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Found 4 structures.
Displayed structures from 1 to 4
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Structure type: oligomer
Trivial name: 1-kestose, inulin
Compound class: fructan, fructo-oligosaccharide
Contained glycoepitopes: IEDB_142488,IEDB_144998,IEDB_146664,IEDB_983931,SB_192
Major fructo-oligosaccharides (FOS) produced by levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 were characterised as 1-kestose and nystose by acid hydrolysis and 13C NMR spectroscopy. The highest yields of 1-kestose (481 mM; 241 g/l) and nystose (81 mM; 54 g/l) were achieved at initial sucrose concentration of 1754 mM (600 g/l), pH 5.5 and 40 C. The synthesized FOS reached 50% (w/w) of total sugars in the reaction mixture, with a conversion efficiency over 70% (w/w) based on the amount of sucrose converted to 1-kestose.
oligosaccharide, structural, analysis, Oligosaccharides, structural analysis, production, 1-kestose, Acetobacter, Acetobacter diazotrophicus, fructo-oligosaccharides, levansucrase, LsdA
Journal NLM ID: 8008051Worldwide interest in oligosaccharides has been increasing ever since they were accorded the prebiotic status. The oligosaccharides of various origin like, bacteria, algae, fungi and higher plants have been used extensively both as food ingredients and pharmacological supplements. The non-digestible oligosaccharides have been implicated as dietary fibre, sweetener, weight controlling agent and humectant in confectioneries, bakeries and breweries. Functional oligosaccharides have been found effective in gastrointestinal normal flora proliferation and pathogen suppression, dental caries prevention, enhancement of immunity, facilitation of mineral absorption, source of antioxidant, antibiotic alternative, regulators of blood glucose in diabetics and serum lipids in hyperlipidemics. Apart from the pharmacological applications, oligosaccharides have found use in drug delivery, cosmetics, animal and fishery feed, agriculture, etc. Keeping in view the importance of the functional oligosaccharides, we present an overview of their natural sources, types, structures, physiological properties. Conventional as well as novel synthesis, purification and analysis methods are summarized. Recent promising developments in this area are presented to facilitate their further exploitation.
Oligosaccharides, microbiota, probiotics, Functional food, Prebiotics
Publication DOI: 10.1007/s11274-010-0558-5Aspergillus phoenicis biofilms on polyethylene as inert support were used to produce fructooligosaccharides (FOS) in media containing 25 % (m/V) of sucrose as a carbon source. The maximum production of total FOS (122 mg/mL), with 68 % of 1-kestose and 32 % of nystose, was obtained in Khanna medium maintained at 30 °C for 48 h under orbital agitation (100 rpm). At high concentrations of sucrose (30 %, m/V), the recovery of FOS was higher than that observed at a low concentration (5 %, m/V). High levels of FOS (242 mg/mL) were also recovered when using the biofilm in sodium acetate buffer with high sucrose concentration (50 %, m/V) for 10 h. When the dried biofilm was reused in a fresh culture medium, there was a recovery of approx. 13.7 % of total FOS after 72 h of cultivation at 30 °C, and 10 % corresponded to 1-kestose. The biofilm morphology, analyzed by scanning electron microscope, revealed a noncompact mycelium structure, with unfilled spaces and channels present among the hyphae. The results obtained in this study show that A. phoenicis biofilms may find application for FOS production in a single-step fermentation process, which is cost-effective in terms of reusability, downstream processing and efficiency.
Biofilm, fructooligosaccharides, Aspergillus sp., kestose, nystose
Journal NLM ID: 9703690In this study, the kinetic for the bioconversion of sucrose to fructooligosaccharides (FOS) by free cells of Aspergillus oryzae N74 was modeled. In addition, the effect of immobilized glucose isomerase (IGI) on FOS production yield was evaluated and considered in the kinetic model. The selected kinetic models were based on a proposed reaction mechanism described by elementary rate equations and modified Michaelis-Menten kinetic equations. The use of IGI allowed to increase the FOS production yield (FOS Yield) and to decrease the glucose/fructose (G/F) ratio. At shake flask scale, the FOS Yield was increased in 4.7 % (final yield 58.3 %), while the G/F ratio was reduced 6.2-fold. At bench scale, the FOS Yield was increased in 2.2 % (final yield 57.3 %), while the G/F ratio was reduced 4.5-fold. The elementary rate equation model was the one that best adjusted experimental data for FOS production using either the fungus biomass or the mixture fungus biomass-IGI, with an overall average percentage error of 7.2. Despite that FOS production yield was not highly improved by the presence of IGI in the reaction mixture, it favored the reduction of residual glucose in the mixture, avoiding the loss of material owe to glucose transformation to fructose that can be used in situ for FOS production by the fructosyltransferase.
fructooligosaccharides, Aspergillus oryzae, fructosyltransferase, immobilized glucose isomerase
NCBI PubMed ID: 22528647Fructooligosaccharides are prebiotics with numerous health benefits within which the improvement of gut microbiota balance can be highlighted, playing a key role in individual health. In this study, an integrated one-stage method for FOS production via sucrose fermentation by Aureobasidium pullulans was developed and optimized using experimental design tools. Optimization of temperature and agitation speed for maximizing the FOS production was performed using response surface methodology. Temperature was found to be the most significant parameter. The optimum fermentation conditions were found to be 32°C and 385 rpm. Under these conditions, the model predicted a total FOS production yield of 64.7 g FOS/g sucrose. The model was validated at optimal conditions in order to check its adequacy and accuracy and an experimental yield of 64.1 (±0.0) g FOS/g sucrose was obtained. A significant improvement of the total FOS production yields by A. pullulans using a one-stage process was obtained.
response surface methodology, Aureobasidium pullulans, fructooligosaccharides, transfructosylation, one-stage fermentation process, product yield
NCBI PubMed ID: 24750929A great demand for prebiotics is driving the search for new sources of fructo-oligosaccharides (FOS) producers and for FOS with differentiated functionalities. In the present work, FOS production by a new isolated strain of Aspergillus ibericus was evaluated. The temperature of fermentation and initial pH were optimized in shaken flask to yield a maximal FOS production, through a central composite experimental design. FOS were produced in a one-step bioprocess using the whole cells of the microorganism. The model (R2 = 0.918) predicted a yield of 0.56, experimentally 0.53 ± 0.03 gFOS.ginitial sucrose −1 was obtained (37.0 °C and a pH of 6.2). A yield of 0.64 ± 0.02 gFOS.ginitial sucrose−1 was obtained in the bioreactor, at 38 h, with a content of 118 ± 4 g.L−1 in FOS and a purity of 56 ± 3%. The chemical structure of the FOS produced by A. ibericus was determined by HPLC and NMR. FOS were identified as 1-kestose, nystose, and 1F-fructofuranosylnystose. In conclusion, A. ibericus was found to be a good alternative FOS producer.
chemical structure, fructo-oligosaccharides, experimental design, Aspergillus ibericus, production yield optimization
Publication DOI: 10.1016/j.lwt.2017.10.015The bifidogenic potential of fructo-oligosaccharides (FOS) produced by a newly isolated strain Aspergillus ibericus was studied. Their activity was compared to FOS produced by Aureobasidium pullulans and to a non microbial commercial FOS sample (Raftilose(center dot) P95). FOS fermentability by a number of probiotic bacteria and their hydrolytic resistance to the simulated harsh conditions of the digestive system was evaluated. Aspergillus ibericus FOS sample effectively promoted probiotic bacteria growth. Overall, microbial-derived FOS promoted greater cellular growth compared to the commercial sample. FOS fermentation was both substrate and strain specific. The FOS structural differences identified may explain their distinct assimilation by the probiotics. [Fru(2→6)Glc] (possibly blastose) and a reducing trisaccharide (possibly [Fru(β2→6)Glc(α1<->β2)Fru], neokestose) were only found in microbial-derived FOS samples, while Raftilosee(center dot) P95 was richer in inulobiose/inulotriose. 1-Kestose and nystose were only slightly hydrolyzed in the presence of gastric and intestinal fluid. FOS synthesized by Aspergillus exhibited great potential as food ingredients with likely prebiotic features.
fructo-oligosaccharides, probiotic, Prebiotics, Aspergillus, glycosidic linkage analysis, neoFOS
Publication DOI: 10.1016/j.jff.2018.05.004Studies on endo-inulinases from yeast are scarce, compared to those from other microbial sources. In this study, a novel endo-inulinase from Lipomyces starkeyi NRRL Y-11557 was identified, expressed in its soluble form, and characterized its physicochemically properties, together with its enzymatic activity and production of fructooligosaccharides (FOSs). A putative endo-inulinase gene inu3 was identified through rational genome mining. Through enzymatic activity and SDS-PAGE analysis, the endo-inulinase putative function of the protein encoded by inu3B gene (INU3B) was confirmed, and its soluble expression was achieved with pET22b (+) in Escherichia coli. INU3B showed effective catalytic activity and high thermostability. To our knowledge, the specific activity of INU3B against inulin reported in this study, 2262.8 ± 82.3 U/mg, at 70 °C and pH 5.0-6.0, is the highest reported to date. When the enzyme catalyzed FOSs production, the main products were DP3, DP4 and DP5. Overall, this report describes a novel yeast-derived endo-inulinase with optimal enzymatic properties, and thus, the reported enzyme has great potential for industrial production of FOSs.
fructooligosaccharides, endo-inulinase, Lipomyces starkeyi, rational genome mining, soluble expression
NCBI PubMed ID: 31233793An efficient system for biotransformation of sucrose to fructooligosaccharides (FOS) was obtained using Aspergillus tamarii NKRC 1229 mycelial fructosyltransferase (m-FTase). Zymographic analysis confirmed mycelial localization of the FTase (36 U/g) and lyophilized fungal pellets were used for bioconversion. m-FTase had molecular weight ~75 kDa with optimum activity at pH 7.0 and 20 °C. FOS production after parametric optimization (sucrose - 50% w/v, m-FTase dose - 4.5% w/v, inoculum age - 48 h and incubation time - 24 h) reached 325 g/L (55% yield) with 14% residual sucrose, 25% glucose and 6% fructose. FTase activity was enhanced after pre-treatment with organic solvents and SDS. FOS was purified in a single step using gel filtration matrix, Bio-Gel P2. FOS was characterized using Diffusion ordered spectroscopy-Nuclear Magnetic Resonance (1H DOSY-NMR) and Fourier-transform infrared spectroscopy (FTIR). Continuous generation of FOS was achieved using recyclable mycelia upto 10 consecutive cycles.
Prebiotics, Aspergillus, fructooligosaccharides (FOS), fructosyltransferase (FTase), zymography, Bio-Gel P2
NCBI PubMed ID: 30827630Starting from a relatively detailed model of a bioprocess producing fructo-oligosaccharides, a set of experimental data collected in batch and fed-batch experiments is exploited to estimate the unknown model parameters. The original model includes the growth of the fungus Aureobasidium pullulans which produces the enzymes responsible for the hydrolysis and transfructosylation reactions, and as such contains 25 kinetic parameters and 16 pseudo-stoichiometric coefficients, which are not uniquely identifiable with the data at hand. The aim of this study is, therefore, to show how sensitivity analysis and quantitative indicators based on the Fisher information matrix can be used to reduce the detailed model to a practically identifiable model. Parametric sensitivity analysis can indeed be used to progressively simplify the model to a representation involving 15 kinetic parameters and 8 pseudo-stoichiometric coefficients. The reduced model provides satisfactory prediction and can be convincingly cross validated.
biotechnology, mathematical modeling, Fisher information matrix, parameter identification
NCBI PubMed ID: 31375963Fructooligosaccharides (FOS) are important ingredients in the functional food industry because they have different biological properties such as decrease level of triglycerides, cholesterol and phospholipids and stimulate growth of probiotics for enhancement of microflora in large intestine. However, current strategies for the FOS production through simple and economical bioprocess has been necessary. The aim of this work was evaluated the capacity of three fungal strains (Aspergillus niger GH1, Aspergillus niger PSH and Aspergillus oryzae DIA-MF) to produce fructooligosaccharides (FOS) using aguamiel from Agave salmiana as an economical substrate. In addition, Czapek Dox medium supplemented with sucrose as carbon source was used as a control medium for the FOS production. A. oryzae DIA-MF was a fungi producer of FOS using aguamiel or Czapek Dox medium as substrate at 24 h of fermentation. However, the yield of FOS was increased two folds (20.30 g/L), with a productivity of 0.84 g FOS/l/h when aguamiel was used as substrate. On the other hand, A. niger GH1 and A. niger PSH showing only hydrolytic activity on sucrose under the studied conditions. In conclusion, this study shown excellent compatibility of A. oryzae DIA-MF using aguamiel as an economical substrate for the FOS production under a simple bioprocess.
