Deciphering the Myrosinase-like Activity of Lactiplantibacillus plantarum WCFS1 among GH1 Family Glycoside Hydrolases.

The hydrolysis of plant glucosinolates by myrosinases (thioglucosidases) originates metabolites with chemopreventive properties. In this study, the ability to hydrolyze the glucosinolate sinigrin by cultures or protein extracts of Lactiplantibacillus plantarum WCFS1 was assayed. This strain possesses myrosinase-like activity as sinigrin was partly hydrolyzed by induced cultures but not by protein extracts. The 11 glycoside hydrolase GH1 family proteins, annotated as 6-phospho-ß-glucosidases, were the proteins most similar to plant myrosinases. The activity of these proteins was assayed against sinigrin and synthetic glucosides. As expected, none of the proteins assayed possessed myrosinase activity against sinigrin or the synthetic ß-thio-glucoside derivative or against the ß-glucoside. However, all 11 proteins were active on the phosphorylated-ß-glucoside derivative. Moreover, only eight of these proteins were active on phospho-ß-thioglucose. These results supported that, in L. plantarum WCFS1, glucosinolates may undergo previous phosphorylation, and GH1 proteins are the glycosidases involved in the hydrolysis of phosphorylated glucosinolates.

Production and Digestibility Studies of ß-Galactosyl Xylitol Derivatives Using Heterogeneous Catalysts of LacA ß-Galactosidase from Lactobacillus Plantarum WCFS1.

The synthesis of ß-galactosyl xylitol derivatives using immobilized LacA ß-galactosidase from Lactobacillus plantarum WCFS1 is presented. These compounds have the potential to replace traditional sugars by their properties as sweetener and taking the advantages of a low digestibility. The enzyme was immobilized on different supports, obtaining immobilized preparations with different activity and stability. The immobilization on agarose-IDA-Zn-CHO in the presence of galactose allowed for the conserving of 78% of the offered activity. This preparation was 3.8 times more stable than soluble. Since the enzyme has polyhistidine tags, this support allowed the immobilization, purification and stabilization in one step. The immobilized preparation was used in synthesis obtaining two main products and a total of around 68 g/L of ß-galactosyl xylitol derivatives and improving the synthesis/hydrolysis ratio by around 30% compared to that of the soluble enzyme. The catalyst was recycled 10 times, preserving an activity higher than 50%. The in vitro intestinal digestibility of the main ß-galactosyl xylitol derivatives was lower than that of lactose, being around 6 and 15% for the galacto-xylitol derivatives compared to 55% of lactose after 120 min of digestion. The optimal amount immobilized constitutes a very useful tool to synthetize ß-galactosyl xylitol derivatives since it can be used as a catalyst with high yield and being recycled for at least 10 more cycles.

Application of sunflower pectin gels with low glycemic index in the coating of fresh strawberries stored in modified atmospheres.

BACKGROUND: This study reports the use of low glycemic sunflower pectin gel, elaborated with calcium and without or with sweeteners (sucrose, stevia and saccharin) as an edible coating and its possible combination with two modified atmosphere packaging (MAP), in order to extend shelf life, maintaining the quality, of strawberries during the storage at 4 °C. RESULTS: This pectin coating, formed with only calcium and/or stevia or saccharin, extended the shelf life of strawberries with respect to uncoating fruits, up to 12 days, keeping the microbial load constant, the firmness and less weight loss. With the same edible coatings, the shelf life of strawberries was extended up to 23 days when they were combined with MAP [10% carbon dioxide (CO2 ), 85% nitrogen (N2 ) and 5% oxygen (O2 )], maintaining the quality of strawberries, while the other MAP, with a higher CO2 concentration (20% CO2 , 75% N2 and 5% O2 ), had no effect. CONCLUSIONS: These results highlight the suitability of the combination of edible pectin coating combined with MAP to obtain an important shelf life extension, maintaining the good quality of the fruit. © 2021 Society of Chemical Industry.

High-Temperature Short-Time and Holder Pasteurization of Donor Milk: Impact on Milk Composition.

