Thermostability Enhancement of GH 62 α-l-Arabinofuranosidase by Directed Evolution and Rational Design.

GH 62 arabinofuranosidases are known for their excellent specificity for arabinoxylan of agroindustrial residues and their synergism with endoxylanases and other hemicellulases. However, the low thermostability of some GH enzymes hampers potential industrial applications. Protein engineering research highly desires mutations that can enhance thermostability. Therefore, we employed directed evolution using one round of error-prone PCR and site-saturation mutagenesis for thermostability enhancement of GH 62 arabinofuranosidase from Aspergillus fumigatus. Single mutants with enhanced thermostability showed significant ΔΔG changes (<-2.5 kcal/mol) and improvements in perplexity scores from evolutionary scale modeling inverse folding. The best mutant, G205K, increased the melting temperature by 5 °C and the energy of denaturation by 41.3%. We discussed the functional mechanisms for improved stability. Analyzing the adjustments in α-helices, ß-sheets, and loops resulting from point mutations, we have obtained significant knowledge regarding the potential impacts on protein stability, folding, and overall structural integrity.

Non-animal protein hydrolysates from agro-industrial wastes: A prospect of alternative inputs for cultured meat.

In light of the growing demand for alternative protein sources, laboratory-grown meat has been proposed as a potential solution to the challenges posed by conventional meat production. Cultured meat does not require animal slaughter and uses sustainable production methods, contributing to animal welfare, human health, and environmental sustainability. However, some challenges still need to be addressed in cultured meat production, such as the use of fetal bovine serum for medium supplementation. This ingredient has limited availability, increases production costs, and raises ethical concerns. This review explores the potential of non-animal protein hydrolysates derived from agro-industrial wastes as substitutes for critical components of fetal bovine serum in cultured meat production. Despite the lack of standardization of hydrolysate composition, the potential benefits of this alternative protein source may outweigh its disadvantages. Future research holds promise for increasing the accessibility of cultured meat.

Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate.

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two ß-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both ß-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.

Influence of cocoa varieties on carbohydrate composition and enzymatic activity of cocoa pulp.

In Brazil, after the witch's broom disease incidence, diverse cocoa hybrids were developed, and variations were reported on their composition and characteristics. Based on this, the present study aimed to evaluate the pulp composition of several cocoa hybrids in order to better understand these variations. Results show that cocoa pulp is composed, on average, of 76 % sugar, and a wide variation (20 %) was observed in sugar content between hybrids. Regarding the sugar profile, a prevalence of reducing sugars was observed. Pod origin also plays an important role in pulp composition, with variations between hybrids from Espírito Santo and Bahia states. In relation to the degree of ripeness, ripe pods showed higher fructose and glucose content, while unripe pods presented mainly sucrose. Similar to sugars, the cello-oligossacharides profile was influenced by the degree of pod ripeness and origin and most ripe samples presented mainly cellobiose, cellotriose and cellotetrose. In addition, the prebiotic potential of cocoa pulp was highlighted by cello-oligossacharides digestion assay which exhibited low rates of degradation. Varying enzymatic activity was observed amongst different pulp hybrids, with polyphenol oxidase showing a higher variation when compared to invertase and polygalacturonase ranging. This study shows that the pod hybrid, origin and ripening degree may change the cocoa pulp composition. Therefore, it is very important to understand and evaluate these variations, in order to obtain better results in pulp utilization either in cocoa fermentation or as a coproduct.

Fractionation of functional oligosaccharides produced from sugarcane straw using serial nanofiltration membranes and their influence on prebiotic potential.

Functional oligosaccharides are non-digestible by human gut enzymes and provide health benefits as fibers and prebiotics. The cello-oligosaccharides (COS) and xylooligosaccharides (XOS) are functional oligosaccharides obtained from xylan and cellulose, respectively, and are present in lignocellulosic material. The serial NF membranes process was performed to investigate the impact of the fractionation process on the prebiotic activity of oligosaccharides from xylan and cellulose. The NP030 (weight cut-off of 500-600 Da) and DK (weight cut-off of 150-300 Da) NF polymeric membranes were employed using defined operational conditions. The diafiltration (DF) was also investigated and it was determined that only a 1-time DF for NP030 was a more suitable strategy and improved the performance indices for short DP oligosaccharides. The short DP fractions obtained favored cell density for probiotic strains, which presented an increase on the optical density of up to 25 % after the fractionating process; enabling the use of short purified fractions in the food and pharmaceutical industry as a prebiotic ingredient.

