Trichoderma as a powerful fungal disease control agent for a more sustainable and healthy agriculture: recent studies and molecular insights.

MAIN CONCLUSION: Molecular studies have elucidated Trichoderma's biocontrol mechanisms. Since fungicides have limited use, Trichoderma could control disease by new metabolic routes and epigenetic alterations. Due to environmental and health hazards, agrochemicals have been a concern since they were introduced in agriculture. Trichoderma, a well-known fungal genus with different mechanisms of action, is an alternative to pesticides and a great tool to help minimize disease incidence. Trichoderma-treated plants mainly benefit from disease control and growth promotion through priming, and these fungi can modulate plants' gene expression by boosting their immune system, accelerating their response to threats, and building stress tolerance. The latest studies suggest that epigenetics is required for plant priming and could be essential for growth promotion, expanding the possibilities for producing new resistant plant varieties. Trichoderma's propagules can be mass produced and formulated depending on the delivery method. Microsclerotia-based bioproducts could be a promising way of increasing the reliability and durability of marketed products in the field, as well as help guarantee longer shelf life. Developing novel formulations and selecting efficient Trichoderma strains can be tiresome, but patent search indicates an increase in the industrialization and commercialization of technologies and an expansion of companies' involvement in research and development in this field. Although Trichoderma is considered a well-known fungal genus, it still attracts the attention of large companies, universities, and research institutes around the world.

An ex vitro hairy root system from petioles of detached soybean leaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.

MAIN CONCLUSION: The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybean leaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.

Disposable electrochemical platform based on solid-binding peptides and carbon nanomaterials: an alternative device for leishmaniasis detection.

Neglected tropical diseases are those caused by infectious agents or parasites and are considered endemic in low-income populations. These diseases also have unacceptable indicators and low investment in research, drug production, and control. Tropical diseases such as leishmaniasis are some of the main causes of morbidity and mortality around the globe. Electrochemical immunosensors are promising tools for diagnostics against these diseases. One such benefit is the possibility of assisting diagnosis in isolated regions, where laboratory infrastructure is lacking. In this work, different peptides were investigated to detect antibodies against Leishmania in human and canine serum samples. The peptides evaluated (395-KKG and 395-G) have the same recognition site but differ on their solid-binding domains, which ensure affinity to spontaneously bind to either graphene oxide (GO) or graphene quantum dots (GQD). Cyclic voltammetry and differential pulse voltammetry were employed to investigate the electrochemical behavior of each assembly step and the role of each solid-binding domain coupled to its anchoring material. The graphene affinity peptide (395-G) showed better reproducibility and selectivity when coupled to GQD. Under the optimized set of experimental conditions, negative and positive human serum samples responses were distinguished based on a cut-off value of 82.5% at a 95% confidence level. The immunosensor showed selective behavior to antibodies against Mycobacterium leprae and Mycobacterium tuberculosis, which are similar antibodies and potentially sources of false positive tests. Therefore, the use of the graphene affinity peptide as a recognition site achieved outstanding performance for the detection of Leishmania antibodies.

An updated review on bacterial community composition of traditional fermented milk products: what next-generation sequencing has revealed so far?

The emergence of next-generation sequencing (NGS) technologies has revolutionized the way to investigate the microbial diversity in traditional fermentations. In the field of food microbial ecology, different NGS platforms have been used for community analysis, including 454 pyrosequencing from Roche, Illumina's instruments and Thermo Fisher's SOLiD/Ion Torrent sequencers. These recent platforms generate information about millions of rDNA amplicons in a single running, enabling accurate phylogenetic resolution of microbial taxa. This review provides a comprehensive overview of the application of NGS for microbiome analysis of traditional fermented milk products worldwide. Fermented milk products covered in this review include kefir, buttermilk, koumiss, dahi, kurut, airag, tarag, khoormog, lait caillé, and suero costeño. Lactobacillus-mainly represented by Lb. helveticus, Lb. kefiranofaciens, and Lb. delbrueckii-is the most important and frequent genus with 51 reported species. In general, dominant species detected by culturing were also identified by NGS. However, NGS studies have revealed a more complex bacterial diversity, with estimated 400-600 operational taxonomic units, comprising uncultivable microorganisms, sub-dominant populations, and late-growing species. This review explores the importance of these discoveries and address related topics on workflow, NGS platforms, and knowledge bioinformatics devoted to fermented milk products. The knowledge that has been gained is vital in improving the monitoring, manipulation, and safety of these traditional fermented foods.

