Article pubs.acs.org/JAFC
Structure of Waxy Maize Starch Hydrolyzed by Maltogenic α‑Amylase in Relation to Its Retrogradation Navneet Grewal,† Jon Faubion,† Guohua Feng,§ Rhett C. Kaufman,# Jeff D. Wilson,# and Yong-Cheng Shi*,† †
Department of Grain Science and Industry, Shellenberger Hall, Kansas State University, Manhattan, Kansas 66506, United States Corbion, 7905 Quivira Road, Lenexa, Kansas 66215, United States # Center for Grain and Animal Health Research, Agricultural Research Service, U.S. Department of Agriculture, 1515 College Avenue, Manhattan, Kansas 66502, United States §
ABSTRACT: Maltogenic α-amylase is widely used as an antistaling agent in bakery foods. The objective of this study was to determine the degree of hydrolysis (DH) and starch structure after maltogenic amylase treatments in relation to its retrogradation. Waxy maize starch was cooked and hydrolyzed to different degrees by a maltogenic amylase. High-performance anion-exchange chromatography and size exclusion chromatography were used to determine saccharides formed and the molecular weight (Mw) distributions of the residual starch structure, respectively. Chain length (CL) distributions of debranched starch samples were further related to amylopectin (AP) retrogradation. Differential scanning calorimetry (DSC) results showed the complete inhibition of retrogradation when starches were hydrolyzed to >20% DH. Mw and CL distributions of residual AP structure indicated that with an increase in %DH, a higher proportion of unit chains with degree of polymerization (DP) ≤9 and a lower proportion of unit chains with DP ≥17 were formed. A higher proportion of short outer AP chains that cannot participate in the formation of double helices supports the decrease in and eventual inhibition of retrogradation observed with the increase in %DH. These results suggest that the maltogenic amylase could play a powerful role in inhibiting the staling of baked products even at limited starch hydrolysis. KEYWORDS: maltogenic amylase, degree of hydrolysis, molecular size distribution, chain length distribution, retrogradation
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starch granules remnants12 are suggested as reasons for bread firming. Maltogenic α-amylase is an antistaling enzyme that is increasingly used in bakery products to improve their shelf life.3,4,11,16 This enzyme is reported to act by shortening the AP side chains, thus preventing AP recrystallization and hindering water immobilization.4,17 This enzyme exhibits both an endo and an exoaction pattern, with a high degree of multiple attack action and with endoaction increasing with increases in temperature.18,19 During baking, maltogenic α-amylase starts to act on starch granules when they begin to gelatinize, thus increasing their flexibility during storage.17 The resulting bread has a softer and more elastic crumb with an extended shelf life. Mode of enzyme action and enzyme activity level play important roles in determining AP molecular structure. A higher degree of multiple attack action and higher enzyme activity levels used for porcine pancreatic α-amylase and Bacillus stearothermophilus maltogenic α-amylase led to a 50% reduction in average chain length of outer AP chains, thus affecting AP retrogradation to a greater extent. In contrast, the endoaction of Bacillus subtilis and Aspergillus oryzae α-amylases, which are used at low enzyme activity levels, had only a limited effect on AP side-chain distribution.20
