Efficient Hydrolysis Research Articles

Optimization of ultrasound-assisted enzymatic hydrolysis Zophobas morio protein and its protective effects against H2O2-induced oxidative stress in RAW264.7 cells.

Zophobas morio protein (ZMP) is a promising protein resource with notable biological properties, and its hydrolysis could unlock enhanced bioactivities. This study investigated ultrasound-assisted enzymatic hydrolysis (UAEH) of ZMP using different enzymes (Alcalase, Neutrase, and Protamex) to determine its effect on the degree of hydrolysis (DH) compared to enzymatic hydrolysis (EH). UAEH showed greater hydrolysis efficiency than EH, with Alcalase exhibiting the highest DH. Response surface methodology (RSM) was applied to optimize UAEH conditions for Zophobas morio protein hydrolysate (ZMPH). Optimal conditions for producing ZMPH with the maximum DH were a substrate concentration of 3.52 % (w/v), enzyme to substrate ratio of 7.64 % (v/v), and pH of 8.35. Under the optimal condition, the maximum DH was 25.03 %. In addition, significant structural changes in the optimized ZMPH compared to ZMP were identified, showing decreased α-helix and β-sheet content, with increased β-turn and unordered coil. Moreover, the optimized ZMPH demonstrated significantly improved ABTS antioxidant activity and attenuated H2O2-induced cell death in RAW264.7 cells compared to ZMP, which was attributed to better mitigation of ROS production. These findings provide an effective enzymatic hydrolysis method for producing ZMPH with significant antioxidant activity, demonstrating the potential of ultrasound-assisted hydrolysis in enhancing the bioactivity of insect proteins.

Characteristics and Biological Activities of Bioactive Peptides Derived from Bulgur Waste

Bulgur is one of the ready or semi-ready to eat cereals produced from wheat specifically Triticum durum variety. Residues of bulgur processing are known as bulgur waste that rich in some food components. Protein is one of the main components of bulgur that may remain in the wastes. This study was carried out to obtain and investigate the properties of bioactive peptides of bulgur waste proteins. Protein isolated from bulgur waste was hydrolyzed enzymatically to bioactive peptides and their potential activity against oxidation stress, microbial inhibition and hypertension control was determined. The bulgur waste proteins extracted from samples were hydrolyzed at different time intervals using pepsin, trypsin, chymotrypsin and protease under the optimum conditions of enzymes and o-phthalaldehyde (OPA) method was used to determine the degree of hydrolyses. The highest rate of hydrolysis efficiency was observed by protease as 10.08% at 240 min treatment while, the highest antioxidant capacity was measured with chymotrypsin (526.35% at 240 min) by 2,2′-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS) and with trypsin (151.93 % at 240 min) by 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. Trypsin hydrolysates showed the highest antibacterial activity against Escherichia coli whereas pepsin hydrolysates exhibited the highest activity against Staphylococcus aureus. It has been observed that trypsin, chymotrypsin and protease hydrolysates have higher antihypertensive effects than protease hydrolyzates. The highest antihypertensive effect was obtained with protein hydrolyzate obtained by hydrolysis with chymotrypsin for 180 minutes. As a result, the novel peptides indicated to offer the selected biological effects, suitable to use as a food additive for different purposes in industrial applications.

Efficient Production of Fuel Ethanol via the Simultaneous Use of Distillery Stillage Biomass and Beet Molasses

The integrated production of ethanol fuel through the simultaneous use of various by-products and waste materials is an intriguing concept, as it maximizes the raw material potential while addressing the challenge of managing waste biomass from different technological processes. The efficient utilization of lignocellulosic waste depends on employing a pretreatment method that enhances the susceptibility of structural polysaccharides to hydrolysis. The aim of the study was to assess the possibility of the simultaneous use of corn stillage biomass and beet molasses as raw materials for the production of ethanol fuel. The research focused on optimizing the process conditions for the acid pretreatment of stillage biomass and the enzymatic hydrolysis of cellulose and evaluating the effectiveness of two fermentation strategies: SHF (Separate Hydrolysis and Fermentation) and SSF (Simultaneous Saccharification and Fermentation). The highest hydrolysis susceptibility was observed in biomass pretreated with 2% v/v H3PO4 for 30 min at 121 °C. The maximum glucose concentration of about 12 g/L (hydrolysis efficiency about 35.5%) was achieved even with the lowest enzyme dose, i.e., 7.5 FPU per gram of biomass. The yeast also showed high fermentation activity in media prepared from stillage biomass and molasses, producing about 50 g/L of ethanol regardless of the fermentation strategy used. The complete fermentation of carbohydrates assimilated by yeast confirmed the complementarity of the two raw materials used to prepare fermentation media, emphasizing the high potential of the proposed technological solution for ethanol fuel production.

