Enzymatic Starch Digestion Research Articles

Inhibiting enzymatic starch digestion by the phenolic compound diboside A: A mechanistic and in silico study

Retarding the starch digestion rate is the key to produce low glycemic index (GI) foods for diabetic patients in prevention of postprandial hyperglycemia. In the search for naturally-occurring starch hydrolase inhibitors as active ingredients for low GI foods, we applied a high throughput assay to screen hundreds of edible botanical materials and discovered that a phenylpropanoid sucrose ester, diboside A (1,3,6′-tri-p-coumaroyl-6-feruloyl sucrose), isolated from the root of wild buckwheat exhibited potent α-amylase inhibitory activity with an IC50 of 26.9μM. Kinetic study revealed that it is an uncompetitive inhibitor of α-amylase with a Ki of 5.1μM. Moreover, diboside A inhibits rat intestinal sucrase as a noncompetitive inhibitor with a Ki of 72.4μM. Remarkably, it has no measurable activity towards maltase. An in silico molecular docking study shows that there are two possible allosteric binding sites between diboside A and enzyme–substrate complex. Binding site one is a more probable binding site because of lower energy of the ternary complex formed by hydrogen bonds and electrostatic interactions. Our results suggest that phenylpropanoid sucrose esters have potential as an active ingredient for low GI foods.

In vitro Starch Digestibility and Nutritional Composition of Improved Rice Varieties from Cameroun

Aims: Resistant starch (RS), kinetics of starch digestion, predicted glycemic index (pGI) and nutritional composition were determined in two improved rice varieties from Cameroun. Place and Duration of Study: Department of Bioresource Engineering, McGill University, Canada between December 2012 and March 2013. Methodology: Non-parboiled and parboiled samples of TOX 3145 and NERICA-3 varieties were involved in this study. An in vitro enzymatic starch digestion method was applied to measure starch digestibility parameters. Standardized methods were adopted for proximate and mineral contents evaluation. Results: The parboiled samples had significantly higher (P<0.05) resistant starch (8.35 11.07%) than the non-parboiled samples (3.81 4.84%). The values for pGI among Original Research Article European Journal of Nutrition & Food Safety, 3(4): 134-145,, 2013 135 samples ranged from 57.57 to 67.78%. Significantly higher values for protein, phosphorus and potassium were found among the parboiled samples (P<0.05). Nutritional composition was positively related to RS while pGI had inverse relationship with protein, ash, fat, phosphorus, potassium and RS. Conclusion: Starch digestibility of these rice varieties was associated to their nutritional composition.

Ethanol fermentation from food processing waste

This study focuses on the use of restaurant waste for production of ethanol. Food wastes (corn, potatoes, and pasta) were converted to ethanol in a two‐step process: a two‐part enzymatic digestion of starch using α‐amylase and glucoamylase and then fermentation of the resulting sugars to ethanol using yeast. Because of the low initial composition of starch in the food waste, low ethanol concentrations were achieved: at best 8 mg/mL ethanol (0.8% by mass). Ethanol concentration increased with increasing enzyme dosage levels. Calculations were conducted to evaluate whether waste heat from restaurant waste could be used to drive flash vaporization to purify ethanol. If the solution produced by fermenting food waste is flashed at a temperature of 99.7°C, 77% of the ethanol is recovered in a vapor stream with 1.14 mol % ethanol (2.87 mass %). Waste heat could provide over a third of the energy for this vaporization process. If 4 mol % ethanol could be produced in the fermentation step by increasing the initial starch content in the waste solution and improving the fermentation process, then a single flash at 98.9°C will recover nearly 99% of the ethanol, giving a mass concentration of ethanol of 10.3%, which is similar to that achieved in industrial grain fermentation. © 2012 American Institute of Chemical Engineers Environ Prog, 32: 1280–1283, 2013

Structural features of two heteroxylan polysaccharide fractions from wheat bran with anti-complementary and antioxidant activities

