Fermentation of xylo-oligosaccharides by Bifidobacterium adolescentis DSMZ 18350: kinetics, metabolism, and β-xylosidase activities

Xylo-oligosaccharides (XOS) are sugar oligomers of β-1,4-linked xylopyranosyl moieties which exert bifidogenic effect and are increasingly used as prebiotics. The kinetics and the metabolism of Bifidobacterium adolescentis DSMZ 18350 growing on XOS and xylose were investigated. The growth rate was higher on XOS, but greater biomass yield was attained on xylose. Unlike other prebiotics, XOS oligomers were utilized simultaneously, regardless of their chain length. Throughout XOS utilization, xylose concentration slightly increased, being not neatly consumed and remaining unfermented. During growth on XOS, β-xylosidase activity was present in the cytosol, but it occurred in the supernatant as well. A β-1,4-xylolytic enzyme was purified from the supernatant of XOS cultures. The enzyme, a homotetramer of a 39-kDa single protein, was capable of complete XOS hydrolysis and exhibited maximum activity at pH?6.0 and 55 °C. Based on the molecular weight, the protein can be ascribable to the product of the gene BAD_1527, the activity of which has been inferred as an endo-β-1,4-xylanase, but has not been characterized so far. This β-1,4-xylolytic enzyme, found to be active in the cultural supernatant, gives a reason for the never explained accumulation of the monosaccharides in the media of bifidobacterial cultures growing on XOS, without excluding the major role of the intracellular hydrolysis of the imported oligomers.     

Production, Purification, and Characterization of a Xylooligosaccharides-forming Xylanase from High-butanol-producing Strain Clostridium sp. BOH3

An endo-acting xylanase is isolated from the culture medium of Clostridium sp. BOH3 when xylan, glucose, xylose, or sugarcane bagasse hydrolysate (SBH) is used as a carbon source. Crude xylanase is purified by using an anionic Q-column with a yield of 39 %. The pure xylanase has a molecular weight of 35.8 kDa, and it shows optimal activity at pH 5 and 60 °C. When beechwood xylan is used as a substrate, this xylanase liberates short oligosaccharides (XOS) predominantly, ranging from xylobiose (X2) to xylopentaose (X5). However, no xylose can be detected, suggesting that this is an endo-β-1,4-xylanase. Kinetic study of this xylanase reveals that K _m and V _max are 1.36 mg/ml and 212 μmol/(min. mg protein), respectively. On the basis of amino acid sequence, this enzyme shows homology to xylanase (xynb) from Clostridium acetobutylicum ATCC 824, but this enzyme has several distinctive characteristics. For example, its activity can be enhanced with the addition of divalent metal ions, and it produces XOS exclusively when xylan is used as a substrate. These unique characteristics suggest that this is a new enzyme.     

Circulating microparticles in patients with coronary heart disease and its correlation with interleukin-6 and C-reactive protein

Microparticles (MPs) are vesicles released from activated or apoptotic cells. MP derive from various cells, most notably platelets, but also leucocytes, lymphocytes, erythrocytes, and endothelial cells. The aim of this study was to investigate endothelial MP (EMP), platelet MP (PMP), lymphocyte MP and monocyte MP and TF-positive MPs (TF⁺ MPs) in patients with coronary heart disease (CHD), and to evaluate the correlation of these MPs with Interleukin-6 (IL-6) and C-reactive protein (CRP). Different cell-derived MPs and TF⁺ MPs were analyzed by flow cytometry in 40 patients with myocardial infarction (MI), 30 unstable angina (UA), 20 stable angina (SA) and 20 healthy individuals, and IL-6 and CRP were determined by ELISA and special protein analyzer, respectively. Compared with SA and control, EMP and PMP was significantly elevated in MI and UA (P < 0.001), and TF⁺ MPs was significantly elevated in MI and UA (P < 0.001). EMP and PMP correlated with IL-6 (r = 0.822, P < 0.001 and r = 0.567, P < 0.001; respectively) or CRP level (r = 0.597, P < 0.001 and r = 0.66, P < 0.001; respectively). Different cell-derived MPs in CHD may indicate the different pathophysiological changes in vessels, and MPs may both participate in the development of thrombosis and enhance the vascular inflammation.     

