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Interaction of Sorghum Tannins with Wheat Proteins and Effect on in Vitro Starch and Protein Digestibility in a Baked Product Matrix Kristen L. Dunn,†,⊥ Liyi Yang,†,⊥ Audrey Girard,†,‡ Scott Bean,§ and Joseph M. Awika*,†,‡ †

Department of Soil and Crop Sciences and ‡Department of Nutrition and Food Science, Texas A&M University, College Station, Texas 77843, United States § USDA-ARS Center for Grain and Animal Health Research, 1515 College Avenue, Manhattan, Kansas 66502, United States ABSTRACT: Carbohydrates contribute the most dietary calories, which makes starchy foods a logical target for modifying calorie intake. This study investigated the interaction of sorghum bran proanthocyanidins (PA) with proteins during wheat flour tortilla processing and impact on in vitro starch digestibility. Brans from wheat, white (low in phenols), brown (high PA), and black (high monomeric flavonoids) sorghum were used. Changes in phenolic profile, starch, and proteins were evaluated. Dough mixing drastically decreased extractable PA (61−72%) but not monomeric phenolics; higher MW PA decreased the most. The high PA bran dough produced the highest insoluble proteins (460 vs 330 mg/g protein for other sorghum brans) at 25% baker’s substitution. The high PA bran tortillas also had higher slow digesting starch and lower rapidly digesting starch than all other bran treatments. Significant sorghum PA-gluten interactions occur during dough mixing that may slow starch digestibility in the baked products. KEYWORDS: condensed tannins, gluten, starch digestibility, wheat, sorghum

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INTRODUCTION Excess calorie intake and associated consequences, for example, obesity, diabetes, and other diseases, are growing global problems and are especially prominent in the United States. Carbohydrates make up a large part of the American diet by contributing about 55% of the total calories;1 a majority of these calories are derived directly from starch. Therefore, starch-based foods such as baked goods and various snacks may serve as a good target to improve caloric profile of foods. Growing consumer interest in natural, high fiber, and other appealing food labels provides opportunity for innovation with natural bioactive compounds. Proanthocyanidins (condensed tannins, PA) are polymeric flavonoid molecules that vary widely in structure and molecular weight depending on botanical origin. PA are especially well investigated for their ability to bind proteins and consequences of such interactions, for example, astringency and nutrient bioavailability.2 PA also have potential health benefits as free radical scavengers and chemoprotective agents, among others.3−5 Certain sorghum varieties are high in the PA; these tannin sorghums have long been known to have reduced feed efficiency and produce negative impact on weight gain of monogastric animals (rats, pigs, poultry, etc.).6−9 The reduced feed efficiency has been largely attributed to PA−protein complexation.10,11 Because of their ability to bind nutrients, the sorghum PA may have a potential as ingredients to lower calorie content of foods.12 Even though the mechanisms of PA binding to proteins and consequences are well established, data are relatively scarce on the ability of the PA to bind to other food macromolecules. We recently demonstrated that under specific conditions, the PA derived from sorghum can interact with starch to form nondigestible complexes.13 The efficiency of the starch−PA complexation depends largely on the degree of polymerization © 2015 American Chemical Society

of the PA as well as the starch composition; higher molecular weight PA and amylose bind most efficiently to each other.14 Thus, the evidence suggests that PA can potentially be used as natural food components to reduce starch digestibility. However, the above data were obtained in pure starch−PA systems. It is not clear how the presence of other macromolecules in a typical food matrix impacts starch−PA interactions. For example, PA affinity for proline-rich proteins with open hydrophobic domains is relatively high10,15 and is thus likely to impede starch−PA interactions. In fact, in contrast to our observations using pure starch model systems,13,14 Lemlioglu-Austin et al.16 and Mkandawire et al.17 reported that the presence of tannins did not significantly affect the rate of starch digestibility in whole grain sorghum porridges; only when the porridges were cooked in extracts relatively high in polyphenols (up to 50% starch weight) were significant reductions in starch digestibility observed. The presence of tannins, however, generally slowed the rate of starch hydrolysis by amylases in sorghum endosperm based porridge.18 It is possible that the proteins in sorghum endosperm prevented the PA interaction with starch. Wheat is the most important cereal grain for food in the West. Wheat proteins are high in proline and during dough mixing form an extensive gluten network with large hydrophobic domains.19 Thus, wheat-based products provide an excellent opportunity to investigate how protein interaction with PA may impact starch digestibility during food processing. Tortillas are an increasingly important baked product; they are popular because of their convenience and versatility as wraps Received: Revised: Accepted: Published: 1234

