Determination of neutral sugars in soil by capillary gas chromatography after derivatization to aldononitrile acetates

We have quantified ribose, rhamnose, arabinose, xylose, fucose, mannose, glucose, and galactose in soil by gas chromatography (GC) simultaneously after converting to aldononitrile acetate derivatives. A recommended single-hydrolytic step by 4 M trifluoroacetic acid (TFA) at 105 °C for 4 h was more effective for releasing soil neutral sugars from non-cellulosic carbohydrates and better suited to our purification procedure compared with the sulphuric acid hydrolysis. Linearity of the GC detection for each neutral sugar was in the range of 10-640 μg ml⁻¹ and the recovery of neutral sugars from the spiked soil samples ranged from 76% to 109.7%. The coefficients of variation of the neutral sugars in four soils were lower than 2.0% for the instrument and 4.6-7.6% for the whole determination procedures. Compared with the trimethylsilyl (TMS) derivatization, the recovery of our newly modified method was more satisfactory and the reproducibility of ribose was improved significantly. Moreover, the aldononitrile acetate derivative was more stable than TMS derivative. Therefore, it is a promising approach suitable for a routine use in the quantitative analysis of soil neutral sugars, since it is fast, sensitive, and reproducible.     

Efficient sugar release by the cellulose solvent-based lignocellulose fractionation technology and enzymatic cellulose hydrolysis

Efficient liberation of fermentable soluble sugars from lignocellulosic biomass waste not only decreases solid waste handling but also produces value-added biofuels and biobased products. Industrial hemp, a special economic crop, is cultivated for its high-quality fibers and high-value seed oil, but its hollow stalk cords (hurds) are a cellulosic waste. The cellulose-solvent-based lignocellulose fractionation (CSLF) technology has been developed to separate lignocellulose components under modest reaction conditions (Zhang, Y.-H. P.; Ding, S.-Y.; Mielenz, J. R.; Elander, R.; Laser, M.; Himmel, M.; McMillan, J. D.; Lynd, L. R. Biotechnol. Bioeng. 2007, 97 (2), 214- 223). Three pretreatment conditions (acid concentration, reaction temperature, and reaction time) were investigated to treat industrial hemp hurds for a maximal sugar release: a combinatorial result of a maximal retention of solid cellulose and a maximal enzymatic cellulose hydrolysis. At the best treatment condition (84.0% H3PO4 at 50 degrees C for 60 min), the glucan digestibility was 96% at hour 24 at a cellulase loading of 15 filter paper units of cellulase per gram of glucan. The scanning electron microscopic images were presented for the CSLF-pretreated biomass for the first time, suggesting that CSLF can completely destruct the plant cell-wall structure, in a good agreement with the highest enzymatic cellulose digestibility and fastest hydrolysis rate. It was found that phosphoric acid only above a critical concentration (83%) with a sufficient reaction time can efficiently disrupt recalcitrant lignocellulose structures.     

氮肥和羊粪对内蒙古典型草原土壤多糖含量及组成的影响

采用三氟乙酸(TFA)水解、糖醇乙酸酯衍生、气相色谱法测定土壤多糖含量,研究了内蒙古羊草草原围栏封育并连续5年施用氮肥和羊粪的表层土壤的多糖含量和组成特征。结果表明:长期施用氮肥显著降低土壤多糖含量6%～19%;施用羊粪显著提高土壤多糖含量20%。施氮肥或羊粪都降低了(半乳糖+甘露糖)(/阿拉伯糖+木糖)(GM/AX)和甘露糖/木糖(M/X)的比例,表明施肥降低了微生物多糖对土壤多糖的贡献,但是施氮肥的土壤降低幅度大于施羊粪的土壤。这表明,长期施氮肥和羊粪都将改变土壤多糖含量和组成。     

Release of D-xylose from wheat straw by acid and xylanase hydrolysis and purification of xylitol

