Hass Avocado (Persea americana Mill) Peel Extract Reveals Antimicrobial and Antioxidant Properties against Verticillium theobromae, Colletotrichum musae, and Aspergillus niger Pathogens Affecting Musa acuminata Colla Species, in Ecuador.

The utilization of agroindustrial residues, such as avocado peel, as a source of bioactive compounds with antioxidant properties has garnered significant attention. In this study, we investigated the antioxidant potential using the DPPH (2,2-diphenyl-1-picrylhydrazyl) and ORAC (oxygen radical absorbance capacity) methods, along with the antimicrobial activity of phenolic compounds extracted from Hass avocado peel. These soluble polyphenols were quantified and identified using high-performance liquid chromatography (HPLC). The research focused on their effects against three fungal pathogens, Verticillium theobromae, Colletotrichum musae, and Aspergillus niger, which significantly impact banana crops, an essential agricultural commodity in Ecuador. The results have revealed that the application of 80% ethanol as an organic solvent led to increased soluble polyphenol content compared to 96% ethanol. Extraction time significantly influenced the phenolic content, with the highest values obtained at 90 min. Interestingly, despite substantial mycelial growth observed across all extract concentrations, the antifungal effect varied among the pathogens. Specifically, V. theobromae exhibited the highest sensitivity, while C. musae and A. niger were less affected. These results underscore the importance of considering both antioxidant and antimicrobial properties when evaluating natural extracts for potential applications in plant disease management.

Prevalence of azole-resistant Aspergillus fumigatus and other aspergilli in the environment from Argentina.

Azole resistance has emerged as a new therapeutic challenge in patients with aspergillosis. Various resistance mutations are attributed to the widespread use of triazole-based fungicides in agriculture. This study explored the prevalence of azole-resistant Aspergillus fumigatus (ARAF) and other aspergilli in the Argentine environment. A collection of A. fumigatus and other aspergilli strains isolated from soil of growing crops, compost, corn, different animal feedstuffs, and soybean and chickpea seeds were screened for azole resistance. No ARAF was detected in any of the environmental samples studied. However, five A. flavus, one A. ostianus, one A. niger and one A. tamarii recovered from soybean and chickpea seeds showed reduced susceptibility to medical azole antifungals (MAA). The susceptibility profiles of five A. flavus isolates, showing reduced susceptibility to demethylase inhibitors (DMIs), were compared with those of 10 isolates that exhibited susceptibility to MAA. Aspergillus flavus isolates that showed reduced MAA susceptibility exhibited different susceptibility profiles to DMIs. Prothioconazole and tebuconazole were the only DMIs significantly less active against isolates with reduced susceptibility to MAA. Although no ARAF isolates were found in the samples analysed, other aspergilli with reduced susceptibility profile to MAA being also important human pathogens causing allergic, chronic and invasive aspergillosis, are present in the environment in Argentina. Although a definitive link between triazole-based fungicide use and isolation of azole-resistant human pathogenic aspergilli from agricultural fields in Argentina remains elusive, this study unequivocally highlights the magnitude of the environmental spread of azole resistance among other Aspergillus species.

This study intended to inform about the prevalence of Aspergillus species showing triazole resistance in the Argentinian environment. Since azole fungicides are used for crop protection, it was expected that azole resistance in this species with cross-resistance to medical azoles could occur.

Biotransformation of rifampicin by Aspergillus niger and antimicrobial activity of proposed metabolites.

Drug biotransformation studies emerges as an alternative to pharmacological investigations of metabolites, development of new drug candidates with reduced investment and most efficient production. The present study aims to evaluate the capacity of biotransformation of rifampicin by the filamentous fungus Aspergillus niger ATCC 9029. After incubation for 312 h, the drug was metabolized to two molecules: an isomer (m/z 455) and the rifampicin quinone (m/z 821). The monitoring of metabolite formation was performed by high-performance liquid chromatography, followed by their identification through ultra-high-performance liquid chromatography coupled to tandem mass spectrometer. In vitro antimicrobial activity of the proposed metabolites was evaluated against Staphylococus aureus microorganism, resulting in the loss of inhibitory activity when compared with the standards, with minimum inhibitory concentration of 7.5 µg/ml. The significant biotransformation power of the ATCC 9029 strain of A. niger was confirmed in this study, making this strain a candidate for pilot studies in fermentation tanks for the enzymatic metabolization of the antimicrobial rifampicin. The unprecedented result allows us to conclude that the prospect of new biotransforming strains in species of anemophilic fungi is a promising choice.

