Impact of enzymatic hydrolysis on the crystal structure, and thermal and textural properties of corn starch

Corn starch has been widely used in biomaterial industry as one of the raw materials. The properties of starch such as annealing and the different ratio of amylose and amylopectin in different starch limit its application in production. These properties play a vital role in establishing relationships between other starches in structures and functions. Normal corn starches were treated with isoamylase for different hours and the gelatinization phenomena was observed with differential scanning calorimetry (DSC). The parameters of fracturability, cohesiveness, springiness and adhesive force were all detected by textural profiles analysis. After treatment with isoamylase, the content of amylose chains was increased, hardness and springiness of starch were all enhanced, while cohesiveness and adhesive presented an opposite trend. It was the different ratio of amylose and amylopectin that led to changes in physicochemical properties.


INTRODUCTION
Starch based polymer materials are seen as biodegradeable polymer matrix reinforced with a natural element and are one of the most often investigated biopolymers which started in the late 1980s (Vilaseca et al., 2007).Many literatures have reported on starch based polymer materials (Fringant et al., 1996;Wang et al., 2004;Rosane et al., 2005;Tzankova et al., 2007;Daniele et al., 2009;Canigueral et al., 2009;Jiirgen, 1998).However, the molecule structure of starch is one of the most important problem, which significantly influences the stress and stiffness properties of starch based biodegradable polymer materials.On the other hand, some literatures have reported that starch degrading enzymes have been used to modify the physicochemical properties of polysaccharides to achieve the desired functional *Corresponding author.E-mail: maying@hit.edu.cn.Tel: +086 0451-86282903; Fax: +086 0451-86282906.
properties (Kyungsoo and Paul, 1997;Karakatsanis and Liakopoulou, 2000;Ma et al., 2006;Olivia et al., 2010;Hieronim et al., 2005).The most important tool in providing a saccharride with a specific composition is the use of starch hydrolyzing enzymes (Sakina et al., 2009).Starch was hydrolyzed with a-amylase (Lohmar, 1954;Sakina et al., 2009), and hydrochloric acid (Wang et al., 2001), which strongly affected the amylose content, molecular size of amylose and amylopectin.Although Atsuo and John (1996) have reported that isoamylase hydrolysis can affect the structure and molecule of starch, little information have reported starch hydrolysis with isoamylase, and the relationships between hydrolyzed time and properties of corn are not clear.Therefore, the objective of this study is to investigate the effects of hydrolyzed time on crystal structure, and thermal and textural properties of corn starch.

Materials
Commercial corn starch was purchased from the Dacheng Ltd.,   10,15,20,25,30,35,40,45,50,55 and 60 h.The reaction was terminated by heating the solution for 15 min.The hydrolyzed starch samples were neutralized to pH 6.5 ~ 7.0 with 1 N NaOH at 60°C according to the method of Cheetham and Tao (1997).Then, the solution was collected by centrifugation at 3000 g for 15 min, and the paste was determined with texture properties by texture analyzer.After texture determination, the remaining paste was dried at 60°C for 36 h to obtain hydrolyzed corn starch.

Determination of amylose
Amylose in the starch samples was determined according to the colorimetric procedure of Chrastil (1987).This is a colorimetric method utilizing the property of amylose to form an iodine complex, which was measured by spectrophotometer.The parameter was set to 620 nm because the absorbance value was maximum at this wavelength.The amylose content in the starch samples was determined from the standard amylose graph prepared.

X-ray diffraction determination
The X-ray patterns and crystallinity (Nara and Komiya, 1983) of hydrolyzed corn starch were obtained with an X-ray diffractometer (Rigaku Co. Ltd., Rint-2000 type, Tokyo, Japan) operated at voltage of 40 kV and current of 30 mA, target was Cu.Hydrolyzed corn starch (ca.8%, w/w, moisture content) were packed tightly into the aluminium sample holder and diffraction data were collected over an diffraction angle (2 ) range from 3 to 60° at 0.02° step size and with a scanning speed of 8.0 °/min.
The pans were sealed hermetically to prevent moisture lose and kept overnight.For all DSC runs, a sealed empty aluminum pan was used as reference.The sample was held isothermally at 20°C for 1 min before being heated from 20 to 140°C at 10°C/min.The onset, peak and conclusion temperatures, and gelatinization enthalpy ( Hg, J/g) of gelatinization were determined.The peak temperature and the enthalpy ( Hg, J/g) associated with the hydrolyzed starch melting peak appearing between 40 and 90°C were calculated.All the DSC measurements were performed in triplicate and the results were presented as mean values.

Textural properties of corn starch
A texture analyzer (TA-XT2, Texture Technologies Corp., UK) with a 50 kg load cell was used to determine the paste texture properties using a two-cycle compression.The analyser was linked to a computer that recorded the data via a software program called Texture Expert Excede Version 1.0 (Stable Micro Systems Software).A two-cycle compression force versus time program was used to compress the samples till 80% of the original gel thickness returned to the original position and again compressed.And then, the paste were compressed with a 6-mm probe at pre-test speed of 1.0 mm/s, test speed and post-test speed of 0.5 mm/s.Parameters recorded from the test curves were hardness and adhesiveness, cohesiveness and stringiness (Gupta et al., 2007).The texture analyses were repeated 10 times per samples.