Impact of adding xylooligosaccharides encapsulated in butter: Microstructural, optical, rheological and sensory aspects.

This study investigated the microstructure, rheological properties, and sensory characteristics of butters produced with free and encapsulated xylooligosaccharides (XOS). Four formulations of butter were processed: BCONT: 0 % w/w XOS (control); BXOS: 20% w/w free XOS; BXOS-ALG: 20% w/w XOS microencapsulated with alginate (XOS-alginate ratio of 3:1 w/w); and BXOS-GEL: 20% w/w XOS microencapsulated with alginate-gelatin (XOS-alginate-gelatin ratio of 3:1:1.5 w/w). The microparticles showed a bimodal distribution, low size and low span values, demonstrating physical stability to be included in emulsions. The XOS-ALG presented surface weighted mean diameter (D3.2) of 90.24 µm, volume-weighted mean diameter (D4.3) of 131.8 µm, and Span of 2.14. In contrast, the XOS-GEL presented D3.2 of 82.80 µm, D4.3 of 141.0 µm, and a Span of 2.46. Products with XOS were characterized by higher creaminess, sweet taste, and lower salty taste than the control. However, the addition form significantly impacted the other evaluated parameters. The utilization of XOS in a free form (BXOS) resulted in smaller droplet sizes (1.26 µm) than encapsulated XOS and control (XOS-ALG = 1.32 µm / XOS-GEL = 1.58 µm, / BCONT = 1.59 µm), and changes in the rheological parameters (higher values of shear stress, viscosity, consistency index, rigidity (J0), and Newtonian viscosity (ηN) and lower elasticity (τ)). Furthermore, it changed the color parameters (more yellow and dark color, lower L* and higher b* values). On the other hand, the utilization of micropaticles of XOS (BXOS-ALG and BXOS-GEL) kept shear stress, viscosity, consistency index, rigidity (J0), and elasticity (τ) more similar to control. The products had a less intense yellow color (lower b* values) and was perceived with more consistency and butter taste. However, the presence of particles was perceived by consumers. The results suggest that consumers were more attentive to reporting flavor-related attributes than texture. In conclusion, adding microparticles of XOS could improve butter's rheological and sensory properties. In conclusion, adding microparticles of XOS could improve butter's rheological and sensory properties.

Hydrothermal pretreatment for the production of prebiotic oligosaccharides from tobacco stem.

Tobacco stem is an abundant and inexpensive renewable source to produce prebiotics by circular economy. In this study, hydrothermal pretreatments were evaluated on the release of xylooligosaccharides (XOS) and cello-oligosaccharides (COS) from the tobacco stem by a central composite rotational design associated with response surface methodology to evaluate the effects of temperature (161.72 to 218.3 °C) and solid load (SL) (2.93 to 17.07%). XOS were the main compounds released to the liquor. Desirability function was performed to maximize the production of XOS and minimize the effects of release of monosaccharides and degradation compounds. The result indicated yield of 96% w[XOS]/w[xylan] for 190 °C-2.93% SL. The highest value for COS and total oligomers content (COS + XOS) was 6.42 g/L and 17.7 g/L, respectively, for 190 °C-17.07% SL. The mass balance for the best yield XOS condition predicted 132 kg of XOS (X2-X6) from 1000 kg of tobacco stem.

Technological and prebiotic aspects of young bamboo culm flour (Dendrocalamus latiflorus) combined with rice flour to produce healthy extruded products.