Wheat bran is a rich source of bioactive substances ascribed to its arabinoxylan component. Two water-soluble arabinoxylans were sequentially extracted from wheat bran. WB1, released during enzymatic digestion of starch and protein, contained medium-branched arabinoxylan (A/X=0.88) consisting of 3-O-substituted (22%), di-substituted (19.8%) and 58% unsubstituted Xylp residues. It was slightly contaminated with (1→3,1→4)-β-glucan and arabinogalactan, and free of protein. WB2 extracted with 0.5% NaOH contained ∼95% arabinoxylan (A/X=1.09). WB2 and two 5% NaOH-extracted arabinoxylans were rich in protein and phenolic compounds. All radical-scavenging assays indicated a relation with the protein and total phenolics contents. The protein-free WB1 displayed the highest hydroxyl radical scavenging effect indicating the distinct role of phenolic acids. The immunomodulatory activity of WB1 was somewhat lower, whereas, that of WB2 higher in comparison to the immunogenic polysaccharide PMII. The arabinoxylans have the potential as immuno-enhancing and antioxidant additives in functional foods.

Digestibility and physicochemical properties of rice (Oryza sativa L.) flours and starches differing in amylose content

Digestibility of starches in four rice samples with amylose content (AC) from 1.7 to 55.4%, including a newly developed high-amylose rice, was investigated. An in vitro enzymatic starch digestion method and an AOAC method were applied to correlate rapidly digestible starch (RDS), slowly digestible starch (SDS), resistant starch (RS), and total dietary fiber (TDF) content with the AC in the samples. SDS content decreased and RS and TDF content increased with the increase in AC. The low-amylose rice (AC=16.1%) had starch granules with weak crystalline structure and was lower in RS and TDF content even though it had a higher AC compared to waxy rice. The digestibility of the starches was not correlated with granule size or degree of crystallinity. The newly developed high-amylose rice starch exhibited a predominant B-type X-ray diffraction pattern, a great proportion of long chains in amylopectin, high gelatinization temperature, and semi-compound starch granules which are attributed to its increased resistance to enzyme digestion.

Starch with High Amylose Content and Low In Vitro Digestibility Increases Intestinal Nutrient Flow and Microbial Fermentation and Selectively Promotes Bifidobacteria in Pigs

Diets containing different starch types can affect enzymatic digestion of starch and thereby starch availability for microbial fermentation in the gut. However, the role of starch chemistry in nutrient digestion and flow and microbial profile has been poorly explained. Eight ileal-cannulated pigs (29.4 ± 0.9 kg body weight) were fed 4 diets containing 70% purified starch (amylose content, <5, 20, 28, and 63%; reflected by in vitro maximal digestion rate; 1.06, 0.73, 0.38, and 0.22%/min, respectively) in a replicated 4 × 4 Latin square. Ileal and fecal starch output, postileal crude protein yield, fecal total SCFA and total butyrate content, and gene copies ofBifidobacterium spp. in feces were higher ( P < 0.05) when pigs consumed the slowly digestible starch diet than the remaining 3 starch diets. The in vitro starch digestion rate had a negative, nonlinear relationship with ileal starch flow ( R 2 = 0.98; P < 0.001). Ileal starch flow was positively related to Bifidobacterium spp. ( R 2 = 0.27; P < 0.01), Lactobacillus group ( R 2 = 0.22; P < 0.01), and total butyrate content ( R 2 = 0.46; P < 0.01) but was not related to Enterobacteriaceae ( R 2 < 0.00; P = 0.92). In conclusion, starch with high amylose content and low in vitro digestibility increased postileal nutrient flow and microbial fermentation and selectively promoted Bifidobacterium spp. in the distal gut.

