Amylolysis of amylopectin and amylose isolated from wheat, triticale, corn and barley starches

Food Hydrocolloids 35 (2014) 686e693

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Amylolysis of amylopectin and amylose isolated from wheat, triticale, corn and barley starches Sabaratnam Naguleswaran a, Thava Vasanthan a, *, Ratnajothi Hoover b, David Bressler a a b

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada Department of Biochemistry, Memorial University of Newfoundland, St. John’s, Newfoundland A1B 3X9, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 January 2013 Accepted 14 August 2013

Amylopectin (AP) and amylose (AM) were isolated from normal (wheat, triticale, corn, and barley), waxy (corn and barley), and high-amylose (corn and barley) starches. The relationship between the molecular characteristics of the above starch polymers and amylolysis was studied in comparison to their native granules. Amylolysis was conducted by using granular starch hydrolyzing enzymes (a mixture of aamylase and glucoamylase) at sub-gelatinization temperatures (<55
C) over a period of 1 to 72 h, and the degree of hydrolysis (DH) was measured as a percentage of reducing value. At the early stages (1 h) of hydrolysis, AP and AM fractions from all starches were significantly hydrolyzed by amylases to a greater extent than native granules. Regardless of starch sources, the DH of AP and AM at 1 h hydrolysis ranged from 71.4 to 86.1 and 66.4 to 81.4%, respectively. Between 1 and 72 h of hydrolysis, the DH of AP was higher than AM in normal and high-amylose starches. Significant correlations were found between the molecular weight (Mw) or molecular size (Rz) of AP, and the DH of native granules at 1 h hydrolysis. The results suggested that a high proportion of short-chains in AP, indicated by its high Mw, high Rz and low average chain-length, were responsible for high DH of native granules. The difference in DH among native granules was mainly influenced by the difference in granular architecture resulting from variations in average chain length of AP. Ó 2013 Published by Elsevier Ltd.

Keywords: Amylolysis Amylose Amylopectin Starch Structure Sub-gelatinization

1. Introduction Starch is the second abundant natural polysaccharide present in higher plants, next to cellulose and it is an inevitable source of energy for animals, including humans. In green plants, starch is deposited as granules and the architecture of a starch granule is built up by two polymers of glucose, essentially a linear amylose (AM) and a heavily branched amylopectin (AP), which are highly organized through intra- and inter-molecular hydrogen bonds resulting in a complex biopolymer. In North America, cereal starches are more frequently used in various food and industrial applications than starches from other sources. One of the current food trends is the consumption of starchy products that are rich in resistant starches (RS). RS can be used as a functional food ingredient for making variety of food products, since some of these can be a source of dietary fiber (i.e. completely resistant to enzyme hydrolysis) and others are known as slowly digestible starches, demonstrated to assist in controlling

* Corresponding author. Tel.: þ1 780 492 2898; fax: þ1 780 492 8914. E-mail address: [email protected] (T. Vasanthan). 0268-005X/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.foodhyd.2013.08.018

sugar metabolism in diabetics (Englyst, Kingman, & Cummings, 1992; Liu, 2005; Mason, 2009). On the other hand, starches are traditionally being used for bioethanol (a renewable source of energy) production (Gomez, Steele-King, & McQueen-Mason, 2008) and in North America, the bioethanol production mainly relies on starches from corn and wheat grains. The ethanol production requires starch granules from grains to be enzymatically hydrolyzed completely to sugars (glucose, maltose and maltotriose), which are subsequently fermented to ethanol by yeast (Chen, Wu, & Fukuda, 2008; Sharma, Rausch, Tumbleson, & Singh, 2007). Despite the extensive collection of starch hydrolysis (by amylases) studies reported in the literature, a substantial research gap yet exists in this area of science. There is no information available on how AM and AP would be hydrolyzed by amylases when isolated from the starch granule. A comparison of the reactivity of amylases towards the intact native granule and its isolated components (AM and AP) would help scientists to understand the role played by AM and AP in starch amylolysis. In general, enzymatic hydrolysis is used as a tool to study the architecture of starch granules (Miao, Zhang, Mu, & Jiang, 2011). Hydrolysis of starch granules with amylases (i.e. amylolysis) occurs in several steps, which include diffusion to the solid surface,

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adsorption, and finally catalysis. The rate of hydrolysis is initially fast but then continues at a slower and more persistent rate (Oates, 1997). Among the amylases, a-amylase and glucoamylase are most commonly used to study the hydrolysis pattern of starch granules. The a-amylases (EC 3.2.1.1) are endo-acting enzymes that internally hydrolyze a-D-(1,4)-glycosidic linkages of both AP and AM yielding soluble products such as oligosaccharides, and branched and low molecular weight a-limit dextrins. However, glucoamylase (EC 3.2.1.3) is an exo-acting enzyme, which depolymerizes both a-(1,4)and a-(1,6)-linkages of starch polymers from their non-reducing ends resulting in the complete conversion of starch into glucose (Sujka & Jamroz, 2007). Thus, a study of the action pattern of amylases is important for understanding the impact of starch structure on physicochemical properties and functionality. The influence of structural properties of native starches such as granule size, granule architecture, and granule porosity on in vitro hydrolysis have been studied (Asare et al., 2011; Dhital, Shrestha, & Gidley, 2010; Liu, Gu, Donner, Tetlow, & Emes, 2007; Naguleswaran, Li, Vasanthan, Bressler, & Hoover, 2012; Naguleswaran, Vasanthan, Hoover, & Bressler, 2013; Salman et al., 2009; Stevnebø, Sahlström, & Svihus, 2006; Sujka & Jamroz, 2007; Uthumporn, Zaidul, & Karim, 2010). However, there is a dearth of information on the extent to which isolated AM and AP are hydrolyzed by a-amylase and glucoamylase. The molecular structural features of AM and AP influence the starch hydrolysis. The molecular characteristics of AP such as molar mass (molecular weight), molecular dimension or size (radius of gyration), molecular density, branching degree, and distribution of short-chains have been shown to influence starch hydrolysis by amylases (Goesaert, Bijttebier, & Delcour, 2010; Miao et al., 2011; Murthy, Johnston, Rausch, Tumbleson, & Singh, 2011). For instance, AP molecules with higher number of short-chains with a greater degree of branching (resulting in more compact structure/ high molecular density, high molar mass and small molecular size of AP molecule) are less susceptible to hydrolysis by amylases (i.e. a property preferred in low-glycemic food production). In contrast, starch granules composed of AP molecules with low molecular density and molar mass could be useful for bioethanol production, as it requires rapid but a complete hydrolysis by amylases. Thus, the objective of this study was to compare the reactivity of amylases towards the intact native granule and isolated AP and AM in order to understand the role played by molecular characteristics of AP and AM on starch amylolysis. 2. Materials and methods 2.1. Materials Two cultivars of wheat grains, Canada prairie spring red (CPS Red) and AC Reed, were provided by Alberta Agriculture and Food in Barrhead (AB, Canada). Triticale grains (Pronghorn and AC Ultima) were obtained from the Field Crop Development Centre of Alberta Agriculture and Rural Development in Lacombe (AB, Canada). Grains from three hull-less barley cultivars (waxy, CDC Candle; normal, CDC McGwire; and high-amylose, SH 99250) were obtained from the Crop Development Center at University of Saskatchewan in Saskatoon (SK, Canada). Commercial corn starches of waxy (Amioca), normal (Melojel) and high-amylose (Hylon VII) were obtained from the National Starch Food Innovation in Bridgewater (NJ, USA). The starches used in this study are categorized into three genotype groups; normal (triticale, wheat, corn, and barley), waxy (corn and barley), and high-amylose (corn and barley), depending on the amylose content in their native form. Granular starch hydrolyzing enzyme, Stargen 002 (570 GAU/g) was a gift from Genencor International in Rochester (NY, USA). All other chemicals and reagents used in this study were of ACS grade.