Effect of Aspergillus niger prolyl endopeptidase in patients with celiac disease on a long-term gluten-free diet.

BACKGROUND: The gluten-free diet (GFD) has limitations, and there is intense research in the development of adjuvant therapies. AIM: To examine the effects of orally administered Aspergillus niger prolyl endopeptidase protease (AN-PEP) on inadvertent gluten exposure and symptom prevention in adult celiac disease (CeD) patients following their usual GFD. METHODS: This was an exploratory, double-blind, randomized, placebo-controlled trial that enrolled CeD patients on a long-term GFD. After a 4-wk run-in period, patients were randomized to 4 wk of two AN-PEP capsules (GliadinX; AVI Research, LLC, United States) at each of three meals per day or placebo. Outcome endpoints were: (1) Average weekly stool gluten immunogenic peptides (GIP) between the run-in and end of treatments and between AN-PEP and placebo; (2) celiac symptom index (CSI); (3) CeD-specific serology; and (4) quality of life. Stool samples were collected for GIP testing by ELISA every Tuesday and Friday during run-ins and treatments. RESULTS: Forty patients were randomized for the intention-to-treat analysis, and three were excluded from the per-protocol assessment. Overall, 628/640 (98.1%) stool samples were collected. GIP was undetectable (< 0.08 µg/g) in 65.6% of samples, and no differences between treatment arms were detected. Only 0.5% of samples had GIP concentrations sufficiently high (> 0.32 µg/g) to potentially cause mucosal damage. Median GIP concentration in the AN-PEP arm was 44.7% lower than in the run-in period. One-third of patients exhibiting GIP > 0.08 µg/g during run-in had lower or undetectable GIP after AN-PEP treatment. Compared with the run- in period, the proportion of symptomatic patients (CSI > 38) in the AN-PEP arm was significantly lower (P < 0.03). AN-PEP did not result in changes in specific serologies. CONCLUSION: This exploratory study conducted in a real-life setting revealed high adherence to the GFD. The AN-PEP treatment did not significantly reduce the overall GIP stool concentration. However, given the observation of a significantly lower prevalence of patients with severe symptoms in the AN-PEP arm, further clinical research is warranted.

Carvacrol nanocapsules as a new antifungal strategy: Characterization and evaluation against fungi important for grape quality and to control the synthesis of ochratoxins.

Fungi are a problem for viticulture as they can lead to deterioration of grapes and mycotoxins production. Despite the widespread use of synthetic fungicides to control fungi, their impact on the agricultural ecosystem and human health demand safer and eco-friendly alternatives. This study aimed to produce, characterize and assess the antifungal activity of carvacrol loaded in nanocapsules of Eudragit® and chia mucilage as strategy for controlling Botrytis cinerea, Aspergillus flavus, Aspergillus carbonarius, and Aspergillus niger. Eudragit® and chia mucilage were suitable wall materials, as both favored the encapsulation of carvacrol into nanometric diameter particles. Fourier Transform Infrared Spectroscopy (FTIR) analysis suggested a successful incorporation of carvacrol into both nanocapsules, which was confirmed by presenting a good encapsulation efficiency and loading capacity. Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) analyses revealed adequate thermal resistance. All fungi were sensible to carvacrol treatments and B. cinerea was the most sensitive compared to the Aspergillus species. Lower concentrations of encapsulated carvacrol than the unencapsulated form were required to inhibit fungi in the in vitro and grape assays. Additionally, lower levels of carvacrol (unencapsulated or encapsulated) were used to inhibit fungal growth and ochratoxin synthesis on undamaged grapes in comparison to those superficially damaged, highlighting the importance of management practices designed to preserve berry integrity during cultivation, storage or commercialization. When sublethal doses of carvacrol were used, the growth of A. niger and A. carbonarius was suppressed by at least 45 %, and ochratoxins were not found. The nanoencapsulation of carvacrol using Eudragit® and chia mucilage has proven to be an alternative to mitigate the problems with fungi and mycotoxins faced by the grape and wine sector.

