D-Glucuronic/D-Galacturonic Acid Assay Kit

Background: The level of available carbohydrates in our diet is directly linked to two major diseases; obesity and Type II diabetes. Despite this, to date there is no method available to allow direct and accurate measurement of available carbohydrates in human and animal foods. Objective: The aim of this research was to develop a method that would allow simple and accurate measurement of available carbohydrates, defined as non-resistant starch, maltodextrins, maltose, isomaltose, sucrose, lactose, glucose, fructose and galactose. Method: Non-resistant (digestible) starch is hydrolysed to glucose and maltose by pancreatic α-amylase and amyloglucosidase at pH 6.0 with shaking or stirring at 37°C for 4 h. Sucrose, lactose, maltose and isomaltose are completely hydrolyzed by specific enzymes to their constituent monosaccharides, which are then measured using pure enzymes in a single reaction cuvette. Results: A method has been developed that allows the accurate measurement of available carbohydrates in all cereal, vegetable, fruit, food, and feed products, including dairy products. Conclusions: A single-laboratory validation was performed on a wide range of food and feed products. The inter-day repeatability (%RSDr) was <3.58% (w/w) across a range of samples containing 44.1 to 88.9% available carbohydrates. The LOD and LOQ obtained were 0.054% (w/w) and 0.179% (w/w), respectively. The method is all inclusive, specific, robust and simple to use. Highlights: A unique method has been developed for the direct measurement of available carbohydrates, entailing separate measurement of glucose, fructose and galactose; information of value in determining the glycemic index of foods.

Fucoidan, a sulphated polysaccharide found in brown seaweeds, has gained attention for its potential as an α-glucosidase inhibitor (AGI), which is significant for managing type 2 diabetes mellitus (T2DM). Although the use of brown algae for fucoidan extraction is becoming more popular, a few challenges remain, such as low extraction yields and a lack of understanding of the structure-activity relationship of fucoidan as an AGI. This study compared fucoidan extraction yields from S. elegans using three novel extraction methods (citrate buffer, EDTA, and enzyme-assisted extraction) and two well-established extraction protocols (hot water and mineral acid extraction). The structure-activity relationships of the fucoidan extracts as potential AGIs were then investigated. The fucoidan extraction yields for enzyme-assisted, citrate buffer, EDTA, hot water extraction and mineral acid extraction were 28%, 20%, 15%, and 2.5% and 3%, respectively. Distinct structural features were noted from the fucoidan extracted by different techniques, according to their chemical profiles confirmed by their carbohydrate content, FTIR and NMR, and physical characteristics. Each extract exhibited unique chemical and structural properties. With IC₅₀ values of 25 and 88 μg/ml, respectively, the fucoidans extracted by water and citrate buffer demonstrated the highest potency as AGIs, whereas the fucoidans extracted with the assistance of enzymes and EDTA showed IC₅₀ values of 186 and 273 μg/ml. In summary, the extraction technology affected the fucoidans' structural and chemical compositions, impacting their biological activity as AGIs. Fucoidan's potent activity as an AGI represents a promising strategy for mitigating the burden of diabetes by reducing or preventing hyperglycaemia. Leveraging fucoidan could contribute meaningfully to developing alternative, natural therapeutic interventions for managing T2DM.

Lignin-carbohydrate esters (LC-esters) formed by glucuronoarabinoxylan and lignin are a key factor for the recalcitrance of corn bran, understanding LC-esters change during pretreatment and enzymatic hydrolysis by glucuronoyl esterases (GEs) is essential to the sustainable utilization of corn bran. Herein, hydrolysis performances of three GEs, SbGE15A, SbGE15B, and SbGE15C from Sordaria brevicollis with different subclades and modularity, and changes in enzyme-reachable LC-esters during different pretreatments of corn bran have been comprehensively compared. F_B enzymes, SbGE15B and SbGE15C showed higher catalytic activity towards model and natural substrates than F_A enzyme, SbGE15A. Particularly, SbGE15C harboring carbohydrate-binding module 1 (CBM1) exhibited much superior catalytic performance and synergistic effect with GH10 endo-xylanase EpXYN1 from Eupenicillium parvum on pretreated residues than SbGE15A and SbGE15B without CBM1. Autohydrolysis and DES (ChCl-LA) pretreatment could decrease the content of enzyme-reachable LC-esters and depolymerize its structure, transitioning from Lignin-(Me)GlcA-Xylan to Lignin-(Me)GlcA-XOS, and eventually to Lignin-(Me)GlcA with increasing pretreatment time. These changes consequently cause a decrease in synergy between SbGE15s and EpXYN1 or commercial enzyme cocktails on pretreated residues. The findings provide new insights into significant changes in enzyme-reachable LC-esters depending on the pretreatment method and intensity and the consequent influence of these changes on the catalytic action of GEs.

