Xylanase Assay Kit (XylX6 Method)

Colourimetric assays were used to measure the activities of six key hydrolases endogenous to barley: β‐glucanase, xylanase, cellulase, α-amylase, beta‐amylase and limit dextrinase. The analysed barley malt samples were previously characterised by 27 conventional malt quality descriptors. Correlations between enzymatic activities and brewing parameters such as extract yield, fermentability, viscosity and filterability were investigated. A single extraction protocol for all six hydrolases was optimised and used for multi‐enzyme analysis using fully automatable assay formats. A regression analysis between malt parameters was undertaken to produce a relationship matrix linking enzyme activities and conventional malt quality descriptors. This regression analysis was used to inform a multi‐linear regression approach to create predictive models for extract yield, apparent attenuation limit, viscosity and filterability using the Small‐scale Wort rapId Filtration Test (SWIFT) and two different mashing protocols – Congress and a modified infusion mash at 65^oC (MIM 65^oC). It was observed that malt enzyme activities displayed significant correlations with the analysed brewing parameters. Both starch hydrolases and cell wall hydrolase activities together with modification parameters (i.e. Kolbach index) were found to be highly correlated with extract yield and apparent attenuation limit. Interestingly, it was observed that xylanase activity in malts was an important predictor for wort viscosity and filterability. It is envisaged that the automatable measurement of enzyme activity could find use in plant breeding progeny selection and for routine assessment of the functional brewing performance of malt batches. This analytical approach would also contribute to brewing process consistency, product quality and reduced processing times.

Stages of the brewing process, such as mash separation to produce wort and beer filtration, can in certain cases prove problematic due to the increased viscosity caused by high levels of the non-starch polysaccharides, primarily β-glucan and arabinoxylan. Of these two polysaccharides, β-glucan has been extensively studied, but arabinoxylan has been somewhat overlooked. The concentration of arabinoxylan present during these process stages is principally expected to be inversely related to the malt endo-1,4-β-xylansase activity that is available to degrade these polysaccharides. The development of a novel method for the measurement of endo-1,4-β-xylansase activity in barley malt extracts is described herein. The method was characterised by two analysts in terms of repeatability (single analyst CVs=2.2% and 2.3%, n=8; interanalyst CV=4.8%, n=16) and sensitivity (LOD=10 U/kg, LOQ=34 U/kg). The assay procedure was then applied to the measurement of xylanase activity in a series of eight standard barley malts and the results obtained were compared with their associated Congress wort viscosities as measured using the conventional EBC Method 4.8, wort viscosity. A highly statistically significant relationship between xylanase activity and wort viscosity was found with a Pearson's correlation coefficient of -0.82 (p-value of 0.007).

endo-1,4-β-Xylanase (EC 3.2.1.8) is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-4⁵-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.

The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.

Onion skins, actually recycled as organic fertilizers, could be used as a substrate in environmental-friendly bioprocesses to recover high-value bioactive compounds and food ingredients. In this work, a bioprospecting method was carried out including 94 bacterial and 45 yeast strains from several agri-food and environmental niches to verify their ability to grow on onion skins as unique nutrients source. Red and yellow onion skins were assessed by newly selected starter-driven liquid submerged fermentation assays mainly aimed at the release and modification of polyphenols through microbial activities. Fermented onion skins were also investigated as a inexpensive favourable source of microbial enzymes (amylases, proteases, lipases, esterases, cellulases, xylanases). In red onion skins, the treatment with Lactiplantibacillus plantarum TB 11-32 produced a slight increase of bioactive compounds in terms of total phenolics, whereas with the yeast strain Zygosaccharomyces mrakii CL 30−29 the quercetin aglycone content increased of about 25% of the initial raw material. In yellow onion skins inoculated, the highest content of phenolic compounds was detected with the yeast strain Saccharomyces cerevisiae En SC, while quercetin aglycone increased of about 60% of the initial raw material in presence of the bacterial strain L. plantarum C 180-34. In conclusion, the proposed microbial pre-treatment method can be a potential strategy to re-use onion skins as a fermentation substrate, and as a first step in the development of a biorefinery process to produce value-added products from onion by-products.

