Starch Hydrolysis Products Research Articles

Study through surveys and fermentation kinetics of the traditional processing of pearl millet ( Pennisetum glaucum) into ben-saalga, a fermented gruel from Burkina Faso

Traditional cereal-based fermented foods are frequently used as complementary foods for infants and young children in Africa. This is the case for ben-saalga, a popular fermented gruel produced from pearl millet ( Pennisetum glaucum) in Burkina Faso. Detailed knowledge of traditional processing is a prerequisite for investigating ways to improve both the nutritional and sanitary qualities of the corresponding foodstuff. In this work, the traditional processing of pearl millet into ben-saalga was investigated in 24 production units, and fermentation kinetics were studied in pilot scale experiments. Processing steps include: washing (optional), soaking of the grains (first fermentation step), grinding and sieving of the wet flour, settling (second fermentation step), and cooking. The soaking step was mainly characterized by alcoholic fermentation whereas lactic acid fermentation occurred during the settling step. Fermentation kinetics during settling indicates a temporal variation of metabolic activity. Initially, both homofermentative and heterofermentative pathways were simultaneously active, and later only a homofermentative pathway was active. The paste produced at the end of settling had a low pH (4.0 ± 0.4) and its microflora was dominated by lactic acid bacteria (LAB) with an amylolytic LAB/LAB ratio of 12%. Sucrose disappeared in the grains during soaking but was not detected in the soaking water, whereas glucose, fructose and maltose appeared transiently. Glucose and fructose were the main substrates observed for lactic acid fermentation during the settling step; however unbalanced fermentation led to the hypothesis that starch hydrolysis products may also serve as substrates for lactic acid formation. At the end of the processing, a 75% and 83% decrease was observed in phytate (IP6) and raffinose, respectively. The sour gruel ben-saalga resulting from cooking the sour paste had inadequate nutritional characteristics with respect to infants' and young children's requirements; it was characterized by fluid consistency (Bostwick flow: 137 mm/30 s) and low energy density (about 30 kcal/100 g of gruel).

Amylopullulanase—A novel enzyme of L. amylophilus GV6 in direct fermentation of starch to L(+) lactic acid

An amylopullulanase enzyme with the ability to hydrolyze different polymeric substrates (starches) was purified from culture supernatants of Lactobacillus amylophilus GV6 during direct fermentation of starch to L(+) lactic acid. The total cell free and cell bound amylase and pullulanase enzyme activities were 0.59 and 0.34 U/(ml min), respectively. Amylopullulanase was active towards soluble starch, raw starch, amylopectin, amylose, glycogen and pullulan. Highest activity was displayed towards the polysaccharide amylopectin. The crude enzyme was purified by ammonium sulphate fractionation, dialysis and Sephacryl S-200 column chromatography. The molecular mass of purified enzyme as estimated by SDS-PAGE and gel filtration chromatography is 90 kDa. Purified enzyme gave an apparent single protein band in SDS-PAGE. This band was found to have both amylase and pullulanase activities. In addition to glucose, maltose and maltotriose were detected as the products of starch hydrolysis by this enzyme. Maximum activity of amylopullulanase was observed at pH 6.5 and temperature 37 °C in acetate buffer. The enzyme activity was observed to be higher at 2.5 mM Ca 2+ in comparison with other metals and surfactants tested. Presence of both amylase and pullulanase activities in amylopullulanase enzyme is a characteristic feature of the fermenting organism, L. amylophilus GV6. This is advantageous for direct fermentation of starch to lactic acid and there are no reports on Lactobacillus spp. producing amylopullulanase enzyme.