Glucuronoyl esterases (GEs) are carbohydrate active enzymes in carbohydrate esterase family 15 which are involved in the hydrolysis of lignin-carbohydrate complexes. They are encoded by a wide range of aerobic and anaerobic fungi and bacteria inhabiting diverse environments. The rumen microbiome is a complex microbial community with a wide array of enzymes that specialize in deconstructing plant cell wall carbohydrates. Enzymes from the rumen tend to show low similarity to homologues found in other environments, making the rumen microbiome a promising source for the discovery of novel enzymes. Using a combination of phylogenetic and structural analysis, we investigated the structure-function relationship of GEs from the rumen bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens, and from the rumen fungus, Piromyces rhizinflata. All adopt a canonical α/β hydrolase fold and possess a structurally conserved Ser-His-Glu/Asp catalytic triad. Structural variations in the enzymes are localized to loops surrounding the active site. Analysis of the active site structures in these enzymes emphasized the importance of structural plasticity in GEs with non-canonical active site conformations. We hypothesize that interkingdom HGT events may have contributed to the diversity of GEs in the rumen, and this is demonstrated by the phylogenetic and structural similarity observed between rumen bacterial and fungal GEs. This study advances our understanding of the structure-function relationship in glucuronoyl esterases and illuminates the evolutionary dynamics that contribute to enzyme diversity in the rumen microbiome.

Considering an ever-growing global population, which hit 8 billion people in the fall of 2022, it is essential to find solutions to avoid croplands competition between human food and animal feed. Agricultural co-products such as soybean meals have become important components of the circular economy thanks to their use in animal feed. Their implementation was made possible by the addition of exogenous enzymes in the diet of monogastric animals, especially fungal carbohydrate-active enzymes (CAZymes). Here, we describe a time-course production and analysis of Aspergillus terreus secretomes for the identification of CAZymes able to enhance the digestibility of soybean meals. Functional assays revealed that the release of nutrients and the degradation of pectins in soybean meals can be tightly interconnected. Using a comparative proteomics approach, we identified several fungal pectin-degrading enzymes leading to increased assimilable nutrients in the soluble fraction of soybean meals. Our results reinforce the importance of deconstructing pectic polysaccharides in feedstuffs and contribute to sharpen our understanding of the fungal enzymatic interplays involved in pectin hydrolysis.IMPORTANCEIn the present study, we developed a strategy to identify the key fungal enzymatic activities involved in the improvement of soybean meal (SBM) digestibility. Our data unravel the importance of pectin degradation for the release of nutrients from SBM and provide some insights regarding the degradation of rhamnogalacturonan-I (RG-I) by ascomycetes. Indeed, the hydrolysis of pectins and RG-I by human microbiota is well documented in the literature, but our knowledge of the fungal CAZymes at play for the degradation of soybean pectins remains hitherto underexplored. Due to its wide use in animal feed, improving the digestibility of SBM by enzymatic treatments is a current challenge for feed additive suppliers. Since non-starch polysaccharides and pectins have often been reported for their anti-nutritional role in SBM, we believe this study will provide new avenues toward the improvement of enzymatic cocktails for animal nutrition and health.

Glucuronoyl esterases (GEs) are serine-type hydrolase enzymes belonging to carbohydrate esterase family 15 (CE15), and they play a central role in the reduction of recalcitrance in plant cell walls by cleaving ester linkages between glucuronoxylan and lignin in lignocellulose. Recent studies have suggested that bacterial CE15 enzymes are more heterogeneous in terms of sequence, structure, and substrate preferences than their fungal counterparts. However, the sequence space of bacterial GEs has still not been fully explored, and further studies on diverse enzymes could provide novel insights into new catalysts of biotechnological interest. To expand our knowledge on this family of enzymes, we investigated three unique CE15 members encoded by Dyadobacter fermentans NS114^T, a Gram-negative bacterium found endophytically in maize/corn (Zea mays). The enzymes are dissimilar, sharing ≤ 39% sequence identity to each other‚ and were considerably different in their activities towards synthetic substrates. Combined analysis of their primary sequences and structural predictions aided in establishing hypotheses regarding specificity determinants within CE15, and these were tested using enzyme variants attempting to shift the activity profiles. Together, the results expand our existing knowledge of CE15, shed light into the molecular determinants defining specificity, and support the recent thesis that diverse GEs encoded by a single microorganism may have evolved to fulfil different physiological functions.

