High level expression of a xyloglucanase from Rhizomucor miehei in Pichia pastoris for production of xyloglucan oligosaccharides and its application in yoghurt.

The xyloglucanase gene (RmXEG12A) from Rhizomucor miehei CAU432 was successfully expressed in Pichia pastoris. The highest xyloglucanase activity of 25,700 U mL-1 was secreted using high cell density fermentation. RmXEG12A was optimally active at pH 7.0 and 65 °C, respectively. The xyloglucanase exhibited the highest specific activity towards xyloglucan (7915.5 U mg-1). RmXEG12A was subjected to hydrolyze tamarind powder to produce xyloglucan oligosaccharides with the degree of polymerization (DP) 7-9. The hydrolysis ratio of xyloglucan in tamarind powder was 89.8%. Moreover, xyloglucan oligosaccharides (2.0%, w/w) improved the water holding capacity (WHC) of yoghurt by 1.1-fold and promoted the growth of Lactobacillus bulgaricus and Streptococcus thermophiles by 2.3 and 1.6-fold, respectively. Therefore, a suitable xyloglucanase for tamarind powder hydrolysis was expressed in P. pastoris at high level and xyloglucan oligosaccharides improved the quality of yoghurt.

Structural characterization of novel arabinoxylan and galactoarabinan from citron with potential antitumor and immunostimulatory activities.

This study aimed to extract polysaccharides from citron and analyze their structures and potential bioactivities. Two novel polysaccharides CM-1 and CM-2 were purified from citron by DEAE-Sepharose Fast Flow and Sephadex G-100 column chromatography. Monosaccharide composition, linkage and NMR data were used to infer their sugar chains composition. The anti-breast cancer cells and immunoregulatory activities of CM-1 and CM-2 were investigated. Results indicated that CM-1 (Mw = 21,520 Da), composed of arabinose, xylose, mannose and glucose in a molar ratio of 10.78:11.53:1.00:1.70, was arabinoxylan (AX) with (1 â†’ 4)-linked ß-d-Xylp skeleton monosubstituted with α-l-Araf units at O-3 position. While CM-2 (Mw = 22,303 Da), composed of arabinose, mannose, glucose and galactose in a molar ratio of 25.46:1.45:1.00:6.57, was galactoarabinan (GA) with (1 â†’ 5)-linked α-l-Araf backbone substituted by ß-d-Galp units at O-2 and/or O-3 positions. Both polysaccharides exhibited potential inhibiting cancer and immunostimulatory activities in vitro, especially CM-1. These results provide a basis for further research on citron polysaccharides.

In Vitro Fecal Fermentation Patterns of Arabinoxylan from Rice Bran on Fecal Microbiota from Normal-Weight and Overweight/Obese Subjects.

Arabinoxylan (AX) is a structural polysaccharide found in wheat, rice and other cereal grains. Diets high in AX-containing fiber may promote gut health in obesity through prebiotic function. Thus, the impact of soluble AX isolated from rice bran fiber on human gut microbiota phylogenetic composition and short-chain fatty acid (SCFA) production patterns from normal-weight and overweight/obese subjects was investigated through in vitro fecal fermentation. Results showed that rice bran arabinoxylan modified the microbiota in fecal samples from both weight classes compared to control, significantly increasing Collinsella, Blautia and Bifidobacterium, and decreasing Sutterella, Bilophila and Parabacteroides. Rice bran AX also significantly increased total and individual SCFA contents (p < 0.05). This study suggests that rice bran AX may beneficially impact gut health in obesity through prebiotic activities.

Isolation, Physicochemical Properties, and Structural Characteristics of Arabinoxylan from Hull-Less Barley.

