Amylolytically-resistant tapioca starch modified by combined treatment of branching enzyme and maltogenic amylase

Tapioca starch was modified using branching enzyme (BE) isolated from Bacillus subtilis 168 and Bacillus stearothermophilus maltogenic amylase (BSMA), and their molecular fine structure and susceptibility to amylolytic enzymes were investigated. By BE treatment, the molecular weight decreased from 3.1 × 10⁸ to 1.7 × 10⁶, the number of shorter branch chains (DP 6–12) increased, the number of longer branch chains (DP >25) decreased, and amylose content decreased from 18.9% to 0.75%. This indicated that α–1,4 linkages of amylose and amylopectin were cleaved, and moiety of glycosyl residues were transferred to another amylose and amylopectin to produce branched glucan and BE-treated tapioca starch by forming α–1,6 branch linkages. The product was further modified with BSMA to produce highly-branched tapioca starch with 9.7% of extra branch points. When subject to digestion with human pancreatic α-amylase (HPA), porcine pancreatic α-amylase (PPA) and glucoamylase, highly-branched tapioca starch gave significantly lowered α-amylase susceptibility (7.5 times, 14.4 times and 3.9 times, respectively), compared to native tapioca starch.     

Structural and thermodynamic properties of rice starches with different genetic background Part 2. Defectiveness of different supramolecular structures in starch granules

High-sensitivity differential scanning microcalorimetry (HSDSC), small-angle X-ray scattering (SAXS), light (LM) and scanning electronic (SEM) microscopy techniques were used to study the defectiveness of different supramolecular structures in starches extracted from 11 Thai cultivars of rice differing in level of amylose and amylopectin defects in starch crystalline lamellae. Despite differences in chain-length distribution of amylopectin macromolecules and amylose level in starches, the invariance in the sizes of crystalline lamellae, amylopectin clusters and granules was established. The combined analysis of DSC, SAXS, LM and SEM data for native starches, as well as the comparison of the thermodynamic data for native and annealed starches, allowed to determine the structure of defects and the localization of amylose chains in crystalline and amorphous lamellae, defectiveness of lamellae, clusters and granules. It was shown that amylose “tie chains”, amylose–lipid complexes located in crystalline lamellae, defective ends of double helical chains dangling from crystallites inside amorphous lamellae (“dangling” chains), as well as amylopectin chains with DP 6–12 and 25–36 could be considered as defects. Their accumulation can lead to a formation of remnant granules. The changes observed in the structure of amylopectin chains and amylose content in starches are reflected in the interconnected alterations of structural organization on the lamellar, cluster and granule levels.     

Structural and thermodynamic properties of rice starches with different genetic background Part 1. Differentiation of amylopectin and amylose defects

A combined DSC - HPAEC-PAD approach, gel permeation chromatography and mild long-term acidic hydrolysis were employed to study the effects of amylopectin chain-length distributional and amylose defects on the assembly structures of amylopectin (crystalline lamellae, amylopectin clusters) in A-type polymorphic starches extracted from 11 Thai cultivars of rice with different amylose level. Joint analysis of the data allowed determining the contributions of different populations of amylopectin chains to the thermodynamic melting parameters of crystalline lamellae. It was shown that amylopectin chains with DP 6-12 and 25or=37 could be related to chains stabilizing these structures. The total effect of amylose and amylopectin defects can be described by means of Thomson-Gibbs' equation. The increase of defects in the assembly structures is accompanied by rise of the rates of acidic hydrolysis of both amorphous and crystalline parts in starches.     

Glucoamylase production by the marine yeast Aureobasidium pullulans N13d and hydrolysis of potato starch granules by the enzyme

Under the optimal conditions, 10 U/ml of glucoamylase was produced by the marine yeast Aureobasidium pullulans N13d. It was noticed that the crude glucoamylase actively hydrolyzed potato starch granules, but poorly digested raw corn starch and sweet potato starch, resulting in conversion of 68.5, 19 and 22% of them into glucose within 6 h of incubation in the presence of 40 g/l of potato starch granules and 20 U/ml of the crude enzyme. When potato starch granules concentration was increased from 10 to 80 g/l, hydrolysis extent was decreased from 85.6 to 60%, while potato starch granules concentration was increased from 80 to 360 g/l, hydrolysis extent was decreased from 60 to 56%. Ratio of hydrolysis extent of potato starch granules to hydrolysis extent of gelatinized potato starch was 86.0% and the hydrolysis extent of potato starch granules by action of the crude glucoamylase (1.0 U/ml) was 18.5% within 30 min at 60 °C. Only glucose was detected during the hydrolysis, indicating that the crude enzyme could hydrolyze both α-1,4 and α-1,6 linkages of starch molecule in the potato starch.     

