Granule Structural, Crystalline, and Thermal Changes in Native Chinese Yam Starch After Hydrolysis with Two Different Enzymes–A-Amylase and Gluco-Amylase
Total Page：16
File Type：pdf, Size：1020Kb
Starch/Sta¨ rke 2011, 63, 75–82 DOI 10.1002/star.201000104 75 RESEARCH ARTICLE Granule structural, crystalline, and thermal changes in native Chinese yam starch after hydrolysis with two different enzymes–a-amylase and gluco-amylase Xia Li1, Wenyuan Gao1, Yanli Wang2, Qianqian Jiang1 and Luqi Huang3 1 School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China 2 Tianjin Press of Chinese Herbal Medicines, Tianjin Institute of Pharmaceutical Research, Tianjin, China 3 Institute of Chinese Materia Medica, China Academy of Chinese Medicinal Sciences, Beijing, China Starch extracted from Chinese yam was characterized by scanning electron microscope Received: August 17, 2010 (SEM), X-ray powder diffractometer (XRD), and differential scanning calorimeter (DSC) in Revised: September 20, 2010 the process of enzymatic hydrolysis. Yam starch was digested by a-amylase and gluco- Accepted: September 23, 2010 amylase for different lengths of time, respectively, and two different enzymatic hydrolysis results were compared. The most notable phenomenon revealed by SEM after a-amylase hydrolysis was the formation of the cavum in the center of the starch granules, while after gluco-amylase hydrolysis, the outer layer of the granules was peeled off and then some granules even broke into pieces. The XRD of the two enzyme hydrolyzed starches revealed the crystal type of the starch changed from typical C-type XRD pattern to the representative A-type pattern in the process of enzymatic hydrolysis. The above results also demonstrated that the partially B-type polymorph was more easily degraded than A-type. The thermal result showed that the modified yam starches by both enzymes exhibited increased peak gelatinization temperatures (Tp) and decreased gelatinization enthalpy (DH). Keywords: a-Amylase / Gluco-amylase / Physicochemical properties / Thermal property / Yam starch 1 Introduction for physico-chemical (e.g., AM content, swelling power, solubility, light transmittance, water binding capacity, and Starch is an abundant natural polysaccharide that is turbidity), morphological (including shape and size), renewable and fully biodegradable. It consists of two major thermal, and crystalline properties in our previous study. components: AM, a mostly linear a-D-(1-4)-glucan and AP, These starches from different cultivars showed different an a-D-(1-4)-glucan, which has a-D-(1-6) linkages at the properties [5–7]. XRD revealed that all the yam starches branch point and contains only glucose as monomer. from different cultivars displayed the typical C-type XRD These two kinds of molecules form a semi-crystalline pattern. It was well known, C-type starch is a mixture of entity. The crystallinity is exclusively associated with the both A-type and B-type. However, further information on AP component, while the amorphous regions mainly the yam starch granule structure is very limited. represent amylase [1–4]. Enzymatic modification could change the physico- Starch separated from different cultivars of Chinese chemical properties involving morphological, crystalline yam (Dioscorea opposita Thunb.) has been investigated properties, and so on [8–11]. Enzyme molecules affect the granules in two ways [12]. First, enzymes erode the outer surface of granule and cause occurring of charac- Correspondence: Professor Wenyuan Gao, Department of teristic fissures and pits (exocorrosion). Second, enzymes Natural Products and TCM, School of Pharmaceutical Science digest channels leading to the granule center which weak- and Technology, Tianjing University, No. 92 Weijin Road, Nankai ens granule integrity and leads consequently to its break- District, Tianjin 300072, P. R. China E-mail: [email protected] down (endocorrosion). A biphasic trend has been Fax: þ86-22-8740-1895 observed with an initial rapid hydrolysis of the amorphous ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com 76 X. Li et al. Starch/Sta¨ rke 2011, 63, 75–82 regions [8, 9, 13] followed by a decreased hydrolysis. Some glucoamylolysis was 20 mmol/L acetate buffer at pH 4.5. researchers demonstrated that the amorphous and crystal- The solution was preheated in a heating mantle until it line regions were hydrolyzed at a similar ratio [14, 15]. The reached the enzyme optimum temperature (508C), and differences in the rate and degree of enzyme hydrolysis of hydrolysis was initiated by the addition of 200 U granular starch have also been attributed to differences in enzyme/g dry starch to the slurry. Aliquots of 5 mL were granular size, extent, and type of chain interactions within taken after 2 h and frequently thereafter until 24 h. At least the granule (i.e., degree and type of crystallinity) and the three slurry samples were prepared for each starch type kind of enzyme (i.e., a-amylase, b-amylase, and gluco- for the enzyme hydrolysis in order to collect duplicate