Anion-exchange Chromatography Analysis Research Articles

Optimization, structural characterization, and biological applications of exopolysaccharide produced by Enterococcus faecium KT990028

The aim of this study was to select the best exopolysaccharide (EPS) producer among the Enterococcus strains to optimize, characterize, and evaluate its biological properties. Among the eleven strains, Enterococcus faecium KT990028 was selected, and the production conditions were optimized: 16.3 % (w/v) sucrose, 0.70 % (w/v) yeast extract, 8.3 % (w/v) reconstituted skimmed milk, at 38 °C in 15 h of incubation, producing 2.880 g/L of EPS. High performance anion exchange chromatography (HPAEC) analysis revealed that the molecular weight was 166.98 kDa. HPAEC, spectroscopy (FTIR), and nuclear magnetic resonance (1H NMR) analyses revealed that the EPS was a heteropolysaccharide composed of galactose (37.74 %), rhamnose (19.79 %), arabinose (17.71 %), glucose (9.50 %), fucose (7.93 %), and mannose (7.33 %). Scanning electron microscopy showed a three-dimensional microstructure in the form of decompressed plates, with wrinkles, and pores. By means of dynamic light scattering (DLS), the EPS showed an average size varying from 135.25 ± 10.56 nm and 410.60 ± 45.20 nm, as the concentration was increased from 0.5 mg/mL to 2.0 mg/mL, respectively. X-ray diffraction revealed that the EPS has an amorphous and crystalline nature, while thermogravimetric analysis indicated stability up to 400 °C. The antioxidant effect (5 mg/mL) against DPPH, ABTS, OH, and O2 was 64.50 ± 0.71 %, 47.50 ± 0.10 %, 68.36 ± 0.59 %, and 44.83 ± 0.86 %, respectively. It was also able to inhibit and biofilm disruption of Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 6057 and had an antimicrobial effect from 50 mg/mL for the strains of against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Listeria monocytogenes ATCC 19117, Staphylococcus aureus ATCC 6538, and Enterococcus faecalis ATCC 6057. Cell cytotoxicity carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the EPS was safe and promoted the proliferation of Vero cells. Thus, the results indicated that the EPS from E. faecium KT990028 is a promising functional biopolymer for possible applications in the food and pharmaceutical fields.

Structural, molecular, and physicochemical properties of starch in high-amylose durum wheat lines

There is growing interest in the development of high-amylose cereals such as wheat due to their functional properties and nutritional value in foods. The increase in amylose content is associated with significant changes in the physicochemical, molecular, and structural characteristics of wheat starch which could affect its processing quality. The present study aimed to analyze the physicochemical, molecular, and structural characteristics of high-amylose starch from three durum wheat near isogenic lines (NILs), each obtained using three different recurrent parents. Morphology, granule size distribution, pasting and thermal properties, X-ray diffraction, and structural features were determined. The NILs showed the highest amylose content (65.3 and 69.8%), granules with an elongated shape, size between the wild-type and the mutant, restricted swelling in the pasting profile, the highest average and final gelatinization temperature, and B-polymorphism. The starches from the NILs showed the highest molar mass of amylopectin (AP) (7.0–7.6 × 107 Da) and the lowest amylose (AM) (1.1–1.8 × 106 Da), an issue associated with the biosynthesis of both components. The study on the debranched sample by high-performance size-exclusion chromatography showed that the high-amylose starch had the highest content of long B2 and B3 chains of AP (23.9–26.6%), which was corroborated with the high-performance anion-exchange chromatography analysis where the chains with degree of polymerization >37 had the highest content (18.3–19.1%). The PC1 is highly associated with the MwAM and the AM fraction in the debranched starch analyzed by HPSEC. Characterizing high-amylose durum wheat starch provides information to determine the structure-function relationship and design strategies to produce crosses with structural characteristics of AM and AP for specific applications.

