Sustainable biorefinery approach by utilizing xylose fraction of lignocellulosic biomass.

Lignocellulosic biomass (LCB) has been a lucrative feedstock for developing biochemical products due to its rich organic content, low carbon footprint and abundant accessibility. The recalcitrant nature of this feedstock is a foremost bottleneck. It needs suitable pretreatment techniques to achieve a high yield of sugar fractions such as glucose and xylose with low inhibitory components. Cellulosic sugars are commonly used for the bio-manufacturing process, and the xylose sugar, which is predominant in the hemicellulosic fraction, is rejected as most cell factories lack the five­carbon metabolic pathways. In the present review, more emphasis was placed on the efficient pretreatment techniques developed for disintegrating LCB and enhancing xylose sugars. Further, the transformation of the xylose to value-added products through chemo-catalytic routes was highlighted. In addition, the review also recapitulates the sustainable production of biochemicals by native xylose assimilating microbes and engineering the metabolic pathway to ameliorate biomanufacturing using xylose as the sole carbon source. Overall, this review will give an edge on the bioprocessing of microbial metabolism for the efficient utilization of xylose in the LCB.

Addressing raw material variability: In-line FTIR sugar composition analysis of lignocellulosic process streams.

For a sustainable economy, biorefineries that use second-generation feedstocks to produce biochemicals and biofuels are essential. However, the exact composition of renewable feedstocks depends on the natural raw materials used and is therefore highly variable. In this contribution, a process analytical technique (PAT) strategy for determining the sugar composition of lignocellulosic process streams in real-time to enable better control of bioprocesses is presented. An in-line mid-IR probe was used to acquire spectra of ultra-filtered spent sulfite liquor (UF-SSL). Independent partial least squares models were developed for the most abundant sugars in the UF-SSL. Up to 5 sugars were quantified simultaneously to determine the sugar concentration and composition of the UF-SSL. The lowest root mean square errors of the predicted values obtained per analyte were 1.02 g/L arabinose, 1.25 g/L galactose, 0.50 g/L glucose, 1.60 g/L mannose, and 0.85 g/L xylose. Equipped with this novel PAT tool, new bioprocessing strategies can be developed for UF-SSL.

Mechanism of Dihydromyricetin-Induced Reduction of Furfural Derived from the Amadori Compound: Formation of Adducts between Dihydromyricetin and Furfural or Its Precursors.

Dihydromyricetin (DMY) was employed to reduce the yield of furfural derived from the Amadori rearrangement product of l-threonine and d-xylose (Thr-ARP) by trapping Thr-ARP, 3-deoxyxyosone (3-DX), and furfural to form adducts. The effect of different concentrations of DMY at different pH values and temperatures on the reduction of furfural production was studied, and the results showed that DMY could significantly reduce furfural production at higher pH (pH 5-7) and lower temperature (110 °C). Through the surface electrostatic potential analysis by Gaussian, a significant enhancement of the C6 nucleophilic ability at higher pH (pH ≥ 5) was observed on DMY with hydrogen-dissociated phenol hydroxyl. The nucleophilic ability of DMY led to its trapping of Thr-ARP, 3-DX, and furfural with the generation of the adducts DMY-Thr-ARP, DMY-3-DX, and DMY-furfural. The formation of the DMY-Thr-ARP adduct slowed the degradation of Thr-ARP, caused the decrease of the 3-DX yield, and thereby inhibited the conversion of 3-DX to furfural. Therefore, DMY-Thr-ARP was purified, and the structure was identified by nuclear magnetic resonance (NMR). The results confirmed that C6 or C8 of DMY and carbonyl carbon in Thr-ARP underwent a nucleophilic addition reaction to form the DMY-Thr-ARP adduct. In combination with the analysis results of Gaussian, most of the DMY-Thr-ARP adducts were calculated to be C6-DMY-Thr-ARP. Furthermore, the formation of DMY-furfural caused furfural consumption. The formation of the adducts also shunted the pathway of both Thr-ARP and 3-DX conversion to furfural, resulting in a decrease in the level of furfural production.

Enhancement mechanism of glycation with l-arabinose and xylose on texture properties of silver carp mince gel.

