Release Of Oligosaccharides Research Articles

Impact of pre-treatments and bioprocessing on the carbohydrate and polyphenol profile of brewers’ spent grain

Brewers’ spent grain (BSG), the solid waste of the brewing industry, is high in fibres, proteins and health-beneficial compounds such as polyphenols. This research investigated bioprocessing with enzymes and microbes to modify the properties of BSG for its utilisation as a food ingredient. Pre-treatment studies showed that wet milling performed better than dry milling, and heat and homogenisation either before or after the enzyme hydrolysis did not significantly influence the release of reducing sugars and free amino nitrogen (FAN). Four treatments were applied to wet-milled BSG: fermentation with Lactiplantibacillus plantarum POM1 with or without the enzyme Ondea pro and enzymatic bioprocessing without any fermentation. Control was the condition without enzyme and starter. Without the enzyme, there was negligible free sugar and FAN, and the starter had limited growth and organic acid production. Only the combination of enzymatic hydrolysis and fermentation reached a pH of 4 and 10 mg/g DW lactic acid. The microbial preference for monosaccharides was evident, and the enzyme influenced the release of oligosaccharides that can have a prebiotic effect. Bioprocessing impacted the phenolic acid composition and microbial consumption, with a significant release of ferulic acid during enzyme hydrolysis.

Coaxial nanofibrous aerogel featuring porous network-structured channels for ovarian cancer treatment by sustained release of chitosan oligosaccharide

Ovarian cancer, the deadliest gynecological malignancy, primarily treated with chemotherapy. However, systemic chemotherapy often leads to severe toxic side effects and chemoresistance. Drug-loaded aerogels have emerged as a promising method for drug delivery, as they can improve drug solubility and bioavailability, control drug release, and reduce drug distribution in non-targeted tissues, thereby minimizing side effects. In this research, chitosan oligosaccharide (COS)-loaded nanofibers composite chitosan (CS) aerogels (COS-NFs/CS) with a porous network structure were created using nanofiber recombination and freeze-drying techniques. The core layer of the aerogel has a COS loading rate of 60 %, enabling the COS-NFs/CS aerogel to significantly inhibit the migration and proliferation of ovarian cancer cells (resulting in a decrease in the survival rate of ovarian cancer cells to 33.70 % after 48 h). The coaxial fiber's unique shell-core structure and the aerogel's porous network structure enable the COS-NFs/CS aerogels to release COS steadily and slowly over 30 days, effectively reducing the initial burst release of COS. Additionally, the COS-NFs/CS aerogels exhibit good biocompatibility, degradability (only retaining 18.52 % of their weight after 6 weeks of implantation), and promote angiogenesis, thus promoting wound healing post-oophorectomy. In conclusion, COS-NFs/CS aerogels show great potential for application in the treatment of ovarian cancer.

Quality evaluation of highly purified human menopausal gonadotropin preparations by means of gel electrophoresis and mass spectrometry.

Human gonadotropins are glycoprotein hormones with a highly complex structure, which demands the application of sophisticated analytical methodologies to assess their quality. The principal objective of this study was a comparative evaluation of gel electrophoretic techniques and mass spectrometry-based methods for the quality study of the two urinary-derived, highly purified, human menopausal gonadotropin preparations, Menopur 75/75 I. U. and Meriofert 75 I. U. Molecular mass (Mr), isoelectric point (pI), and isoform pattern of studied compounds were estimated via SDS-PAGE and 2D gel electrophoresis, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for the downstream characterization of peptides obtained after in-gel tryptic digestion of selected protein spots. Additionally, for the estimation of the glycosylation pattern of these biologics, the enzymatic release of oligosaccharides was performed, and the isoform pattern was studied. Gel electrophoresis showed a typical electrophoretic behaviour for protein biotherapeutics medicines consisting of extremely complex spot patterns migrating at different masses and pIs. MS analysis proved to be a powerful tool for the identification and detailed characterization of the gonadotropins and the relevant peptides were identified with high sequence coverages. The results of this study are not only useful for the quality assessment of this class of complex biopharmaceuticals but may also serve as a supporting platform for further development of biopharmaceuticals based on modulation of the glycosylation pattern to enhance efficacy or reduce side effects.

