Article pubs.acs.org/JAFC
Lipases as Processing Aids in the Separation of Wheat Flour into Gluten and Starch: Impact on the Lipid Population, Gluten Agglomeration, and Yield Sara Melis,* Anneleen Pauly, Lien R. Gerits,§ Bram Pareyt,# and Jan A. Delcour Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 20, Box 2486, B-3001 Leuven, Belgium ABSTRACT: Three lipases with different hydrolysis specificities were tested in a laboratory-scale dough-batter wheat flour separation process in two concentrations. Lipolase specifically hydrolyzed nonpolar flour lipids. At the highest concentration tested, it significantly improved gluten agglomeration and yield, also when combined with a xylanase with hydrolysis specificity toward water-extractable arabinoxylan. We hypothesize that its action is due to the release of adequate levels of free fatty acids, which, because at least a part of them is dissociated, act as anionic surfactants. Lipolase at the lowest concentration, Lecitase Ultra, hydrolyzing both nonpolar and polar lipids, and YieldMAX, which specifically hydrolyzed phospholipids, had no or a negative impact on gluten agglomeration and yield. In conclusion, this study demonstrated that lipases with hydrolysis specificity toward nonpolar lipids can be used as processing aids in wheat flour separation in the absence or presence of added xylanases to maximize gluten agglomeration and yield. KEYWORDS: lipolytic enzymes, wheat flour lipids, gluten-starch separation, Lipolase, lipid binding
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INTRODUCTION Wheat gluten, a coproduct of wheat starch isolation, consists of approximately 75% protein (mainly gliadin and glutenin), up to 8% moisture, and small amounts of starch, lipid, and fiber. It is used in both food and nonfood applications.1 Common industrial wheat starch isolation is through dough (Martin), dough-batter, or batter processes.2 In those involving doughmaking, a viscoelastic gluten network is formed as a result of the high energy input and the relatively low levels of water.3,4 Following extra water addition, separation of gluten from starch and water-soluble components is based on differences in density (e.g., centrifugation) or size (e.g., sieving). Washing removes nonprotein constituents and thus increases the protein content of the recovered gluten phase.2,5−7 Needless to say, the processes are influenced by flour composition and processing aids such as enzymes.2,5,8 Wheat flour contains 2.0−3.0% lipids, which are either nonstarch (60−70%) or starch (30−40%) lipids. Traditionally, nonstarch lipids are further subdivided into free and bound lipids on the basis of their extractability with different organic solvents at room temperature. Free lipids are extracted with nonpolar solvents (e.g., hexane), whereas bound lipids are extracted with more polar solvents [e.g., water-saturated butan1-ol (WSB)] after removal of the free lipids.9−12 Extraction solvent properties relate to the binding forces between lipids and other flour components. Free lipids either do not interact or interact through hydrophobic interactions with other flour components as these interactions can be broken by a nonpolar solvent such as hexane. Bound lipids interact through electrostatic interactions and/or hydrogen bonds or are physically entrapped in a matrix. More polar solvents such as WSB can break these interactions or penetrate the matrix better because of the presence of water, a low molar volume component.13 In wheat flour, the free lipid fraction contains © XXXX American Chemical Society
both nonpolar and polar lipids, whereas the bound lipid fraction consists mainly of polar lipids. Starch lipids are located inside the granular starch structure and are typically extracted after removing nonstarch lipids with a polar solvent [e.g., WSB or isopropanol/water (90:10)] at 90−100 °C.9−12 Interactions between nonstarch lipids and gluten proteins occur during dough making as indicated by a decrease in free and a concomitant increase in bound lipid levels, a phenomenon referred to as “lipid binding”.14,15 Part of the nonpolar and all of the polar free lipids of wheat flour become bound during dough-making.16 In bread-making, because of the ionic and/or amphiphilic nature of polar lipids, lipid binding strengthens the viscoelastic gluten network either by decreasing electrostatic repulsion between gluten polymers17 or by effectively bridging starch and gluten components.18,19 According to Békés et al.,20 lipids mediate the aggregation of gliadin into aggregates of high apparent molecular weight. However, Carcea et al.21 obtained no evidence for lipid-mediated aggregation of gliadin proteins. Although interactions between lipids and gluten proteins may positively contribute to gluten network strength and gluten aggregation, negative roles have been ascribed to (certain) lipids when it comes to separation of wheat flour dough into gluten and starch. Lecithin addition impairs the separation of dough by ultracentrifugation, whereas prior defatting of flour with chloroform improves such separation.5 However, we suspect that chloroform defatting may affect not only lipids but also other flour components. Some enzymes improve the separation of wheat flour into gluten and starch.2 Especially xylanases with substrate Received: November 4, 2016 Revised: February 10, 2017 Accepted: February 12, 2017
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DOI: 10.1021/acs.jafc.6b04955 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
μmol p-nitrophenol/(min·mg EP), respectively, and contained neither amylase, peptidase, nor xylanase side activities (results not shown).30 Shearzyme 2X, a xylanase from Aspergillus aculeatus with specificity toward water-extractable arabinoxylan, was also from Novozymes. It had an endoxylanase activity of 8540 units (U, determined as described below). Solvents, chemical products, and reagents were from Sigma-Aldrich [Bornem, Belgium; p-nitrophenol, p-nitrophenyl palmitate, propan-2-ol, sand, triethyl amine, and tris(hydroxymethyl)aminomethane (Tris)] or Thermo Fisher Scientific (Aalst, Belgium; acetic acid, cellulose filters, chloroform, hydrochloric acid, hexane, and methanol), unless specified otherwise, and of at least analytical grade. All solvents used for lipid extraction and analysis were of highperformance liquid chromatography (HPLC) grade. Wheat Flour Separation. The laboratory-scale wheat flour separation procedure was based on that of Frederix et al.6 with small adaptations. Wheat flour (250 g, 14.0% moisture base) was mixed during 4 min with 140 mL of 15 mmol/L NaCl [VWR Chemicals, Haasrode, Belgium; based on the Farinograph water absorption (AACCI Approved Method 54-21.02, 2011)] in a KitchenAid mixer (KPM5; St. Joseph, MI, USA) equipped with a dough hook. Enzymes were dissolved in 15 mmol/L NaCl and added at the beginning of mixing. Lecitase Ultra, Lipolase, and YieldMAX were added at dosages of 0.5 and 5.0 mg EP/kg flour, and Shearzyme 2X was added at 5125 U/kg flour. A combination of Lipolase (5.0 mg EP/kg flour) and Shearzyme 2X (5,125 U/kg flour) was also tested. The mixed dough was allowed to rest for 8 min and then transformed into batter by adding 250 mL of 15 mmol/L NaCl and stirring with a flat beater for 25 min. Subsequently, 1.0 L of 15 mmol/L NaCl was added, and the suspension was further stirred for 35 min. It was then sieved over a set of vibrating (100 Hz) sieves with a diameter of 20.0 cm and decreasing pore sizes (400, 250, and 125 μm). The retained gluten was washed three times with, in total, 1.0 L of 15 mmol/L NaCl. Gluten fractions were recovered from the three sieves, lyophilized, and ground with a laboratory mill (model A10, IKA-Werke KG, Staufen, Germany) to pass a 250 μm sieve. Separations without (control) and with lipase addition were performed at least in triplicate and separations with Shearzyme 2X and a combination of Lipolase and Shearzyme 2X in duplicate. Analysis of Protein Levels. Protein levels of the isolated gluten fractions were determined in triplicate as above (under Materials). The coefficient of variation of the analysis was
