Changes in wheat lipids during mixing and resting of flour-water doughs

Lipids extracted with water-saturated n-butanol from flour and flour-water doughs were examined to determine the extent of oxidations and other changes which occurred in mixing and resting dough. Extracted lipids were converted to fatty acid methyl esters (FAME) and quantified by gas-liquid chromatography (g.l.c.) using heptadecanoic acid (17 : 0) as internal standard. The original flour or dough and the corresponding solvent-extracted residues were acid hydrolysed, and the hydrolysate lipids converted to FAME for g.l.c. determination of the total lipid and residual unextracted lipid contents. The total flour or dough lipids equalled the extracted lipids + unextracted lipids, except where there were unavoidable autoxidative losses of linoleate (18 : 2) and linolenate (18 : 3). The unextracted flour lipids (13% of total lipids) were not oxidised during dough mixing. There were no changes in any of the extracted lipid classes other than free fatty acids (FFA) and monoglycerides (MG) which showed losses of 18 : 2 and 18 : 3 after aerobic dough mixing. Losses of FFA and MG are attributed to lipoxygenase activity during dough mixing and the period immediately after. The small amount of 18 : 2 in the “free” petroleum ether-extracted FFA appeared to be unaffected by lipoxygenase. Recoveries of FFA other than 18 : 2 or 18 : 3 were constant, indicating no lipolysis of glycerolipids and no general oxidation or degradation of FFA. Experiments with [U-¹⁴C]palmitic acid confirmed that there was no oxidation, degradation or re-esterification of FFA. Much of the non-polar lipids (steryl ester, triglyceride, diglyceride, FFA, MG) and almost all of the polar lipids were bound on dough mixing. Binding was non-selective with regard to fatty acid composition. Triglyceride was the only lipid class bound to a greater extent in anaerobic dough than in aerobic dough. Some selectivity of binding between lipid classes was indicated.     

Studies on the lipids of flour IV.—Factors affecting lipid binding in breadmaking

The binding of specific lipids during the high-speed mixing of doughs and in the resulting bread was studied in relation to mixer atmosphere. the presence of air was found to cause a fall in the linoleic acid content of the triglyceride lipids, an effect apparently related to the reduction in lipid binding. Replacement of the free lipid of flour (the main source of triglyceride linpleate) by extra shortening fat caused a large increase in bound lipid in doughs mixed either in air or in nitrogen, together with the drop in bread quality and volume observed previously. It was concluded that the natural free lipid of flour plays an important part in modern breadmaking processes both in responding to the atmosphere in the dough mixing chamber and in its effect on the binding of shortening triglyceride during dough development.     

Significance of lipid binding on the functional and nutritional profiles of single and multigrain matrices

Lipid fractions and starch- and protein-lipid binding of single and blended oat, rye, buckwheat and wheat flour, dough and bread matrices were investigated, and results correlated with the functional and nutritional properties of the grain matrices during mixing and baking. Non-starch lipid was the most prominent fraction in terms of absolute content and as a percentage of total lipids. Free lipids, starch lipids and bound lipids were, respectively, the major, intermediate and minor lipid fractions in flours, doughs and breads. Great differences in total lipid content due to sampling result in divergences amongst lipid fraction content and distribution, especially for starch and bound lipid fractions. Lipids bound to proteins during dough mixing are translocated and bound to starch during baking. In blended samples, the higher fibre content seems to provoke a reduction of the lipid–protein and lipid–starch linkages due to interactions between fibres and endogenous biopolymers. Starch lipid showed the most significant correlations with parameters related to dough and bread performance during breadmaking, especially over the mixing step. Valuable fresh bread functional characteristics, such as high specific volume and high sensory score for softness and overall acceptability, correspond to a starch lipid’s increase due to mixing. The higher the free and starch lipids decrease by reason of temperature treatment—baking—the larger the starch hydrolysis and the higher β-glucans and total dietary fibre contents.     

