Effect of temperature on the saccharide composition obtained after alpha-amylolysis of starch

The hydrolysis of starch to low-molecular-weight products (normally characterised by their dextrose equivalent (DE), which is directly related to the number-average molecular mass) was studied at different temperatures. Amylopectin potato starch, lacking amylose, was selected because of its low tendency towards retrogradation at lower temperatures. Bacillus licheniformis alpha-amylase was added to 10% [w/w] gelatinised starch solutions. The hydrolysis experiments were done at 50, 70, and 90 degrees C. Samples were taken at defined DE values and these were analysed with respect to their saccharide composition. At the same DE the oligosaccharide composition depended on the hydrolysis temperature. This implies that at the same net number of bonds hydrolysed by the enzyme, the saccharide composition was different. The hydrolysis temperature also influenced the initial overall molecular-weight distribution. Higher temperatures led to a more homogenous molecular weight distribution. Similar effects were observed for alpha-amylases from other microbial sources such as Bacillus amyloliquefaciens and Bacillus stearothermophilus. Varying the pH (5.1, 6.2, and 7.6) at 70 degrees C did not significantly influence the saccharide composition obtained during B. licheniformis alpha-amylase hydrolysis. The underlying mechanisms for B. licheniformis alpha-amylase were studied using pure linear oligosaccharides, ranging from maltotriose to maltoheptaose as substrates. Activation energies for the hydrolysis of individual oligosaccharides were calculated from Arrhenius plots at 60, 70, 80, and 90 degrees C. Oligosaccharides with a degree of polymerisation exceeding that of the substrate could be detected. The contribution of these oligosaccharides increased as the degree of polymerisation of the substrate decreased and the temperature of hydrolysis increased. The product specificity decreased with increasing temperature of hydrolysis, which led to a more equal distribution between the possible products formed. Calculations with the subsite map as determined for the closely related alpha-amylase from B. amyloliquefaciens reconfirmed this finding of a decreased substrate specificity with increased temperature of hydrolysis. Copyright 1999 John Wiley & Sons, Inc.     

FIXED CARBON PARTITIONING IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)

The main products of carbon fixation in the red algae are sulfated cell-wall polysaccharides, floridean starch, and low molecular weight (LMW) carbohydrates, mainly floridoside. In the red microalga Porphyridium sp., sulfated polysaccharide—cell bound and soluble—comprises up to 70% of the algal biomass. The purpose of this study was to elucidate the partitioning of fixed carbon in Porphyridium sp. toward the different products of carbon fixation. Using pulse-chase technique with [¹⁴C]bicarbonate, we followed ¹⁴C flow into the major compounds, namely, cell-wall polysaccharide, floridoside, starch, and protein, under various environmental conditions (i.e. carbon dioxide enrichment and nitrate starvation). ¹³C-NMR and gas chromatography analysis showed the main LMW product in Porphyridium sp. to be floridoside. After the short [¹⁴C]bicarbonate pulse (20 min), 42%–53% of total ¹⁴C uptake was initially found in floridoside. The appearance of ¹⁴C in the soluble polysaccharide was evident immediately at the end of the 20-min [¹⁴C]bicarbonate pulse. The specific radioactivity in the floridoside fraction declined by 80% after the 48-h chase, this decline being accompanied by increased labeling of starch and the soluble polysaccharide. In cells exposed to high CO₂ concentration, larger amounts of ¹⁴C (about twice as much) were channeled into starch and soluble polysaccharide than in cells under low CO₂ concentration. The most significant increase (1500%) in labeling during chase was found in the soluble polysaccharide of the nitrate-deprived cultures. It therefore seems likely that the large amounts of carbon incorporated by Porphyridium sp. cells into floridoside were subsequently used for the synthesis of macromolecular components. The data thus support the premise that floridoside serves as a dynamic carbon pool, which channels the fixed carbon toward polysaccharides and other end products according to the ambient conditions.     

Exopolysaccharide Colanic Acid and Its Occurrence in the Enterobacteriaceae

A study of strains from the genera Salmonella, Escherichia, and Aerobacter has shown that under appropriate conditions many strains produce an exopolysaccharide slime of identical composition, which has been identified as colanic acid on the basis of its chemical composition and its sensitivity to certain bacteriophage-induced depolymerase enzymes. Chemical analysis shows that the polysaccharide contains O-acetyl groups in addition to the sugars glucose, galactose, fucose, and glucuronic acid. Mild acid hydrolysis has led to the isolation of a β-glucosylfucose in addition to glucuronic acid containing oligosaccharides. Many strains were found to synthesize colanic acid under normal conditions of growth or under conditions favoring polysaccharide synthesis, whereas others only synthesized colanic acid when the control mechanism was derepressed by p-fluorophenylalanine.     