August 28, 2014 December 4, 2014 January 9, 2015 January 9, 2015 DOI: 10.1021/jf504112z J. Agric. Food Chem. 2015, 63, 1234−1241

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Journal of Agricultural and Food Chemistry for various foods and condiments.20 Wheat flour tortillas are also a relatively simple food matrix with a short processing time, which thus presents an ideal model for investigating potential impact of gluten-containing food matrix on PA−starch interactions. In this work, we use a tannin sorghum bran to investigate how the presence of PA influences in vitro starch digestibility in a food matrix containing wheat proteins by using wheat flour tortillas as a model. Nontannin sorghum brans with different phenolic profiles and wheat bran were also used to control for effect of other phenolic components in the bran. Interaction of the polyphenols with proteins and the fate of the PA during processing were also investigated.

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Sampling. Samples were retained from four different processing stages: dough, hot-pressed but not baked, immediately after baking (day 0), and after 14 days storage at room temperature. Dough, pressed, and day 0 samples were flash frozen in liquid nitrogen and stored at −20 °C until use. Day 14 samples were frozen at −20 °C after 14 days of room temperature storage. Frozen samples (immediately out of the freezer) were ground in a coffee grinder to reduce the particle size, then dried for 2 h in a hot air oven at 45 °C and further ground with a UDY cyclone sample mill (UDY Corp., Fort Collins, CO) to pass through 0.1 mm screen. For phenolic composition analysis (described below), dried, ground samples were defatted by extracting in hexanes (3:1 hexanes:sample, v/w) for 2 h with shaking at room temperature. Samples were centrifuged, the supernatant was discarded, and the extraction repeated. Defatted samples were air-dried in a fume hood until all remaining hexanes had evaporated. The dried, ground, defatted samples were then stored at −20 °C until analysis. Residual moisture content of the dried samples was determined by a moisture analyzer (Model HB43-S, MettlerToledo, Columbus, OH). Phenolic Composition. Total Phenolic Content. Total phenols were measured according to the Folin−Ciocalteu method.24 Samples were extracted in 1% HCl in methanol with shaking for 2 h. Aliquots were diluted with distilled water and reacted with Folin−Ciocalteu reagent (0.4 mL) and ethanolamine (0.9 mL) for 20 min at room temperature. Absorbance at 600 nm was read on a UV−vis spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD). Results were expressed as mg of gallic acid equivalents (GAE)/g based on a standard curve prepared with serial dilutions of gallic acid in methanol. High-Performance Liquid Chromatography (HPLC) Analysis. Phenolics were profiled using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA). Phenolic acids and 3-deoxyanthocyanins were analyzed following the method described by Awika et al.5 at wavelengths of 280, 330, 360, and 480 nm using a diode array detector. A reverse-phase Luna 5u C182 column (150 mm × 4.6 mm, 5 μm, Phenomenex, Torrence, CA) was used. Samples were extracted in 1% HCl in methanol (0.5 g in 5 mL) for 2 h and filtered (0.45 μm, nylon) before injection (10 μL). The mobile phase was composed of 1% formic acid in water (A) and 1% formic acid in acetonitrile (B). The elution gradient (B) over the 40 min run time was: 0−3 min, 10% isocratic; 3−5 min, 10−18%; 5−10 min, 18−20%; 10−23 min, 20− 26%; 23−25 min, 26−18%; 25−28 min, 28−40%; 28−30 min, 40− 60%; 30−32 min, 60% isocratic; 32−34 min, 60−10%; 34−40 min, 10% isocratic. Flow rate was 1 mL/min. PA were analyzed and separated based on degree of polymerization according to Langer et al.25 A fluorescence detector with excitation and emission wavelengths of 230 and 321 nm, respectively, and a normal phase Develosil Diol column (250 mm × 4.6 mm, 5 μm particle size, Phenomenex, Torrence, CA) were used. Samples were extracted in 2% acetic acid in 70% aqueous acetone (1 g in 5 mL) for 2 h and filtered (0.45 μm, nylon) before injection (10 μL). The elution gradient (B) over the 83 min run time was: 0−3 min, 7% isocratic; 3−57 min, 7− 37.6%; 57−60 min, 37.6−100%; 60−67 min, 100% isocratic; 67−73 min, 100−7%; 73−83 min, 7% isocratic. Flow rate was 0.6 mL/min. Authentic standards (catechin, procyanidins B1 and C1) were used to quantify PA with a degree of polymerization (DP) of 1−3. Quantitative data for PA with a DP greater than or equal to four were based on procyanidin C1 (DP 3) peak response. Results were corrected for molecular weight according to the DP. Starch Analysis. In vitro starch digestibility was determined on fully baked tortilla samples. The dried, ground samples were removed from the freezer and allowed to equilibrate at room temperature for 1 h before starch analysis. Total Starch (TS). The K-TSTA assay kit (determination of total starch content of samples containing resistant starch, but no D-glucose or maltodextrins) from Megazyme (Megazyme International, Wicklow, Ireland) was used to determine TS (AACC International method 76−13.01). The sample (∼100 mg) and 80% (v/v) ethanol (0.2 mL) were added to a glass test tube and mixed on a vortex mixer. A magnetic stirring rod was added to each tube, and the tubes were