Xylitol is a valuable sweetener produced from xylose-rich biomass. Our objective was to optimize conditions for maximum release of D-xylose from wheat straw by acid or enzyme hydrolysis with minimal release of other monosaccharides, and to purify xylitol from three other alditols. Ground straw was treated with 10 parts of 0.2-0.4 M sulfuric acid at 110-130 degrees C for 15-45 min or at reflux with 0.75-1.25 M sulfuric acid for 1.5-3 h. Under optimum conditions of either 0.3 M acid at 123 degrees C for 28 min or 1.0 M acid at 100 degrees C for 3 h, 18 or 19% of D-xylose plus approximately 6% other sugars were produced from straw (dry basis). A 16% yield of D-xylose plus 6% other sugars was obtained when hydrothermally (10% straw, 160 degrees C, 1 h) treated straw was incubated with a commercial xylanase. The lack of enzyme specificity for D-xylose release was attributed to the autohydrolysis of polysaccharides during the pretreatment plus slow hydrolysis of cellulose during enzyme digestion. Xylitol with a purity of 95% was obtained in 10% yield from straw after the reduction of an acid-hydrolyzate followed by fractional crystallization. Purification of the mixture of four alditols by open-column chromatography on a strongly basic anion-exchange resin in hydroxide form gave 7% xylitol crystals with a purity of 99%.     

Involvement of proteins in cloud instability of shamouti orange [Citrus sinensis (L.) Osbeck] juice.

The present study provides evidence for the involvement of protein in cloud instability of natural orange juice. No heat-coagulable proteins were found in the serum. Insoluble cloud matter (ICM) was heat-flocculated following enzymatic pectin degradation (EPD). The degree of flocculation depended on temperature (from approximately 50 to 75 degrees C) and was highest at pH 3.5. The fresh juice contained about 6.5 and 1.8 mg mL(-1) of ICM and alcohol-insoluble solids of the serum (AISS), respectively. The ICM and the AISS contained, respectively, proteins (182+/-14 and 119+/-3 microg mg(-1)), galacturonic acid (37+/-6.6 and 175+/-1 microg mg(-1)), and neutral sugars (350+/-44 and 338+/-22 microg mg(-1)). EPD resulted in removal of a marked portion of the pectin and was accompanied by partial removal of neutral sugars (mainly glucose and galactose) and some proteins from the pectic polymer in both AISS and ICM. Under electrophoresis, proteins of the AISS included bands in the range of 20-52 kDa and 10-14 kDa and those of the ICM at 22 and 50 kDa.     

Manufacture of fermentable sugar solutions from sugar cane bagasse hydrolyzed with phosphoric acid at atmospheric pressure

Sugar cane bagasse, a renewable and cheap bioresource, was hydrolyzed at 100 degrees C using phosphoric acid at different concentrations (2, 4, or 6%) and reaction times (0-300 min) to obtain fermentable sugar solutions, which have a high concentration of sugars (carbon source for microorganism growth) and a low concentration of growth inhibitors (acetic acid and furfural). Xylose, glucose, arabinose, acetic acid, and furfural were determined following the hydrolysis. Kinetic parameters of mathematical models for predicting these compounds in the hydrolysates were obtained. Derived parameters such as efficiency of hydrolysis or purity of hydrolysates were considered to select as optimal conditions 6% phosphoric acid at 100 degrees C for 300 min. Using these conditions, 21.4 g of sugars L(-)(1) and <4 g of inhibitors L(-)(1) were obtained from the hydrolysis with a water/solid ratio of 8 g of water g(-)(1) of sugar cane bagasse on a dry basis.     

Involvement of proteins in cloud instability of valencia orange [Citrus sinensis (L.) Osbeck] juice.

The present study examined the involvement of proteins in cloud flocculation of Valencia orange juice. Marked differences in cloud instability were found between juices of different harvest dates. Heating of enzymatic pectin degraded juice from April and June harvests resulted in development of clumps and their precipitation. Although the juice from both harvesting dates remained hazy, the juice of April harvest was more turbid than that from June. Usually clarification increases as the temperature increases from ambient to 125 degrees C. Clarification occurred at pH 2.5-4.5 and was maximal at pH 3.5. The clarification of the April harvest juice was markedly lower than that of the June harvest. The fresh juice contained about 5.2 and 1.7 mg mL(-1) insoluble cloud matter (ICM) and alcohol-insoluble serum solids (AISS), respectively. The ICM and the AISS, respectively, contained: proteins (244.5+/-8.7 and 132+/-1.8 microg mg(-1)), galacturonic acid (40+/-0 and 120+/-0 microg mg(-1)) and neutral sugars (270+/-39 and 329+/-23 microg mg(-1)). Enzymatic pectin degradation resulted in removal of a marked portion of the pectin, and was accompanied by partial removal of neutral sugars (mainly glucose and galactose) and some proteins from the pectic polymer in both AISS and ICM. Proteins of the AISS included major bands at 10-14, 20, and 28 kDa and those of the ICM bands at 22, 24, 26, and 45 kDa.     