The Rice Endophyte-Derived α-Mannosidase ShAM1 Degrades Host Cell Walls To Activate DAMP-Triggered Immunity against Disease.

Endophytes play an important role in shaping plant growth and immunity. However, the mechanisms for endophyte-induced disease resistance in host plants remain unclear. Here, we screened and isolated the immunity inducer ShAM1 from the endophyte Streptomyces hygroscopicus OsiSh-2, which strongly antagonizes the pathogen Magnaporthe oryzae. Recombinant ShAM1 can trigger rice immune responses and induce hypersensitive responses in various plant species. After infection with M. oryzae, blast resistance was dramatically improved in ShAM1-inoculated rice. In addition, the enhanced disease resistance by ShAM1 was found to occur through a priming strategy and was mainly regulated through the jasmonic acid-ethylene (JA/ET)-dependent signaling pathway. ShAM1 was identified as a novel α-mannosidase, and its induction of immunity is dependent on its enzyme activity. When we incubated ShAM1 with isolated rice cell walls, the release of oligosaccharides was observed. Notably, extracts from the ShAM1-digested cell wall can enhance the disease resistance of the host rice. These results indicated that ShAM1 triggered immune defense against pathogens by damage-associated molecular pattern (DAMP)-related mechanisms. Our work provides a representative example of endophyte-mediated modulation of disease resistance in host plants. The effects of ShAM1 indicate the promise of using active components from endophytes as plant defense elicitors for the management of plant disease. IMPORTANCE The specific biological niche inside host plants allows endophytes to regulate plant disease resistance effectively. However, there have been few reports on the role of active metabolites from endophytes in inducing host disease resistance. In this study, we demonstrated that an identified α-mannosidase protein, ShAM1, secreted by the endophyte S. hygroscopicus OsiSh-2 could activate typical plant immunity responses and induce a timely and cost-efficient priming defense against the pathogen M. oryzae in rice. Importantly, we revealed that ShAM1 enhanced plant disease resistance through its hydrolytic enzyme (HE) activity to digest the rice cell wall and release damage-associated molecular patterns. Taken together, these findings provide an example of the interaction mode of endophyte-plant symbionts and suggest that HEs derived from endophytes can be used as environmentally friendly and safe prevention agent for plant disease control.

Multiple enzymatic approaches to hydrolysis of fungal β-glucans by the soil bacterium Chitinophaga pinensis.

The genome of the soil Bacteroidota Chitinophaga pinensis encodes a large number of glycoside hydrolases (GHs) with noteworthy features and potentially novel functions. Several are predicted to be active on polysaccharide components of fungal and oomycete cell walls, such as chitin, β-1,3-glucan and β-1,6-glucan. While several fungal β-1,6-glucanase enzymes are known, relatively few bacterial examples have been characterised to date. We have previously demonstrated that C.pinensis shows strong growth using β-1,6-glucan as the sole carbon source, with the efficient release of oligosaccharides from the polymer. We here characterise the capacity of the C.pinensis secretome to hydrolyse the β-1,6-glucan pustulan and describe three distinct enzymes encoded by its genome, all of which show different levels of β-1,6-glucanase activity and which are classified into different GH families. Our data show that C.pinensis has multiple tools to deconstruct pustulan, allowing the species' broad utility of this substrate, with potential implications for bacterial biocontrol of pathogens via cell wall disruption. Oligosaccharides derived from fungal β-1,6-glucans are valuable in biomedical research and drug synthesis, and these enzymes could be useful tools for releasing such molecules from microbial biomass, an underexploited source of complex carbohydrates.