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2.2. Grain grinding and starch isolation Triticale, wheat and barley grains were ground into meals in a Retsch mill (Model ZM 200, Haan, Germany) using a ring sieve with an aperture size of 0.5 mm. Pure starch (purity >95%, w/w) was isolated from the grain meal of triticale, wheat and barley using the procedures reported in earlier publications (Gao, Vasanthan, & Hoover, 2009; Kandil, Li, Vasanthan, Bressler, & Tyler, 2011). 2.3. Molecular characteristics determination of amylopectin (AP) and amylose (AM) Starch sample (20 mg, dry basis) was solubilized with the addition of 2 mL of 95% (v/v) DMSO followed by heating (85e90
C) in a water bath for 30 min with vortexing every 5 min. The solubilized starch solution was cooled to room temperature followed by the addition of absolute ethanol (6 mL). The solution was then kept at 4
C for 2 h, centrifuged (6000  g for 10 min) and the pellet washed with cold ethanol (5 mL). The pellet was then re-solubilized by the addition of 2 M KOH (2 mL) followed by mechanical mixing for 1 h in an ice-bath (tubes containing the samples were covered with ice in a styrofoam box) and then for 15 h at room temperature (w22
C). The alkaline solution containing starch molecules was diluted with 0.2 M NaNO3 (15 mL) solution, neutralized by 2 M HCL (pH was adjusted between 6.7 and 6.9) and then made up to volume (20 mL) with 0.2 M NaNO3 (starch polymer concentration was 1 mg/mL) followed by filtration through a nylon membrane filter (1 mm) device (Puradisc 25 NYL, Whatman Inc., NJ, USA). An aliquot (50 mL) of the filtrate was injected into an HPSEC-MALLS-RI system. In order to avoid aggregation of dispersed starch molecules, the solubilized starch in KOH solution was neutralized by HCl instantly before each injection. The HPSEC-MALLS-RI system consisted of an Agilent 1200 HPLC system (Agilent Technologies in Santa Clara, CA, USA) coupled with a multi-angle laser light scattering detector which had a laser wavelength of 658 nm (MALLS, DAWN-HELEOS II, Wyatt Technology in Santa Barbara, CA, USA), and a refractive index detector (RID, Agilent Technologies in Santa Clara, CA, USA). A guard column (UltrahydrogelÔ, 6  40 mm, Waters Corporation in Milford, MA, USA) and an SEC column (UltrahydrogelÔ Linear, 7.8  300 mm, Waters Corporation in Milford, MA, USA) were connected to the HPLC system. The mobile phase used in HPSEC system was aqueous NaNO3 (0.2 M) solution with a flow rate of 0.5 mL/min. The column and RI detector temperatures were maintained at 40
C and 35
C, respectively. Before injection of starch samples, two types of dextran with different molecular weight (Mw) were analyzed to test the chromatography system. The Mw of tested dextrans were (2.45  105 and 1.01  107 g/mol) in agreement with reported values by the manufacturer (SigmaeAldrich Canada Ltd. in Oakville, ON, Canada). Two injections were completed for each starch type. The ASTRA software (Version 5.3.4.20, Wyatt Technology in Santa Barbara, CA, USA) was used to collect and analyze data from the HPSEC-MALLS-RI system. A dn/dc value of 0.146 mL/g for starch was applied in calculations using the Berry extrapolation model with a first-degree polynomial fit (Chen & Bergman, 2007; RollandSabate, Colonna, Mendez-Montealvo, & Planchot, 2007; Yoo & Jane, 2002). The Mw (weight-average molecular weight, g/mol) and Rz (zaverage radius of gyration, nm) of AM and AP were automatically calculated by ASTRA, and the average dispersed-molecular density (r ¼ Mw/R3z in g/mol/nm3) of AP and AM was calculated according to the method of Yoo and Jane (2002). The branching parameters such, average number of branch points (B) (Rolland-Sabate et al., 2007), weight-average degree of polymerization [DPw ¼ Mw/162 (Chen & Bergman, 2007)], average unit chain length [CL ¼ DPw/B (RollandSabate et al., 2007)], and average degree of branching [DB ¼ (B/