INTRODUCTION Bread staling1−3 is one of biggest challenges faced by the baking industry because it leads to substantial economic losses. Starch undergoes structural changes both during and after the baking process that greatly determine the quality of the final product. During cooling and storage of baked products, starch retrogrades. This leads to increased crumb firmness. Longterm firmness of bread is due to the recrystallization of the gelatinized amylopectin (AP) network during storage, which results in increased crumb firmness and decreased crumb resilience,4,5 thus limiting shelf life. Starch granules are partially crystalline in nature and consist of amorphous and crystalline lamella within a radial arrangement of AP clusters. Amorphous lamellae contain a highly branched region, and crystalline lamellae consist of external AP chains (A chains and partial B chains) in the form of double helices.6,7 AP retrogradation depends on exterior chain length, with less retrogradation positively correlated with shorter chains.8,9 Several antistaling amylases are used in the baking industry to prevent the formation of a recrystallized AP network by shortening AP side chains so as to prevent the formation of double helices.5,10,11 Production of low molecular weight dextrins upon enzymatic starch hydrolysis is also believed to inhibit bread staling by interfering with protein− protein and gluten−starch interactions12 and with AP retrogradation.11,13,14 In addition to AP recrystallization, water migration from crumb to crust3,15 and cross-linking between the continuous protein matrix and discontinuous © 2015 American Chemical Society
Received: Revised: Accepted: Published: 4196
December 21, 2014 April 1, 2015 April 5, 2015 April 6, 2015 DOI: 10.1021/jf506215s J. Agric. Food Chem. 2015, 63, 4196−4201
Article
Journal of Agricultural and Food Chemistry In this study, to understand how maltogenic α-amylase affects the retrogradation of AP, we subjected cooked waxy maize starch (WMS), which contained only AP, to different degrees of hydrolysis (DH) by maltogenic α-amylase and examined the structure and retrogradation properties of the resulting hydrolysates. We further related retrogradation properties to the chain length (CL) distributions of debranched starch hydrolysates. Our objective was to study the action of maltogenic α-amylase on AP, relate the structure of the hydrolysates to their retrogradation properties, and determine how much hydrolysis was needed to prevent the retrogradation of AP.
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Differential Scanning Calorimetry (DSC). Each starch hydrolysate and water (40% solid, w/w) were weighed in a DSC pan. The DSC pans were sealed, heated from 10 to 140 °C at 10 °C/min, and analyzed in duplicate using a Pyris-1 DSC (PerkinElmer, Norwalk, CT, USA). An empty pan was used as a reference. The first scan was done on day 0, and the second scan was done on day 7 after storage of the sample at 4 °C. The onset (To), peak (Tp), and conclusion (Tc) temperatures and enthalpy (ΔH) were obtained from the DSC endotherm with DSC software (TA Instruments, New Castle, DE, USA). Debranching of Starch Hydrolysates. Freeze-dried starch samples were dissolved in 0.01 M acetic acid buffer (pH 4.0) with a starch/buffer ratio of 2:1. Starch solutions were cooked at 100 °C to ensure complete dissolution and then cooled to 50 °C. Isoamylase (1% based on dry weight of starch) was added. Debranching was done for 24 h, and the enzyme was denatured by boiling samples at 100 °C for 10 min. The samples were cooled to room temperature before further analysis. CL Distribution Analysis. Debranched AP samples were further analyzed for CL distribution. Part of the samples was directly injected into HPAEC following debranching, and the remaining samples were freeze-dried for MS distribution analysis. For HPAEC, the sample was prepared at a concentration of 1.5 mg/mL in 150 mM sodium hydroxide. Analysis was performed according to the previously cited procedure.22 Statistical Analysis. Each experiment was performed in duplicate. Data were analyzed by analysis of variance (ANOVA) with Tukey’s studentized range (HSD) test using SPSS version 20.0 (IBM Corp., Inc., Armonk, NY, USA). Mean values from the duplicated experiments were reported. The least significant differences (LSD) for comparison of means were computed at p < 0.05.