Selection of Enzymatic Treatments for Upcycling Lentil Hulls into Ingredients Rich in Oligosaccharides and Free Phenolics

In this study, the comprehensive chemical characterization of red lentil hulls obtained from the industrial production of football and split lentils was described. The lentil hulls were rich in dietary fiber (78.43 g/100 g dry weight with an insoluble to soluble fiber ratio of 4:1) and polyphenols (49.3 mg GAE/g dry weight, of which 55% was bound phenolics), which revealed the suitability of this lentil by-product as a source of bioactive compounds with recognized antioxidant and prebiotic properties. The release of oligosaccharides and phenolic compounds was accomplished by enzymatic hydrolysis, microwave treatment and a combination of both technologies. The key role played by the selection of a suitable enzymatic preparation was highlighted to maximize the yield of bioactive compounds and the functional properties of the lentil hull hydrolysates. Out of seven commercial preparations, the one with the most potential for use in a commercial context was Pectinex® Ultra Tropical, which produced the highest yields of oligosaccharides (14 g/100 g lentil hull weight) and free phenolics (45.5 mg GAE/100 g lentil hull weight) and delivered a four-fold increase in terms of the original antioxidant activity. Finally, this enzyme was selected to analyze the effect of a microwave-assisted extraction pretreatment on the yield of enzymatic hydrolysis and the content of free phenolic compounds and oligosaccharides. The integrated microwave and enzymatic hydrolysis method, although it increased the solubilization yield of the lentil hulls (from 25% to 34%), it slightly decreased the content of oligosaccharides and proanthocyanidins and reduced the antioxidant activity. Therefore, the enzymatic hydrolysis treatment alone was more suitable for producing a lentil hull hydrolysate enriched in potential prebiotics and antioxidant compounds.

Quantitative estimation of enzymatic released specific oligosaccharides from Hericium erinaceus polysaccharides using CE-LIF

Polysaccharides exhibit multiple pharmacological activities which are closely related to their structural features. Therefore, quantitatively quality control of polysaccharides based on their chemical characteristics is important for their application in biomedical and functional food sciences. However, polysaccharides are mixed macromolecular compounds that are difficult to isolate and lack standards, making them challenging to quantify directly. In this study, we proposed an improved saccharide mapping method based on the release of specific oligosaccharides for the assessment of Hericium erinaceus polysaccharides from laboratory cultured and different regions of China. Briefly, a polysaccharide from H. erinaceus was digested by β-(1-3)-glucanase, and the released specific oligosaccharides were labeled with 8-aminopyrene-1,3,6-trisulfonic-acid (APTS) and separated by using micellar electrokinetic chromatography (MEKC) coupled with laser induced fluorescence (LIF), and quantitatively estimated. MEKC presented higher resolution compared to polysaccharide analysis using carbohydrate gel electrophoresis (PACE), and provided great peak capacity between oligosaccharides with polymerization degree of 2 (DP2) and polymerization degree of 6 (DP6) in a dextran ladder separation. The results of high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector (HPSEC-MALLS-RI) showed that 12 h was sufficient for complete digestion of polysaccharides from H. erinaceus. Laminaritriose (DP3) was used as an internal standard for quantification of all the oligosaccharides. The calibration curve for DP3 showed a good linear regression (R2 > 0.9988). The limit of detection (LOD) and limit of quantification (LOQ) values were 0.05 μg/mL and 0.2 μg/mL, respectively. The recovery for DP3 was 87.32 (±0.03)% in the three independent injections. To sum up, this proposed method is helpful for improving the quality control of polysaccharides from H. erinaceus as well as other materials.

Leveraging Bioprocessing Strategies to Achieve the Simultaneous Extraction of Full-Fat Chickpea Flour Macronutrients and Enhance Protein and Carbohydrate Functionality

The concurrent extraction of lipids, proteins, and carbohydrates can be achieved by aqueous and enzymatic extraction processes, circumventing the low extractability by mechanical pressing and the use of flammable solvents. The use of alkaline protease, preceded or not by carbohydrase pretreatments, was evaluated on the extractability of oil, protein, and carbohydrates from full-fat chickpea flour and protein functionality. Enzymatic extraction increased oil and protein extractability from 49.8 to 72.0–77.1% and 62.8 to 83.5–86.1%, respectively. Although the carbohydrase pretreatments before the addition of protease did not increase oil and protein extractability, the carbohydrate content of the extracts increased from 7.68 to 9.17−9.33 mg/mL, accompanied by the release of new oligosaccharides in the extracts, as revealed by LC–MS/MS characterization. Enzymatic extraction yielded proteins with significantly higher solubility (25.6 vs. 68.2–73.6%) and digestibility (83.8 vs. 90.79–94.67%). Treatment of the extracts with α-galactosidase completely removed the flatulence-causing oligosaccharides (stachyose and raffinose). This study highlights the effectiveness of environmentally friendly bioprocessing strategies to maximize lipid, protein, and oligosaccharide extractability from full-fat chickpea flour with concurrent improvements in protein solubility and in vitro digestibility, reduction of flatulence related oligosaccharides, and generation of a more diverse pool of oligosaccharides for subsequent prebiotic evaluation.Graphical abstract