The effect of lipoxygenase action on the mechanical development of doughs from fat-extracted and reconstituted wheat flours

The mechanism by which soya lipoxygenase enzyme action improves the Theological properties of wheat flour doughs during mechanical development in air has been investigated further. Free-lipid extraction, reconstitution and replacement experiments have shown that the rheological effect of lipoxygenase action, which is consistent with an oxidative improvement of the dough proteins and may also result in extended mixing tolerance, only occurred in the presence of an oxidisable, polyunsaturated, free-lipid substrate. Addition of this substrate in an oxidised state (produced either by autoxidation or enzyme-oxidation) to doughs mixed from fat-extracted flour under nitrogen resulted only in a small rheological improvement, greater for the autoxidised than the enzyme-oxidised lipid, but in no way comparable with the large rheological effect of lipoxygenase action during dough mixing in air. Furthermore, the presence of an antioxidant, nordihydroguaiaretic acid (NDGA), during dough development, although greatly inhibiting peroxide formation, only marginally impaired the rheological improvement due to lipoxygenase action. Additional evidence is therefore provided for a coupled oxidation mechanism being responsible for the rheological effect, since lipoxygenase-catalysed oxidation actively occurring in the dough during mixing appears to be the fundamental requirement, irrespective of whether the primary oxidation products lead to lipid peroxides or oxidised NDGA.     

Effects of dough mixing and oxidising improvers on free reduced and free oxidised glutathione and protein-glutathione mixed disulphides of wheat flour

Changes in free reduced glutathione (GSH), free oxidised glutathione (GSSG) and protein-glutathione mixed disulphides (PSSG) during dough mixing were monitored. After a rapid decrease in the GSH content and an increase in the GSSG content, the contents of both GSH and GSSG decreased progressively, whereas the PSSG content increased, as a simple flour-water dough was mixed. Total glutathione (GSH plus GSSG plus PSSG) levels in simple flour-water doughs and doughs treated with ascorbic acid or potassium bromate remained essentially constant during dough mixing, indicating that the reactions in which glutathione is involved are simple oxidations of sulphydryl (SH) groups to disulphide (SS) bonds and SH/SS interchange reactions. Yeast contributed high levels of GSH and GSSG to doughs, but analysis of dough aqueous phases (liquors) and the similarity of the PSSG contents of simple and yeasted flour-water doughs suggested that the yeast GSH and GSSG were largely unavailable to react with flour proteins. The GSH content of ascorbic-acid-treated and yeasted dough decreased rapidly on wetting the flour, the magnitude of the decrease indicating that the ascorbate oxidation system oxidised the yeast intracellular GSH as well as the flour GSH. With further mixing, the GSH content of the ascorbate dough remained constant at low levels similar to those of the simple flour-water dough. The GSSG content of the ascorbate dough increased rapidly on wetting the flour, but declined as dough mixing continued. The PSSG content of the doughs increased markedly as dough mixing proceeded to the optimum and then stabilised. The increase in the PSSG content lagged behind the rapid oxidation of GSH to GSSG. Potassium bromate caused a pattern of changes similar to those observed for ascorbate, but the changes in GSH and PSSG contents were smaller in magnitude. The results indicate that the changes in the different glutathione pools and the effects of oxidising bread improvers are rather more complex than envisaged previously, particularly the effects on PSSG.     

