Wheat (Triticum aestivum L.) lipid species distribution in the different stages of straight dough bread making
Graphical abstract
Introduction
Although lipids only make up 1.5 to 2.5% w/w of wheat flour, they strongly impact its bread making quality (Chung, Ohm, Ram, Park, & Howitt, 2009; Hargin & Morrison, 1980; Pareyt, Finnie, Putseys, & Delcour, 2011). Wheat flour lipid structure, classification, and functionality has been a topic of debate over the past century. The first reports on wheat lipids were by Sullivan, Near, and Foley (1936) and Olcott and Mecham (1947). Later, several research groups studied the extraction and separation of wheat lipids (Christie, 1985; Christie, 1986; Christie & Morrison, 1988; Morrison & Coventry, 1985; Prieto, Ebri, & Collar, 1992) and their role in bread making (Chung, Pomeranz, & Finney, 1982; Chung & Tsen, 1975; Fisher, Broughton, Peel, & Bennett, 1964; Hargin & Morrison, 1980; Hoseney, Finney, Pomeranz, & Shogren, 1969; MacRitchie & Gras, 1973; Pomeranz, Chung, & Robinson, 1966). More recently, additional lipid extraction procedures (Hubbard, Downing, Ram, & Chung, 2004; Moreau, Powell, & Singh, 2003), analytical techniques (Finnie, Jeannotte, & Faubion, 2009; Finnie, Jeannotte, Morris, & Faubion, 2010; Finnie, Jeannotte, Morris, Giroux, & Faubion, 2010), or innovative approaches, such as those based on synthetic lipid-like compounds (Selmair & Koehler, 2008; Selmair & Koehler, 2009) or lipases (Gerits, Pareyt, & Delcour, 2014; Gerits, Pareyt, Masure, & Delcour, 2015; Schaffarczyk, Østdal, Matheis, & Koehler, 2016), have allowed further elaborating on wheat lipid structure, classification, and functionality.
Today, wheat lipids are typically classified either as starch lipids, which - as their name implies - occur inside starch granules, or as non-starch lipids (Morrison, 1981). The latter are further subdivided in free lipids (FLs) and bound lipids (BLs) based on their sequential extractability with nonpolar (e.g. hexane) and polar [e.g. water saturated butanol (WSB)] solvents, respectively (Chung et al., 2009; Morrison, 1988; Pareyt et al., 2011). In addition, flour lipids are also classified as either nonpolar or polar. Fig. 1 shows the chemical structures of the most common wheat flour lipid classes. The most abundant flour nonpolar lipids are triacylglycerols (TAGs) and free fatty acids (FFA). Glyceroglycolipids and glycerophospholipids (further referred to as glycolipids and phospholipids, respectively) make up most wheat flour polar lipids. Mono- (MGDG) and digalactosyldiacylglycerols (DGDG) are the main glycolipids, whereas N-acyl phosphatidylethanolamine (NAPE), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and their respective lysoforms lyso-NAPE (NALPE), lyso-PE (LPE), and lyso-PC (LPC) represent the most abundant phospholipid classes (Finnie et al., 2009; Hargin & Morrison, 1980; Pareyt et al., 2011).
Evidently, wheat flour contains a complex mixture of lipids with varying polarities. Some of its lipids play a prominent role in each stage of the bread making process which typically starts by mixing water, flour, yeast, and salt and some nonessential ingredients into viscoelastic dough. During mixing, air is incorporated in the dough (Baker & Mize, 1941; Delcour & Hoseney, 2010). In this process also, gluten proteins interact with one another and form a viscoelastic network while, at the same time, the majority of native flour lipids is redistributed from the surface of starch granules to this gluten network (Chung & Tsen, 1975; Gerits, Pareyt, & Delcour, 2013; Olcott & Mecham, 1947). This phenomenon is known as lipid-binding (Carr, Daniels, & Frazier, 1992; Gerits et al., 2013; Olcott & Mecham, 1947; Ponte, Titcomb, & Cerning, 1964). In the early stages of fermentation, gas cells are embedded in and physically stabilized by the gluten-starch matrix (Gan, Ellis, & Schofield, 1995; Sroan, Bean, & MacRitchie, 2009). However, during late fermentation and early baking, stretching of the gluten network results in discontinuities in the matrix that leave neighboring gas cells only separated by a thin liquid film. From that moment onwards, the stabilization of these gas cells at the air/water interface (Gan et al., 1995; Sroan & MacRitchie, 2009) is taken over by protein and lipid surface-active constituents. During the initial phases of baking, gas cells continue to expand until the liquid films fail to withstand the increase in interfacial area and the gas cells rupture which more or less coincides with the setting of the crumb as a result of starch gelatinization and gluten polymerization. Starch gelatinization provokes (i) migration of water from the gluten to the starch phase which then leads to release of polar lipids from the gluten phase (Eliasson, 1985; Köhler, 2001) and (ii) the in situ dissociation and reformation of amylose-lipid (AM-L) inclusion complexes during bread baking and cooling, respectively (Goderis, Putseys, Gommes, Bosmans, & Delcour, 2014; Kugimiya, Donovan, & Wong, 1980; Putseys, Lamberts, & Delcour, 2010).
