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Determination of volatile compounds in heat-treated straight-grade flours from normal and waxy wheats
Accepted Manuscript Determination of volatile compounds in heat-treated straight-grade flours from normal and waxy wheats Jianteng Xu, Wenbin Zhang, Koushik Adhikari, Yong-Cheng Shi PII:
S0733-5210(16)30447-7
DOI:
10.1016/j.jcs.2017.03.018
Reference:
YJCRS 2319
To appear in:
Journal of Cereal Science
Received Date: 15 November 2016 Revised Date:
15 March 2017
Accepted Date: 21 March 2017
Please cite this article as: Xu, J., Zhang, W., Adhikari, K., Shi, Y.-C., Determination of volatile compounds in heat-treated straight-grade flours from normal and waxy wheats, Journal of Cereal Science (2017), doi: 10.1016/j.jcs.2017.03.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Determination of volatile compounds in heat-treated straight-grade
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flours from normal and waxy wheats
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Jianteng Xu1, Wenbin Zhang1,3, Koushik Adhikari2, Yong-Cheng Shi1*
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University, Manhattan, KS 66506;
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Department of Grain Science and Industry, 2Department of Human Nutrition, Kansas State
Jiangnan University, Wuxi, Jiangsu, China
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*To whom correspondence should be addressed. Telephone: 785-532-6771. E-mail:
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[email protected]
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Abstract Volatile compounds formed during dry heat-treatment of wheat flour influence the application of treated flour. In this study, normal and waxy hard wheat flours before and after
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dry-heat treatment were subjected to headspace analysis by solid-phase microextraction of
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volatiles followed gas chromatography–mass spectrometry (GC/MS). The untreated waxy wheat
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flour contained higher levels of odor-active compounds than normal wheat flour including
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aldehydes, alcohols, furans, and ketones. Lipid oxidation appears to play major role in producing
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such odor compounds. Heat treatments, depending on the severity, alter the profile of volatile
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compounds. Low temperature (100 to 110 °C) treatments effectively eliminated cereal odor
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(aldehyde) and did not introduce additional odors, providing a possible way to produce low-odor
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flours. Heat treatments at 120 °C and higher temperatures elevated the content of pyrazines,
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furans, and sulfur-containing compounds which together gave a roasty aroma to the flours.
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Considering organoleptic properties, treatments of flours at 140 °C was superior to 160 °C. The
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waxy wheat flour was more prone to produce odor-active compounds than normal wheat flour
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during the same heat treatment.
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Keywords: Volatile compounds, waxy wheat flour, GC/MS, SPME, dry heating
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1. Introduction Wheat flour is one of the most important staple food sources in the world. Heat-treated flours
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(HTFs) produced by applying dry heat to base flours under different temperatures and times have
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gained popularity in food production, including coatings, binders, baked foods, and thickeners
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(Neill et al., 2012; Shi, 2009). Heat effectively remove moisture from flour, and in the presence
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of air causes change in the physicochemical properties of flours that can benefit applications. For
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instance, heating at 49-93 °C for 1 h to 10 weeks (Hanamoto and Bean, 1979), 120 °C for 10-120
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min (Nakamura et al., 2008), and 130 °C for 30 min (Neill et al., 2012) result in increased
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volume of high-ratio cakes. A recent patent includes the claim of volume increase of French
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breads made from flours subjected to heat treatment at 143-163 °C for 2-20 min (Upreti et al.,
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2010). Heat treatment conducted at 140-160 °C for 15-60 min also can improve binding and
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thickening properties of various cereal flours, which provides the great potential to replace
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chemically cross-linked starches (Shi, 2009). A heat treated flour in a food can be labeled
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simply as “flour” which creates a more consumer-friendly ingredient profile. Another advantage
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of using HTFs in foods is the elimination of microorganisms contained in raw flour. Even though
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heat treatment has been applied to tailoring the functional properties of flour, its impact on
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volatile compounds that are likely associated with a change in sensory characteristics of flour is
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not fully understood.
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Volatile compounds in foods create odors/aromas, and their types and concentrations
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dictate olfactory and taste perception (Zhou et al., 1999). Despite the fact that volatile
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compounds in unprocessed grains, which are primarily produced from lipid oxidation, are
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presented at a trace level, they nevertheless give a distinct flavor profile to each cereal (Rackis et 3
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al., 1979). Owing to their low lipid contents and possibly to bleaching procedures, raw wheat
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flours have a largely bland flavor but with slight “green” and “grainy” notes. According to a
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systematic study on Kansas-grown wheat grains, climate had a major impact on the volatiles’
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profile in the raw kernels, whereas wheat cultivar seemed to have no significant influence on the
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formation of volatiles (Seitz, 1994). Previous research suggested aldehydes, most of which had a
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carbon chain lengths C6 to C9, represent the major class of odor-active volatile compounds
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present in wheat flour, followed by alcohols and alkenes of similar chain length (Maeda et al.,
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2008; Sayaslan et al., 2000; Seitz, 1994; Zhou et al., 1999).
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Processing conditions of a food may affect its volatiles’ profile. Volatile compounds,
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including pyrazines, pryidines, and furans, are readily generated by Maillard reactions of
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reducing sugar with amino compounds (Hoseney, 1984; BeMiller, 2007). A number of factors
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influence the formation of volatiles such as moisture content, pH, heating temperature and time,
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plus amino acid and sugar profiles (BeMiller, 2007). Volatiles are desirable in baked and
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extruded foods made from flours as they give off “roasty, nutty, and caramel” aromas. However,
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when a HTF is used in certain applications such as a thickener in soups and pie fillings, the HTF
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is expected to be bland without an undesirable aroma. The objectives of this study were to: (1)
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identify the volatile compounds in raw straight-grade flours of normal and waxy hard wheats,
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and (2) assess how the volatile compounds were affected dry heating of those two flours.
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2. Materials and Methods
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2.1. Wheat grains and flours Normal hard (NH) red wheat flour was milled from a mixture of cultivars grown in 2013
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in Kansas obtained from a local grain elevator. Waxy hard (WH) red wheat grain (received in
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2013) was provided by Dr. Robert Graybosch at USDA/ARS, University of Nebraska. The waxy
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wheat grain was inspected by the naked eye to remove foreign material and molded kernels, and
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then sieved on 2 wire (mesh #10)-screen to remove small particles. The moisture content of the
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waxy wheat was determined using a single kernel characterization system (SKCS) and tempered
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to 16% overnight. The tempered waxy wheat was milled into flour with a Bühler MLU-202
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Laboratory mill (Uzwil, Switzerland) adjusted according to AACC method 26-21.02 for hard
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wheat (AACC International, 2009). In the milling of the waxy wheat, the feeding rate was
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lowered to about 100 g/min. Straight-grade flour was collected and stored in an air-tight
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container at 4°C until analysis or heat treatment.
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The moisture content of flour was determined by loss of weight at 105 °C and protein content by a Leco FP-2000 protein analyzer (Leco Corp, St Joseph, MI, USA) according to
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AACC Method 46-30.01 using a conversion factor of 5.7 (AACC International, 2009). Crude
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lipid content was determined gravimetrically by petroleum ether extraction using Soxtech 2045
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Fat Extraction System (FOSS, Denmark) followed by evaporation of solvent. Total starch and
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damaged starch were determined using Megazyme total starch and damaged starch assay kit
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(Wicklow, Ireland) according to AACC Methods 76.13 and 76-31.01, respectively (AACC
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International, 2009).
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2.2. Dry-heat treatment of wheat flours Ten grams of normal and waxy wheat flours (12.5% moisture) were placed separately in
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the bottom of sealed 120 mL pressure bottles (Ace Glass, Vineland, NJ) that were subsequently
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placed in a hot oven equilibrated at 100, 110, 120, 140, and 160 oC (±2 °C). The headspace in the
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bottle above a flour was approximately 11 cm. Heating was allowed to proceed for 30 min after
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the internal temperature of flour reached the targeted temperature. The time for flour to reach a
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target temperature was determined in a preliminary experiment using an embedded thermal
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couple. A treated bottle with flour was cooled at room temperature for 30 min, and the treated
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flour was ground with a mortar and pestle and analyzed by SPME-GC/MS on the same day. In a
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separate experiment,10 grams (as is) of normal hard wheat flour was heated at 160 °C for 2 h in
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order to intentionally produce a higher level of volatile products to assist in compound
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identification. According to our preliminary studies, flours treated at 160 °C for 2 h would be
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acceptable for human consumption, even though slight browning of the flour occurred.