Holder pasteurization (HoP; 62.5 °C, 30 min) is commonly used to ensure the microbiological safety of donor human milk (DHM) but diminishes its nutritional properties. A high-temperature short-time (HTST) system was designed as an alternative for human milk banks. The objective of this study was to evaluate the effect of this HTST system on different nutrients and the bile salt stimulated lipase (BSSL) activity of DHM. DHM was processed in the HTST system and by standard HoP. Macronutrients were measured with a mid-infrared analyzer. Lactose, glucose, myo-inositol, vitamins and lipids were assayed using chromatographic techniques. BSSL activity was determined using a kit. The duration of HTST treatment had a greater influence on the nutrient composition of DHM than did the tested temperature. The lactose concentration and the percentage of phospholipids and PUFAs were higher in HTST-treated than in raw DHM, while the fat concentration and the percentage of monoacylglycerides and SFAs were lower. Other nutrients did not change after HTST processing. The retained BSSL activity was higher after short HTST treatment than that following HoP. Overall, HTST treatment resulted in better preservation of the nutritional quality of DHM than HoP because relevant thermosensitive components (phospholipids, PUFAs, and BSSL) were less affected.

Artichoke pectic oligosaccharide characterisation and virtual screening of prebiotic properties using in silico colonic fermentation.

The aim of this work was to develop a comprehensive workflow to elucidate molecular features of artichoke pectic oligosaccharides (POS) contributing to high potential prebiotic activity. First, obtainment of artichoke POS by Pectinex® Ultra-Olio was optimised using an artificial neural network. Under optimal conditions (pH 6.86; 1.5 h; enzyme dose 520.5 U/g pectin) POS yield was 624 mg/g pectin. Oligosaccharide structures (Mw < 1.3 kDa) were characterised by MALDI-TOF-MS. Then, conformational analysis of glycosidic bonds was performed by replica exchange molecular dynamics simulations and interaction mechanisms between POS and several microbial glycosidases were proposed by molecular modelling. Chemical information was integrated in virtual simulations of colonic fermentation. Highest hydrolysis rate was obtained for GalA-Rha-GalA trisaccharide, while the presence of partial negative charges and high radius of gyration enhance short chain fatty acid formation in distal colon. Established structure-activity relationships could help the rational design of prebiotics and clinical trials.

Biosynthesis of Nondigestible Galactose-Containing Hetero-oligosaccharides by Lactobacillus plantarum WCFS1 MelA α-Galactosidase.

This work describes the high capacity of MelA α-galactosidase from Lactobacillus plantarum WCFS1 to transfer galactosyl residues from melibiose to the C6-hydroxyl group of disaccharide-acceptors with ß-linkages (lactulose, lactose, and cellobiose) or α-linkages (isomaltulose and isomaltose) to produce novel galactose-containing hetero-oligosaccharides (HOS). A comprehensive nuclear magnetic resonance characterization of the transfer products derived from melibiose:lactulose reaction mixtures revealed the biosynthesis of α-d-galactopyranosyl-(1 → 6)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose as the main component as well as the presence of α-d-galactopyranosyl-(1 → 3)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose and α-d-galactopyranosyl-(1 → 6)-α-d-galactopyranosyl-(1 → 6)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose. Melibiose-derived α-galactooligosaccharides (α-GOS), manninotriose and verbascotetraose, were also simultaneously synthesized. An in vitro assessment of the intestinal digestibility of the novel biosynthesized HOS revealed a high resistance of α-galactosides derived from lactulose, lactose, cellobiose, and isomaltulose. According to the evidence gathered for conventional α-GOS and certain disaccharides used as acceptors in this work, these novel nondigestible α-galactosides could be potential candidates to selectively modulate the gut microbiota composition, among other applications, such as low-calorie food ingredients.

Hydrolysis and transglycosylation activities of glycosidases from small intestine brush-border membrane vesicles.

In order to know the catalytic activities of the disaccharidases expressed in the mammalian small intestinal brush-border membrane vesicles (BBMV) high concentrated solutions of sucrose, maltose, isomaltulose, trehalose and the mixture sucrose:lactose were incubated with pig small intestine disaccharidases. The hydrolysis and transglycosylation reactions generated new di- and trisaccharides, characterized and quantified by GC-MS and NMR, except for trehalose where only hydrolysis was detected. In general, α-glucosyl-glucoses and α-glucosyl-fructoses were the most abundant structures, whereas no fructosyl-fructoses or fructosyl-glucoses were found. The in-depth structural characterization of the obtained carbohydrates represents a new alternative to understand the potential catalytic activities of pig small intestinal disaccharidases. The hypothesis that the oligosaccharides synthesized by glycoside hydrolases could be also hydrolysed by the same enzymes was confirmed. This information could be extremely useful in the design of new non-digestible or partially digestible oligosaccharides with potential prebiotic properties.