Identification of plant polysaccharides by MALDI-TOF MS fingerprinting after periodate oxidation and thermal hydrolysis

An autoclave treatment at 121 °C on periodate-oxidized plant polysaccharides and mixes thereof was investigated for the release of oligosaccharides to obtain a generic polysaccharide depolymerization method for polysaccharides fingerprinting. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) analysis of the oligosaccharides released showed that each polysaccharide had a unique oligosaccharides profile, even the same type of polysaccharide derived from different sources and/or having different fine structures (e.g. class of (arabino)xylans, galactomannans, glucans, or pectic materials). Various polysaccharide classes present in a polysaccharide mix could be identified based on significantly different (p < 0.05) marker m/z values present in the mass spectrum. Principal component analysis and hierarchical cluster analysis of the obtained MALDI-TOF MS data highlighted the structural heterogeneity of the polysaccharides studied, and clustered polysaccharide samples with resembling oligosaccharide profiles. Our approach represents a step further towards a generic and accessible identification of plant polysaccharides individually or in a mixture.

Ileal profile of non-starch polysaccharides and oligosaccharides in response to exogenous enzymes in broiler chickens offered wheat- or maize-based diets under subclinical necrotic enteritis challenge.

The present study evaluated the impacts of fibre-degrading enzymes on the profiles of non-starch polysaccharides (NSP) and oligosaccharides (OS) in the ileum of broiler chickens offered wheat- or maize-based diets under subclinical necrotic enteritis (NE) challenge. A 2 × 2 × 4 factorial arrangement of treatments was used. Factors were the following: NE challenge, no or yes; diet type, wheat- or maize-based; and supplemental enzymes, control (no enzyme), family 10 xylanase (XYN10), family 11 xylanase (XYN11) or β-mannanase (MAN). Birds in the challenged group were inoculated with Eimeria on d 9 and Clostridium perfringens on d 14 and 15. A 3-way interaction (P = 0.047) occurred on overall (d 0 to 16) weight gain. When NE was present, all the supplemental enzymes increased weight gain in birds fed the wheat-based diet; whereas in those fed the maize-based diet supplemental XYN10 and XYN11 decreased weight gain. When NE was absent, birds fed the wheat-based diet supplemented with XYN10 or MAN presented increased weight gain compared to non-supplemented birds, but no improvements with enzyme addition were observed in birds fed the maize-based diet. A 3-way interaction (P = 0.002) was observed on insoluble NSP level in the ileum. When NE was absent, all the supplemental enzymes reduced the ileal level of insoluble NSP, regardless of diet type. In the challenged birds, supplementing XYN10 and MAN reduced insoluble NSP level in the ileum, but only in birds fed the wheat-based diet. Ileal soluble NSP level was reduced by supplemental XYN11 and MAN, but only in birds fed the wheat-based diet, resulting in a 2-way diet type × enzyme interaction (P < 0.001). Ileal OS arabinose (P = 0.030) level was highest in birds offered the wheat-based diet supplemented with XYN11. Collectively, supplementation of NSP-degrading enzymes to the wheat-based diet enhanced bird performance regardless of NE challenge, with XYN11 significantly increasing oligosaccharide release. However, enzyme addition did not improve growth performance in birds fed maize-based diet, with supplemental XYN10 and XYN11 impeding weight gain when NE was present.

