RESUMO
Yeast's role in bread making is primarily the fermentative production of carbon dioxide to leaven the dough. Fermentation also impacts dough matrix rheology, thereby affecting the quality of the end product. Surprisingly, the role of ethanol, the other yeast primary metabolite, has been ill studied in this context. Therefore, this study aims to assess the potential impact of ethanol on yeastless dough extensibility and spread and gluten agglomeration at concentrations at which it is produced in fermenting dough, i.e., up to 60 mmol per 100 g of flour. Reduced dough extensibility and dough spread were observed upon incorporation of ethanol in the dough formula, and were more pronounced for a weak than for a strong flour. Uniaxial and biaxial extension tests showed up to 50% decrease in dough extensibility and a dough strength increase of up to 18% for 60 mmol of ethanol/100 g of flour. Ethanol enhanced gluten agglomeration of a weak flour. Sequential extraction of flour in increasing ethanol concentrations showed that better gluten-solvent interaction is a possible explanation for the changed dough behavior.
Assuntos
Pão/análise , Etanol/metabolismo , Saccharomyces cerevisiae/metabolismo , Triticum/microbiologia , Pão/microbiologia , Etanol/análise , Fermentação , Farinha/análise , Farinha/microbiologia , Glutens/análise , Glutens/metabolismo , Triticum/metabolismoRESUMO
Succinic acid (SA) was recently shown to be the major pH determining metabolite produced by yeast during straight-dough fermentation (Jayaram et al., 2013), reaching levels as high as 1.6 mmol/100 g of flour. Here, the impact of such levels of SA (0.8, 1.6 and 2.4 mmol/100 g flour) on yeastless dough properties was investigated. SA decreased the development time and stability of dough significantly. Uniaxial extension tests showed a consistent decrease in dough extensibility upon increasing SA addition. Upon biaxial extension in the presence of 2.4 mmol SA/100 g flour, a dough extensibility decrease of 47-65% and a dough strength increase of 25-40% were seen. While the SA solvent retention capacity of flour increased with increasing SA concentration in the solvent, gluten agglomeration decreased with gluten yield reductions of over 50%. The results suggest that SA leads to swelling and unfolding of gluten proteins, thereby increasing their interaction potential and dough strength, but simultaneously increasing intermolecular electrostatic repulsive forces. These phenomena lead to the reported changes in dough properties. Together, our results establish SA as an important yeast metabolite for dough rheology.
Assuntos
Pão/análise , Saccharomyces cerevisiae/metabolismo , Ácido Succínico/metabolismo , Triticum/química , Fermentação , FarinhaRESUMO
In this paper, the content of all major carbohydrates and the spatial distribution of starch, arabinoxylan and ß-glucan in developing wheat kernels (Triticum aestivum L. var. Homeros) from anthesis until maturity were studied. By combining information from microscopy and quantitative analysis, a comprehensive overview on the changes in storage and structural carbohydrates in developing grains was obtained. In the phase of cell division and expansion, grains were characterized by a rapid accumulation of water and high concentrations of the water-soluble carbohydrates fructan, sucrose, glucose and fructose. During the grain filling phase, starch, protein, ß-glucan and arabinoxylan accumulated, while during grain maturation and desiccation, only a loss of moisture took place. The comprehensive approach of this study allowed finding correlations, which are discussed within the context of grain development. Particular attention was given to the transient presence of high fructan concentrations, which was associated with the most striking compositional changes during grain development.
Assuntos
Sementes/metabolismo , Amido/química , Triticum/metabolismo , Xilanos/química , beta-Glucanas/química , Microscopia , Sementes/química , Sementes/crescimento & desenvolvimento , Amido/metabolismo , Triticum/química , Triticum/crescimento & desenvolvimento , Xilanos/metabolismo , beta-Glucanas/metabolismoRESUMO
The degradation of endogenous wheat grain fructans, oligosaccharides with possible health-promoting potential, during wheat whole meal bread making was investigated, and several strategies to prevent their degradation were evaluated. Up to 78.4 ± 5.2% of the fructans initially present in wheat whole meal were degraded during bread making by the action of yeast ( Saccharomyces cerevisiae ) invertase. The addition of sucrose to dough delayed fructan degradation but had no effect on final fructan concentrations. However, yeast growth conditions and yeast genotype did have a clear impact. A 3-fold reduction of fructan degradation could be achieved when the commercial bread yeast strain was replaced by yeast strains with lower sucrose degradation activity. Finally, fructan degradation during bread making could be prevented completely by the use of a yeast strain lacking invertase. These results show that the nutritional profile of bread can be enhanced through appropriate yeast technology.
