Ashok K. Shrestha

Molecular rearrangement of starch during in vitro digestion: Toward a better understanding of enzyme resistant starch formation in processed starches

Resistant starch (RS) is defined as the fraction of starch that escapes digestion in the small intestine, serving as a fermentation substrate for beneficial colonic bacteria. Several studies have been focused on the description of the RS fractions from different starch varieties, but little attention has been paid to the digestion process itself that, from the present work, seems to play a key role in the generation of enzyme-RS (ERS), as determined in vitro. High-amylose starch samples, extruded at two different processing conditions, have been characterized at different stages of in vitro digestion using scanning electron microscopy (SEM), small-angle X-ray scattering (SAXS), infrared spectroscopy (FT-IR), solid state (13)C NMR spectroscopy, and X-ray diffraction (XRD). Control samples kept for 18 h in the digestion solution without starch hydrolyzing enzymes (alpha-amylase and amyloglucosidase) were used for comparison purposes. An increase in molecular order was favored by the hydrolytic action of the enzymes, reflected in an increase in double helical order observed by NMR, higher crystallinity measured by XRD, and corresponding changes in FT-IR spectra. An increase in the intensity of the scattering objects was also observed by SAXS as a function of digestion. SAXS from the dry ERS fractions reveals the 001 reflection of crystallites formed during the digestion process, corresponding to a characteristic dimension of the resistant crystalline fraction of approximately 5 nm. The changes found suggest that enzyme resistant starch does not refer to a specific structure present in predigested starches, but may in fact be formed during the digestion process through the rearrangement of amylose chains into enzyme-resistant structures of higher crystallinity. Therefore, the resistance to enzyme digestion of a specific processed starch is the result of a competition between the kinetics of enzyme hydrolysis and the kinetics of amylose retrogradation.

Glass Transition Behavior of Spray Dried Orange Juice Powder Measured by Differential Scanning Calorimetry (DSC) and Thermal Mechanical Compression Test (TMCT)

Spray drying behavior of orange juice concentrate with various levels of maltodextrin (DE 6) was studied. Five combinations of orange juice concentrate and maltodextrin (25:75, 30:70, 35:65, 40:60, and 50:50) were spray dried at 160 and 65°C inlet and outlet temperatures, respectively. The product recovered with 50% maltodextrin concentration was sticky and only 20% powder was recovered. The recovery of orange juice powder increased as the amount of maltodextrin in powders increased. The particle size and bulk density remained almost the same in all except in 50% maltodextrin powder which was slightly larger and more dense. The moisture content of spray dried powders was high and desiccated before measuring glass transition temperature. The anhydrous spray dried powders showed increased Tg values with increasing maltodextrin concentration, from 66°C in 50% maltodextrin to 97°C in 75% maltodextrin containing powders. The glass rubber transition (Tg-r) values of all the products measured using novel Thermal Mechanical Compression Test (TMCT) were higher than Tg values measured by DSC; the difference in values increased with increase in maltodextrin concentration.

Folate assay of foods by traditional and tri-enzyme treatments using cryoprotected Lactobacillus casei

Variables affecting the efficacy of the microbiological assay of folate in foods were examined. Spinach, fortified bread and two ready-to-eat breakfast cereals were extracted with or without autoclaving and centrifugation. Autoclaving and centrifugation lowered the yield of total folate in all foods. The food sample, after digestion with protease and α-amylase was deconjugated with chicken pancreas or human plasma (tri-enzyme treatment) or simply with conjugase alone (tradition single enzyme treatment). The tri-enzyme treatment was a significant improvement over the single enzyme treatment only in fortified bread. Deconjugation with chicken pancreas gave a significantly higher folate value than did human plasma in all foods except spinach. Folate assay by cryoprotected frozen Lactobacillus casei was compared with serially sub-cultured inocula. Using the cryoprotected frozen inoculum took a shorter time, was less tedious, gave better reproducibility and was more economical than using the conventional serial culture. The effects of the size of test tubes on the growth of culture and the wavelength at which turbidity was measured to achieve maximum detection were also investigated. L.casei grew faster in small tubes than in larger ones. The absorbance peak at 540 nm was higher than that at 620 nm.

Folate retention in selected processed legumes.

The effect of soaking, boiling and pressure cooking on the retention of folates in whole chickpeas (Cicer arietinum) and field peas (Pisum sativum) was investigated. Pressure-cooking allowed significantly higher (p<0.05) retention of folates compared to boiling in both the legumes. Retention of folates in chickpeas was greater than field peas irrespective of the processing procedure used. Leaching into the cooking medium was identified to be the major cause for losses of folates during soaking and cooking. Leaching losses were greater in field peas compared to chickpeas.

