Molecular Weight Oligosaccharides Research Articles

Streptococcus infantarius 25124 isolated from pozol produces a high molecular weight amylopullulanase, a key enzyme for niche colonization

Abstract Pozol is a beverage made with maize dough that is prepared after boiling the kernels in limewater, causing a decrease in soluble sugars, with starch being the main fermentable carbohydrate in the dough. Previously, Streptococcus infantarius ssp. infantarius 25124 (Sii-25124) was identified as the most amylolytic bacteria isolated in this product. Analysis of Sii-25124 amylolytic enzymes revealed two amylases, a cytoplasmic α-amylase of 55.7 kDa and an extracellular amylopullulanase of 246.3 kDa, with two catalytic domains, one typical of an α-amylase and another typical of a pullulanase/glycogen debranching enzyme. Characterization of the joint activity of both enzymes using Sii-25124 cell lysate supernatant demonstrated stability between 30 °C and 45°C, and pH stability in a range between 6.8 and 8.0. The joint activity of Sii-25124 amylases showed a fast production of reducing sugars when starch was used as the substrate. In contrast, reducing sugar production from amylopectin was lower, but it steadily increased throughout the reaction time. The amylopullulanase produced by Sii-25124 hydrolyzes the starch in the dough to produce low molecular weight oligosaccharides, which may be transported into Sii-25124 cells, so that intracellular α-amylase hydrolyzes them to mono- and disaccharides. Amylopullulanase production by Sii-25124 could be an example of a specialized enzyme that successfully dominates starchy food fermentation.

Gentianose: Purification and structural determination of an unknown oligosaccharide in grape seeds

Grape seeds are among the main constituents of grape pomace, ranging between 20% and 30% of the wet matrix; however, their oligosaccharide composition has not been studied. This paper describes the purification and the identification of low molecular weight oligosaccharides contained in an EtOH/water extract of grape seeds. A sequential two-step purification by size exclusion chromatography was carried out to fractionate compounds according to molecular weights. Chemical characterization of the combined fractions was performed by Magnetic Resonance Spectroscopy and Gas Chromatography-Mass Spectrometry analyses. The separation process gave two fractions abundant in sucrose and glucose. A third fraction containing trisaccharides was acetylated allowing the purification of the main trisaccharide. The structure elucidation of the acetylated product made it possible to identify gentianose, a predominant carbohydrate reserve found in the storage roots of perennial Gentiana lutea. Grape seeds are wine industry by-products and the obtained results suggest the importance of their recovery.

Characterization of a bifunctional alginate lyase as a new member of the polysaccharide lyase family 17 from a marine strain BP-2

ObjectivesBifunctional alginate lyase can efficiently saccharify alginate biomass and prepare functional oligosaccharides of alginate.ResultsA new BP-2 strain that produces alginate lyase was screened and identified from rotted Sargassum. A new alginate lyase, Alg17B, belonging to the polysaccharide lyase family 17, was isolated and purified from BP-2 fermentation broth by freeze-drying, dialysis, and ion exchange chromatography. The enzymatic properties of the purified lyase were investigated. The molecular weight of Alg17B was approximately 77 kDa, its optimum reaction temperature was 40–45 °C, and its optimum reaction pH was 7.5–8.0. The enzyme was relatively stable at pH 7.0–8.0, with a temperature range of 25–35 °C, and the specific activity of the purified enzyme reached 4036 U/mg. A low Na+ concentration stimulated Alg17B enzyme activity, but Ca2+, Zn2+, and other metal ions inhibited it. Substrate specificity analysis, thin-layer chromatography, and mass spectrometry showed that Alg17B is an alginate lyase that catalyses the hydrolysis of sodium alginate, polymannuronic acid (polyM) and polyguluronic acid to produce monosaccharides and low molecular weight oligosaccharides. Alg17B is also bifunctional, exhibiting both endolytic and exolytic activities toward alginate, and has a wide substrate utilization range with a preference for polyM.ConclusionsAlg17B can be used to saccharify the main carbohydrate, alginate, in the ethanolic production of brown algae fuel as well as in preparing and researching oligosaccharides.