Pretreatment and Enzymatic Hydrolysis of Sorghum Bran

Sorghum bran has potential to serve as a low‐cost feedstock for production of fuel ethanol. Sorghum bran from a decortication process (10%) was used for this study. The approximate chemical composition of sorghum bran was 30% starch, 18% hemicellulose, 11% cellulose, 11% protein, 10% crude fat, and 3% ash. The objective of this research was to evaluate the effectiveness of selected pretreatment methods such as hot water, starch degradation, dilute acid hydrolysis, and combination of those methods on enzymatic hydrolysis of sorghum bran. Methods for pretreatment and enzymatic hydrolysis of sorghum bran involved hot water treatment (10% solid, w/v) at 130°C for 20 min, acid hydrolysis (H₂SO₄), starch degradation, and enzymatic hydrolysis (60 hr, 50°C, 0.9%, v/v) with commercial cellulase and hemicellulose enzymes. Total sugar yield by using enzymatic hydrolysis alone was 9%, obtained from 60 hr of enzyme hydrolysis. Hot water treatment facilitated and increased access of the enzymes to hemicellulose and cellulose, improving total sugar yield up to 34%. Using a combination of starch degradation, optimum hot water treatment, and optimum enzymatic hydrolysis resulted in maximum total sugar yield of up to 75%.     

Extending storage life of fresh-cut apples using natural products and their derivatives

Prevention of browning of apples slices has been difficult to achieve because of the rapidity of the enzymatic oxidation of phenolic substrates even under reduced atmospheric pressure storage. Combinations of enzymatic inhibitors, reducing agents, and antimicrobial compounds containing calcium to extend storage life were tested to decrease the browning of Red Delicious apple slices stored at 5 and 10 degrees C under normal atmospheric conditions. Treatments were devised to prevent browning for up to 5 weeks at 5 degrees C with no apparent microbial growth using dipping solutions of compounds derived from natural products consisting of 4-hexylresorcinol, isoascorbic acid, a sulfur-containing amino acid (N-acetylcysteine), and calcium propionate. Analyses of organic acids and the major sugars revealed that the slices treated with the combinations of antibrowning compounds retained higher levels of malic acid and had no deterioration in sugar levels at 5 and 10 degrees C, indicating that higher quality was maintained during storage.     

Characterization of the surface-active components of sugar beet pectin and the hydrodynamic thickness of the adsorbed pectin layer

The fraction of sugar beet pectin (SBP) adsorbed onto limonene oil droplets during emulsification has been isolated, and its chemical and physicochemical characteristics have been determined. While the SBP sample itself was found to contain 2.67 and 1.06% protein and ferulic acid, respectively, the adsorbed fraction contained 11.10% protein and 2.16% ferulic acid. The adsorbed fraction was also found to have a higher degree of acetylation, notably at the C2 position on the galacturonic acid residues, and was also found to contain a higher proportion of neutral sugars, which are present in the ramified side chains of the pectin molecules. The thickness of the layer of SBP adsorbed onto polystyrene latex particles was studied by dynamic light scattering and was found to increase with increasing surface coverage. It was found to have a value of approximately 140 nm at plateau coverage, which closely corresponded to the hydrodynamic diameter of the pectin chains. The adsorbed layer thickness was found to be sensitive to pH and the presence of electrolyte. The thickness at a surface coverage of approximately 20 mg/m(2) in the absence of electrolyte at pH approximately 4 was 107 nm and at pH 8.8 was 70 nm, while at pH approximately 4 in the presence of 10 mM NaCl the thickness was found to be 70 nm. It was concluded that the SBP molecules form multilayers at the surface due to electrostatic interaction between the positively charged protein moieties and the galacturonic acid residues. The removal of calcium from the SBP had no effect on the adsorbed layer thickness; hence, multilayer formation due to calcium ion cross-linking was considered unlikely.     