Biochemical and structural characterization of a novel thermophilic and acidophilic β-mannanase from Aspergillus calidoustus

β-Mannanases hydrolyze lignocellulosic biomass with the release of mannan oligosaccharides, which are considered as renewable resource in higher plants. Here, we cloned, expressed and characterized a novel endo-β-mannanase (ManAC) from Aspergillus calidoustus. Homology alignment analysis indicated that ManAC belonged to glycosyl hydrolase (GH) 5 family members. The analysis of structural homologous model revealed that five residues, Arg116, Asn231, His305, Tyr307, and Trp370, constituted the active site of ManAC. Glu232 and Glu340, proton donor and nucleophile, formed the catalytic residues of ManAC. The recombinant ManAC exhibited maximal activity at pH 2.5 and 70 °C, and it was acid tolerant at a pH range of 2.0–6.0 and thermostable under 60 °C. Meanwhile, the activity of ManAC was not significantly affected by various metal ions, except for Mg2+ and Ag2+. The recombinant ManAC exhibited the highest β-mannanase activity towards locust bean gum (669.7 U/mg) with the Km and Vmax values of 3.4 mg/mL and 982.4 μmol/min/mg, respectively. These thermophilic and acidophilicc characteristics is better than most extreme β-mannanase. As the first reported mannanse from Aspergillus calidoustus (ManAC), these excellent properties of ManAC strongly promote the synthesis of mannooligosaccharides which have potential for food and feed industrial applications.

Structural Profiling of Xyloglucans from Food Plants by High-Performance Anion-Exchange Chromatography with Parallel Pulsed Amperometric and Mass Spectrometric Detection.

Xyloglucans are the dominant hemicelluloses in the primary cell wall of dicotyledonous plants, fulfilling numerous functions. However, routine methods of cell wall analytical chemistry such as methylation analysis are time-consuming and often not adequate to capture the structural diversity of xyloglucans. Here, a xyloglucan profiling method based on the enzymatic release of xyloglucan oligosaccharides by a xyloglucan-specific endo-β-(1→4)-glucanase and subsequent analysis of these oligosaccharides by high-performance anion-exchange chromatography (HPAEC) with parallel pulsed amperometric and mass spectrometric detection was developed. For this purpose, a set of 23 authentic xyloglucan oligosaccharides was generated, structurally characterized by mass spectrometry and NMR spectroscopy, and established as analytical standard compounds. Coupling of HPAEC with parallel electrochemical and MS detection was demonstrated to be an excellent tool to analyze xyloglucan-derived oligosaccharides. The applicability of the method was demonstrated by characterizing the xyloglucan architecture from a set of nine economically relevant food plants from the botanical orders Caryophyllales (rhubarb, buckwheat, amaranth, and quinoa), Cucurbitales (Hokkaido squash), Laurales (avocado), Myrtales (pomegranate), and Sapindales (mango and orange) for the first time. In future studies, this method can ideally be used to monitor structural alterations of xyloglucans as a result of genetic engineering, plant/tissue maturation, and processing of plant material.

Novel Malto‐Oligosaccharide‐Producing Amylase AmyAc from Archangium sp. Strain AC19 and Its Catalytic Properties

AbstractFunctional maltooligosaccharides (MOSs) derived from starch have potential applications in the food industry and medicine due to their specific functional properties. In this study, a novel maltose (G2) and maltotriose (G3)‐producing amylase AmyAc from Archangium sp. strain AC19 is identified. AmyAc is a protein of 874 amino acids, with an N‐terminal signal peptide as well as hydrolase family 13 (GH13) catalytic and C‐terminal binding modules. However, its sequence is not highly similar to any of the reported starch hydrolases. The Vmax of AmyAc is 124 µmol min−1 mg−1 under optimal conditions of 50 °C and pH 7.0. AmyAc catalyzes the conversion of MOSs and soluble starch into maltose and maltotriose as the end‐product with more than 85% purity. AmyAc initially produces G2, G3, and oligosaccharides with even units, following by the release of G2 and oligosaccharides with odd units during later stages of hydrolysis, indicating a distinct catalytic selectivity. The production of MOSs from starch by AmyAc is of significant interest due to potential applications in starch processing.

Bagasse minority pathway expression: Real time study of GH2 β-mannosidases from bacteroidetes.