Solvent-Driven Self-Assembly of Discrete Ni(II)-Azide Complexes: Unraveling Unusual Behavior in Mimicking Jack Bean Urease.

The well-known inhibitory strength of 3d metal Schiff base complexes against urease enzymes has long been acknowledged, but their untapped potential to act as ureolytic mimics of active metallobiosites remained unexplored. To break the new ground, we present pyrrolidine-based mononuclear Ni(II)-azide complex 1 {[NiL(HL)(N3)]·1.5(H2O)} using the N,N,O donor ligand, namely (E)-4-bromo-2-(((2-(pyrrolidin-1-yl)ethyl)imino)methyl)phenol. The initial spectrophotometric analysis showed catalytic inefficiency in methanol since it undergoes unexpected ligand dissociation to generate a new octahedral nickel complex (INC), catalyzing condensation to form BrTz. Notably, complex 1 was subjected to self-assembly in DMF into UV-responsive tetranuclear complex 2 {[NiL(H2O)(N3)]4} and structurally characterized using single-crystal XRD. Furthermore, complex 2 was utilized as a functional urease model, demonstrating catalytic efficiency in urea hydrolysis with the estimation of the liberated ammonia and CO2 in MeOH. The mechanistic pathway was speculated to proceed via the hydrogen bonding of urea with bridging azides, facilitating coordination with the nickel center. Moreover, it significantly inhibited hydrolysis in the presence of external NBPTO [N-(n-butyl)thiophosphoric triamide], guanidine, etc., revealing its potential for precise catalytic control. To the best of our knowledge, this is the first report of the urease-mimicking activity of the tetranuclear Ni(II) complex and elucidating the mechanism through detailed chemical analysis.

Improved enzymatic hydrolysis of corn stover by a low-temperature and low-pressure holding post-treatment.

Lignocellulose is one of the world's most abundant and underutilized biomass resources, and its proper treatment and utilization are critical to environmental issues and sustainable development. However, lignocellulose's inherently compact and intricate structure reduces enzymatic hydrolysis's efficiency, which is still an obstacle to overcome. A new pretreatment method with relatively low-temperature and low-pressure holding (LTLPH) after the traditional extrusion, pulp refining instrument (PFI), and instant catapult steam explosion (ICSE) was proposed to obtain a better output of corn stover saccharification. The chemical composition, SEM, swelling capacity of corn stover before and after treatment, and types and contents of mixed sugars were examined to explore the mechanism for the diversity of the enzymatic hydrolysis effect. It was found that the highest reducing sugar in the optimized compounding pretreatment method of ICSE-LTLPH (LTLPH: 70kPa, 115°C, 30min) could reach 27.96g/L, promoting more than 50%. The cultured single-cell protein content was 8.19% and 7.73% higher than those with glucose and simulated mixed sugar medium, respectively, promisingly replacing commercial sugars for Candida utilis (C. utilis) growth. Therefore, the developed pretreatment method of ICSE-LTLPH could exert better effects on the enzymatic hydrolysis of corn stover, providing a potential for cultivating C. utilis without detoxification of enzymatic hydrolysate.

Unlocking the influence of PNPLA3 mutations on lipolysis: Insights into lipid droplet formation and metabolic dynamics in metabolic dysfunction-associated steatotic liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) covers a range of liver conditions marked by the buildup of fat, spanning from simple fatty liver to more advanced stages like metabolic dysfunction-associated steatohepatitis and cirrhosis. Our in-depth analysis of PNPLA3_WT and mutants (I148M (MT1) and C15S (MT2)) provides insights into their structure-function dynamics in lipid metabolism, especially lipid droplet hydrolysis and ABHD5 binding. Employing molecular docking, binding affinity, MD analysis, dissociation constant, and MM/GBSA analysis, we delineated distinct binding characteristics between wild-type and mutants. Structural dynamics analysis revealed that unbound mutants exhibited higher flexibility, increased Rg and SASA values, and broader energy landscapes, indicating multiple inactive states. Mutations, especially in PNPLA3_MT1, reduced the exposure of the catalytic serine, potentially impairing enzymatic activity and LD hydrolysis efficiency. Altered interaction patterns and dynamics, particularly a shift in ABHD5 binding regions towards the C-terminal domain, underscore its role in LD metabolism. Energy dynamics analysis of the protein complexes revealed PNPLA3_WT exhibited multiple low-energy macrostates, whereas the mutants displayed narrower energy landscapes, suggesting a more stable functional state. PNPLA3_MT1 demonstrated the highest affinity towards ABHD5, highlighting the complex interplay between protein structure, dynamics, and lipid metabolism regulation. PNPLA3_MT1 mutant exhibits the highest flexibility and significantly reduced catalytic serine accessibility, leading to impaired lipolysis. Contrarily, PNPLA3_WT maintains stable catalytic efficiency and effective LD hydrolysis, with PNPLA3_MT2 displaying intermediate behavior. Our research provides valuable insights into the metabolic implications of PNPLA3 mutations, offering a path for potential therapeutic interventions in MASLD.