Applications of Algal Polysaccharides and Derivatives in Therapeutic and Agricultural Fields.

Recently, researchers have given more and more consideration to natural polysaccharides thanks to their huge properties such as stability, biodegradability and biocompatibility for food and therapeutics applications. a number of enzymatic and chemical processes were performed to generate bioactive molecules, such as low molecular weight fractions and oligosaccharides derivatives from algal polysaccharides. These considerable characteristics allow algal polysaccharides and their derivatives such as low molecular weight polymers and oligosaccharides structures to have great potential to be used in lots of domains, such as pharmaceutics and agriculture etc. Conclusion: The present review describes the mains polysaccharides structures from Algae and focuses on the currents agricultural (fertilizer, bio-elicitor, stimulators, signaling molecules and activators) and pharmaceutical (wound dressing, tissues engineering and drugs delivery) applications by using polysaccharides and/or their oligosaccharides derivatives obtained by chemical, physical and enzymatic processes.

Interaction and fragility study in salmon gelatin-oligosaccharide composite films at low moisture conditions

Non mammalian gelatin under low moisture conditions could potentially be used in encapsulation technologies in food and pharmaceutical applications. This work explored the interactions between salmon gelatin (SG) and different molecular weight oligosaccharides (at 0.2, 0.4, 0.6 wt fraction) with ∼5% moisture content. SG and glucose, sucrose and maltodextrin were combined to produce visually transparent composites films. Fourier-transformed infrared spectroscopy showed that changes in secondary structure towards a less ordered configuration occurred upon increasing oligosaccharides weight fraction. This interaction was identified as physical in nature and possibly sustained by hydrogen bonding between components. The oligosaccharides also reduced water sorption as indicated by Guggenheim, Anderson and de Boer monolayer parameters derived from dynamic vapour sorption data. This reduction was higher for glucose, possibly due to reduction in free volume in the composite films in the glassy state. Powder X-ray diffraction showed only diffraction peaks associated to the triple helices of the gelatin, which were reduced significantly upon adding glucose and sucrose. Differential scanning calorimetry data showed reductions in Tg and Tm but higher ΔCp values upon increasing oligosaccharides weight fraction, indicating changes in molecular dynamics. Fragility values indicated strong materials; more likely following an Arrhenius-like dependency between the structure relaxation time and temperature. This work provides valuable information about the nature of the interaction between a non-mammalian gelatin and oligosaccharides with different molecular weight. Our findings could be applied for a number of technological applications including encapsulation.

Highly purified fucosylated chondroitin sulfate oligomers with selective intrinsic factor Xase complex inhibition

Fucosylated chondroitin sulfate (FCS) oligosaccharides of specific molecular weight have shown potent anticoagulant activities with selectivity towards intrinsic factor Xase complex. However, the preparation of FCS oligosaccharides by traditional methods requires multiple purification steps consuming large amounts of time and significant resources. The current study focuses on developing a method for the rapid preparation of FCS oligomers from sea cucumber Pearsonothuria graeffei having 6–18 saccharide residues. The key steps controlling molecular weight (Mw) and purity of these FCS oligomers were evaluated. Structural analysis showed the resulting FCS oligomers were primarily l-Fuc3,4diS-α1,3-d-GlcA-β1,3-(d-GalNAc4,6diS-β1,4-[l-Fuc3,4diS-α1,3-]d-GlcA-β1,3-)nd-anTal-ol4,6diS (n = 1˜5) accompanied by partial de-fucosylation and/or de-sulfation. In vitro and in vivo experiments demonstrate that these FCS oligomers selectively inhibit intrinsic factor Xase complex and exhibit remarkable antithrombotic activity without hemorrhagic and hypotension side effects. This method is suitable for large-scale preparation of FCS oligosaccharides as clinical anticoagulants.