BACKGROUND: Glycation is a green processing technology. Based on our previous studies, glycation with l-arabinose and xylose was beneficial to enhance the texture properties of silver carp mince (SCM) gels. However, the possible enhancement mechanism remained unclear. Therefore, in this study, SCM gels with different types of reducing sugar (glucose, l-arabinose, and xylose) were prepared based on our previous study. The possible mechanism of texture enhancement of SCM gels was analyzed by investigating the changes in water distribution, protein structures, and microstructure in the gel system. RESULTS: The glycation of l-arabinose and xylose enhanced the hardness, cohesiveness, chewiness, and resilience of SCM gels. Hardness increased from 1883.04 (control group) to 3624.54 (l-arabinose group) and 4348.18 (xylose group). Low-field nuclear magnetic resonance (LF-NMR) showed that glycation promoted the tight binding of immobilized water to proteins. Raman spectroscopic analysis showed that glycation increased the surface hydrophobicity and promoted the formation of disulfide bonds. Scanning electron microscopy (SEM) showed that glycation promoted the formation of uniform and dense three-dimensional network structure in SCM gels. CONCLUSION: In summary, glycation enhanced the binding ability of immobilized water to proteins, improved the surface hydrophobicity, promoted the formation of disulfide bonds, and led to a more uniform and dense gel network structure of proteins, thus enhancing the texture properties of SCM gels. This research provided a theoretical basis for a better understanding of the mechanism of the effect of glycation on the quality of gel products and also provided technical support for the application of l-arabinose and xylose in new functional gel foods. © 2024 Society of Chemical Industry.

Effect of alkali treatment on enzymatic hydrolysis of p-toluenesulfonic acid pretreated bamboo substrates.

The comprehensive separation and utilization of whole components of lignocellulosic materials has received extensive attention in present research. This study focused on the efficacy of alkali treatment for enzymatic saccharification of cellulose based on p-toluenesulfonic acid (p-TsOH) pretreated bamboo substrate. The results showed that the cellulose to glucose conversion yield was 94.69 % under optimized conditions of 0.4 g NaOH/g, 160 °C and 4 h (soaked), which after only 6 h enzymatic hydrolysis time. Alkali lignin recovery was 88.51 %, with potential for conversion to lignin derivatives. The yield of hemicellulose in the pretreated filtrate was 51.85 % after the 4th cycling reuse of p-TsOH. This work has borrowed the advantages of p-TsOH pretreatment of depolymerized hemicellulose from bamboo, combined with a low-priced weak alkali secondary treatment method, which can be effectively applied to the co-production of lignin, xylooligosaccharide, xylose and glucose, and the whole process is green and economically sustainable.

Thiamine, cysteine and xylose added to the Maillard reaction of goat protein hydrolysate potentiates the formation of meat flavoring compounds.

A protein hydrolysate of goat viscera added with xylose, cysteine, and thiamine under different pH was used to prepare a meat flavoring. Goat viscera hydrolysate and flavoring were subjected to analysis of physicochemical characteristics, amino acid profile, sugars, fatty acids, and volatile profile. Meat aroma characteristics were initiated in the hydrolysate, in which Strecker's pyrazines and aldehydes were identified, which also had fatty acids and amino acids available for the formation of 96 volatile compounds in the flavorings via lipid manipulation, Maillard occurrence, Strecker manipulation and interactions among these means. Maillard reaction products with intense meat aroma, such as 2-methyl-3-furanthiol, 2-furfurylthiol and, bis(2-methyl-3-furyl) disulfide were isolated only in the flavoring at pH 4. In contrast, the flavoring at pH 6 showed a higher concentration than all the other compounds, providing a lower meat characteristic, but an intense sweet, fatty and goat aroma.

Compositional and temporal division of labor modulates mixed sugar fermentation by an engineered yeast consortium.

Synthetic microbial communities have emerged as an attractive route for chemical bioprocessing. They are argued to be superior to single strains through microbial division of labor (DOL), but the exact mechanism by which DOL confers advantages remains unclear. Here, we utilize a synthetic Saccharomyces cerevisiae consortium along with mathematical modeling to achieve tunable mixed sugar fermentation to overcome the limitations of single-strain fermentation. The consortium involves two strains with each specializing in glucose or xylose utilization for ethanol production. By controlling initial community composition, DOL allows fine tuning of fermentation dynamics and product generation. By altering inoculation delay, DOL provides additional programmability to parallelly regulate fermentation characteristics and product yield. Mathematical models capture observed experimental findings and further offer guidance for subsequent fermentation optimization. This study demonstrates the functional potential of DOL in bioprocessing and provides insight into the rational design of engineered ecosystems for various applications.

Degradation kinetics of sugars (glucose and xylose), amino acids (proline and aspartic acid) and their binary mixtures in subcritical water: Effect of Maillard reaction.