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2.3. Volatile compound extraction
An automated solid-phase microextraction (SPME) was used to extract and concentrate
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the volatile compounds in a flour’s headspace onto the coated fiber. A CombiPAL antosampler
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(CTC Analytics/Leap Technologies, Carrboro, NC) was used to house the SPME fiber, sample
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trays, a bake-out unit and an agitator. The SPME fiber (50/30 µm divinylbenzene/Carboxen on
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polydimethylsiloxane on StableFlex) obtained from Supelco (Bellefonte, PA) was baked at the
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baking station of the CombiPal at 270 °C for 1 h before its use in the study. Each flour sample
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(0.5 g) was accurately weighed into a 10 mL sample vial that had been baked at 105 °C
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overnight and screw capped with teflon-lined silicone septum. Samples in the vials were heated
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at 60 °C (Maeztu et al., 2001) with agitation at 500 rpm while each flour’s headspace was
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exposed to SPME fiber. The SPME fiber was inserted into the injection port of the GC to desorb
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volatile compounds for 5 min at 270 °C. Between each analysis, the fiber was baked at 280 °C
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for 20 min to remove carryover volatiles. Blank runs were carried out using an empty vial and
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were considered as background.
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2.4. GC/MS analysis
A Varian CP-3800 gas chromatograph (Varian Inc., Walnut Creek, CA) fitted with a DB-
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5ms ((5%-phenyl)-methylpolysiloxane) capillary column (30m x 0.25 mm i.d., 0.25 µm film
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thickness, Agilent Technology, Santa Clara, CA) was used to perform volatiles separation. The
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column oven was programmed to start at 40 °C and ramped to 280 °C at 4 °C/min. Helium was
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used as carrier gas and the flow rate was set at 1 mL/min. MS analysis was carried out using a
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Varian Saturn 2200 Ion Trap mass spectrometer (Varian Inc., Walnut Creek, CA). The MS was
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operated in scanning mode with a range of 35-400 amu. Total ion current (TIC) was recorded
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and the TIC peaks were subjected to computerized matching to the National Institute of
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Standards (NIST) mass spectral database. In addition to fragment pattern matching, a C7-C30
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saturated alkane premix (Sigma-Aldrich, St. Louis, MO) was analyzed using the same method,
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and the MS identified compounds were identified by calculating linear retention indices (LRI)
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and comparing the values in the NIST library and other published papers (Cho & Kays, 2013;
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Janes et al., 2010; Maeda et al., 2008; Maeztu et al., 2001; Sayaslan et al., 2000; Ying et al.,
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2012). In the case where a LRI was not found associated with DB-5ms column, similar columns
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(for example – BP-5, HP-5, ZB-5) were used for LRI comparisons. LRI was calculated using the
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following equation: )ܴܽܶ(݃ܮ− )ܴ݊ܶ(݃ܮ )ܴܰܶ(݃ܮ− )ܴ݊ܶ(݃ܮ
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Where n was the number of carbon in the alkane eluted ahead of targeting analyte; RTa, RTn and
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RTN were retention times of the analyte, alkanes eluted ahead and after the analyte, respectively.
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Upon positive identification, sensory descriptors were assigned to each volatile compound (Cho
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and Kays, 2013; Maire et al., 2013). A flowchart of the experiment is depicted in Fig. 1. 2.5. Statistics analysis
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Heat treatments in this study were performed in duplicate and each sample was analyzed
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twice using GC/MS (n=4). For total and damaged starch analysis performed on raw flours, each
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flour sample was analyzed in triplicate (n=3). Principal component analysis (PCA) was
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performed to evaluate the variance in compound classes in the normal and waxy wheat flours.
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Upon heat treatment of flours, the changes in each class of compounds were analyzed by one-
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way ANOVA with heating conditions being the main factors. Tukey’s post-hoc test was used to
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assess the multiple differences of different compound classes at various heating conditions. A
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probability of P < 0.05 was considered significant. All statistical procedures were handled by
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SAS 9.3 package (SAS Institute; Cary, NC).
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3. Results and Discussion 3.1. Volatile compounds in different wheat flours According to GC/MS analysis, the major class of volatile compounds in untreated normal
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hard wheat flour (without dry-heat treatment) was aldehydes. Hexanal constituted over 43% of
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aldehydes by ion current count (Table 1). Other intermediate-length aldehydes, i.e. nonanal,
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decadienal and undecenal, also were identified at appreciable levels in untreated normal hard
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wheat flour (Fig. 2 & Table 1). Untreated waxy hard wheat flour exhibited a profile of volatiles
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similar to the untreated normal hard wheat flour, but the level of hexanal in the waxy flour was
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3-fold greater than in the normal wheat flour (Table 1). Nonanal was higher in the waxy flour,
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whereas decadienal and undecenal were found in normal flour, but not in the waxy flour.
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Hexanal is generally considered the most pronounced secondary autoxidized product of
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linoleic acid formed through homolytic β-scission of linoleic 11-hydroperoxide (Choe and Min,
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2006). Hexanal is characterized by a low threshold odor described as “grassy” and “hay-like”
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(Min et al., 1989; Sayaslan et al., 2000). The higher hexanal content in untreated waxy wheat
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flour compared to normal wheat flour indicate higher extent of lipid oxidation in waxy wheat
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flour (Nielsen and Hansen, 2008), which probably accounts for its “greener” odor. Hexanol was
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found in both normal and waxy wheat flours at a much higher concentration than other alcohols,
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which was as expected because it shares a similar oxidation pathway with hexanal. Hexanol can
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be converted to hexanal by alcohol dehydrogenase (Matoba et al., 1989; Mizutani and
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Hashimoto, 2004).
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Other indicators of lipid autoxidation, i.e. 1-octen-3-ol and 2-pentyfuran, substances exhibiting “mushroom-like” and “beany” odors (Min et al., 1989), also were found higher in 9
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waxy wheat flour (Table 1). The content of ketones in waxy wheat flour was 3-fold greater than
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that in normal wheat flour. In addition, 3-octen-2-one was only detected in the waxy wheat flour.
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An earlier study compared the volatiles present in starches and found that waxy corn starch, both
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commercial and laboratory-prepared, released more lipid oxidation volatiles than the respective
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samples of normal corn starch (Sayaslan et al., 2000). These results on the starches are in
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agreement with our findings on wheat flours in this study.
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Yuan and Chang (2007) reported that soymilk made from lipoxygenase-null soybean
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cultivars had significantly reduced levels of odor-causing compounds including many of the
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aldehydes, ketones, alcohols, and furans found in our wheat flours. It seems reasonable to
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attribute the formation of the higher amount of volatile compounds in waxy wheat flour to the
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joint effect of physical damage to lipid-containing tissue during milling followed by autoxidation
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or enzymatic reactions in flour. In addition, starch in waxy wheat flour is essentially free of
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amylose, a long linear polymer that can form cavities to entrap small flavor-active molecules in
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its helical structure (Arvisenet et al., 2002). Naphthalene (insect repellent) and butylated
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hydroxytoluene (BHT, a synthetic antioxidant) were found in both normal and waxy wheat flours
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(Table 1). Those compounds perhaps were introduced during planting, processing or storage.
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Additional peaks were observed in the GC/MS volatile profiles of heat-treated flours
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compared with the untreated flours. The extensive heat treatment of flours at 160 °C for 2 h
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caused drastic changes in the volatiles’ profiles and provided valuable information regarding
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retention times and MS spectra markers (Fig. 2). Flour treated at 160 °C for 2 h had a notable
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“roasty” aroma, and gave a more distinct volatiles’ profile (Fig. 2b) compared to that without
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dry-heat treatment (Fig. 2a). A number of pyrazine derivatives were detected as the largest group
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of emerging compounds in addition to pyrroles, furans, and sulfur-containing compounds (Table
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1). The volatile compounds of normal and waxy wheat flours before and after heat
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treatments at 100-160 °C were summed in the following classes of organics for each treatment;
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alcohols, ketones, aldehydes, pyrazines, furans, sulfur-containing compounds, alkanes, and
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miscellaneous compounds. The summations of the TIC count of these classes of compounds are
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given in Table 2. Total aldehyde content of both normal and waxy wheat flours exhibited
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somewhat of a “reclining” J-curve in which the lowest point was observed at heat treatment of
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100 oC. Meanwhile, total alcohols and alkenes in heated flours were prone to decrease with
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increasing heating temperature. These data may be explained as follows. Increased temperature
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increases the generation of aldehydes in heat-treated flours, and that increase is not lost during
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grinding and handling of a heated-treated flour. But in the class of alcohols, while heat-treatment
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could cause an increase, the alcohol class is more volatile and is lost during heat treatment before
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analysis.