Aspergillus, submerged fermentation, fructooligosaccharides (FOS), aguamiel
Publication DOI: 10.1016/j.lwt.2018.12.020The β-fructofuranosidase Ffase from the yeast Schwanniomyces occidentalis produces potential prebiotic fructooligosaccharides with health-promoting properties, making it of biotechnological interest. Ffase is one of the highest and more selective known producers of 6-kestose by transfructosylation of sucrose. In this work, production of 6-kestose was simplified by directly using cultures of S. occidentalis and Saccharomyces cerevisiae expressing both the wild-type enzyme and a mutated Ffase variant including the Ser196Leu substitution (Ffase-Leu196). Best results were obtained using yeast cultures supplemented with sucrose and expressing the Ffase-Leu196, which after only 4 h produced ~ 116 g/L of 6-kestose, twice the amount obtained with the corresponding purified enzyme. 6-Kestose represented ~ 70% of the products synthesized. In addition, a small amount of 1-kestose and the neofructoligosaccharides neokestose and blastose were also produced. The Ser196Leu substitution skewed production of 6-kestose and neofructooligosaccharides resulting in an increase of ~ 2.2- and 1.5-fold, respectively, without affecting production of 1-kestose. Supplementing yeast cultures with glucose clearly showed that blastose originates from direct fructosylation of glucose, a property that has not been described for other similar proteins from yeasts. Modeling neokestose and blastose into the Ffase-active site revealed the molecular basis explaining the peculiar specificity of this enzyme.
β-fructofuranosidase, 6-kestose, blastose, prebiotic sugars, Schwanniomyces occidentalis, yeast cultures
NCBI PubMed ID: 30357454Aspergillus thermomutatus produces an extracellular β-D-fructofuranosidase when cultured in Khanna medium with sucrose as additional carbon source at 30°C under agitation for 72 hr. Addition of glucose and fructose in the culture medium affected the production of the enzyme negatively. The optimum hydrolytic activity was achieved at 60°C and pH 5.0, with half-life (T50) of 30 hr at 50°C and 62% of its activity maintained at pH 5.0 for 48 hr. The extracellular extract containing β-D-fructofuranosidase was effective in producing fructooligosaccharides (FOS), mainly 1-kestose. The highest concentration of FOS was obtained at 30°C and 60°C, indicating the existence of at least two enzymes with transfructosylating activity. At 30°C, the maximal FOS concentration was obtained from 48 to 72 hr, while at 60°C, it was achieved only at 72 hr. The best production of FOS (86.7 g/L) was obtained using 500 g/L sucrose as substrate.
Aspergillus, fructooligosaccharide, transfructosylating, β-D-fructofuranosidase
NCBI PubMed ID: 31368547Fructooligosaccharides (FOS) are fructose-based oligosaccharides employed as additives to improve the nutritional and technological properties of foods. The rhizosphere of inulin-accumulating plants from the Cerrado (Brazilian savanna) harbor fungi capable of synthesizing FOS from sucrose through the transfructosylating activity of β-fructosyltransferases and/or β-fructofuranosidases. Here, we investigated the ability of Penicillium janczewskii Zaleski CCIBt 3352, a fungus isolated from the rhizosphere of Chrysolaena obovata (Asteraceae), to produce FOS in a medium supplemented with sucrose concentrations of 30, 100, or 150 g/L . Hydrolytic activity on sucrose was observed in culture filtrates; however, at 150 g/L sucrose, the accumulation of 8 g/L 1-kestose (inulin-type FOS) and 7.3 g/L neokestose (neolevan-type FOS) was observed, the latter being a type of FOS not commonly produced by filamentous fungi. In addition, minor amounts of four unidentified oligosaccharides, with a high degree of polymerization, were detected. The production of FOS was also observed in enzymatic assays, indicating the presence of extracellular enzymes with transfructosylating activity in the culture filtrates. Our findings demonstrate the feasibility of isolating promising microorganisms, for the production of FOS-synthesizing enzymes, from the rhizosphere of fructan-producing plants of the Brazilian Cerrado.
inulin, prebiotic, invertase, transfructosylation, fructans, soluble fiber
NCBI PubMed ID: 30758071Fructooligosaccharides (FOS) are commonly regarded as prebiotics and used as components of functional foods. Currently, the industrial sucrose-to-FOS biotransformation is mainly carried out using the microbial-derived β-fructofuranosidases with transglycosylation activity as catalysts. Evaluation of the ability of a microorganism to produce β-fructofuranosidase is commonly conducted by measuring enzyme activity. However, the traditional method requires several steps to identify strains with high β-fructofuranosidase activity, which is not suitable for high-throughput screening. To facilitate screening of a large number of microbial cultures, this study developed a plate chromogenic assay method based on the glucose oxidase (GOD) - peroxidase (POD) bienzymatic system for screening of β-fructofuranosidase-producing fungal strains and predicting their potential to produce FOS. This method used the amount of glucose released from sucrose as indicator to form clear pink halos around the microbial colonies with β-fructofuranosidase activity. Cultivation conditions for the plate assay were optimized as cultivation time 5 h and spore inoculum concentration 100000000 1/ml. Moreover, the method was applied to screening of an Aspergillus niger ATCC 20611 mutant library. The mutant A11 displaying the largest pink halo was screened out and its β-fructofuranosidase activity was determined to be 1.65 fold than that of the parental strain. Thin layer chromatography (TLC) assay further indicated that A11 with the largest halo possessed the highest FOS synthesis ability. These results demonstrated the potential of this plate chromogyenic assay method in the rapid and effective identification of excellent FOS producers from a large number of strain samples.
β-fructofuranosidase, fructooligosaccharides (FOS), plate chromogenic assay, GOD-POD bienzymatic system, Aspergillus niger ATCC 20611
NCBI PubMed ID: 31614171Fructooligosaccharides (FOSs) are excellent food ingredients or feed additives by stimulating probiotics. In this paper, a CREA gene encoding a glucose repressor in the β-fructofuranosidase producer Aureobasidium melanogenum 33 with high-level FOS biosynthesis was disrupted, and glucose repression in disruptant D28 was relieved. The disruptant D28 produced up to 2100 U/mL of β-fructofuranosidase activity, whereas the enzyme activities produced by parent strain 33 and complemented strain C11 were below 600 U/mL. The whole cells of the disruptant D28 was used to convert cane molasses into FOSs, and 0.58 g of FOSs/g of molasses sugar was synthesized from 350 g/L cane molasses sugar within 4 h. Results demonstrated that the industrial waste cane molasses can be efficiently converted into FOSs by the glucose derepression mutant D28 with high β-fructofuranosidase activity. This low-cost and environmentally friendly bioprocess has great potential applications in bioengineering and biotechnology for FOS production.
Aureobasidium melanogenum, cane molasses, glucose derepression, high-level FOS biosynthesis
NCBI PubMed ID: 31686508Eight strains of Aspergillus spp. were evaluated for their ability to produce intracellular and extracellular fructosyltransferase and fructooligosaccharides from sucrose. To the best of our knowledge, this is the first time that a study evaluates the production of intracellular and extracellular fructosyltransferase simultaneously with its chemometric characterization. High values of intracellular FTase were exhibited by A. oryzae and A. carbonarius and extracellular FTase by A. japonicus and A. ochraceus. The highest concentrations of intracellular FOS were generated by A. carbonarius and A. ochraceus and extracellular FOS by A. carbonarius and A. japonicus. The variations detected between the behavior of intra- and extra-cellular enzymes can be explained by activation of disulfide bridges and N-terminal signal peptides that occur distinctly between these biological structures. 1-kestose and 1-fructofuranosylnystose were the major oligosaccharides detected by HPLC-RID. Thus, transfructosylation activity is the main contributor to the positive correlation of 1-kestose.
fructooligosaccharides, fructosyltransferase, intracellular enzymes, chemometric characterization, extracellular enzymes
Publication DOI: 10.1016/j.biteb.2020.100546Short chain fructo-oligosaccharides (SC-FOS) are the potential prebiotics possessing diverse applications in both food and feed industries. The present study was aimed to extract inulin from chicory roots followed by its conversion into SC-FOS applying endoinulinase from Aspergillus fumigatus. The inulin was extracted from chicory roots through boiling in hot water, followed by precipitation with ethanol at room temperature or freezing condition. Maximum yield (42%) of inulin was obtained with three volumes of chilled absolute ethanol at room temperature. HPLC analysis of enzymatic hydrolysate detected kestose (GF2), nystose (GF3), and other FOS having higher degree of polymerization (DP). Maximum GF2 (5.79 mg/ml) was detected at temperature 50 °C, pH 5.5 with 2 U of enzyme dose after 6 h of hydrolysis; while maximum GF3 (4.33 mg/ml) was recorded at 60 °C, 5.5 pH with 0.5 U enzyme dose after 2 h of hydrolysis. Nevertheless, complete hydrolysis of inulin was noticed with 99% total oligosaccharide yield at 55 °C, 5.5 pH with 0.5 U enzyme dose after 4 h of hydrolysis with negligible amount of mono- and di-saccharides. The present finding demonstrated the process for higher yield of inulin from chicory roots followed by its conversion into SC-FOS applying fungal endoinulinase.
inulin, fungi, prebiotic, endoinulinase, short chain fructo-oligosaccharides
NCBI PubMed ID: 31845198The aim of the study was the fungal production of fructooligosaccharides from solid-state fermentation of different agro-industrial wastes (sugar cane bagasse, coffee husk, pineapple peel, prickle pear peel and banana peel) enriched with maguey sap. An exploratory statistical design with a factorial arrangement (23) was used to identify some parameters that are directly related to the production of FOS. The production was optimized and evaluated through fermentation kinetics in order to determine the time of maximum FOS production. Among the evaluated materials, sugar cane bagasse was the most promising substrate suited for the FOS production reaching a concentration of 7.64 g of FOS per liter of culture medium based on the initial sucrose concentration (21 g/L), a high Yp/s = 0.45 based on sucrose consumed was observed. The results showed that use of sugar cane bagasse enriched with aguamiel is the most economical and excellent alternative source for the FOS production by Aspergillus oryzae DIA-MF.