MATERIALS AND METHODS
Materials. WMS (Amioca) from National Starch LLC (Bridgewater, NJ, USA) was used for the study. Maltogenic α-amylase, Novamyl (BSuA, 3.2.122), was provided by Caravan Ingredients (Lenexa, KS, USA). Enzyme activity was assayed by quantifying the reducing sugars released from soluble starch (1.0% (w/v) solution) (St. Louis, MO, USA) according to the Somogyi−Nelson method21 relative to a maltose standard curve. One enzyme unit is the amount of enzyme that releases 1 μmol of maltose/min at 62 °C and pH 5.0 (100 mM sodium acetate buffer containing 5 mM CaCl2) for Novamyl. Isoamylase (EC 3.2.1.68) was obtained from Hayashibara Biochemical Laboratories, Inc. (Okayama, Japan) and had an enzyme activity of 1.41 × 106 isoamylase activity units (IAU)/g.22 All chemicals and reagents were of analytical grade. Enzymatic Starch Hydrolysis. Starch (1.8 g db) was mixed with 90 mL of 100 mM sodium acetate buffer containing 5 mM CaCl2 (pH 5.0), heated at 120 °C for about 40 min in a pressure bottle (Ace Glass Inc., Vineland, NJ, USA), and cooled to the optimum temperature of 62 °C for Novamyl. Enzyme (9 EU/g starch db) was added to the starch slurry to initiate the enzyme reaction. This enzyme concentration was selected on the basis of previous studies.20,23 Starch was hydrolyzed to ca. 5, 10, 20, 30, 40, and 50% by taking aliquots at different time intervals. Enzyme was denatured in a boiling water bath for 10 min. Starch hydrolysates were cooled to 25 °C and centrifuged at 8000g for 10 min. Part of the supernatant was further analyzed for saccharide composition, and the rest of the samples were freeze-dried for further study. High-Performance Anion-Exchange Chromatography (HPAEC-PAD). Sacchaaride profile analysis of the supernatants from WMS hydrolysates was done using the procedure of Cai and Shi.22 Eluents were (A) 500 mM NaOH and (B) 150 mM NaOH with 0.5 M sodium acetate. The gradient program was as follows: 85% eluent B for 0−0.4 min, 30% at 20 min, 25% at 30 min, 0% at 35 min, 0% at 40 min, 85% at 41 min, and 85% at 55 min. The separations were carried out at 25 °C with a flow rate of 1 mL/min. The concentration of the injected sample was 20 μg/mL in deionized (DI) water. Percentage DH was expressed as the total amount of saccharides released at any given time on the basis of initial dry weight of starch. Size Exclusion Chromatography (SEC). After waxy maize starch (2%) was hydrolyzed by Novamyl, the mixture was heated at 95 °C for 30 min, cooled to 25 °C, and diluted with deionized water to a concentration of 10 mg/mL. Two drops of 0.25 M NaOH was added to further solubilize the sample. Samples were filtered with a 1 μm nylon syringe filter, and 100 μL was injected onto SEC columns. SEC separations were achieved using a HPLC (1200, Agilent, Santa Clara, CA, USA) equipped with OHpak SB-806 M HQ and OHpak SB-805 HQ columns in series with a OHpak SB-G guard column (Shodex, Showa Denko America, New York, NY, USA). The mobile phase, 0.1 M sodium nitrate, was pumped at a 0.5 mL/min flow rate and maintained at 55 °C. Elution was monitored using a multiangle light scattering detector (Dawn Heleos II, Wyatt Technology, Santa Barbara, CA, USA). A refractive index detector (1200, Agilent) was used to determine concentration (dn/dc = 0.147). Astra 6 software was used for data analysis.
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RESULTS AND DISCUSSION Saccharide Composition of Starch Hydrolysates. WMS hydrolysates were analyzed for the saccharides produced and to Table 1. Composition of Saccharides in Waxy Maize Starch Hydrolysates after Different Degrees of Hydrolysis (DH) degree of polymerization targeted %DHa
1
2
3
4
5
6
7
5 10 20 30 40 50
0.5 0.6 0.7 0.8 0.8 1.1
3.3 9.3 16.6 26.1 33.6 40.7
0.3 0.6 0.9 1.1 1.3 1.7
0.3 0.6 2.4 3.6 4.9 5.6
0.1 0.1 0.2 0.3 0.4 0.4
0.0 0.1 0.1 0.1 0.2 0.2
0.0 0.0 0.0 0.0 0.0 0.0
a
Calculated as the total amount of saccharides released at any given time based on initial starch weight.