Statistical analysis
All tests were performed at least in triplicate.Variance analysis (ANOVA) and Duncan multiple-range test were performed by the procedure of SAS 8.0 (SAS Institute, Cary, NC, USA).

Effects of hydrolyzed time on amylose content of corn starch
The amylose content of corn starch is shown in Table 1.
The amylose content of native corn starch was 25.50%, and was 91.80% when hydrolysis was 60 h.The amylose content increased rapidly within 40 h, and then increased slowly with hydrolyzed time.In the hydrolyzed process, the enzyme got into the internal of the corn starch granule, and broke down the corn starch molecule to linear chain, so the amylose content increased, when all the branched molecule in corn starch granule was broken down, the amylose content continued to increase.Enzyme first hydrolyzed the amorphous regions before attacking the crystalline regions and both amylose and amylopectin were hydrolyzed simultaneously to smaller molecular sizes.This is similar to the report of Wang and Wang (2001).Gao and Yang (1993) reported that hydrolyzed process could increase the amylose content of potato in the first hours and then changed slowly.From the earlier results, the optimum hydrolyzed time for corn starch was 40 h, which got the highest amylose content.

X-ray analysis of hydrolyzed corn starch
Figure 1 shows the X-ray patterns of corn starch samples of different hydrolyzed time.Corn starch hydrolyzed for 0 and 5 h, showed an A-type diffraction (Norman et al., 1998), while others cross between A-and V-type patterns (Mua and Jackson, 1997b).Percent crystallinity with different hydrolyzed time was 23.56, 22.20, 19.18, 16.52, 12.01, 10.69 and 9.80%, respectively (Table 2).The crystallinity of corn starch hydrolyzed for 0 h was the largest, the crystallinity decreased with the hydrolyzed time, hydrolyzed process destroyed the amorphous region in the corn granules, the ratio of crystalline region decreased during hydrolyzed process and the crystallinity degree decreased.This result was in accordance with corn starch of different amylose content (Matveev et al., 2001).

Thermal properties of hydrolyzed corn starch
Thermal characteristics (Figure 2), as measured by DSC, was observed for corn starch of different hydrolyzed time, respectively.The thermal parameters were also shown in

Temperature (°C)
. Thermal characteristics of hydrolyzed corn starch determined by DSC.
Table 3. Thermal properties of corn starch with hydrolyzed time.Table 3.The melting temperature (T o ), peak temperature (T p ), conclusion temperature (T c ) and gel enthalpy ( H g ) decreased with hydrolyzed time increase within 30 h, and then changed slowly.Enzyme first hydrolyzed the amorphous regions before attacking the crystalline regions and both amylose and amylopectin were hydrolyzed simultaneously to smaller molecular sizes.Small starch molecule is very easy to gelatinize, and there maybe some big molecule, therefore, the T o , T p , T c and H g decreased with hydrolyzed time.When the hydrolyzed process finished, the smaller molecular sizes (amylose) reached the highest ratio, the T o , T p , T c and H g changed slowly.Gao and Yang (1993) reported the same results when they hydrolyzed potato starch by isoamylase.

Textural properties of hydrolyzed corn starch
The textural parameters of corn starch paste are shown in Table 4. Hardness increased from 15.15 to 26.45 N with hydrolyzed time increase from 0 to 60 h.The  (Mua and Jackson, 1997a), which contributed to the hardness and adhesiveness changes.The cohesiveness and stringiness value had little changes during hydrolysis process, and the molecule structure had not significant effect on cohesiveness and stringiness.

Conclusions
Starch from different hydrolyzed time exhibited different physico-chemical properties after isoamylase treatment and their inherent molecular structures before modification played a very important role in determining the functions.Enzyme treatment first hydrolyzed the amorphous regions before attacking the crystalline regions and both amylose and amylopectin were hydrolyzed simultaneously to smaller molecular sizes.Starch gelation was strongly affected by its amylose content, molecular size of amylose and amylopectin, and short and long branch chains in amylopectin.Starch consisting of amylose with a larger molecular size and amylopectin with a larger amount of long branch chain would produce firmer gels.More work is needed to further probe the reaction pattern of enzymatic hydrolysis towards starch from different origins.
the same column not followed by the same lowercase superscript letter are significantly different (p < 0.05).To = onset temperature; Tp = peak temperature; Tc = conclusion temperature; Hg = gelatinization enthalpy.

Table 1 .
Changes of amylose content with hydrolyzed time.

Table 2 .
Changes of crystallinity degree with hydrolyzed time.

Table 4 .
Textural properties of corn starch with different hydrolysis time.