Assuntos
Pão/microbiologia , Frutanos/metabolismo , Saccharomyces cerevisiae/metabolismo , Triticum/metabolismo , Pão/análise , Microbiologia de Alimentos , Frutanos/análise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Triticum/química , Triticum/microbiologiaRESUMO
Fermenting yeast does not merely cause dough leavening, but also contributes to the bread aroma and might alter dough rheology. Here, the yeast carbon metabolism was mapped during bread straight-dough fermentation. The concentration of most metabolites changed quasi linearly as a function of fermentation time. Ethanol and carbon dioxide concentrations reached up to 60 mmol/100g flour. Interestingly, high levels of glycerol (up to 10 mmol/100g flour) and succinic acid (up to 1.6 mmol/100g flour) were produced during dough fermentation. Further tests showed that, contrary to current belief, the pH decrease in fermenting dough is primarily caused by the production of succinic acid by the yeast instead of carbon dioxide dissolution or bacterial organic acids. Together, our results provide a comprehensive overview of metabolite production during dough fermentation and yield insight into the importance of some of these metabolites for dough properties.
Assuntos
Pão/microbiologia , Saccharomyces cerevisiae/metabolismo , Ácido Succínico/metabolismo , Triticum/microbiologia , Pão/análise , Dióxido de Carbono/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Triticum/metabolismoRESUMO
An improved method for the measurement of fructans in wheat grains is presented. A mild acid treatment is used for fructan hydrolysis, followed by analysis of the released glucose and fructose with high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Not only the amount of fructose set free from fructans but also the released glucose can be quantified accurately, allowing determination of the average degree of polymerization of fructans (DP(av)). Application of the mild acid treatment to different grain samples demonstrated that a correction should be made for the presence of sucrose and raffinose, but not for stachyose or higher raffinose oligosaccharides. The fructan content and DP(av) of spelt flour, wheat flour, and whole wheat flour were 0.6%, 1.2%, and 1.8% of the total weight and 4, 5, and 6, respectively. Validation experiments demonstrate that the proposed quantification method is accurate and repeatable and that also the DP(av) determination is precise.
Assuntos
Frutanos/análise , Frutanos/química , Polímeros/química , Sementes/química , Triticum/química , Frutose/análise , Glucose/análise , Hidrólise , Reprodutibilidade dos TestesRESUMO
Non-catalytic carbohydrate binding on independent carbohydrate-binding modules (CBMs) has been reported frequently for glycoside hydrolases (GHs) and reviewed thoroughly. However, various structural studies of GHs have revealed that non-catalytic carbohydrate binding sites can also occur on the surface of the structural unit comprising the active site. Here, the discovery of these sites, referred to as secondary binding sites (SBSs), and their putative roles in different GHs is reviewed for the first time. The majority of the SBSs have been discovered in starch-active enzymes, but there are also many reports of SBSs in various other enzymes. A wide variety of functions has been ascribed to these sites, including (1) targeting of the enzyme towards its substrate, (2) guiding the substrate into the active site groove, (3) substrate disruption, (4) enhancing processivity, (5) allosteric regulation, (6) passing on reaction products, and (7) anchoring to the cell wall of the parent microorganism. A lot of these putative functions are in agreement with the functions ascribed to non-catalytic binding in CBMs. Contrarily to CBMs, SBSs have a fixed position relative to the catalytic site, making them more or less suitable to take up specific functions.
Assuntos
Metabolismo dos Carboidratos , Glicosídeo Hidrolases/metabolismo , Animais , Sítios de Ligação , Glicosídeo Hidrolases/química , Humanos , Modelos Moleculares , SuínosRESUMO
Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a catalytically incompetent enzyme that allows substrate binding to both the AS and SBS. In the second enzyme, binding to the SBS was impaired by site-directed mutagenesis, whereas in the third enzyme, the AS was blocked using a covalent inhibitor. Both techniques were able to show that AS and SBS have a similar binding affinity.