Changes of lignin phenols and neutral sugars in different soil types of a high-elevation forest ecosystem 25 years after forest dieback

Long-term effects of forest disturbance 25 yr ago on lignin and non-cellulosic polysaccharide pools in an unmanaged high-elevation Norway spruce (Picea abies L. [Karst.]) forest were investigated by comparing three dieback sites with three adjacent control sites with non-infested spruce on identical soils. Samples were taken from the forest floor and the mineral soil; one Ah horizon sample per site was physically fractionated into density and particle size fractions. Additionally, changes in the above- and belowground input of lignin and non-cellulosic polysaccharides after forest dieback were quantified. Lignin and its degree of structural alteration in plant and soil samples were assessed by CuO oxidation and subsequent analysis of the lignin phenols. Non-cellulosic polysaccharides were determined after hydrolysis with trifluoroacetic acid (TFA), derivatisation of their neutral sugar monomers by reduction to alditols, and subsequent acetylation. The total plant-derived input of lignin and non-cellulosic polysaccharides to the soil was similar for the dieback and the control sites. The chemical composition of the input has changed considerably after forest dieback, as shown by significantly higher syringyl/vanillyl (S/V) ratios and significantly lower (galactose+mannose)/(arabinose+xylose) (GM/AX) ratios. This indicates a changed plant input and a higher contribution of microbial sugars. Contents of lignin phenols in the forest floor and coarse particle size fractions of the A horizons were significantly smaller at the dieback sites (p<0.01). Moreover, larger acid-to-aldehyde ratios of vanillyl units (Ac/Al)_v indicated an increased degree of lignin phenol alteration. Also contents of neutral sugars were significantly (p<0.01) smaller in the forest floor, but not in the A horizons of the dieback sites. The GM/AX mass ratios as well as the (rhamnose+fucose)/(arabinose+xylose) (RF/AX) ratios in the forest floor and coarse particle size fractions of the mineral topsoil were significantly (p<0.01) larger after forest dieback, indicating a larger relative contribution of microbial sugars. In general, the lignin phenol and neutral sugar pools of all three soil types exhibited similar response patterns to the changed site conditions. Our results demonstrate that the lignin and neutral sugar pools of humic topsoil horizons are highly sensitive to forest disturbances. However, the two compounds show different patterns in the mineral soil, with the major neutral sugar pool being stabilized against changes whereas the lignin phenol pool decreases significantly.     

几种化学物质对稻草秸秆酶解糖化的影响

在木质纤维素酶解研究领域,高浓度还原糖的获得是实现其能源转化的基础。以稀硫酸预处理后的稻草秸秆为原料,初始酶解物料条件为20%（重量/体积）,木聚糖酶220U.g-（1底物）,纤维素酶6FPU.g-（1底物）,果胶酶50U.g-（1底物）,选取吐温80（Tween80）、MgSO4、FeSO4、聚乙二醇（PEG）和牛血清白蛋白（BSA）作为酶解体系添加物,分别考察了其添加量对还原糖浓度的影响。试验结果表明：在稻草秸秆酶解体系中,Tween80、MgSO4、FeSO4、PEG和BSA5种化学物质各自最佳添加量分别为0.05、0.0005、0.02、0.01g和0.0005g.g-（1底物）;助催化作用强度依次为MgSO4〉Tween80〉BSA〉FeSO4〉PEG。添加MgSO40.0005g.g-（1底物）,48h糖化后,还原糖浓度达到72.45g.L-1,比对照提高了7.98%。试验结果表明添加适量化学物质可以有效提高还原糖浓度。     

Kinetics of hydrolysis of fructooligosaccharides in mineral-buffered aqueous solutions: influence of pH and temperature

High-performance anion exchange chromatography coupled with a pulsed amperometric detection system (HPAEC-PAD) was used to evaluate the extent of chemical hydrolysis of three fructooligosaccharides (FOS) including 1-kestose (beta-D-Fru-(2-->1)(2)-alpha-D-glucopyranoside, GF2), nystose (beta-D-Fru-(2-->1)(3)-alpha-D-glucopyranoside, GF3), and fructofuranosylnystose (beta-D-Fru-(2-->1)(4)-alpha-D-glucopyranoside, GF4). A kinetic study was carried out at 80, 90, 100, 110, and 120 degrees C in aqueous solutions buffered at pH values of 4.0, 7.0, and 9.0. Under each experimental condition, the determination of the respective amounts of reactants and hydrolysis products showed that FOS hydrolysis obeyed pseudo-first-order kinetics as the extent of hydrolysis, which decreased at increasing pH values, increased with temperature. The three oligomers were found to be degraded mainly under acidic conditions, and at the highest temperature value (120 degrees C), a quick and complete acid degradation of each FOS was observed. Using the Arrhenius equation, rate constants, half-life values, and activation energies were calculated and compared with those obtained from sucrose under the same experimental conditions. It appeared that the hydrolysis of FOS took place much more easily at acidic pH than at neutral or basic pH values.     