Alkaline active maltohexaose-forming α-amylase from Bacillus halodurans LBK 34

The gene encoding Amy 34, a maltohexaose-forming α-amylase from Bacillus halodurans LBK 34 isolated from Lake Bogoria, Kenya, was cloned and sequenced. The mature peptide consists of 958 amino acids with a theoretical molecular weight of 107.2 kDa and p I 4.41, respectively. The gene was expressed in Escherichia coli and the recombinant enzyme purified to homogeneity by a combination of metal chelate affinity and size exclusion chromatography. The pure enzyme exhibited optimum activity at 60 °C and pH 10.5–11.5. The enzyme retained over 60% activity after incubation at 55 °C for 4 h and was most stable at pH 9.0. Complete inhibition of enzyme activity was observed in presence of 5 mM Cu 2+, Fe 2+, Fe 3+, Mn 2+ and 5 mM EDTA. The enzyme displayed 80% of its original activity in presence of 1% (w/v) SDS and was stable in presence of up to 5 mM DTT. Maltohexaose (G6) was the main initial product of starch hydrolysis while other products formed were G4 > G2 > G5 > G3 and G1. The main end product of the enzyme's action on amylose, amylopectin and maltodextrin is maltotetraose. Amy 34 could not hydrolyse pullulan, α and β-cyclodextrin but could hydrolyse γ-cyclodextrin to produce glucose, maltose and maltotetraose. Maltotetraose was the smallest α-(1–4) linked maltooligosaccharide that could be hydrolysed by the enzyme.

Immobilization of Aspergillus niger glucoamylase onto a polyaniline polymer

Glucoamylase was immobilized onto chemically synthesized polyaniline after activation with glutaraldehyde (PANIG). The immobilized glucoamylase (PANIG-GA) presented better performance than free enzyme when submitted to heat treatment, showing a 10 °C decrease on the optimal assay temperature. The pH profile of the immobilized and free enzyme revealed similar behaviour, although PANIG-GA exhibited higher stability in the alkaline range (from pH 8.0 to 10). The K M value of free enzyme for starch (4.76 mg mL −1) was higher than that of the immobilized glucoamylase (0.43 mg mL −1) whereas V max was lower for the immobilized glucoamylase (0.95 μmol min −1) preparation than free enzyme (8.58 μmol min −1). Thin layer chromatography (TLC) showed that no changes was observed between free and immobilized enzyme starch hydrolysis products.

ASPECTS OF THE ISOLATION AND CHARACTERIZATON OF THERMOSTABLE &alpha -AMYLASE FROM <i>Alternaria alternata</i>.

A local isolate of Alternaria alternata isolated from soil under the decaying cassava peels heap was screened for the production of a-amylase and hydrolysis of starch. The maximal dextrinizing amylolytic activity of the partially purified enzyme was obtained at temperature of 600C and pH 7.0. Apart from glucose, sucrose and maltose, several other unknown products of cassava starch hydrolysis were obtained at elevated temperatures (60 – 900C). The thermostable property of the enzyme is discussed in relation to its potential in improving the nutritional quality of starchy foods and industrial usefulness in the future. KEY WORDS: Thermostable a Starch hydrolysis products; Alternaria; Starchy foods; Thin Layer Chromatography. Global Jnl Pure & Applied Sciences Vol.10(1) 2004: 75-79

Feasibility Study for Determination of the Dextrose Equivalent (DE) of Starch Hydrolysis Products with Near‐Infrared Spectroscopy (NIRS)

AbstractStarch hydrolysis products such as maltodextrin and glucose syrup have many applications in the pharmaceutical and food industry. Several physical and functional properties, for example sweetness, compressibility and viscosity vary according to the extent of starch hydrolysis which is characterized by the dextrose equivalent (DE) value. The Pharmacopoeias use the titrimetric Lane Eynon method with alkaline Fehling's solution for determination of DE. In order to replace this empirical method, near‐infrared (NIR) calibration models for the reagent‐free determination of DE were developed. The maltodextrin model with a DE of less than 20 showed very good correlation with r = 0.97. The correlation of the extended model of maltodextrin and spray‐dried glucose syrup with a DE up to 44 was calculated to be r = 0.99. The NIR method is a very effective, rapid and non‐destructive alternative to the current pharmacopoeial procedure.

Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme.

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.

Influence of different maltodextrins on properties of O/W emulsions

Maltodextrins, low converted products of starch hydrolysis, are used in various emulsions to provide desirable viscosity, texture and mouthfeel. In order to assess behaviour of such emulsions in process of production (emulsification) as well as end products in application (pouring, spreading) it is necessary to investigate emulsification dynamics, globule size changes and rheological behaviour. In this work emulsification dynamics and the influence of different factors on dispersion and rheological characteristics of sunflower oil in water emulsions stabilized with food grade emulsifier with addition of maltodextrins were studied. Influence of oil and maltodextrin concentration and partial substitution of oil with equivalent quantity of maltodextrin were investigated, too. It was concluded that certain specific maltodextrin characteristics such as DE value (dextrose equivalent-measure of the degree of starch polymer hydrolysis) i.e. size of maltodextrin molecules significantly influence dispersion and viscous properties and stability of emulsions. With increase of oil concentration for the same concentration of maltodextrin in continuous phase emulsions undergo changes in flow type from shear-thinning to thixotropic and antithixotropic. Antithixotropy induced by shear, was result of formation of network due to two competing factors: orientation of deformed oil droplets and maltodextrin molecules and linking of oil droplets through maltodextrin molecules.

Dextrin characterization by high-performance anion-exchange chromatography–pulsed amperometric detection and size-exclusion chromatography–multi-angle light scattering–refractive index detection

Starch hydrolysis products, or dextrins, are widely used throughout the food industry for their functional properties. Dextrins are saccharide polymers linked primarily by α-(1→4) d-glucose units and are prepared by partial hydrolysis of starch. Hydrolysis can be accomplished by the use of acid, enzymes, or by a combination of both. The hydrolysis products are typically characterized by the “dextrose equivalent” (DE), which refers to the total reducing power of all sugars present relative to glucose. While the DE gives the supplier and buyer a rough guide to the bulk properties of the material, the physiochemical properties of dextrins are dependent on the overall oligosaccharide profile. High-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection and size-exclusion chromatography (SEC) with multi-angle light-scattering and refractive index detection were used to characterize dextrins from commercial sources. HPAEC was used to acquire the oligosaccharide profile, and SEC to obtain an overall molar mass distribution. These methods in combination extended our understanding of the relationship between oligosaccharide profile, DE, and the hydrolysis process. Data from the two techniques enabled a method for estimating the DE that gave results in reasonable agreement with the accepted titration method.

Studies on a thermostable alpha-amylase from the thermophilic fungus Scytalidium thermophilum.

An alpha-amylase produced by Scytalidium thermophilum was purified using DEAE-cellulose and CM-cellulose ion exchange chromatography and Sepharose 6B gel filtration. The purified protein migrated as a single band in 6% PAGE and 7% SDS-PAGE. The estimated molecular mass was 36 kDa (SDS-PAGE) and 49 kDa (Sepharose 6B). Optima of pH and temperature were 6.0 and 60 degrees C, respectively. In the absence of substrate the purified alpha-amylase was stable for 1 h at 50 degrees C and had a half-life of 12 min at 60 degrees C, but was fully stable in the presence of starch. The enzyme was not activated by several metal ions tested, including Ca(2+) (up to 10 mM), but HgCl(2 )and CuCl(2) inhibited its activity. The alpha-amylase produced by S. thermophilum preferentially hydrolyzed starch, and to a lesser extent amylopectin, maltose, amylose and glycogen in that order. The products of starch hydrolysis (up to 6 h of reaction) analyzed by thin layer chromatography, showed oligosaccharides such as maltotrioses, maltotetraoses and maltopentaoses. Maltose and traces of glucose were formed only after 3 h of reaction. These results confirm the character of the enzyme studied to be an alpha-amylase (1,4-alpha-glucan glucanohydrolase).