Assuntos
Bacillus subtilis/enzimologia , Calorimetria/métodos , Endo-1,4-beta-Xilanases/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Endo-1,4-beta-Xilanases/antagonistas & inibidores , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Mutagênese Sítio-DirigidaRESUMO
In biomass degradation using simultaneous saccharification and fermentation (SSF), there is a need for efficient biomass degrading enzymes that can work at lower temperatures suitable for yeast fermentation. As xylan is an important lignocellulosic biomass constituent, this study aimed at investigating the possible differences in xylan breakdown potential of endoxylanases using eight different endoxylanases at conditions relevant for SSF. Both solubilising and degrading capacities of the endoxylanases were investigated using water-insoluble and water-soluble oat spelt xylan as model substrates for biomass xylan. Results showed that selecting for combinations of endoxylanases that are efficient at solubilising xylan on the one hand and degrading it to large extent on the other hand, coupled to high specific activities, seems the best option for complete xylan breakdown in lignocellulosic biomass conversion using SSF.
Assuntos
Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Indústria Alimentícia/métodos , Proteínas Fúngicas/metabolismo , Xilanos/metabolismo , Avena , Técnicas de Cultura Celular por Lotes , Biomassa , Endo-1,4-beta-Xilanases/classificação , Fermentação , Microbiologia de Alimentos , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas de Plantas/metabolismo , Solubilidade , Especificidade por Substrato , Temperatura , Xilose/metabolismoRESUMO
Previously, it has been demonstrated that the glycoside hydrolase family 8 xylanase from the psychrophylic bacterium Pseudoalteromonas haloplanktis (XPH) can bind substrate non-catalytically on the surface of its catalytic module. In the present study, the functional relevance of this secondary binding site (SBS) for the enzyme is investigated by site-directed mutagenesis and evaluation of activity and binding properties of mutant variants on a range of structurally different homoxylan and heteroxylan substrates. The SBS had an impact on the activity on insoluble substrates, whereas the activity on soluble substrates remained unaffected. Unexpectedly, the activity on a soluble oligomeric substrate was also affected for some mutants and results on a chromophoric polymeric model substrate were in contrast with the trends observed on the corresponding natural substrate. All in all, results show that the impact of the SBS on the activity of XPH is in part analogous to the functioning of some carbohydrate-binding modules in modular enzymes.
Assuntos
Pseudoalteromonas/enzimologia , Xilosidases/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Pseudoalteromonas/genética , Especificidade por Substrato , Xilosidases/genéticaRESUMO
A new technique to visualize cereal cell walls by fluorescence microscopy was developed. The novel staining technique is based on an inactive fluorescently labeled xylanase binding to arabinoxylan (AX), an important polysaccharide in grain cell walls in terms of the technological and physiological functionalities of grain. The xylanase probe could stain AX in the seed coat, nucellar epidermis, aleurone layer, and starchy endosperm, but not the highly substituted AX of the pericarp layer. The advantage of this new staining technique over the existing immunolabeling techniques is that the staining procedure is clearly faster and less laborious, and uses a smaller probe that can easily be produced by marking a well characterized enzyme with a fluorescent label. In the future, the here proposed technology can be used to develop probes having specificity also for cell wall components other than AX and thus to study plant cell walls further through fluorescence microscopy.
Assuntos
Parede Celular/química , Grão Comestível/química , Microscopia de Fluorescência/métodos , Xilano Endo-1,3-beta-Xilosidase/química , Xilanos/química , Parede Celular/metabolismo , Grão Comestível/metabolismo , Microscopia de Fluorescência/instrumentação , Ligação Proteica , Xilanos/metabolismoRESUMO
Xylanases (EC 3.2.1.8) are enzymes that can hydrolyze the xylan backbone internally. Therefore, they are important for biomass breakdown and they are also often added in various biotechnological applications. In this study, the relationship between their substrate binding affinity and hydrolysis, on the one hand, and their movement over natural substrates, on the other hand, was investigated. Fluorescence recovery after photobleaching (FRAP) experiments using different Bacillus subtilis xylanase A (XBS) mutants were conducted on water-unextractable wheat flour arabinoxylan (WU-AX) and insoluble oat spelt xylan (OSX). To assess the importance of substrate hydrolysis, FRAP of a catalytically inactive mutant was compared to that of the wild-type enzyme. For the wild-type enzyme, substrate binding and a complete recovery of fluorescence after photobleaching was observed on both substrates. For the inactive mutant, however, substrate binding but no fluorescence recovery was observed on WU-AX, while very slow recovery was observed on OSX. Furthermore, the importance of substrate binding to a secondary xylan binding site (SBS) for enzyme mobility was studied by testing two mutants with a modified SBS (N54W-N141Q and G56A-T183A-W185A) that showed different behavior on the tested substrates. On OSX, the two modified enzymes both showed higher mobility than the wild-type enzyme. On WU-AX, in contrast, the N54W-N141Q mutant displayed a lower mobility than the wild-type enzyme, while the G56A-T183A-W185A mutant showed higher mobility. The results clearly demonstrate that both substrate hydrolysis and substrate targeting are key factors for XBS mobility.