Hydrolysis of Lactose and Transglycosylation of Selected Sugar Alcohols by LacA ß-Galactosidase from Lactobacillus plantarum WCFS1.

The production, biochemical characterization, and carbohydrate specificity of LacA ß-galactosidase (locus lp_3469) belonging to the glycoside hydrolase family 42 from the probiotic organism Lactobacillus plantarum WCFS1 are addressed. The ß-d-galactosidase activity was maximal in the pH range of 4.0-7.0 and at 30-37 °C. High hydrolysis capacity toward the ß(1 → 4) linkages between galactose and glucose (lactose) or fructose (lactulose) was found. High efficiency toward galactosyl derivative formation was observed when lactose and glycerol, xylitol, or erythritol were used. Galactosyl derivatives of xylitol were characterized for the first time as 3-O-ß-d-galactopyranosyl-xylitol and 1-O-ß-d-galactopyranosyl-xylitol, displaying high preference of LacA ß-galactosidase for the transfer of galactosyl residues from lactose to the C1 or C3 hydroxyl group of xylitol. These results indicate the feasibility of using LacA ß-galactosidase for the synthesis of different galactosyl-polyols, which could be promising candidates for beneficial and appealing functional and technological applications such as novel prebiotics or hypocaloric sweeteners.

Hydrolysis and transgalactosylation catalysed by ß-galactosidase from brush border membrane vesicles isolated from pig small intestine: A study using lactulose and its mixtures with lactose or galactose as substrates.

Enzymatic transgalactosylation, in different concentrated carbohydrate solutions, was investigated using brush border membrane vesicles (BBMV) from the pig small intestine. When lactulose was incubated with BBMV, the hydrolytic activity of the enzyme towards the disaccharide was observed to be very low compared to that towards the lactose, but the linkage specificity ß-(1 â†’ 3), previously observed in lactose solutions, was not significantly affected. As in the case of lactose, lactulose transgalactosylation by BBMV synthesizes the corresponding 3'-galactosyl derivative (ß-Gal-(1 â†’ 3)-ß-Gal-(1 â†’ 4)-ß-Fru). Fructose released during lactulose hydrolysis was found to be good acceptor for the transgalactosylation reaction, giving rise to the synthesis of the disaccharide ß-Gal-(1 â†’ 5)-Fru. When incubating an 80/20 mixture of lactulose/galactose, the presence of galactose did not affect the qualitative composition of the transglycosylated substrate but enhanced the synthesis of ß-Gal-(1 â†’ 5)-Fru and decreased the synthesis of ß-(1 â†’ 3) glycosidic bonds. The marked tendency for synthesizing this linkage indicates that under hydrolytic conditions, ß-Gal-(1 â†’ 3)-Gal- and ß-Gal-(1 â†’ 5)-Fru glycosidic bonds would be preferentially digested.

Unravelling the carbohydrate specificity of MelA from Lactobacillus plantarum WCFS1: An α-galactosidase displaying regioselective transgalactosylation.

This comprehensive work addresses, for the first time, the heterologous production, purification, biochemical characterization and carbohydrate specificity of MelA, a cold-active α-galactosidase belonging to the Glycoside Hydrolase family 36, from the probiotic organism Lactobacillus plantarum WCFS1. The hydrolytic activity of MelA α-galactosidase on a wide range of p-nitrophenyl glycoside derivatives and carbohydrates of different molecular-weights showed its high selectivity and efficiency towards the α(1 â†’ 6) glycosidic bonds involving the anomeric carbon of galactose and the C6-hydroxyl group of galactose or glucose units. MelA α-galactosidase also presented a high regioselectivity, efficiency and diversity in accommodating donor and acceptor substrates for the synthesis of α-GOS through transgalactosylation reactions. The catalytic mechanism of MelA for the production of α-GOS was elucidated, revealing its great preference for the transfer of galactosyl residues to the C6-hydroxyl group of galactose units to elongate the chain of α-GOS having either a terminal sucrose (raffinose family oligosaccharides, RFOS) or a terminal glucose (melibiose, manninotriose and verbascotetraose). Our findings indicate the feasibility of using MelA α-galactosidase from Lactobacillus plantarum WCFS1 in the hydrolysis of RFOS and in the efficient and versatile synthesis of α-GOS with appealing functional properties in the context of food and nutraceutical applications.