Effect of Starch Hydrolysis Products on the Determination of Limit Dextrinase and Limit Dextrinase Inhibitors in Barley and Malt

Abstract The activity of purified malt limit dextrinase was apparently increased by maltodextrins when enzyme activity was assayed by dyed pullulan substrates. At higher maltodextrin levels (different for each maltodextrin), the enzyme was inhibited. The apparent enzyme activation was not detected when assays were carried out with amylopectin β-limit dextrin, a more natural substrate for limit dextrinase, as substrate. Pullulanase (Aerobacter aerogenes) activity, when assayed with dyed pullulan, was not activated in the presence of maltodextrins. Barley extracts contained material that appeared to increase the activity of limit dextrinase. Results indicated that this material was a mixture of starch dextrins, which lost their activating ability when treated with amyloglucosidase. Treatment of malt extracts with amyloglucosidase prior to assaying for limit dextrinase significantly lowered the apparent limit dextrinase activities (when measured by dyed pullulan) and allowed for a more accurate estimate of both limit dextrinase and limit dextrinase inhibitor levels in the extracts. Enzyme and inhibitor levels in malt extracts, obtained using amyloglucosidase pre-treatments, showed that there was a strong relationship between inhibitor disappearance in malt extracts and the appearance of fully active limit dextrinase. These results confirm earlier conclusions that limit dextrinase is largely ineffective during the mashing stage of brewing because it is inhibited by two barley proteins still present in malt.

Effect of pH control on lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T.

Lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T, a new amylolytic L(+) lactic acid producer, was investigated and compared with starch fermentation by Lact. plantarum A6. At non-controlled pH, growth and lactic acid production from starch by Lact. manihotivorans LMG 18010T lasted 25 h. Specific growth and lactic acid production rates continuously decreased from the onset of the fermentation, unlike Lact. plantarum A6 which was able to grow and convert starch product hydrolysis into lactic acid more rapidly and efficiently at a constant rate up to pH 4.5. In spite of complete and rapid starch hydrolysis by Lact. manihotivorans LMG 18010T during the first 6 h, only 45% of starch hydrolysis products were converted to lactic acid. When pH was maintained at 6.0, lactic acid, amylase and final biomass production by Lact. manihotivorans LMG 18010T increased markedly and the fermentation time was reduced by half. Under the same conditions, an increase only in amylase production was observed with Lact. plantarum A6. When grown on glucose or starch at pH 6.0, Lact. manihotivorans LMG 18010T had an identical maximum specific growth rate (0.35 h(-1)), whereas the maximum rate of specific lactic acid production was three times higher with glucose as substrate. Lactobacillus manihotivorans LMG 18010T did not produce amylase when grown on glucose. Based on the differences in the physiology between the two species and other amylolytic lactic acid bacteria, different applications may be expected.

Synergistic Hydrolysis of Crude Corn Starch by α‐Amylases and Glucoamylases of Various Origins

ABSTRACTFour α‐amylases and two glucoamylases from various sources, in eight combinations, were used to study the synergistic hydrolysis of crude corn starch at various temperatures. At 40 and 50°C, the combinations containing Rhizopus mold glucoamylase enhanced hydrolysis of corn starch compared wth that obtained with the combinations from Aspergillus niger. At 60°C, Rhizopus mold combinations gave low reaction yields as the enzyme was inactivated. The differences observed between α‐amylases are smaller, with the exception of Bacillus licheniformis α‐amylase, which presented more than twice the productivity of the other α‐amylases, at all temperatures. In terms of substrate conversion at 5 hr of hydrolysis, the combination of B. licheniformis α‐amylase with Rhizopus mold glucoamylase at 50°C presents 76% substrate conversion, whereas, with all the other combinations, starch conversion was 13–73%. HPLC analysis of the reaction products obtained at 50°C showed that the main product of corn starch hydrolysis was glucose at 85–100%. Further experiments showed that A. niger glucoamylase and B. licheniformis α‐amylase were the only enzymes that retained their initial activity after incubation at the temperatures studied.