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Pyrazines and furans were detected in steady increasing amounts from 120 to 160 °C in normal wheat flours and from 100 to 160 °C in waxy wheat flours. It appears that waxy wheat
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flour was more prone to formation of these compounds compared to normal wheat flour as
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pyrazines and furans were produced upon heating waxy flour at 100 °C, but not normal flour,
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which required heating at ≥ 110 °C. Moreover, at all heating temperatures, treated waxy flour
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contained more pyrazines and furans than similarly treated normal flour. These differences could
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be explained by the higher level of free sugars commonly associated with waxy wheat (Zhao et
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al., 2009) and by the higher level of damaged starch in waxy wheat flour (Table 3), as also
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previously reported (Bettge et al., 2000; Guan, 2008). Based on the weight of starch, there was
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1.5% more damaged starch in the waxy wheat flour. Damaged waxy wheat starch is probably
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more prone to degradation during thermal process, generating more reducing ends. The increased
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concentration of reducing carbohydrate in waxy flour possibly would lead to greater sugar-amine
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reactions. It is noteworthy that the protein content of the waxy wheat flour in this study was
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higher compared to literature values (Garimella Purna et al., 2011).
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Trace amounts of sulfur-containing compounds including dimethyl trisulfide and 2pentylthiophene were detected in the flours treated at 140 and 160 °C (Table 1). The latter
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compound could be formed from cysteine-involved reactions (Yu and Zhang, 2010). It is
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noteworthy that a sulfur-containing compound typically imparts a very strong aroma, even at
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trace levels.
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3.2. Principal components analysis
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Principal components analysis (PCA) was performed to elucidate which class of volatile compounds within normal and waxy wheat flours changed in level as heating temperature was
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changed (Fig. 3). Principal factor 1 (PC1) explained 59% of the variance in the data and
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differentiated heat-induced compounds of various classes from those compounds in untreated
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flours, whereas principal factor 2 (PC2) mainly distinguished the contribution of aldehydes and
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alkanes and explained 31% of the variance in the data. The PC2 can be reasonably interpreted as
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the strength of “grassiness” from aldehydes because alkanes are odorless.
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PCA clustered the flour samples into three groups according to their volatile profiles (Fig.
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3). Both native flours were separated from the 140-160 °C treated counterparts across PC1 which
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was driven by high concentrations of pyrazines, furans, ketones, and S-containing compounds, 12
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Higher temperature (140-160 °C) treated flours had large positive scores on PC1 and small
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values of PC2, indicating a mix of “roasty" and “green” (lipid oxidation) odors. Interestingly,
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normal hard wheat flour treated at 100, 110, and 120 °C and waxy hard wheat flour treated at
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100 and 110 °C were found to have positive, although small, scores on PC1 and negative scores
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on PC2, which means those treated flours have diminished “green” (cereal) odor and minimal
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“roasty” (heat-treated) odor.
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4. Conclusions
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Volatile compounds in normal and waxy hard wheat flours before and after dry-heat
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treatment were identified with headspace SPME GC/MS techniques. Aldehydes, alcohols, and
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alkanes of various chain lengths were found as major volatile compounds in both waxy and
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normal wheat flours. Waxy flour contained approximately 53% higher total volatiles, which was
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largely caused by the high levels of hexanal and hexanol. Heat treatment at 120 °C or higher
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temperature up to 160 °C led to the formation of pyrazines, furans, ketones, and sulfur-
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containing compounds, particularly in waxy wheat flour. However, the 100 °C to 110 °C
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treatment eliminated “cereal” odor to a great extent without introducing new aromas, indicating
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in the possibility of developing low-odor flour ingredients. The formation of volatiles in heat-
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treated waxy wheat flours may proceed faster than in normal wheat flour, which suggests a lower
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temperature should be considered for waxy wheat flour. Considering the possible disparities
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between instrumental analysis and sensory perception, future work should include the
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determination of sensory properties and its relation to volatiles’ profiles in heat-treated wheat
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flours.
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Acknowledgements
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We thank Dr. Robert Graybosch, USDA-ARS, for providing the waxy wheat and Dr. Paul Seib
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for reviewing the manuscript. This is contribution # 15-170-J from the Kansas Agricultural
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Experiment Station.
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Abbreviations: HTFs, heat-treated flours; NH, normal hard; WH, waxy hard; SKCS, single
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kernel characterization system; SPME, solid-phase microextraction; GC/MS, gas
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chromatography–mass spectrometry; NIST, National Institute of Standards and Technology; LRI,
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linear retention indices; TIC, Total ion current.
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Mizutani, T., Hashimoto, H., 2004. Effect of grinding temperature on hydroperoxide and offflavor contents during soymilk manufacturing process. J. Food Sci. 69, 112–116.
Nakamura, C., Koshikawa, Y., & Seguchi, M., 2008. Increased volume of Kasutera cake
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(Japanese sponge cake) by dry heating of wheat flour. Food Sci. and Technol. Res. 14,
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431–431. 16
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315 316 317 318 319 320 321 322 323 324 325
during storage. Cereal Chem. 85, 716–720.
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Nielsen, M.M., Hansen, Å., 2008. Stability of vitamin E in wheat flour and whole wheat flour
Rackis, J.J., Sessa, D.J., Honig, D.H., 1979. Flavor problems of vegetable food proteins. J. Am. Oil Chem. Soc. 56, 262–271.
Sayaslan, A., Chung, O.K., Seib, P.A., Seitz, L.M., 2000. Volatile compounds in five starches.
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313
for heat treated soft wheat flour. J. Food Eng. 113, 422–426.
Cereal Chem. 77, 248–253.
Seitz, L. M., 1994. Compounds in wheat cultivars from several locations in Kansas. Greece:
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Neill, G., Al-Muhtaseb, A.H., Magee, T.R.A., 2012. Optimisation of time/temperature treatment,
Proceedings of the 8th International Flavor Conference, 37, 2183–2203. Shi, Y.-C., 2009. Non-cohesive waxy flours and method of preparation. U.S. Patent Pub. No. 2009/0041918A1. Washington, DC: U.S. Patent and Trademark Office. Upreti, P., Roberts, J., & Jalali, R. (2010). Heat-treated flour. Patent Pub. No. WO/2010/042825.
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Colombettes, Switzerland: World Intellectual Property Organization. Ying, S., Lasekan, O., Naidu, K., Lasekan, S., 2012. Headspace solid-phase microextraction gas chromatography-mass spectrometry and gas chromatography-olfactometry analysis of
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volatile compounds in pineapple breads. Molecules, 17, 13795–13812.
329 330 331 332
Yu, A.-N., Zhang, A.-D., 2010. Aroma compounds generated from thermal reaction of L-
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ascorbic acid with L-cysteine. Food Chem. 121, 1060–1065.
Yuan, S., Chang, S.K.-C., 2007. Selected odor compounds in soymilk as affected by chemical composition and lipoxygenases in five soybean materials. J. Agric. Food Chem. 55, 426–
431.
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Zhao, R., Wu, X., Seabourn, B. W., Bean, S. R., Guan, L., Shi, Y.-C., Wilson, J. D., Madl, R.,
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Wang, D. 2009. Comparison of waxy vs. nonwaxy wheats in fuel ethanol fermentation.
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Cereal Chem. 86, 145–156. Zhou, M., Robards, K., Glennie-Holmes, M., Helliwell, S., 1999. Analysis of volatile
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compounds and their contribution to flavor in cereals. J. Agric. Food Chem. 47, 3941–
338
3953.