Distinct chemical degradation pathways of AAV1 and AAV8 under thermal stress conditions revealed by analytical anion exchange chromatography and LC-MS-based peptide mapping

Adeno-associated virus (AAV)-based gene therapy is experiencing a rapid growth in the field of medicine and holds great promise in combating a wide range of human diseases. For successful development of AAV-based products, comprehensive thermal stability studies are often required to establish storage conditions and shelf life. However, as a relatively new modality, limited studies have been reported to elucidate the chemical degradation pathways of AAV products under thermal stress conditions. In this study, we first presented an intriguing difference in charge profile shift between thermally stressed AAV8 and AAV1 capsids when analyzed by anion exchange chromatography. Subsequently, a novel and robust peptide mapping protocol was developed and applied to elucidate the underlying chemical degradation pathways of thermally stressed AAV8 and AAV1. Compared to the conventional therapeutic proteins, the unique structure of AAV capsids also led to some key differences in how modifications at specific sites may impact the overall charge properties. Finally, despite the high sequency identity, the analysis revealed that the opposite charge profile shifts between thermally stressed AAV8 and AAV1 could be mainly attributed to a single modification unique to each serotype.

Characterization of Histophilus somni sialic acid uptake mutant (ΔnanP-ΔnanU) using a mouse septicemia and mortality model

Histophilus somni is an important pathogen of the bovine respiratory disease complex, yet the mechanisms underlying its virulence remain poorly understood. It is known that H. somni can incorporate sialic acid into lipooligosaccharide (LOS), and sialylated H. somni is more resistant to phagocytosis and complement-mediated killing by serum compared to non-sialylated bacteria in vitro. However, the virulence of non-sialylated H. somni has not been evaluated in vivo using an animal model. In this study, we investigated the contribution of sialic acid to virulence by constructing an H. somni sialic acid uptake mutant (ΔnanP-ΔnanU) and comparing the parent and mutant strains in a mouse septicemia and mortality model. Intraperitoneal challenge of mice with wildtype H. somni (1 × 108 colony forming units/mouse, CFU) was lethal to all animals. Mice challenged with three different doses (1, 2, or 5 × 108 CFU/mouse) of an H. somni ΔnanP-ΔnanU sialic acid uptake mutant exhibited survival rates of 90 %, 60 %, and 0 % respectively. High-performance anion exchange chromatography analyses revealed that LOS prepared from both parent and the ΔnanP-ΔnanU mutant strains of H. somni were sialylated. These findings suggest the presence of de novo sialic acid synthesis pathway, although the genes associated with de novo sialic acid synthesis (neuB and neuC) were not identified by genomic analysis. The lower attenuation in mice is most likely attributed to the sialylated LOS of H. somni nanPU mutant.

Enhanced phagocytosis and complement-mediated killing of Mannheimia haemolytica serotype 1 following in-frame CMP-sialic acid synthetase (neuA) gene deletion.

Mannheimia haemolytica is the most significant bacterial pathogen associated with the bovine respiratory disease complex. Although sialic acid is a known virulence factor in other members of Pasteurellaceae, such as Histophilus somni and Pasteurella multocida, the significance of sialic acid to the virulence of M. haemolytica is currently unknown. Therefore, the role of sialic acid as a virulence determinant of M. haemolytica was investigated by constructing an in-frame neuA [CMP-N-acetylneuraminic acid (Neu5Ac/sialic) synthetase] mutant, which was shown by high-performance anion exchange chromatographic analysis (HPAEC) to be devoid of sialic acid on the lipopolysaccharide (LPS). Both the neuA mutant and wild-type parent strains exhibited similar growth rates in the growth curve assay. Real-time qPCR and ELISA evaluation showed no differences in proinflammatory cytokine expressions (IL-1β, IL-6, and IL-8) between the neuA mutant and parent strain when peripheral blood mononuclear cells were incubated with LPS. Interestingly, the neuA mutant was three to four logs more sensitive to a whole-blood bacterial killing assay than the parent strain. Similar results were also observed in plasma and serum bacterial killing assays. Flow cytometry analyses showed higher uptake of neuA mutant by phagocytes, compared to the parent strain, in the whole-blood phagocytosis assay; however, no difference in reactive oxygen species production in neutrophils or monocytes was detected for either strain. Taken together, these results indicate that sialylation of M. haemolytica LPS plays a vital role in reducing complement-mediated and phagocytic killing. IMPORTANCE The Gram-negative coccobacillus Mannheimia haemolytica is a natural inhabitant of the upper respiratory tract in ruminants and the most common bacterial agent involved in bovine respiratory disease complex development. Key virulence factors harbored by M. haemolytica are leukotoxin, lipopolysaccharide, capsule, adhesins, and neuraminidase which are involved in evading innate and adaptive immune responses. In this study, we have shown that CMP-sialic acid synthetase (neuA) is necessary for the incorporation of sialic acid onto the membrane, and inactivation of neuA results in increased phagocytosis and complement-mediated killing of M. haemolytica, thus demonstrating that sialylation contributes to the virulence of M. haemolytica.