Sucrose, glucose, and fructose extraction in aqueous carrot root extracts prepared at different temperatures by means of direct NMR measurements

Solutions obtained by heating carrot roots in water (stocks) are widely used in the food industry, but little information is available regarding the metabolites (intermediates and products of metabolism) found in the stock. The effect of treatment temperature and duration on the sugar composition of stocks was investigated directly by quantitative (1)H NMR spectroscopy, to understand the extraction mechanism when processing at 100 degrees C. Stocks prepared at three different temperatures (50, 75, and 100 degrees C) were investigated for up to 36 h. Three sugars (sucrose, glucose, and fructose) were detected and quantified. The concentrations of these three sugars reached a maximum after 9 h when the temperature of treatment was 50 or 75 degrees C. At 100 degrees C, the sucrose concentration reached a maximum after 3 h, whereas the concentration of glucose and fructose was still increasing at that time. Comparison of the kinetic composition of these carrot stocks with that of model sugar solutions leads to the proposal that the changes in stock composition result from sugar diffusion, sucrose hydrolysis, and hydroxymethylfurfural (HMF) formation.     

Reversible conformational change in beta-lactoglobulin A modified with N-ethylmaleimide and resistance to molecular aggregation on heating

beta-Lactoglobulin A (beta LG A) modified with N-ethylmaleimide (NEM-beta LG A) was purified by ion exchange chromatography, and modification of beta LG A by NEM was confirmed by time of flight mass spectrometry and 5,5'-dithiobis(2-nitrobenzoic acid) methods. The fluorescent spectrum of NEM-beta LG A was slightly different from that of native beta LG A. NEM-beta LG A gave no polymerization after heating at 80 degrees C and pH 7.5, as shown by polyacrylamide gel electrophoresis. Conformational change of NEM-beta LG A was observed at 80 degrees C by ultraviolet differential spectra, whereas after cooling it recovered to its original state as before heating, indicating apparent reversible thermal denaturation. Native beta LG A is resistant to pepsin hydrolysis, whereas heated beta LG A was easily hydrolyzed by pepsin. NEM-beta LG A before heating was also resistant to pepsin hydrolysis, and after heating NEM-beta LG A was still resistant to pepsin hydrolysis. These results indicate that NEM-beta LG A maintained a conformation similar to its native form even after heating. Addition of 0.2 M NaCl to the beta LG A heated under salt-free condition induced polymerization of heated beta LG A molecules, but not that of heated NEM-beta LG A. This seemed to indicate that the formation of inter- or intramolecular disulfide linkage made the heat-induced conformational change of beta LG A irreversible.     

Monosaccharide composition of sweetpotato fiber and cell wall polysaccharides from sweetpotato, cassava, and potato analyzed by the high-performance anion exchange chromatography with pulsed amperometric detection method

The cell wall materials (CWMs) from sweetpotato (Ipomoea batatas cv. Kokei 14), cassava (Manihot esculenta), and potato (Solanum tuberosum cv. Danshaku) and commercial sweetpotato fiber as well as their polysaccharide fractions were analyzed for sugar composition by the high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method. The separation of arabinose and rhamnose, and xylose and mannose, by this method has been improved using a CarboPac PA 10 column. Pretreatment of the CWMs and cellulose fractions with 12 M H(2)SO(4) was required for complete hydrolysis to occur. Commercial sweetpotato fiber was found to be mainly composed of glucose (88.4%), but small amounts of other sugars were also detected. Among the root crops, sweetpotato CWM had the highest amount of pectin and galacturonic acid. Fucose was detected only in cassava CWM and its hemicellulose fraction, while galactose was present in the highest amount in potato CWM. Among the polysaccharide fractions, it was only in the hemicellulose fraction where significant differences in the sugar composition, especially in the galactose content, were observed among the root crops.     