Background: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. Results: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L^− 1 after 48 h under oxygen limited condition in bioreactor fermentations. Conclusion: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast’s expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Brown algae are gaining recognition as sources of bio-compounds with diverse properties and potential applications in the food, nutraceutical, and pharmaceutical industries. Compounds such as polyphenols, alginates and fucoidan possess multiple bioactivities, including antidiabetic, antioxidant, anticancer, anti-inflammatory, and antibacterial properties. Conventional extraction methods provide low yields, posing challenges for the industrial applications of biocompounds. However, innovations are rapidly emerging to address these challenges, and one such approach is enzyme-assisted extraction. Furthermore, extracting single compounds undervalues algal biomass as valuable compounds may remain in the waste. Therefore, the aim of our study was to develop a framework for the sequential and enzyme-assisted extraction of various bio-compounds using the same biomass in a biorefinery process. The Ecklonia maxima algal biomass was defatted, and polyphenols were extracted using solid-liquid extraction with aqueous ethanol. The remaining residue was treated with an enzyme combination (Cellic® Ctec 2 and Viscozyme L) to liberate carbohydrates into solution, where an alginate and fucoidan fraction were isolated. A second alginate fraction was harvested from the residue. The phenolic fraction yielded about 11% (dry weight of extract/dry weight of seaweed biomass), the alginate fraction 35% and the fucoidan fraction 18%. These were analysed using a variety of biochemical methods. Structural analyses, including FTIR, NMR and TGA, were performed to confirm the integrity of these compounds. This study demonstrated that a sequential extraction method for various algal bioproducts is possible, which can pave the way for a biorefinery approach. Furthermore, our study primarily employed environmentally and eco-friendly extraction technologies promoting an environmentally sustainable industrial approach. This approach enhances the feasibility and flexibility of biorefinery operations, contributing to the development of a circular bio-economy.

The study involved the development and modeling of a fixed-bed bioreactor for the removal of urea from wines. The reactor, based on the immobilization of acid urease enzyme, was studied under both stationary and non-stationary conditions. The developed model, including internal and external catalyst particle mass transfer, Michaelis-Menten kinetics, convection and dispersion in the liquid along the reactor axis, was able to produce urea concentration profiles in both the solid and liquid phases under various volumetric flow rates and inlet urea concentrations. The experimental results were in good agreement with the model predictions, the mean relative error between simulated and experimental outlet ammonia concentration ranging from 4.1 % to 16.4 %. Model simulations confirmed that in wines the reaction kinetics is of the pseudo-first order and that internal and external catalyst particle diffusion limitations are negligeable. Simulations of the decrease of urea concentration as a function of space velocity for the reactor under study operating in the continuous mode and for three different wines were obtained confirming that urea removal by immobilized urease in wines is more difficult than in sake. The results obtained form the basis for the designing and scaling up of bioreactors for the treatment of wines.

The influence of different extrusion conditions on the cocoa shell (CS) dietary fiber, phenolic compounds, and antioxidant and functional properties was evaluated. Extrusion produced losses in the CS dietary fiber (3-26%), especially in the insoluble fraction, being more accentuated at higher temperatures (160°C) and lower moisture feed (15-20%). The soluble fiber fraction significantly increased at 135°C because of the solubilization of galactose- and glucose-containing insoluble polysaccharides. The extruded CS treated at 160°C-25% of feed moisture showed the highest increase of total (27%) and free (58%) phenolic compounds, accompanied by an increase of indirect (10%) and direct (77%) antioxidant capacity. However, more promising results relative to the phenolic compounds’ bioaccessibility after in vitro simulated digestion were observed for 135°C-15% of feed moisture extrusion conditions. The CS’ physicochemical and techno-functional properties were affected by extrusion, producing extrudates with higher bulk density, a diminished capacity to hold oil (22-28%) and water (18-65%), and improved swelling properties (14-35%). The extruded CS exhibited increased glucose adsorption capacity (up to 2.1-fold, at 135°C-15% of feed moisture) and α-amylase in vitro inhibitory capacity (29-54%), accompanied by an increase in their glucose diffusion delaying ability (73-91%) and their starch digestion retardation capacity (up to 2.8-fold, at 135°C-15% of feed moisture). Moreover, the extruded CS preserved its cholesterol and bile salts binding capacity and pancreatic lipase inhibitory properties. These findings generated knowledge of the CS valorization through extrusion to produce foods rich in dietary fiber with improved health-promoting properties due to the extrusion-triggered fiber solubilization.

Plant polysaccharides have attracted much attention because of their various biological activities. The structure characterization of polysaccharide from durian seed and its neuroprotective effects against Alzheimer's disease in a transgenic Caenorhabditis elegans model were conducted in this study. A water-soluble polysaccharide was obtained using atmospheric pressure plasma treatment, and named DSPP-1. DSPP-1 was composed of rhamnose, galactose and galacturonic acid and its molecular weight was 3.765×10⁵ Da. The study in vitro showed that DPPH radical scavenging activity of DSPP-1 was 79.20% and the inhibitory rate on Aβ_1-42 aggregation was 24.65%. In vivo results showed that DSPP-1 could decrease abnormal Aβ 1-42 aggregation to delay the paralysis process of AD-nematodes. Moreover, DSPP-1 significantly improved the antioxidant enzyme activities and reduced lipid peroxidation in AD-nematodes. Taken together, these results indicated that DSPP-1 could be used as a potential natural source for the prevention and treatment of AD.