Time-kill kinetic of nano-ZnO-loaded nanoliposomes against Aspergillus niger and Botrytis cinerea.

In vitro antimicrobial activity of nano-ZnO-loaded nanoliposomes at different levels of lecithin:nano-ZnO ratio (5:1, 15:1, and 25:1 w/w) against Aspergillus niger (IBRC-M 30095) and Botrytis cinerea (IBRC-M 30162) was evaluated. Nanoliposome formulations containing nano-ZnO were fabricated through thin-layer hydration sonication and heat methods. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of nano-ZnO-loaded nanoliposomes and free nano-ZnO against Aspergillus niger and Botrytis cinerea were determined. The time-kill experiments were performed for each isolate. Results showed that the encapsulation of nano-ZnO in nanoliposome systems significantly enhanced their antimicrobial activities by improving the penetration of ZnO nanoparticles the fungi cell membrane. In vitro antifungal activity of nano-ZnO-loaded nanoliposomes against Aspergillus niger and Botrytis cinerea was increased in thin-layer hydration sonication method compared with the heat method. The log phase for Aspergillus niger and Botrytis cinerea was around 70 h. Adding nano-ZnO-loaded nanoliposomes to the culture medium shortened the log phase for both Aspergillus niger and Botrytis cinerea. The highest antimicrobial activity of nanoliposomes was achieved using nanoliposomes containing the lecithin:nano-ZnO ratio of 25:1 (w/w) as compared to all samples. However, the length of the log phase growth cultures exposed to the nanoliposome formulations prepared by thin-layer hydration sonication method with the lecithin:nano-ZnO ratio of 25:1 (w/w) at MIC and MFC values was 60 and 40 h for both Aspergillus niger and Botrytis cinerea, respectively.

Evaluation of antifungal activity, mechanisms of action and toxicological profile of the synthetic amide 2-chloro-N-phenylacetamide.

Aspergillus niger causes infections such as otitis and pulmonary aspergillosis in immunocompromised individuals. Treatment involves voriconazole or amphotericin B, and due to the increase in fungal resistance, the search for new compounds with antifungal activity has intensified. In the development of new drugs, cytotoxicity and genotoxicity assays are important, as they allow predicting possible damage that a molecule can cause, and in silico studies predict the pharmacokinetic properties. The aim of this study was to verify the antifungal activity and the mechanism of action of the synthetic amide 2-chloro-N-phenylacetamide against Aspergillus niger strains and toxicity. 2-Chloro-N-phenylacetamide showed antifungal activity against different strains of Aspergillus niger with minimum inhibitory concentrations between 32 and 256 µg/mL and minimum fungicides between 64 and 1024 µg/mL. The minimum inhibitory concentration of 2-chloro-N-phenylacetamide also inhibited conidia germination. When associated with amphotericin B or voriconazole, 2-chloro-N-phenylacetamide had antagonistic effects. Interaction with ergosterol in the plasma membrane is the probable mechanism of action.2-Chloro-N-phenylacetamide has favorable physicochemical parameters, good oral bioavailability and absorption in the gastrointestinal tract, crosses the blood-brain barrier and inhibits CYP1A2. At concentrations of 50 to 500 µg/mL, it has little hemolytic effect and a protective effect for type A and O red blood cells, and in the cells of the oral mucosa it promotes little genotoxic change. It is concluded that 2-chloro-N-phenylacetamide has promising antifungal potential, favorable pharmacokinetic profile for oral administration and low cytotoxic and genotoxic potential, being a promising candidate for in vivo toxicity studies.

Development of cassava starch-based films incorporated with phenolic compounds produced by an Amazonian fungus.