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Tables
341
Table 1. Volatile compounds in heat-treated flours (initially 12.5% moisture) milled from normal (NH) and waxy hard (WH) wheats*
Retention
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340
Compound name NH
Heating T (°C) (30 min) ConC
6.41
2,3-Pentanedione
691
7.82
Pyrazine
740
8.29
Pyrrole
752
8.87
Pentanol
770
10.17
Hexanal
801
71
140
109
160
Con
211
139
83
47
933
659
922
156
NH
Heating T (°C) (30 min)
120 min
154
155
100
71
TE D
648
120
EP
2-Butenal
110
AC C
5.31
100
WH
M AN U
time (min)
Total ion count × 103
SC
LRIA
110
88
120
72
140
126
160
164
Odor qualityB
160 264
Flower
448
Cream, butter Pungent, roasted
434 hazelnuts
324
136
134
115
119
133
244
Nutty, sweet
62
Bready, yeasty, Green, fatty, leafy,
1147
1117
808
3301
1060
1693
1685
1425
893
1065
vegetative, fruity, woody
19
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Ripe, nutty, vegetable, 11.00
4-Methylthiazole
836
23
11.23
11.57
2-Methylpyrazine
Furfural
828
68
838
69
235
55
856
853
12.92
1-Hexanol
872
1034
403
202
Xylene
881
13.80
2-Heptanone
889
71
59
61
14.38
Heptanal
907
46
47
76
926 Dimethylpyrazine
15.11
Ethylpyrazine
929
15.87
Vinylpyrazine
947
16.82
2(E)-Heptenal
968
109
2424
6269 roasted, cocoa
64
114
47
79
429
130
662
Bread, almond, sweet
23
Woody, smoky
186
Musty, bready, coffee Fruity and alcoholic,
1070
965
810
sweet, and green 27
Slightly sweet
181
215
185
92
114
188
340
271
661
Cheesy, fruity, creamy
139
160
143
99
72
91
121
169
160
229
Nut-like, fatty, fruity
180
554
42
280
959
4995
61
Nutty, peanut, musty, earthy
AC C
2,514.91
463
128
EP
13.23
180
TE D
2-Furanmethanol
M AN U
pyrrole 12.32
Popcorn, nutty, 89
SC
2-Methyl-1H11.98
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tomato
1598
54
133
20
37
67
156
Roasted cocoa, coffee
64
Nutty, earthy
106
Pungent, green,
ACCEPTED MANUSCRIPT
vegetable Benzaldehyde
982
54
31
60
236
533
302
177
81
17.65
1-Octen-3-ol
982
257
133
76
129
158
193
448
116
Dimethyl 990
68
48
trisulfide Methyl heptenone
991
18.14
2-Pentylfuran
996
18.45
Decane
1000
51
179
247
295
562
49
2-Ethyl-6-methyl 1009 pyrazine 2-Ethyl-518.76
1015
2-Methyl-6-
19.68
2-Ethyl-1-hexanol
1032
19.69
Methylvinylpyrazi
1038
601
146
131
146
81
305
238
Fruity, almond Earthy, green, oily, vegetative and fungal Cooked onion, savory,
47
65
45 meaty, eggy 48
Fruity, apple, musty Fruity, green, earthy,
328
620
765
1255
1090
565 beany
49
96
119
73
86
107
127
144
936
Roasted potato,
91
208
380
87
103
198
328
463
1597
Coffee
169
Hazelnut
AC C
1032 vinylpyrazine
911
131
574
EP
methylpyrazine
19.48
964
TE D
18.55
34
132
46
M AN U
17.86
246
SC
17.77
123
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17.33
72
259
128
126
122
92
Mild, oily, rose, green 87
21
Nutty
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ne
20.14
3-Octen-2-one
1043
21.12
Butyl-benzene
1065
21.60
3-Methylphenol
1080
210
49
30
33
96
45
50
1085
2-Ethyl-2,51087
94
41
154
134
60
45
124
164
69
63
2,51091
36
Undecane
1099
206
53
22.83
Nonanal
1113
589
375
25.14
Pentyl-benzene
1168
25.32
2-Pentylthiophene
1170
25.97
1-(2-
1182
290
AC C
22.53
51
EP
Diethylpyrazine
68
TE D
dimethylpyrazine
22.48
280
138
dimethylpyrazine
22.21
40
100
3-Ethyl-2,521.79
40
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1041
SC
Limonene
M AN U
20.05
360
43
413
196
Earthy, oily, hay and 174
157 mushroom
67
141
84
80
82
64 leather
70
81
327
Cocoa
62
138
Cocoa
58
Nutty, hazelnut
262
Fresh, slightly green
34
258
942
241
274
Aromatic Medical, woody,
60
38
221
Fresh, citrus
55
47
293
383
351
29 31
45
22
58
60
47
Meaty
18
Vegetative, cereal,
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furanmethyl)-1H-
bready
pyrrole Menthol
1190
65
26.41
Dodecane
1199
657
371
226
256
240
69
710
565
648
581
383
93
26.80
Naphthalene
1218
934
365
256
272
245
96
586
212
186
180
157
118
30.06
Tridecane
1299
545
258
191
198
204
51
473
199
138
108
1309
222
52
30
97
1319
142
37
32
69
1365
201
1384
393
1399
260
decadienal 2,4-decadienal 30.70
2-Undecenal Propanoic acid, 2-
EP
methyl-, 3hydroxy-2,4,432.95
esters
33.49
Tetradecane
162
89
103
29
23
Pungent, dry, tarry
Waxy, plastic
249
74
98
Fatty, oily
AC C
trimethylpentyl
203
TE D
(E,E) 32.27
301
Peppermint
M AN U
(Z,Z)2,430.40
SC
133
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26.06
243
115
95
100
73
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Butylated 37.18
hydroxytoluene
1515
216
212
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(BHT)
Contamination
342 343
*
344
A
345
and similar columns
346
B
http://www.thegoodscentscompany.com; (Cho and Kays, 2013; Maire et al., 2013)
347
C
Con: control, raw untreated flour
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Mean values are reported. Statistical analysis was performed and saved as Supplementary Material.
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LRI: Linear retention index calculated from C7-C30 straight chain alkanes; all LRIs were within ±10 of literature values of DB-5
24
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Table 2. Classes of volatile compounds in normal (NH) and waxy hard (WH) wheat flours before and after dry-heat treatment at
349
different temperatures.
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348
Total ion current x 103 Normal hard (30 min)A
Waxy hard (30 min)A
class 120 °C
140 °C
160 °C
Control
M AN U
110 °C
100 °C
110 °C
120 °C
140 °C
160 °C
1732a
691b
325c
465bc
421c
348c
3588a
1450b
1357bc 1178bc
771cd
409d
Ketones
71bc
59c
61c
128b
326a
290a
339abc
216c
278bc
384abc
514a
428ab
Aldehydes
2187ab
1201c
1410bc 1882abc
2443a
1644abc
4518a
1454c
2181b
2382b
2618b
2161bc
Pyrazines
N.D.d
N.D.d
N.D.d
201c
690b
1356a
N.D.d
160cd
189cd
385c
894b
2110a
Furans
179c
247c
295c
562bc
1033a
966ab
305c
328c
620b
765b
1319a
1204a
N.D.b
N.D.b
N.D.b
N.D.b
99a
93a
N.D.d
N.D.d
N.D.d
46c
105b
125a
Alkanes
1718a
844b
506c
557c
518c
149d
1647a
981b
946b
935b
641b
201c
Miscellaneous
1753a
510b
331b
355b
419b
296b
1327a
281b
249b
240b
304b
341b
Total volatiles
7638a
3552c
2928c
4150c
5949ab
5142bc
11725a
4870b
5820b
6315b
7166b
6979b
S-containing
AC C
compounds
TE D
Alcohols
EP
Control 100 °C
SC
Compound
25
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350
A
351
treatments in the same type of wheat flour (P<0.05) (n=4)
352
A
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Means with different lowercase letters a, b, c . . . in the same row indicate significant differences among different temperature
N.D. not detected
AC C
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353
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Table 3. Total starch, damaged starch, and protein contents of the normal and waxy hard wheat flours Protein (%)
Lipid (%)
Starch (%)
Ash (%)
Damaged starch (%)
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Flour
Flour Wt
Starch Wt
10.36a± 0.19
Normal
12.22a± 0.21
1.03a± 0.02
76.59a± 0.67
0.49± 0.02
7.93a± 0.13
Waxy
15.62b± 0.21
1.11a± 0.04
70.87b± 0.36
0.46± 0.02
8.40b± 0.10
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354
11.85b± 0.18
Values expressed as mean± standard deviation (“dry” flour Wt) unless otherwise noted; lowercase letters in the same column indicate
356
significant differences (P<0.05) (n=3)
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Figure Captions:
359
Figure 1. Flow chart of the experiments conducted
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360
Figure 2. Gas chromatogram of headspace volatiles before (a) and after (b) heat treatment at 160 °C for 2h of a straight-grade flour
362
(initial MC 11.5%) from hard winter wheat. The heating of flour was done in a sealed glass bottle, the flour cooled and ground prior to
363
assaying for volatiles. Volatiles in the flour were collected by solid-phase microextration at 60 °C for 20 min, then separated and
364
quantified by GC-MS. Stars (*) represent compounds derived from column bleeds.