Evaluation of the acute antihyperglycemic effects of four selected indigenous plants from Jordan used in traditional medicine

Context: Eryngium creticum Lam. (Umbelliferae), Geranium graveolens L.Her.exn Ait (Geraniaceae), Paronychia argentea Lam. (Caryophyllaceae), and Varthemia iphionoides Boiss (Compositae) have traditionally been used as antidiabetic phytomedicines. However, their alleged benefits and mechanisms remain elusive.Objectives: To evaluate the effect of these plants on in vitro and in vivo enzymatic starch digestion.Materials and methods: In vitro enzymatic starch digestion with acarbose or (1–50 or 100 mg/ml) plants aqueous extracts was assayed using α-amylase and α-amyloglucosidase. Oral starch tolerance tests and oral glucose tolerance tests were determined for the plant extracts at concentrations 125, 250, and 500 mg/kg body weight. Blood glucose levels in rats treated with plant extracts or drugs (acarbose or metformin and glipizide) were measured at −30, 0, 45, 90, and 135 min.Results and discussion: In vitro, acarbose, and water extracts of G. graveolens and V. iphionoides exerted significant dose-dependent dual inhibition of α-amylase and α-glucosidase, with respective IC50s of 1.2 μg/ml, 84.7, and 65.2 mg/ml. Comparable in vivo acute postprandial antihyperglycemic efficacies were obtained for G. graveolens and V. iphionoides in starch-fed rats. E. creticum exhibited substantial acute antihyperglycemic activities in starch-treated rats, despite lacking any favorable in vitro effectiveness. However, P. argentea lacked any inhibitory efficacy. None of the plant extracts qualified for improving the glucose tolerance in fasted rats on glucose loading.Conclusion: G. graveolens and V. iphionoides can be considered as potential candidates for therapeutic modulation of impaired fasting glycemia, impaired glucose tolerance, and type 2 diabetes.

High amylose starch increases post‐ileal nutrient flow, microbial fermentation, and bifidobacteria in pigs

Starch chemistry affects enzymatic digestion of starch and thereby starch availability for microbial fermentation. The effect of starch chemistry on post-ileal nutrient flow and microbial activity was studied in 8 ileal-cannulated pigs (29.4 kg BW) each fed 4 diets containing 70% purified starch (amylose content, < 5, 20, 28 and 63%; reflected by in vitro maximal digestion rate; 1.06, 0.73, 0.38, and 0.22%/min, respectively). Amylose content and in vitro digestion rate of starch quadratically affected (P < 0.001) post-ileal starch flow (4.1, 3.7, 4.1 and 405 mg/g starch fed; respectively, for low to high amylose and rapid to slow digestion rate), fecal total SCFA (10.5, 9.6, 16.2 and 28.7 μmol/g of DM fed) and butyrate concentration (1.1, 1.0, 2.5, and 2.9 μmol/g of DM fed), and fecal bifidobacteria (5.6, 5.2, 5.1, and 6.6 log rDNA gene copies/g). Gene copy numbers of the Lactobacillus group, Enterococcus, Streptpcoccus, Enterobacteriaceae, Bacteroides-Prevotella-Porphyrmona and Clostridial clusters I, IV and XIV in ileal digesta and feces did not differ (P < 0.05) among starches. In conclusion, starch with high amylose and low in vitro digestibility increased post-ileal nutrient flow and microbial fermentation, and selectively promoted growth of bifidobacteria in the distal gut. Substantial changes of starch chemistry (≥45% amylose and ≤0.34%/min in vitro digestion rate) were required to alter the post-ileal variables in pigs. Grant Funding Source: ALMA, Provimi, Alberta Pulse Growers