Background: Pulp refining is an energy consuming, but integral part of paper production with the aim to increase tensile strength and smoothness of paper sheets. Commercial enzyme formulations are used to lower the energy requirements by pre-treatment of pulp before refining. However, a high number of different commercial enzyme products are available on the market containing enzymes of varying origin and composition, which complicates the prediction of their behavior, especially using different pulp types. Results: Endoglucanase-rich enzyme formulations were characterized regarding enzyme activity at different temperatures, resulting in a significant decrease of activity above 70°C. Some enzyme preparations additionally contained arabinosidase, xylanase and β-glucosidase activity consequently resulting in a release of xylose and glucose from pulp as determined by high-performance liquid chromatography. Interestingly, one enzyme formulation even showed lytic polysaccharide monooxygenase (LPMO) activity of 3.05 nkat mg⁻¹. A correlation between enzyme activity using the endoglucanase specific derivatized cellopentaose (CellG5) substrate and enzyme performance in laboratory PFI (Papirindustriens forskningsinstitut) refining trials was observed on softwood pulp resulting in a maximum increase in the degree of refining values from 27.7°SR to 32.7°SR. When added to a purified endoglucanase enzyme (31.6°SR), synergistic effects were found for cellobiohydrolase II (34.7°SR) or β-glucosidase enzymes (35.7°SR) in laboratory refining. Comparison with previously obtained laboratory refining results on hardwood pulp allowed differences in enzyme performance based on varying pulp types to be elucidated. Conclusions: Interestingly, the individual enzymes indeed showed different refining effects on softwood and hardwood pulp. This difference could be predicted after development of an adapted enzyme activity assay by combination of the derivatized cellopentaose CellG5 substrate with either softwood or hardwood sulfate pulp.

This study evaluated the impact of feeding xylo-oligosaccharides (XOS), fermentable fiber in the form of wheat bran (WB), and xylanase (XYL) on laying hen productive performance and nutrient digestibility. The hypothesis was that the WB would provide the microbiota in the hindgut with fermentable dietary xylan, and the XOS and XYL would further upregulate xylan fermentation pathways, resulting in improved nutrient utilization. Isa Brown hens (n = 96) were obtained at 39 wk of age. They were fed 12 dietary treatments, 8 hens per treatment, for 56 d. A commercial laying hen ration was fed, and for half of the treatments 10% of this ration was directly replaced with WB. The diets were then supplemented with either 1) no supplements; 2) XOS 50 g/t; 3) XOS 2000 g/t; 4) XYL (16,000 BXU/kg); 5) XYL + XOS 50 g/t, or 6) XYL + XOS 2,000 g/t. Hen performance and egg quality were measured every 14 d. On d56, ileum digesta samples were collected for determination of starch, nonstarch polysaccharide (NSP), XOS, protein, energy, and starch digestibility. Ceca digesta samples were also collected for analysis of XOS, short chain fatty acid (SCFA), xylanase and cellulase activity and microbial counts. Feeding 2,000 g/t XOS increased ileal protein digestibility. Combined 2,000 g/t XOS and XYL increased cecal Bifidobacteria concentration. This combination also increased cecal xylanase activity in birds fed the control diet. Cecal cellulase activity was improved by feeding WB, XYL, and 2,000 g/t XOS. XYL increased cecal lactate production. Feeding 2,000 g/t XOS with WB increased insoluble NSP degradability and shell breaking strength at d56. In summary, supplementing laying hen diets with fermentable fiber, XYL and XOS increases utilization of dietary xylan, improving nutrient utilization, performance, and gastrointestinal health.