Large scale structure of wheat,rice and potato starch revealed by ultra small angle X-ray diffraction

Rice, wheat, and potato starches were investigated using ultra-small angle X-Ray diffraction (USXRD) in the range of 100–58,000 Å. The results showed trends consistent with the known sizes of starches. However, the observed Rg values for the scattering substances lie in the 100–300 nm range, very much in the low end of the known starch granule size distributions (and below the resolution of the light microscope) suggesting different, perhaps interesting, structures than those observed by light microscopy. Thus what were detected may possibly be the sizes of the crystalline regions postulated to occur in individual starch granules.     

Structural and thermodynamic properties of starches extracted from GBSS and GWD suppressed potato lines

A combined DSC–SAXS approach was employed to study the effects of amylose and phosphate esters on the assembly structures of amylopectin in B-type polymorphic potato tuber starches. Amylose and phosphate levels in the starches were specifically engineered by antisense suppression of the granule bound starch synthase (GBSS) and the glucan water dikinase (GWD), respectively. Joint analysis of the SAXS and DSC data for the engineered starches revealed that the sizes of amylopectin clusters, thickness of crystalline lamellae and the polymorphous structure type remained unchanged. However, differences were found in the structural organization of amylopectin clusters reflected in localization of amylose within these supramolecular structures. Additionally, data for annealed starches shows that investigated potato starches possess different types of amylopectin defects. The relationship between structure of investigated potato starches and their thermodynamic properties was recognized.     

Debranching enzyme concentration effected on physicochemical properties and α-amylase hydrolysis rate of resistant starch type III from amylose rice starch

The effect of debranching enzyme concentration on physicochemical properties and α-amylase hydrolysis rate of resistant starch type III from high amylose rice starch were studied. The pullulanase enzyme (8, 10, 12, 14 and 16 U/g starch) was introduced to modify amylopectin molecules of 15% (w/w) gelatinized rice starches at 55 °C for 16 h. The debranched starches with different degrees of hydrolysis (0.14–5.27%), and having 66.60–98.82% β-amylolysis limit were then induced at 4 °C for 16 h, afterward a one cycle of freeze–thaw process (?10/30 °C) was applied. The results showed that a pullulanase hydrolysis improved the degree of syneresis (51.64–54.85% from 8 to 16 U/g starch). Resistant starch content increased sharply as the amount of the enzyme increased, reaching the highest (19.81%) for a 12 U/g starch and decreased to 13.16% by 16 U/g starch. α-Amylase hydrolysis rate showed that incompletely-debranched had a lower estimated glycemic index than completely debranched rice starches. Microstructure of the selected RS III samples using X-ray diffraction and scanning electron microscopy revealed a crystal pattern change from A- to V-type pattern and formed a coarse honeycomb-like and a filamentous network structure.     

The effects of iodine on kidney bean starch: Films and pasting properties

In the present study the effect of iodine on the structural characteristics (by infrared spectroscopy and X-ray) of films made from kidney bean starch was evaluated. The pasting properties as affected by iodine and glycerol were also evaluated. Kidney bean starch showed C-type (mixture of A- and B-type) crystalline structure, the conversion of starch into films resulted into reduction in intensity of diffractograms. The starch powder FTIR spectra had peaks centered at 1020 and 995 cm^?1 with a higher intensity at 1020 cm^?1, which is consistent with a partially crystalline material since fully crystalline material show similar intensity peaks centered around 1020 and 1006 cm^?1. Films without iodine showed one main peak centered around 1000 cm^?1 consistent with a disordered state similar to that in gelatinised starch. Iodine addition gradually increased the intensity of the bands around 1020 cm^?1 consistent with the formation of more ordered conformation similar to that in the crystalline material. Iodine encourages the formation of helical structures, however, the formation of crystalline material cannot be inferred. The increasing amounts of iodine up to 0.33% level progressively increased the peak-, through- and breakdown-viscosity. Iodine beyond 0.33% level gradually decreased peak-, trough-, breakdown- and setback-viscosity. Pasting temperature gradually increased with the increase in iodine.     