Resistant and Digestible Starch Contents in Sweet Potato Cultivars and Lines

The sweet potato is recognized as a health-promoting food, owing to its richness in functional components such as dietary fiber; however, only a few studies have been conducted on resistant starch (RS) levels in the sweet potato. Now, the sweet potato is expected to have potential as a renewable resource for bio-ethanol production. The enzymatic digestibility of raw starch appears to influence the conversion rate of starch to sugar in the first step of ethanol production. In this study, the RS content of gelatinized starch, digestible starch (DS) content of raw starch and other starch properties were investigated using 21 sweet potato cultivars and lines to assess varietal variations and to estimate relationships among these starch properties. The RS content of gelatinized starch ranged between 1.8 and 9.5%. The DS content of raw starch ranged between 69.3 and 98.3%. Analysis of variance indicated that the varietal differences were significant at the 0.1% level for both the RS content and the DS content. Sweet potato starches with high amylose content and low paste viscosity showed high RS content of gelatinized starch. Starches with low pasting temperature and slow retrogradation showed high DS content of raw starch. A new breeding line, Kyukei 03284-156, contained a unique starch having the highest RS content, highest amylose content, lowest paste viscosity and abnormal granule morphology. The results of this study are expected to provide useful information to food industries that utilize sweet potato starch and to breeders that attempt to improve the RS and DS contents in sweet potato cultivars.

In vitro and in vivo acute antihyperglycemic effects of five selected indigenous plants from Jordan used in traditional medicine

Achillea santolina L., Pistacia atlantica Desf, Rheum ribes L., Sarcopoterium spinosum (L.) Spach and Teucrium polium L. have traditionally been used as herbal antidiabetic medicines. However their alleged benefits and mechanisms remain elusive. This study aimed to evaluate the effect of water extracts of these plants in in vitro and in vivo experiments. In vitro enzymatic starch digestion with aqueous extracts from plants at concentrations of 1, 5, 10, 12.5, 25, 50 and 100 mg/ml was assayed using α-amylase and α-amyloglucosidase. Acarbose was used as control and glucose liberation was determined by glucose oxidase method. Oral starch tolerance test (OSTT) and oral glucose tolerance test (OGTT) were determined for the plant extracts at concentrations 125, 250 and 500 mg/kg b.wt. on Sprague-Dawley rats. Blood glucose levels in rats treated with plant extracts and drugs (acarbose or metformin and glipizide) were measured at -30, 0, 45, 90 and 135 min. Compared to acarbose (IC(50)=1.2 μg/ml), water extracts of Pistacia atlantica, Rheum ribes and Sarcopoterium spinosum exerted significant dose dependent dual inhibition of α-amylase and α-glucosidase in in vitro enzymatic starch digestion bioassay, with IC(50)s; 46.98, 58.9 and 49.9 mg/ml, respectively. Comparable in vivo results were obtained for starch-fed rats, exhibiting significant acute postprandial antihyperglycemic efficacies. While Achillea santolina and Teucrium polium extracts lacked any favourable in vitro anti-α-amylase and anti-α-glucosidase effect, other modes of action can possibly explain their substantial acute antihyperglycemic activities in starch-treated rats. Except for Pistacia atlantica extracts, none of the investigated extracts qualified for improving the glucose intolerance in fasted rats on glucose loading. Pistacia atlantica, Rheum ribes and Sarcopoterium spinosum can be considered as potential candidates for amelioration/management of type 2 diabetes.

Enhancement of fermentative hydrogen production from green algal biomass of Thermotoga neapolitana by various pretreatment methods