Molecular, mesoscopic and microscopic structure evolution during amylase digestion of maize starch granules

Cereal starch granules with high (>50%) amylose content are a promising source of nutritionally desirable resistant starch, i.e. starch that escapes digestion in the small intestine, but the structural features responsible are not fully understood. We report the effects of partial enzyme digestion of maize starch granules on amylopectin branch length profiles, double and single helix contents, gelatinisation properties, crystallinity and lamellar periodicity. Comparing results for three maize starches (27, 57, and 84% amylose) that differ in both structural features and amylase-sensitivity allows conclusions to be drawn concerning the rate-determining features operating under the digestion conditions used. All starches are found to be digested by a side-by-side mechanism in which there is no major preference during enzyme attack for amylopectin branch lengths, helix form, crystallinity or lamellar organisation. We conclude that the major factor controlling enzyme susceptibility is granule architecture, with shorter length scales not playing a major role as inferred from the largely invariant nature of numerous structural measures during the digestion process (XRD, NMR, SAXS, DSC, FACE). Results are consistent with digestion rates being controlled by restricted diffusion of enzymes within densely packed granular structures, with an effective surface area for enzyme attack determined by external dimensions (57 or 84% amylose - relatively slow) or internal channels and pores (27% amylose - relatively fast). Although the process of granule digestion is to a first approximation non-discriminatory with respect to structure at molecular and mesoscopic length scales, secondary effects noted include (i) partial crystallisation of V-type helices during digestion of 27% amylose starch, (ii) preferential hydrolysis of long amylopectin branches during the early stage hydrolysis of 27% and 57% but not 84% amylose starches, linked with disruption of lamellar repeating structure and (iii) partial B-type recrystallisation after prolonged enzyme incubation for 57% and 84% amylose starches but not 27% amylose starch.

Physicochemical and structural properties of maize and potato starches as a function of granule size

Chemical composition, molecular structure and organization, and thermal and pasting properties of maize and potato starches fractionated on the basis of granule size were investigated to understand heterogeneity within granule populations. For both starches, lipid, protein, and mineral contents decreased and apparent amylose contents increased with granule size. Fully branched (whole) and debranched molecular size distributions in maize starch fractions were invariant with granule size. Higher amylose contents and amylopectin hydrodynamic sizes were found for larger potato starch granules, although debranched molecular size distributions did not vary. Larger granules had higher degrees of crystallinity and greater amounts of double and single helical structures. Systematic differences in pasting and thermal properties were observed with granule size. Results suggest that branch length distributions in both amylose and amylopectin fractions are under tighter biosynthetic control in potato starch than either molecular size or amylose/amylopectin ratio, whereas all three parameters are controlled during the biosynthesis of maize starch.

Determination of folate contents in some Australian vegetables

Abstract The undeconjugated and total folate contents of 22 vegetables commonly available in Australia were assayed with Lactobacillus casei after preliminary digestion with chicken pancreas. The effect of conjugate type and extraction technique on the total folate content of two of the vegetables was also investigated. In two test samples, spinach and Chinese broccoli, deconjugation with chicken pancreas gave slightly higher, but statistically not different ( P P

Edible coating materials—their properties and use in the fortification of rice with folic acid

Abstract This study investigated the technical feasibility of adding folic acid on to rice and coating with edible polymers. The coating solutions were cast into film and their properties were investigated. A concentrated premix of rice was prepared in a rotating coating pan by spraying first with folic acid solution, and then with polymer solutions and drying. The fortified rice premixes were evaluated for washing and cooking losses. The loss of folic acid in washing was lowest in rice premixes coated with ethyl cellulose followed by pectin, composite mixtures of locust bean and other coating materials with highest loss in gum arabic coated rice. No edible polymer could satisfactorily retain folic acid during boiling in excess water. Edible polymers failed to mask the yellow color of folic acid and additional masking agent was needed. The premixes had a higher water uptake ratio than raw milled rice had. Triangle tests did not show any significant difference (α=0.05) between the sensory qualities of cooked fortified rice and raw milled rice.

Enzyme protein binding assay for determining folic acid in fortified cereal foods and stability of folic acid under different extraction conditions

The widespread fortification of cereal foods with folic acid has initiated the development of a faster, easier and more reliable method to determine folate in these foods. An enzyme protein binding assay (EPBA) kit developed by a Commercial Diagnostics Company was used for the analysis of folic acid present in folate fortified foods. The food samples were also analyzed by the microbiological assay for comparison of methods. Results from both methods showed no significant difference (p<0.05) in values and showed high correlation (r=0.89, p<0.001). However, the 95% confidence interval for the lower and upper limit of agreement was high. The addition of 1% ascorbic acid to the extraction medium offered better protection of folic acid against oxidation than foods extracted without ascorbic acid. No definite effect on the folate value was observed when extraction was carried out at two different sets of conditions, 10 min at 100 and 121 °C.

Resistant starch in vitro and in vivo : factors determining yield, structure and physiological relevance

Knowledge of the food processing methods and conditions as well as the underlying mechanism that leads to the formation of resistant starch (RS) in foods is of great importance for nutritionists as well as food industries, since it offers the possibility of increasing the RS content in processed foods. RS is normally defined as that portion of dietary starch that escapes digestion in the small intestine of a healthy human. The amount of RS in a given food is determined by many different factors. Food form and structure, botanical source of the starch and its amylose content, other dietary constituents, notably protein and fat, the presence of a-amylase inhibitors and other antinutrients, the methods used to manufacture and store foods, and culinary practices all directly influence starch digestibility in the gut. Although most of the attention that RS has attracted from health professionals relates to its positive effects on bowel health, there is theoretical and mounting experimental evidence that RS has beneficial physiological properties, which extend beyond the gastrointestinal tract. Food scientists have attempted to enhance the level of enzyme-resistant retrograded starch in the diet. Numerous studies are carried out to characterize the molecular and microstructural organization of RS fractions, aiming at unraveling the mechanisms of amylolysis resistance.