Global cocoa fermentation microbiome: revealing new taxa and microbial functions by next generation sequencing technologies.

This review provides an overview of the application of next-generation sequencing (NGS) technologies for microbiome analysis of cocoa beans fermentation. The cocoa-producing regions where NGS has been applied include Brazil, Ghana, Ivory Coast, Cameroon, Nicaragua, and Colombia. The data collected were processed by principal component analysis (PCA) and Venn diagrams to perform a multivariate association between microbial diversity and cocoa-producing regions. NGS studies have confirmed the dominance of three major microbial groups revealed by culture-dependent approaches, i.e., lactic acid bacteria, acetic acid bacteria, and yeasts. However, a more complex microbial diversity has been revealed, comprising sub-dominant populations, late-growing species, and uncultivable microorganisms. A total of 99 microbial genera and species were for the first time reported in cocoa beans fermentation, such as Brevibacillus sp., Halomonas meridiana, Methylobacterium sp., Novosphingobium sp., and Paenibacillus pabuli. PCA and Venn diagrams showed that species composition is rarely fixed and often experiences fluctuations of varying degrees and at varying frequencies between different cocoa-producing regions. Understanding these differences will provide further directions for exploring the functional and metabolic activity of rare and abundant taxa, as well as their use as starter cultures to obtain high-quality cocoa beans.

Bacillus lipopeptides as powerful pest control agents for a more sustainable and healthy agriculture: recent studies and innovations.

MAIN CONCLUSION: Lipopeptides could help to overcome a large concern in agriculture: resistance against chemical pesticides. These molecules have activity against various phytopathogens and a potential to be transformed by genetic engineering. The exponential rise of pest resistances to different chemical pesticides and the global appeal of consumers for a sustainable agriculture and healthy nutrition have led to the search of new solutions for pest control. Furthermore, new laws require a different stance of producers. Based on that, bacteria of the genus Bacillus present a great agricultural potential, producing lipopeptides (LPs) that have high activity against insects, mites, nematodes, and/or phytopathogens that are harmful to plant cultures. Biopesticide activity can be found mainly in three families of Bacillus lipopeptides: surfactin, iturin, and fengycin. These molecules have an amphiphilic nature, interfering with biological membrane structures. Their antimicrobial properties include activity against bacteria, fungi, oomycetes, and viruses. Recent studies also highlight the ability of these compounds to stimulate defense mechanisms of plants and biofilm formation, which is a key factor for the successful colonization of biocontrol organisms. The use of molecular biology has also recently been researched for continuous advances and discoveries of new LPs, avoiding possible future problems of resistance against these molecules. As a consequence of the properties and possibilities of LPs, numerous studies and developments as well as the attention of large companies in the field is expected in the near future.

Current advances in gibberellic acid (GA3) production, patented technologies and potential applications.

MAIN CONCLUSION: Gibberellic acid is a plant growth hormone that promotes cell expansion and division. Studies have aimed at optimizing and reducing production costs, which could make its application economically viable for different cultivars. Gibberellins consist of a large family of plant growth hormones discovered in the 1930s, which are synthesized via the terpenes route from the geranylgeranyl diphosphate and feature a basic structure formed by an ent-gibberellane tetracyclic skeleton. Among them, only four have biological activity, including gibberellic acid (GA3), which acts as a natural plant growth regulator, especially for stem elongation, seed germination, and increased fruit size. It can be obtained from plants, fungi, and bacteria. There are also some reports about microalgae GA3 producers. Fungi, especially Gibberella fujikuroi, are preferred for GA3 production via submerged fermentation or solid-state fermentation. Many factors may affect its production, some of which are related to the control and scale-up of fermentation parameters. Different GA3 products are available on the market. They can be found in liquid or solid formulations containing only GA3 or a mixture of other biological active gibberellins, which can be applied on a wide variety of cultivars, including crops and fruits. However, the product's cost still limits its large and continuous application. New low-cost and efficient GA3 production alternatives are surely welcome. This review deals with the latest scientific and technological advances on production, recovery, formulation, and applications of this important plant growth hormone.