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361
365
Figure 3. Principal component analysis (PCA) of volatile compounds profile of heat-treated flours; NH, normal hard; WH, waxy hard;
367
WHCon and NHCon, not heated.
370 371 372
EP
369
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368
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366
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378 379 380 381
EP
377
AC C
376
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29
382 383
Figure 2.
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385 386 387
Figure 3.
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Highlights
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Volatile profiles in waxy and normal hard red wheat flours were identified after heat treatments. Treatment below 110 °C eliminated much cereal odors and produced little volatile compounds. Waxy wheat flour was more prone to develop aroma-active compounds during heat treatment.
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S0733-5210(16)30447-7
DOI:
10.1016/j.jcs.2017.03.018
Reference:
YJCRS 2319
To appear in:
Journal of Cereal Science
Received Date: 15 November 2016 Revised Date:
15 March 2017
Accepted Date: 21 March 2017
Please cite this article as: Xu, J., Zhang, W., Adhikari, K., Shi, Y.-C., Determination of volatile compounds in heat-treated straight-grade flours from normal and waxy wheats, Journal of Cereal Science (2017), doi: 10.1016/j.jcs.2017.03.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Determination of volatile compounds in heat-treated straight-grade
2
flours from normal and waxy wheats
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1
3
Jianteng Xu1, Wenbin Zhang1,3, Koushik Adhikari2, Yong-Cheng Shi1*
4
1
5
University, Manhattan, KS 66506;
6
3
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Department of Grain Science and Industry, 2Department of Human Nutrition, Kansas State
Jiangnan University, Wuxi, Jiangsu, China
7
*To whom correspondence should be addressed. Telephone: 785-532-6771. E-mail:
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[email protected]
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Abstract Volatile compounds formed during dry heat-treatment of wheat flour influence the application of treated flour. In this study, normal and waxy hard wheat flours before and after
14
dry-heat treatment were subjected to headspace analysis by solid-phase microextraction of
15
volatiles followed gas chromatography–mass spectrometry (GC/MS). The untreated waxy wheat
16
flour contained higher levels of odor-active compounds than normal wheat flour including
17
aldehydes, alcohols, furans, and ketones. Lipid oxidation appears to play major role in producing
18
such odor compounds. Heat treatments, depending on the severity, alter the profile of volatile
19
compounds. Low temperature (100 to 110 °C) treatments effectively eliminated cereal odor
20
(aldehyde) and did not introduce additional odors, providing a possible way to produce low-odor
21
flours. Heat treatments at 120 °C and higher temperatures elevated the content of pyrazines,
22
furans, and sulfur-containing compounds which together gave a roasty aroma to the flours.
23
Considering organoleptic properties, treatments of flours at 140 °C was superior to 160 °C. The
24
waxy wheat flour was more prone to produce odor-active compounds than normal wheat flour
25
during the same heat treatment.
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Keywords: Volatile compounds, waxy wheat flour, GC/MS, SPME, dry heating
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1. Introduction Wheat flour is one of the most important staple food sources in the world. Heat-treated flours
30
(HTFs) produced by applying dry heat to base flours under different temperatures and times have
31
gained popularity in food production, including coatings, binders, baked foods, and thickeners
32
(Neill et al., 2012; Shi, 2009). Heat effectively remove moisture from flour, and in the presence
33
of air causes change in the physicochemical properties of flours that can benefit applications. For
34
instance, heating at 49-93 °C for 1 h to 10 weeks (Hanamoto and Bean, 1979), 120 °C for 10-120
35
min (Nakamura et al., 2008), and 130 °C for 30 min (Neill et al., 2012) result in increased
36
volume of high-ratio cakes. A recent patent includes the claim of volume increase of French
37
breads made from flours subjected to heat treatment at 143-163 °C for 2-20 min (Upreti et al.,
38
2010). Heat treatment conducted at 140-160 °C for 15-60 min also can improve binding and
39
thickening properties of various cereal flours, which provides the great potential to replace
40
chemically cross-linked starches (Shi, 2009). A heat treated flour in a food can be labeled
41
simply as “flour” which creates a more consumer-friendly ingredient profile. Another advantage
42
of using HTFs in foods is the elimination of microorganisms contained in raw flour. Even though
43
heat treatment has been applied to tailoring the functional properties of flour, its impact on
44
volatile compounds that are likely associated with a change in sensory characteristics of flour is
45
not fully understood.
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46
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Volatile compounds in foods create odors/aromas, and their types and concentrations
47
dictate olfactory and taste perception (Zhou et al., 1999). Despite the fact that volatile
48
compounds in unprocessed grains, which are primarily produced from lipid oxidation, are
49
presented at a trace level, they nevertheless give a distinct flavor profile to each cereal (Rackis et 3
ACCEPTED MANUSCRIPT
al., 1979). Owing to their low lipid contents and possibly to bleaching procedures, raw wheat
51
flours have a largely bland flavor but with slight “green” and “grainy” notes. According to a
52
systematic study on Kansas-grown wheat grains, climate had a major impact on the volatiles’
53
profile in the raw kernels, whereas wheat cultivar seemed to have no significant influence on the
54
formation of volatiles (Seitz, 1994). Previous research suggested aldehydes, most of which had a
55
carbon chain lengths C6 to C9, represent the major class of odor-active volatile compounds
56
present in wheat flour, followed by alcohols and alkenes of similar chain length (Maeda et al.,
57
2008; Sayaslan et al., 2000; Seitz, 1994; Zhou et al., 1999).
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Processing conditions of a food may affect its volatiles’ profile. Volatile compounds,
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50
including pyrazines, pryidines, and furans, are readily generated by Maillard reactions of
60
reducing sugar with amino compounds (Hoseney, 1984; BeMiller, 2007). A number of factors
61
influence the formation of volatiles such as moisture content, pH, heating temperature and time,
62
plus amino acid and sugar profiles (BeMiller, 2007). Volatiles are desirable in baked and
63
extruded foods made from flours as they give off “roasty, nutty, and caramel” aromas. However,
64
when a HTF is used in certain applications such as a thickener in soups and pie fillings, the HTF
65
is expected to be bland without an undesirable aroma. The objectives of this study were to: (1)
66
identify the volatile compounds in raw straight-grade flours of normal and waxy hard wheats,
67
and (2) assess how the volatile compounds were affected dry heating of those two flours.
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2. Materials and Methods
69
2.1. Wheat grains and flours Normal hard (NH) red wheat flour was milled from a mixture of cultivars grown in 2013
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70
in Kansas obtained from a local grain elevator. Waxy hard (WH) red wheat grain (received in
72
2013) was provided by Dr. Robert Graybosch at USDA/ARS, University of Nebraska. The waxy
73
wheat grain was inspected by the naked eye to remove foreign material and molded kernels, and
74
then sieved on 2 wire (mesh #10)-screen to remove small particles. The moisture content of the
75
waxy wheat was determined using a single kernel characterization system (SKCS) and tempered
76
to 16% overnight. The tempered waxy wheat was milled into flour with a Bühler MLU-202
77
Laboratory mill (Uzwil, Switzerland) adjusted according to AACC method 26-21.02 for hard
78
wheat (AACC International, 2009). In the milling of the waxy wheat, the feeding rate was
79
lowered to about 100 g/min. Straight-grade flour was collected and stored in an air-tight
80
container at 4°C until analysis or heat treatment.
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The moisture content of flour was determined by loss of weight at 105 °C and protein content by a Leco FP-2000 protein analyzer (Leco Corp, St Joseph, MI, USA) according to
83
AACC Method 46-30.01 using a conversion factor of 5.7 (AACC International, 2009). Crude
84
lipid content was determined gravimetrically by petroleum ether extraction using Soxtech 2045
85
Fat Extraction System (FOSS, Denmark) followed by evaporation of solvent. Total starch and
86
damaged starch were determined using Megazyme total starch and damaged starch assay kit
87
(Wicklow, Ireland) according to AACC Methods 76.13 and 76-31.01, respectively (AACC
88
International, 2009).