Prebiotics, fructooligosaccharides, solid-state fermentation, agricultural by-products
Publication DOI: 10.1016/j.bcab.2020.101704Pectinex Ultra SP-L, a commercial enzyme preparation with fructosyltransferase activity, was successfully immobilized by covalent binding to Fe3O4-chitosan-magnetic nanoparticles. Immobilization carried out according to a 2^3-full factorial design where glutaraldehyde concentration, activation time and time of contact between enzyme and support were selected as the independent variables and immobilization yield as the response. The highest immobilization yield (94.84%) was obtained using 3.0% (v/v) glutaraldehyde and activation and contact times of 180 and 30 min, respectively. The immobilized biocatalyst, which showed for both hydrolytic and transfructosylating activities optimum pH and temperature of 7.0 and 60 °C, respectively, retained 70 and 86% of them after 6 cycles of reuse. A kinetic/thermodynamic study focused on thermal inactivation of the immobilized construct indicated high thermostability at temperatures commonly used for fructo-oligosaccharides (FOS) production. Maximum FOS concentration obtained in lab-scale experiments was 101.56 g/L, with predominant presence of 1-kestose in the reaction mixture. The results obtained in this study suggest that the immobilized-enzyme preparation may be effectively exploited for FOS production and easily recovered from the reaction mixture by action of a magnetic field.
fructo-oligosaccharides, fructosyltransferase, magnetic nanoparticles
NCBI PubMed ID: 32070737Fructooligosaccharides (FOS) have widely used for the manufacture of low-calorie and functional foods, because they can inhibit intestinal pathogenic microorganism growth and increase the absorption of Ca2+ and Mg2+. In this study, the novel fructosyltransferase (FTase) from Aspergillus oryzae strain S719 was successfully purified and characterized. The specific activity of the final purified material was 4200 1/mg with purification ratio of 66 times and yield of 26%. The molecular weight of FTase of A. oryzae S719 was around 95 kDa by SDS-PAGE, which was identified as a type of FTase by Mass Spectrometry (MS). The purified FTase had optimum temperature and pH of 55 °C and 6.0, respectively. The FTase showed to be stable with more than 80% of its original activity at room temperature after 12 h and maintaining activity above 90% at pH 4.0-11.0. The Km and kcat values of the FTase were 310 mmol/L and 2000 1/min, respectively. The FTase was activated by 5 mmol/L Mg2+ and 10 mmol/L Na+ (relative activity of 116 and 114%, respectively), indicating that the enzyme was Mg2+ and Na+ dependent. About 64% of FOS was obtained by the purified FTase under 500 g/L sucrose within 4 h of reaction time, which was the shortest reaction time to be reported regarding the purified enzyme production of FOS. Together, these results indicated that the FTase of A. oryzae S719 is an excellent candidate for the industrial production of FOS.
Aspergillus oryzae, fructosyltransferase, enzyme purification, fructooligosaccharides production
NCBI PubMed ID: 31805395The use of whole-cell biocatalyst for production of fructo-oligosaccharide (FOS) eliminates the need for costly enzyme recovery and purification. In this study, a novel Aureobasidium pullulans strain (FRR 5284) was identified from seven A. pulllulans strains as an efficient whole-cell biocatalyst for FOS production. The strain had a specific intracellular transfructosylating activity of 4.44 ± 0.18 U/mg dry cells, one of the highest transfructosylating activities thus far reported for whole-cell biocatalyst. Under optimal conditions (pH 5.5 and 55 °C), only 5 g/L of dry cells and 3 h reaction time were required to achieve a FOS yield of 61 % from 50 % (w/v) sucrose. Incubation of sugarcane molasses with an invertase-free Saccharomyces cerevisiae prior to addition of A. pullulans whole-cell biocatalyst eliminated glucose inhibition and increased FOS yield from 44 % to 56 % in 1 h. This study has demonstrated that the novel A. pullulans FRR 5284 was an efficient source of whole-cell biocatalyst for FOS production and a promising strategy for transformation of sucrose and sugarcane molasses into a higher-value prebiotic.
Prebiotics, Aureobasidium pullulans, molasses, fructo-oligosaccharide, transfructosylating activity, invertase-free
Publication DOI: 10.1016/j.bej.2020.107747An extracellular fructosyltransferase (Ftase) enzyme with a molar mass of ≈70 kDa from a newly isolated indigenous coprophilous fungus Aspergillus niger sp. XOBP48 is purified to homogeneity and characterized in this study. The enzyme was purified to 4.66-fold with a total yield of 15.53% and specific activity of 1219.17 U/mg of protein after a three-step procedure involving (NH4)2SO4 fractionation, dialysis and anion exchange chromatography. Ftase showed optimum activity at pH 6.0 and temperature 50 °C. Ftase exhibited over 80% residual activity at pH range of 4.0-10.0 and ≈90% residual activity at temperature range of 40-60 °C for 6 h. Metal ion inhibitors Hg2+ and Ag+ significantly inhibited Ftase activity at 1 mmol concentration. Ftase showed Km, vmax and kcat values of 79.51 mmol, 45.04 µmol/min and 31.5 1/min, respectively, with a catalytic efficiency (kcat/Km) of 396 1/µmol/min for the substrate sucrose. HPLC-RI experiments identified the end products of fructosyltransferase activity as monomeric glucose, 1-kestose (GF2), and 1,1-kestotetraose (GF3). This study evaluates the feasibility of using this purified extracellular Ftase for the enzymatic synthesis of biofunctional fructooligosaccharides.
1-kestose, 1, fructooligosaccharides, Aspergillus niger, fructosyltransferase, zymography, 1-kestotetraose
NCBI PubMed ID: 33088656There is a great interest in prebiotics consumptions eminence by their health promoting properties and economic market value. Filamentous fungi have been implicated as the key producers of fructosyltransferase (Ftase) and inulinase for synthesis of fructooligosaccharides (FOSs), which is used as a bioactive ingredient in functional foods. In this study, sixty-one indigenous coprophilous fungal strains were isolated, purified to monoculture and investigated for their potential use in biotransformation of sucrose and inulin into fructooligosaccharides (FOSs) and inulooligosaccharides (IOSs) by producing Ftase and inulinase enzymes, respectively. The molecular identification by 18S rDNA sequencing and morpho-taxonomic keys revealed that axenic fungal strains belonged to the genera Aspergillus, Neocosmospora, Trichoderma, Mucor and Fusarium. The crude enzyme extract from the isolates showed hydrolysis zones of 15–30 mm on the 2,3,5-triphenyltetrazolium chloride (TTC) and Lugol's iodine solution assay plates confirming the Ftase and inulinase activities, respectively. The submerged culture filtrates of eight fungal isolates showed high Ftase activity while six different fungal isolates exhibited high inulinase activity. The reaction products analysed with thin-layer chromatography and high-performance liquid chromatography coupled with refractive index detection indicated the presence of FOSs and IOSs. The strains isolated in this study have potential biotechnological implications to produce FOSs and IOSs.
fructooligosaccharides, fructosyltransferase, coprophilous fungi, inulinase, inulooligosaccharides
Publication DOI: 10.1016/j.bcab.2020.101867Short-chain fructooligosaccharides (ScFOS) are a group of linear fructose oligomers that include 1-kestose, 1-nystose and 1-β-fructofuranosylnystose. ScFOS, which naturally occur at low levels in different plant products, are of high interest as food ingredients because of their prebiotic character, organoleptic characteristics and technological properties. Two different industrial processes are used to achieve large-scale ScFOS production: inulin hydrolysis (enzymatic or chemical hydrolysis) or sucrose biotransformation by transfructosylation (enzymatic synthesis) using specific enzymes like fructosyltransferases and fructofuranosidases. Enzymatic ScFOS synthesis seems to be more advantageous than inulin hydrolysis since it is less expensive, and leads to lower molecular weight FOS. The biotechnological process described to carry out this catalysis includes the production of transfructosylation enzymes, separation, enzyme immobilisation and finally the ScFOS production and purification. Such ScFOS production processes may be conducted under submerged or solid-state fermentation under discontinuous or continuous conditions. Several methodologies with different economic/environmental costs and production yields have been described to carry out these ScFOS production stages, although industrial scale-up needs to be optimised. This review tries to address a revision about enzymatic ScFOS production methods and its scale-up to industrial levels.
submerged fermentation, short-chain fructooligosaccharides, fungal fructosyltransferase, industrial scale-up
Publication DOI: 10.1007/s12393-020-09209-0In present study, statistical optimization of medium composition for endoinulinase production from raw inulin using Aspergillus tritici was performed in shake-flask fermentations. The concentration of four independent variables viz. raw inulin (1.5–3.5%), peptone (0.2–1.8%), (NH4)H2PO4 (0.2–1.8%) and pH (4.5–6.5) was explored to optimize the composition of fermentation medium for maximum endoinulinase production. Fermentation medium containing 2.5% raw inulin, 1% peptone, 1% (NH4)H2PO4 and medium pH adjusted to 5.5 supported maximum (25.39 IU/mL) endoinulinase production. Adequate precision value >4, closeness of coefficient of determination (R2) value to 1, good co-relation between the predicted and experimental values, percentage error <5%, high F-value 164.52 and low Lack of fit F-value 0.19 authenticates the fitness of quadratic model. TLC fingerprints studies confirmed fructooligosaccharides synthesis from inulin by crude fungal endoinulinase. Fructooligosaccharides yield (19.40%) containing 3.70% ketose (GF2), 2.71% nystose (GF3) and 1.42% fructofuranosyl nystose (GF4) were obtained from inulin (10%).
response surface methodology, fructooligosaccharides, endoinulinase, Aspergillus tritici, raw-inulin
Publication DOI: 10.1016/j.biteb.2020.100417Three-step purification technique (isopropanol precipitation, ion-exchange and size-exclusion chromatography) was used for the purification of an endoinulinase from the culture broth of Aspergillus tritici BGPUP6. The molecular mass of purified endoinulinase was found to be 53.45 kDa and 53.70 kDa by denatured protein gel (SDS-PAGE) and size-exclusion (Sephadex G-100) chromatographic analysis, respectively. Higher Km (0.90 mM), Vmax (19.60 mM/min·mg), Kcat (0.0013 1/min) and Vmax/Km ratio (21.77 1/min·mg) of purified endoinulinase for inulin than stachyose depicts its higher affinity towards inulin. Purified enzyme was found stable in a pH range 4.0-7.0 with an optimal pH 5.5. The optimal temperature of purified biocatalyst was 55 °C with thermostability in the range of 50-70 °C. D-value and Z-value for endoinulinase at 55 °C was found to be 100.08 h and 11.62 °C, respectively. Thermodynamics inactivation parameters (ΔG, ΔH and ΔS) of endoinulinase shows its wide range thermal stability. Endoinulinase activity was enhanced by CaCl2 and MnSO4, while CuSO4, CoCl2, AgNO3, CdCl2, NiCl2, ZnSO4, BaCl2, HgCl2 and EDTA inhibited the activity of enzyme. Purified endoinulinase was successfully used for the production of fructooligosaccharides from inulin.
fructooligosaccharides, endoinulinase, Aspergillus tritici, kinetics characterization
NCBI PubMed ID: 32890562A previous study (KL Forsythe, MS Feather [1989] Carbohydr Res 185: 315-319) showed that (13)C nuclear magnetic resonance spectroscopy can be used to detect and identify mixtures of 1-kestose and neokestose after conversion to the acetate derivatives. In this study, unequivocal assignments are made for the anomeric carbon and proton signals for the above two trisaccharide acetates as well as for 6-kestose hendecaacetate and for nystose tetradecaacetate (a 1-kestose-derived tetrasaccharide). A number of oligosaccharide fractions were isolated from several plant species, converted to the acetates, and nuclear magnetic resonance spectra obtained. Using the above reference data, the following information was obtained. The trisaccharide fraction from Dactylis glomerata L. stem tissue and Asparagus officinalis L. roots contain both 1-kestose and neokestose, and the tetrasaccharide fractions contain three components, one of which is nystose. Penta- and hexasaccharide acetates were also isolated from A. officinalis L. roots and were found to contain, respectively, four and at least five components. All components of both of the above species appear to contain a kestose residue and to be produced by the sequential addition of fructofuranosyl units to these. The trisaccharide fraction from Festuca arundinacea is complex, and contains at least five different components, two of which appear to be 1-kestose and neokestose.
NCBI PubMed ID: 16667365The conformational studies of inulin oligomers from G-F_2 to G-F_9, which isolated from Platycodon grandiflorum, suggested a plausible conformational change between G-F_7 and G-F_8 from the trends in their chemical shift patterns and molecular rotation; the oligomers higher than G-F_8 would form some secondary conformations more rigid than shorter oligomers. On the other hand, spin-latice relaxation (T_1) studies of the protons proposed through-space interactions of 2- and 4-H's of glucose moiety in G-F_5,presumably with some atom(s) of the terminal fructose moiety. This would reflect that the inulin molecule adopts a 5/1 helix.
