An Electrochemical Sensor for Reducing Sugars Based on a Glassy Carbon Electrode Modified with Electropolymerized Molecularly Imprinted Poly-o-phenylenediamine Film

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of ethanol, dry extract and reducing sugars on density and viscosity of Brazilian red wines

BACKGROUND Density and viscosity are properties that exert great influence on the body of wines. The present work aimed to evaluate the influence of the alcoholic content, dry extract, and reducing sugar content on density and viscosity of commercial dry red wines at different temperatures. The rheological assays were carried out on a controlled stress rheometer, using concentric cylinder geometry at seven temperatures (2, 8, 14, 16, 18, 20 and 26 degrees C).RESULTSWine viscosity decreased with increasing temperature and density was directly related to the wine alcohol content, whereas viscosity was closely linked to the dry extract. Reducing sugars did not influence viscosity or density. Wines produced from Italian grapes were presented as full-bodied with higher values for density and viscosity, which was linked to the higher alcohol content and dry extract, respectively.CONCLUSIONThe results highlighted the major effects of certain physicochemical properties on the physical properties of wines, which in turn is important for guiding sensory assessments. (c) 2014 Society of Chemical Industry

Reducing acrylamide precursors in raw materials derived from wheat and potato

A review of agronomic and genetic approaches as strategies for the mitigation of acrylamide risk in wheat and potato is presented. Acrylamide is formed through the Maillard reaction during high-temperature cooking, such as frying, roasting, or baking, and the main precursors are free asparagine and reducing sugars. In wheat flour, acrylamide formation is determined by asparagine levels and asparagine accumulation increases dramatically in response to sulfur deprivation and, to a much lesser extent, with nitrogen feeding. In potatoes, in which sugar concentrations are much lower, the relationships between acrylamide and its precursors are more complex. Much attention has been focused on reducing the levels of sugars in potatoes as a means of reducing acrylamide risk. However, the level of asparagine as a proportion of the total free amino acid pool has been shown to be a key parameter, indicating that when sugar levels are limiting, competition between asparagine and the other amino acids for participation in the Maillard reaction determines acrylamide formation. Genetic approaches to reducing acrylamide risk include the identification of cultivars; and other germplasm in which free asparagine and/or sugar levels are low and the manipulation of genes involved in sugar and amino acid metabolism and signaling. These approaches are made more difficult by genotype/ environment interactions that can result in a genotype being "good" in one environment but "poor" in another. Another important consideration is the effect that any change could have on flavor in the cooked product. Nevertheless, as both wheat and potato are regarded as of relatively high acrylamide risk compared with, for example, maize and rice, it is essential that changes are achieved that mitigate the problem.

Sugars in crop plants

We review current knowledge of the most abundant sugars, sucrose, maltose, glucose and fructose, in the world's major crop plants. The sucrose-accumulating crops, sugar beet and sugar cane, are included, but the main focus of the review is potato and the major cereal crops. The production of sucrose in photosynthesis and the inter-relationships of sucrose, glucose, fructose and other metabolites in primary carbon metabolism are described, as well as the synthesis of starch, fructan and cell wall polysaccharides and the breakdown of starch to produce maltose. The importance of sugars as hormone-like signalling molecules is discussed, including the role of another sugar, trehalose, and the trehalose biosynthetic pathway. The Maillard reaction, which occurs between reducing sugars and amino acids during thermal processing, is described because of its importance for colour and flavour in cooked foods. This reaction also leads to the formation of potentially harmful compounds, such as acrylamide, and is attracting increasing attention as food producers and regulators seek to reduce the levels of acrylamide in cooked food. Genetic and environmental factors affecting sugar concentrations are described.