Impact of microbial growth inhibition and proteolytic activity on the stability of a new formulation containing a phytate-degrading enzyme obtained from mushroom.

The development of stable enzymes is a key issue in both the food and feed industries. Consequently, the aim of the current study is to evaluate the impact of various additives (sodium chloride, sodium citrate, mannitol, methylparaben, polyethylene glycol 3350, ethylenediaminetetraacetic acid disodium salt, and a serine protease inhibitor) on the stability of a mushroom phytase produced by solid-state cultivation and recovery. Also observed was the effect of the additives on microbial growth inhibition by monitoring both the change in optical density over 30 days of storage and proteolytic activity. Initially, eight experimental formulations were prepared along with a control. After screening, a 3(2) factorial design was applied to define suitable concentrations of the selected additives. Among the eight formulations tested, the formulation containing NaCl, PEG 3350, and methylparaben retained all of the initial phytase activity after 50 days of storage, with no detected interference from protease activity. Sodium citrate, a metal chelation agent, presented the unusual effect of reducing protease activity in the formulations. Although all formulations presented better phytase stability when compared to the control, NaCl and PEG were both able to prolong the stability of the enzyme activity and also to inhibit microbial growth during storage, making them favorable for application as food and feed additives.

Fumaric acid production by Rhizopus species from acid hydrolysate of oil palm empty fruit bunches.

The hemicellulosic fraction of lignocellulosic biomass is a very important material, due to the significant concentration of pentoses present in its composition and that can be used sustainably in biotechnological processes such as the production of fumaric acid. Research efforts are currently being promoted for the proper disposal and valorization of empty fruit bunches (EFB) from oil palm. In this work, seventeen Rhizopus species were evaluated in a fermentation medium with EFB hydrolyzate, without detoxification, as a carbon source for fumaric acid production. Rhizopus circicans 1475 and Rhizopus 3271 achieved productions of 5.65 g.L-1 and 5.25 g.L-1 of fumaric acid at 30 °C, 120 rpm for 96 h, respectively. The percentage of consumed sugars, mainly pentoses, was 24.88% and 34.02% for R. circicans 1475 and R 3271, respectively. Soy peptone and ammonium sulfate were evaluated as nitrogen sources, where soy peptone stimulated the formation of biomass pellets while ammonium sulfate produced mycelia and clamps.

Effect of forced aeration on citric acid production by Aspergillus sp. mutants in SSF.

Citric acid (CA) is one of the most important products of fermentation in the world. A great variety of agro-industrial residues can be used in solid state fermentation. Aspergillus niger parental strain (CCT 7716) and two strains obtained by mutagenesis (CCT 7717 and CCT 7718) were evaluated in Erlenmeyer flasks and glass columns using citric pulp (CP) as substrate/support, sugarcane molasses and methanol. Best results using glass columns (forced aeration) were found in the fourth day of fermentation: 278.4, 294.9 and 261.1 g CA/kg of dry CP with CCT 7716, CCT 7718 and CCT 7717, respectively. In Erlenmeyer flasks (aeration by diffusion) CA reached 410.7, 446.8 and 492.7 g CA/kg of dry CP with CCT 7716, CCT 7718 and CCT 7717, respectively. The aeration by diffusion improved CA production by the three strains. A data acquisition system specially developed for biotechnological processes analysis was used to perform the respirometric parameters measurement.

Gold-binding peptide as a selective layer for electrochemical detection of SARS-CoV-2 antibodies.