Studies on the lipids of flour. 3. Lipid binding in breadmaking

The proportions of easily extractable (free) lipid and residual bound lipid in dough and bread prepared by three different methods were determined and compared with the proportions of free and bound lipids present in the separate ingredients. Lipid binding increased during mixing to an extent dependent on the rate of dough development employed. The increase in bound lipid observed during dough mixing was due mainly to non-selective binding of the available triglycerides. Diglycerides and free fatty acids were bound in preference to sterol esters and hydrocarbons, while the selective binding of polyunsaturated polar lipid represented only a small proportion of the observed lipid binding. After baking, the binding of polyunsaturated polar lipid accounted for half the observed increase in bound lipid, which may indicate that such lipids are of much greater significance in understanding the behaviour of bread in the oven than has been suspected hitherto. Nevertheless, this selectively bound polar lipid represents only a small proportion of the total bound lipid existing in dough and bread and is therefore not sufficient to affect significantly the overall fatty acid composition of either the total bound lipid or the remaining free lipid. It would appear that the presence of linoleic acid in a lipid is not sufficient in itself to ensure preferential binding during breadmaking.     

Comparison of lipid effects on structural features of hard and soft wheat flour proteins assessed by front-face fluorescence

Front-face fluorescence was used to assess changes in the structural features of proteins in wheat flour dough upon addition of different exogenous lipids. Structural changes resulting from the solvation of proteins and from the mechanical deformation of proteins in dough or dough containing lipids were measured. The effects of lipid type and addition on changes in overall protein surface hydrophobicity were estimated by titrating soft and hard wheat flours, mixed with water and varying the lipid levels, with increasing concentrations of the fluorescence hydrophobic probes 1,8 anilino-napthalene-sulphonate (ANS) and thioflavin T. The lipid type and level modified the exposure of the probe to the solvent. The effects of lipids were more apparent with soft wheat flour having low-affinity hydrophobic sites on the protein surface. The dough was then characterized upon consistent mixing and physical modification in the farinograph at constant water and ANS/thioflavin T concentration, while varying the type and amount of lipids. Lipid-dependent shifts toward longer wavelengths in the probe fluorescent emission with low-protein flour suggest differences in protein coating effects related to lipid structure and protein quality.     

The effects of pure saturated and unsaturated fatty acids bon breadmaking and on lipid binding,using chorleywood bread process doughs containing a ‘model fat’

When a fresh flour dough containing a model fat prepared from pure saturated and unsaturated triglycerides was supplemented with pure oleic or linoleic acids in amounts sufficient to raise the level of unesterified fatty acids to that obtained with a flour badly deteriorated by age, the acids were equally detrimental to loaf volume. Margaric acid added at the same level was not deleterious. Lipid binding studies showed that both unsaturated fatty acids behaved similarly during doughmixing, not only becoming largely ‘bound’ themselves, but also causing increased binding of glyceryl trioleate and flour lipids. Conversely, margaric acid remained largely ‘free’ and did not affect the binding of other dough lipid constituents.     

Studies on the lipids of flour V. —Effect of air on lipid binding

Lipid distribution in mechanically developed doughs was found to be sensitive to relatively small amounts of air present in the dough mixer atmosphere. In doughs mixed to high work levels, less than 5 % air present in the nitrogen-air mixture fed into the mixing bowl was sufficient to cause a significant decrease in bound lipid. Although flour pigments were readily bleached by small amounts of air, lipid peroxides did not increase significantly until at least 50% air was present. Polyunsaturated free fatty acids were most susceptible to peroxidation although, at high work levels in air, peroxides were found in all lipid classes. While lipids bound during nitrogen mixing were readily released by subsequent mixing in air, the effect of air on lipid binding was not reversed by further mixing in nitrogen. Addition of peroxidised lipid to the dough did not prevent lipid binding in nitrogen-mixed doughs and it was concluded that a mechanism of lipoxidase-coupled site oxidation was responsible for the effect of air on lipid binding rather than the direct action of the lipid peroxides themselves.     

