Ellen Fierens

TLXI, a novel type of xylanase inhibitor from wheat (Triticum aestivum) belonging to the thaumatin family.

Wheat (Triticum aestivum) contains a previously unknown type of xylanase (EC 3.2.1.8) inhibitor, which is described in the present paper for the first time. Based on its >60% similarity to TLPs (thaumatin-like proteins) and the fact that it contains the Prosite PS00316 thaumatin family signature, it is referred to as TLXI (thaumatin-like xylanase inhibitor). TLXI is a basic (pI> or =9.3 in isoelectric focusing) protein with a molecular mass of approx. 18-kDa (determined by SDS/PAGE) and it occurs in wheat with varying extents of glycosylation. The TLXI gene sequence encodes a 26-amino-acid signal sequence followed by a 151-amino-acid mature protein with a calculated molecular mass of 15.6-kDa and pI of 8.38. The mature TLXI protein was expressed successfully in Pichia pastoris, resulting in a 21-kDa (determined by SDS/PAGE) recombinant protein (rTLXI). Polyclonal antibodies raised against TLXI purified from wheat react with epitopes of rTLXI as well as with those of thaumatin, demonstrating high structural similarity between these three proteins. TLXI has a unique inhibition specificity. It is a non-competitive inhibitor of a number of glycoside hydrolase family 11 xylanases, but it is inactive towards glycoside hydrolase family 10 xylanases. Progress curves show that TLXI is a slow tight-binding inhibitor, with a K(i) of approx. 60-nM. Except for zeamatin, an alpha-amylase/trypsin inhibitor from maize (Zea mays), no other enzyme inhibitor is currently known among the TLPs. TLXI thus represents a novel type of inhibitor within this group of proteins.

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.

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: 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.

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.

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°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 β-sheets. Much as in the structures of other short-chain thaumatin-like proteins, no α-helix is present. The circular dichroism spectrum of TLXI confirms the absence of α-helices and the presence of antiparallel β-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°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.

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.

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.

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.

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.