Study of galacto‐oligosaccharide formation from lactose using Pectinex Ultra SP‐L

BACKGROUND: Galacto‐oligosaccharides (GOS) are synthesised from lactose by transglycosylation using β‐galactosidase (EC 3.2.1.23) and are recognised as prebiotics. The commercial enzyme preparation Pectinex Ultra SP‐L produced by Aspergillus aculeatus possesses β‐galactosidase activity; however, because its use has been directed towards the formation of 6′‐β‐galactosyl‐lactose, no data have been reported on the formation of other GOS. Since the composition of the oligosaccharide mixture obtained during lactose hydrolysis may affect the prebiotic properties, in this study the influence of various parameters (pH, temperature, time and enzyme and lactose concentrations) on the formation of GOS using Pectinex Ultra SP‐L was investigated. RESULTS: High‐performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD) analysis allowed the detection of disaccharides other than lactose, trisaccharides and minor amounts of higher‐molecular‐weight GOS. The main GOS formed were a trisaccharide identified as β‐D ‐Galp‐(1 → 6)‐Lac (6′‐β‐galactosyl‐lactose) and a disaccharide identified as β‐D ‐Galp‐(1 → 6)‐D ‐Gal (galactobiose). Other GOS detected were tentatively identified as β‐D ‐Galp‐(1 → 6)‐D ‐Glc (allolactose), β‐D ‐Galp‐(1 → 3)‐D ‐Glc and β‐D ‐Galp‐(1 → 3)‐Lac. Trisaccharide formation was favoured by a pH increase from 4.5 to 6.5, whereas the disaccharide content increased as the pH decreased, reaching a level of 11% at pH 4.5. 6′‐β‐Galactosyl‐lactose production increased gradually with increasing temperature, attaining a maximum value of 17% at 60 °C after 7 h, whereas disaccharide formation was optimal at 50 °C, reaching a level of 10% after 24 h. CONCLUSION: The results indicate that Pectinex Ultra SP‐L can be used to obtain GOS mixtures of different composition depending on the operating conditions. It has been shown for the first time that Pectinex Ultra SP‐L can be used for the selective formation of disaccharides. Copyright © 2008 Society of Chemical Industry     

Effect of enzyme‐aided pressing on anthocyanin yield and profiles in bilberry and blackcurrant juices

Bilberries (Vaccinium myrtillus) and blackcurrants (Ribes nigrum) were treated with extensive dosages of commercial cell wall degrading enzyme preparations, i.e. Econase CE, Pectinex Ultra SP‐L, Pectinex Smash, Pectinex BE 3‐L and Biopectinase CCM. The enzymes were dosed based on the polygalacturonase activity. The juice yield was improved in both berries as a result of the enzymatic treatment. The improvement was more pronounced with blackcurrants owing to their thicker cell walls. The impact of the enzymatic treatment on anthocyanins present in the juices was investigated using HPLC‐DAD. The enzyme preparations affected the contents and composition of anthocyanins in the juices. Pectinex Ultra SP‐L, Pectinex Smash, Pectinex BE 3‐L and Biopectinase CCM increased the total content of anthocyanins by 13–41% in the bilberry juices and by 18–29% in the blackcurrant juices. Econase CE, however, produced a dramatic decrease in the total anthocyanin content in the bilberry juice due to its enzyme profile, whereas no such effect was observed with the blackcurrant juice. All the enzyme mixtures tested produced a total or extensive loss of anthocyanidin galactosides in bilberry juice. Commercial enzyme preparations used in the production of berry juices can improve extraction of anthocyanins into the juice. However, they may effectively hydrolyse certain glycosides and thus affect the profile of extracted anthocyanins. Copyright © 2005 Society of Chemical Industry     

Optimising enzymatic maceration in pretreatment of carrot juice concentrate by response surface methodology

In this paper, enzyme preparation for carrot pulp maceration was screened out and enzymatic maceration processing condition of the carrot pulp was optimised by response surface methodology for carrot juice concentrate. Pectinex Smash XXL was the best commercial enzyme preparation than Pectinex Ultra SP‐L, Pectinase FNP‐1 and cellulose FNC‐1 employed in the carrot juice processing in the study. The effect of enzyme concentration and incubation time and their complex interaction on juice β‐carotene content, juice yield and viscosity in the maceration process was studied by using experiments of central composite rotatable design. The results indicated that under the optimal conditions that the enzyme concentration was 100 mL t^?1 and incubation time was 80 min, the juice β‐carotene content was ≥54.2 mg kg^?1, the juice yield ≥63.5% and the juice viscosity ≤2.1 mPa S.     