NMR, conformation, inulin, inulin oligomer, molecular rotation, spin-lattice relaxation time (T1)
Journal NLM ID: 0377775Individual fructan tri-, tetra- and pentasaccharide isomers in neutral, water-soluble extracts from Lolium temulentum were purified and the linkages present in these isomeric oligosaccharides were analysed by combined GC-mass spectrometry of partially methylated alditol acetates. 1-Kestose and neokestose were the most abundant trisaccharides with 6-kestose present in much lower amounts. Analysis of isomers of DP 4 and 5 showed that multiple linkage types were present with structures based on all three trisaccharides. Oligosaccharides based on neokestose but with 2,6 linkages between adjacent fructose residues have not been previously detected in higher plants.
Oligosaccharides, 1-kestose, fructan, sucrose, neokestose, Gramineae, Lolium temulentum
Publication DOI: 10.1016/0031-9422(92)83432-XChembiotech Ltd. Institute of Research and Development, University of Birmingham Research Park, Edgbaston, Birmingham, UK; Birmingham Carbohydrate and Protein Technology Group, Research Laboratory for the Chemistry of Bioactive Carbohydrates and Proteins, School of Chemistry, The University of Birmingham, Edgbaston, Birmingham, UK; School of Chemical Engineering, The University of Birmingham, Edgbaston, Birmingham, UK
Publication DOI: 10.1016/0144-8617(94)90143-0Gomphrena macrocephala St.-Hil. (Amaranthaceae) is a perennial herb that grows spontaneously in the cerrado and is characterized by well-defined phenological phases throughout the year. Soluble carbohydrates are the main reserve compounds of the tuberous root and constitute approximately 50% of the dry weight. These sugars were partially characterized as fructans forming a single homologous series, different from inulin, the most common fructan of dicotyledons. The mean molecular weight of polysaccharides was high and reached 37 kDa in the dormant phase. Fructan spherocrystals were detected in the tuberous root after treatment with ethanol, being associated with the parenchyma of secondary xylem. The content, composition and mean molecular weight of fructans were related to phenology. In late dormancy, there was a marked increase in monosaccharides, particularly fructose, and a concomitant decline of polysaccharides, probably as a result of fructan breakdown. During sprouting and in the vegetative phase, the contents of oligosaccharides and low molecular weight polysaccharides increased. A gradual rise in the molecular weight of polysaccharides occurred during the reproductive phase and at early dormancy, concurrently with decreasing levels of oligosaccharides. The capacity of G. macrocephala to accumulate readily accessible sugars, such as fructans, instead of starch, in response to environmental changes, may be of considerable advantage, since the cerrado is often subjected to seasonal drought and burnings.
fructans, Gomphrena macrocephala St.-Hil., Amaranthaceae, storage carbohydrates, phenology
Publication DOI: 10.1111/j.1365-3040.1993.tb00515.xFructooligosaccharides (FOS) are short-chain sugars that occur naturally and have dietary benefits for humans. They are widely distributed in nature and are a natural part of the human diet. The objective of this study was to determine the concentrations of 1-kestose (GF(2)), nystose (GF(3)), and 1(F)-beta-fructofuranosylnystose (GF(4)) in a variety of common processed and prepared foods. An ion chromatographic method was developed for this purpose in which the sugar concentrations were measured using integrated amperometry. The samples were simply prepared by blending with water and filtering the suspensions through a 10000 Da cutoff centrifugal filter. These samples were then injected into the ion chromatograph, which had been programmed for gradient elution, and the areas of the sugar peaks obtained compared to those of standard sugars on a calibration curve. Selected samples were prepared both with and without standard spikes in order to assess the efficiency of the determination. Of the vegetables investigated, artichokes contained by far the most FOS, followed by onions; bananas contained more FOS than other fruits investigated. The method was shown to be simple, convenient, and relatively fast for the quantitation of FOS in processed and prepared food products.
fructooligosaccharides, food composition, ion exchange chromatography
NCBI PubMed ID: 11087481| b-D-Fruf-(2-1)-b-D-Fruf-(2-1)-b-D-Fruf-(2-1)-a-D-Glcp | Show graphically |
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Show legend Show as text |
Structure type: oligomer
Trivial name: nystose, inulin, 1,1-kestotetraose
Compound class: fructan
Contained glycoepitopes: IEDB_142488,IEDB_144998,IEDB_146664,IEDB_923067,IEDB_983931,SB_192
Major fructo-oligosaccharides (FOS) produced by levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 were characterised as 1-kestose and nystose by acid hydrolysis and 13C NMR spectroscopy. The highest yields of 1-kestose (481 mM; 241 g/l) and nystose (81 mM; 54 g/l) were achieved at initial sucrose concentration of 1754 mM (600 g/l), pH 5.5 and 40 C. The synthesized FOS reached 50% (w/w) of total sugars in the reaction mixture, with a conversion efficiency over 70% (w/w) based on the amount of sucrose converted to 1-kestose.
oligosaccharide, structural, analysis, Oligosaccharides, structural analysis, production, 1-kestose, Acetobacter, Acetobacter diazotrophicus, fructo-oligosaccharides, levansucrase, LsdA
Journal NLM ID: 8008051Worldwide interest in oligosaccharides has been increasing ever since they were accorded the prebiotic status. The oligosaccharides of various origin like, bacteria, algae, fungi and higher plants have been used extensively both as food ingredients and pharmacological supplements. The non-digestible oligosaccharides have been implicated as dietary fibre, sweetener, weight controlling agent and humectant in confectioneries, bakeries and breweries. Functional oligosaccharides have been found effective in gastrointestinal normal flora proliferation and pathogen suppression, dental caries prevention, enhancement of immunity, facilitation of mineral absorption, source of antioxidant, antibiotic alternative, regulators of blood glucose in diabetics and serum lipids in hyperlipidemics. Apart from the pharmacological applications, oligosaccharides have found use in drug delivery, cosmetics, animal and fishery feed, agriculture, etc. Keeping in view the importance of the functional oligosaccharides, we present an overview of their natural sources, types, structures, physiological properties. Conventional as well as novel synthesis, purification and analysis methods are summarized. Recent promising developments in this area are presented to facilitate their further exploitation.
Oligosaccharides, microbiota, probiotics, Functional food, Prebiotics
Publication DOI: 10.1007/s11274-010-0558-5Aspergillus phoenicis biofilms on polyethylene as inert support were used to produce fructooligosaccharides (FOS) in media containing 25 % (m/V) of sucrose as a carbon source. The maximum production of total FOS (122 mg/mL), with 68 % of 1-kestose and 32 % of nystose, was obtained in Khanna medium maintained at 30 °C for 48 h under orbital agitation (100 rpm). At high concentrations of sucrose (30 %, m/V), the recovery of FOS was higher than that observed at a low concentration (5 %, m/V). High levels of FOS (242 mg/mL) were also recovered when using the biofilm in sodium acetate buffer with high sucrose concentration (50 %, m/V) for 10 h. When the dried biofilm was reused in a fresh culture medium, there was a recovery of approx. 13.7 % of total FOS after 72 h of cultivation at 30 °C, and 10 % corresponded to 1-kestose. The biofilm morphology, analyzed by scanning electron microscope, revealed a noncompact mycelium structure, with unfilled spaces and channels present among the hyphae. The results obtained in this study show that A. phoenicis biofilms may find application for FOS production in a single-step fermentation process, which is cost-effective in terms of reusability, downstream processing and efficiency.
Biofilm, fructooligosaccharides, Aspergillus sp., kestose, nystose
Journal NLM ID: 9703690In this study, the kinetic for the bioconversion of sucrose to fructooligosaccharides (FOS) by free cells of Aspergillus oryzae N74 was modeled. In addition, the effect of immobilized glucose isomerase (IGI) on FOS production yield was evaluated and considered in the kinetic model. The selected kinetic models were based on a proposed reaction mechanism described by elementary rate equations and modified Michaelis-Menten kinetic equations. The use of IGI allowed to increase the FOS production yield (FOS Yield) and to decrease the glucose/fructose (G/F) ratio. At shake flask scale, the FOS Yield was increased in 4.7 % (final yield 58.3 %), while the G/F ratio was reduced 6.2-fold. At bench scale, the FOS Yield was increased in 2.2 % (final yield 57.3 %), while the G/F ratio was reduced 4.5-fold. The elementary rate equation model was the one that best adjusted experimental data for FOS production using either the fungus biomass or the mixture fungus biomass-IGI, with an overall average percentage error of 7.2. Despite that FOS production yield was not highly improved by the presence of IGI in the reaction mixture, it favored the reduction of residual glucose in the mixture, avoiding the loss of material owe to glucose transformation to fructose that can be used in situ for FOS production by the fructosyltransferase.
fructooligosaccharides, Aspergillus oryzae, fructosyltransferase, immobilized glucose isomerase
NCBI PubMed ID: 22528647Fructooligosaccharides are prebiotics with numerous health benefits within which the improvement of gut microbiota balance can be highlighted, playing a key role in individual health. In this study, an integrated one-stage method for FOS production via sucrose fermentation by Aureobasidium pullulans was developed and optimized using experimental design tools. Optimization of temperature and agitation speed for maximizing the FOS production was performed using response surface methodology. Temperature was found to be the most significant parameter. The optimum fermentation conditions were found to be 32°C and 385 rpm. Under these conditions, the model predicted a total FOS production yield of 64.7 g FOS/g sucrose. The model was validated at optimal conditions in order to check its adequacy and accuracy and an experimental yield of 64.1 (±0.0) g FOS/g sucrose was obtained. A significant improvement of the total FOS production yields by A. pullulans using a one-stage process was obtained.
response surface methodology, Aureobasidium pullulans, fructooligosaccharides, transfructosylation, one-stage fermentation process, product yield
NCBI PubMed ID: 24750929Studies on endo-inulinases from yeast are scarce, compared to those from other microbial sources. In this study, a novel endo-inulinase from Lipomyces starkeyi NRRL Y-11557 was identified, expressed in its soluble form, and characterized its physicochemically properties, together with its enzymatic activity and production of fructooligosaccharides (FOSs). A putative endo-inulinase gene inu3 was identified through rational genome mining. Through enzymatic activity and SDS-PAGE analysis, the endo-inulinase putative function of the protein encoded by inu3B gene (INU3B) was confirmed, and its soluble expression was achieved with pET22b (+) in Escherichia coli. INU3B showed effective catalytic activity and high thermostability. To our knowledge, the specific activity of INU3B against inulin reported in this study, 2262.8 ± 82.3 U/mg, at 70 °C and pH 5.0-6.0, is the highest reported to date. When the enzyme catalyzed FOSs production, the main products were DP3, DP4 and DP5. Overall, this report describes a novel yeast-derived endo-inulinase with optimal enzymatic properties, and thus, the reported enzyme has great potential for industrial production of FOSs.
fructooligosaccharides, endo-inulinase, Lipomyces starkeyi, rational genome mining, soluble expression
NCBI PubMed ID: 31233793An efficient system for biotransformation of sucrose to fructooligosaccharides (FOS) was obtained using Aspergillus tamarii NKRC 1229 mycelial fructosyltransferase (m-FTase). Zymographic analysis confirmed mycelial localization of the FTase (36 U/g) and lyophilized fungal pellets were used for bioconversion. m-FTase had molecular weight ~75 kDa with optimum activity at pH 7.0 and 20 °C. FOS production after parametric optimization (sucrose - 50% w/v, m-FTase dose - 4.5% w/v, inoculum age - 48 h and incubation time - 24 h) reached 325 g/L (55% yield) with 14% residual sucrose, 25% glucose and 6% fructose. FTase activity was enhanced after pre-treatment with organic solvents and SDS. FOS was purified in a single step using gel filtration matrix, Bio-Gel P2. FOS was characterized using Diffusion ordered spectroscopy-Nuclear Magnetic Resonance (1H DOSY-NMR) and Fourier-transform infrared spectroscopy (FTIR). Continuous generation of FOS was achieved using recyclable mycelia upto 10 consecutive cycles.