The impact of single and dual hydrothermal modifications on the molecular structure and physicochemical properties of normal corn starch

Effect of single and dual hydrothermal modifications with annealing (ANN) and heat-moisture treatment (HMT) on molecular structure and physicochemical properties of corn starch was investigated. Normal corn starch was modified by ANN at 70% moisture at 50 °C for 24 h and HMT at 30% moisture at 120 °C for 24 h as well as by the combination of ANN and HMT. The apparent amylose content and swelling factor (SF) decreased on ANN and HMT, but amylose leaching (AML) increased. These changes were more pronounced on dual modification. The crystallinity (determined by X-ray diffraction), the gelatinization enthalpy (determined by differential scanning calorimetry) and ratio of 1047 cm^?1/1022 cm^?1 (determined by Fourier transform infrared spectroscopy) slightly increased on ANN and decreased on HMT. The ANN and subsequent HMT (ANN-HMT) resulted in the lowest crystallinity, gelatinization enthalpy and ratio of 1047 cm^?1/1022 cm^?1. The gelatinization temperature range decreased on ANN but increased on HMT. However, the gelatinization range of dually modified starches (ANN-HMT and HMT-ANN) was between ANN starch and HMT starch. Birefringence remained unchanged on ANN but slightly decreased on HMT as well as dual modification. Average chain length and amount of longer branch chains (DP ≥ 37) remained almost unchanged on ANN but decreased on HMT and dual modifications (ANN-HMT and HMT-ANN). HMT and dual modifications resulted in highly reduced pasting viscosity. ANN and HMT as well as dual modifications increased RDS content and decreased SDS and RS content.     

Effect of sucrose on dynamic mechanical characteristics of maize and potato starch films

The dynamic mechanical properties of prepared maize and potato starch films were evaluated for mixtures containing 0%, 10% and 15% (w/w) of sucrose at temperatures ranging from 40.0 to 140.0 °C. The spectra of storage modulus (G′), loss modulus (G″), and loss factor (tan δ) of starch films were acquired. Remarkable reduction in the glass transition temperature of maize and potato starch films was observed with the increasing sucrose content. The spectra of storage modulus (G′), loss modulus (G″), and loss factor (tan δ) were measured for the second and third time after two and seven days, respectively. The peaks of loss factor (tan δ) appeared at 59.81 ± 1.86 °C and 95.96 ± 1.67 °C after two-day-storage, but only one peak appeared at 85.46 ± 5.50 °C after seven days. A shifting trend from higher to lower temperature for loss factor was observed after seven days.     

Starch pastes thinning during high-pressure homogenization

High-pressure homogenization induced thinning of potato and cassava starch paste was investigated. The starch slurries at a concentration of 2.0 wt.% were heated at 90 °C for 1 h and then rapidly cooled in tap water. The cooled starch pastes were homogenized at various pressures ranging from 0 to 100 MPa using a lab-scale high-pressure homogenizer. The influence of homogenizing pressure on the temperature, apparent viscosity, electrical conductivity, and percent light transmittance of homogenized starch pastes were determined. Temperatures of homogenized starch pastes increased linearly with the increase of the applied pressure, and the rate was 0.177 °C/MPa and 0.186 °C/MPa for potato and cassava starch pastes, respectively. After high-pressure homogenization, the apparent viscosities of the starch pastes decreased, while the percent light transmittances of them increased. However, the electrical conductivities of starch pastes were not affected by homogenization.     

Optimization of the immobilization of sweet potato amylase using glutaraldehyde-agarose support. Characterization of the immobilized enzyme

A simplified procedure for the preparation of immobilized beta-amylase using non-purified extract from fresh sweet potato tubers is established in this paper, using differently activated agarose supports. Beta-amylase glutaraldehyde derivative was the preparation with best features, presenting improved temperature and pH stability and activity. The possibility of reusing the amylase was also shown, when this immobilized enzyme was fully active for five cycles of use. However, immobilization decreased enzyme activity to around 15%. This seems to be mainly due to diffusion limitations of the starch inside the pores of the biocatalyst particles. A fifteen-fold increase in the Km was noticed, while the decrease of Vmax was only 30% (10.1 U mg^?1 protein and 7.03 U mg^?1 protein for free and immobilized preparations, respectively).     

Changes in starch structure during manufacturing of starch microspheres for use in parenteral drug formulations: Effects of temperature treatment

Starch microspheres were produced by emulsification of a starch dispersion in an aqueous polyethylene glycol (PEG) solution. Crystalline/ordered structure was formed within these starch droplets during incubation at 6 °C for 25 h followed by incubation at 37 °C for 28 h. After incubation at 37 °C the crystalline structure in the samples was of type B. The crystallization process of microspheres was compared with crystallization in a model system. The crystalline structure of the microspheres melted at temperatures almost 20 °C lower than in the model system incubated under the same conditions, as determined by differential scanning calorimetry. It was thus concluded that the crystallization process within microspheres was different than that of bulk starch and the ability of the starch molecules to reorganize themselves within the dispersed starch phase of an aqueous two-phase system at the higher incubation temperature was limited. It was also observed that the presence of PEG or carbonate buffer protected the molecular order formed by the starch molecules during incubation from breakdown during freeze-drying.     