amylase) [16, 17]. Many researchers have reported struc- samples during the course of 24 h. The aliquots were tures and physical properties of A-type and B-type starch as centrifuged at 3000 r/min for 15 min, and the supernatant affected by enzyme hydrolysis, with the B-type crystals was immediately determined for soluble sugars content by being more resistant to enzyme hydrolysis than the A-type using the phenol sulfuric method [21]. Finally the starch [18, 19]. However, there was little information on enzyme was isolated by filtration, and then dried in a forced-air oven hydrolysis of the C-type starch. (508C) for 24 h. Enzymatic modified starch could be very helpful in under- standing the structure of starch granules. In addition, the 2.4 Morphological properties enzymatic hydrolysis of starch granules may be centripetal (from the surface toward the center of the starch granule) or The morphological features of the native starch and enzy- centrifugal (from the center to the periphery) [20]. In this matic hydrolysis starches were observed with a scanning study, the granule morphological, crystalline, and thermal electron microscope (ESEM Philips XL-30). And the properties changes during the enzymatic hydrolysis were images were taken at an accelerating voltage of 20 kV. investigated and compared by SEM, XRD, and DSC. Micrographs were recorded at 1000Â magnification to ensure clear images. 2 Materials and methods 2.5 X-ray diffraction studies 2.1 Materials XRD patterns of native yam starch and enzymatic hydrolysis starches were analyzed using Rigaku D/max The Chinese yam (D. opposita Thunb.) flour was provided by 2500 X-ray powder diffraction (Rigaku, Tokyo, Japan) Henan Wanxi Pharmaceutical Company (Henan Province, with Nickel filtered Cu Ka radiation (l ¼ 1.54056 A˚ )at China). Bacterial a-amylase (20 000 U/g) and gluco-amylase a voltage of 40 kV and current of 200 mA. The scattered were obtained from college of bioengineering, Tianjin radiation was detected in the angular range of 3–408 (2u), University of Science and Technology (Tianjin, China). with a scanning speed of 88 (2u)/min, and step size of 0.068 (2u). 2.2 Starch isolation 2.6 Differential scanning calorimetry (DSC) The yam flour was washed and sieved with a 160 m mesh sifter. After depositing, the supernatant was removed by Thermal properties were assessed by a Perkin-Elmer suction and the settled starch layer was resuspended in Pyris/Diamond DSC-7 DSC (Perkin-Elmer, Norwalk, CT, distilled water. After seven or eight cycles of depositing and USA). The instrument was calibrated with indium and an resuspending repeatedly, the slurry containing starch was empty pan was used for reference. Starch (3.5 mg, db) centrifuged at 3000 r/min for 20 min. The supernatant was was weighed into an aluminum DSC pan and then discarded and the upper non-white layer was removed. moistened with 8.0 L of deionized water using a micro- The white layer was resuspended in distilled water and syringe. The pan was hermetically sealed and allowed to recentrifuged three to five times. The starch suspension stand for 1 h prior to analysis. The sample was scanned obtained was dried in a convection oven at 508C until from 20 to 958C at a rate of 108C/min. The onset (To), peak weight constancy. (Tp), and conclusion (Tc) gelatinization temperature and enthalpy (DHgel) were automatically computed. 2.3 Enzymatic hydrolysis 2.7 Statistical analysis A slurry containing 20 g native yam starch (db) and 75 mL buffer was incubated at 508C in a flask equipped with a The statistical analysis was performed according to the condenser and a mixer. The buffer in a-amylolysis was V13.0 SPSS system. Mean and SEs were used through- 20 mmol/L phosphate buffer at pH 6.9, whereas that of the out, and the statistical significance between the mean ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com Starch/Sta¨ rke 2011, 63, 75–82 Granule structural, crystalline, and thermal changes in native Chinese yam starch 77 values was assessed applying a Duncan’s multiple range were shown in Fig. 3. The native starch showed the pres- tests. A probability of p<0.05 was considered significant. ence of large oval or spherical to small irregular-shape granules. In Fig. 2, with the increase in hydrolysis time, some 3 Results and discussion small starch granules brokedown and there were still some integrated granules existed. When subjected to 12 h of 3.1 Enzymatic hydrolysis of starch granules enzymatic hydrolysis, the starch granules became a cavum due to the heavy degradation of the inner structure Selected results of enzyme hydrolysis of yam starches by a-amylase enzymatic hydrolysis. However, the surface were depicted in Fig. 1. During the course of 24 h hydroly- of the starch granules still remained smooth with little sis, the hydrolysis yield of yam starch after modified by a- fissure. After 24 h of hydrolysis, the starch granules had amylase and glucoamylase increased gradually with the been corroded completely and fell to small pieces under increase in hydrolysis time.