal presence and mycotoxin contamination. Fusarium strains were identified morphologically but the molecular confirmation was performed only for some species. Further differentiation of the chemotypes for trichothecene synthesis by F. graminearum and F. culmorum strains was conducted by PCR assays. Fusarium spp. were present in 62.5% of Syrian samples. 3Acetyl-Deoxynivalenol and nivalenol chemotypes were found in F. culmorum whilst all F. graminearum strains belonged to NIV chemotype. Italian samples were infected with Fusarium spp for 67.4%. 15Ac-DON was the prevalent chemotype in F. graminearum, while 3Ac-DON chemotype was detected in F. culmorum. The 60 Syrian Fusarium strains tested for mycotoxin production by HPLC-MS/MS have shown the prevalence of zearalenone while the emerging mycotoxins were almost absent. The analysis of the different Syrian and Italian samples of wheat kernels for their mycotoxin content showed that Syrian kernels were mainly contaminated with storage mycotoxins, aflatoxins and ochratoxin whilst Italian grains with mainly Fusarium mycotoxins. The aggressiveness of several Syrian F. culmorum isolates was estimated using three different assays: floret inoculation in growth chamber, ear inoculation in the field and a validated new Petri-dish test. The study of the behaviour of different Syrian wheat cultivars, grown under different conditions, has revealed that Jory is a FHB Syrian tolerant cultivar. This is the first study in Syria on Fusarium spp. associated to FHB, Fusarium mycotoxin producers and grain quality.

Association mapping of stem rust resistance in durum wheat at the seedling and adult plant stages

In wheat, stem rust is known to rapidly evolve new virulence to resistance genes. While more than 50 stem rust resistance (Sr) loci have been identified in wheat, only a few remain effective, particularly against the highly virulent race Ug99 (TTKSK race) and a mixture of durum-specific races. An association mapping (AM) study based on 183 durum wheat accessions was utilized to identify resistance loci for stem rust response in Ethiopia over four seasons and artificial inoculation with Ug99 (TTKSK race) and a mixture of durum-specific races under field conditions as well as in greenhouse test at seedling stage under controlled conditions for resistance to four highly virulent stem rust races: TRTTF, TTTTF, (TTKSK (Ug99) and JRCQC. The panel was profiled with 1,253 SSR and DArT markers. Twelve QTL-tagging markers were significant (P < 0.05) across three to four seasons. The role of Sr13, Sr9, Sr14, Sr17, and Sr28 was confirmed. Thirteen significant markers were in regions with no Sr genes/QTLs. The results under controlled conditions showed that 15, 20, 19 and 19 chromosome regions harbored markers that showed significant effects for races TRTTF, TTTTF, TTKSK and JRCQC, respectively. These genomic regions showed marker R2 values ranging from 1.13 to 8.34, 1.92 to 17.64, 1.75 to 23.12 and 1.51 to 15.33% for races TRTTF, TTTTF, TTKSK and JRCQC, respectively. The study demonstrates that stem rust resistance in durum wheat is governed in part by shared loci and in part by race-specific ones. The QTLs identified in this study through AM will be useful in the marker-assisted development of durum wheat cultivars with durable stem rust resistance.

A new global wheat marketmodel (GLOWMM) for the analysis of wheat export prices

Food commodity prices fluctuations have important impacts on poverty and food insecurity across the world. Conventional models have not provided a complete picture of recent price spikes in agricultural commodity markets, while there is an urgent need for appropriate policy responses. Perhaps new approaches are needed in order to better understand international spill-overs, the feedback between the real and the financial sectors and also the link between food and energy prices. In this paper, we present results from a new worldwide dynamic model that provides short and long-run impulse responses of wheat international prices to various real shocks.

Possibilities for the healthy and nutritional improvement of confectionery and sweet products.

The overall objective of this PhD was to investigate the possibility to increase the nutritional value of confectionary products by the use of natural ingredients with healthy functions. The first part of the thesis focused on the possible substitution of the most characteristic component of confectionary products, i.e. refined sugar. Many natural whole sweetening alternatives are available, though not widely used; the use of molasses, the byproduct of sugar beet and cane production, still rich in healthy components as minerals and phytochemicals is hereby discussed; after having verified molasses effectiveness in oxidative stress counteraction on liver cultured cells, the higher antioxidant capacity of a sweet food prepared with molasses instead of refined sugar was confirmed. A second step of the project dealt with another main ingredient of various sweet products, namely wheat. Particularly, the exploitation of soft and durum wheat byproducts could be another sustainable strategy to improve the healthy value of confectionery. The isolation of oligosaccharides with bioactive functions form different fractions of the wheat milling stream was studied and the new ingredients were shown to have a high dietary fiber and antioxidants content. As valid alternative, product developers should consider the appealing and healthy addition of ancient grains flour to sweet baked goods. The possibility of substituting the modern whole durum wheat with the ancient Kamut® khorasan was considered, and the antioxidant and anti-inflammatory effects of these grains were evaluated and compared both in vitro and in vivo on rats. Finally, since high consumption of confectionery is a risk factor for obesity, a possible strategy for the counteraction of this disease was investigated. The ability of three bioactives in inhibiting adipocytes differentiation was investigated. In fact, theoretically, compounds able to influence adipogenesis could be used in the formulation of functional sweet products and contribute to prevent obesity.