Drug-release systems have attracted attention over the last few years since they can be used as a substitute for traditional methods of drug delivery. These have the advantage of being directly administered at the treatment site and can maintain the drug at adequate levels for a longer period, thus increasing their efficacy. Starch-based films are interesting candidates for use as matrices for drug release, especially due to starch's non-toxic properties and its biocompatibility. Endophytic fungi are an important source of bioactive molecules, including secondary metabolites such as phenolic compounds with antioxidant activity. In the present study, cassava starch-based films were developed to act as release systems of phenolic compounds with antioxidant activity. The Amazonian endophytic fungus Aspergillus niger MgF2 was cultivated in liquid media, and the fungal extract was obtained by liquid-liquid partition with ethyl acetate. The starch-based films incorporated with the fungal extract were characterized in regards to their physicochemical properties. The release kinetics of the extract from the film and its antioxidant and cytotoxic properties were also evaluated. The films incorporated with the extract presented maximum release after 25 min at 37 °C and pH 6.8. In addition, it was observed that the antioxidant compounds of the fungal extract maintain their activity after being released from the film, and were non-toxic. Therefore, considering the promising physicochemical properties of the extract-incorporated films, and their considerable antioxidant capacity, the films demonstrate great biotechnological potential with diverse applications in the pharmacological and cosmetic industries.

An innovative multi-enzymatic system for gluconic acid production from starch using Aspergillus niger whole-cells.

The use of multi-enzymatic systems for the industrial production of chemical compounds is currently considered an important green tool in synthetic organic chemistry. Gluconic acid is a multi-functional organic acid widely used in the chemical, pharmaceutical, food, textile, and construction industries. Its industrial production from glucose by fermentation using Aspergillus niger has drawbacks including high costs related to cell growth and maintenance of cell viability. This study presents an innovative one-step multi-enzymatic system for gluconic acid production from starch using Aspergillus niger whole-cells in association with amylolytic enzymes. Using soluble starch as substrate, the following results were achieved for 96 h of reaction: 134.5 ± 4.3 g/L gluconic acid concentration, 98.2 ± 1.3 % gluconic acid yield, and 44.8 ± 1.4 gGA/gwhole-cells biocatalyst yield. Although the process has been developed using starch as raw material, the approach is feasible for any substrate or residue that can be hydrolyzed to glucose.

Evidence of polyamines mediated 2-acetyl-1-pyrroline biosynthesis in aromatic rice rhizospheric fungal species Aspergillus niger.

Rhizosphere soil of aromatic rice inhabits different fungal species that produce many bioactive metabolites including 2-acetyl-1-pyrroline (2AP). The mechanism for the biosynthesis of 2AP in the fungal system is still elusive. Hence, the present study investigates the role of possible nitrogen (N) precursors such as some amino acids and polyamines as well as the enzymes involved in 2AP synthesis in the fungal species isolated from the rhizosphere of aromatic rice varieties. Three fungal isolates were found to synthesize 2AP (0.32-1.07 ppm) and maximum 2AP was synthesized by Aspergillus niger (1.07 ppm) isolated from rhizosphere of Dehradun Basmati (DB). To determine the N source for 2AP synthesis, various N sources such as proline, glutamate, ornithine putrescine, spermine, and spermidine were used in place of putrescine in the synthetic medium (Syn18). The results showed that maximum 2AP synthesis was found with putrescine (1.07 ppm) followed by spermidine (0.89 ppm) and spermine (0.84 ppm). Further, LC-QTOF-MS analysis revealed the mobilization of spermine and spermidine into the putrescine, indicating that putrescine is the key N source for 2AP synthesis. Moreover, higher enzyme activity of DAO, PAO, and ODC as well as higher content of methylglyoxal metabolite in the A. niger NFCCI 5060 as compared to A. niger NFCCI 4064 (control) suggests the prominent role of these enzymes in the synthesis of 2AP. In conclusion, this study showed evidence of the polyamines mediated 2AP biosynthesis in A. niger NFCCI 5060.

Production of a Fungal Punicalagin-Degrading Enzyme by Solid-State Fermentation: Studies of Purification and Characterization.

The present work describes the purification of an enzyme capable of degrading punicalagin. The enzyme was produced by Aspergillus niger GH1 by solid-state fermentation, and the enzyme production was induced by using ellagitannins as the sole carbon source. The purification steps included the concentration by lyophilization, desalting, anionic exchange, and gel filtration chromatography. The enzyme kinetic constants were calculated by using punicalagin, methyl gallate, and sugar beet arabinans. The molecular mass of the protein was estimated by SDS-PAGE. The identified bands were excised and digested using trypsin, and the peptides were submitted to HPLC-MS/MS analysis. The docking analysis was conducted, and a 3D model was created. The purification fold increases 75 times compared with the cell-free extract. The obtained Km values were 0.053 mM, 0.53% and 6.66 mM for punicalagin, sugar beet arabinans and methyl gallate, respectively. The optimal pH and temperature for the reaction were 5 and 40 °C, respectively. The SDS-PAGE and native PAGE analysis revealed the presence of two bands identified as α-l-arabinofuranosidase. Both enzymes were capable of degrading punicalagin and releasing ellagic acid.