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DPw)100% (Rolland-Sabate et al., 2007)] of AP were also computed. 2.4. Amylopectin (AP) and amylose (AM) isolation AP and AM of triticale, wheat, corn and barley starches were isolated according to the protocols described by Charoenkul, Uttapap, Pathipanawat, and Takeda (2006) and Takeda, Hizukuri, and Juliano (1986) with certain modifications. Starch (100 mg, dry basis) was dissolved in 10 mL of 95% dimethyl sulfoxide (DMSO) by heating (85e90
C) in a water bath for 1 h with stirring in a Vortex mixer every 10 min. Dissolved starch was cooled to room temperature (w22
C) followed by addition of 30 mL of anhydrous ethanol and then kept at 4
C for 2 h to settle the starch molecules. The pellet of AP and AM was collected and washed with 10 mL of cool anhydrous ethanol followed by centrifugation (3500  g for 10 min). The starch molecules were then dispersed in 17 mL water at 70e80
C followed by the addition of 1 mL of n-butanol and 1 mL of isopentanol (3-methyl-1-butanol). The mixture in a tightly closed tube was heated (80e85
C) in a water bath for 1 h under a fume hood with stirring (Vortex mixing every 10 min), then cooled and stored in a styrofoam box for 15 h at room temperature and then at 4
C for 24 h. The supernatant liquid (z20 mL) containing AP and the AM pellet were isolated by centrifugation (6000  g for 10 min). To recover AP from the supernatant liquid, 60 mL of cold methanol (absolute) was added. The tubes were then kept at 4
C for 2 h and centrifuged (6000  g for 10 min). After washing with cold methanol (10 mL), the AP pellet was air-dried at room temperature for 24 h. AM pellet (in the form of butanoleamylose complex) was dispersed in 20 mL of cold anhydrous ethanol. The tubes were then kept at 4
C for 2 h and centrifuged (6000  g for 10 min). The AM pellet was washed with cold ethanol (10 mL) followed by cold acetone (10 mL), and airdried at room temperature for 24 h. The isolated AP and AM were stored at room temperature in airtight containers until further analyses. The residual contents of butanol and isopentanol in AM fractions were determined by Gas Chromatography coupled with a Flame-Ionization-Detector (GC-FID) according to the procedure described by Gibreel et al. (2009). An internal standard (0.4% pentanol) and two sample standards (0.4% n-butanol and 0.5% isopentanol) were included in this procedure. The n-butanol and isopentanol contents in isolated amylose samples were in the range from 0.1 to 2% and 0.4 to 1.5%, respectively. 2.5. Amylolysis of isolated AP and AM AP and AM (30 mg, dry basis) were dissolved separately in 2 mL of 1 M KOH and mechanically stirred in an orbit shaker (Lab-line Instruments, Inc., IL, USA) for 1 h (tubes containing the samples were covered with ice in a styrofoam box). The dispersed AP or AM molecules in KOH solution was then diluted with 2 mL of 50 mM sodium acetate buffer and the pH of the solution was adjusted to 4.0 with 1 M HCl. Total volume of the solution was corrected to 10 mL with acetate buffer-enzyme mixture (Stargen 002 enzyme was used at 24 U/g starch) to achieve a sample concentration of 0.3% (w/v). Amylolysis was carried out at 55
C for 1 h and then at 30
C for 72 h in shaking water bath (Model BS-11, Jeio Tech Inc., Korea). The aliquots of hydrolyzed samples were withdrawn at 1, 24, 48, and 72 h for determination of the degree of hydrolysis (DH). DH was expressed as a percentage of reducing value (Bruner, 1964). Control samples for AP and AM were run concurrently without enzyme addition. 2.6. Statistical analysis All analyses and treatments were carried out in duplicate. Analysis of variance using the General Linear Model (GLM)

procedure was performed with the SASÒ Statistical Software, Version 9.3, 2011 (SAS Institute Inc. in Cary, NC, USA). Multiple comparisons of the means were completed by using the Tukey’s Studentized Range (HSD) Test at a ¼ 0.05. The Pearson correlation coefficient (r) using the SASÒ Statistical Software, Version 9.3, 2011 (SAS Institute Inc. in Cary, NC, USA) and Spearman’s rank correlation coefficient (rho) using the Software developed by Wessa (2012) were calculated to measure the statistical dependence between molecular characteristics and degree of amylolysis of starches. Both correlation procedures use the Student t-test to check the significance of the correlation at a ¼ 0.05. 3. Results and discussion 3.1. Molecular characteristics of AP and AM The molecular characteristics of AP and AM such as weightaverage molecular weight (Mw), z-average molecular size (Rz), dispersed-molecular density (r), degree of branching (DB), and average-chain length (CL) are presented in Table 1. The Mw of AP in normal starches ranged between 6.5  106 g/mol (corn) and 24.6  106 g/mol (Pronghorn triticale). The Rz of AP in normal starches was between 63.9 nm (corn) and 72.4 nm (barley). In waxy starches, the Mw and Rz of AP in corn were higher than those of barley AP (Table 1). AP of high-amylose genotypes were much smaller than those of normal and waxy genotypes (Table 1). Regardless of starch sources and genotypes, the AP of most starches was highly branched and the DB varied between 1 and 13%. This could be attributed to the variation in molecular density and average-CL of AP among starches (Table 1). The molecular characteristics of AM were different to those of AP. In comparison to AP, the Mw and molecular density (r) of AM was lower and Rz was higher (Table 1). The Rz of AM isolated from high-amylose corn and barley starches were lower than the Rz of AM isolated from normal types of both corn and barley starches (Table 1). Since Rz is related to the volume occupied by a molecule in a solution, the lower Mw and higher Rz of AM suggest AM chains are loosely packed (i.e. lower in density) with long and unbranched chains. Whereas, the higher Mw and lower Rz of AP suggest AP chains are compactly packed (i.e. higher in density) and highly branched. 3.2. Amylolysis of native granules, AP and AM of normal starches The DH of AP and AM fractions of normal starches are presented in Table 2. The results reported in previous studies (Naguleswaran et al., 2012, 2013) showed that the DH of native granules from normal starches with similar AM content (22.5e26.4%) increased rapidly during first 24 h hydrolysis [CPS Red wheat (91.3%) w Pronghorn triticale (89.3%) > AC Reed wheat (88.6%) w Ultima triticale (87.5%) > barley (81.3%) > corn (59.8%)], and then it was gradual in all starches. The lower DH of native corn starch could be attributed to the morphological characteristics of the granule surface. Hydrolysis of native starch granules has been shown to begin at the granule periphery (due to the presence of surface pores and channels). Pores and channels have been shown to increase the effective surface area for fast enzyme diffusion (Oates, 1997; Tester, Qi, & Karkalas, 2006). Naguleswaran, Li, Vasanthan, and Bressler (2011) have shown that in corn starch, surface pores and channels are blocked by protein and phospholipids and this may have been responsible for the lower DH of corn starch. After 24 h, native starch hydrolysis was gradual in all starches (Naguleswaran et al., 2012, 2013). At the end of 72 h, DH of native granules followed the order: CPS Red wheat (96.9%) w AC Reed wheat (96.2%) > Ultima triticale (92.5%) w Pronghorn triticale (92.4%) > corn (90.6%) > barley (89.8%). The isolated AP and AM

