Impact of aqualysin 1 peptidase from Thermus aquaticus on molecular scale changes in the wheat gluten network during bread baking.

The impact of Aqualysin 1 (Aq1), the thermo-active peptidase of Thermus aquaticus, on wheat albumin, globulin, gliadin and glutenin proteins during heat treatment of wheat dough and bread baking was examined. The level of protein extractable in sodium dodecyl sulfate containing medium under non-reducing conditions (SDS-EP-NR) from wheat dough decreases upon heating to a lesser extent when Aq1 is used than in control experiments. The higher SDS-EP-NR level is caused by the release by Aq1 of peptides from the repetitive gluten protein domains during baking. These peptides are also extractable from bread crumb with salt solution. The resultant thermoset gluten network in bread crumb is mainly made up by protein from non-repetitive gluten domains.

Electrical resistance oven baking as a tool to study crumb structure formation in gluten-free bread.

Gradientless baking by means of ohmic heating was used for the first time in gluten-free (GF) bread making. Combination thereof with in-line measurements of batter height, viscosity and carbon dioxide (CO2) release proved to be powerful for studying structure formation in GF breads. GF breads studied here were based on (i) a mixture of potato and cassava starches and egg white powder (C/P-S+EW), (ii) rice flour (RF) or (iii) a mixture of RF and egg white powder (RF+EW). The work revealed that bread volume and crumb structure rely heavily on the balance between the moment of CO2 release from batter during baking and that of crumb setting. At the moment of CO2 release, C/P-S+EW bread crumb had already (partly) set, while this was not the case for RF bread crumb, resulting in a collapse and thus low volume of the latter. When a part of RF was replaced by egg white powder, the moment of CO2 release was postponed and the batter collapse was less pronounced, leading to a higher volume and a finer crumb. The presence of egg white proteins in C/P-S+EW or RF+EW batters improved gas cell stabilization. Thus, increasing batter stability or altering the moment of crumb setting results in GF breads with higher volume and a finer crumb structure.

Enzymatically Hydrolyzed Wheat Gluten as a Foaming Agent in Food: Incorporation in a Meringue Recipe as a Proof-of-Concept.

There is a growing interest in substituting animal proteins with plant protein sources in food systems. A notable example is the replacement of hen egg white (EW) protein, which is used in a wide range of food products because of its excellent foaming characteristics. Here, enzymatically hydrolyzed wheat gluten, which has greater solubility and better foaming properties than wheat gluten itself, was prepared and incorporated in a classical meringue recipe to investigate its potential as a foaming agent. Meringues based on gluten hydrolysates (GHs) had batters with lower density and greater apparent viscosity than those based solely on EW protein. Furthermore, after baking, these GH containing meringues had greater specific volume than those based on EW protein alone and no notable differences in color or texture between the different samples were noted. These outcomes were related to basic insights in the air-water interfacial behavior of GHs obtained in earlier studies. More specifically, the greater foaming capacity of GH than of EW protein solutions was related to their superior meringue batter (density and apparent viscosity) and product (specific volume) properties. While EW protein solutions had better foam stability than GH solutions (in the absence of sugar), this was apparently less relevant for meringue properties, probably due to the very high viscosity of the sugar rich batter, which could obscure differences in the intrinsic foam stabilizing ability of the samples. PRACTICAL APPLICATION: Replacing animal proteins with plant protein sources in the food industry is desirable from an economic and environmental perspective. Enzymatic hydrolysis serves as a tool to improve the foaming properties of water-insoluble wheat gluten proteins. We conclude that wheat gluten hydrolysates can be a valid functional alternative for egg white proteins in meringues, and possibly other food systems.

Thermo-reversible inhibition makes aqualysin 1 from Thermus aquaticus a potent tool for studying the contribution of the wheat gluten network to the crumb texture of fresh bread.

The thermo-active serine peptidase aqualysin 1 (Aq1) of Thermus aquaticus was applied in bread making to study the relative contribution of thermoset gluten to bread crumb texture. Aq1 is active between 30 °C and 90 °C with an optimum activity temperature of around 65 °C. It is inhibited by wheat endogenous serine peptidase inhibitors during dough mixing and fermentation and starts hydrolyzing gluten proteins during baking above 80 °C when the enzyme is no longer inhibited and most of the starch is gelatinized and contributes to structure formation. Aq1 activity reduced the molecular weight of gluten proteins and significantly increased their extractability in sodium dodecyl sulfate containing medium. While it had no impact on the specific bread volume and only limited impact on hardness, cohesiveness, springiness, resilience and chewiness, it impacted bread crumb coherence. We conclude that starch has a greater impact on crumb texture than thermoset gluten.