Enzymatic treatments as alternative to produce chitin fragments of low molecular weight from Alternaria alternata

ABSTRACTChitin and its oligosaccharides are involved in the plant defense response against fungal pathogens. In most studies, these molecules come from crustacean shell, and there are scarce studies on the use of fungal chitin. Usually the extraction of chitin is by alkaline treatments, which affect the acetylation degree (DA), and the obtaining of oligosaccharides of low molecular weight; so the use of enzymes is proposed as an alternative treatment to obtain chitin oligosaccharides from fungi. The objective of this work was to characterize the chitin fragments of Alternaria alternata extracted by alkaline and enzymatic treatments. The chitin extracted was ultrasonicated and ultrafiltrated to produce chitin fragments. Enzymatic treatments decreased the protein content to 29%, and the bands corresponding to β‐glucans decreased when β‐1,3‐glucanase was applied. Fragments smaller than 1 kDa, DA of 76.7%, and 35.4 μg glucosamine/mg dry weight were obtained by enzymatic treatments. The enzymatic treatments show promising results to extract chitin fragments from A. alternata without severely affect the DA of the molecule. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 47339.

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

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

The alginate lyase from Isoptericola halotolerans CGMCC 5336 as a new tool for the production of alginate oligosaccharides with guluronic acid as reducing end

Alginate oligosaccharides are depolymerization products of alginate by enzymatic degradation or physicochemical treatments. Alginate lyase has received great interests due to its use in oligosaccharide preparation. The substrate specificity of alginate lyase directly determines the type and degree of polymerization of alginate oligosaccharides. In this paper, the degradation products of Alginate lyase from Isoptericola halotolerans CGMCC 5336 were size-fractionated by gel filtration chromatography and the fractions were characterized by TLC, HPLC, MS and 1H NMR spectroscopy. The Mw values for these fractions were 352, 528, 704 Da which represented di-, tri-, and tetrasaccharide fragments, respectively. The 1H NMR revealed that the non-reducing ends of these oligosaccharides were 4-deoxy-L-erythro-hex-4-enepyranosyluronate and the reducing ends of these oligosaccharides were all guluronic acid. Therefore, it is believed that the alginate lyase produced by Isoptericola halotolerans CGMCC 5336 was capable of performing β elimination on guluronic acid residue so that low molecular weight oligosaccharides with guluronic acid on the reducing end could be obtained in this way.

Structural, functional, and biological properties of potato peel oligosaccharides

Enzymatic hydrolysis of water-soluble polysaccharides from potato peel waste (PPPW) generates low molecular weight oligosaccharides with a yield of 63%. The oligosaccharides generated from potato peel polysaccharides (OPPP) were purified by Superdex-30 column. The results showed the presence of 8 peaks (OPPP1–OPPP8). The identification of all the fractions by chromatography analysis (GC-FID) illustrated that the most prominent residues were glucose with traces of galactose, arabinose and rhamnose. Finally, mass spectrometry (MALDI-ToF-ToF) analysis showed that the generated oligosaccharides were heterogeneous and contained different degree of polymerization (DP). Indeed, the obtained oligosaccharides fractions OPPP3, OPPP4, OPPP5, OPPP6 and OPPP7 were compose of the following degree of polymerization DP5; DP4; DP2; DP1 and DP1, respectively. Potato peel oligosaccharides (OPPP) efficiency were tested using different concentrations in functional properties. The results showed good foaming and emulsion properties. This study also aimed to investigate the antioxidant activity of OPPP. The items explored included the DPPH radical-scavenging capacity (IC50 OPPP=2.5mg/mL), reducing power (OD: 0.622±0.032 at a concentration of 20mg/mL), β-carotene bleaching inhibition activity (45.335±3.653%), and also the ABTS radical scavenging activity (14.835±0.1%).These findings indicate that potato peel oligosaccharides have potent antioxidant activities. Hence, one can suggest that these oligosaccharides might contribute as additives in food, pharmaceutical and cosmetic preparations.