Adsorption of apple xyloglucan on cellulose nanofiber depends on molecular weight, concentration and building blocks

Apple xyloglucan (XyG) structural features (molecular weight and building motifs) were studied in relation with the hemicellulose interaction with cellulose nanofiber (CNF) through adsorption isotherm, high performance size exclusion chromatography and high-performance anion exchange chromatography analyses. Up to 75.6 μg of apple XyG adsorbed/mg of CNF. XyG adsorption depended on both Mw and XyG/CNF ratio. The adsorption capacity increased with Mw irrespectively of XyG/CNF ratio. At XyG/CNF ratio > 0.05, the adsorption of high Mw populations (2.0 × 105, 7.9 × 104 g mol−1) gradually ceased, while it kept increasing for low Mw populations (2.6 × 104, 1.2 × 104 g mol−1). The unbranched glucan segments and the galactosylated XLLG motif showed the highest binding affinity to CNF. Most of the XLLG motif was found in “loops and tails”, whereas most of the XyG unbranched glucan segments interact directly with CNF.

Enzymatic Conversion of Different Qualities of Refined Softwood Hemicellulose Recovered from Spent Sulfite Liquor.

Spent sulfite liquor (SSL) from softwood processing is rich in hemicellulose (acetyl galactoglucomannan, AcGGM), lignin, and lignin-derived compounds. We investigated the effect of sequential AcGGM purification on the enzymatic bioconversion of AcGGM. SSL was processed through three consecutive purification steps (membrane filtration, precipitation, and adsorption) to obtain AcGGM with increasing purity. Significant reduction (~99%) in lignin content and modest loss (~18%) of polysaccharides was observed during purification from the least pure preparation (UFR), obtained by membrane filtration, compared to the purest preparation (AD), obtained by adsorption. AcGGM (~14.5 kDa) was the major polysaccharide in the preparations; its enzymatic hydrolysis was assessed by reducing sugar and high-performance anion-exchange chromatography analysis. The hydrolysis of the UFR preparation with Viscozyme L or Trichoderma reesei β-mannanase TrMan5A (1 mg/mL) resulted in less than ~50% bioconversion of AcGGM. The AcGGM in the AD preparation was hydrolyzed to a higher degree (~67% with TrMan5A and 80% with Viscozyme L) and showed the highest conversion rate. This indicates that SSL contains enzyme-inhibitory compounds (e.g., lignin and lignin-derived compounds such as lignosulfonates) which were successfully removed.

Maltogenic α-amylase hydrolysis of wheat starch granules: Mechanism and relation to starch retrogradation

Enzymatic modification is an effective method to inhibit starch retrogradation. However, lack of quantification of relationships between enzymatic modification and starch retrogradation makes the enzymatic improvement unpredictable. In this study, maltogenic α-amylase (MA) was used to treat wheat starch granules to restrain retrogradation, aiming to elucidate the mechanism of MA hydrolysis on wheat starch granules and to establish a quantitative relationship between the degree of hydrolysis (DH) and retrogradation. Scanning electron microscopy and small angle X-ray scattering results showed that MA hydrolyzed starch granules by a “surface pitting” mode simultaneously acting on crystalline and amorphous regions. Debranching and high performance anion exchange chromatography analysis of MA-treated wheat starch granules demonstrated that the amount of short branches with degree of polymerization＜9 increased and the proportion of medium and long branches decreased. Importantly, the extent of impaired short- and long-term retrogradation of MA-treated starch was clearly linearly correlated with the DH. This finding provides a quantitative method for predicting the degree of retrogradation improvement by enzymatic modification.