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2.2. Dry-heat treatment of wheat flours Ten grams of normal and waxy wheat flours (12.5% moisture) were placed separately in
91
the bottom of sealed 120 mL pressure bottles (Ace Glass, Vineland, NJ) that were subsequently
92
placed in a hot oven equilibrated at 100, 110, 120, 140, and 160 oC (±2 °C). The headspace in the
93
bottle above a flour was approximately 11 cm. Heating was allowed to proceed for 30 min after
94
the internal temperature of flour reached the targeted temperature. The time for flour to reach a
95
target temperature was determined in a preliminary experiment using an embedded thermal
96
couple. A treated bottle with flour was cooled at room temperature for 30 min, and the treated
97
flour was ground with a mortar and pestle and analyzed by SPME-GC/MS on the same day. In a
98
separate experiment,10 grams (as is) of normal hard wheat flour was heated at 160 °C for 2 h in
99
order to intentionally produce a higher level of volatile products to assist in compound
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identification. According to our preliminary studies, flours treated at 160 °C for 2 h would be
101
acceptable for human consumption, even though slight browning of the flour occurred.
102
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2.3. Volatile compound extraction
An automated solid-phase microextraction (SPME) was used to extract and concentrate
104
the volatile compounds in a flour’s headspace onto the coated fiber. A CombiPAL antosampler
105
(CTC Analytics/Leap Technologies, Carrboro, NC) was used to house the SPME fiber, sample
106
trays, a bake-out unit and an agitator. The SPME fiber (50/30 µm divinylbenzene/Carboxen on
107
polydimethylsiloxane on StableFlex) obtained from Supelco (Bellefonte, PA) was baked at the
108
baking station of the CombiPal at 270 °C for 1 h before its use in the study. Each flour sample
109
(0.5 g) was accurately weighed into a 10 mL sample vial that had been baked at 105 °C
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overnight and screw capped with teflon-lined silicone septum. Samples in the vials were heated
111
at 60 °C (Maeztu et al., 2001) with agitation at 500 rpm while each flour’s headspace was
112
exposed to SPME fiber. The SPME fiber was inserted into the injection port of the GC to desorb
113
volatile compounds for 5 min at 270 °C. Between each analysis, the fiber was baked at 280 °C
114
for 20 min to remove carryover volatiles. Blank runs were carried out using an empty vial and
115
were considered as background.
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2.4. GC/MS analysis
A Varian CP-3800 gas chromatograph (Varian Inc., Walnut Creek, CA) fitted with a DB-
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5ms ((5%-phenyl)-methylpolysiloxane) capillary column (30m x 0.25 mm i.d., 0.25 µm film
119
thickness, Agilent Technology, Santa Clara, CA) was used to perform volatiles separation. The
120
column oven was programmed to start at 40 °C and ramped to 280 °C at 4 °C/min. Helium was
121
used as carrier gas and the flow rate was set at 1 mL/min. MS analysis was carried out using a
122
Varian Saturn 2200 Ion Trap mass spectrometer (Varian Inc., Walnut Creek, CA). The MS was
123
operated in scanning mode with a range of 35-400 amu. Total ion current (TIC) was recorded
124
and the TIC peaks were subjected to computerized matching to the National Institute of
125
Standards (NIST) mass spectral database. In addition to fragment pattern matching, a C7-C30
126
saturated alkane premix (Sigma-Aldrich, St. Louis, MO) was analyzed using the same method,
127
and the MS identified compounds were identified by calculating linear retention indices (LRI)
128
and comparing the values in the NIST library and other published papers (Cho & Kays, 2013;
129
Janes et al., 2010; Maeda et al., 2008; Maeztu et al., 2001; Sayaslan et al., 2000; Ying et al.,
130
2012). In the case where a LRI was not found associated with DB-5ms column, similar columns
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131
(for example – BP-5, HP-5, ZB-5) were used for LRI comparisons. LRI was calculated using the
132
following equation: )ܴܽܶ(݃ܮ− )ܴ݊ܶ(݃ܮ )ܴܰܶ(݃ܮ− )ܴ݊ܶ(݃ܮ
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= ܫܴܮ100݊ + 100 ×
Where n was the number of carbon in the alkane eluted ahead of targeting analyte; RTa, RTn and
134
RTN were retention times of the analyte, alkanes eluted ahead and after the analyte, respectively.
135
Upon positive identification, sensory descriptors were assigned to each volatile compound (Cho
136
and Kays, 2013; Maire et al., 2013). A flowchart of the experiment is depicted in Fig. 1. 2.5. Statistics analysis
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Heat treatments in this study were performed in duplicate and each sample was analyzed
139
twice using GC/MS (n=4). For total and damaged starch analysis performed on raw flours, each
140
flour sample was analyzed in triplicate (n=3). Principal component analysis (PCA) was
141
performed to evaluate the variance in compound classes in the normal and waxy wheat flours.
142
Upon heat treatment of flours, the changes in each class of compounds were analyzed by one-
143
way ANOVA with heating conditions being the main factors. Tukey’s post-hoc test was used to
144
assess the multiple differences of different compound classes at various heating conditions. A
145
probability of P < 0.05 was considered significant. All statistical procedures were handled by
146
SAS 9.3 package (SAS Institute; Cary, NC).
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148 149
3. Results and Discussion 3.1. Volatile compounds in different wheat flours According to GC/MS analysis, the major class of volatile compounds in untreated normal
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hard wheat flour (without dry-heat treatment) was aldehydes. Hexanal constituted over 43% of
151
aldehydes by ion current count (Table 1). Other intermediate-length aldehydes, i.e. nonanal,
152
decadienal and undecenal, also were identified at appreciable levels in untreated normal hard
153
wheat flour (Fig. 2 & Table 1). Untreated waxy hard wheat flour exhibited a profile of volatiles
154
similar to the untreated normal hard wheat flour, but the level of hexanal in the waxy flour was
155
3-fold greater than in the normal wheat flour (Table 1). Nonanal was higher in the waxy flour,
156
whereas decadienal and undecenal were found in normal flour, but not in the waxy flour.
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Hexanal is generally considered the most pronounced secondary autoxidized product of
158
linoleic acid formed through homolytic β-scission of linoleic 11-hydroperoxide (Choe and Min,
159
2006). Hexanal is characterized by a low threshold odor described as “grassy” and “hay-like”
160
(Min et al., 1989; Sayaslan et al., 2000). The higher hexanal content in untreated waxy wheat
161
flour compared to normal wheat flour indicate higher extent of lipid oxidation in waxy wheat
162
flour (Nielsen and Hansen, 2008), which probably accounts for its “greener” odor. Hexanol was
163
found in both normal and waxy wheat flours at a much higher concentration than other alcohols,
164
which was as expected because it shares a similar oxidation pathway with hexanal. Hexanol can
165
be converted to hexanal by alcohol dehydrogenase (Matoba et al., 1989; Mizutani and
166
Hashimoto, 2004).
167 168
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Other indicators of lipid autoxidation, i.e. 1-octen-3-ol and 2-pentyfuran, substances exhibiting “mushroom-like” and “beany” odors (Min et al., 1989), also were found higher in 9
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waxy wheat flour (Table 1). The content of ketones in waxy wheat flour was 3-fold greater than
170
that in normal wheat flour. In addition, 3-octen-2-one was only detected in the waxy wheat flour.
171
An earlier study compared the volatiles present in starches and found that waxy corn starch, both
172
commercial and laboratory-prepared, released more lipid oxidation volatiles than the respective
173
samples of normal corn starch (Sayaslan et al., 2000). These results on the starches are in
174
agreement with our findings on wheat flours in this study.
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Yuan and Chang (2007) reported that soymilk made from lipoxygenase-null soybean
176
cultivars had significantly reduced levels of odor-causing compounds including many of the
177
aldehydes, ketones, alcohols, and furans found in our wheat flours. It seems reasonable to
178
attribute the formation of the higher amount of volatile compounds in waxy wheat flour to the
179
joint effect of physical damage to lipid-containing tissue during milling followed by autoxidation
180
or enzymatic reactions in flour. In addition, starch in waxy wheat flour is essentially free of
181
amylose, a long linear polymer that can form cavities to entrap small flavor-active molecules in
182
its helical structure (Arvisenet et al., 2002). Naphthalene (insect repellent) and butylated
183
hydroxytoluene (BHT, a synthetic antioxidant) were found in both normal and waxy wheat flours
184
(Table 1). Those compounds perhaps were introduced during planting, processing or storage.