The market for nutraceutical molecules is growing at an impressive pace in all Western countries. A convenient source of bioactive compounds is found in vegetable waste products, and their re-use for the recovery of healthy biomolecules would increase the sustainability of the food production system. However, safe, cheap, and sustainable technologies should be applied for the recovery of these beneficial molecules, avoiding the use of toxic organic solvents or expensive equipment. The soil bacterium Bacillus subtilis is naturally endowed with several enzymes targeting complex vegetable polymers. In this work, a raw bacterial culture supernatant was used to assist in the extraction of bioactives using isothermal pressurization cycles. Besides a wild-type Bacillus subtilis strain, a new strain showing increased secretion of cellulases and xylanases, pivotal enzymes for the digestion of the plant cell wall, was also used. Results indicate that the recovery of compounds correlates with the amount of cellulolytic enzymes applied, demonstrating that the pretreatment with non-purified culture broth effectively promotes the release of bioactives from the vegetable matrix. Therefore, this approach is a valid and sustainable procedure for the recovery of bioactive compounds from food waste.

Refining of cellulose fibers is essential for reaching desired paper properties, yet highly energy demanding. Enzymes like endoglucanases (e.g. EndoC) are increasingly used to reduce energy consumption during pulp refining. However, prediction of the enzyme effect is still a major concern, considering the high variety of commercially available enzyme formulations, containing a range of different enzymes. In this study, synergisms of xylanases and β-glucosidases in combination with endoglucanases purified from enzyme formulations were studied and related to their refining performance. Size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) of carboxymethylcellulose revealed that a narrow size distribution and a high reduction in molecular weight are beneficial characteristics for refining. SEC-MALLS of hardwood pulp resulted in pronounced formation of low molecular weight fractions (log MW 4.3) for most efficient refining enzymes. Application of enzyme formulations and combinations of endoglucanase EndoC with β-glucosidase or xylanase using Fourier-transform infrared spectroscopy (FTIR) revealed synergistic effects that promoted degradation of amorphous parts of cellulose. Laboratory refining trials on hardwood pulp confirmed the increase in degree of refining and tensile index after addition of xylanase and β-glucosidase. Surface plasmon resonance (SPR) analysis resulted in strong binding of endoglucanases to regenerated cellulose, which correlated to refining performance.

The aim of this study was to examine non-starch polysaccharide (NSP) degradation in the gastrointestinal tract of chickens fed a range of commercial-type diets supplemented with a commercial dose of xylanase, a double dose of xylanase or a cocktail of NSP – degrading enzymes. Cobb 500 broilers (n = 1,080) were fed 12 dietary treatments; 4 diets with differing primary grain sources (barley, corn, sorghum, and wheat) and three different enzyme treatments (commercial recommended dose of xylanase (16,000 BXU/kg), a double dose of xylanase (32,000 BXU/kg) or an NSP-degrading enzyme cocktail (xylanase, β-glucanase, cellulase, pectinase, mannanase, galactanase, and arabinofuranosidase at recommended commercial levels). There were 108 pens, approximately 10 birds per pen, 9 replicates per dietary treatment. The diets were fed as 3 phases, starter (d 0-12), grower (d 12-23), and finisher (d 23-35). On bird age d 12, 23, and 35, performance (total pen body weight, feed intake, and feed conversion ratio corrected for mortality [cFCR]), litter and excreta dry matter content, and ileal and total tract soluble and insoluble NSP degradability and free oligosaccharide digestibility was determined. On d 35, the quantity of NSP in the gizzard, jejunum, ileum and excreta was determined. Results from this study showed that the double xylanase dose and NSP-ase cocktail had positive impacts on starter phase performance in birds fed the corn- and wheat-based diets. In the grower phase in birds fed the barley-based diet, these enzyme treatments improved cFCR and increased litter dry matter content. The NSP-ase cocktail had a negative impact on finisher phase cFCR in birds fed the sorghum-based diet. The double xylanase dose induced a positive impact on NSP degradability and free oligosaccharide digestibility. In conclusion, there appears to be advantages to feeding broilers a double xylanase dose, but lack of consistency when using an NSP-ase cocktail containing many enzymes.