Production of xylooligosaccharides in SSF by Bacillus subtilis KCX006 producing β-xylosidase-free endo-xylanase and multiple xylan debranching enzymes

Xylanase and xylooligosaccharides (XOS) are employed in food and feed industries. Though xylanase production from lignocellulosic materials (LCMs) by solid-state fermentation (SSF) is well known, the XOS formed during growth is not recovered due to its conversion to xylose by β-xylosidase and subsequent bacterial metabolism. A new strain, Bacillus subtilis KCX006, was exceptionally found to synthesize β-xylosidase-free endo-xylanase and multiple xylan debranching enzymes constitutively in the presence of LCMs. Absence of β-xylosidase resulted in accumulation of XOS during growth of KCX006 on LCMs. Therefore, this strain was used for simultaneous production of xylanase and XOS from agro-residues in solid-state fermentation (SSF). Partial purification of XOS from culture supernatant using activated charcoal followed by high-performance liquid chromatography (HPLC) analysis showed xylobiose to xylotetraose formed as the major products. Among various LCM substrates, wheat bran and groundnut oil-cake supported highest xylanase and XOS production at 2158 IU/gdw and 24.92 mg/gdw, respectively. The levels of xylanase and XOS were improved by 1.5-fold (3102 IU/gdw) and 1.9-fold (48 mg/gdw), respectively, by optimization of culture conditions.     

Effects of Xylo-Oligosaccharides on Broiler Chicken Performance and Microbiota

In broiler chickens, feed additives, including prebiotics, are widely used to improve gut health and to stimulate performance. Xylo-oligosaccharides (XOS) are hydrolytic degradation products of arabinoxylans that can be fermented by the gut microbiota. In the current study, we aimed to analyze the prebiotic properties of XOS when added to the broiler diet. Administration of XOS to chickens, in addition to a wheat-rye-based diet, significantly improved the feed conversion ratio. XOS significantly increased villus length in the ileum. It also significantly increased numbers of lactobacilli in the colon and Clostridium cluster XIVa in the ceca. Moreover, the number of gene copies encoding the key bacterial enzyme for butyrate production, butyryl-coenzyme A (butyryl-CoA):acetate CoA transferase, was significantly increased in the ceca of chickens administered XOS. In this group of chickens, at the species level, Lactobacillus crispatus and Anaerostipes butyraticus were significantly increased in abundance in the colon and cecum, respectively. In vitro fermentation of XOS revealed cross-feeding between L. crispatus and A. butyraticus. Lactate, produced by L. crispatus during XOS fermentation, was utilized by the butyrate-producing Anaerostipes species. These data show the beneficial effects of XOS on broiler performance when added to the feed, which potentially can be explained by stimulation of butyrate-producing bacteria through cross-feeding of lactate and subsequent effects of butyrate on gastrointestinal function.     

The PMP22 Gene and Its Related Diseases

Peripheral myelin protein-22 (PMP22) is primarily expressed in the compact myelin of the peripheral nervous system. Levels of PMP22 have to be tightly regulated since alterations of PMP22 levels by mutations of the PMP22 gene are responsible for >50 % of all patients with inherited peripheral neuropathies, including Charcot–Marie–Tooth type-1A (CMT1A) with trisomy of PMP22, hereditary neuropathy with liability to pressure palsies (HNPP) with heterozygous deletion of PMP22, and CMT1E with point mutations of PMP22. While overexpression and point-mutations of the PMP22 gene may produce gain-of-function phenotypes, deletion of PMP22 results in a loss-of-function phenotype that reveals the normal physiological functions of the PMP22 protein. In this article, we will review the basic genetics, biochemistry and molecular structure of PMP22, followed by discussion of the current understanding of pathogenic mechanisms involving in the inherited neuropathies with mutations in PMP22 gene.     