Hydrolysis of ginger bagasse starch in subcritical water and carbon dioxide

Ginger bagasse from supercritical extraction was hydrolyzed using subcritical water and CO(2) to produce reducing sugars and other low molecular mass substances. Response surface methodology was used to find the best hydrolysis conditions; the degree of hydrolysis and the yield were the two response variables selected for maximization. The kinetic studies of the hydrolysis were performed at 150 bar and temperatures of 176, 188, and 200 degrees C. The higher degree of hydrolysis (97.1% after 15 min of reaction) and higher reducing sugars yield (18.1% after 11 min of reaction) were established for the higher process temperature (200 degrees C). Different mixtures of oligosaccharides with different molecular mass distributions were obtained, depending on the temperature and on the reaction time. The ginger bagasse hydrolysis was treated as a heterogeneous reaction with a first-order global chemical kinetic, in relation to the starch concentration, which resulted in an activation energy of 180.2 kJ/mol and a preexponential factor of 5.79 x 10(17)/s.     

Nature of soil carbohydrate and its association with soil humic substances

The fulvic acid and alkali-soluble polysaccharide fractions of a sandy loam arable soil of the Countesswells series have been subjected to acid hydrolysis or methylation and the products examined by infra-red and NMR spectroscopy, pyrolysis mass spectrometry and chemical analysis. Infra-red and NMR spectroscopy of the polysaccharide fraction indicated that the substance was predominantly carbohydrate, although sugars accounted for less than one-third of the weight by chemical analysis. Pyrolysis mass spectrometry con-firmed the presence of sugars by sugar anhydride formation, but also showed the presence of ‘secondary’ or ‘pseudo’ polysaccharide. The fulvic acid contained only 2–3% sugars by chemical analysis, whereas a much larger carbohydrate component was suggested by physicochemical analyses. Infra-red and pyrolysis mass spectrometry difference spectra for the residues after acid hydrolysis indicated the release of material with some of the characteristics of glycoprotein. Most of the carbohydrate present in the fulvic acid was of the secondary or pseudo polysaccharide type. The hypothesis that the secondary polysaccharide could be a degraded polysaccharide structure in which some sugar residues have been partly transformed to melanoidins by Maillard reaction is explored.     

Retention of essential amino acids during extrusion of protein and reducing sugars

This research investigates the retention of essential amino acid profiles of products during the extrusion of proteins and reducing sugars. Animal proteins (egg and milk protein at 10 and 30% levels) and reducing sugars (fructose and galactose at 0, 2, and 8% levels), with pregelatinized wheat flour, were extruded at 110 and 125 degrees C product temperatures and feed moistures of 19 and 23.5% for egg protein and 13.75 and 16% for milk protein. The nutritional property analyzed was essential amino acid retention, and sugar retention was also considered to understand the relationship of sugars with retention of amino acids. Lysine showed the lowest retention (up to 40%) of all the essential amino acids. Retention of other essential amino acids varied from 80 to 100% in most situations. Apart from lysine, tryptophan, threonine, and methionine were found to be significantly changed ( P < 0.05) with processing conditions. Increased protein and sugar levels resulted in a significant degradation of lysine. Greater lysine retention was found at a lower temperature and higher feed moisture. Results of sugar retention also showed similar patterns. The products made from fructose had greater lysine retention than products made from galactose with any type of protein. The outcomes of this research suggested that the combination of milk protein and fructose at a lower temperature and higher feed moisture is most favorable for developing high-protein extruded products.     

Enzymatic hydrolysis of chestnut purée: process optimization using mixtures of alpha-amylase and glucoamylase

The enzymatic hydrolysis of starch present in chestnut purée was performed through a one-step treatment with a mixture of a commercial thermostable alpha-amylase (Termamyl 120 L, type S) and glucoamylase (AMG 300 L) at 70 degrees C. The effect of the enzyme concentration and the ratio of both amylases in the reaction mixture was studied by means of a factorial second-order rotatable design, which allowed conditions to be set leading to the total conversion of starch to glucose after 15 min of incubation (60 total enzymatic units g(-1) of chestnut; ratio of alpha-amylase/glucoamylase enzymatic units, 0.35:0.65). At lower enzyme concentration, the delay in the addition of the glucoamylase with regard to the addition of the alpha-amylase allowed a slightly higher hydrolysis percentage to be reached when compared to the simultaneous addition of both amylases at the same low enzyme concentration. The kinetics of liberation of glucose supports the existence of a synergistic effect between these two enzymes only in the first moments of the reaction. Finally, a sequential one-step hydrolysis was assayed, and more concentrated glucose syrups were thus obtained.