Electrochemical immunosensors are excellent alternatives to prepare portable platforms used for rapid and inexpensive diagnostic of infectious diseases such as the recently emerged COVID-19. Incorporating synthetic peptides as selective recognition layers combined with nanomaterials such as gold nanoparticles (AuNPs) can significantly enhance the analytical performance of immunosensors. In the present study, an electrochemical immunosensor based on solid-binding peptide was built and evaluated towards SARS-CoV-2 Anti-S antibodies detection. The peptide used as recognition site has two important portions: one based on the viral receptor binding domain (RBD), capable of recognizing antibodies of the spike protein (Anti-S), and another suitable for interacting with gold nanoparticles. Gold-binding peptide (Pept/AuNP) dispersion was used directly to modify a screen-printed carbon electrode (SPE). The voltammetric behavior of the [Fe(CN)6]3-/4- probe after every construction and detection step was recorded using cyclic voltammetry by assessing the stability of the Pept/AuNP as a recognition layer onto the electrode surface. Differential pulse voltammetry was used as a detection technique, and a linear working range from 75 ng mL-1 to 15 µg mL-1 was established, with 1.059 µA dec-1 of sensitivity and R2 = 0.984. The response selectivity against SARS-CoV-2 Anti-S antibodies was investigated in presence of concomitant species. The immunosensor was used to detect SARS-CoV-2 Anti-spike protein (Anti-S) antibodies in human serum samples, successfully differentiating between negative and positive responses of samples at a 95% confidence level. Therefore, the gold-binding peptide is a promising tool to be applied as a selective layer for antibody detection.

Biofiltration of gasoline and ethanol-amended gasoline vapors.

Assuming the projected increase in use of ethanol as a biofuel, the current study was conducted to compare the biofiltration efficiencies for plain and 25% ethanol-containing gasoline. Two biofilters were operated in a downflow mode for 7 months, one of them being compost-based whereas the other using a synthetic packing material, granulated tire rubber, inoculated with gasoline-degrading microorganisms. Inlet concentrations measured as total hydrocarbon (TH) ranged from 1.9 to 5.8 g m(-3) at a constant empty bed retention time of 6.84 min. Contrary to the expectations based on microbiological considerations, ethanol-amended gasoline was more readily biodegraded than plain hydrocarbons, with the respective steady state elimination capacities of 26-43 and 14-18 gTH m(-3) h(-1) for the compost biofilter. The efficiency of both biofilters significantly declined upon the application of higher loads of plain gasoline, yet immediately recovering when switched back to ethanol-blended gasoline. The unexpected effect of ethanol in promoting gasoline biodegradation was explained by increasing hydrocarbon partitioning into the aqueous phase, with mass transfer being rate limiting for the bulk of components. The tire rubber biofilter, after a long acclimation, surpassed the compost biofilter in performance, presumably due to the 'buffering' effect of this packing material increasing the accessibility of gasoline hydrocarbons to the biofilm. With improved substrate mass transfer, biodegradable hydrocarbons were removed in the tire rubber biofilter's first reactor stage, with most of the remaining poorly degradable smaller-size hydrocarbons being degraded in the second stage.

Molecular characterisation and biomass and metabolite production of Lactobacillus reuteri LPB P01-001: A potential probiotic.

Lactobacillus reuteri LPB P01-001 was isolated from the gastrointestinal tract of wild swine and was characterised by biochemical testing and sequencing of gene 16S rRNA. A simple and low-cost culture medium based on cane sugar (2.5% p/v) and yeast extract (1% p/v) was used in the production of this probiotic. The fermentative conditions were a) pH control at 6.5 and b) no pH control; both were set at 37°C in a 12 L slightly stirred tank bioreactor. Fermentation parameters such as the specific growth rate, productivity and yield of biomass, lactic and acetic acid levels were determined. L. reuteri LPB P01-001 behaves as an aciduric bacteria because it grows better in a low pH medium without pH control. However, the lactic acid production yield was practically half (9.22 g.L(-1)) of that obtained under a constant pH of 6.5, which reached 30.5 g.L(-1) after 28 hours of fermentation. The acetic acid production was also higher under pH-controlled fermentation, reaching 10.09 g.L(-1)after 28 hours of fermentation. These parameters may raise the interest of those committed to the efficient production of a probiotic agent for swine.

Graphene-Binding Peptide in Fusion with SARS-CoV-2 Antigen for Electrochemical Immunosensor Construction.