This study was to examine if it is possible to accelerate sorghum digestion in broiler chickens by targeting fermentation of the xylan. Cobb 500 broilers (n = 960, 80 birds per treatment) were fed 12 sorghum-soybean meal-based dietary treatments fed as 3 phases (starter d 0 to 12, grower d 13 to 23, finisher d 24 to 35), with 8 replicate pens of 10 birds per treatment. For half of the treatments (n = 6), 10% of the sorghum in the diet was directly replaced with 10% wheat bran, as a source of fermentable fibre. The diets were supplemented with either 0, 50 or 2,000 mg/kg xylo-oligosaccharides (XOS), with or without xylanase application. Body weight gain (BWG), feed intake (FI) and feed conversion corrected for mortality (cFCR) was determined at d 0 to 35, and male and female body weight were measured on d 35. On d 35, ileum and caeca samples were collected from 2 birds per pen, for determination of caecal cellulase and xylanase activity, microbiota composition and short chain fatty acid (SCFA) concentration, and ileal XOS concentration. Supplementation with 2,000 mg/kg XOS caused increased BWG at d 0 to 35 (P = 0.007) and enhanced caecal propionic, valeric and succinic acid concentration (P < 0.05). Wheat bran increased FI (P = 0.018) and BWG (P = 0.016), as well as caecal Bifidobacteria concentration (P < 0.001). Ileal XOS concentration was greatest when feeding combined wheat bran, 2,000 mg/kg XOS, and xylanase, resulting in increased caecal total SCFA, acetic acid and butyric acid concentration, and xylanase and cellulase activity (P < 0.05). Results from this study present that feed efficiency in birds fed sorghum-based diets is improved as a consequence of supplementing with fermentable fibre, xylanase and XOS.

A largely defined series of hydrolytic enzymes active during malting and/or mashing, substantially determine the quality, profitability, and efficiency of the brewing process. These enzymes potentially hydrolyze starch, proteins and cell wall non-starch polysaccharides including β-glucan and arabinoxylan. Commercial malts (94) were assayed for the DP enzymes (limit dextrinase, beta/α-amylase), and NSP hydrolyzing enzymes (β-glucanase, xylanase, arabinofuranosidase, β-glucosidase). The levels of enzyme activity were related to conventional measures of malt quality such as extract, fermentability, protein, KI, DP, friability, wort viscosity, FAN, and β-glucan. These parameters were interrelated with less conventional measures of malt quality including coarse extract and fermentability (modified infusion mash 65 °C), lautering efficiency, the Small-scale Wort ‘I’ Filtration Test (SWIFT), and viscosity. Substantial variation was observed between the malt samples for all enzymes assayed. Australian barley, whether malted in Australia (n = 61) or China (n = 24), was observed to be of comparable quality. A limited set of Canadian barley samples (n = 9) were malted in China and produced malts with somewhat higher levels of extract, AAL, and some enzymes. Remarkably, the level of limit dextrinase was observed to be almost double that from previous investigations. Greater levels of steep water aeration were proposed to explain this dramatic increase. The interrelationships between the enzyme activities and malt quality identified, enable potential selection of novel malt quality parameters that are more predictive of a malt’s brewing performance (efficiency and quality) than current measures to provide a malt quality assessment system based on ‘functional’ malt quality.

Arabinoxylan (AX), an important dietary fiber from cereal grains, is mainly metabolised in the large intestine by gut bacteria, especially bifidobacteria. This study investigated the uptake and metabolism of wheat AX by a Bifidobacterium longum strain that could grow well with AX as the sole carbon source. The bacterial growth rate showed a significant correlation to the molecular weight (MW) of AX and its acid hydrolysates. Assessment of the key AX degrading enzymes suggested that the uptake and consumption of AX involved extracellular cleavage of xylan backbone and intracellular degradation of both the backbone and the arabinose substitution. The preference for native or partially hydrolysed AX with single substitutions and a sufficiently high MW suggested the structure-dependant uptake by the bacterial cells. Genetic analysis of B. longum showed the lack of β-xylosidase, suggesting the existence of unknown enzymes or dual/multiple-specific enzymes for hydrolysis of the non-reducing end of xylan backbone.