MATERIALS AND METHODS

Materials. Three different types of sorghum bran were chosen for their diversity in polyphenolic content and composition. White sorghum (ATxArg-1/RTx436, TAMU, College Station, TX, 2008) was decorticated in a PRL mini-dehuller (Nutama Machine Company, Saskatoon, Canada) and separated (Model 6DT4−1, KICE Industries Inc., Wichita, KS) to yield 12% bran. Brown (high tannin) and black sorghum brans were commercial samples (Nu Life Market, Scott City, KS); these two samples were from similar cultivars we previously used.13 The white sorghum was chosen for its low flavonoid and extractable phenol content; the black sorghum is high in monomeric flavonoids, especially 3-deoxyanthocyanins, and the brown sorghum has PA as the dominant polyphenols. Wheat bran (ADM, Decatur, IL) was also used for comparison. The wheat and white sorghum brans were ground using a WonderMill (The WonderMill Company, Pocatello, ID) to reduce the particle size to closely match to the brown and black sorghum brans. Catechin and procyanidin B1 were purchased from Sigma (St Louis, MO); procyanidin B1 and C1 were from Extrasynthese (Genay, France). Tortilla Formulation and Processing. Base wheat flour was substituted with the brans at 10, 15, and 25% (Baker’s, i.e., based on flour weight). The base tortilla flour was made from a 1:1 mixture of high gluten bread flour (C. H. Guenther and Son, Inc., San Antonio, TX) and pastry flour (Cargill Inc., Minneapolis, MN). Control tortillas were made with no bran added. Each batch of tortillas was prepared using 1000 g of flour−bran mixture. To compensate for diluting effect of brans on gluten, 12.0, 19.0, and 31.0 g of vital wheat gluten (Cargill Inc., Minneapolis, MN) were added to the 10, 15, and 25% bran substitution levels, respectively. These values were calculated based on the gluten content of the refined flour blend, which was determined by AACC International method 38−10.01.21 The rest of the tortilla formulation was described previously.22,23 Briefly, the tortilla formulation included 15.0 g of table salt, 5.0 g of sodium stearoyl lactylate, 4.0 g of potassium sorbate (Caravan Ingredients, Inc., Lenexa, KS), 6.0 g of sodium propionate (Niacet Corporation, Niagara Falls, NY), 6.0 g of baking soda (Church and Dwight Co., Inc., Ewing, NJ), 5.8 g of sodium aluminum sulfate (Budenheim USA, Columbus, OH), 3.3 g of encapsulated fumaric acid (Balchem Inc., New Hampton, NY), 60.0 g of all-purpose shortening (Cargill Inc., Minneapolis, MN), and distilled water. Dough moisture content was adjusted to account for increased water absorption of the brans as previously described.20 After mixing, the dough was allowed to rest for 5 min in a proofing chamber (National Manufacturing Co., Lincoln, NE) at 32 °C and 60−70% relative humidity. The dough was then divided and rounded (Duchess Divider and Rounder, Bakery Equipment and Service Co., San Antonio, TX) into 36 pieces, approximately 30 g each. The dough balls were allowed to rest for another 10 min in the proofing chamber under the conditions listed above. Dough balls were hot-pressed (204 °C, 1150 psi, 1.4 s) and baked in a three-tier gas oven (Lawrence Equipment, El Monte, CA) for approximately 40 s at 193−204 °C. Tortillas were cooled to room temperature and packaged in moistureproof bags for storage. 1235