Intestinal anti-inflammatory effects of artichoke pectin and modified pectin fractions in the dextran sulfate sodium model of mice colitis. Artificial neural network modelling of inflammatory markers.

Anti-inflammatory properties of artichoke pectin and modified fractions (arabinose- and galactose-free) used at two doses (40 and 80 mg kg-1) in mice with colitis induced by dextran sulfate sodium have been investigated. Expression of pro-inflammatory markers TNF-α and ICAM-I decreased in groups of mice treated with original and arabinose-free artichoke pectin while IL-1ß and IL-6 liberation was reduced only in mice groups treated with original artichoke pectin. A decrease in iNOS and TLR-4 expression was observed for most treatments. Intestinal barrier gene expression was also determined. MUC-1 and Occludin increased in groups treated with original artichoke pectin while MUC-3 expression also increased in arabinose-free pectin treatment. Galactose elimination led to a loss of pectin bioactivity. Characteristic expression profiles were established for each treatment through artificial neural networks showing high accuracy rates (≥90%). These results highlight the potential amelioration of inflammatory bowel disease on mice model colitis through artichoke pectin administration.

Enzymatic Production and Characterization of Pectic Oligosaccharides Derived from Citrus and Apple Pectins: A GC-MS Study Using Random Forests and Association Rule Learning.

Pectic oligosaccharides (POS) from citrus and apple pectin hydrolysis using ViscozymeL and Glucanex200G have been obtained. According to the results, maximum POS formation was achieved from citrus pectin after 30 min of hydrolysis with ViscozymeL, with a yield of 652 mg g-1 and average molecular mass ( Mw) of 0.8-2.5 kDa, while with Glucanex200G, the yield was 518 mg g-1 and Mw was 0.8-7.1 kDa. Digalacturonic and trigalacturonic acids were identified among other low Mw compounds as di- and tri-POS. In addition, differences in GC-MS spectra of all oligosaccharides found in the hydrolysates were studied by employing random forests and other algorithms to identify structural differences between the obtained POS, and high prediction rates were shown for new samples. Chemical structures were proposed for some influential m/ z ions, and 12 association rules that explain differences according to pectin and enzyme origin were built. This information could be used to establish structure-function relationships of POS.

Trans-ß-galactosidase activity of pig enzymes embedded in the small intestinal brush border membrane vesicles.

This work highlights the utility of brush border membrane vesicles (BBMV) from the pig small intestine as a reliable model for gathering information about the reaction mechanisms involved in the human digestion of dietary carbohydrates. Concretely, the elucidation of the transgalactosylation mechanism of pig BBMV to synthesize prebiotic galacto-oligosaccharides (GOS) is provided, unravelling the catalytic activity of mammalian small intestinal ß-galactosidase towards the hydrolysis of GOS. This study reveals that pig BBMV preferably synthesizes GOS linked by ß-(1 → 3) bonds, since major tri- and disaccharide were produced by the transfer of a galactose unit to the C-3 of the non-reducing moiety of lactose and to the C-3 of glucose, respectively. Therefore, these results point out that dietary GOS having ß-(1 → 3) as predominant glycosidic linkages could be more prone to hydrolysis by mammalian intestinal digestive enzymes as compared to those linked by ß-(1 → 2), ß-(1 → 4), ß-(1 ↔ 1) or ß-(1 → 6). Given that these data are the first evidence on the transglycosylation activity of mammalian small intestinal glycosidases, findings contained in this work could be crucial for future studies investigating the structure-small intestinal digestibility relationship of a great variety of available prebiotics, as well as for designing tailored fully non-digestible GOS.

GC-MS characterisation of novel artichoke (Cynara scolymus) pectic-oligosaccharides mixtures by the application of machine learning algorithms and competitive fragmentation modelling.

Novel artichoke pectic-oligosaccharides (POS) mixtures have been obtained by enzymatic hydrolysis using four commercial enzyme preparations: Glucanex®200G, Pentopan®Mono-BG, Pectinex®Ultra-Olio and Cellulase from Aspergillus niger. Analysis by HPAEC-PAD showed that Cellulase from A. niger produced the greatest amount of POS (310.6 mg g-1 pectin), while the lowest amount was produced by Pentopan®Mono-BG (45.7 mg g-1 pectin). To determine structural differences depending on the origin of the enzyme, GC-MS spectra of di- and trisaccharides have been studied employing three machine learning algorithms: multilayer perceptron, random forest and boosted logistic regression. Machine learning models allowed characteristic m/z ions patterns to be established for each enzyme based on their GC-MS spectra with high prediction rates (above 95% on the test set). Possible chemical structures were given for some m/z ions having a decisive influence on these classifications. Finally, it was observed that several ions could be formed from specific POS structures.