Arabinoxylan (HBAX-60) was fractioned from alkaline-extracted arabinoxylan (HBAX) in the whole grain of hull-less barley (Hordeum vulgare L. var. nudum Hook. f. Poaceae) by 60% ethanol precipitation, which was studied for physicochemical properties and structure elucidation. Highly purified HBAX-60 mainly composed of arabinose (40.7%) and xylose (59.3%) was created. The methylation and NMR analysis of HBAX-60 indicated that a low-branched ß-(1→4)-linked xylan backbone possessed un-substituted (1,4-linked ß-Xylp, 36.2%), mono-substituted (ß-1,3,4-linked Xylp, 5.9%), and di-substituted (1,2,3,4-linked ß-Xylp, 12.1%) xylose units as the main chains, though other residues (α-Araf-(1→, ß-Xylp-(1→, α-Araf-(1→3)-α-Araf-(1→ or ß-Xylp-(1→3)-α-Araf-(1→) were also determined. Additionally, HBAX-60 exhibited random coil conformation in a 0.1 M NaNO3 solution. This work provides the properties and structural basis of the hull-less barley-derived arabinoxylan, which facilitates further research for exploring the structure-function relationship and application of arabinoxylan from hull-less barley.

Valorisation of walnut shell and pea pod as novel sources for the production of xylooligosaccharides.

According to the high interest in agro-industrial waste reutilisation, underutilised lignocellulosic materials, such as walnut shell (WS) and pea pod (PP), come in focus. The aim of this paper was to evaluate WS and PP as sources for the production of xylooligosaccharides (XOS). Hemicelluloses from WS and PP were recovered by combining varying parameters of delignification and alkaline extraction. At optimal recovery conditions, the fractions were further hydrolysed to XOS using GH11 endo-xylanase, by varying time and enzyme concentration. Xylose was predominant in the monomeric composition of the obtained hemicelluloses, building low-branched (arabino)glucuronoxylan, in WS exclusively, while in PP some xyloglucan as well. Delignification was essential for high recovery of total xylose from the materials, up to at least 70 %. High xylan conversions were obtained for 24 h hydrolysis, resulting in xylobiose and xylotriose when using low enzyme concentration, while in xylose and xylobiose with high enzyme concentration.

Extraction and characterization of xylan from sugarcane tops as a potential commercial substrate.

Xylan is the major hemicellulose present in sugarcane stem secondary cell walls. Xylan is composed of xylose backbone with a high degree of substitutions, which affects its properties. In the present study, the xylan from sugarcane tops (SCT) was extracted and characterized. Compositional analysis of xylan extracted from SCT (SCTx) displayed the presence of 74% of d-xylose residues, 16% of d-glucuronic acid residues and 10% of l-arabinose. High performance size exclusion chromatographic analysis of SCTx displayed a single peak corresponding to a molecular mass of ∼57 kDa. The Fourier transform infrared spectroscopic analysis of SCTx displayed the peaks corresponding to those obtained from commercial xylan. FESEM analysis of SCTx showed the granular and porous surface structure. Differential thermogravimetric analysis (DTG) of SCTx displayed two thermal degradation temperatures (Td) of 228°C, due to breakdown of the side chains of glucuronic acid and arabinose and 275°C, due to breakdown of xylan back bone. The presence of arabinose and glucuronic acid as a side chains was confirmed by the DTG and thermogravimetric analysis. The CHNS analysis of SCTx showed the presence of only carbon and hydrogen supporting its purity. The recombinant xylanase (CtXyn11A) from Clostridium thermocellum displayed a specific activity of 1394 ± 51 U/mg with SCTx, which was higher than those with commercial xylans. The thin layer chromatography and electrospray ionization mass spectroscopy analyses of CtXyn11A hydrolysed SCTx contained a series of linear xylo-oligosaccharides ranging from degree of polymerization 2-6 and no substituted xylo-oligosaccharides because of the endolytic activity of enzyme. The extracted xylan from SCT can be used as an alternative commercial substrate and for oligo-saccharide production.

Advance diversity of enzymatically modified arabinoxylan from wheat chaff.

Hydrolysates of arabinoxylan extracted from wheat chaff were prepared using different enzymatic treatments with an emphasis on improvements in their anti-diabetic, antioxidant and functional characteristics. The extracted arabinoxylan was subjected to enzymatic hydrolysis using individual xylanase, arabinofuranosidase, and feruloyl esterase, and their combinations. In all obtained hydrolysates, peaks corresponding to molecular weight lower than 38 kDa were noticed, while non-hydrolysed arabinoxylan had only peaks corresponding to 580 and 38 kDa. Results indicated that applied enzymes could hydrolyse polymeric arabinoxylan while their synergistic actions successfully modified its structure reflecting in lowered viscosity. Besides, it has been observed that the synergistic actions of enzymes improved the biological activities of arabinoxylan more than twice. Chemometric classification analysis showed that synergistic enzymes' actions were predominantly responsible for the improvement of biological activities. It indicated that they might be a useful tool for diversification and enhancement of biological activities of arabinoxylan from wheat chaff.