Use of Biotechnology to increase the content of bioactive compounds in fermented foods of plant origin

The objectives of this PhD research were: i) to evaluate the use of bread making process to increase the content of β-glucans, resistant starch, fructans, dietary fibers and phenolic compounds of kamut khorasan and wheat breads made with flours obtained from kernels at different maturation stage (at milky stage and fully ripe) and ii) to study the impact of whole grains consumption in the human gut. The fermentation and the stages of kernel development or maturation had a great impact on the amount of resistant starch, fructans and β-glucans as well as their interactions resulted highly statistically significant. The amount of fructans was high in kamut bread (2.1g/100g) at the fully ripe stage compared to wheat during industrial fermentation (baker’s yeast). The sourdough increases the content of polyphenols more than industrial fermentation especially in bread made by flour at milky stage. From the analysis of volatile compounds it resulted that the sensors of electronic nose perceived more aromatic compound in kamut products, as well as the SPME-GC-MS, thus we can assume that kamut is more aromatic than wheat, so using it in sourdough process can be a successful approach to improve the bread taste and flavor. The determination of whole grain biormakers such as alkylresorcinols and others using FIE-MS AND GC-tof-MS is a valuable alternative for further metabolic investigations. The decrease of N-acetyl-glucosamine and 3-methyl-hexanedioic acid in kamut faecal samples suggests that kamut can have a role in modulating mucus production/degradation or even gut inflammation. This work gives a new approach to the innovation strategies in bakery functional foods, that can help to choose the right or best combination between stages of kernel maturation-fermentation process and baking temperature.

A multiparental cross population for mapping QTL for relevant agronomic traits in durum wheat (Triticum durum Desf.)

Multiparental cross designs for mapping quantitative trait loci (QTL) in crops are efficient alternatives to conventional biparental experimental populations because they exploit a broader genetic basis and higher mapping resolution. We describe the development and deployment of a multiparental recombinant inbred line (RIL) population in durum wheat (Triticum durum Desf.) obtained by crossing four elite cultivars characterized by different traits of agronomic value. A linkage map spanning 2,663 cM and including 7,594 single nucleotide polymorphisms (SNPs) was produced by genotyping 338 RILs with a wheat-dedicated 90k SNP chip. A cluster file was developed for correct allele calling in the framework of the tetraploid durum wheat genome. Based on phenotypic data collected over four field experiments, a multi-trait quantitative trait loci (QTL) analysis was carried out for 18 traits of agronomic relevance (including yield, yield-components, morpho-physiological and seed quality traits). Across environments, a total of 63 QTL were identified and characterized in terms of the four founder haplotypes. We mapped two QTL for grain yield across environments and 23 QTL for grain yield components. A novel major QTL for number of grain per spikelet/ear was mapped on chr 2A and shown to control up to 39% of phenotypic variance in this cross. Functionally different QTL alleles, in terms of direction and size of genetic effect, were distributed among the four parents. Based on the occurrence of QTL-clusters, we characterized the breeding values (in terms of effects on yield) of most of QTL for heading and maturity as well as yield component and quality QTL. This multiparental RIL population provides the wheat community with a highly informative QTL mapping resource enabling the dissection of the genetic architecture of multiple agronomic relevant traits in durum wheat.

Genetic basis of variation for root traits and response to heat stress in durum wheat

Durum wheat is the second most important wheat species worldwide and the most important crop in several Mediterranean countries including Italy. Durum wheat is primarily grown under rainfed conditions where episodes of drought and heat stress are major factors limiting grain yield. The research presented in this thesis aimed at the identification of traits and genes that underlie root system architecture (RSA) and tolerance to heat stress in durum wheat, in order to eventually contribute to the genetic improvement of this species. In the first two experiments we aimed at the identification of QTLs for root trait architecture at the seedling level by studying a bi-parental population of 176 recombinant inbred lines (from the cross Meridiano x Claudio) and a collection of 183 durum elite accessions. Forty-eight novel QTLs for RSA traits were identified in each of the two experiments, by means of linkage- and association mapping-based QTL analysis, respectively. Important QTLs controlling the angle of root growth in the seedling were identified. In a third experiment, we investigated the phenotypic variation of root anatomical traits by means of microscope-based analysis of root cross sections in 10 elite durum cultivars. The results showed the presence of sizeable genetic variation in aerenchyma-related traits, prompting for additional studies aimed at mapping the QTLs governing such variation and to test the role of aerenchyma in the adaptive response to abiotic stresses. In the fourth experiment, an association mapping experiment for cell membrane stability at the seedling stage (as a proxy trait for heat tolerance) was carried out by means of association mapping. A total of 34 QTLs (including five major ones), were detected. Our study provides information on QTLs for root architecture and heat tolerance which could potentially be considered in durum wheat breeding programs.