It follows from the above that lipids play a decisive role in all stages of bread making. However, because the role of certain lipid classes or even lipid species in the process remains unclear at present, there is a need for a systematic approach for characterizing and analyzing lipids, especially those present in complex matrices (Wenk, 2005) such as wheat flour, dough, or bread crumb. This area of study in general is referred to as lipidomics or lipid profiling (Finnie et al., 2009; Finnie, Jeannotte, Morris, & Faubion, 2010; Finnie, Jeannotte, Morris, Giroux, & Faubion, 2010). Over the last decade, several advanced proteomic techniques [e.g. high-resolution chromatography, mass spectrometry (MS), and nuclear magnetic resonance] have become available (German, Gillies, Smilowitz, Zivkovic, & Watkins, 2007). MS-based lipidomics allows simultaneous identification and quantification of (hundreds of) lipid species in crude lipid extracts (i.e. shotgun lipidomics) [as reviewed by Wenk, 2005, Wenk, 2010 and Dehairs, Derua, Rueda-Rincon, & Swinnen, 2015]. Lipid profiling of wheat whole meal, flour, and starch (Finnie et al., 2009; Finnie, Jeannotte, Morris, & Faubion, 2010; Finnie, Jeannotte, Morris, Giroux, & Faubion, 2010), milling and pearling fractions (González-Thuillier et al., 2015), and flour, dough liquor, and dough liquor foam (Salt et al., 2018) with electrospray ionization tandem MS (ESI-MS/MS) already allowed identification and quantification of different wheat glycolipid and phospholipid classes and the analysis of their distinctive acyl groups. However, detailed knowledge on the distribution of glycolipids and phospholipids during the different phases of bread making is lacking. Furthermore, none of the above studies have included the quantification of NAPE and NALPE, even if they have a unique molecular structure amongst wheat phospholipids because they contain a fatty acid (FA) attached to the sn-3 position.
Recently, a method based on single-run high-performance liquid-chromatography (HPLC) with evaporative light scattering detection (ELSD) was developed at our research group to study wheat lipids (Gerits et al., 2013). Although this method is valuable for studying the role of wheat lipids in bread making (Gerits et al., 2013; Gerits et al., 2014; Gerits et al., 2015), shortcomings include that it is not quantitative and cannot differentiate between individual lipid species within a lipid class. Therefore, we here implemented an MRM-based ESI-MS/MS method for polar lipid profiling, including NAPE and NALPE, thus complementing the HPLC-ELSD lipid analyses, which are still used to assess the distribution of nonpolar lipids. Using this approach, we here set out to study the distribution of polar and nonpolar lipids throughout the entire bread making process by analyzing and comparing lipid extracts from wheat flour, fresh and fermented dough, and bread crumb.
Section snippets
Materials
Wheat flour has a much lower lipase activity than its whole grain counterpart (Almeida, Pareyt, Gerits, & Delcour, 2014). Hence, to minimize the impact of endogenous lipases, wheat flour instead of whole grain flour was used for bread making. Kernels from soft wheat cultivar Claire were from Limagrain (Rilland, The Netherlands) and were conditioned to 16.0% moisture before milling with a Bühler (Uzwil, Switzerland) MLU-202 laboratory mill as in Delcour, Vanhamel, and De Geest (1989). Wheat
Results & discussion
Table 2 lists gravimetrically determined (see section 2.2.3.1) FL, BL, and TL levels of flour, freshly mixed dough, fermented dough, and bread crumb. Polar lipid levels within FL, BL, or TL fractions were calculated by summing up the amounts (expressed in mg/g) of all different lipid species within a given fraction such as determined with ESI-MS/MS. As it was not possible to ionize nonpolar lipids with the ESI-MS/MS set-up used here, their levels were obtained by subtracting the polar lipid
Conclusions
FL extracts from flour contained about 20% polar and 80% nonpolar lipids, while the BL fraction contained no nonpolar lipids. Glycolipids were much more abundant than phospholipids in both the FL and BL fractions of a soft wheat flour studied here. DGDG (36:4) was the most prevalent free glycolipid, whereas BL extracts were dominated by MGDG (36:4) and DGDG (36:4). NAPE (18:2 FA at sn-3 position and acyl group combinations 36:4 and 34:2 at sn-1 and sn-2 positions) was the most abundant free
Acknowledgements
Frederik Janssen would like to thank Sarah Pycarelle and Sara Melis for fruitful discussions. Frederik Janssen, Arno Wouters, and Bram Pareyt gratefully acknowledge the Research Foundation – Flanders (FWO – Vlaanderen, Brussels, Belgium) for positions as doctoral (FJ) and postdoctoral (AW and BP) researchers. Jan A. Delcour is W.K. Kellogg Chair in Cereal Science and Nutrition at KU Leuven. This work is part of the Methusalem program “Food for the Future” at KU Leuven.
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