Concentrations of free amino acids and sugars in nine potato varieties: effects of storage and relationship with acrylamide formation

Acrylamide forms during cooking and processing predominately from the reaction of free asparagine and reducing sugars in the Maillard reaction. The identification of low free asparagine and reducing sugar varieties of crops is therefore an important target. In this study, nine varieties of potato (French fry varieties Maris Piper (from two suppliers), Pentland Dell, King Edward, Daisy, and Markies; and chipping varieties Lady Claire, Lady Rosetta, Saturna, and Hermes) grown in the United Kingdom in 2009 were analyzed at monthly intervals through storage from November 2009 to July 2010. Acrylamide formation was measured in heated flour and chips fried in oil. Analysis of variance revealed significant interactions between varieties nested within type (French fry and chipping) and storage time for most free amino acids, glucose, fructose, and acrylamide formation. Acrylamide formed in chips correlated significantly with acrylamide formed in flour and with chip color. There were significant correlations between glucose or total reducing sugar concentration and acrylamide formation in both variety types, but with fructose the correlation was much stronger for chipping than for French fry varieties. Conversely, there were significant correlations with acrylamide formation for both total free amino acid and free asparagine concentration in the French fry but not chipping varieties. The study showed the potential of variety selection for preventing unacceptable levels of acrylamide formation in potato products and the variety-dependent effect of long-term storage on acrylamide risk. It also highlighted the complex relationship between precursor concentration and acrylamide risk in potatoes.

Effects of nitrogen and sulfur fertilization on free amino acids, sugars, and acrylamide-forming potential in potato

Nitrogen (N) fertilizer is used routinely in potato (Solanum tuberosum) cultivation to maximize yield. However, it also affects sugar and free amino acid concentrations in potato tubers, and this has potential implications for food quality and safety because free amino acids and reducing sugars participate in the Maillard reaction during high-temperature cooking and processing. This results in the formation of color, aroma, and flavor compounds, but also some undesirable contaminants, including acrylamide, which forms when the amino acid that participates in the final stages of the reaction is asparagine. Another mineral, sulfur (S), also has profound effects on tuber composition. In this study, 13 varieties of potato were grown in a field trial in 2010 and treated with different combinations of N and S. Potatoes were analyzed immediately after harvest to show the effect of N and S fertilization on concentrations of free asparagine, other free amino acids, sugars, and acrylamide-forming potential. The study showed that N application can affect acrylamide-forming potential in potatoes but that the effect is type- (French fry, chipping, and boiling) and variety-dependent, with most varieties showing an increase in acrylamide formation in response to increased N but two showing a decrease. S application reduced glucose concentrations and mitigated the effect of high N application on the acrylamide-forming potential of some of the French fry-type potatoes.

The composition of sugarcane juices dervied from burnt cane and whole green cane crop

There has been substantial interest within the Australian sugar industry in product diversification as a means to reduce its exposure to fluctuating raw sugar prices and in order to increase its commercial viability. In particular, the industry is looking at fibrous residues from sugarcane harvesting (trash) and from sugarcane milling (bagasse) for cogeneration and the production of biocommodities, as these are complementary to the core process of sugar production. A means of producing surplus residue (biomass) is to process whole sugarcane crop. In this paper, the composition of different juices derived from different harvesting methods, viz. burnt cane with all trash extracted (BE), green cane with half of the trash extracted (GE), and green cane (whole sugarcane crop) with trash unextracted (GU), were investigated and the results and comparison presented. The determination of electrical conductivity, inorganic composition, and organic acids indicate that both GU and GE cane juice contain a higher proportion of soluble inorganic ions and ionisable organic acids, compared to BE cane juice. It is important to note that there are considerably higher levels of Na ions and citric acid, but relatively low P levels in the GU samples. A higher level of reducing sugars was analysed in the GU samples than the BE samples due to the higher proportion of impurities found naturally in sugarcane tops and leaves. The purity of the first expressed juice (FEJ) of GU cane was on average higher than that of FEJ of BE cane. Results also show that GU juices appear to contain higher levels of proteins and polysaccharides, with no significant difference in starch levels.