Prebiotics, Aspergillus, fructooligosaccharides (FOS), fructosyltransferase (FTase), zymography, Bio-Gel P2
NCBI PubMed ID: 30827630Starting from a relatively detailed model of a bioprocess producing fructo-oligosaccharides, a set of experimental data collected in batch and fed-batch experiments is exploited to estimate the unknown model parameters. The original model includes the growth of the fungus Aureobasidium pullulans which produces the enzymes responsible for the hydrolysis and transfructosylation reactions, and as such contains 25 kinetic parameters and 16 pseudo-stoichiometric coefficients, which are not uniquely identifiable with the data at hand. The aim of this study is, therefore, to show how sensitivity analysis and quantitative indicators based on the Fisher information matrix can be used to reduce the detailed model to a practically identifiable model. Parametric sensitivity analysis can indeed be used to progressively simplify the model to a representation involving 15 kinetic parameters and 8 pseudo-stoichiometric coefficients. The reduced model provides satisfactory prediction and can be convincingly cross validated.
biotechnology, mathematical modeling, Fisher information matrix, parameter identification
NCBI PubMed ID: 31375963Fructooligosaccharides (FOS) are important ingredients in the functional food industry because they have different biological properties such as decrease level of triglycerides, cholesterol and phospholipids and stimulate growth of probiotics for enhancement of microflora in large intestine. However, current strategies for the FOS production through simple and economical bioprocess has been necessary. The aim of this work was evaluated the capacity of three fungal strains (Aspergillus niger GH1, Aspergillus niger PSH and Aspergillus oryzae DIA-MF) to produce fructooligosaccharides (FOS) using aguamiel from Agave salmiana as an economical substrate. In addition, Czapek Dox medium supplemented with sucrose as carbon source was used as a control medium for the FOS production. A. oryzae DIA-MF was a fungi producer of FOS using aguamiel or Czapek Dox medium as substrate at 24 h of fermentation. However, the yield of FOS was increased two folds (20.30 g/L), with a productivity of 0.84 g FOS/l/h when aguamiel was used as substrate. On the other hand, A. niger GH1 and A. niger PSH showing only hydrolytic activity on sucrose under the studied conditions. In conclusion, this study shown excellent compatibility of A. oryzae DIA-MF using aguamiel as an economical substrate for the FOS production under a simple bioprocess.
Aspergillus, submerged fermentation, fructooligosaccharides (FOS), aguamiel
Publication DOI: 10.1016/j.lwt.2018.12.020Aspergillus thermomutatus produces an extracellular β-D-fructofuranosidase when cultured in Khanna medium with sucrose as additional carbon source at 30°C under agitation for 72 hr. Addition of glucose and fructose in the culture medium affected the production of the enzyme negatively. The optimum hydrolytic activity was achieved at 60°C and pH 5.0, with half-life (T50) of 30 hr at 50°C and 62% of its activity maintained at pH 5.0 for 48 hr. The extracellular extract containing β-D-fructofuranosidase was effective in producing fructooligosaccharides (FOS), mainly 1-kestose. The highest concentration of FOS was obtained at 30°C and 60°C, indicating the existence of at least two enzymes with transfructosylating activity. At 30°C, the maximal FOS concentration was obtained from 48 to 72 hr, while at 60°C, it was achieved only at 72 hr. The best production of FOS (86.7 g/L) was obtained using 500 g/L sucrose as substrate.
Aspergillus, fructooligosaccharide, transfructosylating, β-D-fructofuranosidase
NCBI PubMed ID: 31368547Fructooligosaccharides (FOS) are commonly regarded as prebiotics and used as components of functional foods. Currently, the industrial sucrose-to-FOS biotransformation is mainly carried out using the microbial-derived β-fructofuranosidases with transglycosylation activity as catalysts. Evaluation of the ability of a microorganism to produce β-fructofuranosidase is commonly conducted by measuring enzyme activity. However, the traditional method requires several steps to identify strains with high β-fructofuranosidase activity, which is not suitable for high-throughput screening. To facilitate screening of a large number of microbial cultures, this study developed a plate chromogenic assay method based on the glucose oxidase (GOD) - peroxidase (POD) bienzymatic system for screening of β-fructofuranosidase-producing fungal strains and predicting their potential to produce FOS. This method used the amount of glucose released from sucrose as indicator to form clear pink halos around the microbial colonies with β-fructofuranosidase activity. Cultivation conditions for the plate assay were optimized as cultivation time 5 h and spore inoculum concentration 100000000 1/ml. Moreover, the method was applied to screening of an Aspergillus niger ATCC 20611 mutant library. The mutant A11 displaying the largest pink halo was screened out and its β-fructofuranosidase activity was determined to be 1.65 fold than that of the parental strain. Thin layer chromatography (TLC) assay further indicated that A11 with the largest halo possessed the highest FOS synthesis ability. These results demonstrated the potential of this plate chromogyenic assay method in the rapid and effective identification of excellent FOS producers from a large number of strain samples.
β-fructofuranosidase, fructooligosaccharides (FOS), plate chromogenic assay, GOD-POD bienzymatic system, Aspergillus niger ATCC 20611
NCBI PubMed ID: 31614171Fructooligosaccharides (FOSs) are excellent food ingredients or feed additives by stimulating probiotics. In this paper, a CREA gene encoding a glucose repressor in the β-fructofuranosidase producer Aureobasidium melanogenum 33 with high-level FOS biosynthesis was disrupted, and glucose repression in disruptant D28 was relieved. The disruptant D28 produced up to 2100 U/mL of β-fructofuranosidase activity, whereas the enzyme activities produced by parent strain 33 and complemented strain C11 were below 600 U/mL. The whole cells of the disruptant D28 was used to convert cane molasses into FOSs, and 0.58 g of FOSs/g of molasses sugar was synthesized from 350 g/L cane molasses sugar within 4 h. Results demonstrated that the industrial waste cane molasses can be efficiently converted into FOSs by the glucose derepression mutant D28 with high β-fructofuranosidase activity. This low-cost and environmentally friendly bioprocess has great potential applications in bioengineering and biotechnology for FOS production.
Aureobasidium melanogenum, cane molasses, glucose derepression, high-level FOS biosynthesis
NCBI PubMed ID: 31686508Eight strains of Aspergillus spp. were evaluated for their ability to produce intracellular and extracellular fructosyltransferase and fructooligosaccharides from sucrose. To the best of our knowledge, this is the first time that a study evaluates the production of intracellular and extracellular fructosyltransferase simultaneously with its chemometric characterization. High values of intracellular FTase were exhibited by A. oryzae and A. carbonarius and extracellular FTase by A. japonicus and A. ochraceus. The highest concentrations of intracellular FOS were generated by A. carbonarius and A. ochraceus and extracellular FOS by A. carbonarius and A. japonicus. The variations detected between the behavior of intra- and extra-cellular enzymes can be explained by activation of disulfide bridges and N-terminal signal peptides that occur distinctly between these biological structures. 1-kestose and 1-fructofuranosylnystose were the major oligosaccharides detected by HPLC-RID. Thus, transfructosylation activity is the main contributor to the positive correlation of 1-kestose.
fructooligosaccharides, fructosyltransferase, intracellular enzymes, chemometric characterization, extracellular enzymes
Publication DOI: 10.1016/j.biteb.2020.100546Short chain fructo-oligosaccharides (SC-FOS) are the potential prebiotics possessing diverse applications in both food and feed industries. The present study was aimed to extract inulin from chicory roots followed by its conversion into SC-FOS applying endoinulinase from Aspergillus fumigatus. The inulin was extracted from chicory roots through boiling in hot water, followed by precipitation with ethanol at room temperature or freezing condition. Maximum yield (42%) of inulin was obtained with three volumes of chilled absolute ethanol at room temperature. HPLC analysis of enzymatic hydrolysate detected kestose (GF2), nystose (GF3), and other FOS having higher degree of polymerization (DP). Maximum GF2 (5.79 mg/ml) was detected at temperature 50 °C, pH 5.5 with 2 U of enzyme dose after 6 h of hydrolysis; while maximum GF3 (4.33 mg/ml) was recorded at 60 °C, 5.5 pH with 0.5 U enzyme dose after 2 h of hydrolysis. Nevertheless, complete hydrolysis of inulin was noticed with 99% total oligosaccharide yield at 55 °C, 5.5 pH with 0.5 U enzyme dose after 4 h of hydrolysis with negligible amount of mono- and di-saccharides. The present finding demonstrated the process for higher yield of inulin from chicory roots followed by its conversion into SC-FOS applying fungal endoinulinase.
inulin, fungi, prebiotic, endoinulinase, short chain fructo-oligosaccharides
NCBI PubMed ID: 31845198The aim of the study was the fungal production of fructooligosaccharides from solid-state fermentation of different agro-industrial wastes (sugar cane bagasse, coffee husk, pineapple peel, prickle pear peel and banana peel) enriched with maguey sap. An exploratory statistical design with a factorial arrangement (23) was used to identify some parameters that are directly related to the production of FOS. The production was optimized and evaluated through fermentation kinetics in order to determine the time of maximum FOS production. Among the evaluated materials, sugar cane bagasse was the most promising substrate suited for the FOS production reaching a concentration of 7.64 g of FOS per liter of culture medium based on the initial sucrose concentration (21 g/L), a high Yp/s = 0.45 based on sucrose consumed was observed. The results showed that use of sugar cane bagasse enriched with aguamiel is the most economical and excellent alternative source for the FOS production by Aspergillus oryzae DIA-MF.
Prebiotics, fructooligosaccharides, solid-state fermentation, agricultural by-products
Publication DOI: 10.1016/j.bcab.2020.101704Pectinex Ultra SP-L, a commercial enzyme preparation with fructosyltransferase activity, was successfully immobilized by covalent binding to Fe3O4-chitosan-magnetic nanoparticles. Immobilization carried out according to a 2^3-full factorial design where glutaraldehyde concentration, activation time and time of contact between enzyme and support were selected as the independent variables and immobilization yield as the response. The highest immobilization yield (94.84%) was obtained using 3.0% (v/v) glutaraldehyde and activation and contact times of 180 and 30 min, respectively. The immobilized biocatalyst, which showed for both hydrolytic and transfructosylating activities optimum pH and temperature of 7.0 and 60 °C, respectively, retained 70 and 86% of them after 6 cycles of reuse. A kinetic/thermodynamic study focused on thermal inactivation of the immobilized construct indicated high thermostability at temperatures commonly used for fructo-oligosaccharides (FOS) production. Maximum FOS concentration obtained in lab-scale experiments was 101.56 g/L, with predominant presence of 1-kestose in the reaction mixture. The results obtained in this study suggest that the immobilized-enzyme preparation may be effectively exploited for FOS production and easily recovered from the reaction mixture by action of a magnetic field.
fructo-oligosaccharides, fructosyltransferase, magnetic nanoparticles
NCBI PubMed ID: 32070737Fructooligosaccharides (FOS) have widely used for the manufacture of low-calorie and functional foods, because they can inhibit intestinal pathogenic microorganism growth and increase the absorption of Ca2+ and Mg2+. In this study, the novel fructosyltransferase (FTase) from Aspergillus oryzae strain S719 was successfully purified and characterized. The specific activity of the final purified material was 4200 1/mg with purification ratio of 66 times and yield of 26%. The molecular weight of FTase of A. oryzae S719 was around 95 kDa by SDS-PAGE, which was identified as a type of FTase by Mass Spectrometry (MS). The purified FTase had optimum temperature and pH of 55 °C and 6.0, respectively. The FTase showed to be stable with more than 80% of its original activity at room temperature after 12 h and maintaining activity above 90% at pH 4.0-11.0. The Km and kcat values of the FTase were 310 mmol/L and 2000 1/min, respectively. The FTase was activated by 5 mmol/L Mg2+ and 10 mmol/L Na+ (relative activity of 116 and 114%, respectively), indicating that the enzyme was Mg2+ and Na+ dependent. About 64% of FOS was obtained by the purified FTase under 500 g/L sucrose within 4 h of reaction time, which was the shortest reaction time to be reported regarding the purified enzyme production of FOS. Together, these results indicated that the FTase of A. oryzae S719 is an excellent candidate for the industrial production of FOS.
