High Molecular Weight Oligosaccharides Research Articles

Impact of enzymatically derived manno-oligosaccharides on the growth of select lactobacillus strains under aerobic and anaerobic conditions

This study highlights the ability of beta-MOS derived from coconut processing residues (copra meal) to promote the in vitro growth of Lactobacillus probiotics. Enzymatic hydrolysis conditions were manipulated to produce Low Molecular Weight (LMW) and (HMW) Manno-oligosaccharide (MOS) extracts. The LMW MOS contained more mannose and mannobiose, and the HMW MOS contained more mannotriose and higher molecular weight oligosaccharides. The growth of Limosilactobacillus reuteri, Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus GG on the MOS Extracts was evaluated and compared with growth on Inulin, FOS and XOS. There was significant growth of each probiotic on the MOS extracts. Probiotic growth was similar on MOS extracts and on FOS, while growth was much lower on XOS and Inulin. Probiotic growth was greater under anaerobic than under aerobic conditions. The results confirm the value of a bioactive compound from copra meal to support human and animal health, while contributing to a circular bioeconomy.

Pectooligosaccharides rich in galacturonic acid produced from Orange Processing Waste by autohydrolysis: Process optimization and kinetic analysis

Pectooligosaccharides (POS) are notorious for their prebiotics and elicitor activities. In this work, we have optimized the autohydrolysis of Orange Processing Waste (OPW) to obtain POS fractions via a Taguchi approach, maximizing homogalacturonan oligosaccharides yield by considering several process variables: particle diameter, stirring speed, liquid-solid ratio and time, determining that only time had a significant effect in the studied range. 19.38 % w/w POS yield from OPW was obtained in optima conditions. After ensuring no limitation due to mass transfer, the kinetic analysis assumed that homogalacturonan was converted into high, medium and low molecular weight oligosaccharides, and galacturonic acid. Moreover, we considered that there is one pseudo-homogeneous reaction type combined with three irreversible, elemental, of pseudo first global order (first order with respect to the reagent which is hydrolysed) reactions in series. The model successfully reproduced experimental data at constant temperature (150 °C–180 °C) and pH (3.73).

Stabilizing effect of fos during freeze-drying and storage of lactobacillus delbrueckiI SUBSP. Bulgaricus results from a balance between high and low molecular weight oligosaccharides

Stabilizing effect of fos during freeze-drying and storage of lactobacillus delbrueckiI SUBSP. Bulgaricus results from a balance between high and low molecular weight oligosaccharides

Simultaneous production of both high molecular weight pullulan and oligosaccharides by Aureobasdium melanogenum P16 isolated from a mangrove ecosystem

After the compositional change of a pullulan production medium, a molecular weight (Mw) of the pullulan produced by Aureobasidium melanogenum P16 was 2.32×106 and a pullulan titer was 44.4g/L while a Mw of the pullulan produced by A. melanogenum P16 grown in the initial medium was only 3.47×105 and a pullulan titer was 65.3g/L. The increased Mw of the pullulan was due to the decreased activities of α-amylase, glucoamylase and pullulanase while the decreased pullulan titer was related to the decreased transcriptional levels of the genes encoding 6-P-glucose kinase, glucosyltransferase, α-phosphoglucose mutase, UDPG-pyrophosphorylase and pullulan synthetase. During the 10-L fermentation, when the yeast strain P16 was grown in the initial medium, the pullulan and oligosaccharide titers were 65.5g/L and 7.8g/L, respectively and the Mw of the produced pullulan was 4.42×105 while when the yeast strain P16 was grown in the compositionally changed medium, the pullulan and oligosaccharide titers were 46.4g/L and 27.8g/L, respectively and the Mw of the produced pullulan was 2.6×106. Most of the oligosaccharides produced by the yeast strain P16 cultivated in the compositionally changed medium had degree of polymerization of 4 and 5. Therefore, both of the high Mw pullulan and oligosaccharides with high levels were produced by the yeast strain P16.