Synthesis of prebiotic galactooligosaccharides from lactose and lactulose by dairy propionibacteria.

The potential of probiotic bacteria to produce prebiotic oligosaccharides by transgalactosylation has been minimally studied. In this work, we screened the ß-galactosidase (ß-gal) activity of dairy propionibacteria (PAB) isolated from Argentinean foods to select strains for the synthesis of oligosaccharides from lactose (GOS) and lactulose (OsLu). PAB, when grown in a medium with lactose as a carbon source, were disrupted, and the cell-free extracts were assayed for ß-gal activity. Nine strains grew on lactose and showed ß-gal activities from 0.27 to 2.60 U mL-1. Propionibacterium acidipropionici LET 120, the strain with the highest activity, was able to synthesize, using 30% lactose and lactulose at pH 6.5 and 45 °C, 26.8% of LET 120-GOS and 26.1% of LET 120-OsLu after 24 h. When they were tested as carbon sources for growth, P. acidipropionici LET 120 attained higher biomasses, µmax and ß-gal activities at the expense of Aspergillus oryzae-OsLu, Vivinal®-GOS and lactulose compared to lactose or glucose. In addition, LET 120-GOS and LET 120-OsLu synthesized by PAB were prebiotic for some probiotic strains. For the first time, our results show the production of GOS and OsLu by dairy PAB, and these results encourage further studies on the optimization of the synthesis and structure characterization of the obtained oligosaccharides.

High-Temperature Short-Time Pasteurization System for Donor Milk in a Human Milk Bank Setting.

Donor milk is the best alternative for the feeding of preterm newborns when mother's own milk is unavailable. For safety reasons, it is usually pasteurized by the Holder method (62.5°C for 30 min). Holder pasteurization results in a microbiological safe product but impairs the activity of many biologically active compounds such as immunoglobulins, enzymes, cytokines, growth factors, hormones or oxidative stress markers. High-temperature short-time (HTST) pasteurization has been proposed as an alternative for a better preservation of some of the biological components of human milk although, at present, there is no equipment available to perform this treatment under the current conditions of a human milk bank. In this work, the specific needs of a human milk bank setting were considered to design an HTST equipment for the continuous and adaptable (time-temperature combination) processing of donor milk. Microbiological quality, activity of indicator enzymes and indices for thermal damage of milk were evaluated before and after HTST treatment of 14 batches of donor milk using different temperature and time combinations and compared to the results obtained after Holder pasteurization. The HTST system has accurate and simple operation, allows the pasteurization of variable amounts of donor milk and reduces processing time and labor force. HTST processing at 72°C for, at least, 10 s efficiently destroyed all vegetative forms of microorganisms present initially in raw donor milk although sporulated Bacillus sp. survived this treatment. Alkaline phosphatase was completely destroyed after HTST processing at 72 and 75°C, but γ-glutamil transpeptidase showed higher thermoresistance. Furosine concentrations in HTST-treated donor milk were lower than after Holder pasteurization and lactulose content for HTST-treated donor milk was below the detection limit of analytical method (10 mg/L). In conclusion, processing of donor milk at 72°C for at least 10 s in this HTST system allows to achieve the microbiological safety objectives established in the milk bank while having a lower impact regarding the heat damage of the milk.

Enzymatic extraction of pectin from artichoke (Cynara scolymus L.) by-products using Celluclast®1.5L.

The aim of this study was to optimise pectin extraction from artichoke by-products with Celluclast®1.5L using an experimental design analysed by response-surface methodology (RSM). The variables optimised were artichoke by-product powder concentration (2-7%, X1), enzyme dose (2.2-13.3 U g-1, X2) and extraction time (6-24 h, X3). The variables studied were galacturonic acid (GalA) (R2 93.9) and pectic neutral sugars (R2 92.8) content and pectin yield (R2 88.6). In the optimum extraction conditions (X1 = 6.5%; X2 = 10.1 U g-1; X3 = 27.2 h), pectin yield was 176 mgg-1 dry matter (DM). Considering 27.2 h of treatment as the +α value given by the design, the extraction time was increased up to 48 h obtaining a yield of 221 mg g-1 DM. The enzymatic method optimised allows obtaining artichoke pectin with good yield, high GalA (720 mg g-1 DM) and arabinose (127.6mgg-1 DM) contents and degree of methylation of 19.5%.