Extraction of cellulose nanocrystals from date seeds using transition metal complex-assisted hydrochloric acid hydrolysis.

In this study, the role of a transition metal complex in improving hydrolysis efficiency during nanocellulose production was analysed. Cellulose nanocrystals (CNCs) were extracted from date seeds by incorporating a copper metal complex during HCl hydrolysis. In contrast to traditional HCl hydrolysis at moderate conditions, which yielded only microcrystalline cellulose (MCC), this approach resulted in the extraction of CNCs with a 10% improved yield compared to MCC. Morphological analysis using scanning electron microscopy revealed semi-spherical shaped particles, while transmission electron microscopy showed CNCs with a particle size ranging from 70 to 80nm. Dynamic light scattering analysis indicated a significant reduction in average particle size from 900nm to 121nm, highlighting the remarkable efficiency of using the copper metal complex in combination with HCl to improve yield and particle size. Energy dispersive X-ray spectroscopy analysis confirmed the purity of the CNCs, with no residual copper detected. Thermal analysis demonstrated the high stability of the CNCs, with an initial decomposition temperature (Tonset) of 274.02°C and an activation energy (Ea) of 219.90kJ/mol. X-ray diffraction analysis revealed that the CNCs exhibited high degree of crystallinity (Crl=72.03%). Disseminating these research findings will significantly impact the CNCs production industry, facilitating improved yields and the production of nano-sized fibers through the utilization of transition metal complexes alongside hydrolysis solvents.

Efficient Hydrolysis of Fish Parvalbumin by Marine Bacterial Protease VSP2V-280: Allergen Removal.

Parvalbumin is a major allergen in fish. However, there is currently no effective and safe way to remove this allergen from fish. In this study, protease gene VSP2V-280 of marine bacteria Virgibacillus sp. SP2 was cloned and expressed. The protease enzyme showed maximum activity at 50°C and pH 10.0. Ca2+ and Cu2+ promoted the enzyme. The enzyme showed good parvalbumin degradation efficiency in fish. Based on the gel analysis, when 0.3 mg/mL of parvalbumin was incubated with protease VSP2V-280 (30 U/mL) containing 1 mM Ca2+ for 3 h, the parvalbumin removal rate reached 97%. The enzyme was further used for parvalbumin removal from Ctenopharyngodon idella, Pelteobagrus fulvidraco, Parabramis pekinensis, and Carassius auratus. The parvalbumin removal rate reached 93% in 4 h at an enzyme dosage of 72 U/mL. The study showed the potential of VSP2V-280 to remove parvalbumin from aquatic products.

Construction of Brönsted sites on pyrite surface via plasma technology for efficient hydrolysis of microcystins-LR

Construction of Brönsted sites on pyrite surface via plasma technology for efficient hydrolysis of microcystins-LR

Hydrolysis of palm kernel meal fibre using a newly isolated Bacillus subtilis F6 with high mannanase activity

AbstractPalm kernel meal (PKM) presents a challenge for non-ruminant livestock feeding due to its high fibre content predominantly in the form of mannan. Microbial fermentation offers a sustainable solution for fibre hydrolysis in lignocellulosic biomass. In this study, a Bacillus subtilis strain (F6), with high mannanase secretion capability, was isolated from the environment. Fermentation of PKM with B. subtilis F6 resulted in at least a 10% reduction in neutral detergent fibre, decreasing from 78.4 to 60.9% within 24 h. Notably, B. subtilis F6 rapidly responded to PKM, producing significant mannanase activity within 6 h, facilitating quick fibre degradation. Transcriptome analysis identified key enzymes involved in this process, with β-mannanase GmuG showing the highest increase in expression (45.2-fold) after fermentation. Purified recombinant GmuG exhibited strong PKM hydrolysis activity, primarily releasing mannobiose and mannotriose. Characterization of GmuG using locust bean gum as a substrate revealed an optimum temperature of 50–55 °C and pH optima at 5.0 and 9.0. This study highlights the potential of B. subtilis F6 and its mannanase GmuG for efficient PKM fibre hydrolysis, and provides insights into their application in the valorization of mannan-rich bioresources. Graphical Abstract