After being isolated from a sugarcane pile, the bacterium Chitinophaga sp. CB10 demonstrated to be a rich source of carbohydrases, with 350 predicted CAZyme domains. CB10 was able to grow on carbohydrates of different structural complexities: glucose, carboxymethylcellulose, corn starch, galactomannan, Aloe vera gum and sugarcane bagasse. The sugarcane bagasse is a rich source of complex polymers, and the diversity of metabolites released by its enzymatic hydrolysis has an important role for green chemistry, including minority pathways such as the degradation of mannan conjugates. In this sense, CB10 demonstrated considerable levels of gene expression for mannanases, and was stable for a period of 96–144 hours in the presence of sugarcane bagasse as sole carbon source. The bacterium showed respectively 4.8x and 5.6x expression levels for two genes predicted for GH2 β-mannosidase: one located within a gene cluster identified as “polysaccharide utilization loci” (PUL), and another a classic β-mannosidase. These enzymes shared less than 45% of identity with enzymes characterized from the genus Chitinophaga belonging to the phylum Bacteroidetes. The degree of novelty—as demonstrated by the low identity with previously characterized enzymes; the remarkable capability to grow in different substrates; mannanase activity, evidenced by the release of residual oligosaccharides in the cultivation with galactomannan (HPLC-RID, 12.3 mMol); associated to the ability of mannanases expression in a low concentration of inductor conditions (sugarcane bagasse, 0.2%) indicate the high potential for the application of CB10 as a source of enzymes in the production of oligosaccharides from biomass. This capacity might prove to be very valuable for the biorefinery process of pre-biotic precursors and other functional oligosaccharides focused on the food and pharmaceutical industries.

Production of Structurally Defined Chito-Oligosaccharides with a Single N-Acetylation at Their Reducing End Using a Newly Discovered Chitinase from Paenibacillus pabuli.

Partially acetylated chito-oligosaccharides (paCOSs) are bioactive compounds with potential medical applications. Their biological activities are largely dependent on their structural properties, in particular their degree of polymerization (DP) and the position of the acetyl groups along the glycan chain. The production of structurally defined paCOSs in a purified form is highly desirable to better understand the structure/bioactivity relationship of these oligosaccharides. Here, we describe a newly discovered chitinase from Paenibacillus pabuli (PpChi) and demonstrate by mass spectrometry that it essentially produces paCOSs with a DP of three and four that carry a single N-acetylation at their reducing end. We propose that this specific composition of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) residues, as in GlcN(n)GlcNAc1, is due to a subsite specificity toward GlcN residues at the −2, −3, and −4 positions of the partially acetylated chitosan substrates. In addition, the enzyme is stable, as evidenced by its long shelf life, and active over a large temperature range, which is of high interest for potential use in industrial processes. It exhibits a kcat of 67.2 s–1 on partially acetylated chitosan substrates. When PpChi was used in combination with a recently discovered fungal auxilary activity (AA11) oxidase, a sixfold increase in the release of oligosaccharides from the lobster shell was measured. PpChi represents an attractive biocatalyst for the green production of highly valuable paCOSs with a well-defined structure and the expansion of the relatively small library of chito-oligosaccharides currently available.

Production of Bacterial Amylase and Evaluation for Starch Hydrolysis

In starch degrading industry, the first step to be performed is gelatinization of starch granules at elevated temperature. This process tends to form a viscose suspension, thus making mixing and pumping a huge challenge. Therefore, to lower the viscosity of starch paste thermostable enzymes are added for breaking down the polysaccharides into oligosaccharides by a process known as liquefaction. Therefore, the availability of these enzymes can be considered a prerequisite to start successful starch liquefaction and saccharification industry. The main objective of this study was, therefore, to produce efficient microbial amylolytic enzymes for starch hydrolysis. One bacterial isolate designated as BACC 2107 was isolated from locally collected samples. The enzyme has optimum activity at pH of 5.0 and 70°C. The addition of calcium in the reaction mixture was shown to stabilize the enzyme. The isolate BACC 2107 was able to grow under solid-state fermentation (SSF) using wheat bran as a solid substrate. Optimum enzyme production was recorded at 32°C, and at 120 h of incubation and at a moisture content of 75%. Addition of peptone to the SSF medium as an organic nitrogen supplement enhanced enzyme production. The enzyme was used to hydrolyze a 25% (w/v) of starch at 70°C and pH 5.0, and the resulting hydrolysate was analyzed on TLC plates. After 4 h hydrolysis, the TLC chromatogram showed the release of glucose, maltose, maltotriose, and other oligosaccharides, indicating that the enzyme is endo-acting alpha-amylase. The results of this study show that amylase from BACC 2107 has a good potential to be used in starch liquefaction and saccharification processes. The ability of the isolate to grow and produce the enzyme under SSF using cheap agro-industrial wastes could help to greatly reduce the production cost of the enzyme.