Young bamboo culm flour (YBCF) has proved to be a healthy and sustainable ingredient, due to its high fiber content and high yield of bamboo crops. The present study evaluated the effects of YBCF from Dendrocalamus latiflorus on the physicochemical, technological properties and prebiotic activity of rice-based extrudates aiming to expand its application. The extrudates were produced in a twin-screw extruder with different RF:YBCF concentrations (100:0; 95:5, 90:10, and 85:15 %). During the process, the specific mechanical energy increased as YBCF content increased because of the high shear favored by YBCF particles. With increasing RF replacement by YBCF, the extruded products presented a significant (p < 0.05, by the Scott-Knott test) increase in hardness (57.37 to 82.01 N) and water solubility index (12.80 to 34.10 %), as well as a decrease in color luminosity (L*=85.49 to 82.83), expansion index (2.68 to 1.99), and pasting properties. In addition, all extrudate samples presented bifidogenic activity. Therefore, YBCF exhibited attractive technological properties and can be used as an ingredient in the production of healthy and sustainable extruded products.

Evaluation of Saccharomyces cerevisiae modified via CRISPR/Cas9 as a cellulosic platform microorganism in simultaneously saccharification and fermentation processes.

The nonrenewable character and deleterious effects of fossil fuels foster the need for cleaner and more inexhaustible energy sources, such as bioethanol. Especially from lignocellulosic biomasses. However, the economic viability of this product in the market depends on process optimization and cost reduction. This research applied a sequential experimental project to investigate the process of enzymatic saccharification and simultaneous fermentation to produce ethanol with sugarcane bagasse. The differential of the work was the application of the strain of Saccharomyces cerevisiae AGY001 which was improved by evolutionary engineering to become thermotolerant and by a heterologous expression based on genomic integration by CRISPR/Cas9 to produce endoglucanase and ß-glucosidase (AsENDO-AsBGL). The maximum ethanol yield found was 89% of the maximum theoretical yield (released sugars), obtained at temperature concentrations, sugarcane bagasse and inoculum at 40 °C, 16.5%, and 4.0 g/L, respectively (12.5 FPU/g bagasse). The mathematical model obtained can predict approximately 83% of the data set with 95% confidence. Therefore, these findings demonstrated the potential of sugarcane bagasse and S. cerevisiae AGY001 strain (CRISPR/Cas9 modified) in bioethanol production without the need for impractical selection media on an industrial scale, in addition to providing useful insights for the development of SSF processes.

Rational engineering of industrial S. cerevisiae: towards xylitol production from sugarcane straw.

BACKGROUND: Sugarcane hemicellulosic material is a compelling source of usually neglected xylose that could figure as feedstock to produce chemical building blocks of high economic value, such as xylitol. In this context, Saccharomyces cerevisiae strains typically used in the Brazilian bioethanol industry are a robust chassis for genetic engineering, given their robustness towards harsh operational conditions and outstanding fermentation performance. Nevertheless, there are no reports on the use of these strains for xylitol production using sugarcane hydrolysate. RESULTS: Potential single-guided RNA off-targets were analyzed in two preeminent industrial strains (PE-2 and SA-1), providing a database of 5'-NGG 20 nucleotide sequences and guidelines for the fast and cost-effective CRISPR editing of such strains. After genomic integration of a NADPH-preferring xylose reductase (XR), FMYX (SA-1 hoΔ::xyl1) and CENPKX (CEN.PK-122 hoΔ::xyl1) were tested in varying cultivation conditions for xylitol productivity to infer influence of the genetic background. Near-theoretical yields were achieved for all strains; however, the industrial consistently outperformed the laboratory strain. Batch fermentation of raw sugarcane straw hydrolysate with remaining solid particles represented a challenge for xylose metabolization, and 3.65 ± 0.16 g/L xylitol titer was achieved by FMYX. Finally, quantification of NADPH - cofactor implied in XR activity - revealed that FMYX has 33% more available cofactors than CENPKX. CONCLUSIONS: Although widely used in several S. cerevisiae strains, this is the first report of CRISPR-Cas9 editing major yeast of the Brazilian bioethanol industry. Fermentative assays of xylose consumption revealed that NADPH availability is closely related to mutant strains' performance. We also pioneer the use of sugarcane straw as a substrate for xylitol production. Finally, we demonstrate how industrial background SA-1 is a compelling chassis for the second-generation industry, given its inhibitor tolerance and better redox environment that may favor production of reduced sugars.