Exploring the Relationship between Structural and Air-Water Interfacial Properties of Wheat (Triticum aestivum L.) Gluten Hydrolysates in a Food System Relevant pH Range.

The relationship between structural and foaming properties of two tryptic and two peptic wheat gluten hydrolysates was studied at different pH conditions. The impact of pH on foam stability (FS) of the samples heavily depended on the peptidase used and the degree of hydrolysis reached. Surface dilatational moduli were in most, but not all, instances related to FS, implying that, although the formation of a viscoelastic protein hydrolysate film is certainly important, this is not the only phenomenon that determines FS. In contrast to what might be expected, surface charge was not a major factor contributing to FS, except when close to the point-of-zero-charge. Surface hydrophobicity and intrinsic fluorescence measurements suggested that changes in protein conformation take place when the pH is varied, which can in turn influence foaming. Finally, hydrolyzed gluten proteins formed relatively large particles, suggesting that protein hydrolysate aggregation probably influences its foaming properties.

Foam fractionation as a tool to study the air-water interface structure-function relationship of wheat gluten hydrolysates.

Enzymatic hydrolysis of wheat gluten protein improves its solubility and produces hydrolysates with foaming properties which may find applications in food products. First, we here investigated whether foam-liquid fractionation can concentrate wheat gluten peptides with foaming properties. Foam and liquid fractions had high and very low foam stability (FS), respectively. In addition, foam fractions were able to decrease surface tension more pronouncedly than un-fractionated samples and liquid fractions, suggesting they are able to arrange themselves more efficiently at an interface. As a second objective, foam fractionation served as a tool to study the structural properties of the peptides, causing these differences in air-water interfacial behavior. Zeta potential and surface hydrophobicity measurements did not fully explain these differences but suggested that hydrophobic interactions at the air-water interface are more important than electrostatic interactions. RP-HPLC showed a large overlap between foam and liquid fractions. However, a small fraction of very hydrophobic peptides with relatively high average molecular mass was clearly enriched in the foam fraction. These peptides were also more concentrated in un-fractionated DH 2 hydrolysates, which had high FS, than in DH 6 hydrolysates, which had low FS. These peptides most likely play a key role in stabilizing the air-water interface.

Relevance of the Functional Properties of Enzymatic Plant Protein Hydrolysates in Food Systems.

Proteins play a crucial role in determining texture and structure of many food products. Although some animal proteins (such as egg white) have excellent functional and organoleptic properties, unfortunately, they entail a higher production cost and environmental impact than plant proteins. It is rather unfortunate that plant protein functionality is often insufficient because of low solubility in aqueous media. Enzymatic hydrolysis strongly increases solubility of proteins and alters their functional properties. The latter is attributed to 3 major structural changes: a decrease in average molecular mass, a higher availability of hydrophobic regions, and the liberation of ionizable groups. We here review current knowledge on solubility, water- and fat-holding capacity, gelation, foaming, and emulsifying properties of plant protein hydrolysates and discuss how these properties are affected by controlled enzymatic hydrolysis. In many cases, research in this field has been limited to fairly simple set-ups where functionality has been assessed in model systems. To evolve toward a more widely applied industrial use of plant protein hydrolysates, a more thorough understanding of functional properties is required. The structure-function relationship of protein hydrolysates needs to be studied in depth. Finally, test model systems closer to real food processing conditions, and thus to real foods, would be helpful to evaluate whether plant protein hydrolysates could be a viable alternative for other functional protein sources.

Storage of parbaked bread affects shelf life of fully baked end product: a ¹H NMR study.

Full baking of earlier partially baked (parbaked) bread can supply fresh bread to the consumer at any time of the day. When parbaked bread loaves were stored at -25, 4 or 23°C, the extent of crumb to crust moisture migration and amylopectin retrogradation differed with storage temperature, and the firming rate was evidently lowest during frozen storage. The extent of crumb to crust moisture migration during parbaked bread storage largely determined the mass of the fresh finished bread, and its crumb and crust moisture contents. Initial NMR proton mobility, initial resilience, the extent of amylopectin retrogradation and changes in firmness and resilience during storage of fully baked bread were affected by its crumb moisture content. The lowest firming rate was observed for finished bread resulting from parbaked bread stored at -25°C, while the highest firming rate was observed for finished bread from parbaked bread stored at 23°C.

The impact of baking time and bread storage temperature on bread crumb properties.

Two baking times (9 and 24 min) and storage temperatures (4 and 25 °C) were used to explore the impact of heat exposure during bread baking and subsequent storage on amylopectin retrogradation, water mobility, and bread crumb firming. Shorter baking resulted in less retrogradation, a less extended starch network and smaller changes in crumb firmness and elasticity. A lower storage temperature resulted in faster retrogradation, a more rigid starch network with more water inclusion and larger changes in crumb firmness and elasticity. Crumb to crust moisture migration was lower for breads baked shorter and stored at lower temperature, resulting in better plasticized biopolymer networks in crumb. Network stiffening, therefore, contributed less to crumb firmness. A negative relation was found between proton mobilities of water and biopolymers in the crumb gel network and crumb firmness. The slope of this linear function was indicative for the strength of the starch network.