Structural characterization and anti-asthmatic effect of α-l-arabino(4-O-methyl-α-d-glucurono)-ß-d-xylan from the roots of Rudbeckia fulgida.

Many native plant biopolymers or derivatives thereof have interesting biological effects and therefore the search for additional biological activities is important to map their overall effects. A low molecular weight (Mw = 7600 g/mol) hemicellulose polymer α-L-arabino(4-O-methyl-α-D-glucurono)-ß-D-xylan (AGX) was isolated from the crushed roots of the Rudbeckia fulgida medicinal plant by alkaline extractions and anion-exchange chromatography. Analysis of neutral sugars revealed a predominance of xylose (82.3 wt%) and arabinose (6.8 wt%), while other neutral sugars were found only in small amounts as contaminants. The uronic acid content in Rudbeckia AGX was determined to be 8.8 wt%. Pharmacological tests showed that Rudbeckia AGX effectively suppressed cough and the initial amplitude of histamine/methacholine-induced bronchoconstriction in healthy OVA-sensitive guinea pigs. In addition, its effect at a dose of 100 mg/kg was similar to or greater than that of the positive control bronchodilator salbutamol and the antitussive codeine agent. These findings support the fact that Rudbeckia AGX could be a suitable candidate for alternative treatment of allergic asthma.

Bio-based films from wheat bran feruloylated arabinoxylan: Effect of extraction technique, acetylation and feruloylation.

This study demonstrates the potential of feruloylated arabinoxylan (AX) from wheat bran for the preparation of bioactive barrier films with antioxidant properties. We have comprehensively evaluated the influence of the structural features and chemical acetylation of feruloylated AX extracted by subcritical water on their film properties, in comparison with alkaline extracted AX and a reference wheat endosperm AX. The degree of substitution (DS) of AX had a large influence on film formation, higher DS yielded better thermal and mechanical properties. The barrier properties of AX films were significantly enhanced by external plasticization by sorbitol. Chemical acetylation significantly improved the thermal stability but not the mechanical or barrier properties of the films. The presence of bound ferulic acid in feruloylated AX films resulted in higher antioxidant activity compared to external addition of free ferulic acid, which demonstrates their potential use in active packaging applications for the preservation of oxygen-sensitive foodstuff.

Extraction, characterization of xylan from Azadirachta indica (neem) sawdust and production of antiproliferative xylooligosaccharides.

Xylan extracted from neem sawdust gave 22.5%, (w/w) yield. The extracted xylan was composed of xylose and glucuronic acid at a molar ratio of 8:1 and with a molecular mass, ~66 kDa. FTIR and NMR analyses indicated a backbone of xylan substituted with 4-O-methyl glucuronic acid at the O-2 position. FESEM analysis showed a highly porous and granular surface structure of xylan. A thermogravimetric study of xylan showed thermal denaturation at 271 °C. The hydrolysis of xylan by recombinant endo-ß-1,4-xylanase produced a mixture of xylooligosaccharides ranging from degree of polymerization 2-7. Xylooligosaccharides inhibited cell growth of human colorectal cancer (HT-29) cells but did not affect the mouse fibroblast cells confirming its biocompatibility. Western blotting, DNA laddering and flow cytometric analysis displayed inhibition of HT-29 cells by xylooligosaccharides. FLICA staining and mitochondrial membrane potential analyses confirmed the activation of the intrinsic pathway of apoptosis. The study amply indicated that the xylooligosaccharides produced from neem xylan could be potentially used as an antiproliferative agent.

Structural characteristics of oxalate-soluble polysaccharides from Norway spruce (Picea abies) foliage.