Microdialysis clean-up and sampling in enzyme-based methods for the characterisation of starch

Microdialysis was used for sampling enzyme hydrolysis products of starch hydrolysed with β-amylase, pullulanase, and/or isoamylase, to obtain information about the molecular structure of starch. Starches from waxy, normal, and high amylose maize, and from normal and genetically modified potato (amylose deficient) were used, and also commercial potato amyloses. The hydrolysis products were analysed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Simultaneous sampling and sample clean-up were achieved with microdialysis, thus enabling on-line injection into the liquid chromatographic system. The molecular weight cut-off of the membrane allowed for diffusion of small molecules such as oligosaccharides through the membrane, but hindered large molecules, e.g. enzymes and large polysaccharides, from entering the chromatographic system. With microdialysis sampling, it was possible to investigate the short chain fractions of debranched starch in the presence of amylose without pre-fractionation. The microdialysis–HPAEC-PAD system was also used for determination of the A:B chain ratio and the β-limit value. After β-amylolysis, only liberated maltose diffused through the dialysis membrane, which resulted in on-line sample clean-up from branched β-limit dextrin as well as from the enzyme. The proposed method is fast and easy to handle since clean-up of the hydrolysate is achieved on-line with the chromatographic system.

Suitability and limitations of methods for characterisation of activity of malto-oligosaccharide-forming amylases

The suitability and limitations of essential methods and reference substrates used for characterisation of activity of amylolytic enzymes is investigated. Saccharogenic, chromogenic and chromatographic methods are included. The results are discussed in relation to the measurement of reaction rates, determination of action mode and product specificity and the impact on identification and nomenclature of malto-oligosaccharide-forming amylases. An accurate determination of reaction rates using the saccharogenic methods strongly depends on the degree of polymerisation (DP) of the standards used and the hydrolysis products formed by the amylase. Particularly the use of glucose as standard can lead to overestimates due to the differences in the reducing potential of glucose and malto-oligosaccharides. The reliability of the chromogenic methods for determination of action mode depends on the DP of the substrate and the specificity of the amylase. For a characterisation of the starch hydrolysis products and the variation in the DP during hydrolysis, high performance anion-exchange chromatography with pulsed amperometric detection provided a fast and reliable method. A literature survey revealed varying and inconsistent use of nomenclature of malto-oligosaccharide forming amylases. Therefore a systematic approach identifying three main classes of activity is suggested using not only the mode of action and the DP of the major product but also the stage of hydrolysis at which this product is formed.

High-affinity maltose binding and transport by the thermophilic anaerobe Thermoanaerobacter ethanolicus 39E.

Thermoanaerobacter ethanolicus is a gram-positive thermophile that produces considerable amounts of ethanol from soluble sugars and polymeric substrates, including starch. Growth on maltose, a product of starch hydrolysis, was associated with the production of a prominent membrane-associated protein that had an apparent molecular weight of 43,800 and was not detected in cells grown on xylose or glucose. Filter-binding assays revealed that cell membranes bound maltose with high affinity. Metabolic labeling of T. ethanolicus maltose-grown cells with [(14)C]palmitic acid showed that this protein was posttranslationally acylated. A maltose-binding protein was purified by using an amylose resin affinity column, and the binding constant was 270 nM. Since maltase activity was found only in the cytosol of fractionated cells and unlabeled glucose did not compete with radiolabeled maltose for uptake in whole cells, it appeared that maltose was transported intact. In whole-cell transport assays, the affinity for maltose was approximately 40 nM. Maltotriose and alpha-trehalose competitively inhibited maltose uptake in transport assays, whereas glucose, cellobiose, and a range of disaccharides had little effect. Based on these results, it appears that T. ethanolicus possesses a high-affinity, ABC type transport system that is specific for maltose, maltotriose, and alpha-trehalose.

Production and biochemical characterization of an alpha-amylase from the moderate halophile Halomonas meridiana.