Influence of DE-value on the physicochemical properties of maltodextrin for melt extrusion processes

In this study, five different types of maltodextrins (DE-2, DE-6, DE-12, DE-17 and DE-19) were characterized for the physico-chemical properties. TGA, DVS and SEC analyses were carried out and additionally apparent melt-viscosity (in a micro-extruder) and the glass transition temperature (analyzed by DMA) of maltodextrin/plasticizer mixtures were also measured in order to evaluate both the effect of plasticizer nature and content and the effect of the DE-value. For this, three plasticizing agents were compared: water, d-sorbitol and glycerin. The adsorption isotherms showed that depending on the DE-value and the relative humidity they were exposed to, different behavior could be obtained. For example, for relative humidities below 60% RH maltodextrin DE-2 was the least hygroscopic. And on the contrary for relative humidities above 75% RH maltodextrin DE-2 was the most hygroscopic. The rheology measurements showed that the viscosity decreased with the increase of the DE-value and with the plasticizer content, as expected. On the contrary, no direct correlation could be established between the DE-value and the glass transition temperature. These results demonstrated that to predict maltodextrins behavior and to better adapt the process conditions, combined analyses are mandatory as the DE-value alone is not sufficient. The most compelling evidence was obtained by size exclusion chromatography, which pointed out that maltodextrins had a bimodal molecular weight distribution composed of high and low molecular weight oligo-saccharides. Indeed, maltodextrins are highly polydisperse materials (i.e. polydispersity index ranging from 5 to 12) and that should be the reason why such distinct behaviors were observed in some of the physico-chemical analyses that were preformed.

Dextrin, sugar and organic acid profiles of spontaneous and modified gowe: a malted and fermented sorghum beverage from Benin

SummaryGowé is a traditional Beninese fermented red sorghum beverage made from a blend of malted and precooked nonmalted flour. The main objective of this work was to evaluate the degradation of starch during the production of gowé and to characterise the organic acid and sugar profiles of the product obtained using starter cultures. Inoculation was performed with strains of Lactobacillus fermentum or Weissella confusa and of Kluyveromyces marxianus or Pichia anomala used singly or in combination. Size exclusion chromatography revealed that starch was hydrolysed into high molecular weight dextrins (DP over 35), oligosaccharides and glucose, mainly due to the malting and precooking steps. Glucose was the main free sugar, and lactic acid was the main organic acid. The choice of the inoculation strain directly influenced the acidity but also indirectly the sugar content of the resulting gowé and will thus affect consumer acceptability.

The High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects on CD44 Clustering

CD44 is a major cell surface receptor for the glycosaminoglycan hyaluronan (HA). Native high molecular weight hyaluronan (nHA) and oligosaccharides of hyaluronan (oHA) provoke distinct biological effects upon binding to CD44. Despite the importance of such interactions, however, the feature of binding with CD44 at the cell surface and the molecular basis for functional distinction between different sizes of HA is still unclear. In this study we investigated the effects of high and low molecular weight hyaluronan on CD44 clustering. For the first time, we provided direct evidence for a strong relationship between HA size and CD44 clustering in vivo. In CD44-transfected COS-7 cells, we showed that exogenous nHA stimulated CD44 clustering, which was disrupted by oHA. Moreover, naturally expressed CD44 was distributed into clusters due to abundantly expressed nHA in HK-2 cells (human renal proximal tubule cells) and BT549 cells (human breast cancer cell line) without exogenous stimulation. Our results suggest that native HA binding to CD44 selectively induces CD44 clustering, which could be inhibited by oHA. Finally, we demonstrated that HA regulates cell adhesion in a manner specifically dependent on its size. oHA promoted cell adhesion while nHA showed no effects. Our results might elucidate a molecular- and/or cellular-based mechanism for the diverse biological activities of nHA and oHA.

Characterization and application of a detergent-stable alkaline α-amylase from Bacillus subtilis strain AS-S01a

A strain AS-S01a, capable of producing high-titer alkaline α-amylase, was isolated from a soil sample of Assam, India and was taxonomically identified as Bacillus subtilis strain AS-S01a. Optimized α-amylase yield by response surface method (RSM) was obtained as 799.0U with a specific activity of 201.0U/mg in a process control bioreactor. A 21.0kDa alkaline α-amylase purified from this strain showed optimum activity at 55°C and pH 9.0, and it produced high molecular weight oligosaccharides including small amount of glucose from starch as the end product. The Km and Vmax values for this enzyme towards starch were determined as 1.9mg/ml and 198.21μmol/min/mg, respectively. The purified α-amylase retained its activity in presence of oxidant, surfactants, EDTA and various commercial laundry detergents, thus advocating its suitability for various industrial applications.