Aspergillus oryzae, fructosyltransferase, enzyme purification, fructooligosaccharides production
NCBI PubMed ID: 31805395The use of whole-cell biocatalyst for production of fructo-oligosaccharide (FOS) eliminates the need for costly enzyme recovery and purification. In this study, a novel Aureobasidium pullulans strain (FRR 5284) was identified from seven A. pulllulans strains as an efficient whole-cell biocatalyst for FOS production. The strain had a specific intracellular transfructosylating activity of 4.44 ± 0.18 U/mg dry cells, one of the highest transfructosylating activities thus far reported for whole-cell biocatalyst. Under optimal conditions (pH 5.5 and 55 °C), only 5 g/L of dry cells and 3 h reaction time were required to achieve a FOS yield of 61 % from 50 % (w/v) sucrose. Incubation of sugarcane molasses with an invertase-free Saccharomyces cerevisiae prior to addition of A. pullulans whole-cell biocatalyst eliminated glucose inhibition and increased FOS yield from 44 % to 56 % in 1 h. This study has demonstrated that the novel A. pullulans FRR 5284 was an efficient source of whole-cell biocatalyst for FOS production and a promising strategy for transformation of sucrose and sugarcane molasses into a higher-value prebiotic.
Prebiotics, Aureobasidium pullulans, molasses, fructo-oligosaccharide, transfructosylating activity, invertase-free
Publication DOI: 10.1016/j.bej.2020.107747An extracellular fructosyltransferase (Ftase) enzyme with a molar mass of ≈70 kDa from a newly isolated indigenous coprophilous fungus Aspergillus niger sp. XOBP48 is purified to homogeneity and characterized in this study. The enzyme was purified to 4.66-fold with a total yield of 15.53% and specific activity of 1219.17 U/mg of protein after a three-step procedure involving (NH4)2SO4 fractionation, dialysis and anion exchange chromatography. Ftase showed optimum activity at pH 6.0 and temperature 50 °C. Ftase exhibited over 80% residual activity at pH range of 4.0-10.0 and ≈90% residual activity at temperature range of 40-60 °C for 6 h. Metal ion inhibitors Hg2+ and Ag+ significantly inhibited Ftase activity at 1 mmol concentration. Ftase showed Km, vmax and kcat values of 79.51 mmol, 45.04 µmol/min and 31.5 1/min, respectively, with a catalytic efficiency (kcat/Km) of 396 1/µmol/min for the substrate sucrose. HPLC-RI experiments identified the end products of fructosyltransferase activity as monomeric glucose, 1-kestose (GF2), and 1,1-kestotetraose (GF3). This study evaluates the feasibility of using this purified extracellular Ftase for the enzymatic synthesis of biofunctional fructooligosaccharides.
1-kestose, 1, fructooligosaccharides, Aspergillus niger, fructosyltransferase, zymography, 1-kestotetraose
NCBI PubMed ID: 33088656There is a great interest in prebiotics consumptions eminence by their health promoting properties and economic market value. Filamentous fungi have been implicated as the key producers of fructosyltransferase (Ftase) and inulinase for synthesis of fructooligosaccharides (FOSs), which is used as a bioactive ingredient in functional foods. In this study, sixty-one indigenous coprophilous fungal strains were isolated, purified to monoculture and investigated for their potential use in biotransformation of sucrose and inulin into fructooligosaccharides (FOSs) and inulooligosaccharides (IOSs) by producing Ftase and inulinase enzymes, respectively. The molecular identification by 18S rDNA sequencing and morpho-taxonomic keys revealed that axenic fungal strains belonged to the genera Aspergillus, Neocosmospora, Trichoderma, Mucor and Fusarium. The crude enzyme extract from the isolates showed hydrolysis zones of 15–30 mm on the 2,3,5-triphenyltetrazolium chloride (TTC) and Lugol's iodine solution assay plates confirming the Ftase and inulinase activities, respectively. The submerged culture filtrates of eight fungal isolates showed high Ftase activity while six different fungal isolates exhibited high inulinase activity. The reaction products analysed with thin-layer chromatography and high-performance liquid chromatography coupled with refractive index detection indicated the presence of FOSs and IOSs. The strains isolated in this study have potential biotechnological implications to produce FOSs and IOSs.
fructooligosaccharides, fructosyltransferase, coprophilous fungi, inulinase, inulooligosaccharides
Publication DOI: 10.1016/j.bcab.2020.101867Short-chain fructooligosaccharides (ScFOS) are a group of linear fructose oligomers that include 1-kestose, 1-nystose and 1-β-fructofuranosylnystose. ScFOS, which naturally occur at low levels in different plant products, are of high interest as food ingredients because of their prebiotic character, organoleptic characteristics and technological properties. Two different industrial processes are used to achieve large-scale ScFOS production: inulin hydrolysis (enzymatic or chemical hydrolysis) or sucrose biotransformation by transfructosylation (enzymatic synthesis) using specific enzymes like fructosyltransferases and fructofuranosidases. Enzymatic ScFOS synthesis seems to be more advantageous than inulin hydrolysis since it is less expensive, and leads to lower molecular weight FOS. The biotechnological process described to carry out this catalysis includes the production of transfructosylation enzymes, separation, enzyme immobilisation and finally the ScFOS production and purification. Such ScFOS production processes may be conducted under submerged or solid-state fermentation under discontinuous or continuous conditions. Several methodologies with different economic/environmental costs and production yields have been described to carry out these ScFOS production stages, although industrial scale-up needs to be optimised. This review tries to address a revision about enzymatic ScFOS production methods and its scale-up to industrial levels.
submerged fermentation, short-chain fructooligosaccharides, fungal fructosyltransferase, industrial scale-up
Publication DOI: 10.1007/s12393-020-09209-0In present study, statistical optimization of medium composition for endoinulinase production from raw inulin using Aspergillus tritici was performed in shake-flask fermentations. The concentration of four independent variables viz. raw inulin (1.5–3.5%), peptone (0.2–1.8%), (NH4)H2PO4 (0.2–1.8%) and pH (4.5–6.5) was explored to optimize the composition of fermentation medium for maximum endoinulinase production. Fermentation medium containing 2.5% raw inulin, 1% peptone, 1% (NH4)H2PO4 and medium pH adjusted to 5.5 supported maximum (25.39 IU/mL) endoinulinase production. Adequate precision value >4, closeness of coefficient of determination (R2) value to 1, good co-relation between the predicted and experimental values, percentage error <5%, high F-value 164.52 and low Lack of fit F-value 0.19 authenticates the fitness of quadratic model. TLC fingerprints studies confirmed fructooligosaccharides synthesis from inulin by crude fungal endoinulinase. Fructooligosaccharides yield (19.40%) containing 3.70% ketose (GF2), 2.71% nystose (GF3) and 1.42% fructofuranosyl nystose (GF4) were obtained from inulin (10%).
response surface methodology, fructooligosaccharides, endoinulinase, Aspergillus tritici, raw-inulin
Publication DOI: 10.1016/j.biteb.2020.100417Three-step purification technique (isopropanol precipitation, ion-exchange and size-exclusion chromatography) was used for the purification of an endoinulinase from the culture broth of Aspergillus tritici BGPUP6. The molecular mass of purified endoinulinase was found to be 53.45 kDa and 53.70 kDa by denatured protein gel (SDS-PAGE) and size-exclusion (Sephadex G-100) chromatographic analysis, respectively. Higher Km (0.90 mM), Vmax (19.60 mM/min·mg), Kcat (0.0013 1/min) and Vmax/Km ratio (21.77 1/min·mg) of purified endoinulinase for inulin than stachyose depicts its higher affinity towards inulin. Purified enzyme was found stable in a pH range 4.0-7.0 with an optimal pH 5.5. The optimal temperature of purified biocatalyst was 55 °C with thermostability in the range of 50-70 °C. D-value and Z-value for endoinulinase at 55 °C was found to be 100.08 h and 11.62 °C, respectively. Thermodynamics inactivation parameters (ΔG, ΔH and ΔS) of endoinulinase shows its wide range thermal stability. Endoinulinase activity was enhanced by CaCl2 and MnSO4, while CuSO4, CoCl2, AgNO3, CdCl2, NiCl2, ZnSO4, BaCl2, HgCl2 and EDTA inhibited the activity of enzyme. Purified endoinulinase was successfully used for the production of fructooligosaccharides from inulin.
fructooligosaccharides, endoinulinase, Aspergillus tritici, kinetics characterization
NCBI PubMed ID: 32890562A previous study (KL Forsythe, MS Feather [1989] Carbohydr Res 185: 315-319) showed that (13)C nuclear magnetic resonance spectroscopy can be used to detect and identify mixtures of 1-kestose and neokestose after conversion to the acetate derivatives. In this study, unequivocal assignments are made for the anomeric carbon and proton signals for the above two trisaccharide acetates as well as for 6-kestose hendecaacetate and for nystose tetradecaacetate (a 1-kestose-derived tetrasaccharide). A number of oligosaccharide fractions were isolated from several plant species, converted to the acetates, and nuclear magnetic resonance spectra obtained. Using the above reference data, the following information was obtained. The trisaccharide fraction from Dactylis glomerata L. stem tissue and Asparagus officinalis L. roots contain both 1-kestose and neokestose, and the tetrasaccharide fractions contain three components, one of which is nystose. Penta- and hexasaccharide acetates were also isolated from A. officinalis L. roots and were found to contain, respectively, four and at least five components. All components of both of the above species appear to contain a kestose residue and to be produced by the sequential addition of fructofuranosyl units to these. The trisaccharide fraction from Festuca arundinacea is complex, and contains at least five different components, two of which appear to be 1-kestose and neokestose.
NCBI PubMed ID: 16667365The conformational studies of inulin oligomers from G-F_2 to G-F_9, which isolated from Platycodon grandiflorum, suggested a plausible conformational change between G-F_7 and G-F_8 from the trends in their chemical shift patterns and molecular rotation; the oligomers higher than G-F_8 would form some secondary conformations more rigid than shorter oligomers. On the other hand, spin-latice relaxation (T_1) studies of the protons proposed through-space interactions of 2- and 4-H's of glucose moiety in G-F_5,presumably with some atom(s) of the terminal fructose moiety. This would reflect that the inulin molecule adopts a 5/1 helix.
NMR, conformation, inulin, inulin oligomer, molecular rotation, spin-lattice relaxation time (T1)
Journal NLM ID: 0377775Individual fructan tri-, tetra- and pentasaccharide isomers in neutral, water-soluble extracts from Lolium temulentum were purified and the linkages present in these isomeric oligosaccharides were analysed by combined GC-mass spectrometry of partially methylated alditol acetates. 1-Kestose and neokestose were the most abundant trisaccharides with 6-kestose present in much lower amounts. Analysis of isomers of DP 4 and 5 showed that multiple linkage types were present with structures based on all three trisaccharides. Oligosaccharides based on neokestose but with 2,6 linkages between adjacent fructose residues have not been previously detected in higher plants.