Starch Granule Size Distribution in Grains at Different Positions on the Spike of Wheat (Triticum aestivum L.)

The present study was carried out to characterize the effects of soil water status, cultivar and grain position on starch granule size distribution. The starch granule size distribution and protein and starch content were determined from three wheat cultivars with reference to the position of the grain on the spike. The results showed that the contents of A‐, B‐ and C‐type starch granules were affected by soil water, genotype and soil water×genotype interaction. Grain position was significant for A‐ and C‐type starch granules. Genotype×grain position and soil water×genotype×grain position interactions only affected C‐type starch granules. Both basal and distal grains on middle spikelets contained three types of starch granules: smaller C‐type granules with diameters <2.0 μm, medium B‐type granules with diameters 2.0–9.8 μm and large A‐type granules with diameters >9.8 μm at maturity. Both percent volume and percent surface area distribution of starch granules in basal grains showed the typical three populations, and those in distal grains exhibited a two‐peak curve. The volume and surface area percentage of C‐type granules and protein content in basal grains were higher than that in distal grains, but those of A, B‐type granules in basal grains were lower than that in distal grains. Compared with irrigation, the percent volume and surface area of B, C‐type starch granules and protein content in basal and distal grains were significantly increased, and those of A‐type were evidently decreased in rainfed condition.     

Effect of Processing of Sword Beans (Canavalia gladiata) on Physicochemical Properties of Starch

The effect of various processing methods on physico‐chemical properties of sword bean starch was studied. Seed grits and flour were cooked with and without soaking, wet‐autoclaved, and roasted. The changes in starch associated with these processing methods were studied by observing changes in granular structure, water solubility index, water absorption index, molecular size distribution and the degree of gelatinization estimated by differential scanning calorimetry. Intact, ungelatinized starch granules of raw and dry‐heat treated samples were observed under the light microscope. The starch granules were elliptical in shape and had an average length and breadth of 37–40 μm and 27 μm, respectively. Wet‐processed samples had the lowest water solubility, higher water absorption and lower gelatinization enthalpies than the raw bean flour, whereas the dry heat‐treated samples showed higher water solubility and higher gelatinization enthalpies. The starch molecular size distribution pattern showed a higher amount of high molecular size carbohydrates in dry heat‐treated samples and a large fraction of intermediate molecular size carbohydrates in the wet‐processed samples. The low molecular size carbohydrate content was low in wet‐processed samples where processing was done with excess water.     

Composition and Physico-chemical Properties of Starch from Cow Cockle (Saponaria vaccaria L.) Seeds

Starch isolated from two accessions of cow cockle (Saponaria vaccaria L.) seeds consisted of uniform size polygonal granules 0.3–1.5 μm in diameter, having 18% amylose content and a melting peak temperature 68°C. The intact granules gave the A-type X-ray diffraction pattern. The debranched starch exhibited the typical bimodal distribution of amylopectin chains (CL 45 and 12) on Biogel P-10. Lipids associated with the cow cockle starch preparations, involving surface and internal lipids, included triglycerides (45%), free fatty acids (FFA, 39%), lysophospholipids (10%) and diglycerides (6%). The major fatty acids found in polar and FFA fraction were palmitic, oleic and linoleic acids. Cow cockle starch exhibited similar viscoamylograph viscosity, solubility and swelling profiles to those of rice starch. Concentrated starch gels also showed the typical viscoelastic behavior of non-waxy cereal starches. In vitro digestibility studies of cow cockle granular starch with B. Subtilis indicated that this starch, compared to rice, is very susceptible to α-amylolysis presumably because of its small granule size.     

Laboratory Wet‐Milling of Grain Sorghum With Abbreviated Steeping to Give Two Products