Development of a spectroscopic method to determine the content of free radical scavenging compounds and oxidation products in thermally oxidised oils

A simple spectroscopic method using 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) was developed to simultaneously monitor the contents of free radical scavenging antioxidants (FRSs) and oxidised lipid products during oxidation through selection of proper solvents. Validation of the DPPH method was conducted in a stripped oil matrix, and practical applicability of the method was tested with heated oils. The absorbance of DPPH in isooctane simultaneously reflected the changing amounts of both FRSs and oxidised lipid products, whereas the absorbance in methanol mainly reflected changes in FRSs. Total polar materials (TPMs) were found to be representative of oxidised lipid products, which reacted with DPPH in isooctane better than in methanol. Validation parameters including accuracy, precision, linearity, limit of detection and limit of quantification were determined using α‐tocopherol as a model for FRSs and TPMs for oxidised lipid products. The DPPH protocol in isooctane and in methanol is useful for assessing the degree of oxidation in heated oils through comparing the contents of FRSs and oxidised lipids.     

The effect of lipoxygenase action on the mechanical development of wheat flour doughs

The mechanical development of dough has been followed by measurement of the relaxation time of dough samples after mixing to various levels of work input. Parallel determinations of free and bound lipid have also been made. When doughs were mixed in air, addition of full-fat, enzyme-active soya bean flour (subsequently referred to as “soya flour”) resulted in an increase in relaxation time, particularly at higher work levels. The magnitude of this improvement increased with increasing work input and was dependent on the rate of work input. Addition of soya flour also enabled a higher level of mechanical work to be introduced before dough breakdown occurred. When doughs were mixed under nitrogen, or when the soya flour was heat denatured, no change in the rheological properties compared with the respective control doughs was found. The release of bound lipid, which occurred during dough development in air in the presence of soya flour, could also be induced by adding purified lipoxygenase to the dough, together with linoleic acid as a substrate. This resulted in rheological changes similar to those observed using soya flour. However addition of enzymically pre-peroxidised lipid to doughs mixed in nitrogen was without effect on relaxation times. These findings suggest that lipid release is linked with structural changes in dough protein and provide further support for a mechanism of coupled oxidation of protein -SH groups by lipoxygenase.     

Effects of endogenous flour lipids on the quality of short‐dough biscuits

Fractionation and reconstitution techniques were used to study the contribution of endogenous flour lipids to the quality of short‐dough (shortcake type) biscuits. Biscuit flour was defatted with chloroform and baked with bakery fat, but without endogenous lipid. Short‐dough biscuits baked from defatted flour had smaller diameters, and were flatter, denser and harder than control biscuits. Defatted flour shortcake doughs exhibited different rheological behaviour from the control samples, showing higher storage and loss moduli (G′ and G″ values), ie higher viscoelasticity. Functionality was restored when total non‐starch flour lipids were added back to defatted flour. The polar lipid fraction had a positive effect in restoring flour quality whereas the non‐polar lipid fraction had no effect. Both fractions were needed for complete restoration of both biscuit quality and dough rheological characteristics. A study of the microstructure of defatted biscuits revealed that their gluten protein was more hydrated and developed than the gluten of the control biscuits. This conclusion was supported by the higher water absorption of the defatted gluten. Copyright © 2004 Society of Chemical Industry     

Lipid-protein interactions during dough development

Radionuclide-labelled glycerol triolein has been used to follow the fate of triglyceride lipids during the mixing of wheat flour doughs. After removal of residual free lipid by petrol extraction, initial fractionation of freeze-dried dough identified the acetic acid-soluble protein as being the only component involved significantly in work-induced lipid binding during dough development under nitrogen, and also in lipid release on admission of air. Very little labelled lipid was found in either the water-soluble proteins or the starch residue. Sub-fractionation of the acetic acid-soluble protein by ammonium sulphate precipitation from acetic acid-urea-cetyltrimethyl-ammonium bromide (AUC) solvent showed bound lipid to be distributed almost entirely between high-molecular-weight glutenin and protein which remained soluble in the AUC supernatant even in the presence of 20% ammonium sulphate. Precipitated gliadins contained very little labelled lipid. However, significant levels were found in classical ethanol-extracted gliadin, and were traced predominantly to the same supernatant-protein owing to its solubility in aqueous ethanol. Examination of this AUC-supernatant fraction showed it to contain a hitherto unreported protein which had a molecular weight of about 9000, was strongly complexed with tri-glyceride lipid on a 1:1 molar basis and showed a tendency to aggregate in solution. Its amino acid frequency was found to differ significantly from both glutenin and purothionin, the latter containing in particular very much more cysteine than the supernatant-protein. Representing 10% or more of the total gluten, this small, highly interactive protein is responsible for a significant, if not the major, part of lipid binding activity in dough and may well have a fundamental role in the formation of an insoluble glutenin structure through both —SH and hydrophobic interaction. Accordingly, the name ‘Ligolin’ is proposed, from the Latin ligare: to bind, to tie.     