Oligosaccharides, 1-kestose, fructan, sucrose, neokestose, Gramineae, Lolium temulentum
Publication DOI: 10.1016/0031-9422(92)83432-XLeaves of field-grown cheatgrass (Bromus tectorum L.) plants were sampled during early spring when day- and night-time temperatures were relatively cool. Fructan oligomers with a degree of polymerization (DP) 3–6 were extracted and purified using gel and anion-exchange chromatography. The structures of 13 cheatgrass fructans were established. They included two trisaccharides [1-kestotriose (1-kestose) and 6-kestotriose (6-kestose)], four tetrasaccharides [(1.1)-kestotetraose (nystose), (1&6)-kestotetraose (bifurcose), (6,1)-kestotetraose and (6,6)-kestotetraose], three pentasaccharides [(1,1&6)-kestopentaose, (1&6,6)-kestopentaose and (6;1&6)-kestopentaose] plus four hexasaccharides [(1,1,1&6)-kestohexaose (1&6,6,6)-kestohexaose, (6;1&6,6)-kestohexaose and (6;1,6&6)-kestohexaose]. All fructans larger then DP 4 contained a branch point. Each member of the dominant series(1&6)-kestotetraose, (1&6,6)-kestopentaose and (1&6,6,6)-kestohexaose, is a branched fructan in which two fructose moieties are linked to the fructose subunit of each sucrose molecule. Thus the dominant series is built upon (1&6)-kestotetraose. Fructans larger than DP 3 with exclusively 2→1- or 2→6-linkages were absent except for a very small amount of (6,6)-kestotetraose. The unique fructan structures synthesized in cheatgrass, wheat and oats illustrate diversity in the enzymology of fructan biosynthesis among grass species.
synthesis, Structures, fructan, degree of polymerization, Bromus
Publication DOI: 10.1111/j.1469-8137.1993.tb03829.xFructooligosaccharides (FOS) are short-chain sugars that occur naturally and have dietary benefits for humans. They are widely distributed in nature and are a natural part of the human diet. The objective of this study was to determine the concentrations of 1-kestose (GF(2)), nystose (GF(3)), and 1(F)-beta-fructofuranosylnystose (GF(4)) in a variety of common processed and prepared foods. An ion chromatographic method was developed for this purpose in which the sugar concentrations were measured using integrated amperometry. The samples were simply prepared by blending with water and filtering the suspensions through a 10000 Da cutoff centrifugal filter. These samples were then injected into the ion chromatograph, which had been programmed for gradient elution, and the areas of the sugar peaks obtained compared to those of standard sugars on a calibration curve. Selected samples were prepared both with and without standard spikes in order to assess the efficiency of the determination. Of the vegetables investigated, artichokes contained by far the most FOS, followed by onions; bananas contained more FOS than other fruits investigated. The method was shown to be simple, convenient, and relatively fast for the quantitation of FOS in processed and prepared food products.
fructooligosaccharides, food composition, ion exchange chromatography
NCBI PubMed ID: 11087481| b-D-Fruf-(2-6)-b-D-Fruf-(2-1)-a-D-Glcp | Show graphically |
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Structure type: oligomer
Trivial name: 6-kestose
Compound class: fructan
Contained glycoepitopes: IEDB_142488,IEDB_144998,IEDB_146664,IEDB_983931,SB_192
Major fructo-oligosaccharides (FOS) produced by levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 were characterised as 1-kestose and nystose by acid hydrolysis and 13C NMR spectroscopy. The highest yields of 1-kestose (481 mM; 241 g/l) and nystose (81 mM; 54 g/l) were achieved at initial sucrose concentration of 1754 mM (600 g/l), pH 5.5 and 40 C. The synthesized FOS reached 50% (w/w) of total sugars in the reaction mixture, with a conversion efficiency over 70% (w/w) based on the amount of sucrose converted to 1-kestose.
oligosaccharide, structural, analysis, Oligosaccharides, structural analysis, production, 1-kestose, Acetobacter, Acetobacter diazotrophicus, fructo-oligosaccharides, levansucrase, LsdA
Journal NLM ID: 8008051A previous study (KL Forsythe, MS Feather [1989] Carbohydr Res 185: 315-319) showed that (13)C nuclear magnetic resonance spectroscopy can be used to detect and identify mixtures of 1-kestose and neokestose after conversion to the acetate derivatives. In this study, unequivocal assignments are made for the anomeric carbon and proton signals for the above two trisaccharide acetates as well as for 6-kestose hendecaacetate and for nystose tetradecaacetate (a 1-kestose-derived tetrasaccharide). A number of oligosaccharide fractions were isolated from several plant species, converted to the acetates, and nuclear magnetic resonance spectra obtained. Using the above reference data, the following information was obtained. The trisaccharide fraction from Dactylis glomerata L. stem tissue and Asparagus officinalis L. roots contain both 1-kestose and neokestose, and the tetrasaccharide fractions contain three components, one of which is nystose. Penta- and hexasaccharide acetates were also isolated from A. officinalis L. roots and were found to contain, respectively, four and at least five components. All components of both of the above species appear to contain a kestose residue and to be produced by the sequential addition of fructofuranosyl units to these. The trisaccharide fraction from Festuca arundinacea is complex, and contains at least five different components, two of which appear to be 1-kestose and neokestose.
NCBI PubMed ID: 16667365Individual fructan tri-, tetra- and pentasaccharide isomers in neutral, water-soluble extracts from Lolium temulentum were purified and the linkages present in these isomeric oligosaccharides were analysed by combined GC-mass spectrometry of partially methylated alditol acetates. 1-Kestose and neokestose were the most abundant trisaccharides with 6-kestose present in much lower amounts. Analysis of isomers of DP 4 and 5 showed that multiple linkage types were present with structures based on all three trisaccharides. Oligosaccharides based on neokestose but with 2,6 linkages between adjacent fructose residues have not been previously detected in higher plants.
Oligosaccharides, 1-kestose, fructan, sucrose, neokestose, Gramineae, Lolium temulentum
Publication DOI: 10.1016/0031-9422(92)83432-XChembiotech Ltd. Institute of Research and Development, University of Birmingham Research Park, Edgbaston, Birmingham, UK; Birmingham Carbohydrate and Protein Technology Group, Research Laboratory for the Chemistry of Bioactive Carbohydrates and Proteins, School of Chemistry, The University of Birmingham, Edgbaston, Birmingham, UK; School of Chemical Engineering, The University of Birmingham, Edgbaston, Birmingham, UK
Publication DOI: 10.1016/0144-8617(94)90143-0Gomphrena macrocephala St.-Hil. (Amaranthaceae) is a perennial herb that grows spontaneously in the cerrado and is characterized by well-defined phenological phases throughout the year. Soluble carbohydrates are the main reserve compounds of the tuberous root and constitute approximately 50% of the dry weight. These sugars were partially characterized as fructans forming a single homologous series, different from inulin, the most common fructan of dicotyledons. The mean molecular weight of polysaccharides was high and reached 37 kDa in the dormant phase. Fructan spherocrystals were detected in the tuberous root after treatment with ethanol, being associated with the parenchyma of secondary xylem. The content, composition and mean molecular weight of fructans were related to phenology. In late dormancy, there was a marked increase in monosaccharides, particularly fructose, and a concomitant decline of polysaccharides, probably as a result of fructan breakdown. During sprouting and in the vegetative phase, the contents of oligosaccharides and low molecular weight polysaccharides increased. A gradual rise in the molecular weight of polysaccharides occurred during the reproductive phase and at early dormancy, concurrently with decreasing levels of oligosaccharides. The capacity of G. macrocephala to accumulate readily accessible sugars, such as fructans, instead of starch, in response to environmental changes, may be of considerable advantage, since the cerrado is often subjected to seasonal drought and burnings.
fructans, Gomphrena macrocephala St.-Hil., Amaranthaceae, storage carbohydrates, phenology
Publication DOI: 10.1111/j.1365-3040.1993.tb00515.x| -6)-b-D-Fruf-(2- | Show graphically |
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Structure type: homopolymer
Trivial name: levan, levan-type polysaccharide
Compound class: EPS, polysaccharide, fructan
Contained glycoepitopes: IEDB_923066
Major fructo-oligosaccharides (FOS) produced by levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 were characterised as 1-kestose and nystose by acid hydrolysis and 13C NMR spectroscopy. The highest yields of 1-kestose (481 mM; 241 g/l) and nystose (81 mM; 54 g/l) were achieved at initial sucrose concentration of 1754 mM (600 g/l), pH 5.5 and 40 C. The synthesized FOS reached 50% (w/w) of total sugars in the reaction mixture, with a conversion efficiency over 70% (w/w) based on the amount of sucrose converted to 1-kestose.
oligosaccharide, structural, analysis, Oligosaccharides, structural analysis, production, 1-kestose, Acetobacter, Acetobacter diazotrophicus, fructo-oligosaccharides, levansucrase, LsdA
Journal NLM ID: 8008051The acidic exopolysaccharides (EPSs) from 63 strains of mushroom production-associated fluorescent pseudomonads which were mucoid on Pseudomonas agar F medium (PAF) were isolated, partially purified, and characterized. The strains were originally isolated from discolored lesion which developed postharvest on mushroom (Agaricus bisporus) caps or from commercial lots of mushroom casing medium. An acidic galactoglucan, previously named marginalan, was produced by mucoid strains of the saprophyte Pseudomonas putida and the majority of mucoid strains of saprophytic P. fluorescens (biovars III and V) isolated from casing medium. One biovar II strain (J1) of P. fluorescens produced alginate, a copolymer of mannuronic and guluronic acids, and one strain (H13) produced an apparently unique EPS containing neutral and amino sugars. Of 10 strains of the pathogen "P. gingeri," the causal agent of mushroom ginger blotch, 8 gave mucoid growth on PAF. The "P. gingeri" EPS also was unique in containing both neutral sugar and glucuronic acid. Mucoid, weakly virulent strains of "P. reactans" produced either alginate or marginalan. All 10 strains of the pathogen P. tolaasii, the causal agent of brown blotch of mushrooms were nonnmucoid on PAF. Production of EPS by these 10 strains plus the 2 nonmucoid strains of "P. gingeri" also was negative on several additional solid media as well as in two broth media tested. The results support our previous studies indicating that fluorescent pseudomonads are a rich source of novel EPSs.
Pseudomonas, exopolysaccharide, exopolysaccharides, identification, production, fluorescent, mushroom
NCBI PubMed ID: 7574589Burkholderia cepacia is an opportunistic pathogen involved in pulmonary infections related to cystic fibrosis. A clinical strain, BTS13, was isolated and the production of exopolysaccharides was tested growing the bacteria on two different media, one of which was rich in mannitol as carbon source. The primary structure of the polysaccharides was determined using mostly mass spectrometry and NMR spectroscopy. On both media an exopolysaccharide having the following repeating unit was produced: →5)-β-Kdop-(2→3)-β-D-Galp2Ac-(1→4)-α-D-Galp-(1→3)-β-D-Galp-(1→. This polysaccharide has already been described as the biosynthetic product of another Burkholderia species, B. pseudomallei, the microbial agent causing melioidosis. In addition to this, when grown on the mannitol-rich medium, B. cepacia strain BTS13 produced another polysaccharide that was established to be levan: →6)-β-D-Fruf-(2→. The content of levan was about 20% (w/w) of the total amount of polymers. The ability of B. cepacia to produce these two exopolysaccharides opens new perspectives in the investigation of the role of polysaccharides in lung infections.
Burkholderia cepacia, NMR spectroscopy, exopolysaccharide, cystic fibrosis, Structures, MALDIMS
NCBI PubMed ID: 14670727This review is the third update of the original review, published in 1999, on the application of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings the topic to the end of 2004. Both fundamental studies and applications are covered. The main topics include methodological developments, matrices, fragmentation of carbohydrates and applications to large polymeric carbohydrates from plants, glycans from glycoproteins and those from various glycolipids. Other topics include the use of MALDI MS to study enzymes related to carbohydrate biosynthesis and degradation, its use in industrial processes, particularly biopharmaceuticals and its use to monitor products of chemical synthesis where glycodendrimers and carbohydrate-protein complexes are highlighted
carbohydrates, glycosyltransferases, fragmentation, MALDI, glycolipids, glycoproteins, biopharmaceuticals, glycosidases, time-of-flight
NCBI PubMed ID: 18825656Bacillus sp. 3B6, bacterium isolated from cloud water, was incubated on sucrose for exopolysaccharide production. Dialysis of the obtained mixture (MWCO 500) afforded dialyzate (DIM) and retentate (RIM). Both were separated by size exclusion chromatography. RIM afforded eight fractions: levan exopolysaccharide (EPS), fructooligosaccharides (FOSs) of levan and inulin types with different degrees of polymerization (dp 2-7) and monosaccharides fructose:glucose=9:1. Levan was composed of two components with molecular mass ~3500 and ~100kDa in the ratio 2.3:1. Disaccharide fraction contained difructose anhydride DFA IV. 1-Kestose, 6-kestose, and neokestose were identified as trisaccharides in the ratio 2:1:3. Fractions with dp 4-7 were mixtures of FOSs of levan (2,6-βFruf) and inulin (1,2-βFruf) type. DIM separation afforded two dominant fractions: monosaccharides with fructose: glucose ratio 1:3; disaccharide fraction contained sucrose only. DIM trisaccharide fraction contained 1-kestose, 6-kestose, and neokestose in the ratio1.5:1:2, penta and hexasaccharide fractions contained FOSs of levan type (2,6-βFruf) containing α-glucose. In the pentasaccharide fraction also the presence of a homopentasaccharide composed of 2,6-linked βFruf units only was identified. Nystose, inulin (1,2-βFruf) type, was identified as DIM tetrasaccharide. Identification of levan 2,6-βFruf and inulin 1,2-βFruf type oligosaccharides in the incubation medium suggests both levansucrase and inulosucrase enzymes activity in Bacillus sp. 3B6.