Extracellular amylase production by the moderate halophile Halomonas meridiana was optimized and the enzyme was characterized biochemically. The highest amylase production was achieved by growing H. meridiana cultures in media with 5% salts and starch, in the absence of glucose until the end of the exponential phase. The amylase exhibited maximal activity at pH 7.0, being relatively stable in alkaline conditions. Optimal temperature and salinity for activity were 37 degrees C and 10% NaCl, respectively. Moreover, activity at salinity as high as 30% salts was detected. Maltose and maltotriose were the main end products of starch hydrolysis, indicating an alpha-amylase activity.

Production and biochemical characterization of an α-amylase from the moderate halophile Halomonas meridiana

Extracellular amylase production by the moderate halophile Halomonas meridiana was optimized and the enzyme was characterized biochemically. The highest amylase production was achieved by growing H. meridiana cultures in media with 5% salts and starch, in the absence of glucose until the end of the exponential phase. The amylase exhibited maximal activity at pH 7.0, being relatively stable in alkaline conditions. Optimal temperature and salinity for activity were 37°C and 10% NaCl, respectively. Moreover, activity at salinity as high as 30% salts was detected. Maltose and maltotriose were the main end products of starch hydrolysis, indicating an α-amylase activity.

Kinetics and fundamental mechanisms of starch removal by activated sludge: hydrolysis of starch to maltose and maltotriose is the rate-determining step

The mechanisms of hydrolysis and uptake of starch, a polymer in sewage, by activated sludge (AS) were investigated, taking into consideration the mechanisms of the maltose transport system, responsible for the uptake of hydrolysis products of starch, in Gram-negative bacteria. AS was acclimated to dextrin, and the molecular weight distributions of the substrates during dextrin removal are analyzed using a GPC method. The removal rates of substrates such as dextrin, various maltodextrins (maltotriose, etc.), maltose and glucose by AS, and the oxygen uptake rates (OURs) of AS during the removal of these substrates were compared. Low-molecular-weight saccharides such as glucose, maltose and maltotriose were undetectable in the substrates during dextrin removal. The removal rates of maltose and maltotriose were higher than those of dextrin and glucose, and the OURs of AS during the removal of maltose and maltotriose were higher than those for other substrates. Therefore, hydrolysis of starch to maltose (dimer) and maltotriose (trimer), and not to glucose (monomer), appears to be the rate-determining step during the removal of starch, although maltodextrins with molecular weights up to 1152 (maltoheptaose) appear to penetrate the bacterial outer membrane.

The effect of process conditions on the alpha-amylolytic hydrolysis of amylopectin potato starch: An experimental design approach.

The hydrolysis of amylopectin potato starch with Bacillus licheniformis alpha-amylase (Maxamyl) was studied under industrially relevant conditions (i.e. high dry-weight concentrations). The following ranges of process conditions were chosen and investigated by means of an experimental design: pH [5.6-7.6]; calcium addition [0-120 microg/g]; temperature [63-97 degrees C]; dry-weight concentration [3-37% [w/w]]; enzyme dosage [27.6-372.4 microL/kg] and stirring [0-200 rpm]. The rate of hydrolysis was followed as a function of the theoretical dextrose equivalent. The highest rate (at a dextrose equivalent of 10) was observed at high temperature (90 degrees C) and low pH (6). At a higher pH (7.2), the maximum temperature of hydrolysis shifted to a lower value. Also, high levels of calcium resulted in a decrease of the maximum temperature of hydrolysis. The pH, temperature, and the amount of enzyme added showed interactive effects on the observed rate of hydrolysis. No product or substrate inhibition was observed. Stirring did not effect the rate of hydrolysis. The oligosaccharide composition after hydrolysis (at a certain dextrose equivalent) did depend on the reaction temperature. The level of maltopentaose [15-24% [w/w]], a major product of starch hydrolysis by B. licheniformis alpha-amylase, was influenced mostly by temperature.