Influence of physicochemical properties and starch fine structure on the eating quality of hybrid rice with similar apparent amylose content

In this study, we compared the physicochemical properties and starch structures of hybrid rice varieties with similar apparent amylose content but different taste values. In addition to the apparent amylose content, gel permeation chromatography analysis showed that the higher proportions of amylopectin short chains and relatively lower proportions of amylopectin long chains, which could lead to higher peak viscosity and breakdown value, as well as a softer and stickier texture of cooked rice, were the key factors in determining the eating quality of hybrid rice. High-performance anion-exchange chromatography analyses showed that the proportion of amylopectin short chains (degree of polymerization 6–10) and intermediate chains (degree of polymerization 13–24), which might affect the gelatinisation enthalpy and crystallinity, also contributed greatly to the eating quality of hybrid rice. Moreover, this study indicated that a greater diversity of forms and sizes of starch granules might influence the eating quality of hybrid rice.

Improving cellulosic ethanol production using ozonolysis and acid as a sugarcane biomass pretreatment in mild conditions

The efficiency of unpressurized ozonolysis (O) and acid washing (A) was investigated as a simple pretreatment procedure to depolymerize sugarcane biomass for posterior enzymatic hydrolysis and alcoholic fermentation to improve cellulosic ethanol production. The fiber chemical compositional analyses showed that the ozonolysis reduces the lignin content by 25%. High-performance anion-exchange chromatography (HPAEC-PAD) analysis showed that the loss of glucose during treatment was less than 1.5%. After enzymatic hydrolysis, the maximum glucose and xylose yield (310 ± 2 and 122 ± 3 mg g−1, respectively) were achieved from the OA treatment sequence and the fermentation yield was three times more ethanol than the yield from untreated bagasse. This simple treatment may be an alternative for improving cellulosic ethanol production using a mild-operational strategy.

Sialylated O -Glycans from Hen Egg White Ovomucin are Decomposed by Mucin-degrading Gut Microbes.

Ovomucin, a hen egg white protein, is characterized by its hydrogel-forming properties, high molecular weight, and extensive O -glycosylation with a high degree of sialylation. As a commonly used food ingredient, we explored whether ovomucin has an effect on the gut microbiota. O- Glycan analysis revealed that ovomucin contained core-1 and 2 structures with heavy modification by N -acetylneuraminic acid and/or sulfate groups. Of the two mucin-degrading gut microbes we tested, Akkermansia muciniphila grew in medium containing ovomucin as a sole carbon source during a 24 h culture period, whereas Bifidobacterium bifidum did not. Both gut microbes, however, degraded ovomucin O -glycans and released monosaccharides into the culture supernatants in a species-dependent manner, as revealed by semi-quantified mass spectrometric analysis and anion exchange chromatography analysis. Our data suggest that ovomucin potentially affects the gut microbiota through O -glycan decomposition by gut microbes and degradant sugar sharing within the community.

A GH51 \u03b1-l-arabinofuranosidase from Talaromyces leycettanus strain JCM12802 that selectively drives synergistic lignocellulose hydrolysis

BackgroundThe development of sustainable technologies for plant cell wall degradation greatly depends on enzymes with hydrolytic activities against carbohydrates. The waste by-products of agricultural cereals are important biomass sources because they contain large amounts of saccharides. Achieving efficient debranching and depolymerization are two important objectives for increasing the utilization of such renewable bioresources. GH51 α-l-arabinofuranosidases are important in biomass pretreatment because they act synergistically with other enzymes during hemicellulose hydrolysis.ResultsA GH51 α-l-arabinofuranosidase from Talaromyces leycettanus JCM12802 was heterologously expressed in Pichia pastoris GS115 and characterized. The recombinant α-l-arabinofuranosidase, TlAbf51, showed an optimum temperature and pH of 55–60 °C and 3.5–4.0, respectively, and remained stable at 50 °C and pH 3.0–9.0. TlAbf51 showed a higher catalytic efficiency (5712 mM−1 s−1) than most fungal α-l-arabinofuranosidases towards the substrate 4-nitrophenyl-α-l-arabinofuranoside. Moreover, TlAbf51 preferentially removed 1,2- or 1,3-linked arabinofuranose residues from arabinoxylan and acted synergistically with the bifunctional xylanase/cellulase TcXyn10A at an activity ratio of 5:1. The highest yields of arabinose and xylooligosaccharides were obtained when TlAbf51 was added after TcXyn10A or when both enzymes were added simultaneously. High-performance anion-exchange chromatography analyses showed that (i) arabinose and xylooligosaccharides with low degrees of polymerization (DP1–DP5) and (ii) arabinose and xylooligosaccharides (DP1–DP3) were the major hydrolysates obtained during the hydrolysis of sodium hydroxide-pretreated cornstalk and corn bran, respectively.ConclusionsIn contrast to other fungal GH51 α-l-arabinofuranosidases, recombinant TlAbf51 showed excellent stability over a broad pH range and high catalytic efficiency. Moreover, TlAbf51 acted synergistically with another hemicellulase to digest arabino-polysaccharides. These favorable enzymatic properties make TlAbf51 attractive for biomass pretreatment and biofuel production.