Distinct roles of carbohydrate esterase family CE16 acetyl esterases and polymer-acting acetyl xylan esterases in xylan deacetylation

Mass spectrometric analysis was used to compare the roles of two acetyl esterases (AE, carbohydrate esterase family CE16) and three acetyl xylan esterases (AXE, families CE1 and CE5) in deacetylation of natural substrates, neutral (linear) and 4-O-methyl glucuronic acid (MeGlcA) substituted xylooligosaccharides (XOS). AEs were similarly restricted in their action and apparently removed in most cases only one acetyl group from the non-reducing end of XOS, acting as exo-deacetylases. In contrast, AXEs completely deacetylated longer neutral XOS but had difficulties with the shorter ones. Complete deacetylation of neutral XOS was obtained after the combined action of AEs and AXEs. MeGlcA substituents partially restricted the action of both types of esterases and the remaining acidic XOS were mainly substituted with one MeGlcA and one acetyl group, supposedly on the same xylopyranosyl residue. These resisting structures were degraded to great extent only after inclusion of α-glucuronidase, which acted with the esterases in a synergistic manner. When used together with xylan backbone degrading endoxylanase and β-xylosidase, both AE and AXE enhanced the hydrolysis of complex XOS equally.     

高效液相色谱法同时测定保健食品中低聚果糖和低聚木糖

目的 建立高效液相色谱法同时测定保健食品中低聚果糖(包括蔗果三糖、蔗果四糖及蔗果五糖)和低聚木糖（包括木二糖、木三糖、木四糖、木五糖、木六糖及木七糖）的含量。方法 采用键合有酰胺官能团杂化填料的色谱柱,以乙腈和水为流动相,利用亲水保留色谱机制（HILIC）实现对低聚果糖和低聚木糖各单体的有效分离,结合示差检测以外标法定量。该方法同时利用木糖对低聚木糖各单体转换系数进行定量。结果 低聚果糖和低聚木糖各单体线性关系良好,相关系数均大于0.998,平均加标回收率为95%以上,具有较高的准确度和良好的重现性。结论 高效液相色谱法由于采用木糖对照品转换定量低聚木糖含量,减少低聚木糖各单体标准品在日常工作中的使用量,进而降低检测成本。该方法经济可靠,测定结果准确,适合于添加有低聚果糖和低聚木糖的功能性产品检测。     

Thermopressurized diluted phosphoric acid pretreatment of ligno(hemi)cellulose to make free sugars and nutraceutical oligosaccharides

Ligno(hemi)cellulosics (L(h)Cs) as sugarcane bagasse and loblolly pine sawdust are currently being used to produce biofuels such as bioethanol and biobutanol through fermentation of free sugars that are often obtained enzymatically. However, this bioconversion requires a pretreatment to solubilize the hemicellulose fractions, thus facilitating the action of the cellulolytic enzymes. Instead of the main free monosaccharides used in these current models, the modulation of thermopressurized orthophosphoric acid as a pretreatment, in the ranges of 3–12 atm and pH 1.5–2.5, can produce nondigestible oligosaccharides (NDOS) such as xylo-oligosaccharides (XOS) because heteroxylan is present in both types of hardwood and softwood hemicelluloses. A comparative thin-layer chromatographic analysis of the hydrolytic products showed the best conditions for NDOS production to be 7 atm/water, pH 2.25 and 2.50, and 8.5 atm/water for both sources. Particular hydrolysates from 7 atm (171 °C) at pHs 2.25 and 2.50 both for cane bagasse and pine sawdust, with respective oligosaccharide contents of 57 and 59 %, once mixed in a proportion of 1:1 for each plant source, were used in vitro as carbon sources for Bifidobacterium or Lactobacillus. Once both bacteria attained the stationary phase of growth, an unforeseen feature emerged: the preference of B. animalis for bagasse hydrolysates and, conversely, the preference of L. casei for pine hydrolysates. Considering the fact that nutraceutical oligosaccharides from both hemicelluloses correspond to higher value-added byproducts, the technology using a much diluted thermopressurized orthophosphoric acid pretreatment becomes an attractive choice for L(h)Cs.     

Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach

In this study, a precise and reliable ultra-high performance liquid chromatography (UHPLC) method for the simultaneous determination of non-canonical (norvaline and norleucine) and standard amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine) in biopharmaceutical-related fermentation processes was established. After pre-column derivatization with ortho-phthaldialdehyde and 2-mercaptoethanol, the derivatives were separated on a sub-2 μm particle C₁₈ reverse-phase column. Identification and quantification of amino acids were carried out by fluorescence detection. To test method feasibility on standard HPLC instruments, the assay was properly transferred to a core–shell particle C₁₈ reverse-phase column. The limits of detection showed excellent sensitivity by values from 0.06 to 0.17 pmol per injection and limits of quantification between 0.19 and 0.89 pmol. In the present study, the newly established UHPLC method was applied to a recombinant antibody Escherichia coli fermentation process for the analysis of total free amino acids. We were able to specifically detect and quantify the unfavorable amino acids in such complex samples. Since we observed trace amounts of norvaline and norleucine during all fermentation phases, an obligatory process monitoring should be considered to improve quality of recombinant protein drugs in future.     

Production of xylooligosaccharides by xylanase from Pichia stipitis based on xylan preparation from triploid Populas tomentosa

Xylooligosaccharides (XOS) with DP 2-4 are important synbiotics used as food ingredients based on its prebiotic characteristics. In this work, the production of XOS from lignocellulosic material was performed by combined chemical-enzymatic methods. Xylan was prepared from triploid Populas tomentosa, and bioconverted into XOS by crude xylanase solution obtained from Pichia stipitis. The effects of reaction time, temperature, enzyme dosage, and pH value on the production of XOS were fully evaluated. Under the optimal condition (25 U g⁻¹ substrate, pH 5.4 and 50 °C), 36.8% of the xylan preparation was converted to XOS, equivalent to 3.95 mg/mL of the hydrolyzate. Xylobiose, xylotriose and xylotetrose were analyzed to be the main products of the enzymatic hydrolyzate, which together accounted for over 95% of the released oligosaccharides. Meanwhile, the effect of sonication pretreatment on the conversion efficiency of the xylan preparation was also investigated.     

Genetic Variation in Natural Populations of Stipa tenacissima from Algeria

Intermicrosatellite PCR [inter-simple sequence repeat (ISSR)-PCR] markers and cytogenetics criteria were used to assess the level of genetic diversity and genetic structure in 17 populations of Stipa tenacissima (Gramineae) from Algeria. All populations sampled in the steppe area were diploids (2n = 2x = 24), and those sampled in the dry area were hexaploids (2n = 6x = 72). The dendrogram based on ISSR-PCR showed homogeneity within populations and large variability among populations. All individuals of the same population were gathered and formed groups clearly separated in all populations. These groups were separated into two clusters related to biotope, one from the steppe area and the other from the dry area. AMOVA indicated low genetic diversity among populations (30% of variation) and high within populations (70%). This variation pattern would constitute an adaptive strategy to grow in various ecological conditions.     

Low molecular weight derivatives of different carrageenan types and their antiviral activity