Structurally different polymers were derived from Picea abies foliage by successive extraction with water (PAW), HCl solution (PAA) and (NH4)2C2O4 solution (PAO). The P. abies foliage was found to contain basically low-methoxyl pectin extractable with an (NH4)2C2O4 solution. PAW was shown to comprise primarily arabinogalactan-proteins (AGPs); PAA was composed of mixed AGPs and pectic polysaccharides, with the latter prevailing; and polysaccharide PAO isolated in the highest yield included chiefly pectic polysaccharides. The major constituents of PAO were low-methoxyl and low-acetylated 1,4-α-d-galacturonan and partially acetylated RG-I. The sugar side chains of RG-I contained chiefly highly branched 1,5-α-l-arabinan and arabinogalactan type I as a minor constituent. RG-I whose side chains had 1,5-α-l-arabinan represented short regions alternating with non-acetylated and unmethylesterified galacturonan regions. In addition to pectins, polysaccharide PAO contained AGPs, xylanes and glucomannans, indicating that these polysaccharides are in an intimate interaction.

Green Synthesis of Antileishmanial and Antifungal Silver Nanoparticles Using Corn Cob Xylan as a Reducing and Stabilizing Agent.

Corn cob is an agricultural byproduct that produces an estimated waste burden in the thousands of tons annually, but it is also a good source of xylan, an important bioactive polysaccharide. Silver nanoparticles containing xylan (nanoxylan) were produced using an environmentally friendly synthesis method. To do this, we extracted xylan from corn cobs using an ultrasound technique, which was confirmed by both chemical and NMR analyses. This xylan contained xylose, glucose, arabinose, galactose, mannose, and glucuronic acid in a molar ratio of 50:21:14:9:2.5:2.5, respectively. Nanoxylan synthesis was analyzed using UV-vis spectroscopy at kmax = 469 nm and Fourier transform infrared spectroscopy (FT-IR), which confirmed the presence of both silver and xylan in the nanoxylan product. Dynamic light scattering (DLS) and atomic force microscopy (AFM) revealed that the nanoxylan particles were ~102.0 nm in size and spherical in shape, respectively. DLS also demonstrated that nanoxylan was stable for 12 months and coupled plasma optical emission spectrometry (ICP-OES) showed that the nanoxylan particles were 19% silver. Nanoxylan reduced Leishmania amazonensis promastigote viability with a half maximal inhibitory concentration (IC50) value of 25 µg/mL, while xylan alone showed no effective. Additionally, nanoxylan exhibited antifungal activity against Candida albicans (MIC = 7.5 µg/mL), C. parapsilosis (MIC = 7.5 µg/mL), and Cryptococcus neoformans (MIC = 7.5 µg/mL). Taken together, these data suggest that it is possible to synthesize silver nanoparticles using xylan and that these nanoxylan exert improved antileishmanial and antifungal activities when compared to the untreated polysaccharide or silver nitrate used for their synthesis. Thus, nanoxylan may represent a promising new class of antiparasitic agents for use in the treatment of these microorganisms.

Cascade extraction of proteins and feruloylated arabinoxylans from wheat bran.

A cascade process for the sequential recovery of proteins and feruloylated arabinoxylan from wheat bran is proposed, involving a protein isolation step, enzymatic destarching and subcritical water extraction. The protein isolation step combining lactic acid fermentation and cold alkaline extraction reduced the recalcitrance of wheat bran, thus improving the total yields of the subsequent subcritical water extraction. The time evolution of subcritical water extraction of feruloylated arabinoxylan was compared at two temperatures (160 °C and 180 °C). Longer residence times enhanced the purity of target feruloylated arabinoxylans, whereas higher temperatures resulted in faster extraction at the expense of significant molar mass reduction. The radical scavenging activity of the extracted feruloylated arabinoxylans was preserved after the initial protein isolation step. This study opens new possibilities for the cascade valorization of wheat bran into enriched protein and non-starch polysaccharide fractions, which show potential to be used as functional food ingredients.

Facile and green preparation of diverse arabinoxylan hydrogels from wheat bran by combining subcritical water and enzymatic crosslinking.