Biomass of the green algae has been recently an attractive feedstock source for bio-fuel production because the algal carbohydrates can be derived from atmospheric CO 2 and their harvesting methods are simple. We utilized the accumulated starch in the green alga Chlamydomonas reinhardtii as the sole substrate for fermentative hydrogen (H 2) production by the hyperthermophilic eubacterium Thermotoga neapolitana. Because of possessing amylase activity, the bacterium could directly ferment H 2 from algal starch with H 2 yield of 1.8–2.2 mol H 2/mol glucose and the total accumulated H 2 level from 43 to 49% (v/v) of the gas headspace in the closed culture bottle depending on various algal cell-wall disruption methods concluding sonication or methanol exposure. Attempting to enhance the H 2 production, two pretreatment methods using the heat-HCl treatment and enzymatic hydrolysis were applied on algal biomass before using it as substrate for H 2 fermentation. Cultivation with starch pretreated by 1.5% HCl at 121 °C for 20 min showed the total accumulative H 2 yield of 58% (v/v). In other approach, enzymatic digestion of starch by thermostable α-amylase (Termamyl) applied in the SHF process significantly enhanced the H 2 productivity of the bacterium to 64% (v/v) of total accumulated H 2 level and a H 2 yield of 2.5 mol H 2/mol glucose. Our results demonstrated that direct H 2 fermentation from algal biomass is more desirably potential because one bacterial cultivation step was required that meets the cost-savings, environmental friendly and simplicity of H 2 production.

Evaluation of Different Teas against Starch Digestibility by Mammalian Glycosidases

Current work investigated the ability of different tea (green, oolong and black teas) in inhibiting human salivary alpha-amylase (HSA) and mammalian alpha-glucosidase (AGH). The inhibitory profiles were correlated to their major polyphenol content (theaflavins and catechins). The fully fermented black tea was demonstrated to be most potent in inhibiting HSA and AGH (IC50 of 0.42 to 0.67 and 0.56 to 0.58 mg of tea leaves/mL respectively). Its capability in retarding the digestion of a real food system (rice noodle) was further elucidated with an in vitro digestion study. Results indicated that black tea was able to retard starch digestion moderately, thereby allowing a gradual reduction of sugar liberation. Polyphenolic profile analysis suggested that the oxidized catechins, theaflavins, may be responsible for its activity. We have found that refractive index (RI) measurement is a rapid, direct, and highly convenient method for quantifying the degree of enzymatic starch digestion and kinetics. The RI method has good linearity range, limit of detection (0.1596 mg/mL, maltose equivalent) and limit of quantitation (0.6312 mg/mL) and was successfully applied in our study.

Extraction of β‐Glucan from Oat Bran in Laboratory Scale

ABSTRACTEffects of various enzymes and extraction conditions on yield and molecular weight of β‐glucans extracted from two batches of commercial oat bran produced in Sweden are reported. Hot‐water extraction with a thermostable α‐amylase resulted in an extraction yield of ≈76% of the β‐glucans, while the high peak molecular weight was maintained (1.6 × 106). A subsequent protein hydrolysis significantly reduced the peak molecular weight of β‐glucans (by pancreatin to 908 × 103 and by papain to 56 × 103). These results suggest that the protein hydrolyzing enzymes may not be pure enough for purifying β‐glucans. The isolation scheme consisted of removal of lipids with ethanol extraction, enzymatic digestion of starch with α‐amylase, enzymatic digestion of protein using protease, centrifugation to remove insoluble material, removal of low molecular weight components using dialysis, precipitation of β‐glucans with ethanol, and air‐drying.

Effect of particle size on kinetics of starch digestion in milled barley and sorghum grains by porcine alpha-amylase

The influence of milled grain particle size on the kinetics of enzymatic starch digestion was examined. Two types of cereals (barley and sorghum) were ground, and the resulting grounds separated by size using sieving, with sizes ranging from ∼0.1 to ∼3 mm. In vitro enzymatic digestion was performed, using pancreatic alpha-amylase, amyloglucosidase and protease, to determine fractional-digestion rates over 24 h. The resulting glucose production rate data were well fitted by simple first-order kinetics. For each sieve screen size, the digestion rate of barley was always higher than that of sorghum. The rate coefficients for digestion showed a decrease with increasing size, and could be well fitted by an inverse square relationship. This is consistent with the supposition that starch digestion in these systems is controlled by diffusion of enzyme through the grain fragment. Apparent diffusion coefficients of alpha-amylase obtained by fitting the size dependence were 0.76 (sorghum) and 1.7 (barley) × 10 −7 cm 2 s −1, 9 (sorghum) and 4 (barley) times slower than predicted for a molecule of the size of alpha-amylase in water.