A number of low molecular weight (LMW) fractions of carrageenans with different structural types were obtained by free radical depolymerization (H₂O₂), mild acid hydrolysis (HCl), and a specific enzyme. Three samples of carrageenans were depolymerized: kappa-carrageenan from Chondrus armatus, kappa-carrageenan from Kappaphycus alvarezii, and kappa/beta-carrageenan from Tichocarpus crinitus with initial molecular weights of 250, 390, and 400 kDa, respectively. The chemical depolymerization by two methods resulted to LMW derivatives of carrageenans with molecular weight from 1.2 to 3.5 kDa. Oligosaccharides of kappa- and kappa/beta-carrageenans with molecular weight of 2.2 and 4.3 kDa, respectively, were obtained after enzymatic depolymerization by recombinant kappa-carrageenase from Pseudoalteromonas carrageenovora. It was shown that the antiviral activity of high molecular weight carrageenans against tobacco mosaic virus was higher than that of their LWM derivatives independently on the depolymerization method. The method of depolymerization had some influence on the antiviral activity of carrageenan. LMW derivatives of kappa- and kappa/beta-carrageenans obtained by mild acid hydrolysis showed higher antiviral activity than the products of free radical depolymerization. The oligosaccharides prepared by enzymatic degradation possessed the lowest activity.     

Characterization of a tannase from Emericela nidulans immobilized on ionic and covalent supports for propyl gallate synthesis

The extracellular tannase from Emericela nidulans was immobilized on different ionic and covalent supports. The derivatives obtained using DEAE-Sepharose and Q-Sepharose were thermally stable from 60 to 75 °C, with a half life (t₅₀) >24 h at 80 °C at pH 5.0. The glyoxyl-agarose and amino-glyoxyl derivatives showed a thermal stability which was lower than that observed for ionic supports. However, when the stability to pH was considered, the derivatives obtained from covalent supports were more stable than those obtained from ionic supports. DEAE-Sepharose and Q-Sepharose derivatives as well as the free enzyme were stable in 30 and 50 % (v/v) 1-propanol. The CNBr-agarose derivative catalyzed complete tannic acid hydrolysis, whereas the Q-Sepharose derivative catalyzed the transesterification reaction to produce propyl gallate (88 % recovery), which is an important antioxidant.     

Analysis of polyamines in biological samples by HPLC involving pre-column derivatization with o-phthalaldehyde and N-acetyl-l-cysteine

Polyamines (putrescine, spermine and spermidine) play a crucial role in the regulation of cell growth, differentiation, death and function. Accurate measurement of these substances is essential for studying their metabolism in cells. This protocol describes detailed procedures for sample preparation and HPLC analysis of polyamines and related molecules (e.g., agmatine and cadaverine) in biological samples. The method is optimized for the deproteinization of samples, including biological fluids (e.g., 10 μl), plant and animal tissues (e.g., 50 mg), and isolated/cultured cells (e.g., 1 × 10⁶ cells). The in-line reaction of polyamines with o-phthalaldehyde and N-acetyl-l-cysteine yields fluorescent derivatives which are separated on a reversed-phase C₁₈ column and detected by a fluorometer at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The total running time for each sample (including column regeneration on the automated system) is 30 min. The detection limit is 0.5 nmol/ml or 0.1 nmol/mg tissue in biological samples. The assays are linear between 1 and 50 μM for each of the polyamines. The accuracy (the nearness of an experimental value to the true value) and precision (agreement between replicate measurement) of the HPLC method are 2.5–4.2 % and 0.5–1.4 %, respectively, for biological samples, depending on polyamine concentrations and sample type. Our HPLC method is highly sensitive, specific, accurate, easily automated, and capable for the analysis of samples with different characteristics and small volume/amount, and provides a useful research tool for studying the biochemistry, physiology, and pharmacology of polyamines and related substances.     

A novel cell modification method used in biotransformation of glycerol to 3-HPA by Lactobacillus reuteri