Model for on-line moisture-content control during solid-state fermentation

In this study we describe a model that estimates the extracellular (nonfungal) and overall water contents of wheat grains during solid-state fermentation (SSF) with Aspergillus oryzae, using on-line measurements of oxygen, carbon dioxide, and water vapor in the gas phase. The model uses elemental balances to predict substrate dry matter losses from carbon dioxide measurements, and metabolic water production, water used in starch hydrolysis, and water incorporated in new biomass from oxygen measurements. Water losses caused by evaporation were calculated from water vapor measurements. Model parameters were determined using an experimental membrane-based model system, which mimicked the growth of A. oryzae on the wheat grains and permitted direct measurement of the fungal biomass dry weight and wet weight. The measured water content of the biomass depended heavily on the moisture content of the solid substrate and was significantly lower than the estimated values reported in the literature. The model accurately predicted the measured overall water content of fermenting solid substrate during fermentations performed in a 1.5-L scraped drum reactor and in a 35-L horizontal paddle mixer, and is therefore considered validated. The model can be used to calculate the water addition required to control the extracellular water content in a mixed solid-state bioreactor for cultivation of A. oryzae on wheat.     

The study of the reaction of terminated oligomerization in the synthesis of oligo-(beta1-6)-glucosamines

The applicability of terminated oligomerization to the synthesis of oligo-(beta1-6)-glycosamines, fragments of the intercellular polysaccharide adhesin of staphylococci, was studied. The reactions of terminated oligomerization were carried out with mono-, di-, and trisaccharide monomers and N-protected aminopropanol; and spacered mono- and disaccharides as terminating molecules were also attempted. The primary formation of cyclic products of monomer intramolecular glycosylation was observed in almost all the reactions. Only the experiments with the monomer based on the disaccharide bromide under the conditions of the Helferich reaction led to reduced yields (30%) of the cyclic products. However, even in this case, the desired terminated oligosaccharides were generated in approximately 10% yield and mainly were the products of single glycosylation of the terminator by the monomer. These experiments allow the conclusion that, under the examined conditions, the reaction of terminated oligomerization could not result in the synthesis of oligoglucosamines with a high molecular mass.     

Formation of exopolysaccharides by Rhodococcus erythropolis and partial characterization of a heteropolysaccharide of high molecular weight

Summary Polysaccharide formation by Rhodococcus erythropolis was studied using lower mono-, di-and trihydric alcohols, sugars and n-alkanes as carbon sources. Cultural conditions of the organism were examined with regard to polysaccharide production. It was demonstrated that a glycerol substrate, an 30°C incubation temperature and a pH of 7.5 were optimal cultural conditions for polysaccharide formation. Addition of penicillin G in the decelerating growth phase increased the polysaccharide concentration in the culture filtrate to 3.1 g/l. One of the main extracellular heteropolysaccharides formed by Rhodococcus erythropolis consisted of glucose and mannose in the molar ratio 11, a small portion of protein and a trace of glucosamine. The molecular weight was to be 1·14×10⁶.     

Characterization of yeast amylolytic enzymes by HPLC maltooligosaccharides determination

Summary A simple method for determination of starch hydrolysis degree by measurement of maltooligosaccharides using HPLC on SGX C-18 column with deionised water as mobile phase was presented. Separation of seven oligosaccharides in an order from glucose to maltoheptaose illustrated the action of two enzyme systems taking part of starch hydrolysis and following fermentation to ethanol.     

Non-digestible oligosaccharides used as prebiotic agents: mode of production and beneficial effects on animal and human health