An efficient process for obtaining prebiotic oligosaccharides derived from lactulose using isomerized and purified whey permeate.

BACKGROUND: One of the most promising uses of whey permeate (WP) is the synthesis of prebiotic oligosaccharides. Herein, commercial WP was submitted to chemical isomerization catalysed by sodium borate at an alkaline pH and subsequent purification using anion-exchange resins to remove boron. Subsequently, purified mixtures were used to synthesize prebiotic oligosaccharides using ß-galactosidase from Bacillus circulans. RESULTS: Isomerization of concentrated WP (200 g L-1 lactose) gave rise to levels of lactulose up to 155.5 g L-1 after 30 min of reaction (molar ratio of boron/lactose, 1/1; pH 12; 70 °C). Boron was removed from the isomerized WP (IWP) using the combination of a strong acid (IR-120, H+ ) and a weak base (IRA-743) anion-exchange resins, reducing its level to <1 ppm, without loss of lactulose. During the transglycosylation reaction of purified IWP (lactose/lactulose ratio, 1/2.4) maximum content of prebiotic compounds was achieved, i.e. 690 g kg-1 WP after 3 h of reaction. CONCLUSION: This study shows that combined chemical-enzymatic reactions together with the purification of IWP results in an efficient synthesis of prebiotic oligosaccharides. © 2017 Society of Chemical Industry.

Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae ß-Galactosidase.

ß-Galactosidase from Aspergillus oryzae offers a high yield for the synthesis of oligosaccharides derived from lactulose (OsLu) by transgalactosylation. Oligosaccharides with degree of polymerization (DP) ≥ 3 have shown to possess higher in vitro bifidogenic effect than di- and tetrasaccharides. Thus, in this work, an optimization of reaction conditions affecting the specific selectivity of A. oryzae ß-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 production. Assays with ß-galactosidase immobilized onto a glutaraldehyde-agarose support were also carried out with the aim of making the process cost-effective and industrially viable. Optimal conditions with both soluble and immobilized enzyme for the synthesis of OsLu with DP ≥ 3 were 50 °C, pH 6.5, 450 g/L of lactulose, and 8 U/mL of enzyme, reaching yields of ca. 50% (w/v) of total OsLu and ca. 20% (w/v) of OsLu with DP 3, being 6'-galactosyl-lactulose the major one, after a short reaction time. Selective formation of disaccharides, however, was favored at 60 °C, pH 4.5, 450 g/L of lactulose and 8 U/mL of enzyme. Immobilization increased the enzymatic stability to temperature changes and allowed to reuse the enzyme. We can conclude that the use, under determined optimal conditions, of the A. oryzae ß-galactosidase immobilized on a support of glutaraldehyde-agarose constitutes an efficient and cost-effective alternative to the use of soluble ß-galactosidases for the synthesis of prebiotic OsLu mixtures.

Synthesis, optimization and structural characterization of a chitosan-glucose derivative obtained by the Maillard reaction.

Chitosan (Chit) was submitted to the Maillard reaction (MR) by co-heating a solution with glucose (Glc). Different reaction conditions as temperature (40, 60 and 80 °C), Glc concentration (0.5%, 1%, and 2%, w/v), and reaction time (72, 52 and 24h) were evaluated. Assessment of the reaction extent was monitored by measuring changes in UV absorbance, browning and fluorescence. Under the best conditions, 2% (w/v) of Chit, 2% (w/v) of Glc at 60°C and 32 h of reaction time, a chitosan-glucose (Chit-Glc) derivative was purified and submitted to structural characterization to confirm its formation. Analysis of its molecular weight (MW) and the degree of substitution (DS) was carried out by HPLC-Size Exclusion Chromatography (SEC) and a colloid titration method, respectively. FT-IR and (1)H NMR were also used to analyze the functional groups and evaluate the introduction of Glc into the Chit molecule. According to our objectives, the results obtained in this work allowed to better understand the key parameters influencing the MR with Chit as well as to confirm the successful introduction of Glc into the Chit molecule obtaining a Chit-Glc derivative with a DS of 64.76 ± 4.40% and a MW of 210.37 kDa.