Comparative Exploration of Antioxidant Properties of Alcalase- and Trypsin-Hydrolyzed Porcine By-Products and Their Classification for Industrial Use

Porcine by-products have garnered attention as an excellent material for producing antioxidant peptides; however, understanding the antioxidant characteristics of protein hydrolyzates derived from specific parts remains limited. In this study, we compared the antioxidant properties of protein hydrolyzates derived from major porcine organs (heart, kidney, spleen, liver, and lung) and performed classification based on their antioxidative potential. Their chemical composition exhibited significant variations, with a high protein content ranging from 15.90 to 20.30 g/100 g. Alcalase achieved higher hydrolysis efficiency than trypsin, which induced limited degradation of some proteins, such as porcine serum albumin. The hydrolyzates exhibited superior radical scavenging activities compared to the raw materials, although their reducing power remained unaffected or, in some instances, decreased. Hierarchical and k-mean cluster analyses revealed distinct antioxidant profiles and Alcalase-hydrolyzed kidney and trypsin-hydrolyzed lung hydrolyzates were deemed the most promising candidates, with strong radical scavenging activities and reducing power. Our findings indicate that, even when processed in bulk rather than being obtained from specific parts, porcine by-products can produce hydrolyzates rich in antioxidant peptides through enzymatic hydrolysis. However, selectively processing porcine kidneys with Alcalase and lungs with trypsin is recommended to produce premium products with enhanced and balanced antioxidant properties.

Indirect Formation of Peptide Bonds as a Prelude to Ribosomal Transpeptidation.

The catalytic competency of the ribosome in extant protein biosynthesis is thought to arise primarily from two sources: an ability to precisely juxtapose the termini of two key substrates─3'-aminoacyl and N-acyl-aminoacyl tRNAs─and an ability to ease direct transpeptidation by their desolvation and encapsulation. In the absence of ribosomal, or enzymatic, protection, however, these activated alkyl esters undergo efficient hydrolysis, while significant entropic barriers serve to hamper their intermolecular cross-aminolysis in bulk water. Given that the spontaneous emergence of a catalyst of comparable size and sophistication to the ribosome in a prebiotic RNA world would appear implausible, it is thus natural to ask how appreciable peptide formation could have occurred with such substrates in bulk water without the aid of advanced ribozymatic catalysis. Using a combination of fluorine-tagged aminoacyl adenylate esters, in situ monitoring by 19F{1H} NMR spectroscopy, analytical deconvolution of kinetics, pH-rate profile analysis, and temperature-dependence studies, we here explore the mechanistic landscape of indirect amidation, via transesterification and O-to-N rearrangement, as a highly efficient, alternative manifold for transpeptidation that may have served as a prelude to ribosomal peptide synthesis. Our results suggest a potentially overlooked role for those amino acids implicated by the cyanosulfidic reaction network with hydroxyl side chains (Ser and Thr), and they also help to resolve some outstanding ambiguities in the broader literature regarding studies of similar systems (e.g., aminolyzes with Tris buffer). The evolutionary implications of this mode of peptide synthesis and the involvement of a very specific subset of amino acids are discussed.

Exopolygalacturonase Production from the Novel Strain Lichtheimia sp. UV-16 and Enzyme Hydrolysis Properties.

A pectinase-producing strain, Lichtheimia sp. X-8, was isolated from the soil for the first time. Subsequently, Lichtheimia sp. UV-16, with a 1.23-fold increase in pectinase activity, was obtained via UV mutagenesis, and optimization of its liquid fermentation process boosted pectinase activity from 455.6 ± 12.7 to 3202.0 ± 82.1 U/mL. The crude enzyme was purified by salting out and anion exchange resin, with a purification ratio of 2.28-fold and a yield of 36.5%. The optimal reaction temperature for the pure enzyme was 60 °C with an optimal pH of 5.5. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) confirmed that the enzyme was an exopolygalacturonase, achieving over 99% efficiency in pectin hydrolysis. Furthermore, incorporating pure enzymes into juice pulps can substantially enhance the juice yield, which makes this polygalacturonase a promising application in the beverage industry.

