Secoisolariciresinol Diglucoside and Cyanogenic Glycosides in

J. Agric. Food Chem. , 2016, 64 (50), pp 9551–9558. DOI: 10.1021/acs.jafc.6b03962. Publication Date (Web): November 27, 2016. Copyright © 2016 Amer...
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Secoisolariciresinol Diglucoside and Cyanogenic Glycosides in Gluten-free Bread Fortified with Flaxseed Meal Youn Young Shim,*,†,‡,§,∥ Clara M. Olivia,†,∥ Jun Liu,‡,⊥ Rineke Boonen,†,# Jianheng Shen,‡ and Martin J. T. Reaney*,†,‡,§ †

Prairie Tide Chemicals Inc., 102 Melville Street, Saskatoon, Saskatchewan S7J 0R1, Canada Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan S7N 5A8, Canada § Guangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and Engineering, Jinan University, Guangzhou, Guangdong 510632, China # Food Technology Agrobiotechnology Nutrition and Health Science, Wageningen University, Droevendaalsesteeg 4, Wageningen 6708 PB, Netherlands ‡

ABSTRACT: Flaxseed (Linum usitatissimum L.) meal contains cyanogenic glycosides (CGs) and the lignan secoisolariciresinol diglucoside (1). Gluten-free (GF) doughs and baked goods were produced with added flaxseed meal (20%, w/w) then 1, and CGs were determined in fortified flour, dough, and bread with storage (0, 1, 2, and 4 weeks) at different temperatures (−18, 4, and 22−23 °C). 1 was present in flour, dough, and GF bread after baking. 1 was stable with extensive storage (up to 4 weeks) and was not affected by storage temperature. CGs in flaxseed meal and fortified GF samples were analyzed by 1H NMR of the cyanohydrins. Linamarin and/or linustatin were the primary CGs in both flaxseed meal and fortified flour. CGs decreased with storage in dough fortified with flaxseed meal or GF bread after baking. GF bakery food products fortified with flaxseed meal had reduced CGs but remained a good source of dietary 1. KEYWORDS: flaxseed meal, cyanogenic glycosides, secoisolariciresinol diglucoside, gluten-free bread, stability

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lignan content in flaxseed (6100−13300 mg/kg) is principally secoisolariciresinol diglucoside, 1 (Figure 1).19 Small amounts of other lignans such as matairesinol, pinoresinol, lariciresinol, and isolariciresinol are also observed.20 After consumption, 1 is metabolized by colonic microflora to serve as a principal precursor of “mammalian” lignans, enterodiol and enterolactone.21 Enterodiol can be further oxidized to enterolactone. The antioxidant potencies of enterodiol and enterolactone, based on the chemiluminescence of zymosan-activated polymorphonuclear leukocytes, are 5.02 and 4.35, respectively, which are 3.95 and 3.43 times more potent than 1.22 1 is thought to be associated with beneficial health effects of flaxseed-fortified diets that potentially protect against hormonebased cancers, cardiovascular diseases, and adult diabetes.23 However, flaxseed also contains cyanogenic glycosides (CGs), nitrogenous secondary plant metabolites (Figure 2), which are undesirable in food products.24 CGs are glycosides of cyanohydrins, mostly in the form of β-linked D-glucosides such as linamarin (4), linustatin (5), lotaustralin (6), and neolinustatin (7), with a content of 250−550 mg/g dry flaxseed.17,24 CG content and composition in flaxseed are dependent on seed age, cultivar, location, and environmental conditions during growth.24 After consumption, CGs could be liberated as unstable cyanohydrins via intestinal β-glucosidase hydrolysis of glucosidic bonds and further be decomposed in