Relationship between free glutathione content and quality assessment parameters of wheat cultivars (Triticum aestivum L.)

Freshly milled flour samples from ten cultivars of bread wheat were analysed for free reduced glutathione (GSH) and oxidised glutathione (GSSG) by an enzymic method. Dough rheological properties for each flour were assessed by standard Brabender techniques. Flour quality differences were also measured by Pelshenke fermentation and Zeleny sedimentation techniques. Reduced glutathione levels were negatively correlated with oxidised glutathione levels, Farinogram mixing tolerance, Extensogram resistance, Pelshenke time and Zeleny volume. The ratios of oxidised to reduced glutathione (GSSG: GSH) were positively correlated with all dough and flour quality parameters, except Extensogram extensibility. It is concluded that there are differences in the endogenous contents of reduced and oxidised glutathione between flour samples from ten cultivars of wheat. These differences may contribute significantly to variations in the quality assessment parameters established between cultivars in this study.     

Protein–lipid interactions in gluten elucidated using acetic acid fractionation

Protein–lipid interactions in dough have an important impact on the quality of bakery products. Understanding of protein–lipid interactions in gluten can enhance the development of technological solutions to improve the breadmaking quality of flour as well as the functional properties of gluten. In this study, acetic acid at two different concentrations was used for treating and fractionating gluten. The impact of these procedures on the distribution of lipid components was measured. Acetic acid was able to dissociate non-polar lipids from the gluten protein matrix. Upon fractionation monomeric proteins (predominantly gliadins) and phospholipids were high in the 0.01 M acetic acid soluble fraction. The subsequent fractionation step using 0.1 M acetic acid resulted in an increased amount of high-molecular-weight glutenin subunits (HMW-GS) in the soluble fraction, along with more non-polar lipids and glycolipids in both the free and bound lipid extracts. The distribution of lipid classes demonstrates that non-polar lipids are either associated with the glutenin polymeric network through hydrophobic interactions or entrapped within the gluten matrix. The results also indicate that in gluten, glycolipids are likely to be associated with glutenins through both hydrophobic interactions and hydrogen bonds whilst phospholipids preferentially interact with gliadins and lipid binding proteins.     

The association of saturated and unsaturated triglycerides and oleic acid with wheat flour components during dough making

The association of lipids with other flour components after dough making has been examined by aqueous fractionation of flour prior to the extraction of lipids. Glycerol tripalmitate has been shown to be associated with acetic-acid-insoluble protein while glycerol trioleate associates with the glutenin of acetic-acid-soluble protein. Oleic acid divides fairly evenly between these fractions. Changes in lipid and protein distribution in some fractions are associated with the addition of oleic acid to the dough. Some of these changes are at least partially reversed by increasing the triglyceride content of the dough.     

Cooperation of phosphatidylcholine with endogenous lipids of wheat flour for an increase in dough volume

Phosphatidylcholine (PC) increases the gas-retaining ability of dough, the dough volume on fermentation and the loaf volume of bread. The cooperation of wheat flour endogenous lipids with PC was examined. More than 90% of the total wheat flour lipids were extracted with chloroform, the extracted lipids comprising glycolipids (33 wt%), non-polar lipids (56 wt%), and phospholipids (11 wt%). The increase in the specific volume of dough with delipidated wheat flour by the addition of PC was smaller than the increase in the specific volume of dough with native wheat flour. The addition of the extracted lipids to delipidated wheat flour restored the increase in dough volume by the addition of PC. The glycolipid fraction of the extracted lipids was most effective for enhancing the action of PC. The results suggest that interaction of PC with wheat flour glycolipids may synergistically increase foam stability to enhance the gas-retaining stability of dough.     