Feruloylated arabinoxylans demonstrate great potential in developing natural materials through oxidative crosslinking. However, the common alkaline method to extract arabinoxylans causes heavy losses of feruloyl esters thus hinders the crosslinking activity. Highly feruloylated arabinoxylans were fractionated from wheat bran by an alternative method using subcritical water extraction (SWE) and were compared with water- and alkali-extracted arabinoxylans. Various yields, chemical compositions, and molecular weights were obtained as the SWE conditions changed. Methylation analysis showed that the SWE arabinoxylans had a similar linkage pattern as the water-extracted arabinoxylan. Laccase-induced oxidative crosslinking was investigated through dynamic rheology, finding a broad spectrum of gelling ability for the SWE arabinoxylans. Finally, the optimal condition (160 °C, pH 7 for 10 min) was used in a repeated SWE procedure to maximize the yield and crosslinking properties. The combination of subcritical water and enzymatic crosslinking presents as a promising green approach for preparing hydrogels from herbaceous hemicelluloses.

Green synthesis, characterization, antimicrobial and cytotoxic effect of silver nanoparticles using arabinoxylan isolated from Kalmegh.

A green synthesis of silver nanoparticles was synthesized by AgNO3 with arabinoxylan, isolated from green stem of Andrographis paniculata (Kalmegh). The synthesized Ag NPs-arabinoxylan conjugates were characterized by UV-vis spectroscopy, FE-SEM, TEM, XRD, TGA, EDX, and Zeta potential experiments. The Ag NPs formation was established by the surface plasmon resonance band ~410.25 nm. SEM image showed mostly spherical morphology of Ag NPs. The fcc crystalline nature was identified by XRD, SAED and the size were 24.5 and 25 nm from TEM and XRD analysis respectively. The prepared Ag NPs showed dose-dependent antimicrobial activity against Streptococcus pneumonia, Candida albicans and E. coli. The nanoparciles damage 4% hemolysis to human RBCs at 12.5 µg/mL. MTT assay of Ag NPs showed that half of the cell killed at 10 µg/mL and wound healing assay observed effective inhibition cell proliferation.

Structural modifications to water-soluble wheat bran arabinoxylan through milling and extrusion.

Feruloylated arabinoxylan (AX) is one of the most predominant dietary fiber in cereal grains. In recent decades, soluble AX has gained interest, as a result of its well-established health benefits. Apart from enzymatic degradation during cereal storage, food processing causes AX degradation. These reactions lead to structural modifications and influence both the AX functionalities and its health promoting effects. The aim of this study was to investigate the structural modifications and related property changes of health promoting water-extractable (WE) wheat bran AX through grain milling and extrusion. Multi-detector HPSEC revealed a correlation between Mw, conformational changes and the related viscosity behaviour depending on the processing type. Processing caused molecular degradation of insoluble high Mw AX, which increased the solubility significantly. Moreover, extrusion leaded to a more heterogenic AX fine structure. The detailed characterization of processed dietary fiber may help to facilitate the optimized incorporation of AX in health-promoting foods.

Chemopreventive role of arabinoxylan rice bran, MGN-3/Biobran, on liver carcinogenesis in rats.

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and one of the most lethal. MGN-3/Biobran is a natural product derived from rice bran hemicelluloses and has been reported to possess a potent anticancer effect in a clinical study of patients with HCC. The current study examines the mechanisms by which Biobran protects against chemically induced hepatocarcinogenesis in rats. The chemical carcinogen used in this study is N-nitrosodiethylamine (NDEA) plus carbon tetrachloride (CCl4). Rats were treated with this carcinogen, and the animals were pretreated or posttreated with Biobran via intraperitoneal injections until the end of the experiment. Treatment with Biobran resulted in: 1) significant alleviation of liver preneoplastic lesions towards normal hepatocellular architecture in association with inhibition of collagen fiber deposition; 2) arrest of cancer cells in the sub-G1 phase of the cell cycle; 3) increased DNA fragmentation in cancer cells; 4) down-regulated expression of Bcl-2 and up-regulated expression of p53, Bax, and caspase-3; and 5) protection against carcinogen-induced suppression of IkappaB-alpha (IκB-α) mRNA expression and inhibition of nuclear factor kappa-B (NF-κB/p65) expression. Additionally, the effect of Biobran treatment was found to be more significant when supplemented prior to carcinogen-induced hepatocarcinogenesis as compared to posttreatment. We conclude that Biobran inhibits hepatocarcinogenesis in rats by mechanisms that include induction of apoptosis, inhibition of inflammation, and suppression of cancer cell proliferation. Biobran may be a promising chemopreventive and chemotherapeutic agent for liver carcinogenesis.