Enzymatic generation of short chain cello-oligosaccharides from Miscanthus using different pretreatments.

Enzyme combinations producing short-chain cello-oligosaccharides (COS) as major bio-products from cellulose of Miscanthus Mx2779 accessed through different pretreatment methods were compared. Over short hydrolysis times, processive endoglucanase TfCel9a produced a high percentage of cellotetraose and cellopentaose and is synergistic with endoglucanase CcCel9m for producing short oligomers from amorphous cellulose but had low activity on untreated Miscanthus. Hydrolysis of the latter improved when these were combined with a mutant cellobio/triohydrolase OsCelC7(-105) and a lytic polysaccharide monooxygenase TrCel61a, a combination which also produced the highest COS yields from phosphoric acid swollen cellulose. Steam explosion pretreatment of Miscanthus increased COS yields, with/without phosphoric acid swelling, while increased swelling time (from 20 to 45 min) also increased yields but decreased the need for TrCel61a. The highest COS yields (933 mg/g glucan) and most stable product profile were obtained using ionic liquid [C2mim][OAc] pretreatment and the three enzyme mixture TfCel9a, Cel9m and OsCel7a(-105).

Xylo-Oligosaccharide Utilization by Engineered Saccharomyces cerevisiae to Produce Ethanol.

The engineering of xylo-oligosaccharide-consuming Saccharomyces cerevisiae strains is a promising approach for more effective utilization of lignocellulosic biomass and the development of economic industrial fermentation processes. Extending the sugar consumption range without catabolite repression by including the metabolism of oligomers instead of only monomers would significantly improve second-generation ethanol production This review focuses on different aspects of the action mechanisms of xylan-degrading enzymes from bacteria and fungi, and their insertion in S. cerevisiae strains to obtain microbial cell factories able of consume these complex sugars and convert them to ethanol. Emphasis is given to different strategies for ethanol production from both extracellular and intracellular xylo-oligosaccharide utilization by S. cerevisiae strains. The suitability of S. cerevisiae for ethanol production combined with its genetic tractability indicates that it can play an important role in xylan bioconversion through the heterologous expression of xylanases from other microorganisms.

Microalgae-based carbohydrates: A green innovative source of bioenergy.

Microalgae contribute significantly to the global carbon cycle through photosynthesis. Given their ability to efficiently convert solar energy and atmospheric carbon dioxide into chemical compounds, such as carbohydrates, and generate oxygen during the process, microalgae represent an excellent and feasible carbohydrate bioresource. Microalgae-based biofuels are technically viable and, delineate a green and innovative field of opportunity for bioenergy exploitation. Microalgal polysaccharides are one of the most versatile groups for biotechnological applications and its content can be increased by manipulating cultivation conditions. Microalgal carbohydrates can be used to produce a variety of biofuels, including bioethanol, biobutanol, biomethane, and biohydrogen. This review provides an overview of microalgal carbohydrates, focusing on their use as feedstock for biofuel production, highlighting the carbohydrate metabolism and approaches for their enhancement. Moreover, biofuels produced from microalgal carbohydrate are showed, in addition to a new bibliometric study of current literature on microalgal carbohydrates and their use.

Evaluating the addition of xylooligosaccharides into alginate-gelatin hydrogels.

Xylooligosaccharides (XOS) are emerging prebiotic that may improve structural features of biopolymer blends. The investigation around the conformation of XOS into the matrix of alginate and gelatin clarifies the potential applications of this formulation in the food industry as texture modifiers or encapsulation systems. Structural properties verified by flow behavior, SEM, XRD, and FT-IR demonstrated that the add up to 3% XOS into the alginate-gelatin blend formed a cohesive matrix, with smaller pores and crystalline structure, confirming the potential of xylooligosaccharides hydrogels for the development of functional and synbiotic foods.

Multi-omics analysis provides insights into lignocellulosic biomass degradation by Laetiporus sulphureus ATCC 52600.