The effects of coextruded full-fat flaxseed and pulses (FFF; 1:1 wt/wt) mixture on n-3 polyunsaturated fatty acids (PUFA) enrichment in egg yolk, hepatic attributes, apparent retention (AR) of components, and ceca metabolites were evaluated in broiler breeder hens. The diets were as follows: 1) corn–soybean control, 2) control diet plus 18% FFF (FFF−), and 3) FFF plus enzyme supplement (FFF+) containing galactanase, protease, mannanase, glucanase, xylanase, amylase, and cellulase activities. Twenty-six-week-old Cobb 500 broiler breeder hens were allocated to 30 identical cages (2 hens/cage) and given 1-week adaptation period. The 3 diets were assigned to 10 replicate cages based on postadaptation BW and fed based on breeder curve for 30 D. Excreta samples were collected from day 24 to 27 for determination of AR of components, and eggs were collected from day 28 to 30 for yolk polyunsaturated fatty acids analyses. On day 30, birds were weighed, killed via cervical dislocation, liver weighed, and stored for fat analyses. Ceca digesta samples were taken for concentration of short-chain fatty acids. Liver and yolk weights as well as total yolk FA were not influenced by diets (P > 0.05). Control birds had lower yolk concentration of α-linolenic acid than birds fed either FFF− or FFF+ (P < 0.01) corresponding to 7.5, 36.8, and 37.3 mg/g for the control, FFF−, and FFF+, respectively. Control birds also exhibited lower yolk concentration of docosahexaenoic acid (P < 0.01). Control birds had higher hepatic concentration of crude fat and apparent retention of dry matter and crude protein compared with either the FFF− or FFF+ birds (P < 0.05). Birds fed FFF- diet had lower ceca digesta concentration of lactic acid than control and FFF+ (P < 0.05) birds. Results showed broiler breeder hens enriched egg yolk with n-3 polyunsaturated fatty acids without effects on the liver while the supplemental enzyme did not improve the utilization of FFF.

This study aimed to investigate the effect of dietary supplementation with xylanase and probiotics on growth performance and intestinal health of nursery pigs challenged with enterotoxigenic Escherichia coli (ETEC). Sixty-four newly weaned pigs (32 barrows and 32 gilts with 7.9 ± 0.4 kg BW) were allotted in a randomized complete block design (2 × 2 factorial). Two factors were ETEC challenge (oral inoculation of saline solution or E. coli F18⁺ at 6 × 10⁹ CFU) and synbiotics (none or a combination of xylanase 10,000 XU/kg and Bacillus sp. 2 × 10⁸ CFU/kg). All pigs were fed experimental diets following NRC (2012) in two phases (P1 for 10 d and P2 for 11 d). The ETEC was orally inoculated on d 7 after weaning. Feed intake and BW were measured on d 7, 10, 15, and 20. On d 20, pigs were euthanized to collect samples to measure gut health parameters and microbiome. Synbiotics increased (P < 0.05) ADG in phase 1 and ETEC reduced (P < 0.05) ADG and G:F in the post-challenge period. ETEC increased (P < 0.05) the fecal score of pigs from d 7 to 13; however, synbiotics reduced (P < 0.05) it at d 9 and 11 in challenged pigs. ETEC increased (P < 0.05) mucosal MDA, IL-6, Ki-67⁺, and crypt depth, whereas synbiotics tended to reduce TNFα (P = 0.093), protein carbonyl (P = 0.065), and IL-6 (P = 0.064); reduced (P < 0.05) crypt depth and Ki-67⁺; and increased (P < 0.05) villus height. ETEC reduced (P < 0.05) the relative abundance of Bacteroidetes and Firmicutes and increased (P < 0.05) the relative abundance of Proteobacteria. In conclusion, ETEC challenge reduced growth performance by affecting microbiome, immune response, and oxidative stress in the jejunum. Synbiotics enhanced growth performance by reducing diarrhea, immune response, and oxidative stress in the jejunum.