Low-cost homemade cocktails for enzymatic conversion of sugarcane and cassava bagasses.

The agricultural industries generate lignocellulosic wastes that can be modified by fungi to generate high value-added products. This work aimed to analyze the efficiency and the cost-effectiveness of the bioconversion of sugarcane and cassava bagasses using low-cost homemade enzymatic cocktails from Aspergillus niger LBM 134. Both bagasses were pretreated with a soft alkaline solution without any loss of polysaccharides. After the hydrolysis, a 28% of conversion to glucose and 42% to xylose were reached in the hydrolysis of sugarcane bagasse while an 80% of saccharification yield, in the hydrolysis of cassava bagasse using the homemade enzymes. Furthermore, a more disorganised surface and no starch granules were observed in the sugarcane and cassava bagasses, respectively. The bioethanol yield from sugarcane and casava bagasses was predicted to be 4.16 mg mL-1 and 2.57 mg mL-1, respectively. A comparison of the cost of the homemade and the commercial enzymes was carried out. Similar hydrolysis percentages were achieved employing any enzyme; however, it was 1000-2000 times less expensive using the homemade cocktails than using the commercial enzymes. Therefore, the cost of obtaining glucose from bagasses was most expensive when applying the commercial enzymes. Moreover, the hydrolysis of the cassava bagasse was most efficient with the homemade cocktails. The importance and novelty of this work lie in the similar performance and the lower cost of the homemade cocktails from the fungus A. niger LBM 134 compared with the commercial enzymes on the hydrolysis of the sugarcane and cassava bagasses.

Active packaging of poly(lactic acid) nanofibers and essential oils with antifungal action on table grapes.

The table grape is a non-climateric fruit that is very susceptible to fungal contamination, in addition to suffering an accelerated loss of quality during storage. The in vitro and in grape antifungal and antiocratoxigenic effects of the essential oils from Alpinia speciosa and Cymbopogon flexuosus against Aspergillus carbonarius and Aspergillus niger were studied. The oils were encapsulated in poly(lactic acid) (PLA) nanofibers as a potential active packaging to be applied to control the degradation of grapes stored during the post-harvest period. Fungal proliferation and ochratoxin A synthesis in A. carbonarius and A. niger decreased in the presence of the active packaging. However, the nanofiber containing the essential oil from C. flexuosus was more efficient in providing a fungicidal effect against A. carbonarius (10% and 20%) and A. niger (20%). In addition, weight loss and color changes were controlled and the parameters of acidity, °Brix, softening and the texture of the grape were maintained. A very small mass loss of the essential oils encapsulated in nanofibers was observed by thermogravimetric analysis, showing that the nanofiber was efficient in enabling the controlled release. The quality and safety of table grapes were maintained for longer periods of storage in the presence of active packaging, so the incorporation of these oils in nanofibers can be a promising way to increase the shelf life of grapes.

Effect of Shock Waves on the Growth of Aspergillus niger Conidia: Evaluation of Germination and Preliminary Study on Gene Expression.

Shock waves, as used in medicine, can induce cell permeabilization, genetically transforming filamentous fungi; however, little is known on the interaction of shock waves with the cell wall. Because of this, the selection of parameters has been empirical. We studied the influence of shock waves on the germination of Aspergillus niger, to understand their effect on the modulation of four genes related to the growth of conidia. Parameters were varied in the range reported in protocols for genetic transformation. Vials containing conidia in suspension were exposed to either 50, 100 or 200 single-pulse or tandem shock waves, with different peak pressures (approximately 42, 66 and 83 MPa). In the tandem mode, three delays were tested. To equalize the total energy, the number of tandem "events" was halved compared to the number of single-pulse shock waves. Our results demonstrate that shock waves do not generate severe cellular effects on the viability and germination of A. niger conidia. Nevertheless, increase in the aggressiveness of the treatment induced a modification in four tested genes. Scanning electron microscopy revealed significant changes to the cell wall of the conidia. Under optimized conditions, shock waves could be used for several biotechnological applications, surpassing conventional techniques.