High Hydrostatic Pressure Assisted by Celluclast® Releases Oligosaccharides from Apple By-Product

A novel and green procedure consisting of high hydrostatic pressure (HHP) aided by a commercial cellulase (Celluclast®) has been applied to valorise the apple by-product, a valuable source of dietary fibre but mainly composed by insoluble fibre. Optimal conditions for solubilisation of dietary fibre were first determined at atmospheric pressure as 2% (w/v) of substrate concentration and 20 Endo-Glucanase Units of cellulase. Monitoring of polysaccharides and oligosaccharides released from apple by-product was carried out by means of a newly validated HPLC method with refractive index detector. A synergistic effect was observed when the combined HHP plus cellulase treatment was used. Thus, the application of 200 MPa at 50 °C for 15 min enabled a significant increase in the release of water-soluble polysaccharides (1.8-fold) and oligosaccharides (3.8-fold), as well as a considerable decrease in the time required (up to 120-fold), compared to control at 0.1 MPa. Therefore, this technology could be a promising alternative approach to transform an industrial by-product into a novel rich-in-oligosaccharide food ingredient and a step forward into shaping the world of prebiotics.

130 Young Scholar Presentation: Can exogenous carbohydrase supplementation to higher-fiber diets improve gut function, microbiota, and growth performance of weaned pigs?

Abstract There is increasing interest in feeding higher-fiber coproducts to weaned pigs due to their potential benefits on gut function and microbiota. However, young pigs are not efficient at utilizing fibrous coproducts. Exogenous carbohydrases can be used to improve nutrient utilization and growth of pigs fed higher levels of coproducts. Previous results regarding the impact of carbohydrases on performance in pigs have been inconsistent, thus a better understanding of associated mechanisms is needed. Using 460 weaned pigs (6.4 ± 0.1 kg), our first study showed that a carbohydrase enzyme blend (EB) improved ADG of weaned pigs fed higher-fiber diets (with added DDGS and wheat middlings) over a 28-d experimental period (P &lt; 0.05). Pigs fed EB-supplemented diets had lower urinary lactulose:mannitol ratio, decreased plasma IL-8 concentration, and greater ileal CLDN3 (claudin 3) mRNA abundance, compared with those fed diets without EB (P &lt; 0.05). These changes may partly explain the improved growth, providing mode of action evidence for carbohydrase in improving performance of weaned pigs. Carbohydrases may also exert prebiotic effects through release of oligosaccharides from fiber degradation. Thus, carbohydrases and dietary fiber may improve disease resilience of young pigs against bacterial infections, for example, enterotoxigenic Escherichia coli (ETEC) induced post-weaning diarrhea. Our second study evaluated the impact of soluble versus insoluble fiber with or without carbohydrases in newly weaned pigs (n = 60; 6.9 ± 0.07 kg) challenged with F18 ETEC. A diet containing a soluble and highly fermentable fiber from sugar beet pulp with added carbohydrases improved (P &lt; 0.05) ADG, tended to increase (P &lt; 0.10) ileal OCLN (occludin) mRNA, increased (P &lt; 0.05) colonic OCLN mRNA, and tended to decrease (P &lt; 0.10) ileal Escherichia-Shigella compared with ETEC-challenged control. Collectively, appropriate use of exogenous carbohydrases in higher-fiber diets (with the right type and amount of enzyme substrate) is promising in improving gut health and growth performance in weaned pigs.

Delineating thermophilic xylanase from Bacillus licheniformis DM5 towards its potential application in xylooligosaccharides production.