185
Additional peaks were observed in the GC/MS volatile profiles of heat-treated flours
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compared with the untreated flours. The extensive heat treatment of flours at 160 °C for 2 h
187
caused drastic changes in the volatiles’ profiles and provided valuable information regarding
188
retention times and MS spectra markers (Fig. 2). Flour treated at 160 °C for 2 h had a notable
189
“roasty” aroma, and gave a more distinct volatiles’ profile (Fig. 2b) compared to that without
190
dry-heat treatment (Fig. 2a). A number of pyrazine derivatives were detected as the largest group
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191
of emerging compounds in addition to pyrroles, furans, and sulfur-containing compounds (Table
192
1). The volatile compounds of normal and waxy wheat flours before and after heat
194
treatments at 100-160 °C were summed in the following classes of organics for each treatment;
195
alcohols, ketones, aldehydes, pyrazines, furans, sulfur-containing compounds, alkanes, and
196
miscellaneous compounds. The summations of the TIC count of these classes of compounds are
197
given in Table 2. Total aldehyde content of both normal and waxy wheat flours exhibited
198
somewhat of a “reclining” J-curve in which the lowest point was observed at heat treatment of
199
100 oC. Meanwhile, total alcohols and alkenes in heated flours were prone to decrease with
200
increasing heating temperature. These data may be explained as follows. Increased temperature
201
increases the generation of aldehydes in heat-treated flours, and that increase is not lost during
202
grinding and handling of a heated-treated flour. But in the class of alcohols, while heat-treatment
203
could cause an increase, the alcohol class is more volatile and is lost during heat treatment before
204
analysis.
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Pyrazines and furans were detected in steady increasing amounts from 120 to 160 °C in normal wheat flours and from 100 to 160 °C in waxy wheat flours. It appears that waxy wheat
207
flour was more prone to formation of these compounds compared to normal wheat flour as
208
pyrazines and furans were produced upon heating waxy flour at 100 °C, but not normal flour,
209
which required heating at ≥ 110 °C. Moreover, at all heating temperatures, treated waxy flour
210
contained more pyrazines and furans than similarly treated normal flour. These differences could
211
be explained by the higher level of free sugars commonly associated with waxy wheat (Zhao et
212
al., 2009) and by the higher level of damaged starch in waxy wheat flour (Table 3), as also
213
previously reported (Bettge et al., 2000; Guan, 2008). Based on the weight of starch, there was
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1.5% more damaged starch in the waxy wheat flour. Damaged waxy wheat starch is probably
215
more prone to degradation during thermal process, generating more reducing ends. The increased
216
concentration of reducing carbohydrate in waxy flour possibly would lead to greater sugar-amine
217
reactions. It is noteworthy that the protein content of the waxy wheat flour in this study was
218
higher compared to literature values (Garimella Purna et al., 2011).
219
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Trace amounts of sulfur-containing compounds including dimethyl trisulfide and 2pentylthiophene were detected in the flours treated at 140 and 160 °C (Table 1). The latter
221
compound could be formed from cysteine-involved reactions (Yu and Zhang, 2010). It is
222
noteworthy that a sulfur-containing compound typically imparts a very strong aroma, even at
223
trace levels.
224
226
3.2. Principal components analysis
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Principal components analysis (PCA) was performed to elucidate which class of volatile compounds within normal and waxy wheat flours changed in level as heating temperature was
228
changed (Fig. 3). Principal factor 1 (PC1) explained 59% of the variance in the data and
229
differentiated heat-induced compounds of various classes from those compounds in untreated
230
flours, whereas principal factor 2 (PC2) mainly distinguished the contribution of aldehydes and
231
alkanes and explained 31% of the variance in the data. The PC2 can be reasonably interpreted as
232
the strength of “grassiness” from aldehydes because alkanes are odorless.
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PCA clustered the flour samples into three groups according to their volatile profiles (Fig.
234
3). Both native flours were separated from the 140-160 °C treated counterparts across PC1 which
235
was driven by high concentrations of pyrazines, furans, ketones, and S-containing compounds, 12
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Higher temperature (140-160 °C) treated flours had large positive scores on PC1 and small
237
values of PC2, indicating a mix of “roasty" and “green” (lipid oxidation) odors. Interestingly,
238
normal hard wheat flour treated at 100, 110, and 120 °C and waxy hard wheat flour treated at
239
100 and 110 °C were found to have positive, although small, scores on PC1 and negative scores
240
on PC2, which means those treated flours have diminished “green” (cereal) odor and minimal
241
“roasty” (heat-treated) odor.
242
4. Conclusions
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Volatile compounds in normal and waxy hard wheat flours before and after dry-heat
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treatment were identified with headspace SPME GC/MS techniques. Aldehydes, alcohols, and
245
alkanes of various chain lengths were found as major volatile compounds in both waxy and
246
normal wheat flours. Waxy flour contained approximately 53% higher total volatiles, which was
247
largely caused by the high levels of hexanal and hexanol. Heat treatment at 120 °C or higher
248
temperature up to 160 °C led to the formation of pyrazines, furans, ketones, and sulfur-
249
containing compounds, particularly in waxy wheat flour. However, the 100 °C to 110 °C
250
treatment eliminated “cereal” odor to a great extent without introducing new aromas, indicating
251
in the possibility of developing low-odor flour ingredients. The formation of volatiles in heat-
252
treated waxy wheat flours may proceed faster than in normal wheat flour, which suggests a lower
253
temperature should be considered for waxy wheat flour. Considering the possible disparities
254
between instrumental analysis and sensory perception, future work should include the
255
determination of sensory properties and its relation to volatiles’ profiles in heat-treated wheat
256
flours.
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Acknowledgements
258
We thank Dr. Robert Graybosch, USDA-ARS, for providing the waxy wheat and Dr. Paul Seib
259
for reviewing the manuscript. This is contribution # 15-170-J from the Kansas Agricultural
260
Experiment Station.
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Abbreviations: HTFs, heat-treated flours; NH, normal hard; WH, waxy hard; SKCS, single
263
kernel characterization system; SPME, solid-phase microextraction; GC/MS, gas
264
chromatography–mass spectrometry; NIST, National Institute of Standards and Technology; LRI,
265
linear retention indices; TIC, Total ion current.
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267
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Zhao, R., Wu, X., Seabourn, B. W., Bean, S. R., Guan, L., Shi, Y.-C., Wilson, J. D., Madl, R.,
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338
3953.