INTRODUCTION Celiac disease is caused by permanent intolerance to gluten proteins1 and could affect 1−2% of the world’s population.2 A typical symptom of celiac disease is inflammation of the small intestine that leads to malabsorption of food nutrients such as minerals and fat-soluble vitamins (vitamins A, D, E, and K).3 Chronic celiac disease induces extra-intestinal symptoms including anemia,4 skin lesions,5 hypertransaminasemia,6 arthralgia,7 and neurological disorders.8 Treatment of celiac disease involves a strict lifelong gluten-free (GF) diet with no cereals (wheat, barley, rye, triticale, dinkel, and kamut) containing gluten proteins.9 Flours prepared from rice, millet, tapioca, potato starch, and pulses are potential replacements of gluten-rich cereals in GF foods.10 Unfortunately, such flour products lack baking quality as gluten proteins impart unique water absorption capacity, cohesiveness, viscosity, and elasticity on dough and baked goods.11 Interestingly, flaxseed adds texture to GF products, resulting in more appealing, fluffier GF products.12 In addition to adding texture, flaxseed can also improve the nutritional profile of GF food products.13 Flax has been cultivated for more than 5000 years as an oilseed crop.14 Recently, flaxseed (Linum usitatissimum L.) has been used for food and feed purposes due to its high energy density (4.5 kcal/g) and protein content (20%), as well as unique phytochemical nutrient profile.15 It is one of the richest sources of omega-3 fatty acids, especially α-linolenic acid, with remarkable potential health benefits.16,17 Moreover, nonoil flaxseed constituents, such as soluble dietary fiber, minerals, and phenolic compounds, also impart beneficial health effects.15 Particularly, flaxseed is a source of lignan compounds.18 The © 2016 American Chemical Society

Received: Revised: Accepted: Published: 9551

September 4, 2016 November 26, 2016 November 27, 2016 November 27, 2016 DOI: 10.1021/acs.jafc.6b03962 J. Agric. Food Chem. 2016, 64, 9551−9558

Article

Journal of Agricultural and Food Chemistry

Figure 1. Structures of 1−3 residues in flaxseed (average value of n = 3). and sodium hydroxide (NaOH, ACS reagent, ≥97.0%) were obtained from EMD Chemicals Inc. (Gibbstown, NJ, USA). Methanol (MeOH) used in this study was of HPLC grade and purchased from Fisher Scientific International Inc. (Fair Lawn, NJ, USA). 3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TMSP, 98 atom % D) was obtained from Alfa Aesar (Heysham, Lancashire, UK). Flaxseed (L. usitatissimum L.) meal was provided as an “in kind” contribution from Prairie Tide Chemicals Inc. (Saskatoon, SK, Canada). A Milli-Q deionization reversed osmosis (RO) system (Millipore, Bedford, MA, USA) was used to prepare deionized RO water (resistivity was >18.2 MΩ·cm at 25 °C). Sample Preparation for Chemical Analysis. Samples were collected at each step during the preparation of GF bread with or without substitution of rice flour by flaxseed meal. All ingredients present in the standard recipe for GF bread preparation are shown in Table 1.26 For GF bread made without flaxseed meal fortification

Table 1. Ingredients of GF Bread and Flaxseed Meal Fortified GF Bread Figure 2. Structures of CGs of 4−7 in flaxseed. Methyl protons are labeled and colored in the structure (standard integration windows are referred to as bins): bin 1 (pink), bin 2 (green), bin 3 (blue), and bin 4 (red).

neutral or basic conditions to aliphatic ketones and hydrogen cyanide (HCN), which is toxic to the mammalian respiratory, nervous, and endocrine systems.25 In addition, CGs can also act as antinutrients that might influence nutrient absorption from food products.17 Thus, the presence of CGs might limit the exploitation of flaxseed in foods. To design safe GF bakery food products with improved nutritional profiles, flaxseed meal fortified GF bread was prepared as a model. The stability of 1 and CGs during processing and storage of flaxseed meal fortified GF bread was investigated by using HPLC-DAD and 1H NMR, respectively. Results from this study will help to determine the potential for use of flaxseed meal in gluten-free bakery food products with known nutraceutical and toxic potentials.