Rheological activity of peptides,simple disulphides and simple thiols in wheaten dough

The disulphide peptides of flour have been separated into acidic (glutathione) and basic peptides, and their effects on the rheological properties of dough have been investigated. the only peptide normally present in flour to have marked activity in dough was glutathione. The other disulphide peptides of flour were only slightly active in that their sole effect was to diminish the tolerance of the dough to prolonged mixing. Their lack of activity probably results from their disulphide groups being non-reactive at the pH values found in dough. the same peptides in the thiol form were slightly more active in that they decreased development time and maximum resistance. All types of thiols tested were active in dough, but of the disulphides tested, only the acidic peptides GSSG (oxidised glutathione) and bis-γ-glutamylcystine had significant activity. Possible modes of action of disulphide peptides in dough are discussed.     

Changes of chemical composition and dough rheology in two fractions of sieve‐classified Polish spring wheat flour

The study of chemical composition and dough rheology changes in sieve‐classified two fractions (up to 60 and 60–240 μm particles) of wheat flour was the subject of this study. The straight grade flours were obtained by the milling of three Polish varieties of spring wheat, differing in ?? abbreviation? (PSI) values. The flours were separated with the use of a SZ‐1 laboratory sifter. The yield of fine fraction was in the range 50.0–55.7%. The obtained fractions were assayed for the content and composition of free lipids, gluten proteins, damaged starch, ash, water absorption and amylograph viscosity. Dough rheology (extrusion in OTMS cell, alveograph and farinograph tests) and baking trials were also performed. The content of free lipids, including the non‐polar and phospholipids was lower and the content of glycolipids was higher in fine flours. Those fractions were more rich in linoleic acid but the lower content of oleic and linolenic acids resulted in a higher oxidizability index of free lipids. Fine flours contained less ash and significantly more damaged starch. At the same time, they were characterized by a higher content of wet gluten, water absorption, amylograph viscosity and better dough parameters. This was reflected in the bread volume, which was higher by 6.3–10.7%. The influence of the changes in composition and the content of free lipids upon the rheology of the dough after the 90 days flour storage has not been defined unambiguously and requires further research.     

Changes of chemical composition and dough rheology in two fractions of sieve-classified Polish spring wheat flour

The study of chemical composition and dough rheology changes in sieve-classified two fractions (up to 60 and 60-240 microm particles) of wheat flour was the subject of this study. The straight grade flours were obtained by the milling of three Polish varieties of spring wheat, differing in particle size index (PSI) values. The flours were separated with the use of an SZ-1 laboratory sifter. The yield of fine fraction was in the range 50.0-55.7%. The obtained fractions were assayed for the content and composition of free lipids, gluten proteins, damaged starch, ash, water absorption and amylograph viscosity. Dough rheology (extrusion in OTMS cell, alveograph and farinograph tests) and baking trials were also performed. The content of free lipids, including the non-polar and phospholipids was lower and the content of glycolipids was higher in fine flours. Those fractions were more rich in linoleic acid but the lower content of oleic and linolenic acids resulted in a higher oxidizability index of free lipids. Fine flours contained less ash and significantly more damaged starch. At the same time, they were characterized by a higher content of wet gluten, water absorption, amylograph viscosity and better dough parameters. This was reflected in the bread volume, which was higher by 6.3-10.7%. The influence of the changes in composition and the content of free lipids upon the rheology of the dough after the 90 days flour storage has not been defined unambiguously and requires further research.