Carrageenans and carrageenases: versatile polysaccharides and promising marine enzymes

Carrageenans are sulfated polysaccharides isolated from marine red algae that share a common backbone of D-galactose alternately linked by α(1,3) and β(1,4) glycosidic linkages. They are classified based on the number and the position of the sulfate ester groups and the occurrence of a 3,6 anhydro-ring in the α-linked galactose. Accordingly the three most commercially exploited carrageenans are κ-, ι-, and λ-carrageenans. Because of their biocompatibility, exceptional physicochemical features and emulsifying, thickening, gelling and stabilizing abilities, they have found several industrial application, especially in food, pharmaceutical and cosmetic industries. Moreover, carrageenans can be degraded into lower molecular weight oligosaccharides, which have been reported to have promising pharmacological properties and potential therapeutic applications. Enzymes which degrade carrageenans are called carrageenases and are produced only by marine bacterial species. These enzymes all are endohydrolases that hydrolyze the internal β 1,4 linkages in carrageenans and produce a series of homologous even-numbered oligosaccharides with various biological and physiological activities including anti-tumor, anti-inflammation, anti-viral, anti-coagulation, etc. Carrageenase enzymes have also other applications related to the biomedical field, bioethanol production, prevention of red algal bloom, obtaining algal protoplasts, etc. In the first part of this review, general information regarding structure, physicochemical properties, biological activities and potential applications of carrageenans is summarized. The second part deals with research and development works on some aspects of carrageenase enzymes like the source, characterization, the kinetics and biochemical properties and their applications in various industries.

Partial Purification and Antioxidant Activities of Oligosaccharides from Hericium caput-medusae (Agaricomycetes).

Fermentation concentrate of Hericium caput-medusae (HFC) was used in this study and two different molecular weight oligosaccharides (HFCO1, HFCO2) were obtained by dialysis methods. Furthermore, DEAE Sepharose Fast Flow and Sephadex G-25 column chromatography were used to obtain the two purified components (HFCO1-1, HFCO2-1). The two components were partially characterized, and analysis of monosaccharide composition through Fourier-transform infrared spectroscopy was performed. The sugar-containing components in HFC were mainly neutral oligosaccharides, and the monosaccharide composition was mainly glucose, with the configuration of α-pyranose. Their chemical and cellular antioxidant activities were compared, and the results showed that antioxidant activities of HFCO2 were higher than HFCO2-1 and antioxidant activities of crude oligosaccharides were higher than those of neutral oligosaccharides. By both in vitro and in vivo studies of scavenging activities toward the free radicals and increasing the activity of antioxidant enzymes in cells, HFCO2 and HFCO2-1 could suppress H2O2-induced reduction in cell viability.

Prebiotic Oligosaccharides: Special Focus on Fructooligosaccharides, Its Biosynthesis and Bioactivity.

The bacterial groups in the gut ecosystem play key role in the maintenance of host's metabolic and structural functionality. The gut microbiota enhances digestion processing, helps in digestion of complex substances, synthesizes beneficial bioactive compounds, enhances bioavailability of minerals, impedes growth of pathogenic microbes, and prevents various diseases. It is, therefore, desirable to have an adequate intake of prebiotic biomolecules, which promote favorable modulation of intestinal microflora. Prebiotics are non-digestible and chemically stable structures that significantly enhance growth and functionality of gut microflora. The non-digestible carbohydrate, mainly oligosaccharides, covers a major part of total available prebiotics as dietary additives. The review describes the types of prebiotic low molecular weight carbohydrates, i.e., oligosaccharides, their structure, biosynthesis, functionality, and applications, with a special focus given to fructooligosaccharides (FOSs). The review provides an update on enzymes executing hydrolytic and fructosyltransferase activities producing prebiotic FOS biomolecules, and future perspectives.

Thermostable multifunctional GH74 xyloglucanase from Myceliophthora thermophila: high-level expression in Pichia pastoris and characterization of the recombinant protein.