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ingredient

GF bread (control, g)

GF flaxseed meal fortified GF bread (g)

white rice flour potato starch tapioca flour flaxseed meal sugar instant yeast salt milk (45 °C) unsalted butter eggs

846 194 82 0 12 6 7 242 57 146

1054 389 164 637 24 13 14 484 113 293

(control), white rice flour, potato starch, and tapioca flour were thoroughly blended with sugar, yeast, and salt at medium speed using a blender (Mississauga, ON, Canada). Then warm milk (45 °C) and butter were added, and the mixture was completely blended at low speed. Finally, eggs were added one at a time and blended for 3 min at high speed to form the dough that was poured into greased loaf pans, covered, and kept at room temperature (RT) to rise and then baked in an oven at 175 °C for 40 min. The GF bread was taken out of the loaf pan immediately after baking and cooled for 10 min at RT. All control samples, including GF flour, dough, and bread, were analyzed on the day they were first prepared (0 day). Flaxseed meal fortified GF bread was prepared with the same procedure as mentioned above except

MATERIALS AND METHODS

General. Formic acid (HPLC grade) and rutin trihydrate (HPLC grade, ≥90%) were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). Acetonitrile (CH3CN, HPLC grade, ≥99.9%), glacial acetic acid (CH3COOH, ACS reagent, ≥99.7%), 9552

DOI: 10.1021/acs.jafc.6b03962 J. Agric. Food Chem. 2016, 64, 9551−9558

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Journal of Agricultural and Food Chemistry

Table 2. Contenta of 1 in Flaxseed Meal and Its Fortified GF Samples as a Function of Storage Time and Temperature sample

a

week 0

week 1

week 2

week 4

RT (22−23 °C)

bread dough flour meal

1142.9 ± 40.8 725.3 ± 33.9 1251.4 ± 12.7 1224.8 ± 14.7

1124.8 ± 70.9 nd 1230.3 ± 5.20 1144.5 ± 67.1

ndb nd 1155.4 ± 41.6 1157.3 ± 85.7

nd nd 1013.3 ± 103.3 1019.7 ± 97.7

refrigerator (4 °C)

bread dough flour meal

nd nd nd nd

1116.0 ± 117.2 474.0 ± 166.4 1147.7 ± 54.1 1140.5 ± 54.0

1106.7 ± 48.8 569.1 ± 52.8 1152.9 ± 73.3 1117.6 ± 66.4

915.5 ± 109.3 430.7 ± 97.8 1099.5 ± 56.5 1040.8 ± 116.2

freezer (−18 °C)

bread dough flour meal

nd nd nd nd

1188.9 ± 73.4 495.8 ± 219.8 1100.7 ± 52.9 1117.7 ± 59.8

1182.2 ± 55.0 430.9 ± 74.1 1132.7 ± 83.6 1119.0 ± 73.5

1018.9 ± 190.9 345.3 ± 119.3 1121.4 ± 104.1 1055.7 ± 87.8

Content is expressed as integrated 1 peak area (mAU) in HPLC-DAD chromatogram monitored at 214 nm. bnd, not determined.