Assuntos
Endo-1,4-beta-Xilanases/química , Recuperação de Fluorescência Após Fotodegradação/métodos , Substituição de Aminoácidos , Bacillus subtilis/enzimologia , Sítios de Ligação , Biocatálise , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Hidrólise , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Xilanos/química , Xilanos/metabolismoRESUMO
The secondary substrate binding site (SBS) of Bacillus subtilis and Aspergillus niger glycoside hydrolase family 11 xylanases was studied by site-directed mutagenesis and evaluation of activity and binding properties of mutant enzymes on different substrates. Modification of the SBS resulted in an up to three-fold decrease in the relative activity of the enzymes on polymeric versus oligomeric substrates and highlighted the importance of several amino acids in the SBS forming hydrogen bonds or hydrophobic stacking interactions with substrates. Weakening of the SBS increased K(d) values by up to 70-fold in binding affinity tests using natural substrates. The impact that modifications in the SBS have both on activity and on binding affinity towards polymeric substrates clearly shows that such structural elements can increase the efficiency of these single domain enzymes on their natural substrates.
Assuntos
Aspergillus niger/enzimologia , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas Fúngicas/metabolismo , Xilosidases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Xilanos/metabolismo , Xilosidases/química , Xilosidases/genéticaRESUMO
Wheat kernel associated endoxylanases consist of a majority of microbial endoxylanases and a minority of endogenous endoxylanases. At least part of these enzymes can be expected to end up in wheat flour upon milling. In this study, the contribution of both types of these endoxylanases to changes in the arabinoxylan (AX) population during wheat flour breadmaking was assessed. To this end, wheat flour produced from two wheat varieties with different endoxylanase activity levels, both before and after sodium hypochlorite surface treatment of the wheat kernels, was used in a straight dough breadmaking procedure. Monitoring of the AX population during the breadmaking process showed that changes in AX are to a large extent caused by endogenous endoxylanases, whereas the contribution of microbial endoxylanases to these changes was generally very low. The latter points to a limited contamination of wheat flour with microbial enzymes during milling or to an extensive inactivation of these wheat flour associated microbial endoxylanases by endoxylanase inhibitors, present in wheat flour. When all wheat kernel associated microbial endoxylanases were first washed from the kernels and then added to the bread recipe, they drastically affected the AX population, suggesting that they can have a large impact on whole meal breadmaking.
Assuntos
Pão/análise , Endo-1,4-beta-Xilanases/metabolismo , Sementes/enzimologia , Sementes/microbiologia , Triticum/enzimologia , Xilanos/análise , Bacillus subtilis/enzimologia , Farinha/análise , Farinha/microbiologia , Manipulação de Alimentos/métodos , Penicillium/enzimologiaRESUMO
The impact of varying levels of endoxylanase activity in wheat flour on arabinoxylan (AX) in mixed and rested dough was studied using eight industrially milled wheat flour fractions with varying endoxylanase activity levels. Analysis of the levels of reducing end xylose (RX) and solubilized AX (S-AX) formed during mixing and resting and their correlation with the endoxylanase activity in the flour milling fractions showed that solubilization of AX during the mixing phase is mainly due to mechanical forces, while solubilization of AX during resting is caused by endoxylanase activity. Moreover, solubilization of AX during the dough resting phase is more outspoken than that during the mixing phase. Besides endoxylanase activity, there were significant xylosidase and arabinofuranosidase activities during the dough resting phase. The results indicate that wheat flour-associated endoxylanases can alter part of the AX in dough, thereby changing their functionality in bread making and potentially affecting dough and end product properties.