The development of immunosensors to detect antibodies or antigens has stood out in the face of traditional methods for diagnosing emerging diseases such as the one caused by the SARS-CoV-2 virus. The present study reports the construction of a simplified electrochemical immunosensor using a graphene-binding peptide applied as a recognition site to detect SARS-CoV-2 antibodies. A screen-printed electrode was used for sensor preparation by adding a solution of peptide and reduced graphene oxide (rGO). The peptide-rGO suspension was characterized by scanning electron microscopy (SEM), Raman spectroscopy, and Fourier transform infrared spectroscopy (FT-IR). The electrochemical characterization (electrochemical impedance spectroscopy-EIS, cyclic voltammetry-CV and differential pulse voltammetry-DPV) was performed on the modified electrode. The immunosensor response is based on the decrease in the faradaic signal of an electrochemical probe resulting from immunocomplex formation. Using the best set of experimental conditions, the analytic curve obtained showed a good linear regression (r2 = 0.913) and a limit of detection (LOD) of 0.77 µg mL-1 for antibody detection. The CV and EIS results proved the efficiency of device assembly. The high selectivity of the platform, which can be attributed to the peptide, was demonstrated by the decrease in the current percentage for samples with antibody against the SARS-CoV-2 S protein and the increase in the other antibodies tested. Additionally, the DPV measurements showed a clearly distinguishable response in assays against human serum samples, with sera with a response above 95% being considered negative, whereas responses below this value were considered positive. The diagnostic platform developed with specific peptides is promising and has the potential for application in the diagnosis of other infections that lead to high antibody titers.

Improving Cry8Ka toxin activity towards the cotton boll weevil (Anthonomus grandis).

BACKGROUND: The cotton boll weevil (Anthonomus grandis) is a serious insect-pest in the Americas, particularly in Brazil. The use of chemical or biological insect control is not effective against the cotton boll weevil because of its endophytic life style. Therefore, the use of biotechnological tools to produce insect-resistant transgenic plants represents an important strategy to reduce the damage to cotton plants caused by the boll weevil. The present study focuses on the identification of novel molecules that show improved toxicity against the cotton boll weevil. In vitro directed molecular evolution through DNA shuffling and phage display screening was applied to enhance the insecticidal activity of variants of the Cry8Ka1 protein of Bacillus thuringiensis. RESULTS: Bioassays carried out with A. grandis larvae revealed that the LC50 of the screened mutant Cry8Ka5 toxin was 3.15-fold higher than the wild-type Cry8Ka1 toxin. Homology modelling of Cry8Ka1 and the Cry8Ka5 mutant suggested that both proteins retained the typical three-domain Cry family structure. The mutated residues were located mostly in loops and appeared unlikely to interfere with molecular stability. CONCLUSIONS: The improved toxicity of the Cry8Ka5 mutant obtained in this study will allow the generation of a transgenic cotton event with improved potential to control A. grandis.

Use of soybean vinasses as a germinant medium for a Geobacillus stearothermophilus ATCC 7953 sterilization biological indicator.

A novel low-cost medium was developed from by-products and wastes from the ethanol agro-industry to replace commercial media in the production of a steam sterilization biological indicator (BI). Various recovery media were developed using soybean or sugarcane molasses and vinasse to prepare a self-contained BI. Media performance was evaluated by viability and heat resistance (D(121 °C) value) according to regulatory standards. A medium produced with a soybean vinasse ratio of 1:70 (1.4%) (w/v) produced the results, with D(121 °C)=2.9±0.5 min and Usk=12.7±2.1 min. The addition of 0.8% (w/v) yeast extract improved the germination of heat-damaged spores. The pH variation from 6.0 to 7.3 resulted in a gradual increase in the D(121 °C) value. The absence of calcium chloride resulted in a decrease in germination, while no significant differences were observed with starch addition. Soybean vinasses may thus be used as the main component of a culture medium to substitute for commercial media in the production of self-contained biological indicators. The use of ethanol production waste in this biotechnological process realized a reliable performance, minimized the environmental impact, and decreased BI production costs while producing a high quality product.

Designing enzyme cocktails from Penicillium and Aspergillus species for the enhanced saccharification of agro-industrial wastes.