flour mix (rice flour, potato starch, and tapioca flour) was substituted with 20% (w/w) flaxseed meal. All test samples (flaxseed meal and its fortified flour, dough without yeast, and GF bread) were collected and stored in plastic bags (Ziploc, Racine, WI, USA) at RT (22−23 °C), 4 °C (refrigerator), and −18 °C (freezer), respectively. Analysis of test samples was done at 0 days (day of preparation) and then 1, 2, and, 4 weeks thereafter. Extraction of 1. All test samples collected above (2.0 g) were weighed, and the bread samples were preground in a coffee grinder (VWR, Radnor, PA, USA) prior to weighing. 1 was extracted with aqueous MeOH (10.0 mL, 70%, v/v) at 60 °C for 2.0 h by keeping sample tubes in a water bath (VWR, Cornelius, OR, USA). After extraction, sample tubes were cooled at 20 °C for 30 min and centrifuged at 3000g for 10 min (Palo Alto, CA, USA). Supernatant aliquots (500 μL) were collected and mixed with 250 μL of 0.1 M NaOH solution at RT for 1 h to hydrolyze 1 polymers. Rutin solution (75 μL, 1.0 mg/mL) prepared in 50% (v/v) aqueous MeOH was added as an external standard before hydrolysis. The hydrolysis reaction was stopped by adding 500 μL of 0.1 M acetic acid (CH3COOH) solution. The neutralized solution was filtered through PTFE syringe filters (pore size = 0.45 μm and diameter = 13 mm) prior to HPLC-DAD analysis. HPLC Analysis of 1. A 1200 series HPLC system (Agilent Technologies, Mississauga, ON, Canada) equipped with a quaternary pump, an autosampler, a photodiode array detector, and a degasser was employed for 1 analysis. All solvents used for HPLC analysis were passed through a filter-degasser equipped with Teflon filter membranes (Agilent Technologies Canada Inc., Mississauga, ON, Canada). A gradient elution with mobile phases of water (A) and acetonitrile (B), flow rate of 2.0 mL/min, was composed as follows: 0 min, 50% B; 1.8 min, 80% B; 1.9 min, 90% B; 2.0 min, 50% B; and 2.5 min, 50% B. The column used was a 100 mm × 4.6 mm i.d. Chromolith SpeedROD RP-C18 (Merck KGaA, Darmstadt, Germany). Eluting peaks were monitored at 214 nm with a 10 nm bandwidth and against a reference signal at 300 nm (10 nm bandwidth). The sample injection volume was 10 μL, and the column temperature was maintained at 32 °C. Data analysis was conducted using Chemstation LC 3D system software (Agilent Technologies, Mississauga, ON, Canada). Extraction of CGs. CGs in samples collected during processing and storage of flaxseed meal fortified GF bread were extracted according to methods previously described.27 Each sample (10.0 g) was accurately weighed into a 50 mL conical sterile polypropylene centrifuge tube (Fisher Scientific International Inc., Fair Lawn, NJ, USA), whereas the bread sample was preground in a coffee grinder (VWR, Radnor, PA, USA) before weighing. Each sample was thoroughly mixed with 20 mL of 90% (v/v) aqueous MeOH with TMSP (10 mM, 500 μL) as the internal standard. All sample tubes were incubated in a water bath (VWR, Cornelius, OR, USA) to

maintain an extraction temperature of 45 °C for 1 h with vortex agitation for 10 s every 15 min. Subsequently, sample tubes were cooled at 20 °C for 30 min and then centrifuged (Beckman-Coulter Inc., Palo Alto, CA, USA) at 3000g for 10 min to settle the solids. Supernatant was collected and dried under an air stream. Subsequently, dried supernatant fractions were redissolved in deuterium oxide (D2O, 1.75 mL) and filtered through a 0.45 μm nylon membrane. The filtrates were collected and transferred to NMR tubes (Norell Suprasil quartz 5 mm, limit = 500 MHz frequency, Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) for CG analysis. NMR Analysis of CGs. Proton NMR spectra of CGs, including 4− 7, were recorded on a 500 MHz Avance spectrometer (Bruker, Rheinstetten, Germany) equipped with an autosampler and an inverse triple-resonance probe (TXI, 5 mm). Spectra (solvent D2O) were collected at 298 K with 64 scans. Peaks arising from CGs were integrated using ACD/NMR Processor Academic edition software (Advanced Chemistry Development Inc., Toronto, ON, Canada). The concentration of CGs in each of sample was calculated using the equations Cf =

Vi × Ci Vf

(1)

N=

A n

(2)

CCGs =

NCGs × Cf NTMSP

(3)

where Cf is the final concentration of TMSP in sample, Vi is the initial volume of TMSP in the sample, Ci is the initial concentration of TMSP in the sample, Vf is the total volume of the sample, N is the normalized value of integrated peak of interest in 1H NMR spectra, A is the absolute value of integrated peak of interest in 1H NMR spectra, n is the number of nuclei in CGs, CCGs is the concentration of CGs in each sample, NCGs is the normalized value of the integrated peak of CGs in 1H NMR spectra, and NTMSP is the normalized value of the integrated peak of TMSP in 1H NMR spectra. Statistical Analysis. All analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA), and data are presented as the mean ± standard deviation (SD). Differences between mean values were evaluated using one-way analysis of variance (ANOVA) followed by a post hoc least significant difference test or an unpaired Student’s t test. Statistical significance was accepted at p < 0.05.