Table 2. Retrogradation Properties of Waxy Maize Starch Hydrolysates (After Storage at 4 °C for 7 Days) As Determined by Differential Scanning Calorimetrya sample % DHb control 5 10 20 30 40 50
To (°C)
Tp (°C)
Tc (°C)
ΔH (J/g)
38.7 ± 0.9c 38.4 ± 0.3c 41.6 ± 0.7b 46.0 ± 0.2a
54.8 ± 1.2a 56.6 ± 0.7a 57.4 ± 0.1a 59.8 ± 0.5a no endothermic no endothermic no endothermic
79.9 ± 1.6a 78.8 ± 0.2a 73.1 ± 0.3b 73.6 ± 0.6b peak observed peak observed peak observed
9.1 ± 0.8a 7.1 ± 0.2b 3.2 ± 0.2c 0.8 ± 0.1d
a Values with the same letter in the same column are not significantly different (p < 0.05). bDH, degree of hydrolysis.
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DOI: 10.1021/jf506215s J. Agric. Food Chem. 2015, 63, 4196−4201
Article
Journal of Agricultural and Food Chemistry
Figure 1. Molecular weight (Mw) distributions of waxy maize starch and its hydrolysates as determined by size exclusion chromatography: (A) response from refractive index (RI) detection; (B) response from light scattering detection.
Table 3. Average Molecular Weight (Mw) and z-Average Radius of Gyration (Rz) of Waxy Maize Starch (WMS) after Different Degrees of Hydrolysis (DH) As Determined by Size Exclusion Chromatography in Figure 1 peak 1 (23−27.8 min) WMS control 5% DH 10% DH 20% DH 30% DH 40% DH 50% DH
higher than that of other saccharides. The maltose/glucose ratio increased with an increase in %DH, indicating the exoaction of enzyme producing maltose during earlier hydrolysis stages. A change in action pattern of maltogenic amylase from exo to endo with increase in hydrolysis time18 and increase in temperature4 was reported in earlier studies, as was the production of sugars up to DP 7−9, which were further hydrolyzed to maltose by this enzyme.24,25 Thermal and Retrogradation Properties. Retrogradation properties of starch hydrolysates after 7 days of storage at 4 °C are shown in Table 2. Starch hydrolysates produced no endothermic peak during the initial scan before storage (day 0) because the starch crystallinity was completely destroyed during cooking prior to hydrolysis. After 7 days of storage at 4 °C, WMS showed significant retrogradation. AP retrogradation enthalpy decreased to 0.8 J/g for 20% hydrolyzed starch. As the DH increased, the extent of retrogradation decreased, with no retrogradation observed for starches hydrolyzed to >20% DH. We suggest that the shortened outer chains inhibited formation of double helices and prevented AP recrystallization. Another reason for this result might be the increase in low molecular
peak 2 (27.8−35 min)
Mw (kg/mol)
Rz (nm)
Mw (kg/mol)
Rz (nm)
473854 250818 159201 128655 93730 60499 33025
340.8 247.2 202.2 175.1 152.6 141.2 117.6
174075 37583 24316 20198 15981 13135 10435
220.1 115.7 93.1 84.8 75.3 68.9 58.5
determine %DH. Individual sugar percentages (based on initial starch weight) in the reaction mixture are given in Table 1. Saccharides up to degree of polymerization (DP) 6 were formed, and their concentrations continued to increase with increase in hydrolysis time. Maltose was the predominant sugar produced. The rate of formation of maltose was significantly
Figure 2. Schematic presentation of the action of maltogenic α-amylase on amylopectin: (−) 1,4-α-glucan; (→) α-1,6 linkage; (G-G) maltose; (G1− 6) saccharides with 1−6 glucose units. 4198
DOI: 10.1021/jf506215s J. Agric. Food Chem. 2015, 63, 4196−4201
Article
Journal of Agricultural and Food Chemistry
Figure 3. Chain length distributions of waxy maize starch compared to hydrolysates after (A) 5%, (B) 10%, (C) 20%, (D) 30%, (E) 40%, and (F) 50% hydrolysis as determined by high-performance anion-exchange chromatography.
tendency of AP depends on its CL distribution,27−29 with retrogradation rate having a positive correlation with the mole fraction of unit chains of DP 14−24 and a negative correlation with mole fraction of unit chains of DP 6−11.29,30 Using model compounds, other researchers observed no retrogradation when DP was