Short‐time and no‐time steeping were used in the wet‐milling of grain sorghum to give two products, starch in over 78% recovery (starch basis) plus the remaining grain solids. In the wet‐milling process 1.5 parts of fresh water were used per part of grain to compensate for drying and transfer losses. Starting with 100 g (dry solids) of commercial No.2 grain sorghum, steep time (1—3 h), steep temperature (25—60 °C), and coarse‐grinding speed (7, 500—12, 500 rpm with tip speed of 90.4—150.7 km/h) were varied in a model study; starch recovery, starch lightness (L*), and damaged starch were the responses. Grain sorghum was steeped with twice its weight of process water containing 0.2% sulfur dioxide and the steeped kernels were added to an equal volume of process water and the mixture was ground for 6 min in a Waring blender with blunt blades (d = 3.2 cm). The course‐ground material was sieved (opening 1190 μm) to collect the bran/germ, and the throughs were allowed to stand. The sedimented phase was finely ground by one pass through a plate mill, and the fine fiber removed by sieving (opening 73 μm). The slurry was adjusted to a specific gravity of 1.04, and the starch was separated from the protein fraction on a starch table. The protein fraction was combined with the steep‐liquor concentrate (54% solids) plus the bran/fiber and the fine‐fiber fractions to give the co‐product, which contained 70% moisture (wet basis, wb) and 27% protein (dry basis, db). In the surface response study, recovery of starch ranged from 57 to 89%, starch protein content from 0.4 to 0.5%, and lightness (L*) from 90 to 93. Damaged starch content was constant at around 0.4%. Commercial grain sorghum gave the highest starch recovery (90%) after steeping 2 h at 55 °C and coarse‐grinding at 10, 500 rpm; whereas a food‐grade, a white and a red sorghum gave 85, 84, and 80% recoveries, respectively. The four starches had lightness (L*) values of 93—94 and damaged starch contents of 0.4—0.6%. When the commercial grain sorghum was wet‐ground without steeping in 2 parts of water containing 0.2% sulfur dioxide, a 78% recovery of starch was obtained with L* 93.7 and starch damage 0.5%.     

Heat‐Moisture Treatment and Enzymatic Digestibility of Peruvian Carrot,Sweet Potato and Ginger Starches

The aim of this work was to study the effects of heat‐moisture treatment (27% moisture, 100°C, 16 h) and of enzymatic digestion (alpha‐amylase and glucoamylase) on the properties of sweet potato (SP), Peruvian carrot (PC) and ginger (G) starches. The structural modification with heat‐moisture treatment (HMT) affected crystallinity, enzyme susceptibility and viscosity profile. The changes in PC starch were the most pronounced, with a strong decrease of relative crystallinity (from 0.31 to 0.21) and a shift of X‐ray pattern from B‐ to A‐type. HMT of SP and G starch did not change the X‐ray pattern (A‐type). The relative crystallinity of these starches changed only slightly, from 0.32 to 0.29 (SP) and from 0.33 to 0.32 (G). The extent of these structural changes (PC > SP > G) altered the susceptibility of the starches to enzymatic attack, but not in same order (PC > G > SP). HMT increased the starches digestion, probably due to rearrangement of disrupted crystallites, increasing accessible areas to attack of enzymes. The viscosity profiles and values changed significantly with HMT, resulting in higher pasting temperatures, decrease of viscosity values and no breakdown, i.e., stability at high temperatures and shear rates. Changes in pasting properties appeared to be more significant for PC and SP starch, whereas the changes for G starch were small. Setback was minimized following HMT in SP and G starches.     

Studies of Flavor Encapsulation by Agents Produced from Modified Sago and Tapioca Starches

The efficiency of sago and tapioca starch stearates for encapsulating lemon oil were studied and compared to the efficiency of gum arabic. The stearates were prepared by esterification of stearic acid with starch. To accomplish esterification, the stearic acid was first coated on the surface of the starch granules. Then the coated granules were heated at 150 °C for 2 h to obtain sago or tapioca starch stearate (SSS or TSS). SSS or TSS can be prepared as ready‐to‐use products in the form of pregelatinized‐hydrolyzed sago or tapioca starch stearate (PGHSSS or PGHTSS). The resulting modified starches were used for encapsulation of lemon oil. The lemon oil encapsulating efficiency for SSS with DS 0.009 and 0.014 were close to that of gum arabic, whereas the encapsulating efficiency for PGHSSS with DS 0.0052 and 0.016 were higher than that of the gum arabic. The TSS and PGHTSS provided encapsulating efficiencies lower than the gum arabic.     

Comparison of the GFFF and LALLS Methods for the Measurement of Starch Granule Size Distribution in Spring Barley Caryopses

A newly developed method GFFF (Gravitational Field‐Flow Fractionation) and the well known method LALLS (Low Angle Laser Light Scattering) were used to assess starch granule size distribution of ten varieties of spring barley. As a distribution criterion, the ratio of starch granule content larger than 8 μm (type A) and smaller than 8 μm (type B) was chosen. Both methods divided the observed set in a similar way. Varieties Akcent, Forum and Atribut formed a variety set with the highest ratio of large and small starch granules. Varieties Scarlet and Kompakt had intermediate ratios. The remaining five varieties Amulet, Novum, Olbram, Tolar and Krona had the lowest ratios of large and small starch granules. Statistical analysis showed that there was a highly significant correlation between the GFFF and LALLS methods.     