Modelling the flow of cane constituents through the milling process of a raw sugar factory

This thesis reports on an investigation to develop an advanced and comprehensive milling process model of the raw sugar factory. Although the new model can be applied to both, the four-roller and six-roller milling units, it is primarily developed for the six-roller mills which are widely used in the Australian sugar industry. The approach taken was to gain an understanding of the previous milling process simulation model "MILSIM" developed at the University of Queensland nearly four decades ago. Although the MILSIM model was widely adopted in the Australian sugar industry for simulating the milling process it did have some incorrect assumptions. The study aimed to eliminate all the incorrect assumptions of the previous model and develop an advanced model that represents the milling process correctly and tracks the flow of other cane components in the milling process which have not been considered in the previous models. The development of the milling process model was done is three stages. Firstly, an enhanced milling unit extraction model (MILEX) was developed to access the mill performance parameters and predict the extraction performance of the milling process. New definitions for the milling performance parameters were developed and a complete milling train along with the juice screen was modelled. The MILEX model was validated with factory data and the variation in the mill performance parameters was observed and studied. Some case studies were undertaken to study the effect of fibre in juice streams, juice in cush return and imbibition% fibre on extraction performance of the milling process. It was concluded from the study that the empirical relations developed for the mill performance parameters in the MILSIM model were not applicable to the new model. New empirical relations have to be developed before the model is applied with confidence. Secondly, a soluble and insoluble solids model was developed using modelling theory and experimental data to track the flow of sucrose (pol), reducing sugars (glucose and fructose), soluble ash, true fibre and mud solids entering the milling train through the cane supply and their distribution in juice and bagasse streams.. The soluble impurities and mud solids in cane affect the performance of the milling train and further processing of juice and bagasse. New mill performance parameters were developed in the model to track the flow of cane components. The developed model is the first of its kind and provides some additional insight regarding the flow of soluble and insoluble cane components and the factors affecting their distribution in juice and bagasse. The model proved to be a good extension to the MILEX model to study the overall performance of the milling train. Thirdly, the developed models were incorporated in a proprietary software package "SysCAD’ for advanced operational efficiency and for availability in the ‘whole of factory’ model. The MILEX model was developed in SysCAD software to represent a single milling unit. Eventually the entire milling train and the juice screen were developed in SysCAD using series of different controllers and features of the software. The models developed in SysCAD can be run from macro enabled excel file and reports can be generated in excel sheets. The flexibility of the software, ease of use and other advantages are described broadly in the relevant chapter. The MILEX model is developed in static mode and dynamic mode. The application of the dynamic mode of the model is still under progress.

Trehalose Toxicity in Cuscuta reflexa - Sucrose Content Decreases In Shoot Tips Upon Trehalose Feeding

Trehalose, an alpha,alpha-diglucoside, induced a rapid blackening and death of shoot tips of Cuscuta reflexa (dodder) cultured in vitro. The onset of toxic symptom was delayed if any of the several sugars which support the in vitro growth of Cuscuta was supplied with trehalose. The rate of trehalose uptake or its accumulation in the tissue was not affected by sugar cofeeding. The levels of total and reducing sugars declined appreciably in the trehalose-fed shoot tip explants compared to control tissue cultured in absence of a carbon source. This was not due to an increased rate of respiration of the trehalose-treated tissue. In shoot tips cultured in presence of both trehalose and sucrose, the decline in total and reducing sugars was curtailed. There was a marked fall in the level of sucrose; and invertase activity was higher in trehalose-fed shoot tips. The incorporation of label from [14C]glucose into sucrose in the shoot tip explant was reduced as early as 12 h of trehalose feeding. The results suggest that increased utilization of sucrose as well as an inhibition of its synthesis contribute to the drastic fall in the sucrose content upon trehalose feeding.