Impact of amylases on biopolymer dynamics during storage of straight-dough wheat bread.

When Bacillus stearothermophilus α-amylase (BStA), Pseudomonas saccharophila α-amylase (PSA), or Bacillus subtilis α-amylase (BSuA) was added to a bread recipe to impact bread firming, amylose crystal formation was facilitated, leading to lower initial crumb resilience. Bread loaves that best retained their quality were those obtained when BStA was used. The enzyme hindered formation of an extended starch network, resulting in less water immobilization and smaller changes in crumb firmness and resilience. BSuA led to extensive degradation of the starch network during bread storage with release of immobilized water, eventually resulting in partial structure collapse and poor crumb resilience. The most important effect of PSA was an increased bread volume, resulting in smaller changes in crumb firmness and resilience. A negative linear relation was found between NMR proton mobilities of water and biopolymers in the crumb and crumb firmness. The slope of that relation gave an indication of the strength of the starch network.

Biopolymer interactions, water dynamics, and bread crumb firming.

To establish the relationship between biopolymer interactions, water dynamics, and crumb texture evolution in time, proton mobilities in starch and gluten model systems and bread were investigated with NMR relaxometry. Amylopectin recrystallization was observed as an increased amount of fast-relaxing protons, while network strengthening and changes in water levels were noted as a reduced mobility and amount, respectively, of slowly relaxing protons. Amylopectin recrystallization strengthened the starch network with concomitant inclusion of water and increased crumb firmness, especially at the beginning of storage. The inclusion of water and the thermodynamic immiscibility of starch and gluten resulted in local gluten dehydration during bread storage. Moisture migration from crumb to crust further reduced the level of plasticizing water of the biopolymer networks and contributed to crumb firmness at longer storage times. Finally, we noted a negative relationship between the mobility of slowly relaxing protons of crumb polymers and crumb firmness.

Wheat (Triticum aestivum L. and T. turgidum L. ssp. durum) Kernel Hardness: II. Implications for End-Product Quality and Role of Puroindolines Therein.

Wheat kernel hardness is a major quality characteristic used in classifying wheat cultivars. Differences in endosperm texture among Triticum aestivum L. or between T. aestivum and T. turgidum L. ssp. durum cultivars profoundly affect their milling behavior, the properties of the obtained flour or semolina particles, as well as the quality of products made thereof. It is now widely accepted that the presence, sequence polymorphism, or absence of the basic and cysteine-rich puroindolines a and b are responsible for differences in endosperm texture. These proteins show features in vitro, including foaming and lipid-binding properties, which provide them with a potential impact in the production of wheat-based food products, where they may improve gas cell stabilization or modulate interactions between starch, proteins, and/or lipids. We here summarize the impact of wheat hardness on milling properties and bread, cookie, cake, and pasta quality and discuss the role of puroindolines therein.

Wheat (Triticum aestivum L. and T. turgidum L. ssp. durum) Kernel Hardness: I. Current View on the Role of Puroindolines and Polar Lipids.

Wheat hardness has major consequences for the entire wheat supply chain from breeders and millers over manufacturers to, finally, consumers of wheat-based products. Indeed, differences in hardness among Triticum aestivum L. or between T. aestivum L. and T. turgidum L. ssp. durum wheat cultivars determine not only their milling properties, but also the properties of flour or semolina endosperm particles, their preferential use in cereal-based applications, and the quality of the latter. Although the mechanism causing differences in wheat hardness has been subject of research more than once, it is still not completely understood. It is widely accepted that differences in wheat hardness originate from differences in the interaction between the starch granules and the endosperm protein matrix in the kernel. This interaction seems impacted by the presence of either puroindoline a and/or b, polar lipids on the starch granule surface, or by a combination of both. We focus here on wheat hardness and its relation to the presence of puroindolines and polar lipids. More in particular, the structure, properties, and genetics of puroindolines and their interactions with polar lipids are critically discussed as is their possible role in wheat hardness. We also address future research needs as well as the presence of puroindoline-type proteins in other cereals.

Assignments of proton populations in dough and bread using NMR relaxometry of starch, gluten, and flour model systems.