Enzymatic production of linear long-chain dextrin from sago (Metroxylon sagu) starch

Pullulanase (EC.3.2.1.41) was used to generate more linear-chain dextrin from sago starch (24.9% amylose) such that the resulting product could act as a high amylose starch. A starch suspension of 5.0% (w/v) sago starch was heated at 100 °C for 45 min and, after cooling, the gelatinized sago starch was hydrolyzed with 2.0% (v/dry weight starch) pullulanase (Promozyme 400L, Novozymes A/S, Denmark) for 24 h. The linear long-chain dextrin (LLD) content of the hydrolysate after drying, was then compared with the initial LLD content. The surface morphology of the starch granules was observed with a scanning electron microscope (SEM). The effects of gelatinization, time of reaction, pretreatment with different strengths of hydrochloric acid prior to enzyme hydrolysis, and starch and enzyme concentrations were studied. Raw sago starch was resistant to the action of pullulanase, but caused an increase in the LLD of that sago starch from an initial concentration of 24.9–33.2% following gelatinization. The best conditions to maximize the amount of LLD were 5.0% (w/v) sago starch, 2.0% (v/w) enzyme and 12 h reaction time. Acid pretreatment of the sago starch did not cause greater improvement in the accessibility and susceptibility of pullulanase as the LLD content, following pullulanase action did not change significantly. Shrinkage on the surface of the starch granules was observed with the SEM.     

Preparation and structural properties of small‐particle cassava starch

Acid and enzyme hydrolyses followed by ball milling were applied to fracture cassava starch granules. Microscopic and chromatographic evidence suggested different mechanisms of the two hydrolyses. Using the enzyme process, granules with a sponge‐like structure and shells with the interior hydrolysed were produced. Amylose and amylopectin were subjected equally to multiple attacks by enzymes, with no significant change in granule crystallinity. The hydrolysed residues could not be effectively broken down by ball milling, although the crystallinity was destroyed. In contrast, the acid treatment caused superficial external corrosion, mainly at the amorphous lamellae, ie the branch points of amylopectin. Acid‐lintnerised starch granules were mostly of Degree of polymerization, DP 10–15 and exhibited increased crystallinity and brittleness, making them more susceptible to breakdown upon milling. Ball milling, although destroying some degree of crystallinity, could effectively reduce the size of acid‐hydrolysed starch, with no further degradation of amylodextrin molecules. By a combination of lintnerisation and ball milling, smaller particle starch (3–8 µm compared with 3–30 µm for native starch) could be produced. It is clear that removal of the amorphous phase prior to milling is critical for effective rupture of the granules. Copyright © 2003 Society of Chemical Industry     

酶解生产微胶囊胡萝卜速溶粉工艺研究

胡萝卜在我国大部分省市均有种植,其β-胡萝卜素、维生素、矿物质含量丰富。本文利用微胶囊技术将经酶解处理的胡萝卜加工成一种速溶产品。利用液态果胶酶PectinexUltraSP-L和PectinexSMASHXXL酶解胡萝卜浆,提高了胡萝卜汁的产量。结果表明,果胶酶PectinexUltraSP-L最佳用量(以胡萝卜浆重量计)为0.09ml/kg胡萝卜浆液,PectinexSMASHXXL最佳用量0.18ml/kg胡萝卜浆液,最佳温度50℃,酶解时间2h,酶处理后出汁率能提高到82.8%。加入15%玉米油,3%乳化剂,3%酪朊酸钠和40%糊精微胶囊化后喷雾干燥得到胡萝卜速溶粉。该产品既能直接加水冲调饮用,也能作用一种成分加入到其它配方食品中,应用范围得到了进一步的扩大。     

The effect of acid and glucoamylase hydrolysis on partial physicochemical properties and characteristics of Dioscorea alata Linn. starch