In this present study novel endoxylanase producing Bacillus licheniformis DM5 isolated, identified based on 16S rDNA from Garampani hotspring, Assam, India and enzyme was purified. RNA secondary structure predicted the similarity of B. licheniformis DM5 with B. licheniformis ATCC14580. Highest production of xylanase from B. licheniformis DM5 was achieved in the TY medium with cell densities 12g/l and extracellular protein concentration containing xylanase 400mg/l. Partially purified extracellular xylanase displayed optimum pH 6.5 and temperature 50°C. Thermostability of the xylanase at the elevated temperature showed stability between 50 and 60°C retaining its 99% activity. Kinetic parameters of thermophilic xylanase revealed Km 1.5 ± 0.2mg/ml, Vmax 2.7 ± 0.2U/ml and and Kcat 1.8 ± 0.2s-1 against beechwood xylan but ruled out any exo-acting activity against synthetic pNP-xylopyranoside substrate. Time dependent enzymatic hydrolysis of beechwood xylan and preprocessed agrowaste corncob exhibited the release of xylotriose and xylobiose oligosaccharide (XOS) significantly high. Xylobiose and xylotriose exhibited higher binding affinities with BIAXP transporter protein of probiotic bacteria explaining their easy uptake by the cells. Mixed oligosaccharides also exhibited better prebiotic activity by promoting growth of Bifidobacterium infantis and Lactobacillus delbrueckii. Mixed XOS when tested for their cytotoxicity on Hela cell lines in in vitro MTT assay displayed significant lowering of cell viability after 48h and 24h at 100µg/ml to 60% and 50%, respectively. In contrast, cytotoxicity wasn't observed against normal cervical cell line (VK2/E6E7-ATCC-CRL-2616). Therefore, thermophilic endoxylanase from B. licheniformis DM5 could be attributed for the production of prebiotic and anti-inflammatory XOS from agrowaste.

Ulvan lyase from Formosa agariphila and its applicability in depolymerisation of ulvan extracted from three different Ulva species

Members of green macroalgae cause green tides in eutrophicated coastal waters. These green tides pose an environmental issue and an economic burden on coastal municipalities. The biomass from these green tides has a potential to be used as feedstock in biorefinery due to the content of interesting biomacromolecules. Ulvan, an anionic water-soluble polysaccharide, is one of such components, and its depolymerisation to high-value oligosaccharides or fermentable monosaccharides would bring value to green tide biomass which is otherwise left to decompose. However, only a few ulvan depolymerising enzymes are studied to date. Ulvan lyases depolymerise ulvan, via the β-elimination mechanism, leading to release of oligosaccharides with an unsaturated 4‑deoxy‑l‑threo‑hex‑4‑enopyranosiduronic acid at the non-reducing end. In this study, we have identified the presence of two different domains, a catalytic and a non-catalytic, in a putative ulvan lyase from Formosa agariphila KMM 3901. We overexpressed, purified, and biochemically characterised the full-length ulvan lyase, which was found to be most active at a temperature of 45 °C and pH 8.5. It exhibited high specificity for ulvan and did not degrade heparan sulphate, chondroitin sulphate, alginate, pectin or xanthan. Detailed analyses of end products of the enzymatic degradation of ulvan using 1H NMR and LC-MS revealed a disaccharide with an unsaturated uronic acid (∆) linked to 3‑sulphated rhamnose (Rha3S), trisaccharide with xylose (Xyl) flanked by Rha3S (Rha3S-Xyl-Rha3S), tetrasaccharide with an unsaturated uronic acid at the non-reducing end (∆Rha3S-Xyl-Rha3S) and pentasaccharides (Rha3S-Xyl-Rha3S-Xyl-Rha3S and branched ∆Rha3S-Xyl-(∆)Rha3S) as the principal end products. We also found that the catalytic domain that lacks the non-catalytic carbohydrate binding module exhibited higher affinity for the soluble ulvan and efficiently depolymerised it. This study reveals the characteristics of the endolytic ulvan lyase, which is a member of the ulvan utilisation loci in Formosa, and points towards the potential ulvan depolymerisation applications in Ulva biorefinery.