Chemical structure and snake antivenom properties of sulfated agarans obtained from Laurencia dendroidea (Ceramiales, Rhodophyta)

Aqueous and KCl-soluble polysaccharides were extracted from Laurencia dendroidea (Rhodomelaceae, Ceramiales) and their chemical profile was accessed by anion-exchange chromatography, chemical and spectroscopic analyses. The homogeneous agaran DHS-4 (181.3 × 103 g. mol−1, 21.3% of NaSO3) presents A units mostly 2-sulfated (18.9 mol%), nonsubstituted (15.3 mol%) and 6-O-methylated (10.1 mol%), while B units are l-sugars composed predominantly by galactose 6-sulfate precursor units (19.2 mol%) and 3,6-anhydrogalactose (13.8 mol%), besides non-precursor galactose 6-sulfate units bearing d-xylose substituents on C-3 (8.1 mol%). The crude KCl-soluble DHS agaran (20.5% of NaSO3) inhibited proteolysis and hemolysis induced by Lachesis muta and Bothrops jararaca venoms. DHS was able to inhibit up to 75% the L. muta venom hemorrhagic effect and to reduce the lethality displayed by B. jararaca venom, increasing the mice survival time up to 3 times. Therefore, this agaran has high potential to be used as an additional tool to treat snakebite envenomation.

Structural characteristics and functional properties of soluble dietary fiber from defatted rice bran obtained through Trichoderma viride fermentation

Trichoderma viride was utilized to ferment the defatted rice bran (DRB), and soluble dietary fiber was prepared before and after fermentation, named as B-SDF and F-SDF, respectively. Optimum fermentation conditions for yield of F-SDF were determined using response surface methodology (RSM). The yield of F-SDF was significantly increased from 10.5 (w/w, %) to 33.4 (w/w, %) under the optimal conditions (inoculum size = 10%, pH = 5.8 and time = 41 h). Fourier transform infrared spectroscopy (FTIR) demonstrated that both B-SDF and F-SDF had typical characteristics of the infrared spectrum of polysaccharides, and X-ray diffraction (XRD) revealed they were mainly substances of amorphous structure. In addition, F-SDF showed looser structure by scanning electron microscopy (SEM) analysis, increased molecular weight by high-performance gel permeation chromatography (HPGPC) analysis, and more complex monosaccharide composition by high-performance anion exchange chromatography (HPAEC) analysis. Furthermore, functional properties analysis showed that F-SDF exhibited higher water-holding capacity (WHC), oil-holding capacity (OHC), water solubility (WS) and cholesterol absorption capacity (CAC) than B-SDF. In summary, the present study suggested that SDF from defatted rice bran obtained through Trichoderma viride fermentation has a higher potential to be used as a functional ingredient in foods.

Structural characterization of alkali-soluble polysaccharides from Panax ginseng C. A. Meyer.