Dioscorea alata Linn. (D. AL) rich in starch was widely used as food and medicine. This work aimed to compare the effect of acid and glucoamylase on the starch of different reaction time. The physicochemical properties of the native and hydrolyzed starches, such as swelling power (SP), starch solubility (SOL), and paste clarity were also determined in this study. The native and modified starches by acid and glucoamylase were compared and characterized by scanning electron microscope (SEM) and secondary derivative FT‐IR spectroscopy. The morphological characteristics of native D. AL starch granules revealed by SEM varied from round to oval or elliptic with smooth surface. SEM showed that glucoamylase primarily attacked the exterior of starch granules and permeate into the interior by the cracks formed on the surface. Owing to acid hydrolysis, the starch granules were depressed and transformed to bread‐like in shape and then broke into irregular lumps with particles adhered. From secondary derivative FT‐IR spectrum, the intensities of the two peaks from acid and enzyme hydrolysis starches at 1137 and 1066/cm were stronger than native starch. Some new peaks appeared due to hydrolysis. The digested starches exhibited a lower SOL and SP than native starch in water as the temperature increasing. The light transmittance of the digested starches was significantly higher than that of native starch paste and decreased with increase in storage period.     

Ultrastructural analysis of buckwheat starch components using atomic force microscopy

Morphological and structural features of buckwheat starch granules and nanocrystals were examined using atomic force microscopy and dynamic light scattering. Partially digested starch granules revealed a clear pattern of growth rings with the central core revealing lamellar structure. Atomic force microscopy and dynamic light scattering experiments revealed that the buckwheat starch granules were polygonal in shape and were in the range of 2 to 19 μm in diameter. The optimized acid hydrolysis process produced nanocrystals with the shape of spherical structure with lengths ranging from 120 to 200 nm, and the diameter from 4 to 30 nm from aqueous suspensions of buckwheat starch solution. The sorption isotherms on buckwheat starch nanocrystal/glycerol composite exhibited a 3-stage transition of moisture in the blending. The biocompatible nature of buckwheat starch nanocrystals and their structural properties make them a promising green nanocomposite material. PRACTICAL APPLICATION: Buckwheat starches had never been studied on a nanoscale, but we have achieved new understanding of starch granule morphology and concentric growth rings using nanoscale imaging. Since buckwheat is an underutilized crop, we foresee the potential application of buckwheat starch, starch-based nanocrystals, and nanoparticles, to expand markets and encourage producers to expand their buckwheat acreage. The atomic force image analysis suggests that buckwheat starch could be used as a new biopolymer material in food industries.     

Myrosma cannifolia,chemical composition and physicochemical properties of the extracted starch

Myrosma cannifolia Lf (Maranthaceae) is a tuber‐producing plant which is native to Apure State in Venezuela and is consumed by the Pumé Amerindians of the region. Its chemical composition and physical characteristics were determined. The chemical, physicochemical, morphometric characteristics and rheological properties of starch isolated from Myrosma were studied. The starch yield was 438.6 g kg ⁻¹ (DW). Starch granules were oval, elliptical, irregular and polyhedric, with granules of 9.5–21.25 μm wide and 9.6–19.05 μm long. Scanning electron micrographs revealed the presence of smooth surfaces. The starch showed a characteristic ‘A‐pattern’ of cereal starches by X‐ray diffraction, with zones of high crystallinity. The amylographic study showed a rapid gelatinisation with high stability during heating and cooling cycles. The starch might be a potential ingredient for food products manufactured using different temperatures during processing. © 1999 Society of Chemical Industry     

Physico‐chemical Characterization of Floridean Starch of Red Algae

The current work reports on isolation and physico‐chemical characterization of floridean starch from three species of agarophytic macro red algae. As determined by ¹H‐NMR spectroscopy, the average chain length and degree of branching frequency of this starch were 18 and 4.8, respectively. According to its amylopectin chain length distribution obtained by Dionex analysis, the crystalline polymorph of floridean starch from the red alga Gracilariopsis lemaneiformis was deduced to be C‐type and this was further supported from its X‐ray crystallographic pattern. Enzymatic analysis of its glucose 6‐phosphate content showed that floridean starch had a low level of covalently linked phosphate (1 nmol per milligram starch) and this was further confirmed by ³¹P‐NMR. The absorbance peak of floridean starch with iodine occurred at 527—530 nm and the blue value was low (0.1), indicating the absence of amylose, which was confirmed by differential scanning calorimetry (DSC). Floridean starch exhibited low gelatinization temperature, low viscosity, high clarity and little or no retrogradation upon repetitive freeze‐thaw cycles, as studied by DSC and rapid viscosity analysis (RVA). These results are discussed in light of the functional properties and the structure of floridean starch.     