Isolation and purification of Human Milk Oligosaccharides by two‐dimensional chromatography for in vitro and in vivo studies

Human Milk Oligosaccharides (HMO) are a structurally divers group of complex glycans that are highly abundant in human milk but not in infant formula. The structural diversity of HMO, however, makes it difficult to study the molecular mechanisms underlying their potential benefits for the breast‐fed infant. More than 150 different HMO have been described ranging in size from low molecular weight trisaccharides to high molecular weight oligosaccharides with more than 30 monosaccharide monomers and ranging from being neutral to carrying several negative charges. Not all HMO may trigger the same effects. Here, we describe the isolation and purification of HMO subfractions and even individual HMO by two‐dimensional chromatography. In the first dimension we used anion‐exchange chromatography (QAE) to separate pooled HMO in a neutral and 5 negatively charged fractions containing 0 to 5 sialic acids per HMO molecule, respectively. In the second dimension we used automated size exclusion chromatography to separate HMO based on their molecular weight. Structural composition of the generated HMO fractions was analyzed by HPLC and mass spectrometry. Our approach generated structurally defined HMO subfractions and even individual HMO in quantities sufficient for in vitro and in vivo experiments aimed to elucidate HMO structure‐function relationships. (Supported by NIH K99/R00DK078668 to LB)

Minimally invasive monitoring of cellulose degradation by desorption electrospray ionization and laser ablation electrospray ionization mass spectrometry

Minimally invasive desorption electrospray ionization-mass spectrometry (DESI-MS) and laser ablation electrospray ionization-MS (LAESI-MS) were used to look for soluble cellulose degradation products produced by accelerated aging in unsized cotton paper. Soluble extracts from papers aged 144 to 26,856 hours were first analyzed in solution using traditional electrospray ionization-MS (ESI-MS). Results were compared to those from direct analysis of condensed phase degradation products extracted from the absorbent paper substrate using DESI-MS and LAESI-MS. ESI-MS results showed evidence of oligosaccharide degradation products ranging from cellobiose to cellononaose; using DESI-MS and LAESI-MS, products from cellobiose to cellodecaose and glucose to cellooctaose, respectively, were observed. As degradation proceeded, increased quantities of both low and high molecular weight oligosaccharides were observed. The analytical approaches developed in the control study were applied for the detection of degradation products in two naturally-aged books dating from the 19th century, both made from cotton and linen. Oligosaccharides ranging from glucose to cellopentaose were observed.

HPAEC-PAD oligosaccharide analysis to detect adulterations of honey with sugar syrups

A method previously validated in our laboratory to study the oligosaccharide profile of honey was used in this work to detect adulterations of corn syrups (CS) and high fructose corn syrups (HFCS) in genuine honey samples. High molecular weight oligosaccharides (DP3-DP16) of 9 sugar syrups and 25 honey samples were analysed by HPAEC-PAD. Samples were previously treated with activated charcoal to remove mono and disaccharides. This method enabled the detection of honey adulterations with CS down to 5%. Adulterations of honeys with HFCS with different degrees of isomerisation (20% and 40%) were also detected.

Matrix-assisted Laser-desorption/Ionization Time-of-flight Mass Spectrometry and its Application to the Analysis of Fungal Spores

Although much research has been completed on the application of matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS) to the analysis of bacteria, no definitive studies have yet been performed on the analysis of fungi. Preliminary studies on the application of the MALDI-MS methodology, previously developed for the analysis of bacteria, to the analysis of intact fungal spores are described here. MALDI-MS and electrospray mass Spectrometry enable the analysis of high molecular-weight proteins, glycoproteins, oligosaccharides and oligonucleotides. Using MALDI-MS with bacteria has enabled the production of ‘fingerprints» of the intact cells; the ions observed are associated with the proteinaceous components of the cell wall. This study reports the adaptation of this technique to the direct analysis of fungal cells. Because of the large amount of carbohydrate in the fungal cell wall, the ions observed in the mass spectrometric experiments might be of carbohydrate origin. Penicillium spp., Scytalidium dimidiatum and Trichophyton rubrum have been studied in this preliminary investigation and all furnish individually distinctive spectra which seem to provide a profile of the cellular material with discrete peaks being observed over the mass range 2 to 13 kDa. The spectra obtained are reproducible within the method used but, as shown in our previous studies on bacteria, washing might selectively release components from the fungal cell wall.