A systematic kinetic study was conducted in subcritical water medium in the temperature range from 150 to 200 °C for pure glucose, xylose, proline and aspartic acid as well as binary mixtures of sugars + amino acids to understand the reaction kinetics and interactions among biomass components and to discern the influence of Maillard reaction (MR) on the overall reaction kinetics. The main degradation products identified for glucose and xylose were the respective dehydration products, hydroxymethyl furfural and furfural, yielding an increasing solid residue with temperature (15.9 wt% at 200 °C) with an augmented heating value. The degradation of sugars and amino acids in binary systems was faster compared to pure compounds due to MR and the production of dehydration products was delayed when considering total sugar conversion. Higher relative reactivity in MR was observed for xylose over glucose showing also higher antioxidant activity.

Intrinsic Molecular Mechanisms of Transformation between Isomeric Intermediates Formed at Different Stages of Cysteine-Xylose Maillard Reaction Model through Dehydration.

2-Threityl-thiazolidine-4-carboxylic acid (TTCA) and Amadori rearrangement product (ARP), the isomeric intermediates derived from the cysteine-xylose (Cys-Xyl) Maillard reaction model, possessed the ability to produce similar flavor profile during the thermal process, but the flavor formation or browning rate of heated TTCA was significantly lower than that of ARP. Macroscopically, the yield of TTCA reached the maximum when the moisture content of the reaction system just dropped to nearly 0% during the thermal reaction-vacuum dehydration process. During the subsequent dynamic intramolecular dehydration process, the reaction remained at an early stage of the Maillard reaction, and TTCA was the main intermediate. Thereinto, the water activity of the samples decreased with the increased dehydration time. From a molecular perspective, the dissipation of free water promoted the conversion of combined water to immobilized water and free water, increasing the intramolecular dehydration. Instantaneous high-temperature dehydration during the spray drying process revealed a higher efficiency than the thermal reaction-vacuum dehydration process, which facilitated the specific conversion of substrates to intermediates (TTCA, ARP). The loss of free water and immobilized water was a key driving force for the direct formation of TTCA/ARP, regulating the formation stages of MRIs. The increase of the inlet air temperature could alter the ratio of TTCA and ARP at the equilibrium state.

Structural elucidation and targeted valorization of untractable lignin from pre-hydrolysis liquor of xylose production via a simple and robust separation approach.

Effective separation of lignin macromolecules from the xylose pre-hydrolysates (XPH) during the xylose production, thus optimizing the separation and purification process of xylose, is of great significance for reducing the production costs, achieving the high value-added utilization of lignin and increasing the industrial revenue. In this study, a simple and robust method (pH adjustment) for the separation of lignin from XPH was proposed and systematically compared with the conventional acid-promoted lignin precipitation method. The results showed that the lignin removal ratio (up to 60.34 %) of this simple method was higher than that of the conventional method, and the proposed method eliminated the necessity of heating and specialized equipment, which greatly reduced the separation cost. Meanwhile, this simple method does not destroy the components in XPH (especially xylose), ensuring the yield of the target product. On the other hand, the obtained lignin was nano-scale with less condensed structures, which also possessed small molecular weights with narrow distribution, excellent antioxidant activity (8-14 times higher than commercial antioxidants) and UV protection properties. In conclusion, the proposed simple separation method could effectively separate lignin from XPH at low cost, and the obtained lignin had potential commercial applications, which would further enhance the overall profitability of industrial production.

Biochemical characterization of a xylose-tolerant GH43 ß-xylosidase from Geobacillus thermodenitrificans.

Hemicelluloses are the second most abundant polysaccharide in plant biomass, in which xylan is the main constituent. Aiming at the total degradation of xylan and the obtention of fermentable sugars, several enzymes acting synergistically are required, especially ß-xylosidases. In this study, ß-xylosidase from Geobacillus thermodenitrificans (GtXyl) was expressed in E. coli BL21 and characterized. The enzyme GtXyl has been grouped within the family of glycoside hydrolases 43 (GH43). Results showed that GtXyl obtained the highest activity at pH 5.0 and temperature of 60 °C. In the additive's tests, the enzyme remained stable in the presence of metal ions and EDTA, and showed high tolerance to xylose, with a relative activity of 55.4% at 400 mM. The enzyme also presented bifunctional activity of ß-xylosidase and α-l-arabinofuranosidase, with the highest activity on the substrate p-nitrophenyl-ß-d-xylopyranoside. The specific activity on p-nitrophenyl-ß-d-xylopyranoside was 18.33 U mg-1 and catalytic efficiency of 20.21 mM-1 s-1, which is comparable to other ß-xylosidases reported in the literature. Putting together, the GtXyl enzyme presented interesting biochemical characteristics that are desirable for the application in the enzymatic hydrolysis of plant biomass, such as activity at higher temperatures, high thermostability and stability to metal ions.