Effect of partial gelatinization and retrogradation on the enzymatic digestion of waxy rice starch

The effect of partial gelatinization and retrogradation on in vitro enzymatic digestibility of waxy rice starch was investigated, and the relationship between the residual crystallinity and digestibility measured. An aqueous dispersion of starch (5%, dry weight basis) was partially gelatinized by heating at different temperatures (60, 65, or 70 °C for 5 min). The relative melting enthalpy values of the starch samples, based on the melting enthalpy of native starch, were 69.0, 36.7, and 8.5%, respectively. Retrograded starch samples were also prepared by storing a fully gelatinized starch paste (5% starch) at 4 °C for 2, 4, or 7 d, and the relative melting enthalpy values for the starch samples were 36.7, 67.2, and 79.9%, respectively. The partial gelatinization and retrogradation changed the enzymatic digestion behavior of the waxy rice starch samples, and the changes were significant in the initial stage of digestion. The digestion rate was reduced as the melting enthalpy increased. The amounts of slowly digestible starch (SDS) and resistant starch (RS) correlated positively with the relative melting enthalpy of the partially gelatinized or retrograded starch samples. The glycemic index (GI) estimated using an in vitro digestion test correlated negatively with the relative melting enthalpy. At similar melting enthalpy levels, the partially gelatinized starch samples were more resistant to enzymatic digestion than the retrograded starch samples, indicating that the thermal history and the crystalline morphology affected the enzymatic digestion behavior of starch.

Characteristics of native and enzymatically hydrolyzed common wheat (Triticum aestivum) and dicoccum wheat (Triticum dicoccum) starches

The compositional, structural, and enzymatic digestibility of starches isolated from common wheat and dicoccum wheat were determined to find out the possible reason for hypoglycemic nature of dicoccum wheat. The gelatinization temperature range (65±3 °C) as well as the elution profile of both the starches on Sepharose CL-2B gel were comparable, but the peak (PV, 233 RVU) and set back viscosity (SB, 140 RVU) of dicoccum wheat starch were higher than common wheat starch (PV, 211 RVU; SB, 113 RVU) as recorded in rapid visco-analyser (RVA). The degree of crystallinity (DOC: 29%) and the thermal energy (TE: 142.35 J) of dicoccum wheat starch were considerably higher than the DOC (23%) and TE (67.82 J) of the common wheat starch. The starches were digested with alpha-amylase (human salivary), beta-amylase (barley malt), pullulanase (Klebsiella pneumoniae), and amyloglucosidase (Aspergillus niger), and the solubilized fraction was separated from the undigested fraction (residue) by centrifugation. Characterization of the sugars in the solubilized fraction from alpha-amylase were glucose, maltose, and oligosaccharides of 3–7 DP, maltose from beta-amylase, maltotriose from pullulanase and glucose from amyloglucosidase. However, the molecular weight (Mw) of the residues from the enzymatic digestion of dicoccum wheat starch was slightly higher than that of the common wheat starch. The microscopic examination of residues also exhibited a few bigger chunks in case of dicoccum wheat starch and a large number of smaller sized pieces in the case of common wheat starch. Since, starch is the major component of wheat, these parameters may help to explain subtle differences in the digestibility that exist between common wheat and dicoccum wheat.

Physicochemical properties and enzymatic digestibility of starch from edible canna ( Canna edulis) grown in Vietnam

Edible canna starch and other root starches (cassava, potato and sweet potato) extracted from raw roots grown in Vietnam were used to determine physicochemical properties and enzymatic digestibility. The edible canna starch exhibited significantly higher blue value and amylose content than the other root starches. It also had a wide range of gelatinization temperature and a high transition enthalpy. The viscosity of hot paste from edible canna starch was quite low and stable, whereas the cool paste had high viscosity and weak resistance against retrogradation. The paste clarity of edible canna starch was also significantly higher than that of the others. During refrigeration and frozen storage, the paste of edible canna starch released so much expelled and absorbed water, which showed low stability during storage with high net syneresis. In this study, native edible canna starch was also found like potato starch to be highly resistant to hydrolysis by α-amylase.