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Journal of Agricultural and Food Chemistry placed in an ice water bath before 2 M KOH (2 mL) was added to each tube with stirring. The tubes were removed after 20 min, and 1.2 M sodium acetate buffer (8 mL) was added to each tube with stirring. Thermostable α-amylase (0.1 mL; 3,000 U/mL) and amyloglucosidase (0.1 mL; 3,300 U/mL) (Megazyme) were added to each tube. The tubes were incubated in a 50 °C water bath for 30 min with intermittent stirring on a vortex mixer. Samples were diluted to 100 mL with distilled water, centrifuged, and the supernatant was retained for analysis. The K-GLUC (GOPOD format) assay kit from Megazyme was used for the determination of D-glucose using glucose oxidase based on absorbance at 510 nm as read on the UV−vis spectrophotometer. Rapidly Digestible Starch (RDS), Slowly Digestible Starch (SDS), and Resistant Starch (RS). RDS and SDS were determined using the colorimetric version of the procedure described by Englyst et al.26 as modified by Barros et al.13 Dried, ground sample (0.20 g) was used for the analysis. The enzyme mixture was prepared from α-amylase (A3176, Sigma-Aldrich, St. Louis, MO) and purified amyloglucosidase (Cat. No. E-AMGDF, Megazyme International) at concentrations of ∼300 U/mL and ∼95 U/mL, respectively. The K-GLUC (GOPOD format) assay kit from Megazyme was used for the determination of Dglucose using glucose oxidase based on absorbance at 510 nm as read on a UV−vis spectrophotometer (Shimadzu Scientific Instruments). RDS was determined by stopping digestion after 20 min, whereas SDS was based on 120 min of digestion. The K-RSTAR assay kit from Megazyme was used to determine RS (AACC Int. Method 32− 40.01,21). Results were expressed on a TS basis. Protein Analysis. Total Protein Determination. Nitrogen content of dried samples was determined by combustion using a LECO instrument (LECO Corporation, St. Joseph, MI) and converted to total protein by a factor of 5.7. Insoluble Protein (IP) Determination. The soluble protein fraction was extracted as described by Schober et al.27 Samples (100 mg) were extracted three times in 1 mL of 50% 1-propanol for 5 min. This solvent systems was selected because it is known to preserve PA− protein complexes.28,29 The samples were centrifuged after each extraction. The supernatant from each extraction was discarded. The sample remaining after extraction contained the IP fraction. The pellet was freeze-dried, and its protein content was determined by combustion as mentioned previously. In Vitro Protein Digestibility. Protein digestibility was measured using the in vitro pepsin digestibility assay of Mertz et al.30 except that protein was quantified using nitrogen combustion analysis as described above. Statistical Analysis. All procedures were performed in duplicate. Means and standard deviations were determined using Microsoft Office Excel 2010 (Microsoft Corporation, Redmond, WA). Statistical Analysis Software (SAS) version 9.3 (SAS Institute, Cary, NC) was used for data analysis. Differences in treatments were determined at the 5% significance level (α = 0.05) using the general linear model (GLM) and least significant difference (LSD) for mean separation.

Figure 1. Effect of processing step and bran source on extractable phenols (mg GAE/g) in wheat flour tortillas processing and storage. Bars with the same letter within the same bran treatment category are not statistically different (p < 0.05, LSD).