Efficient Cellobiose Hydrolysis over a Sulfonated Carbon Catalyst in a Spatially Separated Microwave Electric- and Magnetic-Field Flow Reactor

Efficient Cellobiose Hydrolysis over a Sulfonated Carbon Catalyst in a Spatially Separated Microwave Electric- and Magnetic-Field Flow Reactor

Elucidation of Domain Function of a Novel Multifunctional Glycoside Hydrolase and Its Use in Efficient Preparation of Oligosaccharides from Kelp Powder.

Kelp contained laminarin, cellulose, and alginate as major polysaccharides and could be utilized as functional oligosaccharides. A new multifunctional glycoside hydrolase CelA was identified and characterized for the efficient degradation of kelp powder. It displayed cellulase (2308.38 U/mg), alginate lyase (578.68 U/mg), and laminarinase (720.97 U/mg) activities. It exhibited maximal activity on both sodium alginate and laminarin at 50 °C and pH 8.0, while it could degrade sodium carboxymethylcellulose (CMC-Na) by maximal activity at 40 °C and pH 7.0. The action mode analysis by thin layer chromatography and electrospray ionization mass spectrometry indicated that CelA adopted an endolytic manner to degrade CMC-Na, sodium alginate, and laminarin releasing oligosaccharides with degrees of polymerization (Dps) of 2-5. According to domain analysis, CelA contained a GH5 module and a PL6 module, and both of them exhibited glycoside hydrolase and polysaccharide lyase activity. The docking results revealed that Glu163 and Glu250 are essential in cellulose and laminarin degradation. As to the degradation of alginate, Asn376, Lys436, Arg464, Asp496, and Asn551 could bind alginate and Tyr492 and Lys529 acted as catalytic sites. CelA displayed high hydrolysis efficiency for cellulose, β-glucan, laminarin, alginate, and kelp powder. Thus, it has strong potential in food and feed industries as a catalyst for bioconversion of algal biomass into value-added products oligosaccharides.

Hydrolysis Process of Red Sorghum Grains for Producing Bioethanol

As part of the effort to continuously enhance the development and utilization of bioenergy/bioethanol and to ensure its sustainability, using sorghum as a raw material for bioethanol production requires a study to obtain efficient hydrolysis conditions and achieve optimal bioethanol conversion. This research method is divided into two main parts: samples with pretreatment and without pretreatment. The pretreatments used NaOH solution (0.05% and 0.1%); soaking temperatures at 35,40 and 45oC; and soaking time at 30, 60, 90, and 120 minutes. The liquefaction and saccharification results with pretreatment showed a greater decrease in viscosity values after both liquefaction and saccharification, and the RS and DE results also demonstrated an increasing trend compared to samples without pretreatment. It is due to the presence of protein bodies in sorghum that inhibited hydrolysis without applying the pretreatment. The best result on the reduced sugar (RS) value achieved was 10.43%, and the DE value was 67.90%. Thus, the optimum operational condition was achieved with a sample with 0.05% NaOH soaking for 90 minutes at 45°C (A9). Subsequently, fermentation was carried out on sample A9 with pretreatment and sample without pretreatment (A0/B0). The alcohol obtained from fermentation with pretreatment was 9.5%, with a fermentation ratio (FR) of 86.97%. Meanwhile, the alcohol yield from fermentation for the sample without pretreatment was 8.9%, with an FR of 83.02%.

The Effect of Polyethylene Glycol Addition on Improving the Bioconversion of Cellulose.

In recent years, many studies have focused on improving the bioconversion of cellulose by adding non-ionic surfactants. In our study, the effect of the addition of a polymer, polyethylene glycol (PEG 4000), on the bioconversion of different cellulose materials was evaluated, focusing on the hydrolysis efficiency and structural changes in pure cellulose after the enzymatic hydrolysis process. The obtained results showed that the addition of non-ionic surfactant significantly improved the digestibility of cellulosic materials. The highest hydrolysis efficiency was observed for Sigmacel 101 (Cel-S101) cellulose, which consists mainly of amorphous regions. In the case of Avicel cellulose (Cel-A), PEG had a lesser effect on the bioconversion's efficiency due to limited access to the crystal structure and limited substrate-cellulase interactions. The consistency of the obtained results is confirmed by qualitative and quantitative analyses (XRD, FTIR, and HPLC). Our findings may be helpful in further understanding the mechanism of the action of surfactants and improving the enzymatic hydrolysis process.

Comparison between detection power of MBT STAR-Carba test and KBM CIM Tris II for carbapenemase-producing bacteria

Abstract Objectives The rapid detection of carbapenemase-producing bacteria is clinically important for selecting appropriate antimicrobial therapy. Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to detect carbapenemase activity. Methods In this study, we evaluated the detection power of MBT STAR-Carba test on identifying carbapenemase-producing bacteria isolated in Kobe city, Japan, compared with that of the KBM CIM Tris II kit using the modified procedure parameters. The obtained results were expressed as normalized logRQ values indicating a measure of hydrolysis efficiency. Results The MBT STAR-Carba test rapidly detected not only major carbapenemases, such as IMP-1 and IMP-6 that are most prevalent in Japan, but also GES-type and OXA-51-like carbapenemases, which are difficult to detect by reaction with inhibitors or KBM CIM Tris II by extending the incubation time. Conclusions The MBT STAR-Carba test will be beneficial in rapid identification of carbapenemases in clinical settings and environmental investigations.

Alkaline Proteases from Rose Snapper (Lutjanus guttatus): Evaluation of Their Stability to Chemical Denaturants and Potential Application to Hydrolyze Seafood Waste Proteins.

Large quantities of by-products are generated after processing of rose snapper (Lutjanus guttatus), such as viscera, head, tail, skin, and bones, which are considered a potential source of valuable molecules. Therefore, the aim of the present study was the biochemical characterization of alkaline proteases isolated from the intestines of L. guttatus and the evaluation of their stability against different chemical denaturants (salts, surfactants/reducing agents, organic solvents, and commercial detergent formulations). In addition, the efficiency to hydrolyze proteins from rose snapper wastes (head, tail, skin, and muscle trimmings) by Alcalase® and alkaline protease extract (APE) isolated from Lutjanus guttatus intestine was evaluated. The APE exhibited a maximum activity at pH 12 and 45°C and high stability at pH and temperature ranges from 9 to 12 and 10 to 40°C, respectively. Assays with specific protease inhibitors indicated that trypsin and chymotrypsin are the main types of proteases in APE. An 80% of the proteolytic activity was retained in the presence of 25% NaCl and was stable in the presence of the reducing agent DTT; however, it lost around 70% of proteolytic activity in the presence of 2-mercaptoethanol. The enzymatic activity of APE was maintained above 60% in methanol, ethanol, and propanol as well as in liquid commercial detergents. Alkaline proteases from rose snapper exhibited higher hydrolytic efficiency, compared to the microbial enzyme Alcalase when protein from L. guttatus wastes were hydrolyzed. According to these results, the integral exploitation of rose snapper could be reached by proper usage of its by-products, creating a baseline to promote circular economy.

Volatile fatty acid production from different spent mushroom substrates via anaerobic fermentation: Hydrolysis and acidogenesis efficiency and bacterial community structure

Converting renewable biomass resources into biofuels or chemical raw materials is expected to help alleviate energy shortage. Selecting suitable anaerobic fermentation substrates is a key factor in determining high yields of volatile fatty acids (VFAs). Spent mushroom substrates (SMSs) rich in polysaccharides, proteins, lignocellulose and micronutrients may be a high-quality feedstock for producing VFAs. However, the hydrolysis and acidogenesis efficacies of different types of SMSs remain unclear. In this study, we investigated the hydrolysis and acidogenesis performances of four SMSs, namely, Hericium erinaceus (HES), Pleurotus ostreatus (POS), Flammulina velutipes (FVS) and Pleurotus eryngii (PES), and analysed the microbial community composition in the anaerobic fermentation system via 16S rRNA sequencing. Results revealed that the PES reactor afforded the maximum VFA yield of 248.48mg/g, followed by HES, FVS and POS. This might be related to the higher content of polysaccharides and proteins in PES compared to the other three types of SMSs. The higher abundance of Bacillus, Sporolactobacillus, Clostridium_Sensu_Stricto_1 and Clostridium_Sensu_Stricto_19 associated with hydrolysis and acidogenesis in the PES anaerobic fermentation system was responsible for its higher VFA production than the other three SMSs. The carbohydrate metabolism and amino acid metabolism driven by microbes were involved in the hydrolysis and acidogenesis process of SMSs. Notably, the expression levels of key functional genes involved in the VFA production pathway with PES as the fermentation substrate were the highest. These findings provide theoretical support for the large-scale industrial application of SMSs in anaerobic digestion.