BACKGROUND: Wood-decay basidiomycetes are effective for the degradation of highly lignified and recalcitrant plant substrates. The degradation of lignocellulosic materials by brown-rot strains is carried out by carbohydrate-active enzymes and non-enzymatic Fenton mechanism. Differences in the lignocellulose catabolism among closely related brown rots are not completely understood. Here, a multi-omics approach provided a global understanding of the strategies employed by L. sulphureus ATCC 52600 for lignocellulose degradation. RESULTS: The genome of Laetiporus sulphureus ATCC 52600 was sequenced and phylogenomic analysis supported monophyletic clades for the Order Polyporales and classification of this species within the family Laetiporaceae. Additionally, the plasticity of its metabolism was revealed in growth analysis on mono- and disaccharides, and polysaccharides such as cellulose, hemicelluloses, and polygalacturonic acid. The response of this fungus to the presence of lignocellulosic substrates was analyzed by transcriptomics and proteomics and evidenced the occurrence of an integrated oxidative-hydrolytic metabolism. The transcriptomic profile in response to a short cultivation period on sugarcane bagasse revealed 125 upregulated transcripts, which included CAZymes (redox enzymes and hemicellulases) as well as non-CAZy redox enzymes and genes related to the synthesis of low-molecular-weight compounds. The exoproteome produced in response to extended cultivation time on Avicel, and steam-exploded sugarcane bagasse, sugarcane straw, and Eucalyptus revealed 112 proteins. Contrasting with the mainly oxidative profile observed in the transcriptome, the secretomes showed a diverse hydrolytic repertoire including constitutive cellulases and hemicellulases, in addition to 19 upregulated CAZymes. The secretome induced for 7 days on sugarcane bagasse, representative of the late response, was applied in the saccharification of hydrothermally pretreated grass (sugarcane straw) and softwood (pine) by supplementing a commercial cocktail. CONCLUSION: This study shows the singularity of L. sulphureus ATCC 52600 compared to other Polyporales brown rots, regarding the presence of cellobiohydrolase and peroxidase class II. The multi-omics analysis reinforces the oxidative-hydrolytic metabolism involved in lignocellulose deconstruction, providing insights into the overall mechanisms as well as specific proteins of each step.

Cello-oligosaccharides production from lignocellulosic biomass and their emerging prebiotic applications.

Cello-oligosaccharides (COS) are linear oligosaccharides composed of ß-1,4-linked glucopyranose units. They comprise a group of important new oligosaccharides of significant interest and potential applications in the pharmaceutical, food, chemical, and feed industries, currently emerging as potential prebiotic compounds. COS from lignocellulosic biomass, specifically the agro-industrial residues and by-products of the forestry industry, constitute a new attractive process that imposes the sustainable use of biomass resources. Two main strategies have been used for the production of COS: acid-based and enzyme-based cellulose hydrolysis. The latter has been considered more attractive due to the use of milder reaction conditions and less production of monomers. This review summarizes that although COS is emerging as a potential prebiotic with also other potential applications, there is a lack of information regarding the large-scale production, which could be associated with the recalcitrant nature of cellulose compared to other polysaccharides, which hinders the hydrolysis of its dense network.

Xylo-oligosaccharide microparticles with synbiotic potential obtained from enzymatic hydrolysis of sugarcane straw.

Synbiotic formulations and microencapsulation techniques have been explored in food industries to guarantee the viability of probiotic organisms; playing an important role in microbiota balance. Microparticles of alginate, gelatin and xylo-oligosaccharides (XOS) were produced by external gelation with the purpose of enhancing the survival rate of the probiotic L. acidophilus. XOS was obtained through enzymatic hydrolysis of xylan extracted from sugarcane straw, achieving more than 70% conversion and used for microparticle preparation. Microparticles containing 3% XOS provided greater cell protection during exposure to the gastrointestinal tract and during refrigerated storage; keeping 97.86 ± 0.44% of viability during 28 days of storage and enabling 87.50 ± 0.02% survival after digestive simulation. However, particles without XOS showed 84.49 ± 0.59% of viability after storage and 68.45 ± 0.03% after digestion assay. These results lead to promising applications in synbiotic and functional food formulations comprised of components requiring extended shelf-life, protection from gastrointestinal conditions and gradual bioactive delivery.