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Tables
341
Table 1. Volatile compounds in heat-treated flours (initially 12.5% moisture) milled from normal (NH) and waxy hard (WH) wheats*
Retention
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340
Compound name NH
Heating T (°C) (30 min) ConC
6.41
2,3-Pentanedione
691
7.82
Pyrazine
740
8.29
Pyrrole
752
8.87
Pentanol
770
10.17
Hexanal
801
71
140
109
160
Con
211
139
83
47
933
659
922
156
NH
Heating T (°C) (30 min)
120 min
154
155
100
71
TE D
648
120
EP
2-Butenal
110
AC C
5.31
100
WH
M AN U
time (min)
Total ion count × 103
SC
LRIA
110
88
120
72
140
126
160
164
Odor qualityB
160 264
Flower
448
Cream, butter Pungent, roasted
434 hazelnuts
324
136
134
115
119
133
244
Nutty, sweet
62
Bready, yeasty, Green, fatty, leafy,
1147
1117
808
3301
1060
1693
1685
1425
893
1065
vegetative, fruity, woody
19
ACCEPTED MANUSCRIPT
Ripe, nutty, vegetable, 11.00
4-Methylthiazole
836
23
11.23
11.57
2-Methylpyrazine
Furfural
828
68
838
69
235
55
856
853
12.92
1-Hexanol
872
1034
403
202
Xylene
881
13.80
2-Heptanone
889
71
59
61
14.38
Heptanal
907
46
47
76
926 Dimethylpyrazine
15.11
Ethylpyrazine
929
15.87
Vinylpyrazine
947
16.82
2(E)-Heptenal
968
109
2424
6269 roasted, cocoa
64
114
47
79
429
130
662
Bread, almond, sweet
23
Woody, smoky
186
Musty, bready, coffee Fruity and alcoholic,
1070
965
810
sweet, and green 27
Slightly sweet
181
215
185
92
114
188
340
271
661
Cheesy, fruity, creamy
139
160
143
99
72
91
121
169
160
229
Nut-like, fatty, fruity
180
554
42
280
959
4995
61
Nutty, peanut, musty, earthy
AC C
2,514.91
463
128
EP
13.23
180
TE D
2-Furanmethanol
M AN U
pyrrole 12.32
Popcorn, nutty, 89
SC
2-Methyl-1H11.98
RI PT
tomato
1598
54
133
20
37
67
156
Roasted cocoa, coffee
64
Nutty, earthy
106
Pungent, green,
ACCEPTED MANUSCRIPT
vegetable Benzaldehyde
982
54
31
60
236
533
302
177
81
17.65
1-Octen-3-ol
982
257
133
76
129
158
193
448
116
Dimethyl 990
68
48
trisulfide Methyl heptenone
991
18.14
2-Pentylfuran
996
18.45
Decane
1000
51
179
247
295
562
49
2-Ethyl-6-methyl 1009 pyrazine 2-Ethyl-518.76
1015
2-Methyl-6-
19.68
2-Ethyl-1-hexanol
1032
19.69
Methylvinylpyrazi
1038
601
146
131
146
81
305
238
Fruity, almond Earthy, green, oily, vegetative and fungal Cooked onion, savory,
47
65
45 meaty, eggy 48
Fruity, apple, musty Fruity, green, earthy,
328
620
765
1255
1090
565 beany
49
96
119
73
86
107
127
144
936
Roasted potato,
91
208
380
87
103
198
328
463
1597
Coffee
169
Hazelnut
AC C
1032 vinylpyrazine
911
131
574
EP
methylpyrazine
19.48
964
TE D
18.55
34
132
46
M AN U
17.86
246
SC
17.77
123
RI PT
17.33
72
259
128
126
122
92
Mild, oily, rose, green 87
21
Nutty
ACCEPTED MANUSCRIPT
ne
20.14
3-Octen-2-one
1043
21.12
Butyl-benzene
1065
21.60
3-Methylphenol
1080
210
49
30
33
96
45
50
1085
2-Ethyl-2,51087
94
41
154
134
60
45
124
164
69
63
2,51091
36
Undecane
1099
206
53
22.83
Nonanal
1113
589
375
25.14
Pentyl-benzene
1168
25.32
2-Pentylthiophene
1170
25.97
1-(2-
1182
290
AC C
22.53
51
EP
Diethylpyrazine
68
TE D
dimethylpyrazine
22.48
280
138
dimethylpyrazine
22.21
40
100
3-Ethyl-2,521.79
40
RI PT
1041
SC
Limonene
M AN U
20.05
360
43
413
196
Earthy, oily, hay and 174
157 mushroom
67
141
84
80
82
64 leather
70
81
327
Cocoa
62
138
Cocoa
58
Nutty, hazelnut
262
Fresh, slightly green
34
258
942
241
274
Aromatic Medical, woody,
60
38
221
Fresh, citrus
55
47
293
383
351
29 31
45
22
58
60
47
Meaty
18
Vegetative, cereal,
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furanmethyl)-1H-
bready
pyrrole Menthol
1190
65
26.41
Dodecane
1199
657
371
226
256
240
69
710
565
648
581
383
93
26.80
Naphthalene
1218
934
365
256
272
245
96
586
212
186
180
157
118
30.06
Tridecane
1299
545
258
191
198
204
51
473
199
138
108
1309
222
52
30
97
1319
142
37
32
69
1365
201
1384
393
1399
260
decadienal 2,4-decadienal 30.70
2-Undecenal Propanoic acid, 2-
EP
methyl-, 3hydroxy-2,4,432.95
esters
33.49
Tetradecane
162
89
103
29
23
Pungent, dry, tarry
Waxy, plastic
249
74
98
Fatty, oily
AC C
trimethylpentyl
203
TE D
(E,E) 32.27
301
Peppermint
M AN U
(Z,Z)2,430.40
SC
133
RI PT
26.06
243
115
95
100
73
ACCEPTED MANUSCRIPT
Butylated 37.18
hydroxytoluene
1515
216
212
RI PT
(BHT)
Contamination
342 343
*
344
A
345
and similar columns
346
B
http://www.thegoodscentscompany.com; (Cho and Kays, 2013; Maire et al., 2013)
347
C
Con: control, raw untreated flour
SC
Mean values are reported. Statistical analysis was performed and saved as Supplementary Material.
AC C
EP
TE D
M AN U
LRI: Linear retention index calculated from C7-C30 straight chain alkanes; all LRIs were within ±10 of literature values of DB-5
24
ACCEPTED MANUSCRIPT
Table 2. Classes of volatile compounds in normal (NH) and waxy hard (WH) wheat flours before and after dry-heat treatment at
349
different temperatures.
RI PT
348
Total ion current x 103 Normal hard (30 min)A
Waxy hard (30 min)A
class 120 °C
140 °C
160 °C
Control
M AN U
110 °C
100 °C
110 °C
120 °C
140 °C
160 °C
1732a
691b
325c
465bc
421c
348c
3588a
1450b
1357bc 1178bc
771cd
409d
Ketones
71bc
59c
61c
128b
326a
290a
339abc
216c
278bc
384abc
514a
428ab
Aldehydes
2187ab
1201c
1410bc 1882abc
2443a
1644abc
4518a
1454c
2181b
2382b
2618b
2161bc
Pyrazines
N.D.d
N.D.d
N.D.d
201c
690b
1356a
N.D.d
160cd
189cd
385c
894b
2110a
Furans
179c
247c
295c
562bc
1033a
966ab
305c
328c
620b
765b
1319a
1204a
N.D.b
N.D.b
N.D.b
N.D.b
99a
93a
N.D.d
N.D.d
N.D.d
46c
105b
125a
Alkanes
1718a
844b
506c
557c
518c
149d
1647a
981b
946b
935b
641b
201c
Miscellaneous
1753a
510b
331b
355b
419b
296b
1327a
281b
249b
240b
304b
341b
Total volatiles
7638a
3552c
2928c
4150c
5949ab
5142bc
11725a
4870b
5820b
6315b
7166b
6979b
S-containing
AC C
compounds
TE D
Alcohols
EP
Control 100 °C
SC
Compound
25
ACCEPTED MANUSCRIPT
350
A
351
treatments in the same type of wheat flour (P<0.05) (n=4)
352
A
RI PT
Means with different lowercase letters a, b, c . . . in the same row indicate significant differences among different temperature
N.D. not detected
AC C
EP
TE D
M AN U
SC
353
26
ACCEPTED MANUSCRIPT
Table 3. Total starch, damaged starch, and protein contents of the normal and waxy hard wheat flours Protein (%)
Lipid (%)
Starch (%)
Ash (%)
Damaged starch (%)
RI PT
Flour
Flour Wt
Starch Wt
10.36a± 0.19
Normal
12.22a± 0.21
1.03a± 0.02
76.59a± 0.67
0.49± 0.02
7.93a± 0.13
Waxy
15.62b± 0.21
1.11a± 0.04
70.87b± 0.36
0.46± 0.02
8.40b± 0.10
SC
354
11.85b± 0.18
Values expressed as mean± standard deviation (“dry” flour Wt) unless otherwise noted; lowercase letters in the same column indicate
356
significant differences (P<0.05) (n=3)
M AN U
355
AC C
EP
TE D
357
27
ACCEPTED MANUSCRIPT
Figure Captions:
359
Figure 1. Flow chart of the experiments conducted
RI PT
358
360
Figure 2. Gas chromatogram of headspace volatiles before (a) and after (b) heat treatment at 160 °C for 2h of a straight-grade flour
362
(initial MC 11.5%) from hard winter wheat. The heating of flour was done in a sealed glass bottle, the flour cooled and ground prior to
363
assaying for volatiles. Volatiles in the flour were collected by solid-phase microextration at 60 °C for 20 min, then separated and
364
quantified by GC-MS. Stars (*) represent compounds derived from column bleeds.
M AN U
SC
361
365
Figure 3. Principal component analysis (PCA) of volatile compounds profile of heat-treated flours; NH, normal hard; WH, waxy hard;
367
WHCon and NHCon, not heated.
370 371 372
EP
369
AC C
368
TE D
366
373 28
ACCEPTED MANUSCRIPT
374
378 379 380 381
EP
377
AC C
376
TE D
M AN U
SC
RI PT
375
29
382 383
Figure 2.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
384 30
385 386 387
Figure 3.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
31
ACCEPTED MANUSCRIPT
Highlights
RI PT
SC M AN U TE D
•
EP
•
Volatile profiles in waxy and normal hard red wheat flours were identified after heat treatments. Treatment below 110 °C eliminated much cereal odors and produced little volatile compounds. Waxy wheat flour was more prone to develop aroma-active compounds during heat treatment.
AC C
•