Toxicity in Cuscuta reflexa Sucrose Content Decreases In Shoot Tips Upon Trehalose Feeding Trehalose

Trehalose, an {alpha},{alpha}-diglucoside, induced a rapid blackening and death of shoot tips of Cuscuta reflexa (dodder) cultured in vitro. The onset of toxic symptom was delayed if any of the several sugars which support the in vitro growth of Cuscuta was supplied with trehalose. The rate of trehalose uptake or its accumulation in the tissue was not affected by sugar cofeeding. The levels of total and reducing sugars declined appreciably in the trehalose-fed shoot tip explants compared to control tissue cultured in absence of a carbon source. This was not due to an increased rate of respiration of the trehalose-treated tissue. In shoot tips cultured in presence of both trehalose and sucrose, the decline in total and reducing sugars was curtailed. There was a marked fall in the level of sucrose; and invertase activity was higher in trehalose-fed shoot tips. The incorporation of label from [14C]glucose into sucrose in the shoot tip explant was reduced as early as 12 h of trehalose feeding. The results suggest that increased utilization of sucrose as well as an inhibition of its synthesis contribute to the drastic fall in the sucrose content upon trehalose feeding

Synthesis of neoglycoconjugates and oligosaccharides with potential anti-Helicobacter pylori activity

The significance of carbohydrate-protein interactions in many biological phenomena is now widely acknowledged and carbohydrate based pharmaceuticals are under intensive development. The interactions between monomeric carbohydrate ligands and their receptors are usually of low affinity. To overcome this limitation natural carbohydrate ligands are often organized as multivalent structures. Therefore, artificial carbohydrate pharmaceuticals should be constructed on the same concept, as multivalent carbohydrates or glycoclusters. Infections of specific host tissues by bacteria, viruses, and fungi are among the unfavorable disease processes for which suitably designed carbohydrate inhibitors represent worthy targets. The bacterium Helicobacter pylori colonizes more than half of all people worldwide, causing gastritis, gastric ulcer, and conferring a greater risk of stomach cancer. The present medication therapy for H. pylori includes the use of antibiotics, which is associated with increasing incidence of bacterial resistance to traditional antibiotics. Therefore, the need for an alternative treatment method is urgent. In this study, four novel synthesis procedures of multivalent glycoconjugates were created. Three different scaffolds representing linear (chondroitin oligomer), cyclic (γ-cyclodextrin), and globular (dendrimer) molecules were used. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc all representing analogues of the tissue binding epitopes for H. pylori. The first synthetic method included the reductive amination of scaffold molecules modified to express primary amine groups, and in the case of dendrimer direct amination to scaffold molecule presenting 64 primary amine groups. The second method described a direct procedure for amidation of glycosylamine modified oligosaccharides to scaffold molecules presenting carboxyl groups. The final two methods that were created both included an oxime-linkage on linkers of different length. All the new synthetic procedures synthesized had the advantage of using unmodified reducing sugars as starting material making it easy to synthesize glycoconjugates of different specificity. In addition, the binding activity of an array of neoglycolipids to H. pylori was studied. Consequently, two new neolacto-based structures, Glcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer and GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer, with binding activity toward H. pylori were discovered. Interestingly, N-methyl and N-ethyl amide modification of the GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer glucuronic acid residue resulted in more effective H. pylori binding epitopes than the parent molecule.

Engineering enzyme systems by recombination

Homologous recombination is a source of diversity in both natural and directed evolution. Standing genetic variation that has passed the test of natural selection is combined in new ways, generating functional and sometimes unexpected changes. In this work we evaluate the utility of homologous recombination as a protein engineering tool, both in comparison with and combined with other protein engineering techniques, and apply it to an industrially important enzyme: Hypocrea jecorina Cel5a.

Chapter 1 reviews work over the last five years on protein engineering by recombination. Chapter 2 describes the recombination of Hypocrea jecorina Cel5a endoglucanase with homologous enzymes in order to improve its activity at high temperatures. A chimeric Cel5a that is 10.1 °C more stable than wild-type and hydrolyzes 25% more cellulose at elevated temperatures is reported. Chapter 3 describes an investigation into the synergy of thermostable cellulases that have been engineered by recombination and other methods. An engineered endoglucanase and two engineered cellobiohydrolases synergistically hydrolyzed cellulose at high temperatures, releasing over 200% more reducing sugars over 60 h at their optimal mixture relative to the best mixture of wild-type enzymes. These results provide a framework for engineering cellulolytic enzyme mixtures for the industrial conditions of high temperatures and long incubation times.