Abdel-Aal and Rabalski33 observed an increase in free phenolic acids in flat bread dough when prepared with whole grain einkorn wheat flour. For black sorghum bran treatment, dough formation did not affect extractable phenol content except at 10% bran substitution, where the net change was modest (Figure 1). In general, it appears that the monomeric 3-deoxyanthocyanidins, which are the dominant extractable phenolics in the black sorghum bran, did not significantly interact with dough components. On the other hand, dough formation dramatically decreased the total phenol content in brown sorghum bran treatments (Figure 1). The brown sorghum bran treatments exhibited 58, 69, and 78% decrease in extractable phenols at 25, 15, and 10% substitutions, respectively, in the dough compared to the dry mix. The large decrease in extractable phenols in dough prepared with brown sorghum bran compared to the other bran treatments suggests that the phenols present in the brown sorghum bran interacted with proteins or starch upon dough formation in a fundamentally different way than the phenols in the other bran treatments. As previously stated, the brown sorghum bran contained the polymeric PA as the dominant polyphenols, which likely accounts for its different behavior. The PA likely formed insoluble complexes with gluten during dough formation. Interestingly, the changes in extractable phenols in the brown sorghum bran treatments were minimal in subsequent steps

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RESULTS AND DISCUSSION Effect of Processing on Total (Folin) Phenolic Content. The content of phenols present in the dry ingredient mix (dry mix) was determined in a mixture of all dry ingredients without the shortening, and final values were calculated to account for the shortening used in the formulations. As expected, the samples with brown and black sorghum brans had much higher phenols in the dry mix than those made with wheat and white sorghum brans (Figure 1). In the treatments with wheat and white sorghum bran, extractable phenol content initially increased upon dough formation and then decreased with processing and storage (p < 0.05) (Figure 1). This may be attributed to increased extractability of phenols from the dough as compared to the bran and flour in the dry mix. Phenolic acids are the predominant phenolic compounds in wheat and white sorghum but are commonly bound to the cell wall.31,32 1236

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respectively. In agreement with the Folin phenolic data (Figure 1), the PA, which are the dominant polyphenols in the brown sorghum bran, dramatically declined during dough mixing as determined by normal-phase HPLC (Table 1). The decline in extractable PA in the dough was in the range of 61−72% compared to dry mixes, for the various brown bran substitutions, with the highest decline observed at the lowest bran substitution level. This suggests that the PA in the brown sorghum bran interacted with gluten during dough mixing and thus became less extractable. We were unable to find any literature reports on interactions of PA with gluten. However, Zhang et al.35 recently reported that a tannic acid of unspecified source and structure reduced free amino groups in dough when added to flour at 10−30 μg/ g. This suggests that the tannic acid participated in cross-linking of the gluten network. It is possible that the PA would participate in similar interactions. Interestingly, after dough mixing, further changes in extractable PA content were relatively small (Table 1), which suggests that most of the “available” or reactive PA constituents in the bran interacted with dough components during mixing. We had previously demonstrated that the PA are relatively stable to thermal processing conditions similar to those used in this study;13,14 thus, changes during tortilla processing are largely attributable to chemical interactions with other components and not thermal degradation. Analysis of the PA MW profile during tortilla processing also demonstrated that the most dramatic changes occurred at the dough mixing stage (Figure 3). The higher MW PA decreased the most during dough mixing, which significantly altered the extractable PA MW profile. This suggests specific PA−protein interactions during dough mixing; high MW PA are known to interact more readily with proteins to form insoluble complexes.10,15 Furthermore, even though the PA can adhere to raw starch and become unextractable,36 such interaction is limited, nonspecific, and does not affect the MW distribution of the PA.13 The fact that extractable PA profile remained relatively similar after dough formation (Figure 3) suggests that the protein preferentially bound the high MW PA in the dough, which limited opportunities for PA to interact further during the baking process. Because the brown sorghum bran itself contained significant amount of proteins, it was important to determine the potential contribution of inherent interactions of the bran PA with the sorghum bran proteins during the dough mixing step to the observed changes in PA profile. The sorghum bran was thoroughly mixed with water (1:0.6, bran:water) and allowed to rest for 2 h at room temperature. The mixture was dried in a convection oven at 45 °C and subjected to PA profiling as described in the Materials and Methods. The PA profile of the wet-mixed sorghum bran was virtually identical to the dry extracted bran (Figures 3 and 4). This suggests that the sorghum bran proteins were not responsible for the dramatic changes in MW profile of the brown sorghum bran dough (Figure 3). More importantly, this provides indirect evidence that the wheat proteins (specifically gluten) are the ones interacting with the PA in the dough. Impact of the above observations on starch and protein properties may provide additional insight. In Vitro Starch Digestibility. Starch digestibility information is most relevant in final product, thus was only measured in tortillas. None of the bran treatments increased resistant starch content in tortillas relative to refined wheat flour control, and