Starch-water, gluten-water, and flour-water model systems as well as straight-dough bread were investigated with (1)H NMR relaxometry using free induction decay and Carr-Purcell-Meiboom-Gill pulse sequences. Depending on the degree of interaction between polymers and water, different proton populations could be distinguished. The starch protons in the starch-water model gain mobility owing to amylopectin crystal melting, granule swelling, and amylose leaching, whereas water protons lose mobility due to increased interaction with starch polymers. Heating of the gluten-water sample induces no pronounced changes in proton distributions. Heating changes the proton distributions of the flour-water and starch-water models in a similar way, implying that the changes are primarily attributable to starch gelatinization. Proton distributions of the heated flour-water model system and those of fresh bread crumb are very similar. This allows identifying the different proton populations in bread on the basis of the results from the model systems.

Biochemical and structural characterization of TLXI, the Triticum aestivum L. thaumatin-like xylanase inhibitor.

Thaumatin-like xylanase inhibitors (TLXI) are recently discovered wheat proteins. They belong to the family of the thaumatin-like proteins and inhibit glycoside hydrolase family 11 endoxylanases commonly used in different cereal based (bio)technological processes. We here report on the biochemical characterisation of TLXI. Its inhibition activity is temperature- and pH-dependent and shows a maximum at approximately 40 degrees C and pH 5.0. The TLXI structure model, generated with the crystal structure of thaumatin as template, shows the occurrence of five disulfide bridges and three beta-sheets. Much as in the structures of other short-chain thaumatin-like proteins, no alpha-helix is present. The circular dichroism spectrum of TLXI confirms the absence of alpha-helices and the presence of antiparallel beta-sheets. All ten cysteine residues in TLXI are involved in disulfide bridges. TLXI is stable for at least 120 min between pH 1-12 and for at least 2 hours at 100 degrees C, making it much more stable than the other two xylanase inhibitors from wheat, i.e. Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibitor protein (XIP). This high stability can probably be ascribed to the high number of disulfide bridges, much as seen for other thaumatin-like proteins.

His22 of TLXI plays a critical role in the inhibition of glycoside hydrolase family 11 xylanases.

Recently, a novel wheat thaumatin-like protein, TLXI, which inhibits microbial glycoside hydrolase family (GH) 11 xylanases has been identified. It is the first xylanase inhibitor that exerts its inhibition in a non-competitive way. In the present study we gained insight into the interaction between TLXI and xylanases via combined molecular modeling and mutagenic approaches. More specifically, site-specific mutation of His22, situated on a loop which distinguishes TLXI from other, non-inhibiting, thaumatin-like proteins, and subsequent expression of the mutant in Pichia pastoris resulted in a protein lacking inhibition capacity. The mutant protein was unable to form a complex with GH11 xylanases. Based on these findings, the interaction of TLXI with GH11 xylanases is discussed.

Xylanase inhibitors bind to nonstarch polysaccharides.

This study is an in-depth investigation of the interaction between polysaccharides and the proteinaceous xylanase inhibitors, Triticum aestivum xylanase inhibitor (TAXI), xylanase inhibitor protein (XIP), and thaumatin-like xylanase inhibitor (TLXI). The binding affinities of all three known types of xylanase inhibitors from wheat are studied by measuring the residual xylanase inhibition activity after incubation of the inhibitors in the presence of different polysaccharides, such as beta-glucans and (arabino)xylans. The binding affinities of all three xylanase inhibitors for (arabino)xylans increased with a decreasing arabinose/xylose ratio (A/X ratio). This phenomenon was observed both with water-extractable and water-unextractable (arabino)xylans. The inhibitors also interacted with different soluble and insoluble beta-glucans. None of the inhibitors tested had the ability to hydrolyze the polysaccharides investigated. The present findings contribute to the unraveling of the function of xylanase inhibitors in nature and to the prediction of the effect of added xylanases in cereal-based biotechnological processes, such as bread making and gluten-starch separation.

Indirect enzyme-antibody sandwich enzyme-linked immunosorbent assay for quantification of TAXI and XIP type xylanase inhibitors in wheat and other cereals.

To quantify Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP) type proteins in cereals in general and wheat ( T. aestivum) in particular, a robust enzyme-linked immunosorbent assay (ELISA) using an uncommon enzyme-antibody sandwich format was developed. Bacillus subtilis glycoside hydrolase family (GH) 11 and Aspergillus oryzae GH 10 xylanases were selected for coating ELISA plate wells to capture TAXI and XIP, respectively, prior to probing with antibodies. The detection threshold of the developed ELISA was much lower than that of the currently used xylanase inhibitor assay and the recently described Western blot approach. Because of its broad dynamic range (TAXI, 30-600 ng/mL, and XIP, 3-60 ng/mL), one proper standard extract dilution can be used for analyzing different wheat varieties, whereas for the currently used colorimetric assay, often different dilutions need to be analyzed. The TAXI ELISA for wheat was successfully adapted for barley ( Hordeum vulgare) and could also be used for other cereals.