Prebiotic agents are food ingredients that are potentially beneficial to the health of consumers. The main commercial prebiotic agents consist of oligosaccharides and dietary fibres (mainly inulin). They are essentially obtained by one of three processes: 1) the direct extraction of natural polysaccharides from plants; 2) the controlled hydrolysis of such natural polysaccharides; 3) enzymatic synthesis, using hydrolases and/or glycosyl transferases. Both of these enzyme types catalyse transglycosylation reactions, allowing synthesis of small molecular weight synthetic oligosaccharides from mono- and disaccharides. Presently, in Europe, inulin-type fructans, characterised by the presence of fructosyl units bound to the beta-2,1 position of sucrose, are considered as one of the carbohydrate prebiotic references. Prebiotics escape enzymatic digestion in the upper gastrointestinal tract and enter the caecum without change to their structure. None are excreted in the stools, indicating that they are fermented by colonic flora so as to give a mixture of short-chain fatty acids (acetate, propionate and butyrate), L-lactate, carbon dioxide and hydrogen. By stimulating bifidobacteria, they may have the following implications for health: 1) potential protective effects against colorectal cancer and infectious bowel diseases by inhibiting putrefactive bacteria (Clostridium perfringens ) and pathogen bacteria (Escherichia coli, Salmonella, Listeria and Shigella ), respectively; 2) improvement of glucid and lipid metabolisms; 3) fibre-like properties by decreasing the renal nitrogen excretion; 4) improvement in the bioavailability of essential minerals; and 5) low cariogenic factor. These potential beneficial effects have been largely studied in animals but have not really been proven in humans. The development of a second generation of oligosaccharides and the putative implication of a complex bacterial trophic chain in the intestinal prebiotic fermentation process are also discussed.     

Photosynthetic preparation and characterization of 13C-labeled carbohydrates in agmenellum quadruplicatum

The blue-green alga Agmenellum quadruplicatum (strain PR6) has been used to prepare photobiosynthetically ¹³C-labeled d-glucose, 2-O-(α-d-glucopyranosyl)-glyceric acid (glucosylglycerate), 2-hydroxy-1-(hydroxymethyl)ethyl α-d-gluco-pyranoside (glucosylglycerol), and α-d-glueopyranosyl β-d-fructofuranoside (sucrose). When grown to a cell density of 4.4 g.L^-1 (dry weight) under nitrate-nitrogen limiting growth conditions for 120 h, the algal cells contained 38% of the dry-cell weight as(1 → 4)-α-d-glucan (amylose). About 1% of the dry-cell weight was glucosylglycerol, glucosylglycerate, and sucrose. Glutamate was obtained, together with carbohydrates of low molecular weight, when the cells were extracted with chloroform-methanol; d-glucose was recovered from the extracted cells by acid hydrolysis of the starch. The algae were grown by using 20 mol% [¹³C] carbon dioxide for preparation of labeled carbohydrates and for cellular component identification by whole-cell n.m.r. spectroscopy.     

Production of maltooligosaccharides from starch and separation of maltopentaose by adsorption of them on activated carbon (I)

For the selective production of maltopentaose (G₅) over other oligosaccharides, enzymatic hydrolysis conditions of starch by commercial α-amylase (Termamyl^®) were investigated. The determined optimum condition was 29.6 KNU (Kilo Novo α-amylase Unit) enzyme loading in 150 mL of 0.3% starch solution under pH level of 5 at 40°C for 30 min. About 40% of G₅ selectivity can be attained using the determined optimum condition. For further enhancing G₅ selectivity, an activated carbon adsorption process has been attached after the enzymatic hydrolysis. From the adsorption process, G₅ can be enriched up to 72% in the solution.     

Structural characterization and immunomodulating activity of a complex glucan from spores of Ganoderma lucidum.

A polysaccharide with a molecular weight of 1.26 x 10(5), obtained from the sporoderm-broken spores of Ganoderma lucidum, was purified by anion-exchange and gel-permeation chromatography. This polysaccharide had a strong effect on suppressing the antibody production and the Con A or LPS induced lymphocyte proliferation in mice. Chemically, the structure of the polysaccharide was identified by methylation analysis, 1 D, 2 D NMR and ESI-MS spectroscopic studies of the native one and of the oligosaccharide fragments generated by partial acid hydrolysis, Smith degradation, and acetolysis. It was concluded that the intact polysaccharide was a complex beta-D-glucan consisting of a (1-->6)-linked backbone chain, in which every other glucosyl residue was substituted at C-3 or C-4 with mono-, di- and trisaccharide branches.     