Exogenous Threonine-Induced Conversion of Threonine-Xylose Amadori Compound to Heyns Compound for Efficiently Promoting the Formation of Pyrazines.

The involvement of exogenous threonine during the degradation of l-threonine-d-xylose Amadori rearrangement product (Thr-ARP) was found to promote the formation of pyrazines. A model including Thr-ARP and 15N-labeled l-threonine was applied to reveal the role of free threonine in Thr-ARP conversion to pyrazines. Quantitative analyses of pyrazines in the model of Thr-ARP/15N-labeled threonine showed a precedence of the endogenous threonine (formed by the degradation of Thr-ARP) over the exogenous threonine in pyrazines formation, and the ratio of 15N to 14N content in pyrazines increased significantly over time. According to the observed occurrence of the Heyns rearrangement products (HRP) derived from 15N-threonine, as well as the sharp decrease of 15N-threonine content and a rapid increase of 14N endogenous threonine at the initial stage of heat treatment, it was proposed that aldimine condensation between exogenous threonine and Thr-ARP followed by the hydrolysis led to the endogenous threonine and the generation of HRP. Then, the HRP underwent dehydration followed by hydrolysis to form exogenous threonine and deoxyxyosones, and the dehydration and hydrolysis of deoxyxyosones to form organic acids was inhibited, but the retro-aldolization of deoxyxyosones was promoted, facilitating the generation of reactive α-dicarbonyl compounds. In this way, exogenous threonine accelerated the release of endogenous threonine and α-dicarbonyl compounds and the pH decline was slowed down, which was favorable for the formation of pyrazines.

Efficient co-production of xylo-oligosaccharides and probiotics from corncob by combined lactic acid pretreatment and two-step enzymatic hydrolysis.

Lactic acid (LA) is efficient in xylo-oligosaccharides (XOS) production from poplar. However, the role of LA in XOS production from corncob has not been carefully elucidated, and the co-production of probiotics of Bacillus subtilis from corncob residue has not been reported. In this study, LA pretreatment was combined with enzymatic hydrolysis to produce XOS and monosaccharides from corncob. An XOS yield of 69.9% was obtained from corncob by combining 2% LA pretreatment and xylanase hydrolysis. Yields of 95.6% glucose and 54.0% xylose were obtained from corncob residue via cellulase, and the resulting cellulase hydrolysate was used to culture Bacillus subtilis YS01. The resulting viable count of the strain was 6.4×108 CFU/mL, and the glucose and xylose utilization were 99.0% and 89.8%, respectively. This study demonstrated a green, efficient, and mild process for producing XOS and probiotics from corncob by combining LA pretreatment and enzymatic hydrolysis.

Structural characterization of corn fiber hemicelluloses extracted by organic solvent and screening of degradation enzymes.

An integrated treatment coupling peracetic acid delignification, dimethyl sulfoxide extraction, and ethanol precipitation were performed to isolate hemicellulose from de-starched corn fiber. Based on chemical composition, molecular weight distribution, methylation, and nuclear magnetic resonance spectroscopy, it is proposed that hemicelluloses in corn fiber were composed of two polysaccharides, glucuronoarabinoxylan (about 80 %) and xyloglucan (about 20 %). Xylose (about 46 %) and arabinose (about 32 %) were the main components in glucuronoarabinoxylan. More than half of the xylose units in the glucuronoarabinoxylan backbone chain were substituted at O-2 and/or O-3 by various monomers or oligomeric side chains. Based on structure analysis, five hemicellulases were selected and added to Penicillium oxalicum MCAX enzymes for enzymatic hydrolysis of corn fiber. The results showed that the addition of hemicellulases increased the sugar yield of corn fiber. These results demonstrate the effectiveness of enzyme consortium constructed by elucidating the chemical structure of hemicellulose in corn fiber for the degradation of corn fiber and also provide a general solution for the rational construction of targeted and efficient enzyme systems for the degradation of lignocellulosic biomass.