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RESULTS AND DISCUSSION Numerous potential health benefits have been associated with flaxseed consumption in the form of flaxseed-fortified foods, especially bakery food products and breakfast cereals. Such 9553

DOI: 10.1021/acs.jafc.6b03962 J. Agric. Food Chem. 2016, 64, 9551−9558

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Journal of Agricultural and Food Chemistry

flour (1251.4 ± 12.7 mAU), and bread (1142.9 ± 40.8 mAU). 1 prepared from flaxseed was added to various bakery products, including rye breads, graham buns, and muffins. 1 was analyzed immediately after baking by HPLC-DAD and found to withstand baking at 225 °C for 15 min in all bakery products. Even longer and hotter baking for 25 min at 250 °C did not decompose 1.40 1 content in the dough (725.3 ± 33.9 mAU) made from flaxseed meal fortified flour was significantly lower than that in flaxseed meal and its fortified samples. This could be caused by the sticky microstructure of dough, which is difficult to mill prior to extraction. Thus, lower extraction efficiency was observed in dough than for meal, flour, and bread as the latter products were dried and ground in a coffee mill prior to extraction. A similar matrix effect was observed by Muir and Westcott41 with a 73−75% recovery of 1 from baked flaxseed fortified goods. Gluten network formation during breadmaking could entrap flaxseed and interfere with 1 extraction, which was thought to be responsible for observed low 1 recovery. Consistently, 1 recovery from flaxseed-fortified macaroni was improved from 40−50 to 80−96% after papain digestion. 1 extraction efficiency was dependent upon the amount of papain added to hydrolyze protein in the gluten matrix. This finding supported the hypothesis that a flaxseed− gluten network entrapped 1.42 The stability of 1 in flaxseed meal fortified GF products was also investigated as a function of storage temperature (−18 °C, 4 °C, and RT) for up to 4 weeks. 1 content in all flaxseed meal fortified products during the processing of GF bread stored at 4 °C and RT was not significantly decreased within 2 weeks (Table 2). A significant decrease of 1 content was observed at the end of week 4, when dough was stored at 4 °C. No significant change in 1 content was observed in test samples until the end of week 4 for samples stored at −18 °C. Lowtemperature (−18 °C) storage could help to inhibit 1 decomposition through enzymatic or acidic hydrolysis of glycosidic bonds. Lower temperatures inhibit microbial growth and could, thereby, increase the stability of 1. 1 was reported to be stable in flaxseed-fortified bakery buns and breads within a storage times of up to 2 months at −25 °C.40 Hydrolysis of 1 glycosidic bonds would produce 2. For example, dried flaxseedfortified macaroni was stored for 32 weeks, but 2 was below detection limits regardless of treatment (0−20% ground whole flaxseed, 15% ground hull flaxseed, and 15% ground steamed whole flaxseed) and drying temperature (40−90 °C), indicating a good stability of 1 in this product.42 1 was also added to milk for nutritional enrichment of dairy products, and no 1 metabolites were detected in milk, yogurt, or cheese during high-temperature pasteurization, fermentation, or renneting. Also, 1 was stable against starter lactic acid bacteria and enzymes present in Edam cheese during ripening over 6 weeks at 9 °C and also stable in acidic yogurt during 21 days of storage at 4 °C. In whey-based drinks up to 75% of added 1 was recovered after storage for 6 months at 8 °C.43 CG Analysis. Methods for CG analysis can be generally separated into two categories: quantitation of cyanide liberated from CGs after enzymatic or chemical hydrolysis by colorimetric measurement and chromatographic analysis of intact CGs after extraction, including thin layer chromatography (TLC), reversed phase liquid chromatography (RPLC), and gas chromatography (GC). Silver nitrate titration, cyanide ion selective electrodes, colorimetry, and high-performance liquid chromatography with ultraviolet detection (HPLC-UV) have been used to quantitate CGs.44 HPLC-UV has the best