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Table 1 Molecular characteristics of amylopectin and amylose of normal, waxy, and high-amylose starches from triticale, wheat, corn and barley. Amylopectin Mwa (x106) Normal Pronghorn Triticale Ultima Triticale AC Reed Wheat CPS Red Wheat Corn Barley Waxy Corn Barley High-amylose Corn Barley

24.6a 14.9d 9.4e 19.7c 6.5f 22.4ab

     

Amylose

rc

Rzb

0.6 1.0 0.2 0.1 0.6 1.1

71.9a 69.5ab 71.4a 66.7ab 63.9bc 72.4a

     

1.3 1.8 1.3 0.7 1.7 2.1

DBd

66.1b 44.4d 25.7e 66.3b 24.8e 59.1c

     

2.0 0.6 0.7 1.7 0.2 2.2

CLe

7.9b 4.1e 1.7f 6.9c 1.2f 6.9c

     

0.2 0.3 0.0 0.0 0.1 0.4

12.7ef 24.1de 59.2c 14.6ef 82.3b 14.5ef

     

0.3 1.6 1.5 0.1 2.2 0.7

16.9d  0.4 6.2f  0.2

69.6ab  1.3 67.0ab  1.4

50.1d  1.6 20.5e  0.5

5.1d  0.1 0.9f  0.0

19.8de  0.4 107.3a  2.5

7.0ef  1.0 20.5bc  0.3

53.1d  1.6 59.9c  0.7

46.8d  2.3 95.5a  1.9

3.3e  0.5 12.8a  0.2

30.5d  1.4 7.8f  0.1

Mw (x105)

Rz

r

11.3e  0.7 22.5cd  0.3 20.6d  0.6 45.5b  0.8 23.8c  0.9 51.1a  0.4

82.4ab  1.6 80.5b  1.3 87.7a  1.8 80.7b  2.0 77.9b  1.1 88.3a  2.3

2.0e  0.0 4.3d  0.1 3.0e  0.1 8.6b  0.5 5.0d  0.0 7.4c  0.5

n/a n/a

n/a n/a

n/a n/a

8.88f  0.2 47.1b  0.3

69.2c  1.6 71.5c  0.8

2.7e  0.1 12.9a  0.4

Values are mean  standard deviation, and values with the same letters in the same column are not significantly different at a ¼ 0.05. a Weight-average molecular weight (g/mol). b z-Average radius of gyration (nm). c Dispersed-molecular density (g/mol/nm3). d Degree of branching (%). e Average chain length.

from all starches (Table 2) were hydrolyzed to a greater extent (72.7e82.7% and 68.7e81.4%, respectively) than native granules (3.5e76.5%; Naguleswaran et al., 2012, 2013) during the initial stage (1 h) of hydrolysis. The difference in DH between native granules and their isolated fractions (AP and AM) is mainly due to granule architecture. As discussed earlier, the first point of enzyme attack is on the granule surface. The granule periphery is highly organized by short-chains of AP clusters that hinder the entry of amylases into the granule interior. This may explain the reduced DH of native granules during the early stage of hydrolysis (Fig. 1A). The DH after 72 h was higher in native granules (89.8e96.9%) than in isolated AP (81.1e87.9%) and AM (73.6e87.0%). This could be attributed to increased flexibility of isolated AP and AM, which facilitates interaction among APeAP, APeAM and AMeAM molecules resulting in the formation of junction zones that become inaccessible to amylases. Between 1 and 72 h of hydrolysis, the difference in DH between isolated AP and AM (AP > AM) could be attributed to the molecular structure of AP. The enzyme cocktail used in this study was composed of both a-amylase (endo-attack on a-(1,4)-glycosidic linkages) and glucoamylase (exo-attack on

both a-(1,4)- and a-(1,6)-linkages). Since AP molecules possess several non-reducing ends, the outer clusters of AP are rapidly hydrolyzed by exo-type glucoamylase (Fig. 1B). The low DH of isolated AM could be attributed to the presence of only one nonreducing end for glucoamylase action (Fig. 1C). Long-chains of AP that connect each cluster are initially hydrolyzed by endo-acting aamylases to release individual clusters (Fig. 1B). Thereafter, clusters composed of short-chains are extensively hydrolyzed by exo-acting glucoamylases to produce sugars. As discussed earlier, a higher number of short-chains in a cluster increases the number of access points for glucoamylase reactions (Fig. 1B). The high DH of native granules of Pronghorn and Ultima triticale, CPS Red wheat and barley starches (Naguleswaran et al., 2012, 2013), and their respective AP molecules (Table 2) is mainly due to the presence of high proportion of AP short-chains (average-CL ¼ 12.7e24.1). The low DH shown by native granules of AC Reed wheat and corn (Naguleswaran et al., 2012, 2013), and their AP (Table 2) at the end of 1 h hydrolysis could be attributed to their AP long average-CL (Table 1). 3.3. Amylolysis of native granules, AP and AM of waxy starches