Panax ginseng C. A. Meyer (ginseng) has been widely used as a herb and functional food in the world. Polysaccharides are the main active components of ginseng. In this paper, the polysaccharides were sequentially extracted by 50 mM Na2CO3, 1 M KOH and 4 M KOH from ginseng roots treated sequentially with hot water, α-amylase and ethylenediaminetetraacetic acid extraction. Na2CO3-soluble ginseng polysaccharide (NGP) was fractionated into one neutral and three acidic fractions by anion exchange and gel permeation chromatography. Fourier transform infrared, NMR and methylation analysis indicated acidic fractions in NGP were highly branched rhamnogalacturonan-I domains, with → 4)-α-GalpA-(1 → 2)-α-Rhap-(1 → disaccharide repeating units as backbone and β-1,4-galactan, α-1,5/1,3,5-arabinan and type II arabinogalactan as side chains. 1-KGP (1 M KOH-soluble ginseng polysaccharide) and 4-KGP (4 M KOH-soluble ginseng polysaccharide) were mainly composed of hemicellulose besides starch-like polysaccharides and minor pectin. Antibody detection, enzymic hydrolysis, high performance anion exchange chromatography and methylation analysis demonstrated xylan was the major component in 1-KGP, while xyloglucan was predominant in 4-KGP. Comparing the polysaccharides obtained by different solvent extractions, we have a comprehensive understanding about total ginseng polysaccharides.

Direct analysis of PI(3,4,5)P3 using liquid chromatography electrospray ionization tandem mass spectrometry

Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a biologically active membrane phospholipid that is essential for the growth and survival of all eukaryotic cells. We describe a new method that directly measures PIP3 and describe the HPLC separation and measurement of the positional isomers of phosphatidylinositol bisphosphate, PI(3,5)P2, PI(3,4)P2 and PI(4,5)P2. Mass spectrometric analyses were performed online using ultra-high performance liquid chromatography (UHPLC)-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the negative multiple-reaction monitoring (MRM) modes. Rapid separation of PIP3 from PI, phosphatidylinositol phosphate (PIP) and PIP2 was accomplished by C18 reverse phase chromatography with the addition of the ion pairing reagents diisopropylethanolamine (DiiPEA) and ethylenediamine tetraacetic acid tetrasodium salt dihydrate (EDTA) to the samples and mobile phase with a total run time, including equilibration, of 12 min. Importantly, these chromatography conditions result in no carryover of PIP, PIP2, and PIP3 between samples.To validate the new method, U87MG cancer cells were serum starved and treated with PDGF to stimulate PIP3 biosynthesis in the presence or absence of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Results generated with the LC/MS method were in excellent agreement with results generated using [33P] phosphate radiolabeled U87MG cells and anion exchange chromatography analysis, a well validated method for measuring PIP3. To demonstrate the usefulness of the new method, we generated reproducible IC50 data for several well-characterized PI3K small molecule inhibitors using a U87MG cell-based assay as well as showing PIP3 can be measured from additional cancer cell lines. Together, our results demonstrate this novel method is sensitive, reproducible and can be used to directly measure PIP3 without radiolabeling or complex lipid derivatization.

Disentangling pectic homogalacturonan and rhamnogalacturonan-I polysaccharides: Evidence for sub-populations in fruit parenchyma systems

The matrix polysaccharides of plant cell walls are diverse and variable sets of polymers influencing cell wall, tissue and organ properties. Focusing on the relatively simple parenchyma tissues of four fruits – tomato, aubergine, strawberry and apple – we have dissected cell wall matrix polysaccharide contents using sequential solubilisation and antibody-based approaches with a focus on pectic homogalacturonan (HG) and rhamnogalacturonan-I (RG-I). Epitope detection in association with anion-exchange chromatography analysis indicates that in all cases solubilized polymers include spectra of HG molecules with unesterified regions that are separable from methylesterified HG domains. In highly soluble fractions, RG-I domains exist in both HG-associated and non-HG-associated forms. Soluble xyloglucan and pectin-associated xyloglucan components were detected in all fruits. Aubergine glycans contain abundant heteroxylan epitopes, some of which are associated with both pectin and xyloglucan. These profiles of polysaccharide heterogeneity provide a basis for future studies of more complex cell and tissue systems.