Cellulase activity of soils

Cellulases are a group of enzymes that catalyze the degradation of cellulose, a polysaccharide built of β-1,4 linked glucose units. This group consists of endo-1,4-glucanase (EC 3.2.1.4), exo-1,4-glucanase (EC 3.2.1.91), and β-d-glucosidase (EC 3.2.1.21). The products of cellulose degradation are glucose, cellobiose, and higher molecular weight oligosaccharides. An accurate and precise method for the assay of cellulase activity in soil was developed. It involves determination of the reducing sugars produced when a soil sample is incubated with acetate buffer (50 mM, pH 5.5), carboxymethyl cellulose (CMC), and toluene at 30°C for 24 h. Results showed that the optimal pH of cellulase activity was 5.5. Reducing sugars produced from cellulase activity increased linearly up to 7 days, and they increased with increasing amounts of soil up to 7 g. The substrate concentration-activity curves of cellulase activity in soils obeyed the Michaelis-Menten equation. In two soils tested, the Michaelis constant (Km) ranged from 9.7 to 21.4 g CMC 1−1. Cellulase activity in soils was maximal at temperatures ranging from 50 to 60°C. The activation energy values of the reaction catalyzed by cellulase in four soils ranged from 21.7 to 28.0 kJ mol−1. Soil cellulase was denatured at temperatures ranging from 60 to 70°C; from 60 to 80%of total cellulase activity remained after heating air-dried soil samples at 100°C for 2 h. The corresponding values for field-moist soils were 18–23%. Toluene treatment markedly increased the cellulase activity values over the nontreated samples. Reducing sugar values increased significantly when air-dried or field-moist soil samples were incubated as described but without CMC, suggesting a role for cellulase in degradation of the native substrates.

Clinical, diagnostic and biochemical features of generalized gylcogenosis type II in brahman cattle

Clinical, diagnostic and biochemical features of generalized glycogenosis are described in 96 Brahman- type calves. Typically the calves were presented when about 6 months of age, with ill-thrift and muscular weakness as the most common signs. Acidic a-glucosidase activity was reduced in peripheral blood lymphocytes and skeletal muscle. Muscle glycogen concentration was consistently higher in affected animals than in clinically normal cattle. Other observations in affected calves included elevation of serum aspartate aminotransferase and creatine kinase activities and excessive amounts of high molecular weight oligosaccharides in urine. Fine cytoplasmic vacuolation of neurones in the brain and spinal cord, skeletal muscle, myocardium and of Purkinje fibers were consistent histological observations. Periodic acid-Schiff staining revealed the presence of glycogen-like material in peripheral blood lymphocytes of all affected calves, indicating that this is a useful aid for the diagnosis of glycogenosis. While 3 of the 96 calves showed somewhat different clinical signs, the similarity of pathology and the biochemical and clinical evidence in the remainder suggested that, in these animals, the disease was expressed as a single syndrome.

Clinical, diagnostic and biochemical features of generalised glycogenosis type II in Brahman cattle

Clinical, diagnostic and biochemical features of generalised glycogenosis are described in 96 Brahman-type calves. Typically the calves were presented when about 6 months of age, with ill-thrift and muscular weakness as the most common signs. Acidic alpha-glucosidase activity was reduced in peripheral blood lymphocytes and skeletal muscle. Muscle glycogen concentration was consistently higher in affected animals than in clinically normal cattle. Other observations in affected calves included elevation of serum aspartate aminotransferase and creatine kinase activities and excessive amounts of high molecular weight oligosaccharides in urine. Fine cytoplasmic vacuolation of neurones in the brain and spinal cord, skeletal muscle, myocardium and of Purkinje fibres were consistent histological observations. Periodic acid-Schiff staining revealed the presence of glycogen-like material in peripheral blood lymphocytes of all affected calves, indicating that this is a useful aid for the diagnosis of glycogenosis. While 3 of the 96 calves showed somewhat different clinical signs, the similarity of pathology and the biochemical and clinical evidence in the remainder suggested that, in these animals, the disease was expressed as a single syndrome.

Isolation and Characterization of Major Urinary Oligosaccharides Excreted by a Patient with Type 3 GM1 Gangliosidosis

Two major oligosaccharides were isolated from the urine of a patient with type 3 GM1 gangliosidosis. From structural studies including compositional sugar analysis, fast-atom bombardment mass spectrometry, direct-inlet chemical ionization mass spectrometry, methylation analysis, chromium trioxide oxidation, and proton magnetic resonance spectroscopy, their structures were deduced to be as follows: [formula: see text] Both oligosaccharides have beta-linked galactose at the non-reducing ends. Oligosaccharide 1 is one of the most common urinary oligosaccharides found in type 1 and type 2 GM1 gangliosidosis. Oligosaccharide 2, lacto-N-difucohexaose II, has not been described in the urine of GM1 gangliosidosis patients. Excretion of oligosaccharide 1 in the type 3 patient was much less than that of a type 2 patient. Thin-layer chromatographic analysis revealed that the excretion of oligosaccharides with higher molecular weight than that of oligosaccharide 1 (octasaccharide) in the type 3 patient was much less than that of a type 2 patient, raising the possibility that the mutant beta-galactosidase of type 3 GM1 gangliosidosis can still act to some extent on higher molecular weight oligosaccharides containing beta-linked galactose at the non-reducing end.