In addition to this work on recombination, we explored three other problems in protein engineering. Chapter 4 describes an investigation into replacing enzymes with complex cofactors with simple cofactors, using an E. coli enolase as a model system. Chapter 5 describes engineering broad-spectrum aldehyde resistance in Saccharomyces cerevisiae by evolving an alcohol dehydrogenase simultaneously for activity and promiscuity. Chapter 6 describes an attempt to engineer gene-targeted hypermutagenesis into E. coli to facilitate continuous in vivo selection systems.

Feasibility study of biofuel production from freshwater fern, Azolla sp in Anzali Wetland

Production of bioethanol through acidic and enzymatic hydrolysis of aquatic Azolla sp., as a new source of bio-mass, has been performed, as a means to control increasing growth and reducing undesirable effects of this plant in Anzali lagoon. After sampling, drying and crushing, Azolla was hydrolyzed, using diluted acid and enzyme. Diluted acid hydrolysis was done using both autoclave and a high-pressure system (Batch Synth® Microwave synthesizer). The effects of temperature and time (in autoclave) and concentration of acid (in both) were compared. Cellubrix®, a ommercial cellulase source, was used for enzymatic hydrolysis process. The amounts of reducing sugars, glucose and furfural, released from hydrolyzate, were measured. To produce alcohol, Sacchromyces cerevisiae (to ferment sixcarbon sugars), Zygowilliopsis californica and Pichia stipitis (to ferment five-carbon and sixcarbon sugars) were used. Maximum amounts of glucose (4.83% w/w) and reducing sugars (14.15% w/w) were obtained using acid hydrolysis in autoclave. In the microwave oven, maximum glucose (5.04% w/w) and reducing sugars (13.27 w/w) were obtained at 180 and 200 °C, respectively. Under these conditions, maximum produced furfural was 1.54 g/L. The difference between amounts of furfural obtained from acid hydrolysis of Azolla in microwave oven compared to autoclave was statistically significant. Amounts of alcohol produced and its yields were 3.99 g/L and 33.13% for S. cerevisiae in 48 hours, 3.73 g/L and 30.45% for Pichia stipites in 48 hours, and 3.73 g/L and 30.45% for Z. californica in 24 hours after inoculation, respectively, with significant differences. Statistical comparison of results showed significant differences (P<0.05) in glucose production, at different conditions. Amounts of reducing sugars and glucose increased after optimization of levels of acid, time, and temperature. The overall optimum released sugar and glucose were obtained with 1.67% (w/v) acid using autoclave. Higher temperatures in microwave oven caused a significant increase (P<0.05) in furfural. Furfural severely inhibits fermentation. Hence, regarding the issues of energy consumption and time, amounts of inhibiting substances and sugar production, autoclave is found to be superior to the high temperature and pressure, generated in microwave oven, for hydrolyzing Azolla. Furthermore, given the amounts of Azolla in Anzali lagoon, it may be recommendable to use this plant as a biomass resource.

Cytochemical changes in the developmental process of Nostoc sphaeroides (cyanobacterium)

There are several apparent developmental stages in the life cycle of Nostoc sphaeroides Kutzing, an edible cyanobacterium found mainly in paddy fields in central China. The cytochemical changes in developmental stages such as hormogonia, aseriate stage, filamentous stage and colony in N. sphaeroides were examined using fluorescent staining and colorimetric methods. The staining of acidic and sulfated polysaccharides increased with development when hormogonia were used as the starting point. Acidic polysaccharides (AP) were most abundant at the aseriate stage and then decreased, while the sulfated polysaccharides (SP) were highest at the colony stage. Quantitatively, along the developmental process from hormogonia to colony, total carbohydrates first increased, then became stable, and then reached their highest level at the colony stage, while reducing sugars were highest at the hormogonia stage and then decreased sharply once development began. SP were not detectable in the hot water soluble polysaccharides (HWSP), and hormogonia had the lowest content of AP, while old colonies had the highest. The AP content of the aseriate stage, filamentous stage and young colony stage were very similar. The evolutionary relationships reflected in the developmental stages of N. sphaeroides are discussed.