Crystal structure of reducing-end xylose-releasing exoxylanase in subfamily 7 of glycoside hydrolase family 30.

TcXyn30A from Talaromyces cellulolyticus, which belongs to subfamily 7 of the glycoside hydrolase family 30 (GH30-7), releases xylose from the reducing end of xylan and xylooligosaccharides (XOSs), the so-called reducing-end xylose-releasing exoxylanase (ReX). In this study, the crystal structures of TcXyn30A with and without xylose at subsite +1 (the binding site of the xylose residue at the reducing end) were determined. This is the first report on the structure of ReX in the family GH30-7. TcXyn30A forms a dimer. The complex structure of TcXyn30A with xylose revealed that subsite +1 is located at the dimer interface. TcXyn30A recognizes xylose at subsite +1 composed of amino acid residues from each monomer and blocks substrate binding to subsite +2 by dimer formation. Thus, the dimeric conformation is responsible for ReX activity. The structural comparison between TcXyn30A and the homologous enzyme indicated that subsite -2 is composed of assembled three stacked Trp residues, Trp49, Trp333, and Trp334, allowing TcXyn30A to accommodate xylan and any branched XOSs decorated with a substitution such as α-1,2-linked 4-O-methyl-d-glucuronic acid or α-1,2- and/or -1,3-linked L-arabinofuranose. These findings provide an insight into the structural determinants for ReX activity of TcXyn30A.

Fast and Selective Degradation of Biomass for Xylose, Glucose and Lignin under Mild Conditions.

The conversion of lignocellulose into valuable chemicals has been recognized as the key technology in green chemistry. However, selective degradation of hemicellulose and cellulose with the production of lignin is still a challenge. Therefore, a two-step process has been developed to degrade corncob into xylose and glucose under mild conditions. At first, the corncob was treated with the lower concentration of zinc chloride aqueous solution (30-55 w%) at 95 °C with a short reaction time (8-12 min) and 30.4 w% (selectivity = 89%) of xylose obtained with a solid residue of the composite of cellulose and lignin. Next, the solid residue was treated with a high concentration of zinc chloride aqueous solution (65-85 w%) at 95 °C for about 10 min, and 29.4 w% (selectivity = 92%) of glucose can be obtained. Combining the two steps, the total yield of xylose is 97%, while glucose is 95%. In addition, high pure lignin can be obtained simultaneously, which was confirmed using HSQC studies. Furthermore, for the solid residue of the first-step reaction, a ternary deep eutectic solvent (DES) (choline chloride/oxalic acid/1,4-butanediol, ChCl/OA/BD) has been used to separate the cellulose and lignin efficiently, and high-quality cellulose (Re-C) and lignin (Re-L) were obtained. Furthermore, it provides a simple method to disassemble the lignocellulose for monosaccharides, lignin, and cellulose.

Production of Designer Xylose-Acetic Acid Enriched Hydrolysate from Bioenergy Sorghum, Oilcane, and Energycane Bagasses.

Xylan accounts for up to 40% of the structural carbohydrates in lignocellulosic feedstocks. Along with xylan, acetic acid in sources of hemicellulose can be recovered and marketed as a commodity chemical. Through vibrant bioprocessing innovations, converting xylose and acetic acid into high-value bioproducts via microbial cultures improves the feasibility of lignocellulosic biorefineries. Enzymatic hydrolysis using xylanase supplemented with acetylxylan esterase (AXE) was applied to prepare xylose-acetic acid enriched hydrolysates from bioenergy sorghum, oilcane, or energycane using sequential hydrothermal-mechanical pretreatment. Various biomass solids contents (15 to 25%, w/v) and xylanase loadings (140 to 280 FXU/g biomass) were tested to maximize xylose and acetic acid titers. The xylose and acetic acid yields were significantly improved by supplementing with AXE. The optimal yields of xylose and acetic acid were 92.29% and 62.26% obtained from hydrolyzing energycane and oilcane at 25% and 15% w/v biomass solids using 280 FXU xylanase/g biomass and AXE, respectively.

Glycomic profiling identifies key-structural differences in three arabinoxylan fractions from sugarcane culms.