In vitro evaluation of starch degradation from feeds with or without various heat treatments

An in vitro method was used to evaluate starch degradation from various feeds with or without heat treatments in four studies. The method was based on incubation of feed samples with a buffered rumen fluid solution and subsequent enzymatic analysis of the remaining starch. In all studies, heat treatment of the feed samples increased rate or extent of starch degradation to glucose. In Study 1, measurements of remaining starch, after 5 h in vitro incubations, demonstrated substantial effects of cooking on starch degradation in potatoes, and a trend to faster degradation from autoclaving peas. Up to 0.60 of the starch remaining after a 5 h of incubation was not recovered by centrifugation at 3000 × g for 10 min. In Study 2, cooking increased in vitro starch degradation rate from isolated potato starch (from 0.038 to 0.197/h). Intact starch in barley and wheat grain had similar rates of degradation (0.117 and 0.109/h, respectively). In Study 3, both autoclaving time (15, 30, 60 min) and temperature (115, 130 and 145°C) affected in vitro starch degradation rates in peas, and, in no case did autoclaving for only 15 min increase degradation rates. For the 30 min autoclaving time, only the highest temperature (145°C) increased the degradation rate of the pea starch compared to the untreated peas (0.175 versus 0.110/h). When autoclaving for 60 min, both 130 and 145°C resulted in a considerable increase in starch degradation rate (0.211 and 0.193/h, compared to 0.110/h for the untreated peas). In Study 4, the proportion of starch degraded at 8 h of in vitro incubation was increased by heat treatment of pure potato starch (0.155 versus 0.870), peas (0.491 versus 0.815), barley (0.686 versus 0.913) and maize (0.351 versus 0.498). Measurements of volatile fatty acid production in the fermentation tubes showed a lower acetate:propionate ratio for the faster fermenting heat-treated feeds. Heat treatment generally increased starch degradation in vitro.     

A photographic approach to the possible mechanism of retrogradation of sweet potato starch

Although the subject of starch retrogradation has been studied for about 20 years, the mechanism of starch retrogradation seems not yet to be completely established. In this paper, the possible retrogradation mechanism of sweet potato starch was postulated from four optical micrographs at the stages of melting of the starch granules, autoclaving treatment and aging. The possible process of retrogradation consists of three stages. Firstly, starch granules was swelled and melted with loss of X-ray crystallinity and formation of both crystalline and amorphous lamellae; secondly, in crystalline lamellae, amylopectin began to form nucleation when they were autoclaved; finally, the nucleus grew up to great rod-like crystals as the result of congregating of amylose on plates which were composed of and prolongated by amylopectin.     

Amylopectin molecular structure reflected in macromolecular organization of granular starch

For lintners with negligible amylose retrogradation, crystallinity related inversely to starch amylose content and, irrespective of starch source, incomplete removal of amorphous material was shown. The latter was more pronounced for B-type than for A-type starches. The two predominant lintner populations, with modal degrees of polymerization (DP) of 13-15 and 23-27, were best resolved for amylose-deficient and A-type starches. Results indicate a more specific hydrolysis of amorphous lamellae in such starches. Small-angle X-ray scattering showed a more intense 9-nm scattering peak for native amylose-deficient A-type starches than for their regular or B-type analogues. The experimental evidence indicates a lower contrasting density within the "crystalline" shells of the latter starches. A higher density in the amorphous lamellae, envisaged by the lamellar helical model, explains the relative acid resistance of linear amylopectin chains with DP > 20, observed in lintners of B-type starches. Because amylopectin chain length distributions were similar for regular and amylose-deficient starches of the same crystal type, we deduce that the more dense (and ordered) packing of double helices into lamellar structures in amylose-deficient starches is due to a different amylopectin branching pattern.     