Effects of mycogenic silver nanoparticles on organisms of different trophic levels.

Biogenic silver nanoparticles (AgNPs) are considered a promising alternative to their synthetic versions. However, the environmental impact of such nanomaterials is still scarcely understood. Thus, the present study aims at assessing the antimicrobial action and ecotoxicity of AgNPs biosynthesized by the fungus Aspergillus niger IBCLP20 towards three freshwater organisms: Chlorella vulgaris, Daphnia similis, and Danio rerio (zebrafish). AgNPs IBCLP20 showed antibacterial action against Klebsiella pneumoniae between 5 and 100 µg mL-1, and antifungal action against Trichophyton mentagrophytes in concentrations ranging from 20 to 100 µg mL-1. The cell density of the microalgae Chlorella vulgaris decreased 40% after 96 h of exposure to AgNPs IBCLP20, at the highest concentration analysed (100 µg L-1). The 48 h median lethal concentration for Daphnia similis was estimated as 4.06 µg L-1 (2.29-6.42 µg L-1). AgNPs IBCLP20 and silver nitrate (AgNO3) caused no acute toxicity on adult zebrafish, although they did induce several physiological changes. Mycosynthetized AgNPs caused a significant increase (p < 0.05) in oxygen consumption at the highest concentration studied (75 µg L-1) and an increase in the excretion of ammonia at the lower concentrations, followed by a reduction at the higher concentrations. Such findings are comparable with AgNO3, which increased the oxygen consumption on low exposure concentrations, followed by a decrease at the high tested concentrations, while impairing the excretion of ammonia in all tested concentrations. The present results show that AgNPs IBCLP20 have biocidal properties. Mycogenic AgNPs induce adverse effects on organisms of different trophic levels and understanding their impact is detrimental to developing countermeasures aimed at preventing any negative environmental effects of such novel materials.

Unveiling the Solubilization of Potassium Mineral Rocks in Organic Acids for Application as K-Fertilizer.

Organic acids produced by soil microorganisms can be useful to promote the release of potassium (K) from potassium mineral rocks (KR), but the complexity of low reactivity minerals limits K solubilization and their use as fertilizer. Here, we investigate the ways that different organic acids (gluconic, oxalic, and citric) can affect the solubilization of potassium minerals, in order to propose process strategies to improve their solubility. For this, evaluations were performed using the model minerals KRpolyhalite (sedimentary mineral), KRfeldspar (igneous mineral), and KCl (commercial fertilizer). For KCl and KRpolyhalite, complete solubilization was achieved using all the organic acids, while for KRfeldspar, the highest K+ solubilization (34.86 mg L-1) was achieved with oxalic acid. The solubility of KRfeldspar was further investigated under submerged cultivation with the filamentous fungus Aspergillus niger, as well as after a mechanochemical grinding treatment. The biotechnological route resulted in solubilized K up to 63.2 mg L-1. The mechanochemical route, on the other hand, increased the release of K by about 8.6 times (993 mg L-1) compared to the natural mineral, due to the greater fragmentation of the particles after the treatment (with a surface area about 2.5 times higher than for the in natura KRfeldspar). These findings demonstrated the potential of applying biotechnological and mechanochemical routes with organic acids to improve the solubilization of K present in low reactivity mineral rocks, indicating the possible use of these minerals in more sustainable agricultural practices.

Production, concentration and partial characterization of an enzymatic extract produced by an Aspergillus niger mutant in solid state fermentation.

An enzymatic extract from Aspergillus niger 3T5B8 was produced by Solid State Fermentation (SSF) in aerated columns, using wheat bran as substrate. A combination of extracts produced using three different process conditions varying temperature, pH and aeration formed the final extract (Mixture). The Mixture was concentrated by an ultrafiltration process that partially purified and provided an efficient recovery of the enzymatic activities of xylanase (88.89%), polygalacturonase (89.3%), ß-glucosidase (93.15%), protease (98.68%) and carboxymethylcellulase (CMCase) (98.93%). SDS-PAGE analysis showed 15 visible protein bands in the crude and concentrated Mixture with molecular weights ranging from 15.1 to 104.6 kDa. Thin layer chromatography confirmed the effective action of ß-glucosidase and xylanase hydrolysis activities over cellobiose and xylan, respectively. A central composite design (CCD) with two variables and four replicates at the center points was used to determine the optimal temperature and pH for CMCase and ß-glucosidase. The optimal temperature was 78.9 °C and pH 3.8 for CMCase and 52.8 °C and pH 4.8 for ß-glucosidase, respectively.