Table 2 Degree of hydrolysis of amylopectin and amylose of normal starches from triticale, wheat, corn and barley. Degree of hydrolysis (%, dry basis) 1 ha Amylopectin Pronghorn Triticale 79.2b Ultima Triticale 82.7a AC Reed Wheat 72.7e CPS Red Wheat 76.0c Corn 73.4de Barley 75.4cd Amylose Pronghorn Triticale 75.6c Ultima Triticale 81.4ab AC Reed Wheat 68.8f CPS Red Wheat 75.1cd Corn 68.7f Barley 74.3cde

24 hb

48 hb

     

1.0 81.6bc  0.8 84.8a  1.1 76.0fg  0.2 78.6de  0.4 79.4cde  0.5 81.5bc 

     

0.8 1.0 0.8 0.4 0.6 0.7

78.5de 83.4ab 71.6h 77.5ef 75.1g 80.7cd

     

1.2 1.3 0.8 0.4 0.7 0.8

83.7b 86.5a 77.9d 80.3c 83.1b 83.8b

72 hb      

0.8 85.3abc  0.6 0.8 87.8a  1.5 0.3 81.1d  1.2 0.8 81.6d  0.8 0.7 86.6ab  0.7 1.0 87.9a  0.7

0.5 81.0c  0.7 0.7 84.7ab  0.4 0.5 72.7e  0.5 0.5 79.8cd  0.6 0.3 79.5cd  0.3 0.5 83.4b  0.8

83.4cd 86.3ab 73.6e 80.9d 84.5bc 87.0ab

     

1.2 0.6 0.3 0.5 1.3 1.0

Hydrolysis carried out at 55
C for 1 h. Hydrolysis carried out at 30
C for 24, 48, and 72 h values are mean  standard deviation, and values with the same letters in the same column are not significantly different at a ¼ 0.05. a

b

Hydrolysis data of AP isolated from waxy corn and waxy barley are presented in Table 3. As both starches were low in AM content (1.1e5.3%), only the AP fraction was isolated for this study. As reported in previous publication (Naguleswaran et al., 2013), native granules of waxy barley starch were hydrolyzed by amylases to a higher extent (49.6%) than waxy corn (29.2%) after 1 h hydrolysis. However, their AP fractions showed an opposite trend in the extent of hydrolysis (Table 3). It is likely that the presence of weaker crystallites (reflected by lower gelatinization temperature) and a lower content of bound lipid (0.5%) in barley starch may have rendered waxy barley starches more susceptible to hydrolysis (Chung et al., 2008; Naguleswaran et al., 2013). In addition, the difference in DH between native granules of waxy corn and barley could be attributed to the higher molecular density (50.1 g/mol/ nm3) of corn AP. In native waxy corn starch granules, the highly branched (Table 1) AP molecules (i.e. higher in density) may have been compactly organized (through intra- and inter-hydrogen bonding) thereby hindering the entry of amylase into the granule interior. The difference in DH between isolated AP of waxy corn and barley starches between 1 h and 48 h (Table 3) suggests that in corn,

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Fig. 1. Reaction pattern of amylases: (A) semicrystalline structure of native granule, (B) dispersed amylopectin molecules, and (C) dispersed amylose molecules. Small-block arrows indicate the access points of amylases towards the native granules (A), and isolated amylopectin (B) and amylose (C) of starches. Amylopectin models shown in this representation are based on the model proposed by Hizukuri (1986).

more non-reducing ends are exposed to the action of glucoamylase as a result of a higher content of short-chains (Table 1). After 24, 48 and 72 h of hydrolysis, the difference in DH between native waxy barley starch (83.8, 88.1 and 89.4%, respectively) and isolated AP (Table 3) was not significant (p > 0.05). This suggests that at later stage of hydrolysis (>24 h), AP structure has no influence on DH. 3.4. Amylolysis of native granules, AP and AM of high-amylose starches The DH results of isolated AP and AM from high-amylose genotypes of corn and barley starches are presented in Table 4. After 1 h hydrolysis, the DH of native granules of high-amylose corn and barley starches were 0.3% and 24.1%, respectively (Naguleswaran et al., 2013). Those values were lower than the DH of isolated AP and AM (Table 4). Native granules of high-amylose corn showed only a DH of 9.4% even after 72 h. This suggests that in highamylose corn starch, AM content (69.7%) had a greater impact than AP structure on DH. The granule size range of high-amylose corn (2e22 mm) and barley (1e20 mm) starches was comparable;

however, their AM content varied greatly (corn > barley). The molecular size (Rz) of AM of high-amylose corn and barley starches was comparable, but was smaller than those of normal corn and barley starches (Table 1). Due to the higher proportion of compactly packed AM chains in high-amylose corn starch, AM chain flexibility would be very low. Consequently, the conformational transformation (chair to half chair) required for hydrolysis of D-glucopyranosyl units would be difficult (Naguleswaran et al., 2013), resulting in a decreased accessibility of amylases towards the glycosidic linkages. It is highly unlikely that AM-lipid complexes could be a factor contributing to difference in DH between starches, since isolated AM from high-amylose corn and barley starches are hydrolyzed nearly to the same extent (Table 4). The higher proportion of short-chains (indicated by high degree of branching and high molecular density) in high-amylose barley AP (Table 1) may have been responsible for the DH of native barley (24.1%) being

Table 4 Degree of hydrolysis of amylopectin and amylose of high-amylose starches from corn and barley. Degree of hydrolysis (%, dry basis)

Table 3 Degree of hydrolysis of amylopectin of waxy starches from corn and barley. Degree of hydrolysis (%, dry basis)

Corn Barley

1 ha

24 hb

48 hb

72 hb

86.1a  0.8 78.0b  1.0

88.3a  0.5 84.0b  1.3

90.8a  0.5 86.6b  0.4

91.4a  0.4 91.3a  1.2

Values are mean  standard deviation, and values with the same letters in the same column are not significantly different at a ¼ 0.05. a Hydrolysis carried out at 55
C for 1 h. b Hydrolysis carried out at 30
C for 24, 48, and 72 h.