Structure of the exopolysaccharide produced by Enterobacter amnigenus

The bacterial species Enterobacter amnigenus was isolated from sugar beets harvested in Finland. It produced an exopolysaccharide rich in l-fucose, which gave viscous water solutions. Its primary structure was determined mainly by NMR spectroscopy and ESIMS of oligosaccharides and a polysaccharide with decreased molecular weight, obtained by Smith degradation of the O-deacetylated native polymer [carbohydrate structure: see text]     

Structural Characterization and Immunomodulating Activity of a Complex Glucan from Spores of Ganoderma lucidum

A polysaccharide with a molecular weight of 1.26×10⁵, obtained from the sporoderm-broken spores of Ganoderma lucidum, was purified by anion-exchange and gel-permeation chromatography. This polysaccharide had a strong effect on suppressing the antibody production and the Con A or LPS induced lymphocyte proliferation in mice. Chemically, the structure of the polysaccharide was identified by methylation analysis, 1D, 2D NMR and ESI-MS spectroscopic studies of the native one and of the oligosaccharide fragments generated by partial acid hydrolysis, Smith degradation, and acetolysis. It was concluded that the intact polysaccharide was a complex β-D-glucan consisting of a (1→6)-linked backbone chain, in which every other glucosyl residue was substituted at C-3 or C-4 with mono-, di- and trisaccharide branches.     

Characterization of the ‘reserve cellulose’ of the endosperm of Carum carvi as a β(1–4)-mannan

The reserve polysaccharide of the endosperm of Carum carvi consists of more than 90% mannose and was characterized as a β(1–4)-mannan. Total or partial acid hydrolysis, enzymatic breakdown or acetolysis of either palm or Carum carvi mannan yielded the same mono- and oligosaccharides, indicating a similar chemical structure of the two reserve polysaccharides. However, Carum carvi contains only traces of the alkali soluble mannan A dominant in the palm endosperm polysaccharide.     

Photolytic depolymerization of alginate

A photochemical reaction has been developed for the partial de-polymerization of sodium alginate, a polysaccharide utilized in medicine, pharmacy, basic sciences and foods. An aqueous solution of sodium alginate was photochemically depolymerized to ∼40% of its average molecular weight using ultraviolet light in the presence of titanium dioxide catalyst at pH 7 over a period of 3 h. The products were separated giving four fractions all having an average molecular weight that was smaller than that of the starting material. Characterization of the guluronate (G) and mannuronate (M) contents, and determination of the M/G ratio of photochemically depolymerized alginate, were accomplished using ¹H NMR spectroscopy. The resulting M/G ratio was compared to that obtained for alginate fractions produced by acid hydrolysis. The M and G content, of each alginate fraction, was also assigned with regards to their occurrence in G-rich, M-rich or M/G heteropolymeric domains. This new depolymerization method might also be applicable in the preparation of alginate oligosaccharides for use in the food and pharmaceutical industries.     

Recrystallisation behaviour of native and processed waxy maize starch in relation to the molecular characteristics

Molecular characteristics were determined for native waxy maize starch and maize starch modified in different way (by mechanical treatment or/and acid hydrolysis). Recrystallisation behaviour was studied. Methods used in this study were MALLS, HPAEC-PAD, NMR, DSC, SEM, light microscopy.

Five starch materials were subjected to storage under the same conditions in the presence of water (70 w/w%). Molecular weight, radius of gyration, initial crystallinity, and degree of polymerisation, degree of branching, chain length distribution profiles, were related to nucleation rate during the recrystallisation process, rate of recrystallisation, thermal stability and amount of obtained crystallinity. This allowed the following connections between the molecular characteristics and kinetic of recrystallisation to be proposed: Amylopectin molecular weight appeared to affect the number of starch crystallites formed and amount of crystallinity but not the stability of the rebuilt crystallites. The stability of rebuilt crystallites can be controlled by degree of polymerization, degree of branching and unit chain length distribution, characteristics which were similar for the starches.

A mixture of two starches, with and without crystalline structure in initial state but with molecular weight in same range, were stored and scanned in order to understand possible cocrystallisation effects.     

ENHANCED BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE) FROM POTATO STARCH BY Bacillus cereus STRAIN 64-INS IN A LABORATORY-SCALE FERMENTER

To decrease the polyhydroxyalkanoate (PHA) production cost by supplying renewable carbon sources has been an important aspect in terms of commercializing this biodegradable polymer. The production of biodegradable poly(3-hydroxyalkanoates) (PHA) from raw potato starch by the Bacillus cereus 64-INS strain isolated from domestic sludge has been studied in a lab-scale fermenter. The bacterium was screened for the degradation of raw potato starch by a starch hydrolysis method and for PHA production by Nile blue A and Sudan black B staining. Shake-flask cultures of the bacterium with glucose [2% (w/v)] or raw potato starch [2% (w/v)] produced PHA of 64.35% and 34.68% of dry cell weight (DCW), respectively. PHA production was also carried out in a 5-L fermenter under control conditions that produced 2.78 g/L of PHA and PHA content of 60.53% after 21 hr of fermentation using potato starch as the sole carbon source. Gas chromatography–mass spectroscopy (GC-MS) analyses confirmed that the extracted PHA contained poly(3-hydroxybutyrate) (PHB) as its major constituent (>99.99%) irrespective of the carbon source used. The article describes, for what we believe to be the first time, PHB production being carried out without any enzymatic or chemical treatment of potato starch at higher levels by fermentation. More work is required to optimize the PHB yield with respect to starch feeding strategies.     