products have gained increasing popularity due to the nutritional components in flaxseed.12,18 Bioactive phytochemicals such as 1, orbitides, and unsaturated fatty acids of oleic acid, linoleic acid, and α-linolenic acid in flaxseed might impart both nutritional and nutraceutical values to foods. Effects of flaxseed incorporation in foods potentially include protection against tumors, blood lipid reduction, improvement in memory, slowed aging, and enhanced immune function.28,29 However, flaxseed lipid oxidation, especially of polyunsaturated fatty acids, serves as another problem during processing and storage of food products that contain flaxseed.12 1 Analysis. Since the first identification of 1 from defatted flaxseed meal by Bakke and Klosterman,30 flaxseed has been confirmed to be, by far, the richest dietary source of 1. An optimized process for extracting lignans from defatted flaxseed was patented31 using aqueous aliphatic alcohol solvents (55− 75%, v/v, methanol or ethanol). 1 was found in amounts of up to 20 mg/g defatted flaxseed. A methanol/water mixture (70/ 30, v/v) was employed for 1 extraction from flaxseed, resulting in a 91.3 ± 1.9% recovery of standard (rutin), which was not significantly different from that (94.9 ± 1.7%) obtained by using methanol/water (80/20, v/v).32 The extraction yield of 1 from flaxseed was improved by increasing the extraction temperature to 60 °C with different alcohol/water ratios for both aqueous methanol and ethanol solvents.33,34 In this study, extraction of 1 from flaxseed meal and samples collected during processing and storage of flaxseed meal fortified GF bread as listed in Table 2 was performed with MeOH aqueous solution (70%, v/v) at a temperature of 60 °C. Studies showed that in defatted flaxseed extracts 1 existed in polymeric form with ester linkages to 3-hydroxy-3-methylglutaric acid, 3 (Figure 1), and/ or to other phenolic compounds such as glycosides of pcoumaric acid and ferulic acid.35−37 Alkaline hydrolysis by 0.1 M sodium hydroxide was performed in this study to release 1 from the macromolecular complex.38,39 The integrated area of 1 chromatographic peak monitored by UV absorbance at 214 nm is proportional to 1 content in test samples. For control samples without flaxseed meal fortification, no 1 was detected (Figure 3). All flaxseed meal fortified samples contained 1, and the contents of 1 were similar in flaxseed meal (1224.8 ± 14.7 mAU), flaxseed meal fortified

Figure 3. HPLC-DAD chromatogram of 1 in flaxseed meal fortified GF bread with rutin as internal standard. 9554

DOI: 10.1021/acs.jafc.6b03962 J. Agric. Food Chem. 2016, 64, 9551−9558

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Journal of Agricultural and Food Chemistry

Figure 4. 1H NMR spectra of CGs in flaxseed meal with TMSP as internal standard: (A) week 0 at RT; (B) week 4 at RT. Spectrum in right corner is magnification of chemical shift between 1.0 and 1.9 ppm. Methyl proton peaks are labeled and colored in the structure (standard integration windows are referred to as bins): bin 1 (pink), bin 2 (green), bin 3 (blue), and bin 4 (red).

The CG content of flaxseed meal fortified samples was determined by 1H NMR as a function of storage time and temperature. Figure 4 shows 1H NMR spectra of flaxseed meal extracts with the internal standard TMSP (singlet signal, Int = 1) at a chemical shift of 0.00 ppm. CGs, including 4−7, were detected and identified by 1H NMR spectroscopy.17,24 Standard NMR integration windows are referred to as bins 1−4. In the

reproducibility and recovery rates, but sensitivity is not satisfactory due to low UV absorption by CGs.45 Thus, it is deemed to be of interest to develop a protocol that is sensitive and more reliable. In this study, analysis of CGs in flaxseed was first reported by using a proton NMR technique, which was demonstrated as a fast, accurate, and sensitive quantitation method. 9555

DOI: 10.1021/acs.jafc.6b03962 J. Agric. Food Chem. 2016, 64, 9551−9558

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Journal of Agricultural and Food Chemistry

Figure 5. Changes of CG content in (A) flaxseed meal and (B) its fortified GF flour as a function of storage time and temperature.