Structure of aBacillus halmapalusfamily 13 α-amylase, BHA, in complex with an acarbose-derived nonasaccharide at 2.1 Å resolution

The enzymatic digestion of starch by alpha-amylases is one of the key biotechnological reactions of recent times. In the search for industrial biocatalysts, the family GH13 alpha-amylase BHA from Bacillus halmapalus has been cloned and expressed. The three-dimensional structure at 2.1 A resolution has been determined in complex with the (pseudo)tetrasaccharide inhibitor acarbose. Acarbose is found bound as a nonasaccharide transglycosylation product spanning the -6 to +3 subsites. Careful inspection of electron density suggests that the bound ligand could not have been formed through successive transglycosylations of acarbose and must also have featured maltose or maltooligosaccharides as an acceptor.

Effect of extrusion, espansion and toasting on the nutritional value of peas, faba beans and lupins

An assessment was made of the effect that different treatments (toasting, expansion, extrusion) have on the nutritional value of protein plants (pea, faba bean, lupin). In a randomized block design, feeds were screened for enzymatic digestibility of starch and protein, N solubility and in vitro protein degradability. Expansion and extrusion cause increased starch enzymatic degradability while toasting produced virtually no effects. In peas this value increased from 11.80% in meal to 39.70% in the extruded product; 85.37% is the percentage for the expanded product, while 10.90% is the starch digestibility value for toasted peas. In faba beans the extrusion process increased starch digestibility from 11.39% to 85.05%, while in extruded lupins a complete starch hydrolysis was obtained, while in the meal the polysaccharide digestion was 54.48%.The expansion and extrusion processes significantly decreased rumen degradability during the first 8 hours of incubation. Toasted peas had lower degradability if compared with controls but not with the other treatments. The only potentially alternative source to soybean is the extruded faba bean. In spite of its lower protein content, this feed is characterized by a considerably lower in vitro protein degradability than soybean. This implies that the digestible food protein content is comparable (124.90 g/kg DM) to that of soybean (109.78 g/kg DM) and definitely higher than that of all other protein plants.

A method for routine measurements of total sugar and starch content in woody plant tissues.

Several extraction and measurement methods currently employed in the determination of total sugar and starch contents in plant tissues were investigated with the view to streamline the process of total sugar and starch determination. Depending on the type and source of tissue, total sugar and starch contents estimated from samples extracted with 80% hot ethanol were significantly greater than from samples extracted with a methanol:chloroform:water solution. The residual ethanol did not interfere with the sugar and starch determination, rendering the removal of ethanol from samples unnecessary. The use of phenol-sulfuric acid with a phenol concentration of 2% provided a relatively simple and reliable colorimetric method to quantify the total soluble-sugar concentration. Performing parallel sugar assays with and without phenol was more useful for accounting for the interfering effects of other substances present in plant tissue than using chloroform. For starch determination, an enzyme mixture of 1000 U alpha-amylase and 5 U amyloglucosidase digested starch in plant tissue samples more rapidly and completely than previously recommended enzyme doses. Dilute sulfuric acid (0.005 N) was less suitable for starch digestion than enzymatic hydrolysis because the acid also broke down structural carbohydrates, resulting in overestimates of starch content. After the enzymatic digestion of starch, the glucose hydrolyzate obtained was measured with a peroxidase-glucose oxidase/o-dianisidine reagent; absorbance being read at 525 nm after the addition of sulfuric acid. With the help of this series of studies, we developed a refined and shortened method suitable for the rapid measurement of total sugar and starch contents in woody plant tissues.