Impact of annealing and heat-moisture treatment on rapidly digestible,slowly digestible and resistant starch levels in native and gelatinized corn,pea and lentil starches

Impact of annealing (ANN) and heat-moisture treatment (HMT) on rapidly digestible starch (RDS), slowly digestible starch (SDS), resistant starch (RS), and expected glycemic index (eGI) of corn, pea, and lentil starches in their native and gelatinized states were determined. ANN was done for 24 h at 70% moisture at temperatures 10 and 15 °C below the onset (T_o) temperature of gelatinization, while HMT was done at 30% moisture at 100 and 120 °C for 2 h. The swelling factor (SF), amylose leaching (AML) and gelatinization parameters of the above starches before and after ANN and HMT were determined. SF and AML decreased on ANN and HMT (HMT > ANN). The gelatinization temperatures increased on ANN and HMT (HMT > ANN). However, the gelatinization temperature range decreased on ANN but increased on HMT. Birefringence remained unchanged on ANN but decreased on HMT. The Fourier transform infrared (FT-IR) absorbance ratio of 1047 cm^?1/1022 cm^?1 increased on ANN but decreased on HMT. ANN and HMT increased RDS, RS and eGI levels and decreased SDS levels in granular starches. HMT had a greater impact than ANN on RDS, RS, and SDS levels. In gelatinized starches, ANN and HMT decreased RDS and eGI, but increased SDS and RS levels. These changes were more pronounced on HMT. This study showed that amylopectin structure and interactions formed during ANN and HMT had a significant impact on RDS, SDS, RS and eGI levels of starches.     

Thermophilic bacterium Caldimonas taiwanensis produces poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from starch and valerate as carbon sources

Caldimonas taiwanensis accumulated polyhydroxybutyrate (PHB) at 55 °C from gluconate, fructose, maltose, and glycerol under nitrogen-limited condition. The PHB content peaked at 14 h after inoculation from gluconate. C. taiwanensis did not grow or accumulate PHA from fatty acids as the sole carbon source; however, it incorporated 3-hydroxyvalerate (3-HV) into PHB polymer from gluconate and valerate as a mixed carbon source. By adjusting the valerate concentration, the molar fraction of 3-HV could be modulated from 10 mol% to 95 mol%. Fatty acid valerate substantially inhibited cell growth and PHA accumulation with the addition of as little as 5 mM to the medium. Supplementing the medium with yeast extract overcame the inhibition, which enhanced not only the yield of biomass but also PHA productivity. The in vivo substrate specificity of PHA synthase ranged from C₄ to C₆. In addition, C. taiwanensis also incorporated a wide range of 3-HV into PHA from soluble starch and valerate as a mixed carbon source. Food-grade starches made from cassava, corn, potato, sweet potato and wheat respectively mixed with valerate were studied for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] production. In this study, C. taiwanensis exhibited high promise for reducing the production cost of P(3HB-co-3HV).     

Physical properties of extruded fish feed with inclusion of different plant (legumes,oilseeds, or cereals) meals

A study was undertaken to evaluate the effect of various ingredients on the physical quality of fish feeds. Eleven fish meal-based diets, formulated to have the same levels of macronutrients, differing in either starch or protein source, were processed in a five section twin-screw extruder. The purified starch, added to reach the nutritional specifications of the diets, was significantly correlated to expansion (r = 0.405, P<0.001), durability (r = 0.276, P=0.012), and hardness (r = 0.494, P<0.001), while such correlations were not seen for the total starch level in the diets. Cellulose, added as filler to reach the same level of NSP in the diets, was negatively correlated to the expansion (r = ?0.603, P<0.001). The specific mechanical energy of the extrusion process was weakly correlated to starch gelatinisation (r = 0.220, P<0.019). The present study showed that traditional parameters and classifications such as chemical composition of plant ingredients are inadequate indicators of processing effects when used in fish diets. The overall conclusion is that processing parameters needed to achieve the desired physical properties of diets, should be based on specific knowledge of each ingredient in the feed.     

Structural basis for the slow digestion property of native cereal starches

Native cereal starches are ideal slowly digestible starches (SDS), and the structural basis for their slow digestion property was investigated. The shape, size, surface pores and channels, and degree of crystallinity of starch granules were not related to the proportion of SDS, while semicrystalline structure was critical to the slow digestion property as evidenced by loss of SDS after cooking. The high proportion of SDS in cereal starches, as compared to potato starch, was related to their A-type crystalline structure with a lower degree of perfection as indicated by a higher amount of shortest A chains with a degree of polymerization (DP) of 5-10. The A-type amorphous lamellae, an important component of crystalline regions of native cereal starches, also affect the amount of SDS as shown by a reduction of SDS in lintnerized maize starches. These observations demonstrate that the supramolecular A-type crystalline structure, including the distribution and perfection of crystalline regions (both crystalline and amorphous lamellae), determines the slow digestion property of native cereal starches.