temperatures (−18, 4, and 22−23 °C) for up to 4 weeks were measured (Figure 5). In both samples on the day they were prepared, 4 and/or 5 were the predominant CGs with contents of 7.63 and 8.59 mM, respectively (week 0). This is in agreement with a previous study that showed 5 was the most abundant CG in flaxseed.24 During storage, flaxseed meal CG content decreased as a function of storage time. CG content was higher in flaxseed meal stored at −18 °C at the end of week 4 (Figure 5A) than that in the same products stored at warmer temperatures. This could be caused by enzymatic hydrolysis of CGs to HCN during storage due to endogenous β-glycosidase. Endogenous enzymatic activity decreased with lower storage temperature, resulting in higher CG content (Figure 5A). However, CG content in flaxseed meal fortified GF flour (Figure 5B) stored at different temperatures was stable over the storage time tested. This could be ascribed to the complex ingredients of flaxseed meal fortified GF flour, which may inhibit or inactivate the β-glycosidase.48 In addition, the introduction of other ingredients into flaxseed meal could decrease the enzyme-to-substrate ratio in flaxseed-fortified flour, resulting in decreased catalytic efficiency.49 Furthermore, water activity might be lower in flaxseed meal fortified GF flour, whereby enzymatic processes that hydrolyze CGs to HCN are

spectra, both triplet (1.03−1.08 ppm) and singlet (1.63−1.66 ppm) resonances labeled as bin 2 (in green) and bin 3 (in blue) were attributed to CH3 protons in the structure of 6 and/or 7, respectively, whereas two singlet resonances (1.66−1.71 ppm) in the spectra arose from CH3 protons (bin 4 in red) in the structure of 4 and/or 5. CG content in the collected samples was calculated using TMSP (bin 1 in pink) as a reference added to samples prior to extraction. CGs were not detected in control samples as no flaxseed meal was added. After baking, CGs were largely eliminated as neither CG nor cyanohydrin resonances were detected by 1H NMR. The effectiveness of different processing methods, including oven heating, single or repeated pelleting, autoclaving, and microwave roasting, was tested for reducing HCN released from flaxseed alone or in a mix with corn or other ingredients. Greater and prolonged exposure of flaxseed to a higher temperatures reduced HCN production potential.46 This is in agreement with studies of the effects of extrusion on cyanogenic compounds, measured as hydrocyanic acid, in full-fat flaxseed. Reduction of CGs was mainly dependent on barrel exit temperature, whereas both screw speed and feed rate had little or no effect.47 In this study, CGs were detected only in flaxseed meal and flaxseed-fortified flour. Here the CG concentrations of flaxseed meal and flaxseed meal fortified GF flour stored under different 9556

DOI: 10.1021/acs.jafc.6b03962 J. Agric. Food Chem. 2016, 64, 9551−9558

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Journal of Agricultural and Food Chemistry diminished.50 All of the mentioned factors might contribute to the higher CG content in flaxseed flour than in flaxseed meal. In conclusion, flaxseed is becoming more popular in the food industry due to its health-promoting properties. Flaxseed is mainly used for its oil, but flaxseed meal also has nutritional benefits. In this study, flaxseed meal was successfully incorporated in GF bakery bread. CGs were detected only in flaxseed meal and flaxseed meal fortified GF flour, with 4 and/ or 5 being observed as the primary CGs. Processing steps during GF bread production reduced and then eliminated CGs. CG content in flaxseed meal decreased over storage time up to 4 weeks under all storage temperatures, whereas CG content stayed constant in flaxseed meal fortified GF flour. However, 1 survived baking during GF bread production and was stable within the storage time tested (4 weeks) and not affected by storage temperature. These findings support the use of flaxseed meal in bakery products, as the health beneficial compounds can survive while the toxic compounds disappear. However, more research should be done to optimize the incorporation of flaxseed meal in GF bakery products.

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AUTHOR INFORMATION

Corresponding Authors

*(Y.Y.S.) Phone: (306) 966-5050. Fax: (306) 966-5015. Email: [email protected]. *(M.J.T.R.) Phone: (306) 966-5027. Fax: (306) 966-5015. Email: [email protected]. ORCID

Youn Young Shim: 0000-0002-5039-8219 Present Address

⊥ (J.L.) Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, No. 17 Qinghua Donglu, Haidian District, Beijing 100083, China.

Author Contributions ∥

Y.Y.S. and C.M.O. equally contributed to the study.

Funding

Financial support for the authors’ work was obtained from Agricultural Development Funds (20080205 and 20120146) of the Saskatchewan Ministry of Agriculture. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED CG, cyanogenic glycoside; GF, gluten-free; TMSP, 3(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt; RT, room temperature

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