The study of the reaction of terminated oligomerization in the synthesis of oligo-(β1-6)-glucosamines

The applicability of terminated oligomerization to the synthesis of oligo-(β1-6)-glucosamines, fragments of the intercellular polysaccharide adhesin of staphylococci, was studied. The reactions of terminated oligomerization were carried out with mono-, di-, and trisaccharide monomers and N-protected aminopropanol; and spacered mono-and disaccharides as terminating molecules were also attempted. The primary formation of cyclic products of monomer intramolecular glycosylation was observed in almost all the reactions. Only the experiments with the monomer based on the disaccharide bromide under the conditions of the Helferich reaction led to reduced yields (30%) of the cyclic products. However, even in this case, the desired terminated oligosaccharides were generated in approximately 10% yield and mainly were the products of single glycosylation of the terminator by the monomer. These experiments allow the conclusion that, under the examined conditions, the reaction of terminated oligomerization could not result in the synthesis of oligoglucosamines with a high molecular mass.     

The xylanase system of Coniophora cerebella

1. The culture filtrate of the fungus Coniophora cerebella grown on poplar 4-O-methylglucuronoxylan as carbon source and enzyme inducer contained an enzyme system that degraded the polysaccharide to xylose, acidic and neutral oligosaccharides and an enzyme-resistant polymer. Free uronic acid was not produced. 2. Cold ethanol fractionation of the culture filtrate yielded two active fractions, one of which had only xylanase (EC 3.2.1.8) and the other both xylanase and xylosidase (EC 3.2.1.37) activities. Further fractionation on DEAE-cellulose resolved the xylanase and xylosidase activities. 3. The xylanase degraded poplar 4-O-methylglucuronoxylan in an essentially random manner, producing oligosaccharides, but some xylose residues in the vicinity of uronic acid side groups were protected from hydrolysis, preventing a truly random attack. The xylosidase attacked the polysaccharide very slowly, releasing xylose, but the oligosaccharides produced by the action of the xylanase were much more susceptible to hydrolysis by the xylosidase. 4. The products of xylanase action were separated into neutral and acidic fractions. The neutral oligosaccharides were separated by chromatography on charcoal-Celite, and the major products were characterized as xylobiose, xylotriose, xylotetraose and xylopentaose. Some of the acidic sugars were branched, having the uronic acid residue attached to a xylose residue other than the terminal non-reducing one. 5. Gel filtration of various xylanase fractions gave values for the molecular weight of the enzyme from 34000 to 38000.     

Application of ultrafiltration for enzyme retention during continuous enzymatic reaction

In most enzymatic reactions, batch or continuous, separation of the enzyme for reuse is difficult if not impossible. A process will be presented in which an Ultrafiltration membrane serves to separate the reaction products from the enzyme and the substrate. In this manner the enzyme may be retained and re-used. Furthermore, under these conditions, the enzyme need only be present in catalytic amounts regardless of the amount of product produced. Under proper operating conditions and proper ultrafiltration membrane selection, a pure solution of α-amylase from Bacillus subtilis may be retained with no loss in enzyme activity over a test period of 30 hr after steadystate has been achieved. In the presence of substrate, the membrane support and ultrafiltration cell serve as the reaction vessel for the hydrolysis of starch. The substrate is continuously pumped into the cell under constant ultrafiltration pressure. The di-, oligo-, and polysaccharides formed from the enzyme reaction then either pass through the membrane as products or are retained. The molecular weight distribution of the products is dependent on the nominal molecular weight cut-off of the membrane, absolute ultrafiltration pressure, enzyme-to-substrate ratio, temperature, and residence time of the substrate in the reactor. In addition to the partial hydrolysis of starch by α-amylase, some preliminary findings on the complete hydrolysis of starch by glucoamylase will also be presented. In these latter studies, the substrate may be completely hydrolyzed to glucose units.