1 ha Amylopectin Corn 71.4b Barley 74.5a Amylose Corn 66.4c Barley 68.3c

24 hb

48 hb

72 hb

 0.9  1.3

78.9a  1.0 77.3ab  1.1

83.0a  0.6 82.2a  0.8

84.7a  0.8 84.8a  0.7

 0.8  1.0

74.9b  0.9 75.7b  1.3

78.5b  1.0 78.4b  0.5

82.9ab  1.0 82.0b  1.0

Values are mean  standard deviation, and values with the same letters in the same column are not significantly different at a ¼ 0.05. a Hydrolysis carried out at 55
C for 1 h. b Hydrolysis carried out at 30
C for 24, 48, and 72 h.

S. Naguleswaran et al. / Food Hydrocolloids 35 (2014) 686e693

higher than that of corn (0.3%) during the early stage (1 h) of hydrolysis. Similarly, isolated barley AP (74.5%) was hydrolyzed to greater extent than corn AP (71.4%) due to the presence of a higher proportion of short-chains (average-CL ¼ 7.8) in the former. However, the difference in DH between high-amylose corn AP and barley AP was insignificant after 24 h (Table 4). Miao et al. (2011), Evans and Thompson (2008) and Sevenou, Hill, Farhat, and Mitchell (2002) reported that native granules of high-amylose corn are resistant to amylase hydrolysis due the presence of a high level of double helical order (determined by Fourier Transform Infrared spectroscopy) of AP short-chains near the vicinity of the granule surface. As shown in Table 4, the DH of isolated corn and barley AM was lower than that of isolated corn and barley AP. This is most likely due to the interactions between AM and AM chains, which hinder amylase hydrolysis (Fig. 1C). Although the molecular characteristics of AM between high-amylose barley and corn were different (Table 1), the difference in DH of AM during the progress of hydrolysis (1e72 h, Table 4) was not significant (p > 0.05). This suggests that the DH of AM was not influenced by the molecular characteristics of AM isolated from high-amylose barley and corn. Since both AP and AM of high-amylose corn and barley were hydrolyzed to the same extent, the difference in DH between native corn and barley probably reflects the denser packing of AM (densely packed AM chains would decrease the accessibility of aamylase towards the glycosidic linkages) within the granule interior of high-amylose corn starch. 3.5. Relations between molecular characteristics and degree of hydrolysis Statistical dependence between molecular characteristics of AP or AM, and DH (at 1 h) of starches was tested using two different correlation statistical procedures and the results are presented in Table 5. The correlation results suggest that the initial rate of hydrolysis of AP and AM are greatly influenced by amylases, when they are within native granules rather than in dispersed solutions. The molar mass (Mw) and molecular size (Rz) of AP significantly correlated to the DH of native granules (p < 0.05, Table 5). A similar trend was seen with AM; however, correlations between molecular characteristics and DH of AM were not significant (p > 0.05, Table 5). The branching parameters of AP have been shown to influence the reaction of amylases on native starch granules (Gilbert et al., 2010; Goesaert et al., 2010; Rolland-Sabate et al., 2007). In this study, the DH of native granules at the initial stages of hydrolysis (at

Table 5 Statistical dependence between molecular characteristics and degree of hydrolysis (at 1 h) of native starches. Molecular characteristics

Correlation coefficients Pearson’s r

Amylopectin Mwa Rzb

r

c

DBd CLe Amylose Mw Rz

*0.66 *0.71 0.24 0.29 0.32

(0.037)f (0.023) (0.502) (0.414) (0.369)

0.25 (0.545) 0.58 (0.130)

*Variables are significantly correlated at a ¼ 0.05. a Weight-average molecular weight. b z-Average radius of gyration. c Dispersed-molecular density. d Degree of branching. e Average chain length. f Probability (p) values are presented in parenthesis.

Spearman’s rho *0.66 *0.70 0.36 0.49 0.50

(0.044) (0.031) (0.313) (0.148) (0.143)

0.29 (0.500) 0.64 (0.096)

691

1 h) was highly influenced by the average-CL, DB, and r of AP. As explained earlier, AP molecules that are composed mainly of shortchains (indicated by low average-CL), showed a higher DH in native granules. Positive correlations of DB and r of AP with DH of native granules (Table 5) were further indicative of the effect of shortchains on hydrolysis. The high DB in AP molecules was found in Pronghorn triticale, CPS Red wheat and barley (normal and highamylose) starches perhaps due to heavily branched and densely packed AP short-chains of the above starches (Table 1). The positive relationships between Mw or Rz and DH (Table 5) and an inverse relationship between average-CL and DH (Table 5) were indicative that large molecules of AP increase the DH of native granules initially due to their higher proportion of short-chains. Molecular characteristics of AP of cereal and tuber starches have been shown to influence amylolysis (Gilbert et al., 2010; Goesaert et al., 2010; Rolland-Sabate et al., 2007). However, none of these studies have reported in detail how AP molecular properties influence starch hydrolysis. According to the present study, the relationship between AP properties and starch hydrolysis could also be explained using the Hizukuri (Hizukuri, 1986) models of AP shown in Figs. 2 and 3. 3.5.1. AP of waxy corn vs. AP of Ultima triticale (Fig. 2) The AP from starches of waxy corn and Ultima triticale had a comparable molecular size (69.5 nm). However, the Mw, DB and density of AP varied between the above starches (Table 1). Waxy corn AP showed a higher molecular-density (50.1 g/mol/nm3) than that of Ultima triticale AP (44.4 g/mol/nm3). The Mw of AP also varied by 2.0  106 g/mol (Table 1) between the above starches, being highest in waxy corn AP (16.9  106 g/mol). In addition, DB of waxy corn AP was higher by 1% than that of Ultima triticale AP. Variation in AP structure between waxy corn and Ultima triticale can be explained using AP models (Fig. 2). The lower DH at 1 h (29.2%) seen with native waxy corn could be attributed to its highly branched AP structure with a high density of short chains present as clusters (A and B1 chains, Fig. 2A), which may have been compactly organized via inter- and intra-molecular hydrogen bonding. In contrast, the higher DH (69.0%) during the same time interval seen with native Ultima triticale suggests a loosely organized AP structure with a lower extent of branching and a less compact arrangement of short chains (Fig. 2B). After separation of AP from native granules, the influence of inter-molecular association (via hydrogen bonding) of starch molecules (AM and AP) on DH is eliminated. However, the larger number of non-reducing ends in short-chains (A and B1 chains) present in waxy corn AP could have played a major role in the difference in DH between waxy corn AP and Ultima triticale AP (waxy corn AP > Ultima triticale AP). 3.5.2. AP of normal barley vs. AP of CPS red wheat (Fig. 3) Although the AP molecules from the above starches had equal DB (6.9%), their Mw, Rz and density (r) were different; Mw and Rz of normal barley were higher and density was lower than that of CPS Red wheat (Table 1). The equal DB, but the difference in Rz indicates that normal barley AP probably has longer B2 and B3 unit-chains than CPS Red wheat AP (Fig. 3). This would result in the size of amorphous lamellae of normal barley starch AP (Fig. 3A) being bigger than AP of CPS Red wheat starch (Fig. 3B). In addition, the higher Mw and lower r of normal barley AP (Table 1) suggests that its structure is different to that of CPS Red wheat AP. Accordingly, there would be more space between AP clusters (Fig. 3A) in normal barley, resulting in a higher susceptibility towards enzymatic hydrolysis when present within native starch granules (DH ¼ 70.6% at 1 h). In contrast, since the clusters of CPS Red wheat AP are compactly organized (Fig. 3B), the AP molecules in the native starch granules of CPS Red wheat may be more resistant to hydrolysis