Bacillus, inulin, levan, cloud water, FOSs, DFA IV
NCBI PubMed ID: 21292244Lactobacillus reuteri strain 100-23 together with a Lactobacillus-free mouse model, provides a system with which the molecular traits underpinning bacterial commensalism in vertebrates can be studied. A polysaccharide was extracted from sucrose-containing liquid cultures of strain 100-23. Chemical analysis showed that this exopolysaccharide was a levan (β-2, 6-linked fructan). Mutation of the fructosyl transferase (ftf) gene resulted in loss of exopolysaccharide production. The ftf mutant was able to colonise the murine gastrointestinal tract in the absence of competition, but colonisation was impaired in competition with the wild type. Biofilm formation by the mutant on the forestomach epithelial surface was not impaired and the matrix between cells was indistinguishable from that of the wild type in electron micrographs. Colonisation of the mouse gut by the wild-type strain led to increased proportions of regulatory T cells (Foxp3+) in the spleen, whereas colonisation by the ftf mutant did not. Survival of the mutant in sucrose-containing medium was markedly reduced relative to the wild type. Comparison of the genomic ftf loci of strain 100-23 with other L. reuteri strains suggested that the ftf gene was acquired by lateral gene transfer early in the evolution of the species and subsequently diversified at accelerated rates. Levan production by L. reuteri 100-23 may represent a function acquired by the bacterial species for life in moderate to high-sucrose extra-gastrointestinal environments that has subsequently been diverted to novel uses, including immunomodulation, that aid in colonisation of the murine gut.
exopolysaccharide, evolution, survival, Lactobacillus reuteri, regulatory T cells
NCBI PubMed ID: 21248858The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic acid and legionaminic acid, respectively. A minor high molecular weight polysaccharide component was recognized as the fructose homopolymer levan, and a glucosylated lipoteichoic acid (LTA) was identified in a micellar fraction. The polysaccharides were conjugated to the CRM197 carrier protein, and the resulting glycoconjugates were used to immunize rabbits. Rabbit immune sera were evaluated for their ability to kill Tx16 in opsonophagocytic assays and in a mouse passive protection infection model. Although antibodies raised against levan failed to mediate opsonophagocytic killing, the other glycoconjugates induced effective opsonic antibodies, with the altruronic acid-containing polysaccharide antisera showing the greatest opsonophagocytic assay activity. Antibodies directed against either novel heteroglycan or the LTA reduced bacterial load in mouse liver or kidney tissue. To assess antigen prevalence, we screened a diverse collection of blood isolates (n = 101) with antibodies to the polysaccharides. LTA was detected on the surface of 80% of the strains, and antigens recognized by antibodies to the two major heteroglycans were co-expressed on 63% of these clinical isolates. Collectively, these results represent the first steps toward identifying components of a glycoconjugate vaccine to prevent E. faecium infection.
antigen, teichoic acid, vaccine, uronic acids, carbohydrate structure, glycoconjugate vaccine, Enterococcus faecium, sialic acids
NCBI PubMed ID: 26109072Burkholderia multivorans C1576 is a Gram negative opportunistic pathogen causing serious lung infection in cystic fibrosis patients. Considering that bacteria naturally form biofilms, and exopolysaccharides are recognized as important factors for biofilm architecture set-up, B. multivorans was grown both in biofilm and in non-biofilm mode on two different media in order to compare the exopolysaccharides biosynthesized in these different experimental conditions. The exopolysaccharides produced were purified and their structure was determined resorting mainly to NMR spectroscopy, ESI mass spectrometry and gas chromatography coupled to mass spectrometry. The experimental data showed that both in biofilm and non-biofilm mode B. multivorans C1576 produced a novel exopolysaccharide having the following structure: [Formula: see text]. About 50% of the 2-linked rhamnose residues are substituted on C-3 with a methyl ether group. The high percentage of deoxysugar Rha units, coupled with OMe substitutions, suggest a possible role for polymer domains with marked hydrophobic characteristics able to create exopolysaccharide junction zones favouring the stability of the biofilm matrix.
NMR, Exopolysaccharide structure, cystic fibrosis, Biofilm, Burkholderia multivorans C1576
NCBI PubMed ID: 25974852OBJECTIVES: A levansucrase from Leuconostoc mesenteroides NTM048 was cloned and expressed and its enzymatic product was characterized. RESULTS: The fructansucrase gene from Leuconostoc mesenteroides was cloned and expressed in Escherichia coli. The recombinant enzyme was purified as a single protein and its properties investigated. The polymer produced by the recombinant enzyme was identified as levan by various means including TLC and NMRs, and the enzyme was identified as a GH68 levansucrase. The enzyme was optimal at pH 5.5-6 and 30 degrees C, and its activity was stimulated by Ca(2+). The levan produced by this strain induced IgA production in mice. CONCLUSION: Leuconostoc mesenteroides, a probiotic strain, possessed levansucrase which catalyzed the produced levan that had immunomodulating activity.
levansucrase, levan, Leuconostoc mesenteroides, Fructansucrase, IgA, Immunomodulating activity
NCBI PubMed ID: 26960415Various microorganisms isolated from polluted environments, such as Pseudomonas sp. and Micrococcus sp. can synthesize exopolysaccharides (EPSs) which are natural, non-toxic and biodegradable polymers. EPSs play a key role in protection of microbial cells under various external influences. For humans, these substances have potential use in many industries. EPSs can be applied as a flavor or a fragrance carrier, an emulsifier, a stabilizer, a prebiotic, an antioxidant or an antitumor agent. In this study, we characterized an environmental microorganism that produces EPS, optimized EPS production by this strain and characterized the EPS produced. Isolate CH-KOV3 was identified as Brachybacterium paraconglomeratum. The sucrose level in the growth medium greatly influenced EPS production, and the highest yield was when the microorganism was incubated in media with 500 g/L, of sucrose. The optimal temperature and pH were 28 degrees C and 7.0, respectively. The nuclear magnetic resonance (NMR) results and GC MS analysis confirmed that the residues were D-fructofuranosyl residues with β-configuration, where fructose units are linked by β-2,6-glycosidic bonds, with β-2,1-linked branches. All these data indicate that the investigated EPS is a levan-type polysaccharide. Thus, it was concluded that Brachybacterium sp. CH-KOV3 could constitute a new source for production of the bioactive polysaccharide, levan.
exopolysaccharides, Polluted environments, Brachybacterium paraconglomeratum
NCBI PubMed ID: 28602989Burkholderia species are a vast group of human pathogenic, phytopathogenic, and plant- or environment-associated bacteria. B. pseudomallei, B. mallei, and B. cepacia complex are the causative agents of melioidosis, glanders, and cystic fibrosis-related infections, respectively, which are fatal diseases in humans and animals. Due to their high resistance to antibiotics, high mortality rates, and increased infectivity via the respiratory tract, B. pseudomallei and B. mallei have been listed as potential bioterrorism agents by the Centers for Disease Control and Prevention. Burkholderia species are able to produce a large network of surface-exposed polysaccharides, i.e., lipopolysaccharides, capsular polysaccharides, and exopolysaccharides, which are virulence factors, immunomodulators, major biofilm components, and protective antigens, and have crucial implications in the pathogenicity of Burkholderia-associated diseases. This review provides a comprehensive and up-to-date account regarding the structural elucidation and biological activities of surface polysaccharides produced by Burkholderia species. The chemical synthesis of oligosaccharides mimicking Burkholderia polysaccharides is described in detail. Emphasis is placed on the recent research efforts toward the development of glycoconjugate vaccines against melioidosis and glanders based on synthetic or native Burkholderia oligo/polysaccharides.
lipopolysaccharides, Burkholderia, capsular polysaccharides, Oligosaccharides, glycoconjugate vaccines, antigens, exopolysaccharides, surface polysaccharide, virulence factor, Biofilm, chemical synthesis, bioterrorism
Publication DOI: 10.1039/C8NP00046HFructan based biopolymers have been extensively characterized and explored for their potential applications. Linear chained biopolymers, like levan-type fructan, have gained attention because they have exhibited unconventional stretchable and unbendable properties along with biodegradable and biocompatible nature. Current study deals with the chemical characterization and cytotoxic analysis of fructose based exopolysaccharide that was extracellularly produced by an indigenously isolated bacterial species (Zymomonas mobilis KIBGE-IB14). Maximum yield of exopolysaccharide (44.7 gL-1) was attained after 72 h of incubation at 30 °C under shaking conditions (180 rpm) when the culture medium was supplemented with 150.0 gL-1 of sucrose as a sole carbon source. This exopolysaccharide displayed high water solubility index (96.0%) with low water holding capacity (17.0%) and an intrinsic viscosity of about 0.447 dL g-1. This biopolymer exhibited a characteristic linear homopolysaccharide structure of levan when characterized using Fourier Transform Infrared (FTIR), Nuclear Magnetic Resonance (NMR) spectroscopy (1H, 13C, TOCSY and NOESY) while, Atomic Force Microscopy (AFM) revealed its pointed and thorny structure. The decomposition temperature of levan was approximately 245 °C as revealed by Thermal Gravimetric Analysis (TGA). X-Ray Diffraction (XRD) results revealed its amorphous nature with crystalline phase. Cytotoxicity of different concentrations of levan was investigated against mouse fibroblast cell lines by measuring their cellular metabolic activity and it was noticed that a higher concentration of levan (2.0 mg ml-1) permitted the normal cell growth of NIH/3T3 cell lines. This non-cytotoxic and biocompatible nature suggests that this levan has the capability to be utilized in food and drug-based formulations as it exhibited biomedical potential.
exopolysaccharide, cytotoxicity, levan, chemical characterization, NIH/3T3 cell line
NCBI PubMed ID: 33342516Levan-type polysaccharides, produced by various organisms, are nontoxic, biocompatible, and biodegradable polymers with a wide range of biological activities. They have high potential for use in medicine, cosmetology, and industry. A large amount of levan (41.1 g L-1) was recovered by ethanol precipitation from a liquid nutrient medium of Paenibacillus polymyxa 88A that contained 15 % w/v sucrose as a carbon source. The levan was fractionated by gel-permeation and anion-exchange chromatography and was analyzed by DRIFT and NMR spectroscopy. It was found that levan was represented by slightly branched chains composed of β-(2→6)-Fruf residues. The average molecular mass of the levan was about 1.9 MDa. When shear stress was applied at different temperatures, aqueous levan solutions showed pseudoplastic behavior. As found by SEM, a freeze-dried powdered levan sample had a microporous structure. The levan had excellent emulsifying activity toward sunflower oil, forming an emulsion with long-term stability. Analysis of the antioxidant activity of the levan showed a higher, dose-dependent activity toward ABTS, as compared with that toward DPPH. Finally, the bioactivity of the levan was examined by MTT assay by using human cervical carcinoma (HeLa) cells
Rheology, levan, antitumor activity, thermal stability, Paenibacillus polymyxa, emulsifying activity
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