after dough formation (Figure 1). This indicates that the most important chemical interactions that impact the phenolics in the brown sorghum largely occur in the dough. By contrast, significant decreases in extractable phenols were observed in the black sorghum bran treatments postdough formation stage. Effect of Tortilla Processing on Bran Polyphenol Profile. In general, the phenolic profiles of the black and white sorghum brans were as previously reported,34 with phenolic acid derivatives dominant in the white sorghum bran treatments and 3-deoxyanthocyanins dominant in the black sorghum bran treatments. The profiles generally did not change during processing, and individual phenolic contents were predictable based on the Folin phenol content trends. An example of 3-deoxyanthocyanin HPLC profile of black sorghum bran treatments is shown in Figure 2. The overall conclusion is

Figure 2. HPLC profile of 3-deoxyanthocyanins in black sorghum bran and dough and tortilla (day 0) made with 25% (baker’s) black sorghum bran recorded at 480 nm. The dough and tortilla chromatograms are offset by 100%. Compounds: a = luteolinidin, b = 7-O-methyl luteolinidin, c = 7-O-methyl apigeninidin, d = 5,7-Odimethyl luteolinidin.

that nonspecific changes in extractability of the monomeric sorghum phenolics are what is impacting the Folin phenols data for white and black sorghum bran treatments and not structural changes (e.g., via thermal degradation) or specific interactions of some of the phenolics in these brans with other ingredients. The brown sorghum bran treatments, however, presented interesting observations during processing. The brown sorghum bran had a PA profile largely dominated by the high molecular weight polymers (Figure 3), which confirms previous reports.13 The PA contents of the dry mixes were 0.76, 1.13, and 1.87 mg/g for the 10, 15, and 25% bran substitutions, 1237

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Figure 3. Effect of tortilla processing step on extractable sorghum PA profile. Profiles were obtained using normal-phase HPLC with fluorescence detection. Numbers denote degree of polymerization; P = polymers with DP > 10. Profiles of tortilla processing steps offset by 15%.

Table 1. Extractable PA in Brown Sorghum Bran Substituted Sample at Various Steps of Tortilla Processing and Storage Relative to Starting Materiala bran substitution (baker’s %)

dry mix

dough

pressed

day 0 tortilla

day 14 tortilla

10% % change from preceding step 15% % change from preceding step 25% % change from preceding step

757 ± 12 a

213 ± 13 b −71.9 336 ± 33 b −70.3 721 ± 102 b −61.4

207 ± 5.0 b −2.7 296 ± 12 b,c −11.8 520 ± 25 c −27.9

191 ± 15 b,c −7.8 304 ± 7.0 b 2.6 553 ± 24 c 6.2

165 ± 6.2 c −13.7 250 ± 2.3 c −17.8 407 ± 10 c −26.3

1132 ± 18 a 1868 ± 30 a

Values are summation of various MW substituents (see Figure 3 for profile), expressed as mean ± standard deviation, μg/g (db), as determined by normal-phase HPLC. Same letter within row not statistically different (p < 0.05, LSD). a

likely precluded starch−PA interactions during baking. This further supports the idea that the gluten proteins are the likely components interacting with the PA in the dough. Contrary to the resistant starch data, significant differences were observed for RDS and the SDS in the tortillas with different bran treatments at 25% substitution level (Table 2). The brown sorghum bran treatment had (as percentage of total starch) the lowest RDS (81.2%) and highest SDS (20.9%) (p < 0.05) among all treatments. The rest of the bran treatments were not significantly different from each other or control for RDS (86.1−89.0%) and SDS content (12.2−15.5%) (Table 2). This observation is interesting and somewhat unexpected. We can only hypothesize that the PA likely formed cross-linked networks with gluten during mixing in a way that interferes with amylase enzyme access to starch, likely by physically shielding and slowing, but not blocking, enzyme degradation of starch. Even though the PA can also interfere with starch digestion via enzyme inhibition, the levels used in this study were too low to produce such effect.17 The rate and extent of digestion of starch in the small intestine has important implications on glycemic response and calorie impact of starch. RDS causes a rapid peak in blood glucose level as compared to SDS, which takes longer to digest and releases glucose over a prolonged amount of time.37 Foods that create a moderate glucose response have been shown to