692

S. Naguleswaran et al. / Food Hydrocolloids 35 (2014) 686e693

Fig. 2. Structure representations of amylopectins with comparable molecular size, isolated from starches of waxy corn (A) and Ultima triticale (B). A, B1, B2, B3 and C in the representations are the unit branch-chains of amylopectin.

(DH ¼ 53.8% at 1 h). As explained earlier, the DH of isolated AP molecules are mainly influenced by the number of non-reducing ends in their short-chains rather than intra-molecular association (by intra-molecular hydrogen bonding) within the molecules. The

marginal difference in DH (at 1 h) between isolated AP of normal barley (75.4%) and CPS Red wheat (76.0%) could be attributed to equal number of non-reducing ends and DB (Table 1) present in both starches.

Fig. 3. Structure representations of amylopectins with equal degree of branching, isolated from starches of normal barley (A) and CPS Red wheat (B).

S. Naguleswaran et al. / Food Hydrocolloids 35 (2014) 686e693

4. Conclusion The study was conducted to understand the relationship between molecular architecture and degree of amylolysis in starches (normal, waxy, and high-amylose genotypes) from wheat and corn (used extensively in food and industrial applications) with two under-utilized starches such as triticale and barley. The molecular characteristics and branching parameters of AP of the above starches varied significantly (p < 0.05), as a function of botanical origin and genotypes. In all starches, isolated AP and AM were hydrolyzed to a higher extent during the initial stages of hydrolysis than native granules. In isolated AP and AM from normal and highamylose starches, AP was hydrolyzed to a greater extent than AM. The difference in DH during the initial stages of amylolysis by a mixture of a-amylase and glucoamylase reflected variations in the average AP chain length. High DH was correlated with a high proportion of short AP branch chains. The relationship between molecular characteristics of AP and amylolysis of native granules suggested that triticale and barley were comparable to corn and wheat with regards to food and industrial applications. For example, triticale and barley starches, which are composed mainly of short AP branch chains, can be utilized to produce both sugar derivatives and bioethanol in which quantitative conversion of starch to sugar is preferred. On the other hand, corn and wheat starches with a lesser proportion of short AP chains and a slower rate of amylolysis are ideal for the development of low-glycemic foods rather than for bioethanol production. Acknowledgments The Natural Sciences and Engineering Research Council (NSERC) of Canada and the Biorefining Conversions Network (BCN) at the University of Alberta in Edmonton, Canada financially supported this research. Graduate student scholarship from the Alberta Innovates-Technology Futures in Edmonton, Canada to Sabaratnam Naguleswaran is also gratefully acknowledged. Dr. Jinqui Lan and Mr. Zhigang Tian from the University of Alberta are acknowledged for their technical assistance. References Asare, E. K., Jaiswal, S., Maley, J., Baga, M., Sammynaiken, R., Rossnagel, B. G., et al. (2011). Barley grain constituents, starch composition, and structure affect starch in vitro enzymatic hydrolysis. Journal of Agricultural and Food Chemistry, 59, 4743e4754. Bruner, R. L. (1964). Determination of reducing value: 3, 5-dinitrosalicylic acid method. In R. L. Whistler, R. J. Smith, J. N. BeMiller, & M. L. Wolform (Eds.), Methods in carbohydrate chemistry (pp. 67e71). New York & London: Academic Press. Charoenkul, N., Uttapap, D., Pathipanawat, W., & Takeda, Y. (2006). Simultaneous determination of amylose content and unit chain distribution of amylopectins of cassava starches by fluorescent labeling/HPSEC. Carbohydrate Polymers, 65, 102e108. Chen, M., & Bergman, C. J. (2007). Method for determining the amylose content, molecular weights, and weight- and molar-based distributions of degree of polymerization of amylose and fine-structure of amylopectin. Carbohydrate Polymers, 69, 562e578. Chen, J., Wu, K., & Fukuda, H. (2008). Bioethanol production from uncooked raw starch by immobilized surface-engineered yeast cells. Applied Biochemistry and Biotechnology, 145, 59e67. Chung, H.-. J., Liu, Q., Donner, E., Hoover, R., Warkentin, T. D., & Vandenberg, B. (2008). Composition, molecular structure, properties, and in vitro digestibility of starches from newly released Canadian pulse cultivars. Cereal Chemistry, 85, 471e479. Dhital, S., Shrestha, A. K., & Gidley, M. J. (2010). Relationship between granule size and in vitro digestibility of maize and potato starches. Carbohydrate Polymers, 82, 480e488. Englyst, H. N., Kingman, S. M., & Cummings, J. H. (1992). Classification and measurement of nutritionally important starch fractions. European Journal of Clinical Nutrition, 46, 33e50.