Structural characterisation of galacto-oligosaccharides (VITAGOS™) sythesized by transgalactosylation of lactose

A galacto-oligosaccharide product (VITAGOS™) was prepared by glycoside hydrolases (β-galactosidase from Aspergillus oryzae and Kluyveromyces lactis) using lactose as substrate. Monosaccharide composition analysis indicated that VITAGOS™ contained 40.3% (w/w, dry basis) glucose and 51.1% (w/w, dry basis) galactose. According to high performance size exclusion chromatography (HPSEC) analysis, VITAGOS™ was a sugar mixture with degree of polymerisation (DP) ranging from 1 to 7, with DP2 and DP3 as the dominant fractions (32% and 33%, w/w). In addition, more than three fractions were observed for each DP pool indicated by the oligosaccharides profiles obtained from high performance anion exchange chromatography (HPAEC) analysis. Six sub-fractions (F1-F6, corresponding to DP1 to DP6 rich fraction, respectively) were then collected using biogel P-2 column, followed by structural characterisation through methylation analysis combined with 1D & 2D NMR spectroscopy. The detailed molecular structures of VITAGOS™ with DP up to 4 are provided.

A glycoproteomic approach reveals that the S-layer glycoprotein of Lactobacillus kefiri CIDCA 83111 is O- and N-glycosylated

In Gram-positive bacteria, such as lactic acid bacteria, general glycosylation systems have not been documented so far. The aim of this work was to characterize in detail the glycosylation of the S-layer protein of Lactobacillus kefiri CIDCA 83111. A reductive β-elimination treatment followed by anion exchange high performance liquid chromatography analysis was useful to characterize the O-glycosidic structures. MALDI-TOF mass spectrometry analysis confirmed the presence of oligosaccharides bearing from 5 to 8 glucose units carrying galacturonic acid. Further nanoHPLC-ESI analysis of the glycopeptides showed two O-glycosylated peptides: the peptide sequence SSASSASSA already identified as a signature glycosylation motif in L. buchneri, substituted on average with eight glucose residues and decorated with galacturonic acid and another O-glycosylated site on peptide 471–476, with a Glc5–8GalA2 structure. As ten characteristic sequons (Asn-X-Ser/Thr) are present in the S-layer amino acid sequence, we performed a PNGase F digestion to release N-linked oligosaccharides. Anion exchange chromatography analysis showed mainly short N-linked chains. NanoHPLC-ESI in the positive and negative ion modes were useful to determine two different peptides substituted with short N-glycan structures. To our knowledge, this is the first description of the structure of N-glycans in S-layer glycoproteins from Lactobacillus species. SignificanceA detailed characterization of protein glycosylation is essential to establish the basis for understanding and investigating its biological role. It is known that S-layer proteins from kefir-isolated L. kefiri strains are involved in the interaction of bacterial cells with yeasts present in kefir grains and are also capable to antagonize the adverse effects of different enteric pathogens. Therefore, characterization of type and site of glycosidic chains in this protein may help to understand these important properties. Furthermore, this is the first description of N-glycosidic chains in S-layer glycoprotein from Lactobacillus spp.

Molecular Structure and Physicochemical Properties of Starches from Rice with Different Amylose Contents Resulting from Modification of OsGBSSI Activity.

OsGBSSI, encoded by the Waxy (Wx) gene, is the key enzyme in the synthesis of amylose chains. Transgenic rice lines with various GBSSI activities were previously developed via site-directed mutagenesis of the Wx gene in the glutinous cultivar Guanglingxiangnuo (GLXN). In this study, grain morphology, molecular structure, and physicochemical properties were investigated in four transgenic lines with modified OsGBSSI activity and differences in amylose content. A milky opaque appearance was observed in low- and non-amylose rice grains due to air spaces in the starch granules. Gel permeation chromatography (GPC) and high-performance anion-exchange chromatography (HPAEC) analyses showed that although OsGBSSI can synthesize intermediate and extra-long amylopectin chains, it is mainly responsible for the longer amylose chains. Amylose content was positively correlated with trough viscosity, final viscosity, setback viscosity, pasting time, pasting temperature, and gelatinization temperature and negatively with gel consistency, breakdown viscosity, gelatinization enthalpy, and crystallinity. Overall, the findings suggest that OsGBSSI may be also involved in amylopectin biosynthesis, in turn affecting grain appearance, thermal and pasting properties, and the crystalline structure of starches in the rice endosperm.