Physical,Thermal and Sorption Profile of Starch Obtained from Tacca leontopetaloides

The purpose of this study was to elucidate functional properties of starch granules obtained from tubers of Tacca leontopetaloides and compare them to a commercially available maize starch. Scanning electron microscopy (SEM), particle size analysis, X‐ray powder diffraction (XRPD), gravimetric moisture sorption, and differential scanning calorimetry (DSC) were used to characterize the samples. Tacca starch exhibited a monomodal distribution of irregularly shaped granules with a mean particle size of 2.64 µm. The spherulites of both samples indicated an A‐type pattern, with the degree of crystallinity estimated to be 35% for tacca starch and 38% for maize starch. The moisture sorption profile of both samples was analyzed according to the Guggenheim, Anderson and de Boer (GAB) equation. GAB analysis estimated the monolayer coverage for tacca and maize starch to be 0.0928 g/g and 0.0856 g/g, respectively. The gelatinization parameters of tacca starch were found to be 65.57 – 68.56 – 73.10°C while that of maize starch were 67.30 – 70.97 – 76.25°C. The results of DSC studies indicate that the associative forces that stabilize the granule structure in tacca starch are weaker than those in maize starch. The results obtained in this study establish the fundamental characteristics of tacca starch and suggest that further exploration of its potential for use in a variety of fields is warranted.     

Effect of Modified Starches on Rheological Properties of Ketchup

The aim of this work was to study the effect of commercial modified starches of different origin on rheological properties of ketchup. The following starches were used to produce the ketchup samples: chemically modified potato (acetylated distarch adipate from potato starch), waxy maize (acetylated distarch adipate from waxy maize starch and hydroxypropyl distarch phosphate from waxy maize starch), and cassava (acetylated distarch adipate from cassava starch) starches and physically modified cassava and waxy maize starches (physically modified cassava starch and physically modified waxy maize starch). The SEM microphotographs revealed that swollen or disrupted starch granules were present in the ketchup samples. As was evaluated by particle size distribution analysis, two peaks characteristic for different starch granule sizes were observed, the first peak at about 100 μm for ketchup thickened with potato starch and the second one at about 50 μm for the rest of the samples. Ketchups showed non-Newtonian, shear-thinning flow with tendency to yield stress. Values of the rheological parameters describing the flow curves significantly correlated with Bostwick consistency. Ketchup samples exhibited different susceptibility for temperature changes, while values of flow activation energy were from 4.18 to 9.00 kJ/mol. On the basis of mechanical spectra, it is noted that values of G′ were higher than these of G″ showing that elastic properties dominated over the viscous ones. Ketchup samples exhibited properties of weak gels which were estimated from the values of G′ and G″ moduli and their relation and from values of tangent of phase angle (tan δ = 0.14???0.37). Principal component analysis revealed both similarities and differences in rheological behavior of the examined ketchup samples thickened with different modified starches.     

Effect of Pullulanase Debranching of Sago (Metroxylon sagu) Starch at Subgelatinization Temperature on the Yield of Resistant Starch

The effects of pullulanase debranching of sago (Metroxylon sagu) starch in the granular state and subsequent physical treatments on the formation and yield of type III resistant starch (RS 3) have been investigated. Sago starch was enzymatically debranched with pullulanase at 60°C and at pH 5.0 using different enzyme concentrations (24, 30, 40, 50 PUN/g dry starch) which was added to 20% (w/v) starch slurry and incubated for 0 to 48 h. Optimum enzyme concentration of 40 PUN/g dry starch and three debranching times (8, 16 and 24 h) have been selected for subsequent preparation of RS. Granule morphology and molecular weight distribution (MWD) of the debranched and resistant starch were examined. Debranched starch samples showed blurred birefringence patterns, a decrease in amylopectin fraction, an increase in low molecular weight fraction and a broadening of MWD. Debranched starch samples with a maximum RS yield of 7% were obtained at 8 h debranching time. Temperature cycling and incubation at certain temperature and storage time enhanced the formation of RS. Under the conditions used in this study, the optimum conditions to obtain the highest RS yield (11.6%) were 8 h of debranching time, followed by incubation at 80°C for seven days. The MWD analysis showed that RS consisted of material with relatively low degree of polymerization. This study showed that pullulanase treatment of starch in the granular state resulted in limited debranching of amylopectin but the subsequent physical treatments (incubation time/temperature) can be manipulated to promote crystallization and enhance formation of RS 3.