The aim of this study was to develop optimized enzyme cocktails, containing native and recombinant purified enzymes from five fungal species, for the saccharification of alkali- and acid-pretreated sugarcane bagasse (SCB), soybean hulls (SBH) and oil palm empty fruit bunches (EFB). Basic cellulases were represented by cellobiohydrolase I (CBH) and endo-glucanase II (EG) from Penicillium verruculosum and ß-glucosidase (BG) from Aspergillus niger. Auxiliary enzymes were represented by endo-xylanase A (Xyl), pectin lyase (PNL) and arabinoxylanhydrolase (AXH) from Penicillium canescens, ß-xylosidase (BX) from Aspergillus japonicus, endo-arabinase (ABN) from A. niger and arabinofuranosidase (Abf) from Aspergillus foetidus. Enzyme loads were 5 mg protein/g dry substrate (basic cellulases) and 1 mg/g (each auxiliary enzyme). The best choice for SCB and EFB saccharification was alkaline pretreatment and addition of Xyl + BX, AXH + BX or ABN + BX + Abf to basic cellulases. For SBH, acid pretreatment and basic cellulases combined with ABN + BX + Abf or Xyl + BX performed better than other enzyme preparations.

A review on enzyme-producing lactobacilli associated with the human digestive process: From metabolism to application.

Complex carbohydrates, proteins, and other food components require a longer digestion process to be absorbed by the lining of the alimentary canal. In addition to the enzymes of the gastrointestinal tract, gut microbiota, comprising a large range of bacteria and fungi, has complementary action on the production of digestive enzymes. Within this universe of "hidden soldiers", lactobacilli are extensively studied because of their ability to produce lactase, proteases, peptidases, fructanases, amylases, bile salt hydrolases, phytases, and esterases. The administration of living lactobacilli cells has been shown to increase nutrient digestibility. However, it is still little known how these microbial-derived enzymes act in the human body. Enzyme secretion may be affected by variations in temperature, pH, and other extreme conditions faced by the bacterial cells in the human body. Besides, lactobacilli administration cannot itself be considered the only factor interfering with enzyme secretion, human diet (microbial substrate) being determinant in their metabolism. This review highlights the potential of lactobacilli to release functional enzymes associated with the digestive process and how this complex metabolism can be explored to contribute to the human diet. Enzymatic activity of lactobacilli is exerted in a strain-dependent manner, i.e., within the same lactobacilli species, there are different enzyme contents, leading to a large variety of enzymatic activities. Thus, we report current methods to select the most promising lactobacilli strains as sources of bioactive enzymes. Finally, a patent landscape and commercial products are described to provide the state of art of the transfer of knowledge from the scientific sphere to the industrial application.

Alternative methods for gibberellic acid production, recovery and formulation: A case study for product cost reduction.

The aim of this work was to develop a new production, recovery and formulation process of gibberellic acid (GA3). Low-cost byproducts - citrus pulp (CP) and soybean hulls (SH) - were employed as substrate for GA3 production by Gibberella fujikuroi in semisolid fermentation. A CP/SH mixture (70%/30%) promoted high productivities both in bubble column reactor (1.66 mg L/h), and in stirred tank reactor (2.13 mg L/h). GA3 production medium cost (US$ 6.70/m3) was reduced by 85% when compared to previously reported synthetic media (US$ 44.96/m3). It was described that GA3 fermented extract has low stability, and that liquid and powder formulation of the fermented extract maintained the biomolecule activity over 6 months. Alginate and alginate/kefiran beads containing GA3 showed encapsulation efficiency of 70% and 60%, respectively. This work supports good perspectives for GA3 production using cheap substrates and simple formulation of clarified extract to favour its use in agricultural countries.

Update and Revalidation of Ghose's Cellulase Assay Methodology.

New studies on cellulolytic enzymes aiming to improve biofuels production lead to a concern over the assaying methods commonly applied to measure their activity. One of the most used methods is Ghose's cellulase and endoglucanase assay, developed by the International Union of Pure and Applied Chemistry in 1987. Carrying out this method demands high volumes of reagents and generation of high amounts of chemical residues. This work aimed to adapt Ghose's methodology to reduce its application cost and residue generation and validate the adjustments. To do so, International and Brazilian laws were applied to validate methodologies. Method's modifications were successfully validated according to all institutions and were considered linear, accurate, precise, and reproducible. It was possible to reduce the volume of reagents and residues in 12 times. Considering the routine work of most laboratories, it is a great reduction on material costs and residue treatment, which reflects in sustainability and environmental impacts.