Figure 4. PA profile of brown sorghum bran after mixing with water to imitate dough mixing. Bran was blended with water (0.6 g H2O/g) and allowed to rest for 2 h before drying in the oven at 45 °C; this was to determine if the PA interaction with the sorghum proteins in the bran contributed to the altered profile observed in the dough (Figure 3). Extraction and analysis followed as described in the Materials and Methods.

all values were below 1% (data not shown). This was contrary to our previous observations using PA and starch in pure systems, where, depending on thermal treatment and starch composition, RS was increased by up to 50% due to interactions with sorghum PA.14 Thus, the extensive interaction or “binding” of PA observed in the dough (Table 1; Figure 3) 1238

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Table 2. In Vitro Starch and Protein Digestibility of Fresh Tortillas Made with 25% (Baker’s) Substitutions of Wheat and Sorghum Bransa treatment control wheat bran white sorghum bran brown sorghum bran black sorghum bran

rapidly digestible starch (%) 87.9 87.3 86.1 81.2 89.0

± ± ± ± ±

1.23 1.39 0.01 1.29 3.44

a a a b a

slowly digestible starch (%) 12.1 14.1 15.5 20.9 12.3

± ± ± ± ±

1.64 2.39 1.60 2.25 0.68

b b b a b

protein content (%) 12.4 14.8 13.1 13.3 13.5

± ± ± ± ±

0.01 0.23 0.35 0.21 0.04

c a b b b

in vitro protein digestibility (%) 93.3 85.3 86.8 82.8 83.5

± ± ± ± ±

0.37 0.29 0.43 0.24 0.34

a b b c c

a

Data reported for day 0 tortillas, all values reported on db. RDS and SDS reported on TS basis. TS and RS were determined independently of RDS and SDS; thus, values may not add up to 100%. All RS values were less than 1% and thus are not reported. Same letter within column not statistically different (p < 0.05, LSD).

Figure 5. Effect of bran source on IP (% of total protein) content of dough, pressed, day 0, and day 14 tortillas substituted at 25%. Bars with the same letter within the processing step are not statistically different (p < 0.05, LSD).

help manage blood glucose levels38 and may contribute to satiety.39,40 Thus, the data suggest that sorghum PA may provide a mechanism to modify enzyme degradation kinetics of starch in wheat-based products. However, the PA content of the brown sorghum bran used in this study was relatively low (8.5 mg/g); this may have partly accounted for the less than “dramatic” effect of sorghum PA on starch digestibility in the tortilla matrix. The tannin content is known to affect the feed efficiency of sorghum in vivo, with a minimum tannin threshold needed to produce an effect.41 In fact, we did not observe any significant difference in starch digestibility among bran substitutions at 15% and 10% levels (data not shown). Thus, it is likely that with a higher level of available PA in the dough formulation, the effect of sorghum PA on starch digestibility would be more significant. Additional work will use purified PA fractions to uncover specific types of interactions of the PA with wheat proteins and how starch digestibility is impacted. Protein Extractability and in Vitro Digestibility. On the basis of observations for starch digestibility, for protein extractability (in 50% 1-propanol) and digestibility (based on in vitro pepsin assay30), only the 25% bran treatments were compared. Protein content of all sorghum bran treatments were similar (Table 2). IP content (as a percent of total proteins) of wheat bran dough was similar to the control but different (p < 0.05) from all the sorghum bran treatments (Figure 5).42 The IP contents of black and white sorghum bran doughs were significantly lower (average 330 mg/g protein) than the wheat bran treatment and control (average 390 mg/g protein) (Figure