Bio-based composite granules with simultaneous biocontrol and phosphorus fertilization roles: Outcomes from a lab-scale in vitro assessment.

The use of phosphate rocks as low-solubility phosphorus fertilizers has been promoted to reduce the environmental impacts of agriculture, but adequate nutrient uptake by plants depends on solubilization of the rock, driven by soil microorganisms. Here, investigation was made of the microbial solubilization of low-solubility phosphate rocks, together with simultaneous bioprotective action involving the biocontrol of microorganisms. The aim was to enhance function and value by delivering two effects using a single bio-based product, in accordance with the concept of a "bioreactor-in-a-granule" system. A composite structure was developed, based on a starch matrix, comprising a combination of Trichoderma asperelloides, as a biocontrol agent, and Aspergillus niger, as an acidulant. A significant increase of up to 150% in P solubilization was achieved, indicating the positive effect of the microorganism-composite interaction. In vitro assays showed that the ability of T. asperelloides to inhibit Fusarium oxysporum mycelial growth was maintained in the presence of A. niger. Moreover, the estimated cost of the composite granule (0.35 US$/kg of product on a dry basis) revealed competitive. The results indicated that the association of T. asperelloides and A. niger is an effective way to increase nutrient availability and to inhibit plant pathogens, opening up possibilities for the design of multifunctional bio-based fertilizer composites.

Preparation of Aspergillus niger 426 naringinases for debittering citrus juice utilization of agro-industrial residues.

Naringin, considered as the main bitter component of grapefruit, requires the use of enzymes to reduce the level of this substance during juice processing. For this reason, it has been the focus of many studies. In this study, to increase the production of naringinase by Aspergillus niger cultivated in solid-state fermentation (SSF), a three-component simplex-centric mixing design along with a response surface methodology (RSM) was applied to generate statistical models and analyze the dataset. First, grapefruit peel, rice bran, and wheat bran were used for substrate selection for naringinase production and, finally, selected the best of the three inducers or their mixtures to remove the bitterness of grapefruit juice. Cultivation with 2.3 g of grapefruit peel, 2.5 g of rice bran, and 5.2 g of wheat bran and medium supplementation with a mixture of naringin, rutin, and hesperidin in the concentration of 2, 5, 4.5, and 3.0 g/L, respectively, resulted in a maximum activity of 28 U/mL. The results indicate that the sequencing procedure, which allowed the definition of an optimal mixture of components, is a new way for microorganisms to have a high naringinase yield, in particular by SSF, since our data showed a 96% increase in the production of naringinase. This dataset can help other researchers apply a mixing design to increase enzyme production.

Sequence Engineering of an Aspergillus niger Tannase to Produce in Pichia pastoris a Single-Chain Enzyme with High Specific Activity.

Tannin acyl hydrolases or tannases (E.C.3.1.1.20) are enzymes that hydrolyze the ester bond of tannins to produce gallic acid and glucose. We engineered the Aspergillus niger GH1 tannase sequence and Pichia pastoris strains to produce and secrete the enzyme as a single-chain protein. The recombinant tannase was N-glycosylated, had a molecular mass after N-deglycosylation of 65.4 kDa, and showed activity over broad pH and temperature ranges, with optimum pH and temperature of 5.0 and 20 °C. Furthermore, the single-chain tannase had an 11-fold increased specific activity in comparison to the double-chain A. niger GH1 tannase, which was also produced in P. pastoris. Structural analysis suggested that the high specific activity may be due to the presence of a flexible loop in the lid domain, which can control and drive the substrate to the active site. In contrast, the low specific activity of the double-chain tannase may be due to the presence of a disordered and flexible loop that could hinder the substrate's access to the binding site. Based on its biochemical properties, high specific activity, and the possibility of its production in P. pastoris, the tannase described could be used in food and beverage processing at low and medium temperatures.