Bacterial Cellulose (BC) is a biopolymer with numerous applications. The growth of BC-producing bacteria, Komagataeibacter intermedius, could be stimulated by Dekkera bruxellensis, however, the effect on BC yield needs further investigation. This study investigates BC production and biochemical changes in the K. intermedius-D. bruxellensis co-culture system. D. bruxellensis was introduced at various concentrations (10³ and 10⁶ CFU/mL) and inoculation times (days 0 and 3). BC yield was ~24% lower when D. bruxellensis was added at 10³ CFU/mL compared to K. intermedius alone (0.63 ± 0.11 g/L). The lowest BC yield was observed when 10³ CFU/mL yeast was added on day 0, which could be compromised by higher gluconic acid production (10.08 g/L). In contrast, BC yields increased by ~88% when 10⁶ CFU/mL D. bruxellensis was added, regardless of inoculation time. High BC yield might correlate with faster sugar consumption or increased ethanol production when 10⁶ CFU/mL D. bruxellensis was added on day 0. These results suggest that cell concentration and inoculation time have crucial impacts on species interactions in the co-culture system and product yield.

Guignardia bidwellii, indigenous to North America, is a significant pathogen of grapes long known in Hungary, infecting only the growing green parts of the vine (leaves, petioles, shoots, and bunches). In the absence of adequate plant protection and extreme weather conditions such as a predominantly humid, warm year, black rot of grapes can be expected. The pathogen can cause high yield losses due to grape rot and reduce wine quality if the infection is severe. The evolution of certain biogenic amine compounds were investigated under the influence of grape black rot. The results obtained showed that they were present in low concentrations from an oenological point of view. Polyphenol composition was consistent with the literature, blackening affected mainly the concentration of catechin. Black rot fungus does not produce β-glucosidase enzyme. In terms of resveratrol content, black rot has no particular effect. However, like Botrytis cinerea, it produces glycerol and, proportionally, gluconic acid in lower concentrations. It can be concluded that black rot of grapes does not cause health problems when introduced into wine processing.

Although there are chemotherapeutic efforts in place for Type 2 diabetes mellitus (T2DM), there is a need for novel strategies (including natural products) to manage T2DM. Fucoidan, a sulphated polysaccharide was extracted from Ecklonia radiata. The integrity of the fucoidan was confirmed by structural analysis techniques such as FT-IR, NMR and TGA. In addition, the fucoidan was chemically characterised and tested for cell toxicity. The fucoidan was investigated with regards to its potential to inhibit α-amylase and α-glucosidase. The fucoidan was not cytotoxic and inhibited α-glucosidase (IC₅₀ 19 µg/mL) more strongly than the standard commercial drug acarbose (IC₅₀ 332 µg/mL). However, the fucoidan lacked potency against α-amylase. On the other hand, acarbose was a more potent inhibitor of α-amylase (IC₅₀ of 109 µg/mL) than α-glucosidase. Due to side effects associated with the use of acarbose, a combination approach using acarbose and fucoidan was investigated. The combination showed synergistic inhibition (>70%) of α-glucosidase compared to when the drugs were used alone. The medicinal implication of this synergism is that a regimen with a reduced acarbose dose may be used, thus minimising side effects to the patient, while achieving the desired therapeutic effect for managing T2DM.

The environmental yeast Cryptococcus neoformans is the most common cause of deadly fungal meningitis in primarily immunocompromised populations. A number of factors contribute to cryptococcal pathogenesis. Among them, inositol utilization has been shown to promote C. neoformans development in nature and invasion of central nervous system during dissemination. The mechanisms of the inositol regulation of fungal virulence remain incompletely understood. In this study, we analyzed inositol-induced capsule growth and the contribution of a unique inositol catabolic pathway in fungal development and virulence. We found that genes involved in the inositol catabolic pathway are highly induced by inositol, and they are also highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. This pathway in C. neoformans contains three genes encoding myo-inositol oxygenases that convert myo-inositol into d-glucuronic acid, a substrate of the pentose phosphate cycle and a component of the polysaccharide capsule. Our mutagenesis analysis demonstrates that inositol catabolism is required for C. neoformans virulence and deletion mutants of myo-inositol oxygenases result in altered capsule growth as well as the polysaccharide structure, including O-acetylation. Our study indicates that the ability to utilize the abundant inositol in the brain may contribute to fungal pathogenesis in this neurotropic fungal pathogen.