A xyloglucanase of the GH74 family was identified in the thermophilic fungus strain Myceliophthora thermophila VKPM F-244, and its gene sequence was optimized for cloning and expression in Pichia pastoris. The recombinant xyloglucanase MtXgh74 exhibited the highest activity toward tamarind seed xyloglucan with a K M value of 0.51±0.06mg/mL. The activities on barley β-glucan and carboxymethylcellulose were about 4 and 2%, respectively, compared to xyloglucan. Maximum xyloglucanase activity was observed at 70-75°C and pH6.5. After pre-incubation at 50°C, pH6.0 for 3h, the enzyme retained 100% of its activity. The half-life of MtXgh74 at 60°C, pH6.0 was 40min. In P. pastoris, MtXgh74 was produced in glycosylated form. The enzyme production in a 1 L bioreactor resulted in a yield of 118U/mL or 5.3g/L after 51 h fermentation. Kinetic studies of the hydrolysis product formation suggest that MtXgh74 has an endo-processive mode of action. The final products were the standard xyloglucan building blocks XXXG, XXLG, XLXG, and XLLG. Additionally, MtXgh74 hydrolyzed various linkages within the xyloglucan building blocks XXXG, XXLG, and XLXG (except XLLG) producing diverse low molecular weight oligosaccharides which may be identified by MALDI-TOF as XG, XX, XXG/GXX/XGX, XXX, LG, LX/XL, XLX/XXL, LLG, GXXXG, GXLLG, XLLGX. The unique combination of different activities within one enzyme along with its high thermostability and specificity toward xyloglucan makes MtXgh74 a promising candidate enzyme for industrial applications.

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

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

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

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

Production of sulfated oligosaccharides from the seaweed Ulva sp. using a new ulvan-degrading enzymatic bacterial crude extract

Green macroalgae of the genus Ulva have complex and hardly degradable polysaccharidic extracellular matrix. One of its main components, the ulvan, is a 3-sulfated rhamnoglucuronan that has a wide range of properties and is a source of rare sugars. The production of mono- and oligo-saccharides from this polysaccharide could motivate its use to an industrial scale. Enzymatic tools to realize this process are still scarce. Here we describe the activity of an enzyme crude extract proceeding from a new Alteromonas species isolated from the gut of Gammarus insensibilis, an amphipod from southern Spanish salt marshes. HPTLC separation and NMR spectrometry allowed describing the formation of low molecular weight oligosaccharides formed principally of unsaturated 3-sulfated rhamnose and uronic acids. The control of hydrolysis kinetics allowed preparing different molecular weight fractions down to a 5kDa oligosaccharides mix. These results highlight the presence of a potentially new β-lyase produced by these hitherto undescribed bacteria. This work identifies a new source of enzymes with potential application for the production of high added value compounds from Ulva sp.

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

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

Structural data and biological properties of almond gum oligosaccharide: Application to beef meat preservation

Enzymatic hydrolysis of almond gum generates low molecular weight oligosaccharides (OAG) with a yield of 33.5%. The generated oligosaccharides were purified and identified. OAG analyses show that the most prominent residues were galactose and arabinose with traces of xylose, rhamnose, glucose and mannose. The glycosyl linkage positions were analyzed using gas chromatography–mass spectrometry showing a main chain composed of galactose units [→3)-Gal-(1→] branched mainly with arabinose residues [Ara-(1→]. The antioxidant and antimicrobial activities of OAG were investigated. As regards the in vitro antioxidant activities, the OAG showed a high total antioxidant activity (347μg ascorbic acid equivalent/mL), an important DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity (IC50=0.64mg/mL) and a high reducing capacity (RP0.5AU=3.6mg/mL). Furthermore, OAG had a high antimicrobial activity against Salmonella thyphimirium, Bacillus cereus, Actinomycetes sp, Klebsiella pneumoniae, Escherichia coli, Alternaria alternate and Candidat albicans. Finally, OAG efficiency was tested using 0.5%; 0.75% and 1% concentrations in beef meat preservation. Microbial growth and lipid oxidation were monitored during 9 days at 4°C. The results showed significant inhibitions (p<0.05) of lipid oxidation and microbial growth in ground beef meat containing OAG.

MALDI Q-TOF CID MS for diagnostic ion screening of human milk oligosaccharide samples.

Human milk oligosaccharides (HMO) represent the bioactive components of human milk, influencing the infant’s gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1–3 or 1–4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI) quadrupole-time-of-flight (Q-TOF) collision-induced dissociation (CID) as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted.