Search Results (184)

Phi Man Long Long Rak Cave, located in Mae Hong Son Province, northern Thailand, is a prehistoric burial site containing ancient wooden coffins that have undergone biodeterioration, likely due to fungal activity. Both culture-dependent and culture-independent approaches were employed to characterize fungal communities and assess their roles in wood degradation. Culture-dependent analysis identified five Aspergillus isolates from the wooden coffins, most of which produced cellulolytic and hemicellulolytic enzymes; some isolates also produced organic acids, indicating significant degradative potential. Culture-independent analysis revealed a community dominated by Aspergillus, together with additional taxa such as Penicillium and Ceriporia that were not detected by cultivation, highlighting greater community diversity and demonstrating the complementarity of the two methods. Functional prediction indicated a predominance of saprotrophic fungi. The presence of shared dominant taxa between soil and coffin-associated substrates suggests ecological connectivity at the soil–coffin interface, although the direction of dispersal cannot be determined from the present data. All tested fungicides inhibited fungal growth, with the highest efficacy observed in the formulation containing the highest proportion of active components. Taken together, these findings provide insights into fungal biodeterioration processes and inform conservation strategies. Full article

Marine endophytic fungi inhabit the internal tissues of seaweed, seagrass, and mangroves without causing harm. These fungi are known to produce extracellular enzymes, including proteases and cellulases, which play crucial roles in various biological processes and have potential applications in diverse industrial sectors. This study aimed to screen the enzymatic potential of marine endophytic fungi, identify selected isolates, and characterize their enzyme activities. A total of 20 fungal isolates were obtained, comprising 16 isolates from seaweed, three from seagrass, and one from mangrove leaves, collected from the coastal areas of the Seribu Islands (Jakarta), Sukabumi (West Java), Nusa Dua (Bali), and the Buton Islands (Southeast Sulawesi). Screening results showed that 50% of the isolates exhibited proteolytic activity on skim milk agar, while 40% demonstrated cellulolytic activity on carboxymethylcellulose (CMC) agar. Two isolates with the highest clear zone indices for protease and cellulase activity were identified as Penicillium citrinum and Fomitopsis sp., with distinct morphological characteristics including velvety colonies and filamentous hyphal structures. The specific activities of the protease and cellulase were 5475.42 ± 2724.25 U/mg protein and 620.77 ± 607.71 U/mg protein, respectively, indicating high catalytic potential. Full article

Table olives, particularly traditionally fermented cracked-style green olives, rely on natural microbial activity without chemical debittering, with fungi playing key roles; in contrast, arbutus berry fermentation remains less characterized in terms of microbial functionality. This study investigated the enzymatic and antibacterial potential of fungal isolates from both systems. A total of 84 isolates belonging to Aureobasidium, Candida, Cryptococcus, Saccharomyces, Pichia, Issatchenkia, Torulaspora, and Sporobolomyces were screened for hydrolytic enzymes (pectinases, amylases, cellulases, xylanases, lipases, proteases, tannases, and β-glucosidases) using selective media, and for antibacterial activity against major foodborne pathogens. Isolates from arbutus fermentation showed no relevant enzymatic or antibacterial ability. In contrast, several isolates from olive fermentation exhibited significant functional traits. Aureobasidium pullulans demonstrated broad enzymatic capacity, producing amylases, esterases, and tannases, along with lipid hydrolysis, but also expressed cellulase, pectinase, and protease abilities. Cryptococcus spp. displayed interesting profiles, with low cellulolytic and pectinolytic capacity and higher phenolase, esterase, and lipase capacities. Antibacterial activity was observed exclusively against Gram-positive bacteria, particularly Staphylococcus aureus and Listeria monocytogenes, mainly among Candida membranifaciens, Cryptococcus spp., and A. pullulans. Overall, table olive fermentation isolates showed promising biotechnological potential for food preservation and quality enhancement, whereas arbutus isolates appeared to have limited functional relevance. Full article

Yeast surface display is a powerful strategy for enzyme immobilization and whole-cell biocatalysis; however, the intracellular processing of heterologous enzymes during secretion and anchoring remains poorly understood. In this study, a GH5 endoglucanase gene (eglS, 1.4 kb) from Bacillus subtilis, originally isolated from a paper mill effluent, was cloned into the pYD1 vector and expressed in Saccharomyces cerevisiae EBY100 using the Aga1–Aga2 surface display system. The recombinant strain produced clear degradation halos on carboxymethyl cellulose (CMC) plates, confirming cellulolytic activity at the whole-cell level. Zymographic analysis revealed multiple active enzyme forms depending on the cellular fraction analyzed. Intracellular extracts displayed active bands ranging from 70 to 57 kDa, consistent with immature or partially processed Aga2 fusion proteins, whereas cell wall-associated fractions showed active bands between 55 and 35 kDa, suggesting proteolytic processing during secretion and surface anchoring. The apparent specific activity of the cytoplasmic fraction was 5.33 ± 0.31 U mg⁻¹, while the cell wall-associated fraction exhibited a higher apparent specific activity (58.4 ± 10.1 U mg⁻¹). Although these values were obtained from non-purified fractions and therefore do not represent intrinsic enzymatic constants, they indicate a relative enrichment of catalytically active enzyme in the cell wall-associated fraction, consistent with functional surface display. The presence of multiple active enzyme forms and the enhanced catalytic efficiency observed in the cell wall-associated fraction suggest that the engineered yeast strain may serve as a promising whole-cell biocatalyst, with potential applications in consolidated bioprocessing (CBP) strategies for lignocellulosic biomass conversion. Full article

Novel ingredients from rowanberry pomace were developed for French-type bread applications via supercritical CO₂ extraction and the enzymatic and ultrasound treatment of the defatted residue (DFR), which contained 6.367% of proteins, 8.36% of soluble, and 43.04% insoluble fiber. Proteolytic enzymes from Bacillus licheniformis and Aspergillus oryzae, and cellulolytic enzyme mixtures Viscozyme L and Celuclast, were used to increase the soluble fraction. Treating DFR with enzymes generated significant amounts of soluble substances containing oligosaccharides, fructose, and glucose, with Viscozyme L being more effective than proteases. Tri-, and tetrapeptides, chlorogenic acids, and dihydroxy coumarins were also present in the soluble extracts of fermented DFR. The antioxidant characteristics of treated DFR were evaluated by the in vitro assays. Substitution of >5% of wheat flour with untreated DFR significantly reduced bread volume and crumb porosity; however, these adverse effects were mitigated by using fermented DFR. The highest bread volume (1845 cm³) and porosity (78.38%) were observed in bread containing 5% pomace that underwent enzymatic hydrolysis and ultrasound treatment. The substitution of flour with DFR significantly increased the antioxidant characteristics of bread samples and the substances generated during the in vitro digestion. It may be concluded that rowanberry pomace ingredients may improve bread nutritional quality and assist in the sustainable use of fruit processing by-products. Full article

The enzymatic saccharification of cellulose remains a key step in biomass conversion processes, often influenced by enzyme stability, distribution, and accessibility at solid–liquid interfaces. Immobilization of cellulolytic enzymes on nanostructured supports has been proposed as a strategy to modulate catalytic behavior; however, the relationship between enzyme loading and catalytic response remains insufficiently understood. In this study, CuO-based nanobiocatalysts were prepared through controlled cellulase immobilization and systematically evaluated under defined experimental conditions. Structural and physicochemical characterization was performed using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and integrated thermal analysis (TGA–DTG–DSC), enabling a comparative assessment of the analyzed systems. SEM analysis showed that the average particle diameter increased from 39.5 ± 14.8 nm (CuO nanoparticles) to 95.6 ± 21.8 nm (NPI10), 106.6 ± 27.7 nm (NPI15), and 113.5 ± 23.1 nm (NPI20), indicating progressive variations in particle organization with increasing enzyme loading. Catalytic performance was evaluated through enzymatic hydrolysis of cellulose filter paper as a model substrate, with products quantified by HPLC at a representative reaction time. The system prepared at lower enzyme loading (NPI10) exhibited product formation comparable to that of the free enzyme, with apparent average glucose formation values of 1.054 and 1.047 mg·mL⁻¹·h⁻¹, respectively. In contrast, higher immobilization levels were associated with reduced catalytic output. Across all systems, glucose was the predominant product, with negligible accumulation of intermediate oligomers under the evaluated conditions. These results indicate that increasing enzyme loading does not correspond to proportional increases in product formation and highlight the influence of enzyme distribution and accessibility within the system. The combined structural and catalytic observations provide a controlled framework for evaluating how immobilization conditions influence system behavior in nanobiocatalytic systems. Full article

Brewer’s spent grain (BSG) is an abundant lignocellulosic by-product whose valorization can support circular bioeconomy strategies. This study evaluated BSG bioconversion by Aspergillus oryzae ATCC 10124 under solid-state fermentation (SSF) to produce lignocellulolytic enzymes and release second-generation (2G) sugars relevant to biorefinery applications. SSF was monitored over 0–10 days, and FPase, endo-cellulase, β-glucosidase, xylanase, mannanase, amylase, and ligninolytic enzyme activities were quantified. Enzymatic crude extracts were further assessed in SDS-PAGE analysis. Glucose, cellobiose, xylose and arabinose release and consumption were tracked throughout fermentation, and substrate transformation was supported by FTIR. The secretome exhibited a predominantly hydrolytic profile, with maximal hemicellulolytic and cellulolytic activity around days 2–4, as well as sustained amylase activity. Ligninolytic activity was not detected. Sugar profiles indicated rapid early hydrolysis of glucose, followed by progressive pentose release. The stabilization and decline were consistent with fungal uptake. Changes in the carbohydrate fingerprint and SDS–PAGE banding supported structural polysaccharide remodeling and hydrolytic protein secretion. Thus, this SSF platform confirmed certain potential for low-cost cellulolytic and hemicellulolytic enzyme generation. However, because sugar accumulation was temporary and followed by consumption, this system is best interpreted as a biological pretreatment and enzyme-generation step that supports subsequent downstream valorization. Full article

Industries seek microorganisms capable of producing all types of cellulases, using low-cost substrate and under adequate process conditions, especially through submerged fermentation. Pleurotus ostreatus “L123” was evaluated as a potential microorganism for cellulase production, assaying total cellulolytic activity (FPase). Fermentation was carried out using a 14L bioreactor, inoculated with 10% (v/v) grown on potato dextrose broth for 4 days. Fermentation media was composed of defatted rice bran (50 g/L), glucose (5 g/L), corn steep liquor (5 g/L) and chloramphenicol (0.25 g/L). Aeration and agitation effects on enzymatic activity were evaluated using a central composite design (CCD) for FPase after 5 days of fermentation. The obtained model was statistically significant, with the interaction of both parameters also being significant and presenting a negative effect. Membrane ultrafiltration (150 kDa MWCO) led to an approximately 3-fold increase in specific activity of permeate (0.6441 vs. 0.2043 FPU/mg of protein), with retention of around 80% of protein content while maintaining enzymatic activity of permeate similar to unfiltered broth (0.0932 vs. 0.0923 FPU/mL). The maximum value obtained experimentally was 0.1444 FPU/mL, which is significantly lower in comparison to commercially used strains and consequently unfeasible for industrial use at current state. However, after further improvements and optimization, Pleurotus ostreatus “L123” can become an alternative for in situ cellulase production through submerged fermentation. Full article

The structural stability of lignocellulosic fibers in crop straw presents a significant challenge to its short-term biodegradation in natural environments, particularly in the cold regions of northern China. To isolate low-temperature straw-degrading bacteria, we selectively enriched microorganisms from straw-amended soils using lignocellulose as the sole carbon source. Three strains were isolated and identified: Stenotrophomonas sp. X24, Flavobacterium sp. X26, and Erwiniaceae bacterium X27. These strains were capable of growth and maize straw degradation within a 4–20 °C range and exhibited key cellulolytic activities (CMCase, FPase, and β-glucosidase). A synthetic three-strain mixture was assembled by combining these isolates in equal proportions. Solid-state fermentation (12 °C, 45 days) was used to assess straw degradation efficacy, while separate enzyme production experiments (12 °C, 3 days) were conducted to evaluate key cellulolytic activities and subsequently optimize culture conditions. The three-strain mixture achieved a net straw degradation rate of 30.93 ± 1.05%. Furthermore, optimization of culture conditions enhanced the carboxymethyl cellulase activity (CMCase) to a maximum of 24.51 ± 0.97 U/mL. The study demonstrates that the three-strain synthetic microbial mixture effectively degrades straw at low temperatures, offering a promising microbial resource to improve straw utilization and soil fertility in cold regions. Full article

In this study, the effects of combined thermal and biochemical pretreatments on the yield and quality of cold-pressed pomegranate seed oil (PSO) were systematically investigated. Convective and microwave roasting were applied individually and in combination with acid or with a commercial pectolytic–cellulolytic enzyme preparation, allowing a comparative evaluation of their synergistic effects under identical cold-pressing conditions. Microwave and convective roasting reduced the seed moisture content from 6.06% to 3–4%, whereas acid pretreatment significantly decreased the seed pH from 4.63 to 3.25–3.33. Lipase activity ranged from 0.061 to 0.191 U/g, with the highest activity in untreated seeds and the lowest in microwave-treated seeds, indicating pretreatment-induced enzyme inactivation. Among all treatments, microwave–acid pretreatment achieved the highest oil yield (11.20%) and the lowest free fatty acid content, whereas microwave–enzyme pretreatment resulted in the lowest peroxide value and the longest oxidative induction period, indicating superior oxidative stability. All pretreatments reduced peroxide value, p-anisidine value, and free fatty acidity compared with the control. Microwave-treated oils exhibited the highest carotenoid content (67.85 mg/kg), while enzyme-treated oils exhibited the lowest carotenoid content (12.05 mg/kg). Total phenolic content was highest in the control oils and decreased following pretreatment. Correlation analysis revealed that oil yield was negatively correlated with seed pH and lipase activity, demonstrating that acid-induced matrix modification and lipase suppression are key mechanisms governing oil recovery. Overall, this study provides new mechanistic insight into the structure enzyme quality relationships in PSO extraction and demonstrates that pretreatment selection should be guided by the intended end use. Microwave–acid pretreatment is most suitable for yield-driven applications (e.g., cosmetic or technical applications), whereas microwave–enzyme pretreatment offers an optimal balance between oxidative stability and quality preservation for food and nutraceutical applications. Full article

The objective of the present study was to ascertain the potential of cellulolytic enzymes produced by selected species of basidiomycetes: Pleurotus ostreatus, Pleurotus eryngii, and Lentinula edodes. In the experimental phase, a selection of basidiomycetes were cultivated on waste substrates containing coffee grounds and wood. During the culture, weekly samples of the substrate were taken, from which enzymes were extracted using citrate buffer (BCA) and purified to obtain cellulolytic preparations. The activity of the obtained preparations was then compared with that of commercial cellulase in a hydrolysis reaction of carboxymethylcellulose (CMC). Statistical analysis demonstrated that the preparations obtained from L. edodes (1.785 mg/mg) and P. ostreatus (0.500 mg/mg) cultures exhibited higher activity compared to commercial cellulase (0.041 mg/mg), while preparations from P. eryngii (0.045 mg/mg) demonstrated comparable activity. The findings of this study demonstrate the viability of utilising a waste substrate comprising coffee grounds and wood for the cultivation of basidiomycetes and the production of enzymes. Full article

Environmental and economic concerns due to the increasing use of fossil-based chemicals, especially fuel, may be alleviated by production of renewable fuels based on plant biomass, in particular, waste. Multistep cascades of enzymatic reactions are being increasingly sought to enhance the effectiveness of sustainable, environment-friendly processes. The biochemical transformation of lignocellulosic biomass and oils into fatty acid esters (“biodiesel”) involves biomass pretreatment, followed by polysaccharide hydrolysis and sugar fermentation to alcohol, either sequentially or simultaneously. Subsequent trans-esterification with waste or non-food-based oils is usually carried out in an organic solvent. Biocatalysis in aqueous emulsion offers significant advantages. This study presents a novel “one-pot” emulsion-based process for transforming unmodified cellulose and castor oil into biodiesel via hybridized yeasts with cellulose-coated micro-particles incorporating cellulolytic enzymes and lipases. The resultant consolidated bioprocess of saccharification, fermentation, and transesterification (cSFT) promotes effective substrate channeling and can potentially serve as a model for emulsion-based “one-pot” transformations of cellulose into valuable chemicals. Full article

Endophytic fungi were isolated from ginger (Zingiber officinale) tubers and identified through molecular characterization of ITS and 28S rRNA regions. Nine species were obtained, belonging to the genera Aspergillus, Penicillium, Plectosphaerella, and Pseudogymnoascus. Several isolates, particularly Penicillium melinii, Aspergillus ustus, and Plectosphaerella cucumerina, exhibited strong antagonistic activity against Botrytis cinerea (up to 98.6% growth inhibition), while moderate effects were observed against Colletotrichum acutatum, Staphylococcus aureus and Klebsiella pneumoniae. All isolates produced at least one extracellular enzyme, with lipolytic and cellulolytic enzymes being the most frequently observed, and showed measurable antioxidant activity (EC₅₀ values ranging from 21.7 to 673.6 µg/mL). P. melinii and P. cucumerina demonstrated the highest radical scavenging capacities. These findings reveal the multifunctional potential of ginger-associated endophytic fungi as sustainable sources of bioactive compounds, with promising applications in biocontrol, food preservation, and industrial biotechnology. Full article

Bacillus safensis strains have emerged as versatile microbial platforms for bioproduction, combining the benefits of probiotic utility and biocontrol. In this study, we describe the isolation and in-depth characterization of a previously unreported B. safensis strain, LG01. The genome of this strain comprises a circular chromosome encoding 13 secondary metabolite biosynthetic gene clusters, 144 carbohydrate-active enzymes, 2 antibiotic resistance loci, and 1 prophage region, indicative of strong antimicrobial and metabolic capacity. Its protein secretion systems support nutrient acquisition, colonization, quorum sensing, and antibiotic synthesis. Our phenotypic assays confirmed the antifungal and antibacterial activity, proteolytic and cellulolytic functions, and robust biofilm formation of the strain. By performing a comparative genomic analysis, we identified 78 strain-specific genes enriched in the bacteriocin immunity and sporulation pathways. Signals of positive selection in the membrane and transcriptional regulator genes further reflect the adaptive evolution underlying the strain’s ecological fitness. Together, these findings advance our understanding of the genomic features of B. safensis LG01 and highlight its promise as a candidate for biocontrol and probiotic applications. Full article

To understand the potential uses of strawberry leaves and their application in the industrial sector, it is important to study their metabolic and lignocellulosic profile. The objective of the study was to characterize the metabolic profile of strawberry leaves grown with biostimulants and to verify whether this by-product can be utilized as a matrix for bioethanol production. The eight treatments studied were the absence and presence of biostimulants, as follows: arbuscular mycorrhizal fungi (AMF), Ascophyllum nodosum (AN), Trichoderma harzianum (TH), AMF + AN, AMF + TH, AN + TH, and AMF + AN + TH. Treatments were applied monthly, either manually (AMF) or with a micropipette (AN and TH), from June 2023 to March 2024. Hydrogen nuclear magnetic resonance (¹H NMR) analysis identified 11 metabolites in the leaves, including 5 amino acids, 4 organic acids, and 2 carbohydrates, which may be of industrial interest. The leaves were used for bioethanol production through saccharification with cellulolytic enzymes, followed by fermentation with Saccharomyces cerevisiae. Enzymatic hydrolysis resulted in a total reducing sugar content of 21.12 g·L⁻¹. Alcoholic fermentation yielded 8.97 g·L⁻¹ of bioethanol in 12 h, which corresponds to 45.48 L·t⁻¹ of dry leaves. In conclusion, regardless of biostimulation, strawberry leaves are an important repository of metabolites that can be used as raw material in different processes. Additionally, the leaves are suitable as raw material for bioethanol production in a biorefinery concept. Full article