Drought Stress Induces Changes in the Non-Structural Carbohydrate Composition of Wheat Stems

The effect of drought stress on the non-structural carbohydrate (NSC) composition and water relations of the wheat (Triticum aestivum L.) stem has been investigated. Five separate parts of the stem were sampled: the unsheathed portion of the peduncle (Stem 1a), the sheathed portion of the peduncle (Stem 1b), the penultimate internode (Stem 2), the lower internodes (Stem 3+4) and the flag-leaf-pulvinus (pulvinus). The NSC was analysed as two fractions, an ethanol-soluble carbohydrate (ESC) fraction containing mono- and di-saccharides and some low molecular weight oligosaccharides, and a water-soluble carbohydrate (WSC) fraction containing mostly fructans and some high molecular weight oligosaccharides. In Stems 1b, 2 and 3 +4, the imposition of drought midway through grain filling resulted in a shift in soluble carbohydrate from the WSC to the ESC fraction indicating hydrolysis of fructans. In Stem 2 on day 29 of grain filling, the WSC/ESC ratio was 7.6 � 1.5 in well watered plants in contrast to 0.5 � 0.1 in droughted plants on day 30, even though NSC concentration did not differ. The NSC content of Stem la and the pulvinus increased threefold under drought, although levels were significantly lower than in the rest of the stem. The WSC/ESC ratio in these tissues was low com- pared to the rest of the stem but still declined noticeably under drought stress. Turgor (P) was fully maintained in Stem 2 and the pulvinus of droughted plants. Under drought, P in the pulvinus was maintained at higher levels (1.9-2.4 MPa) than in Stem 2 (0.8-1.2 MPa).

Appearance of bisected N-acetylglucosamine residue of biantennary sugar chains and decrease of high molecular weight oligosaccharides of human lymphocytic cell membranes during differentiation

Asparagine-linked sugar chains obtained from the plasma membranes of human acute lymphocytic leukemic cells, human B cells, Epstein-Barr virus (EBV)-infected B cell lines, and T cells were quantitatively liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB 3H 4 reduction. The radioactive oligosaccharides were fractionated by high-voltage paper electrophoresis. Their structures were studied by column chromatography and sequential exoglycosidase digestion. The neutral oligosaccharides were of a high mannose type. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Manα→(Manα→)Manβ→GlcNAcβ→(±Fucα→)GlcNAc as their cores. A comparative study of the oligosaccharides of these cells revealed that the biantennary complex-type sugar chain with bisecting N-acetylglucosamine residues was found only in B cells, B lymphoblasts, and B cell lines. High molecular weight oligosaccharides decreased during the differentiation stage of lymphocytes.

Severity of GM1 gangliosidosis and urinary oligosaccharide excretion

Urinary oligosaccharides of GM1 gangliosidosis patients (type 2A, 4 cases; type 2B, 2 cases) were investigated using the Bio Gel system. The levels of urinary oligosaccharide excreted (nmol/mg creatinine) by the type 2A patients were 4.1 times the levels of the type 2B patients. Patients of type 2A excreted high molecular weight oligosaccharides which were not detected in the urine of type 2B, and excreted oligosaccharides with long linkages of repeating structures. Thus, type 2A apparently has biochemically different characteristics from type 2B related to urinary oligosaccharide. Differentiation of type 2A from type 2B can thus be made biochemically. The structures of 5 different kinds of oligosaccharides not reported previously were confirmed.

Production of α-Amylase by Yeast

The enzyme alpha-amylase confers to an organism the enzymatic activity for the degradation of polyglucosides with alpha-1,4 glycosidic bonds such as starch and glycogen which are among the major storage compounds in plants and animals. Most alpha-amylases are single polypeptides of molecular weights around 50,000 dalton. They are generally found in the digestive tract of animals and in germinating seeds. Among the products released upon enzymatic degradation of polyglucosides maltose, a sugar that can be utilized as carbon source by yeast, is a major constituent. A cDNA segment complementary to mouse salivary amylase messenger RNA has been inserted into the yeast expression vector pMA56 behind the promoter of the gene encoding alcohol dehydrogenase I of yeast. Yeast transformants harboring plasmids with the normal orientation of the promoter and the mouse amylase cDNA gene produce amylase and release the enzyme in free form into the culture medium. Approximately 90% of the amylase activity is found in the medium. Yeast strains carrying MAL allele and transformed with a plasmid which directed the synthesis of mouse alpha-amylase were tested on plates containing starch and in batch fermentations using different high molecular weight sugars and oligosaccharides as carbon source. The results of these experimentsmore » will be discussed. (Refs. 21).« less