Heterologous Expression of Lignocellulose-Modifying Enzymes in Microorganisms: Current Status.

Heterologous expression of the carbohydrate-active enzymes in microorganisms is a promising approach to produce bio-based compounds, such as fuels, nutraceuticals and other value-added products from sustainable lignocellulosic sources. Several microorganisms, including Saccharomyces cerevisiae, Escherichia coli, and the filamentous fungi Aspergillus nidulans, have unique characteristics desirable for a biorefinery production approach like well-known genetic tools, thermotolerance, high fermentative capacity and product tolerance, and high amount of recombinant enzyme secretion. These microbial factories are already stablished in the heterologous production of the carbohydrate-active enzymes to produce, among others, ethanol, xylooligosaccharides and the valuable coniferol. A complete biocatalyst able to heterologous express the CAZymes of glycoside hydrolases, carbohydrate esterases and auxiliary activities families could release these compounds faster, with higher yield and specificity. Recent advances in the synthetic biology tools could expand the number and diversity of enzymes integrated in these microorganisms, and also modify those already integrated. This review outlines the heterologous expression of carbohydrate-active enzymes in microorganisms, as well as recent updates in synthetic biology.

Hydrothermal treatment on depolymerization of hemicellulose of mango seed shell for the production of xylooligosaccharides.

Hydrothermal processing is an interesting biorefinery technology for converting lignocellulosic biomass into biofuels and biocompounds. This process is based on the selective solubilization and depolymerization of hemicellulose fraction (xylan) and may be considered beneficial, due to the possibility of obtaining xylooligosaccharides (XOS) with a degree of polymerization (DP) suitable for prebiotic applications. This study evaluated the effect of pressure (2.5 and 10 MPa) in a kinetic study (30 min) of hydrothermal treatment (180 °C) to optimize the extraction of XOS from mango seed shell. Total reducing sugars (TRS) values were close to the maximum in 15 min showing a slower rate for both pressures after this time, but at 10 MPa the value was 20 % lower than at 2.5 MPa. Based on these results, a new extraction was performed at 2.5 MPa and 15 min, and the extracted XOS were quantified, yielding 393.44 mg XOS/g xylan. XOS with a degree of polymerization between X2-X6 corresponded to 82.24 mg/g and XOS with X > 6 (or soluble xylan) corresponded to 311.20 mg/g. A low amount of xylose (8.81 mg/g xylan) was released, resulting in a hemicellulose conversion of 40.2 %. In general, approximately 8.1 kg of total XOS was produced from 100 kg of dried mango seed shell (X2-X6-1.7 kg and X > 6-6.4 kg).

Deconstruction of banana peel for carbohydrate fractionation.

The deconstruction of banana peel for carbohydrate recovery was performed by sequential treatment (acid, alkaline, and enzymatic). The pretreatment with citric acid promoted the extraction of pectin, resulting in a yield of 8%. In addition, xylose and XOS, 348.5 and 17.3 mg/g xylan, respectively, were also quantified in acidic liquor as a result of partial depolymerization of hemicellulose. The spent solid was pretreated with alkaline solution (NaOH or KOH) for delignification and release of residual carbohydrates from the hemicellulose. The yields of xylose and arabinose (225.2 and 174.0 mg/g hemicellulose) were approximately 40% higher in the pretreatment with KOH, while pretreatment with NaOH promoted higher delignification (67%), XOS yield (32.6 mg/g xylan), and preservation of cellulosic fraction. Finally, the spent alkaline solid, rich in cellulose (76%), was treated enzymatically to release glucose, reaching the final concentration of 28.2 g/L. The mass balance showed that through sequential treatment, 9.9 g of xylose, 0.5 g of XOS, and 8.2 g of glucose were obtained from 100 g of raw banana peels, representing 65.8% and 46.5% conversion of hemicellulose and cellulose, respectively. The study of the fractionation of carbohydrates in banana peel proved to be a useful tool for valorization, mainly of the hemicellulose fraction for the production of XOS and xylose with high value applications in the food industry.