693

Evans, A., & Thompson, D. B. (2008). Enzyme susceptibility of high-amylose starch precipitated from sodium hydroxide dispersions. Cereal Chemistry, 85, 480e487. Gao, J., Vasanthan, T., & Hoover, R. (2009). Isolation and characterization of highpurity starch isolates from regular, waxy, and high-amylose hulless barley grains. Cereal Chemistry, 86, 157e163. Gibreel, A., Sandercock, J. R., Lan, J., Goonewardene, L. A., Zijlstra, R. T., Curtis, J. M., et al. (2009). Fermentation of barley by using Saccharomyces cerevisiae: examination of barley as a feedstock for bioethanol production and value-added products. Applied and Environmental Microbiology, 75, 1363e1372. Gilbert, R. G., Gidley, M. J., Hill, S., Kilz, P., Rolland-Sabate, A., Stevenson, D. G., et al. (2010). Characterizing the size and molecular weight distribution of starch: why it is important and why it is hard. Cereal Foods World, 55, 139e143. Goesaert, H., Bijttebier, A., & Delcour, J. A. (2010). Hydrolysis of amylopectin by amylolytic enzymes: level of inner chain attack as an important analytical differentiation criterion. Carbohydrate Research, 345, 397e401. Gomez, L. D., Steele-King, C. G., & McQueen-Mason, S. J. (2008). Sustainable liquid biofuels from biomass: the writing’s on the walls. New Phytologist, 178, 473e485. Hizukuri, S. (1986). Polymodal distribution of the chain lengths of amylopectins, and its significance. Carbohydrate Research, 147, 342e347. Kandil, A., Li, J., Vasanthan, T., Bressler, D. C., & Tyler, R. T. (2011). Compositional changes in whole grain flours as a result of solvent washing and their effect on starch amylolysis. Food Research International, 44, 167e173. Liu, Q. (2005). Understanding starches and their role in foods. In S. W. Cui (Ed.), Food carbohydrates: chemistry, physical properties, and applications (pp. 309e355). Boca Raton, FL., USA: CRC Press, Taylor & Francis Group, LLC. Liu, Q., Gu, Z., Donner, E., Tetlow, I., & Emes, M. (2007). Investigation of digestibility in vitro and physicochemical properties of A- and B-type starch from soft and hard wheat flour. Cereal Chemistry, 84, 15e21. Mason, W. R. (2009). Starch use in foods. In J. BeMiller, & R. Whistler (Eds.), Starch: Chemistry and technology (pp. 745e795). New York, USA: Academic Press of Elsevier Inc. Miao, M., Zhang, T., Mu, W., & Jiang, B. (2011). Structural characterizations of waxy maize starch residue following in vitro pancreatin and amyloglucosidase synergistic hydrolysis. Food Hydrocolloids, 25, 214e220. Murthy, G. S., Johnston, D. B., Rausch, K. D., Tumbleson, M. E., & Singh, V. (2011). Starch hydrolysis modeling: application to fuel ethanol production. Bioprocess and Biosystems Engineering, 34, 879e890. Naguleswaran, S., Li, J., Vasanthan, T., & Bressler, D. (2011). Distribution of granule channels, protein, and phospholipid in triticale and corn starches as revealed by confocal laser scanning microscopy. Cereal Chemistry, 88, 87e94. Naguleswaran, S., Li, J., Vasanthan, T., Bressler, D., & Hoover, R. (2012). Amylolysis of large and small granules of native triticale, wheat and corn starches using a mixture of a-amylase and glucoamylase. Carbohydrate Polymers, 88, 864e874. Naguleswaran, S., Vasanthan, T., Hoover, R., & Bressler, D. (2013). The susceptibility of large and small granules of waxy, normal and high-amylose genotypes of barley and corn starches towards amylolysis at sub-gelatinization temperatures. Food Research International, 51, 771e782. Oates, C. G. (1997). Towards an understanding of starch granule structure and hydrolysis. Trends in Food Science & Technology, 8, 375e382. Rolland-Sabate, A., Colonna, P., Mendez-Montealvo, M. G., & Planchot, V. (2007). Branching features of amylopectins and glycogen determined by asymmetrical flow field flow fractionation coupled with multiangle laser light scattering. Biomacromolecules, 8, 2520e2532. Salman, H., Blazek, J., Lopez-Rubio, A., Gilbert, E. P., Hanley, T., & Copeland, L. (2009). Structure-function relationships in A and B granules from wheat starches of similar amylose content. Carbohydrate Polymers, 75, 420e427. Sevenou, O., Hill, S., Farhat, I., & Mitchell, J. (2002). Organization of the external region of the starch granule as determined by infrared spectroscopy. International Journal of Biological Macromolecules, 31, 79e85. Sharma, V., Rausch, K. D., Tumbleson, M. E., & Singh, V. (2007). Comparison between granular starch hydrolyzing enzyme and conventional enzymes for ethanol production from maize starch with different amylose:amylopectin ratios. Starch-Starke, 59, 549e556. Stevnebø, A., Sahlström, S., & Svihus, B. (2006). Starch structure and degree of starch hydrolysis of small and large starch granules from barley varieties with varying amylose content. Animal Feed Science and Technology, 130, 23e38. Sujka, M., & Jamroz, J. (2007). Starch granule porosity and its changes by means of amylolysis. International Agrophysics, 21, 107e113. Takeda, Y., Hizukuri, S., & Juliano, B. O. (1986). Purification and structure of amylose from rice starch. Carbohydrate Research, 148, 299e308. Tester, R. F., Qi, X., & Karkalas, J. (2006). Hydrolysis of native starches with amylases. Animal Feed Science and Technology, 130, 39e54. Uthumporn, U., Zaidul, I. S. M., & Karim, A. A. (2010). Hydrolysis of granular starch at sub-gelatinization temperature using a mixture of amylolytic enzymes. Food and Bioproducts Processing, 88, 47e54. Wessa, P. (2012). Spearman rank correlation (v1.0.1) in free statistics software (v1.1.23r7). Office for Research Development and Education. http://www.wessa.net/ rwasp_spearman.wasp/ Accessed 09.07.13. Yoo, S., & Jane, J. (2002). Molecular weights and gyration radii of amylopectins determined by high-performance size-exclusion chromatography equipped with multi-angle laser-light scattering and refractive index detectors. Carbohydrate Polymers, 49, 307e314.