The aim of the present study was to develop a new cell modification method to facilitate the cell separation from broth. In order to reduce the transfer limitation of substrate and product caused by general immobilization methods in the following biotransformation of glycerol, the carboxyl-functioned superparamagnetic nanoparticle (MNP) was directly attached to the surface of Lactobacillus reuteri for 3-hydroxypropionealdehyde producing. The modification process could be finished in several minutes by just adding MNP fluid into the bulk fermentation broth. The modified cells could be rapidly separated from the solution with the aid of magnetic field. The interaction between cell and MNP was shown by electron microscopy. The efficiency of the cells attached by MNPs for transformation of various concentrations of glycerol (100–400 mM) was studied at various temperatures (25–40 °C) and pH levels (5.8–7.5) with different cell concentrations (7.5–30 g/L). The 3- hydroxypropionealdehyde (HPA)/glycerol molar conversion under optimal condition (30 °C and pH 7) reached 70 %. The inactive modified cell could be reactivated easily by fresh medium and recovered the ability of glycerol conversion. MNPS distributing on cell surface had little adverse effect on cell activity. The modification method simplified the two-step production of 3-HPA by resting L. reuteri. The method of MNPs attached to cell surface is totally different from the traditional immobilization method in which the cell is attached to or entrapped in big carrier. The results obtained in this study showed that carboxyl-functioned MNP could be directly used as cell modification particle and realized cell recycle with the aid of magnetic field in bioprocess.     

Determination of urinary oligosaccharides by high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry: Application to Hunter syndrome

The reaction of heparan sulfate (HS) and dermatan sulfate (DS) oligosaccharides with 1-phenyl-3-methyl-5-pyrazolone (PMP) yields hydrophobic derivatives that are amenable to separation by reversed-phase high-performance liquid chromatography (RP-HPLC) and analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). We describe here the development of an RP-HPLC-ESI-MS/MS assay for the measurement of di- to pentasaccharides derived from HS and DS in the urine of mucopolysaccharidosis (MPS) type II patients, as PMP derivatives. HPLC separation was performed on a 3-μm Alltima C18-LL column (50 × 2.1 mm) using a gradient elution of up to 25% acetonitrile over 17 min, and an API-4000 mass spectrometer equipped with a turbo-ion-spray source was used in the negative ion multiple reaction monitoring mode for PMP-oligosaccharide determination. Using this method, we found that the derivatization kinetics of the oligosaccharides was influenced by the type of residue present at the reducing end (i.e., N-acetylglucosamine, N-acetylgalactosamine, or uronic acid). The elevation of each of the measured oligosaccharides in MPS II urine enabled complete discrimination of a cohort of MPS II patient urines from unaffected controls. This assay is rapid and reproducible and may be useful for the diagnosis of MPS II, and also for monitoring of disease progression and efficacy of therapy.     

Manufacture and prebiotic potential of oligosaccharides derived from industrial solid wastes

The solid waste obtained in malting industries when dehulling barley grains, which was mainly made up of barley husks, spent grains and grain fragments, was subjected to a double hydrothermal processing under selected conditions. The liquor from the second stage (containing xylooligosaccharides, XOS) was refined by membrane and ion exchange processing (with or without a previous endoxylanase treatment to reduce the XOS molecular weight). Three XOS concentrates with different purity and/or molecular weight distribution were fermented in vitro with faecal inocula to assess their prebiotic potential. Succinate, lactate, formiate, acetate, propionate and butyrate were generated in fermentations, confirming the prebiotic potential of the various products assayed. The purity of XOS concentrates did not play a significant role in fermentation, whereas the sample with shorter average degree of polymerization presented a faster fermentation kinetics and led to the highest concentration of lactic acid.     

Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization

A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC–ESI–MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using l-norvaline as standard. A limit of detection as low as 1 fmol/µl with a linear range of up to 125 pmol/µl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using l-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).     

From lignocellulosic residues to market: Production and commercial potential of xylooligosaccharides

The updated definition of prebiotic expands the range of potential applications in which emerging xylooligosaccharides (XOS) can be used. It has been demonstrated that XOS exhibit prebiotic effects at lower amounts compared to others, making them competitively priced prebiotics. As a result, the industry is focused on developing alternative approaches to improve processes efficiency that can meet the increasing demand while reducing costs. Recent advances have been made towards greener and more efficient processes, by applying process integration strategies to produce XOS from costless lignocellulosic residues and using genetic engineering to create microorganisms that convert these residues to XOS. In addition, collecting more in vivo data on their performance will be key to achieve regulatory claims, greatly increasing XOS commercial value.