Sugarcane is an important food and bioenergy crop, and although the residual biomass is potentially available for biorefinery and biofuels production the complex plant cell wall matrix requires pretreatment prior to enzymatic hydrolysis. Arabinoxylans require multiple enzymes for xylose backbone and saccharide side-branch hydrolysis to release xylooligosaccharides and pentoses. The effect of arabinoxylan structure on xylooligosaccharide release by combinations of up to five xylanolytic enzymes was studied using three arabinoxylan fractions extracted from sugarcane culms by sodium chlorite, DMSO and alkaline treatments. Reducing sugar release and LC-MS detection with chemometric analysis identified different xylooligosaccharide profiles between extracts following enzyme treatments. The position and degree of side-branch decorations are determinants of enzyme activity and xylooligosaccharide diversity with the alkaline and post­sodium chlorite extracts as the most accessible and most recalcitrant, respectively, indicating acetyl substituents as a major recalcitrance factor. The complex xylooligosaccharide profile with the DMSO extract suggests regions with different levels of branching. Chemometric analysis identified GH10 xylanase hydrolysis products that act as substrates for other enzymes, such as α-glucuronidase. The strategy reported here can identify specific enzyme combinations to overcome barriers for biomass processing such as pretreatment selection, recalcitrance to enzyme digestion and optimization of reducing sugar release.

Controlled Selective Formation of Amadori Compounds from α/ε Mono- or Di-glycation of Lysine with Xylose.

Three Amadori rearrangement products (Xyl-α-Lys-ARP, Xyl-ε-Lys-ARP, and diXyl-α,ε-Lys-ARP) were observed in the xylose-lysine (Xyl-Lys) Maillard reaction model. They were separated and characterized by liquid chromatography with tandem mass spectrometry and NMR. The crucial roles of reaction temperature, pH, molar ratio of Xyl to Lys, and reaction time in the formation of different Xyl-Lys-ARPs were investigated. The proportion of Xyl-α-Lys-ARP among all Xyl-Lys-ARPs was increased to 48.41% (its concentration was 25.31 µmol/mL) after the reaction at pH = 5.5 and a molar ratio of 3:1 (Xyl: Lys) for 9 min, while only Xyl-ε-Lys-ARP was generated at a higher pH (7.5) and a lower molar ratio of 1:5. Moreover, the much higher activation energy (84.08 kJ/mol) of diXyl-α,ε-Lys-ARP than Xyl-α-Lys-ARP (34.19 kJ/mol) and Xyl-ε-Lys-ARP (32.32 kJ/mol) indicated a pronounced promoting effect on diXyl-α,ε-Lys-ARP formation by high temperatures. A complete conversion from Xyl-α-Lys-ARP and Xyl-ε-Lys-ARP to diXyl-α,ε-Lys-ARP was achieved through the reaction time prolongation and Xyl concentration increase at a higher temperature; the concentration of diXyl-α,ε-Lys-ARP was 39.05 µmol/mL at a molar ratio of 5:1 for 40 min. Accordingly, the selective preparation of Xyl-α-Lys-ARP, Xyl-ε-Lys-ARP, and diXyl-α,ε-Lys-ARP could be achieved through adjusting the Xyl-Lys ratio, pH, and reaction time.

Characteristics and antioxidant properties of Harpadon nehereus protein hydrolysate-xylose conjugates obtained from the Maillard reaction by ultrasound-assisted wet heating in a natural deep eutectic solvents system.

BACKGROUND: Harpadon nehereus is a high-protein marine fish. A valuable way to add value to H. nehereus is to convert it into protein hydrolysate. The Maillard reaction is an effective way to improve the functional properties of peptides and proteins, which are affected by many factors such as reactant concentration, water activity, pH, temperature, and heating time. However, the traditional Maillard reaction method is inefficient. The purpose of this study was therefore to explore the effect of the ultrasound-assisted wet heating method on the Maillard reaction of H. nehereus protein hydrolysate (HNPH) in a new-type green solvent - a natural hypereutectic solvent (NADES). RESULTS: Harpadon nehereus protein hydrolysate-xylose (Xy) conjugates were prepared via a Maillard reaction in a NADES system using an ultrasound-assisted wet heating method. The effects of different treatment conditions on the Maillard reaction were studied. The optimized glycation degree (DG) of HNPH-Xy conjugates was obtained with a water content of 10%, a reaction temperature of 80 °C, a reaction time of 35 min, and an ultrasonic power level of 300 W. Compared with HNPH, the structure of HNPH-Xy conjugates were significantly changed. Moreover, the functional properties and antioxidant activity of HNPH-Xy were all superior to the HNPH. CONCLUSIONS: An ultrasound-assisted wet-heating Maillard reaction between HNPH and Xy in the NADES system could be a promising way to improve the functional properties of HNPH. © 2023 Society of Chemical Industry.