5). This may have been due to inherent differences in IP composition of sorghum and wheat brans. Interestingly, the brown sorghum bran dough had significantly higher IP content (456 mg/g protein) than all other treatments. This further suggests that the PA in brown sorghum bran bound to the wheat proteins during dough mixing and formed insoluble complexes, which thus agrees with our hypothesis that the drop in higher MW PA extractability was due to PA−protein complexation. The fact that the brown sorghum bran dough had proportionately almost 40% higher IP than the other sorghum bran doughs is particularly revealing given that only 1.87 mg extractable PA/g was present in the brown sorghum bran dry formulation at 25% substitution (Table 1). This suggests a rather significant binding capacity of the sorghum PA with the wheat proteins. On the basis of the fact that approximately 1.15 mg PA/g became unextractable at the dough mixing stage (Table 1), we assume this is the amount of PA that complexed with the proteins. If we further assume that the brown sorghum bran dough had similar protein composition as the other sorghum bran treatments, we can estimate that 1.15 mg of PA resulted in the formation of 126 mg of insoluble protein complexes. Furthermore, it is safe to assume that a portion of the PA complexed with native wheat IP; thus, the amount of PA that produced the 126 mg of “new” IP is likely less than 1.15 mg. This suggests a relatively high binding ratio of sorghum PA with the wheat proteins compared to those reported for other protein sources.3,28 1239

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The large differences in IP between the brown sorghum bran treatment and the other sorghum bran treatments indicate that the PA in the brown sorghum bran were involved in crosslinking with the gluten proteins to form insoluble complexes quantified as IP at the dough mixing stage. This is consistent with the dramatic decline in PA in the brown bran dough previously discussed (Table 1). The fact that the black bran treatment, which had higher Folin phenol content than all other brans and did not differ from white sorghum bran treatment, indicates that the monomeric polyphenols are not involved in protein cross-linking in the dough. Thus, it is apparent that the proline-rich gluten proteins have high affinity for, and readily interact with, the sorghum PA to form insoluble complexes. For all treatments, IP increased with thermal processing (pressing and baking), with the largest increase observed after baking (Figure 5). The changes in IP during 14 day storage was minimal. Increase in IP after baking was expected; heat is known to reduce solubility of wheat gluten proteins, especially glutenins.43 Tortillas prepared with brown sorghum bran also had the highest amount of IP after baking (58.4%) and storage for 14 days (60.1%), but were statistically similar to wheat and white sorghum bran treatments (p < 0.05) (Figure 5). The day 0 and 14 tortilla samples prepared with white and black sorghum bran were statistically similar to the wheat bran and control (p < 0.05). Sorghum proteins, specifically kafirins, have been shown to cross-link upon heat treatment and become insoluble.44,45 This may have contributed to the larger increase in IP of white and black sorghum bran treatments (23 and 19%, respectively) after baking as compared to the control and wheat bran treatment (14 and 16%, respectively). The increase in IP of brown sorghum bran after baking (14%) was not as large as for the other sorghum bran treatments. Some of the proteins that would contribute to this effect due to cross-linking upon heat treatment were likely already interacted with the PA and became insoluble in the dough formation step. In vitro protein digestibility of tortillas was lowest for the brown sorghum bran treatment but not significantly different from the black sorghum treatment (Table 2). The control proteins had the highest (p < 0.05) digestibility, followed by wheat and white sorghum bran treatments. The evidence points to a potential impact of the PA on protein digestibility in the tortillas, but this is not conclusive based on the data. In general, sorghum tannins are known to reduce protein digestibility, but as previously stated, evidence indicates that a minimum amount of the tannins in a matrix is required to produce this effect.12,41,46 In this study, only 2.1 mg/g PA was present in the 25% brown sorghum bran dry formulation; this may have been too low to produce a practically significant effect. The overall data suggest that the interaction of sorghum PA with wheat dough proteins is significant and results in the formation of insoluble complexes. The monomeric polyphenols present in the black and white sorghums do not appear to interact much with gluten. Even more important was the fact that SDS was significantly increased, and RDS decreased, in tortillas made with the sorghum bran with the PA. This suggests that the sorghum PA have a potential as ingredients that can be used to beneficially modify starch digestibility in wheat-based baked foods for populations where excess calorie intake is a problem. We suspect that higher levels or more pure PA inclusion in dough formulation will have a more dramatic effect on starch digestibility profile.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 979-845-2985. Fax: +1 979-845-0456. Present Address ⊥

K.L.D., Pecan Deluxe, 2570 Lone Star Drive, Dallas, Texas 75212, United States. L.Y., Kellogg Company, 2 Hamblin Avenue, Battle Creek, Michigan 49017, United States.

Notes

Names are necessary to report factually on available data; however, the U.S. Department of Agriculture neither guarantees nor warrants the standard of the product, and use of the name by the U.S. Department of Agriculture implies no approval of the product to the exclusion of others that may also be suitable. The authors declare no competing financial interest.

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