Enzyme-aided xylan extraction from alkaline-sulfite pretreated sugarcane bagasse and its incorporation onto eucalyptus kraft pulps.

Hemicellulose-rich substrates produced in the lignocellulose biorefinery context can yield macromolecular xylan structures with assorted application in the chemical industry. Xylan presents natural affinity to cellulose and its incorporation onto fibers increases the physical processability of pulp; however, current studies diverge on how molar mass affects xylan interaction with cellulose. In the current work, xylans with varied structural characteristics were prepared from alkaline-sulfite pretreated sugarcane bagasse with aid of an alkaline-active xylanase and selective precipitations using different ethanol concentrations. Prepared xylan fractions, containing low levels of lignin contamination (4-9%) and molar masses ranging from 2.3 kDa to 34 kDa, were incorporated onto eucalyptus pulp fibers up to 4.7 g xylan/100 g pulp. The efficiency of xylan incorporation onto cellulosic fibers was dependent on the xylan structures, where low molar mass and low substitution degree favored high incorporation levels.

Impact of water extractable arabinoxylan with different molecular weight on the gelatinization and retrogradation behavior of wheat starch.

Water-extractable arabinoxylan (WEAX) could effectively improve the cereal food quality, while its regulatory effect on wheat starch properties has yet to be well-understood. This study selected the WEAX with different molecular weight (Mw) but same branched degree, and comparatively investigated their effects on the gelatinization and retrogradation behavior of wheat starch. The decreased degree of swelling power, solubility and peak viscosity suggested that low Mw WEAX (L-WEAX) could hinder starch gelatinization more evidently compared with high Mw WEAX (H-WEAX), due to the pronounced inhibition effect on amylose leaching and amylose-lipid complex formation. L-WEAX suppressed the recrystallization of amylose and thus the short-term retrogradation. However, H-WEAX mainly retarded the recrystallization of amylopectin, exerting a more significant inhibition effect on the long-term retrogradation. This study could provide a theoretical basis for enhancing the quality and extending the shelf life of starchy foods by selecting the optimum structure of WEAX.

Microgravity Affects the Level of Matrix Polysaccharide 1,3:1,4-ß-Glucans in Cell Walls of Rice Shoots by Increasing the Expression Level of a Gene Involved in Their Breakdown.

The plant cell wall provides each cell with structural support and mechanical strength, and thus, it plays an important role in supporting the plant body against the gravitational force. We investigated the effects of microgravity on the composition of cell wall polysaccharides and on the expression levels of genes involved in cell wall metabolism using rice shoots cultivated under artificial 1 g and microgravity conditions on the International Space Station. The bulk amount of the cell wall obtained from microgravity-grown shoots was comparable with that from 1 g-grown shoots. However, the analysis of sugar constituents of matrix polysaccharides showed that microgravity specifically reduced the amount of glucose (Glc)-containing polysaccharides such as 1,3:1,4-ß-glucans, in shoot cell walls. The expression level of a gene for endo-1,3:1,4-ß-glucanase, which hydrolyzes 1,3:1,4-ß-glucans, largely increased under microgravity conditions. However, the expression levels of genes involved in the biosynthesis of 1,3:1,4-ß-glucans were almost the same under both gravity conditions. On the contrary, microgravity scarcely affected the level and the metabolism of arabinoxylans. These results suggest that a microgravity environment promotes the breakdown of 1,3:1,4-ß-glucans, which, in turn, causes the reduced level of these polysaccharides in growing rice shoots. Changes in 1,3:1,4-ß-glucan level may be involved in the modification of mechanical properties of cell walls under microgravity conditions in space.